airesearch/WangchanThaiMedical · Datasets at Hugging Face

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Introduction {#Sec1} ============ Skeletal muscle tissue represent about 40% of total body weight and is responsible for almost all voluntary movements. It has a high regenerative capacity after injury, which is directly linked to the presence of satellite cells (SCs). These cells are the main muscle stem cells and have a key role in muscle development during embryogenesis^[@CR1]--[@CR3]^. SCs reside between the basal lamina and the muscle membrane^[@CR4]^ and although quiescent in normal adult muscles, SCs can be activated by specific signals upon muscle injury. In diseases characterized by chronic degeneration process, such as muscular dystrophies, SCs are constantly activated, leading to the depletion of the SCs pool and consequent failure of the regenerative process^[@CR5]^. Muscular dystrophies are a heterogeneous group of genetic diseases that cause a progressive loss of motor ability. Defects in components of the dystrophin-glycoprotein complex (DGC) are responsible for different forms of muscular dystrophies. The DGC links the myofiber cytoskeleton proteins and the extracellular matrix, providing mechanical support to sarcolemma during myofiber contraction^[@CR6],[@CR7]^. Mutations in the dystrophin (DMD) gene can lead to Duchenne/Becker muscular dystrophies^[@CR8]^. DGC proteins are linked to extracellular matrix proteins by post-translational glycosylation of α-dystroglycan (α-DG) protein. Mutations in the LARGE1 gene, which encodes a glycosyltransferase that acts in the α-DG, lead to congenital muscular dystrophy type 1D^[@CR9]--[@CR11]^. Identified in nature or generated in laboratory, animal models are an important tool to study neuromuscular disorders. These models generally present physiological alterations observed in human patients and can be used for pathophysiological studies and therapy testing^[@CR12]^. A good genetic and biochemical model for Duchenne muscular dystrophy is the Dmd^mdx^ mouse that bears a nonsense point mutation in exon 23 of the Dmd gene, which causes lack of this protein in skeletal muscle. Regardless of total protein deficiency, this mouse shows a mild phenotype, with comparatively moderate muscle pathology. Muscle histopathology analysis shows a great number of regenerating fibers, variation in fiber size and the presence of central nuclei. Differently from human Duchenne muscular dystrophy patients, muscle degeneration in Dmd^mdx^ mouse is followed by a significant regeneration^[@CR13],[@CR14]^. A model for Congenital muscular dystrophy type 1D is the myodystrophy Large^myd^ mouse, which harbors a mutation in the Large1 gene. Large^myd^ mutant mice develop a progressive severe myopathy, with a maximum lifespan of 39 weeks. Muscle histopathology includes degeneration, loss of striation, variation in fiber size and the presence of central nuclei^[@CR15]^. With the goal of obtaining an animal model for DMD with a severe phenotype, more similar to affected DMD patients, and to better understand the interplay between the DMD protein and LARGE1 glycosyltransferase in muscle function, we generated double-mutant animals deficient in these two proteins^[@CR16]^. The new Dmd^mdx^/Large^myd^ mouse model has a severe phenotype with delayed growth and development, and is unable to reproduce. In addition to the important pathophysiological aspects of these two mutations in muscle formation and function, this model has an important application for testing therapies, integrating functional, molecular and protein studies^[@CR16]^. Aiming to understand the behavior of SCs in dystrophic muscles with different degrees of muscle degeneration, we evaluated histological parameters, as well as gene and protein expression of transcription factors related to SCs, in gastrocnemius muscle of adult male mice with different forms of muscular dystrophies. The results were compared regarding regenerating and degenerating features. Our findings indicate that the pool of SCs of the studied dystrophic strains maintains its proliferative capacity and these cells can contribute to muscle regeneration and new fiber formation. Nevertheless, the maturation of these new fibers is incomplete and do not prevent muscle degeneration. Results {#Sec2} ======= Dystrophic muscles retain their SCs pool {#Sec3} ---------------------------------------- The expression of the transcription factor Pax7, which is related to the maintenance of SCs pool, was measured to investigate a potential reduction in the SCs pool in the dystrophic mice models as compared to wild-type mice. Statistical analysis with Kruskal-Wallis test revealed similar expression of Pax7 in the three dystrophic strains as compared to wild-type mice (Chi-square = 1.05, df = 3, p = 0.790), independently of the degree of muscle degeneration (Fig. [1a](#Fig1){ref-type="fig"}). Protein analysis by Western blot also showed similar expression in the dystrophic strains as compared to wild-type animals (Fig. [1b,c](#Fig1){ref-type="fig"}).Figure 1PAX7 Expression in dystrophic muscles: (a) Boxplot of mRNA relative Pax7 expression and (b) PAX7 protein western blot analysis (full blots figures without contrast adjustments are presented at Supplementary Material). N = 6 animals per strain. (c) Boxplot of densitometric quantification. No significant differences in PAX7 were found between WT and the three dystrophic strains using both approaches (Kruskal-Wallis test, Chi-square = 1.05, df = 3, p = 0.790). \Shows outlier measures. Immunofluorescence analysis using PAX7 antibody in gastrocnemius muscle sections of dystrophic versus wild-type animals showed an apparently higher number of PAX7 positive nuclei per fibers in the three dystrophic models (Fig. [2a](#Fig2){ref-type="fig"}). However, no statistical differences were found among the four groups (p = 0.083, Fig. [2b](#Fig2){ref-type="fig"}). Thus, SCs pool is maintained in spite of the different degrees of degeneration in the dystrophic mouse models.Figure 2Representative images of PAX7-positive SCs in the four mouse strains, magnification 200 × (full immunofluorescence figures are represented at Supplementary Material). (a) Immunofluorescence analysis showing PAX7-positive and DAPI-positive cells in each strain, \~3000 fibers were counted for each animal. White arrows are indicating SCs, purple labelling. (b) Proportion of SCs per fibers in the four strains. No statistical significance was found among wild-type and the three dystrophic strains. N = 4 animals per strain (One-way ANOVA, F(3, 11) = 2.9, p = 0.084). Number of activated satellite cells is elevated in the dystrophic mice, compatible with active regeneration and pool maintenance {#Sec4} -------------------------------------------------------------------------------------------------------------------------------- Using PAX7 and KI67 antibodies, we identified and counted SCs that remained quiescent and the ones that were activated. While PAX7 staining shows SCs population, KI67 staining is a marker of proliferating cells. Therefore, the double staining for PAX7 and KI67 shows activated SCs^[@CR17]^ (Fig. [3a](#Fig3){ref-type="fig"}). A higher number of activated SCs were found in the three dystrophic models, when compared with wild-type, but no differences among the dystrophic strains (Kruskal-Wallis test, Chi-square* = 30.248, df = 3, p \< 0.001). Dystrophic animals showed the following proportions of activated SCs: DMD^mdx^ 17.4%, Large^myd^ 25.1% and DMD^mdx^/Large^myd^ 16,.7% (Fig. [3b](#Fig3){ref-type="fig"}).Figure 3Representative images of PAX7 and KI67 positive SCs, magnification 200X (full immunofluorescence figures are represented at Supplementary Material). (a) Identification of quiescent and activated SCs, using as example the Large^myd^ model. On the merged figure, red arrows indicate quiescent SCs (DAPI+ and PAX7+); green arrows indicate activated SCs (DAPI+, PAX7+ and KI67+). (b) Percentage of activated SCs in wild-type and dystrophic models, N = 200 PAX7+ SCs per strain. Statistical difference was observed between the wild type and each one of three dystrophic models (Kruskal-Wallis test, Chi-square = 30.248, df = 3, p \< 0.001, ^\#\#^p \< 0.01, ^\#\#\#^p \< 0.001). Myogenic regulatory genes are activated in the dystrophic muscle {#Sec5} ---------------------------------------------------------------- We also investigated the relative expression of Myf5, Myod, and Myog, which are important genes of the myogenic program. No significant differences were found among the three dystrophic models and the wild-type regarding the expression of MyoD (Kruskal-Wallis Test - Chi-square = 3.22, df = 3, p = 0.359), nor Myf5 (Chi-square = 3.21, df = 3, p = 0.361). In spite of that, we observed a significant elevation (Chi-square = 17.93, df = 3, p \< 0.001) in Myog expression among the four mice models. Using the post hoc analysis with Bonferroni method of correction, a significant difference on Myog expression was found between wild-type and Large^myd^ (p \< 0.001), wild-type and DMD^mdx^/Large^myd^ (p = 0.0023), and wild-type and DMD^mdx^ (p = 0.0067). No significant differences were found between the dystrophic strains (Fig. [4a](#Fig4){ref-type="fig"}). High Myog expression was confirmed by analysis of protein levels (Fig. [4b,c](#Fig4){ref-type="fig"}).Figure 4Myogenic genes analysis in dystrophic models as compared to wild-type mice: (a) mRNA relative expression of Myf5, MyoD, Myog; Significant differences were observed for the Myog (Kruskal-Wallis test, Chi-square = 17.93, df = 3, p \< 0.001) \Shows outliers animals. (b) MYOG western blot protein analysis (full blots figures without contrast adjustments are represented in Supplementary Material). N = 6 animals per strain, (c) Densitometric analysis of MYOG protein results. Significant differences were observed for the Myog gene ^\#^p \< 0.05 and ^\#\#^p \< 0.01 (Kruskal-Wallis test, Chi-square = 15.20, df = 3, p = 0.002), both at mRNA and protein levels. As data presented great variance in the expression of the myogenic factors, the non-parametric Levene's test was done to verify the equality of variances in samples. We observed that they were not significantly different (Myod, p* = 0.997; Myf5, p = 0.973; Myog, p = 0.190; Pax7, p = 0.890). Dystrophic muscle retains the ability to form new fibers {#Sec6} -------------------------------------------------------- The number and diameter of new muscle fibers were evaluated by detection of developmental myosin heavy chain isoform (dMyHC) (Fig. [5a,b](#Fig5){ref-type="fig"}). Immunofluorescence analysis showed no dMyHC+ fibers in wild-type muscles, while Dmd^mdx^ strain presented the greatest number of dMyHC+ fibers (17%). Large^myd^ and Dmd^mdx^/Large^myd^ showed respectively 7% and 10% of new fibers (Kruskal-Wallis Test, Chi-square = 13.87, df = 3, p = 0.003, Fig. [5c](#Fig5){ref-type="fig"}), which is compatible with an intense regeneration in all dystrophic strains. Interestingly, compared to normal mature fibers size (52 µm+/−10 µm), the diameter of these new fibers was smaller, with a mean of 33 µm ± 13.5 µm in Dmd^mdx^, 30 µm ± 12.1 µm in Large^myd^ and 20 µm ± 9.0 µm in Dmd^mdx^/Large^myd^ (Fig. [5d](#Fig5){ref-type="fig"}), with differences among the dystrophic strains (Kruskal-Wallis test, Chi-square = 53.649, df = 3, p \< 0.001). In addition, the size of these new dMyHC+ fibers was more homogeneous, as shown by the coefficient of variation within normal values (lower than 250) in Dmd^mdx^ (150), Large^myd^ (245) and Dmd^mdx^/Large^myd^ (155). These results suggest that in the dystrophic models, these new fibers remain small and do not complete the regenerative process.Figure 5Representative images of dMyHC+ positive new regenerated fibers, normal and dystrophic muscles (full immunofluorescence figures are represented at Supplementary Material). dMyHC+ fibers are labeled in red/orange. Magnification 200X. (a) Immunofluorescence staining for dMyHC+ in normal WT muscle at time zero, 5 days after induced injury and after 60 days of induced injury, N = 6 animal per time. (b) dMyHC+ fibers in the 3 dystrophic models. N = 6 animals per strain (c) percentage of dMyHC+ fibers related to total fiber number (n = \~1500 fibers/animal). (d) dMyHC+ fiber diameter. Significant differences: ^\#^p \< 0.05 and ^\#\#\#^p \< 0.001 (Kruskal-Wallis test, Chi-square = 53.649, df = 3, p \< 0.001). To confirm our results, the muscles from the different dystrophic strains were compared to regenerating normal muscle subjected to acute lesion by electroporation, at days zero, five, and 60 days post-injury (Fig. [5a](#Fig5){ref-type="fig"}). In wild-type animals, regenerating dMyHC+ fibers appeared five days post-injury and presented variable diameter. The coefficient of variation was very high (484), demonstrating the diversity in fiber diameter. After 60 days, no regenerating fiber was identified, and fiber diameter was more homogeneous^[@CR18]^. Muscle degeneration is persistent in spite of regeneration {#Sec7} ---------------------------------------------------------- The regeneration outcome was evaluated through the quantification of connective tissue substitution, using Sirius red staining (Fig. [6a](#Fig6){ref-type="fig"}). We observed more connective tissue infiltration in dystrophic mice than in wild-type mice (Kruskal-Wallis Test, Chi-square = 81.21, df = 3, p \< 0.001). The differences among the three dystrophic strains were also statistically significant (Kruskal-Wallis Test -- Chi-square = 30.31, df = 2, p \< 0.001), with a higher proportion in the more severely affected muscles: Dmd^mdx^/Large^myd^ \> Large^myd^ \> Dmd^md.^ (Fig. [6b](#Fig6){ref-type="fig"}).Figure 6Connective tissue replacement in muscle degeneration (full Sirius red images are represented at Supplementary Material): (a) Representative images of collagen deposition by Sirius red staining of gastrocnemius muscles of WT, Dmd^mdx^, Large^myd^ and Dmd^mdx^/Large^myd^ mice, N = 6 animals per strain, magnification of 100X. (b) Quantification of percentage area of collagen deposition. Significant differences relative to WT muscle: ^\#\#^p \< 0.01 (Kruskal-Wallis test, Chi-square = 30.31, df = 2, p \< 0.001). Histological analysis of muscles by H&E staining also showed a gradual increase in muscle degeneration and variation in fiber size (Fig. [7a](#Fig7){ref-type="fig"}). An increase in the variation coefficient of fiber size in dystrophic strains was observed (normal: up to 250), in agreement with the degree of muscle affection: 635 in Dmd^mdx^/Large^myd^ \> 444 in Large^myd^ \> 420 in Dmd^mdx^ \> 205 in wild-type (Fig. [7b](#Fig7){ref-type="fig"}).Figure 7Histopathological alterations in the three dystrophic strains and wild-type (full H&E figures, without contrast adjustments, are represented at Supplementary Material). (a) HE staining, magnification 200X. N = 6 animals per strain. (b) Variation coefficient of muscle fiber size (\>250 is considered pathogenic)^[@CR37]^. Discussion {#Sec8} ========== The reservoir of muscle precursor cells, the SCs, is responsible for after-birth growth and muscle regeneration as a response to injuries, either by exercise or disease. Since the first description in 1961 by Mauro^[@CR4]^, SCs have been the target of a huge number of studies, aiming to understand their behavior and action. SCs are able to self-renew and replenish the stem cell pool, and this role is primordial for muscle regeneration. The regenerative response after muscle injury is marked by the exit of SCs from quiescence and their proliferation and fusion, leading to myofiber repair or new myofiber formation^[@CR19]^. At the same time, there is the activation of an inflammatory response, with the infiltration of immune cells, mainly macrophages^[@CR19]^. The dystrophic muscle is persistently injured due to a specific primary mutation in genes encoding for important muscle proteins, and the regenerative process is incessantly activated, recruiting SCs at higher rates than in normal tissue. Nevertheless, progressive replacement of muscle by fibrofatty tissue in dystrophic muscle has been considered as a factor that impairs the complete regeneration process^[@CR20]--[@CR22]^. Many studies have suggested that the regenerative capacity of SCs would be finite, and exhaustion of the pool of these cells has been considered a significant factor that contributes to progressive muscle deterioration in muscular dystrophies, as studied in DMD^[@CR5],[@CR23]^ and in mdx/utrophin^−/−^ mice^[@CR24]^. SCs repair potential, therefore, would not be sufficient to compensate for the persistent degeneration process^[@CR25]^. A recent study showed that deficiency of DMD protein could lead SCs to divide abnormally, resulting in the depletion of myogenic progenitors. In the long term, this condition can impair muscle regeneration^[@CR26]^. In contrast, several studies have shown an increased number of SCs in human and mice dystrophic muscles^[@CR20]--[@CR22],[@CR27]^. The dystrophic niche seems also to be crucial, since removing SCs from a dystrophic milieu revealed that their regenerative capacity remains both intact and similar to SCs derived from healthy muscle^[@CR28],[@CR29]^. Moreover, as observed in dystroglycanopathies, muscle fibrosis increases laminin disorganization, and this alteration in extracellular matrix composition is suggested to impair SCs function and activity^[@CR30]^. To elucidate the players and mechanisms involved in SCs function is of extreme importance, especially for therapeutic strategies. Here, we studied SCs protein and gene expression in different forms of murine muscular dystrophies, with a specific pattern of muscle histopathology, to correlate the impact of muscle degeneration in the population of SCs in these different microenvironments. The expression of the transcription factor PAX7 by SCs has been correlated with the maintenance of these cells in the undifferentiated state, being an important factor for SCs self-renewal that can be expressed both by quiescent and by early activated SCs^[@CR31]^. We studied Pax7 mRNA expression in the muscle of dystrophic mouse models, as compared to wild-type, to determine if there was a reduction in SCs population with the dystrophic process. We also identified and counted PAX7-positive SCs in muscle sections and verified the number of activated cells among them in each mouse model. Our analyses, through mRNA expression, protein quantification, and immunohistochemical evaluation showed similar results in all strains, suggesting the maintenance of a normal pool of SCs despite the active but diverse process of regeneration and degeneration that occurs in the Dmd^mdx^, Large^myd^ and Dmd^mdx^/Large^myd^. These data are in accordance with the hypothesis of the maintenance/increased number of SCs with the dystrophic process^[@CR16],[@CR18],[@CR24]^. The importance of the niche for the regenerative capacity of SCs is well documented in the Large^myd^ mouse, in which SCs were present in a significantly higher number, but with reduced proliferation capacity when in native muscle fibers. However, when removed from their niche, their proliferative capacity in culture was restored^[@CR30]^. This could also be observed in our models, with activated SCs present in the Dmd^mdx^, Large^myd^ and Dmd^mdx^/Large^myd^ in a similar proportion, and higher than in wild-type. This self-renewal capacity of SCs, even in the dystrophic muscle, is extremely important for the replenishment of the stem cell pool, mainly in an attempt to regenerate^[@CR32]^. The molecular players of regeneration are the muscle regulatory factors, including MYF5 and MYOD, responsible for muscle-cell type determination and SCs activation, and MYOG, responsible for muscle differentiation^[@CR33],[@CR34]^. Thus, we measured the relative expression of these genes in our three different dystrophic models. At the mRNA level, we observed similar relative expression of Myf5 and Myod in all strains and higher expression of Myog in the three dystrophic strains as compared to wild-type, suggesting activation of a later stage of muscle regeneration in all of them, independently of the muscle degeneration degree. MYF5 is expressed in both activated and quiescent SCs, but quiescent cells can maintain their quiescence state using a post-transcriptional regulation by miR-31, which sequester Myf5 mRNA in messenger ribonucleoprotein (mRNP) granules. Upon activation, miR-31 levels decrease and Myf5 mRNA is released to translation^[@CR35]^. Therefore, this could explain the similar relative quantification of Myf5 mRNA in all strains. MYOD can be expressed both in first steps of SCs differentiation and by myocytes in the final steps of differentiation^[@CR1],[@CR33],[@CR36]^. Upregulation of MYOD expression leads SCs to exit the cell cycle and to differentiate and fuse, forming myotubes^[@CR33]^. In spite of the normal expression of MYOD in the muscle of these three dystrophic models, they showed regenerating fibers as observed by dMyHC staining (Fig. [5](#Fig5){ref-type="fig"}). This data confirms the maintenance of SCs capacity to follow a myogenic program in the dystrophic muscle and to be able to form new fibers, as already observed in Dmd^mdx\ 28^. In the final step of myogenesis, myogenin expression has a key role in the differentiation of SCs into myotubes. High expression of myogenin initiates myoblast maturation to myocytes, myocytes fusion and myotube formation, a process extremely active in dystrophic muscle^[@CR2],[@CR33]^. We studied MYOG expression in the dystrophic strains to investigate whether myotube formation is impaired in these models. Gene expression analysis showed a significant increase of Myog expression as well as protein expression in the three dystrophic strains, suggesting the occurrence of myoblast maturation to myocytes, fusion induction, and myotube formation in all the dystrophic muscles^[@CR37]^. Newly regenerated muscle fibers have particular characteristics such as central myonuclei, a basophilic pattern on H&E staining, and expression of developmental myosin heavy chain (dMyHC) isoform^[@CR38]^. In our analysis of regenerating fibers expressing dMyHC, we observed a significant number of new fibers in all the dystrophic strains, including the most severely affected double mutant Dmd^mdx^/Large^myd^. The Dmd^mdx^ strain showed the highest number of regenerating fibers, which is compatible with its pronounced regenerative capacity and milder dystrophic phenotype. Large^myd^ and Dmd^mdx^/Large^myd^ strains also presented a significant amount of regenerating fibers in their muscle, in spite of the more severe dystrophic phenotype, suggesting that the formation of new fibers is possible even when the muscle is already significantly degenerated. Contrarily, all dystrophic strains showed, on average, thinner regenerating fibers, especially the double mutant Dmd^mdx^/Large^myd^, as compared to the other strains (Fig. [5d](#Fig5){ref-type="fig"}). These results suggest that in a harsher dystrophic microenvironment, the complete regeneration can be compromised. In addition, the fiber size variation coefficient^[@CR38]^ showed small variation, which suggests the maintenance of these regenerating fibers in an immature state with small diameter, not progressing to a normal mature fiber size. The possibility of fiber branching or splitting fibers cannot be totally ruled out, since it is an alteration commonly found in dystrophic muscles, happening both in degeneration and regeneration conditions. Branched fibers were well described mainly in old fast-twitch EDL muscle approaching the end stage of the dystrophic muscle disease due to dystrophin deficiency^[@CR39]^. During pathological conditions, splitting or branching of large fibers can lead to the formation of many small fibers, increasing fiber diameter variability. The regenerative process can also originate small fibers, but here, we confirmed their immaturity through dMyHC expression. Next, we compared these data with the regeneration process of normal muscle under induced acute lesion by electroporation. At five days post-injury, an intense regeneration was observed with the presence of a high number of dMyHC+ muscle fibers. These new normal fibers presented more variable diameter, leading to a higher variation coefficient. This occurs because these fibers are probably ongoing a normal process of regeneration, reaching an increased size 60 days post-lesion^[@CR18]^. Taken together, these results suggest that the three dystrophic models retain their regenerative capacity, including the maintenance of SCs pool, the proliferation capacity, differentiation marker expression and finally, the formation of new fibers. However, this regenerative process is incomplete, with a possible defect in the maturation characteristics that could be determinant to the dysfunction of these fibers. Remarkably, very interesting data, which are in accordance with our proposal, has been shown by Pipalia et al.^[@CR40]^ in a model of wound repair in larval zebrafish. Two different populations of Pax7+ stem cells with different roles were observed: one, expressing Pax7a, which only initiates fiber formation, and the second, formed by Pax7b cells, which contribute to fiber growth. These two myoblast populations suggest a novel cellular complexity in muscle repair, with the possibility of the presence of distinct populations of myogenic cells contributing differentially to the repair also in other vertebrates. The dystrophic process could alter more specifically the second cell type, associated with fiber growth post-regeneration, and explain the presence of smaller regenerating fibers in the mouse models for muscular dystrophies. As to the degenerative process, we studied histopathological alterations through both connective tissue replacement and variation in fiber size, as a correlation with their regenerative potential. Variability in the coefficient of muscle fiber size \>250 is considered pathogenic^[@CR38]^, and this was observed in all dystrophic strains. We also observed more connective tissue infiltration in dystrophic mice, with a crescent increase in muscles with more weakness, such as Large^myd^ and Dmd^mdx^/Large^myd^. Thus, we conclude that the active regeneration in the dystrophic muscle is not indicative of effective improvement of muscle health, but an insufficient attempt to restore muscle fibers. In conclusion, our findings suggest that dystrophic muscles, independently of the degree of degeneration caused by the primary gene defect, retain the pool of SCs with proliferating capacity and are ready to respond to regenerating stimuli. The potential to repair is preserved by the upregulation of myogenic factors like MYOG and by the formation of new fibers expressing dMyHC. Nonetheless, the maturation of these new fibers is incomplete, since these fibers remain small and do not prevent muscle degeneration, as observed by the intense substitution by connective tissue. In this sense, efforts to improve late muscle regeneration should better contribute to therapeutic approaches. Methods {#Sec9} ======= Animals {#Sec10} ------- Histological, immunofluorescence, gene expression and protein analyses, were examined in the gastrocnemius muscle of adult male mice with 12--14 weeks of age. This muscle has about 51.5% of fiber type IID, 22.7% of fiber type IIA, 18.2% fiber type IIB and 7.6% of fiber type I^[@CR41]^. The studied strains were: DMD^mdx−^ (n = 6), Large^myd^ (n = 6), C57Bl/6 (n = 6) and double mutant DMD^mdx^/Large^myd^ (n = 6, three males and three females). During the experiments, animals were bred and housed in controlled temperature and light in the animal house of the Human Genome and Stem Cell Research Center (HUG-CELL). All experimental protocols and methodologies were performed in accordance with and were approved by CEUA - COMISSÃO DE ÉTICA NO USO DE ANIMAIS (Ethical committee of the use of animal in experiments - <http://www.ib.usp.br/comissoesib/ceua/>), of the Institute of Biosciences, University of Sao Paulo (protocol number 246/2016), which is the licensing committee of our Institution. In addition, the used methods were carried out in accordance with the relevant guidelines and regulations from the Brazilian National Council for Control of Animal Experimentation (Conselho Nacional de Controle de Experimentação Animal -- CONCEA). Tissue collection {#Sec11} ----------------- After euthanasia, the gastrocnemii muscles were collected and frozen in liquid nitrogen to study gene expression, histopathology, immunofluorescence,and protein quantification. RNA extraction, cDNA synthesis, and qRT-PCR {#Sec12} ------------------------------------------- RNA extraction from frozen muscles was performed using the RNeasy Microarray Tissue Mini Kit (Qiagen). RNA was solubilized in RNase-free water (Ambion™) and quantified in a Nanodrop spectrophotometer (Thermo Fisher Scientific). The cDNA synthesis was performed using 1 µg of total RNA according to the M-MLV protocol (Invitrogen). Samples were amplified in triplicates using the MasterMix containing Sybr Green (Applied Biosystems) and primers (Table [1](#Tab1){ref-type="table"}) on a thermocycler 7500 Fast (Applied Biosystems). The fold-change was obtained by the 2^−ΔΔCT^ method, with Tbp gene as normalizer.Table 1Primers sequences.GeneForward sequenceReverse sequencePax75′ GAGTTCGATTAGCCGAGTGC 3′5′ GAGTTCGATTAGCCGAGTGC 3′Myf55′ GAAGGTCAACCAAGCTTTCG 3′5′ GCTCTCAATGTAGCGGATGG 3′MyoD5′ TACAGTGGCGACTCAGATGC 3′5′ TAGGCGGTGTCGTAGCC 3′Myog5′ CTGCACTCCCTTACGTCCAT 3′5′ CCCAGCCTGACAGACAATCT 3′Tbp5′ TGCACAGGAGCCAAGAGTGAA 3′5′ CACATCACAGCTCCCCACCA 3′ Protein analysis {#Sec13} ---------------- Frozen muscle samples were homogenized in RIPA buffer (Sigma-Aldrich) supplemented with 1% of protease inhibitors (Sigma-Aldrich). Samples were kept on ice for 30 minutes and afterward centrifuged at 16.000 g for 20 min at 4 °C, followed by protein quantification using the Pierce BCA Assay kit (Thermo Scientific). Homogenized samples with 30 µg of protein were separated by SDS-PAGE in 4--15% Mini-PROTEAN® TGX™ Gels (BioRad) and transferred to nitrocellulose membranes using iBlot® Gel Transfer Device (Invitrogen) for 7 minutes. The obtained membranes were probed using the antibodies: PAX7 (1:1000 Novus Biologicals; NBP2-34706) and Myogenin (Abcam ab15232)^[@CR42]^. The membranes were then incubated with the appropriated secondary horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies (Thermo Fisher Scientific) and revealed using the Novex™ ECL Chemiluminescent Substrate Reagent kit (Invitrogen). Images were obtained using photo-documenter (ImageQuant TL, GE Healthcare Life Sciences), all images were collected after 450 seconds of standard exposure. For the reaction with the endogenous control, membranes were stripped with a stripping buffer (Thermo Fisher Scientific) and reprobed with GAPDH antibody (1:1000 Cell Signaling 2118)^[@CR43]^. Bands were quantified by densitometry using the software ImageQuant TL (GE Healthcare Life Sciences). The protein of interest and the endogenous protein were probed and analyzed in the same membrane, both for experimental and control samples. Histology and immunofluorescence analyses {#Sec14} ----------------------------------------- ### Hematoxylin-eosin (H&E) {#Sec15} Transverse cross sections of the right gastrocnemius were stained with H&E and histopathological evaluation was performed by measuring muscle fibers to obtain the fiber diameter utilizing ImageJ software (n = 100 fibers/experimental group). The mean fiber diameter of each strain was obtained, and the variability coefficient was calculated according to the formula: Standard Deviation x 1000 ÷ Diameter Mean. Results were represented as mean variation and variability coefficient. The stained sections were observed under a light microscope (Zeiss). ### Sirius red staining {#Sec16} To identify regions with collagen deposition, indicative of connective tissue replacement and infiltration between muscle fibers, the slides were immersed in Bouin fixative for twenty minutes and washed in water. The sections were stained with Sirius red (0.2 g) dissolved in saturated picric acid (100 mL) for 60 minutes, dehydrated and mounted in Canada balsam. The differences were quantified by the positive area of staining, labeled in red, relative to the total area of the section, using ImageJ software (4--5 fields, n = 6 animals/experimental group). The stained sections were observed and photographed under the same exposure conditions (16 ms), using a light microscope (Zeiss) and the Axiovision software Release 4.8.2 SP3 (2013). ### PAX7 and KI67 immunofluorescence {#Sec17} The presence of SCs was determined by double staining with antibodies against PAX7 and laminin to label the basal lamina. Activated SCs were determined by double staining with antibodies against PAX7 and KI67. Primary antibodies used: PAX7 (1:20; DSHB), laminin (1:100; Dako; Z0097) and KI67 (1:200; Abcam AB15580). Secondary antibodies were Cy3-labeled sheep anti-mouse IgG (1:100; Sigma; C2181) and FITC-labeled donkey anti-rabbit IgG (1:100; Amersham; N1034). The 6 µm muscle sections were fixed with 4% formaldehyde solution and submitted to antigen retrieval with hot citric acid buffer. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for one hour at room temperature. The slides were mounted with Vectashield with DAPI to counterstain nuclei. Immunofluorescence micrographs were obtained with a conventional fluorescence microscope (Zeiss). The proportion of PAX7 positive nuclei to the total number of fiber was quantified (\~3000 fibers, n = 4 animals/experimental group). The proportion of double stained PAX7 and KI67 were analyzed in approximately 200 PAX7 positive nuclei. All images were collected using a Zeiss microscope with epi-fluorescence, using specific filters for DAPI, FITC and for Cy3. The images were photographed using the Axiovision software Release 4.8.2 SP3 (2013), under the same exposure conditions (120--130 ms), standardized for the Cy3 staining for identification of dMyHC expression. The same software created merged figures automatically. ### Developmental myosin heavy chain (dMyHC) {#Sec18} The antibody Clone RNMy2/9D2 recognizes a myosin heavy chain (MHC) present during the embryonic and neonatal period in the development of skeletal muscle. The same MHC is expressed during regeneration of muscle fibres. The presence of dMyHC indicates myogenic activity by newly differentiated fibers labeled in red. The 6 µm muscle sections were double labeled with primary antibodies to dMyHC (1:30; Vector; VP- M664)^[@CR44]^ and laminin (1:50; Dako; Z0097)^[@CR45]^, washed, and then incubated for 1 h with secondary antibodies Cy3-labeled sheep anti-mouse IgG (1:100; Sigma; C2181) and FITC-labeled donkey anti-rabbit IgG (1:100; Amersham; N1034), respectively. After washing, the slides were mounted with Vectashield and DAPI, to counterstain nuclei. Immunofluorescence micrographs were obtained with a conventional fluorescence microscope (Zeiss) and the percentage of dMyHC+/total fibers was quantified (4 fields, n = 6 animals/experimental group). The fiber diameter of dMyHC+ fibers of each strain was measured manually, and the variability coefficient was calculated according to the formula: Standard Deviation x 1000 ÷ Diameter Mean^[@CR38]^. All images were collected using a Zeiss microscope with epi-fluorescence, using specific filters for DAPI, FITC and for Cy3. The images were photographed using the Axiovision software Release 4.8.2 SP3 (2013), under the same exposure conditions (120--130 ms), standardized for the Cy3 staining for identification of dMyHC expression. Merged figures were created automatically by the same software. Statistical analysis {#Sec19} -------------------- Differences among the groups were assessed using one-way ANOVA Test, and Kruskal-Wallis Test, for small and non-normally distributed set of data. Results are reported showing means and standard deviations in bar graphs and medians in boxplot graphs. The statistical analyses were calculated using GraphPad Prism version 7 for Windows (San Diego, CA: GraphPad Software, Inc), Minitab 17 Statistical Software (State College, PA: Minitab, Inc) and IBM SPSS Statistics for Windows version 24.0 (Armonk, NY: IBM Corp) software and a p-value equal or less than 0.05 was considered to be significant. For the expression analysis, additional five animals (males) from each strain were added (in a total of eleven animals), to confirm and strengthen the statistical analysis. Supplementary information ========================= {#Sec20} Supplemental information Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= Supplementary information accompanies this paper at 10.1038/s41598-019-48156-7. We would like to thank the help from Leticia Feitosa, Heloisa S. Bueno, Patricia S. Kuriki, and the professionals of the Human Genome and Stem Cells Research Center for helping with facilities. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo - Centro de Pesquisa, Inovação e Difusão (FAPESP-CEPID), Conselho Nacional de Desenvolvimento Cientıfico e Tecnológico (CNPq-INCT), Financiadora de Estudos e Projetos (FINEP). CAPES -- Coordenação de aperfeiçoamento de Pessoal de Nivel Superior. Conceived and designed the experiments: A.F.R.J. and M.V. Implementation of the methodologies: A.F.R.J., R.I., C.F.A., S.A.F., D.A.G., A.L.F.S. and L.S.S. Performed experiments: A.F.R.J. and C.F.A. Contributed reagents/materials/analysis tools: R.I., C.F.A., S.A.F., A.L.F.S. and L.S.S. Prepared and/or revised the manuscript. A.F.R.J., L.S.S., P.O.O., C.F.A. and M.V. The authors declare no competing interests.	{ "pile_set_name": "PubMed Central" }	[ "amount", "gaaggtcaaccaagctttcg", "graphpad", "probabl", "two", "ab", "almost", "control", "myogenin", "work", "origin", "divers", "remain", "program", "induct", "nitrogen", "decreas", "desenvolvimento", "striation", "overnight", "pierc", "compat", "respond", "impair", "rneasi", "collect", "thank", "abfigreftypefig", "gene", "spectrophotomet", "identifi", "basal", "musclescrcrcr", "abcam", "healthcar", "epifluoresc", "mini", "retain", "readi", "mechan", "immunofluorescenceand", "week", "valu", "fix", "hour", "estado", "signal", "split", "gagttcgattagccgagtgc", "÷", "recent", "branch", "restoredcr", "separ", "gener", "use", "mrna", "embryon", "graph", "moder", "myocyt", "nbp", "huge", "ms", "ongo", "support", "final", "supplement", "room", "mhc", "caus", "imag", "complet", "accord", "ny", "cfigreftypefig", "signific", "purpl", "immunohistochem", "probe", "full", "temperatur", "guidelin", "mark", 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Glossary: FRET: fluorescent resonance energy transfer; FRAP: fluorescence recovery after photo-bleaching ZAP-70: ζ-associated protein of 70 KD; a pivotal molecule in membrane proximal T cell receptor signaling; Immune synapse: organized structure that forms at the interface of an antigen-presenting cell and antigen-specific T cell; Lipid rafts: cholesterol-rich plasma membrane microdomains that have been implicated in lymphocyte signaling Images of cells are perplexing. Genetically tinted by the jellyfish fluorescent protein painter kit, cells of all colors and shapes turn serious academic journals into modern art catalogues. After decades of abstract thinking and frequently fruitless interpretation of bands, dots, and their graphic derivatives, the attention of many scientists is now attracted by the opportunity to see almost everything in "real" life. The increased communication value of images is a part of a rapidly evolving cultural tendency demanding real-time event documentation. Nowhere could be more remote from this problem than the caught-in-time Certosa di Pontignano, an old castle near Siena, Italy. Maybe that is why we decided to organize a roundtable discussion about imaging during the 2004 EMBO workshop on Lymphocyte Antigen Receptor and Coreceptor Signaling held at this location ([@bib1]). We asked the experts to reflect on the impact of imaging on a field that has been dominated by biochemical approaches. Our aim was to capture some personal viewpoints on the real and virtual power of imaging and the new challenges the technology brings. Can the traditional craft of cell manipulation match the high speeds that can now be visualized? What new reagents are needed to allow us to take full advantage of imaging in the lymphocyte signaling field? Should imaging data be quantified (definitely, yes!) and if so how? (See also the related Commentary on page 501 of this issue \[[@bib2]\].) At the end, we asked the participants to fantasize about how we might be using imaging technology to study lymphocyte signaling in the more distant future. I regret that we could not present all of the lively discussion, but we have included comments and exchanges on some of the key topics that were covered. We thank all the participants for their willingness to share their insight and opinions. PARTICIPANTS ============ Oreste Acuto: Institut Pasteur, France Mark M. Davis: Stanford University, CA Christopher C. Goodnow: The Australian National University, Australia Claire Hivroz: Institut National de la Sante et de la Recherche Medicale Unite 520, France Bernard Malissen: Centre d\'Immunologie INSERM-CNRS de Marseille-Luminy, France Anjana Rao: Harvard Medical School, MA Michael Reth: Max Planck Institute of Immunobiology, Germany Lawrence E. Samelson: National Cancer Institute, National Institutes of Health, MD Alexander Tarakhovsky: Rockefeller University, NY Salvatore Valitutti: Institut National de la Sante et de la Recherche Medicale Unite 563, France Arthur Weiss: University of California San Francisco, CA Rose Zamoyska: National Institute for Medical Research, UK POWER OF IMAGES =============== Mark M. Davis: If you ask, "What\'s the value of imaging?" one important thing is that it allows us to escape the tyranny of population biochemistry. We\'ve been forced to indulge in the fantasy that if you have a million cells in a tube, and you hit them with something, that they\'re all going to do exactly the same thing at the same time. However, we know that a lot of stochastic processes are going on. If you just grind the cells up at different time points, you\'re averaging over all those processes. It\'s only when you can see an individual cell that you can see it was stimulated, and then went on a particular pathway. And there\'s a level of quantitation we\'ve never seen with biochemistry. We\'re getting much more precise answers to questions like the amount of calcium that\'s being released during T cell activation. We can see that one peptide gives you X amount of calcium, and two give you 2X, and so forth ([@bib3]). Lawrence E. Samelson: With imaging, it\'s not just that you\'re able to overcome the heterogeneity of the cell population; you\'re able to overcome the heterogeneity of the intracellular molecular populations. Many of the molecules that we tag and look at, in terms of intracellular signaling proteins, exist in different pools in the cell: some are cytoplasmic, some are recruited to the membrane, others are associated with the cytoskeleton. It would be impossible to study these different populations without imaging the individual cells and individual molecules. Bernard Malissen: We are also feeling out the speed at which signaling processes are working, and it\'s very fast. Neurobiologists have known that for a very long time. We are just putting our finger on the speed at which a T cell is responding to an antigen. So imaging gives you an idea of the timescale we should be thinking of. Arthur Weiss: I think that imaging in real time is critical. But in addition, FRAP technology allows us to see things that we never imagined, in terms of the turnover and the dynamics of structures that we think are static. Because you see an immune synapse, you think everything is just sitting there and immobile with no turnover. MMD: There are a lot of technical challenges involved in asking questions of a detailed molecular nature, of molecules that are within particular cells, at particular times, and in particular places. But it\'s the future of understanding how these cells work. There are huge challenges in getting markers for events inside the cell. But there are already some that are specifically designed as indicators of a particular kind of phosphorylation. We need many more of those for all the different signals that might be going on. Rose Zamoyska: Single molecule imaging is coming, and it\'s not that far off. That really is going to add another dimension to the way we\'re going to be able to analyze signaling events. IMAGING MEETS BIOCHEMISTRY AND GENETICS ======================================= Oreste Acuto: Nobody would disagree with all that has been said so far. But we should never forget that biochemistry gives us the fastest way to establish protein--protein interactions, and the highest resolution. BM: Ultimately, genetics should allow you to order a pathway without having help of biophysics. If you have powerful genetics, like in the yeast, you can do very nice ordering and epistatic analyses of pathways, and you don\'t need imaging. RZ: One of the problems with biochemistry is that you can never look at the consequences of the signals, because the cells are lysed. And one of the advantages of imaging is that we\'re developing techniques where you\'ll be able to look at the differentiation consequences, downstream of particular signals. Christopher C. Goodnow: Whenever I hear the biochemistry angle, I always think about Arthur Kornberg and how proud he was of DNA polymerase in the tube, until the geneticists knocked it out in bacteria, and they replicated just fine. It\'s very hard to order pathways and I think the triangulation between biochemistry, genetics, and imaging is going to be essential. You need to know where things are in the cell, and their kinetics and the dynamics, which is what the imaging tells you. You need to know what could happen, which is what the test tube experiment tells you. And then the genetics tells you what is actually needed for a given process. As a community, we don\'t put enough emphasis, in our publication and referee processes, on when the genetics and the biochemistry are disconnected. For example, we have a ZAP-70 catalytic domain point mutant mouse, which has a severe problem with thymic positive selection (unpublished data). The genetics is telling us that there is a real problem. But when we look at tyrosine phosphorylation and do old-fashioned calcium analysis, we can\'t see any problem. So the biochemistry is disconnected from the genetics. The question is if you did high speed imaging, would we suddenly be able to connect the genetics with the biochemistry? Michael Reth: The immune synapse is a nice example of how difficult it can be to correlate image analysis data with the biochemical behavior of molecules inside a cell. When the synapse was first discovered, everybody thought that one had finally found the molecular organization of signaling proteins at the activated T cell antigen receptor. Yet when people later on analyzed synapse formation at different time points and used antiphosphotyrosine antibodies ([@bib4]), it became clear that TCR signaling happens much earlier than synapse formation, and that the synapse may have more to do with the secretion of cytokines than with the initiation of the TCR signal. MMD: Absolutely, when we first see things we\'re invariably going to misinterpret them. Seeing may be believing, but it isn\'t always understanding. MR: Another source of artifacts is the use of GFP-tagged proteins to follow the behavior of molecules in a living cell. This is a powerful tool, but it is important to first test whether the fusion to GFP is not drastically changing the localization or biochemical behavior of the fusion proteins inside the cell. Another problem is the use of fluorescent reagents to visualize structures in living cells like lipid rafts. In this respect, it was interesting to see that when FITC-coupled cholera toxin was used to detect raft structures it resulted in raft aggregation ([@bib5]). So you are creating what you want to observe. QUANTITATING IMAGES =================== MR: One problem we have to solve is how to quantify our image analysis. When I see a publication with a beautiful picture of a cell showing the localization of the GFP-tagged protein to a particular membrane structure, I wonder how representative this image is of the behavior of these proteins in the cell population. To overcome the bias generated by our eyes and our brain, we need to apply computer programs for image analysis which, similar to a FACScan, can quantify images and order cells according to the image phenotype. LES: Many of these problems are problems that all cell biologists have. You look at a picture in a journal, and you say, "How do I know this is representative?" That gets into the question of honesty, and ethics, and basically good science. But programs for quantitation exist; you can track movement; you can quantitate velocity; you can quantitate dynamics. So a lot of what we need is there. Salvatore Valitutti: It\'s very possible to quantitate morphology. It\'s very tough. Very time consuming. But it is possible to ask authors for quantification, or even to show the originals from which the quantitation was made. Alexander Tarakhovsky: Do you think that there is an absolute necessity to introduce ways to quantify images? SV: I think it will be in the interests of people doing morphology to somehow enforce measurements of intensity, for instance, at the immune synapse. MMD: The referees always ask us for quantitation: what does the cell population do? How many cells do this? How many cells do that? On some occasions, when the referees have been skeptical about our interpretation of the images, they ask for all the images, and we send all the images on a CD. So the vetting is actually easier than it used to be, in the sense that you can transmit vast amounts of information very quickly, so that people can be sure that you\'re not being subjective. As to a program that Bill Gates would sell us to do this---fine, when it happens. But there are many different questions you can ask using imaging. I can imagine a very simple kind of software being good for some kinds of imaging analysis, but completely useless for other kinds. Claire Hivroz: One of the problems that we have to face is that while these new imaging techniques are really nice, they are sources of artifacts, also. We really need to do the analysis carefully. Biologists are not physicists, and sometimes we don\'t know how to analyze the image, or we don\'t know exactly how to use the software. I think we have to make great efforts to work with physicists, to really understand what we are doing because being sure, for example, that what we are looking at by FRET is really an interaction between two proteins is not that easy. ADVANCING THE TECHNOLOGY ======================== MR: By using GFP-tagged proteins we can employ imaging to localize proteins to certain subcellular structures, but what is really lacking are more techniques to not only measure the localization, but also the activity of a protein. Of course, we can measure the intracellular increase of calcium by fluorescent dyes, but these dyes cannot be localized to certain places inside the cell. What we need are more GFP variants that change their fluorescence upon modification, like calcium binding, phosphorylation, or oxidation. With such techniques, you can then see in real time, not only the localization of molecules in the cell, but also at what time point these proteins become active or modified. Anjana Rao: I\'m dealing with a program of transcriptional regulation that is sensitive to very low intracellular calcium levels---lower than can be measured reliably by the available technology. So I think, in the case of calcium imaging, we\'re clearly going to need some new technology in the picture. MMD: There\'s still a bag of genetic tricks that hasn\'t really been applied to imaging but which could be used. There is development of photo-activatable GFP, for example, and temperature-sensitive mutations. So it might be possible to give cells a burst of a laser, for example, to alter the function of molecules that you had modified. AT: Do you think that one can manipulate structures such as the immune synapse? CCG: The strength of genetics is in the specificity of the dissection. But the limitation is that you just have no temporal control or spatial control. So, in that respect, it\'s a very limited tool. The ideal scenario would be to replace genetics with pharmacology. To have small molecules that you can add in to inhibit signaling pathways, which are as specific as genetic manipulations. But, of course, where we get into trouble is in knowing how specific a compound is. This is where the neurobiologists, and the cardiologists, have it over us as immunologists. They\'ve got a panoply of quite specific agents to dissect processes such as cardiac contractility. AT: Do you think that genetic manipulation could allow conditional protein inactivation at a given time point? CCG: Inactivation has to happen very fast. The only thing would be some adaptation of photo-inactivatable or temperature-sensitive alleles, which as Mark \[MMD\] has pointed out, could allow you to eliminate a wild-type signaling protein both rapidly and in a specific region of a cell. MR: New in vivo imaging studies can follow the dynamic behavior of single lymphocytes in the tissue of a living mouse. This, in combination with genetics, will allow us to understand better the dynamics of cellular interactions inside the immune system. To follow the behavior and activity of single molecules inside a lymphocyte is much more demanding technically, as these are rapid and very dynamic processes. One needs machines with a very fast kinetic image acquisition if one wants to follow these events in real time. Furthermore, if one wants to track single fluorescent molecules, one has to use a lot of laser light to detect them, and this could be another potential source of artifacts. Laser light not only results in photobleaching, but can also generate reactive oxygen species. As many signaling processes inside the cell are highly sensitive to redox changes, it is possible that shining a lot of laser light on the molecules can change their regulation and behavior. AW: One thing I want to stress again is quantitation. And maybe genetics can help there. Chris \[CCG\] talked about a point mutation which doesn\'t show any biochemical difference. We might not be able to validate that our GFP constructs are behaving normally using simple overexpression systems. I would argue that maybe we can use genetic approaches to test the constructs we use to validate the fact that we\'re expressing the right amount of protein, in the right place, at the right time, and that those proteins are regulated properly. Maybe we can harness the power of genetics for proper expression---right place, right time, as well as right level. AR: In terms of technology development, there are two things that I would focus on. Microinjection of mutant proteins into cells that don\'t have that protein would be a very good thing to develop. The protein can be tagged in various ways, with small fluorescent probes for instance. And second, I think electron microscopy will be important. Think about the work that Gunther Blobel did, for instance, with the nuclear pore and with nuclear import and export. He had the biochemistry. He had all the different subunits. And then he used EM to localize them in the basket of the nuclear pore. The stable immune synapse is such a structure that could benefit very greatly from EM analysis. IMAGINING THE FUTURE OF IMAGING =============================== AT: Now I want you to dream. AR: To dream? AT: Yes. So, without discussing limitations, tell me how do you think in ten years your dreams will be fulfilled? \[Laughter\] MR: My dream is a very powerful computer program that is able to generate a virtual cell where we can test our hypothesis of protein signals inside cells. Using the primary sequence data, this program should be able to generate the tertiary structure of the protein not only in its frozen (crystal) stage, but also its normal dynamic stage in solution. With such a program, one could follow the conformational changes of proteins and better understand the dynamic processes of protein--protein interactions during cell signaling. So that is a dream, but I think one has to wait quite a while for such a program. MMD: I don\'t think a computer would be able to know how evolution built a cell. So I think that dream is going to come to a bad end. But in my childhood, there was a movie called "Fantastic Voyage," where there was a tiny, little ship that was built, and miniaturized humans were in it. They cruised around the bloodstream, and they looked at the lymphocytes and other things from this point of view. So a good dream would be one where you miniaturize the ship even more, such that you could go into a cell, through a channel or something, and cruise around the cell and watch different bits of the machinery moving up and down, or doing whatever it does. AR: I think you\'d find what Schrödinger predicted in 1944 ([@bib6]). You\'d find probabilistic movements. I think the whole workings of the cell are governed by probability and stochastic movements that, in turn, are governed by the affinity of interaction. My dream would be to apply the imaging techniques we\'re talking about at the membrane, today, to find out what happens in the nucleus. LES: Mine is another cautionary tale from early twentieth-century physics, and that is the Heisenberg Uncertainty Principle. We have to watch out for what we do to these systems. Certainly by manipulating systems we create artifacts. So that\'s a nightmare. \[Laughter\] OA: My ultimate dream is that we abolish competition. We create a big center and attack very fundamental, basic paradigms in a few systems. It is not strictly necessary to know everything, with everybody working in their own little corner. Why not combine genetics, imaging, and biochemistry in a dedicated center where scientists can perform challenging experiments, like physicists do? That\'s a dream. That\'s something that I think should work. \[Applause\] The editors of the Journal would like to thank the organizers of the 2004 EMBO workshop on Lymphocyte Antigen Receptor and Coreceptor Signalling (Certosa di Pontignano, Siena, Italy)---Cosima Baldari, John Telford, Oreste Acuto, and Gary Koretsky---for allowing us to hold the discussion during the meeting and for their assistance with the organization. We also thank Ingrid Mecklenbrauker for helping us document the discussion. The edited transcript was approved by all participants. We apologize to the participants whose contributions we could not include owing to space limitations. 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1. Introduction {#sec1} =============== Damage to the cornea from chemical burns is a serious clinical problem, accounting for about 7%--10% of eye injuries, of which, alkali-induced injury to cornea is the most dangerous, due to its quick penetration to the eye surface \[[@B1]\]. It can cause corneal infection, ulceration, perforation, neovascularization, and opacification \[[@B2]\] and often leads to permanent visual impairment. Penetrating keratoplasty has been used successfully to treat many corneal diseases; however, several intractable ocular surface diseases, such as chemical burns, are still untreatable using this procedure \[[@B3]\]. The Boston Type 1 keratoprosthesis (KPro), developed at the Massachusetts Eye and Ear Infirmary and approved by the US Food and Drug Administration (1993), is currently the most commonly used keratoprosthesis worldwide \[[@B4]\] and is used in cases at high risk for penetrating keratoplasty failure \[[@B5]\]. It has been successfully used to restore vision in cases of chronic corneal disease and graft failure, including corneal alkali burn \[[@B6]\]. However, despite multiple advances and improvements in prognosis of patients following KPro surgery, several challenges remain. The postoperative development of RPMs is one of the most common complications of keratoprosthesis surgery, affecting between 25% and 65% of patients, and is poorly understood \[[@B7], [@B8]\]. Matrix metalloproteinases (MMPs), zinc endopeptidase proenzymes responsible for the degradation of extracellular matrix components, play a role in many ocular physiological processes, including embryogenesis, angiogenesis, and wound healing \[[@B9]\]. Breakdown of the basement membrane by MMPs is known to contribute to the pathogenic ulceration and perforation of the corneal stroma \[[@B10]\]. The activity of MMPs is closely controlled by a family of natural antagonists, the tissue inhibitors of matrix metalloproteinases (TIMPs), whether MMPs exert normal or abnormal responses depends upon the balance between MMPs and TIMPs. An imbalance between MMPs and TIMPs in favor of inhibitor might provoke the development of fibrosis and in consequence may lead to tissue remodeling \[[@B11]\]. MMP-9 is the enzyme of major importance in the remodeling and degradation of the corneal stromal collagen in both animals and human beings \[[@B12]\]. It was found to be only expressed in rats which underwent corneal injury, instead of the normal corneal stroma \[[@B13]\]. The expression of MMP-9 and its endogenous inhibitor TIMP-1 in the aqueous humor of alkali-burned rabbit cornea before and after KPro implantation and their role in RPMs formation following KPro surgery have previously received limited attention. In this study, we report in greater detail the MMP-9 and TIMP-1 expressions in aqueous humor before and after KPro implantation into the alkali-burned rabbit cornea, the histopathological characteristics of RPMs developed after KPro implantation, and immunohistochemical expression of MMP-9 and TIMP-1 in this RPM. 2. Material and Methods {#sec2} ======================= 2.1. Animals {#sec2.1} ------------ A total of 60 healthy adult New Zealand White rabbits, weighing between 2.5 and 3.0 kg, were obtained from the Animal Experimental Center of Army General Hospital of Shenyang Military Region (Shenyang, China). This study was approved by the Ethics Committee of Shenyang Military Region General Hospital. All experiments were performed on the left eye of animals and all animals and experimental conditions followed laboratory animal regulations of State Science and Technology Commission. 2.2. Experimental Model of Corneal Alkali Burn {#sec2.2} ---------------------------------------------- Corneal alkali burn model was established as previously described with some modification \[[@B14]\]. To create an alkali burn, 50 rabbits were randomly selected and their left eyes were locally anesthetized with 0.5% Alcaine eye drops (Alcon Ophthalmic Products Co. Ltd., Shanghai, China). Then, a 10 mm in diameter Cellulose nitrate filter paper soaked in 1 mol/L NaOH was applied to the cornea for 45 s, followed by a rinse with 10 mL of balanced salt solution. After the corneal alkali burn was created, animals received chlortetracycline eye ointment (Beijing Shuangji Pharmaceutical Co., Ltd., China) and atropine sulphate eye gel (Shenyang Xingqi Pharmaceutical Co., Ltd., China) daily for one week. The severity of conjunctival and corneal injury was determined according to the Roper-Hall (RHG) grading system \[[@B15]\]. Experimental model of corneal alkali burn was successfully established in 42 New Zealand White rabbits, as demonstrated by the presence of complete corneal opacity, corneal neovascularisation involving more than two quadrants of the cornea, and normal anterior chamber 3 months after severe alkali burn. The 36 model rabbits were randomly selected and divided into three groups (n = 12): group I, blank control group, corneal alkali burn without any keratoplasty; group II, control group, cornea alkali burn with penetrating keratoplasty; group III, experimental group, corneal alkali burn with KPro implantation. The 10 untouched rabbits served as allogeneic cornea donors after collection of aqueous humor samples. 2.3. Corneal Implantation {#sec2.3} ------------------------- The surgery was performed as previously described with some modification \[[@B16]\]. The procedures were performed under a surgical microscope (Zeiss, Jena, Germany) by a single investigator (Yinxin Chen), and the surgical time was 45 to 60 min per procedure. Briefly, donor cornea was marked in the periphery with an 8.5 mm diameter biopsy punch (Miltex, Plainsboro, NJ, USA) and then excised with Vannas scissors (Storz Instruments Company, San Damis, CA, USA) after animal euthanasia. The corneas were trephined centrally with a 3 mm diameter punch, slid over the stem of the PMMA front plate, followed by titanium back plate position (KPro device, 8.5 mm in diameter, Boston Keratoprosthesis Operations, MA, USA), and complex was then placed in PBS. Three months after injury, the recipient cornea was marked with an 8.5 mm diameter biopsy punch and excised with Vannas scissors under general and topical anesthesia (120 mg/kg ketamine and 20 mg/kg xylazine). The extracapsular crystalline lens extraction was then performed on eyes of groups II and III and the rabbits were left aphakic with an intact posterior capsule. The lenses of group I eyes were not extracted and the rabbits were left phakic. The donor cornea KPro complex was then positioned in the recipient bed and secured with sixteen interrupted 11.0 nylon sutures (Sharpoint; Angiotech Pharmaceuticals, Vancouver, BC, Canada). A drop of tobramycin and dexamethasone (Tobradex; Alcon, Puurs, Belgium) was applied and a tarsorrhaphy was performed using 8-0 nylon sutures. The tarsorrhaphy was removed 48 h after the procedure, and the same antibiotic and steroid drop was administered once a day for 7 consecutive days following tarsorrhaphy removal. After operation, animals received tobramycin and dexamethasone ophthalmic ointment once daily for three days, atropine sulfate eye gel once daily for one week, chloramphenicol eye drops four times daily for 4 weeks, and chlortetracycline hydrochloride eye ointment once every night for 4 weeks. 2.4. Follow-Up and Clinical Evaluation {#sec2.4} -------------------------------------- RPM referred to membrane growing on the back of the device, all eyes were determined under slit-lamp, and RPMs were diagnosed at the time of explantation. The aqueous humor was obtained by puncturing the anterior chamber using a 45 gauge needle at postoperative days 7, 14, 28, 60, and 90. MMP-9 and TIMP-1 levels in aqueous humor were determined using the commercially available ELISA kits for MMP-9 and TIMP (Shanghai Sunred Biological Technology Co., Ltd., China). Ninety days after implantation, all rabbits were sacrificed by using the air embolism method by injecting 20 mL of air into the ear marginal vein. The presence of complications, like retinal detachment, intraocular infection, and so forth, was assessed by B ultrasound. The RPMs that developed were then excised and histological sections were prepared for further processing. Slit-lamp examination was performed before surgery and at postoperative days 7, 14, 28, 60, and 90, to examine the corneas for signs of inflammation or neovascularization and to assess optical transparency, and photographs were taken. For transmission electron microscopy (TEM), the sections were stained with uranium acetate and lead citrate and observed with JEM-1200 transmission electron microscopy (Tokyo, Japan). Some pieces were fixed in 4% paraformaldehyde in 0.1 mol/L PBS, paraffin embedded, and then sectioned. Hematoxylin and eosin (H&E) staining was performed for routine histopathologic examination. The immunohistochemical staining was performed using rat anti-rabbit polyclonal antibodies against MMP-9 and TIMP-1 and biotin-labeled sheep anti-mouse IgG, all purchased from Beijing Boaosen Biotechnology Co., Ltd., China. Five sections were randomly selected and data were obtained from 50 fields of view (10 fields from each section). 2.5. Statistical Analysis {#sec2.5} ------------------------- Data were expressed as mean ± SD and analyzed using SPSS 19.0 for windows (SPSS Inc., Chicago, Illinois, USA). The One-Way ANOVA procedure was used to determine the difference among groups. A p value of \<0.05 was considered statistically significant. 3. Results {#sec3} ========== 3.1. Clinical Results {#sec3.1} --------------------- Three months after the infliction of alkali wounds, the injured corneas exhibited stromal opacities of varying depth; some corneas completely became opaque, when compared with the normal ones (Figures [1(a)](#fig1){ref-type="fig"} and [1(b)](#fig1){ref-type="fig"}). Slit-lamp examination showed RPMs developed 30 ± 10 days after transplantation of KPro ([Figure 1(c)](#fig1){ref-type="fig"}). After corneal transplantation, 1 rabbit accidently died in group II and 2 in group III. The common symptom in early period of postoperation were eyelid margin redness and swelling, conjunctival injection, tearing, photaesthesia and white secretion from the conjunctival sac. The local irritation lasted between 7 and 14 days in group II and between 7 and 21 days in group III. There was a clear boundary of edema on the cornea graft; then the edema reduced gradually at postoperative day 7 and the corneal grafts became clear ([Figure 2](#fig2){ref-type="fig"}). At the postoperative day 14, there was new vessel growth in the upper corneal limbus, and a large amount of new vessels formed 28 days postoperatively ([Figure 2](#fig2){ref-type="fig"}). At 60 and 90 postoperative days, a reduction in the number of vessels and level of haze was noted relative to earlier examinations ([Figure 2](#fig2){ref-type="fig"}). The postoperative complication in group III included RPMs in 5 eyes ([Figure 2](#fig2){ref-type="fig"}, day 14) and vitreous hemorrhage in two eyes. However, in group II, corneal rejection occurred in 5 eyes 14 days after penetrating keratoplasty, with diffuse edema and opacity of the graft ([Figure 2](#fig2){ref-type="fig"}). The shallow anterior chamber and anterior synechiae developed in 1 case and posterior capsule opacification developed in 3 cases ([Figure 2](#fig2){ref-type="fig"}, day 60). There were no postoperative retinal detachment and endophthalmitis in the both groups II and III. 3.2. Aqueous Humour MMP-9 and TIMP-1 Levels following KPro Implantation {#sec3.2} ----------------------------------------------------------------------- The dynamic expressions of MMP-9 and TIMP-1 in aqueous humour were quantified using commercially available ELISA kit; our results showed that there was a small amount of MMP-9 and TIMP-1 in normal rabbit aqueous humor (12.52 ± 3.05 and 33.49 ± 3.39 ng/mL, resp.). The severe alkali burn employed in this study resulted in a significant increase in both MMP-9 and TIMP-1 levels three months later. After corneal transplantation, aqueous humor MMP-9 in group III showed an increasing trend and peaked at postoperative day 90. By contrast, in group II, MMP-9 in aqueous humour reached peak at postoperative day 14 and then significantly decreased thereafter, although the data at postoperative day 90 was still significantly higher than that of preoperative level and of group I (p \< 0.05, [Table 1](#tab1){ref-type="table"}). However, unlike the increase in MMP-9 postoperatively, aqueous humor TIMP-1 was significantly decreased following operation in both groups II and III. The data reached valley bottom at postoperative day 90 in group III and at postoperative day 14 in group II and then increased until the postoperative day 90 (p \< 0.05 versus preoperative level and blank control, [Table 1](#tab1){ref-type="table"}). The concentration of MMP-9 in group III became higher on the postoperative day 28 when compared with group II, and the difference was statically significant at subsequent points (p \< 0.05). By contrast, TIMP-1 level became lower on the postoperative day 28 and the data became statistically significant thereafter when compared with group II (p \< 0.05). 3.3. Histopathological and Immunohistochemical Analysis of Retroprosthetic Membrane Specimens {#sec3.3} --------------------------------------------------------------------------------------------- In total, five retroprosthetic membrane specimens were obtained following Boston KPro explantation; our hematoxylin-eosin staining showed a large number of fibroblasts and collagen fibers arranged irregularly and inflammatory cell infiltration. There was ingrowth of new blood vessels in the RPMs (Figures [3(a)](#fig3){ref-type="fig"} and [3(b)](#fig3){ref-type="fig"}). Transmission electron microscopy of the retro-KPro fibrous membrane revealed an abundance of elongated collagenous fibers, separated by fibroblasts and granulocyte infiltration. These cells were spindle-shaped with mitochondria and rough endoplasmic reticulum in the cytoplasm. The extracellular space separating these cells was composed of fibroblasts (Figures [3(c)](#fig3){ref-type="fig"} and [3(d)](#fig3){ref-type="fig"}). Immunohistochemical results showed positive staining of retroprosthetic membrane specimens for MMP-9 and TIMP-1, with yellowish-brown cytoplasm and blue nucleus (Figures [3(e)](#fig3){ref-type="fig"}, [3(f)](#fig3){ref-type="fig"}, [3(g)](#fig3){ref-type="fig"}, and [3(h)](#fig3){ref-type="fig"}). 3.4. Aqueous Humor MMP-9 and TIMP-1 Levels in KPro-Implanted Rabbits with or without RPM {#sec3.4} ---------------------------------------------------------------------------------------- To further explore the role of MMP-9 and TIMP-1 expressions in retroprosthetic membrane formation following the KPro implantation, rabbits with KPro implantation were subdivided into RPM and No-RPM groups. Aqueous humor levels of MMP-9 were significantly higher in the RPM group postoperatively when compared with that in the No-RPM group (p \< 0.01 or p \< 0.001). By contrast, Aqueous humor TIMP-1 levels in the RPM group were comparatively lower than that of No-RPM group following the operation (p \< 0.01). There was no significant difference between the two groups in preoperative aqueous humor MMP-9 and TIMP-1 levels ([Figure 4](#fig4){ref-type="fig"}). 4. Discussion {#sec4} ============= Ocular trauma in the form of an alkali burn to the cornea is among the most serious ocular injuries that may cause severe and permanent visual impairment \[[@B17]\]. It can induce a strong inflammatory reaction characterized by cell infiltration and production of proteolytic enzymes and cytokines \[[@B1]\]. Previous studies have indicated an indispensable role for MMPs in the homeostasis and pathophysiology of the cornea \[[@B18]\]. MMPs and TIMPs are present in the aqueous humor \[[@B19]\], and alkali injury enhanced the expression of MMP-9 in mice \[[@B20]\]. Degradation of the basement membrane by proteolytic enzyme MMP-9 may involve the pathogenic process of alkali injury \[[@B21], [@B22]\]. The presence of MMP-9 has been identified in the tear fluid of patients with corneal alkali burn \[[@B23]\]. Consistent with the results of this study, our results showed that alkali insult of rabbit cornea resulted in a significant increase in aqueous humor MMP-9 levels, while the TIMP-1 levels in aqueous humor were found to decrease to some extent three months after corneal alkali burns. After keratoplasty, MMP-9 levels in aqueous humor were further significantly enhanced in experimental rabbits within the postoperative day 90, whereas TIMP-1 levels were downregulated further when compared with that in group I (blank control). All these results showed that evidence suggested the pathophysiological role of MMP-9 in alkali-induced corneal injury and repair. The Boston keratoprosthesis is by far the most commonly used prosthetic cornea in the world \[[@B24]\]. However, there are a number of significant complications that can occur postoperatively. Among which, retroprosthetic membrane is one of the most common postoperative complications encountered \[[@B25]\], occurring in 25% to 65% of patients within a year after KPro implantation, and up to 45% of cases require eventual treatment \[[@B26], [@B27]\]. The formation of retroprosthetic membrane was shown to correlate with the risk of corneal melt \[[@B28]\]. The etiology of retroprosthetic membrane is still largely unknown. However, the presence of this retrokeratoprosthesis fibrous tissue may act as a physical barrier between the cornea and the aqueous humor, which limits the contact between the aqueous and the corneal graft enough, and ultimately result in stromal necrosis and epithelial degeneration in KPro implanted eyes \[[@B29]\]. In our study, retroprosthetic membrane happened in 5 out of 10 KPro-implanted rabbits (50%), indicating the high incidence of retroprosthetic membrane formation following KPro implantation. Previous study showed that chemical injury seems to be an insignificant risk factor for the development of RPM \[[@B5]\], while other study also indicated that chemical burns have higher incidence of RPM development \[[@B30]\]. However, it is not possible to state whether alkali burn used in this series could be a contributing factor in the higher rate of RPM formation reported herein, since, to the best of our knowledge, there seems to be few researches on KPro implantation in animal model of corneal alkali burns up to now. The RPMs have been previously evaluated in patients with KPro implantation and were suggested to vary according to antecedent clinical events of the patients \[[@B7], [@B29]\]. Therefore, we further evaluated the histopathological characteristics of the RPMs obtained, as well as the immunohistochemical expression of MMP-9 and TIMP-1 in these RPMs. Our results showed a large number of fibroblasts and collagen fibers arranged irregularly with inflammatory cells infiltration, as well as ingrowth of new blood vessels in this retrokeratoprosthesis fibrous tissue. Corroborating the results of MMP-9 and TIMP-1 expressions in aqueous humor, our immunohistochemical analysis showed positive staining of retroprosthetic membrane specimens for MMP-9 and TIMP-1. Moreover, aqueous humor MMP-9 levels were found to be significantly higher in KPro-implanted eyes with RPM developed when compared with that of No-RPM developed ones. By contrast, aqueous humor TIMP-1 levels were comparatively lower than that of No-RPM group. All these results suggested that higher expression of MMP-9 may contribute to retroprosthetic membrane formation following KPro implantation. There are several limitations in our study. The most important one is that the lens status was different among the black control group (group I), control group (group II), and KPro group (group III). Lens status has been reported to be a confounding factor on MMP-9 and TIMP-1 levels \[[@B31], [@B32]\], which may partially contribute to the elevated MMP-9 and decreased TIMP-1 levels observed in our study. However, despite the potential effect of lens status, our study did show the potential pathophysiological role of MMP-9 in RPMs formation, as demonstrated by significantly different aqueous humor MMP-9 and TIMP-1 levels between KPro-implanted eyes with and without RPM development. In conclusion, the finding of our study showed the upregulated expression of MMP-9 and downregulated endogenous MMP-9 inhibitor, TIMP-1, after alkali-induced injury to the rabbit corneas and following KPro implantation, and to the best of our knowledge this study is the first to show the potential pathophysiological role of MMP-9 in RPMs formation following KPro implantation in a rabbit model of corneal alkali burn. MMP-9: : Matrix metalloproteinases 9 TIMP-1: : Tissue inhibitors of matrix metalloproteinase 1 KPro: : Boston Type 1 keratoprosthesis RPM: : Retroprosthetic membrane. Conflict of Interests ===================== The authors declare that there is no conflict of interests regarding the publication of this paper. ![Slit-lamp appearance of anterior segment of the rabbit eyes before alkali burn (a), three months after alkali burn (b), and after implantation of Boston Type 1 keratoprosthesis (KPro), with the retroprosthetic membranes developed (c).](JOPH2016-1094279.001){#fig1} ![Clinical observation of alkali-burned rabbit corneas following Boston Type 1 keratoprosthesis (KPro) implantation or penetrating keratoplasty. Photographs taken immediately after operation and at postoperative days 7, 14, 28, 60, and 90.](JOPH2016-1094279.002){#fig2} ![Histopathology of retroprosthetic membrane extracted from the explanted keratoprosthesis in the corneal alkali-burned rabbits. H&E staining of normal cornea ((a) ×100) and retroprosthetic membrane ((b) ×100); transmission electron microscopy of normal cornea ((c) ×4000) and fibrous retroprosthetic membrane ((d) ×4000); immunohistochemical analysis of MMP-9 ((e) ×200) and TIMP-1 ((f) ×200) expressions in normal cornea (MMP-9: (e) ×200; TIMP-1: (g) ×200) and retroprosthetic membrane (MMP-9: (f) ×200; TIMP-1: (h) ×200).](JOPH2016-1094279.003){#fig3} ![Aqueous humor levels of MMP-9 and TIMP-1 in Boston Type 1 keratoprosthesis (KPro) implanted rabbits with or without RPM formation. Pre: preoperation; Day: postoperative day; RPM: retroprosthetic membrane. ^∗∗^ p \< 0.01, ^∗∗∗^ p \< 0.001 versus the No-RPM group.](JOPH2016-1094279.004){#fig4} ###### Dynamic expressions of MMP-9 and TIMP-1 in aqueous humor of alkali-burned rabbit cornea following Boston Type 1 keratoprosthesis (KPro) implantation. Time MMP-9 (ng/mL) TIMP-1 (ng/mL) -------------- --------------------- ------------------ -------------- --------------------- ------------------ -------------- Preoperation 22.85 ± 4.20 22.52 ± 4.03 22.39 ± 3.36 29.42 ± 1.05 29.44 ± 1.08 29.37 ± 1.01 Day 7 33.48 ± 7.29^∗\#^ 43.54 ± 4.39^\#^ 22.47 ± 4.13 27.44 ± 1.16^\#^ 26.57 ± 1.94^\#^ 29.46 ± 1.14 Day 14 40.55 ± 8.43^∗\#^ 75.48 ± 5.20^\#^ 22.41 ± 3.56 24.51 ± 1.98^∗\#^ 21.45 ± 3.59^\#^ 29.51 ± 1.31 Day 28 56.42 ± 3.56^\#^ 52.39 ± 6.83^\#^ 22.40 ± 4.15 21.44 ± 3.57^\#^ 23.50 ± 2.77^\#^ 29.52 ± 1.13 Day 60 69.55 ± 3.52^∗\#^ 45.44 ± 6.02^\#^ 22.39 ± 4.16 19.42 ± 1.96^∗\#^ 25.41 ± 1.96^\#^ 29.54 ± 1.30 Day 90 83.42 ± 7.63^∗\#^ 38.53 ± 3.54^\#^ 22.36 ± 3.55 14.49 ± 4.36^∗\#^ 28.46 ± 6.84 29.58 ± 1.37 Group III: corneal alkali-burned rabbits implanted with Boston Type 1 keratoprosthesis (KPro); group II: corneal alkali-burned rabbits with penetrating keratoplasty; group I: corneal alkali-burned rabbits without keratoplasty. ^∗^ p \< 0.05 versus group II, ^\#^ p \< 0.05 versus group I. [^1]: Academic Editor: Sachin Mulik	{ "pile_set_name": "PubMed Central" }	[ "efigreftypefig", "bc", "amount", "treatment", "zinc", "two", "control", "refer", "remain", "decreas", "fibrou", "administr", "penetr", "impair", "abund", "quick", "collect", "biopsi", "donor", "employ", "identifi", "alcain", "risk", "select", "detach", "miltex", "alkaliinduc", "yellowishbrown", "perfor", "week", "valu", "fix", "multipl", "paraffin", "may", "back", "neovascularis", "piec", "cjophfig", "embryogenesi", "imbal", "pb", "dexamethason", "proteolyt", "paraformaldehyd", "separ", "use", "gener", "etiolog", "germani", "world", "bottom", "caus", "complet", "accord", "conjunctiv", "cfigreftypefig", "signific", "immunohistochem", "establish", "mark", "suggest", "elisa", "process", "nucleu", "retrokpro", "happen", "ffigreftypefig", "also", "growth", "state", "humour", "mgkg", "cell", "conflict", "perform", "commerci", "pmma", "chemic", "graft", "transmiss", "us", "normal", "sutur", "steroid", "forth", 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Introduction ============ The London Psychosocial Group has been funded by Cancer Research UK to implement and evaluate a psychoeducational intervention to promote early presentation amongst older women with breast cancer. The intervention is designed to be radiographer-delivered to women receiving their final invited mammogram with the National Health Service Breast Screening Programme. Objective ========= To report the experiences of research radiographers in delivering the 10-minute intervention to women in breast screening clinics. Methods ======= Reflective diaries and supervision records were used to collate the experiences of radiographers delivering the intervention as part of a research trial and to discuss emerging issues and challenges for widening the role of diagnostic radiographers across the National Health Service. Results ======= The main challenges included learning and rehearsing a structured interview, delivering complex health messages within time constraints using behavioural change techniques, and dealing with unexpected events, including emotional distress, during interviews. Benefits included enhanced interaction with women attending screening and extending the usual role of the radiographer. Conclusion ========== It was beneficial yet demanding for radiographers to extend their role in this way. Not all radiographers may be motivated or suitable to undertake this work. Training and ongoing supervision are essential to support radiographers who deliver the intervention.	{ "pile_set_name": "PubMed Central" }	[ "research", "evalu", "cancer", "work", "clinic", "messag", "collat", "rehears", "part", "method", "includ", "discuss", "trial", "attend", "fund", "suitabl", "yet", "uk", "receiv", "minut", "diari", "may", "unexpect", "reflect", "mammogram", "promot", "interact", "use", "across", "constraint", "conclus", "invit", "final", "ongo", "support", "diagnost", "within", "psychoeduc", "radiograph", "result", "introduct", "intervent", "enhanc", "implement", "motiv", "demand", "emot", "women", "screen", "deal", "chang", "essenti", "nation", "object", "undertak", "older", "role", "widen", "challeng", "distress", "emerg", "design", "extend", "amongst", "event", "record", "way", "breast", "deliv", "supervis", "benefit", "servic", "psychosoci", "experi", "complex", "time", "structur", "present", "group", "london", "main", "train", "health", "programm", "techniqu", "benefici", "behaviour", "report", "issu", "interview", "radiographerdeliv", "earli", "learn", "usual" ]	22,177	[ "Introduction", "London", "Psychosocial", "Group", "funded", "Cancer", "Research", "UK", "implement", "evaluate", "psychoeducational", "intervention", "promote", "early", "presentation", "amongst", "older", "women", "breast", "cancer", "intervention", "designed", "women", "receiving", "final", "invited", "mammogram", "National", "Health", "Service", "Breast", "Screening", "Programme", "Objective", "report", "experiences", "research", "radiographers", "delivering", "intervention", "women", "breast", "screening", "clinics", "Methods", "Reflective", "diaries", "supervision", "records", "used", "collate", "experiences", "radiographers", "delivering", "intervention", "part", "research", "trial", "discuss", "emerging", "issues", "challenges", "widening", "role", "diagnostic", "radiographers", "across", "National", "Health", "Service", "Results", "main", "challenges", "included", "learning", "rehearsing", "structured", "interview", "delivering", "complex", "health", "messages", "within", "time", "constraints", "using", "behavioural", "change", "techniques", "dealing", "unexpected", "events", "including", "emotional", "distress", "interviews", "Benefits", "included", "enhanced", "interaction", "women", "attending", "screening", "extending", "usual", "role", "radiographer", "Conclusion", "beneficial", "yet", "demanding", "radiographers", "extend", "role", "way", "radiographers", "may", "motivated", "suitable", "undertake", "work", "Training", "ongoing", "supervision", "essential", "support", "radiographers", "deliver", "intervention" ]
INTRODUCTION {#sec1-1} ============ The artificial urinary sphincter (AUS) has been considered the standard of care for men with severe stress urinary incontinence (SUI) and/or radiotherapy-related SUI.[@ref1][@ref2] The hydraulically controlled AMS 800™ AUS (Boston Scientific, Minnetonka, MN, USA) remains the most commercially successful and effective sphincteric device since it was developed in 1972.[@ref3] This ingenious urinary device consists of three components: a control pump placed in the scrotum, an inflatable cuff which is usually implanted around the proximal bulbar urethra, and a pressure-regulating balloon in the retropubic space, which serves dual functions, both a pressure regulator and a fluid reservoir. While long-term clinical outcomes, safety profile, and mechanical durability of the current model of the AMS 800 device are well documented, it is not without its limitations and complications such as prosthetic infection and mechanical failure rate.[@ref4] While the clinical use of AMS 800 AUS in a standard male patient with "uncomplicated" SUI history is well established, there are limited high-quality studies pertaining to AUS outcomes in the selected high-risk groups such as neurogenic bladder dysfunction, revision cases, men who require concurrent penile prosthesis implant for erectile dysfunction, and finally, in female SUI. This article explores the clinical outcomes of AUS in these special populations. MATERIALS AND METHODS {#sec1-2} ===================== A critical review of all relevant publications pertaining to AMS 800 AUS was conducted in PubMed and Embase databases, using keywords "urinary incontinence," "AMS 800," "artificial urinary sphincter," "neurogenic urinary incontinence," "revision," "female stress urinary incontinence," and "concurrent penile prosthesis implant." Full surgical descriptions related to AUS implant and/or revision surgery were excluded in this review. The discussion of each category of special populations also includes a brief review of surgical challenge and a practical action-based set of recommendations on surgical options ([Table 1](#T1){ref-type="table"}). ###### Special populations and recommended practical action-based strategies ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Special populations Main issues to considered Surgical strategies References -------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ -------------------------------------------------------------------------------------------------------------------------- Neurogenic Need for self-catheterization (higher risk of erosion with bulbar urethral cuff) Cuff placement at bladder neck [@ref5][@ref6][@ref7][@ref8][@ref9][@ref10][@ref11][@ref12][@ref13] Underlying bladder dysfunction Need for greater intraoperative and postoperative care Higher nonmechanical failure and revision rate Lower continence rate Revision (urethral atrophy) Ischemic changes to previous cuff area\ Replace urethral cuff in a new location Downsizing the urethral cuff size [@ref14][@ref15][@ref16][@ref17][@ref18][@ref19][@ref20][@ref21][@ref22][@ref23][@ref24][@ref25] More difficult surgical dissection Higher risk of infection and nonmechanical failure Tandem or second urethral cuff Transcorporal cuff placement Revising pressure-regulating balloon to high pressure Concurrent erectile dysfunction for penile prosthesis implant Synchronous or staged (delayed) AUS and penile prosthesis implant Synchronous surgery [@ref26][@ref27][@ref28][@ref29][@ref30][@ref31][@ref32][@ref33][@ref34][@ref35][@ref36][@ref37] More difficult surgery and cautious corporal dilatation near the urethral cuff Higher complication rates and the difficulty of manipulating the two scrotal pumping devices Higher risk of infection Staged or sequential (delayed) surgery Cost issue Higher surgery attention to avoid damaging existing implant\ Additional surgical cost Female stress urinary incontinence Usually as salvage option in failed slings Preserving integrity of the vesicovaginal surgical plane during cuff placement at bladder neck [@ref38][@ref39][@ref40][@ref41][@ref42][@ref43][@ref44][@ref45][@ref46][@ref47][@ref48][@ref49][@ref50][@ref51][@ref52] More difficult surgical dissection Higher risk of infection and nonmechanical failure Avoid future vaginal delivery (cuff erosion) ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- AUS: artificial urinary sphincter DISCUSSION {#sec1-3} ========== {#sec2-1} ### Neurologic SUI {#sec3-1} Neurogenic bladder dysfunction due to spinal cord injury (SCI) can result in urinary incontinence, renal impairment, urinary tract infection, bladder or renal stones, and poor quality of life. Most patients will require careful bladder management to ensure a low-pressure bladder, complete bladder emptying, and adequate urinary continence with the use of anticholinergic medications and clean intermittent catheterization (CIC) as first-line therapy, prior to more invasive procedures such as neuromodulation or bladder augmentation. In many SCI patients with persistent urinary incontinence, the AUS provides an ideal continence therapy to address underlying intrinsic sphincter insufficiency, without resorting to urinary diversion. In contrast to the traditional placement of AUS cuff in the proximal bulbar urethra for the treatment of male SUI, the cuff is often placed at the bladder neck or periprostatic tissue to ensure a lower rate of cuff erosion from frequent urethral instrumentations. Furthermore, patients with SCI-related urinary incontinence should be counseled regarding higher risks of nonmechanical device failure and revision surgery and that their overall long-term continence rates may be poorer compared to the nonneurogenic group. Chartier Kastler et al.[@ref5] published a 10-year retrospective series on AUS in neurogenic male urinary incontinence and showed that 74% of the patients had no or moderate incontinence between CICs that spanned at least 4 h, at a mean 83-month follow-up period. The majority of these men had SCI and 39% of men with detrusor overactivity underwent concomitant bladder augmentation. Similarly, Guillot-Tantay et al.[@ref6] reported the 5-, 10-, 15-, and 20-year explantation-free survival rates of 85.7%, 62.3%, 52.0%, and 39.0%, respectively, in SCI men who underwent AMS 800 implantation. The revision-free survival rates were 78.6% (at 5 years), 42.9% (at 10 years), 28.6% (at 15 years), and 7.1% (at 20 years). Approximately 50% of men were continent at recent follow-up visit (mean: 18.3 years). Three native devices were still in place, eight were revised (four of them were secondarily explanted), and three were explanted due to erosion or infection. Comparing the clinical outcomes between AUS cuff placement at bulbar urethra and bladder neck, Khene et al.[@ref7] found a trend favoring bladder neck over peribulbar cuff placement with median revision-free device survival at 14.3 and 11.7 years, respectively (P = 0.73), while the median explantation-free device survival was 24.5 and 18.5 years, respectively (P = 0.08). On multivariate analysis, CIC was the only predictor of AUS device failure. In a study that directly compares the AUS implantation for neurogenic and nonneurogenic incontinence, Murphy et al.[@ref8] found higher revision rates in the neurogenic group (11 out of 13 patients) and only three remained completely dry at a mean 6-year follow-up. In contrast, seven of 17 patients in the nonneurogenic group did not require revision, and eleven were completely dry. The nonmechanical failure rate of the AUS was significantly higher in the neurogenic group. Gonzalez et al.[@ref9] published a retrospective study on AUS and bladder augmentation surgery and reported no significant difference in clinical success between the timing of AUS placement and bladder augmentation. However, the two patients who had sphincter erosions had an injury of the augmented bladder during surgery or belonged to the simultaneous surgery group, highlighting the importance of adequate bowel preparation and maintenance of sterile urine during the AUS surgery. Using a modified surgical implant of a port instead of a pump, Bersch et al.[@ref10] reported that this technique was effective and safe at the reported cure rate at 90% with a 35% revision rate after 8-year follow-up. Robotic-assisted AUS implantation was reported in the early 2010s and Yates et al.[@ref11] showed no complications in six SCI patients. However, longer-term safety and cost efficacy of this novel technique will need to be conducted to compare with traditional open surgical methods. In the younger population, Ruiz et al.[@ref12] found that patients with bladder exstrophy and many previous bladder procedures are more exposed to complications such as device erosion compared with patients with epispadias or anorectal malformation. In another series, Spiess et al.[@ref13] reported that 19 out of 30 (63%) boys were completely dry, 6 (20%) were slightly wet, and 5 (17%) were incontinent, with the mean lifetime of all AUS devices at 4.7 years with no statistically significant difference in device survival between cuff placed at bladder neck or bulbar urethra (4.6 and 4.9 years, respectively). However, survival analysis revealed a sharp drop after 100 months with only 8.3% of the sphincters implanted lasting beyond this point. There were a total of 32 revisions performed in 17 patients constituting a 0.164 revision rate per patient-years. ### Revision group secondary to urethral atrophy {#sec3-2} A common cause of recurrent or delayed SUI following AUS implantation is tissue atrophy resulting in loss of circumferential urethral compression and luminal occlusion. In fact, tissue atrophy is probably the most common cause of nonmechanical failure and the most common cause for AUS revision. Patients who were initially continent with the device usually complain of gradually increasing urinary incontinence over months or even years and report having to squeeze the pump more often to deflate the cuff to void. Withdrawal urethral pressure profile can be conducted with the cuff in inflated and deflated modes to show the difference in luminal pressure while cystourethroscopic evaluation will show ischemic changes to the bulbar urethra and poor mucosal coaptation when the cuff is fully inflated.[@ref14] Surgical options for AUS revision in urethral atrophy include replacement of the urethral cuff in a new location,[@ref15] downsizing the urethral cuff size,[@ref16] placement of a second (tandem) urethral cuff,[@ref17] transcorporal cuff placement,[@ref18] or revising the pressure-regulating balloon to a higher pressure.[@ref19] In a recent publication comparing primary versus AUS revision cases, Suh et al.[@ref20] showed that nonmechanical failure (70.7%) was a dominant etiology of reoperation, and at longer-term follow-up, men with primary AUS performed better than those with AUS revision. While the immediate success rates of primary AUS without reoperation (pAUS) and AUS revision without secondary reoperation (rAUS) groups were 88.6% and 79.2% (P = 0.352), respectively, at a median follow-up of 45.1 (range: 9--126) months, the social continence rate was higher in patients with pAUS (92.1%) than with rAUS (62.5%) (P = 0.001). In an ex vivo culture study, Ziegelmann et al.[@ref21] reported that the two most common reasons for AUS revision were urethral atrophy (n = 31, 39%) and mechanical failure (n = 49, 52%). Excluding patients undergoing revision for infection or erosion, positive culture swabs were identified in 37/200 components (19%), including 25/86 cuffs (29%), 7/56 pumps (13%), and 5/58 reservoirs (9%), with the majority being skin organisms such as Staphylococcus species (57%), Propionibacterium (10%), and Aerococcus (5%). More than a third (39%) of patients had at least one positive component culture, and those patients were more likely to have a history of radiation (65% vs 33%, P = 0.006). Positive AUS component bacterial swab cultures were found in 39% of patients undergoing AUS revision in the absence of clinical infection. Those patients with positive cultures were more likely to have a history of pelvic radiation. These results suggest that bacterial colonization of organisms with low virulence may not lead to device infection. In another large series, Raj et al.[@ref22] showed nonmechanical failure accounted for the majority of AUS revision compared to mechanical failure. Of the 119 patients undergoing secondary implantation, 91 (76.5%) needed no additional surgical intervention, while 28 (23.5%) required a total of 40 surgical revisions for new mechanical (15 \[37.5%\]) and nonmechanical (25 \[62.5%\]) problems. Five-year durability outcomes for primary and secondary AUS implantation were comparable at 80% and 88%, respectively. This study confirmed that outcomes for secondary AUS reimplantation remained comparable to those of primary AUS implantation and that salvage surgery can still produce a good clinical outcome, even following multiple prior revisions and cuff erosion. Linder et al.[@ref23] compared the clinical outcomes between cuff downsize versus tandem cuff placement in AUS revision and concluded that there was no significant difference in the overall device survival in patients undergoing single cuff downsizing or tandem cuff placement during AUS revision for urethral atrophy. Of the 69 revision surgeries for urethral atrophy, 56 (82%) were tandem cuff placements, 12 (18%) were single cuff downsizings, and one was the relocation of a single cuff. Furthermore, there was no difference in 3-year overall device survival compared between the single cuff and tandem cuff revisions (60% vs 76%, P = 0.94). In another retrospective study, O'Connor et al.[@ref24] reported a significantly higher rate of complete continence and improvement in the urinary continence score seen in men with double-cuff compared with single-cuff devices. Daily pad use decreased from 7.7 to 1.1 in patients treated with a single-cuff AUS and from 7.8 to 0.7 in patients with a double-cuff AUS (P = 0.25) and complete continence was reported in 3 (11%) of 28 men with single-cuff and 12 (43%) of 28 men with double-cuff sphincters (P = 0.008). Five complications were reported in the single-cuff recipients and four in the double-cuff patients. In a multicenter study, Eswara et al.[@ref25] reported the outcomes after various revision of AUS strategies and found that tandem cuff placement was associated with a lower rate of incontinence failure (P = 0.02), whereas cuff repositioning was associated with a higher rate of incontinence failure (P = 0.02). An increased rate of mechanical failure was observed with cuff downsizing (P = 0.01). ### Coexisting erectile dysfunction for concurrent penile prosthesis implant surgery {#sec3-3} In patients who have both SUI and erectile dysfunction (ED), it may be necessary to counsel these men regarding synchronous or sequential (delayed) AUS and penile prosthesis implantation. Candidates for dual implants are usually patients who have failed conservative management for both conditions and wish to undergo a single surgery. The theoretical concerns of synchronous prostheses implant are potential higher complication rates and the difficulty of manipulating the two scrotal pumping devices in some patients especially in the early stage. On the other hand, a 2-stage procedure requires more attentive surgery in order to avoid damaging the components of the existing implant and possibly operating in less well-defined tissue planes. In either situation, patients need to be counseled on the pros and cons of dual versus staged implants and technical considerations for the device placement will vary depending on the sequence of device implant and the surgeon preferences. Most surgeons usually advocate the dissection and mobilization of the proximal bulbar first as inadvertent urethral injury would require abandoning or modifying the procedure without discarding any of the prostheses. Corporal dilatation must proceed cautiously in the proximal part of corpora near the cuff. At the level of the bulbar urethra, the corporal bodies have already diverged and therefore the likelihood of injuring the cuff or urethra is lower. Single-step dilatation may be a better option than sequential dilatation to avoid injuring the urethra. The publication of trans-scrotal insertion of AUS implant[@ref26] has led to synchronous dual implants performed through a single incision. If the implants are not placed synchronously, the AUS is usually placed first followed by penile prosthesis for the reasons cited above. In a sequential device implant, the greatest concern is accidental damage to the preexisting device and its tubing. Great care needs to be taken to avoid damaging the AUS cuff when subsequently placing the penile cylinders. Some authors proposed that malleable penile prosthesis is utilized in a sequential implant because of lesser components required and that the implant could be performed more distally on the corporal bodies and hence avoid encountering the AUS device. While it is expected that all implants should have ipsilateral tubing and reservoir placement, surgeons should exercise precaution such as reviewing previous surgical records or organizing preoperative imaging study to avoid intraoperative surprises and the placement of the second implant can be planned for the pump and reservoir at the contralateral side. The incision should be performed away from the existing device, with meticulous dissection and the use of cutting currents to avoid damage to any components of the first device. Prosthetic infection is the most dreaded complication and generally occurs due to the inadvertent introduction of skin organisms at the time of surgery and early postoperative bacteremia. Shortening the operative time would seem a logical way of decreasing this incidence, but published literature on synchronous and sequential dual implants has not revealed any higher incidence of prosthetic infection, probably due to the low incidence of prosthesis infection. In the event of an infection, the concern is that it will spread to all components necessitating their removal and making revision surgery more technically challenging. In order to salvage an infected prosthesis, one needs to determine which prosthetic device is infected and whether it is possible to leave the components of the unaffected device intact. Bhalchandra et al.[@ref27] argued that in this situation, provided that the infected device is identified early, and if one acts promptly to locate the components affected, it is possible to salvage the components of the unaffected device. While device erosion without infection may make salvaging one of the devices possible, in the context of device infection, it is often difficult to ascertain which device is affected, confidently exclude the possibility of only one device being infected and therefore it would be safer to remove both devices. However, Wilson et al.[@ref26] showed that it is possible to preserve one device when the other infected device was removed. The feasibility of salvage surgery was also conferred by Bryan et al.[@ref28] where three patients who had simultaneous dual implant had their device successfully salvaged and one patient underwent two dual salvage procedures. In a multi-institutional study on the dual implant, Kendirci et al.[@ref29] reported no prosthetic infection postoperatively among the 22 men over a mean follow-up of 17 months following a single upper transverse scrotal approach. The overall revision rate was 14%, mostly due to complications associated with the AUS device such as cuff erosion. In a cost analysis study, Sellers et al.[@ref30] showed that dual prosthetic implantation in a single-stage procedure significantly reduced the operative time (24.7%, P \< 0.05) and was associated with approximately a \$7000 cost savings compared with individual procedures, with no report of infective complications or device erosion over the 16 months of follow up. Rolle et al.[@ref31] showed no difference in the pain score, postoperative hospital stays (2.5 days after the double-implant procedure and 2.4 days after AUS alone), and continence rate (65% vs 68%). Furthermore, when given options, all patients stated that they would have preferred synchronous surgery. Mancini et al.[@ref32] also reported encouraging outcomes in patient satisfaction, ease of use, and functionality in the dual implantation group that are similar to those observed in the individual prosthesis group. In fact, most patients (94%) stated that they would recommend the dual implantation procedure to others and have the procedure done again. In their series of 55 combined procedures compared to the single insertion of 336 inflatable penile prostheses and 279 artificial urinary sphincters over a 12-year period, Segal et al.[@ref33] reported that men treated with combined implantation had a greater mean age and were at greater risk for prostate cancer diagnosis and treatment, and at lesser risk for Peyronie\'s disease than men who received an inflatable penile prosthesis alone (each P \< 0.05). Although the operative time was significantly longer for the combined procedure than for the inflatable penile prosthesis alone and the AUS alone (mean: 218.1 vs 145.9 and 114.7 min, respectively, P \< 0.0001), the rate of device infection, erosion, or malfunction was not increased in combined or staged procedures (P \> 0.05). Based on the SPARCS (New York State Department of Health Statewide Planning and Research Cooperative) database for men who underwent inflatable penile prosthesis and/or artificial urinary sphincter insertion between 2000 and 2014, Patel et al.[@ref34] found that combined inflatable penile prosthesis and artificial urinary sphincter insertion portends a higher likelihood of inflatable penile prosthesis reoperation at 1 year (OR: 2.08, 95% CI: 1.32--3.27, P \< 0.01) and 3 years (OR: 2.60, 95% CI: 1.69--3.99, P \< 0.01), while the artificial urinary sphincter outcomes remain comparable. The literature on patients who underwent dual implants after radiotherapy is limited. Kendirci et al.[@ref29] reported that the only erosions following synchronous insertion of AUS and PP occurred in the irradiated patients postradical prostatectomy. The higher AUS revision rates in patients who received previous radiation were also confirmed by Martins and Boyd.[@ref35] The poorer surgical outcomes in irradiated population are likely attributed to several factors such as poorer quality of urethral tissue, difficult surgical dissection with delayed recognition of urethral injury as well as improper cuff sizing, and higher rates of cuff atrophy. In a more complex patient cohort such as those with neobladders, Loh-Doyle et al.[@ref36] concluded that an AUS and PPI can be performed without an increased risk of device-related complications when compared to a control group of nonneobladder patients. Furthermore, among the five prosthetic infection cases, the infection was confined to a single device and salvage surgery was feasible. Dual implantation of AUS and penile prosthesis is feasible, safe, efficient, and cost-effective for the surgical treatment of PPI and ED. The decision to perform synchronous or delayed surgery for concurrent urinary and penile prosthetic implants is dependent on whether concurrent surgery increases the complication rate and is cost-effective in the longer term. Pertinent points to discuss with prospective patients interested in dual implants include counseling on the complexity of the surgery and potential complications and having enough manual dexterity to recycle either pump as required.[@ref37] In a carefully selected group of patients, there is no doubt that most patients are satisfied with the outcomes and highly appreciative of the improvements in their quality of life. From the surgeon point of view, the dual implant of urinary and penile devices is technically challenging, and the surgeon should be competent at placing either device independently before contemplating dual implantation. Dual implantation of urinary and penile prostheses is feasible and safe provided that strict adherence to prosthetic surgical protocols are adhered to and has been shown to be cost-effective and highly efficacious to treat men with PPI and ED. ### Female stress urinary incontinence {#sec3-4} Synthetic sling surgery has gained widespread acceptance as the standard of care in female SUI, but recent negative press related to synthetic mesh has placed considerable concerns among surgeons and patients. In women with multiple continence surgeries, the placement of AUS potentially offers a better continence rate through circumferential compression and coaptation of the "scarred" urethra and remains a safe and effective salvage option in a carefully selected patient cohort. The limited number of publications on the outcomes of urinary continence following placement of AUS in women is likely related to the complexity of AUS surgery and lack of awareness of AUS as an effective treatment option for female AUS. Among all surgical procedures for female SUI, AUS implantation is thought to have the highest long-term (\>5 years) success rate in a recent web-based survey.[@ref38] However, the responding international urologists and gynecologists also think that AUS implantation has the highest risk of complications and/or revisions. Unfortunately, at present, there is no randomized head-to-head trial between minimally invasive synthetic sling surgery and AUS. The earliest publication on AUS implantation for women with SUI was by Scott,[@ref39] who reported more than 90% of patients with socially acceptable continence and 66% of patients completely dry among the 139 female patients with variable ages and mixed etiologies of urinary incontinence when followed up to 6 years. Over the last three decades, published literature shows that AUS surgery is safe and clinically effective in properly selected women with genuine SUI.[@ref40][@ref41][@ref42][@ref43][@ref44][@ref45][@ref46] In fact, the comparison of the clinical outcomes between AUS in men and women were not too dissimilar in terms of continence rate and patient satisfaction.[@ref45] Similar continence rates were also reported in women who received laparoscopic AUS implantation.[@ref47][@ref48] More recently, robotic-assisted AUS implantation has been shown to be equally effective and safe.[@ref49][@ref50] In a pilot study comparing robotic-assisted and open approaches, Peyronnet et al.[@ref51] reported that the change in surgical trend from an open approach (2008--2012) to a robot-assisted approach (2013--2014) was associated with a lower intraoperative complication rate (37.5% vs 62.5%; P = 0.25), decreased blood loss (17 ml vs 275 ml; P = 0.22), and shorter length of stay (3.5 vs 9.3 days; P = 0.09) in the robot-assisted group with comparable continence rates in both groups (75% vs 68.8%; P = 0.75). Most published literature pertaining to AUS implantation in female patients has a shorter-term follow-up of \<5 years. A longer-term follow-up study by Chung and Cartmill[@ref46] showed that more than 80% of AUS devices remained functioning after 100 months and currently has the longest mean follow-up period of 13.5 years. It is difficult to deduce from most published literature with regard to the role of AUS as a salvage surgical option due to the indiscriminate reporting of the outcomes for patients with or without previous anti-incontinence surgeries; incomplete data collection; and the loose definition of continence rates whether it is pad free or with minimal usage of pads. Most published studies on AUS span over the different generations of AUS and advances in technology such as durable kink-proof tubing, lockout valve, as well as the pressure-regulating balloon may have increased the durability of the AUS device.[@ref52] In recent years, there has been a shift in the treatment paradigm for female stress urinary incontinence toward minimally invasive surgery. Currently, the placement of AUS in female patients is only performed in few major teaching centers. Despite the few existing indications for AUS in women, AUS can potentially be a suitable treatment option for females with SUI secondary to intrinsic sphincter deficiency. Perioperative injury remains the most significant risk factor for AUS explantation. Most studies stressed the importance of preserving the integrity of the vesicovaginal surgical plane as the key to successful implantation. In addition, women of child-bearing age should be warned of the danger of cuff erosion during vaginal delivery with advice ranging from no future pregnancy, elective caesarean delivery, and deactivation of the AUS in the final trimester, as recommended to minimize the risk of cuff erosion. Deactivation during labor and delivery is imperative. With proper selection and strict adherence to surgical techniques, AUS is safe, highly effective, and demonstrated excellent long-term outcomes. Perhaps the role of AUS in distressed and socially restricted women with persistent SUI or recurrent urinary incontinence following anti-incontinence surgeries is underutilized and should be given due consideration. CONCLUSION {#sec1-4} ========== Over the past four decades, advances in mechanical design, applications of new technology, and lessons learned from the past clinical experiences have made AMS 800 AUS the standard of care in men with SUI. While the current AMS 800 device provides an effective, safe, and durable therapeutic option, it is not without its limitations and complications, especially with regard to its utility in some of high-risk populations. Increased understanding of the pathophysiology of various SUI cases, coupled with effective strategies, further improves AUS clinical outcomes. However, the emergence of novel therapies such as a nanotechnology-driven device and stem cell therapy may one day circumvent traditional AUS surgery. COMPETING INTERESTS {#sec1-5} =================== The author declared no competing interests.	{ "pile_set_name": "PubMed Central" }	[ "cord", "treatment", "probabl", "khene", "two", "control", "refer", "given", "divers", "remain", "singlestep", "doubleimpl", "decreas", "sui", "respond", "robotassist", "impair", "lifetim", "discard", "collect", "longterm", "bowel", "exist", "identifi", "aerococcu", "length", "risk", "select", "ruiz", "neobladd", "mechan", "neck", "intermitt", "urinari", "locat", "multipl", "deflat", "unaffect", "may", "ppi", "recent", "poorer", "yate", "circumferenti", "toward", "raj", "use", "gener", "etiolog", "accident", "moder", "final", "score", "transcorpor", "complet", "caus", "imag", "prior", "paradigm", "signific", "safer", "depart", "establish", "prosthes", "constitut", "full", "bacteri", "martin", "suggest", "relev", "suirefrefrefrefrefrefref", "excel", "also", "doublecuff", "intervent", "directli", "state", "shift", "transvers", "multivari", "difficult", 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"reported", "prosthetic", "infection", "postoperatively", "among", "men", "mean", "months", "following", "single", "upper", "transverse", "scrotal", "approach", "overall", "revision", "rate", "mostly", "due", "complications", "associated", "AUS", "device", "cuff", "erosion", "cost", "analysis", "study", "Sellers", "et", "al", "showed", "dual", "prosthetic", "implantation", "procedure", "significantly", "reduced", "operative", "time", "P", "associated", "approximately", "cost", "savings", "compared", "individual", "procedures", "report", "infective", "complications", "device", "erosion", "months", "follow", "Rolle", "et", "al", "showed", "difference", "pain", "score", "postoperative", "hospital", "stays", "days", "procedure", "days", "AUS", "alone", "continence", "rate", "vs", "Furthermore", "given", "options", "patients", "stated", "would", "preferred", "synchronous", "surgery", "Mancini", "et", "al", "also", "reported", "encouraging", "outcomes", "patient", "satisfaction", "ease", "use", "functionality", "dual", "implantation", "group", "similar", "observed", "individual", "prosthesis", "group", "fact", "patients", "stated", "would", "recommend", "dual", "implantation", "procedure", "others", "procedure", "done", "series", "combined", "procedures", "compared", "single", "insertion", "inflatable", "penile", "prostheses", "artificial", "urinary", "sphincters", "period", "Segal", "et", "al", "reported", "men", "treated", "combined", "implantation", "greater", "mean", "age", "greater", "risk", "prostate", "cancer", "diagnosis", "treatment", "lesser", "risk", "disease", "men", "received", "inflatable", "penile", "prosthesis", "alone", "P", "Although", "operative", "time", "significantly", "longer", "combined", "procedure", "inflatable", "penile", "prosthesis", "alone", "AUS", "alone", "mean", "vs", "min", "respectively", "P", "rate", "device", "infection", "erosion", "malfunction", "increased", "combined", "staged", "procedures", "P", "Based", "SPARCS", "New", "York", "State", "Department", "Health", "Statewide", "Planning", "Research", "Cooperative", "database", "men", "underwent", "inflatable", "penile", "prosthesis", "artificial", "urinary", "sphincter", "insertion", "Patel", "et", "al", "found", "combined", "inflatable", "penile", "prosthesis", "artificial", "urinary", "sphincter", "insertion", "portends", "higher", "likelihood", "inflatable", "penile", "prosthesis", "reoperation", "year", "CI", "P", "years", "CI", "P", "artificial", "urinary", "sphincter", "outcomes", "remain", "comparable", "literature", "patients", "underwent", "dual", "implants", "radiotherapy", "limited", "Kendirci", "et", "al", "reported", "erosions", "following", "synchronous", "insertion", "AUS", "PP", "occurred", "irradiated", "patients", "postradical", "prostatectomy", "higher", "AUS", "revision", "rates", "patients", "received", "previous", "radiation", "also", "confirmed", "Martins", "Boyd", "poorer", "surgical", "outcomes", "irradiated", "population", "likely", "attributed", "several", "factors", "poorer", "quality", "urethral", "tissue", "difficult", "surgical", "dissection", "delayed", "recognition", "urethral", "injury", "well", "improper", "cuff", "sizing", "higher", "rates", "cuff", "atrophy", "complex", "patient", "cohort", "neobladders", "et", "al", "concluded", "AUS", "PPI", "performed", "without", "increased", "risk", "complications", "compared", "control", "group", "nonneobladder", "patients", "Furthermore", "among", "five", "prosthetic", "infection", "cases", "infection", "confined", "single", "device", "salvage", "surgery", "feasible", "Dual", "implantation", "AUS", "penile", "prosthesis", "feasible", "safe", "efficient", "surgical", "treatment", "PPI", "ED", "decision", "perform", "synchronous", "delayed", "surgery", "concurrent", "urinary", "penile", "prosthetic", "implants", "dependent", "whether", "concurrent", "surgery", "increases", "complication", "rate", "longer", "term", "Pertinent", "points", "discuss", "prospective", "patients", "interested", "dual", "implants", "include", "counseling", "complexity", "surgery", "potential", "complications", "enough", "manual", "dexterity", "recycle", "either", "pump", "required", "carefully", "selected", "group", "patients", "doubt", "patients", "satisfied", "outcomes", "highly", "appreciative", "improvements", "quality", "life", "surgeon", "point", "view", "dual", "implant", "urinary", "penile", "devices", "technically", "challenging", "surgeon", "competent", "placing", "either", "device", "independently", "contemplating", "dual", "implantation", "Dual", "implantation", "urinary", "penile", "prostheses", "feasible", "safe", "provided", "strict", "adherence", "prosthetic", "surgical", "protocols", "adhered", "shown", "highly", "efficacious", "treat", "men", "PPI", "ED", "Female", "stress", "urinary", "incontinence", "Synthetic", "sling", "surgery", "gained", "widespread", "acceptance", "standard", "care", "female", "SUI", "recent", "negative", "press", "related", "synthetic", "mesh", "placed", "considerable", "concerns", "among", "surgeons", "patients", "women", "multiple", "continence", "surgeries", "placement", "AUS", "potentially", "offers", "better", "continence", "rate", "circumferential", "compression", "coaptation", "scarred", "urethra", "remains", "safe", "effective", "salvage", "option", "carefully", "selected", "patient", "cohort", "limited", "number", "publications", "outcomes", "urinary", "continence", "following", "placement", "AUS", "women", "likely", "related", "complexity", "AUS", "surgery", "lack", "awareness", "AUS", "effective", "treatment", "option", "female", "AUS", "Among", "surgical", "procedures", "female", "SUI", "AUS", "implantation", "thought", "highest", "years", "success", "rate", "recent", "survey", "However", "responding", "international", "urologists", "gynecologists", "also", "think", "AUS", "implantation", "highest", "risk", "complications", "revisions", "Unfortunately", "present", 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1. Introduction {#sec1} =============== First synthesized in 1891 and with current production estimated at 4 billion kilograms each year globally \[[@B1]\], bisphenol A (BPA) is a multipurpose compound that is widely used in the modern industrial world. BPA was initially investigated for its potentially therapeutic estrogenic properties in the 1930s; when diethylstilbestrol (DES) was found to be more potent, however, BPA was temporarily cast aside. Its commercial value was reassessed in the 1950s with the introduction of BPA as a fundamental component in the manufacturing of some plastics. As its primary use currently, BPA is a key monomer in the production of the most common form of clear and shatter-proof polycarbonate plastic, but it has also been incorporated into a variety of everyday goods. Questions regarding the safety and side effects of BPA began to emerge in the late 1990s when BPA was found to leech out of plastics and into experimental animal subjects, resulting in an increased incidence of chromosomal anomalies in offspring \[[@B2]\]. There has since been ongoing discussion in both scientific and political spheres about the potential for harm resulting from human BPA exposure and potential bioaccumulation. An overview of the literature regarding the effects of BPA on human health is provided, followed by a presentation of data from 20 subjects whose blood, urine, and sweat were tested for BPA. Results and discussion regarding BPA bioaccumulation and elimination are presented for consideration. 2. Background {#sec2} ============= Currently, BPA is most commonly found as a component in polycarbonates (\~74% of total BPA produced) and in the production of epoxy resins (\~20%). As well as being found in a myriad of products including plastic food and beverage containers (including baby and water bottles), BPA is also commonly found in various household appliances, electronics, sports safety equipment, adhesives, cash register receipts, medical devices, eyeglass lenses, water supply pipes, and many other products. It is also frequently used as an adjunct in the production of brominated flame retardants and brake fluid \[[@B1]\]. Moreover, BPA derivatives, such as bisphenol A-glycidyl methacrylate and bisphenol A-dimethacrylate, have recently been incorporated into the dental industry and used in dental fillings and sealants. The widespread use of this compound is receiving increasing scrutiny as concerns about BPA effects on human health have recently emerged. The main mechanism by which the population is exposed to BPA is through leaching from plastic products. This results from either the release of unpolymerized monomers or the slow decay of polymer bonds in polycarbonates leading to monomer release into proximal foods and liquids. Occupational exposures are also present where plastics are burned and manufactured, and thus BPA may be inhaled by workers \[[@B3], [@B4]\]. An analysis of Chinese employees in factories where BPA and epoxy resins are produced, for example, revealed that over 90% of exposed workers have notable levels of BPA in their serum and urine \[[@B5]\]. A plethora of recent studies affirms that the majority of the population (91--99%) does indeed have detectable levels of BPA, but the level and the toxicological relevance of current exposure levels is a subject of intense academic and public health debate \[[@B6]--[@B13]\]. An extensive review conducted in 2007 concluded that BPA levels in human blood and/or urine are within the range shown to be dangerous in animals and are therefore likely to be biologically active in humans \[[@B6]\]. (As will be discussed, however, blood and urine testing may underestimate the full extent of exposure and bioaccumulation.) Conversely, an industry-sponsored literature review from 2008 declared that daily human consumption was far below dangerous levels and is therefore of minimal concern \[[@B7]\]. Sources of BPA ingestion may vary. In infants and children, baby and beverage bottles used by most individuals in the pediatric population provide ongoing daily sources of BPA \[[@B14]--[@B22]\]. Le et al. found that at room temperature, leaching of BPA occurred into the contained fluid, which increased 55-fold if boiling water was added \[[@B14]\]. Another study found that exposure levels increased not only with temperature, but also with repeated use of a container \[[@B15]\]. Other common sources of ingestion include foods stored in food cans, which are lined with BPA epoxy resin films to prevent corrosion \[[@B23]--[@B28]\], thermal printing paper commonly used in cash register receipts \[[@B29]--[@B31]\], and BPA containing dental composites and sealants \[[@B32]--[@B34]\]. Medical equipment is also raising concerns about BPA levels as a study of newborns found that those who regularly spent time in a neonatal intensive care unit had significantly higher serum BPA levels than the general population---thought to be due to exposure to plastics in medical devices \[[@B35]\]. Likewise, dialysis patients appear to have higher rates of exposure, which may be attributable to circulating solvents which expedite the leaching of BPA from polycarbonate hemodialysis equipment \[[@B36], [@B37]\]. When ingested, unconjugated BPA---the biologically active form of BPA---has historically been thought to be rapidly conjugated in the liver and then excreted through bile or urine, with a half life of approximately 5.3 hours \[[@B38]--[@B40]\]. This rapid excretion has been the basis of reassuring safety evaluations and declarations given by some public health authorities worldwide. However, within many tissues, particularly the lungs, livers and kidneys in rats, and the placenta of animals and humans, β-glucuronidase enzyme is present at detectable concentration. This enzyme is able to deconjugate BPA and thus release its active form again \[[@B41]\]. This is of great significance, as it is plausible that, in pregnancy, the conjugated form of BPA will circulate through the placenta, undergo deconjugation, and cause subsequent fetal exposure in utero. This may also result in bioaccumulation of some portion of BPA after exposure. In fact, recent evidence suggests that at low concentrations, while most plasma BPA (about 95%) is bound to serum proteins, BPA has lipophilic affinity with a fat: blood coefficient of 3.3 \[[@B42]\]. Furthermore, BPA appears to have a disproportionate affinity to fat in comparison to other tissues such as kidney, muscle, and other sites; "in fat, the accumulation of BPA was about three times higher than in other tissues" \[[@B42]\]. With evidence of potential bioaccumulation, BPA has the prospect of exerting ongoing metabolic effects. 2.1. Potential Implications of BPA Exposure {#sec2.1} ------------------------------------------- BPA is thought to wield its effects through endocrine disruption, epigenetic modification, cytokine release, and oxidative stress. When first discovered, BPA was investigated for its estrogenic properties, as it is thought to alter the synthesis of estradiol and testosterone and interfere with receptor binding \[[@B43], [@B44]\]. Consequently, exposure to BPA has been linked with a number of developmental and reproductive pathologies in both animal models and human subjects. These include abnormalities in reproductive organ function (irregular cycles, multiple ovarian cysts, reduction in primordial follicles \[[@B45]--[@B49]\]), placental dysfunction \[[@B50]\], increased incidence of miscarriage and neonatal mortality \[[@B50], [@B51]\], precocious puberty \[[@B52]\], and sexual dysfunction such as erectile dysfunction, decreased libido, and ejaculation difficulties \[[@B53]--[@B55]\]. Moreover, interference with the production and signaling of sex hormones has led to neurological impairment \[[@B56]--[@B60]\]. Synapse formation during development is regulated by estrogen and androgens; however with exposure to BPA, a recent study found that levels deemed safe by the US Environment Protection Agency, completely abolish the response of synapses to estrogen in the prefrontal cortex and hippocampus \[[@B61]\]. Epigenetic effects of BPA have been associated with an increased risk of cancer, particularly breast and prostate malignancies \[[@B62]--[@B69]\]. The exposure of breast epithelial cells to BPA was found to alter gene expression of 170 genes and increase their vulnerability to other carcinogens \[[@B62], [@B63]\]. Additionally, there was silencing of lysosomal-associated membrane protein 3, as occurs in ERα-positive breast cancer \[[@B62]\]. Similar effects have been seen with respect to prostatic disease, as exposure to BPA has been repeatedly shown to modify methylation of implicated genes \[[@B64]--[@B66]\]. Low dose BPA exposure at weaning during the perinatal period has also been found to increase adipogenesis in female animals \[[@B70]\]. As it is hypothesized that adult body weight may be programmed during early life, these results are noteworthy with regards to the childhood obesity pandemic and the action of endocrine disruptors as determinants of obesity \[[@B70]\]. BPA exposure appears to have widespread impact as it has also been linked by various researchers and studies throughout the world with a whole host of other health problems, including metabolic syndrome, obesity, non-insulin-dependent diabetes mellitus, allergies and asthma, ADHD, autism, cognitive decline, memory impairment, depression, and anxiety \[[@B71]--[@B92]\]. With the rise of sensitivity-related illness, there is also concern that BPA may be a determinant of this recently recognized causative mechanism of disease and source etiology of assorted clinical conditions \[[@B93]\] by stimulating the release of proinflammatory adipokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) from human adipose tissue \[[@B71]\]. As a result of all the emerging attention in the scientific literature, various governments have also embarked on research and policy decisions relating to BPA. In 2010, for example, the Minister of Health for the Canadian Government declared the results of a four-year study indicating, "The Government of Canada is a world leader in chemicals management. Our science indicated that Bisphenol A may be harmful to both human health and the environment" \[[@B94]\]. With emerging information of concern about BPA, the Canadian government became the first to prohibit the sale of BPA-containing polycarbonate baby bottles. France, Denmark and several American states have since implemented similar regulations. 2.2. Limitations of Toxicant Biomonitoring {#sec2.2} ------------------------------------------ It is often assumed that after exposure, BPA is rapidly metabolized to a hormonally inactive metabolite and efficiently excreted in toto from the body \[[@B95]--[@B97]\]. As a result, there has been little concern about bioaccumulation. The question arises, however, that if the compound is rapidly excreted without any accrual, why is there increasing evidence that BPA exposure is anything but innocuous and is able to cause potentially serious problems in animal and human organisms? While some believe that only unremitting ongoing exposure to BPA generates risk, concern has been raised that unrecognized bioaccumulation of some fraction of BPA may occur in some exposed individuals. How does one monitor to determine if toxicant compounds bioaccumulate and thus remain within the human organism? Throughout the world, blood and urine sampling are the general modalities used to biomonitor levels of most toxicant compounds including toxic elements, synthetic compounds, petrochemical compounds, biologic toxicants such as mycotoxins, and xenobiotics sometimes produced as byproducts from processing of parent compounds \[[@B98]\]. There is increasing evidence, however, that relying on blood and urine measurements as indicators of bioaccumulation can be very flawed \[[@B99]\]. Many compounds sequester in tissues and do not remain in blood; testing of whole blood or serum may miss toxicants which have exited the blood compartment and are being stored primarily in tissues such as bone, muscle, or adipose compartments. Levels of toxicant compounds can also rapidly fluctuate with changes in immediate status such as caloric restriction, level of hydration, underlying nutrient status, thermal changes, or exercise \[[@B98], [@B100]\]. Reliance on blood or urine testing for assessment of the body burden of many toxicants may thus be less than reliable clinically or for public health purposes. As a result, attempts to biomonitor toxicant levels by sampling other tissues and bodily excretions have been explored, including hair sampling, salivary testing, stool sampling, perspiration testing, breath analysis, provocation testing, and biopsies of adipose tissue through needle aspiration into fat pads under the skin. It is evident, however, that there are limitations with each of these approaches. Hair samples, for example may only reflect what has been in the blood stream for the last few weeks, while stool samples only assess what is being eliminated though fecal waste---these do not measure the body burden. As detailed toxicokinetics for many xenobiotic compounds are not fully understood, it is difficult to know which proportion of parent compounds and their metabolites accrue within various bodily compartments. Some researchers have recently commenced doing fat biopsies as a tool to biomonitor toxicant levels---this technique involves taking a sample of fat, usually from the abdominal or gluteal area and sending the sample for analysis. Recent evidence, however, confirms that toxicants sequester differently even within specific compartments such as adipose tissue; one adipose tissue site may display toxicant concentrations that are totally different than concentrations at another site \[[@B101]\]. So the toxicant concentration in brain adipose tissue, for example, may be very different than that found in breast or abdominal wall adipose tissue. In review, attempts to biomonitor the levels of toxicant compounds, including BPA, in humans using a single modality such as blood or urine are inadequate at best. This is a challenging realization as most population studies on toxicant compounds reported in the scientific literature as well as most ongoing biomonitoring research is based on blood or urine testing. 2.3. Study Objective {#sec2.3} -------------------- BPA exposure is generally assessed by measuring urine levels of this compound. In this research, we endeavor to determine the relative concentrations of BPA in blood, urine, and sweat. By assessing BPA levels in these three compartments, the possibility of identifying retained BPA will be explored as well as the potential for induced perspiration as a means to eradicate this compound. 3. Methods {#sec3} ========== 3.1. Participant Recruitment {#sec3.1} ---------------------------- 9 males and 11 females with mean ages 44.5 ± 14.4 years and 45.6 ± 10.3 years, respectively, were recruited to participate in the study after appropriate ethical approval from the Health Research Ethics Board of the University of Alberta. 10 participants were patients with various clinical conditions and 10 were otherwise healthy adults. Participants with health issues were recruited from the first author\'s clinical practice by invitation. Each participant in the study provided informed consent and volunteered to give one 200 mL random sample of blood, one sample of first morning urine, and one 100 mL sample of sweat. Demographic and clinical characteristics of all research participants are provided in [Table 1](#tab1){ref-type="table"}. 3.2. Samples Collection {#sec3.2} ----------------------- All blood samples were collected at one Dynalife laboratory site in Edmonton, Alberta, Canada through vacutainer blood collection equipment (BD Vacutainer, Franklin Lakes, NJ, 07417, USA) using 21 gauge stainless steel needles which were screwed into the "BD Vacutainer One-Use Holder" (REF 364815). The 10 mL glass vacutainer was directly inserted into the holder and into the back end of the needle. This process and the use of glass were used to preclude contamination. Blood was collected directly into plain 10 mL glass vacutainer tubes, allowed to clot, and spun down 30 minutes later. After serum was separated off, samples were picked up by ALS Laboratories (about 3 kilometres from the blood collection site) for storage pending analysis. When received at ALS, serum samples were transferred to 4-mL glass vials and stored in a freezer at −20°C, pending transfer to the analytical laboratory. We chose to analyze BPA in serum rather than in whole blood, based on the fact that matrix effect of serum is much lower than whole blood. For urine collection, participants were instructed to collect a first morning urine sample directly into a provided 500 mL glass jar container with Teflon-lined lid on the same day that blood samples were collected. Urine samples were delivered by the participants directly to Edmonton ALS Laboratories. Samples were transferred to 4-mL glass vials and stored in a freezer at −20°C, pending transfer. For sweat collection, participants were instructed to collect perspiration from any site on their body directly into the provided 500 mL glass jar container with Teflon-lined lid---by placing the jar against their prewashed skin when actively sweating or by using a stainless steel spatula against their skin to transfer perspiration directly into the glass jar. (Stainless steel---made up primarily of iron, chromium, and nickel---was chosen as it is the same material as the needles used in standard blood collections and is reported not to off-gas or leach at room or body temperature.) Excess of 100 mL of sweat was provided in all but one case. Each of the glass bottles used for sampling in this study was provided by ALS laboratories and had undergone extensive cleaning and rinsing. The containers were deemed appropriate for sweat collection with negligible risk of contamination: laboratory-grade phosphate-free detergent wash; acid rinse; multiple hot and cold deionized water rinses; oven-dried; capping and packing in quality controlled conditions. Sweat was collected within 1 week before or after doing the blood work. No specifications were given as to how long sweating had commenced before collection. 10 participants collected sweat inside an infrared sauna; 7 collected inside a regular steam sauna, and 3 collected during and immediately after exercise---no specific instruction was given regarding the type or location of exercise. Sweat was delivered by the participants directly to ALS laboratories. Samples were transferred to 4 mL glass vials and stored in a freezer at −20°C, pending analysis. No preservatives were used in the jars provided for sweat and urine collection, nor in the serum storage vials. 3.3. Analytical Methods {#sec3.3} ----------------------- Human serum was analyzed for levels of bisphenol A (BPA) at ALS Canada following the general procedures presented by the Centres for Disease Control and Prevention \[[@B8], [@B102]\]. Briefly, samples were fortified with 12.5 nanograms of isotopically labelled phthalate metabolites, 50 nanograms of labeled bisphenol-A, 250 nanograms of 4-methylumbelliferone glucuronide, 300 microliters of ammonium acetate buffer (pH 6.5), and 10 microliters of β-glucuronidase (Escherichia coli K12, Roche Biomedical). The samples were mixed and incubated at 37°C overnight to allow for the deglucuronidation. Following enzymatic hydrolysis, a 20 uL aliquot of the sample is added to 70 uL of HPLC-grade water and 10 ng of labelled 4-methylumbelliferone to determine deglucuronidation efficiency (done once every 100 samples). The remaining sample is loaded onto a Zymark Rapid Trace Station for automated solid phase extraction (SPE). The 60 milligram/3 mL Oasis-HLB cartridges were conditioned with HPLC-grade methanol (2 mL) and 0.1 M formic acid (2 mL). The samples were diluted with 5 mL of 0.1 M formic acid and loaded onto the SPE cartridge at a rate of 1.0 mL/min. The cartridge was washed with water (1 mL) and 10% methanol in water (2 mL) at a flow rate of 1 mL/min. The samples were eluted with 1.0 mL of acetonitrile at a flow rate of 0.5 mL/min. The eluate was evaporated to dryness under a stream of dry nitrogen and the residue was resuspended in 85% methanol in water (200 microliters) and transferred to glass autosampler vials. Quality control of the analysis was maintained by analysing a method blank (calf serum) and two spiked calf serum samples (20 ng/mL, all analytes) along with every 17 samples. The detection limit (0.2 ng/mL) was based upon our lower calibration standard (0.5 ng/mL) which gave an instrument signal to noise response of 3 : 1. Analysis was performed using an API 4000 liquid chromatograph/tandem mass spectrometer. 4. Results and Discussion {#sec4} ========================= Demographic characteristics as well as results for each individual participant are summarized in [Table 1](#tab1){ref-type="table"}. All concentrations are in nanograms per milliliter. The fact that some subjects showed undetectable levels confirms that generalized contamination of these samples is not likely. Furthermore, the levels of BPA were similar to those recently published in studies from Italy and Greece \[[@B103]\] and are comparable with the serum levels found (0.79--7.12 ng/mL) in a recent study by Cobellis et al. in 2009 \[[@B104]\]. The rather low percentage detection among the serum samples in North America is hard to compare as (to our knowledge) there is only one study in the literature that documents BPA levels in blood of North Americans \[[@B105]\]. In that study, using the same extraction method as the methodology used in this study, the authors reported BPA levels in the range of \<0.5 (detection limit) to 22.3 ng/mL in the blood plasma of 40 pregnant American women in the state of Michigan. However, the authors did not report in how many of these 40 women that BPA was detected above their current limit of detection of 0.5 ng/mL. In general, there is major variability in the range of concentrations of BPA detected in blood, and this may be explained by the fact that detection methods vary widely and the specific populations studied also vary considerably \[[@B106]\]. For the urine samples, the percentage detection in the current study (70%) is lower than that of the large scale Canadian biomonitoring study (90.7%) also known as the Canadian Health Measures Survey as reported by Bushnik et al. \[[@B10]\]. However, the geometric mean level of urine BPA in this study is generally higher than those in other biomonitoring studies in North America. Comparative levels for urine BPA as found in the literature are presented in [Table 2](#tab2){ref-type="table"}. For example, Bushnik et al. reported a geometric mean of 1.15 ng/mL urine BPA in Canada for a sample size of 5462 \[[@B10]\], Calafat et al. found a value of 2.4 ng/mL for urine BPA among 950 American adults aged between 20 and 59 \[[@B9]\] and more recently Mendolia et al. reported on 375 American males with a geometric mean value of 1.50 ng/mL of urine BPA \[[@B107]\]. As far as sweat data is concerned, comparison across studies is impossible as to our knowledge this is the first study which attempts to quantify BPA in sweat. One obvious qualitative interpretation of the data from the cohort of 20 study participants is that BPA is rarely detected in blood, and this is probably why most large scale biomonitoring studies, such as the NHANES (National Health and Nutrition Examination Survey) and CHMS (Canadian Health Measures Survey), use urine as the human sample of choice to determine exposure levels in populations. In an attempt to summarize the findings on the distribution of BPA in the 3 different body fluids, we give three 2 × 2 tables in [Figure 1](#fig1){ref-type="fig"}. As discussed earlier, the 2 discordant pairs urine+/serum− and sweat+/serum−, with 12 and 14 in their respective grids show clearly that serum is not the appropriate body fluid to test if BPA biomonitoring in humans is to be characterised. Although there seems to be high correlation between urine and sweat in terms of the presence/absence of BPA in these media, with 12 individuals in the urine+/sweat+ concordant pair, what is more surprising is that there are 4 individuals for which BPA was detected in sweat but undetectable in urine ([Figure 1](#fig1){ref-type="fig"}C). In an attempt to compare the excretion efficiencies of urine and sweat for BPA, we calculated the ratio of BPA concentration in sweat versus urine for those 12 individuals who are in the urine+/sweat+ concordant pair. As shown in [Table 1](#tab1){ref-type="table"}, with the exception of 2 individuals (participants 4 and 11) where urine concentration of BPA is slightly higher than in sweat, in general the ratio is higher than 1, suggesting that induced sweating may be an efficient method for eliminating BPA from the body. This is not surprising in the light of the findings of Csanády et al. whereby they found a preferential partitioning of BPA in adipose tissue compared to blood with a ratio of 3.3 \[[@B42]\]. This suggests that the BPA in blood which is then conjugated and excreted in the urine may only represent about one-third of the body burden of BPA. It is not surprising therefore that induced sweating in saunas can mobilise BPA in adipose tissue thus leading to enhanced excretion in sweat. Given that BPA in body fat is mostly unconjugated, further studies looking at the ratio of free BPA to conjugated BPA in sweat will help to confirm whether the BPA excreted in sweat comes from adipose tissue. Given that in normal circumstances the daily volume of urine is much higher than sweat, urine remains an important mode of elimination of BPA from the human body. An important consideration in response to the results is why there are two participants with evidence of BPA in their urine with no positive level in their sweat. Presumably, the sweat level may be reflective of accrued toxicants in tissue, whereas urine may reflect in part at least recent exposure which the body is endeavoring to eliminate. It may be that BPA begins to bioaccumulate only after a threshold of exposure is reached or once the detoxification mechanisms of the organism are unable to completely eliminate the BPA load that presents after exposure. Further research is required in order to clarify, but this result may indicate that the two individuals have had some recent exposure, but no significant level of stockpiled toxicant. Similarly, the marked variation in the range of the sweat/urine ratio may once again be reflective of different phenomenon: the sweat results may represent transcutaneous excretion of accured BPA toxicant, while the urine results are perhaps reflecting recent BPA exposure in addition to some release of accrued BPA that the body is able to eliminate through renal mechanisms. It is also noteworthy that there was no statistically significant difference (P-value \>0.05) in sweat BPA levels depending on the method of sweat collection whether through exercise, infrared sauna, or regular sauna. Although this study sheds light on the importance of sweat as a pathway of elimination for BPA from the body, it has some limitations. First, given the small study size (n = 20) it is not possible to extrapolate the findings to the general population. Secondly, samples were analysed for total BPA, instead of unconjugated BPA and conjugated BPA separately. Thirdly, other rare forms of BPA such as chlorinated and sulfated BPA were not analysed for. 5. Conclusion {#sec5} ============= As a result of increased scrutiny of health sequelae associated with human BPA exposure, it is apparent that this endocrine-disrupting compound has potentially negative consequences for the human organism. With new evidence for the possibility of BPA accrual within the body, interventions to facilitate elimination of this toxic compound have clinical relevance with regards to the prevention and treatment of adverse outcomes associated with BPA bioaccumulation. The results of this study suggest that: (i) Sweat testing may be an additional tool for BPA bio-monitoring; and (ii) Induced sweating appears to be a clinically useful tool to facilitate the release of BPA through the skin in order to eliminate this toxicant from the human body. Notable findings and implications of data from this study(i) BPA is excreted in sweat(ii) Sweat BPA concentrations are consistently much higher than urine(iii) Only 2/20 participants had BPA in serum, while 16/20 had BPA in sweat(iv) The data suggests that BPA likely bioaccumulates to some degree in humans(v) The data suggests that BPA retained in tissues (likely adipose) excretes via sweat(vi) The finding in some individuals that little or no BPA is excreted in urine while considerable levels are found in sweat suggests that current biomonitoring via serum (as done in Europe) or urine (as done in North America) may not provide a reliable indication of the BPA toxicant burden(vii) With the recognition that BPA has the potential for hormonal dysregulation, the significance of accrued BPA remains to be conclusively elucidated Conflict of Interests {#sec6} ===================== There is no conflict of interests. No funding has been received for any part of this work. The authors thank Jarrod Roberts for his technical work and assistance with analysis of samples. ![2 × 2 tables indicating the presence of BPA in specific body compartments. Each cell represents number of study participants.](JEPH2012-185731.001){#fig1} ###### Participant results for BPA in three body compartments: serum, urine, and sweat. Participant Gender Age Clinical diagnosis Serum conc. Urine conc. Sweat conc. Sweat/urine ratio Technique used for sweat collection ------------- -------- ----- --------------------------------- ------------- ------------- ------------- ------------------- ------------------------------------- 1 M 61 Diabetes, obesity, hypertension 0 4 82 20.5 Exercise 2 F 40 Rheumatoid arthritis 0 22 24 1.1 Steam sauna 3 M 38 Addiction disorder 0 20 22 1.1 Steam sauna 4 F 25 Bipolar disorder 0 40 22 0.6 Steam sauna 5 F 47 Lymphoma 0 10 24 2.4 Steam sauna 6 F 43 Fibromyalgia 0 32 0 n/a Steam sauna 7 F 48 Depression 0 0 16 n/a Steam sauna 8 F 40 Chronic fatigue 0 0 22 n/a Infrared sauna 9 F 68 Diabetes, fatigue, obesity 0 0 10 n/a Steam sauna 10 M 49 Chronic pain, cognitive decline 0 8 10 1.3 Exercise 11 M 53 Healthy 10 32 20 0.6 Exercise 12 M 23 Healthy 0 30 46 1.5 Infrared sauna 13 M 21 Healthy 30 4 10 2.5 Infrared sauna 14 F 47 Healthy 0 8 12 1.5 Infrared sauna 15 M 53 Healthy 0 4 35 8.8 Infrared sauna 16 F 43 Healthy 0 0 12 n/a Infrared sauna 17 F 51 Healthy 0 0 0 n/a Infrared sauna 18 M 46 Healthy 0 42 0 n/a Infrared sauna 19 M 57 Healthy 0 0 0 n/a Infrared sauna 20 F 50 Healthy 0 8 22 2.8 Infrared sauna ###### Comparison of urine BPA levels across published studies. Urine levels of BPA (ng/mL)-comparison across studies ------------------------------------------------------- --------- ------- ----- ------------------ ------ ---------------------- ----------------------------- This study Canada LC-MS 0.2 20 adults 70 AM:13 Median 8 0--42 Bushnik et al. \[[@B10]\] Canada GC-MS 0.2 5462 (age 6--79) 90.7 GM 1.16 (1.08--1.24) N/A Calafat et al. \[[@B9]\] USA LC-MS 0.4 950 adults N/A GM 2.4 N/A Calafat et al. \[[@B8]\] USA GC-MS 0.1 394 adults 95 GM 1.33 Median 1.28 0.1--5.18 (95th Centile) Moors et al. \[[@B108]\] Germany GC-MS 3 15 adults 60 ND-55 Mendiola et al. \[[@B107]\] USA LC-MS 0.4 375 males 90 GM 1.50 \<0.4--6.5 (95th Centile) DL: detection limit; AM: arithmetic mean; GM: geometric mean; ND: non detect; N/A: not available. LC-MS: liquid chromatography-mass spectrometry. GC-MS: gas chromatography-mass spectrometry. 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Introduction ============ Liver abscesses contain pus and develops due to bacteria or amoebae. In some cases, they can be fatal, so appropriate treatment is required to obtain a favorable prognosis. In the past, this disease had a high mortality rate. It was treated either conservatively by administering antibiotics and observing the course of the disease or by performing laparotomy drainage, and the mortality was 9-80% ([@B1]). However, in the last 40 years, the management of liver abscess has greatly changed. With the progress of imaging techniques, in addition to laparotomy drainage, percutaneous transhepatic drainage or drainage by endoscopic retrograde cholangiopancreatography (ERCP) has been developed. Percutaneous transhepatic drainage can be performed safely under computed tomography (CT) or ultrasound guidance, but it is an invasive procedure. In recent years, broader antibiotics have also been developed and are being used to treat bacterial liver abscess, but no criteria have been established for deciding whether it is possible to rely on conservative treatment with antibiotics alone or whether drainage is required. In this study, we examined the efficacy of contrast-enhanced ultrasonography (CEUS) for deciding whether or not liver abscess can be treated conservatively with antibiotics only. Materials and Methods ===================== We performed a retrospective analysis of 21 patients who had been diagnosed with liver abscess via a biochemical examination of the blood, conventional ultrasonography (US), CEUS, and contrast-enhanced CT (CECT) in the gastroenterology department of our hospital from July 2011 to December 2015. The diagnostic criteria included hypoechoic to hyperechoic lesions and the detection of internal echoes reflecting debris or septation on US/CEUS and round lesions with central hypoattenuation, peripheral rim enhancement or surrounding edema on CECT. US/CEUS and CECT were performed for the initial diagnosis. The ultrasonic devices used for conventional US/CEUS were a Aplio500, Xario (Toshiba, Tokyo, Japan), LOGIQ E9, LOGIQ E9 XD Clear 2.0 (GE Healthcare, Chicago, USA), and Ascendus (Hitachi, Tokyo, Japan). The contrast agent was Sonazoid^Ⓡ^ \[common name perfluorobutane; Dai-Ichi Sankyo Seiyaku (Tokyo, Japan)\]. Sonazoid^Ⓡ^ was administered intravenously at 0.01 mL/kg, and after flushing with 10 mL of saline, an evaluation was made in the arterial-predominant phase (10-30 seconds following administration of the contrast agent) and the post-vascular phase (10 min after injection and lasting for 1 hour or more) ([@B2]). In the conventional US and CEUS examinations, videos were recorded, and the abscess size, stain area rate relative to the whole abscess area in the arterial-predominant phase and defect rate of the whole abscess in the post-vascular phase were calculated ([Fig. 1](#g001){ref-type="fig"}). In cases with multiple liver abscess, we evaluated the largest abscess using CEUS. The stain area rate was evaluated in the maximum parting plane of the abscess. CEUS was performed for the initial diagnosis by a skilled Ultrasound Physician who was a supervisor of the Japan Society of Ultrasonics in Medicine with 20 years of experiences (C.O.). The diagnosis and analysis were performed by two doctors who are board-certified hepatologists of the Japan Society of Hepatology. The strategy in our hospital is to start antibiotics at the time of the diagnosis of hepatic abscess, with drainage treatment added at the judgment of the attending physician if exacerbation was observed in the clinical course. ![Results of an examination with conventional US (a) and CEUS in the arterial-predominant phase (b). CEUS revealed a clear boundary between the necrotic area and normal liver cells.](1349-7235-59-0471-g001){#g001} Patients were evaluated for their age, gender, presence of dementia, diabetes mellitus, cancer, outcome, hospitalization, period, whether or not drainage was performed (including catheter drainage, needle aspiration and surgical operation) and whether or not antithrombotic drugs were being taken. They were divided into two groups: the vascular phase enhancement (VE) group, in which ≥50% or more of the whole abscess was enhanced in the arterial-predominant phase, and the vascular phase non-enhancement (VNE) group, in which \<50% of the whole abscess was enhanced in the arterial-predominant phase ([Fig. 2](#g002){ref-type="fig"}), and the proportion of improvement by the use of antibiotics only and patient characteristics were recorded ([Table 1](#t001){ref-type="table"}). We compared the stain rate in the arterial-predominant phase with the improvement with antibiotics only. We also compared the defect rate in the post-vascular phase with the improvement with antibiotics only. ![Images of the VE and VEN groups. (a) ≥50% of the whole abscess was enhanced in the arterial-predominant phase. (b) \<50% of the whole abscess was enhanced in the arterial-predominant phase.](1349-7235-59-0471-g002){#g002} ###### Characteristics of the Patients with Improvement with Antibiotics Only and Those with No Improvement by Conservative Therapy. ----------------------------------------------------------------------------------------- Improvement by\ No improvement by\ antibiotics only 12 cases conservative therapy 9 cases ------------------------------ --------------------------- ------------------------------ Sex (male/ female) 7/5 3/6 Age 74.4 (57-92) 65.6 (53-83) Comorbidities Dementia 4 1 Diabetes mellitus 4 5 Cancer (colon cancer) 9 (1) 4 (2) Biliary infection 5 3 Antithrombotic drugs 5 2 Laboratory test White blood cell count (/μL) 9,903 (4,300-18,800) 13,951 (5,940-37,700) CRP 12.7 (3.53-21.25) 22.6 (8.7-35.38) Platelet count (×10^4^/μL) 19.8 (10.4-48.9) 20.4 (8-42.2) DIC 0 5 ----------------------------------------------------------------------------------------- All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics. The chi-square or Fisher\' s exact tests were applied to evaluate the differences in the categorical variables. Continuous data were presented as the mean, and Student\' s t-test was used to evaluate the difference in continuous variables. The statistical analyses were performed with a two-tailed significance level of 0.05. Results ======= The 21 patients were 10 men and 11 women, the mean age was 70.6±13.0 years old (mean ± standard deviation). The average abscess diameter was 55.2 mm, and in all 21 cases, the cause was bacterial. Amoeba cases were not included in this study. Regarding the outcomes, 12 patients improved with antibiotics only, while 9 did not. Of the 21 patients, the VE group consisted of 12 patients (average diameter 41.1 mm), and the VNE group consisted of 9 patients (average diameter 73.9 mm). In the VE group, 11 of 12 patients (91.7%) improved with antibiotics only, whereas in the VNE group, only 1 of 9 (11.1%) improved with antibiotics only, showing a significantly higher proportion of improvement with antibiotics only in the VE group (p\<0.001). The patient in the VE group who did not improve with antibiotics only had a rupture on the liver surface ([Table 2](#t002){ref-type="table"}). ###### Demographics and Abscess Characteristics in the Patients with Improvement with Antibiotics Only and Those with No Improvement by Conservative Therapy. ---------------------------------------------------------------------------------------------------------- Improvement by\ No improvement by\ antibiotics only 12 cases conservative therapy 9 cases ----------------------------------------------- --------------------------- ------------------------------ Abscess size (mm) 39.5 (8-64.7) 76.1 (45-132) Diameter ≤5cm 8 2 Abscess location(right/left lobe) 9/5 6/4 Single / Multiple 7/5 8/1 Unilocular / Multilocular 10/2 5/4 Stain rate in the arterial-predominant phase\ 1/11 8/1 \<50% (VNE group) / ≥50% (VE group) Hospitalization 25.6 (13-50) 37.6 (2-57) ---------------------------------------------------------------------------------------------------------- The mean hospitalization period in the VE group was 47 days when drainage was performed and 28 days without drainage, whereas that in the VNE group was 41.3 days when drainage was required and 14.5 days when drainage was not required (including 1 case who died of other causes). All seven patients with a stain rate of ≥71% improved with antibiotics only (7 out of 7 patients, 100%). In contrast, patients with a stain rate of ≤ 30% did not improve with antibiotics only \[6 out of 7 (85.7%) underwent drainage, and 1 patient had no drainage but died from other causes\] ([Fig. 3](#g003){ref-type="fig"}). ![The number of patients that showed an improvement by antibiotics alone, and those that showed no improvement by conservative therapy were analyzed regarding the stain area rate in the arterial-predominant phase. All patients who obtained an enhancement of ≥71% for the whole abscess in the arterial-predominant phase were treated by conservative therapy, but those who obtained an enhancement of ≦30% for the whole abscess in the arterial-predominant phase did not demonstrate any improvement by conservative therapy.](1349-7235-59-0471-g003){#g003} Considering the defect rate in the post-vascular phase and improvement rate with antibiotics only, 16 out of 21 patients (76.2%) showed a defect rate of ≥71%. Both groups of patients who demonstrated an improvement whether they took antibiotics or not, showed a high defect rate in the post-vascular phase. As a result, it was considered to be difficult to determine the optimal treatment approach base on the post-vascular phase ([Fig. 4](#g004){ref-type="fig"}). ![The number of patients who showed an improvement by antibiotics alone, and those who demonstrated no improvement by conservative therapy were analyzed regarding the defect area rate in the post-vascular phase. Both groups who improved by antibiotics or not, showed high defect rate in the post-vascular phase. As a result, evaluations using the defect area rate in the post-vascular phase are therefore not considered to be useful.](1349-7235-59-0471-g004){#g004} Of the 21 cases, 33.3% (7 of 21) were receiving antithrombotic drugs, 23.8% (5 of 21) had dementia, and 4.8% had both (1 of 21). The presence of either antithrombotic drug treatment or dementia was noted in 52.4% (11 of 21). A total of 61.9% (13 of 21) of patients had malignant disease, so a substantial proportion of patients required special care when performing drainage. The relationship between the abscess size and improvement rate with antibiotics only was also examined. Eight of 10 patients (80.0%) with an abscess size of ≤ 50 mm and 11 of 13 patients with an abscess size of ≤ 60 mm improved with antibiotics only ([Fig. 5](#g005){ref-type="fig"}). ![Relationship between the abscess size and improvement rate with conservative treatment. The rate of improvement in abscesses measuring ≥61 mm in size was not good.](1349-7235-59-0471-g005){#g005} Discussion ========== Liver abscesses are usually composed of a viscous fluid in an intrahepatic partition caused by inflammation as a defense reaction against infection. Conventional US is used to assess the condition of the normal liver, perform an accurate diagnosis in real time, and perform repeated evaluations at a low cost, and this modality is well tolerated without radiation exposure. However, for the diagnosis of liver abscesses, it is limited. Although CECT is extremely useful for evaluating the extent of abscesses and necrosis, it carries a risk of radiation exposure and contrast agent allergy, and it is difficult to perform in patients with renal dysfunction. It also cannot be used repeatedly to evaluate the treatment effect. CEUS solves the above problems, since it can be used without the risk of affecting the kidney function and can be used in real time without a risk of radiation exposure. The utility of CEUS for examining liver abscesses has been reported to be comparable to that of CECT and magnetic resonance imaging (MRI) ([@B3]-[@B5]). The European Federation for Ultrasound in Medicine and Biology (EFSUMB) and World Federation for Ultrasound in Medicine and Biology (WFUMB) guidelines specify ultrasonic patterns for liver abscesses. Conventional US findings of liver abscess are a low-echoic mass with a thick irregular wall and interior partitions, sometimes including gas (presenting as a bright dotted echo with a shadow behind). These are the most frequently observed findings, but they are not specific. Regarding the findings of CEUS, mature abscesses typically show marginal enhancement in the arterial-predominant phase, occasionally with enhancement of septae followed by venous hypoenhancement ([@B2], [@B6], [@B7]). In the present study, the evaluation of the defect rate in the post-vascular phase was not recognized as a useful marker for deciding whether conservative treatment of liver abscess was possible or whether drainage treatment was necessary. However, even if only a few normal liver cells remain, they are likely to be stained in the arterial-predominant phase, so the evaluation of the arterial-predominant phase, where there is a clear boundary from the necrotic area, where normal liver cells are not present, would be a useful marker for deciding whether conservative treatment is possible or whether drainage treatment is required. Several types of ultrasound contrast agents are currently in use, but Sonazoid^Ⓡ^, a second-generation contrast ultrasonic agent, can evaluate the blood flow dynamics of the post-vascular phase as well as that of the arterial-predominant phase, so a more precise diagnosis has become possible ([@B8]). Sonazoid^Ⓡ^ is used clinically in Japan ([@B9]) and in recent years has come to be used in China, South Korea, Norway and other countries as well; however, in countries where Sonazoid^Ⓡ^ is not approved for use, the evaluation in the post-vascular phase is impossible. Thus far, the treatment approach for liver abscess was determined by the number and size of the abscesses. In cases of a single lesion with a diameter of ≤ 5 cm, percutaneous catheter drainage or needle aspiration is often performed. Drainage catheters should remain in place until drainage is minimal (usually up to seven days). Repeat needle aspiration may be required in up to half of cases if a catheter is not left in situ. If the lesion is ≤ 5 cm, the prognosis is expected to be good, regardless of a puncture or catheter being used ([@B10]-[@B13]). For a single, large abscess \>5 cm in diameter, catheter drainage is preferred over needle aspiration. According to Zerem et al., in 60 cases of antibiotics + catheter drainage vs. antibiotic + needle aspiration, in abscesses of ≤ 5 cm, treatment was successful in all patients, irrespective of catheter drainage or needle aspiration. However, in cases with an abscess diameter \>5 cm, catheter drainage was successful in all cases, whereas needle aspiration succeeded in only 50% of cases ([@B13]). Even very large abscesses (\>10 cm) can be successfully managed with catheter drainage, although the risk of treatment failure and other complications is substantial ([@B14], [@B15]). According to Ahmed et al., in Singapore, in 44 cases of hepatic abscesses exceeding 10 cm, 25% of 39 patients who underwent drainage therapy suffered complications such as death, sepsis, and pleural infiltration, and frequent drainage therapy was necessary ([@B15]). However, some reports have claimed that percutaneous transhepatic drainage therapy does not work with abscesses \>5 cm in size ([@B16]). In a retrospective analysis of 80 liver abscesses exceeding 5 cm, the failure rate of percutaneous transhepatic drainage was greater than that of surgical drainage (28% vs. 7%). However, there was no significant difference in the mortality, morbidity, fever period, or incidence of complications. Surgical drainage is usually preferred in the following circumstances: multiple abscesses, loculated abscesses, abscesses with viscous contents obstructing the drainage catheter, underlying disease requiring primary surgical management, and inadequate response to percutaneous drainage within seven days ([@B10], [@B11], [@B17], [@B18]). In our study, 13 of 21 patient had malignant disease. In some reports, liver abscess has been described as occasionally complicated with colon cancer ([@B19]). General screening, especially that of the colon tract, should be performed in hepatic abscess patients. A considerable proportion of pyogenic liver abscesses follow one or more episodes of portal vein pyemia, usually related to bowel leakage and peritonitis. Another important route is the direct spread from biliary infection. Underlying biliary tract disease, such as gallstones or malignant obstruction, is present in 40% to 60% of cases ([@B20]-[@B22]). Occasionally, abscesses arise from surgical or penetrating wounds, including injury from migration of an ingested foreign body ([@B23], [@B24]). Recently, in aging societies, cases with various complications have increased, and patients taking antithrombotic drugs at the diagnosis cannot be punctured. In the present study as well, five patients were taking antiplatelet agents, and two were taking anticoagulants. In the future, the proportion of high-risk patients in whom puncture is difficult will increase. For this reason, it was considered very useful to identify a predictor of conservative treatment based on noninvasive contrast echography at an early stage. In the present study, we found that the stain rate of the abscess in the arterial-predominant phase of contrast ultrasound was useful as a marker for conservative treatment. If the arterial-predominant phase has a high stain rate, there is a strong likelihood that risky drainage treatment can be avoided without prolonging the hospital stay. Although the identification of pathogenic bacteria was not possible partially due to the fact that drainage was not performed, it may nevertheless be possible to successfully detect pathogenic bacteria in blood cultures and thereby choose the optimal sensitive antibiotics. If pathogenic bacteria are not detected in a blood culture and the patient\' s medical condition is getting worse, then drainage treatment should be carefully considered, including the identification of the causative bacteria and the selection of appropriate antibiotics. In addition, regarding the abscess size, 8 of 10 cases with an abscess size of ≤ 50 mm, and 11 of 13 cases with an abscess size of ≤ 60 mm improved with antibiotics only. Since it becomes difficult to form an abscess cavity as the abscess diameter shrinks, this is thought to be correlated with the stain rate in the arterial-predominant phase. Although the diameter of the abscess is useful as a marker, it is thought that the stain rate may be more useful, as in this study, 11 of the 12 patients in the VE group improved with antibiotics only. In the VE group, the hospitalization period was not extended, even when drainage was not performed. Due to the fact that the non-necrotic area was deemed to be indicated for conservative treatment and the area had still not liquefied, the benefits obtainable by drainage were therefore thought to be negligible. In the VE group, one patient who did not improve with conservative therapy had an abscess that burst near the liver surface. When an abscess is near the liver surface, the risk of rupture should always be considered. Our study was limited by the small sample size and the use of only a single contrast agent that is not available in many countries. Conclusion ========== Although decision-making in cases of liver abscess is difficult based on the post-vascular phase of CEUS, the enhancement rate in the arterial-predominant phase can predict the response to conservative treatment. The authors state that they have no Conflict of Interest (COI). 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1.. Introduction ================ Lung cancer is the leading cause of cancer deaths in both men and women in the United States, with an overall 5-year survival rate of 10--15% that has persisted for decades \[[@b1-cancers-03-02975]\]. There are two types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). About 85% of all lung cancers are NSCLC, which can be further divided into three main types: large cell carcinoma; squamous cell carcinoma; and adenocarcinoma of the lung. Adenocarcinoma of the lung is the single most common type of lung cancer; it accounts for about 40% of all lung cancers. Although NSCLC grows more slowly and is less aggressive than SCLC, the most efficient treatment for NSCLC is considered to be surgical resection of the tumor, while SCLC is remarkably sensitive to chemotherapy and radiation treatments. The low survival rate of NSCLC patients is related to the late presentation of patients with an unresectable tumor due to the lack of a validated screening approach for early detection. By early detection of precancerous microscopic lesion, patients can avoid developing clinically diagnosable cancer and high-risk healthy individuals can be prevented from developing cancer by timely chemoprevention. Therefore, early detection of lung cancer, followed by appropriate treatment, will significantly increase the survival rate. Low-dose computerized tomography (CT) scan has suggested a promising possibility to detect lung cancer earlier. Compared to chest X-rays, CT screening significantly reduced mortality in a high-risk population by 20.3% based on the recently reported National Lung Screening Trial (NLST), a large randomized trial funded by the National Cancer Institute (NCI)\[[@b2-cancers-03-02975]\]. Although it is promising, the limited impact of CT screening on mortality may be because it cannot distinguish non-calcified nodules (NCNs) that progress to cancer from the benign ones. It also has limited ability to differentiate between benign and malignant lesions of tumors, especially the centrally located ones \[[@b3-cancers-03-02975]\], demanding a complementary screening tool to increase specificity of detection for cancer. False-positive CT screening results will lead to unnecessary invasive procedures, which can cause stress and economic burden. Therefore, there is still a demand for non-invasive, sensitive biomarkers in order to accurately identify the high-risk population for optimal treatments and minimize unnecessary invasive procedures caused by false-positive CT screening results. These biomarkers will be also useful to increase sensitivity for the detection of centrally located lung cancer and to determine the strategy for the therapeutic management of clinically uncertain nodules that are detected by CT screen. Tobacco smoking is the single, most important risk factor for lung cancer. An estimated 90% of lung cancer is due to cigarette smoking, which also causes at least 18 other types of cancer \[[@b4-cancers-03-02975]\]. It is well accepted that the repeated exposure of the respiratory tracts of smokers to tobacco-related carcinogens causes molecular genetic and epigenetic abnormalities that culminate in lung cancer. Consequently, smoking may create a field of molecular injury throughout the airway epithelium exposed to cigarette smoke and increase the susceptibility of an entire field or area to carcinogenesis (field carcinogenesis) \[[@b5-cancers-03-02975]\]. This suggests that the smoke-induced molecular abnormalities found in the respiratory tract (regardless of its exact location) may mirror the progression of cancer and therefore can be used as potential biomarkers to detect lung tumor at an early stage. Thus, sputum that contains exfoliated airway epithelial cells is an ideal specimen type for the discovery of biomarkers for lung cancer. Molecular analysis of sputum has been an active area for the investigation of lung cancer biomarkers for several reasons. Sputum is the most easily accessible body fluid that contains the pathogenically relevant cell type, namely, bronchial epithelial cells. These cells are typically exfoliated from the central airways, where the CT scan seems to have a blind spot when the cancer arises from here. This also suggests that it is possible to identify tumor cells in sputum. Additionally, the other airway epithelial cells found in sputum may also harbor important molecular information, supported by the field cancerization theory \[[@b5-cancers-03-02975]\], which reflects the local milieu of the lung. High-risk smokers typically produce increased amount of sputum, which makes it feasible to obtain enough material for analysis. Finally, collecting sputum is non-invasive, fast, and economical, which are important characteristics to be an ideal specimen type for large-scale population screening. In this review, we discuss various sputum-based molecular biomarkers and how each one has developed during the last decade. We then explore the strengths and weaknesses of each marker and discuss ways to overcome the limitations. 2.. Sputum-Based Molecular Biomarkers ===================================== 2.1.. Cytology -------------- Sputum cytology is the oldest technique that utilizes sputum in detecting lung diseases. Identified and developed by Saccomannao \[[@b6-cancers-03-02975]\], sputum cytology was shown to detect premalignant changes in high-risk groups several years before a clinical diagnosis of lung cancer. This finding is supported by the concept that changes in the cytology of bronchial epithelial cells reflect the progress from inflammation to cancer \[[@b7-cancers-03-02975]\]. However, it is not considered to be sensitive enough mainly because it requires skills that can identify subtle nuclear changes in cells that often comprise less than 5% of the sputum samples. The "subjectivity" of the pathologist performing the cytopathological classification raises concerns about its reliability and reproducibility due to the lack of standardization. The results of two large, randomized clinical trials support this notion \[[@b8-cancers-03-02975],[@b9-cancers-03-02975]\]. In these studies, they compared annual chest X-ray (CXR) plus sputum cytology with CXR only. After nine years of follow-up, they noticed the mortality due to lung cancer was slightly lower in the double-screened group. However, it is arguable whether it could have been occurred by chance. To overcome this limitation of low sensitivity and reliability, sputum cytology has often been used as a supplement to other screening methods. Lam et al. \[[@b10-cancers-03-02975]\] reported that among the high-risk patients with sputum atypia (47%), who were followed by bronchoscopy and CT scans, 15% of these patients had lung cancer. Notably, approximately half of these patients had early-stage cancer. It was reported that the overall sensitivity of sputum cytology in combination with bronchoscopy and CT scan in detecting lung cancer was 71% for all histological types and 100% for squamous cell lung cancer located in the central airways. On the other hand, combining sputum atypia with FISH assay for the detection of gene copy number changes of specific cancer-related genes did not improve the diagnostic performance compared to FISH screening alone \[[@b11-cancers-03-02975]\]. These inconsistent results demand further improvement in the application of sputum cytology for cancer screening. Automated image cytometry for objective and quantitative cytopathological imaging may greatly improve the sensitivity. An automated imaging system quantifying the DNA content had greater sensitivity (80%) than conventional cytology (4.2%) for the diagnosis of lung cancer \[[@b12-cancers-03-02975]\]. Kemp et al. \[[@b13-cancers-03-02975]\] performed multicenter validation trial to compare the automated sputum analysis using DNA cytometry to the conventional one and found that the automated method detected lung cancer with 75% sensitivity while conventional cytology only identified 16%. These studies suggest that an objective, automated imaging system in combination with adequate molecular biomarkers may be a promising tool for the screening of lung cancer. 2.2.. Allelic Alteration ------------------------ Microsatellites are stretches of DNA in which a short motif (usually one to five nucleotides long) is repeated ten to hundred times. Microsatellites are prone to mutations during replication due to the transient split of the two helical strands and slippage of the DNA polymerase complex at reannealing, which generates an insertion or deletion loop depending on the slippage direction \[[@b14-cancers-03-02975]\]. Microsatellite instability (MSI) is a situation in which these gained or lost repeat units as a consequence of the mutation result in a somatic change in length. MSI is known to be associated with defective DNA mismatch repair machinery \[[@b15-cancers-03-02975]\]. In hereditary non-polyposis colorectal carcinoma (HNPCC), in which MSI was first observed and is found frequently, MSI is caused by germline mutations in DNA mismatch-repair genes \[[@b16-cancers-03-02975]\], suggesting the phenomenon of MSI is associated with tumorigenesis. Furthermore, changes in the microsatellite repeats are known to correlate with altered gene expression \[[@b17-cancers-03-02975],[@b18-cancers-03-02975]\]. Thus, MSI in tumor is likely to lead to mutations and deregulated gene expression compared to normal tissue, which may be critical for understanding tumorigenesis. Another form of allelic alteration that can be revealed by MSI analysis is the loss of heterozygosity (LOH). LOH represents the loss of one allele of a gene whereby the other allele is already inactivated. LOH is caused by a variety of genetic mechanisms, including physical deletion of chromosome nondisjunction, mitotic nondisjunction followed by republication of the remaining chromosomes, mitotic recombination, and gene conversion \[[@b19-cancers-03-02975]\]. The frequent allelic loss at particular chromosomal regions in tumors is indicative of the presence of a tumor suppressor gene. Consequently, detection of LOH has been used to identify novel tumor suppressor genes. Furthermore, it is shown that some LOH patterns are associated with different tumor types, pathological stages, and progression \[[@b20-cancers-03-02975],[@b21-cancers-03-02975]\]. Allelic imbalance in lung cancer has been actively investigated. Individual LOH or MSI is rarely used as a stand-alone biomarker because there are numerous of them existing through the human genome and the frequency of the occurrence of each marker hardly provides statistical significance. Therefore, the key is to find the right combination of MSI and LOH to increase the sensitivity and specificity. A few commonly chosen regions for assessing MSI and LOH include, but are not limited to, chromosome 3p and 9p \[[@b22-cancers-03-02975]-[@b26-cancers-03-02975]\]. Notably, 3p deletions are detected in almost 100% of SCLC and more than 90% of NSCLC cell lines \[[@b27-cancers-03-02975]\]. Three bona fide lung cancer tumor suppressor genes, namely RBSP3 (AP20 region), NPRL2 and RASSF1A (LUCA region), were identified in the 3p21.3 region. In addition, the FHIT gene, of which inactivation is frequently found in various human cancers including lung cancer \[[@b28-cancers-03-02975]\], is located at chromosome 3p14. Chromosome 9p21, which was reported to be most frequently deleted in lung cancer cell lines \[[@b29-cancers-03-02975]\], contains two tumor suppressor genes, CDKN2A and CDKN2B. The frequency of individual or combination of several MSIs or LOHs in lung cancer is typically lower than 70% \[[@b22-cancers-03-02975],[@b23-cancers-03-02975],[@b25-cancers-03-02975]\]. However, this low prevalence may be increased by combining with other types of molecular markers, for example, methylation \[[@b26-cancers-03-02975]\] and sputum cytology \[[@b24-cancers-03-02975]\]. 2.3.. Methylation ----------------- DNA methylation is a common epigenetic mechanism that regulates gene expression. It involves the addition of a methyl group to the 5^th^ position of the cytosine base located 5′ to a guanosine in a CpG dinucleotide \[[@b30-cancers-03-02975]\]. A CpG island, a contiguous stretch of DNA of at least 200 bp up to 4 kb, contains high CpG contents \[[@b31-cancers-03-02975]\] and is often found in the promoter regions. Promoter hypermethylation is accompanied by histone modifications such as acetylation, methylation or phosphorylation of histone tails within the island \[[@b32-cancers-03-02975]\]. This series of epigenetic alterations leads to a conformational change of the chromatin in the promoter regions, which prevents the RNA polymerase and other transcriptional regulatory proteins from accessing this region \[[@b32-cancers-03-02975]\]. As a consequence, transcription is blocked and the gene becomes silenced. About 50% of human gene promoters contain CpG islands, which are generally unmethylated in the normal genome. However, in cancer, it is well established that CpG hypermethylation in the promoter region of genes that function in various pathways (such as DNA repair, cell cycle regulation, apoptosis and RAS signaling) is tightly associated with tumorigenesis \[[@b31-cancers-03-02975]\]. There are several genes that are frequently silenced by promoter hypermethylation in lung cancer. P16 (CDKN2A) is a tumor suppressor gene that is hypermethylated at a prevalence of up to 67% in adenocarcinoma and 70% in squamous cell carcinoma of the lung \[[@b33-cancers-03-02975]\]. It regulates the cell cycle and prevents excessive cell growth by regulating the tumor suppressor gene RB1 and its protein, pRb. pRb prevents the cell from replicating damaged DNA by inhibiting its progression along the cell cycle through G1 into S. Intriguingly, inactivation of P16 has been proposed as an early step to immortalization \[[@b34-cancers-03-02975],[@b35-cancers-03-02975]\], supporting its potential to be a useful biomarker for the early detection of cancer. MGMT (O^6^-Methylguanine DNA methyltransferase) is a DNA repair enzyme that is inactivated in 24--48% of lung adenocarcinomas \[[@b33-cancers-03-02975]\]. It antagonizes the genotoxic effects of alkylating agents. MGMT rapidly reverses alkylation, including methylation, at the O^6^ position of guanine by transferring the alkyl group to the active site of the enzyme \[[@b36-cancers-03-02975]\]. Inactivation of MGMT in the cell allows the accumulation of O^6^-alkylguanine adducts in the DNA and can lead to transition mutations in genes such as KRAS and TP53 \[[@b33-cancers-03-02975]\]. RASSF1A (Ras-association domain family member 1A) is a tumor suppressor that is silenced in 40--60% and 100% of NSCLC and SCLC, respectively \[[@b33-cancers-03-02975]\]. RASSF1A lacks apparent enzymatic activity but contains a Ras association (RA) domain and is potentially an effector of the Ras oncoprotein. RASSF1A modulates multiple apoptotic and cell cycle checkpoint pathways. Current evidence supports the hypothesis that it serves as a scaffold for the assembly of multiple tumor suppressor complexes and may relay pro-apoptotic signaling byK-Ras \[[@b37-cancers-03-02975]\]. DAPK (death-associated protein kinase) is a pro-apoptotic, calcium/calmodulin-regulated serine/threonine protein kinase that is hypermethylated in both adenocarcinoma and squamous cell carcinoma at prevalences ranging from 30-48% \[[@b33-cancers-03-02975]\]. DAPK mediates interferon-γ-mediated apoptosis and participates in a number of additional apoptosis-inducing pathways downstream of CD95 (Fas), tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) \[[@b38-cancers-03-02975]\]. Additionally, DAPK inactivation reduces the induction of p19^ARF^/p53, thus inactivating the p53-dependent pathway for apoptosis, suggesting that attenuation of p53 by loss of DAPK may be an important factor in transformation in vivo \[[@b39-cancers-03-02975]\]. These four genes along with a few other genes, including PAX5 and GATA, have been the major focus for examining the sensitivity/specificity of promoter methylation as sputum-based biomarkers for lung cancer detection. Among these, P16 is reported to be the most frequently hypermethylated gene in sputum samples at prevalence ranging from 25--74% \[[@b40-cancers-03-02975]-[@b42-cancers-03-02975]\]. The combination of promoter methylation of more than one gene and/or with other types of biomarkers such as gene mutations or cytology seems to increase the sensitivity and specificity for cancer detection \[[@b40-cancers-03-02975],[@b41-cancers-03-02975],[@b43-cancers-03-02975]\]. More importantly, hypermethylation of genes, particularly P16, may also be useful as biomarker for the prediction of the development of lung cancer among healthy high-risk smokers. Several studies have reported that hypermethylation in a panel of genes can be found in cancer-free smokers and is associated with increased risk of lung cancer \[[@b34-cancers-03-02975],[@b44-cancers-03-02975]-[@b47-cancers-03-02975]\]. The earlier occurrence of epigenetic abnormality than genetic modification such as gene mutation detected in histologically normal cells suggests that hypermethylation may be an early event during tumorigenesis. This may also explain the higher frequency of p16 hypermethylation than mutations in the TP53 gene and/or the KRAS gene in sputum (see below) \[[@b47-cancers-03-02975],[@b48-cancers-03-02975]\]. Gene methylation can be detected by methylation-specific PCR (MSP) \[[@b49-cancers-03-02975]\] using sodium bisulfite, which converts all unmethylated, but not methylated, cytosines to uracil. Thus, bisulfite treatment introduces specific changes in the DNA sequence, which depends on the methylation status of individual cytosine residues. In the subsequent PCR reaction, primer pairs are designed to be "methylation-specific" by including sequences complementing only unconverted 5-methylcytosines. Recently, a modified version of MSP has been reported to increase the sensitivity. It involves a two-step nested PCR, which can improve the sensitivity by \>50-fold compared to the conventional MSP \[[@b46-cancers-03-02975]\]. One advantage of promoter hypermethylation as a biomarker compared to other genetic biomarkers (e.g., gene mutations, microsatellite abnormalities) is its reversibility by demethylating agent. Clinical trials for the treatment of myeloid neoplasms demonstrated that demethylating agents in combination with histone deacetylation inhibitor could demethylate certain genes in responding patients \[[@b50-cancers-03-02975]\]. Thus, the reversibility of promoter hypermethylation in sputum biomarkers can be a powerful monitoring tool for evaluating the efficacy of demethylation therapy for lung cancer patients. 2.4.. Mutation -------------- There has been significant progress in the understanding of the genetic basis underlying lung cancer. Accumulation of mutations in genes that are involved in cell differentiation and growth leads to lung tumorigenesis \[[@b51-cancers-03-02975]\]. Naturally, genetic mutations have been the area of focus for biomarker discovery. TP53 and KRAS are two of the most frequently mutated genes that are found in lung tumors and believed to be important in lung cancer pathogenesis. Thus, they are most well-studied genetic markers for lung cancer. TP53 is a tumor suppressor and "guardian of the genome" \[[@b52-cancers-03-02975]\]. Hence, mutations in the TP53 gene are among the commonest in most cancers, including lung cancer \[[@b53-cancers-03-02975]\]. A recent study found that about 65% of lung adenocarcinomas harbor mutations in TP53, which identifies TP53 as the most frequently mutated gene in lung cancer \[[@b54-cancers-03-02975]\]. TP53 functions as an emergency brake by regulating tumor-preventing apoptosis and cell cycle progression. It modulates the transcription of genes that govern the major defense against tumor growth, which includes cell cycle arrest, apoptosis, maintenance of genetic integrity, inhibition of angiogenesis and cellular senescence \[[@b55-cancers-03-02975]\]. Most mutations are missense and single-base substitutions distributed throughout the core DNA binding domain from exon 5 to exon 8, which abolish its function as a transcription factor \[[@b56-cancers-03-02975]\]. KRAS is an oncogene that belongs to the Ras family. RAS genes encode a family of membrane-bound guanosine triphosphate (GTP)-binding proteins that regulate cell growth, differentiation, and apoptosis by interacting with multiple effectors, including those in the mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT), and phosphoinositide 3-kinase (PI3K) pathways \[[@b57-cancers-03-02975]\]. RAS proteins acquire transforming potential when an amino acid at position 12, 13, or 61 is replaced as a result of a point mutation in the gene \[[@b58-cancers-03-02975]\]. These activating mutations result in the production of RAS protein that has impaired GTPase activity, leading to constitutive activation of RAS. KRAS mutations are frequently found in human malignancies and notably up to 60% in lung adenocarcinoma \[[@b54-cancers-03-02975]\], being the second most frequently mutated gene in lung cancer. The fact that mutations in the KRAS gene occur almost exclusively at two hot spots (codons 12 and 13) facilitates the assessment of the presence of mutations at these codons by mutant allele enrichment method (See below). Epidermal growth factor receptor (EGFR) belongs to a family of receptor tyrosine kinases (TKs) that transduce important growth factors signaling from the extracellular milieu to the cell. Structurally, each receptor is composed of an extracellular ligand binding domain, a transmembrane domain and an intracellular domain \[[@b59-cancers-03-02975]\]. The receptor, which exists as an inactive monomer, undergoes conformational change upon the binding of a ligand such as EGF and forms a homo- or heterodimer. This event is then followed by autophosphorylation of the key tyrosine residues in the catalytic domain. Subsequently, proteins involved in downstream signaling events that control multiple cellular processes, including proliferation and survival, get recruited to the activated receptor via adaptor molecules \[[@b60-cancers-03-02975]\]. Intracellular signaling is mediated mainly through the RAS-RAF-MEK-MAPK pathway, the PI3K-PTEN-AKT pathway, and the signal transducer and activator of transcription (STAT) pathway \[[@b61-cancers-03-02975]\]. Two independent studies first reported the existence of somatic mutations in the TK domain of EGFR; the mutations are characterized by short deletions in exon 19 and point mutations (G719S, L858R, and L861Q) in exons 19 and 21 \[[@b62-cancers-03-02975],[@b63-cancers-03-02975]\]. The mutations have been classified into three types. Class I mutations include short in-frame deletions that result in the loss of four to six amino acids (E746 to S752) encoded by exon 19. Class II mutations are single-nucleotide substitutions that may occur throughout exons 18 to 21. Class III mutations are in-frame duplications and/or insertions that occur mostly in exon 20 \[[@b64-cancers-03-02975]\]. These mutations destabilize the kinase domain conformation and lead to constitutive activation, followed by uncontrolled activation of downstream signaling pathways including proliferation and survival. The occurrence of mutations in EGFR and KRAS, which encodes a GTPase downstream of EGFR (see above), is mutually exclusive in lung cancers and they exhibit many contrasting characteristics such as clinical background, pathological features of patients harboring each mutation, and prognostic or predictive implications. Clinico-pathological features that correlate with EGFR-activating mutations include East Asian ethnicity, adenocarcinoma histology, female gender, and a history of never having smoked \[[@b64-cancers-03-02975]\], while KRAS mutations are present in those with significant tobacco exposure. Lung cancers harboring the EGFR mutations are remarkably sensitive to EGFR tyrosine kinase inhibitors such as gefitinib or erlotinib, which has been a very important discovery impacting the clinical treatment of lung cancer. Because it is tightly associated with never-smokers, EGFR mutations are common targets for biomarker discovery for lung cancers in never-smokers but not in smokers. Considering the great heterogeneity of cell types and low number of tumor cells found in sputum, it is crucial to employ a sensitive detection method that will distinguish the true positive signal of tumor cells from the vast majority of contaminating normal cells. Traditionally, to detect KRAS mutation at the 12^th^ and 13^th^ codons, the PCR-mutant allele enrichment (PCR-MAE) method was utilized, which has been reported to improve the sensitivity \[[@b65-cancers-03-02975]\] compared to conventional PCR. However, this method is only suitable for the detection of specific known mutations because it requires the use of PCR primers that are complementary to the mutated sequence. For the detection of multiple and unknown mutations in TP53, the PCR-single stranded conformation polymorphism (PCR-SSCP) method is commonly used. Although this method may increase the sensitivity compared to conventional PCR, optimal conditions to sensitively separate alleles harboring various mutations have to be determined empirically, which can be tricky and time-consuming. As a result, studies employing similar methods had reported mutation frequencies ranging from lower than 10% frequency \[[@b25-cancers-03-02975],[@b48-cancers-03-02975]\] to about 50% \[[@b65-cancers-03-02975],[@b66-cancers-03-02975]\]. Notably, the rapid development of technology such as high-throughput sequencing now allows the detection of extremely rare mutant alleles in a highly heterogeneous sample. High-throughput sequencing is a method that parallelizes the sequencing process, producing thousands or millions of sequences at once. Choi et al. successfully identified two novel mutations in EML4-ALK gene in the sputum sample from a lung cancer patient using deep sequencing \[[@b67-cancers-03-02975]\]. The high-throughput nature of this system will also expand the capacity of screening, facilitating the examination of mutations in multiple genes simultaneously. Recently genome-wide screening studies to identify frequently mutated genes in lung cancer (e.g., STK11, LRP1B, LPHN3) have provided an exciting new insight into the discovery of potentially novel gene candidates for lung cancer in addition to TP53 and KRAS \[[@b54-cancers-03-02975],[@b68-cancers-03-02975]\]. The findings of these studies are promising because they offer additional and perhaps better target (or combination of targets) for the early detection of lung cancer. Moreover, identification of the functions of these candidate genes and the consequences of mutations during the early stage of tumorigenesis will provide rationale for the use of mutations of these candidate genes for the early detection of cancer. In summary, genes that harbor limited number of hot spot mutations (e.g., KRAS mutations exclusively occur on codon 12 or 13) will be more practical for large-scale screening than genes (e.g., TP53) with numerous mutations scattered throughout the gene, which will complicate the screening strategy. In addition, since TP53 mutations are found in almost every type of human cancer, an ideal biomarker for lung cancer should be a gene with mutation that is specifically associated with lung cancer. Lastly, the use of high-throughput sequencing for evaluating mutations in these new candidate genes in addition to TP53 and KRAS in sputum will make gene mutation screening a powerful tool for lung cancer detection. 2.5.. microRNAs --------------- MicroRNAs (miRNAs) are a class of short (∼22 nucleotides long) and highly conserved non-coding RNAs involved in numerous developmental processes. They regulate gene expression by incomplete base-pairing to a complementary sequence in the 3′ untranslated region of a target mRNA, which leads to mRNA degradation or translation repression \[[@b69-cancers-03-02975]\]. Deregulation of miRNAs has been linked to cancer initiation and progression, indicating that miRNAs may act as tumor suppressor genes or oncogenes and can target apoptosis \[[@b70-cancers-03-02975]\]. It is believed that miRNAs regulate signal transduction pathways and oncogenic pathways leading to lung cancer. Interestingly, several studies have identified differential miRNA expression profiles associated with lung cancers \[[@b71-cancers-03-02975]-[@b74-cancers-03-02975]\], which suggests that miRNA signatures are potential biomarkers for lung cancers. Although miRNA has recently emerged as powerful molecular biomarkers for the detection of cancer, its potential as a sputum-based biomarker has not been fully explored. Jiang et al. recently reported two studies evaluating differential miRNA expression in sputum samples from lung cancer patients \[[@b74-cancers-03-02975],[@b75-cancers-03-02975]\]. Although they demonstrated that miRNAs existed stably in sputum and could be amplified robustly \[[@b76-cancers-03-02975]\], the studies are somewhat limited because the candidate miRNA molecules were initially discovered from tissue, not sputum, followed by validation of the selected candidates in sputum. Although this strategy can still provide valuable information, screening the entire miRNA transcriptome in sputum would be more desirable for the discovery of novel sputum-based miRNA biomarkers. However, one of the practical hindrances to the latter is that the normalization methods for miRNA, especially in an extremely heterogeneous sample like sputum, have not been fully established yet, which makes it challenging to assess the validity of miRNA profiling data. In addition, due to the inevitable variability in the sputum cell composition among samples, which is a major confounding factor, interpreting any differential miRNA expression can be challenging. Therefore, although sputum-based miRNAs have great potential as biomarkers for lung cancer, a vigorous system for evaluating the differential miRNA expression in sputum has to be developed. 3.. Conclusions and Future Directions ===================================== Although biomarker discovery in sputum has been promising, the clinical applications have been limited, mainly because of the inconsistent results and low sensitivity. One key to tackling these issues is obtaining more bronchial epithelial cells (BECs) in sputum, which is typically less than 5% \[[@b77-cancers-03-02975],[@b78-cancers-03-02975]\]. Increasing the number of BECs will eventually result in more cancer cells in sputum. One way to achieve this might be using sputum induction instead of spontaneous collection. However, the effect of different collection methods on the resultant percentage of bronchial epithelial cells in sputum has not been actively discussed in the literature. In addition, published studies frequently omit the detailed description of collection methods for sputum, which makes it difficult to deduce the effect collection methods on cell populations from these studies. Therefore, careful assessment of the sputum cell composition collected by different methods is urgently required. More importantly, standardized sputum collection and processing protocols should be established to minimize the inconsistencies resulting from different laboratories using different methods. Enriching the bronchial epithelial cells after collection is another strategy to increase the percentage of bronchial epithelial cells and get rid of the potentially confounding cell types such as macrophages and neutrophils. A few different enrichment methods have been proposed, including laser microdissection and magnetic-assisted cell sorting (MACS). When epithelial cells in lung cancer sputum samples were selectively captured by microdissection, the sensitivity for the detection of mutations in TP53 and KRAS increased about three times (∼46%) compared to using the entire sputum cells \[[@b79-cancers-03-02975]\]. Furthermore, the same research group was able to detect mutations in TP53 (14.1%) and KRAS (1.1%) in lung cancer-free, healthy high-risk group using laser microdissection \[[@b80-cancers-03-02975]\]. MACS is another common cell selection method. Typically, bronchial epithelial cells are negatively selected by using anti-CD14 and anti-CD16 magnetic beads to deplete the macrophages and the neutrophils, respectively, which together account for the majority of the cell populations in sputa. It has been reported that bronchial epithelial cells can be enriched from 1.1% (unsorted) up to 40% after MACS, producing an average of 43% purity in the processed samples and resulting in an average 36-fold enrichment \[[@b81-cancers-03-02975]\]. These studies suggest that by selectively enriching the bronchial epithelial cells, molecular abnormalities in sputum samples could be detected with higher sensitivity. In spite of the efforts to enrich for BECs in sputum, the rare presence of cancer cells in sputum strongly argues for biomarkers that are present in non-cancerous epithelial cells in early stage cancer or in high-risk smokers. Thus, examining molecular abnormalities, which have been identified in tumor tissue, in sputum samples may not be very successful. This requires the discovery of novel sputum-based biomarkers that are caused by lung cancer risk factors, namely tobacco smoking, and are harbored in pre-malignant cells. An exciting example is a recent study by the Backman group, which described abnormal nanostructure architecture in microscopically normal-appearing buccal epithelial cells from lung cancer patients using a novel optical technology, partial wave spectroscopic microscopy, which is exquisitely sensitive to the nanoarchitectural manifestation of the genetic/epigenetic alterations of field carcinogenesis \[[@b82-cancers-03-02975]\]. This suggests the possibility that the histologically normal airway epithelium harbors potentially powerful biomarkers that await discovery. Thus, the combination of epithelium cell enrichment, more sensitive detection methods and novel field cancerization biomarkers will lead to the discovery of more accurate and reliable sputum-based biomarkers that can be used for the early detection of lung cancer.	{ "pile_set_name": "PubMed Central" }	[ "amount", "unmethyl", "treatment", "nanostructur", "destabil", "noncalcifi", "saccomannao", "discov", "two", "fish", "singlenucleotid", "phenomenon", "almost", "control", "defens", "remain", "induct", "tumor", "eg", "inevit", "compris", "ncn", "respond", "ligand", "impair", "neoplasm", "collect", "ten", "exist", "timeconsum", "gene", "employ", "identifi", "length", "methyltransferas", "risk", "classif", "block", "pathogenesi", "lastli", "rais", "∼", "fund", "mgmt", "mechan", "select", "sort", "nondisjunct", "convers", "mitogenactiv", "locat", "multipl", "signal", "may", "split", "republ", "theori", "recent", "imbal", "acquir", "separ", "use", "gener", "chest", "mrna", "await", 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Introduction {#sec0005} ============ Meningococcal disease may present as meningococcal meningitis, meningococcal meningitis with meningococcemia, and isolated meningococcemia. It is a disease caused by the bacterium Neisseria meningitidis. Even though there are 12 bacterial serotypes, invasive meningococcal disease is associated with six, classified based on the polysaccharide capsule (A, B, C, W-135, X, Y) \[[@bib0005]\]. The bacterium colonizes the nasopharynx of asymptomatic carriers up to about 5--10 % of the population, being transmitted from person to person by respiratory secretions \[[@bib0010]\]. In Brazil, meningococcal disease is endemic with periodic outbreaks of epidemics in several municipalities. The incidence coefficients have remained stable in recent years, with approximately 1.8 cases per 100,000 inhabitants \[[@bib0015]\]. In São Paulo city, in 2018, 204 cases were confirmed, with 36 deaths, with incidence coefficient being 1.74 and mortality 0.31 \[[@bib0020]\]. The lethality of the disease in our country has been around 18--20% in recent years, and it may achieve coefficients of almost 50 % in septic presentations \[[@bib0015]\]. Serotypes B and C cause disease predominantly in Europe and the Americas, being the most common in Brazil \[[@bib0015]\]. Although the most common clinical manifestations are fever and acute and rapidly progressive petechial and/or purpuric rash, progressing to septic shock within a few hours, there are some chronic, initially more benign presentations, such as chronic meningococcemia \[[@bib0025]\]. This form represents less than 5% of all cases of meningococcemia, with incidence \<0.05 cases per 100,000 inhabitants per year in developed countries \[[@bib0030]\]. It is a rare manifestation of meningococcal disease without meningitis, of prolonged course of more than one week, with intermittent or continuous fever, arthralgia and cutaneous vasculitis \[[@bib0005]\]. Few cases of this presentation have been reported, both in previously healthy and immunocompromised patients \[[@bib0025],[@bib0035]\]. A case of chronic meningococcemia diagnosed in a vertically infected adolescent with human immunodeficiency virus (HIV) infection will be described, providing a review of the literature on the topic. Its importance lies in the description of a rare disease with atypical manifestation, which is difficult to suspect, and which may be life-threatening if not diagnosed and treated promptly and adequately. This case report was approved by local ethics committee; the patient signed the consent form, with permission to disclose images. Clinical case {#sec0010} ============= Adolescent, 19 years old, male, vertically HIV-infected, followed up since 3 months of age at our Pediatric Infectious Disease Clinical Care Center. Not adherent to the antiretroviral treatment, he has presented several clinical intercurrences during his life. At the time of this event, there was significant viral replication (HIV viral load 87,802 copies/μL) and severe immunosuppression (CD4+ count 17 cells/μL). He came to our medical center with 5 days of prostration, epigastric pain, sparse petechiae, joint pain, ocular pain and conjunctival hyperemia. Fever at the beginning for three days. Initially, he sought another service and the laboratory tests performed detected leukocytosis 19,600/ul (81 % neutrophils), and thrombocytopenia 77,000/ul. He received intravenous hydration and was discharged with symptomatic medication. In our service, he was in good general condition, pale, presenting conjunctival hyperemia, some petechiae on the trunk and limbs, without other signs (see Pictures [1](#fig0005){ref-type="fig"}[--](#fig0010){ref-type="fig"}[3](#fig0015){ref-type="fig"} ). Laboratory tests were performed: leukocytes 4330/ul (53 % neutrophils, 33 % lymphocytes) and improved thrombocytopenia 124,000/uL. As there is epidemiological conditions for arboviruses in our country, serology and blood viral research by polymerase chain reaction (PCR) were done for dengue, zika and chikungunya -- all tested negative; serology tested non-reactive for toxoplasmosis and syphilis; for cytomegalovirus serology tested IgG reagent IgM non-reactive. He received symptomatic treatment with partial clinical improvement, keeping skin lesions unchanged. At this time, he was afebrile.Picture 1Right eye conjunctival hyperemia.Picture 1Picture 2Petechiae on the soles of the feet.Picture 2Picture 3Petechia on the arms.Picture 3 Sixteen days after the onset of symptoms, he presented a return of prostration, fever and disseminated hemorrhagic suffusions (see [Pictures 4 and 5](#fig0020){ref-type="fig"}, [Pictures 4 and 5](#fig0020){ref-type="fig"}). He was hospitalized in septic shock, and during the diagnostic investigation, two blood cultures were positive for Neisseria meningitidis C, eighteen days since disease onset.Pictures 4 and 5Hemorrhagic suffusions on the limbs.Pictures 4 and 5 He presented normal cerebrospinal fluid examination with negative bacterioscopy and culture. The patient evolved with acute respiratory distress syndrome and acute renal failure, requiring endotracheal intubation and hemodialysis. He was hospitalized for 46 days, received 10 days of ceftriaxone 2 g 12/12 h. During hospitalization, HIV viral load 382,863 copies/μL and CD4+ count 13 cells/μL. He progressively evolved with improvement, being discharged in good clinical conditions, receiving combined antiretroviral therapy. The vaccine card was checked, he had received two doses of the meningococcal conjugate vaccine C before this clinical complication: 15 years before with HIV viral load 146,000 copies/μL and CD4+ count 762 cells/μL and 3 years before with HIV viral load 130,214 copies/μL and CD4+ count 75 cells/μL. Currently, the patient presents good adherence to the antiretroviral medication, with an improvement in CD4+ cell count (445 cells/μL) and a decrease in viral load (155 copies/μL); he has cutaneous scars derived from meningococcal vasculitis (see [Picture 6](#fig0025){ref-type="fig"})Picture 6Cutaneous scars on the limbs.Picture 6 Discussion {#sec0015} ========== We describe a case of a vertically HIV-infected adolescent with chronic meningococcal disease. Chronic meningococcemia is a disease caused by the bacterium Neisseria meningitidis, characterized by a period of more than one week of intermittent or continuous fever, arthralgia and skin lesions without meningitis \[[@bib0005]\]. Initial erythema develops to petechiae and purpura due to dermal microvascular thrombosis and perivascular hemorrhage \[[@bib0040]\]. Other clinical manifestations may occur, such as myalgia, abdominal pain, weight loss, iritis, retinitis \[[@bib0045]\]. It may be a self-limiting disease, but meningitis and death may occur as late complications \[[@bib0050]\]. These manifestations mimic several other infections, namely arboviruses, which was a confounding factor in our patient, delaying the diagnosis. The gold standard diagnostic method is culture isolation of Neisseria meningitidis in sterile material \[[@bib0055]\]. Even if the course seems to be self-limited, the fact that the patient may remain as an asymptomatic carrier and thus develop an invasive disease or spread the agent has tended to favor antimicrobial treatment \[[@bib0060]\]. The suggested treatment is based on the use of beta-lactams, namely 3rd generation cephalosporin, used in our patient \[[@bib0005],[@bib0030]\]. The reason why these less severe forms of the disease occur is unknown; the susceptibility of the host and the virulence of the bacterium are possible explanations \[[@bib0005],[@bib0065]\]. Although there seems to be a greater association with serotype B \[[@bib0005]\], serotype C was isolated in our patient. The disease can occur in previously healthy individuals or with some immunodeficiency \[[@bib0025],[@bib0035],[@bib0045]\]. After a literature review, we identified only four cases of chronic meningococcemia in horizontally HIV-infected adult patients since 1990, none with this serious evolution \[[@bib0030],[@bib0045],[@bib0070]\]. A recent study found a substantial increased risk of meningococcal disease among adults with HIV infection that met AIDS criteria; in this study they observed a similar clinical presentation and outcomes of meningococcal disease compared to those without HIV infection \[[@bib0075]\]. The increased relative risk observed for meningococcal disease was similar to those observed for HIV-infected individuals in New York City, South Africa and England, ranged from 3.4--6.6 per 100,000 (relative risk = 5--13 compared with HIV-uninfected persons) \[[@bib0075], [@bib0080], [@bib0085], [@bib0090]\]. Among HIV-infected persons, a low CD4 count or high viral load were associated with an increased risk \[[@bib0085]\]. Considering this increased risk, in 2016 the US Advisory Committees on Immunization Practices approved a recommendation for routine vaccination of HIV-infected person \[[@bib0095]\]. In Brazil, meningococcal conjugate vaccine type C is routinely recommended for HIV-infected patients. With this clinical case we intend to highlight the heterogeneity and low specificity of the symptoms that meningococcal disease may present, especially in immunocompromised hosts, leading to a possible diagnostic delay of a potentially fatal disease. The association between Neisseria meningitidis infection and HIV infection is not yet well defined. Due to the potential for progression and the risk of N. meningitidis transmission, a better understanding of the association between HIV infection and meningococcal disease is important to prevention strategies. Ethical approval {#sec0020} ================ Written informed consent was obtained from the patient for publication of this case report and accompanying images. This case report was approved by local ethics committee. Funding {#sec0025} ======= This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CRediT authorship contribution statement {#sec00005} ======================================== Rita S.B. Cardona: Data curation, Formal analysis, Writing - original draft. Fabiana Bononi do Carmo: . Suenia Vasconcelos Beltrão: . Aída de Fátima T. Barbosa Gouvêa: . Reinaldo Salomão: . Regina Célia de Menezes Succi: Supervision, Writing - review & editing. Daisy Maria Machado: Conceptualization, Writing - review & editing. Declaration of Competing Interest ================================= The authors have no conflict of interests, personal or financial, to declare. The authors would like to acknowledge the entire multidisciplinary team that took care of the patient during hospitalization.	{ "pile_set_name": "PubMed Central" }	[ "treatment", "two", "almost", "origin", "intub", "remain", "cours", "decreas", "salomão", "identifi", "discharg", "risk", "written", "grant", "fund", "intermitt", "authorship", "week", "hour", "may", "keep", "team", "recent", "suspect", "cardona", "gener", "use", "hyperemia", "caus", "imag", "conjunctiv", "signific", "bacteri", "suggest", "right", "epidemiolog", "suenia", "endotrach", "difficult", "vaccin", "cell", "conflict", "perform", "eighteen", "popul", "commerci", "evolv", "horizont", "criteria", "transmiss", "meningococcemia", "us", "normal", "though", "syndrom", "medic", "neutrophil", "old", "even", "england", "h", "rel", "recommend", "highlight", "perivascular", "distress", "weight", "rd", "chronic", "machado", "onset", "lie", "promptli", "base", "achiev", "sector", "author", "andor", "regina", "leukocyt", "hiv", 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Saliva research =============== The need to advance saliva research is strongly recognized by the Strategic Plan of the National Institute of Dental and Craniofacial Research.^[@bib1]^ The National Cancer Institute has also recognized saliva as a promising cancer biomarker source.^[@bib2]^ The ability to monitor health status, disease onset, progression, recurrence and treatment outcome through non-invasive means is highly important to advancing health care management. Saliva (oral fluid) is a perfect medium to be explored, offering the potential for a non-invasive, easy to obtain means for detecting and monitoring disease. The adoption of saliva testing would allow a patient to collect their own specimens at home, yielding savings in health costs, convenience for the patient and facilitating multiple sampling. Specimen collection is less objectionable to patients than in the case of other bodily fluids and easier in children and older individuals. The analysis of saliva can thus provide a cost-effective approach for the screening of large populations. Due to these significant advantages, developing biomarkers in saliva for the detection of serious illnesses such as oral and systemic cancers has been on the national healthcare agenda for several years (Government Performance & Results Act 2008).^[@bib3]^ One mandate formulated in the Government Performance & Results Act report is that by the year 2013 proof of principle will be obtained for the ability of saliva to monitor health and diagnose for one systemic disease. Challenges and opportunities ============================ A vast amount of saliva omics data has been generated by recent studies using high throughput technologies.^[@bib4],[@bib5],[@bib6],[@bib7]^ However, there are still barriers which researchers must overcome before such data can be exploited, such as lack of computationally accessible salivary data and information, and inability to cross-reference the salivaomics data that could potentially be made available through different proteomics, transcriptomics, genomics and metabolomics studies. For these reasons, there is an urgent need to create the Salivaomics Knowledge Base (SKB), a data management system and web resource constructed to support saliva diagnostics research, and we present below the informatics advances brought about through the SKB and through the associated tools and resources. Biomedical ontology =================== Ontologies are controlled structured vocabularies designed to provide consensus-based means to ensure consistent description of data by scientists working in disparate domains. As applied in the biomedical domain, ontology plays a key role in providing consensus-based controlled vocabularies serving the consistent annotation of biological and medical data and information, most conspicuously within the framework of the Gene Ontology^[@bib8]^ and now of its sister ontologies within the Open Biomedical Ontologies Foundry (<http://obofoundry.org>). The Basic Formal Ontology (BFO) is a formal ontological framework developed by Barry Smith, Pierre Grenon and others, which serves as the starting point for some 100 ontology projects primarily in the biomedical domain (<http://www.ifomis.uni-saarland.de/bfo/>). The BFO framework can be readily extended to the treatment of families of ontologies of other types, above all to the treatment of relations between ontologies of different levels of granularity, from genes to species and from a single patient to epidemics at a geographical scale (combining applications of BFO to the medical and to the geographical domain). The framework may also be used as a tool for dealing with the relations between distinct perspectives on the biomedical domain, including culturally generated perspectives of the sort which are studied by linguists and anthropologists.^[@bib9]^ Two BFO-based ontologies of special significance for our work here are the Ontology for Biomedical Investigations (OBI)^[@bib10]^ and the Ontology for General Medical Sciences. OBI addresses the need for a controlled vocabulary to support integration of experimental data. The OBI is an ontology designed to serve the coordinated representation of designs, protocols, instrumentation, materials, processes, data and types of analysis in all areas of biological and biomedical investigation. Ontology for General Medical Science is an ontology of the entities involved in the clinical encounter. Thus, it includes very general terms that are used across medical disciplines, including: 'disease\', 'disorder\', 'disease course\', 'diagnosis\', 'patient\' and 'healthcare provider\'.^[@bib11]^ A dental research ontology consortium ===================================== To advance the consistency of data in the dental research community, Smith et al.^[@bib12]^ propose an approach to building a consensus-based ontology to support dental research (ODR). In analogy to efforts in other fields,^[@bib11]^ a consortium of research groups specializing in different areas of study would undertake such an effort, each building different components of ontology to support dental research. Initial efforts in this direction, by scientists in dental research and biomedical ontology at University at Buffalo and University of California, include work on the ontology of oral pathology, oral maxillofacial anatomy, dental disease and dental procedures, and as we discuss below, the Saliva Ontology.^[@bib13]^ Integral to his work is a plan to allow a seamless connection between the use of ontology to support dental research in the dental domain and the use of existing ontology resources developed in other areas of biology and medicine, by reusing elements and strategies from them. The anatomy work is based on the Foundational Model of Anatomy. The ontology of dental procedures use the framework established by OBI.^[@bib14]^ The work on dental diseases is carried out in conjunction with the development of Ontology for General Medical Science.^[@bib11]^ Saliva ontology =============== The Saliva Ontology (SALO) ([Figure 1](#fig1){ref-type="fig"}) is a detailed ontology of this bodily fluid that is optimized to meet the needs of both the clinical diagnostic community and the cross-disciplinary community of omics researchers. The SALO is created through cross-disciplinary interaction with saliva experts, protein experts, diagnosticians and ontologists. To aid development and testing of SALO, we develop a corpus of saliva-relevant literature in SKB to assist in characterizing core terms and synonyms within the ontology and to provide links between SALO content and relevant items in PubMed. SKB will also incorporate the results of experiments in data and text mining using the ontology. SALO will incorporate links to existing ontologies and terminology resources involving treatment of saliva-relevant phenomena. We will also identify and represent within SALO relationships to saliva-relevant types represented in ontologies such as the Gene Ontology, the Protein Ontology^[@bib15]^ and the Chemical Entities of Biological Interest ontology,^[@bib16]^ and also provide links to corresponding SNOMED CT terms where available. SALO is a public domain resource and entirely web-based. Each term in the ontology has its own URL which points to a webpage providing definitions, PubMed sources, references to annotations to SKB and to external databases. SALO and Blood Ontology^[@bib17]^ are the foundation for a unified body fluids ontology resource---Body Fluids Ontology.^[@bib18]^ BioMart ======= BioMart is a free, open-source, federated database system. It is cross-platform and supports many popular relational database managements systems, including MySQL, Oracle, PostgreSQL, SQL Server and DB2. The software is data-agnostic, and can therefore be easily adapted to existing data sets. It is expandable and customizable through a plug-in system, and is open-source so the community can participate in deeper development. Furthermore, BioMart can seamlessly connect geographically disparate databases, facilitating collaboration between different groups. These features have catalyzed the creation of BioMart Central Portal, a first of its kind community-supported effort to create a single access point integrating many different, independently administered biological databases. Anybody can contribute an independently maintained resource to the Central Portal, allowing it to be exposed to and shared with the research community, and linking it with the other resources in the portal. Users can take advantage of the common interface to quickly utilize different sources without learning a new system for each. The system also simplifies cross-database searches that might otherwise require several complicated steps. Several integrated tools streamline common tasks, such as converting between ID formats and retrieving sequences. The combination of a wide variety of databases, an easy-to-use interface, robust programmatic access and the array of tools make Central Portal a one-stop shop for biological data querying.^[@bib19]^ SDxMart---a BioMart portal for salivaomics data =============================================== SDxMart is a BioMart data portal that hosts salivary proteomic, transcriptomic, metabolomic and microRNA data and offers access to the data by using the BioMart interface and querying environment. The SDxMart is designed to provide a variety of queries to facilitate saliva biomarker discovery including complex queries that integrate genomic, clinical and functional information. The SDxMart holds data from projects of oral diseases and systemic diseases including oral cancer, Sjögren\'s syndrome, pancreatic cancer and breast cancer. The types of datasets are: (i) proteomics; (ii) transcriptomics; (iii) microRNA; and (iv) metabolomics. In addition, the SDxMart is imported with several public databases including Ensembl genome database (Ensembl release 37),^[@bib20]^ and the number of resources is continuously growing. Summary ======= The SKB is being created to facilitate researchers using salivary data from multiple perspectives. It is being built in tandem with the SALO and SDxMart which will allow the SKB to interoperate with other omics databases as part of a general strategy to facilitate integration of heterogeneous and disparate data sources that enable system biology approaches. Either SALO or SDxMart is a first and only resource of its kind in the field of dentistry. This work was supported by the US National Institutes of Health (grant number U01DE017790), the Felix & Mildred Yip Endowed Professorship and the Barnes Family Trust Fund. ![A fragment of the basic Saliva Ontology in its current form.](ijos201226f1){#fig1}	{ "pile_set_name": "PubMed Central" }	[ "unifi", "amount", "treatment", "formijosffig", "two", "corpu", "customiz", "control", "refer", "work", "cours", "collect", "resourcebodi", "exist", "gene", "interoper", "identifi", "healthcar", "sort", "albib", "kind", "grant", "fund", "take", "task", "multipl", "may", "id", "readili", "recent", "mildr", "gener", "use", "mine", "support", "collabor", "easier", "signific", "db", "establish", "represent", "build", "relev", "process", "phenomena", "proof", "also", "pierr", "scientist", "item", "catalyz", "perform", "popul", "screen", "chemic", "us", "anatomi", "featur", "syndrom", "medic", "nation", "agenda", "first", "felix", "speci", "tool", "popular", "involv", "array", "oracl", "vocabulari", 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Moriuchi et al. reported a comprehensive reclassification of bacterial strains from the genera Cupriavidus and Ralstonia based on percentage of conserved proteins (POCP), average nucleotide identity (ANI), multilocus sequence analysis and 16S rRNA gene sequence. In the study, conflicting results were repeatedly observed for the taxonomic classification of strain PBA that was initially identified as Ralstonia sp. PBA based on 16S rRNA gene sequence (Gan et al., [@B4]; Moriuchi et al., [@B8]). Strain PBA was isolated as a co-culture with Hydrogenophaga intermedia PBC from textile wastewater a decade ago. The co-culture could grow on 4-aminobenzenesulfonate (4-ABS), a recalcitrant dye intermediate (Wagner and Reid, [@B12]), as the sole nitrogen, carbon, and sulfur source to a relatively high cell density (Gan et al., [@B4]). In this syntrophic relationship, strain PBA is the sole provider of p-aminobenzoate, an essential vitamin required for the growth of H. intermedia PBC, the main 4-ABS degrader (Gan et al., [@B2], [@B3]). In light of new genomic resources, the initial taxonomic assignment of strain PBA has also been previously questioned by Kim and Gan ([@B6]) given its closer phylogenetic affiliation to the genus Cupriavidus than to the genus Ralstonia. Unfortunately, both recent genome-based taxonomic classifications of strain PBA (Kim and Gan, [@B6]; Moriuchi et al., [@B8]) suffered from incomplete and biased taxon sampling (restricted mostly to members from the genus Ralstonia and Cupriavidus) that can result in the misinterpretation of evolutionary relationships (Heath et al., [@B5]). The taxonomic affiliation of strain PBA should be inferred from a comprehensive phylogenomic analysis that includes all genera with genome availability from the family Burkholderiaceae. A total of 428 Burkholderiaceae (including strain PBA) and 15 non-Burkholderiaceae genome assemblies were obtained from the NCBI RefSeq database (accessed on 30th May 2019). The genomes were processed using two microbial phylogenomic analysis pipelines e.g., GToTree v1.2.1 (Lee, [@B7]) and PhylophlAN v0.99 (Segata et al., [@B11]) that identify single copy bacterial genes (GToTree: n = 203, Betaproteobacteria HMM set; PhylophIAN: n = 400) and produce concatenated protein alignment. Maximum likelihood tree construction from the protein alignments used IQTree v.1.6.8 with 1,000 ultrafast bootstrap replicates (Nguyen et al., [@B9]). In both phylogenomic trees, the Ralstonia and Cupriavidus clusters received maximal support and are sister taxa to the exclusion of strain PBA ([Figures 1A,B](#F1){ref-type="fig"}). The updated phylogenomic placement of strain PBA in light of extensive taxon sampling precludes its genus assignment to the genus Ralstonia or Cupriavidus and suggests that it is a member of a hitherto undescribed genus within the family Burkholderiaceae. Within the Genome Taxonomy Database (Parks et al., [@B10]) that inferred standardized bacteria taxonomy from conserved proteins present in 143,512 bacterial genomes (GTDB release R04-RS89), strain PBA was still assigned to its own genus (g\_\_AKCV01) despite an even more extensive taxon sampling of 4,378 genomes from the family Burkholderiaceae (<https://gtdb.ecogenomic.org/tree?r=g__AKCV01> accessed on 1st August 2019). ![Genome-based phylogeny of Burkholderiaceae. Construction of the phylogenomic trees used protein alignments generated from (A) PhyloPhlAN and (B) GToTree. Numbers in brackets indicate number of taxa in the collapsed clades. GCF codes preceding the species names are RefSeq Assembly numbers. The positions of strain PBA in both trees are shown by black arrows. Branch lengths and labels indicate the number of substitutions per site and IQTree ultrafast bootstrap support values, respectively. The uncollapsed trees are available in the Zenodo database (<http://doi.org/10.5281/zenodo.3258920>).](fmicb-10-02011-g0001){#F1} Given the concordance observed from these independent analyses, the taxonomic assignment of strain PBA has been updated from Ralstonia sp. PBA to Burkholderiaceae sp. PBA in the NCBI database (Bioproject: PRJNA78957; BioSample: SAMN02471424) (Gan et al., [@B1]) pending future genus description. To facilitate future strain description and comparison, strain PBA has been deposited in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) under the accession number [DSM 106616](DSM106616). Furthermore, the concatenated alignments, uncollapsed phylogenomic trees and genome information are also made available in the Zenodo database (<http://doi.org/10.5281/zenodo.3258920>). Author Contributions {#s1} ==================== HG performed data analysis and wrote the manuscript. Conflict of Interest Statement ------------------------------ The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author is grateful to the Deakin Centre of Integrative Ecology for financial support. Funding. This research was supported by the Deakin Centre of Integrative Ecology. [^1]: Edited by: Iain Sutcliffe, Northumbria University, United Kingdom [^2]: Reviewed by: Roberta Fulthorpe, University of Toronto Scarborough, Canada; Zhang Yong, Southwest University, China; Jorgen Johannes Leisner, University of Copenhagen, Denmark [^3]: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology	{ "pile_set_name": "PubMed Central" }	[ "research", "ncbi", "ani", "black", "vitamin", "roberta", "incomplet", "two", "ab", "nucleotid", "posit", "given", "toronto", "figur", "arrow", "biosampl", "respect", "eg", "nitrogen", "grate", "paminobenzo", "resourc", "restrict", "financi", "submit", "likelihood", "concaten", "conduct", "decad", "collect", "gtdb", "dsm", "textil", "deakin", "china", "cluster", "sole", "gene", "burkholderiacea", "identifi", "yong", "length", "v", "dye", "includ", "genera", "classif", "new", "data", "comprehens", "iain", "park", "fund", "assign", "august", "maxim", "valu", "lee", "moriuchi", 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Key messages {#Sec1} ============ What is already known about this subject?o The phenotype of adult-onset Still's disease appears to be dichotomized into systemic or chronic articular forms.o Biological treatments are dominated by IL-1 or IL-6 receptor inhibitors.What are the new lines of research suggested by this study?o The presence of arthritis and a chronic articular phenotype may be associated with a preferential response to tocilizumab, whereas the systemic form may be associated with a preferential response to anakinra.o Tocilizumab increases the likelihood of corticosteroid withdrawal regardless of delays in its initiation or when it is initiated relative to other treatment in the overall therapeutic strategyHow might this impact clinical practice?o These results should help us better codify the treatment strategy for adult-onset Still's disease. Introduction {#Sec2} ============ Adult-onset Still's disease (AOSD) is a rare systemic inflammatory disorder. Its annual incidence is estimated to be between 0.16 and 0.62 per 100,000 persons globally \[[@CR1]\], and its prevalence approximately 1--24 per million \[[@CR2]\]. AOSD is an autoinflammatory disease resulting from multifactorial causes such as genetic factors and immunological imbalances involving macrophage, neutrophil, and inflammasome hyperactivation or dysfunction \[[@CR3]\]. Recent studies suggest that there is a dichotomy between two major phenotypes of AOSD: a systemic pattern that presents with an acute onset associating high fever, skin rash, hematopoietic signs, and sometimes visceral damage; and a chronic articular profile in which polyarthritis prevails \[[@CR4], [@CR5]\]. This phenotypic dichotomy is highlighted by recent studies showing that there are different cytokine imbalances in these two phenotypes. Although results may vary, this imbalance would sometimes favor interleukin (IL)-1β, IL-18, and interferon (IFN)-γ production in the systemic form, whereas in the chronic articular form, IL-6, IL-8, tumor necrosis factor (TNF)-α, and IL-17 production would predominate \[[@CR6]--[@CR12]\]. The prognosis of AOSD is unpredictable as it can manifest with polyarticular erosive disease or life-threatening manifestations such as reactive hemophagocytic lymphohistiocytosis (rHLH). The disease course is also very polymorphic, ranging from a monophasic flare to polycyclic or chronic forms. This clinical and prognostic polymorphism presents a major challenge and makes it difficult to predict the outcome at the time of diagnosis and to determine appropriate management. Therapy is therefore usually empirical, and beside methotrexate, biologics have played an increasing role in the chronic or refractory forms. A recent meta-analysis suggested that IL-1 and IL-6 receptor inhibitors are the most effective biotherapies for AOSD \[[@CR13]\] and have a strong steroid-sparing effect. The systemic phenotype has been suggested to respond preferentially to IL-1 receptor inhibitors, such as anakinra, while the chronic articular form shows better responses to IL-6 receptor inhibitors, such as tocilizumab \[[@CR6], [@CR7], [@CR14]--[@CR17]\]. This exploratory study therefore aimed to identify initial clinical and/or biological factors that were predictive of a preferential response to anakinra or tocilizumab. Secondary objectives were to investigate whether the choice of biotherapy (anakinra or tocilizumab) and their delay in initiation influenced the likelihood of steroid discontinuation and to describe the clinical and biological phenotypes of patients with AOSD. Methods {#Sec3} ======= Study design and data collection {#Sec4} -------------------------------- A multicenter exploratory retrospective study of all patients diagnosed with AOSD at Bordeaux teaching hospital and Pau and Dax peripheral hospitals until 2018 was conducted. Patients included had to be 16 years old or older, to be diagnosed with AOSD, to meet the Yamaguchi and/or Fautrel criteria, to have a chronic or refractory disease course, and to receive or have received a biological disease-modifying antirheumatic drugs (bDMARD). Exclusion criteria were age younger than 16 years old, a diagnosis of systemic-onset juvenile idiopathic arthritis (SoJIA), an exclusion of AOSD diagnosis during disease course, Yamaguchi and Fautrel criteria not met, lack of data, and absence of bDMARD during disease course. Patients with AOSD were identified via ICD-10 code, via a diagnostic grid that automatically references autoimmune and inflammatory diseases at Bordeaux University Hospital, via the bioinformatics services of each hospital, and via direct contact with the specialists treating the patients. Data regarding clinical and biological characteristics at diagnosis and treatment details were collected using a standard data extraction form. All the information was double-checked for any discrepancies. The following demographic and clinical AOSD-related characteristics were collected: sex, date of birth, age at diagnosis, year of diagnosis, duration of disease, duration of disease at first bDMARD initiation, smoking habits, personal or familial history of rheumatic autoimmune inflammatory or neoplastic disease, type of Still's disease-related rash if present, arthralgia, arthritis, hands affected or not, early morning stiffness, myalgia, fever characteristics, general deterioration and weight loss \> 4 kg, lymphadenopathy, pharyngitis, angina, and visceral involvement (pleuritis, pericarditis, pulmonary hypertension). The following biological characteristics were recorded: leukocytosis \> 10,000/mm^3^, neutrophilia \> 8000/mm^3^, eosinophilia \> 500/mm^3^, anemia with hemoglobin \< 12 g/dl, thrombocytosis \> 400,000/mm^3^, increased C-reactive protein (CRP) levels, maximum CRP at diagnosis, decreased albumin, increased liver enzymes, increased creatine kinase, increased lactate dehydrogenase (LDH), increased ferritin, decreased glycosylated ferritin, autoimmunity markers (antinuclear antibodies (ANA), antideoxyribonucleic acid, anti-extractable nuclear antigen, rheumatoid factor, antiβ2GP1, lupus anticoagulant, anticardiolipin), disseminated intravascular coagulation markers, and rHLH markers (cytopenia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis in the bone marrow). Patients were classified as having two distinct disease patterns: the systemic form or the chronic articular form. In the absence of consensual criteria in this regard, this classification was based on expert opinions that was often spelled out in the medical reports and was confronted to disease course outcome, which was available due to the retrospective nature of this study. Outcome measurements {#Sec5} -------------------- The effectiveness of treatment was defined as follows: effective treatment was considered when all initial symptoms and biological anomalies had resolved, partially effective treatment was considered when all but one initial symptom or biological anomaly had resolved, and ineffective treatment was considered when two or more initial symptoms or biological anomaly persisted. The attending doctors assessed disease flares and relapses. Treatment lines were assessed as follows: nonsteroidal anti-inflammatories (NSAIDs) were not considered a treatment modality for chronic and refractory AOSD, but corticosteroids, conventional synthetic disease-modifying antirheumatic drugs (csDMARD) and especially methotrexate (both galenic forms taken together) and cyclosporine, and bDMARD (anakinra, tocilizumab, canakinumab, infliximab, and etanercept) were considered treatment lines. The outcome of interest for the univariate analysis of predictive factors was the full effectiveness of the treatment as defined above, including both systemic and articular manifestations. Statistical analysis {#Sec6} -------------------- Quantitative variables are presented as mean with standard deviation or as median with interquartile range, and qualitative variables are presented as percentages. Analysis was performed on the following variables: all the clinical and biological manifestations included in the Yamaguchi or Fautrel criteria, common demographic characteristics, and all the symptoms or biological abnormalities usually observed during AOSD according to the literature and our personal experience. The nonparametric Mann-Whitney test was used to perform a univariate analysis of quantitative variables, and the Fisher exact test was used for qualitative variables. Calculations were performed with STATA 13.1 SE software. P values less than 0.05 were considered significant. Results {#Sec7} ======= Patients' characteristics {#Sec8} ------------------------- Twenty-seven patients were included in this study: 21 from Bordeaux teaching hospital, 4 from Dax hospital, and 2 from Pau hospital. Patient characteristics at diagnosis are described in Table [1](#Tab1){ref-type="table"}.Table 1Characteristics of the patients included in the study All patients fulfilled both the Yamaguchi and Fautrel criteria. No patients had any personal or familial history of rheumatic, autoimmune, inflammatory, or neoplastic disease. All patients presented with a fever above 38 °C, mainly intermittent and vesperal fevers. The next most prevalent symptoms were arthralgia (96%), arthritis (52%), skin rash (81%), general deterioration (72%), and ear-nose-throat (ENT) symptoms (69%). Eleven (40.7%) patients were classified as systemic form of disease, and 16 (59.3%) as chronic articular form. All patients presented with elevated CRP levels. The next most commonly observed anomalies were neutrophilia (96%), low albumin (75%), and increased ferritin (73%). No patients presented with increased creatine kinase (CK), positive extractable nuclear antigen (ENA), positive antineutrophil cytoplasmic antibody (ANCA), positive anticitrullinated protein antibody (ACPA), or positive anticardiolipin. Nine patients were either ANA-positive (five, 1/160), anti-DNA positive (one), or rheumatoid factor (RF)-positive (three) at diagnosis, but these results were judged clinically insignificant because lower than two times the upper normal range and never associated with symptoms concordant with SLE, APLS, or RA and were all controlled to be negative. Furthermore, all patients included in the study met both Yamagushi and Fautrel criteria. The median duration of disease was 5 years (IQR 2--7) and 6.5 years (IQR 5--9) for the systemic and chronic articular forms, respectively. All patients presented a chronic refractory form of the disease, which required the off-label use of bDMARD because of failures of corticosteroids and csDMARD or necessity to reduce or stop corticosteroids. Biological treatment efficacy {#Sec9} ----------------------------- Table [2](#Tab2){ref-type="table"} presents the detailed responses to anakinra and tocilizumab among the patients included in the study. Twenty-six of 27 patients (96.3%) achieved remission with either anakinra or tocilizumab. The median delay in the introduction of the first biotherapy was 5 months (interquartile range (IQR), 1--21 months): this delay was median 1 month (IQR, 0--6) in the systemic form and median 17 months (IQR, 4--38) in the chronic articular form.Table 2Delay in corticosteroids withdrawal and detailed responses to anakinra and tocilizumab among the patients included in the study A total of 15 patients received anakinra; 5 as a first-line treatment, 4 as a second-line treatment, 5 as a third-line treatment, and 1 as a fourth-line treatment. The median delay between diagnosis and the introduction of anakinra was 1.5 months (IQR, 0--14). Thirteen (86.7%) patients were responders. The 2 anakinra nonresponders received anakinra as a third-line treatment: one had previously failed to respond to tocilizumab, and the other later responded to tocilizumab and even managed to stop it. A total of 17 patients received tocilizumab, 2 as a first-line treatment, 6 as a second-line treatment, 5 as a third-line treatment, and 4 as a fourth-line treatment. The median delay between diagnosis and the introduction of tocilizumab was 17 months (IQR, 4--39 months). Fourteen patients (82.4%) were responders. Among the 3 tocilizumab nonresponders, one presented with a systemic form and received tocilizumab as a first-line treatment but later responded to anakinra, one presented with a systemic form and received tocilizumab as a fourth-line treatment after responding to anakinra but was switched because of a suspicion of anakinra-induced toxidermia, and one presented with a chronic articular form and received tocilizumab as a fourth-line treatment after failure of anakinra. The difference of median duration of disease at the introduction of the first bDMARD in the anakinra-treated patients group and in the tocilizumab-treated patients group was statistically significant (p = 0.4457): median 2 months (IQR, 0.25--12 months) in the anakinra-treated patients and median 14.5 (IQR, 3.25--38.25 months) in the tocilizumab-treated patients. Eight patients (29.6%) managed to stop their biological treatment without relapse at the last known visit which ranged from 6 to more than 24 months after biological treatment discontinuation: 3 of 13 (23%) anakinra responders and 5 of 14 (35.7%) tocilizumab responders. The difference in delays in initiation of anakinra or tocilizumab was not statistically significant between those who managed to stop their biotherapy without relapse versus those who could not stop their biotherapy or those who relapsed, with a median delay of 3 months (IQR, 1.25--13.75) versus 8.5 months (IQR, 1--23.25), respectively (p = 0.61). Five patients received both biotherapies. Two of these patients presented with a systemic form and were tocilizumab nonresponders but anakinra responders, one presented with a chronic articular form and was anakinra nonresponder but tocilizumab responder, one presented with a chronic articular form and did not respond to either biotherapy, and one patient presented with a systemic form and responded to both biotherapies; this patient first responded to anakinra but corticosteroids could not be tapered so he was switched to tocilizumab in order to successfully stop corticosteroids. One patient received canakinumab and failed to respond but was anakinra responder. Three patients received antitumor necrosis factor (TNF) agents but these treatments were not efficacious (2 etanercept, 1 infliximab): two of them presented a chronic articular form and one a systemic form. One of 27 (3.7%) patients did not respond to either biotherapy (anakinra then tocilizumab) and died of a probably infectious-induced AOSD flare. Predictive factors for preferential responses to anakinra or tocilizumab {#Sec10} ------------------------------------------------------------------------ Patients who responded to either biotherapies or no biotherapy were excluded from this analysis (n = 2). Table [3](#Tab3){ref-type="table"} describes the univariate analysis for potential predictive factors of a good therapeutic response to anakinra or tocilizumab. The outcome of interest was the full effectiveness of the treatment, defined as the resolution of all initial systemic and articular symptoms and biological anomalies.Table 3Univariate analysis of predictive factors for a good therapeutic response to anakinra or tocilizumab (n = 25) A chronic articular phenotype and the presence of arthritis were associated with a substantial response to tocilizumab: 12 of the 14 patients presenting a chronic articular phenotype responded to tocilizumab whereas 3 of the 13 patients presenting a systemic phenotype responded to tocilizumab (p = 0.0009, OR 36 \[2.6--1703\]) and 10 of the 14 patients presenting arthritis responded to tocilizumab whereas 3 of the 13 patients without arthritis responded to tocilizumab (p = 0.017, OR 10 \[1.22--92.6\]). A systemic form and the absence of arthritis were associated with a substantial response to anakinra: 1 of the 14 patients presenting a chronic articular phenotype responded to anakinra whereas 9 of the 13 patients presenting a systemic phenotype responded to anakinra (p = 0.0009, OR 36 \[2.6--1703\]) and 3 of the 14 patients presenting arthritis responded to anakinra whereas 9 of the 13 patients without arthritis responded to anakinra (p = 0.017, OR 10 \[1.22--92.6\]). No difference in biological variables was statistically predictive of treatment response, although the presence of thrombocytosis seemed to be preferentially associated with a better response to tocilizumab than to anakinra (n = 8/14 vs. n = 3/13, p = 0.123). Likelihood of steroid withdrawal depending on the specific biotherapy and delays in initiation of biotherapy {#Sec11} ------------------------------------------------------------------------------------------------------------ All patients received corticosteroids at diagnosis. Among the 26 patients in remission, 14 (53.8%) achieved corticosteroid withdrawal within a mean delay of 27 months, 10 (71.4%) were on tocilizumab, and 4 (28.6%) were on anakinra (Table [2](#Tab2){ref-type="table"}). Table [4](#Tab4){ref-type="table"} presents the likelihood of corticosteroid withdrawal depending on treatment with anakinra and tocilizumab: tocilizumab was associated with an increased likelihood of corticosteroid withdrawal (p = 0.029), whereas anakinra did not seem to increase the likelihood of corticosteroid withdrawal (p = 1.000).Table 4Likelihood of corticosteroid withdrawal stratified by biotherapy A shorter delay before the initiation of anakinra or tocilizumab was not associated with a greater likelihood of corticosteroid withdrawal among biotherapy responders. Among anakinra responders, the difference in the median delay from diagnosis to anakinra initiation between those who achieved steroid withdrawal (n = 4) and those who did not (n = 10) was not statistically significant (p = 0.5767): the median delay was 2.5 months (IQR, 0--8 months) among those who did not stop corticosteroids versus 2 months (IQR, 2--14 months) among those who stopped corticosteroids. Among tocilizumab responders, the difference in the median delay from diagnosis to tocilizumab initiation between those who achieved steroid withdrawal (n = 11) and those who did not (n = 5) was not statistically significant (p = 0.9548): the median delay was 17 months (IQR, 9--21 months) among the tocilizumab responders who did not stop corticosteroids versus 17 months (IQR, 4--38 months) among those who stopped corticosteroids. Initiating tocilizumab as the first-line treatment rather than as a second-line or later treatment did not improve the likelihood of corticosteroid withdrawal (n = 1/2 vs. n= 11/15, p = 1.000). In addition, initiating tocilizumab as a first- or second-line treatment rather than as a third-line or later treatment also did not improve the likelihood of steroid withdrawal (n = 6/8 vs. n = 5/9, p = 0.619); similarly, initiating tocilizumab as a first-, second- or third-line treatment did not improve the likelihood of steroid withdrawal compared to that from tocilizumab used as a fourth-line treatment (n = 9/13 vs. n = 2/4, p = 0.584). Discussion {#Sec12} ========== This retrospective study was conducted to evaluate bDMARD strategies in the management of AOSD, with a special attention to predictive factors of their efficacy and their effect on corticosteroid sparing. All but one patient achieved remission with either anakinra or tocilizumab. Our results support the dichotomy of AOSD in two phenotypes: treatment responses to bDMARD seemed to depend on disease phenotype, the presence of arthritis, and a chronic articular phenotype were associated with a substantial response to tocilizumab, whereas the systemic form was associated with a substantial response to anakinra. In our cohort, anakinra did not increase the likelihood of corticosteroid withdrawal but tocilizumab did, regardless of delays in initiation, or when it was initiated relative to other treatment in the overall therapeutic strategy. The clinical and biological characteristics of the patients included in this study seem similar to those of AOSD patients reported in the literature \[[@CR18]\], with the exception of life-threatening conditions, which have been estimated to affect as many as 15--20% of AOSD patients, while only 8% were affected in this study. Multivariate analysis was not performed because of the sample size, a limitation inherent to rare diseases such as AOSD. As a consequence, we lacked patients who received both biotherapies in each phenotype. But rather than aiming to look for significant differences and to draw definite conclusions, the goal of this study was to establish a reference for future large-scale studies and compile data for registries to confirm the results. The distinction between systemic and chronic articular forms is not always straightforward, and no consensual criteria exist to distinguish these two phenotypes. For example, a systemic form progressing for several years, who received corticosteroids for a long time and who benefited from bDMARD late in disease course, might be confused for a chronic articular form. These results highlight the excellent efficacy of anakinra and tocilizumab in AOSD, regardless of the phenotype, but show some differences between them. However, the retrospective nature of the study is a major limitation to compare these two drugs. The choice of biotherapy may have been different depending on their availability on the year of diagnosis: anakinra was launched on the market several years before tocilizumab and the effects of anakinra and tocilizumab on AOSD were not simultaneously described. One could argue that clinicians may have influenced the choice of biotherapy by preferentially prescribing tocilizumab to patients presenting with arthritis or a chronic articular form, analogous to rheumatoid arthritis, and therefore distorted the results. Comparing these two bDMARDs is impossible outside a randomized controlled trial (RCT), and none ever demonstrated that tocilizumab should be preferentially prescribed to AOSD patients with arthritis \[[@CR5], [@CR19], [@CR20]\]. Yet the following results reinforce our findings. All but three of the patients included in this study were diagnosed in 2009 or later, and these three received anakinra or tocilizumab after both their efficacies were described in AOSD. A major result of our study is that anakinra responders more commonly did not have arthritis, as the absence of arthritis was statistically associated with a better response to anakinra (p = 0.017). Tocilizumab also showed less efficiency on the systemic form. Although our results cannot imply definitive conclusions on the fact that one biotherapy is more effective than the other in each phenotypes because of the lack of patients who received both biotherapy in each phenotype, this result supports our clinical feeling and the notion that even if clinicians' choices may have been influenced by analogy to rheumatoid arthritis, their insights seem to be founded. While there is no doubt about anakinra's dramatic efficacy on AOSD in general and particularly on the systemic phenotype \[[@CR2], [@CR18], [@CR19], [@CR21]--[@CR27]\], whether anakinra is effective for joint involvement is a subject of debate as data seem to be inconsistent \[[@CR4], [@CR21]--[@CR24], [@CR28], [@CR29]\]. Some studies reported a substantial efficacy \[[@CR5], [@CR23], [@CR24]\], when others suggest otherwise \[[@CR21], [@CR28], [@CR30]\]. The largest retrospective study investigating anakinra in AOSD showed a significant improvement on articular manifestations (DAS28 falling from 4.7 ± 1.2 to 1.7 ± 0.9 after 12 months) but 14.4% of the patients still presented arthritis after 12 months of treatment \[[@CR24]\]. Notably, they found no difference in delay of response to therapy between the two phenotypes and did not observe any difference in the type of response in the two patterns of the disease. In our study, 5 patients presenting with a chronic articular form received anakinra: 3 responded to anakinra and never received tocilizumab, one was an anakinra nonresponder but responded to tocilizumab, and one responded to anakinra but had to be switched to tocilizumab in order to stop the corticosteroid. Definitive conclusions about anakinra's efficacy on AOSD's joint involvement therefore seem still premature. The absence of demonstrated steroid-sparing with anakinra in this study is questionable as it demonstrated a steroid-sparing effect in several other studies \[[@CR22], [@CR23], [@CR25]\]. First, the evaluation in our study was based on the rate of steroid withdrawal, not on a steroid-sparing effect as prednisone dosage was not available. Secondly, a channeling bias exists: the acute onset of the systemic phenotype and its potential life-threatening complications might cause the clinicians to be more cautious while tapering corticosteroids, and the tocilizumab-induced fall in CRP might reassure them. Furthermore, anakinra was made available before tocilizumab: the progressively known dramatic efficacy of anakinra may have reassured the clinicians and entice them into a faster corticosteroids tapering when tocilizumab was made available. In addition, three patients presenting a systemic form in our study were able to taper corticosteroids down to 5 mg daily but the clinicians opted to maintain this dosage as a long-term therapy. Both the management and the place of corticosteroids in AOSD treatment strategy should maybe differ based on the differences between the two phenotypes. No patients in this study responded to anti-TNF blockers, whatever the phenotype. The efficacy of these medications in AOSD is controversial, but some studies suggest that they have a place in the treatment of the chronic articular form \[[@CR31]--[@CR33]\], while others have showed rather disappointing results \[[@CR19], [@CR20], [@CR34]\]. The excellent efficacy and clear steroid-sparing effect of tocilizumab determined in our study, concurring with the findings of a recent meta-analysis showing that AOSD patients treated with tocilizumab had a pooled remission rate of approximately 85% regardless of the phenotype \[[@CR35]\], and of the first RCT investigating tocilizumab in AOSD \[[@CR36]\], should make clinicians prioritize tocilizumab to anti-TNF blockers for patients with a chronic articular form of AOSD. Canakinumab showed some inconsistent results in AOSD \[[@CR37]--[@CR39]\] but is currently being investigated for its efficacy on AOSD's joint involvement (NCT02204293). No patient in this study received abatacept or rituximab; the data are very limited but some authors reported efficacy in patients who fail to respond to TNF, IL-1, or IL-6 inhibition \[[@CR40]--[@CR45]\]. Physiopathological data may highlight the efficacy of IL-17 or IL-18 blockade in AOSD. A recent study showed that IL-18 inhibition using the recombinant human IL-18-binding protein tadekinig alpha is a therapeutic option in patients with AOSD in a phase 2 open-label trial \[[@CR46]\]. IL-17 \[[@CR47]\] and INF-γ \[[@CR48]\] blockade have not been studied yet but should be in the near future. Janus kinase (JAK) inhibitors may also be of interest, particularly in the chronic articular form of AOSD. The two main goals when initiating a bDMARD in AOSD should be a complete remission and corticosteroids withdrawal or significant tapering. This study does not reveal definitive answers regarding whether an early introduction of biologics improves the general prognosis of AOSD. We believe that the early use of biologics in AOSD patients is worth considering. It is our impression that the earlier biologics are used, the shorter and smoother the disease course is. Similarly, other authors have suggested that biotherapies should be initiated earlier in the course of AOSD \[[@CR13], [@CR18]\], arguing that earlier initiation may improve the prognosis mainly via a steroid-sparing effect. Notably, the results of this study suggest that the steroid-sparing effect of biologics is not influenced by delays in initiation of biological therapy. One-quarter to one-third (29.6%) of our patients managed to stop biologics despite chronic or refractory forms of AOSD, raising the question of how and when to stop biologics in these patients. A channeling bias exists, as no predefined corticosteroids tapering strategy could be set due to the retrospective nature of this study but patients who managed to stop anakinra had all achieved remission for at least 6 to 12 months, had initially stopped corticosteroids, and then all underwent progressive reduction in their biological treatments before definitive discontinuation. Among patients who managed to stop tocilizumab, corticosteroids had always previously been stopped, and tocilizumab dosage was progressively decreased when it had been administered by perfusion or tapered when it had been administered by injections. The high percentage of patients who successfully discontinued biological treatments in our study should encourage clinicians to consider tapering biologics among patients without visceral involvement who are in clinical and biological remission for 6 to 12 months. The first evidence-based clinical practice guidelines for the management of AOSD \[[@CR49]\] emphasized the use of IL-6 inhibitors in the treatment of AOSD, but IL-1 inhibitors were judged to be "weakly recommended as a therapeutic agent against refractory AOSD" although one RCT and several case reports suggest otherwise. A proposal may be to treat a first flare of AOSD with corticosteroids and to introduce biologics in cases of steroid-refractory flares or as early as the second flare of AOSD. In that case, anakinra should be preferred when faced with the systemic form of AOSD and tocilizumab in patients with the articular form. The well-known idea of a "window of opportunity" in rheumatoid arthritis may also be valid in AOSD, with the objective being to prevent the development of a self-perpetuating "cytokine storm." We need large-scale studies, RCTs, and international collaborative registries regarding this multifaceted disease. As emphasized in a recent editorial, these studies will have to be more precise about evaluating the patients' disease course and particularly their phenotypes in order to concentrate research on the subgroup of patients with a chronic or refractory form of the disease \[[@CR50]\]. A national French cohort, the French Adult and Childhood Onset Still Disease Cohort (RaDiCo-ACOSTILL), was recently opened for inclusion and will surely help to shed light on these issues in the near future. Conclusion {#Sec13} ========== This study supports the dichotomy of AOSD in two phenotypes and introduces the idea that treatment response to bDMARD may depend on disease phenotype. The presence of arthritis and a chronic articular phenotype seem to be associated with a substantial response to tocilizumab, whereas the systemic form seems to be associated with a substantial response to anakinra. Although further research is needed, this exploratory study highlights the therapeutic implications of the phenotypic dichotomy of AOSD. These results should help us better codify treatment strategies for AOSD. ANA : Antinuclear antibodies ANCA : Antineutrophil cytoplasmic antibody ACPA Positive anticitrullinated protein antibody Anti-DNA : Antideoxyribonucleic acid Anti-ENA : Anti-extractable nuclear antigen AOSD : Adult-onset Still's disease APLS : Anti-phospholipid syndrome bDMARD : Biological disease-modifying antirheumatic drugs CK : Creatine kinase CRP : C-reactive protein csDMARD : Conventional synthetic disease-modifying antirheumatic drugs ENT : Ear-nose-throat IL : Interleukin INF : Interferon IQR : Interquartile range JAK : Janus kinase LDH : Lactate dehydrogenase NSAIDs : Nonsteroidal anti-inflammatories RCT : Randomized controlled trial RF : Rheumatoid factor rHLH : Reactive hemophagocytic lymphohistiocytosis SLE : Systemic lupus erythematosus SoJIA : Systemic-onset juvenile idiopathic arthritis TNF : Tumor necrosis factor The authors thank the patients who were involved in this study and the individuals from the rheumatology and internal medicine departments who took part in the care of these patients. Funding {#FPar1} ======= This study was not funded. Availability of data and materials {#FPar2} ================================== The authors state that they had full access to all the data in the study and they take responsibility for the integrity of the data and the accuracy of the data analysis as well as the decision to submit them for publication. FV and MT contributed to the conception and design of the study. FV contributed to the acquisition of the data. FV, TB, and MT contributed to the analysis and interpretation of the data. FV, MT, TB, FL, ES, EL, BF, CR, and TS drafted the manuscript. FV, MT, AB, XD, FL, ES, EL, BF, CR, and TS contributed to the critical revision of the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== Not applicable Consent for publication {#FPar4} ======================= The authors state that they had full access to all the data in the study and they take responsibility for the integrity of the data and the accuracy of the data analysis as well as the decision to submit them for publication. Competing interests {#FPar5} =================== François Vercruysse has no relevant relationship conditions or circumstances that present a potential conflict of interest regarding this study. Thomas Barnetche has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Estibaliz Lazaro has received consultancy fees from Roche, Novartis, and UCB. Emilie Shipley has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. François Lifermann has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Alexandre Balageas has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Xavier Delbrel has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Bruno Fautrel, Marie-Elise Truchetet, Christophe Richez, and Thierry Schaeverbeke have received consultancy fees and/or research funding from Abbvie, Bristol-Myers Squibb, Pfizer, Roche, Lilly, Novartis, and UCB. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	{ "pile_set_name": "PubMed Central" }	[ "treatment", "roch", "probabl", "choic", "two", "anaposit", "ab", "idiopath", "control", "refer", "shipley", "openlabel", "remain", "tumor", "cours", "decreas", "bristolmy", "respond", "polyarthr", "collect", "thank", "longterm", "inf", "exist", "identifi", "classif", "antiphospholipid", "rais", "fund", "controversi", "intermitt", "take", "confront", "fautrel", "valu", "hematopoiet", "may", "exploratori", "anakinrao", "recent", "thirteen", "imbal", "use", "gener", "ineffect", "judg", "univari", "support", "collabor", "final", "ever", "caus", "complet", "accord", "pleuriti", "stiff", "discrep", "vercruyss", "signific", "depart", "establish", "diseasemodifi", "full", "mt", "guidelin", "suggest", "relev", "excel", "market", "also", "spare", "anakinrainduc", "state", "anakinra", "multivari", 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Introduction {#s1} ============ Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) [@pone.0061089-Castello1]. The viral genome encodes a single polyprotein precursor of approximately 3000 amino acids, which is cleaved by cellular and viral proteases into three structural proteins (Core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [@pone.0061089-Levrero1]. The HCV core protein, a component of the viral capsid, is initially split from the viral polyprotein between amino acid residues 191 and 192 by a signal peptidase within the endoplasmic reticulum (ER) lumen. Subsequently, this full-length core protein (amino acids 1--191, 191 a.a.) is further cleaved into a mature form (amino acids 1--173, 173 a.a.) by a signal peptide peptidase (SPP) [@pone.0061089-Liu1], [@pone.0061089-Weihofen1]. Further processing of the core protein in the C-terminal region produces a more truncated form, which can only be produced from limited HCV genotypes [@pone.0061089-Lo1]. The full-length core protein localizes in the cytoplasm, whereas truncated core proteins (amino acids 1--173 and 1--151) localize in the nucleus [@pone.0061089-Liu1], [@pone.0061089-Lo1], [@pone.0061089-Suzuki1]. Some cytoplasmic and nuclear proteins have been reported to interact with HCV core protein [@pone.0061089-Jin1]--[@pone.0061089-Tsutsumi1]. Recently, Moriishi et al. have reported that HCV core protein binds to the proteasome activator PA28γ in the nucleus, which induces liver steatosis and hepatocarcinogenesis through a PA28γ-dependent pathway in core transgenic mice [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2]. These studies suggest that HCV core as a regulatory protein may be involved in hepatocarcinogenesis during chronic HCV infection. MicroRNAs are small, endogenous non-coding RNA molecules that regulate the expression of at least one-third of human genes by inhibiting mRNA translation or inducing its degradation depending on the degree of complementarity [@pone.0061089-Lewis1], [@pone.0061089-Brodersen1]. Studies have shown that cellular microRNAs play an important role in various physiological and pathological processes including human cancers [@pone.0061089-EsquelaKerscher1], [@pone.0061089-Gaur1]. It has been reported that some cellular microRNAs are misregulated in HCV-related HCC [@pone.0061089-Varnholt1], [@pone.0061089-Jiang1]. Recently, HCV-specific effects on the modulation of cellular microRNAs have been shown in full-length HCV genome-expressing HepG2 cells [@pone.0061089-Braconi1]. These studies suggest that HCV proteins-modulated microRNAs may function as oncogenes or as tumor suppressor genes. The misregulation of p21^Waf1/Cip1^ gene expression is frequently observed in human cancers [@pone.0061089-Abbas1]. Hepatocarcinogenesis requires continuous cellular stresses such as viral replication, oxidative stress, inflammation, and continued cell death after regeneration, to receive DNA damage in hepatocytes [@pone.0061089-Castello1], [@pone.0061089-Koike1]. The up-regulation of p21^Waf1/Cip1^ gene expression by cellular stresses may prevent hepatocytes from transformation by inducing a sufficient G1 span to trigger apoptosis or repair DNA damage [@pone.0061089-Abbas1]. Although p21^Waf1/Cip1^ deficiency may not be sufficient to hepatocarcinogenesis, p21^Waf1/Cip1^ gene misregulation may be involved in multistep hepatocarcinogenesis. Moreover, p21^Waf1/Cip1^ may be a target for cancer therapy [@pone.0061089-Abbas1]. Curcumin, a potential anticancer agent, has been used in preclinical in vitro and in vivo models of HCC [@pone.0061089-Darvesh1]. Curcumin exerts its effect on anticancer, at least in part, by triggering apoptosis. Curcumin may induce apoptosis through p21^Waf1/Cip1^-dependent pathway [@pone.0061089-Srivastava1]. Recently, curcumin has been reported to induce apoptosis in human hepatoma cell lines [@pone.0061089-Wang1]. The HCV core protein is considered to be involved in hepatocarcinogenesis [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2], [@pone.0061089-Moriya1]. HCV has been reported to regulate cellular microRNAs [@pone.0061089-Braconi1]. Moreover, the core protein can regulate p21^Waf1/Cip1^ expression [@pone.0061089-Lee1]--[@pone.0061089-Ohkawa1]. In this study, we analyzed the expression profiles of cellular microRNAs in core-overexpressing human hepatoma cells compared to cells nonexpressing core. The HCV core protein was able to up-regulate microRNA-345 (miR-345) expression in human hepatoma cells. The HCV core-induced miR-345 suppressed endogenous p21^Waf1/Cip1^ gene expression through targeting its 3′ untranslated region (3′UTR) in HepG2 cells and curcumin-stimulated Huh7 cells. In addition, the core protein inhibited curcumin-induced apoptosis through p21^Waf1/Cip1^-targeting miR-345 in Huh7 cells. Materials and Methods {#s2} ===================== Antibodies, MicroRNA mimics, Mutant microRNA mimic, Small interfering RNA, MicroRNA inhibitor, and Chemicals {#s2a} ------------------------------------------------------------------------------------------------------------ Anti-HA epitope antibody (catalog no. 11583816001) was purchased from Roche Applied Science, anti-p21^Waf1/Cip1^ antibody (catalog no. sc-6246) was from Santa Cruz Biotechnology, and anti-β-actin antibody (catalog no. A1978) was from Sigma-Aldrich. Anti-HCV core (catalog no. ab2740) and anti-HCV NS5B (catalog no. ab35586) antibodies were purchased from Abcam. Human microRNA-345 (hsa-miR-345, MIMAT0000772) and microRNA-93 (hsa-miR-93, MIMAT0000093) mimics were purchased from Thermo Scientific. Mutant microRNA-345 mimic was synthesized by Thermo Scientific. The sequence was 5′- GCU[CUGA]{.ul}CCUAGUCCAGGGCUC-3′. The four mutated sites are underlined. Human p21^Waf1/Cip1^ small interfering RNA (siRNA, catalog no. sc-29427) was purchased from Santa Cruz Biotechnology. Human miR-345 inhibitor (MH12733) was purchased from Applied Biosystems. Curcumin (catalog no. C7727) and dimethyl sulfoxide (DMSO, catalog no. D2650) were purchased from Sigma-Aldrich. Curcumin was dissolved in DMSO to 100 mM and stored at −20°C until use. Plasmid Construction {#s2b} -------------------- Plasmid pT-REx-HA-Core was generated by cloning the N-terminus HA-tagged HCV core coding sequence (genotype 1b strain) [@pone.0061089-Chen1] into pT-REx-DEST30 vector (Invitrogen) according to the manufacturer\'s instructions. HA-Core was amplified by PCR using pcDNA3-HA-Core191 (amino acids 1--191) as a template. Primers used for cloning HA-Core191 were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAAGCGGAAGCTGGGATGG-3′. Primers used for cloning HA-Core173 (amino acids 1--173) were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAAGAGCAACCGGGCAGAT-3′. Primers used for cloning HA-Core153 (amino acids 1--153) were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAATGTGCCAGGGCTCTGG-3′. A control vector, pT-REx-Mock, was created by deleting HCV core coding sequence from the above construct using BsrGI restriction enzyme. Wild-type human p21^Waf1/Cip1^ 3′UTR was amplified by PCR from human genomic DNA and cloned into pGL3-Control vector (Promega) immediately downstream of luciferase reporter gene but upstream of poly (A) signal using XbaI restriction enzyme to generate pGL3-Control-p21 3′UTR Sense (S) and pGL3-Control-p21 3′UTR Antisense (AS) vectors. Primers used for cloning human p21^Waf1/Cip1^ 3′UTR were forward 5′-CCC[TCTAGA]{.ul}TCCGCCCACAGGAAGCCTGC-3′ and reverse 5′-CCC[TCTAGA]{.ul}AAAGTCACTAAGAATCATTT-3′. The XbaI site is underlined. The mutant p21^Waf1/Cip1^ 3′UTR in the seed sequence of hsa-miR-345 was generated by PCR and ligation of two pieces of DNA fragments, and then cloned into pGL3-Control vector to yield a pGL3-Control-p21 3′UTR Mutant vector. Primer pairs used for cloning mutated p21^Waf1/Cip1^ 3′UTR were forward 5′-CCC[TCTAGA]{.ul}TCCGCCCACAGGAA-3′ and reverse 5′-CCTTGTTCCGCTGCTAATCA-3′, and were forward 5′-TCAGAGACATTTTAAGATGGTGGC-3′ and reverse 5′-CCC[TCTAGA]{.ul}AAAGTCACTAAGAA-3′. The XbaI site is underlined. Full-length HCV Replicon {#s2c} ------------------------ A genotype 1b strain of full-length HCV replicon (HCV-N) [@pone.0061089-Beard1] was kindly provided by Dr. Michael M.C. Lai at Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C.. A control replicon HCV-A357T, which expresses only the initial five amino acids of the core protein due to introduction of a termination codon, was created by the change of one nucleotide at HCV nt 357 in pHCV-N ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, upper panel) using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer\'s instructions. ![HCV core protein up-regulates miR-345 and miR-93 expression in human hepatoma cells.\ (A) Huh7 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1--191, 1--173 and 1--153, respectively. At 48 hours after transfection, the expression of HCV core protein was analyzed by immunoprecipitation. (B) Huh7 cells were transiently transfected with empty vector and two HCV core gene-expressing vectors, pT-REx-HA-Core191, and pT-REx-HA-Core173, for core protein with amino acids 1--191 and 1--173, respectively. At 48 hours after transfection, cellular microRNA profiling was analyzed by TaqMan low density array. Three microRNAs, miR-21, miR-345 and miR93, of thirty-one microRNAs were indicated. (C) Huh7 and HepG2 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1--191, 1--173 and 1--153, respectively. At 48 hours after transfection, relative expression of miR-345 or miR-93 was determined by TaqMan real-time qPCR in Huh7 cells (left upper panel) and HepG2 cells (right upper panel). The expression of HCV core protein was analyzed by Western blotting (left lower and right lower panels). (D) The genotype 1b strain of full-length HCV replicon (HCV-N) and control replicon HCV-A357T which expresses only the initial five amino acids of the core protein due to introduction of a termination codon, was created by the change of one nucleotide at HCV nt 357 in pHCV-N (upper panel). HCV core and NS5B gene expression in full-length HCV replicon-expressing cells was analyzed by immunoprecipitation followed by Western blotting and Western blotting only respectively (left lower panel). The relative expressions of miR-345 and miR-93 were determined by TaqMan real-time qPCR in full-length HCV replicon-expressing Huh7 cells (right lower panel). Data was shown as the means ± S.D. from triplicate experiments. \*P\<0.05, \\P\<0.001.](pone.0061089.g001){#pone-0061089-g001} In Vitro Transcription of HCV RNA {#s2d} --------------------------------- pHCV-N DNA was linearized with XbaI, purified by phenol/chloroform extraction and ethanol precipitation [@pone.0061089-Beard1], and then the linearized DNA was used as template to transcribe full-length HCV RNA using Riboprobe in vitro Transcription Systems (Promega, catalog no. P1440) following the manufacturer\'s protocol. The integrity of RNA transcripts was determined by agarose gel electrophoresis and ethidium bromide staining. Cell Culture and Transfection {#s2e} ----------------------------- Human hepatoma cell lines, Huh7 and HepG2 cells, were cultured at 37°C in a humidified incubator containing 5% CO~2~ in Dulbecco\'s modified Eagle\'s medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone). Plasmid DNA was transfected into cells using GenJet In Vitro DNA Transfection Reagent Ver. II (SignaGen Laboratories) following the manufacturer\'s protocol. To establish the full-length HCV replicon-expressing system in Huh7 cells, HCV RNA was transfected into cells using TransIT-mRNA Transfection Kit (Mirus Bio LLC) according to the manufacturer\'s instructions. The siRNA, microRNA mimic and inhibitor were transfected into cells using GenMute siRNA & DNA Transfection Reagent (SignaGen Laboratories) following the manufacturer\'s protocol. RNA Extraction {#s2f} -------------- Total RNA was extracted using miRNeasy Mini Kit (Qiagen) according to the manufacturer\'s protocol. The trace quantities of genomic DNA were further removed using Turbo DNA-free Kit (Applied Biosystems) following the manufacturer\'s instructions. TaqMan Low Density Array for MicroRNA {#s2g} ------------------------------------- TaqMan low density array was performed using Applied Biosystems 7900HT Fast Real-Time PCR System according to the manufacturer\'s protocol. Briefly, cDNA templates were synthesized using TaqMan MicroRNA Megaplex RT Kit (Applied Biosystems) following the manufacturer\'s instructions. A total reaction mixture containing RT products and TaqMan Universal PCR Master Mix (Applied Biosystems) was added to each line of TaqMan low density array card after gentle vortex mixing. Thermal cycler conditions were as follows: 50°C for 2 minutes, 94.5°C for 10 minutes, and then 40 cycles of 97°C for 30 seconds and 59.7°C for 1 minute. Cycle threshold was automatically given by SDS software v2.2 (Applied Biosystems) using automatic baseline settings and a threshold of 0.2. MammU6 was used as an internal control to normalize the amount of individual microRNA in each sample. Significant difference of relative microRNA expression was determined using the 2^−ΔΔCt^ method [@pone.0061089-Livak1]. TaqMan Real-time qPCR for Single MicroRNA Assay {#s2h} ----------------------------------------------- cDNA template for single microRNA was synthesized using TaqMan MicroRNA RT Kit (Applied Biosystems) according to the manufacturer\'s protocol. Expression of specific microRNA was determined by real-time qPCR using TaqMan MicroRNA qPCR Kit (Applied Biosystems) following the manufacturer\'s instructions. MammU6 was used as an internal control to normalize the amount of specific microRNA in each sample. Significant difference of relative microRNA expression was determined as described above. Real-time qPCR {#s2i} -------------- cDNA template was synthesized using ImProm-II Reverse Transcription System (Promega) according to the manufacturer\'s protocol. Expression of human p21^Waf1/Cip1^ mRNA was determined by real-time qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems) following the manufacturer\'s instructions. The β-actin mRNA was used as an internal control to normalize the amount of human p21^Waf1/Cip1^ mRNA in each sample. Primer sequences for human p21^Waf1/Cip1^ and β-actin mRNAs were described previously [@pone.0061089-Wu1]. Significant difference of relative gene expression was determined as described above. Luciferase Reporter Assay {#s2j} ------------------------- Cells were transfected with luciferase reporter vector in combination with microRNA mimic. All transfections included pRL-TK vector (Promega) for normalization. Luciferase activity was analyzed by Dual-Glo Luciferase Assay System (Promega) according to the manufacturer\'s instructions. Renilla luminescence, expressed from pRL-TK vector, was used as an internal control to normalize the luciferase activity. Immunoprecipitation {#s2k} ------------------- Cell lysates were extracted using RIPA buffer (Sigma-Aldrich) supplemented with Halt Protease Inhibitor Cocktail (Thermo Scientific). The supernatants were incubated for 2 hours at 4°C with anti-HA epitope or anti-HCV core antibody, and then overnight at 4°C with Protein A-agarose beads (Roche Applied Science, catalog no. 11719408001). Following four washes with RIPA buffer, proteins were eluted for 10 minutes at 100°C with 2× Laemmli sample buffer (Sigma-Aldrich), electrophoresed on SDS-PAGE and electroblotted onto PVDF membranes (Millipore). Western Blotting {#s2l} ---------------- Cell lysates were extracted as described above. Cell lysates were electrophoresed on SDS-PAGE and electroblotted onto PVDF membranes. Anti-HA epitope, anti-HCV core, anti-HCV NS5B, anti-p21^Waf1/Cip1^, and anti-β-actin antibodies were used in Western blotting according to the manufacturer\'s protocol. β-actin was used as an internal control. Relative protein expression was quantified by BioSpectrum Imaging System (UVP). DNA Fragmentation Analysis {#s2m} -------------------------- Genomic DNA from cells was extracted using Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer\'s instructions. DNA ladder was determined by agarose gel electrophoresis and ethidium bromide staining. Annexin V-FITC Apoptosis Assay {#s2n} ------------------------------ Apoptotic cells were detected and quantified by fluorescence microscopy and FACS Calibur (Becton Dickinson) respectively, using Annexin V-FITC Apoptosis Detection Kit (BioVision) following the manufacturer\'s protocol. Cell Cycle Analysis {#s2o} ------------------- Cells were subjected to serum starvation for 24 hours to arrest cell growth, and then cultured in fresh serum-containing medium for 24 hours for cell cycle re-entry. Cells were harvested after trypsinization in serum-containing medium, centrifuged and suspended in phosphate buffered saline (PBS). The absolute ethanol was added drop-wise and cells were maintained overnight at −20°C to complete fixation. Cells were centrifuged, resuspended in PBS plus RNase A and propidium iodide (Sigma-Aldrich), and incubated at 37°C for 30 minutes. Fluorescence was measured and analyzed by FACS Calibur. Cell Proliferation Assay (MTT assay) {#s2p} ------------------------------------ Cell proliferation was determined using CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer\'s instructions. To analyze cell proliferation, Huh7 cells were seeded in 96-well culture plates at 3000 cells per well. After 24 hours, the medium was replaced with fresh medium supplemented with increased amount of curcumin. At 24 hours after treatment, Dye Solution was added to each well and cells were incubated at 37°C for 2 hours. Solubilization/Stop Solution was then added to each well and the absorbance was measured at 570 nm using an ELISA reader. Relative cell number was calculated by normalizing the absorbance to untreated cells. Relative cell viability was compared to untreated cells. Statistical Analysis {#s2q} -------------------- Data was shown as the means ± SD from triplicate experiments. The two-sided Student\'s t-test was used for comparisons between experimental groups. P\<0.05 was considered statistically significant. Results {#s3} ======= MicroRNA-345 and microRNA-93 are overexpressed in HCV core-overexpressing human hepatoma cells {#s3a} ---------------------------------------------------------------------------------------------- It has been reported that the full-length HCV core protein (amino acids 1--191) is further cleaved into truncated forms (amino acids 1--173 and 1--153) [@pone.0061089-Liu1], [@pone.0061089-Lo1]. Although the truncated core protein localizes in the nucleus [@pone.0061089-Liu1], [@pone.0061089-Lo1], [@pone.0061089-Suzuki1], only a small quantity of the core protein localizes to the nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [@pone.0061089-Moriya1], [@pone.0061089-Falcn1], [@pone.0061089-Yamaguchi1]. In this study, three core gene-expressing vectors for core protein with amino acids 1--191, 1--173 and 1--153, respectively, were transfected into Huh7 cells. Immunoprecipitation followed by Western blotting was used to determine HCV core expression. The results showed that three forms of HCV core protein were overexpressed in Huh7 cells ([Fig. 1A](#pone-0061089-g001){ref-type="fig"}). Interestingly, a small quantity of a product with lower molecule weight was observed when the full-length core gene was overexpressed in Huh7 cells ([Fig. 1A](#pone-0061089-g001){ref-type="fig"}, lane 2). This product may suggest a truncated core protein trimmed at the C-terminal region due to the intracellular processing of the full-length core protein. To investigate the effect of the full-length and mature core proteins on the modulation of cellular microRNAs, two core gene-expressing vectors for core protein with amino acids 1--191 and 1--173, respectively, were transfected into Huh7 cells, and then the expression profiles of cellular microRNAs was determined by TaqMan low density array at 48 hours after transfection. The result showed that thirty-one microRNAs exhibited a greater than 2-fold up- or down-regulation in full-length core (191 a.a.) or mature core (173 a.a.)-overexpressing Huh7 cells compared to cells nonexpressing core ([Fig. 1B](#pone-0061089-g001){ref-type="fig"}). The HCV core protein can regulate p21^Waf1/Cip1^ expression [@pone.0061089-Lee1]--[@pone.0061089-Ohkawa1]. Recently, Wu et al. have reported that human p21^Waf1/Cip1^ gene expression can be inhibited by twenty-eight microRNAs in HEK 293 cells [@pone.0061089-Wu1]. In this study, we indicated that two p21^Waf1/Cip1^-targeting microRNAs, microRNA-345 (miR-345) and microRNA-93 (miR-93), were up-regulated in core-overexpressing Huh7 cells ([Fig. 1B](#pone-0061089-g001){ref-type="fig"}). Array data were further confirmed by TaqMan real-time qPCR for miR-345 and miR-93 in Huh7 and HepG2 cells. Three core gene-expressing vectors for core protein with amino acids 1--191, 1--173 and 1--153, respectively, were transfected into cells, and then the relative expression of miR-345 and miR-93 was determined at 48 hours after transfection. The results showed that miR-345 and miR-93 were overexpressed with more than 3.5- and 2-fold changes, respectively, in mature core (173 a.a.) and more truncated core (153 a.a.)-overexpressing Huh7 cells but not in full-length core (191 a.a.)-overexpressing cells compared to cells nonexpressing core ([Fig. 1C](#pone-0061089-g001){ref-type="fig"}, left upper panel). Similar results also indicated in mature core and more truncated core-overexpressing HepG2 cells but not in full-length core-overexpressing cells ([Fig. 1C](#pone-0061089-g001){ref-type="fig"}, right upper panel). To further verify the expressions of cellular microRNAs in full-length HCV replicon-expressing system, the full-length HCV replicon was transfected into Huh7 cells. At 96 hours after transfection, immunoprecipitation followed by Western blotting was used to determine HCV core expression, and Western blotting only for NS5B expression ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, left lower panel, lane 2). The relative expressions of miR-345 and miR-93 were determined by TaqMan real-time qPCR. The results showed that miR-345 induction was not significantly affected in full-length HCV replicon-expressing system in Huh7 cells compared to control cells and untreated cells ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, right lower panel, third bar pair). Indeed, there is no detectable or little, if any, amount of truncated form of HCV core protein expression in full-length HCV replicon-expressing system in Huh7 cells ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, left lower panel, lane 2). Furthermore, miR-93 overexpression in full-length HCV replicon-expressing cells but not in full-length core-overexpressing cells suggested that other HCV proteins might up-regulate miR-93 expression. Together, these results demonstrate that truncated HCV core proteins (amino acids 1--173 and 1--153) up-regulate cellular miR-345 and miR-93 expression in human hepatoma cells. MicroRNA-345 down-regulates p21^Waf1/Cip1^ gene expression in human hepatoma cells {#s3b} ------------------------------------------------------------------------------------ It has been reported that human p21^Waf1/Cip1^ gene expression can be inhibited by miR-345 and miR-93 in HEK 293 cells [@pone.0061089-Wu1]. Because the different cell types might generate different results, the effects of miR-345 and miR-93 on human p21^Waf1/Cip1^ gene expression were examined in human hepatoma cells. Two luciferase reporter vectors which bear the sense and antisense 3′UTRs from human p21^Waf1/Cip1^ gene, respectively, were used in luciferase reporter assay ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, upper panel). The relative luciferase activity was determined in Huh7 and HepG2 cells at 24 hours after transfection. The results showed that the treatment with miR-345 mimic led to a significant reduction of luciferase activity in p21 3′UTR Sense construct-transfected Huh7 cells, but treatment with miR-93 mimic had no significant inhibition in luciferase activity ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower panel, second bar cluster). Moreover, treatment with a mixture of miR-345 and miR-93 mimics also had no double reduction of luciferase activity ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower panel, second bar cluster). As expected, miR-345 and miR-93 mimics had no effect in control vector-transfected and p21 3′UTR Antisense construct-transfected Huh7 cells ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower panel, first and third bar clusters). Similar results also indicated in HepG2 cells ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, right lower panel). We further examined human p21^Waf1/Cip1^ gene expression at protein level when miR-93 mimic was transfected into HepG2 cells. As the results in luciferase reporter assay ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower and right lower panels), p21^Waf1/Cip1^ expression at protein level was not suppressed by miR-93 in HepG2 cells ([Fig. S1](#pone.0061089.s001){ref-type="supplementary-material"}). These results demonstrated that human p21^Waf1/Cip1^ gene may be not a target of miR-93 in human hepatoma cells. We further focused on miR-345 to identify the seed sequence in human p21^Waf1/Cip1^ 3′UTR by using computational tools, miRanda (<http://www.microrna.org/microrna/home.do>) and TargetScan (<http://www.targetscan.org/>) ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, left panel). To further verify the seed sequence of miR-345, two luciferase reporter vectors which bear the wild-type and mutant (mutated in the seed sequence of miR-345) 3′UTRs from human p21^Waf1/Cip1^ gene, respectively, were used in luciferase reporter assay ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, left panel, mutated sites are underlined). The results showed that wild-type miR-345 mimic can significantly inhibit the luciferase activity in wild-type p21 3′UTR construct-transfected Huh7 cells but not in mutant p21 3′UTR construct-transfected cells ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, right panel, second and third bar pairs). However, an additional experiment with mutant miR-345 mimic ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, left panel, mutated sites are underlined) and mutant p21 3′UTR with restoring complementarity was able to show a significant inhibition of luciferase activity ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, right panel, fourth bar pair). These results showed that miR-345 may down-regulate human p21^Waf1/Cip1^ gene expression through targeting its 3′UTR in human hepatoma cells. To further verify the down-regulation of endogenous p21^Waf1/Cip1^ gene expression by miR-345, p21^Waf1/Cip1^ mRNA and protein levels were examined in HepG2 cells at 24 hours after transfection with miR-345 mimic. As expected, p21^Waf1/Cip1^ gene expression at mRNA and protein levels was suppressed with the increased amount of miR-345 mimic in HepG2 cells ([Fig. 2C](#pone-0061089-g002){ref-type="fig"}, left and right panels). Together, these results demonstrate that miR-345 down-regulates p21^Waf1/Cip1^ gene expression through targeting its 3′UTR in human hepatoma cells. ![MicroRNA-345 down-regulates p21^Waf1/Cip1^ gene expression through targeting its 3′UTR but not microRNA-93 in human hepatoma cells.\ (A) Two luciferase reporter vectors, pGL3-Control-p21 3′UTR Sense (S) and pGL3-Control-p21 3′UTR Antisense (AS) which bear the sense and antisense 3′UTRs from human p21^Waf1/Cip1^ gene, respectively, were constructed (upper panel). Huh7 cells and HepG2 cells were transfected with p21 3′UTR sense or antisense luciferase reporter vector in combination with miR-345 mimic, miR-93 mimic or a mixture of miR-345 and miR-93 mimics. At 24 hours after transfection, Huh7 cells (left lower panel) and HepG2 cells (right lower panel) were collected for luciferase reporter assay. (B) Base-pairing between mature hsa-miR-345 and target site in human p21^Waf1/Cip1^ 3′UTR is shown (left panel). One luciferase reporter vector, pGL3-Control-p21 3′UTR Mutant (mutated in the seed sequence of miR-345), was constructed. Mutant miR-345 mimic was synthesized. The mutated site is underlined (left panel). Huh7 cells were transfected with wild-type p21 3′UTR sense or mutant p21 3′UTR luciferase reporter vector in combination with wild-type or mutant miR-345 mimic. At 24 hours after transfection, Huh7 cells were collected for luciferase reporter assay. The experiment with mutant miR-345 mimic and mutant p21 3′UTR with restoring complementarity was performed (right panel). (C) HepG2 cells were transiently transfected with the increased amount of miR-345 mimic for 24 hours. The p21^Waf1/Cip1^ gene expression was analyzed by real-time qPCR (left panel) and Western blotting (right panel). β-actin served as an internal control. Data was shown as the means ± S.D. from triplicate experiments. \*P\<0.05.](pone.0061089.g002){#pone-0061089-g002} MicroRNA-345 inhibits curcumin-induced apoptosis through down-regulation of p21^Waf1/Cip1^ gene expression in Huh7 cells {#s3c} -------------------------------------------------------------------------------------------------------------------------- Curcumin, a potential anticancer agent, has been used in preclinical in vitro and in vivo models of HCC [@pone.0061089-Darvesh1]. Recently, curcumin has been reported to induce apoptosis in Huh7 cells [@pone.0061089-Wang1]. In this study, we showed that p21^Waf1/Cip1^ gene expression at protein level was enhanced with increased amount of curcumin in Huh7 cells ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, upper panel). To investigate the functional relevance of p21^Waf1/Cip1^ up-regulation and curcumin treatment in Huh7 cells, annexin V-FITC apoptosis assay, MTT assay, and cell cycle analysis were performed. The results showed that curcumin induced apoptosis and inhibited cell viability in Huh7 cells in a dose-dependent manner ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, middle and left lower panels). Furthermore, the result also showed that S-phase entry of cell cycle was slightly inhibited in Huh7 cells in response to high doses of curcumin ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, right lower panel). To further verify that the up-regulation of p21^Waf1/Cip1^ gene expression involved in curcumin-induced apoptosis in Huh7 cells, curcumin-stimulated cells were transfected with increased amount of p21^Waf1/Cip1^ siRNA, and then apoptosis was analyzed by using DNA fragmentation analysis at 24 hours after transfection. The result showed that apoptosis was inhibited as presented in DNA ladder disappeared when p21^Waf1/Cip1^ gene expression at protein level was suppressed in curcumin-stimulated Huh7 cells ([Fig. 3B](#pone-0061089-g003){ref-type="fig"}), indicating that curcumin induced apoptosis through up-regulation of p21^Waf1/Cip1^ gene expression in Huh7 cells. Similar results were also showed in [Figure 3C](#pone-0061089-g003){ref-type="fig"}. Curcumin had no effect on the modulation of cellular miR-345 expression in Huh7 cells ([Fig. S2](#pone.0061089.s002){ref-type="supplementary-material"}). As expected, p21^Waf1/Cip1^ gene expression at protein level was suppressed by miR-345 mimic in curcumin-stimulated Huh7 cells in a dose-dependent manner ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, upper panel). Curcumin-induced apoptosis was inhibited with increased amount of miR-345 mimic ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, left middle panel). Moreover, the introduction of miR-345 mimic enhanced cell viability in curcumin-stimulated Huh7 cells ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, right middle panel). The number of apoptotic cells (early and late apoptotic cells) had a maximum reduction of about 55% when curcumin-stimulated cells were transfected with 50 nM miR-345 mimic ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, left lower and right lower panels). In HepG2 cells, we also showed that curcumin induced apoptosis, but curcumin had no effect on the modulation of p21^Waf1/Cip1^ gene expression. The results indicated that curcumin was able to induce apoptosis through a p21^Waf1/Cip1^-independent pathway in HepG2 cells ([Fig. S4](#pone.0061089.s004){ref-type="supplementary-material"}). Together, these results demonstrate that miR-345 inhibits curcumin-induced apoptosis through down-regulation of p21^Waf1/Cip1^ gene expression in Huh7 cells. ![MicroRNA-345 inhibits curcumin-mediated apoptosis through down-regulation of p21^Waf1/Cip1^ gene expression in Huh7 cells.\ (A) Huh7 cells were treated with different doses (6.25, 12.5, 25 and 50 µM) of curcumin for 24 hours. DMSO served as control (labeled with 0 µM). The p21^Waf1/Cip1^ gene expression at protein level was determined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was determined by annexin V-FITC apoptosis assay (middle panel). Cell proliferation was analyzed by MTT assay (left lower panel). Cell cycle distribution was examined by cell cycle analysis (right lower panel). (B) Huh7 cells were transfected with the increased amount of p21^Waf1/Cip1^ siRNA in response to curcumin stimulation (50 µM) for 24 hours. Apoptosis was analyzed by DNA fragmentation analysis. The p21^Waf1/Cip1^ gene expression at protein level was examined by Western blotting. (C) Huh7 cells were transfected with the increased amount of miR-345 mimic in response to curcumin stimulation (50 µM) for 24 hours. The p21^Waf1/Cip1^ gene expression at protein level was examined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was analyzed by fluorescence microscopy (left middle panel) and FACS Calibur (left lower and right lower panels) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Cell proliferation was analyzed by MTT assay (right middle panel). Data was shown as the means ± S.D. from triplicate experiments. \*P\<0.05, \\P\<0.001.](pone.0061089.g003){#pone-0061089-g003} HCV core-induced microRNA-345 inhibits p21^Waf1/Cip1^ gene expression in HepG2 cells and curcumin-stimulated Huh7 cells {#s3d} ------------------------------------------------------------------------------------------------------------------------- It has been reported that the mature form (amino acids 1--173) of HCV core protein in the nucleus suppresses p21^Waf1/Cip1^ gene expression in HepG2 cells [@pone.0061089-Yamanaka1]. To determine that HCV core-induced miR-345 down-regulated endogenous p21^Waf1/Cip1^ gene expression, core expressing HepG2 cells were transfected with or without miR-345 inhibitor, and then p21^Waf1/Cip1^ gene expression at protein level was determined at 24 hours after transfection. As expected, the mature form (amino acids 1--173) of the core protein suppressed p21^Waf1/Cip1^ gene expression ([Fig. 4A](#pone-0061089-g004){ref-type="fig"}, lane 3). However, the suppression of p21^Waf1/Cip1^ gene expression was attenuated in the presence of increased amount of miR-345 inhibitor ([Fig. 4A](#pone-0061089-g004){ref-type="fig"}, lane 3, lane 6 and lane 7). The full-length core protein (amino acids 1--191) slightly suppressed p21^Waf1/Cip1^ gene expression ([Fig. 4A](#pone-0061089-g004){ref-type="fig"}, lane 2), this result may be due to the expression of a small quantity of the truncated core protein ([Fig. 1A](#pone-0061089-g001){ref-type="fig"}, lane 2). We have shown that only little amount of p21^Waf1/Cip1^ protein can be detected in Huh7 cells ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, upper panel, lane 1). Therefore, curcumin was used to induce endogenous p21^Waf1/Cip1^ gene expression to investigate the inhibition of p21^Waf1/Cip1^ gene expression by HCV-core-induced miR-345 in Huh7 cells. Similar results were also showed in [Figure 4B](#pone-0061089-g004){ref-type="fig"}. As expected, the mature form (amino acids 1--173) of the core protein suppressed p21^Waf1/Cip1^ gene expression in curcumin-stimulated Huh7 cells ([Fig. 4B](#pone-0061089-g004){ref-type="fig"}, upper panel, lane 3). Similarly, the suppression of p21^Waf1/Cip1^ gene expression was attenuated when mature core-expressing cells were transfected with miR-345 inhibitor ([Fig. 4B](#pone-0061089-g004){ref-type="fig"}, upper panel, lane 3, lane 6 and lane 7). Additionally, the suppression of curcumin-induced apoptosis by mature core protein (amino acids 1--173) was significantly attenuated in the presence of increased amount of miR-345 inhibitor ([Fig. 4B](#pone-0061089-g004){ref-type="fig"}, middle and lower panels). Together, these results demonstrate that HCV core-induced miR-345 inhibits endogenous p21^Waf1/Cip1^ gene expression in HepG2 cells and curcumin-stimulated Huh7 cells. ![HCV core-induced microRNA-345 inhibits p21^Waf1/Cip1^ gene expression in HepG2 cells and curcumin-stimulated Huh7 cells.\ (A) HCV core (amino acid 1--191 or 1--173)-expressing HepG2 cells were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, p21^Waf1/Cip1^ gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control. (B) HCV core (amino acid 1--191 or 1--173)-expressing Huh7 cells in response to curcumin stimulation were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, p21^Waf1/Cip1^ gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control (upper panel). Apoptosis was analyzed by fluorescence microscopy (middle panel) and FACS Calibur (lower panel) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Data was shown as the means ± S.D. from triplicate experiments. \*P\<0.05, \\P\<0.001.](pone.0061089.g004){#pone-0061089-g004} Discussion {#s4} ========== In this study, we determine the effect of a mature HCV core protein on miR-345 induction in human hepatoma cells. Moreover, HCV core-induced miR-345 suppresses p21^Waf1/Cip1^ gene expression in HepG2 cells, and inhibits curcumin-mediated apoptosis through down-regulation of p21^Waf1/Cip1^ gene expression in Huh7 cells. HCV core protein has an effect on cellular microRNA regulation. HCV core protein is initially separated from HCV polyprotein by a signal peptidase. The full-length core protein (amino acids 1--191) localizes in the cytoplasm, which mainly functions as a component of the viral capsid. HCV core protein is considered to play a crucial role in hepatocarcinogenesis [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2], [@pone.0061089-Moriya1]. Many studies have reported that HCV core protein can interact with cytoplasmic and nuclear proteins [@pone.0061089-Jin1]--[@pone.0061089-Tsutsumi1]. Chen et al. has shown that HCV core protein interacts with Dicer, an RNase enzyme that generates mature microRNAs, in the cytoplasm, and then inhibits the function of Dicer [@pone.0061089-Chen2]. This inhibitive effect may contribute to HCV replication [@pone.0061089-Pedersen1]. The mature form (amino acids 1--173) of HCV core protein cleaved by SPP, which lacks the 174--191 peptide for attachment to ER membrane, enables subcellular distribution of the core protein [@pone.0061089-Liu1]--[@pone.0061089-Suzuki1]. Unlike full-length core protein (amino acids 1--191), mature form (amino acids 1--173) has been reported to mainly localize to nucleus [@pone.0061089-Liu1], [@pone.0061089-Lo1], [@pone.0061089-Suzuki1]. Some studies have shown that a small quantity of HCV core protein localizes to the nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [@pone.0061089-Moriya1], [@pone.0061089-Falcn1], [@pone.0061089-Yamaguchi1]. In this study, we observed a small quantity of a product with lower molecule weight when full-length HCV core gene was expressed in cultured Huh7 cells. These studies suggest that the nuclear localization or truncated form of the core protein may play an important role in chronic HCV infection. Moriishi et al. has reported that mature (amino acids 1--173) and more truncated (amino acids 1--151) forms of HCV core protein can bind to PA28γ in the nucleus and hence induces liver steatosis and HCC development through a PA28γ-dependent pathway in core gene-transgenic mice [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2], suggesting that the nuclear localization of the truncated core proteins may function as a transcriptional factor or regulator. In this study, up-regulation of miR-345 expression by mature form (amino acids 1--173) of the core protein may be also associated with its nuclear localization. Indeed, we also demonstrated that more truncated core protein (amino acids 1--153) which deletes more residues of hydrophobic C-terminal region up-regulated miR-345 expression, indicating that the up-regulation of miR-345 expression may be associated with the nuclear localization of HCV core protein. MiR-345 is down-regulated in full-length HCV genome-expressing cells as reported by Braconi et al. [@pone.0061089-Braconi1]. Our result was not consistent with the observation from Braconi\'s group, since the different expressing system and different hepatoma cells were used in the experiments. There is no detectable or little, if any, amount of truncated form of HCV core protein expression in our full-length HCV replicon-expressing system. However, truncated form of HCV core protein has been demonstrated to localize in nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [@pone.0061089-Moriya1], [@pone.0061089-Falcn1], [@pone.0061089-Yamaguchi1]. The up-regulation of miR-345 expression by HCV core protein may be associated with the demethylation of its promoter. Tang et al. has demonstrated that miR-345 with a CpG island in the promoter is a methylation-sensitive microRNA and is highly induced by demethylating agent in human colorectal cancer cell lines [@pone.0061089-Tang1]. Recently, miR-21 has been identified to indirectly down-regulate DNA methyltransferase 1 (DNMT1) expression by directly targeting human RASGRP1 gene, a known critical regulator of the upstream Ras-MAPK pathway of DNMT1 [@pone.0061089-Pan1]. In this study, we also determined the up-regulation of miR-21 expression in HCV core-overexpressing Huh7 and HepG2 cells ([Fig. S3](#pone.0061089.s003){ref-type="supplementary-material"}). The up-regulation of miR-21 expression by mature form (amino acids 1--173) of the core protein may contribute to miR-345 promoter hypomethylation. Up-regulation of miR-21 expression has been reported in HCV infectious clone-infected Huh7.5 cells and patients with chronic HCV infection [@pone.0061089-Marquez1]. In our study, the mature form (amino acids 1--173) of the core protein can enhance miR-21 expression, however, the full-length core protein had no effect on miR-21 induction. This finding suggested that the mature form (amino acids 1--173) of the core protein was relevant to the regulation of cellular microRNAs in chronic HCV infection. In fact, we also initially observed the modulation of most of cellular microRNAs by mature form (amino acids 1--173) of the core protein ([Fig. 1B](#pone-0061089-g001){ref-type="fig"}). Further confirmation will be needed. Up-regulation of miR-345 expression may involve in cancer development. Some studies have described the up-regulation of miR-345 expression in human cancers including oral squamous cell carcinomas and malignant mesothelioma [@pone.0061089-Cervigne1], [@pone.0061089-Guled1]. A recent study also reveals that some tumor-related microRNAs including miR-345 are up-regulated from 28 published tumor studies by analyzing microRNA expression microarray datasets, indicating that miR-345 may be an oncomiR in human cancers including HCC [@pone.0061089-Wang2]. MiR-345 has been reported to down-regulate BAG3 gene expression, an anti-apoptosis gene, as a tumor suppressor microRNA in human colorectal cancer [@pone.0061089-Tang1]. However, in our study, miR-345 down-regulates p21^Waf1/Cip1^ gene expression as an oncomiR in human hepatoma cells. These two studies suggest that the same microRNA may function as an oncogene or as a tumor suppressor gene depending on cell types and microenvironment. Our study shows that the up-regulation of miR-345 expression may be related to hepatocarcinogenesis during chronic HCV infection. In conclusion, our study demonstrates that mature or more truncated HCV core protein (amino acids 1--173 or 1--153) can up-regulate the expression of miR-345 which then suppresses p21^Waf1/Cip1^ gene expression in human hepatoma cells. We also show that mature HCV core-induced miR-345 can suppress endogenous p21^Waf1/Cip1^ gene expression in HepG2 and curcumin-stimulated Huh7 cells. Furthermore, we demonstrate that mature HCV core-induced miR-345 involves in anti-apoptosis through down-regulation of p21^Waf1/Cip1^ gene expression in curcumin-stimulated Huh7 cells. In conclusion, it is the first time that HCV core protein has ever been demonstrated to inhibit human p21^Waf1/Cip1^ gene expression through miR-345 targeting. Supporting Information {#s5} ====================== ###### MicroRNA-93 cannot down-regulate endogenous *p21^Waf1/Cip1^* gene expression in human hepatoma cells. HepG2 cells were transiently transfected with the increased amount of miR-93 mimic for 24 hours. The p21^Waf1/Cip1^ gene expression at protein level was determined by Western blotting. β-actin served as an internal control. (TIF) ###### Click here for additional data file. ###### Curcumin has no effect on endogenous miR-345 expression. Huh7 cells were treated with curcumin in dose- (left panel) and time- (right panel) dependent manners. The relative expression of miR-345 was determined by TaqMan real-time qPCR. Data was shown as the means ± S.D. from triplicate experiments. \*P\<0.05. (TIF) ###### Click here for additional data file. ###### HCV core protein up-regulates miR-21 expression in human hepatoma cells. Huh7 and Hepg2 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1--191, 1--173 and 1--153, respectively. At 48 hours after transfection, relative expression of miR-21 was determined by TaqMan real-time qPCR in Huh7 cells (left panel) and HepG2 cells (right panel). Data was shown as the means ± S.D. from triplicate experiments. \*P\<0.05, \\P\<0.001. (TIF) ###### Click here for additional data file. ###### Curcumin induces apoptosis through p21^Waf1/Cip1^-independent pathway in HepG2 cells. HepG2 cells were treated with different doses (6.25, 12.5, 25, and 50 µM) of curcumin for 24 hours. DMSO served as control (labeled with 0 µM). The p21^Waf1/Cip1^ gene expression at protein level was determined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was determined by annexin V-FITC apoptosis assay (lower panel). Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. (TIF) ###### Click here for additional data file. We thank King-Song Jeng and Michael M.C. Lai at Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C. for reading the manuscript and providing helpful suggestions and the full-length HCV replicon of a Japanese genotype 1b strain (pHCV-N). [^1]: Competing Interests:The authors have declared that no competing interests exist. [^2]: Final approval of the version to be published: SMH TYH. Conceived and designed the experiments: TYS TYH. Performed the experiments: TYS SMH. Analyzed the data: YLS HCC WKC. 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INTRODUCTION ============ Helicobacter pylori infection affects more than 50% of the world's human population and is associated with gastritis, peptic ulcer disease, and gastric cancer \[[@b1-kjim-2019-139]\]. In Korea, the H. pylori infection rate in adults is approximately 60% and gastric cancer is the second most frequently diagnosed malignancy \[[@b2-kjim-2019-139],[@b3-kjim-2019-139]\]. Successful eradication of H. pylori could be beneficial in alleviating H. pylori-related gastroduodenal diseases and reduce the risk of gastric cancer in countries like Korea where the prevalence of both H. pylori infection and gastric cancer are high. Until now, the triple therapy combining proton pump inhibitor (PPI) with two antibiotics (clarithromycin and amoxicillin) has been the standard first-line treatment for H. pylori eradication. Nonetheless, the eradication rate of this regimen has decreased to \< 80% \[[@b4-kjim-2019-139]-[@b7-kjim-2019-139]\]. The primary factor associated with eradication failure is clarithromycin resistance. In Korea, the clarithromycin resistance rate gradually increased from 17.2% to 23.7% during 2003 to 2008 \[[@b8-kjim-2019-139]\]. The second factor related to eradication failure is increased rates of antibiotic-associated adverse events, which could result in poor patient compliance \[[@b9-kjim-2019-139]\]. In areas with high clarithromycin resistance, bismuth-containing quadruple or non-bismuth quadruple therapies are recommended. Additionally, alternative treatment strategies that increase the eradication rate and reduce adverse events should be developed. Probiotics supplementation with the standard triple therapy might be a candidate to satisfy this purpose. Administration of probiotics have reportedly improved H. pylori eradication rates and reduced adverse events associated with the triple therapy \[[@b10-kjim-2019-139]-[@b12-kjim-2019-139]\]. However, the inhibitory effect of probiotics on H. pylori infection remains controversial. Recently, two large meta-analyses showed that Saccharomyces boulardii reduced overall adverse events and increased eradication rates \[[@b13-kjim-2019-139]\]. Sulforaphane extracted from broccoli is another supplementary candidate for H. pylori eradication therapy. Broccoli sprout extract containing sulforaphane (BSES) exhibits cellular anti-oxidative, anti-inflammatory, and anti-cancer effects \[[@b14-kjim-2019-139]-[@b16-kjim-2019-139]\]. Sulforaphane is a potent bacteriostatic agent against H. pylori strains and also exhibits bactericidal effects in human epithelial cells \[[@b17-kjim-2019-139]\]. Although no statistically significant effects have been demonstrated in the previous study, we have identified a tendency to show positive results in some urea breath test (UBT) results; further studies have been presented on the effects of using the combination with standard triple therapy \[[@b18-kjim-2019-139]\]. This study excluded patients with cultures resistant to clarithromycin and registered only those subjects with infections not resistant to clarithromycin. Based on our literature review, to our knowledge, this is the first study designed to eliminate clarithromycin resistance as the affecting bias of H. pylori eradication failure. We aimed to determine whether S. boulardii probiotics or sulforaphane supplementation could increase the H. pylori eradication rate and/or reduce antibiotic-associated adverse events in a Korean population. METHODS ======= Patients and study protocol --------------------------- All patients with H. pylori-positive chronic gastritis or peptic ulcer disease were recruited at the Kyung Hee University Hospital in Seoul, Korea, from August 2014 to April 2016. All participants were diagnosed with H. pylori infection using rapid urease or C^13^-UBTs. Patients with gastric cancer, previous gastrectomy, and severe underlying systemic diseases, or those who had received antibiotics or PPI within the previous month were excluded. Data including age, gender, cigarette smoking, alcohol intake, salty food consumption, family history of gastric cancer, body mass index (BMI), hypertension, and diabetes were collected. All patients underwent clarithromycin resistance testing and subjects with clarithromycin-resistant infections were excluded from the study. Finally, subjects were registered and a computer program was used to randomly assigned the subjects into three treatment groups: triple therapy only (group A), triple therapy plus probiotics (group B), and triple therapy plus sulforaphane (group C) ([Fig. 1](#f1-kjim-2019-139){ref-type="fig"}). Group A received 40 mg of pantoprazole, 1 g of amoxicillin, and 500 mg of clarithromycin twice daily for 7 days. Groups B and C received the same PPI-based triple therapy for 7 days. Additionally, one capsule with either probiotics or sulforaphane was provided three times daily for 4 weeks to patients in groups B and C, respectively. All patients provided written informed consent prior to enrolment. This study was approved by the Institutional Review Board of the Kyung Hee University Hospital (KMC IRB: 1427-01) and registered with ClinicalTrials. gov (NCT03220542). All authors had access to the study data and reviewed and approved the final manuscript. Probiotics and broccoli sprout extract containing sulforaphane -------------------------------------------------------------- S. boulardii capsules (3 × 10^10^ colony-forming units/g, Bioflor, Kuhnil Pharm., Seoul, Korea) were administered as the probiotic. BSES capsules containing 250 mg standardized broccoli sprout, yielding 1,000 μg of sulforaphane (Oregon Health, Phoenix, AZ, USA), were administered. The doses from broccoli and probiotics were determined to meet the doses presented in several studies, including previous studies of us \[[@b18-kjim-2019-139]\]. Assessment of H. pylori eradication and adverse events -------------------------------------------------------- C^13^-UBTs were performed 4 weeks after the last dose of the study medication. One week after completing treatment, information on adverse events and compliance was collected from the outpatient department through telephone interviews. Compliance was considered satisfactory when patients had taken \> 85% of the study medications. Clarithromycin resistance tests ------------------------------- Mutation of the 23S rRNA results in increased antibiotic resistance \[[@b19-kjim-2019-139]\]. Clarithromycin resistance in H. pylori in the present study was determined by evaluating for the presence of the A2142G or A2143G mutations in 23S rRNA. H. pylori genomic DNA was extracted from two biopsy specimens from the antrum and body collected during endoscopy. The DNA was amplified using the Seeplex home-brew primer mix from the Seeplex ClaR-H. pylori polymerase chain reaction (PCR) kit (Seegene Inc., Seoul, Korea) that was developed using a dual-priming oligonucleotide system to detect the A2142G or A2143G mutations. CYP2C19 genotyping ------------------ The genotyping of exons 4 and 5 in CYP2C19 was conducted by PCR with restriction fragment length polymorphism (PCR-RFLP). The polymorphism of CYP2C19 was detected as previously described \[[@b20-kjim-2019-139]\]. The patients were classified into three groups according to CYP2C19 genotype: rapid metabolizers (RM) had exon 5, wildtype/exon 4, wild-type; intermediate metabolizers (IM) had exon 5, wild-type/exon 4, heterozygote or exon 5, heterozygote/exon 4, wild-type; and poor metabolizers (PM) had both mutated alleles. Data analysis ------------- Eradication was evaluated using intention-to-treat (ITT) and per-protocol (PP) analyses. Continuous variables are expressed as means ± standard deviation, and categorical variables as percentages. Student's t test, chi-square test, and two-factor analysis of variation (ANOVA) were used. PASW Statistics for Windows version 22.0 (IBM Co., Armonk, NY, USA) was used for all statistical analyses. All statistical tests were two-tailed and p values of \< 0.05 were assumed to indicate statistical significance. RESULTS ======= Baseline characteristics of the study population ------------------------------------------------ A total of 217 patients with H. pylori-positive chronic gastritis or peptic ulcer disease were recruited during study periods. Of these, 34 subjects with clarithromycin-resistant infections were excluded from the study. The rate of clarithromycin resistance in this study was 17.3% (34/217 cases). Two cases were dropped. Finally, 183 patients with clarithromycin-sensitive infections were randomly assigned the subjects into three treatment groups: group A (n = 61), B (n = 61), and C (n = 61). The clinical parameters such as age, gender, cigarette smoking, alcohol intake, family history of gastric cancer, salty food consumption, and BMI, were similar among the three groups. Endoscopic finding and CYP2C19 genotype did not differ significantly between the three groups ([Table 1](#t1-kjim-2019-139){ref-type="table"}). Eradication rates ----------------- The eradication rates according to ITT and PP analyses were 85.2% and 89.6% in group A, respectively. ITT and PP analyses showed 81.9% and 89.2% eradication rates in group B and 86.8% and 96.3% in group C, respectively. The eradication rates according to ITT and PP analyses were not significantly different across the groups based on two-group comparison tests (ITT analysis: A vs. B, p = 0.744; A vs. C, p = 1.000) (PP analysis: A vs. B, p = 0.313; A vs. C, p = 0.273). The eradication rate in group C was slightly higher compared to group A. However, the difference between the two groups (A vs. C) was not statistically significant ([Table 2](#t2-kjim-2019-139){ref-type="table"}). Adverse events -------------- In order of frequency, the adverse events included taste disturbance, diarrhea, headache, epigastric pain, nausea, and urticaria. The frequency of overall adverse events in the three groups was not significantly different (A vs. B, p = 0.574; A vs. C, p = 1.000). The frequencies of gastrointestinal disturbances such as diarrhea, epigastric pain, and nausea were lower in group B compared to group A, but the difference was not statistically significant (p = 0.339) ([Table 3](#t3-kjim-2019-139){ref-type="table"}). CYP2C19 genotypes ------------------- Analysis of CYP2C19 genotypes revealed the RM genotype in 16.4%, 24.6%, and 21.3% of patients in groups A, B, and C, respectively. The frequency of the RM genotype did not differ significantly across the three groups as per the results of the multiple group comparison test (p = 0.532) ([Table 1](#t1-kjim-2019-139){ref-type="table"}). DISCUSSION ========== The Maastricht V/Florence Consensus Report stated that PPI-clarithromycin-amoxicillin triple therapy without prior susceptibility test should be reconsidered in regions with a clarithromycin resistance rate above 15% \[[@b21-kjim-2019-139]\]. Based on our literature review, ours is the first study to exclude patients resistant to clarithromycin, which minimized the most significant bias associated with eradication failure. In our study, 17.3% of the patient infections were clarithromycin-resistant. In another recent Korean study, the clarithromycin resistance rate was \> 20% \[[@b22-kjim-2019-139]\]. This difference may be attributable to different tests used for assessing clarithromycin resistance as well as our study including a large number of relatively heathy subjects. Nonetheless, considering that the clarithromycin resistance rate was \> 15%, alternative regimens are needed for first-line treatment in Korea. Bismuth quadruple (PPI, bismuth, tetracycline, and metronidazole) or concomitant non-bismuth quadruple (PPI, amoxicillin, clarithromycin, and metronidazole) therapies are recommended as first-line treatments \[[@b21-kjim-2019-139]\]. However, these therapies are associated with poor patient compliance and increased adverse events owing to antibiotic overdose. Although not highly evident, supplemental therapy with probiotics or sulforaphane could increase eradication rates and decrease antibiotic-associated adverse events. A meta-analysis of 10 clinical trials on the efficacy of probiotics in H. pylori treatment showed that Lactobacillus-containing and Bifidobacterium-containing probiotic compound preparations increased the overall eradication rate and decreased the overall adverse events \[[@b23-kjim-2019-139]\]. However, another meta-analysis of 33 randomized controlled trials (RCTs) could confirm this finding for only four individual strains (Lactobacillus acidophilus, L. casei, L. gasseri, and Bifidobacterium infantis) \[[@b24-kjim-2019-139]\]. This study also performed sub-group analysis according to the effectiveness of eradication regimens. In that study, the less effective the antibiotic therapies, the more useful the probiotic supplementation was: for eradication rates \< 60%, the pooled risk ratio (RR) was 1.28 (1.12 to 1.45), for eradication rates of 60% to 69%, the pooled RR was 1.18 (1.10 to 1.27); for eradication rates of 70% to 79%, the pooled RR was (1.11; 1.06 to 1.17); and supplementation had no effect for eradication rates \> 80% (pooled RR, 1.01; 0.96 to 1.77). The authors also reported a significant difference between the probiotics supplement and triple therapy only groups in the overall incidence of side effects (RR, 0.735; 95% confidence interval, 0.598 to 0.902) \[[@b24-kjim-2019-139]\]. However, the result of side effects was only confirmed for non-blinded trials. One double-blind RCT showed that Bacillus clausii significantly reduced gastrointestinal adverse events such as nausea, epigastric pain, and diarrhea but insignificantly increased eradication rates \[[@b25-kjim-2019-139]\]. Recently, a meta-analysis of 11 RCTs showed that S. boulardii significantly increased eradication rates but that the rates were still below the desired level of success \[[@b13-kjim-2019-139]\]. Probiotics reduce antibiotic-associated adverse events such as nausea, diarrhea, and abdominal pain, thus helping the completion of eradication therapy. Therefore, probiotics can improve the H. pylori eradication rate by reducing adverse events rather than by directly eradicating H. pylori. The proposed mechanism of probiotics is to counteract the harmful effects of antibiotics on gut microbiota. However, the opposite conclusions were drawn regarding side effects in three studies reporting no significant reduction in overall side effects with probiotics use \[[@b26-kjim-2019-139]-[@b28-kjim-2019-139]\]. Our study showed that probiotic supplementation did not significantly increase the eradication rate. This may be because of several factors. Firstly, the number of subjects was small. Secondly, the eradication rate was \> 80% and so likely did not contribute an additive advantage, similar to that reported by Dang et al. \[[@b24-kjim-2019-139]\]. Overall, the rate of adverse events was lower in the probiotics supplement group B (triple therapy plus probiotics group) compared to group A (standard triple therapy only group). However, the difference was not statistically significant. Sulforaphane is a molecule within the isothiocyanate group of organosulfur compounds. It can be obtained from cruciferous vegetables such as broccoli, Brussels sprouts, and cabbages. Sulforaphane enhances the protection and repair of the gastric mucosa against oxidative stress in vitro and has been shown to have anti-inflammatory effects on H. pylori-infected gastric mucosae in mice and human subjects \[[@b29-kjim-2019-139]\]. Sulforaphane has a potent bacteriostatic and bactericidal component against H. pylori in vitro \[[@b17-kjim-2019-139]\]. Furthermore, it is highly effective against a large number of clinical isolates of H. pylori, which are highly resistant to conventional antibiotics \[[@b17-kjim-2019-139]\]. Recently, sulforaphane was reported to reduce colonization and attenuate gastritis in H. pylori-infected mice and humans \[[@b30-kjim-2019-139]\]. However, a Japanese epidemiological investigation of the relationship between the prevalence of H. pylori-induced chronic atrophic gastritis (CAG) and broccoli consumption found no association between frequent broccoli consumption and a low prevalence of CAG. In addition, our previous study did not show the inhibitory effect of sulforaphane on H. pylori colonization density in the gastric mucosa of patients with H. pylori-positive gastritis \[[@b18-kjim-2019-139]\]. In the present study, the eradication rate in the sulforaphane supplementation group was slightly higher than that in group A (standard triple therapy only group), but the difference was not statistically significant. The overall rate of adverse events was also similar to group A. Our study has some limitations. First, we included relatively small number of subjects in each group. However, this study was a single center study, and it had limitation to recruit enough patients during study period. Instead, during the study period, all patients who agreed to participate were enrolled by our study. In our study, probiotic supplementation and sulforaphane did not significantly increase the eradication rate nor decrease the adverse events. Nevertheless, the eradication rate in sulforaphane group was slightly higher than that in only triple therapy group. A further study including large number of subjects is needed in the future. Second, we used only S. boulardii capsules as the probiotic. The probiotics most commonly used include Lactobacillus spp., Bacillus spp., and yeast such as S. boulardii. The choice of the S. boulardii was determined by the effectiveness as well as being the most commonly used in most studies. In conclusion, we found that supplement therapy with probiotics or sulforaphane for H. pylori eradication was ineffective at increasing eradication rates and decreasing adverse events. KEY MESSAGE =========== 1\. Probiotic or sulforaphane supplementation with triple therapy for Helicobacter pylori infection did not significantly increase the eradication rate nor reduced the adverse events. 2\. An appropriate treatment strategy and new drawn up guidelines on treatment of H. pylori are necessary for the effective and safety eradication of H. pylori. We appreciate the excellent support of our nursing team at the Kyung Hee University Hospital-Digestive Endoscopy Center. No potential conflict of interest relevant to this article was reported. ![Study enrolment flowchart.](kjim-2019-139f1){#f1-kjim-2019-139} ###### Baseline characteristics of the study population Variable Group A (n = 61) Group B (n = 61) Group C (n = 61) p value ------------------------ ------------------ ------------------ ------------------ ----------- Age, yr 53.20 ± 10.97 55.6 ± 10.65 55.77 ± 10.05 0.315 Sex, male/female 33/28 30/31 30/31 0.821 Smoking habits 14 (23.0) 13 (21.3) 10 (16.4) 0.644 Alcohol intake 28 (45.9) 23 (37.7) 19 (31.1) 0.244 Family history of GC 6 (9.8) 8 (13.1) 7 (11.5) 0.851 Salty food consumption 16 (26.2) 27 (44.3) 26 (42.6) 0.076 BMI 24.10 ± 3.56 23.75 ± 2.47 23.77 ± 2.94 0.756 Endoscopic findings Gastric ulcer 3 (4.9) 5 (8.2) 4 (6.6) 0.765 Atrophy 35 (57.4) 32 (52.5) 36 (59.0) 0.749 Intestinal metaplasia 12 (19.7) 12 (19.7) 15 (24.6) 0.746 CYP2C19 genotype RM 10 (16.4) 15 (24.6) 13 (21.3) 0.532 IM 44 (72.1) 40 (65.6) 44 (72.1) 0.660 PM 7 (11.5) 6 (9.8) 4 (6.6) 0.635 Values are presented as mean ± SD or number (%). GC, gastric cancer; BMI, body mass index; RM, rapid metabolizer; IM, intermediate metabolizer; PM, poor metabolizer. ###### Helicobacter pylori eradication rates among the three treatment groups Variable Eradication rate p value^[a](#tfn1-kjim-2019-139){ref-type="table-fn"}^ p value^[b](#tfn2-kjim-2019-139){ref-type="table-fn"}^ ----------------- ------------------ ---------------------------------------------------------- ---------------------------------------------------------- ------- ------- Eradicated 52 50 53 Failed 6 6 2 Follow-up loss 1 5 6 Dropped out 2 0 0 Poor compliance 0 0 0 ITT analysis 52/61 (85.2) 50/61 (81.9) 53/61 (86.8) 0.744 1.000 PP analysis 52/58 (89.6) 50/56 (89.2) 53/55 (96.3) 0.313 0.273 Values are presented as number (%). ITT, intention to treat; PP, per protocol. p was determined by two-group (A vs. B) comparison test. p was determined by two-group (A vs. C) comparison test. ###### Adverse events in the three treatment groups Variable Group A (n = 61) Group B (n = 61) Group C (n = 61) p value^[a](#tfn3-kjim-2019-139){ref-type="table-fn"}^ p value^[b](#tfn4-kjim-2019-139){ref-type="table-fn"}^ --------------------------------------------------------------- ------------------ ------------------ ------------------ ---------------------------------------------------------- ---------------------------------------------------------- Taste disturbance 30 29 28 1.000 1.000 Diarrhea 9 5 8 0.396 1.000 Headache 5 2 6 0.440 0.758 Epigastric pain 4 3 3 1.000 1.000 Nausea 3 2 1 1.000 0.619 Urticaria 3 1 1 0.619 0.619 GI disturbance^[c](#tfn5-kjim-2019-139){ref-type="table-fn"}^ 13 8 12 0.339 0.482 Total 36 31 34 0.574 1.000 GI, gastrointestinal. p was determined by two-group (A vs. B) comparison test. p was determined by two-group (A vs. C) comparison test. Diarrhea, epigastric pain, and nausea. 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"frequencies", "gastrointestinal", "disturbances", "diarrhea", "epigastric", "pain", "nausea", "lower", "group", "B", "compared", "group", "difference", "statistically", "significant", "p", "Table", "table", "genotypes", "Analysis", "genotypes", "revealed", "RM", "genotype", "patients", "groups", "B", "C", "respectively", "frequency", "RM", "genotype", "differ", "significantly", "across", "three", "groups", "per", "results", "multiple", "group", "comparison", "test", "p", "Table", "table", "DISCUSSION", "Maastricht", "Consensus", "Report", "stated", "triple", "therapy", "without", "prior", "susceptibility", "test", "reconsidered", "regions", "clarithromycin", "resistance", "rate", "Based", "literature", "review", "first", "study", "exclude", "patients", "resistant", "clarithromycin", "minimized", "significant", "bias", "associated", "eradication", "failure", "study", "patient", "infections", "another", "recent", "Korean", "study", "clarithromycin", "resistance", "rate", "difference", "may", "attributable", "different", "tests", "used", "assessing", "clarithromycin", "resistance", "well", "study", "including", "large", "number", "relatively", "heathy", "subjects", "Nonetheless", "considering", "clarithromycin", "resistance", "rate", "alternative", "regimens", "needed", "treatment", "Korea", "Bismuth", "quadruple", "PPI", "bismuth", "tetracycline", "metronidazole", "concomitant", "quadruple", "PPI", "amoxicillin", "clarithromycin", "metronidazole", "therapies", "recommended", "treatments", "However", "therapies", "associated", "poor", "patient", "compliance", "increased", "adverse", "events", "owing", "antibiotic", "overdose", "Although", "highly", "evident", "supplemental", "therapy", "probiotics", "sulforaphane", "could", "increase", "eradication", "rates", "decrease", "adverse", "events", "clinical", "trials", "efficacy", "probiotics", "pylori", "treatment", "showed", "Lactobacillus", "Bifidobacterium", "probiotic", "compound", "preparations", "increased", 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"clausii", "significantly", "reduced", "gastrointestinal", "adverse", "events", "nausea", "epigastric", "pain", "diarrhea", "insignificantly", "increased", "eradication", "rates", "Recently", "RCTs", "showed", "boulardii", "significantly", "increased", "eradication", "rates", "rates", "still", "desired", "level", "success", "Probiotics", "reduce", "adverse", "events", "nausea", "diarrhea", "abdominal", "pain", "thus", "helping", "completion", "eradication", "therapy", "Therefore", "probiotics", "improve", "pylori", "eradication", "rate", "reducing", "adverse", "events", "rather", "directly", "eradicating", "pylori", "proposed", "mechanism", "probiotics", "counteract", "harmful", "effects", "antibiotics", "gut", "microbiota", "However", "opposite", "conclusions", "drawn", "regarding", "side", "effects", "three", "studies", "reporting", "significant", "reduction", "overall", "side", "effects", "probiotics", "use", "study", "showed", "probiotic", "supplementation", "significantly", 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1. Introduction {#sec1-molecules-25-02234} =============== Recently, water has become more and more important subject of studies, as evidenced by the increasing number of water-related publications in various fields of science \[[@B1-molecules-25-02234],[@B2-molecules-25-02234],[@B3-molecules-25-02234],[@B4-molecules-25-02234]\]. Water is fundamental to life---it is one of the essential and widely distributed components of biologic systems, which can be considered as a biomolecule in its own right \[[@B5-molecules-25-02234],[@B6-molecules-25-02234]\]. Different scientific fields have been studying water from different aspects, trying to reach better understanding of its properties, structure and functions, yet still, our picture of water as a substance is rather incomplete. Spectroscopy methods, which are based on interaction of light and matter, have contributed a lot to our understanding of water. Recently, established, scientific discipline of aquaphotomics aims to integrate the knowledge these methods acquired about water based on its interaction with light, into one "omics" discipline which relates the structure of water with its functionality, and whose ultimate objective is better understanding of water as a matrix of aqueous and biologic systems \[[@B7-molecules-25-02234]\]. In this regard, aquaphotomics have made significant novel discoveries, and utilized the properties of water in various application fields ranging from water and food quality monitoring, microbiology, to biomeasurements, biodiagnostics and biomonitoring \[[@B8-molecules-25-02234],[@B9-molecules-25-02234],[@B10-molecules-25-02234]\]. Owing to the wide range of aqueous systems and biologic systems aquaphotomics studied, some novel, surprising insights have been discovered, which place focus on importance of water structure, hydrogen bonding and temperature. The effect of temperature has always been one of the most studied phenomena in spectroscopy with respect to its influence on water structure. The very terms "structure-makers" and "structure-breakers" are coined to describe substances which induce changes in the water structure and consequently its spectrum, comparable to decrease in temperature and increase of temperature, respectively. Effects of salts or sugars are commonly described using those terms \[[@B11-molecules-25-02234],[@B12-molecules-25-02234],[@B13-molecules-25-02234]\]. The known influence of temperature on hydrogen bonding in water is something that can be used as an etalon for better understanding of how substances affect the structure of water in various solutions. Among many others, principal component analysis and two-dimensional correlation spectroscopy \[[@B14-molecules-25-02234]\] or multivariate curve resolution-alternating least squares technique \[[@B15-molecules-25-02234]\] were used to describe the effects of temperature perturbations on the NIR spectra of water in terms of hydrogen bonding either alone or in comparison to salt perturbation. Aquaphotomics studies made some further steps uncovering that various phenomena, related to biomolecules or functionality of living organisms and aqueous systems can be described as related to specific water molecular structure and presented using spectral pattern. For example, in one work, concerned with the detection of UV-induced damage on DNA structure, it was found that the aqueous solutions of UVC-damaged DNA caused increase in hydrogen bonded water---i.e., that damaged DNA was a structure-making element causing changes of water similar to low temperature \[[@B16-molecules-25-02234]\]. In another study, which explored cold tolerance ability of different soybean cultivars, it was found that the water structure in the leaves of those cultivars with higher cold tolerance ability even at low temperatures preserved the water in less-hydrogen bonded state compared to those cultivars who are more susceptible, as if the environmental temperature would have been in fact higher \[[@B17-molecules-25-02234]\]. What these novel insights into the water structure and analogy with temperature influence provided is a novel knowledge and better understanding of the water functionality at different levels of organization of biologic systems. One of the main visualization and analytical tools of aquaphotomics, is the so-called aquagram \[[@B8-molecules-25-02234],[@B18-molecules-25-02234]\] which provides a comprehensible demonstration of the ratios of different water species present in a sample. Aquagrams have been found very useful in many applications, such as diagnosis of estrus in giant pandas \[[@B19-molecules-25-02234]\], orangutans \[[@B20-molecules-25-02234]\] and cows \[[@B21-molecules-25-02234]\] and showing the different water spectral patterns of probiotic and non-probiotic bacteria strains \[[@B22-molecules-25-02234]\], or for example, revealing different types of water in the soft contact lenses, in a completely nondestructive manner \[[@B23-molecules-25-02234],[@B24-molecules-25-02234],[@B25-molecules-25-02234]\]. The cited references demonstrated the usefulness of the presentation of the water spectral patterns in aquagrams. In order to unify the relation between water species depicted by aquagrams and related water functionalities we propose to use temperature as a common denominator to express changes of light absorbance at each of the water vibrational frequencies as changes caused by the most influential perturbation for water---temperature. Considering the advantages of better understanding of the functionality of water structure if the analogy is made with the influence of temperature, the objective of this work was to develop a novel method and a visualization tool, so called temperature-based aquagram which translates the effects of any type of perturbation of water structure in aqueous or biologic systems to the equivalent effects that would have been caused by the temperature changes and expressed in temperature units. The need to introduce the temperature-based aquagrams arose from the experiments on more complex systems, such as previously described, where it was observed that certain phenomena, (caused by solutes for example) have effects on the water molecular structure of the system and contribute to its functionality in the way analog to the changes in temperature. In a study concerned with classification of bacteria based on the probiotic strength it was found that the strong probiotic bacteria, compared to the moderately strong strains or non-probiotic strains, create less hydrogen-bonded water species ([Figure 1](#molecules-25-02234-f001){ref-type="fig"}) \[[@B22-molecules-25-02234]\]. On the other hand, the spectra of pure water at different temperatures show shift of the main band towards the shorter wavelengths with increasing temperature \[[@B14-molecules-25-02234]\], i.e., showing that increase in temperature results in breaking of the hydrogen bonds. If the two cases are compared, it can be seen that probiotic bacteria affect surrounding water similarly to the influence of temperature increase, as if they are a structure-breaking element \[[@B22-molecules-25-02234]\]. This conclusion supports the novel insight into molecular mechanisms of how probiotics work--they increase solubility of substances in water \[[@B26-molecules-25-02234]\], just like the increase in temperature of the water would contribute to better solubilization. From this example, it can be seen that comparisons with effect of temperature can be useful and contribute to better intuitive understanding of the functionality of the studied system. Recalling the soybean cultivars example, the role of many substances that plants accumulate in response to cold stress, becomes more obvious---their function is to provide a certain water molecular structure in the leaves as if the temperature of the environment is different \[[@B17-molecules-25-02234],[@B27-molecules-25-02234]\]. The concept of the novel visualization tool---a temperature-based aquagram---was developed similarly to the one Bernal and Fowler introduced long ago---the so called "structural temperature" concept \[[@B28-molecules-25-02234]\]. The "structural temperature" is the respective temperature at which pure water would have effectively the same molecular structure as the water of the aqueous system under study. At the time of introduction, no adequate method that could describe that structure was found; there were propositions that it can be estimated based on the measurements of viscosity, Raman spectra or X-ray diffraction. In addition, the intended purpose was mainly concerned with the applications in analysis of electrolyte solutions, and since the concept did not offer the possibility to separate effects of the individual ions, the entire idea was more or less abandoned \[[@B29-molecules-25-02234]\]. However, the structural temperature concept fits well into the framework of aquaphotomics, which places the water spectral pattern and the respective water functionality of the system in the central place. In contrast to "reductionistic omics methods" which are focused on isolating the biomolecules and separation of elements of the system, the aquaphotomics views each aqueous or biologic system as a whole, where all the components of the system exert their influence on the water matrix, whose structure is directly related to the function of the system \[[@B9-molecules-25-02234],[@B10-molecules-25-02234]\]. Just like the temperature is the macroscopic, measurable characteristic, arising from the molecular structure of the system, so is the light absorbance at each water specific vibrational frequency, and one can benefit from expressing one in the terms of another. The purpose of this study is to introduce a novel visualization tool, which expresses the effects of any type of perturbation of water molecular structure in aqueous or biologic systems to the equivalent effects that would have been caused by the temperature and expressed in temperature units. To illustrate this, we have chosen a simple salt solution and a concentration as a major perturbation. Salt is chosen as it is not near infrared active substance, hence the changes in absorbance of the solutions are only due to the changes in water molecular structure \[[@B15-molecules-25-02234],[@B30-molecules-25-02234]\]. Following the steps provided in the study, one can easily replicate the experiment, develop the temperature-based aquagrams and use it further for specific purposes. While in this study, the analysis and the results are presented for only 1st overtone of water region, the same methodology is applicable for any region of the water absorbance spectra. This new method provides numerical results on a clearly defined scale with confidence intervals. It enables the comparison of results across time and different experiments and provides information about the statistical significance of the found differences. 2. Results and Discussion {#sec2-molecules-25-02234} ========================= The spectral data in the wavelength interval of 1300 to 1600 nm for the two experiments were separately subjected to principal component analysis (PCA). The PCA score plots demonstrated the multidimensional patterns of the spectral data. Specifically, the highest variations displayed in the first principal components (PC1) were related to temperature and concentration of potassium chloride, representing 99.3% and 99.1% of the spectral variation in case of the temperature and the potassium chloride experiments, respectively. The PCA results did not show outliers either in the temperature or the potassium chloride datasets. The raw and 2nd derivative spectra of the two experiments were analyzed separately to discover the wavelengths exhibiting the largest changes caused by the temperature and salt perturbations. 2.1. Results of Temperature Experiment {#sec2dot1-molecules-25-02234} -------------------------------------- The raw and 2nd derivative absorbance (logT^−1^) spectra in the spectral range of 1300 to 1600 nm of Milli-Q water in the temperature range of 20 to 70 °C are shown in [Figure 2](#molecules-25-02234-f002){ref-type="fig"}. The main feature of the NIR spectrum of water is a broad peak around 1450 nm, comprised of several overlapping bands, described mainly as the 1st overtone of the OH stretching vibration \[[@B31-molecules-25-02234]\]. This observation is confirmed by the second derivative spectra which indicated very intense bands at 1412 and 1462 nm. These bands are well known as bands associated with free water molecules \[[@B32-molecules-25-02234],[@B33-molecules-25-02234],[@B34-molecules-25-02234]\] and strongly hydrogen-bonded water \[[@B14-molecules-25-02234],[@B35-molecules-25-02234]\], respectively. From a spectroscopic point of view, an increase in temperature has been interpreted as a decrease of number of hydrogen bonds \[[@B14-molecules-25-02234],[@B34-molecules-25-02234]\], while others explain it via weakening of hydrogen bonds \[[@B36-molecules-25-02234]\]. There is agreement, regardless of one's preferred theory, that the change of temperature causes alteration in hydrogen-bonding configurations of water as described in more details previously \[[@B37-molecules-25-02234]\]. [Figure 2](#molecules-25-02234-f002){ref-type="fig"} shows a "blue shift", i.e., movement of the main band towards the shorter wavelengths with increasing temperature and an isosbestic (temperature invariant) point. These phenomena are well studied in scientific literature \[[@B14-molecules-25-02234],[@B34-molecules-25-02234],[@B38-molecules-25-02234]\]. Evidence clearly suggest that less hydrogen-bonded water is predominant at higher temperature, while spectra acquired at low temperature are represented mostly in the more hydrogen-bonded area where water molecules form species with one (S1), two (S2), three (S3) and four (S4) hydrogen bonds \[[@B39-molecules-25-02234]\]. Despite the competing theories about the water structure, aquaphotomics identifies water-specific absorbance bands with higher variations caused by respective perturbation for the system of interest and uses them to depict the unique spectral pattern as an integrated marker of the system--perturbation interaction. The presentation of the 12 specific water coordinates \[[@B7-molecules-25-02234]\] of the spectra of Milli-Q water acquired in the temperature range of 20 to 70 °C together with the 95% confidence intervals is given in [Figure 3](#molecules-25-02234-f003){ref-type="fig"}a,b, calculated with the temperature-based aquagram calculation method. In the case of working with more complex system, it would be advisable to follow the protocol of aquaphotomics analysis which can provide more thorough examination of activated water absorbance bands and not necessarily limit the presentation to only the 12 coordinates as is chosen here. The aquagram provides an easy-to-comprehend presentation of the phenomena described above, i.e., the change of the strongly and weakly hydrogen-bonded patterns, based on [Figure 2](#molecules-25-02234-f002){ref-type="fig"}. The movement of the higher absorbance towards shorter wavelengths with increasing temperature is convincing. The scales of the aquagrams ([Figure 3](#molecules-25-02234-f003){ref-type="fig"}a,b) express the effect of perturbation occurring at the 12 coordinates (i.e., in the defined wavelength ranges) in degrees Celsius equivalent. Therefore, in this specific example the radial values show the temperatures corresponding to the signal acquisitions (20 to 70 °C). The plotted dashed and dotted lines represent the upper and lower confidence levels of the aquagram values, respectively, for each single analyzed temperature step. There was no overlapping of the confidence intervals (95%) of the Milli-Q samples measured at different temperature observed, meaning that 2 °C temperature changes caused statistically significant effects on the individual coordinates (i.e., in the defined wavelength ranges). The stability of the temperature-based aquagram calculation is presented in [Figure 3](#molecules-25-02234-f003){ref-type="fig"}b, by depicting only three selected temperature levels (20, 30 and 40 °C). Though the spectral dataset used to calculate the temperature-based aquagram of [Figure 3](#molecules-25-02234-f003){ref-type="fig"}b is only a subset of the dataset used to calculate [Figure 3](#molecules-25-02234-f003){ref-type="fig"}a, the shape and the range are the same in both cases. This type of stability was not possible with the "classic" aquagram ([Figure 3](#molecules-25-02234-f003){ref-type="fig"}c,d). These examples present the applicability and the additional benefits of the newly developed temperature-based aquagram for the evaluation and presentation of the water species in the 12 coordinates (representing defined wavelength ranges) through the evaluation of a well-known perturbation, i.e., the effect of temperature on the water spectral changes. 2.2. Results of Potassium Chloride Experiment {#sec2dot2-molecules-25-02234} --------------------------------------------- The raw and 2nd derivative absorbance (logT^−1^) spectra of the aqueous solutions of 0.001 to 1 M potassium chloride salt in the 1300--1600 nm wavelength range (OH first overtone) are presented in [Figure 4](#molecules-25-02234-f004){ref-type="fig"}. The main component of the aqueous solution, other than water, is KCl, which has no absorption in the NIR region; thus, it is not surprising that the spectra show a broad peak around 1450 nm. The second derivative spectra also provide similar patterns to those of the Milli-Q water acquired at different temperature, indicating bands at 1412 and 1462 nm. The trend of the shift in the peak position was similar to that observed in the temperature-perturbed pure water (i.e., the peak moves towards lower wavelengths as temperature is increased), but the actual peak locations were different from those for the pure water. The effect of low concentrations of salts diluted in water has been illustrated by the changes in the OH bonding of water molecular systems in many experiments \[[@B15-molecules-25-02234],[@B40-molecules-25-02234],[@B41-molecules-25-02234],[@B42-molecules-25-02234]\]. As salts do not absorb NIR light, accurate measurement of even low concentration of salts, published in the above-mentioned papers, means salts change the surrounding water molecular structure according to the number of the solvent molecules in the solution which still can restructure the water molecular system. This phenomena is not new; Bernal and Fowler \[[@B28-molecules-25-02234]\] showed that the addition of electrolytes changes the spectrum of water in the infrared overtone region. Lin and Brown \[[@B43-molecules-25-02234]\] also analyzed the effects of different salts on water spectra, and the authors were able to build accurate regression models using the spectral range of 1490 to 1610 nm for salt content prediction. The spectra shown in [Figure 4](#molecules-25-02234-f004){ref-type="fig"} imply that the increasing concentration of KCl causes "blue shift", i.e., a shift towards the spectral range referring to the band of less hydrogen-bonded water molecules. Our findings, hence show a good agreement with the results of previous research \[[@B15-molecules-25-02234],[@B40-molecules-25-02234],[@B41-molecules-25-02234],[@B42-molecules-25-02234]\]. A more detailed evaluation of the proposed 12 specific water coordinates \[[@B7-molecules-25-02234]\] of the spectra of the aqueous solutions of potassium chloride compared to the spectra of water is provided in [Figure 5](#molecules-25-02234-f005){ref-type="fig"}. The plots show the aquagrams of the single concentration levels together with the respective 95% confidence intervals calculated with the "classic" method ([Figure 5](#molecules-25-02234-f005){ref-type="fig"}a) and the new temperature-based aquagram calculation method ([Figure 5](#molecules-25-02234-f005){ref-type="fig"}b). The relative position of the patterns of the single concentration levels shows some similarity for the two methods. Furthermore, in both methods, spectral patterns of the highest concentration range (100--1000 mM) show the highest difference from the spectra of the lower ranges and Milli-Q samples (the latter one in black). This pattern explains the same phenomena which were found based on the raw spectra, i.e., a higher concentration of salt create water with less hydrogen bonds and decreases the number of water species to more hydrogen bonds. For the higher concentration ranges the aquagrams of the individual concentrations do not overlap, i.e., addition of 0.01 or 0.1 M KCl caused statistically significant change at the individual coordinates. However, the results also show an increasing tendency of the C7 coordinate (1432--1444 nm) with increasing concentration of KCl, which can be assigned to water molecules with one hydrogen bond (S1), meaning that increase in concentration of salt leads to increase in the number of these molecules \[[@B39-molecules-25-02234]\]. The dominantly higher effect of the higher concentrations on the spectral pattern hides the pattern of the lower concentrations; therefore, the calculation of the aquagrams were performed using the spectral data set for the two lowest concentration ranges only ([Figure 6](#molecules-25-02234-f006){ref-type="fig"}). The phenomena of the higher concentrations on the water spectral pattern, namely the higher concentration being more dominantly a structure breaker, can be observed in case of the concentration of samples at the concentration range of 10 to 100 mM. It is interesting to note the similarities to the pattern of the aquagrams calculated on all the three ranges ([Figure 5](#molecules-25-02234-f005){ref-type="fig"}b). The aquagrams of the higher concentrations (10--100 mM) show consistency with the previous findings regarding the structure-breaking characteristic as displayed in [Figure 6](#molecules-25-02234-f006){ref-type="fig"}. More specifically, the higher concentration samples present higher absorbance values in the range between 1342 and 1374 nm, i.e., C01-C03 that refer to the 1st overtone of free OH stretch (OH--(H~2~O)~n~, n = 1...4) \[[@B44-molecules-25-02234],[@B45-molecules-25-02234]\] and 1440 and 1452 nm, i.e., C07-C08 that are known as the bands of water hydration \[[@B35-molecules-25-02234]\] and water molecule connected to another water molecule (S~1~) \[[@B14-molecules-25-02234],[@B46-molecules-25-02234]\] and the symmetric and asymmetric stretching of first overtone of water \[[@B35-molecules-25-02234],[@B46-molecules-25-02234],[@B47-molecules-25-02234]\]. However, in the range between 1476 and 1512 nm, i.e., C10-C12, the higher concentration samples show lower values and these wavelengths are usually assigned to strongly hydrogen bonded water \[[@B14-molecules-25-02234],[@B35-molecules-25-02234]\] and aqueous protons (\[H^+^•(H~2~O)~6~\]--H~2~O in H~5~O~2~^+^ symmetric stretch) \[[@B48-molecules-25-02234]\]. These findings also mean decreasing concentration of salt causes a shift towards longer wavelengths, i.e., the range referring to the band of more hydrogen bonded water molecules. Using the additional benefit of the temperature-based aquagram calculation method, further results can be achieved from [Figure 6](#molecules-25-02234-f006){ref-type="fig"}. The addition of, for instance, 0.1 M potassium chloride to Milli-Q water results in water structural changes equivalent to changes caused by temperature of about 0.65, 0.6, 0.3, 0.1, 0.2 0.6, 1.8, 1.2, 0.2, −0.1, −0.3 and −0.6 °C at C01, C02, C03, C04, C05, C06, C07, C08, C09, C10, C11 and C12 coordinates, respectively. Furthermore, having calculated the confidence intervals, the statistical significance of the differences is also available. For example, calculations showed that addition of 0.02 M KCl to Milli-Q caused change equivalent to the change due to 0.33 and 0.23 °C temperature increase at coordinates C07 and C08 when compared to pure Milli-Q, which was found significant (p = 0.05), too. The aquagrams of the lowest concentration range (1--10 mM) calculated with the temperature-based aquagram method is shown in [Figure 7](#molecules-25-02234-f007){ref-type="fig"} to further evaluate the findings of the effects of decreasing concentrations of salt. The results suggest that we can observe concentration levels of 5 to 10 mM that cause a shift towards longer wavelengths, i.e., the range referring to the band of more hydrogen-bonded water molecules compared to the spectrum of Milli-Q water. These findings imply that salts can also have structure-breaker and structure-maker effects on water structure similar to the behavior of sugars \[[@B13-molecules-25-02234]\]. However, the aquagrams of the even lower concentrations (1 to 5 mM) also show alteration with nearly the same deviation, but towards shorter wavelengths. This may suggest that changing between structure-breaker and structure-maker properties of salt exist at such a low concentration, but the discernment of these minor changes would require very accurate measurements. 3. Materials and Methods {#sec3-molecules-25-02234} ======================== 3.1. Samples {#sec3dot1-molecules-25-02234} ------------ Two experiments with different sources of perturbations were conducted to demonstrate the procedure of temperature-based aquagram development and show the advantages in comparison to the representation of spectral data using classic aquagrams. In both experiments, pure Milli-Q water was used as a sample (Milli-Q purification system (Millipore, Molsheim, France, resistance = 18 MΩ) and in the first experiment perturbation of the sample was caused by changes in temperature, while in the second one, by changes in the concentration of salt. 3.2. The Temperature Experiment {#sec3dot2-molecules-25-02234} ------------------------------- The effect of temperature perturbation on water near-infrared spectra has been well-studied and is thoroughly described in the literature \[[@B14-molecules-25-02234],[@B34-molecules-25-02234],[@B49-molecules-25-02234]\]. Therefore, the experiment was performed on Milli-Q water in the temperature range of 20 to 70 °C to acquire spectra which could be used for the evaluation of the aquagram methods. The Milli-Q water was produced by a Milli-Q purification system (Millipore, Molsheim, France, resistance = 18 MΩ). The spectral acquisition was performed at 2 °C increments, resulting in 26 temperature steps in the range of 20 to 70 °C. 3.3. The Potassium Chloride Experiment {#sec3dot3-molecules-25-02234} -------------------------------------- The addition of salt to water at different concentrations is also an often evaluated perturbation \[[@B42-molecules-25-02234],[@B50-molecules-25-02234]\]. Therefore, an experiment was performed with different concentrations of aqueous solutions of potassium chloride. Potassium chloride (KCl, M = 74.56 g mol^−1^, purity min. 99.0% mass/mass) was purchased from Wako Pure Chemical Industries, Ltd. (Kobe, Japan). Aqueous solutions were prepared in different concentrations of KCl, in the range of 1 to 1000 mM. Three concentration ranges were prepared: Range A, from 100 to 1000 mM concentration, in steps of 100 mM; Range B from 10 to 100 mM, in steps of 10 mM and finally, Range C, in 1 to 10 mM in 1 mM concentration steps. Each dilution was prepared by serial dilution from the stock samples with the highest concentration in a given range (A, B or C) and prepared in two replicates, resulting in two independently prepared sets of samples. Stock solutions were prepared and further serially diluted with added Milli-Q water step-by-step to reach the appropriate concentrations---a solution created in each step was further diluted to prepare the next lower concentration. 3.4. NIR Spectral Acquisition {#sec3dot4-molecules-25-02234} ----------------------------- A FOSS-XDS spectrometer (FOSS NIRSystems, Inc., Hoganas, Sweden) equipped with a Rapid Liquid Analyzer module including a temperature-controlled 1 mm pathlength cuvette holder was used to measure transmittance spectra (logT^−1^) of the Milli-Q samples for the temperature experiment and of the aqueous solutions for the KCl experiment. Spectral acquisition was performed by saving three consecutive spectra in the range of 400--2500 nm at 0.5 nm spectral steps. Each saved spectrum was the average of 32 successive scans. A thermal bath with continuous water circulation was attached to the Rapid Liquid Analyzer module to ensure the required temperature of the sample during scanning in the range of 20 to 70 °C at 2 °C increments. The same apparatus was used to provide a constant temperature of 28 °C where each aqueous solution of potassium chloride was incubated for 90 s to equilibrate to the required temperature before scanning. Milli-Q water samples were measured as every fifth sample during the KCl experiment to provide environmental controls. The total number of spectra for the temperature experiment was 78 (26 temperature steps × 3 consecutive scans) and for the KCl experiment was 330 (30 concentrations × 2 repeats × 3 consecutive scans + 150 Milli-Q control scans) ([Appendix A](#app1-molecules-25-02234){ref-type="app"} Dataset). The FOSS-XDS instrument was operated using VISION 3.5 software (FOSS NIRSystems, Inc., Hoganas, Sweden). In both experiments, reference spectra were recorded before every sample. 3.5. Statistical Data Analysis {#sec3dot5-molecules-25-02234} ------------------------------ Only the wavelength interval of 1300 to 1600 nm, corresponding to the first overtone of the O--H stretching band \[[@B51-molecules-25-02234]\] was used for the evaluations. For the purpose of explaining methodology of how to develop temperature-based aquagrams, in this study, the focus is placed on this particular part of the water absorbance spectra, because it is best understood so far in the terms of the water molecular species whose absorbance bands are well-resolved and their assignments known \[[@B7-molecules-25-02234]\]. In the first overtone of water as a result of systematization of experimental work done on many different systems, not only different aqueous solutions, but also a great variety of biologic systems under different perturbations 12 water absorbance bands termed WAMACs (Water Matrix Coordinates)--each range from 6 to 12 nm width, were discovered \[[@B7-molecules-25-02234]\]. The great body of evidences in scientific literature provided the meaning to these ranges---i.e., it was possible to connect each of these 12 coordinates to specific water molecular species. In aquaphotomics studies the 12 WAMACs are called, coordinates because they represent windows in the spectra through which water structure of the system under study can be observed. In this study, the 12 WAMACs will be used to develop aquagrams, but it should be noted that the methodology explained is applicable for any region of the water absorbance spectra; it is not necessarily limited to the coordinates chosen here. Here, we chose for the reason of simplicity and because they are well-understood to use only those. However, depending on the system under study and the range of the spectra used, the reader is advised to follow the aquaphotomics protocol of analysis for extraction of the "activated water absorbance bands--WAMACs \[[@B8-molecules-25-02234]\] and then follow the further instructions to develop temperature-based aquagrams as described below. Before the development of aquagrams, exploratory analysis was performed. First, Principal component analysis (PCA) \[[@B52-molecules-25-02234]\] was used to describe multidimensional patterns in the spectral data and to discover outliers. The raw and 2nd derivative spectra were plotted to visualize the spectral changes induced by temperature perturbation and by the perturbation of salt concentration on the spectra of Milli-Q water and aqueous solutions of potassium chloride, respectively. The 2nd derivative spectra were calculated using a Savitzky--Golay filter \[[@B53-molecules-25-02234]\] using the 2nd order polynomial and 21 points. ### 3.5.1. Calculation Protocol of "Classic" Aquagram {#sec3dot5dot1-molecules-25-02234} The absorbance values at specific water matrix coordinates (WAMACs) \[[@B7-molecules-25-02234]\] define the water spectral pattern (WASP), which is different for different perturbations. The WASP can be visualized by a chart called the aquagram \[[@B18-molecules-25-02234],[@B19-molecules-25-02234]\]. This representation of the WAMACs was first introduced by Tsenkova \[[@B18-molecules-25-02234]\]. This aquagram (from now on called the "classic" aquagram) displays the multiplicative-scatter-corrected (MSC) (or standard-normal-variate (SNV)) transformed, normalized and averaged absorbance values of different samples or sample groups at 12 specific characteristic wavelengths. As, mentioned, these specific wavelengths were experimentally discovered as absorbance bands of specific water molecular species in previous studies and are later confirmed by overtone calculations of already reported water absorbance bands in the infrared range \[[@B7-molecules-25-02234]\]. The aforementioned water absorbance bands cover various form of water molecular species and are thus useful to depict characteristic spectral patterns in the first overtone region of water. This calculation can be summarized by Equation (1). $$A_{\lambda}^{\prime} = \frac{A_{\lambda} - \mu_{\lambda}}{\sigma_{\lambda}}$$ where, $A_{\lambda}^{\prime}$---value on aquagram for a given wavelength; A~λ~---absorbance after MSC applied on 1st overtone region of OH (i.e., 1300--1600 nm); μ~λ~---mean of all spectra for the examined group at a given wavelength (after MSC applied); σ~λ~---SD of all spectra for the examined group at a given wavelength (after MSC applied); λ---12 wavelengths (1342, 1364, 1374, 1384, 1412, 1426, 1440, 1452, 1462, 1476, 1488, 1512 nm) \[[@B7-molecules-25-02234]\]. The classic aquagram shows the relative fingerprint, i.e., the WASPs, in the context of all spectra in the examined group \[[@B18-molecules-25-02234],[@B19-molecules-25-02234],[@B21-molecules-25-02234],[@B54-molecules-25-02234]\]. Recently, this aquagram calculation method was extended by adding the possibility to observe the statistical significance of the differences presented on the aquagrams. Therefore, besides the average spectra of the individual groups used to plot the aquagrams, the respective confidence intervals are also calculated using the so-called Bootstrap method \[[@B55-molecules-25-02234]\]. This improvement makes it possible to plot the aquagrams together with their upper and lower 95% confidence interval limits. ### 3.5.2. Calculation Protocol for Newly Developed (Temperature-Based) Aquagram {#sec3dot5dot2-molecules-25-02234} The newly developed temperature-based aquagram calculation algorithm presents the respective water matrix coordinates in units equivalent to change in temperature and includes the respective confidence intervals. Therefore, it gives rise to the possibility to compare the WAMACs across time and also across different experiments and it provides information about the statistical significance of the differences. The new calculation method is based on the comparison of the areas (under the ranges of the above-mentioned 12 specific water coordinates \[[@B7-molecules-25-02234]\]) of the respective test sample spectra and the spectra of Milli-Q water. This method aims to express the spectral pattern changes in units equivalent to the change of temperature that would cause the observed change. The calculation of this aquagram concept (from now called the temperature-based aquagram) can be summarized in the following steps. Note that the calculation steps are explained for one coordinate (C01 representing spectral range between 1336 and 1348 nm, as an example) out of the 12 coordinates (C01-C12, i.e., defined wavelength ranges) to give an easily understandable description, but the same steps have to be repeated for each of the 12 coordinates. The main calculation steps are summarized in a chart to provide an overview of the developed method ([Appendix B](#app2-molecules-25-02234){ref-type="app"} [Figure A1](#molecules-25-02234-f0A1){ref-type="fig"}). 1. The dataset of the temperature experiment (i.e., the spectra of the Milli-Q water samples acquired during the temperature experiment in the temperature range between 20--70 °C) is defined as the reference dataset. The dataset of the experiment of interest is defined as the experimental dataset. 2. The average spectra of the consecutive scans are calculated for each temperature step, yielding 26 single, unique spectra in the reference dataset. The average spectra of the groups of interest (in this case, salt concentration levels) are calculated in the experimental dataset together with their respective confidence intervals using the Bootstrap method \[[@B55-molecules-25-02234]\], yielding as many single, unique spectra as there are groups are in the experimental dataset (plus their upper and lower 95% confidence interval limits). 3. The area under the spectrum for every single average spectrum---in the reference dataset and in the experimental dataset---at the wavelength range of 1336 to 1348 nm (C01) is calculated taking into account the baseline estimated by linear fitting on the two edges of the first overtone region (i.e., 1300 and 1600 nm). In case of the experimental dataset, the areas of the respective confidence interval limits are also calculated in addition to the area of the average spectrum. [Figure 8](#molecules-25-02234-f008){ref-type="fig"} provides graphical interpretation of the relevant areas and the wavelength regions used for the 12 coordinates. 4. The ratio of the area under the curve for each single coordinate is calculated with respect to the full area under the curve for the first overtone OH region (i.e., the area of C01 is divided by the full area under the spectrum in the range of 1300 to 1600 nm). This is done for every single average spectrum, in the reference dataset and the experimental dataset (together with the respective confidence interval limits for the experimental dataset). This calculation step provides normalized values and avoids possible differences due to scattering and/or pathlength effects. 5. Based on the reference dataset, a continuous array of values for the relative area of C01 (as calculated in Step 1) is calculated for a continuous temperature range from 20 to 70 °C using local polynomial regression. This is an essential step in order to accommodate the data from an experiment performed at specific temperature---see Step 6. 6. The basic principle of the temperature-based aquagram method is to compare the effect of the perturbation used on the system under study which resulted in a certain water spectral pattern to the effect the temperature changes would induce in pure water. Thus, any perturbation can be expressed as an equivalent temperature effect on a Milli-Q water sample. It is necessary to perform a "local calibration" with the reference dataset around the temperature of the experimental dataset. Therefore, in this step, the temperature calibration range is defined. This range is used to express the effect of perturbation in degrees Celsius equivalent. For this, a symmetrical scale is defined from the reference dataset (calculated at Step 5) using two degrees, plus and minus around the temperature of the experiment (hence, a span of 4 °C). For example, if the experiment was performed at 25.0 °C, then the calibration range of 23.0 to 27.0 °C would be used. 7. The temperature calibration equation, the relationship between the change of the temperature and change of the area of C01 at the temperature of the experiment, is determined based on the calculation performed in Step 5 on the reference dataset. (It is known how the area of C01 changes as a function of temperature described by a linear function). Therefore, it is easy to compare the changes for areas for C01 for the experimental dataset (calculated at Step 3) to the changes of the area of C01 caused by temperature, i.e., to express the changes in C01 in units of temperature (degrees Celsius) equivalent. 8. The calculated temperature (degrees Celsius) equivalent value for every group of the experimental dataset is finally visualized together with the respective 95% confidence intervals in a radar chart, where the units of the axes are in degrees Celsius. 9. The calculation and visualization of the results were performed using the R programing language \[[@B56-molecules-25-02234],[@B57-molecules-25-02234]\]. 4. Conclusions {#sec4-molecules-25-02234} ============== Recently, aquaphotomics has been introduced to focus on water as a key component for providing information about the function of the entire system. Non-destructive NIR spectroscopy and aquaphotomics have been applied to explain new phenomena in the field of life sciences. In contrast to reductionistic methods in which biomolecules and other elements are analyzed separately from the system, aquaphotomics studies the aqueous systems through its water matrix. In the present study, a newly developed temperature-based aquagram calculation method is presented as an additional tool to express the changes of water molecular structure in aqueous systems which are caused by perturbations different from temperature. Although the need to introduce temperature-based aquagrams originated from experiments on complex systems, the successful application of the new aquagram calculation method was demonstrated through the evaluation of the results of well-known phenomena. The results of temperature and salt perturbations on Milli-Q water are demonstrated in the present study. The effect of temperature on the spectral pattern of Milli-Q water acquired in the temperature range of 20 to 70 °C is presented with the temperature-based aquagram calculation method. The method provides the presentation of the phenomena demonstrating blue shift in the first OH overtone range with increasing temperature. Furthermore, the scale of the aquagram expressed the effect of perturbation in degrees Celsius equivalent. Aqueous solutions of potassium chloride were chosen for the experiment as KCl has no absorption in the NIR region. Thus, its effect on water spectral patterns can be clearly evaluated and presented as caused by temperature changes at respective wavelengths. Furthermore, it demonstrates that the method is invariably applicable for evaluation the effects of all types of solutes. The new temperature-based aquagram calculation method provided further information about the magnitude of the change. In other words, the results were displayed on a degree-Celsius scale that showed how much a given sample would have needed to have been warmed up or cooled down in each of the single coordinates (C01 to C12) to achieve the same results as the actual measurement, while all measurements were performed at precisely the same temperature. Adding 0.1-M potassium chloride to Milli-Q water resulted in structural changes equivalent to an approximately 0.6 °C temperature increase in the less hydrogen-bonded, and 0.3 °C temperature decrease in the more hydrogen-bonded areas of the OH first overtone spectral region. The examples presented here confirm the applicability and the additional benefits of the temperature-based aquagram calculation method. They provide a demonstration of the ratio of the different water species existing in different aqueous and biologic systems. Additionally, this new type of aquagram calculation displayed the spectral patterns in a meaningful scale and stable pattern independent of any modification of the evaluated dataset, which gives rise to the opportunity to compare results not only within a single chart, but also across time and different experiments. This newly developed tool is especially suitable for visualizing water structure evolution and phase transitions, for example, in the food preservation industry, pharmaceutical development, material science and related applications. The presented new chemometric tool, developed in R-Project, is freely accessible as an R-package from GitHub repository \[[@B58-molecules-25-02234]\] and can be used in various fields of NIR spectroscopy and water research. Authors are grateful to Professor David Funk for his help to revise the manuscript from language editing and from scientific point of view. Sample Availability: Samples of the compounds KCl are available from the authors. All authors contributed to this research. The design of the experiments was performed by Z.K., B.P. and R.T., Z.K. and B.P. did the recording and the processing of the NIRS data, as well as the result of the evaluation and implementing the concept in R-project. The manuscript was written by Z.K., B.P., G.B. and J.M., R.T. assisted in writing the study and revised it. Z.K., R.T., G.B. and J.M. contributed to designing the research and revise the manuscript. The work presented in the study was conceived within research projects led by R.T. and Z.K. All authors have read and agreed to the published version of the manuscript. Authors acknowledge the financial support of the ÚNKP-19--4 New National Excellence Program of the Ministry for Innovation and Technology (Z.K.), János Bolyai Research Scholarship of the Hungarian Academy of Sciences (Z.K.) and by the European Union and co-financed by the European Social Fund through project No. EFOP-3.6.3-VEKOP-16--2017--00005. JM gratefully acknowledges the financial support provided by Japanese Society for Promotion of Science (P17406). The authors declare no conflict of interest. Dataset. Raw spectral data used for the calculations presented in this study. Both data of temperature experiment and potassium chloride experiment is included in this dataset where the column experiment describes which spectra belong to which experiment. ![Workflow of the calculation protocol of temperature-based aquagram.](molecules-25-02234-g0A1){#molecules-25-02234-f0A1} ![Water spectral pattern presented on aquagrams shows different water structure in different bacteria strains (Reprinted with permission from Slavchev, A., Kovacs, Z., Koshiba, H., Nagai, A., Bázár, G., Krastanov, A., Kubota, Y. and Tsenkova, R. 2015. \[[@B22-molecules-25-02234]\]).](molecules-25-02234-g001){#molecules-25-02234-f001} ![Raw and 2nd derivative (calculated with Savitzky--Golay filter using 2nd order polynomial and 21 points) absorbance (logT^−1^) spectra in the spectral range of 1300--1600 nm (OH first overtone) of Milli-Q water in the temperature range of 20--70 °C (n = 78).](molecules-25-02234-g002){#molecules-25-02234-f002} ![Aquagrams of Milli-Q water in the temperature range of 20--70 °C, (a,b) with 95% confidence intervals calculated with temperature-based aquagram calculation method, (c,d) calculated with the classic calculation method, (a,c) all the 26 temperature steps (n = 78) and (b,d) on three selected temperature steps (n = 9) to show the stability of the methods (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g003){#molecules-25-02234-f003} ![Raw and 2nd derivative (calculated with Savitzky--Golay filter using 2nd order polynomial and 21 points) absorbance (logT^−1^) spectra in the range of 1300--1600 nm (OH first overtone) of 0.001--1 M KCl solutions (n = 180).](molecules-25-02234-g004){#molecules-25-02234-f004} ![Aquagrams with 95% confidence intervals of Milli-Q water (n = 150) and 0.001--1 M KCl solutions (n = 180) calculated with the classic (a) or the temperature based aquagram (b) calculation methods on the individual concentrations (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g005){#molecules-25-02234-f005} ![Aquagrams with 95% confidence intervals of Milli-Q water (n = 150) and 0.001--0.1 M KCl solutions (n = 120) calculated with the temperature based aquagram calculation method on the individual concentrations (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g006){#molecules-25-02234-f006} ![Aquagrams with 95% confidence intervals of Milli-Q water (n = 150) and 0.001--0.01 M KCl solutions (n = 60) calculated with the temperature based aquagram calculation method on the individual concentrations (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g007){#molecules-25-02234-f007} ![Scheme of the calculation for Area under the curve (AUC) aquagram method. Spectrum of pure water with highlighted subranges of the 12 specific water matrix coordinates (WAMACs).](molecules-25-02234-g008){#molecules-25-02234-f008}	{ "pile_set_name": "PubMed Central" }	[ "unifi", "discov", "lifeit", "two", "github", "ab", "control", "krastanov", "refer", "given", "work", "origin", "program", "tendenc", "decreas", "compris", "ie", "holder", "purpos", "someth", "kubota", "cow", "exist", "identifi", "savitzkygolay", "kovac", "spectrum", "classif", "pathlength", "written", "select", "systemperturb", "fund", "bappmoleculesreftypeapp", "mechan", "bernal", "fossxd", "take", "reprint", "valu", "locat", "signal", "may", "exploratori", "move", "necessarili", "potassium", "graphic", "theori", "recent", "biomolecul", "squar", "fifth", "live", "acquir", "toward", "separ", "use", "moder", "support", "score", "proton", "final", "focus", "convinc", "bath", "languag", "caus", "complet", "accord", "lcllower", "signific", "focu", "structuremak", "establish", "build", 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What is your diagnosis ([Figures 1](#f1-dp0504a10){ref-type="fig"}[](#f2-dp0504a10){ref-type="fig"}--[3](#f3-dp0504a10){ref-type="fig"})? ========================================================================================================================================= A. Verruca vulgaris B. Actinic keratosis C. Bowenoid papulosis D. Seborrheic keratosis E. Acanthoma Answer ====== - C\) High-grade squamous intraepithelial lesion of the oral commissure (bowenoid papulosis) Discussion ========== Bowenoid papulosis (BP) is characterized clinically by one or more small, verrucous papules usually located on the genitalia or thighs of younger patients \[[@b1-dp0504a10]\]. Rarely, BP has been reported on extragenital sites with or without concomitant genital lesions \[[@b2-dp0504a10],[@b3-dp0504a10],[@b4-dp0504a10]\]. On the genitalia, BP clinically resembles condyloma acuminatum or lichen planus; however, histopathologically, BP is indistinguishable from squamous cell carcinoma in situ or Bowen's disease, hence, its designation "bowenoid papulosis," a term coined by Wade, Kopf and Ackerman, in 1978 \[[@b1-dp0504a10]\]. BP has been shown to be associated primarily with the high-risk human papillomavirus (HPV) infection subtypes, 16 and 18; however, other high-risk sub-types, such as 31, 32 \[[@b5-dp0504a10]\], 33, 35, 39, 53, and 67, have also been reported. Many lesions of BP resolve with or without therapy and behave in a clinically benign fashion despite their malignant histology \[[@b7-dp0504a10]\]; however, cases of squamous cell carcinoma in-situ and invasive squamous cell carcinoma have been reported to occur in association with lesions of BP, particularly in immunosuppressed patients \[8,9.10\]. Risk of penile squamous cell carcinoma in men may be as high as 30% in patients with BP and increases incrementally with the duration of the disease \[[@b11-dp0504a10]\]. Patients with BP have also been reported to have concurrent HPV-associated dysplasia of the vulva and uterine cervix, of various degrees, including high-grade dysplasia, VIN 3 and CIN 3, respectively. Our patient, a 22-year-old man, presented with verrucous papules centrally located within a central lichenified plaque on the oral commissure. The lesion was clinically thought to be a wart or a lesion of lichen simplex chronicus. Histopathologically, the lesion showed full thickness epithelial atypia demonstrating increased numbers of mitotic figures, loss of polarity, and nuclear pleomorphism compatible with squamous cell carcinoma in situ ([Figures 1](#f1-dp0504a10){ref-type="fig"}[](#f2-dp0504a10){ref-type="fig"}--[3](#f3-dp0504a10){ref-type="fig"}). In situ hybridization was positive for the high-risk subtypes 16/18 ([Figure 4](#f4-dp0504a10){ref-type="fig"}). p16 immunoperoxidase stain demonstrated strong diffuse staining in the lower portion of the lesion with individually positive cells extending into the upper reaches of the epithelium ([Figure 5](#f5-dp0504a10){ref-type="fig"}). Oral BP is exceeding rare with only nine cases, to our knowledge, reported in the medical literature \[[@b11-dp0504a10]--[@b19-dp0504a10]\]. Men were more commonly affected than women, and the ages ranged from 20 to 40 years. Clinically, reported cases of oral lesions of BP in the medical literature are similar to those occurring on genital sites, namely, small verrucous papules \[[@b16-dp0504a10]\]; however, erythematous velvety plaques \[[@b20-dp0504a10]\], raised solitary nodules \[[@b11-dp0504a10]\], leukoplakia or macules resembling candidiasis \[[@b13-dp0504a10]\] have also been described. Histopathologically, lesions of oral BP were indistinguishable from squamous cell carcinoma in situ. A computerized search of the files of the Ackerman Academy of Dermatopathology in New York, NY, from July 1999 through August 31, 2013, yielded 560 biopsies diagnosed as BP; however, only three patients with extragenital BP were identified, including the present case. This finding parallels that of the medical literature and indicates that extragenital BP is exceedingly rare. All three extra-genital lesions from the Ackerman Academy were oral: two occurred on the lip and one, the present case, was located on the oral commissure. There were two males and one female, 22, 64 and 40 years of age, respectively. Clinically, the lesions were thought to be lichen simplex chronicus, verruca vulgaris or pemphigus vulgaris, and bowenoid papulosis, respectively. Histopathologically all lesions were indistinguishable from squamous cell carcinoma in situ. In 2012, a Consensus Panel of the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology recommended a change in terminology for BP from "bowenoid papulosis" to "high-grade squamous intraepithelial lesion." They noted that bowenoid papulosis could be added to the diagnosis in parentheses if it could be verified that the lesion was small and had been excised. If verification could not be accomplished but the clinical setting was one of small papules, they wrote that, "a note stating that the differential diagnosis includes Bowenoid papulosis may be warranted." They also stated that, "Bowenoid papulosis may have a lower risk of progression to cancer than cutaneous HSIL \[high-grade squamous intraepithelial lesion\] found in larger plaques (Bowen disease)." \[[@b21-dp0504a10]\] Treatment for oral BP is similar as that for genital BP and includes intralesional, topical or oral medication as well as surgical excision. Fluoropyrimidine TS-1 (prodrug of 5-FU, gimestat (CDHP), and oteracil potassium (Oxo)), 100 mg daily for three weeks was administered in one elderly woman with HPV-16 positive oral BP and resulted in regression of the lesion \[[@b16-dp0504a10]\]. Intralesional interferon alpha followed by topical imiquimod has also been reported as successful \[[@b17-dp0504a10]\]. Other treatments that have been used for genital BP and may be appropriate in oral lesions include 5-FU, podophylin, retinoic acid, and cidofovir. Surgical modalities include simple excision, cryosurgery, laser vaporization, and electrodessication of small lesions. Podophyllin is toxic in large amounts but has been used successfully for the treatment of oral hairy leukoplakia and could potentially be used to treat refractory BP \[[@b22-dp0504a10],[@b23-dp0504a10]\]. Our patient was treated successfully with 5-fluorouracil cream followed by imiquimod cream each applied five times per week for five weeks with two weeks between each medication. He remains lesion-free five months post treatment. ![Scanning magnification of the lesion on the oral commissure. \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g001){#f1-dp0504a10} ![Higher magnification of the oral lesion. \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g002){#f2-dp0504a10} ![Higher magnification of the slightly verrucous lesion shows hyperplasia with full-thickness atypia of squamous epithelial cells, evidence of loss of polarity, nuclear crowding, nuclear pleomorphism and increased mitotic figures indicative of high-grade squamous intraepithelial lesions. \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g003){#f3-dp0504a10} ![Human papillomavirus in situ hybridization showing positive staining for the high-risk subtypes, 16/18 (red nuclear staining). \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g004){#f4-dp0504a10} ![p16 immunoperoxidase stain shows strong diffuse positive staining of the lower portion of the lesion with individually positive cells extending into the upper reaches of the epithelium (brown staining). \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g005){#f5-dp0504a10}	{ "pile_set_name": "PubMed Central" }	[ "larg", "amount", "cervic", "treatment", "york", "nine", "find", "pathologist", "parenthes", "two", "small", "cin", "howev", "concomit", "benign", "terminolog", "posit", "papulosi", "cancer", "figur", "remain", "clinic", "without", "respect", "hybrid", "hsil", "larger", "elderli", "kupetski", "one", "macul", "compat", "well", "cryosurgeri", "wart", "gimestat", "five", "acanthoma", "knowledg", "interferon", "lesionfre", "american", "biopsi", "affect", "identifi", "differenti", "lichen", "includ", "extragenit", "risk", "new", "discuss", "associ", "rais", "juli", "resolv", "particularli", "highgrad", "loss", "describ", "august", "diffus", "week", "podophyllin", "locat", "show", "verifi", "fluoropyrimidin", "may", "henc", "vapor", "potassium", "variou", "cream", "invas", "slightli", "lip", "increment", "treat", "b", 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Plain English summary {#Sec1} ===================== Disordered Eating Behaviours (DEB) start to appear during adolescence and bring serious consequences. Previous studies showed DEB is becoming common in China, and even more prevalent than that is in Western countries. However, limited research has been conducted among adolescents in rural China. The current study aimed to establish the prevalence of DEB and associated psychosocial factors among rural adolescents. Results from the current research showed a high prevalence rate of DEB among rural adolescents and associated psychosocial factors of DEB. Background {#Sec2} ========== Disordered Eating Behaviours (DEB) are abnormal eating attitudes and behaviours which have much in common with diagnosed eating disorders \[[@CR1]\].These can have serious implications which relate to depression, substance abuse, suicidal behaviours, social isolation and which may develop into an eating disorder \[[@CR2], [@CR3]\]. DEB has long been regarded as strongly related to Western cultures but according to a review in the most recent published epidemiologic data relevant to eating disorders in Asia and the Pacific, abnormal eating behaviours found to be highly prevalent in China \[[@CR4]\]. Traditional Chinese culture considers plumpness as beautiful and slimness as reflecting poverty. However, China has been dramatically influenced by Western cultures for the past 39 years due to the 'open-door policy'. The thin-ideal has come to overshadow standards for beauty which contributes to body dissatisfaction and DEB among females \[[@CR5]\]. Accordingly, a number of Chinese studies report a high prevalence of DEB \[[@CR6]--[@CR13]\]. Specifically, female adolescents living in Hong Kong have been found to have the highest prevalence of DEB (10.8%), followed by those from Shenzhen (5.2%) and from Hunan (2.5%) \[[@CR6]\]. Two school-based studies in Taiwan revealed DEB in 17.1% and 10.4% of high school female adolescents \[[@CR7], [@CR8]\]. Further, a school-based study in Hong Kong revealed that DEB were present in 3.9% of adolescent males and 6.5% of adolescent females \[[@CR9]\]. Three school-based studies in Mainland China have also reported that the prevalence of DEB varied from 2.3% to 17% among middle school and college students \[[@CR10]--[@CR12]\]. Western studies have revealed DEB in 29.4% of female adolescents, specifically 14.9% of male adolescents in Germany, and 56% of female adolescents and 28% of male adolescents in US \[[@CR14], [@CR15]\]. However, few studies in DEB have included male adolescents in China \[[@CR9], [@CR13]\]. The variations in measuring instrument, sample characteristics, methodological approaches and study areas, however, may limit the comparability of prevalence across studies. The updated epidemiologic review in 2016 suggested that fat concern and weight control behaviours are prevalent among Chinese young people and replication epidemiological studies in eating disturbances are needed due to ongoing rapid modernization and socio-economic change \[[@CR4]\]. Psychosocial risk factors are strongly associated with a high prevalence of DEB among adolescents \[[@CR13], [@CR16]\]. For instance, longitudinal studies conducted in Southern China found body dissatisfaction, negative affect, perceived sociocultural pressures, and high socioeconomic status (SES) all have significant associations with DEB. \[[@CR13], [@CR17]\]. A comparative study among female adolescents in three different socioeconomically communities of China documented that those in rural areas had fewer concerns about body fat and demonstrated a lower prevalence rate of DEB than those in developed cities \[[@CR6]\]. Socioeconomic development in China is growing very fast nowadays and the rapid ongoing socioeconomic transition has influenced rural China to a large extent. Rural populations now have more opportunities to access the mass media, which may relate to a higher risk of DEB. T Jackson and H Chen have reported that Chinese middle adolescent girls reported higher levels of appearance experiences related to mass media and DEB \[[@CR18]\], and pressure from mass media related to DEB have been observed in Chinese adolescent boys \[[@CR19]\]. There are some studies examining the epidemiology of eating disorders in rural China \[[@CR17], [@CR20], [@CR21]\], but little research has focused on epidemiology of DEB in rural China. In more than 20 years, there has been only one study which has focused on DEB among Chinese female adolescents in a rural area \[[@CR6]\]. Further, given that little attention has been paid to DEB in both genders in rural China, it is necessary to re-investigate the situation under current influences. The present study, therefore, aimed to examine prevalence and associated psychosocial factors of DEB among male adolescents and female adolescents at a Chinese middle school in a rural area. This may contribute to the updated information in the epidemiology of DEB in China and also respond to the updated review's request for replication studies, that is this review suggested further replication epidemiological studies in eating disturbances due to ongoing rapid modernization and socio-economic change \[[@CR4]\]. Methods {#Sec3} ======= Participants and study procedure {#Sec4} -------------------------------- All students from a middle school -- a secondary high school -- in Dongfanghong town (a less economically developed rural area), Heilongjiang province were invited to participate in the study. In 2015, the per capita annual income in our sample area was 30,977 RMB (or \$4509). The school that was studied was the only middle school in this area. Four hundred and sixty-six students aged 11--18 years old were invited to complete an anonymous, self-reported questionnaire covering a wide range of topics including DEB and related psychosocial behaviours on a voluntary basis. Before starting the research, schoolteachers were trained by researchers and schoolteachers explained the study to students and were able to answer students' questions. During the study, the confidentiality and the anonymity of participants were assured. With the assistance of schoolteachers, the questionnaires were administered in September 2015 -- at the beginning of autumn semester. The class teachers decided the time when questionnaires were conducted. They usually selected self-learning class (students studied by themselves) and the time of self-learning class was different among class-rooms. The students used around 1 h -2 h to finish the questionnaires. The class teachers collected the questionnaires following completion and returned them to the researcher. Of 466 questionnaires that were issued, 389 valid questionnaires were returned (a response rate of 83.5%). Outcome variables {#Sec5} ----------------- SCOFF: The SCOFF questionnaire is an effective tool that can screen DEB quickly \[[@CR22]\]. It includes five items addressing the main features of DEB \[[@CR22]\]. See the Additional file [1](#MOESM1){ref-type="media"}: Table S1 for the descriptive statistics of each item. One point for every "yes"; a score of \> = 2 indicates a likely case of DEB. The scale has been validated and has demonstrated effective specificity in identifying DEB \[[@CR23]\]. The Chinese version of the SCOFF questionnaire has been proved to be effective for detecting potential DEB \[[@CR24]\]. Independent variables {#Sec6} --------------------- Body Mass Index (BMI) was computed from self-reported measures of height and weight. Self-reported BMI has been demonstrated to be a reliable measure of BMI \[[@CR25]\]. The classification of BMI was made according to BMI cut-offs by the Working Group on Obesity in China (WGOC) \[[@CR26]\]: ≤ 18.49 kg/m^2^ (underweight), 18.50--23.99 kg/m^2^ (normal weight), 24--27.99 (overweight), ≥28 (obesity). We combined the overweight group and the obesity group into one category due to the small number size in the obesity group. Participants were asked to describe their weight perceptions. Responses were ranged into three groups: underweight, average /normal, and overweight. Compared weight perceptions with actual BMI, weight misconception was ranged into four groups: misconception of underweight, overweight, normal weight and correct weight conception. See the Additional file [2](#MOESM2){ref-type="media"}: Table S2 for the association between BMI and perceived weight categories. Body dissatisfaction was examined using a Body Part Satisfaction Scale with each response from 1 = extremely dissatisfied to 6 = extremely satisfied \[[@CR27]\]. The scale consists of eight items rating the individual's level of satisfaction with the following body areas: face, upper torso, middle torso, lower torso, muscle tone, weight, height and overall appearance. A high score indicates a higher level of body satisfaction. The scale has shown reliable psychometrics \[[@CR27]\]. The Chinese version of this scale has been tested and has strong reliability \[[@CR18]\]. The internal consistency of the scale was 0.90. Depressive symptoms were measured by the Positive and Negative Affect Schedule \[[@CR28]\]. This schedule is a 20-item test with 10 items for negative affect and positive affect, respectively. Participants were asked to describe the extent to which they had varying feelings and emotions in the past month from 1 = very slightly or not at all to 5 = extremely. The scale has shown good validity and acceptable psychometric properties \[[@CR28]\]. The original scale has been tested on Chinese samples \[[@CR19]\]. The Perceived Sociocultural Pressure Scale was used to assess pressures to be thin from family, friends, dating partners and media \[[@CR18]\]. The questionnaire has 10 items that investigate perceived sociocultural pressures to be thin. An example of an item is: "I've noticed a strong message from my family to have a thin body". Three items are for family and friends, respectively. Two items are for dating partners and media, respectively. Responses ranged from 1 = not at all to 5 = extremely. Higher scores indicate more perceived pressure. Internal consistency (α = 0.83), test-retest reliability (r = 0.93) and predictive validity of this scale have been documented \[[@CR29]\]. The scale has been tested and used in Chinese samples \[[@CR19]\]. Media use was assessed by a media exposure questionnaire. Participants were asked: whether they watch television and DVDs, whether they use the Internet, whether they use chat rooms and play computer games, as well as how long they spent on media. The questionnaire was developed from a Chinese study \[[@CR5]\]. Parental socioeconomic status (SES) was determined on the adolescents' report of parents' occupation. Occupations were categorized according to the classification by the Chinese National Bureau of Statistics \[[@CR30]\] with nine items to be selected. Four groups were created based on the salary of occupations: professional, service, labourers, and unemployed. The variables of mother's job and father's job were merged into one variable (parents' occupation), recording the higher of the two values. Age and sex were also recorded. Statistical analysis {#Sec7} -------------------- Chi-square analysis was used to compare differences in prevalence of DEB and for comparing perceived weight categories to BMI categories. To examine associated factors of DEB, first, a univariate logistic regression was employed to examine the association between each of the independent variable and DEB, and then multiple logistic analysis was performed. A forward stepwise multiple logistic regression was performed to determine factors significantly associated with DEB, including independent variables based on univariate logistic regression results (cut-off point p ⩽ 0.25). Age and gender were controlled. The final model was built based on this forward stepwise multiple logistic regression (p ⩽ 0.05). The multi-collinearity among associated factors was examined with collin command in Stata (mean variance inflation factor (VIF) = 1.43, correlation matrix = 0.0528). Secondly, Model fit to the data (validity and reliability) was assessed by the likelihood ratio χ^2^ test, Hosmer-Lemeshowgoodness-of-fit test and receiver operating characteristics (ROC) curve. P ⩽ 0.05 was regarded as statistically significant and odds ratio (OR) with 95% confidence intervals (95% CI) were calculated. Data analyses were conducted with the Statistical Package for the Social Sciences (SPSS) 22.0 and StataSE 14. Ethical clearance {#Sec8} ----------------- The study was approved by the Regional Committee for Medical Research Ethics in Norway, and the Committee for the middle school in Dongfanghong, China. Results {#Sec9} ======= Descriptive summary of the study participants {#Sec10} --------------------------------------------- Table [1](#Tab1){ref-type="table"} presents the descriptive summary of study participants (N = 389).Table 1Descriptive summary of the study participants (N = 389)VariablesNPercentMsdFemale19048.8SCOFF total score (five tiems)1.01.1 017244.2 110527.0 26717.2 3328.2 4123.1 510.3Age(year)15.11.7 Early Adolescence \[[@CR11]--[@CR13]\]9524.4 Middle Adolescence \[[@CR14]--[@CR16]\]19850.9 Late Adolescence \[[@CR17], [@CR18]\]9624.7BMI(kg/m^2^)20.54.7 Underweight14036.0 Normal Weight16943.4 Overweight7719.8Perceived Weight Underweight6115.7 Normal Weight21455.0 Overweight11429.3Weight Misconception Misconception of Underweight184.6 Misconception of Normal Weight10226.2 Misconception of Overweight5714.7 Correct Weight Conception20753.2Parents' Occupation Professional11529.6 Service7218.5 Labourers4812.3 Unemployed15439.6With DEB11228.8Pressure Family1.91.1 Friend1.81.0 Dating1.51.0 Media1.61.1Watch TV32383.0Watch DVD7419.0Surfing Online35390.7Chat Online31681.2Play Online25365.0Hours on Media average - 0-4.9 h31581Hours on Media Total14.19.1Body Satisfaction31.18.4 Face4.01.3 Upper torso4.01.3 Middle torso4.01.4 Lower torso4.01.4 Muscle tone3.71.4 Height3.81.4 Weight3.71.5 Overall appearance4.11.2Positive Affect29.67.5Negative Affect21.27.2N number, M mean, sd standard deviationPressure perceived sociocultural pressure to thin body and weight lossHours on Media Total time on Internet working days + time on Internet weekends + time on TV working days + time on TV weekendsHours on Media average Hours on Media Total ÷ 4 Prevalence of disordered eating behaviours {#Sec11} ------------------------------------------ Table [2](#Tab2){ref-type="table"} presents the prevalence of DEB according to the participants' general information by using chi-square analysis. The prevalence of DEB among adolescents was 28.8%. The prevalence of DEB among male adolescents and female adolescents was 27.1% and 30.5%, respectively; no significant differences were found based on gender. No significant differences of prevalence of DEB were found across different age groups. Significant differences in the prevalence of DEB were found in different BMI groups, χ^2^ (2) = 14.896, p = .001. Overweight adolescents (42.9%) had a significantly higher prevalence of DEB as compared to underweight adolescents (18.6%). Significant differences in the prevalence of DEB were also found in different perceived weight groups, χ^2^ (2) = 51.524, p \< .001. Adolescents in the perceived overweight group (54.4%) had a significantly higher prevalence of DEB than those among the perceived underweight group (18.0%). Significant differences in the prevalence of DEB were found in different weight misconception groups, χ^2^ (2) = 43.399, p \< .001. Adolescents with overweight misconception (59.4%) had a significantly higher prevalence of DEB than those without overweight misconception. There were no significant differences in the prevalence of DEB among students across different SES groups. Associations between misconception of over−/under-weight and DEB by gender is shown in the Additional file [3](#MOESM3){ref-type="media"}: Table S3.Table 2Prevalence of disordered eating behaviours across participants' general informationWith DEBWithout DEBChi-square% (N)% (N)StatisticGender0.545 Female30.5 (58)69.5 (132) Male27.1 (54)72.9 (145)Age4.919 Early Adolescent \[[@CR11]--[@CR13]\]24.2 (23)75.8 (72) Middle Adolescent \[[@CR14]--[@CR16]\]26.8 (53)73.2 (145) Late Adolescent \[[@CR17], [@CR18]\]37.5 (36)62.5 (60)BMI14.896\\ Underweight18.6 (26)81.4 (114) Normal Weight30.8 (52)69.2 (117) Overweight42.9 (33)57.1 (44)Perceived Weight51.524\\\* Underweight18.0 (11)82.0 (50) Normal Weight18.2 (39)81.8 (175) Overweight54.4 (62)45.6 (52)Weight Misconception41.521\\\* Misconception of Underweight22.2 (4)77.8 (14) Misconception of Normal Weight13.7 (14)86.3 (88) Misconception of Overweight61.4 (35)38.6 (22) Correct Weight Conception27.5 (57)72.5 (150)Parents' Occupation2.218 Professional33.9 (39)66.1 (76) Service25.0 (18)75.0 (54) Labourers27.1 (13)72.9 (35) Unemployed27.3 (42)72.7 (112)N number of participants\*p \< 0.05, \\p \< 0.01, \\\*p \< 0.001 Psychosocial factors independently predicting disordered eating behaviours {#Sec12} -------------------------------------------------------------------------- Table [3](#Tab3){ref-type="table"} presents results from univariate and multiple logistic regression analyses reporting psychosocial associated factors of DEB. The final multiple regression model included perceived weight, body dissatisfaction, negative affect and watching TV. The risk of having DEB among the perceived overweight group was 2.8 times higher than among those in the perceived underweight group. Students with higher levels of body dissatisfaction were significantly more likely to develop DEB compared to those with lower levels of dissatisfaction. Students with a higher negative affect were significantly more likely to develop DEB compared with those with a lower negative affect. Students watching TV were almost three times more likely to report DEB than students who did not watch TV. The corresponding coefficients and standard errors in the multiple logistic regression model are shown in the Additional file [4](#MOESM4){ref-type="media"}: Table S4.Table 3Multiple logistic regression model showing factors associated with disordered eating behavioursUnivariate Logistic Regression Analysis per Independent VariableForward Multivariate Stepwise Logistic Regression AnalysisOR (95% CI)POR (95% CI)PBMI Underweight1---- Normal Weight1.95 (1.14--3.33).015\\---- Overweight3.29 (1.77--6.11)\<.001\\\\----Perceived Weight Underweight11 Normal Weight1.01 (0.48--2.12).9730.79 (0.34--1.86).589 Overweight5.42 (2.56--11.47)\<.001\\\\2.80 (1.05--7.48).041\\Weight Misconception Correct Weight Conception1---- Misconception of Underweight0.75 (0.24--2.38).628---- Misconception of Normal Weight0.42 (0.22--0.80).008\\\---- Misconception of Overweight4.19 (2.27--7.74)\<.001\\\\----Pressure from family2.17 (1.75--2.70)\<.001\\\\----Pressure from friends2.48 (1.93--3.18)\<.001\\\\----Pressure from dating1.86 (1.48--2.34)\<.001\\\\----Pressure from media1.90 (1.53--2.36)\<.001\\\\----Watch TV No11 Yes1.80 (0.94--3.46).076\2.40 (1.11--5.18).026\\Watch DVD No1---- Yes1.67 (0.98--2.85).058\----Chat Online No1---- Yes2.35 (1.21--4.57).011\\----Play Online No1---- Yes1.34 (0.84--2.14).23\----Body Satisfaction0.92 (0.89--0.95)\<.001\\\\0.96 (0.93--0.99).019\\Positive Affect0.96 (0.93--0.99).005\\\----Negative Affect1.10 (1.06--1.14)\<.001\\\\1.07 (1.03--1.12)\<.001\\\\*OR odds ratio, CI confidence intervalPressure perceived sociocultural pressure to thin body and weight loss\*p ⩽ 0.25, \\p \< 0.05, \\\*p \< 0.01, \\\\p \< 0.001 The reliability and the validity of the multiple logistic regression model was assessed by employing post-estimation statistics (likelihood ratio χ^2^ test significant, χ^2^ = 107.925, degrees of freedom (df) = 9, p \< 0.001; Hosmer-Lemeshow χ^2^ test = 10.196, df = 8, p = 0.252). The Hosmer-Lemeshow test of the goodness-of-fit suggests the model is a good fit to the data (small Chi-squared values with larger p-value closer to 1). Area under the ROC curve (AUC) was 0.825 with 95% confidence interval (0.781, 0.869), which is a prediction power for the model. Discussion {#Sec13} ========== The main findings of this school-based study reveal a high prevalence rate of DEB among male adolescents (27.1%) and female adolescents (30.5%). The study also found that perceived weight, negative affect, body dissatisfaction, and watching TV were significantly associated with DEB among adolescents. The prevalence estimates of the current study are among a wide range of previous school-based studies conducted in China (2.26%--58.3%) \[[@CR10], [@CR31]--[@CR33]\]. The available information on DEB in rural China has been reported by Sing Lee and Antoinette M. Lee \[[@CR6]\]. In their study, 266 school female adolescents from Grade 11 from Rural Hunan were included with a prevalence rate of 2.5%. In the present study, prevalence was much higher. This may be attributed to different screening instruments for DEB. The study by Sing Lee and Antoinette M. Lee \[[@CR6]\] used EAT-26 as the screening instrument, while we used SCOFF. The sensitivity and the specificity between the two instruments are different and SCOFF has been reported to have a tendency toward overinclusion \[[@CR34]\]. It is further comparable to reports from Western countries; in spite of variation in measuring instruments, study methodologies and sample compositions: for instance 29.4% of female adolescents and 14.9% of male adolescents in Germany, and 56% of female adolescents and 28% of male adolescents in US \[[@CR14], [@CR15]\]. Given the harmful consequences related to DEB and the high prevalence of DEB, it is vital for public health officials to increase public awareness by introducing relevant and important information about DEB at individual, school and society level. In this way, health organisations can aim at intervening and preventing harmful eating behaviours. So far, there has been no such programs for DEB in the local region. This study did not show significant gender differences in prevalence of DEB. Most Chinese and Western studies report that prevalence of DEB was significantly higher among female adolescents than among male adolescents \[[@CR9], [@CR14], [@CR15]\]. The prevalence of DEB for males is possibly rising and the incidence of DEB for males may have been underrated due to cultural stigma \[[@CR35]\]. It may also due to changing lifestyles: increased access to TV, social media, Internet and gaming. It is possible that female adolescents and male adolescents have a similar risk for developing DEB. More attention, therefore, should be also be paid to male adolescents who are at risk of DEB. The present study found perceived weight as a significant associated factor of DEB. Results are consistent with previous observations on perceived weight status in Chinese adolescents \[[@CR6]\] and Western samples \[[@CR36], [@CR37]\]. It has been suggested that one's perceived weight has a greater influence than one's actual weight when engaging in DEB \[[@CR38]\]. In addition, a considerable number of adolescents in this study misperception with regard to weight status, which is consistent with previous Chinese research \[[@CR39]\]. Furthermore, separate logistical regression in this study showed that students with the misconception of being overweight were more likely to report DEB. This is in line with previous research \[[@CR38]\] and it indicates that one possible avenue for prevention programs may be a focus upon helping students develop accurate weight perception. Along the same lines, body dissatisfaction was a significant associated factor for DEB. Similar results have been reported in both Chinese and Western research \[[@CR13], [@CR16]\]. The thin-ideal image contributes to perfectionism and body dissatisfaction. Male adolescents wish to become more muscular and achieve the male-ideal of V-shaped figure. On the other hand, female adolescents wish to become thinner and achieve the female-ideal of extremely thin. They put much emphasis upon having a perfect body shape, though they have a clinically healthy weight. Perfectionism and body dissatisfaction are both highly correlated with DEB \[[@CR40]\]. Body dissatisfaction can increase dieting behaviours \[[@CR41]\] and negative affect \[[@CR16]\], which can, in turn, lead to poor eating habits and increased eating disturbances. Negative affect was significantly associated with DEB in this study. The result is in agreement with previous Chinese and Western research \[[@CR13], [@CR16]\]. One explanation is that negative urgency (i.e., the tendency to act rashly in response to negative affect) is significantly associated with disordered eating \[[@CR42]\]. Another explanation can be derived from escape theory - people tend to escape negative emotions by abnormal eating (overeating) and they believe or have learned that overeating can relieve negative emotions \[[@CR43], [@CR44]\]. Moreover, negative affect and disordered eating may be interacted mutually, where the feeling of shame and guilty caused by disordered eating may result in negative self-perceptions among female adolescents \[[@CR44]\]. In addition, this study found television access as a significant associated factor of DEB. A small number of adolescents did not have access to TV, possibly related to high academic expectations, family economic situations or personal preference. Media consumption can significantly predict female adolescents' DEB and male adolescents' interest in dieting \[[@CR45]\]. Such an effect of media can also be found in other non-western studies. For example, research in Fiji \[[@CR46]\], Pakistan \[[@CR47]\], Japan \[[@CR48]\], and Korea \[[@CR49]\] have documented significant associations between media exposure and DEB. Becker \[[@CR50]\] has also discussed how Western media introduced by TV has caused thin-ideal internalization among Fijian females. Garfinkel and Garner suggest: "The media have capitalized upon and promoted this image (of thinness) and through popular programming have portrayed the successful and beautiful protagonists as thin. Thinness has thus become associated with self-control and success." (p.145) \[[@CR51]\]. Other media variables in this study did not show significant associations with DEB. It is possible that specific media exposure may contribute to thin body and eating disturbances instead of other general media variables. Although BMI did not contribute to the multiple logistical regression model in this study, BMI was significantly positively associated with DEB from univariate logistical regression analysis. Previous research showed there may exist a U-shaped relation between DEB and BMI \[[@CR52], [@CR53]\]. That is, disordered eating behaviours were more problematic at the two extreme ends of BMI (underweight and obesity). In addition, there is a non-significant trend of increasing prevalence of DEB with age in this study; late adolescents reported highest prevalence of DEB. This may be related to an increased social and emotional stress. Late adolescents, face an exceptional amount of pressure to succeed in college entrance examinations and achieve parental expectations \[[@CR54]\] and experience peer competition, copious homework and reduced social support. These stressors may be associated with an increased risk for DEB \[[@CR55]\]. This study adds to a growing literature on epidemiology of DEB among adolescents in China. It, therefore, provides current knowledge on the epidemiology of DEB in rural China, which helps to capture more attention to DEB in rural China. Since DEB may develop into more severe forms of DEB or eating disorders without appropriate intervention strategies, it is important to design early interventions against DEB. Secondly, this study included both male adolescents and female adolescents which has provided valuable insights into DEB in both genders. Thirdly, this study found risky behaviours associated with DEB among adolescents, which may contribute to better identification of high-risk groups and may be helpful for early prevention and intervention programs. For instance, adolescents with inaccurate weight perception and body dissatisfaction could be a targeted group for eating problems' prevention programs. A school-based program could be carried out by helping students reduce weight-related teasing, improve self-esteem and body satisfaction. This study has some limitations, however. Firstly, the cross-sectional nature of this study could cause difficulty for examining cause-and-effect relationships between DEB and associated factors. Secondly, self-report measures in this study may lead to under- or overestimation of prevalence of DEB and associated factors. For example, self-reported BMI may overestimate measured BMI at the low end of the BMI scale and underestimate measured BMI at the high end \[[@CR56]\]. Thirdly, unknown response bias may exist that can reduce the reliability and validity of the study. For example, students may respond untruthfully or give answers that they felt the researchers wanted to hear. Fourthly, the limitation of the SCOFF questionnaire needs to be considered. Since it has shown low sensitivity in a validation population-based study \[[@CR57]\]. Fifthly, information on students' reading level was not available. Since low reading levels may lead to misunderstanding of the questionnaires, the lack of data on reading levels may reduce the reliability of the study. Lastly, this study was school-based and thus access to participants was limited by those who were attending school. No accurate statistical information about school dropout rate was available. According to a literature review in 2015 \[[@CR58]\], cumulative dropout rate in rural secondary schools was as high as 63%, while dropout rate in cities was less than 10%. There have been no statistics for students attending vocational and technical schools in China. This sample may be biased towards adolescents with higher education, therefore. The results would be more representative by conducting a population- or community-based study. Conclusion {#Sec14} ========== The present study has re-examined DEB among Chinese adolescents in a rural area. It suggests that DEB among rural Chinese adolescents are prevalent. Perceived weight, negative affect, body dissatisfaction and watching TV are significant associated factors for DEB among adolescents. This may provide valuable implications for prevention and intervention strategies in DEB. Furthermore, longitudinal population-based studies are recommended to produce a more comprehensive understanding of causes and risk factors of DEB among Chinese adolescents. Additional files ================ {#Sec15} Additional file 1: Table S1.Descriptive statistics of each item of SCOFF. (DOCX 11 kb) Additional file 2: Table S2.Cross-tabulation of BMI and perceived weight categories among adolescents. (DOCX 11 kb) Additional file 3: Table S3.Associations between misconception of over−/under-weight and DEB by gender. (DOCX 11 kb) Additional file 4: Table S4.Multiple logistic regression model showing factors associated with disordered eating behaviours by coefficient and standard errors. (DOCX 13 kb) AUC : Area under the ROC curve BMI : Body Mass Index CI : Confidence intervals DEB : Disordered eating behaviours df : Degrees of freedom M : Mean N : Number OR : Odds ratio ROC curve : Receiver operating characteristics curve sd : Standard deviation SES : Socioeconomic status SPSS : Statistical package for the social sciences VIF : Variance inflation factor Electronic supplementary material The online version of this article (10.1186/s40337-017-0175-x) contains supplementary material, which is available to authorized users. The authors sincerely thank Li Chunying, Zhao Yanhua and Gong Chengzhi for help during research in the first middle school. The authors thank teachers' and students' in the first middle school for their cooperation. Funding {#FPar1} ======= This was a self-financed study. Availability of data and materials {#FPar2} ================================== The data that support the findings of this study are available from the corresponding author upon reasonable request. TF made substantial contributions to study design, acquisition of data, analysis and interpretation of data, and writing the manuscript. DSA made substantial contributions to study design, analysis and interpretation of data, revising the manuscript critically for important intellectual content and general supervision of the research. Both authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== The study was approved by the Regional Committee for Medical Research Ethics in Norway, and the Committee for the first middle school in Dongfanghong, China. Consent for publication {#FPar4} ======================= Not applicable. Competing interests {#FPar5} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	{ "pile_set_name": "PubMed Central" }	[ "amount", "reexamin", "two", "heilongjiang", "affectneg", "causeandeffect", "almost", "control", "teacher", "freedom", "given", "work", "origin", "program", "tendenc", "remain", "ie", "respond", "collect", "thank", "exist", "employ", "identifi", "put", "korea", "incom", "risk", "classif", "dvd", "lastli", "select", "multicollinear", "fund", "student", "valu", "hour", "hosmerlemeshow", "multipl", "may", "÷", "theori", "recent", "debwithout", "septemb", "live", "toward", "separ", "use", "gener", "friend", "dropout", "socioeconom", "forward", "univari", "germani", "ongo", "invit", "score", "room", "final", "focus", "interven", "support", "teas", "complet", "imag", "accord", "caus", "signific", "focu", "establish", "cover", "li", "becom", "mother", "exposur", "overinclus", "suggest", 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Angiogenesis, the development of new blood vessels, is a feature common to many carcinomas and has been proposed as a hallmark of cancer ([@bib6]). However, morphological assessment of lung carcinomas has revealed that they may be subdivided into angiogenic and relatively non-angiogenic tumours ([@bib21]); the latter co-opt pre-existing vasculature to support an alveolar pattern of growth. Nevertheless, the majority of non-small cell lung carcinomas (NSCLCs) are angiogenic and express vascular endothelial growth factor-A (VEGF) ([@bib12]), the predominant pro-angiogenic ligand that is exploited by tumours ([@bib14]). Animal models ([@bib15]; [@bib7]; [@bib18]) and human studies ([@bib28]) have shown that blocking VEGF signalling results in tumour shrinkage, associated with the pruning of angiogenic (proliferative, leaky and immature (i.e., without associated pericytes)) blood vessels. This observation led to the development of a humanised anti-VEGF monoclonal antibody, bevacizumab, which has shown an improved progression-free survival benefit in numerous tumour types ([@bib8]; [@bib19]; [@bib23]). In recurrent or advanced NSCLC, the addition of bevacizumab to carboplatin and paclitaxel chemotherapy (E4599 study) resulted in a 2-month improvement in median overall survival ([@bib24]). When combined with cisplatin and gemcitabine (AVAiL study) in NSCLC, bevacizumab showed no improvement in overall survival, but demonstrated improved progression-free survival ([@bib22]). The E4599 study excluded patients with brain metastases due to concerns that bevacizumab might exacerbate the incidence of tumour-associated brain haemorrhage ([@bib24]). Several analyses now suggest that bevacizumab is safe in patients with brain metastases ([@bib26]; [@bib1]), but there is no evidence regarding the efficacy of bevacizumab in this indication. Indeed, there is little published on the vascular phenotype, VEGF expression, hypoxia and proliferation of NSCLC brain metastases, which may inform efficacy. To date, no valid biomarkers of the survival benefit of bevacizumab have been identified. Objective response rates have been shown not to predict benefit from bevacizumab in colorectal cancer ([@bib5]), and there are concerns that changes in the MRI appearance of glioblastoma following bevacizumab treatment do not predict clinical benefit ([@bib3]). Efforts to identify novel in situ biomarkers of efficacy have focused on available tissue samples from primary tumour resections ([@bib9]; [@bib11]), to make inferences concerning the treatment of metastatic disease. However, while there is evidence to support this for metastatic colorectal cancer ([@bib16]), there is little evidence to suppose that this is a valid approach to inform the biology of NSCLC brain metastases. In summary, a greater understanding of the vascular phenotype of NSCLC brain metastases may better inform the use of anti-angiogenic agents in this indication. The aim of this study was to assess the relative expression of VEGF, hypoxia, proliferation, microvessel density, vascular pattern and vascular maturity in a series of matched primary NSCLCs and brain metastases. MATERIALS AND METHODS ===================== Tissue ------ Formalin-fixed paraffin-embedded blocks for 15 cases of primary NSCLC with matched resected brain metastases were retrieved from the pathology archives at the Catholic University Hospital, Rome. Ethical approval for this research was granted by the local research ethics committees. Immunohistochemistry -------------------- Characterisation of the antibodies and immunohistochemical methods for VEGF ([@bib27]), CA9 ([@bib13]), Ki67 ([@bib25]), CD34 ([@bib17]) and CD34 and smooth muscle actin (SMA) (double stain; QBEnd/10 and 1A4; Dako, Ely, UK) ([@bib20]) have been described in detail elsewhere. Scoring ------- The maximum intensity of VEGF expression in \>10% of tumour cells was recorded on a semiquantitative scale from 0 (no expression) to 3 (very strong expression). The percentage of tumour cells with membranous expression of CA9 was estimated by a pathologist. The percentage of tumour cells with nuclear positivity for Ki67 was counted by a pathologist. Microvessel density analysis was performed using the Chalkley method; at low power ( × 40), five vascular hotspots in CD34-labelled sections were selected. Microvessel density was assessed by counting the number of CD34-labelled vessels that overlapped with dots on a 25-point Chalkley eyepiece graticule in each high-power field ( × 200) ([@bib2]). The five counts were then added together to provide the density score. Vascular maturity is defined by the coverage of endothelial cells (CD34 positive) by pericytes (SMA positive) and was calculated by counting the proportion of CD34-labelled vessels that were surrounded, at least in part, by SMA-labelled cells from 10 high-power fields ( × 400). Each case was classified according to the predominant vascular pattern (alveolar, basal, diffuse or papillary) described elsewhere ([@bib21]). All scoring was performed blind to the patients\' identities. Statistical analyses -------------------- Student\'s t-test was used to assess the difference between subsets of continuous data and Spearman\'s correlation coefficient was used to assess covariance. The χ^2^ test was used to evaluate associations between categorical data and Cohen\'s κ statistic was used to determine agreement. RESULTS ======= Chalkley counts in primary NSCLCs and brain metastases were not significantly correlated (r=0.148, P=0.60) ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). Although, mean Chalkley counts were similar in primary and secondary cancers (42.5 vs 43.5, respectively, P=0.77). The proportion of mature vessels was on average 63.2% greater in brain metastases than their matched primary NSCLCs (mean 25.7% vs 88.9%, respectively, P=0.004) ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). The proportions of mature vessels in primary and secondary cancers were not significantly correlated (r=−0.46, P=0.09). Differences were also observed in vascular patterns, with a predominance of alveolar, basal and diffuse patterns in primary tumours and a predominance of diffuse and papillary patterns in secondary tumours ([Table 1](#tbl1){ref-type="table"}). The percentage of CA9-positive tumour cells was similar to that previously reported ([@bib4]) and was closely correlated between primary NSCLCs and brain metastases (r=0.825, P=0.0002), suggesting that there were similar levels of hypoxia in matched cases from this series ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). The mean percentage of cells positive for CA9 was not statistically significantly different in primary and secondary cancers (14.0 vs 10.7, respectively, P=0.27). Vascular endothelial growth factor expression was seen in all primary and secondary NSCLCs; however, there was no agreement between the scores in matched pairs (κ=−0.25) and the association was not statistically significant (P=0.15) ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). The proliferative fraction (percentage of Ki67-labelled cells) was also not significantly correlated between primary and secondary cancers (r=0.179, P=0.524), though the mean Ki67 was significantly higher in the brain metastases than the primary lung cancers (35.7% vs 19.3%, respectively, P=0.018) ([Table 1](#tbl1){ref-type="table"}). DISCUSSION ========== This is the first report comparing VEGF expression, CA9 expression, proliferation, microvessel density, vascular pattern and vascular maturity in matched primary NSCLCs and brain metastases. The data show that brain metastases have a significantly greater proliferation rate and vascular maturity than their matched primaries. Neither the proliferation rate, vascular maturity, expression of VEGF, vascular pattern nor microvessel density could be predicted in the brain metastasis from examination of the primary NSCLC. Only CA9 expression showed a strong correlation between primary and secondary NSCLCs. These observations are important for the following reasons: First, anti-VEGF therapies were thought to increase the risk of cerebral haemorrhage in patients with brain metastases, but recent data suggest that bevacizumab is safe in this indication. Our data provide a biological explanation for this observation, showing that the vasculature of brain metastases is stable, and (extrapolating from observations in rectal cancer) ([@bib28]) is unlikely to regress following anti-VEGF therapy, minimising the risk of associated haemorrhage. Second, mature vasculature is less sensitive to anti-VEGF therapy, suggesting that while patients with primary NSCLCs may benefit from anti-VEGF therapy, those with brain metastases may not. Moreover, preclinical data suggest that when endothelial regression is seen in mature vessels targeted by anti-VEGF therapy, a pericyte scaffold remains and permits rapid re-angiogenesis following cessation of therapy ([@bib18]). Thus, a rebound effect may be expected if anti-VEGF therapy is used in brain metastases and continuous, rather than intermittent, therapy might show greater efficacy. No efficacy data exist for bevacizumab in brain metastases, but [@bib26] have stated that 'patients with treated brain metastases will likely derive similar benefit from bevacizumab as patients without brain metastases\' without any biological, pathological or clinical evidence. The data herein are an important counterbalance to this claim. Third, non-angiogenic NSCLCs ([@bib21]) that show an alveolar pattern of growth may metastasize, and do not merely co-opt existing vasculature in the brain, but instead develop a more angiogenic phenotype with haphazardly arranged vessels. Moreover, even non-angiogenic, alveolar tumours express VEGF, are under the influence of hypoxia and are supported by an often immature, dense vasculature. Therefore, one may expect non-angiogenic tumours to potentially respond to anti-VEGF therapy at their site of origin. Finally, retrospective analyses of archived primary tumours from trials of anti-VEGF therapy have thus far failed to identify a biomarker of efficacy ([@bib10]). The data presented herein suggest that the vascular phenotypes of primary and secondary cancers are very different. Therefore, when evaluating anti-angiogenic treatments in the metastatic setting, biomarker studies should also be conducted on metastases and not the primary tumours. The limitations of this study include the sample size, which is small in statistical terms, but it is large for a series of matched brain metastases, provides informative data and reports novel findings. Furthermore, while one may make inferences regarding the applicability of these observations to anti-VEGF therapy using preclinical data ([@bib7]; [@bib18]) and clinical data from other tumours ([@bib28]), only serial biopsies of brain metastases from patients receiving bevacizumab will provide a definitive answer. To date, the two reported series of NSCLC brain metastases treated with bevacizumab in the literature are small, n=36 ([@bib1]) and n=115 ([@bib26]) and provide no histopathological data. Moreover, it is unlikely that clinicians would want to subject terminally ill patients to repeated invasive procedures to provide such material. In conclusion, brain metastases of NSCLCs have a vascular phenotype that is distinct from the primary tumours. This has implications for the use of anti-VEGF therapy in this setting and should be taken into consideration when designing clinical trials to assess the efficacy of such drugs and biomarkers that may predict benefit. We thank all patients and clinicians who provided material for this research. Dr Adrian Jubb is the recipient of a Career Development Fellowship from the Pathological Society of Great Britain and Ireland. This study was supported by Cancer Research UK, Pathological Society of Great Britain and Ireland and the NIHR Biomedical Research Centre, Oxford. Dr Adrian M Jubb received a speaker\'s honorarium in 2010 from Genentech Inc., which is developing anti-angiogenic therapies. ![(A) A scatter plot of CD34 Chalkley counts in matched primary (lung) and secondary (brain) cancers. (B) A line plot showing differences in the percentage of blood vessels covered by pericytes in matched primary and secondary cancers. (C) A scatter plot of the percentage of matched primary and secondary cancers positive for carbonic anhydrase 9. (D) A line plot showing differences in the VEGF score in matched primary and secondary cancers. (E, F) Double-labelled immunohistochemistry for CD34 (blue) and smooth muscle actin (brown) in a matched primary (E) and secondary (F) cancer.](bjc2011147f1){#fig1} ###### Scoring frequencies Case VEGF intensity score (0 to 3) CA9 (% positive) Ki67 (% positive) *Chalkley count (n)* Vascular maturity (% mature) Vascular pattern ---------- ----------------------------------- ---------------------- ----------------------- -------------------------- ---------------------------------- ---------------------- ---------- -------- -------- ---------- ----------- ----------- 1 2 2 0 20 10 50 34 51 36 96 Basal Papillary 2 2 1 0 0 50 50 45 43 21 88 Basal Basal 3 2 3 0 0 10 30 39 35 23 97 Basal Papillary 4 2 3 0 0 0 15 44 62 4 97 Alveolar Papillary 5 2 2 30 20 60 80 44 47 4 93 Diffuse Basal 6 1 2 5 0 20 15 45 44 14 95 Alveolar Diffuse 7 2 3 0 0 40 30 33 53 27 94 Basal Basal 8 2 2 10 15 0 70 24 18 9 58 Diffuse Diffuse 9 2 2 0 0 40 40 57 45 61 86 Papillary Papillary 10 2 1 0 0 0 20 40 56 30 88 Alveolar Diffuse 11 1 2 30 20 10 5 43 25 30 97 Diffuse Diffuse 12 2 3 80 50 30 10 63 55 28 77 Alveolar Papillary 13 2 1 50 30 5 40 43 39 40 89 Basal Diffuse 14 1 2 5 5 5 40 48 35 12 94 Diffuse Papillary 15 3 1 0 0 10 40 36 44 47 84 Basal Diffuse Median 2 2 0 0 10 40 43 44 27 93 NA NA IQR 2--2 1.5--2.5 0--20 0--20 5--35 17.5--45 37.5--45 37--52 13--33 87--95.5 NA NA Abbreviations: CA9=carbonic anhydrase 9; IQR=interquartile range; NA=not applicable; VEGF=vascular endothelial growth factor.	{ "pile_set_name": "PubMed Central" }	[ "anhydras", "treatment", "sma", "two", "fellowship", "origin", "remain", "speaker", "ie", "respond", "ligand", "prune", "thank", "exist", "biopsi", "identifi", "basal", "risk", "block", "grant", "select", "intermitt", "student", "mri", "signal", "may", "cisplatin", "recent", "use", "support", "score", "final", "focus", "smalabel", "accord", "signific", "immunohistochem", "cover", "carbon", "suggest", "also", "growth", "formalinfix", "state", "cell", "preexist", 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Background ========== The evolutionary arms race between bacteria and their phages, giving rise to resistance and counter resistance mechanisms, shapes the bacterial and phage population size and composition as well as the evolutionary success of these two entities \[[@B1],[@B2]\]. The major phage resistance mechanisms exhibited by bacteria and the strategies adopted by phages to counter these resistance mechanisms have been extensively treated in two recent reviews \[[@B1],[@B2]\]. Understanding phage resistance mechanisms is critical in developing phage therapy applications. Bacillus anthracis, a category A biothreat agent, is a spore-forming Gram-positive bacterium of the Bacillus cereus sensu lato group. It is a zoonotic soil bacterium that infects animals and occasionally humans, causing the disease anthrax. The October-November 2001 bioterrorism attacks using mail laced with anthrax spores brought about renewed interest in research on B. anthracis. The prospect of biothreats using B. anthracis and the possibility of naturally emergent or deliberately created antibiotic resistant B. anthracis call for highly integrated and enhanced technological platforms for diagnosis and detection of this organism. This need is best illustrated in the case of a bacterial bioterror attack, where timely detection and intervention with countermeasures such as antibiotic therapy are paramount in preventing fatal consequences. Bacteriophages have been and still remain useful tools for bacterial species and strain differentiation \[[@B3]-[@B6]\], although evidence of successful application of phage therapy is still sparse in Western medicine \[[@B7]\]. Recently, the inherent binding specificity and lytic action of bacteriophage-encoded enzymes called lysins have been exploited for the rapid detection and killing of B. anthracis\[[@B8]\]. These authors demonstrated that the PlyG lysin isolated from the γ phage of B. anthracis specifically kills B. anthracis isolates and other members of the B. anthracis "cluster" of bacilli in vitro and in vivo. Both vegetative cells and germinating spores were shown to be susceptible. The lytic specificity of PlyG was also exploited as part of a rapid method for the identification of B. anthracis\[[@B8]\]. Thus, PlyG is a useful tool not only for the treatment but also for the detection of B. anthracis. The standard diagnostic tests for suspected B. anthracis as recommended by the Centers for Disease Control and Prevention (CDC) are as follows: presumptive identification to genus level (Bacillus family of organisms) requires Gram stain and colony identification. Presumptive identification to species level (B. anthracis) requires tests for motility, lysis by γ phage, capsule production and visualization, hemolysis, wet mount and malachite green staining for spores. Confirmatory identification of B. anthracis may include lysis by γ phage, capsular staining, and direct fluorescent antibody (DFA) testing on capsule antigen and cell wall polysaccharide. Thus, testing for γ phage sensitivity is an integral part of B. anthracis identification \[[@B9]\]. γ phage exhibits a fairly narrow host range but several B. cereus strains (e.g., ATCC 4342) have been shown to be sensitive to infection by this phage \[[@B8],[@B10]-[@B12]\]. Several phages (CP51, CP54 and TP21) isolated from B. cereus and B. thuringiensis strains have been successfully used for transducing chromosomal markers and plasmids between B. anthracis strains \[[@B13]-[@B17]\]. However, their utility as B. anthracis diagnostic phages is limited because of their broad host range. Bacteriophage AP50 was isolated from soil using B. anthracis Sterne as the host \[[@B18]\]. Originally it was thought to be an RNA phage but was later shown to contain double stranded DNA and phospholipid \[[@B19]\]. AP50 was also shown to have a narrow host range; only one third of the 34 B. anthracis strains and none of the 52 strains belonging to 6 different Bacillus species were susceptible to infection by AP50 \[[@B20]\]. Nine major structural proteins were identified on SDS-PAGE gels. The molecular weight of the phage DNA was estimated to be 9 × 10^6^ daltons \[[@B21]\]. Treatment with organic solvents such as chloroform (5%) and ether (25%) for 30 minutes inactivated the phage to a survival of about 1 × 10^-4^\[[@B22]\]. In a recent study, we described the genetic characterization of AP50c, which is a derivative of AP50 \[[@B23]\]. In contrast to the original report \[[@B20]\] on AP50, AP50c exhibited a much narrower host range, infecting 111 of the 115 B. anthracis strains (97%) and none of the 100 B. cereus sensu lato strains. We also determined the genome sequence by 454 pyrosequencing and compared it to other Tectiviridae phages \[[@B23]\]. The genome size was determined to be 14,398 bp in length and shown to be highly similar to other Tectiviridae phages infecting Gram-positive bacteria in genetic organization and encoded proteins. We identified two mutations in the AP50 genome that were responsible for the control of lysogenic/lytic lifestyles and consequently the phenotypic shift from turbid to clear plaque morphology. Based on these properties, we proposed that AP50c could be used as an additional diagnostic and therapeutic tool for B. anthracis. Before embarking on phage therapy using AP50c, we sought to characterize the phage resistance mechanisms in B. anthracis in response to AP50c infection. In order to address this problem, we took an approach that began with whole genome scanning and concluded with genetic validation. Initially, we isolated a number of B. anthracis mutants that were spontaneously resistant to killing by AP50c and observed that these mutants produced an extracellular material that coats the cell surface \[[@B23]\]. We hypothesized that this may be a bacterial adaptive resistance mechanism masking the bacterial receptor of AP50c. In the current study, we have mapped multiple bacterial mutations responsible for spontaneous AP50c resistance. Historically, mapping mutations responsible for specific phenotypes has involved transfer of the mutation with a closely linked selectable marker via one of the three conventional methods: transduction, conjugation or transformation. Physical mapping has involved cloning and sequencing of the genetically mapped gene using traditional cloning vectors and methods. The recent development of rapid, cost-effective sequencing of bacterial genomes using second-generation sequencers or array-based comparative genome resequencing has accelerated the mapping of causal variations in bacterial as well as in other model system genomes \[[@B24]-[@B26]\]. We recently demonstrated the use of 454- generated draft sequences to map mutations conferring various phenotypes, and in that study a putative mutation associated with AP50c resistance was mapped to csaB\[[@B27]\], although the link between genotype and phenotype was not verified in that report. CsaB (for cell surface anchoring) protein is involved in pyruvylation of the peptidoglycan-associated polysaccharide, and this modification is necessary for binding of the S-layer homology (SLH) domain. Thus, CsaB is critical for the non-covalent anchoring of SLH-domain containing proteins onto the cell surface \[[@B28]\]. In the current study, we explore the relevance of the preliminary finding of a potential linkage between the csaB gene and AP50c phage resistance by isolating and characterizing additional spontaneous mutants and, by using a recently described method \[[@B29]\], validating the WGS data by recreating csaB point and deletion mutations in a different genetic background and showing phage AP50c resistance. Furthermore, we restored sensitivity to AP50c infection in the mutants by complementing the defect in the ΔcsaB mutant in cis as well as in trans, thereby establishing an essential role of csaB in phage infectivity. Results and discussion ====================== Whole genome pyrosequencing and identification of putative variants in AP50^R^ mutants -------------------------------------------------------------------------------------- We took a whole genome sequencing (WGS) approach to map the mutations and identify the gene(s) responsible for the phage resistant phenotype of the AP50^R^ mutants. We have shown previously that the draft sequences generated by Roche/454 shotgun pyrosequencing can be used efficiently to map mutations responsible for various phenotypes \[[@B27]\]. We generated whole genome sequence data of five spontaneous AP50^R^ mutant strains and compared them to that of the AP50c sensitive parent strain. We reasoned that the causal variation of phage resistance might occur in the same gene (for example in the gene encoding the bacterial receptor of the phage) in all five mutants (S-R1, S-R2, S-R3, S-R4, and S-R6), assuming that there is a single pathway to resistance. Hence, we searched for a common variation in all five mutants that is not present in the phage-sensitive parent strain. The phage-sensitive parent strain (Sterne 34F~2~) and the five mutants were sequenced by FLX or Titanium protocol. Some of the basic statistics of the genomic sequences are presented in Table [1](#T1){ref-type="table"}. The estimated average coverage of the genomes ranged from 12X to 46X, and the number of high-quality variations (see Methods) ranged from 46--52 (Table [1](#T1){ref-type="table"}). The number of contigs was generally proportional to the Q39 scores; i.e., the lower the Q39 score the lesser the number of contigs. Among the high-confidence variations, the most likely true positive variations (high-quality) were selected for by applying the following criterion: variations that had a percentage concordance of more than 75% variant base in the reads spanning any given position. Figure [1](#F1){ref-type="fig"} shows the variations as a function of the percentage concordance of the variant reads. There were a total of 242 high-quality variations across all the five AP50^R^ mutants, and of these 215 showed 75% or more concordance. Among these, 200 were identity-by-descent variations. The genomic locations of the 15 unique variations along with their annotations are shown in Table [2](#T2){ref-type="table"}. Among all the genes encoding variations, only two were mutated in all five resistant strains and not in the sensitive strain. These were BAS0840 (csaB), which encodes a cell-surface anchoring protein \[[@B28]\] and BAS3946, which encodes a 2, 3-diketo-5-methylthiopentyl-1-phosphate enolase. ###### *Statistics of genome sequence of wild type and mutantB. anthracisstrains* Strain Runs Run type Length of assembled genome (bp) % ≤ Q39 Mean read length (bp) Number of contigs Depth of Coverage *Number of putative HQ\ variants** ------------ ---------- -------------- ------------------------------------- ------------- --------------------------- ----------------------- ----------------------- -------------------------------------- 34F~2~ 1 FLX 5,356,131 0.06 274 101 23 48 S-R1 1 FLX 5,346,471 0.37 245 227 12 47 S-R2 2 FLX 5,355,072 0.02 274 60 46 50 S-R3 1 FLX 5,355,679 0.04 283 92 28 46 SR-4 1 FLX 5,347,433 0.06 282 102 21 47 SR-6 1 FLX 5,349,716 0.05 277 98 26 52 JB220 1 Titanium 5,349,773 0.39 264 111 23 43 J-R1 1 Titanium 5,348,852 0.59 269 128 22 45 \HQ, high confidence and high concordance (≥75%). ![Filtration of true positive variations from false positive variations by percent concordance and coverage.* The graph displays 260 high quality (HQ) variations in the genome sequences (34F~2~ and its derivatives) produced by GS Reference Mapper software as a function of percentage concordance. The graph indicates all the high-quality variations in the six genomic sequences obtained in this work. The variations from the six strains are indicated by different symbols. AP50 R1, AP50 R2, AP50 R3, AP50 R4 and AP50 R6 are S-R1, S-R2, S-R3, S-R4 and S-R6 respectively.](1743-422X-9-246-1){#F1} ###### *Location of true positive variations in genomes of mutantB. anthraciscompared to reference genome* Strain Start position Stop position Reference allele Sample allele Locus description Coverage Percent Concordance ------------ -------------------- ------------------- ---------------------- ------------------- --------------------------------------------------- -------------- ------------------------- S-R1 894,910 894,910 \- G CsaB protein 10 100 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 8 100 S-R2 210,679 210,679 T G intergenic 29 97 895,097 895,097 \- G CsaB protein 38 82 2,139,026 2,139,026 G T sensor histidine kinase 34 76 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 24 100 S-R3 895,097 895,097 \- G CsaB protein 23 100 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 19 100 S-R4 895,066 895,066 A \- CsaB protein 18 100 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 19 100 S-R6 160,743 160,743 A \- pX01 58 86 895,083 895,083 \- GCTTA CsaB protein 17 88 1,330,746 1,330,746 G CGGT intergenic 4 75 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 14 100 4,303,652 4,303,652 \- AC solute-binding family 5 protein 20 100 J-R1 68,981 68,981 A \- hypoxanthine-guanine phosphoribosyltransferase 33 97 895,436 895,436 G T CsaB protein 23 96 Mutation in csaB is responsible for phage AP50c resistance ------------------------------------------------------------ Of the two mutated genes present in the AP50^R^ strains identified by WGS, BAS0840 (csaB) was determined to be the most probable candidate gene for phage resistance based on the following criteria: 1) In each of the five mutants (S-R1, S-R2, S-R3, S-R4 and S-R6) for which WGS was performed, a frame-shift mutation (∇G46 in S-R1, ΔA203 in S-R4, ∇GCTTA219 in S-R6, ∇G234 in S-R2 and S-R3) (Figure [2](#F2){ref-type="fig"}) was identified, which in all cases resulted in truncation of CsaB protein and possibly inactivation of its function. 2) Four additional spontaneous resistant mutants from the same genetic background (34F~2~) were screened by csaB PCR followed by Sanger sequencing of the csaB amplicons and were found to carry mutations in csaB as well (C385T, T274G, ΔC129, C254T). Similar to the findings in the first set of mutants, the C385T and ΔC129 mutations result in a truncated CsaB protein, whereas the T274G and C254T mutations result in a change from a noncharged (tyrosine) to a charged amino acid (aspartic acid) and a polar hydrophilic to a nonpolar hydrophobic amino acid, respectively. 3) A spontaneous AP50^R^ mutant generated in a different genetic background (JB220) was subjected to WGS and found not to carry the enolase (BAS3946) mutation, but it did carry a mutation in csaB (G573T), which results in a change from a positively charged (arginine) to a noncharged (serine) residue. 4) Seven additional spontaneous phage resistant mutants in the JB220 background were screened by the csaB PCR method described above and also found to carry mutations in csaB (G421T, C292T, G796C, T258G, C767A, and ∇G234, the latter of which was found in 2 different mutants). Interestingly, two of these mutations result in a noncharged residue being mutated to a charged residue, and the others result in a truncated csaB. These results are summarized in Figure [2](#F2){ref-type="fig"} and Table [3](#T3){ref-type="table"}. 5) Targeted introduction of mutations in csaB in a strain (7702) not previously exposed to AP50c resulted in phage resistance (see below and Table [4](#T4){ref-type="table"}). 6) Finally, BAS3946 was deemed unlikely to be involved in phage resistance because the mutation causes a non-synonymous change at G1024A leading to G342S in all five mutants (S-R1, S-R2, S-R3, S-R4 and S-R6) and thus does not result in the loss of the protein. PCR amplification and Sanger sequencing of BAS3946 from all the AP50^R^ mutants showed that the mutation is present only in seven out of nine mutants arising from 34F~2~ background and the parent and not in any of the eight mutants of JB220 background. Additionally, a targeted deletion of BAS3946 or the G1024A point mutation in BAS3946 in a different genetic background (strain 7702) in a strain not exposed to AP50c had no effect on AP50c phage sensitivity (Figure [3](#F3){ref-type="fig"}). ![*Genetic map ofcsaoperon, DNA and protein sequences ofcsaB.a) Genetic map of the csaB* region on B. anthracis chromosome. The block arrows indicate the genes and the direction of transcription of the different genes. b) Nucleotide sequence of the wild type csaB gene and the location and nucleotide changes in the AP50^R^ mutants. Insertions and deletions are indicated by the red arrowheads and inverted arrowheads respectively. The various strains are indicated next to the mutated sites: derivatives of Sterne 34F~2~ and JB220 are indicated by the prefixes S and J respectively. The mutant loci labeled in black were discovered by WGS and those in green by Sanger sequencing of csaB PCR fragments. The altered bases are indicated in blue with the changed base above the wild type. A693C and H270A mutations were genetically engineered mutants. The sequences at the beginning and end of the gene marked in pink are the junctions the csaB deletion mutant. c) Amino acid sequence of wild type and mutant CsaB proteins. The sites of truncation of CsaB protein due to frame-shift and nonsense mutations are indicated by blue downward arrows. The amino acid changes in non-synonymous mutants are indicated in blue above the wild type residue. The amino acid sequences of the frame-shift and truncated proteins are shown. The residues in red are the additional residues added due to the frame-shift before truncation of the mutant proteins. d) DNA and protein sequence of the csaB gene in csaB deletion mutant.](1743-422X-9-246-2){#F2} ###### Results of Sanger verification of the various spontaneous AP50^R^mutants ----------------------------------------------------------------------------------------------------------------------------------------------- Sanger run ID Strain Sanger data Mutation 454 data Implication BAS3946 ------------------- --------------- ----------------------------------- -------------- -------------- --------------------------- ------------- csaB 1 34F~2~ parent WT None WT \- G1024A csaB 2 S-R1 ∇ G between positions 46 and 47 Frame-shift Same Truncation of CsaB G1024A csaB 3 S-R2 ∇ G between positions 233 and 234 Frame-shift Same Truncation of CsaB G1024A csaB 4 S-R3 ∇ G between positions 233 and 234 Frame-shift Same Truncation of CsaB G1024A csaB 5 S-R4 Δ A at position 203 Frame-shift Same Truncation of CsaB G1024A csaB 6 S-R5 C385T Nonsense ND Truncation of CsaB G1024A csaB 7 S-R6 ∇ GCTTA between position 219-220 Frame-shift Same Truncation of CsaB G1024A csaB 8 S-R7 T274G Y92D ND Noncharged to charged (−) G1024A csaB 9 S-R8 Δ C at position 129 Frame-shift ND Truncation of CsaB WT csaB 10 S-R9 C254 T T85M ND Polar hydrophilic to\ WT nonpolar hydrophobic csaB 11 JB220 parent WT None WT \- WT csaB 12 J-R1 G at position 573 to T R191S Same Charged (+) to noncharged WT csaB 13 J-R2 ∇ G between positions 233 and 234 Frame-shift ND Truncation of CsaB WT csaB 14 J-R3 G421T Nonsense ND Truncation of CsaB WT csaB 15 J-R4 C 292T Nonsense ND Truncation of CsaB WT csaB 16 J-R5 G796C G266R ND Noncharged to charged (+) WT csaB 17 J-R6 T258G S86R ND Noncharged to charged (+) WT csaB 18 J-R7 ∇ G between positions 233 and 234 Frame-shift ND Truncation of CsaB WT csaB 19 J-R8 C767A Nonsense ND Truncation of CsaB WT ----------------------------------------------------------------------------------------------------------------------------------------------- ∇ and Δ indicate insertion and deletion of base(s) respectively at the indicated position from the start of the gene. ND- Not Done. WT- Wild type; G1024A indicates a SNP in BAS3946 at position 1024 from the start of gene from G to A. ###### Strains and plasmids used in this study Strain Description Source/Reference ---------------------------------------- --------------------------------------------- ---------------------- Escherichia coli SM10 \- \[[@B39]\] Bacillus anthracis 34F~2~ Sterne pXO1^+^pXO2^-^ Hanna lab Bacillus anthracis BA663 7702 Sterne pXO1^+^pXO2^-^ Koehler lab Bacillus anthracis JB220 7702 ΔBAS2245 Calendar lab Bacillus anthracis S-R1 through S-R9 Spontaneous AP50R mutants This study Bacillus anthracis J-R1 through J-R8 Spontaneous AP50R mutants This study Bacillus anthracis BAP350 BA663 ΔcsaB This study Bacillus anthracis BAP356 BA663 (AP50R-3)-csaB ∇G234 This study Bacillus anthracis BAP366 BA663 (AP50R-1)-csaB ∇G46 This study Bacillus anthracis BAP411 BA663 (AP50R-6)-csaB ∇GCTTA219 This study Bacillus anthracis BAP435 BA663 (AP50R-4)-csaB ΔA203 This study Bacillus anthracis BAP503 BA663 csaB H270A This study Bacillus anthracis BAP533 BAP350 csaB A693C creating an XmaI site This study Bacillus anthracis BAP553 BA663 ΔBAS3946 This study Bacillus anthracis BAP560 BA663 with BAS 3946 G1024A mutation This study Bacillus anthracis BGBN 001 BAP350/pBGBN1003 This study Plasmid pRP1028 Mutant construction This study Plasmid pSS4332 Mutant construction This study Plasmid pSW4 E. coli- B. anthracis shuttle vector \[[@B37]\] Plasmid pBGBN1001 pSW4 NotI This study Plasmid pBGBN1002 pSW4 oriT This study Plasmid pBGBN1003 pSW4 oriT csaB This study Plasmid pGOv4 pUC19 Amp^R^, Kan^R^ Gene Oracle Plasmid pNV36 pGOv4 ΔBAS3946 Gene Oracle Plasmid pNV37 pGOv4 BAS3946 G1024A Gene Oracle ∇-insertion and Δ-deletion of base (s) at any given position from the start of the gene; e.g., csaB ∇G46 indicates insertion of G base after position 46 (between 46 and 47) from the start of the gene, and csaB ΔA203 means deletion of A at position 203 from the start of gene. ![Phage sensitivity spot test on Sterne 7702 and various derivatives. For each row the left spot was spotted with medium (M), the middle spot received AP50c (A) and the right spot was spotted with γ (G). The clearing in the bacterial spot indicates infection and killing of bacteria. In rows 3 through 6, the γ spots completely cleared the bacterial spots. The various strains are as follows: 1) and 2) BA663 (wild type); 3) BAP350 (ΔcsaB); 4) BAP356 (csaB ∇G234); 5) BAP366 (csaB ∇G46); 6) BAP411 (csaB ∇GCTTA219); 7) BAP435 (csaB ΔA203); 8) BAP503 (csaB H270A); 9) BAP533 (csaB A693C); 10) BAP553 (ΔBAS3946); 11) BAP560 (BAS3946 G1024A); 12) BGBN 001 (ΔcsaB/pBGBN1003)](1743-422X-9-246-3){#F3} Reconstructed csaB point mutants and in-frame deletion of csaB result in mucoid colony morphology and phage resistance -------------------------------------------------------------------------------------------------------------------------- In order to unequivocally link the phage resistance phenotype to csaB and to rule out the involvement of other mutations that may be present in the original AP50^R^ mutants, we reconstructed four of the point mutations in a different genetic background, in Sterne strain 7702, which was not previously exposed to AP50c (see Methods). All four mutants (∇G46, ΔA203, ∇GCTTA219, ∇G234) gave rise to mucoid colony morphology and phage resistant phenotype similar to the original spontaneous mutants, indicating that csaB indeed is involved in phage sensitivity. In addition to these mutants, we created a scarless, markerless in-frame deletion of csaB in 7702 background, which also showed mucoid and phage resistant phenotypes (Figure [3](#F3){ref-type="fig"}). Functional CsaB is needed for phage sensitivity ----------------------------------------------- Having shown that inactivation of csaB results in phage resistance, we wished to rule out the possibility that this result is due to a polar effect of the csaB mutations on downstream gene(s) or due to an alteration of regulatory elements in the deletion mutant. csaA and csaB form an operon upstream of sap and eae (Figure [2a](#F2){ref-type="fig"}), which have been shown to be involved in the formation of the cell surface associated S-layer (Sap) or an alternate S-layer (EA1) respectively \[[@B28]\]. In order to test the hypothesis that a functional CsaB is needed for phage sensitivity, we sought to inactivate the protein by mutating a critical amino acid residue while retaining the full-length protein and all the potential upstream and downstream control elements. A protein sequence alignment of 163 known orthologs revealed several conserved residues within CsaB protein (Figure [4](#F4){ref-type="fig"} and Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1) of which the histidine at position 270 is 100% conserved. We mutated histidine 270 to an alanine, and the resulting mutant was found to be mucoid and phage resistant (Figure [3](#F3){ref-type="fig"}), suggesting that a functional CsaB protein is necessary for phage infectivity, and inactivation of the CsaB protein either by truncation or by mutating a critical amino acid residue gives rise to phage resistance. ![*Amino acid sequence alignment ofcsaBorthologs.* BAS0840 was aligned with the Pfam PF04230 seed alignment, and percent conservation was determined as described in Methods. Only the alignment of the amino acids in the vicinity of histidine 270 of CsaB protein is shown. The full-length alignment is provided in Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1.](1743-422X-9-246-4){#F4} Complementation of csaB mutants with wild type copy of csaB --------------------------------------------------------------- We took two parallel approaches to test whether complementation of ΔcsaB mutant with a wild type copy of csaB restores phage sensitivity. First, we introduced a copy of csaB in its native locus in the csaB deletion strain. In this case, however, we engineered the gene to carry a silent mutation (A693C) creating an XmaI site in order to distinguish this strain from the phage-sensitive parent strain. The resulting csaB-XmaI mutant strain was verified by PCR to carry the XmaI mutation (Figure [5a](#F5){ref-type="fig"}) and was found to be non-mucoid and phage sensitive. Thus, restoring the wild type allele in its native locus restored the phage sensitive phenotype. Second, complementation was done in trans by cloning the wild type csaB on a multicopy plasmid (Figure [5b](#F5){ref-type="fig"}) and transferring the recombinant plasmid (pNBGD1003) into the ΔcsaB strain via conjugation. In this plasmid, B. anthracis pagA promoter was used to drive the constitutive expression of the cloned csaB. The resulting recombinant strain was found to be non-mucoid and phage sensitive (Figure [3](#F3){ref-type="fig"}). ![*Complementation ofcsaBmutation.a) PCR verification of XmaI mutant csaB. The wild-type sequence of csaB* was restored in the ΔcsaB strain with the addition of a silent mutation creating an XmaI site (see Methods). The region was amplified by PCR, and PCR products were purified and digested with XmaI. The lane labeled BA663 indicates the csaB PCR product from wild type strain (7702) after digestion with XmaI, whereas the lane labeled BAP533 is the PCR product from the mutant showing the two bands after digestion with XmaI. b) Genetic map of csaB complementing plasmid pNBGD1003. pNBGD1003 is a shuttle vector derived from pSW4. The locations of the Gram positive and Gram negative replicons (from pUB110 and ColE1, respectively), antibiotic resistance genes aadD and bla, origin of conjugational transfer oriT, constitutively expressed B. anthracis pagA promoter (labeled PA promoter) and the csaB gene downstream of the promoter are indicated.](1743-422X-9-246-5){#F5} Transmission electron microscopic analyses of csaB mutants and complemented strains ------------------------------------------------------------------------------------- In an earlier study we found that the spontaneous AP50^R^ mutants produced an extracellular matrix and that AP50c phage failed to attach to the mutant bacterial surface \[[@B23]\]. In the current study, we confirmed these observations by transmission electron microscopy (Figure [6a](#F6){ref-type="fig"}-d). As evident from these images, AP50c phage particles do not attach to the surface of the ΔcsaB mutant and consequently fail to infect. Phage adsorption and infectivity were regained when ΔcsaB was complemented in cis or in trans with the wild type copy of csaB (Figure [6e](#F6){ref-type="fig"}-h). The extracellular secretion is seen only in the mutants and is absent in the wild type and the complemented mutants. The TEM images also revealed that there may be a defective or improperly assembled S-layer, or the S-layer may have been masked by the presence of the extracellular matrix. Further confirmation of the adsorption defect and reversion of this defect in complemented strains was obtained in phage adsorption assays. In the csaB mutants (BAP350, BAP356, BAP366, BAP411, BAP435, BAP503) \~62-100% of the phage particles were left in the supernatant whereas in wild type (BA663), ΔcsaB complemented strains (BAP533 and BGBN001) and the BAS3946 mutants (BAP553, BAP560) most of the phage (\>99%) particles were adsorbed and very few particles (\~0.1-0.65%) were left in the supernatant. Thus, the defect in phage adsorption was rectified in the complemented strains. ![Transmission electron microscopic images of wt and ∆csaB mutants and the complemented strains.a) BA663 (csaB+); b) BA663+AP50c; c) BAP350 (∆csaB); d) BAP350 (∆csaB)+AP50c; e) BAP533 (csaB+XmaI); f) BAP533+AP50c; g) BAP350 (∆csaB)+ pBGBN 1003 (csaB+); h) BAP350 (∆csaB)+ pBGBN 1003 (csaB+)+AP50c. The scale bar is in the bottom right corner of each figure.](1743-422X-9-246-6){#F6} csaB mutants are sensitive to γ phage --------------------------------------- Earlier studies have shown that the cell surface protein GamR, which carries a LPXTG motif, is the cellular receptor of the γ phage \[[@B12]\]. We asked whether the cell surface-anchoring defect associated with csaB mutants affects GamR in any way, thereby preventing γ adsorption and subsequent infection. Consistent with earlier findings \[[@B30]\], all the mutants and the complemented strains allow infection of γ phage, indicating that mutations in csaB do not affect gamR expression or bacterial surface localization of GamR protein (Figure [3](#F3){ref-type="fig"}). Thus, csaB-mediated resistance is specific to AP50 phage and is not a general phage resistance mechanism. Conclusions =========== Phage resistance mechanisms are an integral part of the evolutionary arms race between phage and bacteria that has been waged for millions of years. Generally two types of bacterial defense mechanisms are recognized: active (restriction/modification systems, CRISPR/cas, and abortive infections) and passive host adaptations \[[@B2]\]. In the latter category, at least two, if not three, types of host adaptations interfering with the phage life cycle can be hypothesized: 1) phage entry, 2) phage growth after entry, and 3) potentially lysogeny can be considered to be the first line of defense against phage infection since lysogens acquire resistance rather quickly and at very high percentage upon infection with a turbid plaque AP50t phage \[[@B23]\]. Mutants of phage that make clear plaques arise in the phage population; these mutants were found to carry two mutations within the phage genome---one in a promoter region and another in a gene of unknown function \[[@B23]\]. Although the clear plaque mutants do not seem to regain the infectivity of a lysogen in the case of AP50 (Sozhamannan, unpublished data), spontaneous resistant strains to AP50c phage arise at very low frequencies in bacterial populations. In mutants that are bacterial surface modified, phage resistance could be caused by masking of the bacterial receptor or by bacterial receptor mutations. Other mutations might affect subsequent steps after the phage DNA enters the cell such as DNA replication, particle assembly, or even lysis of the host. In an effort to understand the passive defense mechanisms, we isolated spontaneous phage resistant bacterial mutants. These mutants secreted an extracellular matrix that appeared to protect the bacterium from phage infection. We hypothesized that this extracellular layer might mask the actual cellular receptor, thereby preventing phage adsorption, since it is a gain of new function due to a mutation and is absent in the wild type strain. Genetic mapping of a mutation entails linking the phenotype to a genetic locus. Conventional genetic methods use phage-mediated transduction, transformation or conjugation, by which the mutated gene is co-transferred with a known marker and mapped with reference to the known marker. Recombinant DNA and cloning techniques have been used to isolate and identify the gene responsible for a given phenotype. These conventional techniques are time-consuming and are not feasible in many organisms due to the lack of well-defined genetic systems. Recent developments in second-generation sequencing technologies have allowed us to obtain whole genome sequences of mutants and compare the sequences to that of the wild type strain to determine the causal variations. By taking a whole genome sequencing approach, we have traced the causal variation of the phage resistant phenotype to csaB. There are two caveats to this approach. First, the sequencing technology used here (Roche/454) is highly error-prone, resulting in approximately 50--100 false-positive variant calls per bacterial genome sequence. Empirically, we have determined that by applying a filter based on the coverage and percentage of variant reads in a given position, positions with 75% or above tend to be true positive variations. We have used a second filter in this study: a biological replicate strategy for ascertaining causal variations. A second caveat to this approach is that we assumed that the phenotype is due to a monogenic trait and that there is a single pathway to give rise to that phenotype. However, despite these limitations, in the current study, we were able to correctly associate the causal variation of the phage resistance phenotype to csaB and to further validate this prediction by reconstructing various csaB mutants and showing the linkage between phage resistance and csaB mutations. Mesnage et al. have characterized the csaAB operon of B. anthracis extensively \[[@B28]\]. These authors have proposed a role for csaB in cell surface anchoring of S-layer homology (SLH)-containing proteins via pyruvylation of cell wall polysaccharides. Consequently, mutants lacking csaB appear to be defective in cell surface anchoring of various SLH proteins including EA1 and Sap. In support of this idea, in csaB mutants, Sap and EA1 are conspicuously absent from the cellular fraction and are primarily found in the secretome. These authors also observed additional aberrant cellular morphologies that were replicated in this study, including the accumulation of cell wall material on the cell surface. These authors have attributed this phenotype to the lack of autolysis function in csaB mutants \[[@B28]\]. Recently, the role of yet another gene, tagO, has been implicated in S-layer protein assembly via binding to secondary cell wall polysaccharide \[[@B31]\]. Given the known function of CsaB in the pyruvylation pathway of polysaccharides, its most probable role in AP50 life cycle may be indirect: in the proper cell surface anchoring of phage receptor protein. The most probable candidates for the bacterial receptor may be Sap and EA1.It is possible that Sap or EA1 is the AP50 receptor, and csaB mutants fail to support AP50 adsorption because of the defect in proper cell surface anchoring of the S-layer. The transmission electron micrographs support the idea that the csaB mutants lack a well-organized S-layer. The spontaneous phage resistant mutants isolated in this study carry different types of mutations: SNPs and small insertions leading to nonsense codons, frame-shifts and non-synonymous changes. While a vast majority of these mutations cause CsaB truncation, some other mutants carry non-synonymous changes in the full-length protein. The essentiality of these amino acid residues in the overall structure and function of CsaB is not readily apparent since these mutations fall in regions of very low conservation in the protein alignment (Figure [4](#F4){ref-type="fig"} and Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1), but it is possible that the individual amino acid changes may affect protein folding and therefore function. The AP50^R^ mutants derived from 34F~2~ also carried a mutation in BAS3946 besides csaB. We have clearly established that the mutation in BAS3946 does not contribute to phage resistance in any way, and it is probably a 'hitch-hiker' mutation that was carried in the immediate parent prior to the selection for phage resistance. This result also highlights the power of the next generation sequencers for unbiased mapping of all variations arising in a given bacterial strain under selection pressure or mutagenesis, unlike conventional genetic techniques, which scan a focused area of the genome for mutations. Another intriguing question that remains is why spontaneous AP50 resistant mutants were not found in the actual phage receptor gene itself, assuming Sap or EA1 is the AP50 phage receptor. It is possible that there are differences in the mutability or essentiality of the genes, although we have already shown that sap is not an essential gene for B. anthracis viability (Plaut et al., unpublished data). Further characterization of additional genes involved in AP50c life cycle identified by this study is underway. The fact that γ phage and AP50 use different receptors is useful in phage therapy experiments, since development of resistance against both phages would be kept under check when used in combination. Methods ======= Bacterial strains, plasmids and oligonucleotide primers ------------------------------------------------------- Strains and plasmids used in this study are listed in Table [4](#T4){ref-type="table"}. The three Sterne strains used in this study were 34F~2~ (obtained from Philip C. Hanna), 7702 (obtained from Theresa M. Koehler) and JB220 (obtained from Richard Calendar). Strain 7702 is designated as BA663 in this study. Derivatives of 34F~2~ that were spontaneously resistant to AP50 are designated S-R1 through S-R9, and those of JB220 are designated J-R1 through J-R8. When necessary, strains were grown in the presence of spectinomycin (100 μg/ml for E. coli; 250 μg/ml for B. anthracis) or kanamycin (20 μg/ml for E. coli; 25 μg/ml for B. anthracis). The primers used in various plasmid constructions and Sanger sequencing of PCR amplicons are listed in Additional file [2](#S2){ref-type="supplementary-material"}: Table S1. Genome sequencing and variation detection ----------------------------------------- Whole-genome sequencing (WGS) was performed in the Genome Sequencer FLX (454 Life Sciences/Roche) using FLX or FLX Titanium reagents according to the manufacturer\'s protocols and instructions \[[@B32]\]. Signal processing of FLX data was performed on the sequencer itself, whereas signal processing of Titanium data was performed off-rig on a Linux cluster of 10 nodes connected via gigabit ethernet. Each node contained eight 64-bit processing cores running at 2.3 GHz with 8 GB of RAM. De novo or reference-guided assembly of either FLX or Titanium sequences, preliminary annotation of the genome using the DIYA pipeline \[[@B33]\] and identification of variations were performed using the above-described computer cluster and Sterne (NC 005945) as reference genome as described \[[@B27]\]. A variant flagged by GS Reference Mapper was defined as high confidence if it met the following criteria: a) at least three non-duplicated reads showed the same variation; b) at least one read in each of the forward and reverse orientations showed this variation, unless there are at least five reads with quality scores over 20 (or 30 if the difference involves a 5-mer or higher). A more detailed description of differences is available in the Genome Sequencer Data Analysis Software manual. In this study, a high quality variation is defined as a high confidence (as defined by 454/Roche) and high concordance (≥75% reads have the variation) variation. Culture methods and phage assays -------------------------------- All bacterial strains were grown in Luria Broth or Brain Heart Infusion medium, and the phage assays were performed according to published protocols. Phage spot tests were performed as follows: first, triplicate spots of 50 μl of an overnight culture were made on large phage assay agar plates and allowed to dry. Five μl of phages (\~10^8^ PFU) AP50c and γ was spotted on two bacterial spots while the third spot received 5 μl of medium. The spots were dried at room temperature and the plates were subsequently incubated at 37°C overnight. Phage adsorption assay was performed as follows: One ml of log phase wild type and mutant B. anthracis cells was spun and the pellet was resuspended in 100 μl of AP50c phage and incubated for 30 minutes at room temperature. After addition of 900 μl of phage assay broth, the bacteria-phage complex was filtered through a 0.45 μm syringe filter. The plaque forming units (PFU) in the filtrate was titred using 7702 as the indicator bacterium. The percentage adsorption was calculated against the same volume of unadsorbed phage filtered through the syringe filter. Construction of mutants ----------------------- Two plasmids were engineered to facilitate allelic exchange in B. anthracis, as improvements to a published system \[[@B29]\]. Details of the improvements will be published elsewhere. Briefly, pRP1028 and pSS4332 serve the functions of pBKJ236 and pBKJ223, respectively, and contain many of the same features. In pRP1028, the erythromycin resistance of pBKJ236 was replaced with spectinomycin resistance, and in pSS4332, the tetracycline resistance of pBKJ223 was replaced with kanamycin resistance. Allelic exchange constructs derived from pRP1028 were transferred to B. anthracis, and plasmid integrants were isolated following a temperature shift \[[@B29]\]. Introduction of pSS4332 into the integrant led to I-SceI-mediated cleavage of the integrated plasmid, stimulating the second crossover event. The presence of the desired changes was confirmed by PCR and sequencing. Specifically, to reconstruct the point mutations found in the spontaneous AP50^R^ mutants, the mutations and approximately 500 bp of flanking homology on each side were PCR-amplified from the B. anthracis strains and cloned into pRP1028. To delete csaB, standard cloning methods were used to engineer a construct with the sequence GTG CGG CCCGGG GGA TCT TAA, representing START+one codon+XmaI site+two codons+STOP, along with approximately 500 bp of flanking homology. To restore the wild-type allele to the csaB deletion strain, a derivative of pRP1028 was engineered with the csaB sequence with the adenosine at position 693 changed to cytosine (creating the XmaI site CCCGGG without changing the amino acid sequence), along with approximately 1kb of flanking homology. The change was confirmed by PCR and sequencing, as well as by digestion of the PCR product with XmaI (Figure [5a](#F5){ref-type="fig"}). To identify conserved residues of CsaB, the nucleotide sequence (BAS0840) was aligned with the 163 proteins in the seed alignment of Pfam family PF04230 (polysaccharide pyruvyl transferase) \[[@B34]\] using ClustalW \[[@B35]\]. The resulting alignment was processed with WebLogo \[[@B36]\] to calculate the consensus sequence and percent conservation at each position (Figure [4](#F4){ref-type="fig"} and Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1). The histidine at position 270 of BAS0840 was found to be 100% conserved in these proteins. Standard cloning techniques were used to engineer a derivative of pRP1028 in which the histidine codon (CAT) was changed to alanine (GCT) (Figure [2](#F2){ref-type="fig"}), flanked by approximately 850 bp of homology. The presence of the H270A change was confirmed by PCR and sequencing. DNA sequences used to generate ΔBAS3946 and BAS3946 G1024A were synthesized (Gene Oracle, Mountain View, CA). For ΔBAS3946, the sequence included the first 6 codons of the gene, followed by the last 5 codons of the gene and the STOP codon, all flanked by approximately 500 bp of homology. For BAS3946 G1024A, the sequence included the mutation flanked by 500 bp of homology. In each case, DNA was cloned into pRP1028, allelic exchange was performed as described above, and the presence of the desired changes was confirmed by PCR and sequencing. Construction of pNBGD1003 and complementation of ΔcsaB ------------------------------------------------------ The starting plasmid for pNBGD1003 was pSW4 \[[@B37]\] which was modified by inverse PCR to include a NotI-NdeI site on either side of the pagA promoter using primers 1004 and 1005. The pagA promoter was amplified using primers 1006 and 1026, and fragments were digested with NotI and NdeI and cloned, giving rise to pNBGD1001. Plasmid pNBGD1002 was derived from pNBGD1001 by introducing an origin of transfer, oriT, as a NotI fragment at the NotI site. The oriT fragment was PCR amplified from pIN297, which is a derivative of pPL2 \[[@B38]\] using primers 1064 and 1065. Plasmid pNBGD1003 was derived from pNBGD1002 by cloning a PmlI- BglII fragment carrying the csaB gene sequences into NdeI digested, blunted and BamHI digested pNBGD1002. Plasmid pNBGD1003 was electroporated into SM10 and subsequently transferred into B. anthracis ΔcsaB mutant by conjugation, selecting for polymyxinB and kanamycin resistance as described previously \[[@B29]\]. The resulting ex-conjugants were purified and checked for phage adsorption and infection. PCR and Sanger verification of the csaB and BAS3946 genes from AP50^R^ mutants ------------------------------------------------------------------------------ The DNA sequences spanning the csaB (1385 bp) and BAS3946 (1445 bp) genes were PCR amplified from wild type and various mutants (both 34F~2~ and JB220 derivatives), and the PCR amplicons were purified and sequenced on both strands using primers indicated in Table S-1 (BGBN14 through BGBN21). The resulting nucleotide sequences were aligned to the wild type sequences of these two fragments to determine the mutational changes. Transmission electron microscopy -------------------------------- Bacterial pellets were fixed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate pH 7.4 overnight at 4°C. Following buffer rinse, samples were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate for 1 hr on ice in the dark. After a brief distilled H~2~O rinse samples were placed in 2% aqueous uranyl acetate (0.22 um filtered) for 1 hour at room temperature in the dark. Following en-bloc staining, cells were dehydrated through a graded series of ethanol to 100% ethanol, transferred through propylene oxide, embedded in Spurr's low viscosity resin (Polysciences), and cured at 60°C for two days. Sections were cut on a Riechert Ultracut E with a Diatome Diamond knife. Eighty nm sections were picked up on formvar coated 1 × 2 mm copper slot grids and stained with uranyl acetate followed by lead citrate. Grids were viewed on a Hitachi 7600 TEM operating at 80 kV and digital images captured with an AMT 1 K × 1 K CCD camera. Competing interests =================== The authors declare no competing interests. Authors' contributions ====================== KABL, KMW, AB, SD, TL, and MG carried out the whole genome sequencing. KABL, PEC, AA, TDR, and SSo conducted nextgen and Sanger sequence analysis. RDP and SSt carried out deletion and complementation studies. CC performed primer design and microbiological work. AM, VM, and RC participated in the study's design and coordination and helped draft the manuscript. SSo conceived of the study, participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ###### Additional file 1 Figure S1. Sequence alignment of csaB and orthologs. BAS0840 was aligned with the Pfam PF04230 seed alignment and percent conservation was determined as described in Methods. ###### Click here for file ###### Additional file 2 Table S1. List of primers used in this study. ^a^ Restriction sites are underlined. ###### Click here for file Acknowledgments and disclaimers =============================== The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. The BDRD, NMRC authors are employees of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. Â§105 provides that "Copyright protection under this title is not available for any work of the United States Government." Title 17 U.S.C. Â§101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person\'s official duties. We would like to thank Michael J Delannoy (JHU EM facility) for help with the electron microscopy.	{ "pile_set_name": "PubMed Central" }	[ "peptidoglycanassoci", "treatment", "pnv", "roch", "cleavag", "probabl", "discov", "two", "cereu", "ab", "control", "viabil", "refer", "given", "work", "origin", "adsorb", "remain", "defens", "phage", "eg", "referenceguid", "overnight", "ie", "phagesensit", "thank", "timeconsum", "dalton", "lysogen", "gene", "put", "identifi", "length", "charg", "block", "indicatedxf", "retain", "select", "apc", "mapper", "mechan", "gamr", "hypoxanthineguanin", "take", "indirect", "hour", "narrow", "fix", "lysi", "locat", "enter", "multipl", "signal", "may", "id", "necessarili", "therebi", "readili", "attack", "recent", "pbkj", "γ", "q", "suspect", "acquir", "capsular", "ea", "use", "gener", "graph", "sciencesroch", "copyright", "forward", "cat", "score", "final", "support", "symbol", "focus", "bottom", "room", "caus", "complet", "fairli", "atcc", "imag", "prior", 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1. Case history {#sec1-1} =============== A 45-year-old married Hindu female, non-smoker, non hypertensive and non diabetic, was diagnosed with paranoid schizophrenia, for which she was maintaining well on tablet haloperidol 20 mg/day for the last 5 years. The total duration of illness was around 7 years, with a drug naive period of approximately 2 years. Around three months ago, the patient's symptoms had become exaggerated as she had not taken her antipsychotic medication for approximately 8-10 days because she was out of town and did not have her medicines with her. Her complaints were sleep disturbance, referential and persecutory ideas with intermittent auditory hallucinations and episodes of irritability with aggressive behavior. The patient presented to the hospital after which haloperidol was started again at 10 mg/day, increased to 20 mg/day and maintained for the next month. However no significant improvement was reported this time using haloperidol. Olanzapine 5 mg/day was added and increased to 10 mg/day within 2 weeks. One month after adding Olanzapine, the patient developed symptoms of nausea, anorexia and weakness. However there were no episodes of vomiting or diarrhea. Also, there was no significant decrement in diet. The symptoms exacerbated over the next 2 weeks along with muscle cramps, unsteady gait and fluctuating orientation. The patient was admitted to emergency services for evaluation. Her physical examination revealed tachycardia (pulse - 118/min), low blood pressure (94/60 mm hg), dehydration, and disorientation to time. Her systemic examination was unremarkable. Her blood tests revealed low serum sodium of 120 mmol/l (135-150 mmol/l). The results of the remaining tests were within normal limits (including renal function tests, liver function tests, serum concentrations of potassium, blood sugar, total proteins and lipids). Treatment was started on the diagnosis of probable normovolemic hyponatremia as physical examination did not reveal any signs of fluid overload. The electrocardiography revealed only sinus tachycardia and plasma osmolarity was reported to be 288 mOsm/kg H~2~O (285--295mOsm/kgH~2~O). She was started on 3% NaCl infusion over the initial few hours until achieving full consciousness followed by normal saline. Fluid restriction and oral salt supplement was advised. The patient recovered in 2 days with serum sodium levels of 140 mmol/l. However, the exact reason for hyponatremia could not be established even after detailed investigations (including thyroid function tests). Olanzapine continued to be administered, except for the day emergency treatment was given. Upon detailed assessment, the patient's diet was found to be consistently good over these years, and even during the last 2 weeks. No specific diet/salt restriction was reported. A week later, the patient again had similar episode of hyponatremia (serum sodium of 116 mmol/l) while continuing on the antipsychotics. She had to be managed in the emergency room with similar procedures and protocols. With no evident organic cause for the hyponatremia, a literature search was carried out and Olanzapine was postulated as a potential agent. It was planned to stop Olanzapine and observe the patient, while continuing medical management for hyponatremia. Within 3 days of stopping Olanzapine, the patient improved and never had a recurrence of hyponatremia over the next 6 months follow up. She was maintained on haloperidol 20 mg/day and risperidone 4 mg/day thereafter. Naranjo algorithm^\[[@ref1]\]^ established a "probable" causal relation between the drug and adverse event, with a total score of 8 (suggestive of a probable relationship). 2. Discussion {#sec1-2} ============= Olanzapine is a commonly used atypical antipsychotic for patients with schizophrenia and other psychotic disorders. In the CATIE study (Clinical Antipsychotic Trials of Intervention Effectiveness), Olanzapine was found to have the lowest discontinuation rate, possibly due to fewer incidences of acute side effects.^\[[@ref2]\]^ Still, life threatening adverse effects such as hyponatremia may limit its safe and effective use. A systematic review of evidence for antipsychotic induced hyponatremia from 1974 to 2003, found more cases of hyponatremia with typical compared to atypical antipsychotics.^\[[@ref3]\]^ Hyponatremia is the commonest electrolyte imbalance encountered in medical emergencies.^\[[@ref4]\]^ It is characterized by a plasma sodium concentration less than 135 mEq/L^\[[@ref5]\]^ and manifests clinically with neurological and systemic symptoms such as nausea, vomiting, anorexia, headache, disorientation, confusion, irritability and lethargy.^\[[@ref6]\]^ Profound hyponatremia may manifest as severe mental status changes including confusion, delirium, seizure, coma, and death. The most common causes of hyponatremia are fluid loss and dehydration, congestive heart failure, liver and renal failure, and Syndrome of inappropriate anti-diuretic hormone. Hyponatremia as a potential adverse event from the use of antipsychotics has not been greatly researched. Only a few case reports/series are available.^\[[@ref7],[@ref8]\]^ Meulendijks and colleagues did a systematic review of evidence for antipsychotic induced hyponatremia from 1974 to 2003 and found that both typical (58 case reports) and atypical antipsychotics (10 case reports) were implicated in hyponatremia and the association was independent of age or gender or dose.^\[[@ref3]\]^ Many antipsychotics (both typical and atypical) like chlorpromazine, fluphenazine, haloperidol, trifluoperazine, amisulpride, olanzapine and risperidone have been implicated.^\[[@ref3],[@ref9]\]^ Hyponatremia is postulated to be caused by blockade of inhibitory effect of dopamine on release of anti-diuretic hormone (ADH) by antipsychotics.^\[[@ref10]\]^ Clozapine, because of its low affinity for D2 receptors, is less prone to causing hyponatremia and may improve associated polydipsia. However on the contrary, it has been demonstrated in rats that dopamine could have a stimulatory effect on ADH release.^\[[@ref11]\]^ Therefore, the exact mechanism of antipsychotic-induced hyponatremia is still unclear. In the index case, hyponatremia and olanzapine were temporally associated, still it is difficult to establish the exact offending medication. Haloperidol is also a known culprit that is described in some case reports. However, haloperidol alone did not produce hyponatremia nor did it in combination with risperidone suggesting that olanzapine may have a stronger causal relation with hyponatremia. Also, Naranjo algorithm established a "probable" causal relationship with olanzapine. Another possible mechanism is that olanzapine precipitated hyponatremia in a predisposed individual taking haloperidol, and the causality may be attributed to the combined effect of both olanzapine and haloperidol. Hyponatremia is a rare yet clinically important adverse reaction to treatment with psychotropic drugs. It is a potentially dangerous medical complication in psychiatric patients. Psychogenic polydipsia is an important differential diagnosis, as it is characterized by similar symptoms of polydipsia and polyuria. Also, psychogenic polydipsia is commonly found in psychiatric patients, more likely in schizophrenia.^\[[@ref12]\]^ Multiple other factors such as diet, salt intake, ageing comorbid conditions (smoking, diabetes) or concurrent medications may also play a role and should be considered.^\[[@ref13]\]^ There is a complete and rapid recovery of drug induced hyponatremia once the offending agent is discontinued, as happened with the index case, without any further recurrences. Clinicians need to be aware of this dangerous complication and exercise caution when prescribing psychotropic drugs. Funding statement No funding support was obtained for preparing this case report. Conflict of interest statement The authors declare that they have no conflict of interest related to this manuscript Informed consent The patient and his guardian signed an informed consent form and agreed to the publication of this case report. ![](sap-29-177-g001.gif) Dr. Ankur Sachdeva obtained his MBBS degree from Vardhman Mahavir Medical College and Safdarjang Hospital, New Delhi in 2009 and an MD (Psychiatry) degree from the Post Graduate Institute of Medical Education and Research, at the Dr. Ram Manohar Lohia Hospital in 2013. He subsequently received the Diplomate of the National Board (D.N.B) in psychiatry in 2014. He is presently working as Assistant Professor (Psychiatry) in ESIC medical college, Faridabad, Haryana, India.	{ "pile_set_name": "PubMed Central" }	[ "treatment", "probabl", "given", "work", "remain", "disorient", "fund", "mechan", "intermitt", "take", "week", "hour", "antipsychot", "multipl", "may", "potassium", "imbal", "normovolem", "use", "room", "score", "supplement", "support", "caus", "complet", "intak", "signific", "lipid", "establish", "hindu", "full", "becom", "suggest", "ram", "recov", "happen", "also", "intervent", "difficult", "conflict", "antidiuret", "clozapin", "normal", "syndrom", "medic", "aggress", "nation", "even", "organ", "safe", "tempor", "assist", "pressur", "assess", "achiev", "author", "anorexia", "weak", "common", "physic", "profound", "compar", "dr", "servic", "carri", "time", "protein", "initi", "post", "osmolar", "culprit", "found", "confus", "ankur", "consideredref", "ago", "vomit", "detail", "gender", "india", "potenti", "known", "statu", "mahavir", 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1. Introduction {#sec1} =============== South Africa\'s HIV epidemic remains the largest in the world, with an estimated 5.6 million people living with HIV in 2009 \[[@B1]\]. Young people in particular have the fastest-growing infection rates \[[@B2], [@B3]\]. They are expected to delay sexual activity and avoid unplanned pregnancy and sexually transmitted infection to safeguard their sexual health \[[@B4]\]. Yet, the complexity of sexual behaviour has been underscored \[[@B5]\] given that HIV risk behaviour is influenced by various factors. Condom use and negotiation of safer sex are reported to be the most effective means of HIV risk reduction \[[@B6]\]. Often, young people struggle to exert control over their emotions when they engage in sexual activity \[[@B7]\], resulting in risky sexual behaviours that lead to HIV infection. The concept of self-efficacy is defined as having confidence in one\'s ability to perform a particular behavior, and it has been regarded as an important component of health-related behavioral change \[[@B7]\]. The AIDS risk reduction model in which self-efficacy plays an important role \[[@B8]\] emphasize three stages where a young person has knowledge about a particular safer sex behavior (i.e., using condoms), they then have to think that the behavior is socially acceptable (norms/attitudes) and to believe that they would be able to practice the behavior (self-efficacy) before they actually engage in the behavior \[[@B8]\]. Studies of HIV risk behaviours and sexual health have consistently shown that high self-efficacy for condom use is strongly associated with behaviours of condom use with recent partners and consistent use \[[@B6]\]. A study conducted among young people age 15--24 years reported very high levels of self-efficacy when it came to believing that they could discuss condoms with their partners and refusing sex when they did not want to, but this was not always matched with their actual behaviour \[[@B9], [@B10]\]. Even though a decline in HIV prevalence has been reported among South African youth 15--24 from 10.3% in 2005 to 8.6% in 2008, the prevalence remains disproportionately high for females overall in comparison to males \[[@B2]\]. The burden of HIV on women, however, varies considerably by region but is heaviest in sub-Saharan Africa with 1.4 times more adult women than men who were living with HIV in 2010 \[[@B1]\]. In South Africa, for example, young females have 3 to 4 times the prevalence of HIV than their male peers \[[@B1]\]. Thus, it is crucial to take into account the gender differences of HIV prevalence \[[@B11]\] and HIV risk behaviours. There is still a dearth of studies that investigate factors that may influence HIV risk reduction self-efficacy by gender. Therefore, this study examines factors associated with HIV risk reduction self-efficacy among South African youth age 15--24 years as part of an evaluation of the impact of loveLife, a youth focused HIV prevention programme. 2. Method {#sec2} ========= 2.1. Sample and Procedures {#sec2.1} -------------------------- A cross-sectional population-based household survey was conducted using a multi-stage stratified cluster sampling approach. In each household all eligible household members were invited to participate and interviewed. The survey included persons of ages 18 to 24 years living in South African households of the four (out of nine) selected provinces, KwaZulu-Natal, Mpumalanga, Eastern Cape and Gauteng Province, providing an urban-rural representation of South Africa. The selection of the provinces was guided by selecting two provinces with the highest HIV prevalence in the country, KwaZulu-Natal and Mpumalanga, and one most urban province (Gauteng) and one rural province (Eastern Cape). Ethical approval for the study was obtained from the HSRC Research Ethics Committee. Participants signed informed consent forms. 2.2. Measures {#sec2.2} ------------- Age, gender, educational and employment status were assessed. Contraceptive knowledge was assessed with 7 items, for example, "Have you heard about the Pill that women can take every day to avoid becoming pregnant?" Response options were "Yes," or "No." Cronbach alpha for this contraception knowledge index was 0.70 in this sample. HIV knowledge was assessed with two items: (1) How many people living with HIV do you personally know? And (2) How many people have you personally known (in your lifetime) that have died from AIDS? Responses of the two questions were added up and coded as 0= 0, 1= knows any person living with HIV (PLHIV) or who has died from AIDS, and 2= knows any PLHIV and a person who has died from AIDS. Cronbach alpha for this 2-item HIV knowledge index was 0.73 in this sample. "Has goals in life" was assessed with 6 items such as "I have a plan for the future"; response options were agree or disagree. "Has goals in life" was classified as those who indicated to all 6 items to have any goals. Cronbach alpha for this "goals in life" index was 0.63 in this sample. Partner risk reduction self-efficacy was assessed with 4 items such as "Would you be able to avoid sex any time you did not want it?" Response options were no, probably no, probably yes, yes. Cronbach alpha for this partner risk reduction self-efficacy index was 0.73 in this sample. Peer pressure was assessed with 2 items; (1) "How much pressure you get from your friends to have sexual intercourse, would you say...?" (Response options ranged from 1: "No pressure at all" to 4: "A lot of pressure") and (2) "I feel pressure from friends to do things I do not want to." (Response options ranged from 1: "Very often" to 4: "Never"). Low peer pressure was coded 2, medium 3--5, and high 6--8. Cronbach alpha for this peer pressure index was 0.61 in this sample. Poverty was assessed with 6 items on the availability or nonavailability of shelter, fuel or electricity, clean water, medicines or medical treatment, food, and cash income in the past week. Response options ranged from 1: "Not one day" to 4: "Every day of the week" Poverty was defined as higher scores on non-availability of essential items. Cronbach alpha for this poverty index was 0.70 in this sample. HIV communication was assessed with 10 items, specifying 10 different source persons or institutions, for example, "Have you ever talked to your mother or female primary care giver/guardian about HIV/AIDS issues" Responses options were 1: Yes or 2: No. Cronbach alpha for this HIV communication index was 0.75 in this sample. Further, 1 item assessed "Did you talk about using a condom with your latest sexual partner in the last 12 months?" Responses options were 1: Yes or 2: No. Accessibility of condoms was assessed with the question, "How easy is it for you to get condoms if you needed or wanted them?" Response options ranged from 1: very easy to 4: very difficult. Relationship controlwas assessed with 4 items (for those never in a relationship, they were asked imagine to be) such as "Your partner has more control than you do in important decisions that affect your relationship." Response options ranged from 1: "Strongly disagree" to 4: "Strongly agree." Higher scores on lack of relationship power were defined as lack of relationship control. Cronbach alpha for this relationship control index was 0.81 in this sample. Risk behavior various questions were asked to assess risk behaviour. These included number of lifetime transactional sexual partners, early sexual debut (below 15 years, ever forced to have sex, concurrent sexual relationships, sex with someone who is much older, sexual intercourse frequency, and length of last relationship). Alcohol use was assessed with the Alcohol Use Disorder Identification Test (AUDIT-C) questionnaire \[[@B12]\], a measure of consumption of alcohol (i.e., the frequency of drinking, the quantity consumed at a typical occasion), and the frequency of heavy episodic drinking (i.e., consumption of five standard drinks, 60 gram alcohol, or more on a single occasion). Because AUDIT is reported to be less sensitive at identifying risk drinking in women \[[@B13]\], the cutoff points of binge drinking for women (4 units) were reduced by one unit as compared with men (5 units), as recommended by Bush et al. \[[@B14]\]. Using a cut off score of 5 or more hazardous or harmful drinking was defined \[[@B15], [@B16]\]. Cronbach alpha reliability coefficient was in this study for the AUDIT-C 0.91. HIV status was assessed by self-report. Prevention programme exposure was assessed with the following items. Exposure to ever loveLife face to face programmes was assessed with 24 items, for example, Gone to a loveLife clinic, Participated in a loveLife Community Dialogue, or Gone to a loveLife Youth Centre. LoveLife exposure to face-to-face programmes was summed up and coded as 0, 1-2, 3-4, or 5 or more programme exposures. Further, longer-term participation was assessed with having participated in loveLife programmes for at least one year. In addition, loveLife multi-media exposure was assessed with 9 items, for example, "Have you ever watched a loveLife television show?" "Contacted loveLife on facebook" "Heard a loveLife advert on radio." "Read UNCUT (loveLife) youth magazine." Response options were 1: Yes or 2: No. The 9 multi-media programmes were summed up and coded as 1: 0-1 media exposures, 2: 2--4, and 3: 5--9 media exposures. 2.3. Data Analysis {#sec2.3} ------------------ Both descriptive and inferential statistics were applied in data analysis. Sample weights were calculated after editing the data, and STATA software was used for the analysis. STATA software (svy methods) was used to obtain the estimates of key indicators, significance values (P values), and confidence intervals (95% CI) that take into account the complex design and individual sample weights. Computed estimates and odds ratios are reported with 95% confidence intervals and a two-side P value of 0.05 used as the cut-off point for statistical significance. Data were checked for normality distribution and outliers. For nonnormal distribution nonparametric tests were used. The Chi-square test was used for comparing nominal variables. Logistic regression analysis was used to study the association between key outcomes (high self-efficacy) and predictor variables. All variables statistically significant at the P \< .05 level in bivariate analyses were included in the multivariable models. 3. Results {#sec3} ========== 3.1. Survey Response Rate {#sec3.1} ------------------------- A total of 5 768 households were sampled and approached for the interview. Only 94.8% households were valid and among the valid households 93.6% agreed to be interviewed. Only households that indicated that they had a person aged 18 to 24 years were eligible for an individual questionnaire administration. Of the eligible and valid households 47.2% were eligible for an individual interview, 1.3% refused the individual interview, and 2.3% individuals were absent from the household so that the individual interview response rate was 96.4%. 3.2. Sample Characteristics {#sec3.2} --------------------------- The total samples of young people in the study were 3123, aged 18--24, from four of nine provinces (Eastern Cape, Gauteng, KwaZulu-Natal, and Mpumalanga) in South Africa. From the total sample 1127 and 1007, men and women, respectively, reported to have ever had sex. Overall, among those who had been tested for HIV and indicated their test result 9.3% of the women and 6.2% of the men were HIV positive. Youth programme exposure was assessed in terms of face-to-face participation and media programme exposure, 32.6% had participated in one or more loveLife face-to-face programmes, while more than 80% had been exposed to 2 or more loveLife HIV prevention media programmes (see [Table 1](#tab1){ref-type="table"}). 3.3. Self-Efficacy and Condom Use Consistency in South African Females and Males {#sec3.3} -------------------------------------------------------------------------------- Among males who indicated that they were not able to avoid sex any time they did not want it, 56.5% use condoms consistently. Males who indicated that they are able to avoid sex any time they did not want it, 58.9% use condoms consistently. These findings were statistical significant. For women though, no statistical significance was reported when asked "would you be able to avoid sex any time you did not want it." When asked "would you be able to talk about using condoms with your partner?", of those women who answered no, only 26% are using condoms consistently (versus 41.8% of males) (see [Table 2](#tab2){ref-type="table"}). Generally, across the self-efficacy scale items, more males than females were more likely to use condoms consistently. 3.4. Predictors of Self-Efficacy {#sec3.4} -------------------------------- Univariate analysis found that for males, having been tested for HIV, concurrent sexual partnerships, transactional sex partner, low HIV risk perception, Talked to partner about condom use in the past 12 months, difficulty in getting condoms, acceptable to have coerced sex, high relational control, current tobacco, ever drugs, and participating in few face-face loveLife programmes (1-2) to be associated with high self-esteem. In the case of females, ever forced to have sex, concurrent sexual partnerships, transactional sex partner in lifetime, low HIV risk perception, talked to partner about condom use in the past 12 months, high peer pressure, stigma, ever drugs and having life goals were associated with high self-efficacy in univariate analysis. Multivariate analysis found the following variables to be significantly associated with high self-efficacy for males: having been tested for HIV, concurrent sexual partners, transactional sex partners in life time, low HIV risk perception, difficulty of getting condoms, acceptable to have coerced sex, high relationship control, and participating in 1-2 loveLife face-face programmes. With regards to females, low HIV risk perception, HIV/AIDS stigma, ever drugs and having life goals were associated with high self-efficacy in multivariate analysis (see [Table 3](#tab3){ref-type="table"}). 4. Discussion {#sec4} ============= The findings indicate that, for males, high self-efficacy is significantly associated with knowing your HIV status but only half of the men reported ever testing for HIV. In the case of females, the present study found a significant association between high self-efficacy and having life goals. This finding is similar with a study done among South African youth 15 to 24 years that also found high self-efficacy to be associated with having life goals but in this study it was reported for both young men and women \[[@B6]\]. A significant association was also found for young males finding it acceptable to have coerced sex. This probably reflects the higher percentage reported by females than their male counterparts (7.3% versus 1.1%) ever being forced to have sex against their will. This reflects some sense of disempowerment of young females in preventing themselves against HIV infection \[[@B17]\]. As a result it perpetuates social and cultural forces that shape young people\'s sexual behaviour \[[@B18]\]. Indeed, Varga \[[@B19]\] found that young men seemed to have more freedom to be involved with more than one sexual partner at a time while young women are expected to be faithful to their (one) partner. Thus Varga in her study found that a young man involved with multiple sexual partners was regarded as "isoka" (the act of "ubusoka") to indicate the young man\'s popularity with women something that seemed to be desirable while a young woman with more than one sexual partner was perceived as lacking self-respect \[[@B19]\]. Jewkes et al. \[[@B17]\] would have argued that HIV prevention programmes which emphasize male condoms and male circumcision may further perpetrate young men\'s freedom in sexual decision making, thus overlooking the vulnerability of young women. This brings into question the effectiveness of existing risk reduction programmes that specifically focus on risk reduction through behavioural interventions for adolescents. Ross \[[@B20]\] paints a rather bleak picture of the efficacy of behavioural intervention. In an update of a recent systematic review of HIV prevention interventions focusing on trials that have included HIV as an outcome, Ross \[[@B20]\] noted that the behavioural interventions conducted through trials were disappointing in terms of the impact on HIV even though some trials showed an impact on other sexual and reproductive health outcomes. The interventions tested were inherently ineffective partly because they were inadequately implemented, or there were problems with the measurement of effectiveness. Perhaps, particular attention needs to be given to the social and sexual norms of the general population among whom the particular target group in this case, young people, live and interact \[[@B20]\]. The present study found that, for males, there was a significant association between high self-efficacy and having concurrent sexual partners as well as having had a transactional sex partner in lifetime. Of grave concern is that high self-efficacy was also significantly associated with those males having difficulty in getting condoms. This is an indication that, even though males reported higher consistent condom use than females, many young men still engage in unprotected sex thus putting themselves and their partners at risk. Varga \[[@B21]\] noted that a predominant theme has been the powerful positive symbolism attached to unprotected sex and negative connotations of condom use. Many women feel stigmatized when insisting on using a condom during sexual intercourse as this is a sign of distrust in their partner \[[@B6]\] or an indication that they are HIV positive. A review done by Marston and King \[[@B18]\] on identifying key themes from various qualitative findings highlighted one theme that specifically relates to this aspect; "condoms are stigmatizing and associated with lack of trust" \[[@B18]\]. Despite an increase in awareness of HIV status in the general population between 15 and 49 year old doubling from 11.9% in 2005 to 24.7% in 2008 \[[@B11]\], unprotected sex with intimate personal partners is nearly always chosen over condom use \[[@B21]\]. A partial explanation for the sexual dynamics perhaps lies in a sociocultural context which has not fostered sexual negotiation skills. A renewed focus on programmes related to self-efficacy programmes in young people is urgently needed. Given the significant finding that amongst males, high self-efficacy is associated with exposure to at least 1 to 2 face-to-face loveLife programmes, evidence do exist that similar intervention strategies focusing on self-efficacy has a positive effect. The results further confirm loveLife\'s emphasis on normative and social factors as being one of the issues that young people have to contend with in their constructions of sexuality, and this seems to be particularly true of young women \[[@B22], [@B23]\]. For instance, more young women report having had sex with a much older partner. This is consistent with the findings from findings in the general population survey \[[@B11]\] where young people reported having partners five or more years older than themselves, and there was a substantive increase from 9.6% in 2005 to 14.5% in 2008. This makes them more vulnerable to HIV infection because they find it difficult to negotiate sex with their older partner \[[@B24]\]. If indeed young women\'s sexual partnerships with older men are solely aimed at economical gain, initiatives aimed at young women\'s economic emancipation need further support. 5. Limitations {#sec5} ============== One of the limitations of that study was that it was a cross-sectional study. Furthermore, it was only done in four provinces; thus, the results cannot be generalized. This paper was a collaborative project between loveLife and the Human Sciences Research Council and was made possible by the Henry J. Kaiser Family Foundation. The authors would like to acknowledge loveLife and the anonymous review committee for their comments and suggestions. They would also like to thank the research participants who took part in the study. ###### Individual, social, structural, contraception, and prevention programme exposure of study sample by gender. ---------------------------------------------------------------------------------------------------------------------------------------------------------------- Male (N = 1127) Female (N = 1007) -------------------------------------------------------------------------------------------------------------------- --------------------- --------------------- Sociodemographics N (%) or M (SD) N (%) or M (SD) Poverty index (range 6--24) 8.3 (3.0) 7.9 (2.6) ≤Grade 10 218 (14.7) 171 (18.0) Grade 11 233 (17.3) 201 (18.5) Grade 12 or more 670 (68.0) 632 (63.5) Student 459 (42.3) 361 (44.0) Employed 217 (23.0) 114 (11.1) Unemployed 370 (34.8) 440 (45.0) Knowledge Contraceptive knowledge (range 0--7) 4.7 (1.6) 5.1 (1.5) Knows person living with HIV and/or died from AIDS 0 393 (40.0) 272 (36.3) 1 = knows PLHIV or died from AIDS 252 (23.5) 215 (18.2) 2 = knows PLHIV and died from AIDS 475 (36.6) 514 (45.5) Ever HIV test 556 (48.9) 779 (80.4) Prior sexual experiences Early sex (\<15) 145 (17.8) 65 (6.9) Ever forced sex 14 (1.1) 58 (7.3) Sex with much older partner 70 (4.3) 232 (19.3) Concurrent sexual partners 126 (6.8) 53 (3.1) Transactional sex partner in lifetime 103 (7.8) 50 (5.0) Outcome expectancies Low HIV risk perception 294 (23.6) 354 (30.2) Believes that condom use is a sign of not trusting the partner 259 (15.9) 229 (17.5) Sociostructural factors Peer pressure Low 350 (41.0) 465 (54.6) Medium 447 (42.3) 398 (37.5) High 206 (16.6) 121 (7.9) Talked with partner about condoms in past 12 months 820 (94.1) 769 (90.7) HIV communication (range 0--10) 5.6 (2.5) 5.6 (2.2) HIV/AIDS stigma 118 (5.5) 75 (3.8) Difficulty of getting condoms (range 1--4) 1.22 (0.6) 1.13 (0.5) Lack of relationship control (range 4--16) 8.5 (2.3) 8.3 (2.5) Agreed with statement "It is acceptable to have sex with my sex partner even though my partner does not want to." 60 (4.0) 43 (2.9) Hazardous or harmful alcohol use 430 (34.0) 160 (16.4) Ever drug use 162 (25.4) 32 (2.6) Current tobacco use 363 (25.4) 69 (5.6) Has goals in life 728 (72.4) 628 (63.6) HIV status Diagnosed HIV positive\ 35 (6.7)\ 64 (9.5)\ Diagnosed HIV negative 493 (93.3) 654 (90.5) HIV risk reduction self-efficacy 462 (52.7) 488 (56.6) Programme exposure One year or more loveLife participation 315 (22.4) 250 (21.5) loveLife face-to-face participation 0 700 (70.4) 639 (62.6) 2--4 219 (12.2) 193 (22.1) 5--9 100 (8.0) 71 (4.3) 5 or more 98 (9.3) 101 (11.0) loveLife multimedia programme exposure 0-1 174 (18.1) 180 (20.7) 2--4 577 (50.8) 458 (48.2) 5--9 340 (31.1) 331 (31.0) ---------------------------------------------------------------------------------------------------------------------------------------------------------------- ###### Self-efficacy scale responses and condom use consistency. -------------------------------------------------------------------------------------------------------------------------------------- Men Women -------------------------------------------------------------------------------- ----- ------- ------- ------- ------- ------- ------- Would you be able to avoid sex any time you did not want it? No\ 43.5\ 56.5\ 0.000 57.9\ 42.1\ 0.352 Yes 41.1 58.9 54.4 45.6 Would you be able to use a condom every time you have sexual intercourse? No\ 46.1\ 53.9\ 0.000 66.8\ 33.2\ 0.000 Yes 40.4 59.6 51.4 48.6 Would you be able to refuse to have sex if your partner will not use a condom? No\ 47.3\ 52.7\ 0.000 65.7\ 34.3\ 0.000 Yes 38.4 61.6 49.8 50.2 Would you be able to talk about using condoms with your partner? No\ 58.2\ 41.8\ 0.000 74.0\ 26.0\ 0.000 Yes 32.2 67.8 44.0 56.0 -------------------------------------------------------------------------------------------------------------------------------------- ^∗^Scale items response options were No, Probably No, Probably Yes, Yes; "No" was coded "No, Probably No, Probably Yes." ###### Association between individual, social and structural variables, loveLife exposure, and high self-efficacy. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Men Women --------------------------------------------------------------------------------------------------------------------- ------------------------ ------------------------ ------------------------ ----------------------- Sociodemographics Education 1.02 (0.84--1.24) 1.07 (0.78--1.46) 1.27 (0.99--1.62) 1.16 (0.82--1.64) Student 1.00 1.00 1.00 1.00 Employed 2.35 (0.79--6.96) 2.03 (0.62--6.01) 0.75 (0.35--1.58) 0.66 (0.22--1.95) Unemployed 0.72 (0.49--1.07) 0.67 (0.33--1.37) 1.13 (0.59--2.18) 1.33 (0.60--2.94) Poverty index 1.04 (0.95--1.13) 1.11 (0.99--1.25) 1.06 (0.86--1.31) 1.15 (0.91--1.45) Knowledge Knows person living with\ HIV and/or died from AIDS 0 1.00 --- 1.00 --- 1 = knows PLHIV or died from AIDS 0.41 (0.12--1.40) 0.97 (0.54--1.77) 2 = knows PLHIV and died from AIDS 0.72 (0.45--1.15) 0.72 (0.37--1.39) Have been tested for HIV ever? 2.38 (1.41--4.02)^∗∗∗^ 2.54 (1.38--4.67)^∗∗^ 0.88 (0.55--1.40) --- Contraceptive knowledge 1.11 (0.75--1.65) --- 1.01 (0.90--1.13) --- Prior sexual experiences Early sex (\<15) 2.72 (0.72--10.2) --- 0.94 (0.36--2.44) --- Ever forced sex 0.62 (0.10--3.91) --- 0.31 (0.11--0.86)^∗^ 0.27 (0.03--2.03) Concurrent sexual partners 0.23 (0.12--0.47)^∗∗∗^ 0.28 (0.12--0.65)^∗∗^ 0.30 (0.11--0.84)^∗^ 0.49 (0.16--1.51) Sex with much older partner 0.34 (0.11--1.12) --- 0.72 (0.45--1.17) --- Transactional sex partner in lifetime 0.10 (0.05--0.20)^∗∗∗^ 0.10 (0.03--0.29)^∗∗∗^ 0.09 (0.03--0.27)^∗∗∗^ 0.70 (0.17--2.83) Outcome expectancies Low HIV risk perception 1.41 (1.13--1.76)^∗∗^ 1.45 (1.04--2.02)^∗^ 1.85 (1.47--2.34)^∗∗∗^ 2.26 (1.25--4.09)^∗∗^ Beliefs condom use is a sign of not trusting\ 0.79 (0.42--1.46) --- 0.66 (0.36--1.20) --- the partner Sociostructural factors Talked to partner about condom use in the past 12 months 2.50 (1.25--5.00)^∗∗^ 1.21 (0.46--2.75) 2.93 (1.55--5.53)^∗∗∗^ HIV communication 1.07 (0.89--1.30) --- 0.97 (0.83--1.14) --- Peer pressure Low 1.00 --- 1.00 1.00 Medium 0.35 (0.14--0.87) 0.68 (0.40--1.15) 0.93 (0.51--1.67) High 0.23 (0.05--1.13) 0.28 (0.14--0.55)^∗∗∗^ 0.89 (0.33--2.30) Difficulty of getting condoms 0.60 (0.37--0.97)^∗^ 0.60 (0.38--0.98)^∗^ 0.81 (0.63--1.03) --- Agreed with statement "It is acceptable to have sex with my sex partner even though my partner does not want to."\ 0.23 (0.08--0.67)^∗∗^ 0.26 (0.07--0.98)^∗^ 0.76 (0.23--2.49) --- (Acceptable to have coerced sex) HIV/AIDS stigma 0.68 (0.25--1.82) --- 0.24 (0.11--0.50)^∗∗∗^ 0.20 (0.06--0.64)^∗∗^ High relationship control 0.46 (0.27--0.79)^∗∗^ 0.24 (0.10--0.58)^∗∗^ 1.10 (0.72--1.69) --- Hazardous or harmful drinking (AUDIT \> 4) 0.70 (0.44--1.14) --- 0.70 (0.21--2.33) --- Current tobacco use 0.48 (0.27--0.87)^∗^ 0.58 (0.26--1.28) 0.60 (0.13--2.64) --- Ever drugs 0.35 (0.18--0.66)^∗∗∗^ 0.45 (0.17--1.31) 0.10 (0.03--0.39)^∗∗∗^ 0.12 (0.02--0.74)^∗^ Goals Has life goals 2.78 (0.98--7.86) --- 1.85 (1.16--2.96)^∗^ 2.98 (1.16--7.63)^∗^ Programme exposure One year or more loveLife participation 0.85 (0.43--1.71) --- 1.15 (0.63--2.08) --- loveLife face-to-face participation 0 1.00 1.00 1.00 --- 1-2 0.42 (0.22--0.81)^∗∗^ 0.38 (0.16--0.89)^∗^ 0.48 (0.14--1.72) 3-4 0.87 (0.30--2.47) 0.50 (0.22--1.13) 0.62 (0.22--1.74) 5 or more 1.70 (0.47--6.20) 2.11 (0.46--9.59) 0.81 (0.28--2.33) loveLife multimedia programme exposure 0-1 1.00 --- 1.00 --- 2--4 0.88 (0.40--1.90) 0.63 (0.34--1.17) 5--9 0.70 (0.37--1.34) 1.33 (0.54--3.25) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ^∗∗∗^ P \< .001; ^∗∗^ P \< .01; ^∗^ P \< .05. [^1]: Academic Editors: K. Ikuta and J. 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"associated", "life", "goals", "study", "reported", "young", "men", "women", "significant", "association", "also", "found", "young", "males", "finding", "acceptable", "coerced", "sex", "probably", "reflects", "higher", "percentage", "reported", "females", "male", "counterparts", "versus", "ever", "forced", "sex", "reflects", "sense", "disempowerment", "young", "females", "preventing", "HIV", "infection", "result", "perpetuates", "social", "cultural", "forces", "shape", "young", "sexual", "behaviour", "Indeed", "Varga", "found", "young", "men", "seemed", "freedom", "involved", "one", "sexual", "partner", "time", "young", "women", "expected", "faithful", "one", "partner", "Thus", "Varga", "study", "found", "young", "man", "involved", "multiple", "sexual", "partners", "regarded", "isoka", "act", "ubusoka", "indicate", "young", "popularity", "women", "something", "seemed", "desirable", "young", "woman", "one", "sexual", "partner", "perceived", "lacking", "Jewkes", "et", "al", "would", 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Introduction {#s1} ============ The nuclear envelope (NE) delineates the boundary of the nucleus in eukaryotic cells and is composed of two membranes enclosing a luminal space. Because chromosomes are located in the nucleus, cells must enable the access of spindle microtubules to the nucleoplasm in order to segregate the daughter genomes during mitosis. Cells undergoing open mitosis achieve this by breaking down the NE in prophase and reassembling it again in telophase. Alternatively, a number of species segregate chromosomes within the nucleus in the process known as closed mitosis. Like most fungi, the fission yeast Schizosaccharomyces pombe elaborates the bipolar intranuclear mitotic spindle with the minus ends of microtubules anchored at specialized microtubule-organizing organelles, the spindle pole bodies (SPBs). The SPBs are functionally analogous to metazoan centrosomes and undergo a duplication and separation cycle generally correlated with the cell division cycle. The SPB of S. pombe spends most of interphase in the cytoplasm, adjacent to the NE \[[@pbio-0050170-b001],[@pbio-0050170-b002]\]. However, it appears to be securely anchored at the outer NE as suggested by live-imaging studies of the SPB and the NE behavior in interphase \[[@pbio-0050170-b003],[@pbio-0050170-b004]\]. As the SPB duplicates and matures, daughter SPBs remain connected by a bridge. Upon mitotic entry, a portion of the NE underlying the SPB pair invaginates, forming an opening (fenestra), and the SPBs settle into it. Each SPB initiates intranuclear microtubules, and they eventually separate and move to opposite sides of the nucleus while enclosed in their individual fenestrae that are somewhat larger than the SPBs themselves. In anaphase, the fenestrae contract to become about the same size as the SPBs. Late in mitosis, the fenestrae close, and the SPBs are extruded back into the cytoplasm \[[@pbio-0050170-b001]\]. These observations may indicate the existence of specialized and cell cycle--specific structures that function to anchor the SPB at the NE. Interestingly, the NE adjacent to the SPB itself shows some degree of specialization, including local thickening of the NE and presence of electron-dense material between the SPB and the NE \[[@pbio-0050170-b001],[@pbio-0050170-b005]\]. Also, γ-tubulin material localizes to the dark-staining region underlying the SPB in interphase \[[@pbio-0050170-b001]\]. Thus, it seems that there is a complex of molecular associations that defines a structure, including both the SPB and an adjacent portion of the NE. At least one membrane-spanning protein, Cut11p, is known to participate in SPB anchorage during early mitosis \[[@pbio-0050170-b006]\]. An interesting corollary of closed mitosis is the requirement for regulated NE division in order to prevent chromosome entanglement during spindle elongation and to ensure proper NE and endoplasmic reticulum (ER) inheritance. The elongating spindle is a stiff structure that is capable of exerting considerable force on the NE. For instance, assembly of microtubule polymers within liposomes induces striking shape changes, indicating that microtubules are capable of deforming the cellular membranes \[[@pbio-0050170-b007],[@pbio-0050170-b008]\]. The NE of S. pombe is a double-layered protein-containing structure that is likely more rigid than artificial phospholipid membranes. However, the NE is still capable of undergoing severe deformations due to forces exerted by microtubules, as suggested by three-dimensional reconstructions of monopolar spindles in cut11 ^ts^ mutant cells \[[@pbio-0050170-b006]\] and live NE imaging during the process of spindle assembly (unpublished data) or in interphase \[[@pbio-0050170-b003]\]. Notably, the NE behaves in a radically different manner during mitosis, when it follows an ordered progression of shape changes resulting in accurate division. The NE remains relatively spherical throughout metaphase and anaphase A. As cells proceed to anaphase B, the NE first elongates to become an ellipse with spindle minus ends located at its poles. It eventually assumes a dumbbell shape, after which it undergoes fission resulting in the formation of two daughter nuclei. Unlike microtubules polymerizing within liposomes \[[@pbio-0050170-b007],[@pbio-0050170-b008]\], the elongating mitotic spindle contacts the membrane at specific sites, including the SPBs and, likely, the underlying specialized section of the NE. It is thus possible that the presence of these rigid structures could allow the NE to sustain the pushing forces produced by the elongating spindle. This way, the SPB could be considered not only a microtubule-organizing center, but also a supporting structural feature allowing NE division during closed mitosis. Here, we describe experimental evidence supporting this hypothesis. While investigating mitotic functions of the transforming acid coiled coil (TACC)-related protein, Mia1p, in fission yeast, we found that cells overexpressing Mia1p failed to separate the SPBs, but frequently assembled elongating bipolar spindles. Under these circumstances, spindle poles deformed the NE, and NE division failed. Chromosome segregation was often unsuccessful, and DNA masses were confined within the undivided NE. Laser microsurgery in wild-type cells together with experiments on dis1Δ cells that exhibit faulty microtubule/kinetochore attachment, but normal spindle elongation, confirmed that the pole-located SPBs were required for NE division. Strikingly, genetic ablation of the NE during mitosis in Mia1p-overexpressing cells allowed sister chromatid segregation by acentrosomal spindles. Results {#s2} ======= Cells Overexpressing Mia1p Arrest in Mitosis, with Fully Elongated Spindles and Nonsegregated Chromosomes {#s2a} --------------------------------------------------------------------------------------------------------- Previous experiments suggested that the fission yeast TACC-related protein, Mia1p, is required for organizing and anchoring microtubule minus ends in interphase and for proper spindle function during mitosis \[[@pbio-0050170-b004],[@pbio-0050170-b009],[@pbio-0050170-b010]\]. To further our understanding of Mia1p function, we created S. pombe cells carrying a pREP1-based plasmid in which the mia1+ open reading frame was expressed under the control of the inducible nmt1 promoter \[[@pbio-0050170-b011]\]. We found that overexpression of Mia1p in wild-type cells resulted in severe lethality ([Figure S1](#pbio-0050170-sg001){ref-type="supplementary-material"}, see also \[[@pbio-0050170-b010]\]). Overexpression of Mia1p in strains carrying various green fluorescent protein (GFP)-tagged cell cycle protein markers revealed that cells arrested during late mitosis. First, the centromere I marker, Cen1-GFP \[[@pbio-0050170-b012]\], was often found in two distinct and well-separated dots, indicating that anaphase promoting complex (APC) activation occurred and chromosome cohesion was lost ([Figure 1](#pbio-0050170-g001){ref-type="fig"}A and [1](#pbio-0050170-g001){ref-type="fig"}B). We confirmed this by examining the localization of the general centromere/kinetochore marker, Mis6p-GFP \[[@pbio-0050170-b013]\], upon Mia1p overexpression. Mis6p-GFP labeled either two distinct kinetochore clusters, similar to wild-type control, or localized to six closely positioned dots, likely representing nonsegregated anaphase kinetochores ([Figure S2](#pbio-0050170-sg002){ref-type="supplementary-material"}). We also found that the Cdc14p-like phosphatase, Clp1p \[[@pbio-0050170-b014],[@pbio-0050170-b015]\], localized to intranuclear spindles in the majority of cells, consistent with their mitotic state ([Figure 1](#pbio-0050170-g001){ref-type="fig"}A and [1](#pbio-0050170-g001){ref-type="fig"}B). Moreover, most Mia1p-overexpressing cells exhibited actomyosin division rings, as judged by localization of the myosin II light chain, Rlc1p-GFP \[[@pbio-0050170-b016]\] ([Figure 1](#pbio-0050170-g001){ref-type="fig"}A and [1](#pbio-0050170-g001){ref-type="fig"}B). Because the mitotic checkpoint protein, Mad2p-GFP \[[@pbio-0050170-b017]\], largely localized to the NE, it appeared that most Mia1p-overexpressing cells overcame the spindle assembly checkpoint and entered anaphase ([Figure S3](#pbio-0050170-sg003){ref-type="supplementary-material"}) (even though approximately 12% of cells exhibited Mad2p-GFP on kinetochores, indicative of some aspect of spindle malfunction). The component of the septation initiation network, Cdc7p-GFP \[[@pbio-0050170-b018]\], localized to a single SPB, both in control and Mia1p-overexpressing cells (unpublished data). ![Fission Yeast Cells Overexpressing Mia1p Arrest in Mitosis with Fully Extended Spindles\ (A) The centromere markers, Cen1-GFP, Clp1-GFP, and Rlc1p-GFP, exhibit late mitotic localization in cells overexpressing Mia1p. Shown are single maximum-intensity reconstructions of live cells.\ (B) Graph quantifying the proportion of cells exhibiting mitotic localization of marker proteins (n = 100).\ (C) Immunofluorescence images of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies and the DNA dye, DAPI.\ (D) Graph quantifying the proportion of mitotic cells exhibiting wild-type or abnormal spindle architecture (n = 300). MT, microtubule.](pbio.0050170.g001){#pbio-0050170-g001} We performed immunofluorescence analyses of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies and the DNA dye, DAPI. All anaphase spindles in wild-type cells corresponded to two DNA masses located at the spindle poles, indicative of normal mitotic function ([Figure 1](#pbio-0050170-g001){ref-type="fig"}C and [1](#pbio-0050170-g001){ref-type="fig"}D). Cells overexpressing Mia1p exhibited mitotic phenotypes that could be roughly divided into three groups: 10% of cells showed a wild-type morphology with respect to chromosome segregation; 7% of cells exhibited aster-like monopolar spindles and highly condensed chromosomes, and the rest displayed extended anaphase spindles and nonsegregated DNA masses ([Figure 1](#pbio-0050170-g001){ref-type="fig"}C and [1](#pbio-0050170-g001){ref-type="fig"}D). Taken together, our data indicated that Mia1p-overexpressing cells were predominantly arrested in anaphase, with fully extended spindles and nonsegregated chromosomes. The SPBs Are Displaced from Spindle Poles, and Nuclear Envelope Division Fails in Mia1p-Overexpressing Cells {#s2b} ------------------------------------------------------------------------------------------------------------ The SPBs are thought to be required for mitotic spindle assembly and function in fungi, and are always found at the spindle poles ([Figure 2](#pbio-0050170-g002){ref-type="fig"}A, wild type). We overexpressed Mia1p in S. pombe strains carrying the core SPB marker, Pcp1p-GFP or the peripheral SPB protein, Sad1p-GFP. Strikingly, we found that only 20% of Mia1p-overexpressing Pcp1p-GFP anaphase cells exhibited spindles with pole-located SPBs. Nonseparated SPB pairs located at one, but not the other, spindle pole were found in 25% of cells, whereas the rest of Mia1p-overexpressing cells exhibited duplicated SPBs that were completely mispositioned with respect to either spindle pole ([Figure 2](#pbio-0050170-g002){ref-type="fig"}A and [2](#pbio-0050170-g002){ref-type="fig"}B). Overexpression of Mia1p in Sad1p-GFP cells confirmed that while the SPBs were duplicated and spindles elongated, the SPBs were often displaced from the spindle poles ([Figure S4](#pbio-0050170-sg004){ref-type="supplementary-material"}). ![The SPBs Are Displaced from the Spindle Poles, and NE Division Fails in Cells Overexpressing Mia1p\ (A) Immunofluorescence images of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies, Pcp1p-GFP, and the DNA dye, DAPI.\ (B) Graph quantifying the proportion of mitotic Mia1p-overexpressing cells exhibiting normal or aberrant spindle architecture (n = 100). Figures over each bar schematically represent position of the SPBs (black dots) with respect to the spindle (grey line).\ (C) Mia1p overexpression induces panhandle-shaped deformations of the NE. Shown are single maximum-intensity reconstructions of live wild-type and Mia1p-overexpressing cells containing the NE marker proteins Cut11p-GFP and Uch2p-GFP.\ (D) Graph quantifying the proportion of cells exhibiting the panhandle-shaped NE deformations (n = 100).\ (E) Time-lapse sequence of Cut11p-GFP--expressing wild-type cells undergoing mitosis. Numbers refer to the time, in minutes.\ (F) Time-lapse sequence of Mia1p-overexpressing Cut11p-GFP cells undergoing mitosis. Note an elongating protrusion of the NE and the SPB pair on the opposite side of the nucleus. Numbers refer to the time, in minutes.\ (G) The SPBs largely localize away from the NE protrusion tips in cells overexpressing Mia1p. Note that in wild-type cells, the SPBs seem to lead the NE division. Shown are single maximum-intensity reconstructions of live wild-type and Mia1p-overexpressing cells containing the NE marker protein, Uch2p-GFP, and the SPB protein, Sad1p-DSRed.](pbio.0050170.g002){#pbio-0050170-g002} Microscopy analyses of the NE markers, Cut11p-GFP \[[@pbio-0050170-b006]\] and Uch2p-GFP \[[@pbio-0050170-b019]\], suggested that the extending intranuclear spindles often deformed the NE, with most cells exhibiting striking panhandle-shaped protrusions with chromosomes "trapped" in the central compartment ([Figure 2](#pbio-0050170-g002){ref-type="fig"}C and [2](#pbio-0050170-g002){ref-type="fig"}D). To visualize NE dynamics, we performed time-lapse analyses of Cut11-GFP behavior in wild-type cells and cells overexpressing Mia1p. In the wild type, the NE went through dramatic changes after the onset of anaphase B. As the spindle elongated, the NE assumed an ovoid shape with the SPBs positioned at its leading edges. The dividing nucleus eventually underwent medial constriction. Finally, the remaining link between the two daughter nuclei was resolved ([Figure 2](#pbio-0050170-g002){ref-type="fig"}E and [Video S1](#pbio-0050170-sv001){ref-type="supplementary-material"}). By contrast, in cells overexpressing Mia1p, we observed the appearance and extension of the NE protrusions at one ([Figure 2](#pbio-0050170-g002){ref-type="fig"}F and [Video S2](#pbio-0050170-sv002){ref-type="supplementary-material"}) or both sides of the nucleus, and eventual failure of nuclear division. Microscopy analysis of Mia1p-overexpressing Uch2p-GFP Sad1p-DSRed cells revealed that the nonseparated SPB pairs were usually found at the base of the protrusions or at the opposite side of the nucleus ([Figure 2](#pbio-0050170-g002){ref-type="fig"}G). To gain insight into the intranuclear architecture upon overexpression of Mia1p, we performed electron microscopy serial-section analyses of rapidly frozen and freeze-substituted cell samples, followed by three-dimensional image reconstruction \[[@pbio-0050170-b020]--[@pbio-0050170-b022]\]. We confirmed that the SPBs were duplicated, but not separated ([Figure 3](#pbio-0050170-g003){ref-type="fig"}A and [3](#pbio-0050170-g003){ref-type="fig"}B, and [Video S3](#pbio-0050170-sv003){ref-type="supplementary-material"}). In the example shown, the spindle is positioned between the SPB pair at the base of the protrusion and the projection tip. The free end of the spindle deforms the double-layered NE ([Figure 3](#pbio-0050170-g003){ref-type="fig"}A and [Video S3](#pbio-0050170-sv003){ref-type="supplementary-material"}). ![The SPBs Are Duplicated but Not Separated in Cells Overexpressing Mia1p\ (A) Electron microscopy images of serial sections of a Mia1p-overexpressing cell showing the abnormal panhandle-shaped protrusion of the NE. Note that microtubules extend throughout the protrusion (indicated by indented arrowheads, panel 4). The SPB pair is located at the base of protrusion. The NE protrusion and position of the SPBs are indicated on panel 4. Scale bar represents 1 μm.\ (B) Higher magnification image of the duplicated SPB pair. Mitochondrion is labeled as M, nucleus as N. Scale bar represents 0.2 μm.](pbio.0050170.g003){#pbio-0050170-g003} Thus, we concluded that (1) the SPBs were duplicated, but not separated; (2) the SPBs were often disassociated from spindle poles; and (3) the SPB-free, or acentrosomal, spindle poles deformed the NE during spindle extension in Mia1p-overexpressing cells. Spindles in Mia1p-Overexpressing Cells Are Bipolar and Require the Microtubule-Crosslinking Protein, Ase1p, for Their Assembly {#s2c} ------------------------------------------------------------------------------------------------------------------------------ Given that the overexpression of Mia1p induced assembly of elongating spindles even though SPBs were not separated, we were interested in determining the polarity of microtubules in these structures. The BimC-related tetrameric kinesin, Cut7p, is essential for spindle formation and exhibits a complex cell cycle--specific pattern of localization \[[@pbio-0050170-b023],[@pbio-0050170-b024]\]. In metaphase wild-type cells, Cut7p-GFP was concentrated at spindle poles and localized along the entire spindle length. As the cell proceeded into anaphase, Cut7p-GFP mostly redistributed along the spindle to eventually concentrate in the broad midzone area, consistent with its function in spindle elongation. It also exhibited a weaker association with the SPBs ([Figure 4](#pbio-0050170-g004){ref-type="fig"}A, ten out of ten cells). Interestingly, Cut7p-GFP exhibited a largely bipolar localization in Mia1p-overexpressing cells, somewhat similar to wild type. Time-lapse analyses of elongating spindles indicated that they were clearly bipolar until early anaphase B (eight out of 11 cells). As spindles elongated, Cut7p-GFP redistributed to the spindle midzone and was eventually almost completely depleted from one of the spindle poles while being enriched on the other one ([Figure 4](#pbio-0050170-g004){ref-type="fig"}B). Fluorescence microscopy analyses utilizing Cut7p-mCherry, Uch2p-GFP, and Pcp1p-GFP marker proteins indicated that Cut7p was enriched on acentrosomal spindle poles in cells overexpressing Mia1p (93% of cells, n = 100, see [Figure 4](#pbio-0050170-g004){ref-type="fig"}C). Thus, even though the Cut7p-GFP localization was not symmetric in late anaphase spindles, it defined the spindle midzone, suggesting that orientation of microtubules in Mia1p-induced spindles was antiparallel with plus ends in the center of the spindle, similar to wild type. ![Spindle Structures in Mia1p-Overexpressing Cells Are Bipolar and Antiparallel\ (A) Time-lapse sequence of Cut7p-GFP--expressing wild-type cells undergoing spindle elongation. Numbers refer to the time, in minutes.\ (B) Time-lapse sequence of Mia1p-overexpressing Cut7p-GFP cells undergoing mitosis. Note that Cut7p-GFP clearly localizes to both poles early in anaphase and is progressively depleted from one of them as the cell progresses through mitosis. The midzone localization is similar to wild type. Shown are single maximum-intensity reconstructions of z-stacks. Numbers refer to the time, in minutes.\ (C) Cut7p-mCherry is enriched on acentrosomal spindle poles as judged by double fluorescence microscopy analysis with the SPB core marker, Pcp1p-GFP. Shown are single maximum-intensity reconstructions of z-stacks.\ (D) The γ-TuRC marker, Alp4p-GFP, is present on acentrosomal poles in Mia1p-overexpressing cells, where it co-localizes with Cut7p-mCherry. Shown are single maximum-intensity reconstructions of z-stacks.\ (E) The chromosomal passenger complex protein, Ark1p-GFP, defines the spindle midzone in acentrosomal spindles. Shown are single maximum-intensity reconstructions of z-stacks.\ (F) Ase1p-GFP localizes to the spindle poles and spindle midzone in wild-type and Mia1p-overexpressing cells. Shown are single maximum-intensity reconstructions of z-stacks.\ (G) Graph quantifying the proportion of cells exhibiting monopolar spindles upon Mia1p overexpressing in wild-type and ase1Δ genetic backgrounds (n = 300). Inset, immunofluorescence image of the monopolar spindle in a Mia1p-overexpressing ase1Δ cell using anti--α-tubulin antibodies and the DNA dye, DAPI. MTs, microtubules.](pbio.0050170.g004){#pbio-0050170-g004} Consistently, the γ-tubulin ring complex (γ-TuRC) component, Alp4p-GFP \[[@pbio-0050170-b025]\], concentrated at both spindle poles in the control and Mia1p-overexpressing cells, as judged by double fluorescence microscopy analyses using Cut7p-mCherry as a spindle marker ([Figure 4](#pbio-0050170-g004){ref-type="fig"}D). Also, although in control cells, the Alp4p-GFP signal always coincided with the SPB marker, Sad1p-DSRed, we observed the presence of Alp4p-GFP-positive, Sad1p-DSRed-negative structures upon Mia1p overexpression indicative of γ-TuRC--containing acentrosomal poles ([Figure S5](#pbio-0050170-sg005){ref-type="supplementary-material"}). We further evaluated spindle polarity by overexpressing Mia1p in cells containing spindle midzone markers. We found that the member of the chromosomal passenger complex aurora kinase, Ark1p-GFP \[[@pbio-0050170-b026]\], localized to kinetochores in metaphase (\~1% of cells) and to the spindle midzone during anaphase (\~5% of cells) in control cells. Ark1p-GFP defined the spindle midzone in 43% of Mia1p-overexpressing cells, and we found it localizing to kinetochores in 15% of cells ([Figure 4](#pbio-0050170-g004){ref-type="fig"}E). The microtubule-binding protein Ase1p is another spindle midzone marker that has been suggested to contribute to proper midzone formation and the structural integrity of the bipolar spindle \[[@pbio-0050170-b027],[@pbio-0050170-b028]\]. Although Ase1p is not essential, spindles in ase1Δ cells tend to collapse, but maintain bipolarity, and occasionally break during anaphase elongation \[[@pbio-0050170-b027]\]. Ase1p-GFP localized to the region of the spindle midzone and spindle poles in Mia1p-overexpressing cells ([Figure 4](#pbio-0050170-g004){ref-type="fig"}F). Strikingly, we found that Ase1p was required for the assembly of acentrosomal bipolar spindles upon Mia1p overexpression. The number of cells exhibiting typical aster-like monopolar spindles dramatically increased when Mia1p was overexpressed in the ase1Δ genetic background ([Figure 4](#pbio-0050170-g004){ref-type="fig"}G). Control cells lacking Ase1p, but not overexpressing Mia1p, exhibited a very low incidence of monopolar spindles (less than 0.5%). Thus, we concluded that Mia1p-overexpressing cells could assemble bipolar and antiparallel spindle structures despite failure of SPB separation. Elongation of Acentrosomal Spindles Results in Protruding Deformations of the Nuclear Envelope and Failure of Nuclear Division {#s2d} ------------------------------------------------------------------------------------------------------------------------------ Spindle structures assembled in cells overexpressing Mia1p failed to segregate chromosomes and to ensure NE division. A striking feature of Mia1p-induced spindles was a conspicuous absence of the SPBs at one or both spindle poles. We sought to determine whether the SPBs were in fact required for providing a structural rigidity that could allow spindle poles to push against the NE without deforming it. To test this hypothesis, we severed the SPBs from early anaphase spindles in α-tubulin-GFP Cut11p-GFP ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A and [5](#pbio-0050170-g005){ref-type="fig"}B), and in α-tubulin-GFP Uch2p-GFP (unpublished data) expressing wild-type cells using laser microsurgery \[[@pbio-0050170-b029]\]. When both SPBs were cut off, spindles continued elongation due to sliding forces produced in the spindle midzone \[[@pbio-0050170-b029]\]. These spindle remnants readily formed panhandle-shaped protrusions at opposite sides of the nucleus ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A), evocative of the phenotype observed in Mia1p-overexpressing cells ([Figure 2](#pbio-0050170-g002){ref-type="fig"}C), and similar to what has been previously noted \[[@pbio-0050170-b029]\]. When the SPB was severed from one spindle pole, spindles deformed the NE at this location, whereas no NE deformation was observed at the opposite spindle pole still tethered to the other SPB ([Figure 5](#pbio-0050170-g005){ref-type="fig"}B). ![NE Division Fails When the SPBs Are Not Positioned at Elongating Spindle Poles\ (A) Time-lapse sequence of a Cut11p-GFP α-tubulin-GFP--expressing wild-type cell when both SPBs were severed from the elongating anaphase spindle by laser microsurgery.\ (B) Time-lapse sequence of a Cut11p-GFP α-tubulin-GFP--expressing wild-type cell when one SPB was cut off the spindle. Note that the NE is forming the panhandle-shaped protrusions at the sites of contact with the elongating spindle remnants. DIC images at the end of the sequence show normally proceeding septation. Shown are single maximum-intensity reconstructions of z-stacks. Numbers refer to the time, in minutes and seconds.\ (C) Time-lapse fluorescence microscopy analyses of the NE and spindle dynamics in Uch2p-GFP α-tubulin-GFP--expressing wild-type, dis1Δ, and Mia1p-overexpressing cells. Formation of membrane tethers in dis1Δ and Mia1p-overexpressing cells is indicated by arrowheads. Shown are single maximum-intensity reconstructions of z-stacks. Numbers refer to the time, in minutes.](pbio.0050170.g005){#pbio-0050170-g005} It is possible that the presence of either the SPB or of chromosomes at the poles, or both, is required for maintaining shape of the nucleus during anaphase movement. To distinguish between these possibilities, we performed time-lapse analyses of cells lacking the MAP215-related protein, Dis1p, and expressing the NE marker, Uch2p-GFP, and the microtubule marker, α-tubulin-GFP. It has previously been shown that at the restrictive temperature of 20 °C, Dis1p is required for kinetochore/microtubule attachment although the spindle elongates to the full extent \[[@pbio-0050170-b030],[@pbio-0050170-b031]\]. We found that initial elongation of the NE proceeded normally in dis1Δ cells as compared to wild-type control, and eventually, nuclei of both cell types assumed an ovoid shape ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). However, as the spindle in dis1Δ cells elongated further, the NE abruptly collapsed, resulting in the formation of the panhandle-shaped protrusion ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). We then performed imaging of Mia1p-overexpressing cells that also contained Uch2p-GFP α-tubulin-GFP during the same stages of mitosis. In the presence of overexpressed Mia1p, constitutive expression of α-tubulin-GFP failed to generate fluorescent bipolar spindles (unpublished data). Therefore, we simultaneously induced expression of Mia1p from the nmt1 promoter and α-tubulin-GFP from the weak nmt81 promoter. We found that although there was still a considerable increase in the number of cells with monopolar spindles, we could observe few cells with properly assembled elongating spindles. The NE in these cells was abnormally deformed immediately after spindle elongation, without going through the initial expansion and elongation phase ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). Based on these experiments, we concluded that the SPBs positioned at spindle poles were essential for NE division and that successful segregation of the NE was sustained by keeping daughter chromosomes attached to the SPBs throughout anaphase B. Genetic Ablation of the Nuclear Envelope Allows Chromosome Segregation in Mia1p-Overexpressing Cells {#s2e} ---------------------------------------------------------------------------------------------------- As shown above, Mia1p overexpression is lethal, largely due to the failure in nuclear division and chromosome segregation. Because most Mia1p-overexpressing cells bypassed the spindle assembly checkpoint and arrested in anaphase, we wondered whether, in part, it was the NE division defect that interfered with chromosome segregation due to entrapment of DNA masses. Thus, we tested whether the Mia1p-induced spindles could segregate DNA at least at some frequency in an experimental situation in which cells underwent mitosis in the absence of the NE. The temperature-sensitive mutant in the Ran-GEF, pim1--1, has been reported to induce NE fragmentation upon passage through mitosis \[[@pbio-0050170-b032]\]. Time-lapse analyses of spindle and NE dynamics in α-tubulin-GFP Uch2p-GFP pim1--1 cells at the permissive ([Figure 6](#pbio-0050170-g006){ref-type="fig"}A and [Video S4](#pbio-0050170-sv004){ref-type="supplementary-material"}) and restrictive ([Figure 6](#pbio-0050170-g006){ref-type="fig"}B and [Video S5](#pbio-0050170-sv005){ref-type="supplementary-material"}) temperatures suggested that lack of Pim1p function induced an irreversible NE fragmentation in early anaphase B. Spindles continued elongating ([Figure 6](#pbio-0050170-g006){ref-type="fig"}B and [Video S5](#pbio-0050170-sv005){ref-type="supplementary-material"}), and cells eventually underwent cytokinesis, arresting at this point in the cell cycle (\[[@pbio-0050170-b032]\] and [Figure 6](#pbio-0050170-g006){ref-type="fig"}D). We induced overexpression of Mia1p in pim1--1 cells containing the integral SPB marker, Pcp1p-GFP, and the NE marker, Uch2p-GFP, at either permissive or restrictive temperature. Panhandle-shaped NE protrusions could be induced in pim1--1 cells at 24 °C at a rate similar to control Uch2p-GFP cells ([Figure S6](#pbio-0050170-sg006){ref-type="supplementary-material"}). Only 10% of Pcp1p-GFP Uch2p-GFP pim1--1 cells overexpressing Mia1p segregated DNA in two masses when the NE was intact ([Figure 6](#pbio-0050170-g006){ref-type="fig"}C), consistent with the above experiments. When the NE was disassembled upon shifting cells to the restrictive temperature, the proportion of binucleate cells increased considerably (32%) ([Figure 6](#pbio-0050170-g006){ref-type="fig"}C). ![DNA Segregation Occurs in Mia1p-Overexpressing Cells When the NE Is Fragmented in Mitotic Cells Harboring the pim1--1 Mutation\ (A) Time-lapse sequence of an Uch2p-GFP α-tubulin-GFP--expressing pim1--1 cell undergoing anaphase at the permissive temperature of 24 °C.\ (B) Time-lapse sequence of an Uch2p-GFP α-tubulin-GFP--expressing pim1--1 ^ts^ cell undergoing anaphase at the restrictive temperature of 36 °C. Cells were shifted to the restrictive temperature 3 h prior to imaging to allow inactivation of Pim1p protein. Note that the NE is fragmented shortly after anaphase B onset. Shown are single maximum-intensity reconstructions of z-stacks. Numbers refer to the time, in minutes.\ (C) Graph quantifying the proportion of binucleate Pcp1p-GFP Uch2p-GFP pim1--1 cells overexpressing Mia1p at 24 °C and 36 °C (n = 300).\ (D) Graph quantifying the proportion of uninucleate and binucleate Mia1p-overexpressing Pcp1p-GFP Uch2p-GFP pim1--1 cells that do not separate the SPBs at 36 °C (n = 100). Insets, examples of the scored phenotypes. Epifluorescence images of fixed Mia1p-overexpressing pim1--1 cells containing the core SPB marker, Pcp1p-GFP, and the outer NE marker, Uch2p-GFP, at 36 °C. DNA is stained with DAPI. The NE is fragmented. Shown are single maximum-intensity reconstructions of z-stacks.\ (E) Epifluorescence images of Cen1-GFP Sad1-DSRed--containing pim1--1 cells, with and without Mia1p overexpression. Shown are single maximum-intensity reconstructions of z-stacks. Cartoons outline the phenotypes scored. Circle indicates the SPB; star indicates the centromere I.](pbio.0050170.g006){#pbio-0050170-g006} We found that few cells with spindles lacking the SPBs at least at one spindle pole exhibited two or more closely positioned DNA masses when the NE was intact (4% of cells, [Figure S7](#pbio-0050170-sg007){ref-type="supplementary-material"}). However, when the NE was fragmented, we observed an appearance of divided cells with DNA segregated to daughter compartments. Upon the shift to the restrictive temperature, 27% of cells exhibiting duplicated, but not separated, SPBs on one side of the septum showed presence of chromosomes in both daughter cells (n = 100, [Figure 6](#pbio-0050170-g006){ref-type="fig"}D). We repeated this experiment using cells expressing the centromere I marker, Cen1-GFP, and the SPB marker, Sad1p-DSRed. We found that 32% of cells (n = 68) with acentrosomal spindles properly segregated sister chromatids when the NE was fragmented, suggesting that failure in NE division could hinder DNA segregation ([Figure 6](#pbio-0050170-g006){ref-type="fig"}E). Taken together, our results suggest that (1) overexpression of the TACC-related protein, Mia1p, results in the assembly of bipolar and moderately functional spindles that lack properly positioned SPBs; (2) one of the essential functions of the SPB in fission yeast could be to facilitate NE division during closed mitosis; and (3) failure to divide the NE could result in restriction of DNA masses movement and, ultimately, in a chromosome segregation defect. Discussion {#s3} ========== In this study, we show that anaphase elongation of acentrosomal spindles during closed mitosis in fission yeast causes NE division failure and associated chromosome segregation defects. Mia1p belongs to a family of microtubule-associated proteins named after the human transforming acid coiled coil protein, TACC, which has been implicated in cancer development (for review, see \[[@pbio-0050170-b033]\]). TACC proteins participate in spindle pole focusing and organization in metazoans \[[@pbio-0050170-b034],[@pbio-0050170-b035]\]. Fission yeast cells lacking Mia1p exhibit a variety of microtubule defects throughout the cell cycle. Mitotic mia1Δ cells lack astral microtubules \[[@pbio-0050170-b009]\] and show signs of defective kinetochore/microtubule attachment \[[@pbio-0050170-b010]\]. During interphase, mia1Δ cells fail to establish and maintain properly organized microtubule arrays, likely due to a molecular defect in microtubule attachment to the original nucleation sites \[[@pbio-0050170-b004]\]. These functions correlate well with the Mia1p localization pattern. Indeed, Mia1p-GFP is mainly concentrated at the microtubule-organizing centers, though it is also found at plus ends of microtubules \[[@pbio-0050170-b004],[@pbio-0050170-b009],[@pbio-0050170-b010]\]. We found that cells overexpressing Mia1p were capable of organizing ectopic spindle poles and acentrosomal spindles. Although formation of intranuclear microtubule bundles in interphase S. pombe cells has previously been observed \[[@pbio-0050170-b036]\], overexpression of Mia1p led to assembly of mitotic spindles exhibiting normal microtubule arrangement ([Figures 1](#pbio-0050170-g001){ref-type="fig"} and [4](#pbio-0050170-g004){ref-type="fig"}). These spindles contained the γ-TuRC complexes and the BimC kinesin, Cut7p, at acentrosomal spindle poles, and exhibited a well-defined spindle midzone ([Figure 4](#pbio-0050170-g004){ref-type="fig"}). Curiously, unlike the case of normal wild-type spindles, formation of these structures entirely depended on the presence of the microtubule-crosslinking protein, Ase1p ([Figure 4](#pbio-0050170-g004){ref-type="fig"}G). Mia1p-GFP expressed at high levels localized to spindle poles, where it formed large fluorescent structures, and elsewhere in the cytosol (unpublished data). Interestingly, upon overexpression of Mia1p, Ase1p-GFP aggregated at the spindle poles in addition to its normal localization to the spindle midzone ([Figure 4](#pbio-0050170-g004){ref-type="fig"}F). The γ-TuRC component, Alp4p-GFP, also localized to the acentrosomal spindle poles ([Figure 4](#pbio-0050170-g004){ref-type="fig"}D and [Figure S5](#pbio-0050170-sg005){ref-type="supplementary-material"}), suggesting that they could indeed contain a host of microtubule-associated factors. The fact that some proteins required for proper spindle assembly might be sequestered to these abnormal structures could also explain the reason why the SPBs failed to separate in cells overexpressing Mia1p. TACC proteins contain extensive coiled coil regions and can likely form oligomers. This, in combination with their microtubule binding properties, could provide a framework for clustering and focusing of microtubule minus ends observed in vivo. We envisage that accumulation of the γ-TuRC complexes and microtubule-crosslinking proteins near the SPBs, followed by subsequent sliding off through the force produced by the growing spindle, could lead to the formation of acentrosomal spindle poles. A particularly interesting phenotype of Mia1p overexpression was the formation of long, panhandle-shaped NE protrusions upon spindle elongation and the fact that NE division failure was reproducibly accompanied by DNA segregation defects. One possible explanation for such a phenotype could be the lack of microtubule/kinetochore attachment in these cells, which is not particularly surprising given that Mia1p normally functions in this process. We did observe an occurrence of trailing chromosomes in some cells overexpressing Mia1p when the SPBs delineated both spindle poles (for example, see [Figure 2](#pbio-0050170-g002){ref-type="fig"}A). Alternatively, the presence of the SPB inserted into the specialized region of the NE could prevent the formation of membrane tethers during spindle elongation. We found that most acentrosomal spindle poles in Mia1p-overexpressing cells distorted the NE, whereas most poles associated with the SPBs did not exhibit this behavior ([Figure 2](#pbio-0050170-g002){ref-type="fig"}). We also performed a series of laser microsurgery experiments in which we severed either one or both SPBs from the rest of the elongating anaphase spindle ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A and [5](#pbio-0050170-g005){ref-type="fig"}B). We found that, similar to Mia1p overexpression, the free spindle poles deformed the NE, and these cells failed to divide their nuclei despite normally timed septation ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A and [5](#pbio-0050170-g005){ref-type="fig"}B). We sought to distinguish between the possibilities outlined above by following the NE dynamics in cells lacking the MAP215 protein, Dis1p, at the restrictive temperature of 20 °C. dis1Δ cells do not attach sister chromatids to the spindle microtubules, but proceed with spindle elongation \[[@pbio-0050170-b030],[@pbio-0050170-b031]\]. Because spindles in dis1Δ cells contained the SPBs, we could assess relative contributions of chromosomes and SPBs to NE division. We found that initially, the NE in dis1Δ cells went through normal elongation and assumed the ovoid shape typical of wild-type nuclei ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). However, during subsequent spindle elongation, the NE abruptly collapsed, suggesting that positioning of chromosomes at the poles was essential for maintaining the shape of the dividing nucleus ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C, indicated by an arrowhead). It is possible that once the membrane reservoir (see below) is exhausted during the initial elongation stage, a mass of chromosomes provides a physical block to the NE division. Contrary to the dis1Δ phenotype, spindles immediately produced nuclear membrane tethers in Mia1p-overexpressing cells ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C, emerging protrusion is indicated by an arrowhead), and nuclei did not assume the elliptical shape. We concluded that both the SPBs and the spindle pole--positioned chromosomes were essential for successful NE division. The SPB of S. pombe undergoes striking changes in morphology and cellular localization during the cell cycle. Because the SPBs move in and out of the nuclear membrane, depending on the mitotic phase, this process likely relies on a tightly regulated membrane-anchoring system. It has been suggested that Cut11p, a transmembrane protein similar to budding yeast Ndc1p, might participate in SPB anchoring during mitosis. In cells lacking Cut11p function, the SPBs are frequently found floating in the nucleoplasm, in addition to the pronounced SPB separation defect \[[@pbio-0050170-b006]\]. Cut11p localizes to the nuclear pores throughout the cell cycle and is recruited to the SPBs at the onset of mitosis, which would be consistent with its proposed function in the SPB anchorage. Interestingly, at least one computer-generated reconstruction of a defective spindle in Cut11p-deficient cells shows an image very similar to spindles assembled in Mia1p-overexpressing cells. The spindle is bipolar, but only one spindle pole is connected to the SPB. There is no SPB at the other pole that forms a herniated protrusion on the NE \[[@pbio-0050170-b006]\]. However, the SPB localization of Cut11p is lost in mid-anaphase B despite the fact that the SPBs still appear to lead the nuclear movement (\[[@pbio-0050170-b006]\] and unpublished data). Thus, it is possible that there is an additional mechanism executing proper SPB tethering and NE reinforcement at the site of insertion. The integral NE protein and SPB component, Sad1p, could possibly function in this process \[[@pbio-0050170-b037]\]. Sad1p-related proteins from other organisms have been involved in anchoring centrosomes to the NE \[[@pbio-0050170-b038]--[@pbio-0050170-b040]\]. Also, the intranuclear system of Mlp filaments might be a good candidate for reinforcing the site of SPB attachment to the NE. Mlps are coiled coil filamentous proteins localized to the nucleoplasmic side of the NE \[[@pbio-0050170-b041]\]. Both budding yeast Mlp1p and Mlp2p localize to the NE crescent region underlying the SPB, with Mlp2 forming discernible foci. Interestingly, Mlp2p interacts with the core components of the SPB and is required for proper SPB assembly and function \[[@pbio-0050170-b042]\]. It is possible that a large structure such as the SPB has to be anchored into the underlying assembly of Mlp filaments. It is also possible that the Mlp assembly might structurally reinforce the NE in order to maintain the stiffness and the appropriate curvature of the membrane during closed mitosis. It will be of significant interest to address potential roles of Sad1p and Mlp proteins in NE division in future studies. Panhandle-shaped protrusions of the nuclear membrane produced in our experiments can be compared to membrane tethers produced by microtubule polymerization or application of laser tweezers in artificial lipid vesicles \[[@pbio-0050170-b007],[@pbio-0050170-b043]\]. Upon force application, the membrane develops regions of negative curvature and collapses into narrow tubes around the spindle. Interestingly, formation of these long tethers may suggest the existence of a functional membrane reservoir at this stage of the cell cycle \[[@pbio-0050170-b044]\]. We performed time-lapse analyses of wild-type cells (n = 7) expressing the NE marker, Uch2p-GFP, and α-tubulin-GFP, and measured the nuclear diameter at metaphase (3.76 ± 0.14 μm) and anaphase (3.12 ± 0.12 μm). Assuming nuclei to be spherical, the combined surface area of the two daughter nuclei is estimated to be approximately 1.37 times larger than that of the mother nucleus. This apparent increase in surface area could result from membrane contribution from the ER or unraveling of nuclear membrane folds. At the same time, it is conceivable that the nuclear envelope behaves like a rigid network of locally deformable components. Studies performed on the NE of mammalian cells suggest that it could be organized as a heterogeneous meshwork consisting of regular lattice domains and interconnecting filaments \[[@pbio-0050170-b045]\]. Under mechanical stress, the meshwork would expand resulting in vacancies of lamin A. Although fungal nuclear envelopes do not contain lamins, it is possible that other proteins underlying the inner membrane, for instance, Mlp filaments, could perform similar function in connecting the network. We reasoned that should the undivided NE restrict the movement of chromosomes in cells overexpressing Mia1p, the removal of this obstacle could enable some degree of chromosome segregation through the action of acentrosomal spindles. Cells harboring a mutation (pim1--1) in a gene encoding for the RCC1-related protein, Pim1p, have been shown to undergo NE fragmentation upon passage through mitosis, and arrest with condensed DNA following hyperseptation (\[[@pbio-0050170-b032]\], see also [Figure 6](#pbio-0050170-g006){ref-type="fig"}A and [6](#pbio-0050170-g006){ref-type="fig"}B, and [Videos S4](#pbio-0050170-sv004){ref-type="supplementary-material"} and [S5](#pbio-0050170-sv005){ref-type="supplementary-material"}). We indeed found that a failure of DNA segregation in Mia1p-overexpressing cells was considerably relieved in the pim1--1 genetic background ([Figure 6](#pbio-0050170-g006){ref-type="fig"}C and [6](#pbio-0050170-g006){ref-type="fig"}D). Interestingly, a significant proportion of these cells could accurately segregate sister chromatids, suggesting that acentrosomal spindles were functional although less efficient than normal ones ([Figure 6](#pbio-0050170-g006){ref-type="fig"}D). It is likely that a combination of factors contributes to the low efficiency of Mia1p-induced acentrosomal spindles in chromosome segregation. First, as outlined above, Mia1p overexpression could interfere with kinetochore attachment to spindle microtubules. Secondly, it is possible that attachment of microtubules to the SPB contributes to the increased accuracy of chromosome segregation, similar to what has been suggested for the case of centrosomes in animal cells \[[@pbio-0050170-b046]\]. There is an enormous structural diversity of spindles in eukaryotes \[[@pbio-0050170-b047],[@pbio-0050170-b048]\]. Apart from the well-established open and closed mitosis categories, spindles can assemble as extra- or intranuclear ones \[[@pbio-0050170-b047]\]. Although in some organisms the NE remains intact, in others, it might partially break down \[[@pbio-0050170-b049],[@pbio-0050170-b050]\] or be forcibly stripped off \[[@pbio-0050170-b051]\]. Similarly, various structural modifications throughout evolution resulted in the appearance of various types of SPBs. Here, we suggest that the highly structured SPB of fission yeast, and likely of other organisms in which chromosome segregation occurs strictly within the nuclear membrane, functions not only as a microtubule-organizing center, but also serves as a structural feature that enables NE division. Materials and Methods {#s4} ===================== S. pombe strains, antibodies, reagents, and constructs. {#s4a} ------------------------------------------------------- S. pombe strains used in this study and their genotypes are listed in [Table S1](#pbio-0050170-st001){ref-type="supplementary-material"}. EMM2 medium with suitable supplements was used for vegetative growth. For live-imaging experiments Mia1p overexpression was routinely induced for 18 h at 24 °C in EMM2 lacking thiamine. Genetic crosses and sporulation were performed on YPD agar plates. Plasmids were constructed using standard techniques of molecular cloning. The anti--α-tubulin antibody, TAT-1, was a gift from Dr. K. Gull. Immunofluorescence techniques. {#s4b} ------------------------------ Cells were fixed with 3.7% formaldehyde for 12 min and then spheroplasted using lysing enzymes and zymolyase in 1.2 M sorbitol in PBS. Permeabilization was performed in 1% Triton X-100 in PBS. Spheroplasts were washed with PBS. PBAL buffer (1 mM sodium azide, 1% BSA, 100 mM lysine hydrochloride, 50 μg/ml carbenicilin in PBS) was used for blocking and for incubation with primary and secondary antibodies. Imaging was done on a Zeiss Axiovert 200M microscope (Carl Zeiss, <http://www.zeiss.com>) with appropriate sets of filters, and images were generated using a CoolSnap camera (Photometrics, <http://www.photomet.com>) and Metamorph software package (Universal Imaging/Molecular Devices, <http://www.moleculardevices.com>). Image processing was done in Adobe Photoshop 7.0.1 (Adobe Systems, <http://www.adobe.com>). Time-lapse fluorescent microscopy. {#s4c} ---------------------------------- Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with CSU10 confocal optical scanner, 488-nm laser illumination source, and CoolSnap digital camera (Photometrics). Alternatively, we performed imaging in epifluorescence mode using a mercury lamp as an illumination source together with appropriate sets of filters, CoolSnap camera (Photometrics), and Uniblitz shutter driver (<http://www.uniblitz.com>) under the control of Metamorph software package (Universal Imaging). Typically, we acquired image stacks that consisted of five or six sections at 0.5-μm spacing. Imaging at 20 °C was performed using a homemade, cooled microscope housing. Laser microsurgery. {#s4d} ------------------- Laser microsurgery was conducted on a custom-assembled microscopy workstation centered around a Nikon TE2000-E2 microscope (Nikon Instruments, <http://www.nikon.com>). Laser pulses (532 nm for 8 ns) were generated by Q-switched Nd:YAG laser (Diva II; Thales Lasers, <http://thales.nuxit.net>) run at a 20-Hz repetition rate. Collimated laser beam was expanded to approximately 8 mm to fill the aperture of a 100× 1.4 NA PlanApo lens and delivered through an dedicated epi-port. Fluorescence images were recorded with a Cascade512B back-illuminated EM-CCD camera (Photometrics) attached to the left microscope port (100% transmission) in confocal mode (spinning disk confocal; Perkin-Elmer, <http://www.perkinelmer.com>) as three-dimensional datasets at 0.25-μm Z steps. Differential interference contrast (DIC) images were recorded with a CoolSnap CF CCD camera attached to the right port of the microscope (modified to 100% transmission). All light sources were shuttered by either fast mechanical shutters (Vincent Associates, <http://www.uniblitz.com>) or AOTF (Solamere Technology Group, <http://www.solameretech.com>) so that cells were exposed to light only during laser operations and/or image acquisition. Electron microscopy. {#s4e} -------------------- Cells were rapidly frozen by high-pressure freezing (BAL-TEC HPM-010; Technotrade International, <http://www.technotradeinc.com/>) and freeze-substituted at −90 °C in 2% glutaraldehyde plus 0.1% uranyl acetate in acetone for 96 h in an EM-AFS device (Leica, <http://www.leica-microsystems.com>). The cells were warmed for more than 3 h to −60 °C and then infiltrated with HM20 (Electron Microscopy Sciences, <http://www.emsdiasum.com>) resin over a period of 3 d. The cells were embedded under UV light at −60 °C in HM20 for 3 d and then warmed to room temperature over a 6-h period. Serial sections (60 nm) of embedded cells were picked up on Formvar-coated slot grids, stained with 1% aqueous uranyl acetate and lead citrate, and imaged in a Philips Tecnai TF20 FEG electron microscope (<http://www.fei.com/>) operating at 80 keV. Serial sections were aligned and modeled using the IMOD software package. Supporting Information {#s5} ====================== ###### Overexpression of Mia1p in S. pombe Is Lethal Cells carrying either an empty pREP1 vector or the pREP1-based plasmid containing mia1^+^ open reading frame (ORF) were grown overnight in EMM2 medium containing thiamine to the optical density at 595 nm (OD~595~) equal to 0.4. They were spotted onto EMM2 plates with or without thiamine at increasing 10× dilutions and grown for 4 d at 30 °C. (961 KB TIF) ###### Click here for additional data file. ###### Localization of Mis6p-GFP in Wild-Type and Mia1p-Overexpressing Cells The centromere/kinetochore marker, Mis6p-GFP, labeled either two distinct kinetochore clusters (upper panel), similar to wild-type control, or localized to six closely positioned dots, likely representing nonsegregated anaphase kinetochores (lower panel). Shown are single maximum-intensity reconstructions of live cells. (447 KB TIF) ###### Click here for additional data file. ###### The Majority of Mia1p-Overexpressing Cells Exhibit Mad2p-GFP on the NE Graph quantifying the proportion of cells with Mad2p-GFP on kinetochores in wild type and cells overexpressing Mia1p (n = 100). Inset displays typical appearance of Mad2p-GFP in Mia1p-overexpressing cells, either on kinetochores or broadly spread around the NE. Shown are single maximum-intensity reconstructions of live cells. (555 KB TIF) ###### Click here for additional data file. ###### The SPBs Are Displaced from the Spindle Poles in Cells Overexpressing Mia1p \(A\) Immunofluorescence images of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies, Sad1p-GFP, and the DNA dye, DAPI. \(B\) Graph quantifying proportion of mitotic Mia1p-overexpressing cells exhibiting normal or aberrant spindle architecture (n = 100). (996 KB TIF) ###### Click here for additional data file. ###### The γ-TuRC Complex Protein, Alp4p-GFP, Localizes to Acentrosomal Spindle Poles in Mia1p-Overexpressing Cells Double fluorescence microscopy images of control and Mia1p-overexpressing Alp4p-GFP Sad1p-DSRed cells. Shown are single maximum-intensity reconstructions of live cells. (489 KB TIF) ###### Click here for additional data file. ###### Overexpression of Mia1p Induces Abnormal NE Deformations in pim1--1 Cells at the Permissive Temperature \(A\) Epifluorescence images of the NE labeled by Uch2p-GFP in wild-type and Mia1p-overexpressing pim1--1 cells at 24 °C. Shown are single maximum-intensity reconstructions of live cells. \(B\) Graph representing the proportion of cells forming the panhandle-shaped protrusions in both genetic backgrounds. (298 KB TIF) ###### Click here for additional data file. ###### Epifluorescence Images of Fixed Mia1p-Overexpressing pim1--1 Cells Containing the Core SPB Marker, Pcp1p-GFP, and the Outer NE Marker, Uch2p-GFP, at 24 °C Rarely, we could observe Mia1p-overexpressing pim1--1 cells with acentrosomal spindles that exhibited two or more closely positioned DNA masses at the permissive temperature. DNA is stained with DAPI. Shown are single maximum-intensity reconstructions of z-stacks. (492 KB TIF) ###### Click here for additional data file. ###### List of S. pombe Strains Used in This Study (49 KB DOC) ###### Click here for additional data file. ###### Time-Lapse Sequence of a Cut11p-GFP--Expressing Wild-Type Cell Undergoing Mitosis Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with CSU10 confocal optical scanner, 488-nm laser illumination source, and CoolSnap digital camera. Image stacks consisted of six sections at 0.5-μm spacing taken every 30 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (584 KB MOV) ###### Click here for additional data file. ###### Time-Lapse Sequence of a Mia1p-Overexpressing Cut11p-GFP Cell Undergoing Mitosis Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with CSU10 confocal optical scanner, 488-nm laser illumination source, and CoolSnap digital camera. Image stacks consisted of six sections at 0.5-μm spacing taken every 30 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (353 KB MOV) ###### Click here for additional data file. ###### QuickTime Video Was Assembled Based on Electron Microscopy Data Presented in [Figure 3](#pbio-0050170-g003){ref-type="fig"} Serial sections were aligned and modeled using the IMOD software package. The NE is in green, the SPBs are in purple, and microtubules are in yellow. (1.4 MB MOV) ###### Click here for additional data file. ###### Time-Lapse Sequence of an Uch2p-GFP α-Tubulin-GFP--Expressing pim1--1 Cell Undergoing Anaphase at the Permissive Temperature of 24 °C Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope in epifluorescence mode. Image stacks consisted of five sections at 0.5-μm spacing taken every 60 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (126 KB MOV) ###### Click here for additional data file. ###### Time-Lapse Sequence of an Uch2p-GFP α-Tubulin-GFP--Expressing pim1--1 Cell Undergoing Anaphase at the Restrictive Temperature of 36 °C Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope in epifluorescence mode. Image stacks consisted of five sections at 0.5-μm spacing taken every 60 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (618 KB MOV) ###### Click here for additional data file. Accession Numbers {#s5a} ----------------- The National Center for Biotechnology Information (NCBI) (<http://www.ncbi.nlm.nih.gov>) accession numbers for the proteins discussed in this paper are Alp4p (BAA77269), Ark1p (O59790), Ase1p (CAC21482), Cdc7p (CAA55382), Clp1p (CAB76271), Cut11p (CAB11272), Cut7p (CAA40738), Dis1p (CAA19278), Mad2p (CAA16846), Mia1p (Q9URY2), Mis6p (BAA20458), Pcp1p (CAB03608), Pim1p (CAB60670), Rlc1p (CAB54151), Sad1p (BAA87133), and Uch2p (CAB52608). We are most grateful to M. Balasubramanian for suggesting the NE ablation experiment and allowing us to use strains from his S. pombe collection. E. Makeyev, V. Wachtler, and M. Balasubramanian kindly commented on the manuscript. Many thanks are due to R. Thadani, A. Tolstikov, J. R. McIntosh, and members of the Cell Dynamics group at the Temasek Life Sciences Laboratory for discussions. We would also like to thank Dr. K. Gull for his gift of TAT-1 antibody. Author contributions. SO conceived and designed the experiments and wrote the paper. LZ, CS, VM, and SO performed the experiments. All authors analyzed the data. LZ, CS, AK, and SO contributed reagents/materials/analysis tools. Funding. This work was supported by National Institutes of Health and Biotechnology Resources grant RR00592 to the National Resource for 3D Microscopy, a National Institutes of Health grant GM59363 to AK, and intramural funds from the Temasek Life Sciences Laboratory. Competing interests. The authors have declared that no competing interests exist. 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Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder of the central nervous system, clinically characterized by memory impairment and cognitive dysfunction. The pathological hallmarks of AD in the brain are massive cerebral accumulation of senile plaques largely composed of β-amyloid (Aβ) peptide, intracellular neurofibrillary tangles and neuronal loss[@b1][@b2][@b3][@b4]. Recently, several epidemiological studies have revealed that mutations of particular genes can confer susceptibility to the pathogenesis of AD. APP, PSEN1, PSEN2 gene mutations are considered to be the main causes of familial AD, and play an important role in the early-onset of AD[@b5]. While in late-onset AD, the ε4 allele of the Apolipoprotein E gene (ApoEε4) is known as the major genetic risk factor[@b6]. Moreover, the mutations of genes such as APBB2, GAB2, PRNP and SORL1 have also been reported to be associated with the risk of AD[@b7][@b8][@b9][@b10][@b11]. The ATP-binding cassette subfamily B member 1 (ABCB1) gene in the chromosome 7q21 region spans 209-kb and contains 29 exons[@b12]. To date, several ABCB1 genetic variants have been identified to be involved in various kinds of diseases through re-sequencing, including 3435C \> T (rs1045642), 2677G \> T/A (rs2032582), 1236C \> T (rs1128503) polymorphisms. The 3435C \> T single nucleotide polymorphism (SNP) was the most commonly studied polymorphism, which was previously reported to be related to the susceptibility to the osteonecrosis of the femoral head (ONFH), leukemia, breast cancer and hepatocellular carcinoma[@b13][@b14][@b15][@b16]. The 2677G \> T/A polymorphism is associated with glucocorticoid-induced avascular necrosis of the femoral head (GANFH) and cancer[@b17][@b18]. The 1236C \> T polymorphism may contribute to the tumor response to chemotherapy in cancers in Asians[@b19][@b20]. P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an integral component of the blood-brain barrier (BBB)[@b21]. P-gp pumps various drugs and toxicants out of the brain and Aβ is an endogenous substrate for P-gp, suggesting that P-gp is able to mediate the clearance of Aβ through BBB[@b22]. Therefore, mutations of ABCB1 gene possibly result in an aberrant function of P-gp, and thus promote the pathogenesis of AD. Recently, some epidemiological studies have focused on the association between ABCB1 gene variants and AD susceptibility, and these variants include the 3435C \> T polymorphism, the 2677G \> T/A polymorphism and the 1236C \> T polymorphism[@b23][@b24][@b25][@b26][@b27][@b28][@b29][@b30][@b31]. The 3435C \> T polymorphism of ABCB1, which is a C to T transformation in exon 26 with no change in the amino acid encoded, is not related to AD risk in most studies[@b24][@b25][@b27][@b29][@b30][@b31]. However, Fehér et al.[@b23] and van Assema et al.[@b26] suggested a positive association between the 3435C \> T polymorphism and AD susceptibility. The 2677G \> T/A SNP is a missense mutation in exon 21, leading to an alanine to threonine or serine substitution. The association between the 2677G \> T/A SNP and the risk of AD is also controversial[@b23][@b24][@b26][@b27][@b31]. The ABCB1 1236C \> T polymorphism is a nucleotide change in exon 12 with no change in the amino acid encoded, and van Assema et al.[@b26] suggested 1236C \> T polymorphism might contribute to the progression of Aβ deposition in brain. Because of multiple sites in linkage disequilibrium, haplotypes analysis is necessary to consider the impact of genetic information and guarantee the risk assessment process. In this study, we carried out a meta-analysis to investigate the associations between ABCB1 gene polymorphisms and haplotypes and AD risk. Results ======= Characteristics of eligible studies ----------------------------------- Fourty-nine potential studies were identified after an initial search from the Pubmed, Embase, Web of Science, Medline and Alzgene databases. After further screening, nine studies were finally enrolled in our meta-analysis (2366 cases and 2256 controls). The flow diagram of the search process was shown in [Fig. 1](#f1){ref-type="fig"}. The ethnicities of the populations involved in these studies were Caucasian, Asian and mixed. AD was diagnosed according to the criteria set by the Diagnostic and Statistical Manual of Mental Disorders (DSM) or National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS--ADRDA)[@b32]. Most studies used the Mini Mental State Examination (MMSE) to identify healthy controls[@b33]. The median age of AD cases in these studies ranged from 64 to 82.5 years and the percentage of female patients ranged from 29.4 \~ 74.1%. The methods for gene identification consisted of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), a direct sequencing method and the TaqMan assay. Three common polymorphisms in the ABCB1 gene (3435C \> T, 2677G \> T/A, 1236C \> T) and three haplotypes (1236T/2677T/3435T, 1236T/2677T/3435C, 1236C/2677T/3435T) were assessed. The genotype frequencies of controls were all in Hardy-Weinberg Equilibrium (HWE) (P \> 0.05). The key information of the studies on ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms and AD risk is summarized in [Table 1](#t1){ref-type="table"}. The Magliulo's study[@b28] was not included in [Table 1](#t1){ref-type="table"}, because this study only performed the ABCB1 haplotype analysis. Meta-analysis: ABCB1 3435C \> T SNP ----------------------------------- Eight studies investigated the association between the 3435C \> T SNP and AD susceptibility. The pooled summary crude odds ratios (ORs) and corresponding 95% confidence intervals (CIs) in all the genetic models were as follows: TT vs. CC: OR = 1.04, 95% CI = 0.76--1.41, P = 0.82; CT vs. CC: OR = 1.18, 95% CI = 0.97--1.45, P = 0.10; CT + TT vs. CC: OR = 1.14, 95% CI = 0.91--1.42, P = 0.26; TT vs. CT + CC: OR = 0.91, 95% CI = 0.75--1.11, P = 0.36; T vs. C: OR = 1.01, 95% CI = 0.87--1.17, P = 0.90. A moderate heterogeneity was found between the individual studies (TT vs. CC: χ^2^ = 15.51, P = 0.03, I^2^ = 54.9%; CT vs.CC: χ^2^ = 9.56, P = 0.22, I^2^ = 26.8%; CT + TT vs. CC: χ^2^ = 12.5, P = 0.09, I^2^ = 44%; TT vs. CT + CC: χ^2^ = 11.18, P = 0.13, I^2^ = 37.4%; T vs. C: χ^2^ = 14.64, P = 0.04, I^2^ = 52.2%). We conducted a sensitivity analysis by excluding each study, and found the Fehér's study[@b23] could interpret the heterogeneity, because the statistical power of this study was higher than the others (OR \> 1.25, P \< 0.05). Moreover, after the exclusion of the Fehér's study[@b23], the I^2^ decreased to 0% with a different but statistically significant pooled estimate in the homozygote comparison model (CT vs. CC: OR = 1.24, 95% CI = 1.06--1.45, P = 0.01, [Fig. 2A](#f2){ref-type="fig"}) and the dominant model (CT + TT vs. CC: OR = 1.21, 95% CI = 1.04--1.41, P = 0.01, [Fig. 2B](#f2){ref-type="fig"}), indicating that the Fehér's study[@b23] was not robust. These results suggested that there might be an association between ABCB1 3435C \> T SNP and AD susceptibility. Additionally, subgroup analysis by ethnicity showed a significant association between 3435C \> T SNP and an increased risk of AD among Caucasians (CT vs.CC: OR = 1.23, 95% CI = 1.02--1.47, P = 0.03, [Fig. 2C](#f2){ref-type="fig"}; CT + TT vs. CC: OR = 1.20, 95% CI = 1.01--1.42, P = 0.04, [Fig. 2D](#f2){ref-type="fig"}), but not among Asians or mixed. Pooled ORs and 95% CIs for the association of ABCB1 3435C \> T SNP with AD risk are shown in [Table 2](#t2){ref-type="table"}. Funnel plots were adopted to assess the publication bias, and an evidence of asymmetry was observed (CT vs.CC: P = 0.04, [Fig. 3A](#f3){ref-type="fig"}; CT + TT vs. CC: P = 0.07, [Fig. 3B](#f3){ref-type="fig"}). This result was further supported by the analysis using Egger's test (TT vs.CC: P = 0.28; CT vs.CC: P = 0.01; CT + TT vs. CC: P = 0.01; TT vs. CT + CC: P = 0.61; T vs. C: P = 0.39). However, the funnel plots after the adoption of trim and fill method had perfect symmetry and no significant difference was found between the ORs and 95% CIs before and after, suggesting that there were no change within the crude and adjusted estimates ([Fig. 3C,D](#f3){ref-type="fig"} for CT vs. CC model and CT + TT vs. CC model, respectively). These findings indicated that the meta-estimates for 3435C \> T SNP were robust to confounders and methods of exposure assessment. Therefore, there appeared to be an association between the ABCB1 3435C \> T SNP and an increased AD risk according to our analysis. Meta-analysis: ABCB1 2677G \> T/A SNP ------------------------------------- Five studies investigated the association of 2677G \> T/A SNP with the AD risk. No significant association between 2677G \> T/A SNP and AD susceptibility was detected in the total populations (for TT + AA vs. GG: OR = 0.99, 95% CI = 0.59--1.67, P = 0.97, for GT + GA vs. GG: OR = 1.20, 95% CI = 0.93--1.56, P = 0.16, for GT + TT + GA + AA + TA vs. GG: OR = 1.15, 95% CI = 0.90--1.46, P = 0.27, for TT + TA + AA vs. GT + GA + GG: OR = 0.86, 95% CI = 0.57--1.32, P = 0.50, [Fig. 4A](#f4){ref-type="fig"}, for T + A vs. G: OR = 1.01, 95% CI = 0.80--1.29, P = 0.91). An obvious heterogeneity was observed in most models (for TT + AA vs. GG: χ^2^ = 8.29, P = 0.08, I^2^ = 51.8%, for GT + GA vs. GG: χ^2^ = 1.89, P = 0.76, I^2^ = 0.0%, for GT + TT + GA + AA + TA vs. GG: χ^2^ = 3.72, P = 0.45, I^2^ = 0.0%, for TT + TA + AA vs. GT + GA + GG: χ^2^ = 7.4, P = 0.12, I^2^ = 45.9%, for T + A vs. G: χ^2^ = 7.21, P = 0.13, I^2^ = 44.6%). However, subgroup analysis according to the ethnicity in the recessive model showed a significant association between 2677G \> T/A SNP and a decreased AD risk among Caucasians (TT + TA + AA vs. GT + GA + GG: OR = 0.68, 95% CI = 0.47--0.98, P = 0.04) with no heterogeneity (χ^2^ = 1.33, P = 0.72, I^2^ = 0.0%), but not in mixed ([Fig. 4B](#f4){ref-type="fig"}). Begg's funnel plot and Egger's linear regression test were used to assess the publication bias. All P values of Begg's funnel plots were more than 0.05 (for TT + AA vs. GG: P = 0.81, for GT + GA vs. GG: P = 0.46, for GT + TT + GA + AA + TA vs. GG: P = 0.81, for TT + TA + AA vs. GT + GA + GG: P = 0.81, for T + A vs. G: P = 0.81), as well as the Egger's linear regression test (for TT + AA vs. GG: P = 0.92, for GT + GA vs. GG: P = 0.27, for GT + TT + GA + AA + TA vs. GG: P = 0.66, for TT + TA + AA vs. GT + GA + GG: P = 0.64, for T + A vs. G: P = 0.97), suggesting that there was no publication bias in this meta-analysis. Pooled ORs and 95% CIs for the association of ABCB1 2677G \> T/A SNP with AD risk are shown in [Table 2](#t2){ref-type="table"}, and the funnel plot for the recessive model is demonstrated in [Fig. 5](#f5){ref-type="fig"}. Meta-analysis: ABCB1 1236C \> T SNP ----------------------------------- Four studies were selected to estimate the association of the ABCB1 1236C \> T SNP with the risk of AD. As a result, no statistically significant difference was found in all the genetic models. Subgroup analysis for the 1236C \> T SNP showed no significant differences in overall ORs ([Table 2](#t2){ref-type="table"}). Meta-analysis: ABCB1 haplotypes ------------------------------- Four studies investigated the role of ABCB1 haplotypes in AD, including the 1236T/2677T/3435T (TTT) haplotype, the 1236T/2677T/3435C (TTC) haplotype and the 1236C/2677T/3435T (CTT) haplotype ([Table 3](#t3){ref-type="table"}). The pooling estimates showed the TTC genotype was associated with a significant increase in the risk of AD (OR = 1.99, 95% CI = 1.24--3.18, P = 0.00). The TTT and CTT haplotypes exhibited no association with the AD risk (CTT: OR = 0.58, 95% CI = 0.31--1.08, P = 0.09; TTT: OR = 0.90, 95% CI = 0.56--1.44, P = 0.65, [Table 4](#t4){ref-type="table"}). Discussion ========== As one of the most common forms of dementia, AD affects millions of people worldwide. Recently, several studies have revealed that mutations of ABCB1 can be responsible for the pathogenesis of AD by promoting the accumulation of Aβ via P-gp[@b22][@b23][@b24][@b26]. Although ABCB1 gene variants such as 3435C \> T (rs1045642), 2677G \> T/A (rs2032582), 1236C \> T (rs1128503) polymorphisms have been reported to be associated with AD risk[@b23][@b24][@b26], several researchers have shown opposite results[@b25][@b27][@b28][@b29][@b30][@b31]. To investigate if ABCB1 polymorphisms are associated with the susceptibility to AD, we conducted this meta-analysis to study the relationship between ABCB1 polymorphisms and AD risk. Fehér et al.[@b23] reported that the ABCB1 3435C \> T SNP was associated with AD. Van Assema et al.[@b26] supported that ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T SNPs were related to changes in P-gp function at the BBB in AD patients, and thus may contribute to the pathogenesis of AD. In this study, we found a positive association between the ABCB1 3435C \> T polymorphism and AD susceptibility using a heterozygous comparison model and a dominant model. Sensitivity analysis was applied to ascertain the potential source of the heterogeneity between the studies. After excluding the Fehér's study[@b23] due to its high OR, homogeneity across studies was achieved, and a statistically significant association between the 3435C \> T SNP and AD risk was found. Moreover, subgroup analysis by ethnicity showed a significant association between the 3435C \> T SNP and an increased risk of AD among Caucasians, but not in Asians or mixed races. Trim and fill analysis for 3435C \> T SNP meta-estimates demonstrated no evidence of publication bias in these studies, suggesting a definite association between the ABCB1 3435C \> T SNP and AD risk. Consistent with the report by Cascorbi et al.[@b24], we found that there was an association between the ABCB1 2677G \> T/A SNP and a decreased AD susceptibility among Caucasian, but not in total population, implying that the 2677G \> T/A SNP may play a potential role in the AD pathogenesis. Moreover, our results demonstrated that there was no association between the ABCB1 1236C \> T SNP and AD risk. Due to the existence of multiple genes in linkage disequilibrium, haplotype analysis was useful to identify other indirect SNPs at the same region. As for the ABCB1 gene, the TTT, TTC and CTT haplotypes were most investigated for their roles in AD susceptibility. The studies by Fehér et al.[@b23], Cascorbi et al.[@b24] and Frankfort et al.[@b31] supported that there were no associations between these haplotypes and AD risk. However, Magliulo et al.[@b28] found that the TTT haplotype might play a role in donepezil disposition and clinical outcome. In the present study, we found no association between TTT, CTT haplotypes and the risk of AD, consistent with most studies[@b23][@b24][@b31]. In contrast, although no studies have identified a positive association between the TTC haplotype and AD risk, our meta-analysis showed the TTC haplotype was significantly associated with an increased AD susceptibility. In addition, no heterogeneity was found in the overall models, suggesting that the results of this meta-analysis were statistically robust. A recent Genome-Wide Association Study (GWAS) by Lambert et al.[@b11] found that no SNPs in the ABCB1 gene are genome-wide significant. In this study, we found that the 3435C \> T SNP and 2677G \> T/A SNP were significantly associated with AD risk. Different ethnicities may contribute to the difference between our study and the GWAS, because the GWAS consisted of individuals of European ancestry, while our study included Caucasians and Asians participants. Moreover, the GWAS focused on the possible susceptibility loci for late-onset AD, whereas our study evaluated the association between ABCB1 SNPs and early-onset and late-onset AD. There are some limitations in this meta-analysis. First, although we collected all the eligible publications, studies included in our meta-analysis were still limited. Randomized controlled clinical trials with larger sample sizes are required to confirm our conclusion. Second, AD is a multifactorial disease, and the potential interactions among gene-gene and gene-environment should take into account. Many other factors such as ApoEε4 may participate in the association between ABCB1 polymorphisms and AD risk. However, we did not carry out a subgroup analysis based on it due to limited data. Third, since some studies have not provided informations such as age, gender, diagnostic criteria and sequencing method, we could not carry out a subgroup analysis to study the effects of these parameters on the association between ABCB1 polymorphisms and AD risk. To better understand the role of ABCB1 polymorphisms or haplotypes in AD susceptibility, large-scale, high-quality studies with multi-ethnic populations are needed. In conclusion, based on the published studies, our meta-analysis confirmed that the ABCB1 3435C \> T SNP and TTC haplotype were significantly associated with an increased AD risk, while the 2677G \> T/A SNP may contribute to a lower susceptibility of AD. Our study, for the first time, found that there were associations between ABCB1 polymorphisms and haplotypes and AD risk. Our findings provide a better understanding of AD pathogenesis, and ABCB1 polymorphisms and haplotypes may serve as important targets for the prevention and diagnosis for AD. Methods ======= Selection of studies -------------------- To identify studies eligible for the meta-analysis, a comprehensive search strategy was applied using the electronic databases including Pubmed, Embase, Web of Science, Medline and Alzgene. The following keywords were used: ABCB1 or ATP-binding cassette subfamily B member 1, and polymorphism or rs1045642 or rs2032582 or rs1128503, and Alzheimer's disease or AD. All selected articles were published from January 2001 to March 2016. Other relevant studies were identified by hand searching the references of included articles. Data extraction --------------- Studies included in this meta-analysis met the following criteria: (1) the study had to be focused on the 3435C \> T, 2677G \> T/A, 1236C \> T SNP or haplotypes within the ABCB1 gene using original data; (2) the study should be case-control-designed or cohort-designed; (3) all patients must meet the diagnostic criteria of AD; (4) both AD patients and controls were included; and (5) the frequencies of individual genotypes in cases and controls were reported or provided from the authors. Exclusion criteria included studies with no relevance to ABCB1 polymorphisms and AD risk, overlapping data, and review articles. Each article was checked with inclusion and exclusion criteria independently by two investigators (X.Z. and X.-H.S.). Statistical analysis -------------------- To assess the association between ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms and AD risk, ORs and corresponding 95% CIs were calculated using homozygote comparison (BB vs. AA), heterozygous comparison (AB vs. AA), dominant model (BB + AB vs. AA), recessive model (BB vs. AB + AA), and allele comparison (B vs. A) (A stands for major allele, B stands for minor allele). The significance of the pooled OR was determined by Z-test, and P \< 0.05 was considered as statistically significant. We adopted the random-effects model to calculate the pooled ORs when a significant heterogeneity existed in the genetic models in the initial overall analysis, otherwise a fixed-effects model should be applied. The between-study heterogeneity was estimated using Cochran's Q statistic and the I^2^ statistic. P \> 0.10 in Q-test[@b34] and I^2^ \< 25%[@b35] indicated no heterogeneity among studies. The HWE in the controls was measured by the Pearson's Chi-square test, and the significance level was maintained at P \< 0.05[@b36]. We assessed publication bias by Begg's funnel plot and Egger's linear regression test[@b37], and adjusted for possible publication bias by means of the trim and fill method[@b38]. Moreover, subgroup analysis was conducted to explain the high heterogeneity in the studies according to the ethnicity. All statistical tests were performed using STATA 12.0 software (Stata Corporation, College Station, TX, USA). Additional Information ====================== How to cite this article: Zhong, X. et al. Association between ABCB1 polymorphisms and haplotypes and Alzheimer's disease: a meta-analysis. Sci. Rep. 6, 32708; doi: 10.1038/srep32708 (2016). This study was supported by grants from Program for Liaoning Innovation Research Team in University (NO.LT2014016); Liaoning Province Scientific Research Foundation (2014226033); Key Laboratory Foundation from Shenyang S&T Projects (F16-094-1-00). This study was also supported in part by National Natural Science Foundation of China (No. 81501098) and National Science and Technology Major Projects for "Significant New Drugs Development" (2013ZX09103001-003). The funders have no roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions X.Z. and M.-J.W. conceived and designed the experiments; X.Z. and X.-H.S. searched databases and collected full-text papers; X.Z. and M.-Y.L. extracted and analyzed data; X.Z. wrote the manuscript; M.-J.W. reviewed the manuscript. ![Flow diagram of search strategy and study selection for meta-analysis.](srep32708-f1){#f1} ![Forest plots for the association between the ABCB1 3435C \> T SNP and AD risk.\ (A) heterozygous comparison model for overall populations; (B) dominant model for overall populations; (C) heterozygous comparison model for ethnicity subgroup; (D) dominant model for ethnicity subgroup.](srep32708-f2){#f2} ![Funnel plot analysis and the trim and fill method to detect publication bias of ABCB1 3435C \> T SNP.\ (A) funnel plot analysis in heterozygous comparison model; (B) funnel plot analysis in dominant model; (C) trim and fill method in heterozygous comparison model; (D) trim and fill method in dominant model. Circles represent the weight of the studies and boxes stand for the added studies.](srep32708-f3){#f3} ![Forest plots for the association between ABCB1 2677G \> T/A SNP and AD risk in the recessive model.\ (A) for overall populations; (B) for ethnicity subgroup.](srep32708-f4){#f4} ![Funnel plot analysis to detect publication bias of ABCB1 2677G \> T/A SNP in the recessive model.](srep32708-f5){#f5} ###### Characteristics of individual studies on ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms and AD risk included in the meta-analysis. First Author Year Country Ethnicity Diagnostic criteria MRI/CT MMSE Genotyping Sample size Mean age Female (%) Case Control P~HWE~ ------------------------------------- ------ ----------------- ----------- --------------------- -------- ------ ------------ ------------- ------------------------- ------------ -------- ----------------- ------------------------------ --------- ----------------- ------------------------------ ------ Fehér, Á. 2014 Hungary Caucasian NINCDS/ADRDA No Yes PCR-RFLP 234/225 75.6 ± 6.8/74.8 ± 7.2 67.9/66.7 66 119 49 43 109 73 0.84 Cascorbi, I. 2013 Germany Caucasian --- No No PCR-RFLP 71/81 78.4 ± 10.7/69.5 ± 11.4 54.9/40.7 9 42 20 17 33 31 0.15 Van Assema, D.M. 2012 Dutch Caucasian NINCDS/ADRDA Yes Yes PCR-RFLP 32/17 64 ± 7/47 ± 17 29.4/43.8 2 9 6 8 15 9 0.73 Liu, H.C. 2012 China Asian --- No No --- 111/114 82.5 ± 8.4/77.3 ± 6.6 63.1/52.6 44 52 15 49 44 21 0.06 Kohen, R. 2011 USA Mixed NINCDS/ADRDA No Yes TaqMan 286/240 68.2 ± 9.4/52.5 ± 19.5 48/50 63 138 83 66 114 60 0.44 Li, H. 2008 Canada Caucasian DSM-IV No Yes Sequencing 753/736 77.8 ± 8.6/73.4 ± 7.9 57.6/64.4 158 352 185 168 339 183 0.66 Reiman, E.M. 2007 USA/Netherlands Caucasian --- No No Sequencing 861/550 74.9 ± 6.6/77.4 ± 7.3 --- 188 426 238 139 257 152 0.15 Frankfort, S.V. 2006 Netherlands Caucasian NINCDS/ADRDA Yes Yes Sequencing 48/41 81 ± 5.5/81.9 ± 5.7 68.8/65.9 5 26 17 9 18 14 0.49 2677G \> T/A(rs2032582) GG GT + GA TT + TA + AA G/G GT + GA TT + TA + AA Fehér, Á. 2014 Hungary Caucasian NINCDS/ADRDA No Yes PCR-RFLP 234/225 75.6 ± 6.8/74.8 ± 7.2 67.9/66.7 69 132 33 62 117 46 0.50 Cascorbi, I. 2013 Germany Caucasian --- No No PCR-RFLP 71/81 78.4 ± 10.7/69.5 ± 11.4 54.9/40.7 25 35 11 27 33 21 0.10 Van Assema, D.M. 2012 Dutch Caucasian NINCDS/ADRDA Yes Yes PCR-RFLP 32/17 64 ± 7/47 ± 17 29.4/43.8 3 8 6 10 10 10 0.07 Kohen, R. 2011 USA Mixed NINCDS/ADRDA No Yes TaqMan 286/240 68.2 ± 9.4/52.5 ± 19.5 48/50 73 147 66 78 119 43 0.84 Frankfort, S.V. 2006 Netherlands Caucasian NINCDS/ADRDA Yes Yes Sequencing 48/41 81 ± 5.5/81.9 ± 5.7 68.8/65.9 13 24 13 12 16 11 0.26 1236C \> T(rs1128503) CC CT TT CC CT TT Fehér, Á. 2014 Hungary Caucasian NINCDS/ADRDA No Yes PCR-RFLP 234/225 75.6 ± 6.8/74.8 ± 7.2 67.9/66.7 56 130 48 54 119 52 0.39 Cascorbi, I. 2013 Germany Caucasian --- No No PCR-RFLP 71/81 78.4 ± 10.7/69.5 ± 11.4 54.9/40.7 23 36 12 26 38 17 0.65 Van Assema, D.M. 2012 Dutch Caucasian NINCDS/ADRDA Yes Yes PCR-RFLP 32/17 64 ± 7/47 ± 17 29.4/43.8 3 8 6 11 11 10 0.08 Frankfort, S.V. 2006 Netherlands Caucasian NINCDS/ADRDA Yes Yes Sequencing 48/41 81 ± 5.5/81.9 ± 5.7 68.8/65.9 12 25 11 12 20 9 0.90 MRI/CT: Magnetic Resonance Imaging/Computerized Tomography; P~HWE~: P value of HWE in control. ###### Meta-analysis of the ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms with AD risk. 3435C \> T(rs1045642) N TT vs.CC CT vs. CC CT + TT vs. CC TT vs. CT + CC T vs. C ------------------------------------- --- ---------------------------- ---------------------------- ------------------------------------------------------- ------------------------------------------------------- ------------------------- ---------------------------- -------------- ------- -------------- ---------------------------- -------------- ------- ------ ------------ ------ ------ ------ ------------ ------ ------ Total 8 1.17 0.97--1.40 0.10 0 1.24 1.06--1.45 0.01 0 1.21 1.04--1.41 0.01 0 1.01 0.87--1.16 0.94 0 1.08 0.98--1.18 0.12 0 Ethnicity Caucasian 5 1.15 0.94--1.41 0.17 0 1.23 1.02--1.47 0.03 0 1.20 1.01--1.42 0.04 0 0.99 0.84--1.16 0.89 0 1.06 0.96--1.17 0.26 0 Asian 2 0.80 0.37--1.73 0.56 0 1.32 0.74--2.33 0.35 0 1.15 0.67--1.95 0.61 0 0.69 0.34--1.42 0.32 0 0.97 0.66--1.42 0.86 0 Mixed 1 1.45 0.90--2.34 0.13 0 1.27 0.83--1.94 0.27 0 1.33 0.89--1.98 0.16 0 1.24 0.84--1.83 0.28 0 1.21 0.95--1.54 0.12 0 2677G \> T/A(rs2032582) TT + AA vs. GG GT + GA vs. GG GT + TT + GA + AA + TA vs. GG TT + TA + AA vs. GT + GA + GG T + A vs. G Total 5 0.99 0.59--1.67 0.97 51.8 1.20 0.93--1.56 0.16 0 1.15 0.90--1.46 0.27 0 0.86 0.57--1.32 0.50 45.9 1.01 0.80--1.29 0.91 44.6 Ethnicity Caucasian 4 0.73 0.48--1.12 0.15 0 1.13 0.81--1.58 0.48 0 1.00 0.73--1.37 0.98 0 0.68 0.47--0.98 0.04 0 0.88 0.72--1.08 0.22 0 Mixed 1 1.64 1.00--2.70 0.05 0 1.32 0.88--1.97 0.17 0 1.41 0.96--2.05 0.08 0 1.37 0.89--2.11 0.15 0 1.28 1.00--1.63 0.05 0 1236C \> T(rs1128503) TT vs.CC CT vs. CC CT + TT vs. CC TT vs. CT + CC T vs. C Total 4 0.97 0.64--1.47 0.87 0 1.13 0.80--1.60 0.48 0 1.08 0.78--1.50 0.64 0 0.89 0.63--1.25 0.49 0 0.99 0.81--1.21 0.91 0 ###### Characteristics of studies on ABCB1 1236C \> T-2677G \> T/A-3435C \> T haplotypes and AD risk included in the meta-analysis. First Author Year Country Ethnicity Case Control Haplotype distribution ----------------- ------ ------------- ----------- ------ --------- ------------------------ ---- ----- ---- ---- ----- Fehér, Á. 2014 Hungary Caucasian 234 225 12 25 141 23 10 164 Cascorbi, I. 2013 Germany Caucasian 71 81 1 1 28 2 1 30 Magliulo, L. 2011 Italy Caucasian 54 285 0 17 22 3 37 120 Frankfort, S.V. 2006 Netherlands Caucasian 48 41 3 0 43 1 1 33 ###### Meta-analysis of the ABCB1 1236C \> T-2677G \> T/A-3435C \> T haplotypes and AD risk. 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ANNOUNCEMENT {#s1} ============ Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV), which leads to mild to severe watery diarrhea, vomiting, and dehydration in pigs of all ages and a high mortality rate in neonatal piglets ([@B1], [@B2]). PEDV is an enveloped single-stranded RNA virus that belongs to the genus Alphacoronavirus of the family Coronaviridae ([@B3], [@B4]). Although PED has been reported in China since the 1980s, it was sporadic and regional before 2010 ([@B1]). In 2010, a large-scale outbreak of PED with high morbidity and mortality swept across China, leading to enormous economic losses ([@B5][@B6][@B7]). In February 2018, a stool sample obtained from a naturally infected piglet from Zhejiang Province in China was processed and determined to be positive for PEDV by reverse transcription-PCR (RT-PCR) with primers targeting the N gene. A PEDV strain named ZJ/ZX2018-C10 was isolated using Vero cell cultures as previously described ([@B8]). Viral RNA was extracted from the cell supernatant using the Macherey-Nagel viral RNA purification kit (Macherey-Nagel GmbH & Co. KG, Germany) according to the manufacturer's instructions. To determine the complete genomic sequence of the PEDV isolate of strain ZJ/ZX2018-C10, the extreme 5′ and 3′ termini were acquired by rapid amplification of cDNA ends (RACE, Clontech, Japan). Subsequently, a total of 13 overlapping fragments were amplified by RT-PCR using PrimeSTAR hot-start (HS) DNA polymerase (TaKaRa, China) with primers ([Table 1](#tab1){ref-type="table"}) based upon the sequences of PEDV strain CV777 (GenBank accession number [AF353511](https://www.ncbi.nlm.nih.gov/nuccore/AF353511)). The PCR products were analyzed and confirmed on 1% agarose gel and cleaned with a DNA fragment purification kit (TaKaRa, China). Purified PCR products were cloned into the pMD19-T vector and sequenced in both directions with the Sanger sequencing method (Biosune Biotechnology Shanghai Co., China) on the ABI 3730xl DNA analyzer platform (Life Technologies). For each amplification, three to five individual clones were sequenced to determine the consensus sequence of any given genomic region. Sequence contigs with the consensus sequence were assembled into the full-length genome for the PEDV isolate of strain ZJ/ZX2018-C10 using Lasergene version 7.10 (DNAStar, Inc., USA). ###### Primers used for amplifications of the PEDV genomic fragments Primer name Sequence (5′ to 3′) Position[^a^](#ngtab1.1){ref-type="table-fn"} Product size (no. of nucleotides) ------------- ------------------------------ ------------------------------------------------- ----------------------------------- 1F GCGTTCCGTCGCCTTCTACA 190--209 2,562 1R CAGGAATCTGGAAGACACTTGCA 2751--2729 2F GTATTATGCCACCAGTGTCCCA 2663--2684 2,295 2R CAGTTGCCAGCAGGCACTGT 4957--4938 3F ACCAGCGGTGCATTGCTTGA 4887--4906 2,589 3R CAATGTGCTCTTGCAATCCTGCA 7475--7453 4F CTGTTAAGTTAGTGGACTCAGCGT 7327--7350 2,549 4R ACTAGCGCCTTCAACTTGCA 9875--9856 5F GCGCTTGTGGTTCACCTGGT 9712--9731 2,547 5R GGATCCACAGCGAAAGCGCA 12259--12240 6F ACGCTTGCAGGCTGGTAAACA 12182--12202 2,281 6R TGGGCAGTGCTCTATCGCACT 14462--14442 7F ATACTAGGGGCGCTTCGGTT 14322--14341 2,459 7R GTCAGGGTGCACAGGAATGAA 16780--16760 8F GTATGTGTGCCCTTAAGCCTGAT 16662--16684 2,341 8R GTAAGTGGACGTTCGGCTTCATA 19002--18980 9F TGTATGCCAAGCGTAAGGTAGGAC 18888--18911 1,816 9R ATCTTGTGGTAGGCTGAGTGTTGG 20703--20680 10F GAAGAATGGTAAGTTGCTAGTGCGTA 20563--20588 2,130 10R GGCTAACAACTGTCCAGAATCAGATG 22683--22658 11F AAAGGTGAGTTGATTACTGGCACG 22498--22521 2,164 11R CTAGTAATGACACAACAAAGATGAGAAC 24664--24637 12F GTGTACGATCCTGCAAGTGGC 23272--23292 2,439 12R TCACCTCATCAACGGGAATAGA 25715--25694 13F TCGTCCAATTGGTTAATCTGTGC 25535--25557 2,306 13R TACCGTTGTGTGCAAGACCAA 27840--27820 5′ RACE TCCACTAGCGGCGGCCTCAGAATA 410--387 185 3′ RACE TCAACGAGATCTTCGATACAGGAA 27671--27694 193 Positions correspond to strain CV777 (GenBank accession number [AF353511](https://www.ncbi.nlm.nih.gov/nuccore/AF353511)). The complete genome sequence of strain ZJ/ZX2018-C10 is 28,035 nucleotide (nt) bases long, excluding the poly(A) tail, with a G+C content of 41.72%. The open reading frames for ZJ/ZX2018-C10 were predicted using Genome Annotation Transfer Utility with PEDV strain CV777 as the reference genome. The genomic organization of the virus is in the following order: 5′ untranslated region (UTR), nucleotide 1 to 292; replicase polyprotein, nucleotide 293 to 12616 for 1a and nucleotide 12616 to 20637 for 1b; spike (S) protein, nucleotide 20634 to 24791; open reading frame 3 (ORF3), nucleotide 24791 to 25465; envelope (E) protein, nucleotide 25446 to 25676; membrane (M) protein, nucleotide 25684 to 26364; nucleocapsid (N), nucleotide 26376 to 27701; and 3′ UTR, nucleotide 27702 to 28035. The complete genome sequence of ZJ/ZX2018-C10 shares 98.4%, 97.1%, and 96.3% nucleotide sequence identities with strain AH2012 (GenBank accession number [KC210145](https://www.ncbi.nlm.nih.gov/nuccore/KC210145)), strain virulent DR13 (GenBank accession number [JQ023161](https://www.ncbi.nlm.nih.gov/nuccore/JQ023161)), and vaccine strain CV777 (GenBank accession number [KT323979](https://www.ncbi.nlm.nih.gov/nuccore/KT323979)), respectively. Interestingly, strain ZJ/ZX2018-C10 possesses one unique deletion site compared to strain AH2012 (nucleotide 24225 to 24227) in the S gene, resulting in a single-amino-acid deletion (^1198^T). Of note, the deletion site is in the S2 domain of the S protein. These findings suggest that ZJ/ZX2018-C10 is a novel PEDV variant. The genome information of strain ZJ/ZX2018-C10 will promote a better understanding of the evolutionary characteristics and molecular epidemiology of PEDV in China. Data availability. {#s1.1} ------------------ The genome sequence of PEDV strain ZJ/ZX2018-C10 has been deposited in GenBank under the accession number [MK250953](https://www.ncbi.nlm.nih.gov/nuccore/MK250953). This work was supported by the Zhejiang Science and Technology Key Research and Development Project (2018C02028) and the National Natural Scientific Foundation of China (31802235). [^1]: Citation Su F, Yu B, Li J, Xu L, Yuan X. 2019. Complete genome sequence of variant porcine epidemic diarrhea virus strain ZJ/ZX2018-C10, isolated in Zhejiang, China, in 2018. 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Correction to: Scientific Reports 10.1038/s41598-017-14467-w, published online 25 October 2017 The original version of this Article contained a repeated typographical error in the Abstract and Introduction where, 'Gg' now reads: 'Tg' This has now been corrected in the PDF and HTML versions of the Article.	{ "pile_set_name": "PubMed Central" }	[ "contain", "correct", "repeat", "origin", "tg", "gg", "onlin", "scientif", "articl", "sw", "introduct", "abstract", "error", "pdf", "report", "publish", "read", "version", "html", "octob", "typograph" ]	22,198	[ "Correction", "Scientific", "Reports", "published", "online", "October", "original", "version", "Article", "contained", "repeated", "typographical", "error", "Abstract", "Introduction", "reads", "corrected", "PDF", "HTML", "versions", "Article" ]
Introduction {#Sec1} ============ Medieval Central Asia (ca. AD 2^nd^--16^th^ c.) was a locus of extensive cultural and economic interaction between East Asia, the Middle East, and Europe through a vast network of overland trade routes, commonly called the 'Silk Roads'^[@CR1]--[@CR4]^. Urban centers, located in fertile oases and often described by archaeologists and historians alike as cosmopolitan cities^[@CR5],[@CR6]^, helped anchor Silk Road exchange and foster early globalization across Asia^[@CR7],[@CR8]^. Although settled communities provided a substantial economic foundation through agricultural output and the manufacture of valuable craft commodities such as metals, ceramics, glass, and textiles^[@CR9]--[@CR14]^, mobile pastoralists also had strong influence on the trade system as operators of highland pathways that were based on seasonal movements for herding livestock^[@CR15]^. While transfers of objects and materials have long represented the intensity and scope of Silk Road exchange^[@CR16]^, we still lack detailed data about the way food systems, which reflect sustained engagements with the environment and broader community dynamics, were influenced by these far-reaching economic networks. Medieval Central Asia was defined by unusually diverse multicultural intersections, sudden social upheavals, and frequent demographic movements (Supplementary Information [1](#MOESM1){ref-type="media"}), but fundamental and perhaps durable dealings with food remain unclear. Food is culturally expressive of ecological adaptation, social relations, ideology, and economy^[@CR17]--[@CR21]^. Resolving dietary diversity in ancient Central Asian foodways provides an opportunity to investigate subsistence models of 'nomadic' and 'urban' communities that, together, played key roles in transcontinental interaction across the Silk Roads. In Central Asia, which is characterized by strong seasonal climate and uneven distribution of resources on the landscape^[@CR22],[@CR23]^, there is high potential for dietary diversity among pastoralist communities. Mobile pastoralists heavily subsist on livestock herding but also draw from a variety of food resources beyond domesticated animal products, including cultivated cereals, wild plants, fishes, and hunted game, which are exploited and consumed at varying intensities depending on environment, seasonal availability, mobility, and social networks^[@CR24]--[@CR28]^. On the other hand, medieval urban food systems were strongly invested in cereal agriculture, food storage, and sedentism^[@CR29],[@CR30]^, and were subject to powerful political and religious institutions^[@CR7]^, which may have generated less diverse dietary repertoires. However, dynamic commercial, political, and social activities between population centers and peripheral settlements, in addition to transactions with pastoralists, could have greatly expanded food availability and choice, as people and provisions, such as grains and live animals, regularly moved between urban and nomadic domains^[@CR31]--[@CR35]^. We consider community-level dietary breadth over long periods of human life history to be a marker of dietary connectivity, which represents the cultural integration of food production, distribution, and consumption among individuals. Through globalization processes, which involve growing economic networks between increasingly distant communities, cultural differences diminish as groups cooperate and synchronize their consumption patterns, whether of foods, styles, or ideas^[@CR36]^. In contemporary societies, globalized food economies expand with production standardization and increased dietary uniformity^[@CR37],[@CR38]^. Along these lines, high intra-community dietary variability indicates that community members maintained divergent connections to food resources that express individual dietary preferences and group partitioning. Conversely, low dietary variability within communities signals converged trajectories of foods that reflect shared dietary practices and socio-economic coordination. In order to establish diversity in human dietary intake in Central Asian urban and nomadic communities, we analysed the carbon and nitrogen stable isotopic composition of bone collagen from human remains of 74 individuals (Table [1](#Tab1){ref-type="table"}). Carbon and nitrogen stable isotope ratios (δ^13^C and δ^15^N) provide an integrative measure of dietary intake^[@CR39],[@CR40]^, and human bone collagen reflects a 10--15 year rolling average of protein consumption^[@CR41]--[@CR43]^. We model isotopic niches in bi-variate isotopic space (δ-space) to estimate the breadth and structure of community-level diets based on intra-group variation across both dimensions of isotopic ratios simultaneously^[@CR44],[@CR45]^. Food resources with distinct isotopic content, which are incorporated into individual diets in various proportions, drive community dietary breadth^[@CR46]^. The isotopic niche modelling uses Bayesian inference to fit standard ellipses to data points that are then expressed as probability distributions of area (‰^2^) and position in δ-space^[@CR47]^. Crucially, we performed redundancy analysis to unravel isotopic diversity driven by culturally defined food choices from environmental variables that influence the isotopic composition of food resources.Table 1Geographic and archaeological information about analysed sites, including number of human samples. Detailed archaeological information for each site is provided in Supplementary Information [3](#MOESM1){ref-type="media"}. \See methods.CountryRegionSiteElevation (m.a.s.l.)ChronologynArchaeological contextReferencesUzbekistanWest Pamir-AlayTashbulak21009th--11th c.4Highland urban complex of the Qarakhanid Empire; citadel, metal workshops, necropolis; 7 ha^[@CR86],[@CR120]^Alyntepe47510th--13th c.1Provincial city with fortified walls and surrounding settlements; industrial scale brick and ceramic production; 40 ha^[@CR121]^Frinkent53010th--13th c.4Fortress complex with cemetery of unique Zoroastrian burials in large ceramic vessels; 14 ha^[@CR122],[@CR123]^Ferghana ValleyChor Dona58011th--13th c.4Fortress mound with associated grain processing facility and ancillary ancient settlement of Andijan; estimated 10--15 ha^[@CR124],[@CR125]^Chartok54512th c.11Context information is not available^[@CR126]^Tashkent OasisUturlik27012th c.9Large city with diverse economic production of crafts; along trade routes with Otrar; 60 ha^[@CR34],[@CR127]^KhoresmTok-kala609th--12th c.9Urban fortress and surrounding settlements that functioned as a regional centre of political and economic influence; estimated 10--15 ha^[@CR128]^KazakhstanOtrar OasisKonyr-tobe I1805th--7th c.9Cemetery platform raised 2.5 m above ground level on the outskirts of a fortress; burials suggest nomadic traditions^[@CR81],[@CR129]^Temirlanovka3202nd--4th c.4Cemetery unassociated with a settlement containing burials of nomadic individualsSI 3Zhetysu (Semirech'ye)Turgen II10402nd--6th c.7Settlement and burial complex with nomadic occupations from the late Bronze Age to medieval period; estimated \< 5 ha^[@CR79]^Butakty II115010th--12th c.6Settlement and burial complex with nomadic occupations from the late Bronze Age to medieval period; estimated \< 5 ha^[@CR80],[@CR130],[@CR131]^Karatal6208th--11th c.3Cemetery complex in use from the late Bronze Age to historical period; numerous nomadic encampment structures^[@CR84]^; SI 3TurkmenistanDehistan PlainGeotchik Depe130Iron Age\2Urban complex with occupation from the early Iron Age to late Islamic period; 5.5 ha^[@CR102],[@CR132],[@CR133]^Misrijan17011th--12th c.1One of numerous large villages in the Misrijan Oasis; precise site is not reported in the literature^[@CR102],[@CR132],[@CR133]^ Isotopic variation in Central Asia {#Sec2} ---------------------------------- We sampled 14 cemeteries associated with medieval Silk Road communities that span a long transect of Central Asian geography to include present-day Kazakhstan, Uzbekistan, and Turkmenistan (Fig. [1](#Fig1){ref-type="fig"}). The human individuals analysed in this study represent urban and non-urban consumers, who potentially had access to substantial food options that were available through prolific agricultural systems and marketplaces that drew in people and foodstuffs from oasis, desert, steppe, and highland environments (Supplementary Information [2](#MOESM1){ref-type="media"}). Diverse food remains were recovered from the sites represented in this study, which included cereals, legumes, fruits, fish, and livestock (Supplementary Information [3](#MOESM1){ref-type="media"}).Figure 1Map of Central Asia showing sites and regions with human stable isotopic data (δ^13^C and δ^15^N) analysed in this paper. Uzbekistan: 1) Tok-kala, 2) Uturlik, 3) Chor Dona, 4) Chartok, 5) Tashbulak, 6) Altyntepe, 7) Frinkent; Turkmenistan: 8) Geoktchik Depe, 9) Misrijan; Kazakhstan: 10) Konyr-Tobe, 11) Temirlanovka, 12) Turgen, 13) Butakty, 14) Karatal. Map generated with Quantum GIS, version 2.18.2 (<https://www.qgis.org>), using public domain data from Natural Earth (<http://www.naturalearthdata.com>). Pronounced variation in the regional environments and topographies, combined with strong seasonality, in which these sites are situated, confers high isotopic variation in Central Asian foodwebs accessed by people. Vegetation communities range from cool montane meadows and forests consisting of largely C~3~ taxa to hot lowland deserts that support both C~3~ and C~4~ plants^[@CR48]--[@CR51]^, which exhibit (pre-modern^[@CR52],[@CR53]^) δ^13^C values of −25 ± 2--5‰ and −11 ± 1‰, respectively^[@CR54]--[@CR57]^. Food crops exploited in medieval Central Asia included the full spectrum of Eurasian domesticates including C~3~ taxa, such as wheat, barley, rice, nuts, and fruits, and C~4~ taxa, such as millets^[@CR58]^. Although nitrogen isotopic variation in vegetation communities in Central Asia is under-characterized, research in ecosystems comparable to those of Central Asia, such as the Gobi steppe-desert^[@CR59]^, the steppe deserts of the Caspian Depression^[@CR60]^, and semi-arid western Loess Plateau^[@CR61]^, demonstrate wide variation in plant δ^15^N values, ranging from −5 to 14‰, due to differences in local soil nitrogen pools and animal stocking rates^[@CR62]--[@CR65]^. In general, fish exhibit high δ^15^N values relative to terrestrial fauna^[@CR66],[@CR67]^, and in Central Asia, fish exhibit an apparent continuum of δ^13^C values from ca. −11.5‰ to −27‰^[@CR68]^. Results {#Sec3} ======= Human remains in this study represent two chronological intervals of the medieval period. The bulk of the dataset (n = 63) dates to a 'mid-late' period of 6^th^--13^th^ c., which were recovered from sites in southern Kazakhstan, Uzbekistan, and western Turkmenistan. A small sample (n = 11) dates to an 'early' period of nomadic occupation at sites in southern Kazakhstan, which allows for a diachronic comparison of nomadic dietary intake within this region. Overall, human isotopic values range from ca. −20‰ to −10.5‰ for δ^13^C and 9‰ to 15‰ for δ^15^N (Fig. [2a](#Fig2){ref-type="fig"}). Isotope values cluster in both δ^13^C and δ^15^N for urban communities on a regional basis, while pastoralist communities from later periods exhibit wide distributions of δ^13^C values and relatively narrow ranges of δ^15^N values. Subsequent analyses using Bayesian inference clarified isotopic differences among communities while factoring in uncertainty due to small sample sizes. Summary statistics of isotopic data are provided in Supplementary Figure [S1](#MOESM1){ref-type="media"} and Table [S1](#MOESM1){ref-type="media"}; raw isotopic data are provided in Supplementary Tables [S2](#MOESM1){ref-type="media"}--[S4](#MOESM1){ref-type="media"}.Figure 2(a) Human carbon and nitrogen isotopic ratios from medieval Central Asia; (b--c) Posterior probability distributions of isotopic means obtained by Bayesian bootstrapping (mean~b~) from medieval urban communities in Uzbekistan and Turkmenistan and (d-e) from medieval nomadic communities in southern Kazakhstan. Human isotopic variation across medieval Central Asia {#Sec4} ----------------------------------------------------- Significant distinctions in the dietary intake of medieval urban and nomadic communities across Central Asia are revealed by Bayesian means (mean~b~) of δ^13^C and δ^15^N values. Sharp dissimilarities are observed between urban communities located south of the Syr-Darya river, which provides a rough environmental boundary between the semi-arid/mountain steppe landscapes in southern Kazakhstan and sandy desert landscapes interspersed with oases and foothill zones to the east. Significant differences in the 95% CIs of isotopic values from each southerly community, with the exception of West Pamir-Alay and Tashkent, are present with mean~b~ δ^13^C values of −19.9‰ for Dehistan, −18.1‰ for West Pamir-Alay, −17.5‰ for Tashkent, −16.3‰ for Ferghana, and −13.3‰ for Khoresm (Fig. [2b](#Fig2){ref-type="fig"}; Information 4). Regional communities in Uzbekistan and Turkmenistan fall into in three trophic groups, each separated by ca. 2‰ in mean~b~ δ^15^N (Fig. [2c](#Fig2){ref-type="fig"}). A diachronic shift in the dietary intake of nomadic communities located in southern Kazakhstan is indicated by a significant 3‰ decrease in mean~b~ δ^13^C values between early and late Otrar (Fig. [2d](#Fig2){ref-type="fig"}; Supplementary Information [4](#MOESM1){ref-type="media"}) and an increase of ca. 2‰ in mean~b~ δ^13^C between early and late Zhetysu. However, mean~b~ δ^13^C for late Zhetysu has a wide 95% CI between −15.9 and −12.3‰, which precludes a reliable estimate of the change. Overall, mean~b~ δ^15^N for communities in southern Kazakhstan are more mutually similar to each other than that in southerly regions (Fig. [2c,e](#Fig2){ref-type="fig"}). Early Otrar displays mean~b~ δ^15^N that is spaced apart by slightly less than ca. 1.7‰ from that of early Zhetysu, while in the later period the regions have identical mean~b~ δ^15^N of ca 12‰. Environment and isotopic variation {#Sec5} ---------------------------------- Isotopic patterns observed at individual archaeological sites are not driven by environmental inputs. Redundancy analysis between mean δ^13^C and δ^15^N values per cemetery site and 25 environmental parameters of ecologically relevant rainfall and temperature variations, in addition to elevation and soil properties, did not result in statistically significant relationships. Multiple linear regressions were also performed, which further failed to generate statistically significant relationships. (See Supplementary Information [5](#MOESM1){ref-type="media"} for methods and results.) Isotopic niche modelling {#Sec6} ------------------------ Central Asian medieval communities display isotopic niches that cluster in two distinct size ranges (Fig. [3](#Fig3){ref-type="fig"}; Supplementary Information [6](#MOESM1){ref-type="media"}). Group 1 exhibits small areas between 0.1 and 3.7‰^2^ for urban communities in Uzbekistan and Turkmenistan, as do nomadic communities in southern Kazakhstan from the early medieval period. Over the next several hundred years, dietary diversity among individuals in Zhetysu and the Otrar Oasis (Group 2) appears to have radically increased, with isotopic niches estimated between 2.7 and 15.7‰^2^. Notably, the greatest intra-community dietary diversity is visible in late Zhetysu, indicated by a 95% CI exceeding that from Group 1 communities, except early Otrar, which slightly overlaps by 0.7‰^2^.Figure 3Community-level dietary diversity of medieval humans represented by posterior distributions of core isotopic niche area (‰^2^) by sites and regions in Central Asia. Isotopic niches were calculated by fitting standard ellipses to cover ca. 39% of the δ^13^C and δ^15^N data points using Bayesian inference. Black dots indicate area means, and the shaded boxes, from dark to light, represent the 50%, 75%, and 95% credible intervals. While all narrow, modelled isotopic niches for medieval urbanites in Uzbekistan are highly unique in orientation and position in δ-space (Fig. [4a](#Fig4){ref-type="fig"}). For late Zhetysu and Otrar, the diffuse spacing of isotopic values indicates exceedingly varied dietary intake. Individuals in late Zhetysu, which represent two archaeological sites, fall within the isotopic niches for Khoresm, Ferghana and Tashkent. Likewise, individuals in late Otrar, which represent one site, span all four urban isotopic niches from Uzbekistan. In contrast, there is substantial proportional overlap between the small isotopic niches from early Zhetysu and Ferghana and also early Otrar and Khoresm, respectively (95% CI: 0.13--0.64 and 0.17--0.69; Supplementary Fig. [S6](#MOESM1){ref-type="media"}), which indicates a high likelihood of dietary parity.Figure 4(a) Medieval urban isotopic niches from Uzbekistan are displayed as probability clouds, and individual isotopic values from southern Kazakhstan (nomadic communities) and western Turkmenistan (urban community) are represented as points. (b) Isotopic niche overlap analysis for urban communities in Uzbekistan. Standard ellipses covering 95% of δ^13^C and δ^15^N values were modelled using Bayesian inference. Overlapping areas for each pairwise comparisons in δ-space were visualized as probability clouds with underlying isotopic data points superimposed (lower left). Area overlap of total isotopic niche area for each pair was plotted as probability distributions (upper right). Isotopic niches from urban communities in Uzbekistan illustrate strong dissimilarity from each other (Fig. [4b](#Fig4){ref-type="fig"}). The highest proportion of niche overlap occurs between West Pamir-Alay and Ferghana (95% CI: 0.05--0.36) and between West Pamir-Alay and Tashkent (95% CI: 0.01--0.32), suggesting that a maximum of one-third of individuals in these communities had similar dietary intake. However, this niche overlap could be as low as 1--5%. The remaining pairwise comparisons of urban communities show negligible occurrence of overlap (Supplementary Information [7](#MOESM1){ref-type="media"}). Discussion {#Sec7} ========== Taken together, urban and nomadic communities display distinctive but wide-ranging isotopic values that are strongly suggestive of diverse dietary intake across medieval Central Asia. A large overall range of human δ^13^C values from ca. −20 to −11‰ is likely due to individuals consuming mostly C~3~ crops (wheat, barley, and rice) or C~4~ millets at sustained intensities. Among Eurasian cultigens, millets are isotopically distinct with high ^13^C concentration. Millets thrive in hot and arid climate and exhibit fast-growing and drought-tolerant adaptations^[@CR69]^, traits which would have provided Central Asian farmers and mobile pastoralists opportunities for low risk, low-investment cultivation in marginal agricultural areas^[@CR70],[@CR71]^. The natural abundance of C~4~ vegetation is substantially higher in the desert zones of Central Asia^[@CR50]^, where livestock, as part of urban or nomadic subsistence, could have accessed enriched δ^13^C biomass and provided human consumers with protein-dense foods (meat and milk) with high δ^13^C values. A lack of correlations between human isotopic values and site environmental parameters suggests that dietary intake across the region was shaped primarily by food choice. Medieval agriculture in Central Asia, which used complex crop schedules and large irrigation works^[@CR71]--[@CR76]^, likely enabled productivity to overcome environmental constraints on crop variety in order to meet the inter-connected dietary demands of consumers. Modelled isotopic niches indicate that nomadic communities exploited a wide variety of dietary resources, while urban communities engaged in more limited dietary repertoires. The small isotopic niche sizes documented among urban communities in Uzbekistan and Turkmenistan suggest food channels that were shaped via consistent and insular dietary connectivity. On the other hand, large isotopic niches in southern Kazakhstan from a relatively contemporaneous period indicate that a multitude of food acquisition strategies were in use, by which communities tied more closely with pastoral nomadic lifeways comprised individuals with assorted dietary relationships that led to sustained differences in isotopic variability. The dietary connectivity for these nomadic groups may have fostered group partitioning through unsynchronized food interactions among different community members. The small isotopic niches for nomadic communities in early Otrar and Zhetysu, which in this case substantially overlap with that for Khoresm and Ferghana, suggesting more restricted dietary intake by pastoralists, emphasize the subsistence plasticity of pastoral nomads who readily contour their own food production and interaction networks in response to dynamic social and natural landscapes^[@CR24],[@CR26],[@CR77]^. The diachronic shift in isotopic niche size between early and late nomadic communities also highlights two distinct scales of dietary variability that illustrate the importance of multi-resource pastoralism to Silk Road interactions. In southern Kazakhstan, the early medieval period is marked by a growth of urban centres, villages, and agricultural economies^[@CR78]--[@CR81]^, which is also historically associated with frequent conflict among nomadic confederacies that instigated socio-political turmoil^[@CR1],[@CR82]^. In order to mitigate risk and take advantage of economic opportunities presented by these newly founded centres, nomadic communities likely participated in coordinated subsistence interactions with settled populations over short distances, which would have effectively limited access to diverse food resources and thus narrowed their dietary breadth. During the strengthening of Turkic empires several centuries later^[@CR1],[@CR3],[@CR31],[@CR83]^, Silk Road trans-regional trading expanded to include bulk commodities and raw materials^[@CR32]^, and nomadic communities across southern Kazakhstan expressed wide dietary breadth, as indicated by large isotopic niche sizes. One explanation for greater inter-individual dietary diversity during this later medieval period is that nomadic communities tapped into growing trade economies as agents of food exchange and broke out of insular urban subsistence channels. Recent excavations of nomadic encampments in the foothill zones of West Pamir-Alay and Zhetysu illustrate highly variable levels of economic interaction between pastoralists and urban centres. At these sites, cultural materials associated with 8^th^--13^th^ c. radiocarbon chronologies include cotton fabrics^[@CR84]^ and variable mixtures of ceramics ranging between standardized wheel-spun food storage vessels from distant oases communities and locally produced 'handmade' coarsewares, which are rare in urban contexts^[@CR85],[@CR86]^. The presence of hybrid ceramic assemblages and cotton, a woven trade good associated with oasis production centers^[@CR87],[@CR88]^, suggests that complex and non-uniform relationships with urban economies coincided with intra-group dietary diversity in mobile pastoralist communities^[@CR25]^. Accordingly, at the community level, a second scale of dietary variability that is representative of multi-resource pastoralism is observed at late Otrar and Zhetysu, which display wide ranges of δ^13^C values (Fig. [4a](#Fig4){ref-type="fig"}). Some individuals in late Zhetysu have δ^13^C values similar to those commonly observed in humans from prehistoric millet-based farming societies in China, where millets were domesticated^[@CR89]--[@CR91]^, while individuals in late Zhetysu and Otrar had low δ^13^C values, typical of Neolithic and early Bronze Age humans before millet spread to the C~3~-dominant Central Asian steppe^[@CR92]^. Both communities in late Otrar and Zhetysu display similar δ^15^N distributions, though individuals exhibit differences in dietary intake of relative proportions of meat and dairy products (Fig. [2e](#Fig2){ref-type="fig"}). Together, these findings suggest that dietary connectivity at the steppe margins was associated with an ecumene of diverse food consumption, in which individuals maintained separate subsistence strategies as they simultaneously participated in a common nomadic ethos. Compared to urbanites, mobile pastoralists likely maintained closer control of food production and distribution, allowing them to eat according to food preferences, which may have been less important for maintaining social ties than in urban contexts. The combination of narrow dietary niches with isotopic distinction in human remains from medieval urban communities south of the Syr-Darya river is due to trophic-level variation in the intensity of meat and cereal consumption between communities as well as differences in the contribution of millet to urban diets, either eaten directly or indirectly obtained from meat and dairy of animals foddered with millet. Isotopic niche overlap was highest for urban communities in close geographic proximity to one another, a pattern that suggests neighbouring communities either participated in similar food traditions or agricultural practices that were confined to small catchments. These communities may have participated in limited inter-regional trading of staple foods, which likely would have moved through established subsistence channels as if locally produced. There is also the possibility that dietary connectivity in urban contexts was subject to bureaucratic intermediaries, which exerted influence through land tenure and taxation^[@CR1],[@CR32],[@CR93]^. Alternatively, in the absence of top-down control, food exchange networks may have steadily channelled provisions to urbanites as a reflection of other economic networks that inevitably developed to be streamlined towards cultural insiders in cosmopolitan contexts. While the consensus among historians and archaeologists is that urbanites in medieval Central Asia dwelled in rich multicultural settings^[@CR5],[@CR7],[@CR16]^, there appears to be a limit to this diversity in dietary intake as revealed through isotopic niche modelling. Distinctions in food choice and diet between urban communities suggest regional food repertoires were narrowly circumscribed, at least between C~3~ and C~4~ crops and also between animal and plant protein. Regional patterns in diet imply that cultural differences surrounding foods may have been surprisingly diminished within urban communities, which runs counter to the notion of collective cosmopolitanism in medieval Central Asia. Medieval urbanites in Central Asia maintained inward-focused dietary connectivity that likely generated a localized social cohesion through culturally integrated supply chains for consumers. Scholars also associate Silk Road activity with early globalization processes^[@CR7],[@CR8]^, in which urban centres are viewed as the main drivers of cultural influence and outward economic connectivity, while 'nomads' are interpreted as antagonists to ancient civilization^[@CR94]--[@CR96]^. Yet, through multi-resource subsistence strategies, nomadic communities likely wielded flexible economic engagements that traversed open landscapes of contact with people who facilitated far-reaching connectivity. In this sense, nomadic individuals may have been more culturally interoperable and able to participate in, disengage from, and influence cultural spheres more easily than urban populations. Indeed, many of the pan-regional turnovers in religion, language, and political authority that resulted in changes in architecture, technologies, and other commodity classes in the medieval period are historically described as nomadic innovations^[@CR1],[@CR2],[@CR31]^, and essential routes that connected Silk Road sites in the highland regions of Central Asia were likely shaped by nomadic mobility^[@CR15]^. This study takes a new step toward resolving the complex interplay between urban and nomadic societies that are rarely available through archaeological datasets. Establishing dietary diversity provides an emerging understanding of food and connectivity along Central Asia's Silk Roads that highlights the significance of ancient nomadic pastoralists in bridging seemingly insulated urban centres. Materials and Methods {#Sec8} ===================== Human remains {#Sec9} ------------- We performed new analyses on human remains from Uzbekistan and Kazakhstan, which were selected based on 1) medieval chronology from ca. 8^th^--13^th^ c., 2) information on archaeological context, and 3) minimum age estimation of young adult. The majority of human remains analysed from Uzbekistan (n = 38) were excavated at various times over the past 80 years and do not include associated post-cranial elements. Human remains from Tashbulak (n = 4) were excavated in 2015 and are represented by complete inventories of skeletal elements. Human remains from Uzbekistan are stored in the Institute of Archaeology of the Uzbek Academy of Sciences in Samarkand, under the auspices of the Archaeology of the Qarakhanids Project (Co-PIs: Farhod Maksudov and Michael Frachetti). Human remains from Karatal (n = 3) were excavated in 2006 and 2008 and include incomplete skeletal elements due to ancient burial looting and modern erosion (Supplementary Information [3](#MOESM1){ref-type="media"}). Human remains from Karatal are stored at the Central State Museum of Kazakhstan in Almaty, under the auspices of the Dzhungar Mountain Archaeology Project (Co-PIs: Alexei Mar'yahshev and Michael Frachetti). A list of human samples is provided in Supplementary Tables [S2](#MOESM1){ref-type="media"}--[S4](#MOESM1){ref-type="media"}. Isotopic analysis {#Sec10} ----------------- Approximately 1--2 cm^3^ of sample were cut from dense cortical bone. Collagen extraction was performed in the Archaeological Stable Isotope Laboratory of Kiel University following Tuross et al.^[@CR97]^. Mass spectrometry for δ^13^C and δ^15^N was performed at the Boston University Stable Isotope Laboratory using a EuroVector Euro EA elemental analyser coupled with a GVI IsoPrime in continuous flow mode with an analytical error of 0.1‰ and 0.2‰ for δ^13^C and δ^15^N, respectively. Isotopic values are reported in permil (‰) relative to the Vienna Pee Dee Belemnite (VPDB) standard for δ^13^C and atmospheric nitrogen (AIR) for δ^15^N. Collagen samples with an elemental C:N ratio less than 2.9 or greater than 3.6 were considered diagenically altered and unsuitable for inclusion^[@CR98]--[@CR100]^. Failed samples (n = 3) are reported in Supplementary Table [S5](#MOESM1){ref-type="media"}. Previously published δ^13^C and δ^15^N values of human bone collagen from southern Central Asia were analysed (Supplementary Tables [S3](#MOESM1){ref-type="media"}-[S4](#MOESM1){ref-type="media"}). Isotopic data from medieval southern Kazakhstan included 13 samples from the Otrar Oasis (3^rd^--7^th^ c.) and 13 samples from southern Zhetysu (2^nd^--12^th^ c.)^[@CR101]^. Data from the Dehistan Plain in western Turkmenistan included two samples from the Iron Age (ca. 1300 BC) and one sample from the medieval period (ca. 11^th^--12^th^ c.)^[@CR102]^. These data were lumped together irrespective of chronology due to low sample size for the region and similar isotopic values, which were unique in our dataset. Human collagen is δ^13^C enriched by ca. 1--3‰ relative to the carbon isotope composition of consumed foods^[@CR103]^, whereas herbivores are enriched in δ^13^C by ca. 5‰ relative to consumed vegetation^[@CR104],[@CR105]^. Human nitrogen isotope values reflect the intensity of cereal versus meat consumption, as there is a 2--5‰ trophic enrichment in δ^15^N with each step up in the food web^[@CR40],[@CR106],[@CR107]^, but also reflect variation in food production systems that introduce exogenous nitrogen, usually in the form of manure, to the floral base of the food web, potentially imparting considerable nitrogen isotopic variation in agricultural and livestock food^[@CR62],[@CR63],[@CR65],[@CR108],[@CR109]^. In attempts to estimate the relative contribution of specific foods in diets, a common approach in archaeological dietary studies is to sample ancient and modern plants and animals to assess possible isotopic variability in food sources that result from various environmental and anthropogenic factors^[@CR110]^. For the purposes of establishing human dietary diversity at the community level, however, knowledge of the isotopic content of ancient food remains is unnecessary. Statistical analysis {#Sec11} -------------------- All statistical analyses were performed using R, version 3.4.0^[@CR111]^. Due to low sample sizes per site (1 ≤ n ≤ 11), δ^13^C and δ^15^N values were analysed by geographic region (3 ≤ n ≤ 15) using Bayesian techniques to quantify uncertainty and overcome issues with non-parametric data distributions, which cannot be reliably analysed with frequentist methods. The means of isotopic values for each region were calculated with a Bayesian bootstrapping method at 5000 iterations using the package bayesboot^[@CR112]^. Posterior distributions of means for each group were considered statistically different from one another if the 95% credible intervals (CI) did not overlap. Isotopic niche modelling {#Sec12} ------------------------ Isotopic niches were analysed using SIBER (Stable Isotope Bayesian Ellipses in R), version 2.1.3^[@CR47]^. The Markov chain Monte Carlo simulation was run with uniform priors 2,000,000 times, with the first 10,000 results discarded (burn-in), followed by a 1:100 thinning. The fitted ellipses express a posterior probability distribution of area (‰^2^) and position in δ-space. This technique is statistically advantageous for analysing communities of consumers, as fitted ellipses are less sensitive to sample size than other spatial metrics, such as convex hulls^[@CR47]^, and uncertainty is factored into estimates, such as sample size, which has previously challenged studies using a finite number of specimens from the archaeological record. Isotopic niches were compared among regional groups by using the Bayesian standard ellipse areas (SEA~b~) and the proportion of SEA~b~ overlap as total SEA~b~ pairwise for each region. Overlapping areas of isotopic niches indicate similarity in isotopic inputs and thus comparable resource exploitation^[@CR113]--[@CR115]^. Environmental modelling and GIS {#Sec13} ------------------------------- Interactive effects between human stable isotope values and environmental parameters at each site in 10 km and 50 km spatial buffers were explored using redundancy analysis (RDA) with the R package vegan, version 2.4--5^[@CR116]^. Environmental parameters included elevation, bioclim data^[@CR117]^, which are derived from monthly temperature and rainfall values from 1970--2000, and soil parameters obtained from the Soil Grid Project^[@CR118]^. (See Supplementary Information [5](#MOESM1){ref-type="media"} for RDA methods and results.) Data were mapped using Quantum GIS, version 2.18.2^[@CR119]^. Base imagery in Fig. [1](#Fig1){ref-type="fig"} was obtained from Natural Earth (<http://www.naturalearthdata.com>). Electronic supplementary material ================================= {#Sec14} Supplementary Information Electronic supplementary material Supplementary information accompanies this paper at 10.1038/s41598-018-22995-2. Publisher\'s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. We especially thank Amridin Berdimuradov, the director of the Institute of Archaeology in Samarkand (National Academy of Sciences of Uzbekistan) for his continued support and enthusiasm for this project and international collaborations in general. We also thank Dima Voyakin for providing access to archaeological reports on Temirlanovka and for his help facilitating research in Kazakhstan. Recognition is also due to Marc Meyer and Mellissa Murphy for providing osteological data for the human individuals excavated from the Karatal cemetery complex. A special thank you goes to Malte Rühlemann for assisting with the redundancy analysis. Funding for this research was provided by the Graduate School 'Human Development in Landscapes' at Kiel University for the doctoral research of Taylor Hermes. Research at Tashbulak was funded by the National Geographic Society, the Max van Berchem Foundation, and Washington University in St. Louis. We are grateful for support from the Open Access Publikationsfonds of Land Schleswig-Holstein. Finally, we thank two anonymous reviewers for their suggestions on improving this paper. T.R.H. designed and initiated the research. T.R.H., E.A.B., F.M., and S.M. conducted sample collection. E.A.B. performed the osteological analysis of human remains and, with T.R.H., described sites in Uzbekistan for the supplementary information. T.R.H. wrote the remaining supplementary information. T.R.H. prepared specimens for stable isotope analysis and conducted the data analysis. T.R.H. and C.A.M., and M.D.F. interpreted the results. T.R.H., M.D.F., and C.A.M. wrote the manuscript. Competing Interests {#FPar1} =================== The authors declare no competing interests.	{ "pile_set_name": "PubMed Central" }	[ "efigreftypefig", "bc", "wheat", "vienna", "contextscrcr", "probabl", "interspers", "choic", "two", "fish", "bioclim", "commod", "transect", "control", "kazakhstan", "sandi", "carlo", "divers", "remain", "auspic", "hybrid", "nitrogen", "individualssi", "decreas", "compris", "inevit", "webcrcrcr", "textilescrcr", "contextreferencesuzbekistanwest", "purpos", "abund", "collect", "discard", "overland", "thank", "interoper", "network", "osteolog", "spectrum", "risk", "regularli", "herm", "rais", "select", "fund", "altyntep", "take", "valu", "convers", "narrow", "areascrcr", "locat", "bridg", "multipl", "signal", "may", "oas", "move", "taylor", "readili", "recent", "sudden", "live", "foster", "ea", "toward", "separ", "gener", "use", "settingscrcrcr", "socioeconom", "constraint", "‰figur", "support", "languag", "enthusiasm", "collabor", "herbivor", "final", 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"Laboratory", "using", "EuroVector", "Euro", "EA", "elemental", "analyser", "coupled", "GVI", "IsoPrime", "continuous", "flow", "mode", "analytical", "error", "respectively", "Isotopic", "values", "reported", "permil", "relative", "Vienna", "Pee", "Dee", "Belemnite", "VPDB", "standard", "atmospheric", "nitrogen", "AIR", "Collagen", "samples", "elemental", "C", "N", "ratio", "less", "greater", "considered", "diagenically", "altered", "unsuitable", "Failed", "samples", "n", "reported", "Supplementary", "Table", "media", "Previously", "published", "values", "human", "bone", "collagen", "southern", "Central", "Asia", "analysed", "Supplementary", "Tables", "media", "media", "Isotopic", "data", "medieval", "southern", "Kazakhstan", "included", "samples", "Otrar", "Oasis", "samples", "southern", "Zhetysu", "Data", "Dehistan", "Plain", "western", "Turkmenistan", "included", "two", "samples", "Iron", "Age", "ca", "BC", "one", "sample", "medieval", "period", "ca", "data", "lumped", "together", "irrespective", "chronology", "due", "low", "sample", "size", "region", "similar", "isotopic", "values", "unique", "dataset", "Human", "collagen", "enriched", "ca", "relative", "carbon", "isotope", "composition", "consumed", "whereas", "herbivores", "enriched", "ca", "relative", "consumed", "Human", "nitrogen", "isotope", "values", "reflect", "intensity", "cereal", "versus", "meat", "consumption", "trophic", "enrichment", "step", "food", "also", "reflect", "variation", "food", "production", "systems", "introduce", "exogenous", "nitrogen", "usually", "form", "manure", "floral", "base", "food", "web", "potentially", "imparting", "considerable", "nitrogen", "isotopic", "variation", "agricultural", "livestock", "attempts", "estimate", "relative", "contribution", "specific", "foods", "diets", "common", "approach", "archaeological", "dietary", "studies", "sample", "ancient", "modern", "plants", "animals", "assess", "possible", "isotopic", "variability", "food", "sources", "result", 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Introduction {#Sec1} ============ Bone age assessment (BAA) is crucial in the evaluation of endocrine disorders and in the prediction of adult height when hormone therapy is the treatment \[[@CR1]\]. Radiographic examination is an easy and cost-effective method for the assessment of bone age. BAAs are performed using various body parts such as the hand, elbow, knee, cervical vertebra or pelvis \[[@CR2]\]. Hand radiography is certainly the most widely used examination \[[@CR3]\]. Several methods have been proposed, including Greulich-Pyle (GP) atlas, Tanner-White (TW) score and Gilsanz-Ratibin (GR) atlas, which are qualitative \[[@CR4]\]. The TW method is based on the level of maturity for 20 regions of interest (ROIs), including the epiphysis, metaphysis and diaphysis in the radius, ulna, first, third and fifth fingers and the carpal bones, so it is complex and time-consuming. The GR method is another technique that uses the new digital atlas that includes reference images of the left hand taken at 6-month intervals from birth to age 6 years, and at 1-year intervals from age 7 to 17 years. However, this subjective method relies on matching of the hand radiograph of the subject with the GR atlas. In the GP method, bone age is determined by matching the left-hand radiograph of the subject with reference radiographs from the atlas. This explains why the GP method is generally easier and quicker to use, so it is more often selected over other methods \[[@CR5]--[@CR8]\]. One disadvantage of the GP method is the characteristics inherent in subjective analysis, and the interobserver reproducibility of BAA with the GP method has been controversial \[[@CR5], [@CR7]\]. A quantitative evaluation method with objective parameters is needed. BAA by using the capitate and hamate, which are the first bones to develop embryologically, has been recommended by a previous study but not performed as of yet \[[@CR9]\]. In addition, validation of BAA across different ethnicities would be necessary for a more reliable method \[[@CR10]\]. Therefore, the aim of this study was to determine if the area of the capitate and hamate bones measured on hand radiography could be a reliable indicator of bone age accuracy, precision, and reproducibility between the planimetry and GP methods. Materials and methods {#Sec2} ===================== Populations {#Sec3} ----------- In this study, all data were collected from the electronic medical records of patients. From January 2014 to December 2015, 423 patients (age 1--180 months) presented to the emergency department. The study population was selected according to the following inclusion criteria: PA radiography of both hands, age younger than 15 years and belonging to a single ethnic group. The following patients were excluded: those with either only right-hand (n=42, 30 boys and 12 girls) or only left-hand (n=33, 24 boys and nine girls) PA radiograph, one with acute leukaemia (one boy) one with idiopathic hypertrophic pyloric stenosis (one boy) and those with other ethnicities (one Chinese, one Japanese and one American) \[[@CR1], [@CR10]\]. Finally, a total of 391 consecutive patients (242 boys and 149 girls) were enrolled in this study. In total, we analysed 782 hands of 391 patients (242 boys and 149 girls). The chronological age, sex and ethnicity of the patients were recorded. The mean patient age was 5 years and 8.52 months (age range 1--180 months). The patients complained of contusion (n=222), laceration (n=175) and/or fracture (finger, 40; humerus, 54). The absence or presence of capitate and/or hamate bones of the hand was registered in all patients. According to the growth chart published by the Korea Centers for Disease Control and Prevention (CDC) in 2007, among patients aged 1 month to 3 years, those whose height was between the 50th percentile of a child who is 1 month younger and the 50th percentile of a child who is 1 month older were selected \[[@CR11]\]. Among patients aged 3--14 years, those whose height was between the 50th percentile of a child who is 6 months younger and the 50th percentile of a child who is 6 months older were selected \[[@CR12]\]. A total of 109 children (mean age 113.07 months; standard deviation 36.07) were included in the 50th percentile group. Equipment and imaging protocol {#Sec4} ------------------------------ Hand radiography was performed using Innovion-SH (DK Medical Systems, Seoul, Korea). The patients were seated alongside or facing a table, and examined in the PA position. The protocols used in this study for taking the PA radiographs of both hands are described in Table [1](#Tab1){ref-type="table"}. Both hands were in neutral position with no flexion, extension or deviation, and were placed palm down on the cassette with the fingers extended \[[@CR13]\].Table 1Imaging protocol of both hands posterior-anterior (PA) x-rayProjectionPosterior-anterior bilateral projectionDetector size24×30 cmOrientationLandscapeExposure50--60Kvp/2--5mAsGridNoSID100 cmCentral rayBetween the two hands at the level of the metacarpophalangeal jointsCollimationTo include one-third of the distal radius and ulna\ to include soft tissuesSID source to image-receptor distance To assess reliability of measurements, calibration was performed using a coin (real value of the coin diameter, 26.51 mm; measured value of the coin diameter, 26.53 mm; relative error, 0.08 %). In this process, the source to image-receptor distance (SID) was set to 100 cm (Table [1](#Tab1){ref-type="table"}). To prevent projection errors, the x-ray source was directed perpendicular to the cassette (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1(a) The diameter of a coin measured using a digital micrometre was 26.51 mm. (b) The diameter of the coin on a digital radiograph was measured as 26.53 mm on a Picture Archiving and Communication System (PACS) Image analysis {#Sec5} -------------- Four radiologists (three with 21, 18 and 12 years experience in radiology, respectively, and one first-year resident), blinded to the clinical data, analysed the images on a Picture Archiving and Communication System (PACS; G3, Infinitt Healthcare, Seoul, Korea). For planimetric analysis, the first and second radiologists (with 18 and 12 years experience in radiology, respectively) independently measured the area of the capitate and the hamate on PACS, respectively. ROIs were drawn manually on plain radiographs to encompass the entire capitate and hamate separately. Area measurements of the capitate and hamate were performed for both right and left hands (Fig. [2](#Fig2){ref-type="fig"}).Fig 275-month (6 years and 3 months)-old boy. (a and b) The areas of the bilateral capitate bones (solid line) and bilateral hamate bones (dotted line) were measured by drawing two separate regions of interest in the posterior-anterior (PA) radiograph of both hands When the ROI values were \> 10 % different between the two reviewers, another series of measurements were performed by the same two reviewers to reach a consensus. Differences \< 10 % were considered negligible, and the average was reported \[[@CR7]\]. The CH planimetry measurement was calculated by summing the area of the capitate and that of the hamate as follows: CH planimetry = area of capitate + area of hamate. The values of CH planimetry were obtained from both hands. For the analysis of accuracy and reproducibility for BAA, the third and fourth reviewers (with 21 years experience in radiology and the first-year resident, respectively) measured the area of the capitate and hamate of children who were included in the 50th percentile group. Each reviewer independently assessed the bone age of children using the CH planimetry method. On the basis of the GP method, the reviewers estimated the bone age of the 50th percentile group at 2 weeks after the planimetry assessment. Statistical analysis {#Sec6} -------------------- All statistical analyses were conducted using SPSS (version 20; IBM Corporation). Linear regression analysis was used to analyse the relationship between the planimetry measurement of each hand and the chronological age of each sex. Correlation coefficients of ≤ 0.3 were considered to indicate a weak relationship; 0.30--0.70, moderate relationship; and \> 0.70, strong relationship \[[@CR14]\]. A z-test was used to compare two correlation coefficients. Differences in the planimetry measurements between the right and left CH and in the accuracy and precision between two reviewers were analysed by using a paired t-test. A p-value of \< 0.05 was considered statistically significant. To compare the two methods, Deming regression analysis and accuracy determination were performed. Accuracy was calculated using the difference percentage between the estimated bone age and the chronological bone age of the 50th percentile group. To assess the reproducibility of the measurements between the third and fourth reviewers, precision, Bland-Altman analysis and Lin's concordance correlation coefficient (ρ~c~) were used. Precision was defined as the percentage difference in the estimated bone ages between two reviewers. The strength of ρ~c~ was categorized as follows: ρ~c~ \< 0.90, poor agreement; 0.9 ≤ ρ~c~ ≤ 0.95, moderate; 0.95 \< ρ~c~ ≤ 0.99, substantial; ρ~c~ \> 0.99, almost perfect agreement. Results {#Sec7} ======= The age and sex distribution of the Korean subjects are listed in Table [2](#Tab2){ref-type="table"}. There were 242 boys (mean age 75.33 months; age range 1--180 months) and 149 girls (mean age 57.46 months; age range 1--178 months).Table 2Age and sex distribution of the individualsAge (months)/SexBoys (n=242)Girls (n=149)1--1113912--23373124--35331736--47212648--59161360--7114672--83171284--956596--107135108--119107120--13143132--143184144--155143156--16795168--17916318010Data represent number of patients For 241 boys (99.59 %) and 149 girls (100 %), irrespective of age, left and right capitate and hamate bones were demonstrated on the radiographs of both hands. The right capitate and bilateral hamates were not demonstrated on the radiograph in only one boy (0.41 %) aged 1 month (Fig. [3](#Fig3){ref-type="fig"}).Fig. 3One-month-old boy. (a and b) Left capitate bone (arrow) is demonstrated but right capitate bone and bilateral hamate bones are not demonstrated on the posterior-anterior (PA) radiograph of both hands There were no significant differences in areas between the left and right capitate and hamate of both sexes (Table [3](#Tab3){ref-type="table"}). Additionally, there was also no significant difference in the sum of the area of the capitate and the area of the hamate between right and left hands.Table 3Differences in the values of planimetry of boys and girlsGenderLocationRight (95 % CI)Left (95 % CI)p-value\Boy\ (n=242)Capitate130.52 (117.22--142.64)129.94 (117.23-142.64)0.3049Hamate87.52 (78.48--96.57)87.00 (78.07-95.93)0.1691Capitohamate218.04 (196.32--239.76)216.94 (195.38-238.50)0.1333Girl\ (n=149)Capitate95.70 (84.00--107.41)94.70 (83.17-106.22)0.3611Hamate64.69 (56.66--72.71)64.16 (56.23-72.10)0.2545Capitohamate160.39 (140.75--180.02)158.86 (139.53-178.20)0.1779CI* confidence interval\A paired t-test was used to assess the difference of the means The planimetry curves of the capitate and hamate and the sum of the CH areas demonstrated increased slopes in both sexes (Fig. [4](#Fig4){ref-type="fig"}). There was a strong positive correlation between chronological age and the CH planimetry measurement (Table [4](#Tab4){ref-type="table"}). Moreover, strong correlations were found between chronological age and the capitate area of both sexes, as well as between the chronological age and hamate area (Table [4](#Tab4){ref-type="table"}).Fig. 4High positive relationships between chronological age and planimetries of capitates, hamates and CHs of the right (a) and left (b) hands of boys and right (c) and left (d) hands of girls. CH* capitohamateTable 4Differences in the correlation coefficients between planimetry and chronological age of boys and girlsGenderLocationRight (95 % CI)Left (95 % CI)z-statisticBoyCapitate0.9711 (0.9629--0.9775)0.9729 (0.9652--0.9789)0.9729Hamate0.9607 (0.9496--0.9693)0.9597 (0.9484--0.9686)0.8886Capitohamate0.9702 (0.9618--0.9768)0.9709 (0.9627--0.9774)0.8951GirlCapitate0.9659 (0.9531--0.9752)0.9509 (0.9327--0.9643)0.1118Hamate0.9552 (0.9386--0.9674)0.9526 (0.9351--0.9655)0.8052Capitohamate0.9664 (0.9538--0.9756)0.9580 (0.9424--0.9695)0.3313CI confidence interval In the linear equation y = a + bx, y stands for capitate, hamate and CH area; x stands for age in months; and constants (a and b) were calculated. The equations of planimetry for the left hands of boys and girls are as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\displaystyle \begin{array}{l}\mathrm{CH}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{boy}\right)=3.0571\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-13.3391\\ {}\mathrm{CH}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{girl}\right)=2.5856\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-10.2867\\ {}\mathrm{Capitate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{boy}\right)=1.8057\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-6.6278\\ {}\mathrm{Capitate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{girl}\right)=1.5303\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-6.7608\\ {}\mathrm{Hamate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{boy}\right)=1.2514\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-7.2632\kern0.5em \\ {}\mathrm{Hamate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{girl}\right)=1.0552\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-3.5258\end{array}} $$\end{document}$$ By using the CH planimetry and GP methods, the third and fourth reviewers estimated the bone age of 109 patients who were classified into the 50th percentile group. None of the mean areas of the capitate, hamate and sum of the capitate and hamate were significantly different between the two reviewers. The mean value of chronological age of 109 children (mean age 113.07 months; standard deviation 36.07) was not significantly different from that of the estimated bone age measured by both CH planimetry and GP methods (p ≥ 0.1378). There was no statistically significant difference in the mean value of bone age between the two reviewers using CH planimetry. However, the mean values of bone age were significantly different between the two reviewers assessing the bone age according to the GP atlas (Table [5](#Tab5){ref-type="table"}). The mean value of bone age estimated using the GP method was lower than that measured using CH planimetry (p \< 0.0001).Table 5Descriptive statistics of the area and bone age estimated by two reviewers in the 50th percentile groupReviewer 3Reviewer 4p-value\Area (mm^2^) Capitate185.94 ± 65.42184.11 ± 67.970.1128 Hamate121.45 ± 44.63120.25 ± 46.230.2487 Sum307.39 ± 108.92304.36 ± 112.820.0729BAA (months) CH planimetry118.58 ± 39.52117.43 ± 40.960.0687 GP method105.49 ± 39.00109.49 ± 39.380.0003Data are means ± standard deviation\A paired t test was used to assess the difference of the meansBAA bone age assessment, CH capitohamate, GP Greulich-Pyle atlas method When assessing bone age based on the CH planimetry and GP methods, there was no significant difference in the accuracy between the third (CH planimetry, 84.39 % ± 22.33 %; GP method, 85.15 % ± 13.78 %) and fourth (CH planimetry, 84.46 % ± 21.29 %; GP method, 87.66 % ± 12.34 %) reviewers (p ≥ 0.0867) (Fig. [5](#Fig5){ref-type="fig"}). Deming regression analysis showed no statistically significant difference in the results between the CH planimetry and GP methods, as the standard errors can be determined from the 95 % confidence intervals (CIs) of intercept (standard error, 4.0028; 95 % CI −4.9522--10.9164) and slope (standard error, 0.03955; 95 % CI, 0.9917--1.1485) including the values 0 and 1, respectively (Fig. [6](#Fig6){ref-type="fig"}).Fig. 5Graph showing accuracy of CH planimetry and GP method in assessing the bone age of 50th percentile group between two reviewers. A paired t-test demonstrated no statistically significant differences between two methods and between two reviewers (p-value ≥ 0.0867). Accuracy was defined as the difference percentage between the estimated bone age and the chronological bone age of 50 percentile group. CH capitohamate, GP Greulich-PyleFig. 6(a) Deming regression analysis for comparison of CH planimetry and GP atlas method in assessing bone age. (b and c) Bland-Altman plots of two reviewers show the interobserver reproducibility for CH planimetry and GB method for bone age assessment. The 95 % limits of agreement were narrower in CH planimetry than in GP method. CH capitohamate, GP Greulich-Pyle We analysed the interobserver variability of the CH planimetry and GP methods. The values of ρ~c~ were substantial for both methods for assessing bone age (CH planimetry, 0.9862; GP method, 0.9553) between the two reviewers. However, the precision of CH planimetry (4.42 % ± 3.87 %) was significantly different from that of the GP method (8.45 % ± 8.52 %) (p \< 0.001). The Bland-Altman analysis demonstrated that CH planimetry had narrower 95 % limits of agreement (−10.5 to 13.4 months) than the GP method (−29.5 to 21.1 months) (Fig. [6](#Fig6){ref-type="fig"}). These results suggest that the interobserver reproducibility of CH planimetry may be greater than that of the GP method. Discussion {#Sec8} ========== In our study, there were no significant differences noted in the areas between the right and left capitates and between the right and left hamates. Patterson et al. \[[@CR15]\] reported that the volume of carpal bones was not significantly different between the left and right hands, using three-dimensional (3D) computed tomography (CT) volumetry. However, hand PA radiography for bone assessment is usually performed on the left side rather than on the right side, and perhaps because most people are right-handed, the right hand is more prone to get injured and therefore may have deformity. In the early 1900s, a physical anthropologists meeting determined that body measurements should be performed on the left \[[@CR3]\]. The right capitate and bilateral hamates were not demonstrated on the radiograph in only one boy (0.41 %) aged 1 month. The capitate and hamate are known to emerge most frequently and simultaneously between the first and fourth months from birth; however, occasionally, the capitate emerges first, followed by the hamate. As seen in the GP atlas, the nuclei of the capitate and hamate were more likely to be present earlier in girls than in boys \[[@CR16]\]. There was also a strong positive correlation between chronological age and the CH planimetry measurement (Table [3](#Tab3){ref-type="table"}). As shown in Fig. [3](#Fig3){ref-type="fig"}, there were increased slopes in the planimetry curves of the capitates and hamates of boys and girls. Canovas et al. \[[@CR17]\] reported that there was a strong correlation between chronological age and volumes of the carpal bones of the hands measured using 3D CT volumetry. The strongest correlations were found with the triquetrum, capitate and hamate bones, which were present in all 20 hands. There was no significant difference between the two methods in accuracy for estimating bone age (CH planimetry, 84.39--84.46 %; GP method, 85.15--87.66 %). The research performed by Roche et al. showed that the median bone ages estimated using the GP method were similar to that of the chronological ages \[[@CR18]\]. On the contrary, some researchers reported that the difference between chronological age and bone age estimated using the GP method was −0.6 to 2.15 years \[[@CR10], [@CR19]--[@CR21]\]. The accuracy of BAA with the GP method has been reported to vary depending on the patient's age or sex and ethnicity \[[@CR22]\]. To date, few studies have evaluated the percentage accuracy of BAA using the GP method. Lee et al. \[[@CR23]\] reported that a deep learning system for BAA achieved 57.32 % and 61.40 % accuracies for the female and male cohorts when age was precisely matched. The accuracy of matching bone age within 1 year was 90.39 % and 90.39 %, respectively. In our study, the mean bone age measured using the GP method (105.49--109.49 months) was significantly lower than the mean bone age measured using CH planimetry (117.43--118.58 months). This result may be attributed to the difference in growth patterns among different ethnicities because the GP method was established in Caucasians \[[@CR3]\]. Some researchers reported that bone age measured with the GP method tended to be lower than the chronological age or the bone age measured with the TW3 method or the Korean bone standard method, although there was no significant difference between the estimated bone age and chronological age \[[@CR21], [@CR24]\]. These previous studies may have shown no statistically significant difference because of the small number of children or targeting children of a limited age range. In the second part of our study, the interobserver reproducibility of CH planimetry was greater than that of the GP method. The values of ρ~c~ were substantial for both methods (CH planimetry, 0.9862; GP method, 0.9553). In Bland-Altman analysis, the 95 % limit of agreement of CH planimetry was −10.5 to 13.4 months and that of the GP method was −21.1 to 29.5 months (Fig. [6](#Fig6){ref-type="fig"}). The precision of CH planimetry (4.42 % ± 3.87 %) was significantly different from that of the GP method (8.45 % ± 8.52 %) (p \< 0.001). To date, there have been many comparative studies on the interobserver reproducibility of BAA with the GP method by using various statistical tests \[[@CR5], [@CR7]\]. Bull et al. \[[@CR7]\] reported that the 95 % CI of the GP method (−2.46 to 2.18 years) was greater than that of the TW2 method (−1.48 to 1.43 years). King et al. \[[@CR5]\] reported that the average spread of intra-observer variation was 0.96 years for the GP method. These results are similar to those of our study. By contrast, the percentage precision of the GP method was 65.5−88.5 % \[[@CR8], [@CR25]\]. Improving the observer variability of manual methods is a motivation for developing automated methods of BAA \[[@CR26]\]. To date, computer-assisted or artificial intelligence-based methods have attempted to make accurate measurements, with discrepancies from 0.39 to 2.41 years \[[@CR27]\]. This study has several limitations. First, it was performed retrospectively at a single institution. We excluded children with chronic illnesses, nutrition deficiency or growth problems from this study population. However, to our knowledge, this planimetric study is the largest series assessing the areas of the capitate and hamate. Second, CT scans provide 3D measurements, whereas radiography demonstrates the 2D projected images of 3D objects; therefore, volumetry can be more accurate than planimetry, and may determine bone age more objectively \[[@CR17]\]. However, CT volumetry has some disadvantages such as high cost and radiation exposure. In contrast, x-ray planimetry has the advantages of lower costs and lower radiation doses \[[@CR28]\]. Moreover, planimetry is an easy-to-use method with an area measurement tool integrated in PACS without any separate software \[[@CR29], [@CR30]\]. Third, manually drawing ROIs along the cortical margin of the capitate and hamate may cause interobserver variation, and problems in planimetric analysis. To minimize this variation, another series of consensus measurements was performed in cases in which the differences between the reviewers exceeded 10 % \[[@CR7], [@CR31]\]. Fourth, quantitative analyses undertaken using digital radiographs displayed on a PACS monitor could be subject to measurement errors \[[@CR32]\]. To avoid this error, to ensure the accuracy of the absolute measurement value, calibration was performed using a coin prior to our study \[[@CR33]\]. Different equipment and protocols among different institutions may cause measurement errors. In the future, a multicentre prospective study should be conducted while avoiding this error. Finally, there is no gold standard for BAA \[[@CR29]\]. We set a reference standard with a range \< 1 standard deviation of the Korea CDC growth chart. Children were enrolled in the reference standard only if their heights were between the 50th percentile of the pervious age stage and the 50th percentile of the later age stage, so that the height ranges do not overlap \[[@CR12]\]. Moreover, as the height of Korean children has changed since the growth chart was published in 2007, it may be necessary to adjust the age range of the reference heights \[[@CR11]\]. In conclusion, the CH planimetry method may be useful for BAA. In the future, if automated CH planimetry can be integrated in PACS with validation for ethnicity and sex, it may save time and allow more precise BAA. CH : Capitohamate GP : Greulich-Pyle TW : Tanner-White GR : Gilsanz-Ratibin PACS : Picture Archiving and Communication System ROI : Region of interest This research was supported by the Hallym University Research Fund (HUFR-2017-27). Guarantor {#FPar1} ========= The scientific guarantor of this publication is Young Chul Kim. Conflict of interest {#FPar2} ==================== The authors of this manuscript declare no relationships with any companies whose products or services may be related to the subject matter of the article. Statistics and biometry {#FPar3} ======================= One of the authors has significant statistical expertise (Young Chul Kim). Ethical approval {#FPar4} ================ Institutional Review Board approval was obtained. Informed consent {#FPar5} ================ Written informed consent was waived by the Institutional Review Board. 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All relevant data are within the paper. Introduction {#sec001} ============ Proper neuronal function necessitates a highly regulated extracellular environment in the brain. Accumulation of interstitial solutes, such as amyloid β and toxic compounds \[[@pone.0229702.ref001]\] may lead to degenerative diseases, such as Alzheimer's disease \[[@pone.0229702.ref002],[@pone.0229702.ref003]\] or even autism \[[@pone.0229702.ref004]\]. Although the Blood Brain Barrier is thought to be the primary mechanism involved in controlling the brain blood-exchange, the existence of a fluid driven transport system (so-called glymphatic system) via cerebrospinal fluid (CSF) or interstitial fluid (ISF) has been proposed recently as a waste clearance system through the perivascular and interstitial spaces in the brain \[[@pone.0229702.ref001],[@pone.0229702.ref005]\]. Hypothetically, CSF crosses the astrocyte end-feet bound to arteries in the perivascular space \[[@pone.0229702.ref006],[@pone.0229702.ref007]\]. After washing the interstitial space, the resulting ISF is flushed back outside the brain via veins in the perivascular space. Several factors play a crucial role in the modulation of this clearance system activity, notably sleep and anesthesia, perhaps via a modulation of brain blood volume and pressure \[[@pone.0229702.ref008]--[@pone.0229702.ref011]\]. This scheme gives astrocytes a crucial role in controlling water movements between the blood and the brain, through a mechanism dependent on Aquaporin-4 (AQP4), a membrane-bound water channel expressed at their end-feet \[[@pone.0229702.ref012],[@pone.0229702.ref013]\]. AQP4 is involved in the rapid volume regulation of astrocytes \[[@pone.0229702.ref014]\] and deletion of the AQP4 gene suppresses the clearance of soluble amyloid β \[[@pone.0229702.ref001]\]. Although most studies have been performed in vitro, some studies have shown astrocyte volume changes in vivo using 2-photon microscopy. Acute osmotic and ischemic stress induce astrocyte volume changes in vivo mice \[[@pone.0229702.ref015]\], and Thrane et al showed that the astrocyte volume change induced by osmotic stimulation was inhibited in AQP4 KO mice \[[@pone.0229702.ref016]\]. Overall, such studies suggest that dynamic volume change of astrocytes, through water flux mediated by AQO-4 channels may be associated with CSF flow regulation. Actually, astrocytes end-feet are involved in the CSF/ISF exchanges in perivascular space during sleep/awake cycle \[[@pone.0229702.ref017]\]. Recently MRI has been proposed as a more versatile approach to investigate the glymphatic system in vivo, using intrathecal or intravenous injections of gadolinium-based contrast agents (GBCAs) as tracers \[[@pone.0229702.ref007],[@pone.0229702.ref018]\]. However, this approach remains invasive and, paradoxically, gadolinium has been shown to deposit in the brain \[[@pone.0229702.ref019],[@pone.0229702.ref020]\] possibly in relation to a lack of brain drainage \[[@pone.0229702.ref010]\]. Therefore, alternative methods are needed to investigate the glymphatic system non-invasively, especially in the human brain. Fluid-dynamics driven and BOLD fast MRI have the potential to evaluate CSF pulsations in the ventricles and hemodynamics \[[@pone.0229702.ref011],[@pone.0229702.ref021],[@pone.0229702.ref022]\], while IVIM and diffusion MRI have been shown as promising methods for the evaluation of the ISF \[[@pone.0229702.ref007],[@pone.0229702.ref023]--[@pone.0229702.ref027]\]. Those considerations led us to investigate whether diffusion MRI was sensitive to astrocyte activity and, in turn, could become a marker of the overall glymphatic system. Diffusion MRI is exquisitely sensitive to changes in tissue microstructure, notably cell swelling \[[@pone.0229702.ref028]\]. Diffusion MRI is, for instance, sensitive to astrocyte swelling induced in rodents \[[@pone.0229702.ref029]\]. Hence, we hypothesized that dynamic changes in astrocytes activity and related volume changes could be monitored directly and non-invasively with diffusion MRI. To test this hypothesis we monitored variations of new diffusion MRI markers, namely the Sindex and the sADC, which have been tailored to increase sensitivity to tissue microstructure through water diffusion hindrance \[[@pone.0229702.ref030],[@pone.0229702.ref031]\] upon acute inhibition of astrocyte AQP4 channels in a mouse brain model using 2-(nicotinamide)-1,3,4-thia-diazole (TGN-020), a compound that blocks AQP4 channels in vivo in the mouse brain \[[@pone.0229702.ref032]\]. Material and methods {#sec002} ==================== Animal preparation {#sec003} ------------------ Thirty-two male C57BL6 mice (16--28 g, 4--10 weeks, Charles River, France) were allocated to two groups. The choice of a mouse brain model was motivated by prospect of using our protocol later to AQP-4 knock-out mice. First, for the TGN-020 group, 16 mice received an intra-peritoneal injection of 250mg/kg TGN-020 diluted in a gamma-cyclodextrine solution (10 mM) in order to increase its solubility. Second, for the control group, sixteen mice received an intra-peritoneal injection of the vehicle solution only (10 mM gamma-cyclodextrine in saline). The mice were housed on a 12-hour light-dark cycle and fed standard food ad libitum. Anesthesia was induced using 3% isoflurane in a mix of air and oxygen (air: 2 L/min, O2: 0.5 L/min). Then, 0.015 mg/kg of dexmedetomidine was administered intraperitoneally and followed by a continuous infusion of 0.015 mg/kg/h via subcutaneous catheter and maintained isoflurane at 0.8%. Throughout the acquisition, the animals' body temperature was maintained between 36.5 and 37.0 °C using heated water (Grant TC120, Grant Instruments, Shepreth, UK). To avoid motion-related artifacts the head was immobilized using a bite bar and ear pins. The respiration rate was monitored and stable (60--90 /min) throughout the experiment. All animal procedures used in the present study were approved by an institutional Ethic Committee (Comité d'Ethique en Expérimentation Animale, Commissariat à l'Energie Atomique et aux Énergies Alternatives, Direction des Sciences du Vivant (Fontenay-aux-Roses, France)) and by Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (France) (reference APAFIS\#8462-2017010915542122v2) and were conducted in strict accordance with the recommendations and guidelines of the European Union (Directive 2010/63/EU). This manuscript is in compliance with the ARRIVE guidelines (Animal Research: Reporting in Vivo Experiments) on how to REPORT animal experiments. MRI experiments {#sec004} --------------- The MRI experiments were conducted on a Bruker 11.7T scanner (Bruker BioSpin, Ettlingen, Germany) equipped with a gradient system allowing a maximum gradient strength of 760 mT/m. A cryo-cooled mouse brain coil was used. Animal positioning was performed using multi-slice fast low angle shot imaging (FLASH, TE/TR = 2.3/120 ms). A global first and second order shim was achieved followed by a local second order shim over the brain parenchyma. Structural (anatomical) images were acquired with the following parameters: T2 TurboRARE sequence, TE/TR = 11.15/2500 ms. Diffusion-weighted echo-planar imaging (DW-EPI) data sets were acquired with the following parameters: 150x150x250 μm^3^ resolution, 3 b-values (0, 250, 1750) s/mm^2^ along 6 directions, NA = 4, TE/TR = 36.3/2300 ms, 18 slices, diffusion time = 24 ms, total scan time = 5 min. The area covered by the 18 slices of the scans encompassed 5 mm in the axial plane with the center of the slab positioned at the middle of the brain. Six DW-EPI sets were first acquired (baseline) before TGN-020 or vehicle was injected. Then, after the injection, 12 additional DW-EPI data sets were acquired ([Fig 1](#pone.0229702.g001){ref-type="fig"}) every 5 minutes. ![Schematic figure of MRI experiment protocol.\ The vertical arrow indicates the saline or TGN-020 injection. DWI, diffusion weighted MRI; NR, number of repetitions. Horizontal arrow indicates the timeline.](pone.0229702.g001){#pone.0229702.g001} Data processing {#sec005} --------------- ### Preprocessing {#sec006} Regions of interest (ROIs) were originally created using the Allen Brain Atlas \[[@pone.0229702.ref033]\] and registered to a mouse brain MRI template to create a labeled atlas. We referred to previous studies based on microscopy \[[@pone.0229702.ref034]\] to assess the vascular distribution within the selected brain structures. The atlas was then co-registered with the b = 0 images of the first scans for each subject, and not the other way around to avoid changing raw voxel signals. Those preprocessing steps, also including radiofrequency bias field correction \[[@pone.0229702.ref035]\] and denoising were performed using ANTs (<https://stnava.github.io/ANTs/>) \[[@pone.0229702.ref036]\] ([Fig 2](#pone.0229702.g002){ref-type="fig"}). We assumed geometric distortion to be negligible; this condition was qualitatively checked on several mice for all b values. Furthermore, the standard-deviation of the signal intensity across scans was evaluated for each b value and each voxel (after Gaussian smoothing) was estimated as a marker of motion or instability. A threshold of 4% was used to flag unstable voxels. Data sets were at least one of the ROIs contained flagged voxels were discarded.\[[@pone.0229702.ref037]\]. ![Example of ROI registration with ANTs on a mouse brain.](pone.0229702.g002){#pone.0229702.g002} ### DWI analysis {#sec007} First, a shifted ADC (sADC) was computed using signal acquired at two key b values (instead of the standard values of 0 and 1000s/mm^2^), chosen to optimize signal sensitivity to both Gaussian and non-Gaussian diffusion which is more sensitive to tissue microstructure through hindrance effects \[[@pone.0229702.ref030],[@pone.0229702.ref031]\]. $$sADC = \frac{\ln\left( \frac{S_{Lb}}{S_{Hb}} \right)}{Hb - Lb}$$ where Lb is the low key b value (250s/mm^2^), Hb the high key b value (1750 s/mm^2^), S~Lb~ the signal at low key b value and S~Hb~ the signal at high key b value. Second, data were analyzed using the Sindex method \[[@pone.0229702.ref031]\]. The Sindex diffusion marker has been designed to identify tissue types or conditions based on their microstructure \[[@pone.0229702.ref031]\]. The Sindex was calculated from the direction-averaged, normalized signals, S~V~(b) in each voxel, as the algebraic relative distance between the vector made of these signals and those of 2 signature tissue signals S~A~ in condition A, and S~B~ in condition B, at each key b value as \[[@pone.0229702.ref031]\]: $$SI(V) = \left\{ {{\max\left( {\frac{\left\lbrack {{dS}_{V}\left( {Hb} \right) - {dS}_{V}\left( {Lb} \right)} \right\rbrack}{\left\lbrack {{dS}_{B}\left( {Hb} \right) - {dS}_{B}\left( {Lb} \right)\rbrack} \right.},0} \right)} - \left\lbrack {{\max\left( \frac{\left\lbrack {{dS}_{V}\left( {Hb} \right) - {dS}_{V}\left( {Lb} \right)} \right\rbrack}{\left\lbrack {{dS}_{A}\left( {Hb} \right) - {dS}_{A}\left( {Lb} \right)} \right\rbrack} \right.},0} \right)} \right\}$$ with dS~V~,~A,B~(b) = \[S~V~,~A,B~(b)- S~N~(b)\]/S~N~(b). S~N~ is taken as an intermediate signal between S~A~ and S~B~. SI was then linearly scaled as Sindex = (SI+1)\25+25 to be centered at 50. A tissue with status similar to condition A has Sindex* = 75, while for a status similar to condition B one has Sindex = 25. The library of the 2 reference diffusion MRI signals was built in advance from previously acquired data, one representing a generic mouse brain tissue (B) and another derived by simulating a moderate increase in diffusion hindrance (A) using the Kurtosis diffusion model \[[@pone.0229702.ref031]\]. For this study, S~A~(Lb) = 0.858; S~A~(Hb) = 0.370; S~B~(Lb) = 0.855; S~B~(Hb) = 0.317. Obviously the Sindex is expected to vary widely across brain regions according to their degree of diffusion hindrance ([Fig 3](#pone.0229702.g003){ref-type="fig"}), for instance white matter regions have higher Sindex values than gray matter regions. However, our focus was on the local changes in Sindex values induced by the injection of TGN-020, reflecting local changes in the degree of diffusion hindrance (a decrease in Sindex reflecting a decrease in hindrance). Sindex and sADC were calculated on a voxel-by-voxel basis to generate parametric maps. Calculation was also performed on a ROI level, averaging signals from all voxels within the ROI. ROIs were placed over the cerebral cortex, the striatum and the hippocampus (whole hippocampus, CA3 region and dentate gyrus, DG). The hippocampus is known to be reached in AQP4 expression \[[@pone.0229702.ref038],[@pone.0229702.ref039]\]. ![Sindex (first row) maps and sADC (second row) maps for a representative mouse from vehicle group (first column) and from TGN-020 group (right column). These maps were obtained by averaging the 6 last time points.](pone.0229702.g003){#pone.0229702.g003} Values obtained before (6 first scans) and after injection (6 last scans) were averaged in each ROI for each animal. The parameter time course was also calculated by averaging 3 successively acquired datasets (resulting in six time points with a resolution of 15 minutes) for each animal. Before averaging across individual subjects, we performed an outlier exclusion using a z-score filter (z\>3) calculated over the subject's parameter values for each time point. Statistical analysis {#sec008} -------------------- The statistical tests were performed in python (Python Software Foundation. Python Language Reference, version 3.7. Available at [http://www.python.org](http://www.python.org/)). We performed a paired two sample t-test between pre and post-injection data in each group and a pair-wise t-test with a posthoc correction for multiple comparison to compare the post-injection data between two groups and the pre-injection data between two groups. For the time course data, we performed a two sample t-test between the two groups for each time point. Results {#sec009} ======= Averaged Sindex and sADC following vehicle or TGN-020 injection {#sec010} --------------------------------------------------------------- [Fig 3](#pone.0229702.g003){ref-type="fig"} shows brain maps of Sindex and sADC for a representative mouse following vehicle and TGN-020 injection. Differences between the two conditions are readily visible with a decrease in Sindex and an increase in sADC following TGN-20 injection. Those changes are quantitatively assessed in [Fig 4](#pone.0229702.g004){ref-type="fig"}. [Fig 4A--4C](#pone.0229702.g004){ref-type="fig"} show the Sindex averaged over six scans before and after the injection in different locations, i.e., cerebral cortex, hippocampus and striatum, in vehicle and the TGN-020 group. TGN-020 injection resulted in significant decrease in Sindex in the cortex (p = 0.0061) and the hippocampus (p = 0.00069) but not in the striatum (p = 0.26). No significant change of Sindex was observed following vehicle injection in those locations (p = 0.94 in cortex, p = 0.85 in hippocampus and p = 0.14 in striatum). [Fig 4D--4F](#pone.0229702.g004){ref-type="fig"} show the sADC averaged over six scans before and after the injection in the same locations as for the Sindex, in vehicle and TGN-020 group. TGN-020 injection resulted in significant increase in sADC in the cortex (p = 0.0064) and the hippocampus (p = 0.00068) but not in the striatum (p = 0.26). No significant change of sADC was observed following vehicle injection in any of the ROIs considered (p = 0.91 in cortex, 0.78 in hippocampus and 0.14 in striatum). We also investigated subregions of the hippocampus CA3 and DG ([S1 Fig](#pone.0229702.s001){ref-type="supplementary-material"}). The TGN-020 injection significantly decreased Sindex and increased sADC values both in the DG and the CA3 (Sindex in DG: -11.8, p = 4.8e-7 and in CA3: -4.8, p = 0.00012; sADC in CA3: +10.2, p = 0.00013, sADC in DG: +22.5, p = 7.14e-7). However, the baseline for Sindex and sADC in DG was found noisy and not stable ([S1B and S1F Fig](#pone.0229702.s001){ref-type="supplementary-material"}) due to the smaller size of DG resulting in a very low voxel count compared to the other ROIs (voxel count for DG, CA3, hippocampus, striatum, cortex were 85, 181, 763, 2069 and 8270, respectively). ![Boxplot of Sindex and sADC in the cortex (a: Sindex, d: sADC), the striatum (b: Sindex, e: sADC) and hippocampus (c: Sindex, f: sADC). Left, Vehicle group; right TGN-020 group in each figure. Pre, pre-injection (light blue); post, post-injection (dark blue) of vehicle or TGN-020 group. \: p\<0.05, \\: p\<0.01, \\\: p\<0.001 are the result for the paired t-test between pre and post-injection for each group. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 are the result for a t-test with posthoc correction for multiple comparison between post-injection of the two groups. Diamonds represent outliers, a point is defined as an outlier if its value is below Q1--1.5×IQR or above Q3 + 1.5×IQR, where Q1 is the first quartile, Q3 the third quartile and IQR the interquartile range.](pone.0229702.g004){#pone.0229702.g004} Sindex and sADC time courses following vehicle or TGN-020 injection {#sec011} ------------------------------------------------------------------- We then investigated the time course of the Sindex and sADC for each group. The same ROIs were used as for the averaged Sindex and sADC values. [Fig 5A--5C](#pone.0229702.g005){ref-type="fig"} represents the Sindex time course for each ROI. A decrease of Sindex was observed after the injection of TGN-020 but not with vehicle injection in all ROIs. The Sindex significantly decreased (around 9% in the cortex) following TGN-020 injection in all ROIs and continued until the end of the scanning period. [Fig 5D--5F](#pone.0229702.g005){ref-type="fig"} show the sADC time course with an opposite trend compared to Sindex in all ROIs. ![Time course of Sindex and sADC in the cortex (a: Sindex, d: sADC), the striatum (b: Sindex, e: sADC) and hippocampus (c: Sindex, f: sADC). Blue line is vehicle group and orange line is TGN-020 group. Error bar shows standard deviation. The dashed line represents the injection time. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 by two sample t-test between the two groups for each time point.](pone.0229702.g005){#pone.0229702.g005} Discussion {#sec012} ========== Several studies have underlined the potential of Diffusion MRI to investigate the glymphatic system \[[@pone.0229702.ref007],[@pone.0229702.ref023]--[@pone.0229702.ref025]\]. Astrocytes are considered to play a key role in brain waste clearance through membrane AQP4 channels expressed at their end-feet \[[@pone.0229702.ref012]\]. The ADC has been proposed as a biomarker of AQP gene expression, as earlier studies have demonstrated that the ADC obtained with high b values was correlated with the amount of increasing expression of AQP1 in glioblastoma cell lines \[[@pone.0229702.ref040]\]. In this study, we have investigated whether two diffusion MRI markers more sensitive to tissue microstructure than the ADC, namely the Sindex and sADC, could reveal changes in astrocyte activity induced by TGN-020. Following AQP4 channel inhibition with a TGN-020 solution a decrease in Sindex and increase in sADC were readily observed in the cortex, more in the hippocampus, but not in the striatum, reflecting local differences in astrocyte \[[@pone.0229702.ref041]\] and vascular density \[[@pone.0229702.ref034]\]. In the hippocampus changes in Sindex and sADC were larger in DG than in CA3 layer, presumably in line with differences in AQP4 channels expression on astrocytes \[[@pone.0229702.ref039]\]. However, further validation is required as the baseline Sindex and sADC values were not stable due to the small count in the DG ROI (see [Result](#sec009){ref-type="sec"}). Histological studies would also confirm differences in astrocyte density and volume. Nonetheless, those results suggest that diffusion MRI is sensitive to astrocyte activity and, indirectly, to the status of the glymphatic system. Those diffusion MRI markers provide a higher sensitivity to small changes in tissue features by encompassing in a single marker Gaussian and non-Gaussian diffusion effects. Furthermore they are easy to calculate and are not diffusion signal model dependent, such as the kurtosis model \[[@pone.0229702.ref031]\]. The low key b value used in this study was high enough (250 s/mm^2^) to make perfusion-related IVIM effects negligible. Hence the observed changes in the diffusion markers reflect genuine tissue microstructure related diffusion effects and not perfusion effects (cerebral blood flow) which have already been reported observed with TGN-020 \[[@pone.0229702.ref042]\]. Indeed, acquisition of perfusion-driven IVIM data could be valuable to get information on the vascular distribution which is known to vary across brain structures \[[@pone.0229702.ref034]\], but this was not possible within our protocol timeframe to guaranty animal stability. The Sindex decrease and the sADC increase jointly point out to a decrease in the amount of hindrance for water diffusion in astrocyte rich areas (cortex and hippocampus) under acute AQP4 channel inhibition induced by TGN-20. Based on established diffusion MRI mechanisms \[[@pone.0229702.ref028]\] this hindrance decrease suggests an astrocyte volume reduction \[[@pone.0229702.ref043],[@pone.0229702.ref044]\] associated with an increase of the ISF (were diffusion is tortuous) \[[@pone.0229702.ref045]\] or an increase in astrocyte membrane permeability and water exchange. Indeed, astrocytes rapidly regulate their volume throughout AQP4 channels. Those results obtained by acute AQP4 inhibition contrast earlier reports using chronic models, such as an ADC decrease observed during AQP4 inhibition with interfering RNA \[[@pone.0229702.ref046]\] or change in ADC in AQP4 knockout mice \[[@pone.0229702.ref047]\]. Beside the higher sensitivity to tissue microstructure of the sADC over the ADC, such discrepancy could possibly result from the modified astrocyte phenotype associated with a long-term inhibition of AQP4 expression found in those previous studies \[[@pone.0229702.ref048]\]. The sub-acute or chronic inhibition of AQP4 activity by AQP4-antibodies or small interfering RNA duplexes alter astrocyte morphology and decrease water permeability \[[@pone.0229702.ref049],[@pone.0229702.ref050]\] which could result in an ADC decrease. Also, the effects we observed in baseline conditions should be distinguished from those obtained in conditions of neuronal activation for which AQP4 inhibition by extracellular acidification results in astrocyte swelling, capillary lumen expansion and Virchow-Robin space reduction \[[@pone.0229702.ref051]\]. Further studies should aim at precising the mechanisms involved in the observed effects, however histology would require the brain to be fixed, preventing astrocyte volume changes to be monitored before and after TGN-020/saline injection within the same animal. In vivo fluorescent imaging could be used, but only in the cortex. Clearly, the detailed mechanisms underlying acute AQP4 channel inhibition by TGN-20 and chronic AQP4 knockout mice models are lacking. Diffusion MRI has the potential to clarify those mechanisms, notably through non-Gaussian diffusion markers, such as the Sindex and the sADC. Fractional anisotropy (FA) measurements results were not reported as they are relevant only to areas exhibiting diffusion anisotropy, ie, white matter given the spatial resolution of our images. Beside, due to noise FA values are highly corrupted and often reflect mainly variations in underlying ADC (here sADC) \[[@pone.0229702.ref052]\]. Another issue to consider is that our studies were obviously performed under anesthesia. Anesthetic drugs are known to impact intracranial pressure, which could interfere with CSF-ISF exchanges. For instance, dexmedetomidine and ketamine enhance CSF influx alongside perivascular spaces \[[@pone.0229702.ref053]\]. Moreover, similar to what happens during sleep, anesthesia is associated with a substantial increase in both perivascular and extracellular space volume \[[@pone.0229702.ref054]\]. Thus, it would be interesting to check whether the effects we have observed with TGN-20 persist in awake animals. A recent study has shown that, while the baseline ADC did not change between anesthetic and awake conditions, DOTA-Gd accumulation was significantly suppressed during anesthesia in contrast enhanced MRI \[[@pone.0229702.ref055]\]. Conclusion {#sec013} ========== Work remains to better understand the mechanisms involved in brain waste clearance through a so-called glymphatic system and the contribution of astrocytes to this system. The reference method to investigate the glymphatic system in preclinical settings relies on the intracisternal injection of gadolinium in the cisterna magna \[[@pone.0229702.ref056]\]. Our results show that changes in astrocyte activity thought to regulate CSF-ISF exchanges, an important component of glymphatic system functionality, could be monitored non-invasively with diffusion MRI, especially through its Sindex metric. Further studies will be required to more directly establish the potential of diffusion MRI to monitor the glymphatic system. Due to its complete non-invasiveness, this new approach could be used for clinical studies to confirm or infirm the existence of such a glymphatic system in humans \[[@pone.0229702.ref024]\]. Supporting information {#sec014} ====================== ###### Boxplot and time course of Sindex in the DG (a: boxplot, b: time course) and in the CA3 (c: boxplot, d: time course). Boxplot and time course of sADC in the DG (e: boxplot, f: time course) and in the CA3 (g: boxplot, h: time course). Left, Vehicle group; right TGN-020 group in each figure. Pre, pre-injection (light blue); post, post-injection (dark blue) of vehicle or TGN-020 group in boxplot. \: p\<0.05, \\: p\<0.01, \\\: p\<0.001 are the result for the paired t-test between pre and post-injection for each group. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 are the result for a t-test with posthoc correction for multiple comparison between post-injection of the two groups. Diamonds represent outliers, a point is defined as an outlier if its value is below Q1--1.5×IQR or above Q3 + 1.5×IQR, where Q1 is the first quartile, Q3 the third quartile and IQR the interquartile range. Blue line is vehicle group and orange line is TGN-020 group. Error bar shows standard deviation. The dashed line represents the injection time. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 by two sample t-test between the two groups for each time point. Note: The voxel count in DG was very small (85 versus 181 in CA3) resulting in very noisy data. The apparent significant difference in sADC and Sindex values between the TGN-020 and saline groups one point before baseline (b, f) might result from an underestimation of the standard-deviation. The difference becomes largely more significant after injections, overcoming any uncertainty in standard-deviation estimates. (TIF) ###### Click here for additional data file. 10.1371/journal.pone.0229702.r001 Decision Letter 0 Jiang Quan Academic Editor © 2020 Quan Jiang 2020 Quan Jiang This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 3 Mar 2020 PONE-D-20-03675 Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor. PLOS ONE Dear Pr. Le Bihan, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. How do you know that the observed diffusion changes are related to the glymphatic system?How do you explain the DG change, given the mentioned AQP4 abundant?Do you have the data to identify regions with high vascular presence to look at the Si and sADC changes on those area?In addition to sindex, the authors could use FA for comparison.Response to reviewer's questions. We would appreciate receiving your revised manuscript by Apr 17 2020 11:59PM. When you are ready to submit your revision, log on to <https://www.editorialmanager.com/pone/> and select the \'Submissions Needing Revision\' folder to locate your manuscript file. 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Thank you for including your ethics statement: \"All animal procedures used in the present study were approved by an institutional Ethic Committee and government regulatory agency (reference APAFIS\#8462-2017010915542122v2) and were conducted in strict accordance with the recommendations and guidelines of the European Union (Directive 2010/63/EU). This manuscript is in compliance with the ARRIVE guidelines (Animal Research: Reporting in Vivo Experiments) on how to REPORT animal experiments.\". i\) Please amend your current ethics statement to include the full name of the ethics committee that approved your specific study. ii\) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the "Ethics Statement" field of the submission form (via "Edit Submission"). For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to <http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research> \[Note: HTML markup is below. Please do not edit.\] Reviewers\' comments: Reviewer\'s Responses to Questions Comments to the Author 1\. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer \#1: Yes Reviewer \#2: Yes \\\\\\\\\\ 2\. Has the statistical analysis been performed appropriately and rigorously? Reviewer \#1: Yes Reviewer \#2: Yes \\\\\\\\\\ 3\. Have the authors made all data underlying the findings in their manuscript fully available? The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing) requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data---e.g. participant privacy or use of data from a third party---those must be specified. Reviewer \#1: No Reviewer \#2: Yes \\\\\\\\\\ 4\. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer \#1: Yes Reviewer \#2: Yes \\\\\\\\\\ 5\. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer \#1: Authors presented a very interesting study, evaluating the utility of diffusion MRI to map glymphatic function, through the modification of the AQP4 expression. This is of high scientific and clinical value, very timely and of high impact. The experiment is well designed, and the execution is sound. This reviewer has below comments regarding the validity and conclusions. Figures quality/dpi are low! Major comments: 1\. The main limitation is interpreting diffusion measures as glymphatic system function. AQP4 is also involved in brain inflammation and regulation of extracellular space volume. How do you know that the observed diffusion changes are related to the glymphatic system? Even though authors were cautious about the interpretation of their results, they should probably discuss other potential explanations for their findings and adjust their main conclusion accordingly. 2\. Authors reported significant differences in both CA3 and DG of the TGN-020 group. How do you explain the DG change, given the mentioned AQP4 abundant? 3\. Regarding above comment: Do you have the data (for example SWI or high resolution T2) to identify regions with high vascular presence to look at the S_i and sADC changes on those area? That could potentially help identify the underlying AQP4 involvement. 4\. Based on ref \[36\]: "AQP4 protein levels were highest in the cerebellum with lower expression in the cortex and hippocampus." This suggests that the cerebellum should have been a major ROI in this study. Why cerebellum is not included? 5\. Figures have extremely low quality (for example Figures 4 and 5). I could only guess the labels and axes. 6\. It seems rather strange to have (significant) differences prior to injection (e.g. Fig 5.h)? This needs to be addressed. One would expect to see no difference whatsoever. 7\. In favor with reported findings, below paper showed that diffusion MRI is affected by perivascular space fluid presence, which should be cited: Sepehrband, F., Cabeen, R. P., Choupan, J., Barisano, G., Law, M., Toga, A. W., & Alzheimer\'s Disease Neuroimaging Initiative. (2019). Perivascular space fluid contributes to diffusion tensor imaging changes in white matter. NeuroImage, 197, 243-254. 8\. Not in favor of reported finding, below study reported "we failed to detect a significant change in the brain extracellular water volume using diffusion weighted imaging in awake and anesthetized mice." This paper also should be cited/discussed: Gakuba, C., Gaberel, T., Goursaud, S., Bourges, J., Di Palma, C., Quenault, A., \... & Gauberti, M. (2018). General anesthesia inhibits the activity of the "glymphatic system". Theranostics, 8(3), 710. 9\. Another paper that links diffusion changes to glymphatic system is below, which also could be cited: Thomas, C., Sadeghi, N., Nayak, A., Trefler, A., Sarlls, J., Baker, C. I., & Pierpaoli, C. (2018). Impact of time-of-day on diffusivity measures of brain tissue derived from diffusion tensor imaging. Neuroimage, 173, 25-34. A minor comment: 1\. Page 11, line 251: "peculiar to this this brain" -\> "peculiar to this brain" 2\. Please report the age of the mice, in each group (maybe it is reported somewhere, but I couldn't find it). Reviewer \#2: In this paper the dynamic changes of astrocyte activity were investigated using DWI in 32 mice by inhibiting AQP4 channels with a TGN-020 solution. Two novel DWI measures were used to study the results which show a significant decrease in the Sindex, a diffusion marker of tissue microstructure, and a significant increase of the water diffusion coefficient (sADC) in cerebral cortex and hippocampus compared to saline injection. Developing reliable non-invasive biomarkers for the glymphatic system is important for translational studies. This study has done a great job to introduce such biomarkers. However, it may need some more work to improve the study. Major points: 1- In addition to sindex, the authors could use FA for comparison or at least confirming (in a few sentences) that FA is not showing the trend seen in sindex. 2- Why mice were chosen? Rats have bigger brain and therefore the imaging could be easier. 3- Histology analyses of the mice after imaging could be so helpful in confirming the study outcomes. Minor points: Line 16: "We assumed no change of position among the different scans during acquisitions for each mouse and geometric distortion with b values to be negligible; this condition was qualitatively checked on several mice." Why not using motion correction to compensate for animal movement during the imaging? Line 119: "Furthermore, signal instabilities were quantitatively evaluated for each subject and subjects exhibiting instabilities above 4% for most voxels were eliminated." Could you please explain more? Line 156: Please mention in Fig3 caption the time point of the displayed maps? I assume the maps correspond to the averaged 6 time points of the pre injection and the last 6 time points after the injection. Line 251: There is an extra "this". \\\\\\\\\\ 6\. PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy). Reviewer \#1: No Reviewer \#2: No \[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link \"View Attachments\". If this link does not appear, there are no attachment files to be viewed.\] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, <https://pacev2.apexcovantage.com/>. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at <[email protected]>. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0229702.r002 Author response to Decision Letter 0 10 Apr 2020 Editor comment 1\. How do you know that the observed diffusion changes are related to the glymphatic system? The existence of the glymphatic system is controversial. Our results do not aim at demonstrating the existence or not of a glymphatic system. However, if we admit that such a system exists it has been strongly suggested in the literature (REF XXX) that astrocytes must play a major role, and, in turn, AQP4 channels expressed by astrocytes. The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all. The Discussion and Conclusion have been revised to make this point clear. 2\. How do you explain the DG change, given the mentioned AQP4 abundant? The density of AQP4 receptors in DG is higher than in the other regions we have investigated. Indeed, the larger sADC and Sindex change observed in DG under TGN-020 administration is an indirect proof that TGN-020 acted on astrocyte AQP4 channels. The Discussion has been revised accordingly. 3\. Do you have the data to identify regions with high vascular presence to look at the Si and sADC changes on those area? A relevant parameter could be, indeed, the density of small vessels. Such vessels are not visible even within the high resolution T2-weighted images we acquired using Turbo RARE (see Methods and Figure 2). Such vessels would require the additional acquisition of perfusion MRI data, such as IVIM MRI, which could not be done during the acquisition time window our anesthetic protocol permitted. Hence, we relied on previous studies reporting the vascular distribution across brain structures using microscopy (ref 40). 4\. In addition to sindex, the authors could use FA for comparison. FA is relevant to assess diffusion anisotropy which is present only in whiter matter at the resolution of our images (not in the cortex). The Sindex (and the sADC) is a much more relevant parameter. Furthermore, as explained in a recent article (Iima at al. European Radiology, 2020), FA values are highly sensitive to noise and, in turn, to ADC (or sADC) values. Variations in FA would thus reflect the changes we have observed in sADC values more than genuine changes in diffusion anisotropy. Journal Requirements Please ensure that your manuscript meets PLOS ONE\'s style requirements, including those for file naming. The PLOS ONE style templates can be found at <http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf> and <http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf> 2\. Thank you for including your ethics statement: i\) Please amend your current ethics statement to include the full name of the ethics committee that approved your specific study. ii\) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the "Ethics Statement" field of the submission form (via "Edit Submission"). For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to <http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research> We initially refrained to provide information which could potentially conflict with an anonymous review. Full details have been added in the Methods section. Response to reviewers Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer \#1: Authors presented a very interesting study, evaluating the utility of diffusion MRI to map glymphatic function, through the modification of the AQP4 expression. This is of high scientific and clinical value, very timely and of high impact. The experiment is well designed, and the execution is sound. This reviewer has below comments regarding the validity and conclusions. Figures quality/dpi are low! Major comments: 1\. The main limitation is interpreting diffusion measures as glymphatic system function. AQP4 is also involved in brain inflammation and regulation of extracellular space volume. How do you know that the observed diffusion changes are related to the glymphatic system? Even though authors were cautious about the interpretation of their results, they should probably discuss other potential explanations for their findings and adjust their main conclusion accordingly. The existence of the glymphatic system is controversial. Our results do not aim at demonstrating the existence or not of a glymphatic system. However, if we admit that such a system exists it has been strongly suggested in the literature (Illif et al, Sci Transl Med, 2012,) that astrocytes must play a major role system (Mestre et al, eLife, 2018), and, in turn, AQP4 channels expressed by astrocytes (Ikeshima-Kataoka et al, Int J Mol Sci, 2016). The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all. Further studies will be required to establish the potential of diffusion MRI to monitor the glymphatic system. The Discussion and Conclusion have been revised to make this point clear. 2\. Authors reported significant differences in both CA3 and DG of the TGN-020 group. How do you explain the DG change, given the mentioned AQP4 abundant? The density of AQP4 receptors in DG is higher than in the other regions we have investigated (Hsu et al, Neuroscience, 2011; Hubbard et al, ASN Neuro, 2015). Indeed, the larger sADC and Sindex change observed in DG under TGN-020 administration is an indirect proof that TGN-020 acted on astrocyte AQP4 channels. However, as we explain in the response to comment\#6, the baseline of DG was not stable due to the smaller voxel size. We need higher resolution image for the validation of Sindex changes in DG. The Discussion has been revised accordingly. 3\. Regarding above comment: Do you have the data (for example SWI or high resolution T2) to identify regions with high vascular presence to look at the S_i and sADC changes on those area? That could potentially help identify the underlying AQP4 involvement. We agree that a relevant parameter could be, indeed, the density of small vessels. Such vessels are not visible even within the high resolution T2-weighted images we acquired using Turbo RARE (see Methods and Figure 2). Such vessels would require the additional acquisition of perfusion MRI data, such as IVIM MRI, which could not be done during the acquisition time window our anesthetic protocol permitted. Hence, we relied on previous studies reporting the vascular distribution across brain structures using microscopy (Xiong et al, Front Neuroanat, 2017). 4\. Based on ref \[36\]: "AQP4 protein levels were highest in the cerebellum with lower expression in the cortex and hippocampus." This suggests that the cerebellum should have been a major ROI in this study. Why cerebellum is not included? We did not have a full brain coverage including the cerebellum due to the limitation of the scanning time (scanning time is determined by number of slices). Instead, we investigated the hippocampus because AQP4 expresses abundantly in this region (Hsu et al, Neuroscience, 2011; Hubbard et al, ASN Neuro, 2015). 5\. Figures have extremely low quality (for example Figures 4 and 5). I could only guess the labels and axes. We are sorry for the very low quality of Figure 4 and 5 which probably resulted from the PDF conversion. Revised figures were uploaded after checking quality with the digital diagnostic tool of PLOS: , <https://pacev2.apexcovantage.com/>. 6\. It seems rather strange to have (significant) differences prior to injection (e.g. Fig 5.h)? This needs to be addressed. One would expect to see no difference whatsoever. Because the volume of DG is smaller than cerebral cortex and striatum, the Sindex and sADC in DG were noisier rather than these regions. (The number of voxel size in DG, CA3, hippocampus, striatum, cortex is respectively: 85, 181, 763, 2069 and 8270) This is the reason why baseline of Sindex and sADC in DG was not stable. We decided to move DG and CA3 from the manuscript to supplementary data, instead we have used whole hippocampal region for the main figure. We described the instability in DG and CA3 in the Results section. 7\. In favor with reported findings, below paper showed that diffusion MRI is affected by perivascular space fluid presence, which should be cited: Sepehrband, F., Cabeen, R. P., Choupan, J., Barisano, G., Law, M., Toga, A. W., & Alzheimer\'s Disease Neuroimaging Initiative. (2019). Perivascular space fluid contributes to diffusion tensor imaging changes in white matter. NeuroImage, 197, 243-254. We have added this reference in Introduction (Reference 26). 8\. Not in favor of reported finding, below study reported "we failed to detect a significant change in the brain extracellular water volume using diffusion weighted imaging in awake and anesthetized mice." This paper also should be cited/discussed: Gakuba, C., Gaberel, T., Goursaud, S., Bourges, J., Di Palma, C., Quenault, A., \... & Gauberti, M. (2018). General anesthesia inhibits the activity of the "glymphatic system". Theranostics, 8(3), 710. This article compared the ADC and intracerebral accumulation of DOTA-Gd injected in intracisternal site in awaked and anesthetized state. The suppressed accumulation of DOTA-Gd was observed in anesthetized state compared with awaked state, while ADC was not changed between awaked and anesthetized sate. The DOTA-Gd increased the SNR when they accumulated for long time (30-60 min for accumulation in the study) and acquisition has been done under anesthesia. In contrast, diffusion MRI has been performed in awaked and anesthetized condition. The diffusion MRI could be influenced by the head motion and respiration, which could be more significant in awaked state. This potentially interferes ADC measurement to reduce the sensitivity. Further study to improve the sensitivity is required to assess the potential of diffusion MRI to detect the awaked and anesthetized state. We have described this article in Discussion. 9\. Another paper that links diffusion changes to glymphatic system is below, which also could be cited: Thomas, C., Sadeghi, N., Nayak, A., Trefler, A., Sarlls, J., Baker, C. I., & Pierpaoli, C. (2018). Impact of time-of-day on diffusivity measures of brain tissue derived from diffusion tensor imaging. Neuroimage, 173, 25-34. We thank for these valuable references. We agree that those references would be helpful for the readers to better understand and appreciate our results. This has been cited as reference 27. A minor comment: 1\. Page 11, line 251: "peculiar to this this brain" -\> "peculiar to this brain" We have corrected this typo. 2\. Please report the age of the mice, in each group (maybe it is reported somewhere, but I couldn't find it). We used the mice aged 4-10 weeks in each group. The age of mice has been added into the Material and Methods part of the manuscript. Reviewer \#2: In this paper the dynamic changes of astrocyte activity were investigated using DWI in 32 mice by inhibiting AQP4 channels with a TGN-020 solution. Two novel DWI measures were used to study the results which show a significant decrease in the Sindex, a diffusion marker of tissue microstructure, and a significant increase of the water diffusion coefficient (sADC) in cerebral cortex and hippocampus compared to saline injection. Developing reliable non-invasive biomarkers for the glymphatic system is important for translational studies. This study has done a great job to introduce such biomarkers. However, it may need some more work to improve the study. Major points: 1- In addition to sindex, the authors could use FA for comparison or at least confirming (in a few sentences) that FA is not showing the trend seen in sindex. FA is relevant to assess diffusion anisotropy which is present only in whiter matter at the resolution of our images. The Sindex (and the sADC) is a much more relevant parameter. Furthermore, as explained in a recent article (Iima at al. European Radiology, 2020), FA values are highly sensitive to noise and, in turn, to ADC (or sADC) values. Variations in FA would thus reflect the changes we have observed in sADC values more than genuine changes in diffusion anisotropy. 2- Why mice were chosen? Rats have bigger brain and therefore the imaging could be easier. We chose mice instead of rats because our diffusion MRI protocol could be applicable in the future to investigate AQP-4 KO mice. There is no such transgenic model for rats. Also, our MRI setup includes a cryoprobe dedicated to mouse head imaging, which increases the SNR and contrast. This specific coil is too small for rat heads. 3- Histology analyses of the mice after imaging could be so helpful in confirming the study outcomes. We agree that histology would have been a nice addition to confirm volume changes of astrocytes in the different groups (TGN-020 group and vehicle group). However, such tests require the brain to be fixed, hence they cannot be performed to check volume changes before and after TGN-020 (or saline) injection within the same animal. Another method to monitor astrocyte volume could be in vivo fluorescent imaging, but this method can be applied only in the cortex. Those issues have been added to the Discussion. Minor points: Line 16: "We assumed no change of position among the different scans during acquisitions for each mouse and geometric distortion with b values to be negligible; this condition was qualitatively checked on several mice." Why not using motion correction to compensate for animal movement during the imaging? Motion correction is tricky when considering quantitative diffusion MRI as motion algorithms work on the reconstructed images. Especially, as acquisitions are performed with specific orientations of the diffusion-probing gradient pulses in space any rotation in the reconstructed images would not reflect genuine signal changes which would have occurred if the diffusion-probing gradient pulses had rotated in the same way. Instead, we decided to simply reject data sets were motion was too large (\>4%) using a semi-quantitative index based on signal stability across successive scans. We agree that the text was not clear and we have revised it. Line 119: "Furthermore, signal instabilities were quantitatively evaluated for each subject and subjects exhibiting instabilities above 4% for most voxels were eliminated." Could you please explain more? The standard-deviation of the signal intensity across scans was evaluated for each b value and each voxel (after Gaussian smoothing) was estimated. A threshold of 4% was used flag unstable voxels. . Data sets were one of the ROIs have only flagged voxels were discarded. Line 156: Please mention in Fig3 caption the time point of the displayed maps? I assume the maps correspond to the averaged 6 time points of the pre injection and the last 6 time points after the injection. We have added in the legend of the figure 3 that the maps correspond to the mean of the 6 last time points. Line 251: There is an extra "this". We have corrected this typo. ###### Submitted filename: 200303-responses_to_reviewers-FINAL.docx ###### Click here for additional data file. 10.1371/journal.pone.0229702.r003 Decision Letter 1 Jiang Quan Academic Editor © 2020 Quan Jiang 2020 Quan Jiang This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 12 Apr 2020 PONE-D-20-03675R1 Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor. PLOS ONE Dear Pr. Le Bihan, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. 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This letter should be uploaded as separate file and labeled \'Response to Reviewers\'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled \'Revised Manuscript with Track Changes\'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled \'Manuscript\'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Quan Jiang, Ph,D. Academic Editor PLOS ONE \[Note: HTML markup is below. Please do not edit.\] Reviewers\' comments: Reviewer\'s Responses to Questions Comments to the Author 1\. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the "Comments to the Author" section, enter your conflict of interest statement in the "Confidential to Editor" section, and submit your \"Accept\" recommendation. Reviewer \#1: (No Response) Reviewer \#2: All comments have been addressed \\\\\\\\\\ 2\. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer \#1: Yes Reviewer \#2: Yes \\\\\\\\\\ 3\. Has the statistical analysis been performed appropriately and rigorously? Reviewer \#1: I Don\'t Know Reviewer \#2: Yes \\\\\\\\\\ 4\. Have the authors made all data underlying the findings in their manuscript fully available? The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing) requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data---e.g. participant privacy or use of data from a third party---those must be specified. Reviewer \#1: No Reviewer \#2: Yes \\\\\\\\\\ 5\. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer \#1: Yes Reviewer \#2: Yes \\\\\\\\\\ 6\. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer \#1: Authors responded to the raised issues and addressed reviewer's concern. One point that remained unaddressed is the general assumption. The author states that "The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all." But throughout the paper the text suggests that that's not all. For example, the abstract says "The Glymphatic System (GS) has been proposed as a mechanism to clear brain tissue from waste. Its dysfunction might lead to several brain pathologies, including the Alzheimer'disease. A key component of the GS and brain tissue water circulation is the astrocyte which is regulated by acquaporin-4 (AQP4), a membrane-bound water channel on the astrocytic end-feet." Well, clearly the author is relating this to glymphatic system. Yet, relating this to glymphatic system is not the main concern. If one can measure astrocyte activity, it's not unreasonable to relate it to glymphatic system. The main concern is that AQP-4 is not only involved in modulation of astrocyte activity. It is also involved in the brain inflammatory response and also in the regulation of the extracellular volume. Both of these could affect diffusion signal. Blocking AQP-4 could lead to inflammation or changes in the extracellular fluid which affect the diffusion signal. Therefore, what you observe here may have nothing to do with the astrocyte activity. This limitation should be addressed. PS. Regarding DG, if the data is noisy, it should lead to higher standard deviation not a systematic group mean difference. Reviewer \#2: The authors resolved well my concerns about their work and made the manuscript more clear. I have no other question to add. \\\\\\\\\\ 7\. PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? 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If you encounter any issues or have any questions when using PACE, please email us at <[email protected]>. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0229702.r004 Author response to Decision Letter 1 28 Apr 2020 Reviewer \#1: Authors responded to the raised issues and addressed reviewer's concern. One point that remained unaddressed is the general assumption. The author states that "The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all." But throughout the paper the text suggests that that's not all. For example, the abstract says "The Glymphatic System (GS) has been proposed as a mechanism to clear brain tissue from waste. Its dysfunction might lead to several brain pathologies, including the Alzheimer'disease. A key component of the GS and brain tissue water circulation is the astrocyte which is regulated by acquaporin-4 (AQP4), a membrane-bound water channel on the astrocytic end-feet." Well, clearly the author is relating this to glymphatic system. Yet, relating this to glymphatic system is not the main concern. If one can measure astrocyte activity, it's not unreasonable to relate it to glymphatic system. The main concern is that AQP-4 is not only involved in modulation of astrocyte activity. It is also involved in the brain inflammatory response and also in the regulation of the extracellular volume. Both of these could affect diffusion signal. Blocking AQP-4 could lead to inflammation or changes in the extracellular fluid which affect the diffusion signal. Therefore, what you observe here may have nothing to do with the astrocyte activity. This limitation should be addressed. PS. Regarding DG, if the data is noisy, it should lead to higher standard deviation not a systematic group mean difference � The statement about the Glymphatic System in the abstract and the introduction is only to explain our general motivation to conduct our study of the potential of diffusion MRI to monitor astrocyte activity. It is not a claim that the GS exists (we believe we have been careful in the manuscript about this fact). We only briefly review the literature to shade light on the context of the study and indicate why investigating astrocyte activity might be important. We are aware that TGN-020 effects are not specific to astrocytes, as stated in the manuscript. Effects on the brain inflammatory response can be discarded given the time course of the effects we have observed. Regarding the extracellular space (ISF) effects on the diffusion MRI would result only from changes in diffusion hindrance/tortuosity (fluid flow is well too slow to result in IVIM effects, this has been shown by other groups, furthermore the Sindex was purposely calculated using the lowest key b value 250s/mm² to make residual IVIM effects completely negligible). Hence, variations in extracellular space shape and volume only reflect changes in the local cellular background (a kind of negative imprint). The decrease in Sindex (and sADC increase) points out to decrease in the amount of diffusion hindrance from astrocytes (which is by far the dominant cell type in the regions where we observed the largest response). As stated in the discussion "Based on established diffusion MRI mechanisms \[28\] this hindrance decrease suggests an astrocyte volume reduction \[43,44\] associated with an increase of the ISF (were diffusion is tortuous) \[45\] or an increase in astrocyte membrane permeability and water exchange". We believe that this statement about the mechanism leading to our observation is correct and broad enough, and the manuscript was not changed. � Regarding the DG, the data are, indeed, very noisy given the very small voxel count. The apparent group difference may just be coincidental as the standard-deviation cannot be accurately estimated precisely given the small voxel count. We realize that this might be puzzling, but, for scientific integrity, we decided not to "hide" those results but to show them. A note has been added to the caption of SuppL Fig.1. ###### Submitted filename: 200303-responses_to_reviewers-REV.docx ###### Click here for additional data file. 10.1371/journal.pone.0229702.r005 Decision Letter 2 Jiang Quan Academic Editor © 2020 Quan Jiang 2020 Quan Jiang This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 29 Apr 2020 Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor. PONE-D-20-03675R2 Dear Dr. Le Bihan, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. 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Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer \#1: Yes \\\\\\\\\\ 3\. Has the statistical analysis been performed appropriately and rigorously? Reviewer \#1: Yes \\\\\\\\\\ 4\. Have the authors made all data underlying the findings in their manuscript fully available? The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing) requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data---e.g. participant privacy or use of data from a third party---those must be specified. Reviewer \#1: No \\\\\\\\\\ 5\. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer \#1: Yes \\\\\\\\\\ 6\. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer \#1: This reviewer was not convinced by authors respond, claiming extracellular changes will not affect Sindex. However this does not damper reviewers enthusiasm about this interesting work. I congratulate the authors and wish them well. \\\\\\\\\\ 7\. PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy). Reviewer \#1: No 10.1371/journal.pone.0229702.r006 Acceptance letter Jiang Quan Academic Editor © 2020 Quan Jiang 2020 Quan Jiang This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 4 May 2020 PONE-D-20-03675R2 Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor. Dear Dr. Le Bihan: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Quan Jiang Academic Editor PLOS ONE [^1]: Competing Interests:The authors have declared that no competing interests exist.	{ "pile_set_name": "PubMed Central" }	[ "control", "ie", "respond", "purpos", "longterm", "round", "kind", "grant", "fracslbshb", "week", "dotagd", "signal", "back", "acquir", "enthusiasm", "discrep", "modul", "journal", "process", "deposit", "orang", "happen", "shift", "state", "lb", "conflict", "context", "authorbillingplosorg", "higher", "even", "front", "gbca", "h", "na", "slow", "pace", "perivascular", "multislic", "pairwis", "preprocess", "chronic", "linearli", "duplex", "govern", "whether", "flux", "sblb", "cortex", "strongli", "upcom", "thu", "uncertainti", "center", "intracistern", "gaussian", "protein", "tgnsalin", "util", "striatum", "whatsoev", "significantli", "timeofday", "chose", "vivant", "copyedit", "abundantli", "row", "consid", "endfeet", "potenti", "statu", "turn", "caption", 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"throughout", "paper", "text", "suggests", "example", "abstract", "says", "Glymphatic", "System", "GS", "proposed", "mechanism", "clear", "brain", "tissue", "waste", "dysfunction", "might", "lead", "several", "brain", "pathologies", "including", "key", "component", "GS", "brain", "tissue", "water", "circulation", "astrocyte", "regulated", "water", "channel", "astrocytic", "Well", "clearly", "author", "relating", "glymphatic", "system", "Yet", "relating", "glymphatic", "system", "main", "concern", "one", "measure", "astrocyte", "activity", "unreasonable", "relate", "glymphatic", "system", "main", "concern", "involved", "modulation", "astrocyte", "activity", "also", "involved", "brain", "inflammatory", "response", "also", "regulation", "extracellular", "volume", "could", "affect", "diffusion", "signal", "Blocking", "could", "lead", "inflammation", "changes", "extracellular", "fluid", "affect", "diffusion", "signal", "Therefore", "observe", "may", "nothing", "astrocyte", "activity", "limitation", "addressed", "PS", "Regarding", "DG", "data", "noisy", "lead", "higher", "standard", "deviation", "systematic", "group", "mean", "difference", "statement", "Glymphatic", "System", "abstract", "introduction", "explain", "general", "motivation", "conduct", "study", "potential", "diffusion", "MRI", "monitor", "astrocyte", "activity", "claim", "GS", "exists", "believe", "careful", "manuscript", "fact", "briefly", "review", "literature", "shade", "light", "context", "study", "indicate", "investigating", "astrocyte", "activity", "might", "important", "aware", "effects", "specific", "astrocytes", "stated", "manuscript", "Effects", "brain", "inflammatory", "response", "discarded", "given", "time", "course", "effects", "observed", "Regarding", "extracellular", "space", "ISF", "effects", "diffusion", "MRI", "would", "result", "changes", "diffusion", "fluid", "flow", "well", "slow", "result", "IVIM", "effects", "shown", "groups", "furthermore", "Sindex", "purposely", "calculated", "using", "lowest", "key", "b", "value", "make", "residual", "IVIM", "effects", "completely", "negligible", "Hence", "variations", "extracellular", "space", "shape", "volume", "reflect", "changes", "local", "cellular", "background", "kind", "negative", "imprint", "decrease", "Sindex", "sADC", "increase", "points", "decrease", "amount", "diffusion", "hindrance", "astrocytes", "far", "dominant", "cell", "type", "regions", "observed", "largest", "response", "stated", "discussion", "Based", "established", "diffusion", "MRI", "mechanisms", "hindrance", "decrease", "suggests", "astrocyte", "volume", "reduction", "associated", "increase", "ISF", "diffusion", "tortuous", "increase", "astrocyte", "membrane", "permeability", "water", "exchange", "believe", "statement", "mechanism", "leading", "observation", "correct", "broad", "enough", "manuscript", "changed", "Regarding", "DG", "data", "indeed", "noisy", "given", "small", "voxel", "count", "apparent", "group", "difference", "may", "coincidental", "accurately", "estimated", "precisely", "given", "small", "voxel", "count", "realize", "might", "puzzling", "scientific", "integrity", "decided", "hide", "results", "show", "note", "added", "caption", "SuppL", "Submitted", "filename", "Click", "additional", "data", "file", "Decision", "Letter", "Jiang", "Quan", "Academic", "Editor", "Quan", "Jiang", "Quan", "Jiang", "open", "access", "article", "distributed", "terms", "Creative", "Commons", "Attribution", "License", "permits", "unrestricted", "use", "distribution", "reproduction", "medium", "provided", "original", "author", "source", "credited", "Apr", "Diffusion", "MRI", "reveals", "vivo", "changes", "astrocyte", "function", "induced", "inhibitor", "Dear", "Dr", "Le", "Bihan", "pleased", "inform", "manuscript", "judged", "scientifically", "suitable", "publication", "formally", "accepted", "publication", "complies", "outstanding", "technical", "requirements", "Within", "one", "week", "receive", "containing", 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"publication", "information", "please", "contact", "onepress", "kind", "regards", "Quan", "Jiang", "Ph", "Academic", "Editor", "PLOS", "ONE", "Additional", "Editor", "Comments", "optional", "comments", "Responses", "Questions", "Comments", "Author", "authors", "adequately", "addressed", "comments", "raised", "previous", "round", "review", "feel", "manuscript", "acceptable", "publication", "may", "indicate", "bypass", "Comments", "Author", "section", "enter", "conflict", "interest", "statement", "Confidential", "Editor", "section", "submit", "recommendation", "Reviewer", "comments", "addressed", "manuscript", "technically", "sound", "data", "support", "conclusions", "manuscript", "must", "describe", "technically", "sound", "piece", "scientific", "research", "data", "supports", "conclusions", "Experiments", "must", "conducted", "rigorously", "appropriate", "controls", "replication", "sample", "sizes", "conclusions", "must", "drawn", "appropriately", "based", "data", "presented", 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Background ========== It is widely acknowledged that breastfeeding provides the ideal nutrition for infants, with authorities worldwide recommending exclusive breastfeeding for the first six months for children living in developed countries \[[@B1],[@B2]\]. Despite such endorsements, studies have shown that physician knowledge on breastfeeding is far from ideal \[[@B3]-[@B8]\], as they receive relatively little formal training on breastfeeding \[[@B5]\], and often fail to follow evidence-based medicine (EBM) guidelines \[[@B9]\]. However, it can be difficult for physicians to keep up-to-date with the high volume of constantly changing, sometimes conflicting, information. It has been suggested that a physician needs to read 17 articles each day to keep up with current medical literature, yet they have limited time to assess the information they receive \[[@B10]\]. Additionally, studies have shown that access to EBM resources is variable \[[@B11]-[@B16]\], and even when resources are available, the ability of physicians to locate relevant information and assess its validity appears limited \[[@B11],[@B12],[@B17]\]. It is therefore unsurprising that physicians may turn to specialist information services for help. Such services include, UKMi (a UK National Health Service \[NHS\] funded medicines information service that has specialist centres taking medicine calls on pregnancy and breastfeeding) \[[@B18]\] and the Motherisk Program (a Canadian-based teratogen information service) \[[@B19]\]. These services tend to be aimed at healthcare professionals, who report high levels of satisfaction with the information provided \[[@B20],[@B21]\]. Equivalent information services for consumers are limited, but the Breastfeeding Network (BfN) (a UK-based charity established in 1997) aims to be an independent source of support and information for breastfeeding women and others, including healthcare professionals \[[@B22]\]. One of the BfN services is the provision of 'Supporterline'; a service where information and support is provided to mothers who are breastfeeding that is complementary to that of health visitors (a health visitor is a UK qualified nurse whose role promotes health in the whole community, particularly involved with families who have children under five), midwives and other healthcare professionals. In 2006 the Supporterline took over 20,000 calls, many specifically related to medicine use. Consequently, in 2007 a national 0800 (free) telephone service for medicine-related enquiries was established, The Drugs in Breastmilk Helpline, which has seen year-on-year increases in enquiry numbers (e.g. 576 in 2007, which had risen to 2215 in 2010). To cope with this increased demand, the service now operates seven days a week and when not manned takes calls via an answer machine. The majority of enquiries are answered by a pharmacist, with support from a trained BfN worker. The Helpline has access to standard medical reference texts (e.g. The British National Formulary \[[@B23]\]) and specialist breastfeeding references, for example Hale\'s Medications and Mother's Milk\[[@B24]\], LactMed (an online database from The National Library of Medicine) \[[@B25]\], Briggs et al\'s Drugs in Pregnancy and Lactation\[[@B26]\] and Martindale The Extra Pharmacopoeia\[[@B27]\]. Given the growth in user numbers, the aim of this study was to assess consumer and healthcare professional opinion, analyse the enquiries taken and to ascertain how information provided was used. Methods ======= Enquirer opinion was surveyed via a structured telephone interview or self-administered survey. A sample size calculator \[[@B28]\] indicated that 90 participants were required to achieve 95% confidence (with a 10% margin for error) so that the study results reflect the results expected from the total enquirer population to the Helpline (in 2009, n = 1388). Analysis of workload data and factoring in a 40% drop-out rate indicated that the study duration would be approximately two months. Participant recruitment began in December 2010. Enquiries were generated either by telephone call or email. All enquirers who contacted the service were eligible to be enrolled in to the study, provided they were English speakers. For telephone enquiries, prior to the BfN call handler concluding the conversation, the enquirer was informed of the study and asked if they would be happy to be contacted in 2 to 4 weeks time to discuss the call further. For those people who agreed to be contacted, their details, along with the enquiry and answer provided by the Helpline, were sent to researchers at the University of Wolverhampton. Each caller was then telephoned and interviewed. Interviews followed a semi-structured format and took approximately 10 min to complete. For email replies, information relating to the study was included at the bottom of the written emailed reply, along with an invitation to complete the self-administered survey. Return of the email was taken as the person giving consent to participate. Questions used for both the interviews and surveys were drawn and adapted from UKMi surveys used to quality assure their service. Questions asked differed depending on whether the enquirer was a member of the public or a healthcare professional. However, the questions asked to each respective group were the same for both the telephone interview and self-administered survey. Data collected fell broadly into three sections: demographic information; evaluation of the Helpline; and usefulness of the information. Questions consisted of a mixture of open, closed and semantic differential scales (e.g. 5 point Likert scales). Prior to data collection starting, a two-week pilot study was undertaken on the study population to determine the data collection tools' content validity and reliability. Feedback from the pilot study showed that no changes were required. Research ethics committee approval was granted by The Behavioural Sciences Research Ethics Committee at Wolverhampton University. NHS ethics approval was not required as the work was assessed as evaluation and not research. Data analysis ============= Responses were collated, stored and analysed using survey software (SNAP for Windows) and spreadsheets (Microsoft Excel 2003 for Windows). Data entry were performed by two research assistants and the validity of data entry assured by a member of the research team (PR) who was not involved in data entry. PR undertook random checks of the data (equating to approximately 20% of all data). Simple descriptive statistics were used to summarise respondents' data. Free text answers were reviewed and categorised by PR. Data were managed using Excel and common themes generated that were agreed upon by both authors. Results ======= One hundred and forty-two enquirers were contacted. On follow up, twenty-one declined to participate and twenty were lost, leaving 101 replies for analysis: 77 from women (64 telephone calls and 13 emails); and 24 from healthcare professionals (10 telephone calls and 14 emails). Data from breastfeeding women ----------------------------- ### Demographic characteristics Most women were under the age of forty (88%, n = 68/77), had either 1 or 2 children (87%, n = 67/77), undertook formal education beyond A levels (84%, n = 63/75) (i.e. further education colleges or university) and were Caucasian (92%, n = 70/76). No calls were received from any person under the age of twenty-five. Demographic data are shown in Table [1](#T1){ref-type="table"}. ###### Characteristics of participants Demographic characteristics of the breastfeeding women enquirers ---------------------------------------------------------------------- ------------------------------------------ ------- ------- Age (n = 77) 25-29 18 23 30-34 29 38 35-39 21 27 over 40 9 12 Which child is this in your family? (n = 77) first 46 60 second 21 27 third 8 10 fourth 1 1 fifth 1 1 Ethnic origin (n = 76) white 70 92 mixed 2 3 Asian/Asian British 3 4 other 1 1 School leaving age (n = 75) 16 or under 8 11 17 4 5 18 12 16 19 or over 51 68 Healthcare professional occupation (n = 24) n % Infant feeding co-ordinator 8 33 Health visitor 6 25 Midwife 4 17 Physician 1 4 Pharmacist 1 4 Community staff nurse 1 4 Community breastfeeding leader 1 4 Hospital nurse specialising in lactation 1 4 Not stated 1 4 ### Breastfeeding women's opinion on the helpline Their experience with the service was very positive. Seventy (91%) women were either 'very satisfied' or 'satisfied' with how quickly their call was answered and stated that the answer they received provided sufficient information to enable them to make a decision regarding their enquiry (90%, n = 69/77). These findings are reflected in the level of satisfaction noted, where 72 (94%) were either 'very satisfied' or 'satisfied' with the overall service offered (Table [2](#T2){ref-type="table"}). Only three women felt that phoning the Helpline had not helped them. Analysis of these three calls showed that two involved complex calls, involving persistent nipple and breast pain that were being managed unsatisfactorily by their physician. The third case involved use of a topical nasal decongestant whilst breastfeeding. Advice given was that the woman could use the product, so it is unclear as to why this person said the advice did not provide enough information to make a decision. ###### Questions asked of breastfeeding women and healthcare professionals Healthcare professionals (n = 24) Breastfeeding women (n = 77) ------------------------------------------------------------------------------------ --------------------------------------- ---------------------------------- ---- ----- How did you find out about the telephone number? Birth to Five 0 0 12 16 given it by someone 6 26 23 30 internet 8 35 27 35 leaflet 8 35 16 21 other 1 4 0 0 Have you called the Drugs in Breastmilk Helpline before? Yes 17 71 13 17 No 7 30 64 83 Would you recommend this service to others? Yes 22 92 77 100 No 2 9 0 0 Did the information provide you with enough information to make a decision? Completely 20 87 69 90 Partly 3 13 5 7 Not at all 0 0 3 4 How satisfied were you with the response to your query, with respect to speed? Very satisfied N/A N/A 64 83 Satisfied N/A N/A 6 8 Neither N/A N/A 5 7 Dissatisfied N/A N/A 2 3 Very dissatisfied N/A N/A 0 0 Overall, how satisfied were you with the service? Very satisfied N/A N/A 69 90 Satisfied N/A N/A 3 4 Neither N/A N/A 4 5 Dissatisfied N/A N/A 1 1 Very dissatisfied N/A N/A 0 0 Despite these enquirers not having been given information to enable them to make a decision, they, as well as all other women, said they would recommend the service to other people (Table [2](#T2){ref-type="table"}). Most women (83%, n = 64) had not contacted the Helpline before and found out about the service primarily through the internet (35%, n = 27) or had been given the telephone number (30%, n = 23). Twelve (16%) had also been made aware by the 'Birth to Five' information booklet (an NHS information booklet distributed to all parents on the health, wellbeing and development of children from birth to five years old) (Table [2](#T2){ref-type="table"}). ### Content of enquiries Seventy enquiries directly related to medicine taking and seven involved giving general advice (Table [3](#T3){ref-type="table"}). Four of the latter involved issues of infant feeding whilst the mother was being treated with antifungal medication. Sixty-five enquiries involved the use of proposed medicines. These were predominantly to manage acute conditions, with self-management of minor illness (n = 11), anti-infectives (n = 14) and pain relief (n = 9) most frequently enquired about. Seventeen enquiries involved medicines that could be used for the management of long-term illness. These covered many medicine classes, with only medicines for depression (n = 4) and Crohn's disease (n = 3) receiving more than one enquiry. ###### Type of medicine-related enquiry received from participants Proposed medicines Currently taken medicinesN (%) Advice givenn (%) ----------------------------------- ------------------------ ------------------------------------ ----------------------- --------- Healthcare professionals (n = 24) 13 (54%) 5 (21%) 2 (8%) 4 (16%) Breastfeeding women (n = 77)\* 50 (65%) 17 (22%) 5 (7%) 7 (9%) \* Two enquiries involved medicines for the treatment of proposed acute and chronic illness. Five calls involved medicines that were currently being taken. Two concerned the effect medicines had on breastmilk (amitriptyline and penicillin), whilst the other three related to continuation of prescribed medication (asthma inhalers, azathioprine and propylthiouracil). The latter three enquirers had already spoken to a healthcare professional. In the calls involving azathioprine and propylthiouracil the women had been told by hospital physicians that they should not take the medicine, but this advice had been contradicted by pharmacists. In both instances the women wanted clarification on what to do. The Helpline advisor agreed with the pharmacist that the medicines could still be taken whilst breastfeeding. The call involving asthma inhalers centred on the woman wanting reassurance that the advice (to continue taking salbutamol and beclometasone inhalers) given by a practice nurse was correct, which it was. The Helpline stated that of the 70 enquiries involving medicines, 68 could be taken whilst breastfeeding. All women followed the guidance given by the Helpline. In the two enquiries where the Helpline did not advise to take the medicine, both involved referral back to the physician on grounds of potential misdiagnosis. Fifty-three women contacted a healthcare professional before phoning the Helpline; 49 of these enquiries were about the suitability of taking a medicine (Figure [1](#F1){ref-type="fig"}). General practitioners (n = 12) and hospital doctors (n = 8) were most frequently asked for advice; other healthcare professionals were infrequently asked (pharmacists, n = 2; midwife n = 2; health visitor n = 2; practice nurse n = 1). In 10 instances women asked more than one healthcare professional for advice (in 7 cases two were asked, and in 3 cases three were asked). The need for reassurance, and receiving conflicting information were two common reasons why women called the Helpline. Typical comments were: ![Handling of medicine-related enquiries from breastfeeding women by healthcare professionals (HCPs) and the Drugs in Breastmilk Helpline.](1746-4358-7-6-1){#F1} 'Conflicting information between different sources, wasn\'t sure which information was correct.' (participant 70, telephone interview) 'Conflicting information between the physician, pharmacist and drug information.' (participant 40, telephone interview) 'Instinctively I felt the drug would be fine, but just wanted to double check.' (participant 5, email survey) 'I had to know whether such an extended period of not breastfeeding was good for my baby. You were the only group offering advice tailored to breastfeeding mums.' (participant 9, email survey) Women also often spoke of distrust of the advice given by a healthcare professional or lack of confidence in their answer: 'Lack of confidence in the doctor's advice as he sounded confused and had to be reminded that I was breastfeeding.' (participant 51, telephone interview) 'Conflicting advice off the internet and didn\'t trust the consultant's advice.' (participant 31, telephone interview) 'Pharmacist wasn\'t sure; wanted to see if ok to use while breastfeeding.' (participant 33, telephone interview) In 11 of the 49 enquiries, the healthcare professional (seven of whom were physicians) was unsure of the answer and unable to tell the person if the medicine could be taken whilst breastfeeding. For 10 of these 11 cases, staff from the Helpline, after consulting reference sources, were able to advise the women that the medicine could be safely taken. In the remaining enquiry, information was supplied to the woman but no definitive answer on whether the medicine could be taken was given. This enquiry was answered by the BfN worker and, following protocol, is only able to provide information and not advice on whether the medication can be taken. Just 17 of the remaining 38 enquiries saw agreement between the healthcare professional and Helpline staff; in all these enquiries both parties agreed that the medicine could be taken. In the 21 enquiries where there was disagreement, healthcare professionals, on 10 occasions, said the medicine should not be taken, but information from the Helpline was that it was safe to take. In one enquiry the healthcare professional stated the medicine could be taken, whereas Helpline staff recommended the mother to speak again to the physician or a breastfeeding expert about her symptoms, even though the medicine was safe to use. In the remaining 10 enquiries women received conflicting advice from two or more healthcare professionals; in all cases the Helpline staff advised that the medicine could be taken. For all enquiries where the healthcare professional was either unsure or provided information contradictory to the Helpline, women chose to follow the Helpline advice. Table [4](#T4){ref-type="table"} highlights examples of the enquiries received during the study. ###### Examples of enquiries received and answers given --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Enquiry Helpline response Resources used by Helpline --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 'At around 37 weeks pregnant I had assumed that as I had been told to continue my medication (antidepressant) during pregnancy that it would be ok to breastfeed too, but I raised the query with the registrar at my clinic just to double check. He said he thought it would be fine but would check in a book; he did this while I waited and then told me the book said I should not breastfeed. He said he would check with the hospital pharmacy and let me know. I received a letter informing me it was ok for me to breastfeed whilst taking the medication about 4 weeks after my baby was born. Luckily enough I had contacted BfN rather than wait for the hospital to let me know!!' Information of studies carried out were supplied and an explanation was given that it was important for the patient to continue to take the medication so that she remained well, and that the benefits of breastmilk to her child far outweighed any risk to the baby through her taking medication. The amount of drug passing to the baby was discussed with the mother along with any potential side effects to look out for. Information was provided using: · Hale· Lactmed (<http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?LACT>) 'Got a bad back and wanted to know if it was safe to take naproxen or co-codamol (combination paracetamol/codeine product). Both the doctor and chemist said it was ok to take the medicines'. The mother was provided with information from Hale on the half lives of the drugs and how long the drug would remain in the mother's breastmilk. Naproxen has a half life of 12--15 hours and the amount passing into breastmilk is more than other non steroidal anti-inflammatory drugs such as ibuprofen or diclofenac. This makes it less preferable if the baby is under 6 weeks of age before which time hepatic and renal function is lower. There have been concerns raised about the safety of co-codamol during breastfeeding following the death of a baby in Canada. The mother was counselled to watch for signs of unusual drowsiness and poor feeding in the baby. If she noticed these responses she was advised to stop the drug and seek medical advice. Information was provided using: · Hale · Lactmed (<http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?LACT>) · <http://www.breastfeedingnetwork.org.uk/pdfs/Analgesia_and_breastfeeding_March_2010.pdf>\ <http://www.breastfeedingnetwork.org.uk/pdfs/Codeine-in-breastfeeding-June-08.pdf> Have bleeding cracked nipples, slight inversion so using nipple shields. Want something for pain and also want to know what formula to use as concerned about milk supply. Spoke with GP, pharmacist and nurse but none gave a very clear answer. Discussed moist wound healing and the need to feed or express milk more frequently. Tried to arrange a home visit from a breastfeeding supporter to help with optimising attachment of the baby at the breast, thereby preventing further nipple trauma. Mother was contacted again that evening; she was applying Vaseline to the cracks to prevent scab formation and taking paracetamol. She reported feeling better and will go back to the breast support clinic the following week Used information from:\ · BfN leaflet on moist wound healing plus breastfeeding support\ <http://www.breastfeedingnetwork.org.uk/pdfs/Cracked_Nipples_and_Moist_Wound_Healing_2002.pdf> Patient called about general anaesthetic as she was scheduled to have an ovarian cyst removed (planned operation not emergency). Consultant said to stop breastfeeding for 48 hours. Baby was 5 months old. Mother was informed about short half life of drugs used in general anaesthetic and that some of the drug would remain in fat cells of the body, to be released slowly over the following 48 hours, which might make her baby drowsy. It was suggested that she breastfeed as soon as she felt awake enough to do so. Information on the effect of any anti-emetics, which might be given was also discussed (could increase milk supply). Also discussed how the mother would manage feeds whilst in hospital, and whether she needed to have a breast pump available. Information was provided using:\ ·Hale\ ·Lactmed (<http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?LACT>)\ <http://www.breastfeedingnetwork.org.uk/pdfs/Day_Surgery_and_Breastfeeding_March_2009.pdf> --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Healthcare professional data ============================ Enquiries were taken predominantly from people whose role it is to support new mothers (Table [1](#T1){ref-type="table"}). All (100%, n = 24) healthcare professionals believed the Helpline response either completely or partially answered their enquiry, with 92% (n = 22/24) finding the information 'useful' or 'very useful'; consequently, the majority (92%, n = 22/24) would recommend the service to others. Table [2](#T2){ref-type="table"} shows that, in contrast to enquiries from breastfeeding women, the majority (71%, n = 17/24) of healthcare professionals had used the Helpline previously and preferred to email their enquiry (58%, n = 14/24 compared to only 17%, n = 13/77 of women), even though telephone responses were answered more quickly (100% within 24 hours, compared to 79%). Like women, the internet (35%, n = 8/23) and recommendation by another person (26%, n = 6/23) were common ways that they found out about the Helpline, although healthcare professionals also stated leaflets (35%, n = 8/23) were frequently the source of the information. Analysis of the 24 enquiries showed that 18 were for proposed medicines, 4 involved advice giving, and just 2 were for medicines that were being currently taken (Table [3](#T3){ref-type="table"}). Of the 18 medicines for proposed use, 13 (72%) were to treat acute problems. Medicines involved in these cases were wide ranging, with only anti-infectives (n = 2) and domperidone (n = 2) occurring more than once. The 5 enquiries received for proposed medicines to treat chronic conditions were equally diverse, for example medicines to treat schizophrenia, Attention Deficit Hyperactivity Disorder and Cystic Fibrosis. The two enquiries that concerned medicines being currently taken for chronic conditions were montelukast for asthma and levothyroxine for hypothyroidism. Fourteen (58%) had consulted at least one reference source before contacting the Helpline. The most frequently used reference text was Hale (a specialist text on the safety of drugs in breastfeeding) (n = 9) or the British National Formulary (BNF - a standard text used in the UK for prescribing information) (n = 6). In two cases, a hospital drug information pharmacist was also consulted. Five of the six people that consulted the BNF went on to say that the information it contained was not detailed enough to answer the problem. One of these respondents also stated they did not have access to Hale and thus called the Helpline. Hale was most frequently consulted, yet despite it being more informative than the BNF, 66% of respondents still contacted the Helpline as they believed they would get a more definitive answer based on prior use of the service. Where healthcare staff contacted a specialist drug information pharmacist, the information they provided was deemed insufficient by the enquirer to answer their question. Four of the 10 healthcare professionals stated that previous positive experiences of the Helpline prompted them to contact the service rather than consult reference sources and two called for reassurance (the other enquirers never specified a reason). All enquirers said the information provided by the Helpline either completely (n = 20/23, 87%) or partly (n = 3/23, 13%) provided them with enough information to make a decision. Once in possession of the information, 22 went on to say how they used the information. Thirteen passed on the information to the patient, five gave the information to the relevant prescriber and four used it to make a treatment decision. Discussion ========== The findings from this study show that the predominately pharmacist-led service provided by the BfN is well received by both healthcare professionals and breastfeeding women, and is consistent with user satisfaction surveys of pharmacist-led drug information services \[[@B20],[@B21]\]. Unlike most information services, the BfN Helpline actively encourages the public to contact the service, and this was reflected in a higher proportion of enquiries taken from the public compared to healthcare professionals. Most women were previous non-users of the service in contrast to healthcare professionals, where the majority had contacted the service before. The healthcare professionals predominantly had a specialist role associated with maternal services and this may explain why they were aware of the service. Provision of the Helpline seems well-founded given the level of conflicting information given by two or even three healthcare staff, and the lack of confidence or distrust women had in answers they provided. The disparity seen between the answers given by the Helpline and the responses from healthcare staff further reinforces the decision by their patients to seek out specialist advice. The fact that over 75% of healthcare staff did not know the answer, or gave a conflicting answer compared to that given by Helpline staff, suggests that healthcare staff may not have sufficient knowledge or resources in this field; which echoes previous findings \[[@B2]-[@B7],[@B29],[@B30]\]. Where Helpline staff contradicted healthcare staff advice, breastfeeding women chose to follow Helpline advice. This was not unexpected as many women had contacted the Helpline as a 'last resort'. However, in clinical practice the contradiction of one healthcare professional by another can be difficult, especially when one is perceived as more authoritative than the other (e.g. lactation consultant doctor versus a pharmacist). In these situations, it would seem most prudent for the contradicting person to talk with the person who gave the initial advice, so that a consistent message is given to the breastfeeding women. This did not routinely appear to happen in this study. Findings from this study also raise questions about the suitability of 'generalist' healthcare staff to handle breastfeeding enquiries; in particular, physicians, as they were most consulted. A study by Wallace and Kosmala-Anderson reinforces this view, as they found that general practitioners in the UK saw updates on prescribing for breastfeeding mothers as a low priority, with just 19% highlighting this as an area of need, despite only 34% describing themselves as competent or expert in this field \[[@B31]\]. Only just over half of healthcare professionals had consulted a reference source, and those that did found standard texts, such as the BNF (which almost all UK healthcare staff have easy access to), to be inadequate, as data contained within it lacked sufficient detail. The deficiency of the BNF has been highlighted by the National Institute for Health and Clinical Excellence (NICE) and advocates that healthcare professionals consult supplementary sources of information \[[@B32]\]. Whilst some healthcare staff did consult other sources (i.e. the specialist text by Hale), they still felt unsure on how to interpret the information in a way that was useful to the mother, and sought guidance from the Helpline. This lack of confidence in interpretation of appropriate reference sources suggests a greater need for training. It further supports the use of a specialist, given their access to more resources (as evidenced from Table [4](#T4){ref-type="table"}) and ability to interpret the data to provide informed answers on medicine suitability. A study by Akus also found that healthcare practitioners were using outdated sources for making safety recommendations to their patients \[[@B33]\]. Whilst this cannot be substantiated in this study, other studies \[[@B34]\] have found similar results to Akus and it seems unlikely that healthcare staff in practice are using the most up-to-date reference sources when evaluating medicine suitability for breastfeeding mothers. Enquiries predominantly related to proposed medicines to treat acute self-limiting problems. Advice given by healthcare staff to patients erred on the side of caution; that is they were told to avoid taking the medicine. Whilst this negates any possible adverse events to the child, it does not necessarily equate to appropriate clinical care where decisions should be evidence-based and an assessment of the risk versus benefits to the baby made \[[@B35],[@B36]\]. In defence of healthcare staff, the lack of manufacturer data on the effects of medicines passing in to breastmilk makes recommendation of products problematic. Without safety data, manufacturers generally advocate avoidance. If products are then recommended, prescribers do so outside of the product licence, and liability will fall on that individual and not the company. Amir and colleagues looked at knowledge, attitude and practices of Australian GPs in obtaining information for medicines for breastfeeding women, and found that medico-legal concerns were common, and 76% stated that this was important in their decision-making \[[@B37]\]. What is not in question is that patients will continue to require medication during breastfeeding. Studies have shown that this is relatively commonplace \[[@B38]-[@B40]\], and medicines play an important part in a woman's decision to start and continue breastfeeding, with women frequently hesitant to combine the two, choosing either to stop breastfeeding or attempt to limit exposure \[[@B40]-[@B42]\]. Therefore, given healthcare professional uncertainty and caution in advice-giving, it was interesting to note that the Helpline was able to give definitive answers in almost all cases relating to medicine taking. Given that other countries do experience similar problems with under-skilled healthcare staff in this field, then the establishment of similar Helplines could be considered. The costs of running the BfN Helpline was initially modest, but as call numbers have increased the Helpline has had to rely on greater levels of goodwill from the people that answer the enquiries. From our experience it would be prudent, when starting such a Helpline, to factor in additional resources and money to meet increased demand. Limitations =========== It is acknowledged that this study provides a 'snapshot' of the service, and the number of participants was relatively low. In addition, the study had a high drop-out rate/lost to follow-up, which may have affected the data through non-response bias. Lastly, the findings may not be broadly generalisable to other countries due to differing healthcare structures. Conclusion ========== The Breastfeeding Networks' Drugs in Breastmilk Helpline provides an important service to patients and healthcare staff to make informed decisions on medicine-taking whilst breastfeeding. The relatively high level of healthcare professional uncertainty or incorrect advice given to patients, coupled with patient distrust, signals that healthcare professional education needs improving and that greater use of specialist services should be encouraged. Competing interests =================== The work was supported by the Breastfeeding Network to employ Miss Seera and Miss Matharu to conduct the telephone interviews. Wendy Jones is employed by the Breastfeeding Network to answer calls, but was not involved in data collection or analysis (except for agreeing qualitative themes). Authors' contributions ====================== PR and WJ constructed the data collection tool. WJ answered the majority of the calls. PR oversaw the telephone interviews and verified data capture. PR undertook the analysis. PR and WJ jointly wrote the manuscript. Acknowledgements ================ Thanks to Rinisha Seera and Summet Matharu in data collection and entry. The role of Mrs Ruth Rhodes in answering calls to the Drugs in Breastmilk Helpline and collection of data from callers is gratefully acknowledged, as is the support of the Breastfeeding Network in funding the operation of the Drugs in Breastmilk Helpline.	{ "pile_set_name": "PubMed Central" }	[ "amount", "treatment", "incorrect", "born", "two", "almost", "went", "refer", "given", "work", "origin", "beclometason", "program", "remain", "eg", "divers", "machin", "speaker", "ie", "respond", "someth", "collect", "thank", "longterm", "ovarian", "employ", "healthcar", "network", "written", "risk", "grant", "rais", "lastli", "fund", "take", "week", "convers", "twentyf", "hour", "locat", "occas", "signal", "may", "back", "luckili", "keep", "team", "necessarili", "therebi", "supporterlin", "wellfound", "httptoxnetnlmnihgovcgibinsishtmlgenlact", "thirteen", "contradict", "fifth", "live", "use", "dropout", "gener", "cystic", "invit", "support", "visitor", "bottom", "complet", "anaesthet", "prior", 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Introduction ============ Dental implants have always undergone different modifications in order to adapt them as much as possible to the morphology of the bone where they will be placed ([@B1]). In this regard, it is important to reduce the diameter of implants placed in areas with limited space for prosthetic reconstruction, such as between adjacent crowns, as conventional-diameter implants may cause damage to the periodontal ligament of adjacent teeth o compromising aesthetics ([@B2]-[@B4]). Narrow implants (those with a diameter less than 3.4 mm) ([@B5]) are particularly useful when the bone crest width does not allow for placement of a conventional diameter implant without the use of regeneration techniques ([@B6]-[@B8]). There is currently no clear scientific evidence regarding the success rate of narrow implants ([@B9]-[@B11]). This study's main purpose is to evaluate the bone loss (BL) around narrow implants after 2 years of implant loading and compare it to that of conventional¬ diameter implants. BL were also evaluated by taking into account where implants were placed, type of loading, whether implants were placed in areas that had previously undergone bone regeneration, or whether patients had previously suffered from periodontal disease. Material and Methods ==================== -A retrospective case-control study was carried out. 82 implants (31 narrow implants and 51 conventional implants) placed in 40 patients were selected. To avoid statistical bias, a cluster of one implant per patient was randomly selected (20 narrow implants and 20 conventional implants). Randomization was carried out by numbering the implants present in the patient and rolling a die to identify the selected implant. The group "Cases" described as patients of either gender or age, carriers exclusively narrow implants (StraumannTM, grade 4 titanium, SLA surface, 3.3 millimeters in diameter, length 10-12 mm, Switzerland) and having a minimum 2 years of follow- up. 20 patients with these characteristics were found from 2009 until 2014. The group "Control", sought by matching for gender and age other 20 patients, who will carry only standard implants of the same brand, with identical lengths and identical tracking system but with 4.1 millimeters in diameter. To evaluate the changes resulting from BL around the implants, a total of 80 panoramic radiographs were used of the 40 patients. The first panoramic image of each patient was taken when the implants were loaded and the second one 2 years after implant placement, using a Planmeca ProMax® orthopantomograph, serial number RDX309857, with a magnification of 1-1. The patients' head and lip position in the orthopantomograph was controlled when taking the radiographs. All BL measurements were taken by a calibrated examiner. This calibration consisted in having a second examiner retake all measurements and compare them. The evaluator calculated a correlation coefficient of 0.925, which implies a coincidence percentage higher than 90%. -Inclusion and exclusion criteria: The patients included in this study had implants (3.3 or 4.1 mm) placed in either the anterior or posterior region of the mandible and maxilla. The patients excluded in this study were treated with 8-mm or smaller implants, whose implants were loaded for less than 2 years, with more than two years of follow-up but no radiography at 2 years (the initial total number of patients with narrow implants was 22, but the final number of patients studied is 20), patients taking medication that affects bone metabolism (corticosteroids, bisphosphonates), patients with inflammatory bone diseases or genetic diseases, patients with active periodontal disease (must be under control after 6 months of periodontal treatment). No early failures were recorded before two years. -Studied variables: In the present study, BL (main variable) was measured (each implant was assigned to a category) using Lagervall & Jansson's index ([@B12]) modified by Corcuera Flores et al. ([@B8]) to add a fifth category, using panoramic radiographs of each patient to compare the panoramic images taken after implant loading with the ones taken two years later. The implants used in this study were loaded with single crowns, fixed implant-borne prostheses that require splinting with other implants or overdentures. The variables tobacco smoking, age and gender were collected from the medical records of patients enrolled in the study. Periodontitis was pre-diagnosed when patients exhibited insertion loss and/or a probing depth of 4 mm or more and bleeding on probing ([@B13]). An osteoconductive biomaterial (cancellous bone) and equine collagen membranes (Bio- Gen® and Bio-Teck®, BIOTECK S.p.A., Arcugnano VI Italy) were used when bone grafting was necessary. If this type of bone graft was used, it was noted in the patient's medical record. -Statistical study. All statistical procedures were carried out using SPSS 19.0 for Windows (IBM, USA). Frequency and percentage were used to describe qualitative variables. The correlation coefficient was measured using the Spearman test. Qualitative data were compared using the Chi-squared test and applying Haberman's post hoc test. Quantitative ordinal data (BL) were compared using the Mann-Whitney U test if the independent variable had 2 categories, and the Kruskal-Wallis test was used if the independent variable had more than 2 categories. The statistical significance can be found in the Tables, while the normal ranges are displayed in the text. Regarding the power of the study, this requires an expected difference between groups who was estimated at 0.6 points in relation to the primary endpoint (BL). Under these conditions and considering a margin of error of 5% alpha, the power of the test is higher than 78% (less than 22% beta error). The statistical significance was p \< 0.05. Results ======= The study's sample consisted of 40 patients (23 women and 17 men) with an average age of 66.97 years (range 58-72). A total of 82 implants were placed in these patients. Data on the patients studied and implants placed can be seen in [Table 1](#T1){ref-type="table"} and [Table 2](#T2){ref-type="table"}. If the sample was restricted to one implant per patient, only 40 implants were analyzed ([Table 2](#T2){ref-type="table"}). No significant differences in BL were found when comparing narrow implants with conventional implants after 2 years of implants loading. Table 1Descriptive data of the sample. Table 2Data from the selected implants for case-control study. The BL variable was analyzed in addition to its correlation with other variables (bone regeneration, previous periodontal disease, type of prostheses, age...) using one implant per patient, and by analyzing only narrow implants ([Table 3](#T3){ref-type="table"}). No significant differences in BL were found when factoring for age or for patients with previous cases of periodontitis. Significant differences were found when comparing BL with patient tobacco use (p \< 0.05), in the sense that all implants placed on patients who used tobacco had some kind of BL. Table 3Comparison of BL (Lagervall & Janson) in selected implants. Data that compare BL of narrow implants, conventional implants or booth using or not bone regeneration didn't show any significant differences. No significant differences were found when comparing bone loss of narrow implants with that of conventional implants. There were also no significant differences found when comparing BL with type of implant prostheses ([Table 3](#T3){ref-type="table"}). Discussion ========== Both periapical ([@B14],[@B15]) and panoramic ([@B8]) radiographs can be used to measure BL. Measurements can be taken directly in millimeters, comparing the position of the peri-implant bone in relation to the implant shoulder ([@B16]), or by dividing the implant into 3 equal parts from the implant shoulder to the apex and observing which third of the implant length the bone resides in ([@B12]). Although measurement in millimeters provide greater accuracy and greater statistical power, the complexity of measuring the BL in panoramic radiographs could subtract accurately measure. The use of Langervall-Jansson's ([@B12]) scale for this study was selected because it had been validated in previous publications ([@B8]). This could be of interest when comparing the results of this study with those of other researchers. Limited bucco-lingual bone crest width has always been one of the main indications for placement of narrow implants ([@B17]-[@B23]). The BL of narrow implants without bone regeneration was compared with the BL of conventional implants that used bone regeneration to gain bucco-lingual crest width. No significant differences in rates of bone loss were found between both types of implants studied. With the limitations of the present study, both procedures appear to present similar predictability, although of course the use of narrow implants is less traumatic for the patient ([@B24],[@B25]). The present study used narrow implants to rehabilitate all types of prosthesis (single crowns, splinted crowns, and overdentures). No significant differences in BL were found. However, it should be noted that given the sample size of the present study, further studies focusing on this point would be necessary. In the present study, no early or late failures were recorded in narrow implants. Regarding the survival rate of narrow implants, implant fracture is a frequent complication when using this type of implants ([@B9],[@B10]). The present study found no incidence of fracture in the implants studied. Wang et al. ([@B19]) determined a survival rate of 93.5% based on 31 narrow implants observed over one year. On the other hand, Anitua et al. ([@B2]) placed 89 narrow implants, using prostheses similar to those in the present study (overdentures, single crowns and implants splinted) and evaluating the implants after 4 years. The results showed that only one implant was lost, resulting in an overall survival rate of 98.9%. It should be noted that in the study by Anitua et al., the follow-up radiograph carried out after 2 years (similar to the final radiographs in the present study), the average BL was 1.26 mm, or less than one-third of the implant length ([@B2]). The majority of the narrow implants in the present study fell within this range (51%). The present study fails to find statistical differences when comparing the BL of narrow implants placed in patients who had previously suffered periodontitis with implants placed in patients who had not. There is scientific evidence regarding the link between periodontitis and periimplantitis ([@B26]). However, in the present study, this evidence is not conclusive when BL is compared against to prior cases of periodontitis that were successfully treated. Despite this, there are other studies that support the findings indicating a link between the two variables ([@B8],[@B27]). As in other studies, in our data we also found a higher BL in smokers, both narrow implants and standard ([@B27],[@B28]). Within the confines of the present study, no significant differences in BL around implants were found when comparing narrow implants with conventional implants after 2 years of implants loading. In this sense, no differences were found when bone regeneration techniques had been used or not, or between types of prosthesis load supporting the implants. Smoking appears to increase BL around the implants studied, thus, practitioners should take greater precautions when placing implants in smokers. Ackcnowledgements None declared. \${Availability of data and materials} None declared. Authors contributions None declared. Ethics None declared. Conflicts of interest The authors have declared that no conflict of interest exist. 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Introduction {#Sec1} ============ A vast majority of currently published research on microsaccades, as well as almost all public discourse on them, refers to these tiny fixational eye movements, which occur when we attempt to fix our gaze, as involuntary or spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}). However, a multitude of evidence from the literature actually points in the opposite direction. For example, likelihoods of microsaccade direction, amplitude, and frequency are systematically modulated under a variety of conditions^[@CR1]--[@CR17]^, and microsaccade generation is causally affected by activity in the midbrain superior colliculus (SC)^[@CR18]--[@CR21]^ and the cortical frontal eye fields (FEF)^[@CR22]^, both involved in voluntary eye movement control^[@CR23]^. Microsaccades can also be suppressed either voluntarily^[@CR24],[@CR25]^ or when perceptually challenging discrimination stimuli are expected^[@CR7],[@CR8]^, and they precisely re-align gaze on the fixated stimulus^[@CR26]--[@CR29]^, even when behavioral tasks require peripheral-stimulus monitoring^[@CR3]--[@CR5]^. Finally, two highly experienced human subjects were described as being able to generate voluntary horizontal or vertical saccades as small as microsaccades^[@CR30]^. The persistence of microsaccade descriptions as being involuntary and spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}), despite the above evidence, stems from a severe lack of explicit demonstration that these eye movements, like larger saccades, can be generated by naïve subjects at will, based on abstract task-defined instructions, and without visual guidance. The strongest such evidence^[@CR30]^, based on a small number of expert observers and only with cardinal movement directions, has gone without direct follow-up for more than 40 years. However, such explicit demonstration is critically needed, particularly in the present day, given the resurgence of the field^[@CR4],[@CR6],[@CR20]^ (Supplementary Fig. [1](#MOESM1){ref-type="media"}), and given that the occurrence of any individual microsaccade is now known to strongly alter even peripheral visual performance^[@CR3]--[@CR5],[@CR31]--[@CR35]^, providing an almost-deterministic link between covert visual attentional effects and the execution of such tiny eye movements^[@CR3]--[@CR5]^. Here, we provide such demonstration; by adapting, in a novel way, a behavioral task exercising voluntary eye movement control to the realm of microsaccades, we show in both naïve human subjects and monkeys that tiny eye movements overwhelmingly thought to be involuntary (Supplementary Fig. [1](#MOESM1){ref-type="media"}) can be generated at will, based on arbitrary instruction, and without any visual guidance. We also show that these eye movements are governed by highly specific SC neural circuitry. Results {#Sec2} ======= Memory-guided microsaccades are generated at will {#Sec3} ------------------------------------------------- We first asked three male rhesus macaque monkeys to perform a novel memory-guided microsaccade task, which was adapted from the known memory-guided saccade task^[@CR36],[@CR37]^ (Methods). The monkeys were initially trained to maintain fixation while a brief (58 ms) flash was presented at an eccentricity ≥5^o^, greater than an order of magnitude larger than microsaccade amplitudes (most frequently defined as \< 1^o^, ref. ^[@CR38]^, but often significantly smaller, ref. ^[@CR18]^). The monkeys had to remember flash location for 300--1100 ms, after which the fixation spot was removed. The monkeys used fixation spot disappearance as the abstract instruction to voluntarily execute a timely eye movement toward the remembered flash location, and the target only reappeared after the eye movement had ended (Methods); there was no specific instruction to re-fixate the reappearing target, but the monkeys often did so nonetheless. Our task therefore required voluntary eye movement generation based on an arbitrary instruction that we enforced by task design (fixation spot disappearance) and without any visual guidance^[@CR36],[@CR37]^; corrective re-fixation saccades were visually guided. After initial training with large saccades, we tested the monkeys on pseudorandomly chosen stimulus locations, including at small eccentricities typically associated with microsaccades. We specifically interleaved small and large eccentricities across trials within any given session, and we also sampled different visual field locations. We did so in order to avoid overtraining any specific locations or eccentricities. We were struck by the fact that the monkeys very easily generated memory-guided movements even for target eccentricities as low as 6 min arc (0.1^o^), and without any need for special training. They simply generalized the rule that they had learned for eccentricities ≥5^o^. Figure [1a](#Fig1){ref-type="fig"} demonstrates this by way of an example trial from monkey N. The monkey fixated steadily between flash onset (at a location 6 min arc left of and 12 min arc below fixation) and the instruction to trigger a movement (the go signal). The monkey then successfully generated an oblique downward--leftward microsaccade approximately 206 ms after the go signal (fixation spot disappearance). After target reappearance, the monkey corrected the remaining eye position error (due to overshoot in the memory-guided microsaccade) with a second visually guided microsaccade. Note how the reaction time of the corrective, visually guided microsaccade (from target reappearance) was comparable to that of the voluntarily generated memory-guided microsaccade (from fixation spot disappearance), and that the latter movement was directionally accurate (directed towards the remembered foveal flash location). Thus, spatially and temporally accurate microsaccades can be triggered at will, based on arbitrary instruction and without visual guidance (also see Supplementary Movies [1](#MOESM5){ref-type="media"}, [2](#MOESM6){ref-type="media"}).Fig. 1Memory-guided microsaccades in monkeys and humans. a Eye position measurements from an example trial in monkey N. Upward deflections in each trace mean rightward or upward eye position displacements, respectively, and the position scale bar denotes 15 min arc. A brief target flash appeared 6 min arc to the left of and 12 min arc below fixation. After a memory interval, the fixation spot disappeared, instructing a memory-guided eye movement towards the remembered flash location. The monkey generated a spatially accurate memory-guided microsaccade after a reaction time (RT) of approximately 206 ms from fixation spot disappearance. When the target reappeared, a smaller, corrective movement (which was now visually guided) was triggered after a shorter reaction time. b Similar observations from an example trial in a human subject. In this case, the target flash occurred at 18 min arc to the right of and 6 min arc above fixation. Note that the human data were recorded with a video-based eye tracker; monkey N data were obtained from a scleral search coil (Methods). Figures [2](#Fig2){ref-type="fig"}--[4](#Fig4){ref-type="fig"} and Supplementary Figs. [2](#MOESM1){ref-type="media"}--[4](#MOESM1){ref-type="media"} demonstrate the robustness of the observations in this figure across the population, and Figs. [6](#Fig6){ref-type="fig"}--[7](#Fig7){ref-type="fig"} and Supplementary Fig. [5](#MOESM1){ref-type="media"} highlight underlying neural mechanisms Timely voluntary memory-guided microsaccade generation was consistent in all three monkeys, and also in seven human subjects (Methods; Fig. [1b](#Fig1){ref-type="fig"} shows an example trial from one human subject). For all flash eccentricities \< 1^o^ (the most typically cited amplitude threshold for microsaccades^[@CR18],[@CR38]^; Methods), we plotted histograms of all microsaccade latencies after the go signal (fixation spot disappearance; Fig. [2a, d, g, j](#Fig2){ref-type="fig"}). This allowed us to evaluate the reaction times of instructed, memory-guided microsaccades. We also plotted the histograms of microsaccade latencies after target reappearance (Fig. [2m, p, s, v](#Fig2){ref-type="fig"}), for evaluating the reaction times of corrective, visually guided microsaccades. In all cases, memory-guided microsaccades had reaction times consistent with these movements being successfully triggered after task instruction^[@CR39],[@CR40]^. Moreover, the latency distributions were similar to those of larger memory-guided saccades with flash locations \>1^o^ (Supplementary Fig. [2](#MOESM1){ref-type="media"}). Corrective, visually-guided microsaccades expectedly had shorter reaction times than memory-guided microsaccades (monkey N: 146 ms vs. 203 ms; monkey M: 160 ms vs. 212 ms; monkey P: 189 ms vs. 245 ms; humans: 189 ms vs. 252 ms; all p \< 10^−50^, t-test). Therefore, timely, voluntary microsaccades were generated at will after the abstract instruction to do so (fixation spot disappearance).Fig. 2Memory-guided microsaccades had reaction times and success rates consistent with being genuine responses to task instruction. a Reaction time distribution for memory-guided microsaccades in monkey N. The data shows all trials with target eccentricities \<1^o^ (Methods). The monkey had a reaction time distribution typical of saccadic responses. Error bars denote 95% confidence intervals, and the histogram was normalized by the total number of trials. b Cumulative probability of the same data, demonstrating a plateau of \>80% success rate; the monkey successfully generated a memory-guided microsaccade (that is, within a reasonable reaction time; Methods) on the great majority of trials. Error bars denote 95% confidence intervals. c Same data as in b but separated by target eccentricity (inset legend). Even the smallest target eccentricities were associated with a majority of successful reactions. d--f Similar observations from monkey M. g--i Similar observations from monkey P. j--l Similar observations from our human subjects (Methods). m--x We repeated the same analyses above, but now for corrective, visually guided microsaccades occurring after target reappearance. Reaction times (m, p, s, v) were faster than for memory-guided microsaccades, and often exhibited express reactions (e.g. arrow in m). However, success rates were significantly lower than in the instructed memory-guided microsaccades (n, q, t, w; the horizontal blue line in each panel, and associated 95% confidence interval, replicates the plateau success rate from the corresponding panel in b, e, h, k). Success rates of corrective, visually-guided microsaccades also depended on target eccentricity (o, r, u, x) similarly to memory-guided microsaccades. n = 781, 1,602, 2,235, 7,402 memory-guided trials in monkey N, monkey M, monkey P, and humans, respectively. n = 2774, 4471, 3461, 10,866 visually guided trials in monkey N, monkey M, monkey P, and humans, respectively It may be argued that while memory-guided microsaccades did occur in a timely fashion, their likelihood of occurrence (what we defined as the success rate) was simply low and random as opposed to reflecting a deterministic, voluntary reaction to an explicit go instruction. After all, irrelevant abrupt onsets can reflexively reset microsaccade generation rhythms^[@CR41]--[@CR43]^, suggesting that some aspects of microsaccade behavior may appear to be reflexive in nature. However, plotting cumulative probabilities of memory-guided microsaccades after the go signal (i.e. all trials with target eccentricity \<1^o^), we found that almost every single instructed trial was associated with a response (Fig. [2b, e, h, k](#Fig2){ref-type="fig"}; erroneous trials were primarily ones with reaction times larger than our upper limit for acceptance, Methods). That is, using solely reaction time as the success criterion (Methods), the great majority of memory-guided microsaccade trials were successful. This was not the case for, say, microsaccades after flash onset at the beginnings of trials when monkeys were instructed to maintain fixation (Supplementary Fig. [3a-k](#MOESM1){ref-type="media"}). These early flash-induced microsaccades were not instructed, and therefore did not happen on every single trial (e.g. they were missing in the example trials of Fig. [1](#Fig1){ref-type="fig"}); their occurrence depended on a variety of other factors related to the quality of fixation control^[@CR3],[@CR5]^. More importantly, success rate for the instructed memory-guided microsaccades (i.e. the fraction of trials in which a movement was successfully generated within a reasonable reaction time; Methods; Fig. [2b, e, h, k](#Fig2){ref-type="fig"}) was equal to or significantly higher than that for the corrective, visually guided microsaccades, which were not instructed (Fig. [2n, q, t, w](#Fig2){ref-type="fig"}) (monkey N: p = 0.028; monkey M: p = 0.5; monkey P: p \< 10^−8^; humans: p \< 10^−50^, χ^2^-test). This indicates that task instruction was a strong impetus to generate the voluntary microsaccadic eye movements in our task. Expectedly, the lowest success rate for both memory- and visually guided (corrective) microsaccades was for the smallest target eccentricities (Fig. [2c, f, i, l, o, r, u, x](#Fig2){ref-type="fig"}). Overall, these results indicate that memory-guided microsaccades can indeed be generated explicitly following abstract instruction (i.e. one that is learned from task design), and even more successfully than uninstructed corrective, visually-guided movements. These are hallmarks of voluntary behavior. Interestingly, when visually-guided microsaccades were explicitly instructed in a separate condition (the delayed, visually guided saccade task; Methods), their success rates were also less than those of the memory-guided microsaccades (Supplementary Fig. [4a, d](#MOESM1){ref-type="media"}). This is largely due to the fact that the monkeys reflexively fixated the visually persistent target in this task paradigm even before the go signal. Memory-guided microsaccades are spatially accurate {#Sec4} -------------------------------------------------- High success was also evident in the directional accuracy of memory-guided microsaccades and not just in their reaction times (Fig. [2](#Fig2){ref-type="fig"}). For the same data in Fig. [2](#Fig2){ref-type="fig"}, we now plotted the angular directional difference between memory-guided microsaccades and target locations (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}). That is, we plotted the direction error of the movements. We also plotted the same measure for the uninstructed corrective, visually guided microsaccades (Fig. [3c, g, k, o](#Fig3){ref-type="fig"}); in this case, we compared corrective microsaccade direction to the direction of the reappearing target relative to gaze position after the memory-guided microsaccade had ended (Methods). Once again, in both monkeys and humans, memory-guided microsaccades were significantly more directionally accurate than the uninstructed corrective, visually guided movements. We confirmed this statistically by comparing the distribution of absolute directional errors for memory-guided microsaccades to that of absolute directional errors for corrective, visually guided microsaccades. In monkey N, the former was 20.4° ± 5.9° 95% c. i. and the latter was 27.1° ± 1.3° 95% c. i. (p \< 10^−4^, Watson-Williams test, circular analog of the two-sampled t-test); for monkey M, the results were 23.4^o^ ± 2.5° 95% c. i. and 31.5° ± 1.1° 95% c. i. (p \< 10^−10^); and for monkey P, they were 33.8° ± 2.4° 95% c. i. and 51.2° ± 1.4° 95% c. i. (p \< 10^−50^). Similarly, for humans, the directional errors were 23.8° ± 0.8° 95% c. i. versus 43.1° ± 0.9° 95% c. i. (p \< 10^−50^). When instructed visually guided microsaccades were tested separately (during the delayed, visually guided saccade task; Methods), directional accuracy was restored to the levels of memory-guided microsaccades (Supplementary Fig. [4b, e](#MOESM1){ref-type="media"}), consistent with prior results^[@CR26]^. Therefore, we observed high success in both reaction time (Fig. [2](#Fig2){ref-type="fig"}) and directional accuracy (Fig. [3](#Fig3){ref-type="fig"}) of instructed memory-guided microsaccades.Fig. 3Memory-guided microsaccades had better directional accuracy than either microsaccades during fixation or corrective, visually-guided microsaccades. a We plotted the angular distribution of microsaccade direction error (that is, the difference between target direction and microsaccade direction; Methods). In this measure, if a microsaccade has the same direction as the target direction, then the movement has zero direction error. In this panel, this measure is shown for all microsaccades from monkey N occurring in the final 250 ms of fixation before fixation spot disappearance. Microsaccades could occur either towards or away from the remembered target location. The histogram was normalized by the total number of observations. b The same monkey exhibited highly accurate microsaccade directions toward the remembered target location when explicitly instructed to do so. c After target reappearance, corrective, visually-guided microsaccades were also target-directed, but their direction errors were significantly more variable than the instructed movements in b (see statistics in text). Thus, instructed memory-guided microsaccades were highly directionally accurate. d Each group of colored arrows shows the directions of 40 randomly selected memory-guided microsaccades when remembered target directions were drawn from the underlying corresponding-colored faint cone. Directionally accurate microsaccades could be generated for all oblique target directions. e--h Similar observations for monkey M. i--l Similar observations for monkey P. m--p Similar observations for our human subjects. In all cases, directional accuracy was the highest for the instructed, memory-guided movements and not for fixational microsaccades (a, e, i, m) or corrective, visually guided ones (c, g, k, o); also see Supplementary Fig. [3](#MOESM1){ref-type="media"} for flash-induced microsaccades. For the leftmost column, n = 1,432, 2,875, 4,870, 2,831 microsaccades for monkey N, monkey M, monkey P, and the human subjects, respectively. The numbers of trials for memory-guided or corrective, visually guided microsaccades are the same as in Fig. [2](#Fig2){ref-type="fig"} It may also be argued that the directional accuracy described above was not necessarily a result of a voluntary effort of will to follow task instruction, but instead an outcome of covert attentional shifts. For example, monkey^[@CR7]^ and human^[@CR1],[@CR2]^ microsaccades exhibit direction biases in covert attentional tasks, and it could be the case that the requirement to remember target location was enough to bias microsaccade directions near the ends of trials (perhaps due to sustained covert attention). However, if so, such a bias should have also appeared during the memory interval itself. We therefore analyzed microsaccade directions in the final 250 ms of the memory interval, during instructed fixation (Fig. [3a, e, i, m](#Fig3){ref-type="fig"}). Directional accuracy, relative to target location, was much worse than in the instructed memory-guided microsaccades (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}) (p \< 10^−50^ for comparing absolute directional error in memory-guided microsaccades and microsaccades at the end of the memory interval in each of the monkeys and also in the human data; Watson-Williams test). In fact, the fixational microsaccades (at the end of the memory interval) were biased either away (monkeys) or towards (humans) the target location. This difference in directional bias between species (for the same trial-duration distributions) likely reflects expected differences in the temporal dynamics of microsaccadic oscillations between humans and monkeys^[@CR3],[@CR33],[@CR42]^. We also analyzed microsaccades immediately after target flash onset, to compare potential reflexive stimulus-induced effects (perhaps due to exogenous covert attentional effects after target flash onset) to instructed movements after fixation spot disappearance (Supplementary Fig. [3c, f, i, l](#MOESM1){ref-type="media"}): other than an expected minority of express visually driven reactions immediately after flash onset^[@CR5]^, the directions of flash-induced microsaccades were much more variable than the directions of instructed memory-guided microsaccades. Therefore, even transient, exogenous covert attention shifts^[@CR1],[@CR2]^ are not sufficient to explain the directional accuracy of the instructed memory-guided microsaccades (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}). It is also important to note that voluntary, memory-guided microsaccades could be triggered in all directions. Thus, directional accuracy was not restricted to, say, only horizontal or only vertical directions, one limitation in the original Haddad and Steinman study exploring voluntary triggering of small eye movements^[@CR30]^. To illustrate this, Fig. [3d](#Fig3){ref-type="fig"} shows raw plots of example memory-guided microsaccade direction vectors, color-coded by target locations in each of the four quadrants from monkey N. The other two monkeys and the human subjects also generated directionally accurate memory-guided microsaccades in all directions (Fig. [3h, l, p](#Fig3){ref-type="fig"}). Thus, not only did all subjects and monkeys successfully generate untrained voluntary eye movements for small-eccentricity target locations, but these eye movements were also highly directionally accurate. The directional accuracy was, once again, better than in the uninstructed corrective, visually-guided movements (Fig. [3c, g, k, o](#Fig3){ref-type="fig"}). We next investigated memory-guided microsaccade amplitudes. We found that such amplitudes also scaled with remembered target eccentricity, consistent with the notion that there was voluntary generation of spatially accurate (in both direction and amplitude) microsaccadic eye movements in the task. For example, in Fig. [4a, c, e, g](#Fig4){ref-type="fig"}, we plotted the amplitude of memory-guided microsaccades as a function of remembered target eccentricity (this figure also included larger saccades and eccentricities for comparison). We used logarithmic scaling to magnify the small eccentricities associated with microsaccades^[@CR19]^. There was a small overshoot in movement amplitude for the smallest target eccentricities, but the amplitude of eye movements systematically increased with increasing eccentricity of the remembered target location. That is, despite the initial overshoot, there was scaling of microsaccade amplitudes with remembered target eccentricity. This scaling of memory-guided microsaccade amplitude was true in all 3 monkeys (Fig. [4a, c, e](#Fig4){ref-type="fig"}), as well as in the human subjects (Fig. [4g](#Fig4){ref-type="fig"}). Interestingly, when the monkeys or subjects made uninstructed corrective, visually-guided microsaccades in the same task, the amplitudes of these visually-guided movements (relative to target eccentricity) were worse than for the instructed memory-guided microsaccades (i.e. did not scale appropriately; Fig. [4b, d, f, h](#Fig4){ref-type="fig"}). Larger corrective movements were accurate in amplitude. Similarly, when visually-guided microsaccades were instructed (in the delayed, visually-guided saccade task; Methods), the amplitudes were more accurate (as expected^[@CR26]^), and overshoot did not occur as strongly for the tiniest of eccentricities as in the case of the memory-guided microsaccades (Supplementary Fig. [4c, f](#MOESM1){ref-type="media"}). These results are consistent with all other results above, demonstrating willful generation of individual eye movements of microsaccadic sizes, based on abstract task-defined instruction to do so (fixation spot disappearance).Fig. 4Memory-guided microsaccade amplitudes scaled better with target eccentricity than corrective, visually-guided microsaccades. a For all target eccentricities in monkey N, we plotted memory-guided movement amplitude as a function of target eccentricity. The data below 1^o^ target eccentricity (vertical dashed line) are from the same trials described in Figs. [1](#Fig1){ref-type="fig"}--[3](#Fig3){ref-type="fig"} for reaction times, success rates, and directional accuracy. Here, we show their amplitude scaling. Memory-guided microsaccades increased in amplitude with increasing target eccentricity, consistent with them being genuine responses to task instruction, but they showed systematic overshoot (e.g. Figure [1](#Fig1){ref-type="fig"}). The overshoot disappeared for larger eye movements (also see Fig. [5](#Fig5){ref-type="fig"} for a potential explanation). Target eccentricities were binned at the following non-overlapping bin-center values: 0.05, 0.15, 0.25, 0.35, 0.55, 0.75, 1.05, 1.45, 2, 3, 5, 7.5, 10, 15; error bars denote 95% confidence intervals. b For similar small target eccentricities, corrective, visually-guided microsaccades overshot the target more than the instructed memory-guided microsaccades, and their amplitudes did not scale with target eccentricity as well as the memory-guided movements (despite the presence of a visual target). Larger corrective movements were more accurate. c, d Similar observations in monkey M. e, f Similar observations in monkey P. g, h Similar observations in the human subjects. In all cases, despite the amplitude overshoot for the smallest memory-guided microsaccades (see Fig. [5](#Fig5){ref-type="fig"}), instructed memory-guided microsaccades exhibited better amplitude scaling with increasing target eccentricity than corrective, visually-guided microsaccades (also see Fig. [5](#Fig5){ref-type="fig"}). n = 2,947, 4,931, 3,972, 10,890 for memory-guided movements of all amplitudes in monkey N, monkey M, monkey P, and the human subjects; n = 2,528, 4,086, 2,674, 7,002 for corrective, visually-guided movements of all amplitudes in monkey N, monkey M, monkey P, and the human subjects It is also interesting that the latency distributions for the corrective, visually-guided microsaccades in the same task (Fig. [2m, p, s, v](#Fig2){ref-type="fig"}) showed suggestive evidence of the oculomotor system being implicitly aware of residual errors after the previous memory-guided saccades or microsaccades (e.g. potential overshoot). Specifically, there was a distribution of express microsaccades with very short reaction times of \<120 ms after target reappearance. Since express microsaccades are most likely when a movement plan is already well on its way at the time of stimulus onset^[@CR5]^, the occurrence of such express microsaccades in the corrective, visually-guided movements after target reappearance indicates that the previously-occurring memory-guided movements were indeed internally accounted for. This provides further evidence that memory-guided microsaccades are voluntarily triggered, and it is similar to larger saccades^[@CR44]^. Foveal working-memory expansion even with manual responses {#Sec5} ---------------------------------------------------------- Despite the timeliness (Fig. [2a, d, g, j](#Fig2){ref-type="fig"}), success rate (Fig. [2b, e, h, k](#Fig2){ref-type="fig"}), and directional accuracy (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}) of memory-guided microsaccades, their amplitude overshoots for some eccentricities (Fig. [4a, c, e, g](#Fig4){ref-type="fig"}) may be interpreted as a failure to make genuine target-directed voluntary eye movements of small amplitude. However, an alternative possibility is that the overshoots were instead a property of the encoding of foveal locations in spatial working memory, and not an execution limitation by the oculomotor system. For example, this could be a result of foveal magnification of neural tissue, which also exists in the SC to a much larger extent than previously thought^[@CR45]^. To investigate this alternative, we ran our human subjects on two control tasks not explicitly requiring an eye movement to report the perceived location of the remembered target flash (Methods). Rather, we asked the subjects to perceptually report the remembered target location, and they did so via button or computer-mouse presses (Methods). In one variant, the button-press task, subjects were free to move their eyes while responding, and they had up to 20 s to finalize their response; in another variant, the mouse pointer task, subjects pointed to the remembered location (with mouse cursor) while still maintaining fixation. In both cases, amplitude overshoot for the smallest target eccentricities was still clearly evident (Fig. [5](#Fig5){ref-type="fig"}), much like when eye movements were used to perform the task (the memory-guided microsaccade task). We should also note here that directional accuracy was also very high in the memory-guided manual tasks, similar to Fig. [3](#Fig3){ref-type="fig"}. Therefore, memory-guided microsaccade amplitude overshoot (Fig. [4a, c, e, g](#Fig4){ref-type="fig"}) was not a failure of the oculomotor system to voluntarily generate small eye movements. Instead, it reflected an intriguing foveal expansion in the representation of space during working memory (Fig. [5](#Fig5){ref-type="fig"}). Consistent with this, and as stated above, microsaccade amplitude overshoot was less evident with instructed delayed, visually-guided microsaccades (Supplementary Fig. [4c, f](#MOESM1){ref-type="media"}).Fig. 5Overshoot was not restricted to microsaccades; perceptual localization of foveal remembered targets was also expanded in an illusory manner. a In humans, we tested perceptual localization of remembered target locations without explicitly requiring an eye movement response. In the button-press task, subjects used a button box to move a displayed cursor to the remembered target location (Methods). We analyzed their perceptual reports similarly to eye movement reports. The same overshoot for small target eccentricities was observed. Error bars denote 95% confidence intervals. b We also ran a variant of the task (the mouse pointer task; Methods), in which we also maintained a visual reference at fixation (the fixation spot). The same overshoot for foveal target eccentricities was still observed. Therefore, the overshoot of memory-guided microsaccades in Fig. [4](#Fig4){ref-type="fig"} was likely a general property of representing foveal space in spatial working memory, as opposed to an intrinsic inability to generate small memory-guided microsaccades. n = 6,405 and 9,062 trials for the button-press and mouse pointer tasks, respectively Superior colliculus bursts for memory-guided microsaccades {#Sec6} ---------------------------------------------------------- The strongest evidence that we obtained for voluntary control over microsaccades emerged when we recorded SC neural activity. In monkeys N and M, we recorded visual, visual-motor, and motor (saccade-related) SC neurons (Methods) during the memory-guided saccade task. We specifically aimed to sample sites in the rostral SC, in which microsaccade-related activity is expected^[@CR18],[@CR19],[@CR21]^, but we also sampled slightly more caudally (i.e. more eccentrically) for completeness (Methods). We inserted multi-electrode linear arrays into the SC, and we used offline sorting to isolate individual putative single units (Methods). Our first goal was to ask whether SC neurons responded at all during memory-guided microsaccades. For example, some SC neurons do not emit saccade-related responses in the absence of a visual target^[@CR46]--[@CR48]^, and it is not clear whether this can happen for microsaccades or not. Individual SC neurons did indeed emit saccade-related motor bursts for memory-guided microsaccades. For example, in Fig. [6a](#Fig6){ref-type="fig"}, we plotted neural activity from a sample neuron from monkey M. We identified a set of memory-guided microsaccades of amplitude \<1^o^ and direction toward the neuron's movement response field (RF), shown in Fig. [6b](#Fig6){ref-type="fig"} (neural RF's were assessed as described in Methods, and the movements included in Fig. [6a](#Fig6){ref-type="fig"} were directed towards the RF hotspot location and with angular direction within ±90^o^ from the vector connecting gaze center to this location). We plotted radial eye position aligned on microsaccade onset and also firing rate (individual rows of tick marks indicate action potential times, with each row representing an individual movement; this formatting is similar to that in Fig. [1c](#Fig1){ref-type="fig"} of ref. ^[@CR18]^). Memory-guided microsaccade-related SC discharge was clearly present even for memory-guided movements as small as 6 min arc, and similar to expected saccade-related discharge in general. Therefore, individual voluntary, memory-guided microsaccades are associated with SC neural discharge.Fig. 6Superior colliculus (SC) activity during memory-guided microsaccades. a The top panel shows radial eye position aligned on microsaccade onset (for memory-guided microsaccades \<1^o^). All traces were aligned to start (on the vertical axis) from the same eye position. Most movements had a radial amplitude \<30 min arc (0.5^o^). These movements were selected relative to the response field (RF) as assessed from the analysis in b. The bottom panel shows microsaccade-aligned firing rate. Classic SC microsaccade-related activity^[@CR18]^ was observed for memory-guided microsaccades. Each row of tick marks shows the neuron's discharge for an individual movement from the top panel, and the rows are sorted by memory-guided microsaccade amplitude (black for \<30 min arc; gray for 30--60 min arc). Even the smallest memory-guided microsaccades were associated with SC microsaccade-related discharge^[@CR18]^. The gray bar on the x-axis indicates the analysis window used in b. Error bars denote 95% confidence intervals. b Movement RF of the same neuron. For each movement displacement, plotted in log-polar coordinates^[@CR19]^, we plotted mean firing rate from the measurement interval in a. The neuron, from the right SC, exhibited a movement RF on the contralateral side along an oblique upward direction. The neuron clearly responded for memory-guided microsaccades as small as 0.1° (6 min arc). The origin of the log-polar plot denotes 0.03° radial amplitude. The inset shows spike waveforms from every 100^th^ spike recorded in the task (gray; 682 waveforms); red shows the average spike waveform (and 95% confidence intervals) across all recorded spikes Not only did we find microsaccade-related responses for memory-guided microsaccades (Fig. [6](#Fig6){ref-type="fig"}), but there were also neurons that discharged exclusively for memory-guided microsaccades and not for similarly sized corrective, visually-guided microsaccades. Figure [7a](#Fig7){ref-type="fig"} shows an additional example neuron from the same monkey as in Fig. [6](#Fig6){ref-type="fig"}, now demonstrating this unexpected phenomenon. We plotted an estimate of the neuron's movement field on log-polar coordinates up to 1° eccentricity. In the left panel, we plotted the movement field of the neuron when memory-guided microsaccades were generated. Clear discharge for specific movement vectors was evident. In the right panel, we plotted the movement field of the same neuron when corrective, visually-guided microsaccades of similar amplitudes and directions were generated. The neuron now did not emit any movement-related discharge at all (compare to the left panel), even though movements of similar sizes were executed. These results were also obvious when we plotted firing rates as a function of time from microsaccade onset in the two conditions (Fig. [7b](#Fig7){ref-type="fig"}). Activity was present when memory-guided microsaccades were executed, but not when visually-guided movements of the same amplitude and direction range were generated. Activity for these latter movements looked indistinguishable from activity during baseline fixation before trial onset (Fig. [7b](#Fig7){ref-type="fig"}, black curve; Methods define baseline intervals). Therefore, not only was there strong behavioral evidence that naïve monkeys and humans can generate voluntary, memory-guided microsaccades (Figs. [1](#Fig1){ref-type="fig"}--[4](#Fig4){ref-type="fig"}), but there were also individual SC neurons showing exclusive responses only for memory-guided microsaccades but not for similarly sized visually guided ones.Fig. 7Double dissociation of SC microsaccade-related discharge for visually- and memory-guided microsaccades. a Movement RF's of an example SC neuron. Left shows peri-movement discharge (like in Fig. [6b](#Fig6){ref-type="fig"}) for memory-guided microsaccades; right shows the same for corrective, visually-guided microsaccades. The neuron was silent for the visually-guided movements, but not for similar-sized memory-guided ones. b Peri-movement discharge of the same neuron under the two conditions. The black curve (when visible) shows the neuron's activity during fixation before trial onset; the neuron only discharged for memory-guided, but not visually guided, microsaccades. Error bars denote 95% confidence intervals. c, d Same as a, b but for an example neuron only showing microsaccade-related discharge for visually guided movements (similar to visually dependent saccade-related, VDSR, neurons^[@CR46]^). The inset shows (as an example) the analyzed eye movements (like Fig. [6a](#Fig6){ref-type="fig"}) in the two conditions, demonstrating clear overlap (similar overlap was present in a, b). e Distribution of the different neuron types (V = Visual; VM = Visual-motor; M = Motor; VDSR = Visually-dependent saccade-related; ER = Exclusively responding for memory-guided microsaccades). f Movement RF hotspots for the neurons. ER-Neurons tended to be more eccentric than other neurons exhibiting microsaccade-related discharge, consistent with a foveal expansion for memory-guided microsaccades (see text and Supplementary Fig. [5](#MOESM1){ref-type="media"}). g, h Peri-stimulus (g) or peri-microsaccadic (h) discharge for all of our encountered neuron types during microsaccade generation. Each neuron's response was first normalized before averaging responses across neurons. V- and VM-Neurons exhibited strong visual responses; VM- and M-Neurons showed movement-related activity regardless of stimulus; VDSR-Neurons only showed movement-related activity in the presence of a visual target; ER-Neurons only showed movement-related activity in the absence of a visual target. In all cases, we analyzed neural activity for microsaccades (\<1^o^) toward the RF. Error bars denote 95% confidence intervals We found 41 SC neurons exhibiting exclusive responses for memory-guided microsaccades; these constituted 22% of SC neurons with saccade-related discharge in either memory or visual condition (Fig. [7e, f](#Fig7){ref-type="fig"}). Our criteria for including neurons in this accounting was the observation of visual and/or movement discharge for eccentricities \<1° (Methods). We referred to these neurons as ER-Neurons, for "Exclusively Responding" (Methods). Their average firing rate modulations for foveal visual target onsets and for microsaccades are shown in Fig. [7g, h](#Fig7){ref-type="fig"} (rightmost column), and these modulations are consistent with those in the example neuron of Fig. [7a, b](#Fig7){ref-type="fig"}. Interestingly, for larger saccades, ER-Neurons did exhibit saccade-related activity for visually guided eye movements (Supplementary Fig. [5](#MOESM1){ref-type="media"}), suggesting that memory-guided microsaccades may have recruited additional neural resources from SC saccade-related neurons that would normally be recruited for slightly larger eye movements when visual targets are present (Supplementary Fig. [5](#MOESM1){ref-type="media"}). Consistent with this, RF hotspots for ER-Neurons tended to be more eccentric than other neuron types in our database (Fig. [7f](#Fig7){ref-type="fig"}). Interestingly, we also found all other classic saccade-related SC responses to be present as well for memory-guided microsaccades. For example, we found visual-only neurons responding to foveal flash onset in the beginning of a trial but not during the actual memory-guided (or visually guided) eye movements (Fig. [7g, h](#Fig7){ref-type="fig"}, leftmost column). We also found visual-motor neurons, exhibiting both a visual response and either a memory- or visually-guided microsaccade response (Fig. [7g, h](#Fig7){ref-type="fig"}, second column). Motor neurons that responded for both memory- and visually guided microsaccades, but not to flash or target onset, were also present (Fig. [7g, h](#Fig7){ref-type="fig"}, third column). Finally, in addition to the ER-Neurons (e.g. Fig. [7a, b](#Fig7){ref-type="fig"}), we discovered neurons exhibiting the exact opposite profile: these were visually-dependent microsaccade-related neurons, extending earlier findings of SC visually-dependent saccade-related neurons (VDSR-Neurons^[@CR46]--[@CR48]^) to the realm of microsaccades. One example such neuron is shown in Fig. [7c, d](#Fig7){ref-type="fig"}. The neuron exhibited microsaccade-related activity exclusively during visually-guided microsaccades, but not during similarly sized memory-guided microsaccades. This neuron type (Fig. [7g, h](#Fig7){ref-type="fig"}, fourth column) was not described previously in refs. ^[@CR18],[@CR19]^. Overall, in the memory-guided microsaccade task, the different classes of neurons that we encountered (Fig. [7g, h](#Fig7){ref-type="fig"}) were distributed in our database (Methods) as shown in Fig. [7e](#Fig7){ref-type="fig"}. Our results demonstrate that memory-guided microsaccades are generated at will: based on abstract task-defined instruction, without visual guidance, with high fidelity in spatial accuracy, and with underlying SC circuitry supporting all different aspects of memory- or visually-guided behavior. These results demonstrate that overwhelming descriptions of microsaccadic eye movements as being involuntary and spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}) are too simplistic and can mask the significance of these tiny eye movements in modulating perceptual and attentional performance. Discussion {#Sec7} ========== We were motivated by a frustrating conundrum in the field of microsaccade research. Haddad and Steinman^[@CR30]^ showed in 1973 that two expert human observers were able to voluntarily trigger a microsaccade along one of the cardinal directions. Their study has become virtually forgotten, especially after the resurgence of the field of microsaccade research from its almost-complete death at the end of the previous century^[@CR6]^ (Supplementary Fig. [1](#MOESM1){ref-type="media"}). We decided to revisit this particular study, but avoid some of its methodological constraints (such as the use of only two expert subjects, the limited amount of data, and the lack of neurophysiological insights), and we were able to demonstrate clear voluntary control over individual microsaccades even in naïve human subjects and monkeys. In doing so, we were also able to additionally demonstrate an ability for microsaccades to be driven by foveal visual working memory, in a novel adaptation of the memory-guided saccade task. We were struck by how easily memory-guided microsaccades could be generated, with no need for special training. Monkeys simply generalized the task rule that they had learned for larger saccades, and humans were only verbally instructed to "look at the remembered target location". We believe that our results overturn an overwhelming discourse on microsaccades, even in public understanding of these eye movements, as being involuntary and spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}). Indeed, and as stated above, despite strong supportive evidence like that cited above and in Introduction, a surprisingly large fraction of references to microsaccades in the literature (especially in the last two decades) asserts that these eye movements are involuntary eye movements (Supplementary Fig. [1](#MOESM1){ref-type="media"}). We believe that explicit demonstration of behavioral and neural evidence for voluntary control over individual microsaccades, in our novel adaptation of the memory-guided saccade paradigm, is absolutely necessary, especially in the present day when there is both a renewed interest in microsaccades as well as continued intense debates about their functional roles in influencing brain activity and behavior. Consider, for example, the field of covert visual attention. A long history of research has assumed for several decades that microsaccades either do not happen in these tasks at the time of task-relevant events, or that they happen in a random and spontaneous manner that should not influence the interpretation of results. However, it is now known that this view is too simplistic^[@CR1]--[@CR5],[@CR31]--[@CR34]^. For this important revelation to become appreciated more widely, and in other fields related to visual and cognitive neuroscience, explicit demonstration of voluntary control over individual microsaccades, like we have shown in this study, is firmly needed. The strongest evidence that we found for voluntary control over individual microsaccades is related to how the SC is involved in generating memory-guided versions of these eye movements. We were particularly intrigued by the ER-Neurons (Fig. [7](#Fig7){ref-type="fig"}). These neurons are SC neurons that discharge exclusively for memory-guided microsaccades but not for similarly sized visually-guided microsaccades. It would be highly interesting to investigate the mechanisms associated with these ER-Neurons in more detail in future studies. For example, the fact that these neurons discharge (i.e. exhibit saccade-related activity) for larger visually-guided saccades might suggest an expansion of RF's towards the fovea when spatial working memory is engaged (Supplementary Fig. [5](#MOESM1){ref-type="media"}). Prior work is inconclusive about how SC RF's change during spatial working memory tasks^[@CR47],[@CR49]^, and there is no systematic investigation, to our knowledge, of foveal spatial representation in working memory anyway. Therefore, this issue needs to be investigated in future follow-up studies. This can allow understanding the entire transformation from visual input (flash onset) to memory representation in the memory interval, and finally to movement generation. Relevant questions could include whether RF's change during the memory interval as well, or only at the time of memory-guided microsaccade generation (like in Fig. [7a,b](#Fig7){ref-type="fig"}, Supplementary Fig. [5](#MOESM1){ref-type="media"}). Importantly, studying this in the SC would clarify a sub-cortical involvement in working memory, which is a topic that is generally investigated more strongly from a cortical perspective^[@CR36],[@CR50]^. Studying this in the SC would also link SC activity to mechanisms of spatial updating of memory representations across eye movements^[@CR51],[@CR52]^. We were equally intrigued by the VDSR-Neurons^[@CR46]--[@CR48]^ that we found (Fig. [7c, d, g, h](#Fig7){ref-type="fig"}). In the past approximately four decades since they were first reported on in the primate SC, very few studies have mentioned these neurons, which only exhibit saccade-related discharge if saccades are directed towards a visual target. Moreover, these few mentions were only made from the perspective of larger saccades. Here, we found that VDSR-Neurons also exist in the rostral SC generating^[@CR18]^ microsaccades. That is, we found microsaccade-related neurons that only showed movement-related discharge if there was a visual target guiding microsaccade generation; the same neurons did not discharge when the microsaccades were generated toward a blank. We find these neurons interesting because they suggest that the oculomotor system, even for microsaccades, incorporates information about visual features at the time of saccade execution. It would be interesting to relate such incorporation of visual information in saccade commands to cortical interactions between saccades and visual representations^[@CR53]^ and to shape guidance of saccades^[@CR53]^ and microsaccades^[@CR42]^. Because SC neurons exhibit visual-feature tuning in visual responses^[@CR54]--[@CR56]^, it would also be interesting to explore how these VDSR-Neurons behave during saccades to different visual objects or features. In all, our neural results with visual, visual-motor, and motor neurons all being involved in microsaccade generation confirm views that microsaccades are essentially indistinguishable from larger saccades in terms of neural mechanisms^[@CR20]^. This is consistent with a variety of neurophysiological evidence in multiple areas^[@CR19],[@CR21],[@CR22],[@CR57],[@CR58]^, and it also gives further credence to the notion that these eye movements are indeed voluntary movements, despite the opposite description in a large portion of the literature (Supplementary Fig. [1](#MOESM1){ref-type="media"}). The visual responses, in particular, are worth further investigation, both for understanding SC foveal vision^[@CR45]^ in general, and also for exploring the speeded reactions to target reappearance in the corrective, visually-guided microsaccades (Fig. [2m, p, s, v](#Fig2){ref-type="fig"}). Behaviorally, there was a remarkable difference between instructed and uninstructed movements in general. Most strikingly, the corrective, visually-guided microsaccades in the memory-guided task were both less successful (i.e. less often generated) and also sloppier in direction and amplitude (e.g. Figures [2](#Fig2){ref-type="fig"}--[4](#Fig4){ref-type="fig"}) than the instructed memory-guided movements, even though these latter movements did not have explicit visual guidance. Only when we instructed visually guided saccades (in the delayed, visually guided saccade task) did visually guided microsaccades appear more spatially precise (Supplementary Fig. [4b, c, e, f](#MOESM1){ref-type="media"}). This (perhaps expected) observation supports our interpretation that our memory-guided microsaccades were generated voluntarily. Further support for this conclusion is the presence of some express microsaccades after target reappearance (Fig. [2](#Fig2){ref-type="fig"}). As stated earlier, because express microsaccades are more likely when a movement plan is already under way^[@CR5]^, the presence of these express microsaccades after target reappearance indicates that the oculomotor system was already planning a corrective movement for any residual errors from previous memory-guided microsaccades. Evidence for such rapid correction also exists in larger saccades (see, for example, ref. ^[@CR44]^). Naturally, all of the above may be related to questions of awareness. Specifically, and as stated elegantly by Haddad and Steinman^[@CR30]^, labels of involuntary, spontaneous, and reflexive may have arisen in association with microsaccades exactly because people are generally unaware of making these movements. However, we are also very frequently unaware of making larger saccades. Moreover, these same authors demonstrated^[@CR30]^ that they could be aware of their own self-triggered microsaccades on a surprisingly large fraction of trials. Therefore, awareness on its own is not sufficient to argue that microsaccades are fundamentally different from larger saccades. Finally, our results pave the way for interesting investigations of spatial working memory at the microscopic scale of the fovea. A wealth of research has been performed on the mechanisms of working memory^[@CR36],[@CR50]^ in general, and there were even recent findings that accessing visual working memory engages the oculomotor system^[@CR59]^ (similar to how preparing a manual response in monkeys caused them to generate microsaccades to the four possible previous stimulus locations in ref. ^[@CR7]^). However, just like subcortical mechanisms are less investigated, how foveal space is represented in spatial working memory remains unknown. For example, our perceptual control experiments (Fig. [5](#Fig5){ref-type="fig"}) suggest that the overshoot in microsaccade amplitudes that we observed (Fig. [4](#Fig4){ref-type="fig"}) was not a deficit in oculomotor control per se. Instead, it likely reflected an expansion of foveal space when working memory was engaged (Fig. [5](#Fig5){ref-type="fig"}). This might be a complement of foveal biases in peripheral visual perception^[@CR60]--[@CR62]^. Specifically, because of foveal magnification of neural tissue^[@CR63]^, it could be that engaging spatial working memory in the fovea causes distortions in the representation of space, such that foveal locations get mislocalized outward (i.e. overshoot) whereas peripheral locations get mislocalized inward (i.e. undershoot). Interestingly, perceptual mislocalizations around microsaccades^[@CR31]^ are also consistent with outward distortions in the fovea and inward distortions in peripheral vision. This might suggest that there may be generalized underlying mechanisms of perceptual distortions of different kinds (whether due to eye movements, working memory, attention, or otherwise), likely being caused by how individual brain circuits represent visual space in anatomical tissue, and how multiple such circuits (with potentially different levels and kinds of representational magnification) may interact with each other^[@CR64]^. Methods {#Sec8} ======= Ethics approvals {#Sec9} ---------------- All monkey experiments were approved by ethics committees at the Regierungspräsidium Tübingen. The experiments were in line with European Union directives, and German laws, governing animal research. Human experiments were approved by ethics committees at the Medical Faculty of Tuebingen University. Subjects provided written, informed consent, and they were financially compensated for their participation. Our experiments were in accordance with the Declaration of Helsinki. Laboratory setups {#Sec10} ----------------- Monkey experiments were performed in the same laboratory as that described recently^[@CR54],[@CR55],[@CR65]^. Human experiments were done in the laboratory described in refs. ^[@CR31],[@CR64]^. Eye movements were recorded at 1 kHz using magnetic induction^[@CR66],[@CR67]^ (for monkeys N and M) or video-based eye tracking (for monkey P and the human subjects). Head position in the human subjects was stabilized using a custom head-holder^[@CR31]^. Animal preparation {#Sec11} ------------------ We collected behavioral data from three adult, male rhesus macaques (macaca mulatta). Monkeys N and M (aged 10 and 7 years, respectively, and weighing 11.5 and 8 kg, respectively) were implanted with scleral search coils to allow measuring eye movements using the magnetic induction technique^[@CR66],[@CR67]^. The eye movements of Monkey P (aged 10 years and weighing 8.6 kg) were recorded using a video-based eye tracker (EyeLink 1000, SR Research). All three monkeys were implanted with a head holder to stabilize head position during experiments, with details on all implant surgeries provided earlier^[@CR65],[@CR68]^. We also recorded SC neural activity from monkeys N and M. The SC was approached through recording chambers cranially-implanted on the midline and aimed at a point 1 mm posterior of and 15 mm above the inter-aural line. The chambers were tilted backwards from vertical by an angle of 35° in monkey N and 38° in monkey M^[@CR32],[@CR34],[@CR55]^. Monkey behavioral tasks {#Sec12} ----------------------- Monkeys performed a memory-guided saccade task^[@CR36],[@CR37]^. In each trial, a central white fixation spot (72 cd per m^2^ for monkey N; 86 cd per m^2^ for monkey M; 48.1 cd per m^2^ for monkey P) was presented over a uniform gray background (21 cd per m^2^ for monkey N; 29.7 cd per m^2^ for monkey M; 4.4 cd per m^2^ for monkey P). The fixation spot was a square of 5.3 × 5.3 min arc dimensions. After 300--1,200 ms, a brief target flash (identical to the fixation spot) occurred at some location on the display (duration: \~58 ms), while the fixation spot remained on; this flash represented the location to be memorized by the monkeys for an upcoming saccadic eye movement. After the flash, the fixation spot remained visible for 300--1100 ms; this period constituted the memory interval of the task, after which the fixation spot disappeared. Fixation spot disappearance was the abstract instruction for the monkeys to generate a spatially accurate saccadic eye movement to the remembered flash location. Because of the disappearance of the fixation spot and the flash before it, the saccade that was triggered was generated without any visual guidance. Monkeys had to trigger this saccade within \<700 ms from fixation spot disappearance (reaction times were actually much shorter as shown by the distributions in Fig. [2](#Fig2){ref-type="fig"}, Supplementary Fig. [2](#MOESM1){ref-type="media"}). After gaze entered within a virtual window around the true target location by 300 ms, the target reappeared for 400--500 ms. During this time, a visually-guided saccade, which we also referred to as a corrective saccade, was sometimes triggered to correct for any remaining error between the memory-guided saccade and the actual target position. This corrective movement happened readily, and we did not specifically instruct the monkeys to generate it. It was, however, useful for analysis because individual trials often had both a memory movement as well as a control visually-guided one. The radius of the virtual window around the invisible target position was \<3° and was varied depending on target eccentricity. The monkeys were rewarded with water or apple juice at the end of every trial. If they broke fixation during the fixation interval or failed to remain within the virtual window around target location (after the instruction to make the memory-guided saccade), the trial was aborted and repeated. Across trials, we varied target location (i.e. flash location) between 6 min arc and 16^o^ throughout the entire display. During SC recordings, we made sure to include within our sampling of target locations positions that were in and around the response fields (RF's) of neurons around our recording electrodes, as assessed online using our neurophysiological data acquisition system (see below). We collected a total of 6428 trials in this task from monkey N, 9631 from monkey M, and 14,666 from monkey P. In monkeys N and M, we also compared memory-guided saccades to those obtained in a task with explicit visual guidance. In this delayed, visually-guided saccade task, stimulus events were identical to those described above, except that the target flash was now replaced by target persistence until trial end. In other words, the target remained on during the memory interval (now called the delay interval) and also after fixation spot disappearance. Thus, the instructed saccade (i.e. after the offset of the fixation spot) was visually-guided. We collected 6147 trials in this task from monkey N and 5871 trials from monkey M. Human behavioral tasks {#Sec13} ---------------------- Human subjects performed the same memory-guided saccade task described above. Target locations were chosen, across trials, from a total of 480 possible pre-defined display positions ranging in eccentricity from 6 min arc to 12°. 288 of these 480 possible target positions were within a rectangle of ±0.8^o^ × ±0.8^o^ size in order to allow us to explore memory-guided microsaccades in particular detail; the remaining locations were equally spaced in the rest of the display. Seven subjects (four naïve and three untrained authors) performed this experiment, with each subject completing 480--600 trials per session and 4--5 sessions (total of 16,844 trials overall). Ten practice trials (with target eccentricities \>1^o^) were performed at the beginning of every session. This task allowed us to relate the monkey behavioral and neurophysiological results to human performance. The subjects were aged 23--39, and three were female. We also ran two additional control experiments in the humans, in order to understand whether intricacies of memory-guided microsaccade behavior (e.g. Fig. [4](#Fig4){ref-type="fig"}) were specific to a particular motor modality (eye movements) or more related to the engagement of spatial working memory per se. We therefore required a perceptual assessment of remembered flash location without the use of an instructed eye movement. In the first control task, which we refer to as the button-press task, trials were identical to the memory-guided saccade task described above up until the end of the memory interval. At this point, instead of fixation spot disappearance, the fixation spot was replaced with a cross-shaped cursor of 59 min arc dimensions. Subjects used a response box with four buttons allowing them to move the cursor at individual pixel resolution (1.46 min arc per click) rightward, leftward, upward, or downward. Individual button presses moved the cursor by individual pixels; prolonged button presses moved the cursor at one of two faster speeds depending on the duration of the press. Once subjects were satisfied with the cursor position as reflecting, as accurately as possible, the remembered flash location, they pressed a fifth button confirming their choice. The target then reappeared at its true position with the cursor disappearing simultaneously. Subjects were free to move their eyes at will when the cursor was visible, and they were given up to 20 s to move the cursor and decide on their perceptual localization of the remembered target location. Therefore, this task allowed assessing the quality of target recall independent of making an instructed memory-guided saccade. Seven subjects (ages: 25--39 years; one female) participated in this experiment, four of which had performed the original saccade version of the task. Each subject participated in 5--6 sessions (250--350 trials per session), for an overall total of 11,249 trials across subjects. The second control experiment, called the mouse pointer task, was similar to the button-press task except that the cursor was replaced with a computer mouse pointer, which appeared at a random position 1°--5° away from the true flashed target location in every trial. Critically, in this version of the control task, we maintained the fixation spot on during the manual response interval (i.e. until subjects pressed a mouse button), so that an explicit foveal reference frame was always present when subjects were pointing to the remembered target location (similar to how eye movements presumably relied on the initial reference frame of fixation for localization). Subjects were instructed to fixate the fixation spot throughout the response interval until they pressed a mouse button, and they were given up to 15 s to make their response. This task tested whether an explicit foveal reference frame altered the overall pattern of behavior from the button-press task or from the original memory-guided saccade task (Fig. [5](#Fig5){ref-type="fig"}). Seven subjects (ages: 25--39 years; one female) performed this task, four of which had also participated in the button-press and memory-guided saccade versions of it. We collected 380--500 trials per session for each subject. We ran five sessions per subject, with the exception of one participant who completed only four sessions (for a total of 16,213 trials overall). Monkey neurophysiological recordings {#Sec14} ------------------------------------ We recorded SC activity from monkeys N and M using multi-electrode linear arrays (16-channel V-Probes with 150 μm inter-electrode spacing, Plexon). We performed 25 experiments in monkey N and 16 experiments in monkey M. In each experiment, we advanced either one or two V-probes toward SC surface. We monitored the deepest electrode contact in a probe and compared physiological landmarks picked up on this contact to anatomical landmarks in MRI's obtained from the same animals before implant surgeries. Once the deepest electrode contact was in the SC, we advanced the probes further, such that as many electrode contacts as possible were recording SC activity. We used online spike sorting to guide our approach and maximize the number of channels that were recording SC activity. When we inserted two simultaneous V-Probes (23/25 sessions in monkey N), one probe was in the right rostral SC (rostral being the region where microsaccade-related activity is expected^[@CR18],[@CR19]^) and the other was at a slightly more caudal position in the left SC (to allow sampling multiple neuronal preferred eccentricities from the SC's topographic map). When we inserted a single probe, it could be in the right or left SC and at a variety of rostral and slightly more caudal sites. Once the probes were in the SC, we ran a series of experimental tasks, including the memory-guided and delayed, visually-guided saccade tasks described above. We also ran a variety of other tasks that were not directly relevant to the present study. Our neurophysiology system sampled electrical activity in each contact at 40 kHz. Spike times during online sorting were sampled at 1 kHz. Monkey and human behavioral analyses {#Sec15} ------------------------------------ We detected saccades and microsaccades using established methods in our laboratory^[@CR68],[@CR69]^. We manually inspected all trials to correct for false alarms or misses by the automatic algorithms. We also marked blinks or noise artifacts (which were more likely in video-based eye tracking data) for later removal. We analyzed all eye movements occurring in the interval between fixation onset and trial end in the memory-guided and delayed, visually-guided saccade tasks. In the button-press control task, subjects were free to move their eyes after cross-shaped cursor appearance, so we only analyzed eye movements up to such appearance. Individual subjects had idiosyncratic patterns of cursor trajectories and accompanying eye movements that did not appear related in systematic ways to task performance; we thus elected not to analyze eye movements during the response interval in this task. In the mouse pointer task, we monitored eye movements after cursor appearance to ensure that subjects maintained fixation while responding. In this paper, we report each individual monkey's behavioral performance separately. For the human data, we combined results from all subjects into aggregate measurements. We felt justified in doing so because the human effects exhibited very strong similarities to the monkey behavior, which was a primary reason for performing the human experiments in the first place. Moreover, trial numbers were uniformly distributed across the individual human subjects as much as possible; therefore, no single subject biased the pooled results. In all tasks, we first identified valid trials that we accepted for subsequent analysis. Valid trials did not have any saccades larger than 1^o^ during the memory or delay interval (to ensure that fixation was maintained properly), and also no blinks or noise artifacts within ±100 ms from target flash time (or target onset time in the delayed, visually-guided version of the task). This latter criterion ensured that subjects could see the target flash well in the memory version of the tasks. We also removed trials if there were blinks or noise artifacts from −100 to +400 ms from the instruction to generate a memory- or visually guided saccade. Because microsaccades alter visibility and modulate visual sensitivity in general^[@CR3],[@CR33],[@CR35],[@CR70]^, we wanted to ensure that subjects could detect target flash (or onset) and fixation spot disappearance (or other instruction to respond) with high sensitivity. We therefore required that valid trials also did not have any microsaccades (or large saccades) within ±50 ms from flash (target) onset or fixation spot disappearance (or any other instruction to respond). Finally, memory-guided saccades had to land within 2° of the target; this constraint was irrelevant for memory-guided microsaccades (which were much smaller), but it helped ensure that the eye movements were accurate when large target eccentricities were used. Based on the detected saccades and microsaccades, we computed measures of saccade amplitude, latency (reaction time), direction, starting point, and endpoint. We calculated the starting point as the average eye position in the interval 30 ms before the start of the saccade. The end point was simply the very last sample of the detected saccade. To obtain saccade direction error (i.e. saccade direction relative to target direction from initial fixation spot position), we computed the counter-clockwise angular difference between saccade and target direction. For corrective saccades after target reappearance in the memory-guided saccade task, the target reappeared at a position that was variable relative to gaze position (depending on the landing error of the previous memory-guided movement). We therefore redefined the position of the reappearing target relative to corrective saccade starting eye position. We then calculated saccade direction error as above. We used a similar procedure for target eccentricity. Note that after large memory-guided saccades, target position error for the corrective movements was often small (i.e. large memory-guided saccades were relatively accurate as expected). We therefore had significantly more data for corrective, visually-guided microsaccades than for memory-guided microsaccades (e.g. see the numbers of trials in the legend of Fig. [2](#Fig2){ref-type="fig"}). In the memory-guided saccade task, we defined as the memory-guided saccade (or memory-guided microsaccade) of interest as the first saccade occurring with a latency of at least 50 ms after fixation spot disappearance and less than 50 ms after target reappearance. Similarly, the corrective, visually guided saccade was defined as the first saccade that was executed with latency \>50 ms after target reappearance. Because most studies define microsaccades as smaller than 1° in amplitude and we were interested in comparing these to voluntarily generated movements, we treated trials with target eccentricities less than 1° as memory-guided microsaccade trials. The above analyses were also applied in the delayed, visually-guided saccade task. For the control tasks, we treated button and mouse press locations similarly to how we analyzed saccade endpoints. In all analyses, we defined trials with target eccentricities \<1° as the trials involving microsaccades, as stated above. Corrective, visually-guided saccades were also classified as microsaccades when target eccentricity (relative to eye position at target reappearance) was \<1°. As stated above, we used this threshold because it was a typical threshold used in the literature^[@CR18],[@CR38]^, but our results (also from other studies) confirm that there is indeed a continuum of saccade generation across different amplitudes. Spike sorting for neurophysiological data analyses {#Sec16} -------------------------------------------------- For all neurophysiological data analyses, we performed offline sorting to isolate individual single units. We isolated single units from wide-band (40 kHz) electrode data by adapting the Klusta toolbox^[@CR71]^. As an additional input to the toolbox, we recreated the electrode geometry (16 channels with linear spacing) such that neighboring channels could share spike timing and waveform information (as in, virtual tetrodes). From filtered neural data (750--5000 Hz), the toolbox performed automated spike detection and classification. We inspected the resulting cluster assignments (putative units) using the Kwik-gui interface included in the Klusta toolbox. Based on auto- and cross-correlograms, as well as waveform shapes, we manually curated each cluster in order to obtain well-isolated units. Clusters that showed no characteristic auto-correlogram, or that produced atypical spike waveforms, were clustered together, labeled as noise, and subsequently excluded from further analysis. We estimated single unit isolation quality of all resulting units using established metrics^[@CR72],[@CR73]^. Specifically, we computed an estimate of the false positive rate based on refractory period violations (inter-spike interval violations). Furthermore, we obtained an isolation distance measure for each cluster, by calculating the Mahalanobis distance between any two units. All isolated units that exceeded an estimated false positive rate of 10% or had an isolation distance below 30 were excluded from further analysis; all other units are hereafter termed putative neurons (297 neurons in total; we selected for analysis all 188 neurons that showed clear SC visual and/or saccade-related RF's encompassing microsaccadic amplitude ranges, as assessed by the procedures described below). Spike trains of all putative neurons were then convolved with an appropriately-scaled Gaussian (σ = 10 ms) to obtain an estimation of the neuron's firing rate in spikes per s. Neurophysiological data analyses {#Sec17} -------------------------------- We classified SC neurons as visual, visual-motor, or motor depending on visual and saccade-related responses. We defined several 100-ms intervals based on key trial events. The visual response interval comprised the time from 50 to 150 ms after target onset. The motor interval was set to be 100 ms centered around saccade onset. We also measured this saccade-related interval for corrective saccades in the memory-guided saccade task. Since we were especially interested in characterizing microsaccade-related discharge, we classified movement-related aspects of neurons based on responses associated with saccades \<1° in amplitude. To estimate baseline activity during fixation without any target or saccade onset, we used the100-ms steady fixation interval before target onset. We avoided contamination of baseline and visual neural activity by saccades or microsaccades; we excluded all trials in which such movements occurred during the measurement intervals. Similarly, we excluded trials in which there was more than one movement within the motor interval. In all cases, we measured average firing rate within a given measurement interval, which resulted in a population of measurements across trials; we only included neurons with \>10 repetitions per measurement interval. We compared each of visual, memory-guided microsaccade, corrective microsaccade, or visually-guided microsaccade (in the delayed version of the task) activity to baseline activity using one-tailed Wilcoxon-ranksum tests (α = 0.05). We then grouped neurons into five categories: (1) Visual (V-Neuron), possessing only a visual increase relative to baseline; (2) Visual-motor (VM-Neuron), possessing a visual increase and either memory- or corrective-(visual) saccade response; (3) Motor (M-Neuron), exhibiting both memory- and corrective-(visual) saccade response but no visual response; (4) Visually dependent (VDSR-Neuron), possessing a saccade-related response only when a visual target was present for the eye movement^[@CR46]--[@CR48]^; (5) Exclusively-responding (ER-Neuron), exhibiting a microsaccade-related response only in the memory-guided microsaccade task but not for similarly sized visually-guided microsaccades. RF hotspot locations (e.g. Fig. [7f](#Fig7){ref-type="fig"}) were estimated based on all of the sampled saccade vector amplitudes (and corresponding flash locations for visual responses) in the memory-guided saccade task (even \> 1°); our estimates may therefore include neurons that may potentially have responded even more strongly had we obtained even larger eccentricities (at the very least, all neurons in Fig. [7f](#Fig7){ref-type="fig"} were related in one form or another to microsaccades). For every isolated neuron that had a motor response, we also estimated the saccadic amplitudes that caused the neuron to discharge, also known as the neuron's movement field. For every neuron, we compared the population of baseline values as described above to the trial-by-trial responses for saccades. We then calculated the movement field by a linear-interpolation of all z-transformed saccade-related responses. Locations with z-scores \> 1.96 were considered to be part of the movement field. We plotted movement fields using log-polar coordinates, to magnify small eccentricities associated with microsaccades, as described previously^[@CR19]^. Movement-related hotspots were identified based on the stronger of either the memory-guided or the visually guided movement-related responses. Statistical analyses {#Sec18} -------------------- We performed statistical tests (behavioral and neural) with an α level of 0.05 unless otherwise noted. When the assumptions of normality were not met for a two-sampled t-test, we used a Wilcoxon ranksum test instead. For tests related to saccade directions, we used the CircStat toolbox^[@CR74]^. When comparing proportions (for example, for success rate), we used a χ^2^-test. Literature meta-analysis {#Sec19} ------------------------ We searched for and read articles published between 1965 and November 2017. We used the Web of Science search database, and we employed the keyword "microsaccade\". We then selected, out of 467 research articles in the global results, a total of 342 articles that were genuinely related to microsaccades and that were also written in English. Any article that referred to microsaccades as being "voluntary" (20/31 articles), "controlled" (6/31 articles), or "suppressed voluntarily" (5/31 articles) was classified in the "voluntary" category (31 articles). Even though "suppressed voluntary" is conceptually different from the voluntary control that we were interested in for the current study, we nonetheless included this description in the voluntary category to give this category the benefit of the doubt in our meta-analysis when comparing to "involuntary" descriptions. In contrast to 31 articles, there were 148 articles in the "involuntary" category (almost 5 times as many). For the "involuntary" category (148 articles), we included any article using the following descriptors for microsaccades: "involuntary" (134/148 articles), "spontaneous" (7/148 articles), "unaware" (3/148 articles), "automatic" (2/148 articles), "unconscious" (1/148 articles), "stochastic" (1/148 articles). Reporting summary {#Sec20} ----------------- Further information on research design is available in the [Nature Research Reporting Summary](#MOESM3){ref-type="media"} linked to this article. Supplementary information ========================= {#Sec21} Supplementary Information Peer Review File Reporting Summary Description of Additional Supplementary Files Supplementary Movie 1 Supplementary Movie 2 Peer review information: Nature Communications* thanks Jochen Braun, Emilio Salinas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Konstantin F. Willeke, Xiaoguang Tian, Antimo Buonocore. Supplementary information ========================= Supplementary Information accompanies this paper at 10.1038/s41467-019-11711-x. We were funded by the Werner Reichardt Centre for Integrative Neuroscience (CIN). The CIN is an Excellence Cluster (EXC307) funded by the Deutsche Forschungsgemeinschaft (DFG). We were also supported by the Hertie Institute for Clinical Brain Research at Tuebingen University, and DFG-funded Research Unit (FOR1847; project: HA6749/2--1). X.T., A.B., Z.M.H. collected neurophysiological data. X.T., J.B., Z.M.H. collected monkey behavioral data. K.F.W., A.R-C. collected human behavioral data. K.F.W., A.B., Z.M.H. analyzed data. Z.M.H. wrote paper. K.F.W., X.T., A.B., A.R-C., Z.M.H. edited paper. All data presented in this paper are stored in institute computers and are available upon reasonable request. The authors declare no competing interests.	{ "pile_set_name": "PubMed Central" }	[ "rightmost", "ofigreftypefig", "visualmotor", "ab", "control", "visualfeatur", "ie", "respond", "holder", "collect", "ten", "abfigreftypefig", "frontal", "kind", "signal", "memor", "squar", "fifth", "logarithm", "systemcr", "idiosyncrat", "constraint", "microsaccadesfig", "caus", "cfigreftypefig", "represent", "probe", "modul", "circuit", "visuallyguid", "happen", "shift", "state", "attempt", "faster", "timeli", "fals", "realign", "interaur", "dimens", "salina", "medic", "higher", "representationscr", "even", "monkeycr", "remov", "herti", "laboratorycrcr", "h", "zero", "se", "govern", "sr", "whether", "strongli", "craniallyimpl", "upcom", "thu", "rhesu", "center", "overwhelmingli", "gaussian", "randomli", "gi", "rt", "significantli", "steinman", "determinist", "row", "consid", "potenti", "despit", "store", "miss", "vs", "explicitli", "posit", "respect", "combin", "financi", "conceptu", "stem", 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Sir, Basal cell carcinoma (BCC) is the most common malignancy of skin worldwide. Many clinical morphologies of BCC exist. Clinical diagnosis depends on the awareness and knowledge of the varied presentations of BCC. This not only facilitates an early diagnosis of uncommon morphologies of the malignancy, but also ensures prompt intervention and treatment. The morphological variants include nodular, cystic, morpheaform, infiltrative, micronodular, superficial, pigmented, polypoid, pore-like, aberrant, and fibroepithelioma of Pinkus (FEP).\[[@ref1]\] A 60-year-old man presented with a solitary, nonhealing fragile mass over the abdomen of 6 months duration. To start with, the lesion was an asymptomatic pea-sized nodule, which gradually increased in size to attain the present dimension. There was history of bleeding from the lesion---both spontaneously and on minor trauma. Past medical, surgical, and family history were unremarkable. There was no history of trauma, chronic arsenical exposure, or exposure to radiation. Cutaneous examination revealed bright-red, dome-shaped fragile tumor that had a glistening moist surface, covered with hemorrhagic crust \[[Figure 1](#F1){ref-type="fig"}\]. There was no associated lymphadenopathy of inguinal or cervical region. Hairs, nails, and mucosae were absolutely normal, and systemic examination was noncontributory. Pyogenic granuloma, squamous cell carcinoma, and nodular melanoma were considered in clinical differentials. Routine biochemistry panel, including serology for Hepatitis B virus, Venereal Disease Research Laboratory test, and Human immunodeficiency virus (HIV) were nonreactive. The tumor was excised and sent for histopathology. There was basaloid cell proliferation in nodular pattern with peripheral palisading of cells and cleft-like space around it at places. The cells were elongated with an elongated oval nucleus and scanty cytoplasm. The cells showed increased pigmentation in some areas \[Figures [2](#F2){ref-type="fig"}--[4](#F4){ref-type="fig"}\]. Histopathological findings were diagnostic of BCC. ![Nodular growth with moist surface and covered with hemorrhagic crust](IDOJ-7-446-g001){#F1} ![Proliferation of basaloid cells showing peripheral palisading (H and E, 100×)](IDOJ-7-446-g002){#F2} ![Higher magnification (H and E × 400)](IDOJ-7-446-g003){#F3} ![Increased pigmentation in tumor mass (H and E × 400)](IDOJ-7-446-g004){#F4} BCCs are the most common cutaneous tumors, accounting for approximately 70% of all malignant diseases of the skin. It is slow growing, locally invasive malignancy, which arises from pleuripotent cells within the basal layer of the epidermis or follicular structure. The malignancy predominantly affects fair-skinned individuals above the age of 40 years. Usually it is caused by a combination of cumulative ultraviolet (UV) exposure and intense, occasional UV exposure, especially on areas of chronic sun exposure, such as the face, head, and neck. Other known risk factors include chronic exposure to arsenic, chronic inflammation, and genetic conditions such as xeroderma pigmentosum, albinism, and basal cell nevus syndrome. However, occurrence of BCC in covered areas and also in persons without any of the aforementioned predisposing factors, as exemplified in our case indicates that etiopathogenesis is still elusive and more studies are needed to identify other contributing factors. Uncommonly, BCC may develop over atypical locations such as axilla, nipple, scrotum, pubis, and perianal region.\[[@ref2][@ref3]\] According to some authors, patients with truncal BCCs were significantly at a higher risk of multiple BCCs.\[[@ref4]\] There are different clinical and histological variants of BCC. The clinical variants are nodular (most common), pigmented, morpheaform, superficial, and FEP. Some may manifest as nodules, patches, plaques, ulcers, or rarely, a pyogenic granuloma like.\[[@ref5]\] Very few cases of BCCs mimicking pyogenic granuloma have been reported. A unique case of polypoidal BCC on the face of an elderly lady masquerading as pyogenic granuloma, was reported from India.\[[@ref5]\] Besides, Kim et al. reported a case of BCC on the dorsolateral side of the ring finger, which clinically simulated pyogenic granuloma.\[[@ref6]\] The treatment in such cases essentially consists of surgical excision and a systemic workup to exclude any metastatic deposit, followed by periodic follow-up. Thus, a rare presentation of BCC along with uniqueness in the site prompted us to report the case. Our report highlights the noteworthiness of taking into consideration BCC in the clinical differentials of lesions, which have a tendency to bleed, even if the classical features of BCC-like pearly border, telangiectasia, and others are absent. Financial support and sponsorship {#sec2-1} ================================= Nil. Conflicts of interest {#sec2-2} ===================== There are no conflicts of interest.	{ "pile_set_name": "PubMed Central" }	[ "research", "basaloid", "treatment", "cervic", "cumul", "find", "manifest", "howev", "nippl", "figur", "uncommon", "clinic", "tumor", "combin", "inflamm", "without", "elderli", "tendenc", "financi", "usual", "masquerad", "absolut", "period", "knowledg", "infiltr", "local", "hemorrhag", "surfac", "exist", "affect", "granulomaref", "differenti", "basal", "uv", "condit", "identifi", "aberr", "includ", "risk", "fairskin", "oval", "associ", "layer", "follicular", "classic", "neck", "take", "fep", "fibroepithelioma", "show", "factor", "locat", "multipl", "person", "may", "radiat", "idojgf", "abdomen", "pattern", "occurr", "uncommonli", "brightr", "invas", "face", "elong", "porelik", "b", "intens", "system", "rare", "squamou", "year", "unremark", "cystic", "skin", "domeshap", "serolog", "regionrefref", "still", "support", "immunodefici", "scanti", "pubi", 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Introduction ============ The paraoxonase (PON)[^3^](#FN4){ref-type="fn"} family consists of three members: PON1, PON2, and PON3 ([@B1][@B2][@B3]). PON1 was the first family member described and was named for its ability to degrade the organophosphate paraoxon. PON1 was subsequently found to inhibit low density lipoprotein oxidation and be important for cardiovascular disease ([@B4]). In addition, analysis of PON1 single-nucleotide polymorphisms (SNPs) has linked PON1 to the pathogenesis of numerous other human disorders including atherosclerosis, diabetes, cerebrovascular disease, Alzheimer disease, amyotrophic lateral sclerosis, organophosphate susceptibility, and Parkinson disease ([@B5][@B6; @B7; @B8; @B9; @B10][@B11]). Despite these numerous association studies, the mechanism(s) underlying the role of PON in disease pathogenesis remains to be fully determined, in part because of uncertainty regarding the endogenous or natural substrate of PON. However, the native enzyme activity of PON was recently found to be as a lactonase ([@B12]), suggesting that despite its known enzymatic promiscuity for other substrates including esters and phosphotriesters, its endogenous substrates are lactones. Importantly, all three PON family members have conserved their lactonase activity, but individual PON family members have widely disparate enzymatic activities toward other substrates. One specific lactone-containing molecule that PONs can degrade is the bacterial quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC12-HSL) ([@B13][@B14; @B15][@B16]). 3OC12-HSL is an acyl-homoserine lactone that Pseudomonas aeruginosa, a common cause of hospital-acquired infections and an important cause of pulmonary morbidity and mortality in cystic fibrosis, uses to control biofilm formation and virulence factor production ([@B17], [@B18]). Of the three PON family members, PON2 has the greatest lactonase activity toward 3OC12-HSL ([@B13], [@B19]). In contrast to PON1 and PON3, PON2 is not present in serum, and it has minimal arylesterase and paraoxonase activity. Human airway epithelial cells degrade 3OC12-HSL ([@B19], [@B20]), and murine PON2-deficient airway epithelial cells have an impaired ability to inactivate 3OC12-HSL ([@B19]). In addition, we have recently found that PON1-transgenic Drosophila melanogaster are protected from organophosphate poisoning and P. aeruginosa lethality ([@B21]). PON2 activity in airway epithelial cells may represent a novel antibacterial defense mechanism against invading pathogens that utilize acyl-homoserine lactones for quorum sensing and virulence regulation. Genetic variation in human genes can have a substantial impact on host responses, and SNP analysis may be used in anticipating responses and outcomes or in adjusting drug dosing. In this study we asked whether a common PON2 SNP, a serine to cysteine amino acid change at codon 311 in PON2 ([@B2]), alters PON2 lactonase activity for 3OC12-HSL. This SNP is fairly common in the general population and is strongly conserved evolutionarily. We chose this PON2 SNP because PON2 has the greatest lactonase activity of PON family members, phylogenetic analysis suggests that PON2 is the oldest PON family member ([@B11]), and several PON1 SNPs are in linkage disequilibrium with PON2 SNPs. We found that recombinant PON2 Cys^311^ exhibits an impaired ability to inactivate 3OC12-HSL and that airway epithelial cells from humans homozygous for Cys/Cys at PON2 amino acid position 311 also have an impaired ability to degrade 3OC12-HSL. EXPERIMENTAL PROCEDURES ======================= ### #### Recombinant PON2 Cys^311^ or Ser^311^ Expression Chinese hamster ovary (CHO) cells were used for transfection studies because these cells have no endogenous PON or lactonase activity ([@B13]). CHO cells were cultured as monolayers in plastic dishes and transfected with plasmids expressing hPON2 Cys^311^ or Ser^311^ using Lipofectamine 2000 (Invitrogen), according to the manufacturer\'s protocol, and allowed to express for 24--48 h. #### 3OC12-HSL Degradation by Airway Epithelia and CHO Cells For epithelial cell lysate experiments, epithelia were first washed with cold phosphate-buffered saline (containing calcium and magnesium), and then 50 μl of lysis buffer (50 m[m]{.smallcaps} Tris·HCl, pH 6.9, 150 m[m]{.smallcaps} NaCl, 10 μ[m]{.smallcaps} leupeptin, 10 μ[m]{.smallcaps} aprotinin, 1 μ[m]{.smallcaps} pepstatin A, 1 m[m]{.smallcaps} phenylmethylsulfonyl fluoride, 0.1 mg/ml benzamidine) was added to the apical surface. After 20 min of rocking in lysis buffer at 4 °C, the cells were scraped free from the membrane with a pipette tip and lysed by sonication (10 pulses, pulse duration of ∼1 s) (Branson Sonifier 250, Danbury, CT). The cellular debris was cleared from the lysate by centrifugation (4500 × g for 30 s at 4 °C). The relative protein concentrations were determined by the Bio-Rad protein assay (500-0006). Lysate preparations (10--20%) were diluted in phosphate-buffered saline (containing calcium and magnesium) and incubated in the presence of 3OC12-HSL (RTI International, Research Triangle Park, NC) at 37 °C. 3OC12-HSL in acidified ethyl acetate was dried under a nitrogen gas stream and then dissolved in phosphate-buffered saline (containing calcium and magnesium) to achieve a final concentration of 10 μ[m]{.smallcaps} 3OC12-HSL. At various time points, 6-μl aliquots were collected, added to 100 μl of ethyl acetate, and stored in airtight glass vials at −20 °C. #### 3OC12-HSL and 3OC12-HSL Quantitative Assay 3OC12-HSL was obtained from RTI International. 3OC12-HSL was measured in a quantitative bioassay as previously described using Escherichia coli MG4 (pKDT17) ([@B22]). #### Tunicamycin Treatment of Transfected CHO Cells PON2 chemical deglycosylation was performed by first transfecting CHO cells with a plasmid expressing PON2 Cys^311^ or Ser^311^. 30 min later, tunicamycin was added to the cell culture medium at a final concentration of 0.5 μg/ml. 36--48 h later cell lysates were harvested, and SDS-PAGE was performed for PON2. #### Deglycosylation of PON2 with Peptide-N-glycosidase F and Endoglycosidase H Digestion Peptide-N-glycosidase F (PNGase F) (Sigma) digestion was performed according to the manufacturer\'s directions. Briefly, cell lysates were collected in lysis buffer (50 m[m]{.smallcaps} Tris·HCl, pH 7.5, 138 m[m]{.smallcaps} NaCl, 5 m[m]{.smallcaps} EDTA, 1 m[m]{.smallcaps} EGTA, 1 m[m]{.smallcaps} NaF, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) and incubated with PNGase F (10 units) for 4 h at 37 °C. The reaction was stopped by incubating at 100 °C for 5 min. Deglycosylation was then assessed with SDS-PAGE. Endoglycosidase H (Endo H) (Sigma) digestion was performed according to the manufacturer\'s directions. The cell lysates were incubated with reaction buffer and denaturation solution and heated for 5 min at 100 °C. Following cooling of the sample, Endo H was added to the samples and incubated for 4 h at 37 °C. Digestion was then determined using SDS-PAGE. #### Western Blot Analysis CHO cell lysates were made by incubation in lysis buffer (50 m[m]{.smallcaps} Tris·HCl, pH 7.5, 138 m[m]{.smallcaps} NaCl, 5 m[m]{.smallcaps} EDTA, 1 m[m]{.smallcaps} EGTA, 1 m[m]{.smallcaps} NaF, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) with 1% Triton X-100 for 20 min at 4 °C on a rocker. The cells were scraped from the tissue culture plates and transferred to a microcentrifuge tube. Sonication was performed for 10 s, and cell debris was cleared by centrifugation at 4,500 × g for 30 s at 4 °C. The lysates were then combined with loading buffer and separated by SDS-PAGE. Protein was then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA). Polyvinylidene difluoride membranes were blocked either overnight at 4 °C or for 2 h at room temperature in 5% bovine serum albumin in phosphate-buffered saline. The membranes were then incubated with the PON2 antibody (1:1000; rabbit, polyclonal anti-human PON2; Orbigen, San Diego, CA) for 2 h at room temperature and then washed three times in 1× TTBS (137 m[m]{.smallcaps} NaCl, 2.7 m[m]{.smallcaps} KCl, 2.5 m[m]{.smallcaps} Tris, 0.05% Tween 20). Secondary antibodies were conjugated to horseradish peroxidase (Amersham Biosciences) and used at 1:10,000 for 1 h. Following three washes with 1× TTBS, immunoreactive bands were detected with SuperSignal solution (Pierce) and exposed to film. #### Construction of N-Linked Glycosylation Mutants Site-directed mutagenesis was performed with the Stratagene QuikChange™ kit according to the manufacturer\'s standard protocol (Stratagene, Cedar Creek, TX). Double-stranded DNA template was prepared by a standard megaprep protocol (Qiagen). Mutant strand synthesis was performed with the following primers for N226Q (5′-GATTCAGCAAATGGGATCCAGATTTCACCTGATGATAAG-3′), for N254Q (5′-GTTTTGGAAAAACACACTAATATGCAGTTAACTCAGTTGAAGG-3′), for N269Q (5′-TGGATACACTGGTGGATCAGTTATCTATTGATCCTTCC-3′), for N323Q (5′-ACTACAGTTTATGCCAACCAGGGGTCTGTTCTCCAAGG-3′), for N254A (5′-GTTTTGGAAAAACACACTAATATGGCTTTAACTCAGTTGAAGG-3′), and for N323A (5′-ACTACAGTTTATGCCAACGCTGGGTCTGTTCTCCAAGG-3′). The reaction products were then treated with the restriction endonuclease DpnI to enrich for multiply mutated single-stranded DNA. This reaction mixture was then transformed into XL10-Gold® Ultracompetent cells and spread on Luria-Bertani ampicillin agar plates (containing 80 ng/ml 5-bromo-4-chloro-3-indolyl-β-[d]{.smallcaps}-galactopyranoside (X-gal) and 20 m[m]{.smallcaps} isopropyl β-[d]{.smallcaps}-thiogalactopyranoside). The colonies were picked, and site-specific mutations were confirmed by DNA sequencing (DNA Facility, University of Iowa). #### Immunofluorescence Localization of PON2 in Transfected CHO Cells CHO cells were plated on collagen-coated four-well chamber slides and transfected with a plasmid encoding either human PON2 Cys^311^ or Ser^311^. At 36 h following transfection, the cells were fixed with 1% paraformaldehyde in methanol, blocked in 2% bovine serum albumin in SuperBlock (Pierce), and stained with anti-human PON2 antibody (rabbit polyclonal, Orbigen) and a monoclonal antibody specific for protein-disulfide isomerase to localize the endoplasmic reticulum (Assay Designs, Ann Arbor, MI). Confocal images were acquired with a Bio-Rad MRC-1024 laser scanning confocal system mounted on a Nikon E600 microscope. #### Primary Airway Epithelial Cell Culture Model Primary human airway epithelial cells were isolated from the trachea and bronchi of human donor lungs. The isolated cells were seeded onto collagen-coated, semi-permeable membranes (0.6-cm^2^ Millicell-HA; Millipore, Bedford, MA) and grown at the air-liquid interface as previously described ([@B23]). After genotypic identification of PON2 SNPs in donor fibroblasts, primary or passage 1 cryopreserved airway epithelial stocks were thawed and expanded for the described studies. All of the experiments were performed on well differentiated human airway epithelia (more than 14 days after seeding of cells). The samples were collected with the approval of the University of Iowa Institutional Review Board. #### Identification of PON Polymorphisms and Determination of Genotypic Frequencies DNA samples were genotyped for the presence of several common coding and noncoding polymorphisms, including PON2 Cys^311^ → Ser. PCR primers for amplifying the sequences containing the SNP were designed using Primer 3. The PCR primers are listed in the [supplemental materials](http://www.jbc.org/cgi/content/full/M109.051706/DC1). Sense and antisense probes used for fluorescence polarization-single base extension detection of SNPs were designed using Primer Premier. The sense and antisense probes for genotyping the PON2 Cys^311^ → Ser SNP are: 5′-CTG TAG TCA CTG TAG GCT TCT CA-3′ and 5′-CCG CAT CCA GAA CAT TCT AT-3′, respectively. Each genotyping reaction consisted of a polymerase chain reaction followed by a single base pair extension reaction using either the sense or antisense probe and dideoxynucleotides labeled with carboxytetramethylrhodamine (TAMRA) or R110 for the alternative alleles. The fluorescence signal was detected by fluorescence polarization using the analyst HT (Molecular Devices, Sunnyvale, CA), and genotypes were determined based on the plot of TAMRA versus R110 signal values. #### Statistical Analysis All of the experiments were performed in at least triplicate, and the data are presented as the means ± S.E. of the mean (S.E.). Comparisons between two groups were made with Student\'s t test. RESULTS ======= ### #### Cysteine Substitution at Amino Acid Position 311 Impairs PON2 Lactonase Activity We cloned PON2 and asked whether mutating the 311 amino acid, from a serine to cysteine, would affect PON2 lactonase activity. Lysates from CHO cells expressing both PON2 Cys^311^ and Ser^311^ inactivated 3OC12-HSL over time, but PON2 Cys^311^ lysates had an impaired ability compared with PON2 Ser^311^ lysates ([Fig. 1](#F1){ref-type="fig"}A). These data suggest that cysteine substitution at amino acid 311 impairs PON2 function through mechanisms other than alternative splicing, linkage disequilibrium, or promoter differences. ![Recombinant PON2 Cys^311^ has impaired lactonase activity. A, 3OC12-HSL degradation by CHO cell lysates following transfection with PON2 Cys^311^ or PON2 Ser^311^. n = 3--5 experiments/group. Lysates (20% volume) of CHO cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL and after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of 3OC12-HSL inactivation. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. The data are the means ± S.E. \, p* \< 0.05. B, CHO cells were transfected with PON2 Cys^311^ or Ser^311^ and 48 h later lysed for gel electrophoresis. Subsequently, Western immunoblot analysis for PON2 was performed as described under "Experimental Procedures." C, densitometric analysis of PON2 Cys^311^ and Ser^311^ protein levels in CHO cell lysates at 48 h following transfection. ImageJ software was used for densitometric analysis of PON2 immunoreactive bands. GAPDH levels were used for normalization. n = 3. The data are the means ± S.E.](zbc0021098990001){#F1} To test whether the PON2 Cys^311^ variant expresses similar PON2 protein levels as PON2 Ser^311^, PON2 immunoblotting was performed in CHO cell lysates 48 h following transfection. [Fig. 1](#F1){ref-type="fig"}B shows that in PON2 Cys^311^ samples we observed a single immunoreactive band migrating slightly higher than 37 kDa, a molecular mass consistent with PON2. In PON2 Ser^311^ samples, we also observed a similarly sized upper band as in PON2 Cys^311^, but noted an additional band with greater electrophoretic mobility several kDa lower than the upper PON2 band. Densitometric analysis and quantification showed no difference in total PON2 protein levels between PON2 Cys^311^ and Ser^311^ groups ([Fig. 1](#F1){ref-type="fig"}C). These findings suggest that the 311 amino acid change does not alter PON2 protein stability. The smaller, second band could result from alternative splicing (although this seems highly unlikely to occur using cDNA) ([@B2]), from PON2 aggregation ([@B24]), or most likely from differential post-translational modification or proteolysis. #### An Altered Core Glycosylation Pattern Occurs in the PON2 Cys^311^ Variant PON2 has four putative N-linked glycosylation sites at asparagine residues. Purified PON1 is ∼15.8% carbohydrate by weight ([@B25]). To investigate the glycosylation of PON2 and determine whether PON2 Cys^311^ and Ser^311^ are differentially glycosylated, we first expressed PON2 Cys^311^ or Ser^311^ in CHO cells and then treated with tunicamycin, an antibiotic from the bacterium Streptomyces iysosuperficus that inhibits GlcNAc phosphotransferase, one of the initial steps required for glycoprotein synthesis. Two days later, cell lysates were harvested, and Western blot analysis showed that, in PON2 Cys^311^ samples, the predominant immunoreactive band following tunicamycin treatment had a greater electrophoretic mobility compared with the PON2 band in untreated cells ([Fig. 2](#F2){ref-type="fig"}A). This finding is consistent with N-linked core glycosylation of PON2. Similar findings were observed with PON2 Ser^311^ ([Fig. 2](#F2){ref-type="fig"}B). After tunicamycin treatment there was no difference in the appearance of the PON2 band between Cys^311^ and Ser^311^ variants. This suggests that the lower band in PON2 Ser^311^ samples represents differential or incomplete glycosylation compared with the upper band in PON2 Cys^311^ and Ser^311^. ![Effect of chemical and enzymatic deglycosylation of PON2. CHO cells were transfected with a plasmid expressing human PON2 Cys^311^ (A) or PON2 Ser^311^ (B). Tunicamycin was added shortly after transfection, and cell lysates were harvested 48 h later. In separate experiments, enzymatic deglycosylation with either PNGase F or Endo H, as described under "Experimental Procedures," was also performed on cell lysates 36--48 h following transfection.](zbc0021098990002){#F2} Because tunicamycin has a number of effects on the cell including induction of the unfolded protein response, we also treated cell lysates with PNGase F at 48 h following transfection. PNGase F is an enzyme that cleaves N-linked glycoproteins between the innermost GlcNAc and asparagine residues. N-Linked deglycosylation with PNGase F caused both PON2 Cys^311^ and Ser^311^ migration patterns to change as observed with tunicamycin treatment, and after PNGase F treatment there was no difference between the Cys and Ser variants ([Fig. 2](#F2){ref-type="fig"}). PON2 was also sensitive to treatment with Endo H, an endoglycosidase that cleaves the chitobiose core of high mannose and oligosaccharides from N-linked glycoproteins. When proteins are correctly processed through the endoplasmic reticulum and Golgi complex, they become resistant to Endo H. Therefore sensitivity to Endo H indicates the presence of proteins that have not been processed beyond the ER. We observed qualitatively similar results when comparing Endo H, tunicamycin, and PNGase F treatments in both PON2 Cys^311^ and Ser^311^ samples ([Fig. 2](#F2){ref-type="fig"}). From these results, we conclude that PON2 undergoes N-linked core glycosylation, and incomplete glycosylation is likely responsible for the additional band observed when PON2 Ser^311^ is expressed in CHO cells. #### PON2 Undergoes N-Linked Core Glycosylation at Asparagine Residues 254 and 323 Based on the two different bands of glycosylated PON2, we predicted that at least two of the four putative N-linked glycosylation sites are glycosylated in PON2. To determine which asparagine residues of the Asn-Xaa-(Ser/Thr) consensus sequence are N-glycosylated in PON2 Cys^311^ and Ser^311^ variants, putative N-linked glycosylation sites were disrupted by substituting glutamine for asparagine individually and also in combination (quadruple mutant). Western blotting for PON2 revealed one immunoreactive band in wild-type PON2 Cys^311^ and the N226Q and N269Q mutants. Cell lysates from the N254Q and N323Q mutants also showed one PON2 immunoreactive band, but with a greater electrophoretic mobility than wild-type PON2 Cys^311^ ([Fig. 3](#F3){ref-type="fig"}A). Lysate from the quad mutant had a PON2 immunoreactive band with a smaller molecular mass than either N254Q or N323Q. This shows that, for PON2 Cys^311^, two of the four putative N-linked glycosylation sites undergo oligosaccharide addition: asparagine residues 254 and 323. ![*Effect of asparagine to glutamine site-specific mutations in predicted N-linked glycosylation sites of PON2.* CHO cells expressing wild-type (WT) and glutamine site-specific glycosylation mutants of PON2 Cys^311^ (A) and PON2 Ser^311^ (B) were lysed, electrophoresed, and analyzed by Western blot analysis for PON2 immunoreactive bands. PON2 Cys^311^ (C) and PON2 Ser^311^ (D) immunoblots following site-directed mutagenesis and PNGase F or Endo H treatment are shown.](zbc0021098990003){#F3} We next hypothesized that the upper band in PON2 Ser^311^ also represents glycosylation at both asparagine 254 and 323, whereas the lower electrophoretic mobility band could be glycosylation at either one of these asparagine residues or a mixture of individually glycosylated PON2 proteins. Alternatively, PON2 Ser^311^ could use different asparagine residues for core glycosylation. Similar to the PON2 Cys^311^, only asparagine 254 and 323 were glycosylated ([Fig. 3](#F3){ref-type="fig"}B). The immunoreactive band in both PON2 Cys^311^ and Ser^311^ quad mutants had a similar molecular mass compared with PON2 treated with PNGase F or Endo H ([Fig. 3](#F3){ref-type="fig"}, C and D). These substitution experiments allowed us to investigate which site is glycosylated in the lower band of PON2 Ser^311^. The N254Q mutant had an unglycosylated band as well as an intermediate glycosylated band, suggesting that the lower PON2 Ser^311^ band is glycosylated at asparagine 254. This is confirmed by the finding that the N323Q substitution results in a single intermediate band ([Fig. 3](#F3){ref-type="fig"}B). Substitution at asparagine 323 caused loss of the upper band, but the lower band remained in place. These data show that the upper band in PON2 Cys^311^ and Ser^311^ is glycosylated at asparagine 254 and 323, whereas the lower band in PON2 Ser^311^ is only glycosylated at asparagine 254. #### Glycosylation at Asparagine Residue 323 Is Required for PON2 Lactonase Function We hypothesized that differential glycosylation of PON2 Cys^311^ and Ser^311^ accounts for the differences in lactonase activity between the 311 variants, so we initially asked whether glycosylation is important for PON2 inactivation of 3OC12-HSL. Lysates from CHO cells transfected with the PON2 Cys^311^ or Ser^311^ with the N254G mutation had preserved 3OC12-HSL lactonase activity ([Fig. 4](#F4){ref-type="fig"}, A and B). We expected that if the lower electrophoretic band, only glycosylated at asparagine 254, in wild-type PON2 Ser^311^ accounts for its enhanced 3OC12-HSL inactivation, then glycosylation at asparagine 323 must inhibit PON2 lactonase activity. In contrast, we found that the N323Q mutant had impaired lactonase activity, showing that glycosylation at asparagine 323 is important for PON2 function. Similarly, lysates from CHO cells transfected with the PON2 Cys^311^ or Ser^311^ quad mutant (N226Q/N254Q/N269Q/N323Q) were unable to degrade 3OC12-HSL, also showing that glycosylation might be important for PON2 3OC12-HSL inactivation. ![*Effect of mutating N-linked glycosylation sites on lactonase activity.* A and B, PON2 Cys^311^ (A) or PON2 Ser^311^ (B) transfected CHO cell lysates from either wild-type (WT), N254G, N323G, or N226/254/269/323G (Quad) mutants were tested for 3OC12-HSL inactivating ability. Lysates (20% volume) of CHO cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL and after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of inactivation of 3OC12-HSL. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. \* denotes p \< 0.05 for lactonase activity between wild-type samples and N323G and quad mutant. C, CHO cells expressing wild-type and alanine site-specific glycosylation mutants of PON2 Cys^311^ and PON2 Ser^311^ were lysed, electrophoresed, and analyzed by Western blot analysis for PON2 immunoreactive bands. D and E, PON2 Cys^311^ (D) or PON2 Ser^311^ (E) transfected CHO cell lysates from either wild-type, N254A, N323A, or N254A/N323A mutants were tested for 3OC12-HSL inactivating ability. Lysates (20% volume) of CHO cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL; after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of inactivation of 3OC12-HSL. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. The data are the means ± S.E. \* denotes p \< 0.05 for lactonase activity between wild-type samples and N323A and double mutant.](zbc0021098990004){#F4} Mutation of asparagine residues to glutamine could have other effects on PON2 structure and activity besides disrupting N-linked glycosylation. Therefore, we constructed additional mutants of the PON2 Cys^311^ and Ser^311^ variants by replacing asparagine with alanine individually at positions 254 and 323, as well as a double mutant, N254A/N323A. Compared with the glutamine mutants, we found similar patterns of PON2 immunoreactive bands in the N254A, N323A, and N254A/N323A mutants ([Fig. 4](#F4){ref-type="fig"}C). In addition, the N323A and N254A/N323A mutants had an impaired ability to inactivate 3OC12-HSL ([Fig. 4](#F4){ref-type="fig"}, D and E). From these data we conclude that: 1) PON2 undergoes N-linked core glycosylation at asparagine residues 254 and 323; 2) the upper band in PON2 Ser^311^ is glycosylated at asparagine 254 and 323, whereas the lower band is only glycosylated at asparagine 254; 3) glycosylation at asparagine 323 is required for PON2 lactonase activity; and 4) differential glycosylation between the PON2 Cys^311^ and Ser^311^ variants does not account for greater lactonase activity by PON2 Ser^311^. #### PON2 Localizes to the Endoplasmic Reticulum Our data show that PON2 is Endo H-sensitive and does not undergo complex glycosylation,[^4^](#FN5){ref-type="fn"} suggesting that PON2 resides in the ER. Moreover, recent work by Horke et al. ([@B26]) demonstrated PON2 localization to the ER in endothelial cells. To test for changes in PON2 cellular localization as a mechanism of disrupted lactonase activity between PON2 Cys^311^ and Ser^311^ variants, immunohistochemical staining for PON2 was performed in CHO cells following transfection with either PON2 Cys^311^- or Ser^311^-expressing plasmids. A similar pattern of staining for both PON2 Cys^311^ and Ser^311^ was observed with the most intense staining corresponding to an ER location ([Fig. 5](#F5){ref-type="fig"}). ![Immunohistochemical localization of PON2 Cys^311^ and Ser^311^ variants. CHO cells were transiently transfected with a plasmid encoding human PON2 Cys^311^ or Ser^311^. 24 h later cells were fixed, stained with human anti-PON2 and an ER-specific antibody, and examined with confocal microscopy. A, PON2 Cys^311^-expressing CHO cells. B, PON2 Ser^311^-expressing CHO cells. Scale bar, 30 μm.](zbc0021098990005){#F5} #### Genotypic Analysis of Human Airway Donor Samples for PON2 S311C SNP To test whether the PON2 311 SNP also affects lactonase activity in vivo, we asked whether naturally occurring PON2 mutations at position 311 would affect the ability of primary human airway epithelial cells to degrade 3OC12-HSL. We first isolated DNA from 200 human donor lung samples and performed genotyping by polymerase chain reaction allele-specific oligonucleotide hybridization assays for the PON2 S311C polymorphism and a number of other common PON1, PON2, and PON3 allelic variants including coding (PON1 Q192R, PON1 L55M, PON3 C133A, and PON3 G99A) and promoter (PON1 G-907C) polymorphisms ([supplemental Table S1](http://www.jbc.org/cgi/content/full/M109.051706/DC1)). For PON2, we found that 62% of human donor samples were homozygous for Ser/Ser, 35% heterozygous for Ser/Cys, and 3% homozygous for Cys/Cys at amino acid 311 ([Fig. 6](#F6){ref-type="fig"}A). These findings are consistent with the HapMAP-CEU European SNP data base showing 57% of individuals homozygous for Ser/Ser, 38% heterozygous for Ser/Cys, and 5% homozygous for Cys/Cys. ![Impaired airway lactonase activity in humans homozygous for cysteine at amino acid 311 in PON2. A, percentage of human subjects with genotype of Cys/Cys, Cys/Ser, or Ser/Ser at amino acid position 311 in PON2. B, 3OC12-HSL degradation by human airway epithelia. n = 5 human donor samples for Ser/Ser, 2 for Ser/Cys, and 4 for Cys/Cys. Lysates (20% volume) of human airway epithelial cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL, and after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of 3OC12-HSL inactivation. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. The data are the means ± S.E. \, p* \< 0.005 comparing lactonase activity between Cys/Cys and Ser/Ser donor epithelia.](zbc0021098990006){#F6} #### Human Airway Epithelial Cells from PON2 Cys/Cys^311^ Donors Have an Impaired Ability to Inactivate 3OC12-HSL To test whether differences in the common PON2 311 variant affect airway epithelia inactivation of 3OC12-HSL, human airway epithelial cells from four individuals homozygous for cysteine, five homozygous for serine, and two heterozygous for cysteine/serine at amino acid position 311 were grown at the air-liquid interface. We chose these donors based upon their PON2 311 SNP and similarities in other described SNPs for PON1 and PON3. Lysates from airway epithelial cells homozygous for serine degraded 3OC12-HSL with ∼25% active 3OC12-HSL remaining after 60 min of exposure ([Fig. 6](#F6){ref-type="fig"}B). In contrast, airway epithelial cell samples from donors homozygous for cysteine had an impaired ability to inactivate 3OC12-HSL, and samples from heterozygotes (Ser/Cys) demonstrated an intermediate phenotype for 3OC12-HSL inactivation compared with Ser/Ser and Cys/Cys samples ([Fig. 6](#F6){ref-type="fig"}B). These data show that the common PON2 311 polymorphism alters the endogenous ability of human airway epithelia to degrade 3OC12-HSL. DISCUSSION ========== The human PON2 S311C polymorphism is common and has been linked to several disorders including coronary artery disease, abnormal plasma lipoprotein levels, ischemic stroke, and Alzheimer dementia ([@B2], [@B7], [@B27][@B28][@B29][@B30]--[@B31]), but the mechanisms underlying these potential associations remain unknown. The serine allele is ancestral, has relatively little variation across human populations (Single Nucleotide Polymorphism database (dbSNP) data), and is strongly conserved in evolution. We hypothesized that the cysteine for serine substitution at amino acid position 311 in PON2 would disrupt PON2 lactonase activity. We found that the PON2 Cys^311^ variant had an impaired ability to inactivate 3OC12-HSL. More importantly, human airway epithelial cells from Cys/Cys homozygous individuals had an impaired ability to degrade 3OC12-HSL, compared with cells from Ser/Ser homozygous individuals. The impaired lactonase activity by Cys/Cys airway epithelia was not due to changes in promoter activity, alternative splicing, or linkage disequilibrium with other PON SNPs or genes because transfection of CHO cells with a plasmid expressing PON2 Cys^311^ also showed similar results as primary human airway epithelia. Linkage studies of monogenic diseases have resulted in the identification of genes responsible for the pathogenesis of several diseases including cystic fibrosis and Huntington disease. In contrast, association studies of complex genetic diseases often result in the identification of SNPs that may represent false positives or linkage to other genetic mutations. This becomes particularly troubling when the SNPs are found in proteins whose function are poorly understood, when the effect of the SNP on protein function is not known, and when there is no obvious physiologic connection between the gene and the pathogenesis of the studied disease. The identification of a physiologic consequence of a SNP becomes important in understanding association studies. A great example is the work of Richter and Furlong ([@B32]) in their studies determining the "PON1 status" of an individual. These investigators use a functional genomic approach and are able to directly measure, from plasma samples, the PON1 function of an individual based upon hydrolysis of both paraoxon and diazoxon. By using this method, any polymorphisms that might affect PON1 function are taken into account. In addition, hydrolysis of diazoxon quantifies the PON1 levels of an individual, allowing for comparisons in PON1 function to be determined. This assay allows for accurate identification of PON1 phenotype, predicts PON1 E192R status (Glu/Glu, Glu/Arg, or Arg/Arg), and can account for any other yet unidentified SNPs or those changes that may be secondary to a second SNP in linkage disequilibrium with the SNP of interest. This functional genomic approach was utilized by Jarvik et al. ([@B33]) to study the effects of PON1 polymorphisms on risk of vascular disease, an association that has been inconsistently linked. Examination of PON1 192 or 55 genotypes alone failed to predict the risk of vascular disease of the carotid arteries. However, when genotype status was combined with the rates of substrate analysis, as described above, vascular disease risk was predicted accurately, thus demonstrating the importance of including a functional analysis in association studies. A similar approach was used to test organophosphate hydrolysis by subjects with sporadic amyotrophic lateral sclerosis (ALS) ([@B34]). As with vascular disease, discrepant results regarding a linkage between PON1 polymorphisms and ALS exist. Wills et al. ([@B34]) reported an increased frequency of the PON1 Arg/Arg^192^ variant in ALS patients but similar levels of organophosphate hydrolysis by plasma samples from control and ALS patients. These findings were important because they suggested that if a linkage does exist between the PON1 192 SNP and ALS, etiologies other than organophosphate toxicity are responsible for the association, including disturbances in metabolism of other yet unidentified toxic substances or an effect secondary to linkage disequilibrium with either additional PON variants or other proteins. Application of this functional genetic analysis for PON2 is more difficult because PON2 is primarily an intracellular enzyme, as opposed to PON1, which is found in the serum, and no simple assay exists to detect a functional consequence of the PON2 311 SNP in human samples. Furthermore, the natural substrate and in vivo physiological function of PON2 are not yet fully elucidated. However, by performing genetic analysis of donor human lung samples and testing 3OC12-HSL degradation by human airway epithelia, we were able to show that the Cys allele at PON2 311 impairs lactonase activity. The in vivo physiological consequence of this effect remains to be determined, but our previous data showing that PON2 deficiency in airway epithelial cells impairs the ability of these cells to inactivate 3OC12-HSL suggest that despite the intracellular location of PON2, it may be important in the host response to quorum sensing-dependent bacteria. Differing molecular masses of PON2 immunoreactive bands between the PON2 311 alloenzymes were observed, so we investigated the N-linked glycosylation pattern of PON2. In our study, tunicamycin, Endo H, and PNGase F treatment of PON2 Cys^311^ and Ser^311^ showed that PON2 undergoes N-linked core oligosaccharide addition and that following deglycosylation PON2 Cys^311^ and Ser^311^ immunoreactive bands appeared similar in molecular mass. Using directed evolution, Harel et al. ([@B35]) reported a crystal structure of PON1 and suggested that of the four corresponding PON1 asparagine residues, 254 and 323 are most likely core glycosylated because of their location on surface loops. Asparagine 226 and 269 are in the central tunnel of the β-propeller and are predicted not to be glycosylated. These predictions are in agreement with our findings. We found that PON2 Cys^311^ has a single molecular mass protein glycosylated at asparagine residues 254 and 323, whereas PON2 Ser^311^ had one isoform identical to PON2 Cys^311^ (glycosylated at asparagine 254 and 323) and another with a smaller molecular mass only glycosylated at 323. Based upon our findings and others ([@B15], [@B36], [@B37]), it appears that differential glycan processing occurs between PON family members and that differential N-linked glycosylation occurs between the PON2 311 alloenzymes. Furthermore, even though we showed that glycosylation is important for PON2 3OC12-HSL degradation, it does not explain the differences seen in PON2 Cys^311^ lactonase activity. Although we hypothesized that glycosylation at asparagine 323 would inhibit PON2 activity (based upon the differential glycosylation pattern and lactonase activity between PON2 Cys^311^ and Ser), this was not the case. Several possible explanations exist for both differential glycosylation and lactonase activity between PON2 Cys^311^ and Ser^311^. First, although unlikely, the Cys^311^ mutation may affect PON2 function, which in turn affects the glycosylation pathway of other proteins. Second, changes in glycosylation and PON2 function may be completely unrelated. Finally, whereas differential glycosylation may not account for the impaired ability of the PON2 Cys^311^ airway epithelia to degrade 3OC12-HSL, the two findings might be related. We hypothesize that the most likely underlying mechanism common to both findings is due to differences in disulfide bond formation. This might result from changes in which cysteine residues undergo disulfide bond formation, changing the number of disulfide bonds formed, or changes in which intermolecular disulfide bonds between cysteine residues of different PON2 molecules occur. For example, the free cysteine residue at position 284 in PON1 is required for its lactonase activity ([@B38]), and disruption of PON1 disulfide-linked Cys residues 41 or 352 impaired catalytic activity ([@B39]). Similarly, we found that treatment of both PON2 Cys^311^ and Ser^311^ with the reducing agent dithiothreitol (10 m[m]{.smallcaps}) impaired lactonase activity (data not shown). These data also suggest that disulfide bonds are important for PON2 activity. Alternatively, an altered protein structure or differences in substrate binding to the PON2 active site might account for the phenotype of PON2 Cys^311^ mutants. Therefore, it appears that the altered glycosylation profile observed between the PON2 311 alloenzymes is a marker of altered lactonase activity but is not the mechanism for this difference. In summary, we found that human airway epithelia homozygous for PON2 Cys^311^ have an impaired ability to inactivate 3OC12-HSL. There have been few successes to date in identifying the specific etiologic variant influencing a common disease when an association has been confirmed ([@B40]), mostly because of inadequate functional assay testing. This is the first time for PON2 that a functional consequence for the 311 SNP has been demonstrated in humans. Moreover, these findings suggest three important speculations. First, infections with quorum sensing-dependent bacteria should have a differential phenotype in patients with the PON2 Cys^311^ variant. Second, diseases that associate genetically with this PON2 311 SNP may have an infectious etiology. Third, some of these diseases may be caused by an as yet unidentified metabolic pathway that requires lactonase activity. This work was supported, in whole or in part, by National Institutes of Health Grants DK54759, AI076671, HL61234-09. This work was also supported by the Cystic Fibrosis Foundation. The on-line version of this article (available at <http://www.jbc.org>) contains [supplemental Table S1](http://www.jbc.org/cgi/content/full/M109.051706/DC1). D. A. Stoltz, T. J. Recker, and J. Zabner, unpublished data. The abbreviations used are: PONparaoxonaseSNPsingle-nucleotide polymorphism3OC12-HSLN-3-oxododecanoyl homoserine lactonePNGase Fpeptide-N-glycosidase FEndo Hendoglycosidase HALSamyotrophic lateral sclerosisCHOChinese hamster ovaryERendoplasmic reticulum. We thank Nick Gansemer, Debbie Hunter, Phil Karp, Peter Kiss, Jan Launspach, Thomas Moninger, Peter Taft, and Geri Traver for excellent assistance and Drs. Jeff Murray and Michael Welsh for insightful discussions. [^1]: Supported by a Parker B. 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"association", "confirmed", "mostly", "inadequate", "functional", "assay", "testing", "first", "time", "functional", "consequence", "SNP", "demonstrated", "humans", "Moreover", "findings", "suggest", "three", "important", "speculations", "First", "infections", "quorum", "bacteria", "differential", "phenotype", "patients", "variant", "Second", "diseases", "associate", "genetically", "SNP", "may", "infectious", "etiology", "Third", "diseases", "may", "caused", "yet", "unidentified", "metabolic", "pathway", "requires", "lactonase", "activity", "work", "supported", "whole", "part", "National", "Institutes", "Health", "Grants", "work", "also", "supported", "Cystic", "Fibrosis", "Foundation", "version", "article", "available", "http", "contains", "supplemental", "Table", "http", "Stoltz", "Recker", "Zabner", "unpublished", "data", "abbreviations", "used", "N", "homoserine", "lactonePNGase", "N", "FEndo", "Hendoglycosidase", "HALSamyotrophic", "lateral", "sclerosisCHOChinese", "hamster", 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Introduction ============ Although there is a relatively large body of knowledge assessing postoperative physical therapy (post-op PT),[@b1-jpr-9-493]--[@b6-jpr-9-493] there is limited literature investigating preoperative physical therapy (pre-op PT) on pain intensity and disability after musculoskeletal surgery. The main goal of pre-op PT treatment is to reduce postoperative complications and improve postoperative outcomes.[@b7-jpr-9-493] However, previous literature shows that isolating the real impact of a preoperative rehabilitation is not easy, mainly because of the heterogeneity in the type of the physical therapy (PT) intervention.[@b8-jpr-9-493] In cases, such as cardiac, thoracic, and abdominal surgeries, the main goal of pre-op PT is to improve the physiological capacity of the individual. In this case, it seems that strengthening the inspiratory muscle prior to surgery may result in fewer postoperative complications and shorter postoperative length of stay, enhance functional capacity,[@b9-jpr-9-493],[@b10-jpr-9-493] improve the quality of life, and reduce postoperative pain and complications.[@b11-jpr-9-493] The reported effects of pre-op PT intervention on postoperative outcomes in patients having elective musculoskeletal surgeries are conflicting. A systematic review including functional measures as outcome measures concluded that preoperative exercise programs were not effective on postoperative outcomes on total knee or total hip arthroplasty.[@b12-jpr-9-493] On the other hand, preoperative level of function is predictive of postoperative outcome after lower limb arthroplasty, where patients with better function preoperatively had better function postoperatively.[@b13-jpr-9-493] Moreover, the authors[@b8-jpr-9-493],[@b14-jpr-9-493] have recommended a multidisciplinary preoperative rehabilitation program before total hip and knee arthroplasty to reduce hospital length of stay and modify discharge conditions. Reported evidence on the effects of pre-op PT intervention on general postoperative outcomes for musculoskeletal pain is inconclusive,[@b15-jpr-9-493] and few studies have investigated this issue for shoulder arthroscopy. Arthroscopy of the shoulder is a common musculoskeletal surgery and has become widely accepted as a diagnostic and therapeutic tool in the management of shoulder disorders[@b1-jpr-9-493],[@b16-jpr-9-493],[@b17-jpr-9-493]; however, data describing postoperative outcomes following pre-op PT interventions are scarce. This topic is important because whether pre-op PT intervention influences postoperative outcomes may assist in decision making for optimal timing of rehabilitation in the operative management of shoulder pain. Therefore, the purposes of this prospective study were to 1) identify differences in demographic, clinical, surgical type, and psychological characteristics between patients having and not having pre-op PT treatment and 2) determine whether having pre-op PT influences the length of post-op PT treatment (number of sessions) and 3-month and 6-month postsurgical outcomes (pain intensity and disability). We hypothesized that 1) there would be a difference between patients having and not having pre-op PT in demographic, clinical, surgical types and psychological characteristics and 2) having pre-op PT would favorably influence the length of post-op PT treatment and 3-month and 6-month postsurgical outcomes. Patients and methods ==================== Patients -------- The University of Florida's Institutional Review Board for human participants approved this study. This prospective design includes data from consecutive patients seeking operative treatment of shoulder pain, where procedures were limited to arthroscopy of the glenohumeral joint or distal clavicle. Specific operative procedures included rotator cuff repair, adhesive capsulitis, acromioplasty, and labral repair ([Table 1](#t1-jpr-9-493){ref-type="table"}). Patients were recruited by participating physicians and provided written informed consent before participating in this study. ### Inclusion criteria The inclusion criteria for being a participant were as follows: 1) aged between 18 years and 85 years; 2) complained of pain limited to anterior, lateral, or posterior shoulder; 3) documented or suspected rotator cuff tendinopathy (evidence from clinical examination or imaging studies), including small (\<1 cm), medium (1--3 cm), and large (3--5 cm) tears; 4) documented or suspected adhesive capsulitis (evidence from clinical examination or imaging studies); 5) documented or suspected superior labrum from anterior to posterior lesion (evidence from clinical examination or imaging studies); 6) shoulder instability (anterior or posterior labral lesions); and 7) scheduled for arthroscopic surgery. ### Exclusion criteria The exclusion criteria were as follows: 1) current complaints of pain lasting longer than the past 3 months involving neck, elbow, hand, low back, hip, knee, or ankle; 2) massive rotator cuff tear (\>5 cm); 3) documented shoulder osteoarthritis or rheumatoid arthritis; 4) prior shoulder surgery within the past year or currently complaining of pain from prior shoulder surgery; 5) current shoulder fracture, tumor, or infection; 6) previously diagnosed chronic pain disorder (including, but not limited to, irritable bowel syndrome, fibromyalgia, temporomandibular disorders, and chronic low back pain); 7) current psychiatric management; and 8) current gastrointestinal or renal illness.[@b18-jpr-9-493] Overall procedure ----------------- After signing the informed consent, study participants completed a standard intake information form. Patients underwent baseline assessments, which included psychological questionnaires, and self-reported pain intensity and disability 24--48 hours before shoulder surgery. Patients underwent an arthroscopic procedure affecting the glenohumeral joint and/or distal clavicle. Specific operative procedures included acromioplasty, subacromial bursectomy, labral repair, rotator cuff repair, and others (which included, but was not limited to, capsulorrhaphy, coracoacromial ligament release, stabilization, and Bankart). In addition, the size of the rotator cuff tear was recorded from the operative report, ranging from "no tear" to "massive tear". Four different surgeons operated on all the patients in this cohort. After 3 months and 6 months from the surgical procedure, patients were reassessed on all measures. Measures -------- ### Demographic and historical information Study participants completed a standard intake information form. Demographic data collected at initial evaluation included sex, age, employment status, litigation status, marital status, educational level, and health history. Historical data included the type of onset of symptoms, the length of time of the symptoms, the number of previous episodes of musculoskeletal pain, and previous PT treatment for their shoulder. ### Pre-op PT and post-op PT measurements Study participants were asked if they had had previous PT treatments (under the supervision of a licensed physical therapist) for their shoulder condition in the last 6 months. Participants who attended presurgical treatment were classified as "having pre-op PT treatment", and those who did not attend were classified as "not having pre-op PT treatment". Since the main goal of this study was to pragmatically determine whether having or not having pre-op PT intervention would influence postsurgical outcome, the frequency, intensity, and type of the pre-op PT were not tracked. At 3 months and 6 months, patients were asked for the total number of sessions (and weeks) of post-op PT treatment that they had after shoulder surgery. ### Shoulder pain intensity Shoulder pain intensity was assessed with the Brief Pain Inventory (BPI),[@b19-jpr-9-493] which includes a numerical rating scale for pain intensity. Patients rated their pain intensities over the following three conditions: the present pain intensity, the worst pain intensity over the past 24 hours, and the least pain intensity over the past 24 hours. These three ratings were summed and divided by three for use in data analyses.[@b20-jpr-9-493] Studies have shown that this aggregate measure has sufficient psychometric strengths.[@b20-jpr-9-493],[@b21-jpr-9-493] ### Shoulder disability Disability was assessed with the Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire.[@b22-jpr-9-493] The DASH questionnaire includes 30 items to measure the extent to which patients' pain or limited activity affects their ability to perform certain functions, to sleep, and to carry on routine daily activities and social activities. The DASH questionnaire has been validated for the assessment of shoulder disorders.[@b23-jpr-9-493]--[@b25-jpr-9-493] ### Psychological factors A previous study from our group has shown that pain catastrophizing and depressive symptoms were relevant psychological factors in predicting the postoperative clinical pain intensity in a population with similar characteristics.[@b18-jpr-9-493] Therefore, for our current study, we selected the same psychological factors to explore if pre-op PT has an influence on psychological factors. #### Depressive symptoms Self-reports of depressive symptoms were measured using the patient health questionnaire (PHQ-9).[@b26-jpr-9-493] The PHQ-9 is a 9-item self-report questionnaire designed to evaluate the presence of depressive symptoms during the prior 2 weeks. As a severity measure, scores can range from 0 (absence of depressive symptoms) to 27 (severe depressive symptoms). Each of the nine items, asking for each of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) diagnostic criteria, can be scored from 0 (not at all) to 3 (nearly every day). As a diagnostic measure, major depression is diagnosed if five or more of the nine depressive symptom criteria have been present at least "more than half the days" (a score of 2) in the past 2 weeks, and one of the symptoms is depressed mood or anhedonia. A previous study supports the PHQ-9's validity, feasibility, and capacity to detect changes in depressive symptoms over time.[@b27-jpr-9-493] #### Pain catastrophizing Pain catastrophizing was measured by the pain catastrophizing scale (PCS).[@b28-jpr-9-493] The PCS has 13 descriptions of pain experience assessing catastrophic cognitions, for example, "I feel I can't go on", and "There's nothing I can do to reduce the intensity of the pain". Patients were asked to indicate whether they agreed with these statements by using a five-point rating scale (0, "not at all" to 4, "all the time") to rate the frequency of these cognitions. A PCS sum score was calculated for all items (range, 0--52), with a high score indicating a high level of pain catastrophizing. Data analysis ------------- Data analysis was conducted with SPSS, Version 18.0. Significance levels were set a priori at P\<0.05 for all comparisons. Descriptive statistics (mean and standard deviation) were calculated for all variables for baseline, 3 months, and 6 months between groups (patients who had pre-op PT treatment and those who did not). The proportion of patients and the sex distribution between each group were determined. The distributions of variables were tested for normality by visual examination and with Kolmogorov--Smirnov test before conducting analyses. Analysis of variance (ANOVA) models and the Mann--Whitney U test were performed to identify differences across all measures for patients who had pre-op PT treatment and those who did not. Multivariate ANOVA models were performed to examine outcome differences at 3 months and 6 months (pain intensity and disability) and the length of post-op PT (number of PT sessions after shoulder surgery) on patient characteristics observed to be significantly different between groups. Results ======= This study included 124 consecutive patients having arthroscopic surgery with a completed 6-month follow-up. Descriptive statistics for the demographic and psychological characteristics, clinical pain, and medical history between patients who had pre-op PT treatment and those who did not is summarized in [Table 1](#t1-jpr-9-493){ref-type="table"}. All continuously dependent variables were found to approximate a normal distribution by visual examination and were deemed appropriate for our planned analyses and ANOVAs. Differences between preoperative groups --------------------------------------- No significant baseline differences (P\>0.05) in age, pain intensity, disability, and psychological factors between patients having and not having pre-op PT treatment were observed ([Table 1](#t1-jpr-9-493){ref-type="table"}). However, sex significantly differed (P=0.01) between patients having and not having pre-op PT treatment. Proportionally, more men were in the not having pre-op PT treatment group (59%), and more women were in the having pre-op PT treatment group (65%). Size of rotator cuff tear (P=0.56) had no significant difference between patients having and not having pre-op PT treatment. Differences in surgical type were further explored between patients having and not having pre-op PT treatment. Significantly, more subacromial bursectomies were performed (37.7%) in patients having pre-op PT treatment compared to patients not having pre-op PT treatment (20.6%) ([Table 1](#t1-jpr-9-493){ref-type="table"}). Influence of pre-op PT on outcomes ---------------------------------- There was no significant difference in the length of post-op PT treatment at 3 months (F\[1,96\]=0.37; P=0.54) and at 6 months (F\[1,81\]=0.26; P=0.61) between patients having and not having pre-op PT treatment. Repeated measures of ANOVA examined changes in clinical pain intensity and disability between patients having and not having pre-op PT treatment. Results showed no time × group interaction for clinical pain intensity (F\[2,180\]=0.33; P=0.72); as expected, there was a significant main effect of clinical pain over time (F\[2,180\]=64.41; P\<0.001), where pain significantly decreased from baseline (mean =3.33; SD =2.33) to 3 months (mean =1.52; SD =1.61) and from 3 months to 6 months (mean =1.17; SD =1.45) ([Figure 1](#f1-jpr-9-493){ref-type="fig"}). In addition, no time × group interaction for disability was found (F\[2,192\] =0.69; P=0.49); as expected, there was a significant main effect of disability over time (F\[2, 192\]=73.16; P\<0.001), where disability significantly decreased from baseline (mean =34.18; SD =17.08) to 3 months (mean =24.67; SD =14.51) and from 3 months to 6 months (mean =13.75; SD =10.39) ([Figure 2](#f2-jpr-9-493){ref-type="fig"}). Exploratory analysis -------------------- Since significant differences at baseline were found for the distribution of sex and subacromial bursectomy between patients having and not having pre-op PT treatment, an exploratory analysis of these results was performed. Differences in demographic, clinical pain, medical history, and psychological characteristics between sex and subacromial bursectomy are summarized in [Table 2](#t2-jpr-9-493){ref-type="table"}. Significant differences were found in baseline clinical pain intensity (BPI) (t\[118\]=2.57; P=0.01) and baseline disability (DASH) (t\[122\]=4.05; P\<0.01) between males and females, where females had higher mean in both clinical outcome measures (BPI mean =4.09; DASH mean =42.97) compared to males (BPI mean =2.97; DASH mean =30.05) ([Table 2](#t2-jpr-9-493){ref-type="table"}). Patients who had bursectomy were significantly older (t\[122)=--3.37; P\<0.01) and predominantly females (41.8% for females vs 22% for males). Repeated measures of ANOVA were used to assess whether pre-op PT treatment differences in sex or subacromial bursectomy affected postsurgical outcomes. No significant interaction was found between pre-op PT treatment, sex, and pain intensity (baseline, 3 months, and 6 months) (F\[2,176\]=0.06; P=0.93) or between pre-op PT treatment, sex, and disability (F\[2,188\]=1.38; P=0.26), suggesting that the potential effect of pre-op PT treatment on the length of post-op PT and clinical outcomes did not differ between males and females. The same trend of results was found between pre-op PT treatment, bursectomy, and pain intensity or disability, suggesting that differences in surgical intervention do not influence 3-month or 6-month postsurgical outcomes. Discussion ========== The main goal of pre-op PT treatment is to reduce postoperative complications and improve postoperative outcomes.[@b8-jpr-9-493] This study identified differences in demographic, clinical, and psychological characteristics between patients having and not having pre-op PT treatment and determined that pre-op PT treatment did not influence postoperative outcomes. The only pre-op PT treatment differences observed in this cohort were that males had less participation in pre-op PT treatment than females, and significantly more subacromial bursectomies were performed in patients having pre-op PT treatment. The results of this study also indicated that pre-op PT treatment did not influence the length of post-op PT treatment or postoperative pain and disability outcomes. Our study is a new addition to the small body of literature investigating this topic in elective arthroscopic shoulder surgery and extends the work from different surgical cohorts.[@b29-jpr-9-493]--[@b31-jpr-9-493] In addition, the larger sample size and prospective design are other strengths of this study. The results from several studies have showed the positive effects of pre-op PT treatment on postsurgical outcomes in patients undergoing cardiac surgery and in patients after lung volume reduction surgery.[@b32-jpr-9-493],[@b33-jpr-9-493] However, the results from studies looking at the effect of pre-op PT on postsurgical outcomes after musculoskeletal or joint replacement surgery have been contradictory.[@b8-jpr-9-493] For example, Beaupre et al[@b34-jpr-9-493] showed no differences in knee measurements (range of motion \[ROM\] and strength), pain, function, or quality of life between patients having a preoperative exercise/education program and patients having a total knee arthroplasty. Rooks et al[@b35-jpr-9-493] evaluated the effect of a short preoperative exercise intervention on the functional status, pain, and muscle strength of patients before and after total joint arthroplasty. They found that hip and knee arthroplasty patients responded differently in the preoperative and immediate postoperative periods. D'Lima et al[@b30-jpr-9-493] compared the effects of pre-op PT of general cardiovascular conditioning exercises with the routine procedure of no pre-op PT in patients undergoing primary total knee replacement. All three groups showed significant improvement postoperatively; however, neither type of preoperative exercise added to the degree of improvement after surgery. In contrast, Crowe and Henderson[@b36-jpr-9-493] showed the effectiveness in reducing the length of stay after an individually tailored rehabilitation program for patients undergoing hip or knee arthroplasty. Our results add to this literature by suggesting that in a cohort of patients undergoing shoulder arthroscopy, there appears to be no benefit of pre-op PT treatment on postsurgical pain and disability outcomes. Overall, this finding is in-line with previous studies,[@b29-jpr-9-493]--[@b31-jpr-9-493] indicating that a beneficial effect of pre-op PT treatment is more likely to occur after major surgeries.[@b14-jpr-9-493] One potential explanation for the differences in the benefits of pre-op PT is that the recovery period after invasive and major surgeries involves bed rest and sometimes long periods of physical inactivity, which induces deconditioning and postoperative complications. Therefore, it is reasonable to assume that increasing a patient's physical activity and physiological parameters prior to a surgical intervention will increase the patient's functional capacity with an increase in quality of life. Previous studies have shown that poor fitness before the surgery results in a significantly longer stay in hospital after the surgery[@b37-jpr-9-493],[@b38-jpr-9-493] and greater surgical complications.[@b8-jpr-9-493],[@b37-jpr-9-493] However, the "standard of care" pre-op PT intervention for shoulder conditions would not target the increment in physiological parameters or fitness improvement; therefore, the chance of overall fitness improvement is low. Even though an ambulatory surgery such as a shoulder arthroscopy may not involve bed rest or long periods of inactivity, there is often a period of immobility considered as a "relative rest" that could eliminate any potential benefits from pre-op PT. Looking at sex differences, Charousset et al[@b39-jpr-9-493] described that female sex was negatively associated with the improvement of clinical results. Other studies[@b40-jpr-9-493],[@b41-jpr-9-493] have reported that sex is not associated with postoperative outcomes. Our results are consistent with earlier findings, because even though males had less participation in pre-op PT treatment than females, baseline sex differences did not influence the length of post-op PT treatment, pain intensity, and disability outcomes. Psychological factors are believed to play a role in the patient's response to treatment and final outcome.[@b42-jpr-9-493]--[@b44-jpr-9-493] However, there is little evidence in the literature assessing the potential influence of psychological characteristics on seeking PT care. Our data show that depression and pain catastrophizing did not differ at baseline between patients having or not having pre-op PT treatment. This is an interesting finding because we would expect that patients with greater psychological distress would be more likely to utilize pre-op PT; however, this was not the case for this cohort. Therefore, this is preliminary evidence that pre-op PT is not associated with common measures of psychological distress that have been associated with an increased health care utilization for musculoskeletal pain. A previous study[@b45-jpr-9-493] has shown that after a conservative treatment for subacromial impingement syndrome has failed, there is a greater rate of successful outcome with a specific exercise protocol (strengthening eccentric exercises for the rotator cuff and concentric/eccentric exercises for the scapula stabilizers in combination with manual mobilization) compared to a control exercise group. In addition, a significantly lower proportion of patients in the specific exercise group subsequently chose to undergo surgery. Lee et al[@b5-jpr-9-493] show that pain, range of motion, muscle strength, and function significantly improved after arthroscopic rotator cuff repair, regardless of early postoperative rehabilitation protocols. The goal of this study was to evaluate the broad influence of having pre-op PT on postoperative outcomes and not to test the effectiveness of any specific exercise approaches used during PT. These results still advance the field by showing that pain intensity and disability significantly potentially decrease after shoulder arthroscopy, regardless of having or not having preoperative rehabilitation. Future research using more highly controlled designs is needed, however, to determine if specific exercise approaches have measurable impact on postoperative outcomes. There are some limitations to this study that should be recognized. First, the operational definition of PT treatment was pragmatically determined, so it does not include treatment dosage or type, so we cannot account for the intensity, frequency, or nature of the PT delivered in this study. This is a relevant issue because the nature of the preoperative intervention may be an important consideration, so that future prospective studies can better measure the application of pre-op PT. Furthermore and consistent with the pragmatic nature of the analysis, there was no control over how the pre-op PT was selected or administered, and there could be other relevant factors in the patient decision making (eg, protocol selection, access to care, socioeconomic status, dosage, and content of treatment) that were not included in our analysis. Second, by recruiting patients who are having surgery, we may be missing patients who already succeeded with conservative treatment approaches; therefore, our sample may be less likely to have had a positive response to PT treatment in general. A more comprehensive future study including a wider range of patients to determine whether pre-op PT is preventative of future surgery is needed, as this study was not designed to answer that particular question. Despite these limitations, this study represents a new contribution to the literature by investigating the influence of pre-op PT treatment on 3-month and 6-month postsurgical outcomes. In summary, having pre-op PT was not associated with the length of post-op PT treatment or postsurgical pain and disability. Conclusion ========== This prospective cohort study provides no evidence of benefit for pre-op PT on post-op PT treatment or postsurgical outcomes. Receiving pre-op PT treatment did not influence the length of post-op PT treatment or pain and disability outcomes at 3 months and 6 months. Females or patients receiving certain surgical procedures are more likely to undergo pre-op PT treatment. However, these differences did not influence postsurgical outcomes in this cohort. The authors thank Warren Greenfield III for his assistance with clinical participant screening and recruitment. Doctor Deenesh T Sahajpal provided patients from his clinic. The authors also wish to thank Lindsay Kindler for assistance with testing. This study was supported by Grant \#AR055899 from NIAMS/NIH. Disclosure The authors report no conflicts of interest in this work. ![Clinical pain over time between patients having pre-op PT treatment and patients not having pre-op PT treatment.\ Abbreviations: pre-op PT, preoperative physical therapy; NRS, numerical rating scale.](jpr-9-493Fig1){#f1-jpr-9-493} ![Disability over time between patients having pre-op PT treatment and patients not having pre-op PT treatment.\ Abbreviations: pre-op PT, preoperative physical therapy; DASH, Disabilities of the Arm.](jpr-9-493Fig2){#f2-jpr-9-493} ###### Demographic and psychological characteristics and summary of medical history between patients having and not having pre-op PT treatment Sample characteristics Having pre-op PT (n=61), mean (SD) Not having pre-op PT (n=63), mean (SD) P-value ------------------------------------------- ------------------------------------ ---------------------------------------- ----------- Age (years) 44.79 (16.28) 44.32 (19.11) 0.88 Sex Female 28 (45.9%) 15 (23.8%) 0.01 Male 33 (54.1%) 48 (76.2%) Dominant side Right 54 (88.5%) 55 (87.3%) 0.84 Left 7 (11.5%) 8 (12.7%) Ethnicity Hispanic or Latino 5 (8.2%) 5 (7.9%) 0.98 Non-Hispanic or Latino 54 (88.5%) 56 (88.9%) Unknown or not reported 2 (3.3%) 2 (3.2%) Race Asian 0 (0%) 0 (0%) 0.78 Native Hawaiian or others 0 (0%) 1 (1.6%) Black or African 4 (6.6%) 3 (4.8%) American White 52 (85.2%) 55 (87.3%) More than one race 3 (4.9%) 3 (4.8%) Unknown or not reported 2 (3.3%) 1 (1.6%) Surgery side Right 35 (57.4%) 32 (50.8%) 0.46 Left 26 (42.6%) 31 (49.2%) Pain duration (weeks) 68.75 (90.81) 68.14 (103.35) 0.42 BPI baseline 3.47 (2.43) 3.24 (2.23) 0.59 BPI 3 months 1.58 (1.71) 1.45 (1.36) 0.67 BPI 6 months 1.24 (1.31) 1.24 (1.47) 0.99 DASH baseline 35.58 (17.15) 33.51 (18.75) 0.52 DASH 3 months 26.48 (14.34) 22.52 (14.39) 0.15 DASH 6 months 16.04 (11.95) 12.94 (10.76) 0.17 PHQ-9 baseline 4.37 (4.19) 3.34 (4.12) 0.18 PHQ-9 3 months 3.67 (3.89) 2.68 (3.66) 0.19 PHQ-9 6 months 3.21 (4.06) 2.58 (3.07) 0.36 PCS baseline 11.23 (8.57) 10.22 (8.41) 0.51 PCS 3 months 7.53 (7.89) 7.75 (8.28) 0.89 PCS 6 months 6.47 (6.83) 6.82 (7.19) 0.80 Size of rotator cuff tear No tear 32 (52.5%) 30 (47.6%) 0.56 Partial tear \<50% 10 (16.4%) 11 (17.5%) Partial tear \>50% 4 (6.6%) 2 (3.2%) Small 5 (8.3%) 3 (4.8%) Medium 2 (3.3%) 7 (11.1%) Large 2 (3.3%) 4 (6.3%) Massive 6 (9.8%) 5 (7.9%) Unknown 0 1 (1.6%) Acromioplasty 47 (37.9%) 24 (39.3%) 23 (36.5%) 0.75 Subacromial bursectomy 36 (29.0%) 23 (37.7%) 13 (20.6%) 0.03 Labral repair 49 (39.5%) 24 (39.3%) 25 (39.7%) 0.97 Other operative procedures 86 (69.4%) 41 (67.2%) 45 (71.4%) 0.61 Post-op PT sessions at 3 months 18.82 (9.82) 20.92 (22.04) 0.54 Post-op PT sessions at 6 months 7.67 (9.85) 6.56 (9.84) 0.61 Weeks in post-op PT treatment at 3 months 11.18 (2.59) 9.19 (4.26) \<0.01 Weeks in post-op PT treatment at 6 months 4.64 (6.18) 4.77 (6.39) 0.93 Abbreviations: BPI, brief pain inventory; DASH, Disabilities of the Arm, Shoulder, and Hand; PCS, pain catastrophizing scale; PHQ-9, patient health questionnaire; post-op PT, postoperative physical therapy; pre-op PT, preoperative physical therapy; SD, standard deviation. ###### Exploratory analyses -- differences in demographic and psychological characteristics between sex and subacromial bursectomy Sample characteristics Females (n=61), mean (SD) Males (n=63), mean (SD) P-value Bursectomy (n=36), mean (SD) Not having bursectomy (n=88), mean (SD) P-value --------------------------------- --------------------------- ------------------------- ----------- ------------------------------ ----------------------------------------- ----------- Age (years) 48.11 (15.44) 41.71 (18.82) 0.04 52.61 (13.58) 41.25 (18.19) 0.001 Sex Female -- -- -- 18 (50.0%) 25 (28.4%) 0.02 Male 18 (50.0%) 63 (71.6%) BPI baseline 4.14 (2.30) 2.89 (2.23) 0.004 3.67 (2.27) 3.22 (2.35) 0.34 BPI 3 months 1.54 (1.41) 1.51 (1.58) 0.93 1.56 (1.38) 1.50 (1.60) 0.84 BPI 6 months 1.41 (1.66) 1.09 (1.33) 0.33 1.13 (1.39) 1.29 (1.51) 0.62 DASH baseline 42.97 (15.95) 30.32 (17.42) \<0.01 34.91 (16.55) 34.37 (18.56) 0.88 DASH 3 months 28.35 (14.03) 22.07 (14.14) 0.03 23.97 (14.62) 24.64 (14.46) 0.83 DASH 6 months 17.05 (14.26) 12.40 (9.45) 0.08 12.95 (9.57) 15.21 (12.14) 0.38 PHQ-9 baseline 4.16 (4.68) 3.83 (4.65) 0.71 3.86 (4.66) 3.83 (3.98) 0.97 PHQ-9 3 months 2.47 (2.96) 3.46 (4.06) 0.20 3.27 (4.07) 3.12 (3.71) 0.85 PHQ-9 6 months 2.86 (4.31) 3.04 (3.65) 0.82 3.33 (4.27) 2.70 (3.32) 0.43 PCS baseline 10.91 (8.91) 10.72 (8.29) 0.38 10.27 (7.64) 10.90 (8.82) 0.71 PCS 3 months 6.72 (7.85) 8.16 (8.22) 0.61 7.83 (8.58) 7.55 (7.89) 0.87 PCS 6 months 6.23 (7.44) 6.98 (6.78) 0.77 5.79 (6.54) 7.00 (7.16) 0.43 Post-op PT sessions at 3 months 22.25 (15.31) 19.31 (17.05) 0.43 19.92 (16.94) 19.83 (17.06) 0.98 Abbreviations: BPI, brief pain inventory; DASH, Disabilities of the Arm, Shoulder, and Hand; PCS, pain catastrophizing scale; PHQ-9, patient health questionnaire; post-op PT, postoperative physical therapy; SD, standard 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Chemical context {#sec1} ================== Non-covalent interactions concern a broad range of attractive effects with an equally varied energy contribution to bonding. An interesting group of interactions is one formed by the stabilizing weak polar contacts such C---H⋯ X (X = O, F, Cl, Br, I), C---H⋯π hydrogen bonds and offset π--π interactions. These interactions are involved in biological, materials, supramolecular chemistry and crystal engineering (Desiraju, 1989[@bb5]; Desiraju & Steiner, 1999[@bb6]; Lehn, 1995[@bb14]; Steed & Atwood, 2000[@bb22]). Naphthalimide is a highly fluorescent moiety that has been used as a construction block in the design of receptors and sensors that recognize charged species and other guests (Landey-Álvarez et al., 2016[@bb13]). Aromatic imides show a highly efficient photo-induced electron transfer (PET) process that can be used as a signaling method in the building of sensors or on--off molecular switches. In this sense, some researchers have proposed one approximation that involves the use of two different fluorescent units linked via a suitable spacer group characterized by PET or singlet--singlet energy transfer mechanisms (SSET) called dyads: such units are naphthalimide and dansyl amide. In a former study, these moieties were linked by methylene units as a bridging group and only the photon-induced fluorescence switching was studied (Abad et al., 2005[@bb1]). Later, interactions with different metallic ions were investigated (Shankar & Ramaiah, 2011[@bb18]). Actually, we have studied by single-crystal X-ray diffraction the molecular structure of a naphthalimide-dansyl amide dyad and its interaction in solution with anions and aromatic molecules (Claudio-Catalán et al., 2016[@bb3]). The ability of the dyad to function as a receptor of electron-rich guests and such aromatic compounds and anions are being studied by UV--Vis, fluorescence and NMR experiments. We have found that the dyad could interact with the guests tested through the aryl C---H⋯anion and aryl C---H⋯π interactions. In our ongoing research on naphthalimides as anion receptors, we report herein the synthesis and crystal structure of the title compound, a 1,2-dichlorobenzene solvate, C~56~H~50~N~6~O~8~S~2~·3C~6~H~4~Cl~2~, (I)[](#scheme1){ref-type="chem"}, which has been shown to be inert to the presence of anions or neutral molecules in solution probably due to high stability acquired by the overlap of the aromatic rings. Structural commentary {#sec2} ======================= The asymmetric unit of the title compound (I)[](#scheme1){ref-type="chem"} contains two half-molecules of the parent molecule (A and B), both having crystallographic inversion symmetry \[(i) −x, −y + 2, −z + 1 for (A) and (ii) −x, −y + 2, −z + 2 for (B)\], and three 2,3-dichlorobenzene molecules of solvation (Fig. 1[▸](#fig1){ref-type="fig"}). The N,N-naphthalenediimide \[N2/C13--C19 (A); N5/C41--C47 (B)\] and aromatic dansyl groups \[C1--C10 (A) and C29--C38 (B)\] are almost planar with r.m.s. deviations of 0.0055, 0.0183, 0.0664 and 0.0698 Å, respectively. The two molecules are conformationally similar with dihedral angles between the central naphthalenediimide ring and the peripheral naphthalene and benzyl rings, respectively, of 2.43 (7), 81.87 (7)° (A) and 3.95 (7), 84.88 (7)° (B). The conformations of A and B are stabilized by the presence of intramolecular aromatic ring-stacking with distances of 3.5795 (8) and 3.5640 (8) Å for Cg1..Cg2 and Cg3⋯ Cg4, respectively \[Cg1 and Cg3 are the centroids of naphathaleneimides C13--C17/N2 (A) and C41--C45/N5 (B) and Cg2 and Cg4 are the centroids of phenyl rings C1--C5/C10 (A) and C29--C33/C38 (B)\] (Fig. 2[▸](#fig2){ref-type="fig"}). Supramolecular features {#sec3} ========================== In the crystal, four C---H⋯O hydrogen bonds link the molecules into infinite supramolecular chains extending along the c axis (Fig. 3[▸](#fig3){ref-type="fig"}, Table 1[▸](#table1){ref-type="table"}). The chains are interconnected through C---H⋯π and offset π--π interactions, generating channels which are filled by solvent molecules (Fig. 4[▸](#fig4){ref-type="fig"}). The C---H⋯π interactions are between the benzyl groups with distances C48⋯Cg5′ = 3.6180 (17) and C20⋯Cg6′ = 3.6054 (17) Å (Cg5′ and Cg6′ are the centroids of the phenyl rings C21--C26 and C49--C54, respectively) (Fig. 5[▸](#fig5){ref-type="fig"}). The weak offset π--π interaction is between adjacent phenyl rings with Cg6⋯Cg6′(−x, −y + 1, −z + 1) = 4.0277 (10) Å (Cg6 is the centroid of the C49--C54 phenyl ring). In addition, the dansyl groups show C---H⋯π interactions, with distances C27⋯Cg7′ = 3.585 (2) and C55⋯Cg8′ = 3.632 (2) Å (Fig. 6, Table 1[▸](#table1){ref-type="table"}) where Cg7′ and Cg8′ are the centroids of naphthyl ring systems C1--C10 and C29--C38, respectively. In the channel, the N⋯N distance is 12.5 Å. The solvent molecules are interconnected by C71---H71⋯Cg(C63--C68)\] and C68---H68⋯Cl1 interactions and are also linked to the channel by C72---H72⋯O6 and C60---H60⋯O2 interactions (Table 1[▸](#table1){ref-type="table"}). In the crystal, there are also short Cl4⋯O4(−x, 1 − y, 1 − z) interactions \[3.0923 (12) Å\] and 22.6 Å^3^ solvent-accessible voids. Database survey {#sec4} ================= A search of the Cambridge Structural Database (Version 5.37; Groom et al., 2016[@bb9]) revealed the existence of 324 deposited naphthalenediimide structures. Amongst those, 94 structures are metal complexes. Supramolecular constructs based on naphthalenediimide moieties with potential applications have been reported; for example PUNPAR (Wu et al., 2015[@bb23]) and NUXJEX (Liu et al., 2014[@bb15]) exhibit the formation of supramolecular nanotubes through cooperative \[C---H⋯O=C\] interactions. In the same way, pseudorotaxanes BALVIU and GUNPEL (Colquhoun, et al., 2010[@bb4]) and catenanes IVUNUI (Fallon, et al., 2004[@bb8]), SUJZIG (Hamilton et al., 1998[@bb10]) and WATYAR (Hansen et al., 2000[@bb11]) have been prepared. Naphthalenediimides have also been used in molecular recognition \[HIRLAX (Schneebeli et al., 2013[@bb17]), MUVJUJ (Shimizu, 2010[@bb21]), PUBPAE (Koshkakaryan et al., 2009[@bb12]) and RULWUS (Ono et al., 2015[@bb16])\]. Synthesis and crystallization {#sec5} =============================== The title compound (I)[](#scheme1){ref-type="chem"} was prepared from 2,7-bis(2-benzylaminoethyl)naphthalenediimide (II), which was synthesized as follows. To a stirred solution of 1,4,5,8-naphthalenetetracarboxylic dianhydride (0.5 g, 1.86 mmol) in toluene (25 mL) was added N-benzylethylendiamine (0.56 mL, 0.56 g, 3.73 mmol) followed by the addition of triethylamine (0.52 mL, 0.377 g, 3.73 mmol). The reaction mixture was heated to reflux with azeotropic removal of water using a Dean--Stark trap, for 24 h. The solution was cooled and the solvent was removed under reduced pressure. The resultant oil was purified by column chromatography on silica gel (CH~2~Cl~2~--MeOH 95:05). Compound (II) was obtained as a yellow solid (0.777 g, 78%). M.p. 482--484 K. IR (neat): 3314, 2817, 1700, 1655, 1579, 1454 cm^−1^. RMN ^1^H (400 MHz, CDCl~3~) δ: 1.59 (s, 2H, NH), 3.03 (t, J = 6.4 Hz, 4H, CH~2~NH), 3.84 (s, 4H, CH~2~Ph), 4.38 (t, J = 6.4 Hz, 4H, NCH~2~), 7.16--7.30 (m, 10H, H~aromatic~), 8.74 (s, 4H, H~aromatic~). RMN ^13^C (100 MHz, CDCl~3~) δ: 40.6 (2C, NCH~2~), 47.0 (2C, CH~2~NH), 53.7 (2C, CH~2~Ph), 126.8 (4C), 126.9 (2C), 127.1 (2C), 128.3 (4C), 128.5 (4C), 131.2 (4C), 140.4 (2C), 163.2 (C=O). MS (FAB^+^): m/z (%) 533 (37) \[M\]; HRMS (FAB^+^): calculated for C~32~H~29~O~4~N~4~ \[M\], m/z 533.2189; found for \[M\], m/z 533.2142. Synthesis of N,N′-bis\[2-((benzyl){\[5-(dimethylamino)naphthalen-1-yl\]sulfonyl}amino)ethyl\]naphthalene-1,8:4,5-tetracarboximide 1,2-dichlorobenzene trisolvate (I)[](#scheme1){ref-type="chem"}. A mixture of 2,7-bis(2-benzylaminoethyl)naphthalenediimide (II) (0.5 g, 0.937 mmol), dansyl chloride (0.505 g, l.874 mmol) and K~2~CO~3~ (0.259 g, 1.874 mmol) in chloroform/water (4:1) (10 mL) was stirred at room temperature for 20 h. The organic layer was extracted with dichloromethane (2 x 20 mL), dried over anhydrous Na~2~SO~4~, filtered and concentrated under reduced pressure. Further purification was performed by flash chromatography on silica gel (CH~2~Cl~2~--MeOH 95:5). After treatment with diethyl ether, the unsolvated title compound was obtained as a yellow solid (0.936 g, 100%). Crystallization from a chloroform:1,2-dichlorobenzene mixture afforded suitable crystals of the solvated compound (I)[](#scheme1){ref-type="chem"} for the X-ray crystallographic analysis. M.p. 516--517 K. IR (neat): cm^−1^. RMN ^1^H (400 MHz, CDCl~3~) δ: 2.59 \[s, 12H, (CH~3~)~2~N\], 3.69 (t, J = 5.4 Hz, 4H, CH~2~NSO~2~), 4.28 (t, J = 5.4 Hz, 4H, CH~2~NCO), 4.85 (s, 4H, CH~2~Ph), 6.73 (d, J = 7.2 Hz, 2H, H~aromatic~), 7.18 (dd, J = 8.4, 7.2 Hz, 2H, H~aromatic~), 7.22--7.29 (m, 12H, H~aromatic~), 7.89 (d, J = 8.4 Hz, 2H, H~aromatic~), 7.97 (t, J = 8.8 Hz, 2H, H~aromatic~), 8.08 (dd, J = 7.2, 1.2 Hz, 2H, H~aromatic~), 8.42 (s, 4H, H~aromatic~). RMN ^13^C (100 MHz, CDCl~3~) δ: 37.5 (2C, CH~2~NCO), 43.1 (2C, CH~2~NSO~2~), 45.3 (4C, (CH~3~)~2~N), 49.9 (2C, CH~2~Ph), 114.4 (2C), 119.1 (2C), 123.0 (2C), 125.9 (4C),126.3 (2C), 128.1 (2C), 128.3 (2C), 128.9 (4C), 129.1 (4C), 129.2 (2C), 129.7 (2C), 130.1 (2C), 130.5 (2C), 130.6 (4C), 134.9 (2C), 135.6 (2C), 151.3 (2C), 162.9 (C=O). MS (FAB^+^): m/z (%) 999 (31) \[M + H\]^+^; HRMS (FAB^+^): calculated for C~21~H~19~O~2~N~2~ \[M + H\]^+^, m/z 999.3210; found for \[M + H\]^+^, m/z 999.3365. UV/Vis three bands CH~3~Cl: λ nm (∊, M^−1^ cm^−1^): 350 (24698), 362 (25362), 383 (29604). Refinement {#sec6} ============ Crystal data, data collection and structure refinement details are summarized in Table 2.[▸](#table2){ref-type="table"} Aromatic, methylene and methyl H atoms were positioned geometrically and were constrained using the riding-model approximation \[C---H = 0.95--0.98 Å with U ~iso~(H) = 1.5U ~eq~(C-methyl) and 1.5U ~eq~(C) for other H atoms\]. Supplementary Material ====================== Crystal structure: contains datablock(s) I. DOI: [10.1107/S2056989016015188/zs2369sup1.cif](http://dx.doi.org/10.1107/S2056989016015188/zs2369sup1.cif) Structure factors: contains datablock(s) I. DOI: [10.1107/S2056989016015188/zs2369Isup2.hkl](http://dx.doi.org/10.1107/S2056989016015188/zs2369Isup2.hkl) CCDC reference: [1506790](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1506790) Additional supporting information: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?zs2369&file=zs2369sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?zs2369sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?zs2369&checkcif=yes) This work was supported by CONACyT, project CB2011/168952. The authors thank the LANEM Laboratory for the use of spectrometric instruments. Crystal data {#tablewrapcrystaldatalong} ============ --------------------------------------- ---------------------------------------- C~56~H~50~N~6~O~8~S~2~·3C~6~H~4~Cl~2~ F(000) = 1492 M~r~ = 1440.11 D~x~ = 1.431 Mg m^−3^ Triclinic, P1 Melting point = 516--517 K a = 12.17737 (14) Å Cu Kα radiation, λ = 1.54184 Å b = 17.2876 (2) Å Cell parameters from 29431 reflections c = 17.8916 (2) Å θ = 2.9--72.6° α = 110.9544 (12)° µ = 3.44 mm^−1^ β = 96.2760 (11)° T = 100 K γ = 103.5159 (10)° Prism, orange V = 3341.91 (8) Å^3^ 0.20 × 0.10 × 0.05 mm Z = 2 --------------------------------------- ---------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== ------------------------------------------------------------------ --------------------------------------- Agilent SuperNova, Dual, Cu at zero, EosS2 diffractometer 13086 independent reflections Radiation source: sealed X-ray tube, SuperNova (Cu) X-ray Source 11914 reflections with I \> 2σ(I) Mirror monochromator R~int~ = 0.026 Detector resolution: 8.0769 pixels mm^-1^ θ~max~ = 73.0°, θ~min~ = 2.7° ω scans h = −15→15 Absorption correction: multi-scan (CrysAlis PRO; Agilent, 2014) k = −21→21 T~min~ = 0.874, T~max~ = 1.000 l = −21→22 54279 measured reflections ------------------------------------------------------------------ --------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ------------------------------------- ----------------------------------------------------------------------------------------------- Refinement on F^2^ Primary atom site location: structure-invariant direct methods Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites R\[F^2^ \> 2σ(F^2^)\] = 0.034 H-atom parameters constrained wR(F^2^) = 0.097 w = 1/\[σ^2^(F~o~^2^) + (0.056P)^2^ + 1.406P\] where P = (F~o~^2^ + 2F~c~^2^)/3 S = 1.03 (Δ/σ)~max~ = 0.001 13086 reflections Δρ~max~ = 0.74 e Å^−3^ 869 parameters Δρ~min~ = −0.45 e Å^−3^ 0 restraints ------------------------------------- ----------------------------------------------------------------------------------------------- Special details {#specialdetails} =============== -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. 1. Fixed Uiso At 1.2 times of: All C(H) groups, All C(H,H) groups At 1.5 times of: All C(H,H,H) groups 2.a Secondary CH2 refined with riding coordinates: C12(H12A,H12B), C11(H11A,H11B), C20(H20A,H20B), C39(H39A,H39B), C48(H48A, H48B), C40(H40A,H40B) 2.b Aromatic/amide H refined with riding coordinates: C2(H2), C18(H18), C4(H4), C23(H23), C19(H19), C3(H3), C22(H22), C9(H9), C25(H25), C8(H8), C73(H73), C7(H7), C74(H74), C24(H24), C26(H26), C71(H71), C72(H72), C47(H47), C32(H32), C30(H30), C62(H62), C46(H46), C51(H51), C36(H36), C50(H50), C37(H37), C31(H31), C61(H61), C54(H54), C35(H35), C60(H60), C53(H53), C52(H52), C59(H59), C65(H65), C67(H67), C68(H68), C66(H66) 2.c Idealised Me refined as rotating group: C28(H28A,H28B,H28C), C27(H27A,H27B,H27C), C56(H56A,H56B,H56C), C55(H55A,H55B, H55C) -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords} ================================================================================================== ------ --------------- -------------- -------------- -------------------- -- x y z U~iso~\/U~eq~ S1 0.19073 (3) 0.75685 (2) 0.24683 (2) 0.01493 (8) Cl6 0.67288 (4) 0.57176 (3) 0.54497 (3) 0.03455 (10) Cl5 0.69686 (4) 0.73661 (3) 0.50338 (3) 0.04428 (13) O3 −0.08861 (9) 0.89558 (7) 0.24342 (6) 0.0210 (2) O1 0.19908 (9) 0.77111 (7) 0.17289 (6) 0.0212 (2) O4 −0.08934 (9) 0.72885 (6) 0.39083 (6) 0.0194 (2) O2 0.26566 (9) 0.71343 (7) 0.27162 (6) 0.0197 (2) N2 −0.08971 (10) 0.81240 (7) 0.31685 (7) 0.0149 (2) N1 0.05830 (10) 0.70359 (7) 0.24087 (7) 0.0152 (2) C13 −0.07211 (12) 0.89229 (9) 0.31046 (9) 0.0157 (3) C1 0.21767 (11) 0.86101 (9) 0.32637 (9) 0.0148 (3) C2 0.22425 (12) 0.92925 (9) 0.30290 (9) 0.0185 (3) H2 0.2112 0.9190 0.2464 0.022\ N3 0.35385 (11) 1.06595 (8) 0.61154 (8) 0.0203 (3) C15 −0.01802 (11) 0.96231 (9) 0.46334 (8) 0.0133 (3) C18 −0.01783 (12) 0.87352 (9) 0.54171 (9) 0.0170 (3) H18 −0.0290 0.8181 0.5437 0.020\* C4 0.27689 (12) 1.02957 (9) 0.44374 (9) 0.0181 (3) H4 0.2967 1.0873 0.4834 0.022\* C16 −0.03523 (11) 0.88034 (9) 0.46729 (9) 0.0139 (3) C23 −0.14700 (13) 0.41817 (10) 0.06594 (10) 0.0226 (3) H23 −0.2258 0.3848 0.0445 0.027\* C17 −0.07277 (11) 0.80072 (9) 0.39066 (9) 0.0148 (3) C19 0.01643 (12) 0.94848 (9) 0.61479 (9) 0.0177 (3) H19 0.0270 0.9435 0.6661 0.021\* C12 −0.13094 (12) 0.73496 (9) 0.23968 (9) 0.0169 (3) H12A −0.1931 0.7419 0.2043 0.020\* H12B −0.1642 0.6838 0.2517 0.020\* C5 0.27561 (11) 0.96116 (9) 0.47012 (9) 0.0148 (3) C10 0.23834 (11) 0.87377 (9) 0.41091 (9) 0.0140 (3) C21 −0.00149 (12) 0.54144 (9) 0.17070 (9) 0.0157 (3) C14 −0.03475 (12) 0.97082 (9) 0.38777 (9) 0.0150 (3) C3 0.25032 (13) 1.01428 (9) 0.36250 (10) 0.0202 (3) H3 0.2495 1.0608 0.3463 0.024\* C6 0.30788 (11) 0.97820 (9) 0.55544 (9) 0.0166 (3) C22 −0.11631 (13) 0.49109 (9) 0.13944 (9) 0.0186 (3) H22 −0.1741 0.5065 0.1683 0.022\* C9 0.22245 (12) 0.80530 (9) 0.43852 (9) 0.0168 (3) H9 0.1960 0.7468 0.4000 0.020\* C11 −0.03476 (12) 0.71921 (9) 0.19387 (8) 0.0155 (3) H11A −0.0676 0.6685 0.1412 0.019\* H11B −0.0020 0.7702 0.1815 0.019\* C25 0.05176 (14) 0.44374 (10) 0.05536 (10) 0.0241 (3) H25 0.1097 0.4273 0.0272 0.029\* C8 0.24521 (13) 0.82367 (10) 0.52047 (9) 0.0202 (3) H8 0.2306 0.7774 0.5384 0.024\* C73 0.43748 (14) 0.73761 (12) 0.62140 (11) 0.0321 (4) H73 0.3867 0.7718 0.6371 0.039\* C7 0.28998 (13) 0.90977 (10) 0.57924 (9) 0.0201 (3) H7 0.3079 0.9205 0.6357 0.024\* C74 0.51894 (16) 0.75901 (11) 0.57842 (11) 0.0300 (4) H74 0.5243 0.8078 0.5647 0.036\* C69 0.59299 (14) 0.70853 (10) 0.55541 (10) 0.0243 (3) C20 0.03168 (12) 0.62007 (9) 0.25087 (9) 0.0163 (3) H20A −0.0326 0.6166 0.2802 0.020\* H20B 0.1002 0.6189 0.2853 0.020\* C70 0.58373 (13) 0.63686 (10) 0.57470 (10) 0.0226 (3) C24 −0.06324 (14) 0.39416 (10) 0.02411 (10) 0.0234 (3) H24 −0.0842 0.3441 −0.0256 0.028\* C28 0.35179 (15) 1.08222 (11) 0.69698 (10) 0.0276 (3) H28A 0.2750 1.0524 0.7009 0.041\* H28B 0.3692 1.1447 0.7286 0.041\* H28C 0.4098 1.0606 0.7189 0.041\* C26 0.08184 (13) 0.51720 (9) 0.12751 (9) 0.0199 (3) H26 0.1603 0.5515 0.1478 0.024\* C71 0.50229 (16) 0.61617 (12) 0.61816 (11) 0.0308 (4) H71 0.4965 0.5673 0.6318 0.037\* C27 0.47087 (14) 1.10772 (12) 0.60510 (11) 0.0308 (4) H27A 0.5270 1.0855 0.6283 0.046\* H27B 0.4904 1.1706 0.6354 0.046\* H27C 0.4729 1.0948 0.5474 0.046\* C72 0.42945 (15) 0.66686 (13) 0.64163 (11) 0.0346 (4) H72 0.3738 0.6530 0.6717 0.042\* S2 0.17486 (3) 0.76401 (2) 0.74465 (2) 0.01531 (8) Cl1 0.74045 (3) 0.73768 (3) 0.03177 (3) 0.03251 (10) Cl2 0.67800 (3) 0.56374 (2) 0.06086 (3) 0.03107 (10) O7 −0.11632 (9) 0.89353 (7) 0.74506 (6) 0.0215 (2) O5 0.17643 (9) 0.78095 (7) 0.67160 (6) 0.0225 (2) O8 −0.10021 (9) 0.72829 (6) 0.89241 (6) 0.0199 (2) O6 0.25383 (9) 0.72127 (7) 0.76537 (7) 0.0212 (2) N5 −0.10660 (10) 0.81110 (8) 0.81829 (7) 0.0157 (2) N4 0.04457 (10) 0.70822 (7) 0.74075 (7) 0.0154 (2) C47 0.03877 (12) 0.94984 (9) 1.11372 (9) 0.0171 (3) H47 0.0585 0.9454 1.1646 0.020\* C41 −0.09349 (12) 0.89094 (9) 0.81190 (9) 0.0163 (3) C38 0.23320 (11) 0.87835 (9) 0.91056 (9) 0.0141 (3) C44 −0.03418 (11) 0.88033 (9) 0.96747 (9) 0.0144 (3) C49 −0.00293 (12) 0.54617 (9) 0.67090 (9) 0.0161 (3) C32 0.26568 (12) 1.03448 (9) 0.94680 (9) 0.0175 (3) H32 0.2866 1.0918 0.9874 0.021\* C29 0.20294 (11) 0.86685 (9) 0.82676 (9) 0.0149 (3) N6 0.36162 (11) 1.06867 (8) 1.11139 (8) 0.0201 (3) C30 0.20231 (12) 0.93567 (9) 0.80535 (9) 0.0184 (3) H30 0.1833 0.9263 0.7493 0.022\* C43 −0.02205 (11) 0.96206 (9) 0.96352 (8) 0.0136 (3) C57 0.61803 (13) 0.70800 (10) 0.06827 (9) 0.0209 (3) C62 0.54740 (14) 0.76180 (10) 0.08531 (10) 0.0250 (3) H62 0.5645 0.8131 0.0750 0.030\* C46 −0.00313 (12) 0.87448 (9) 1.04134 (9) 0.0161 (3) H46 −0.0101 0.8194 1.0432 0.019\* C45 −0.08202 (11) 0.80018 (9) 0.89182 (9) 0.0151 (3) C42 −0.05138 (12) 0.96965 (9) 0.88864 (9) 0.0153 (3) C39 −0.05221 (12) 0.72071 (9) 0.69431 (9) 0.0161 (3) H39A −0.0843 0.6695 0.6419 0.019\* H39B −0.0233 0.7719 0.6814 0.019\* C51 −0.14298 (14) 0.42729 (10) 0.55784 (10) 0.0250 (3) H51 −0.2212 0.3968 0.5306 0.030\* C48 0.02496 (12) 0.62465 (9) 0.75062 (9) 0.0169 (3) H48A −0.0395 0.6180 0.7793 0.020\* H48B 0.0951 0.6266 0.7858 0.020\* C36 0.25694 (13) 0.82636 (9) 1.01796 (9) 0.0196 (3) H36 0.2471 0.7796 1.0351 0.024\* C40 −0.14825 (12) 0.73367 (9) 0.74141 (9) 0.0178 (3) H40A −0.2126 0.7394 0.7069 0.021\* H40B −0.1783 0.6821 0.7536 0.021\* C50 −0.11737 (13) 0.50007 (9) 0.63098 (10) 0.0202 (3) H50 −0.1784 0.5184 0.6538 0.024\* C34 0.31473 (11) 0.98141 (9) 1.05462 (9) 0.0163 (3) C37 0.22504 (12) 0.80914 (9) 0.93661 (9) 0.0168 (3) H37 0.1974 0.7509 0.8976 0.020\* C58 0.59165 (13) 0.63204 (10) 0.08181 (9) 0.0203 (3) C31 0.22989 (12) 1.02016 (9) 0.86658 (9) 0.0191 (3) H31 0.2236 1.0670 0.8521 0.023\* C61 0.45182 (14) 0.74021 (11) 0.11743 (10) 0.0273 (3) H61 0.4038 0.7772 0.1298 0.033\* C54 0.08498 (13) 0.51758 (9) 0.63686 (10) 0.0209 (3) H54 0.1633 0.5485 0.6633 0.025\* C33 0.27227 (11) 0.96537 (9) 0.97068 (9) 0.0148 (3) C56 0.37005 (14) 1.08286 (11) 1.19736 (10) 0.0267 (3) H56A 0.2963 1.0521 1.2048 0.040\* H56B 0.3882 1.1450 1.2306 0.040\* H56C 0.4314 1.0610 1.2146 0.040\* C35 0.30409 (13) 0.91200 (10) 1.07700 (9) 0.0194 (3) H35 0.3288 0.9219 1.1328 0.023\* C60 0.42596 (14) 0.66501 (11) 0.13160 (11) 0.0275 (3) H60 0.3605 0.6507 0.1539 0.033\* C53 0.05946 (15) 0.44438 (10) 0.56466 (10) 0.0251 (3) H53 0.1202 0.4250 0.5425 0.030\* C52 −0.05465 (15) 0.39939 (10) 0.52484 (10) 0.0257 (3) H52 −0.0721 0.3496 0.4751 0.031\* C59 0.49533 (14) 0.61065 (10) 0.11340 (10) 0.0243 (3) H59 0.4770 0.5587 0.1225 0.029\* C55 0.47316 (14) 1.11330 (12) 1.09971 (11) 0.0323 (4) H55A 0.5340 1.0921 1.1187 0.048\* H55B 0.4914 1.1758 1.1312 0.048\* H55C 0.4682 1.1017 1.0415 0.048\* Cl4 0.30187 (3) 0.43343 (3) 0.69251 (3) 0.03212 (10) Cl3 0.40852 (4) 0.62180 (3) 0.83265 (3) 0.03876 (11) C63 0.49571 (14) 0.55818 (10) 0.79349 (10) 0.0242 (3) C65 0.52000 (14) 0.42501 (11) 0.70186 (11) 0.0269 (3) H65 0.4878 0.3685 0.6599 0.032\* C64 0.44887 (13) 0.47549 (10) 0.73244 (10) 0.0226 (3) C67 0.68559 (14) 0.54154 (12) 0.79409 (12) 0.0313 (4) H67 0.7667 0.5641 0.8151 0.038\* C68 0.61463 (15) 0.59036 (11) 0.82303 (11) 0.0302 (4) H68 0.6469 0.6473 0.8641 0.036\* C66 0.63803 (15) 0.45730 (12) 0.73272 (12) 0.0322 (4) H66 0.6870 0.4227 0.7126 0.039\* ------ --------------- -------------- -------------- -------------------- -- Atomic displacement parameters (Å^2^) {#tablewrapadps} ===================================== ----- -------------- -------------- -------------- -------------- -------------- -------------- U^11^ U^22^ U^33^ U^12^ U^13^ U^23^ S1 0.01687 (16) 0.01485 (15) 0.01071 (16) 0.00375 (12) 0.00225 (12) 0.00316 (13) Cl6 0.0310 (2) 0.0289 (2) 0.0372 (2) 0.01418 (16) 0.00131 (17) 0.00403 (18) Cl5 0.0503 (3) 0.0319 (2) 0.0516 (3) 0.00476 (19) 0.0321 (2) 0.0162 (2) O3 0.0307 (6) 0.0202 (5) 0.0120 (5) 0.0091 (4) 0.0009 (4) 0.0061 (4) O1 0.0241 (5) 0.0239 (5) 0.0123 (5) 0.0032 (4) 0.0045 (4) 0.0057 (4) O4 0.0258 (5) 0.0134 (5) 0.0168 (5) 0.0037 (4) 0.0036 (4) 0.0050 (4) O2 0.0198 (5) 0.0201 (5) 0.0182 (5) 0.0089 (4) 0.0034 (4) 0.0046 (4) N2 0.0169 (5) 0.0141 (5) 0.0111 (6) 0.0045 (4) 0.0008 (4) 0.0026 (5) N1 0.0160 (5) 0.0129 (5) 0.0151 (6) 0.0031 (4) 0.0000 (4) 0.0053 (5) C13 0.0171 (6) 0.0164 (7) 0.0126 (7) 0.0062 (5) 0.0016 (5) 0.0043 (6) C1 0.0128 (6) 0.0142 (6) 0.0134 (7) 0.0019 (5) 0.0005 (5) 0.0030 (5) C2 0.0194 (7) 0.0205 (7) 0.0145 (7) 0.0039 (5) 0.0002 (5) 0.0079 (6) N3 0.0178 (6) 0.0194 (6) 0.0144 (6) 0.0007 (5) 0.0001 (5) 0.0001 (5) C15 0.0117 (6) 0.0154 (6) 0.0132 (7) 0.0053 (5) 0.0029 (5) 0.0053 (6) C18 0.0202 (7) 0.0145 (6) 0.0178 (7) 0.0059 (5) 0.0042 (5) 0.0077 (6) C4 0.0170 (6) 0.0138 (6) 0.0199 (7) 0.0023 (5) 0.0014 (5) 0.0048 (6) C16 0.0127 (6) 0.0150 (6) 0.0136 (7) 0.0050 (5) 0.0027 (5) 0.0047 (5) C23 0.0232 (7) 0.0163 (7) 0.0226 (8) 0.0006 (6) −0.0023 (6) 0.0066 (6) C17 0.0139 (6) 0.0166 (7) 0.0136 (7) 0.0049 (5) 0.0025 (5) 0.0055 (6) C19 0.0229 (7) 0.0189 (7) 0.0125 (7) 0.0070 (6) 0.0029 (5) 0.0073 (6) C12 0.0188 (7) 0.0148 (6) 0.0123 (7) 0.0036 (5) −0.0007 (5) 0.0016 (5) C5 0.0113 (6) 0.0165 (6) 0.0147 (7) 0.0033 (5) 0.0022 (5) 0.0046 (6) C10 0.0118 (6) 0.0156 (6) 0.0138 (7) 0.0044 (5) 0.0020 (5) 0.0050 (5) C21 0.0211 (7) 0.0130 (6) 0.0136 (7) 0.0058 (5) 0.0022 (5) 0.0060 (5) C14 0.0151 (6) 0.0161 (6) 0.0139 (7) 0.0063 (5) 0.0022 (5) 0.0051 (6) C3 0.0224 (7) 0.0156 (7) 0.0225 (8) 0.0038 (5) 0.0009 (6) 0.0096 (6) C6 0.0122 (6) 0.0192 (7) 0.0139 (7) 0.0031 (5) 0.0013 (5) 0.0028 (6) C22 0.0201 (7) 0.0163 (6) 0.0195 (7) 0.0057 (5) 0.0034 (6) 0.0070 (6) C9 0.0191 (7) 0.0150 (6) 0.0139 (7) 0.0036 (5) 0.0018 (5) 0.0044 (6) C11 0.0192 (6) 0.0144 (6) 0.0111 (6) 0.0046 (5) −0.0002 (5) 0.0041 (5) C25 0.0292 (8) 0.0214 (7) 0.0215 (8) 0.0097 (6) 0.0094 (6) 0.0058 (6) C8 0.0245 (7) 0.0202 (7) 0.0179 (7) 0.0057 (6) 0.0041 (6) 0.0104 (6) C73 0.0235 (8) 0.0393 (10) 0.0249 (9) 0.0132 (7) 0.0023 (7) 0.0013 (8) C7 0.0220 (7) 0.0249 (7) 0.0119 (7) 0.0066 (6) 0.0020 (5) 0.0064 (6) C74 0.0367 (9) 0.0268 (8) 0.0255 (9) 0.0121 (7) 0.0035 (7) 0.0084 (7) C69 0.0245 (7) 0.0234 (7) 0.0206 (8) 0.0029 (6) 0.0062 (6) 0.0058 (6) C20 0.0217 (7) 0.0132 (6) 0.0133 (7) 0.0049 (5) 0.0026 (5) 0.0049 (6) C70 0.0207 (7) 0.0224 (7) 0.0187 (8) 0.0052 (6) −0.0005 (6) 0.0033 (6) C24 0.0339 (8) 0.0147 (7) 0.0160 (7) 0.0048 (6) 0.0017 (6) 0.0021 (6) C28 0.0292 (8) 0.0279 (8) 0.0150 (8) 0.0050 (7) 0.0011 (6) −0.0006 (6) C26 0.0205 (7) 0.0174 (7) 0.0197 (7) 0.0048 (6) 0.0033 (6) 0.0056 (6) C71 0.0371 (9) 0.0308 (9) 0.0239 (9) 0.0053 (7) 0.0069 (7) 0.0131 (7) C27 0.0218 (8) 0.0295 (8) 0.0254 (9) −0.0051 (6) 0.0004 (7) 0.0023 (7) C72 0.0275 (8) 0.0441 (10) 0.0253 (9) 0.0039 (8) 0.0111 (7) 0.0085 (8) S2 0.01726 (16) 0.01544 (16) 0.01142 (16) 0.00420 (12) 0.00325 (12) 0.00358 (13) Cl1 0.02762 (19) 0.0374 (2) 0.0291 (2) 0.00037 (16) 0.01108 (16) 0.01363 (18) Cl2 0.02618 (19) 0.02378 (19) 0.0357 (2) 0.01054 (15) 0.00128 (16) 0.00270 (17) O7 0.0302 (6) 0.0203 (5) 0.0139 (5) 0.0094 (4) 0.0009 (4) 0.0064 (4) O5 0.0268 (5) 0.0241 (5) 0.0130 (5) 0.0027 (4) 0.0052 (4) 0.0060 (4) O8 0.0265 (5) 0.0138 (5) 0.0184 (5) 0.0043 (4) 0.0055 (4) 0.0062 (4) O6 0.0199 (5) 0.0214 (5) 0.0211 (5) 0.0095 (4) 0.0042 (4) 0.0050 (4) N5 0.0172 (5) 0.0146 (5) 0.0140 (6) 0.0051 (4) 0.0020 (4) 0.0043 (5) N4 0.0172 (6) 0.0133 (5) 0.0147 (6) 0.0039 (4) 0.0009 (5) 0.0055 (5) C47 0.0200 (7) 0.0192 (7) 0.0141 (7) 0.0073 (5) 0.0032 (5) 0.0081 (6) C41 0.0170 (6) 0.0169 (7) 0.0154 (7) 0.0066 (5) 0.0027 (5) 0.0060 (6) C38 0.0119 (6) 0.0157 (6) 0.0138 (7) 0.0037 (5) 0.0022 (5) 0.0052 (5) C44 0.0132 (6) 0.0153 (6) 0.0155 (7) 0.0055 (5) 0.0039 (5) 0.0060 (6) C49 0.0216 (7) 0.0132 (6) 0.0141 (7) 0.0049 (5) 0.0024 (5) 0.0065 (6) C32 0.0168 (6) 0.0136 (6) 0.0197 (7) 0.0026 (5) 0.0031 (5) 0.0053 (6) C29 0.0140 (6) 0.0141 (6) 0.0144 (7) 0.0030 (5) 0.0024 (5) 0.0040 (5) N6 0.0185 (6) 0.0192 (6) 0.0152 (6) 0.0015 (5) 0.0011 (5) 0.0017 (5) C30 0.0191 (7) 0.0205 (7) 0.0159 (7) 0.0045 (5) 0.0016 (5) 0.0090 (6) C43 0.0123 (6) 0.0153 (6) 0.0145 (7) 0.0056 (5) 0.0040 (5) 0.0060 (6) C57 0.0193 (7) 0.0250 (7) 0.0135 (7) 0.0009 (6) 0.0015 (6) 0.0060 (6) C62 0.0282 (8) 0.0222 (7) 0.0227 (8) 0.0051 (6) −0.0008 (6) 0.0099 (6) C46 0.0187 (6) 0.0145 (6) 0.0175 (7) 0.0061 (5) 0.0050 (5) 0.0080 (6) C45 0.0144 (6) 0.0167 (7) 0.0159 (7) 0.0059 (5) 0.0054 (5) 0.0070 (6) C42 0.0153 (6) 0.0165 (7) 0.0151 (7) 0.0057 (5) 0.0039 (5) 0.0065 (6) C39 0.0190 (7) 0.0148 (6) 0.0120 (7) 0.0043 (5) 0.0002 (5) 0.0039 (5) C51 0.0270 (8) 0.0178 (7) 0.0230 (8) 0.0012 (6) −0.0033 (6) 0.0059 (6) C48 0.0220 (7) 0.0143 (6) 0.0145 (7) 0.0053 (5) 0.0030 (5) 0.0061 (6) C36 0.0235 (7) 0.0191 (7) 0.0177 (7) 0.0066 (6) 0.0027 (6) 0.0093 (6) C40 0.0188 (7) 0.0158 (6) 0.0138 (7) 0.0038 (5) −0.0008 (5) 0.0022 (6) C50 0.0215 (7) 0.0173 (7) 0.0216 (8) 0.0059 (6) 0.0031 (6) 0.0079 (6) C34 0.0125 (6) 0.0182 (7) 0.0143 (7) 0.0034 (5) 0.0020 (5) 0.0031 (6) C37 0.0192 (7) 0.0146 (6) 0.0150 (7) 0.0043 (5) 0.0022 (5) 0.0050 (6) C58 0.0199 (7) 0.0205 (7) 0.0154 (7) 0.0053 (6) −0.0010 (6) 0.0031 (6) C31 0.0217 (7) 0.0153 (6) 0.0218 (8) 0.0048 (5) 0.0025 (6) 0.0100 (6) C61 0.0243 (8) 0.0297 (8) 0.0248 (9) 0.0115 (7) 0.0003 (6) 0.0065 (7) C54 0.0221 (7) 0.0170 (7) 0.0217 (8) 0.0050 (6) 0.0036 (6) 0.0062 (6) C33 0.0113 (6) 0.0167 (6) 0.0153 (7) 0.0033 (5) 0.0030 (5) 0.0056 (6) C56 0.0289 (8) 0.0264 (8) 0.0150 (8) 0.0059 (6) −0.0005 (6) −0.0002 (6) C35 0.0210 (7) 0.0241 (7) 0.0125 (7) 0.0068 (6) 0.0009 (5) 0.0072 (6) C60 0.0198 (7) 0.0350 (9) 0.0254 (8) 0.0055 (6) 0.0071 (6) 0.0104 (7) C53 0.0325 (8) 0.0192 (7) 0.0247 (8) 0.0101 (6) 0.0118 (7) 0.0067 (6) C52 0.0399 (9) 0.0148 (7) 0.0169 (8) 0.0050 (6) 0.0035 (7) 0.0026 (6) C59 0.0245 (7) 0.0243 (8) 0.0231 (8) 0.0028 (6) 0.0023 (6) 0.0119 (7) C55 0.0239 (8) 0.0291 (8) 0.0276 (9) −0.0054 (7) 0.0026 (7) 0.0017 (7) Cl4 0.01841 (17) 0.0384 (2) 0.0331 (2) 0.00178 (15) 0.00127 (15) 0.01215 (18) Cl3 0.0389 (2) 0.0291 (2) 0.0496 (3) 0.01544 (17) 0.0146 (2) 0.0123 (2) C63 0.0263 (8) 0.0249 (8) 0.0262 (8) 0.0083 (6) 0.0062 (6) 0.0149 (7) C65 0.0296 (8) 0.0228 (8) 0.0301 (9) 0.0058 (6) 0.0090 (7) 0.0130 (7) C64 0.0190 (7) 0.0243 (7) 0.0264 (8) 0.0035 (6) 0.0025 (6) 0.0146 (7) C67 0.0168 (7) 0.0407 (10) 0.0444 (11) 0.0047 (7) 0.0024 (7) 0.0296 (9) C68 0.0305 (8) 0.0249 (8) 0.0304 (9) −0.0012 (7) −0.0021 (7) 0.0138 (7) C66 0.0270 (8) 0.0356 (9) 0.0481 (11) 0.0154 (7) 0.0151 (8) 0.0267 (9) ----- -------------- -------------- -------------- -------------- -------------- -------------- Geometric parameters (Å, º) {#tablewrapgeomlong} =========================== ----------------------------- -------------- ------------------------------- -------------- S1---O1 1.4397 (11) O7---C41 1.2168 (18) S1---O2 1.4361 (10) O8---C45 1.2146 (17) S1---N1 1.6303 (12) N5---C41 1.3991 (18) S1---C1 1.7789 (14) N5---C45 1.4049 (18) Cl6---C70 1.7313 (16) N5---C40 1.4677 (18) Cl5---C69 1.7308 (16) N4---C39 1.4694 (17) O3---C13 1.2181 (18) N4---C48 1.4853 (17) O4---C17 1.2124 (17) C47---H47 0.9500 N2---C13 1.3952 (18) C47---C46 1.404 (2) N2---C17 1.4065 (18) C47---C42^ii^ 1.3808 (19) N2---C12 1.4706 (17) C41---C42 1.478 (2) N1---C11 1.4701 (17) C38---C29 1.433 (2) N1---C20 1.4834 (17) C38---C37 1.4186 (19) C13---C14 1.4812 (19) C38---C33 1.4292 (19) C1---C2 1.376 (2) C44---C43 1.4139 (19) C1---C10 1.432 (2) C44---C46 1.380 (2) C2---H2 0.9500 C44---C45 1.483 (2) C2---C3 1.409 (2) C49---C48 1.5117 (19) N3---C6 1.4189 (19) C49---C50 1.394 (2) N3---C28 1.456 (2) C49---C54 1.391 (2) N3---C27 1.476 (2) C32---H32 0.9500 C15---C15^i^ 1.413 (3) C32---C31 1.367 (2) C15---C16 1.4118 (19) C32---C33 1.4208 (19) C15---C14 1.410 (2) C29---C30 1.376 (2) C18---H18 0.9500 N6---C34 1.4169 (19) C18---C16 1.376 (2) N6---C56 1.456 (2) C18---C19 1.407 (2) N6---C55 1.474 (2) C4---H4 0.9500 C30---H30 0.9500 C4---C5 1.4190 (19) C30---C31 1.411 (2) C4---C3 1.367 (2) C43---C43^ii^ 1.416 (3) C16---C17 1.4841 (19) C43---C42 1.408 (2) C23---H23 0.9500 C57---C62 1.388 (2) C23---C22 1.395 (2) C57---C58 1.391 (2) C23---C24 1.384 (2) C62---H62 0.9500 C19---H19 0.9500 C62---C61 1.386 (2) C19---C14^i^ 1.3787 (19) C46---H46 0.9500 C12---H12A 0.9900 C42---C47^ii^ 1.3808 (19) C12---H12B 0.9900 C39---H39A 0.9900 C12---C11 1.522 (2) C39---H39B 0.9900 C5---C10 1.4285 (19) C39---C40 1.527 (2) C5---C6 1.437 (2) C51---H51 0.9500 C10---C9 1.4194 (19) C51---C50 1.394 (2) C21---C22 1.392 (2) C51---C52 1.384 (2) C21---C20 1.5134 (19) C48---H48A 0.9900 C21---C26 1.393 (2) C48---H48B 0.9900 C14---C19^i^ 1.3787 (19) C36---H36 0.9500 C3---H3 0.9500 C36---C37 1.368 (2) C6---C7 1.374 (2) C36---C35 1.410 (2) C22---H22 0.9500 C40---H40A 0.9900 C9---H9 0.9500 C40---H40B 0.9900 C9---C8 1.365 (2) C50---H50 0.9500 C11---H11A 0.9900 C34---C33 1.437 (2) C11---H11B 0.9900 C34---C35 1.378 (2) C25---H25 0.9500 C37---H37 0.9500 C25---C24 1.390 (2) C58---C59 1.387 (2) C25---C26 1.386 (2) C31---H31 0.9500 C8---H8 0.9500 C61---H61 0.9500 C8---C7 1.412 (2) C61---C60 1.384 (2) C73---H73 0.9500 C54---H54 0.9500 C73---C74 1.382 (3) C54---C53 1.388 (2) C73---C72 1.380 (3) C56---H56A 0.9800 C7---H7 0.9500 C56---H56B 0.9800 C74---H74 0.9500 C56---H56C 0.9800 C74---C69 1.391 (2) C35---H35 0.9500 C69---C70 1.384 (2) C60---H60 0.9500 C20---H20A 0.9900 C60---C59 1.384 (2) C20---H20B 0.9900 C53---H53 0.9500 C70---C71 1.385 (2) C53---C52 1.388 (2) C24---H24 0.9500 C52---H52 0.9500 C28---H28A 0.9800 C59---H59 0.9500 C28---H28B 0.9800 C55---H55A 0.9800 C28---H28C 0.9800 C55---H55B 0.9800 C26---H26 0.9500 C55---H55C 0.9800 C71---H71 0.9500 Cl4---C64 1.7274 (15) C71---C72 1.383 (3) Cl3---C63 1.7278 (17) C27---H27A 0.9800 C63---C64 1.388 (2) C27---H27B 0.9800 C63---C68 1.390 (2) C27---H27C 0.9800 C65---H65 0.9500 C72---H72 0.9500 C65---C64 1.388 (2) S2---O5 1.4391 (11) C65---C66 1.383 (2) S2---O6 1.4347 (11) C67---H67 0.9500 S2---N4 1.6323 (12) C67---C68 1.361 (3) S2---C29 1.7815 (14) C67---C66 1.409 (3) Cl1---C57 1.7341 (15) C68---H68 0.9500 Cl2---C58 1.7293 (15) C66---H66 0.9500 O1---S1---N1 109.76 (6) C39---N4---S2 117.29 (9) O1---S1---C1 106.07 (7) C39---N4---C48 117.42 (11) O2---S1---O1 118.32 (7) C48---N4---S2 120.32 (9) O2---S1---N1 107.61 (6) C46---C47---H47 119.8 O2---S1---C1 108.46 (6) C42^ii^---C47---H47 119.8 N1---S1---C1 105.96 (6) C42^ii^---C47---C46 120.35 (13) C13---N2---C17 125.20 (12) O7---C41---N5 120.01 (13) C13---N2---C12 116.53 (11) O7---C41---C42 122.72 (13) C17---N2---C12 118.27 (11) N5---C41---C42 117.27 (12) C11---N1---S1 117.28 (9) C37---C38---C29 124.03 (13) C11---N1---C20 117.11 (11) C37---C38---C33 118.94 (13) C20---N1---S1 121.14 (9) C33---C38---C29 117.03 (12) O3---C13---N2 120.21 (13) C43---C44---C45 119.78 (12) O3---C13---C14 122.57 (13) C46---C44---C43 120.51 (13) N2---C13---C14 117.22 (12) C46---C44---C45 119.70 (12) C2---C1---S1 116.75 (11) C50---C49---C48 120.56 (13) C2---C1---C10 121.69 (13) C54---C49---C48 120.66 (13) C10---C1---S1 121.45 (10) C54---C49---C50 118.77 (14) C1---C2---H2 119.9 C31---C32---H32 119.3 C1---C2---C3 120.12 (13) C31---C32---C33 121.38 (13) C3---C2---H2 119.9 C33---C32---H32 119.3 C6---N3---C28 115.76 (13) C38---C29---S2 121.66 (10) C6---N3---C27 113.39 (12) C30---C29---S2 116.46 (11) C28---N3---C27 109.56 (13) C30---C29---C38 121.72 (13) C16---C15---C15^i^ 119.37 (16) C34---N6---C56 115.73 (13) C14---C15---C15^i^ 119.12 (15) C34---N6---C55 113.73 (12) C14---C15---C16 121.51 (13) C56---N6---C55 109.64 (13) C16---C18---H18 120.0 C29---C30---H30 120.0 C16---C18---C19 120.10 (13) C29---C30---C31 120.10 (13) C19---C18---H18 120.0 C31---C30---H30 120.0 C5---C4---H4 119.2 C44---C43---C43^ii^ 119.12 (16) C3---C4---H4 119.2 C42---C43---C44 121.49 (13) C3---C4---C5 121.53 (13) C42---C43---C43^ii^ 119.39 (16) C15---C16---C17 119.85 (12) C62---C57---Cl1 119.23 (12) C18---C16---C15 120.56 (13) C62---C57---C58 120.14 (14) C18---C16---C17 119.59 (12) C58---C57---Cl1 120.62 (12) C22---C23---H23 119.8 C57---C62---H62 120.2 C24---C23---H23 119.8 C61---C62---C57 119.58 (15) C24---C23---C22 120.31 (14) C61---C62---H62 120.2 O4---C17---N2 121.06 (13) C47---C46---H46 119.9 O4---C17---C16 122.35 (13) C44---C46---C47 120.15 (13) N2---C17---C16 116.59 (12) C44---C46---H46 119.9 C18---C19---H19 119.9 O8---C45---N5 120.90 (13) C14^i^---C19---C18 120.23 (13) O8---C45---C44 122.48 (13) C14^i^---C19---H19 119.9 N5---C45---C44 116.62 (12) N2---C12---H12A 109.2 C47^ii^---C42---C41 119.85 (13) N2---C12---H12B 109.2 C47^ii^---C42---C43 120.46 (13) N2---C12---C11 112.25 (11) C43---C42---C41 119.68 (12) H12A---C12---H12B 107.9 N4---C39---H39A 109.1 C11---C12---H12A 109.2 N4---C39---H39B 109.1 C11---C12---H12B 109.2 N4---C39---C40 112.29 (11) C4---C5---C10 119.27 (13) H39A---C39---H39B 107.9 C4---C5---C6 121.13 (13) C40---C39---H39A 109.1 C10---C5---C6 119.57 (13) C40---C39---H39B 109.1 C5---C10---C1 117.12 (12) C50---C51---H51 120.0 C9---C10---C1 123.99 (13) C52---C51---H51 119.9 C9---C10---C5 118.88 (13) C52---C51---C50 120.10 (15) C22---C21---C20 120.26 (13) N4---C48---C49 114.11 (11) C22---C21---C26 118.75 (13) N4---C48---H48A 108.7 C26---C21---C20 120.98 (13) N4---C48---H48B 108.7 C15---C14---C13 119.63 (12) C49---C48---H48A 108.7 C19^i^---C14---C13 119.76 (13) C49---C48---H48B 108.7 C19^i^---C14---C15 120.61 (13) H48A---C48---H48B 107.6 C2---C3---H3 120.1 C37---C36---H36 119.3 C4---C3---C2 119.80 (13) C37---C36---C35 121.49 (13) C4---C3---H3 120.1 C35---C36---H36 119.3 N3---C6---C5 117.79 (13) N5---C40---C39 111.48 (11) C7---C6---N3 123.13 (13) N5---C40---H40A 109.3 C7---C6---C5 119.05 (13) N5---C40---H40B 109.3 C23---C22---H22 119.8 C39---C40---H40A 109.3 C21---C22---C23 120.39 (14) C39---C40---H40B 109.3 C21---C22---H22 119.8 H40A---C40---H40B 108.0 C10---C9---H9 120.0 C49---C50---H50 119.8 C8---C9---C10 119.91 (13) C51---C50---C49 120.50 (14) C8---C9---H9 120.0 C51---C50---H50 119.8 N1---C11---C12 112.44 (11) N6---C34---C33 118.05 (13) N1---C11---H11A 109.1 C35---C34---N6 122.96 (13) N1---C11---H11B 109.1 C35---C34---C33 118.94 (13) C12---C11---H11A 109.1 C38---C37---H37 120.0 C12---C11---H11B 109.1 C36---C37---C38 119.94 (13) H11A---C11---H11B 107.8 C36---C37---H37 120.0 C24---C25---H25 120.0 C57---C58---Cl2 121.08 (12) C26---C25---H25 120.0 C59---C58---Cl2 119.07 (12) C26---C25---C24 120.04 (15) C59---C58---C57 119.85 (14) C9---C8---H8 119.2 C32---C31---C30 119.85 (13) C9---C8---C7 121.58 (13) C32---C31---H31 120.1 C7---C8---H8 119.2 C30---C31---H31 120.1 C74---C73---H73 119.8 C62---C61---H61 119.8 C72---C73---H73 119.8 C60---C61---C62 120.36 (15) C72---C73---C74 120.47 (16) C60---C61---H61 119.8 C6---C7---C8 120.58 (14) C49---C54---H54 119.6 C6---C7---H7 119.7 C53---C54---C49 120.74 (14) C8---C7---H7 119.7 C53---C54---H54 119.6 C73---C74---H74 120.2 C38---C33---C34 119.53 (13) C73---C74---C69 119.51 (16) C32---C33---C38 119.41 (13) C69---C74---H74 120.2 C32---C33---C34 121.04 (13) C74---C69---Cl5 119.49 (13) N6---C56---H56A 109.5 C70---C69---Cl5 120.55 (13) N6---C56---H56B 109.5 C70---C69---C74 119.96 (15) N6---C56---H56C 109.5 N1---C20---C21 113.67 (11) H56A---C56---H56B 109.5 N1---C20---H20A 108.8 H56A---C56---H56C 109.5 N1---C20---H20B 108.8 H56B---C56---H56C 109.5 C21---C20---H20A 108.8 C36---C35---H35 119.7 C21---C20---H20B 108.8 C34---C35---C36 120.66 (13) H20A---C20---H20B 107.7 C34---C35---H35 119.7 C69---C70---Cl6 120.85 (13) C61---C60---H60 120.0 C69---C70---C71 120.14 (15) C59---C60---C61 120.07 (15) C71---C70---Cl6 119.01 (13) C59---C60---H60 120.0 C23---C24---C25 119.59 (14) C54---C53---H53 119.9 C23---C24---H24 120.2 C52---C53---C54 120.14 (15) C25---C24---H24 120.2 C52---C53---H53 119.9 N3---C28---H28A 109.5 C51---C52---C53 119.75 (15) N3---C28---H28B 109.5 C51---C52---H52 120.1 N3---C28---H28C 109.5 C53---C52---H52 120.1 H28A---C28---H28B 109.5 C58---C59---H59 120.0 H28A---C28---H28C 109.5 C60---C59---C58 119.98 (15) H28B---C28---H28C 109.5 C60---C59---H59 120.0 C21---C26---H26 119.6 N6---C55---H55A 109.5 C25---C26---C21 120.89 (14) N6---C55---H55B 109.5 C25---C26---H26 119.6 N6---C55---H55C 109.5 C70---C71---H71 120.1 H55A---C55---H55B 109.5 C72---C71---C70 119.83 (16) H55A---C55---H55C 109.5 C72---C71---H71 120.1 H55B---C55---H55C 109.5 N3---C27---H27A 109.5 C64---C63---Cl3 120.99 (12) N3---C27---H27B 109.5 C64---C63---C68 119.41 (15) N3---C27---H27C 109.5 C68---C63---Cl3 119.61 (13) H27A---C27---H27B 109.5 C64---C65---H65 120.2 H27A---C27---H27C 109.5 C66---C65---H65 120.2 H27B---C27---H27C 109.5 C66---C65---C64 119.70 (16) C73---C72---C71 120.08 (16) C63---C64---Cl4 121.09 (12) C73---C72---H72 120.0 C65---C64---Cl4 118.60 (13) C71---C72---H72 120.0 C65---C64---C63 120.31 (14) O5---S2---N4 109.36 (6) C68---C67---H67 120.2 O5---S2---C29 105.88 (7) C68---C67---C66 119.59 (15) O6---S2---O5 118.74 (7) C66---C67---H67 120.2 O6---S2---N4 107.79 (6) C63---C68---H68 119.5 O6---S2---C29 108.16 (6) C67---C68---C63 121.05 (16) N4---S2---C29 106.27 (6) C67---C68---H68 119.5 C41---N5---C45 125.05 (12) C65---C66---C67 119.94 (16) C41---N5---C40 116.49 (12) C65---C66---H66 120.0 C45---N5---C40 118.46 (11) C67---C66---H66 120.0 S1---N1---C11---C12 −132.54 (10) Cl2---C58---C59---C60 179.14 (13) S1---N1---C20---C21 −94.83 (13) O7---C41---C42---C47^ii^ 1.3 (2) S1---C1---C2---C3 −177.24 (11) O7---C41---C42---C43 −177.36 (13) S1---C1---C10---C5 171.10 (10) O5---S2---N4---C39 −29.11 (12) S1---C1---C10---C9 −9.81 (19) O5---S2---N4---C48 125.32 (11) Cl6---C70---C71---C72 −179.28 (14) O5---S2---C29---C38 −165.99 (11) Cl5---C69---C70---Cl6 −1.77 (19) O5---S2---C29---C30 9.43 (13) Cl5---C69---C70---C71 178.36 (13) O6---S2---N4---C39 −159.49 (10) O3---C13---C14---C15 179.55 (13) O6---S2---N4---C48 −5.06 (12) O3---C13---C14---C19^i^ −0.6 (2) O6---S2---C29---C38 −37.72 (13) O1---S1---N1---C11 −32.20 (12) O6---S2---C29---C30 137.70 (11) O1---S1---N1---C20 123.41 (11) N5---C41---C42---C47^ii^ −178.82 (13) O1---S1---C1---C2 8.55 (13) N5---C41---C42---C43 2.53 (19) O1---S1---C1---C10 −167.82 (11) N4---S2---C29---C38 77.78 (12) O2---S1---N1---C11 −162.21 (10) N4---S2---C29---C30 −106.80 (11) O2---S1---N1---C20 −6.60 (12) N4---C39---C40---N5 61.25 (15) O2---S1---C1---C2 136.63 (11) C41---N5---C45---O8 177.01 (13) O2---S1---C1---C10 −39.74 (13) C41---N5---C45---C44 −2.55 (19) N2---C13---C14---C15 0.36 (19) C41---N5---C40---C39 77.33 (15) N2---C13---C14---C19^i^ −179.77 (13) C38---C29---C30---C31 −1.2 (2) N2---C12---C11---N1 61.88 (15) C44---C43---C42---C47^ii^ −179.98 (13) N1---S1---C1---C2 −108.08 (11) C44---C43---C42---C41 −1.3 (2) N1---S1---C1---C10 75.55 (12) C49---C54---C53---C52 0.9 (2) C13---N2---C17---O4 −179.49 (13) C29---S2---N4---C39 84.76 (11) C13---N2---C17---C16 1.17 (19) C29---S2---N4---C48 −120.81 (11) C13---N2---C12---C11 77.16 (15) C29---C38---C37---C36 179.38 (13) C1---S1---N1---C11 81.92 (11) C29---C38---C33---C32 7.81 (19) C1---S1---N1---C20 −122.47 (11) C29---C38---C33---C34 −173.81 (12) C1---C2---C3---C4 4.5 (2) C29---C30---C31---C32 4.9 (2) C1---C10---C9---C8 179.50 (13) N6---C34---C33---C38 174.87 (12) C2---C1---C10---C5 −5.09 (19) N6---C34---C33---C32 −6.8 (2) C2---C1---C10---C9 173.99 (13) N6---C34---C35---C36 −179.89 (13) N3---C6---C7---C8 −179.25 (13) C43---C44---C46---C47 1.3 (2) C15^i^---C15---C16---C18 0.4 (2) C43---C44---C45---O8 −175.86 (13) C15^i^---C15---C16---C17 −179.83 (14) C43---C44---C45---N5 3.70 (18) C15^i^---C15---C14---C13 179.77 (15) C43^ii^---C43---C42---C47^ii^ −0.8 (2) C15^i^---C15---C14---C19^i^ −0.1 (2) C43^ii^---C43---C42---C41 177.83 (15) C15---C16---C17---O4 −179.57 (13) C57---C62---C61---C60 −0.8 (2) C15---C16---C17---N2 −0.24 (18) C57---C58---C59---C60 −0.2 (2) C18---C16---C17---O4 0.2 (2) C62---C57---C58---Cl2 179.84 (12) C18---C16---C17---N2 179.56 (12) C62---C57---C58---C59 −0.8 (2) C4---C5---C10---C1 7.51 (19) C62---C61---C60---C59 −0.3 (3) C4---C5---C10---C9 −171.62 (13) C46---C44---C43---C43^ii^ −0.2 (2) C4---C5---C6---N3 −7.2 (2) C46---C44---C43---C42 178.94 (13) C4---C5---C6---C7 170.94 (13) C46---C44---C45---O8 3.4 (2) C16---C15---C14---C13 0.5 (2) C46---C44---C45---N5 −177.06 (12) C16---C15---C14---C19^i^ −179.38 (13) C45---N5---C41---O7 179.39 (13) C16---C18---C19---C14^i^ −1.2 (2) C45---N5---C41---C42 −0.5 (2) C17---N2---C13---O3 179.57 (13) C45---N5---C40---C39 −102.07 (14) C17---N2---C13---C14 −1.2 (2) C45---C44---C43---C43^ii^ 179.00 (14) C17---N2---C12---C11 −103.74 (14) C45---C44---C43---C42 −1.8 (2) C19---C18---C16---C15 0.6 (2) C45---C44---C46---C47 −177.95 (13) C19---C18---C16---C17 −179.24 (13) C42^ii^---C47---C46---C44 −1.3 (2) C12---N2---C13---O3 −1.41 (19) C39---N4---C48---C49 61.96 (16) C12---N2---C13---C14 177.80 (12) C48---N4---C39---C40 71.72 (15) C12---N2---C17---O4 1.49 (19) C48---C49---C50---C51 −179.45 (13) C12---N2---C17---C16 −177.84 (11) C48---C49---C54---C53 178.52 (13) C5---C4---C3---C2 −1.9 (2) C40---N5---C41---O7 0.03 (19) C5---C10---C9---C8 −1.4 (2) C40---N5---C41---C42 −179.86 (12) C5---C6---C7---C8 2.7 (2) C40---N5---C45---O8 −3.64 (19) C10---C1---C2---C3 −0.9 (2) C40---N5---C45---C44 176.80 (11) C10---C5---C6---N3 174.66 (12) C50---C49---C48---N4 −93.61 (16) C10---C5---C6---C7 −7.2 (2) C50---C49---C54---C53 −0.2 (2) C10---C9---C8---C7 −3.2 (2) C50---C51---C52---C53 −0.3 (2) C14---C15---C16---C18 179.66 (13) C37---C38---C29---S2 −10.89 (19) C14---C15---C16---C17 −0.54 (19) C37---C38---C29---C30 173.93 (14) C3---C4---C5---C10 −4.2 (2) C37---C38---C33---C32 −171.33 (13) C3---C4---C5---C6 177.62 (13) C37---C38---C33---C34 7.05 (19) C6---C5---C10---C1 −174.30 (12) C37---C36---C35---C34 2.9 (2) C6---C5---C10---C9 6.57 (19) C58---C57---C62---C61 1.3 (2) C22---C23---C24---C25 −0.7 (2) C31---C32---C33---C38 −4.4 (2) C22---C21---C20---N1 −105.22 (15) C31---C32---C33---C34 177.27 (13) C22---C21---C26---C25 −0.9 (2) C61---C60---C59---C58 0.8 (2) C9---C8---C7---C6 2.5 (2) C54---C49---C48---N4 87.67 (16) C11---N1---C20---C21 60.82 (16) C54---C49---C50---C51 −0.7 (2) C73---C74---C69---Cl5 −178.72 (13) C54---C53---C52---C51 −0.7 (2) C73---C74---C69---C70 0.8 (3) C33---C38---C29---S2 170.02 (10) C74---C73---C72---C71 −0.7 (3) C33---C38---C29---C30 −5.2 (2) C74---C69---C70---Cl6 178.69 (13) C33---C38---C37---C36 −1.5 (2) C74---C69---C70---C71 −1.2 (2) C33---C32---C31---C30 −2.1 (2) C69---C70---C71---C72 0.6 (3) C33---C34---C35---C36 2.7 (2) C20---N1---C11---C12 70.86 (15) C56---N6---C34---C33 161.71 (13) C20---C21---C22---C23 −179.45 (13) C56---N6---C34---C35 −15.7 (2) C20---C21---C26---C25 178.09 (13) C35---C36---C37---C38 −3.5 (2) C70---C71---C72---C73 0.4 (3) C35---C34---C33---C38 −7.6 (2) C24---C23---C22---C21 1.3 (2) C35---C34---C33---C32 170.71 (13) C24---C25---C26---C21 1.5 (2) C52---C51---C50---C49 1.0 (2) C28---N3---C6---C5 161.65 (13) C55---N6---C34---C33 −70.03 (17) C28---N3---C6---C7 −16.4 (2) C55---N6---C34---C35 112.59 (17) C26---C21---C22---C23 −0.5 (2) Cl3---C63---C64---Cl4 0.69 (19) C26---C21---C20---N1 75.81 (16) Cl3---C63---C64---C65 −179.71 (13) C26---C25---C24---C23 −0.7 (2) Cl3---C63---C68---C67 179.29 (13) C27---N3---C6---C5 −70.49 (17) C64---C63---C68---C67 −1.2 (2) C27---N3---C6---C7 111.47 (17) C64---C65---C66---C67 −0.9 (3) C72---C73---C74---C69 0.1 (3) C68---C63---C64---Cl4 −178.84 (12) S2---N4---C39---C40 −133.10 (10) C68---C63---C64---C65 0.8 (2) S2---N4---C48---C49 −92.44 (13) C68---C67---C66---C65 0.5 (3) S2---C29---C30---C31 −176.58 (11) C66---C65---C64---Cl4 179.88 (13) Cl1---C57---C62---C61 −177.56 (12) C66---C65---C64---C63 0.3 (2) Cl1---C57---C58---Cl2 −1.31 (18) C66---C67---C68---C63 0.5 (3) Cl1---C57---C58---C59 178.02 (12) ----------------------------- -------------- ------------------------------- -------------- Symmetry codes: (i) −x, −y+2, −z+1; (ii) −x, −y+2, −z+2. Hydrogen-bond geometry (Å, º) {#tablewraphbondslong} ============================= Cg, Cg5\', Cg6\', Cg7\' and Cg8\' are the centroids of atoms C63--C68, C21--C26, C49--C54, C1--C10 and C29--C38, respectively. --------------------------- --------- --------- ------------- --------------- D---H···A D---H H···A D···A D---H···A C8---H8···O5 0.95 2.53 3.1926 (19) 127 C19---H19···O7 0.95 2.55 3.2615 (19) 132 C31---H31···O3^i^ 0.95 2.59 3.3818 (19) 141 C47---H47···O3^iii^ 0.95 2.58 3.2148 (19) 125 C60---H60···O2 0.95 2.54 3.335 (2) 142 C68---H68···Cl1^iii^ 0.95 2.79 3.5922 (19) 142 C72---H72···O6 0.95 2.50 3.293 (2) 141 C71---H71···Cg 0.95 2.99 3.813 (2) 145 C55---H55A···Cg8′^iv^ 0.98 2.94 3.632 (2) 129 C27---H27A···Cg7′^v^ 0.98 3.03 3.585 (2) 117 C20---H20B···Cg6′^vi^ 0.99 3.12 3.6054 (17) 111 C48---H48B···Cg5′^vi^ 0.99 3.13 3.6180 (17) 112 --------------------------- --------- --------- ------------- --------------- Symmetry codes: (i) −x, −y+2, −z+1; (iii) x, y, z+1; (iv) −x+1, −y+2, −z+2; (v) −x+1, −y+2, −z+1; (vi) −x, −y+1, −z+1. ![The molecular structure of (I)[](#scheme1){ref-type="chem"}, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level and H atoms are omitted for clarity. The inversion-related halves of molecules A and B are generated by symmetry operations (i) −x, −y + 2, −z + 1 and (ii) −x, −y + 2, −z + 2, respectively.](e-72-01503-fig1){#fig1} ![Molecules A and B showing intramolecular aromatic stacking. Dashed lines indicate the interactions between naphathaleneimide centroids Cg1 \[C13--C17/N2 (A)\] and Cg3 \[C41--C45/N5 (B)\] and aryl centroids Cg2 \[C1--C5/C10 (A)\] and Cg4 \[C29--C33/C38 (B)\]. Benzyl and methyl groups and H atoms are omitted.](e-72-01503-fig2){#fig2} ![A view of the supramolecular chain extending along the c axis, generated by C---H⋯O hydrogen bonds (dashed lines).](e-72-01503-fig3){#fig3} ![A perspective view along the c axis of the supramolecular nanotube generated by cooperative C---H⋯π and offset π--π interactions, showing filling by 1,2-dichlorobenzene molecules.](e-72-01503-fig4){#fig4} ![(a) A view of the C---H⋯π and offset π--π interactions between adjacent benzyl groups; (b) A view of additional C---H⋯π interactions between dansyl amide moieties. Hydrogen atoms not involved in the hydrogen-bonding interactions are omitted.](e-72-01503-fig5){#fig5} ###### Hydrogen-bond geometry (Å, °) Cg, Cg5′, Cg6′, Cg7′ and Cg8′ are the centroids of atoms C63--C68, C21--C26, C49--C54, C1--C10 and C29--C38, respectively. D---H⋯A D---H H⋯A D⋯A D---H⋯A -------------------------- --------- ------- ------------- ------------- C8---H8⋯O5 0.95 2.53 3.1926 (19) 127 C19---H19⋯O7 0.95 2.55 3.2615 (19) 132 C31---H31⋯O3^i^ 0.95 2.59 3.3818 (19) 141 C47---H47⋯O3^ii^ 0.95 2.58 3.2148 (19) 125 C60---H60⋯O2 0.95 2.54 3.335 (2) 142 C68---H68⋯Cl1^ii^ 0.95 2.79 3.5922 (19) 142 C72---H72⋯O6 0.95 2.50 3.293 (2) 141 C71---H71⋯Cg 0.95 2.99 3.813 (2) 145 C55---H55A⋯Cg8′^iii^ 0.98 2.94 3.632 (2) 129 C27---H27A⋯Cg7′^iv^ 0.98 3.03 3.585 (2) 117 C20---H20B⋯Cg6′^v^ 0.99 3.12 3.6054 (17) 111 C48---H48B⋯Cg5′^v^ 0.99 3.13 3.6180 (17) 112 Symmetry codes: (i) ; (ii) ; (iii) ; (iv) ; (v) . ###### Experimental details -------------------------------------------------------------------------- -------------------------------------------------- Crystal data Chemical formula C~56~H~50~N~6~O~8~S~2~·3C~6~H~4~Cl~2~ M ~r~ 1440.11 Crystal system, space group Triclinic, P Temperature (K) 100 a, b, c (Å) 12.17737 (14), 17.2876 (2), 17.8916 (2) α, β, γ (°) 110.9544 (12), 96.2760 (11), 103.5159 (10) V (Å^3^) 3341.91 (8) Z 2 Radiation type Cu Kα μ (mm^−1^) 3.44 Crystal size (mm) 0.20 × 0.10 × 0.05 Data collection Diffractometer Agilent SuperNova, Dual, Cu at zero, EosS2 Absorption correction Multi-scan (CrysAlis PRO; Agilent, 2014[@bb2]) T ~min~, T ~max~ 0.874, 1.000 No. of measured, independent and observed \[I \> 2σ(I)\] reflections 54279, 13086, 11914 R ~int~ 0.026 (sin θ/λ)~max~ (Å^−1^) 0.620 Refinement R\[F ^2^ \> 2σ(F ^2^)\], wR(F ^2^), S 0.034, 0.097, 1.03 No. of reflections 13086 No. of parameters 869 H-atom treatment H-atom parameters constrained Δρ~max~, Δρ~min~ (e Å^−3^) 0.74, −0.45 -------------------------------------------------------------------------- -------------------------------------------------- Computer programs: CrysAlis PRO (Agilent, 2014[@bb2]), SHELXS97 (Sheldrick, 2008[@bb19]), SHELXL2014/7 (Sheldrick, 2015[@bb20]), DIAMOND (Brandenburg, 1999[@bb24]) and OLEX2 (Dolomanov et al., 2009[@bb7]).	{ 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Introduction {#s1} ============ Soybean meal (SBM) is an excellent plant protein source for fish feed [@pone.0058115-Lilleeng1]. However, high levels of soybean protein caused a poor growth rate in juvenile Jian carp (Cyprinus carpio var. Jian) [@pone.0058115-Zhang1] and juvenile tilapia (Oreochromis niloticus×O. aureus) [@pone.0058115-Lin1]. In addition, SBM has been shown to decrease intestinal protease activity in juvenile tilapia [@pone.0058115-Lin1] and reduce intestinal alkaline phosphatase (AKP) activity in Jian carp [@pone.0058115-Zhang2]. These negative influences were partly due to the anti-nutritional factors present in SBM, such as β-conglycinin, glycinin, and trypsin inhibitor [@pone.0058115-Buttle1]. Among these anti-nutrients, β-conglycinin was suggested as being one of the key anti-nutritional factors [@pone.0058115-Ostaszewska1]. A recent study has reported that β-conglycinin reduced weight gain in pigs [@pone.0058115-Zhao1]. However, little attention has been given to the potential negative effects of β-conglycinin on the growth, digestive and absorptive ability of fish. The reductions in digestive and absorptive ability may be associated with a diminished growth of the intestine; it has been reported that β-conglycinin decreased the villus height in piglet intestines [@pone.0058115-Hao1]. Protein not only provides architectural support for cells but also serves vital roles in maintaining their function and survival, and protein deposition is a result of protein synthesis and degradation [@pone.0058115-Liu1]. It was demonstrated that the target of rapamycin (TOR) signaling pathway plays an important role in protein synthesis and degradation in terrestrial animals, and the eIF4E-binding protein (4E-BP) is one of the major downstream targets of TOR protein [@pone.0058115-Schmelzle1]. Our laboratory was the first to clone the cDNA of TOR (GenBank accession no. FJ899680) and 4E-BP2 (GenBank accession no. HQ010440) of Jian carp and to show that the expressions of these two genes were influenced by choline [@pone.0058115-Wu1] and arginine [@pone.0058115-Chen1]. Accordingly, further investigation is warranted into whether β-conglycinin causes poor growth of digestive organs via changing the TOR and 4E-BP gene expression. The decreased growth, development and function of the digestive organs may be partly due to the fact that β-conglycinin damages the integrity of the intestine. Previous studies have shown that β-conglycinin causes pathological disruption in the intestines of rats [@pone.0058115-Guo1] and piglets [@pone.0058115-Hao1]. Enterocytes are the primary cells lining the intestine [@pone.0058115-AlHussaini1], and increasing levels of β-conglycinin damaged the integrity of mouse enterocytes, leading to LDH releasing into the culture medium [@pone.0058115-Xu1]. Thus, we hypothesize that β-conglycinin may also injure the intestine and its enterocytes in fish, a theory that requires further investigation. It has been shown in pigs that the intestinal injury caused by β-conglycinin results from the effect of this allergen as an inducer of stress [@pone.0058115-Chen2]. Allergies usually cause inflammatory disorders [@pone.0058115-Li1]; for example, it has been reported that β-conglycinin promoted the secretion of inflammatory cytokines (IL-8) in mouse enterocytes [@pone.0058115-Xu1]. In addition, transforming growth factor β (TGF-β) was identified as an important factor in the prevention of intestinal mucosal inflammation in humans and mice [@pone.0058115-Strober1]. A study of Atlantic salmon (Salmo salar L.) showed that the TGF-β gene level was significantly decreased by dietary SBM [@pone.0058115-Lilleeng2]. However, little attention has been given to the potential negative effects of β-conglycinin on these inflammatory factors in fish. Inflammation is always accompanied by lipid oxidation, the end products of which are generated during inflammation in mice [@pone.0058115-West1]. Thus, it appears that β-conglycinin-induced intestinal damage may be involved in lipid peroxidation. In addition, lipid peroxidation may bring about protein damage due to its end products [@pone.0058115-Bhor1]. Protein carbonyl content is the most widely used biomarker for oxidative damage to proteins and reflects cellular damage [@pone.0058115-Baltacolu1]. Like other organisms, fish combat elevated levels of oxidative stress with protective enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione-dependent enzymes [@pone.0058115-Bhor1]. However, studies referring to the effects of β-conglycinin on the antioxidant system are scarce. To our knowledge, only this study has examined the activities and mRNA levels of antioxidant enzymes in assessing the antioxidant defenses challenged with β-conglycinin. To this context, we hypothesize that β-conglycinin may reduce fish growth through damaging digestive organ which was in part attributed to antioxidant disturbance. Thus, efficient antioxidant becomes of great important. Glutamine (Gln), a conditionally essential amino acid, appears to be a key nutrient for the gut in mammalian [@pone.0058115-Satoh1]. Our laboratory has demonstrated that dietary Gln supplementation increased the intestinal weight and alkaline phosphosphate activity, and thus improved fish intestinal structure and function in Jian carp [@pone.0058115-Lin2]. Furthermore, Gln serves as a metabolic precursor for glutathione (GSH) which can efficiently reduce ROS [@pone.0058115-Johnson1], [@pone.0058115-Wasa1]. It can protect fish enterocytes against H~2~O~2~-induced oxidative damage [@pone.0058115-Chen3]. Thus, we set up a treatment that administrated Gln along with β-conglycinin to study the hypothesis that Gln can protect fish against β-conglycinin toxicity. Together, this study investigated the hypothesis that β-conglycinin might decrease the growth of fish via dysfunction of digestion and absorption which was in part attributed to the digestive organ damage induced by β-conglycinin-mediated inflammation and oxidative stress. To our knowledge, this study provides the first insight into the underlying mechanism by which β-conglycinin decreases digestive and absorptive ability and damages intestinal integrity in fish. The potential protective effect of Gln against β-conglycinin toxicity was also investigated. Materials and Methods {#s2} ===================== Growth Trial (in vivo) {#s2a} ------------------------ Purified β-conglycinin was kindly donated by the China Agricultural University (Patent No. 200410029589·4, China) and was analyzed following the method of [@pone.0058115-Hao1] to be 80% pure. The test diets are presented in [Table S1](#pone.0058115.s001){ref-type="supplementary-material"}. The control diet, devoid of soybean protein, contained fish meal, gelatin and casein as the dietary protein sources. β-conglycinin (80 g β-conglycinin/kg diet) was substituted for the casein in the corresponding experimental diets, following the method of [@pone.0058115-Sun1]. β-conglycinin accounts for about 30% of the total soybean proteins [@pone.0058115-Utsumi1]. Our previous study found that diet with 517.8 g/kg of dehulled soybean meal (CP = 47%) (approximately 73 g/kg of β-conglycinin) significantly depressed the growth, feed efficiency, intestinal weight, length, length index and folds height and the intestinal AKP and γ-GT activities in Jian carp [@pone.0058115-Zhang1], [@pone.0058115-Zhang2]. Therefore, to investigate the toxic effects and potential toxic mechanisms of β-conglycinin in fish, 80.0 g/kg of β-conglycinin was used in this study, which was proved to be enough to induce toxicity in fish by a preliminary experiment (unpublished data). β-conglycinin with 12.0 g/kg of the Gln group was established as a positive control. The concentration of Gln was the optimal dose for the growth of Jian carp [@pone.0058115-Lin2]. All diets were made isonitrogenous with the addition of appropriate amounts of glycine according to the method of [@pone.0058115-Lin2]. Lysine, methionine, threonine, pyridoxine, pantothenic acid, inositol, thiamin, riboflavin, zinc and iron were prepared to meet the nutrient requirements of juvenile Jian carp according to our laboratory studies [@pone.0058115-Zhou1]--[@pone.0058115-Ling1]. The levels of other nutrients met the requirements of common carp (Cyprinus carpio L.) according to the NRC (1993) [@pone.0058115-NationalResearch1]. Diets were prepared by thoroughly mixing all the ingredients. Distilled water (approximately 40%, w/w) was added to the premix dry ingredients and further thoroughly mixed. Slow sinking pellets were wet-extruded through a laboratory-scale, single screw extruder. The machine was continually cooled by a cooling wet paper tower. Thus, the temperature of wet-extruded pellets was controlled to be between 55 and 65°C. The noodle-like diets were immediately freeze-dried at −55°C. After freeze-drying, the pellets were stored at −20°C until feeding according to the method described by [@pone.0058115-Salze1]. The Animal Care and Use Committee of Sichuan Agricultural University approved all experimental procedures. Juvenile Jian carp (Cyprinus carpio var. Jian) purchased from Tong Wei Hatchery (Sichuan, China) were used in this experiment. Fish were acclimatized to the experimental environment for 4 weeks, and were fed with a carp feed (without soybean protein, prepared in our laboratory, crude protein = 34%, crude lipid = 4.6%). Four hundred and fifty fish, with a mean initial weight of 5.36±0.02 g, were randomly assigned to each of 9 experimental aquaria (90 L×30 W×40 H cm). Aquaria were supplied with flow-through water at a rate of 1.2 L/min; water was drained through biofilters to remove solid substances and reduce ammonia concentration. Water temperature was maintained at 23±1°C. For the feeding trial, each of the diets was fed to a triplicate of fish six times per day for the first 4 weeks and four times per day from the 5^th^ to 6^th^ week, a feeding rhythm that was established in previous study [@pone.0058115-Xiao1]. The fish were fed their respective diets to apparent satiation. Uneaten feed was removed by siphoning thirty minutes after feeding, dried and weighed to calculate feed intake. Fish from each aquarium were counted and weighed at the beginning and end of the experiment. At the end of the experiment, fish were anaesthetized in benzocaine bath (50 mg/L) 12 h after the last feeding as described by [@pone.0058115-Bohne1]; the intestines and hepatopancreas were removed quickly, weighed, frozen in liquid nitrogen and stored at −70°C until analysis. In vitro Experiment {#s2b} --------------------- The procedures for cell isolates and cultures were based on those of [@pone.0058115-Jiang2] and [@pone.0058115-Bicho1], with slight modifications. Healthy Jian carp were maintained for approximately 24 h without feeding and were killed by decapitation. The intestines were rapidly removed from the carcass, opened and rinsed with Hanks Balanced Salt Solution (HBSS). The enterocytes were isolated by enzymatic dissociation. Cells were suspended in Dulbecco's Modified Eagle's Media (DMEM) and washed with the same medium 5 times to remove enzymes. Isolated enterocytes were plated in high-glucose DMEM supplemented with 10% FBS, 0.02 mg transferring/mL (Sigma, St. Louis, MO, USA), 0.01 mg insulin/mL (Sigma, St. Louis, MO, USA), 100 IU benzyl penicillin/mL (Sigma, St. Louis, MO, USA) and 100 µg streptomycin sulfate/mL (Sigma, St. Louis, MO, USA). The cells (2×10^3^ cells per well) were seeded in 24-well culture plates (Falcon, Franklin Lake, NJ, USA) that had been previously coated with collagen (Sigma, St. Louis, MO, USA), as previously described by [@pone.0058115-Jiang3]. Cultures were kept at 26±0.5°C. The β-conglycinin treatment was performed by adding fresh medium containing 0, 0.25, 0.6, 0.9, 1.2 and 2.5 mg/mL for 36 h. Four replicate wells were prepared for each concentration of β-conglycinin (n = 4). The second in vitro trial was based on the first in vitro trial and was performed similarly, except for the concentrations of β-conglycinin (0, 1.2, 2.5, 3.5, 4.5 and 5.5 mg/mL). Analysis and Measurement Methods {#s2c} -------------------------------- ### Investigated indexes {#s2c1} The in vivo experiment investigated the hepatopancreas weight (HW), protein content (HPC), hepatopancreas somatic index (HSI) and intestinal weight (IW), protein content (IPC), length (IL), intestinal length index (ILI), intestinal somatic index (ISI) and folds height. In addition, digestive ability (intestinal trypsin, chymotrypsin, lipase and amylase activities) and absorptive function (creatinkinase, Na^+^,K^+^-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase (γ-GT) in the proximal (PI), mid (MI) and distal intestine (DI)) were studied. Furthermore, malondialdehyde (MDA), protein carbonyls (PC), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in the hepatopancreas and intestine were also investigated. Finally, gene expression of TOR and 4E-BP in the PI, MI and DI and interleukin-1 (IL-1), IL-8, tumor necrosis factor-α (TNF-α) and TGF-β genes in the distal intestine (DI) as well as the whole intestinal GPx1a, GPx1b, GR, CuZnSOD, MnSOD and CAT were investigated. The first in vitro trial investigated the cell viability (MTT), protein retention (PR), glutamate oxaloacetate transaminase (GOT) in enterocytes and LDH, GOT and glutamate pyruvate transaminase (GPT) activities in culture medium. The second in vitro trail investigated the oxidative damage indexes (including LDH and MDA in medium and cellular PC) and the cellular functional indexes (AKP) as well as cell antioxidant indexes (including SOD, CAT, GPx, GST, GR and GSH). ### Digestive and absorptive analysis {#s2c2} At the end of the growth trail, the intestines of 5 fish from each aquarium were sampled and fixed with formalin (10%), sectioned and stained with H-E stain for histological analysis (height of intestinal folds) according to the method described by Lin and Zhou [@pone.0058115-Lin2]. Samples of the intestine and hepatopancreas were each homogenized in 10 volumes (w/v) of ice-cold physiological saline solution and centrifuged at 6,000×*g* for 20 min at 4°C, respectively. After centrifugation, the supernatant was used for determination of trypsin [@pone.0058115-Hummel1], chymotrypsin [@pone.0058115-Hummel1], lipase [@pone.0058115-Furne1], amylase [@pone.0058115-Refstie1], alkaline phosphatase (AKP) [@pone.0058115-Krogdahl1] and Na^+^, K^+^-ATPase activities [@pone.0058115-Ilenchuk1]. Gamma-glutamyl transpeptidase (γ-GT) activities in the intestine were determined by using a γ-GT Assay Kit (Sigma, St Louis, MO). Protein content was determined following the Bradford method [@pone.0058115-Bradford1]. ### MTT cell viability assay {#s2c3} Cell proliferation was determined by assaying the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to formazan, following the method described by [@pone.0058115-Daly1]. Briefly, at the end of the experiment, the medium was removed and 500 µL of MTT working solution was added to the cultures. After incubation for 4 h, the MTT working solution was removed and replaced with 500 µL of dimethyl sulphoxide (Sigma) to dissolve the formazan precipitates. The amount of formazan was determined by measuring the optical density (OD) at 595 nm on a plate reader (Thermo Labsystems Oy, Helsinki, Finland). ### Cell injury analysis {#s2c4} The amount of LDH released by the cells was measured following the method of [@pone.0058115-Mulier1]. Lipid peroxidation was analyzed as described by [@pone.0058115-Zhang3] and measured in terms of malondialdehyde (MDA) equivalents using the thiobarbituric acid (TBA) reaction. In brief, samples were mixed with trichloroacetic acid and centrifuged. Then, TBA was added to the supernatant. The mixture was heated in water at 95°C for 40 minutes. MDA forms a red adduct with TBA, which has an absorbance of 532 nm. The cellular protein carbonyl (PC) content was determined according to the method described by [@pone.0058115-Baltacolu1], with a minor modification using the 2,4-dinitrophenylhydrazine (DNPH) reagent. The carbonyl content was calculated from the peak absorbance at 340 nm, using an absorption coefficient of 22,000/M/cm. ### Antioxidant enzyme activities and GSH content {#s2c5} The abilities of the anti-superoxide anion (ASA) and the anti-hydroxyl radical (AHR) (OH-scavenging ability) were determined following the instructions of the specific kits (Jiancheng Bioengineering Ltd., Nanjing, China). Briefly, superoxide radicals (O~2~ ^−^) were generated by the action of xanthine and xanthine oxidase. With the addition of an electron acceptor, a coloration reaction (absorbance at 550 nm) was developed using the gross reagent. Vitamin C was used as the standard agent. AHR was then assayed based on the Fenton reaction (Fe^2+^+H~2~O~2~→Fe^3+^+OH^−^+•OH). A coloration reaction (absorbance at 550 nm) was also developed using the gross reagent. CAT activities were measured, as have previously described [@pone.0058115-Jiang4]. The superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were assayed as described by [@pone.0058115-Zhang3]. The glutathione-S-transferase (GST) activity was measured by monitoring the formation of an adduct between GSH and 1-chloro-2,4-dinitrobenzene (CDNB) [@pone.0058115-Lushchak1]. GSH content was determined by the method described by [@pone.0058115-Vardi1] with a minor modification. ### Analysis of gene expression {#s2c6} The total RNA of samples was extracted using RNAiso Plus (TaKaRa Biotechnology, Dalian Co. Ltd., China, D9108B), according to the manufacturer's instructions. RNA quantity and quality were assessed by electrophoresis on 1% agarose gels and by spectrophotometry at 260 and 280 nm. Subsequently, cDNA was synthesized using a PrimeScript™ RT reagent Kit (Takara, Dalian, Liaoning, China), according to manufacturer's instructions. Briefly, oligo dT primers (50 µM) were used to reverse transcribe respective RNAs in the presence of PrimeScript™ RT enzyme Mix I, 5×PrimeScript™ buffer, Random 6 mers (100 µM) and RNase free dH~2~O at 37°C for 15 min, following inactivation at 85°C for 5 s. Specific primers for genes were designed with Primer Premier software (Premier Biosoft International, Palo Alto, CA, USA), according to the Jian carp sequences (except for IL-1, IL-8, TNF-α and TGF-β, the other genes were cloned and submitted to NCBI by our laboratory) ([Table S2](#pone.0058115.s002){ref-type="supplementary-material"}). Real-time PCRs were performed with a chromo 4™ continuous fluorescence detector (Bio-Rad Laboratories, Inc.) using a SYBR PrimeScriptTM RT-PCR Kit II, according to standard protocols. The expression levels of these genes were normalized to the expression levels of a housekeeping gene, β-actin ([Table S2](#pone.0058115.s002){ref-type="supplementary-material"}). Each assay was performed with 5 replications. The concentrations of the target genes were calculated based on the threshold cycle number (CT). The CT for each sample was determined by using MJ Option Monitor Software (version 3.1; Bio-Rad, Hemel Hempstead, Herts, UK). In addition, the cDNA concentration in each sample was determined according to gene-specific standard curves. Standard curves were generated for both the target and endogenous control genes based on 10-fold serial dilutions. All standard curves exhibited correlation coefficients higher than 0.99, and the corresponding real-time PCR efficiencies ranged from 0.90--1.10. Statistical Analysis {#s2d} -------------------- All data are presented as the mean ± standard deviation (SD). The data were subjected to a one-way analysis of variance (ANOVA). If significant differences were found (P\<0.05), Duncan's multiple range tests were used to rank the groups. TNF-α and TGF-β mRNA levels were evaluated with Student's t-test. All statistical analyses were performed using the SPSS 13.0 for Windows (SPSS Inc, Chicago, IL, USA). Results {#s3} ======= In vivo Experiment {#s3a} -------------------- Initial body weight (IBW), final body weight (FBW), special growth ratio (SGR), survival rate, feed intake (FI), feed efficiency (FE), hepatopancreas weight (HW), protein content (HPC) and somatic index (HSI) and intestinal weight (IW), intestinal protein content (IPC), intestinal length (IL), intestinal length index (ILI) and intestinal somatic index (ISI) and folds height in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) are presented in [Table 1](#pone-0058115-t001){ref-type="table"}. β-conglycinin alone significantly reduced FBW by approximately 15.1% compared with the control, but co-treatment with Gln completely blocked the reduction of FBW caused by β-conglycinin. Similarly, β-conglycinin alone also significantly reduced SGR compared with the unexposed control (P\<0.05). The SGR significantly increased by co-treatment with Gln compared with that of fish exposed to β-conglycinin alone (P\<0.05); the value was equivalent to the control. Survival was not affected by dietary treatments. The FI was lowest in fish exposed to β-conglycinin alone, and significantly recovered by co-treatment with Gln. The FE was higher in fish fed the control diet than in fish fed with β-conglycinin alone. HW was highest in fish fed the control diet, followed by fish co-fed with Gln and β-conglycinin, and was lowest in fish fed with β-conglycinin alone. HPC was higher in fish co-treated with Gln and β-conglycinin than in fish fed with β-conglycinin alone. Fish exposed to dietary β-conglycinin experienced a decrease in HSI compared with the control. There was no significant difference between the combinations and the β-conglycinin alone. IPC was lower in fish exposed to dietary β-conglycinin alone than in other groups. IW and IL were significantly reduced by dietary β-conglycinin alone compared with the unexposed control but was partly recovered by supplementation with dietary Gln. In addition, ILI was also significantly reduced by β-conglycinin alone compared with the unexposed control. However, there was no significant alteration in ISI among the treatments. The folds height in the PI was significantly reduced by dietary β-conglycinin alone compared with the unexposed control, but was partly recovered by supplementing the diet with Gln (P\<0.05). The folds height in the MI and DI were lower in fish exposed to β-conglycinin alone than in that of the control. 10.1371/journal.pone.0058115.t001 ###### Initial body weight (IBW), final body weight (FBW), special growth ratio (SGR), survival rate, feed intake (FI), feed efficiency (FE), hepatopancreas weight (HW), protein content (HPC) and somatic index (HSI) and intestinal weight (IW), intestinal protein content (IPC), intestinal length (IL), intestinal length index (ILI) and intestinal somatic index (ISI) and folds height (µm) in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of juvenile Jian carp (Cyprinus carpio var. Jian) exposed to dietary β-conglycinin for 42 days (in vivo experiment). ![](pone.0058115.t001){#pone-0058115-t001-1} Indexes Control β-conglycinin alone β-conglycinin plus Gln ------------------ ----------------- --------------------- ------------------------ IBW (g/fish) 5.37±0.04^a^ 5.36±0.01^a^ 5.36±0.01^a^ FBW (g/fish) 28.68±0.92^a^ 24.34±0.86^b^ 27.15±0.60^a^ SGR 3.99±0.09^a^ 3.60±0.08^b^ 3.86±0.05^a^ Survival rate 100.00±0.00^a^ 100.00±0.00^a^ 100.00±0.00^a^ FI (g/fish) 30.21±0.05^a^ 26.42±0.07^c^ 28.79±0.01^b^ FE 87.69±3.50^a^ 81.60±3.47^b^ 86.01±2.35^ab^ HW (g/fish) 1.18±0.22^a^ 0.60±0.12^c^ 0.73±0.24^b^ HPC 4.15±0.20^ab^ 4.01±0.32^b^ 4.36±0.19^a^ HSI 3.56±0.40^a^ 3.07±0.34^b^ 3.39±0.86^ab^ IW (g/fish) 1.10±0.20^a^ 0.62±0.15^c^ 0.75±0.19^b^ IPC 2.35±0.16^a^ 1.99±0.13^b^ 2.41±0.21^a^ IL (cm/fish) 17.93±2.34^a^ 14.10±1.58^c^ 15.09±2.08^b^ ILI 170.22±21.54^a^ 160.42±13.04^b^ 164.49±18.85^ab^ ISI 3.23±0.27^a^ 3.13±0.39^a^ 3.25±0.36^a^ PI folds heights 700.44±70.72^a^ 536.80±22.42^c^ 633.53±37.97^b^ MI folds heights 443.58±42.94^a^ 378.74±23.12^b^ 425.99±46.47^ab^ DI folds heights 524.14±46.60^a^ 447.93±45.50^b^ 482.20±34.71^ab^ Data represent means ± S.D. Values within the same row with different superscripts are significantly different (P\<0.05). SGR = 100× (ln final weight - ln initial weight)/number of days. FE = 100× (g weight gain/g feed intake). HPC = 100×(g hepatopancreas protein/g wet hepatopancreas weight). HSI = 100×(g wet hepatopancreas weight/g wet body weight). IPC = 100×(g intestine protein/g wet intesitine weight). ILI = 100× (cm intestine length/cm total body length). ISI = 100×(g wet intestine weight/g wet body weight). Intestinal trypsin, chymotrypsin, amylase and lipase activities are presented in [Table 2](#pone-0058115-t002){ref-type="table"}. The intestinal trypsin of fish fed with β-conglycinin and Gln was significantly increased compared with that of the group fed β-conglycinin alone (P\<0.05); the value was equivalent to fish not exposed to β-conglycinin. The intestinal chymotrypsin and lipase activities reflected the same pattern in regard to intestinal trypsin activity. No significant differences were observed in intestinal amylase activities among the treatments. 10.1371/journal.pone.0058115.t002 ###### Intestinal digestive enzyme activity of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (in vivo experiment). ![](pone.0058115.t002){#pone-0058115-t002-2} Indexes Control β-conglycinin alone β-conglycinin plus Gln --------------------------- ------------------ --------------------- ------------------------ Trypsin (U/g tissue) 2.41±0.07^a^ 2.14±0.24^b^ 2.36±0.08^a^ Chymotrypsin (U/g tissue) 2.85±0.26^a^ 1.38±0.15^b^ 2.65±0.30^a^ Lipase (U/g tissue) 373.29±45.57^a^ 176.56±29.76^b^ 322.84±49.43^a^ Amylase (U/g tissue) 1309.52±52.42^a^ 1295.24±71.52^a^ 1323.81±56.18^a^ Data represent means ± S.D. of six replicates. Values within the same row with different superscripts are significantly different (P\<0.05). CK, Na^+^, K^+^-ATPase, AKP and γ-GT activities in the PI, MI and DI are presented in [Table 3](#pone-0058115-t003){ref-type="table"}. β-conglycinin alone caused significant decreases in CK and Na^+^, K^+^-ATPase activity in all intestinal segments compared with the unexposed control but was completely recovered by a diet combined with Gln (P\<0.05). Similarly, the AKP activity in the PI, MI and DI were lower in fish exposed to β-conglycinin alone than that of the other groups. Conversely, the gamma-glutamyl transpeptidase (γ-GT) activity in all intestinal segments was increased by β-conglycinin. Only the β-conglycinin-induced increase of γ-GT activity in the MI was partly prevented by Gln. 10.1371/journal.pone.0058115.t003 ###### Creatine kinase (CK, µmol of phosphorus released/g tissue/h), Na^+^, K^+^-ATPase (µmol of phosphorus released/g tissue/h), alkaline phosphatase (AKP, mmol of nitrophenol released/g tissue/h) and gamma-glutamyl transpeptidase (γ-GT, mmol of 5-amino-2-nitrobenzoate released/g tissue/min) activity in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (in vivo experiment). ![](pone.0058115.t003){#pone-0058115-t003-3} Indexes Intestines Control β-conglycinin alone β-conglycinin plus Gln ------------------------ ------------ ----------------- --------------------- ------------------------ CK PI 47.14±4.60^a^ 41.62±2.17^b^ 46.52±3.90^a^ MI 55.77±5.74^a^ 43.36±5.63^b^ 50.68±4.66^a^ DI 22.38±2.37^b^ 16.60±2.49^c^ 30.94±3.12^a^ Na^+^, K^+^-ATPase PI 289.06±31.54^a^ 237.63±42.39^b^ 298.83±35.00^a^ MI 220.70±30.23^a^ 158.85±24.29^b^ 195.31±16.01^a^ DI 207.68±36.26^a^ 144.53±54.07^b^ 203.13±45.29^a^ AKP PI 26.23±3.19^a^ 21.44±2.52^b^ 25.82±3.38^a^ MI 17.28±2.09^a^ 13.43±2.47^b^ 16.53±1.83^a^ DI 3.30±0.24^a^ 2.83±0.30^b^ 3.28±0.32^a^ γ-GT PI 38.37±4.85^b^ 89.77±22.69^a^ 82.91±14.52^a^ MI 65.47±9.02^c^ 214.30±37.74^a^ 134.42±17.60^b^ DI 80.81±11.07^b^ 180.23±26.50^a^ 160.82±23.32^a^ Data represent means ± S.D. of six replicates. Values within the same row with different superscripts are significantly different (P\<0.05). The oxidative status of the hepatopancreas and intestines and their antioxidant levels are presented in [Table 4](#pone-0058115-t004){ref-type="table"}. The MDA and PC content in the hepatopancreas and intestine were lower in fish fed the control diet than in the other groups (P\<0.05). There was no change between the β-conglycinin group and the Gln plus β-conglycinin group. In addition, except for GST, the antioxidants (SOD, CAT, GPx and GR activities and GSH content) in the hepatopancreas and intestine were reduced by β-conglycinin compared with the unexposed control. The decreases in these antioxidants were completely recovered by supplementation with dietary Gln (P\<0.05). There was no significant change in GST activity in the hepatopancreas among the treatments. In addition, GST activity in the intestine was reduced by β-conglycinin (P\<0.05), but no significant difference was observed between the β-conglycinin alone group and the Gln plus β-conglycinin group. 10.1371/journal.pone.0058115.t004 ###### Oxidative status (malondialdehyde (MDA, nmol/mg protein), protein carbonyl content (PC, nmol/mg protein) and antioxidative enzyme activities (superoxide dismutase (SOD, U/mg protein), catalase (CAT, U/mg protein), glutathione-S-transferase (GST, U/mg protein), glutathione peroxidase (GPx, U/mg protein), glutathione reducase (GR, U/g protein) activities and glutathione (GSH, mg/g protein) content) in the hepatopancreas and intestine of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (in vivo experiment). ![](pone.0058115.t004){#pone-0058115-t004-4} Organs Indexes Control β-conglycinin alone β-conglycinin plus Gln -------------------- --------- ---------------- --------------------- ------------------------ Hepatopancreas MDA 1.12±0.09^b^ 1.38±0.12^a^ 1.29±0.08^a^ PC 0.71±0.13^b^ 1.08±0.14^a^ 0.96±0.15^a^ SOD 41.66±9.02^a^ 21.25±5.67^b^ 28.91±5.58^b^ CAT 11.40±1.36^a^ 8.78±1.14^b^ 10.76±1.65^a^ GST 32.49±5.13^a^ 27.80±4.31^a^ 29.36±3.79^a^ GPx 404.0±31.3^a^ 313.7±51.0^b^ 358.6±43.0^ab^ GR 39.56±7.13^a^ 25.03±4.48^b^ 34.54±7.57^a^ GSH 24.31±2.66^a^ 19.30±1.75^b^ 20.97±2.95^b^ Intestine MDA 2.83±0.31^b^ 3.58±0.40^a^ 3.42±0.18^a^ PC 1.16±0.18^b^ 1.72±0.38^a^ 1.56±0.16^a^ SOD 46.13±7.69^a^ 30.61±4.58^b^ 41.69±2.76^a^ CAT 11.03±0.94^a^ 8.90±1.09^b^ 10.13±0.82^a^ GST 83.42±10.46^a^ 51.98±7.93^b^ 62.84±12.21^b^ GPx 174.9±23.7^a^ 143.1±13.2^b^ 170.8±26.1^a^ GR 91.09±12.86^a^ 67.48±12.26^b^ 82.00±11.08^ab^ GSH 38.21±2.99^a^ 29.82±2.78^b^ 35.49±6.85^ab^ Data represent means ± S.D. of six replicates. Values within the same row with different superscripts are significantly different (P\<0.05). The TOR and 4E-BP gene levels in the PI, MI and DI are presented in [Table 5](#pone-0058115-t005){ref-type="table"}. β-conglycinin significantly reduced the levels of TOR mRNA in the PI and MI (P\<0.05). However, Gln was found to completely block the reductions in TOR expression caused by β-conglycinin in the PI and partly block them in the MI. The TOR gene expression levels in the DI were not significantly different among the three groups, only showing a tendency to decrease with β-conglycinin. The results indicated that β-conglycinin caused an increase in 4E-BP gene expression in the DI, which could partly be reversed by Gln. However, the levels of 4E-BP gene expression in the PI and MI showed no significant differences among the three groups. 10.1371/journal.pone.0058115.t005 ###### Relative expression of TOR and 4E-BP genes in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (in vivo experiment). ![](pone.0058115.t005){#pone-0058115-t005-5} Genes Intestines Control β-conglycinin alone β-conglycinin plus Gln ----------- ------------ ------------------ --------------------- ------------------------ TOR PI 0.012±0.0032^a^ 0.0075±0.0017^b^ 0.012±0.0022^a^ MI 0.033±0.0047^a^ 0.0086±0.0014^c^ 0.016±0.0026^b^ DI 0.0617±0.0128^a^ 0.0538±0.0152^a^ 0.0573±0.0178^a^ 4E-BP PI 0.060±0.018^a^ 0.082±0.025^a^ 0.091±0.025^a^ MI 0.0029±0.0005^a^ 0.0024±0.0004^a^ 0.0026±0.0008^a^ DI 0.0041±0.0012^c^ 0.015±0.0036^a^ 0.011±0.0033^b^ Data represent means ± S.D. of five replicates. Values within the same row with different superscripts are significantly different (P\<0.05). To investigate the effects of β-conglycinin on the intestinal inflammatory response in fish, the expressions of IL-1, IL-8, TNF-α and TGF-β genes in the DI were determined ([Table 6](#pone-0058115-t006){ref-type="table"}). The highest levels of IL-1, TNF-α and TGF-β genes were observed in the β-conglycinin with Gln group (P\<0.05). The relative expressions of IL-8 were reduced by β-conglycinin (P\<0.05). However, the mean values of TNF-α and TGF-β mRNA levels among the three groups are largely different. The TNF-α and TGF-β were not significantly affected by β-conglycinin treatment when Duncan's multiple range tests were used ([Table 6](#pone-0058115-t006){ref-type="table"}). However, TNF-α ([Figure 1a](#pone-0058115-g001){ref-type="fig"}) and TGF-β ([Figure 1b](#pone-0058115-g001){ref-type="fig"}) were significantly increased by β-conglycinin treatment when Student's t test was used. ![Student's t-test of TNF-α (a) and TGF-β mRNA (b) levels.\ Data represent means ± S.D. of five replicates. Values with \* are significantly different (P\<0.05).](pone.0058115.g001){#pone-0058115-g001} 10.1371/journal.pone.0058115.t006 ###### Relative expression of interleukin-1 (IL-1), IL-8, tumor necrosis factor-α (TNF-α) and transforming growth factor β (TGF-β) genes in the distal intestine (DI) of juvenile Jian carp (Cyprinus carpio var. Jian) exposed to dietary β-conglycinin for 42 days (in vivo experiment). ![](pone.0058115.t006){#pone-0058115-t006-6} Genes Control β-conglycinin alone β-conglycinin plus Gln ------- ------------------ --------------------- ------------------------ IL-1 0.0021±0.0004^b^ 0.0014±0.0009^b^ 0.0036±0.0009^a^ IL-8 0.0020±0.0004^a^ 0.00060±0.0002^b^ 0.00040±0.0001^b^ TNF-α 0.0002±0.0001^b^ 0.0012±0.0003^b^ 0.015±0.0021^a^ TGF-β 0.019±0.0045^b^ 0.045±0.012^b^ 0.57±0.19^a^ Data represent means ± S.D. of five replicates. Values within the same row with different superscripts are significantly different (P\<0.05). [Table 7](#pone-0058115-t007){ref-type="table"} shows the relative expressions of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR genes in the whole intestine of fish exposed to dietary β-conglycinin. The expressions of CuZnSOD, MnSOD, CAT and GPx1b genes were all increased by β-conglycinin (P\<0.05). However, only MnSOD gene expression was completely recovered with Gln supplementation. The levels of GPx1a and GR gene expressions showed similar trends, but there were no significant differences among the three groups. 10.1371/journal.pone.0058115.t007 ###### Relative expression of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR genes in the whole intestine of juvenile Jian carp (Cyprinus carpio var. Jian) exposed to dietary β-conglycinin for 42 days (in vivo experiment). ![](pone.0058115.t007){#pone-0058115-t007-7} Genes Control β-conglycinin alone β-conglycinin plus Gln --------- ------------------ --------------------- ------------------------ CuZnSOD 0.026±0.0086^b^ 0.037±0.0034^a^ 0.042±0.011^a^ MnSOD 0.0064±0.0020^b^ 0.013±0.0038^a^ 0.0065±0.0016^b^ CAT 0.054±0.0067^b^ 0.095±0.024^a^ 0.075±0.017^ab^ GPx1a 0.32±0.097^a^ 0.36±0.047^a^ 0.34±0.092^a^ GPx1b 0.020±0.0055^b^ 0.057±0.015^a^ 0.050±0.016^a^ GR 0.019±0.0058^a^ 0.020±0.0061^a^ 0.014±0.0041^a^ Data represent means ± S.D. of five replicates. Values within the same row with different superscripts are significantly different (P\<0.05). In vitro Experiment {#s3b} --------------------- ### First in vitro trial {#s3b1} The effects of β-conglycinin on MTT OD, protein retention (PR) and glutamic-oxaloacetic transaminase (GOT) activity in enterocytes, and the activities of LDH, GOT and GPT in medium are presented in [Table 8](#pone-0058115-t008){ref-type="table"}. MTT OD was significantly reduced with higher levels of β-conglycinin, and the lowest MTT OD was observed in cells treated with 2.5 mg/mL of β-conglycinin (P\<0.05). Cellular PR was highest in cells incubated in the control medium, and lowest in cells exposed to 2.5 mg/mL of β-conglycinin. Cellular GOT activity was lower in cells exposed to 1.2 and 2.5 mg/mL of β-conglycinin than cells in the other groups. LDH in medium was significantly higher in 2.5 mg/mL of β-conglycinin than LDH in the control group. GOT activities in medium were higher in 1.2 and 2.5 mg/mL of β-conglycinin than in the other treatments. GPT activities in medium were highest in 2.5 mg/mL of β-conglycinin, followed by 1.2 and 0.9 mg/mL in the β-conglycinin groups, and lowest in the 0--0.6 mg/mL of β-conglycinin groups. It should be noted that the cellular PR, cellular GOT, and LDH, GOT and GPT in medium, were not significantly different among the treatments of 0, 0.25, 0.6 and 0.9 mg/mL of β-conglycinin. Thus, we deleted those doses in the second in vitro trial. 10.1371/journal.pone.0058115.t008 ###### Effects of β-conglycinin (mg/mL) on MTT OD, protein retention (PR, µg) and glutamic-oxaloacetic transaminase (GOT, U/mg protein) activity in enterocytes and the activities of lactate dehydrogenase (LDH, U/L), GOT (U/L) and glutamic-pyruvic transaminase (GPT, U/L) in medium (the first in vitro experiment). ![](pone.0058115.t008){#pone-0058115-t008-8} β-conglycinin MTT OD PR GOT LDH GOT GPT --------------- ----------------- --------------- ------------- ------------ --------------- --------------- 0.00 0.115±0.002^a^ 53.5±2.6^a^ 20.0±3.7^a^ 243±7.6^b^ 6.93±0.53^b^ 6.51±0.92^c^ 0.25 0.108±0.002^b^ 44.3±15.4^ab^ 17.1±4.5^a^ 255±16^ab^ 7.11±1.05^b^ 6.75±0.71^c^ 0.60 0.104±0.002^bc^ 43.9±5.7^ab^ 17.6±6.6^a^ 250±23^ab^ 7.35±1.09^b^ 6.69±0.63^c^ 0.90 0.103±0.002^c^ 40.0±4.6^ab^ 14.4±10^a^ 271±37^ab^ 7.47±0.20^b^ 7.59±0.82^bc^ 1.20 0.101±0.002^c^ 43.6±4.7^ab^ 12.6±4.6^b^ 262±13^ab^ 9.80±1.15^a^ 8.25±0.50^b^ 2.50 0.093±0.007^d^ 35.4±10.7^b^ 12.0±3.4^b^ 280±15^a^ 10.72±0.42^a^ 10.24±0.46^a^ Data represent means ± S.D. of four replicates. Values within the same column with different superscripts are significantly different (P\<0.05). ### Second in vitro trial {#s3b2} AKP and PC in cells and LDH and MDA in medium are presented in [Table 9](#pone-0058115-t009){ref-type="table"}. AKP activity in cells decreased with higher levels of β-conglycinin, up to 4.5 mg/mL (P\<0.05), with no difference shown between 4.5 and 5.5 mg/mL (P\>0.05). LDH and MDA content in medium significantly increased with increasing levels of β-conglycinin, up to 4.5 mg/mL (P\<0.05), and no differences were found with a further increase in β-conglycinin concentration (P\>0.05). Cellular PC increased with higher levels of β-conglycinin, up to 4.5 mg/mL (P\<0.05), with no difference found between 4.5 and 5.5 mg/mL of β-conglycinin (P\>0.05). 10.1371/journal.pone.0058115.t009 ###### Effects of β-conglycinin (mg/mL) on the activity of alkaline phosphatase (AKP, U/g protein) in enterocytes, lactate dehydrogenase (LDH, U/L) in medium, content of malondialdehyde (MDA, nmol/mL) in medium and protein carbonyls (PC, nmol/mg protein) in enterocytes (the second in vitro experiment). ![](pone.0058115.t009){#pone-0058115-t009-9} β-conglycinin AKP in cell LDH MDA PC in cell --------------- --------------- ---------------- --------------- --------------- 0.00 1.97±0.18^a^ 9.13±2.03^d^ 1.30±0.05^d^ 0.50±0.08^a^ 1.20 1.79±0.09^ab^ 10.14±2.34^cd^ 1.39±0.12^d^ 0.53±0.09^a^ 2.50 1.59±0.14^bc^ 18.26±4.06^bc^ 1.58±0.03^c^ 0.66±0.09^b^ 3.50 1.56±0.17^c^ 24.34±6.62^ab^ 1.67±0.16^bc^ 0.85±0.07^c^ 4.50 1.22±0.17^d^ 30.43±7.77^a^ 1.72±0.09^a^ 1.13±0.09^cd^ 5.50 1.02±0.09^d^ 30.42±7.72^a^ 1.81±0.02^a^ 1.15±0.14^d^ Data represent means ± S.D. of four replicates. Values within the same column with different superscripts are significantly different (P\<0.05). Cellular SOD, CAT, GPx, GST and GR activities and GSH content are shown in [Table 10](#pone-0058115-t010){ref-type="table"}. SOD activities decreased with higher levels of β-conglycinin, up to 3.5 mg/mL (P\<0.05), and plateaued thereafter (P\>0.05). A dose-dependent decrease in CAT activities was observed in the presence of β-conglycinin and was lowest when β-conglycinin reached 5.5 mg/mL (P\<0.05). GPx activities were highest in the group containing no β-conglycinin, followed by 1.2--4.5 mg/mL, with GPx being lowest in the presence of 5.5 mg/mL of β-conglycinin (P\<0.05). GST activity in cells treated with 5.5 mg/mL of β-conglycinin significantly decreased by approximately 34.4%, compared with the cells incubated with no β-conglycinin (P\<0.05). GR activity and GSH content also decreased with higher levels of β-conglycinin, up to 2.5 mg/mL (P\<0.05), and no differences were found with a further increase in β-conglycinin concentrations (P\>0.05). 10.1371/journal.pone.0058115.t010 ###### Activities of SOD (U/mg protein), CAT (U/mg protein), GPx (U/mg protein), GST (U/mg protein), GR (U/g protein) and content of GSH (mg/g protein) in enterocytes exposed to β-conglycinin (mg/mL) (the second in vitro experiment). ![](pone.0058115.t010){#pone-0058115-t010-10} β-conglycinin SOD CAT GPx GST GR GSH --------------- -------------- --------------- ----------- --------------- -------------- --------------- 0.00 12.2±1.3^a^ 1.68±0.13^a^ 594±31^a^ 108±13^a^ 63.1±10.4^a^ 12.13±0.52^a^ 1.20 12.0±1.0^a^ 1.63±0.18^a^ 490±19^b^ 91.7±21.6^ab^ 51.8±4.5^ab^ 12.12±0.51^a^ 2.50 9.95±0.94^b^ 1.35±0.20^b^ 461±32^b^ 91.7±7.1^ab^ 47.9±8.7^b^ 11.22±0.50^b^ 3.50 6.04±0.62^c^ 1.26±0.03^bc^ 444±47^b^ 89.2±15.5^ab^ 46.0±8.4^b^ 10.54±0.58^b^ 4.50 5.93±0.73^c^ 1.13±0.07^c^ 443±29^b^ 87.2±10.2^ab^ 46.6±8.5^b^ 10.92±0.51^b^ 5.50 6.02±1.06^c^ 0.87±0.07^d^ 347±25^c^ 70.5±21.0^b^ 48.0±8.7^b^ 10.88±0.25^b^ Data represent means ±S.D. of four replicates. Values within the same column with different superscripts are significantly different (P\<0.05). Discussion {#s4} ========== Soybean is a source of high-quality protein due to its relatively well-balanced composition of amino acids [@pone.0058115-Friedman1]. However, soybean is also a dietary allergic source, which threatens individuals [@pone.0058115-Sun1]. β-conglycinin is a primary storage protein and it has been identified as one of the major allergenic proteins in soybean [@pone.0058115-Hao1]. Thus, to define mechanisms whereby excessive soybean induces growth depression in animals; this study investigated the role of purified soybean β-conglycinin, separating its effects from the effects of other anti-nutritional factors. Firstly, the present study observed that fish fed diet with β-conglycinin exhibited poor weight gain, which was also observed in piglets [@pone.0058115-Zhao1], [@pone.0058115-Hao1] and rats [@pone.0058115-Han1]. However, survival was not influenced by the dietary treatments. No reports exist concerning the effects of β-conglycinin on survival rate in fish. Similar findings were observed that replacement of dietary fishmeal with more than 41% of soy protein isolate did not influence the survival, but indeed reduced the growth performance in Pacific white shrimp (Litopenaeus vannamei) [@pone.0058115-GamboaDelgado1]. A reduction in feed intake was regarded as the primary factor responsible for the depressed growth observed in rats [@pone.0058115-Jiang1]. In the present study, the feed intake in the β-conglycinin group was significantly lower than that of the control group, which was in accordance with the results for rats [@pone.0058115-Nishi1]. This result suggested that the growth reduction caused by β-conglycinin was most likely attributed to the suppression of feed intake. However, there was no information referring to how β-conglycinin leads to the suppression of feed intake in fish. In rats, it has been reported that β-conglycinin peptone stimulating endogenous cholecystokinin (CCK) release suppresses food intake by inducing a feeling of satiety and by reducing gastric emptying [@pone.0058115-Nishi1]. Besides feed intake, feed utilization efficiency also influences growth performance. In the present study, β-conglycinin depressed feed efficiency, indicating that the reduction of fish growth was partly attributed to the fact that β-conglycinin decreased feed efficiency. Interestingly, when fish were administered Gln with β-conglycinin, growth performances were close to or equal to those in the control group, showing that Gln might mitigate the poor growth caused by β-conglycinin. As an initial step in understanding how β-conglycinin reduced the growth of fish, we examined the digestive and absorptive ability of the fish. In this study, β-conglycinin alone significantly decreased the activities of trypsin, chymotrypsin, lipase, creatine kinase, Na^+^, K^+^-ATPase and alkaline phosphatase in the intestine and its enterocytes. Although no reports exist concerning the effects of β-conglycinin on digestive and absorptive enzyme activity in fish, similar findings were observed that diets containing soybeans might decrease the activity of protease in tilapia [@pone.0058115-Lin1], alkaline phosphatase in Atlantic salmon [@pone.0058115-BakkeMckellep1] and creatine kinase in Atlantic halibut (Hippoglossus hippoglossus) [@pone.0058115-Murray1]. In rats, it has been reported reported that β-conglycinin peptone in the lumen stimulating endogenous cholecystokinin (CCK) release [@pone.0058115-Nishi1] which exerts a negative-feedback regulation on pancreatic enzyme secretion. It is well known that feed intake is suppressed by a feeling of satiety and by reducing gastric emptying in rats [@pone.0058115-Nishi1]. Gastric emptying is associated with the digestive and absorptive ability. Those suggested that the depressed digestive ability in this study may be in part contributed to the suppressed feed intake, which needs further investigations. In addition, when compared with those in the control, the digestive and absorptive enzyme activities in the Gln with β-conglycinin group showed no significant differences. These results indicated that dietary Gln exerted a mitigating role against the disruption in intestinal function caused by β-conglycinin. The reduction of digestive and absorptive capacity is usually due to poor growth of the digestive organs. The exocrine pancreas is the main site for digestive enzyme synthesis and secretion in fish [@pone.0058115-Guillaume1]. The present results showed that the weight and index of the hepatopancreas decreased with β-conglycinin. However, there is a lack of information in regard to the effects of β-conglycinin on the hepatopancreas in animals. Similar results were observed in cobia (Rachycentron canadum) that were fed diets with soybean products [@pone.0058115-Salze1]. In addition, the disruption of absorptive capacity in fish has been traced to the growth suppression of the intestine, which was reflected by the decrease of the intestinal length index in Atlantic salmon [@pone.0058115-Refstie1]. The present results showed that the intestinal weight, length, length index and folds height in the intestine was decreased with β-conglycinin alone. In the present in vitro study, β-conglycinin decreased the MTT OD values of enterocytes in a dose-dependent way. Although there are no reports concerning the effects of β-conglycinin on the growth and development of fish intestine, similar results in regard to decreased jejunum villus height induced by β-conglycinin were observed in rats [@pone.0058115-Han1]. Additionally, when fish were fed with Gln and β-conglycinin, the poor growth of the hepatopancreas and intestine caused by β-conglycinin was partly mitigated. The reduced growth and development of the digestive organs may be partly due to a reduction in protein deposition. This study showed that protein content in the intestine and in its enterocytes was decreased by β-conglycinin. As mentioned above, protein syntheses are regulated by TOR signaling [@pone.0058115-Schmelzle1]. Interestingly, the expressions of the TOR gene in the PI, MI and DI followed similar patterns to the protein content. The patterns of 4E-BP2 mRNA levels showed opposite tendencies to those of the TOR mRNA levels in the PI and DI, indicating that β-conglycinin increased the inhibition of translation. However, this study is the first to investigate the effects of β-conglycinin on the TOR/4E-BP pathway. Thus, the underlying mechanism by which β-conglycinin affects the expression of TOR and 4E-EP genes is largely unknown and needs to be further investigated. Gln can regulate TOR activity in HeLa cells [@pone.0058115-Nicklin1]. Interestingly, fish fed Gln with β-conglycinin presented a partly mitigated reduction of the TOR/4E-BP pathway caused by β-conglycinin. The impairment of intestinal growth and function by β-conglycinin might result from a destruction of intestinal integrity. Damage to the integrity of the intestine is a common phenomenon in animals consuming soybean proteins [@pone.0058115-Sun1]. Recently, β-conglycinin has been shown to result in intestinal epithelium damage in rats [@pone.0058115-Han1]. Moreover, β-conglycinin was shown to cause apoptosis and structural damage to enterocytes in mice [@pone.0058115-Xu1]. When the cell membrane is damaged, the important metabolic enzymes, such as LDH, GOT and GPT are released from the cells [@pone.0058115-Chen3]. Thus, the activity of LDH, GOT and GPT in the culture medium are often used to assess cell damage [@pone.0058115-Xu1], [@pone.0058115-Chen3]. This study showed that when the β-conglycinin level exceeded 2.5 mg/mL, the activities of LDH, GOT and GPT in the culture medium increased, indicating severe damage to the cellular membrane, which agrees with the results of the study on mouse intestinal epithelium cells [@pone.0058115-Xu1]. The intestinal injury caused by β-conglycinin may result from inflammation. It is well known that soybean protein might cause inflammation of the distal intestine in fish [@pone.0058115-Krogdahl1], [@pone.0058115-BakkeMckellep1]. IL-1 and TNF-α are indicated as pro-inflammatory, whereas TGF-β is indicated as an anti-inflammatory cytokine [@pone.0058115-Urn1]. The expression of the TNF-α gene in the β-conglycinin alone group is above the control level ([Figure 1a](#pone-0058115-g001){ref-type="fig"}), suggesting that β-conglycinin induced an inflammatory response in the DI of fish. It is well known that inflammation may lead to increase of water content, which may be an explanation of the decreased intestinal protein content caused by β-conglycinin. However, the expression of IL-8 gene was decreased by β-conglycinin. The reasons for these differences are largely unknown and may be partly associated with the sample period; it was reported that common carp fed SBM sampled at 0, 1, 3 and 5 weeks showed different patterns of IL-1, TNF-α and TGF-β mRNA levels in the DI [@pone.0058115-Urn1]. However, fish fed with Gln and β-conglycinin obtained the highest values of IL-1, TNF-α and TGF-β mRNA levels. TGF-β plays an important role in the prevention of mucosal inflammation [@pone.0058115-Lilleeng2]. Thus, the up-regulation of TGF-β levels by Gln may be involved in protecting against β-conglycinin-induced inflammation. However, these observations need more attention and additional measurements need to be made in other intestinal compartments to explain their significance. Intestinal inflammation may invoke a subsequent peroxidative damage due to excessive ROS production [@pone.0058115-Mahmood1]. In addition, the N-glycan structure of β-conglycinin is essential for the formation of dityrosine bridges, which is a ubiquitous process primarily responsible for oxidative stress [@pone.0058115-Baudry1]. MDA is one of the most easily assayed end products of lipid peroxidation [@pone.0058115-Requena1]. The current results showed that β-conglycinin elevated MDA content in the hepatopancreas and intestine as well as in its enterocytes, supporting the hypothesis that β-conglycinin may induce oxidative damage in these organs. It is known that the end products of lipid peroxidation may bring about protein damage [@pone.0058115-Bhor1]. Protein carbonyls (PC) are the oxidative product of amino acid residue and can be used as a biomarker of oxidative damage to protein [@pone.0058115-Baltacolu1]. In this study, the protein oxidations in the intestine and its enterocytes exhibited a similar trend to those of MDA. The study showed that Gln might protect fish enterocytes against H~2~O~2~-induced oxidative damage [@pone.0058115-Chen3]. However, in this study, lipid peroxidation (MDA) and protein oxidation (PC) in the hepatopancreas and intestine of fish fed with Gln and β-conglycinin only showed reduction trends when compared with those in the β-conglycinin alone group. The reasons for this result are still unknown. It may be partly because the dose of Gln used in this study was not large enough to inhibit β-conglycinin-induced oxidative damage. Several studies had indicated that oxidative damage was often accompanied by the reduction of antioxidant capacity, reflected by GSH content and the inhibition of antioxidant activity, including that of SOD, CAT, GPx, GST and GR [@pone.0058115-Chen3], [@pone.0058115-Jiang2]. Therefore, in the current study, non-enzymatic and enzymatic antioxidant responses were also measured. The current study demonstrated that the content of GSH in the hepatopancreas, intestine and its enterocytes was reduced by β-conglycinin. It has been reported that the gut was one of the major sites for GSH synthesis in rats [@pone.0058115-Cao1]. GSH synthesis in endothelial cells was through a process that required the activity of γ-GT [@pone.0058115-Moellering1]. Thus, in this study, the increase in γ-GT activity may correspond to a first attempt to overcome oxidative stress by producing a high amount of GSH. In agreement with this result, an increase of γ-GT activity was observed in the liver and white muscle of Nile tilapia exposed to ammonia [@pone.0058115-Hegazi1]; however, we do not know why GSH decreased when γ-GT activity increased in this study. We hypothesize that GSH was consumed during the scavenging process for the oxidation induced by β-conglycinin. Moreover, our results showed that with dietary β-conglycinin treatment alone, the activities of SOD, CAT, GPx and GR in the hepatopancreas and intestine and the activity of intestinal GST decreased significantly when compared with those in the control group. Moreover, significant reductions in SOD, CAT, GPx, GST and GR activity were also observed in enterocytes treated with a certain level of β-conglycinin. However, in contrast to the reduced activities of these enzymes, the mRNA levels of the CuZnSOD, MnSOD, CAT and GPx1b genes increased with β-conglycinin. β-conglycinin-induced increases in the mRNA expression of antioxidant enzymes may be related to an adaptive mechanism against stress. Concurring with this hypothesis, an over-expression of cardiac-specific CAT prevents injury to the heart following oxidative damage induced by exposure to the anticancer agent doxorubicin [@pone.0058115-Kang1]. Thus, perhaps these antioxidant enzymes were largely consumed during the defense against the oxidation caused by β-conglycinin. In addition, in the Gln with β-conglycinin group, most antioxidant enzyme activities and gene expressions are close or equal to those values in the control group. Our previous study showed that in enterocytes treated with Gln in the presence of H~2~O~2~, antioxidant enzyme activities were higher than treatment with H~2~O~2~ alone and were close to the values in the control group [@pone.0058115-Chen3], proving that β-conglycinin damages the antioxidant system of fish. Gln is a precursor of nucleotides, glucose, other amino acids and serves as a major fuel for intestinal epithelial cells in mammals [@pone.0058115-Windmueller1]. Recent studies have demonstrated that dietary Gln supplementation could improve growth performance, intestinal structure and function in carp [@pone.0058115-Lin2], as well as enhance the antioxidant defense in hybrid sturgeon (Acipenser schrenckii ♀×Huso dauricus ♂) [@pone.0058115-Zhu1]. According to these findings, we investigated the potential protection of Gln against β-conglycinin toxicity in the present study. As results, compared with the β-conglycinin alone group, dietary supplementation with Gln significantly increased SGR, feed intake, feed efficiency, hepatopancreas protein, hepatopancreas weight, intestinal protein, intestinal weight, intestinal length, intestinal length index and foregut folds height, and also significantly increased the activities of intestinal digestive enzymes including trypsin, lipase and chymotrypsin, absorptive enzymes including creatinkinase, Na^+^, K^+^-ATPase and alkaline phosphatase in the PI, MI and DI, as well as antioxidant enzymes including CAT and GR in hepatopancreas and SOD, CAT, GPx in the intestine. Moreover, SGR, intestinal length index, activities of intestinal trypsin, lipase, chymotrypsin, creatinkinase, Na^+^,K^+^-ATPase, alkaline phosphatase and GPx were restored to have no significant difference compared with the control. These results indicated that dietary Gln exerted a protective role against the growth suppression, intestinal integrity damage and function disruption induced by β-conglycinin. Conclusion {#s4a} ---------- The potential action pathway of β-conglycinin-induced poor growth of fish is presented in [Figure 2](#pone-0058115-g002){ref-type="fig"}. Briefly, this study indicates that β-conglycinin can cause oxidative damage (and impair antioxidant system in animals), and thus lead to damage and poor growth of digestive organs, subsequent by dysfunction of digestion and absorption, and finally reduce fish growth, which may provide some information to the mechanism of β-conglycinin-induced negative effects in fish. Moreover, since Gln was observed to mitigate negative influences of β-conglycinin, it is reasonable for us to recommend supplementation with Gln when high levels of SBM (β-conglycinin) are used in fish diets. ![The potential action pathway of β-conglycinin-induced depression of fish growth.](pone.0058115.g002){#pone-0058115-g002} Supporting Information {#s5} ====================== ###### Ingredients and nutrient content of the experimental diets (in vivo experiment). (DOC) ###### Click here for additional data file. ###### Real-time PCR primer sequences. (DOC) ###### Click here for additional data file. [^1]: Competing Interests:The authors have declared that no competing interests exist. [^2]: Primary responsibility for final content: X-QZ. Read and approved the final manuscript: J-XZ L-YG LF W-DJ S-YK YL KH JJ S-HL LT X-QZ. Conceived and designed the experiments: X-QZ LF. Performed the experiments: J-XZ L-YG W-DJ KH JJ S-HL. Analyzed the data: S-YK YL LT. 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"recovered", "Gln", "supplementation", "levels", "GR", "gene", "expressions", "showed", "similar", "trends", "significant", "differences", "among", "three", "groups", "Relative", "expression", "CuZnSOD", "MnSOD", "CAT", "GR", "genes", "whole", "intestine", "juvenile", "Jian", "carp", "Cyprinus", "carpio", "var", "Jian", "exposed", "dietary", "days", "vivo", "experiment", "Genes", "Control", "alone", "plus", "Gln", "CuZnSOD", "MnSOD", "CAT", "GR", "Data", "represent", "means", "five", "replicates", "Values", "within", "row", "different", "superscripts", "significantly", "different", "P", "vitro", "Experiment", "First", "vitro", "trial", "effects", "MTT", "OD", "protein", "retention", "PR", "transaminase", "GOT", "activity", "enterocytes", "activities", "LDH", "GOT", "GPT", "medium", "presented", "Table", "table", "MTT", "OD", "significantly", "reduced", "higher", "levels", "lowest", "MTT", "OD", "observed", "cells", "treated", "Cellular", "PR", "highest", "cells", "incubated", "control", "medium", "lowest", "cells", "exposed", "Cellular", "GOT", "activity", "lower", "cells", "exposed", "cells", "groups", "LDH", "medium", "significantly", "higher", "LDH", "control", "group", "GOT", "activities", "medium", "higher", "treatments", "GPT", "activities", "medium", "highest", "followed", "groups", "lowest", "groups", "noted", "cellular", "PR", "cellular", "GOT", "LDH", "GOT", "GPT", "medium", "significantly", "different", "among", "treatments", "Thus", "deleted", "doses", "second", "vitro", "trial", "Effects", "MTT", "OD", "protein", "retention", "PR", "µg", "transaminase", "GOT", "protein", "activity", "enterocytes", "activities", "lactate", "dehydrogenase", "LDH", "GOT", "transaminase", "GPT", "medium", "first", "vitro", "experiment", "MTT", "OD", "PR", "GOT", "LDH", "GOT", "GPT", "Data", "represent", "means", "four", "replicates", "Values", "within", "column", "different", "superscripts", "significantly", "different", "P", "Second", "vitro", "trial", "AKP", "PC", "cells", "LDH", "MDA", "medium", "presented", "Table", "table", "AKP", "activity", "cells", "decreased", "higher", "levels", "P", "difference", "shown", "P", "LDH", "MDA", "content", "medium", "significantly", "increased", "increasing", "levels", "P", "differences", "found", "increase", "concentration", "P", "Cellular", "PC", "increased", "higher", "levels", "P", "difference", "found", "P", "Effects", "activity", "alkaline", "phosphatase", "AKP", "protein", "enterocytes", "lactate", "dehydrogenase", "LDH", "medium", "content", "malondialdehyde", "MDA", "medium", "protein", "carbonyls", "PC", "protein", "enterocytes", "second", "vitro", "experiment", "AKP", "cell", "LDH", "MDA", "PC", "cell", "Data", "represent", "means", "four", "replicates", "Values", "within", "column", "different", "superscripts", "significantly", "different", "P", "Cellular", "SOD", "CAT", "GPx", "GST", "GR", "activities", "GSH", "content", "shown", "Table", "table", "SOD", "activities", "decreased", "higher", "levels", "P", "plateaued", "thereafter", "P", "decrease", "CAT", "activities", "observed", "presence", "lowest", "reached", "P", "GPx", "activities", "highest", "group", "containing", "followed", "GPx", "lowest", "presence", "P", "GST", "activity", "cells", "treated", "significantly", "decreased", "approximately", "compared", "cells", "incubated", "P", "GR", "activity", "GSH", "content", "also", "decreased", "higher", "levels", "P", "differences", "found", "increase", "concentrations", "P", "Activities", "SOD", "protein", "CAT", "protein", "GPx", "protein", "GST", "protein", "GR", "protein", "content", "GSH", "protein", "enterocytes", "exposed", "second", "vitro", "experiment", "SOD", "CAT", "GPx", "GST", "GR", "GSH", "Data", "represent", "means", "four", "replicates", "Values", "within", "column", "different", "superscripts", "significantly", "different", "P", "Discussion", "Soybean", "source", "protein", "due", "relatively", "composition", "amino", "acids", "However", "soybean", "also", "dietary", "allergic", "source", "threatens", "individuals", "primary", "storage", "protein", "identified", "one", "major", "allergenic", "proteins", "soybean", "Thus", "define", "mechanisms", "whereby", "excessive", "soybean", "induces", "growth", "depression", "animals", "study", "investigated", "role", "purified", "soybean", "separating", "effects", "effects", "factors", "Firstly", "present", "study", "observed", "fish", "fed", "diet", "exhibited", "poor", "weight", "gain", "also", "observed", "piglets", "rats", "However", "survival", "influenced", "dietary", "treatments", "reports", "exist", "concerning", "effects", "survival", "rate", "fish", "Similar", "findings", "observed", "replacement", "dietary", "fishmeal", "soy", "protein", "isolate", "influence", "survival", "indeed", "reduced", "growth", "performance", "Pacific", "white", "shrimp", "Litopenaeus", "vannamei", "reduction", "feed", "intake", "regarded", "primary", "factor", "responsible", "depressed", "growth", "observed", "rats", "present", "study", "feed", "intake", "group", "significantly", "lower", "control", "group", "accordance", "results", "rats", "result", "suggested", "growth", "reduction", "caused", "likely", "attributed", "suppression", "feed", "intake", "However", "information", "referring", "leads", "suppression", "feed", "intake", "fish", "rats", "reported", "peptone", "stimulating", "endogenous", "cholecystokinin", "CCK", "release", "suppresses", "food", "intake", "inducing", "feeling", "satiety", "reducing", "gastric", "emptying", "Besides", "feed", "intake", "feed", "utilization", "efficiency", "also", "influences", "growth", "performance", "present", "study", "depressed", "feed", "efficiency", "indicating", "reduction", "fish", "growth", "partly", "attributed", "fact", "decreased", "feed", "efficiency", "Interestingly", "fish", "administered", "Gln", "growth", "performances", "close", "equal", "control", "group", "showing", "Gln", "might", "mitigate", "poor", "growth", "caused", "initial", "step", "understanding", "reduced", "growth", "fish", "examined", "digestive", "absorptive", "ability", "fish", "study", "alone", "significantly", "decreased", "activities", "trypsin", "chymotrypsin", "lipase", "creatine", "kinase", "alkaline", "phosphatase", "intestine", "enterocytes", "Although", "reports", "exist", "concerning", "effects", "digestive", "absorptive", "enzyme", "activity", "fish", "similar", "findings", "observed", "diets", "containing", "soybeans", "might", "decrease", "activity", "protease", "tilapia", "alkaline", "phosphatase", "Atlantic", "salmon", "creatine", "kinase", "Atlantic", "halibut", "Hippoglossus", "hippoglossus", "rats", "reported", "reported", "peptone", "lumen", "stimulating", "endogenous", "cholecystokinin", "CCK", "release", "exerts", "regulation", "pancreatic", "enzyme", "secretion", "well", "known", "feed", "intake", "suppressed", "feeling", "satiety", "reducing", "gastric", "emptying", "rats", "Gastric", "emptying", "associated", "digestive", "absorptive", "ability", "suggested", "depressed", "digestive", "ability", "study", "may", "part", "contributed", "suppressed", "feed", "intake", "needs", "investigations", "addition", "compared", "control", "digestive", "absorptive", "enzyme", "activities", "Gln", "group", "showed", "significant", "differences", "results", "indicated", "dietary", "Gln", "exerted", "mitigating", "role", "disruption", "intestinal", "function", "caused", "reduction", "digestive", "absorptive", "capacity", "usually", "due", "poor", "growth", "digestive", "organs", "exocrine", "pancreas", "main", "site", "digestive", "enzyme", "synthesis", "secretion", "fish", "present", "results", "showed", "weight", "index", "hepatopancreas", "decreased", "However", "lack", "information", "regard", "effects", "hepatopancreas", "animals", "Similar", "results", "observed", "cobia", "Rachycentron", "canadum", "fed", "diets", "soybean", "products", "addition", "disruption", "absorptive", "capacity", "fish", "traced", "growth", "suppression", "intestine", "reflected", "decrease", "intestinal", "length", "index", "Atlantic", "salmon", "present", "results", "showed", "intestinal", "weight", "length", "length", "index", "folds", "height", "intestine", "decreased", "alone", "present", "vitro", "study", "decreased", "MTT", "OD", "values", "enterocytes", "way", "Although", "reports", "concerning", "effects", "growth", "development", "fish", "intestine", "similar", "results", "regard", "decreased", "jejunum", "villus", "height", "induced", "observed", "rats", "Additionally", "fish", "fed", "Gln", "poor", "growth", "hepatopancreas", "intestine", "caused", "partly", "mitigated", "reduced", "growth", "development", "digestive", "organs", "may", "partly", "due", "reduction", "protein", "deposition", "study", "showed", "protein", "content", "intestine", "enterocytes", "decreased", "mentioned", "protein", "syntheses", "regulated", "TOR", "signaling", "Interestingly", "expressions", "TOR", "gene", "PI", "MI", "DI", "followed", "similar", "patterns", "protein", "content", "patterns", "mRNA", "levels", "showed", "opposite", "tendencies", "TOR", "mRNA", "levels", "PI", "DI", "indicating", "increased", "inhibition", "translation", "However", "study", "first", "investigate", "effects", "pathway", "Thus", "underlying", "mechanism", "affects", "expression", "TOR", "genes", "largely", "unknown", "needs", "investigated", "Gln", "regulate", "TOR", "activity", "HeLa", "cells", "Interestingly", "fish", "fed", "Gln", "presented", "partly", "mitigated", "reduction", "pathway", "caused", "impairment", "intestinal", "growth", "function", "might", "result", "destruction", "intestinal", "integrity", "Damage", "integrity", "intestine", "common", "phenomenon", "animals", "consuming", "soybean", "proteins", "Recently", "shown", "result", "intestinal", "epithelium", "damage", "rats", "Moreover", "shown", "cause", "apoptosis", "structural", "damage", "enterocytes", "mice", "cell", "membrane", "damaged", "important", "metabolic", "enzymes", "LDH", "GOT", "GPT", "released", "cells", "Thus", "activity", "LDH", "GOT", "GPT", "culture", "medium", "often", "used", "assess", "cell", "damage", "study", "showed", "level", "exceeded", "activities", "LDH", "GOT", "GPT", "culture", "medium", "increased", "indicating", "severe", "damage", "cellular", "membrane", "agrees", "results", "study", "mouse", "intestinal", "epithelium", "cells", "intestinal", "injury", "caused", "may", "result", "inflammation", "well", "known", "soybean", "protein", "might", "cause", "inflammation", "distal", "intestine", "fish", "indicated", "whereas", "indicated", "cytokine", "expression", "gene", "alone", "group", "control", "level", "Figure", "fig", "suggesting", "induced", "inflammatory", "response", "DI", "fish", "well", "known", "inflammation", "may", "lead", "increase", "water", "content", "may", "explanation", "decreased", "intestinal", "protein", "content", "caused", "However", "expression", "gene", "decreased", "reasons", "differences", "largely", "unknown", "may", "partly", "associated", "sample", "period", "reported", "common", "carp", "fed", "SBM", "sampled", "weeks", "showed", "different", "patterns", "mRNA", "levels", "DI", "However", "fish", "fed", "Gln", "obtained", "highest", "values", "mRNA", "levels", "plays", "important", "role", "prevention", "mucosal", "inflammation", "Thus", "levels", "Gln", "may", "involved", "protecting", "inflammation", "However", "observations", "need", "attention", "additional", "measurements", "need", "made", "intestinal", "compartments", "explain", "significance", "Intestinal", "inflammation", "may", "invoke", "subsequent", "peroxidative", "damage", "due", "excessive", "ROS", "production", "addition", "structure", "essential", "formation", "dityrosine", "bridges", "ubiquitous", "process", "primarily", "responsible", "oxidative", "stress", "MDA", "one", "easily", "assayed", "end", "products", "lipid", "peroxidation", "current", "results", "showed", "elevated", "MDA", "content", "hepatopancreas", "intestine", "well", "enterocytes", "supporting", "hypothesis", "may", "induce", "oxidative", "damage", "organs", "known", "end", "products", "lipid", "peroxidation", "may", "bring", "protein", "damage", "Protein", "carbonyls", "PC", "oxidative", "product", "amino", "acid", "residue", "used", "biomarker", "oxidative", "damage", "protein", "study", "protein", "oxidations", "intestine", "enterocytes", "exhibited", "similar", "trend", "MDA", "study", "showed", "Gln", "might", "protect", "fish", "enterocytes", "oxidative", "damage", "However", "study", "lipid", "peroxidation", "MDA", "protein", "oxidation", "PC", "hepatopancreas", "intestine", "fish", "fed", "Gln", "showed", "reduction", "trends", "compared", "alone", "group", "reasons", "result", "still", "unknown", "may", "partly", "dose", "Gln", "used", "study", "large", "enough", "inhibit", "oxidative", "damage", "Several", "studies", "indicated", "oxidative", "damage", "often", "accompanied", "reduction", "antioxidant", "capacity", "reflected", "GSH", "content", "inhibition", "antioxidant", "activity", "including", "SOD", "CAT", "GPx", "GST", "GR", "Therefore", "current", "study", "enzymatic", "antioxidant", "responses", "also", "measured", "current", "study", "demonstrated", "content", "GSH", "hepatopancreas", "intestine", "enterocytes", "reduced", "reported", "gut", "one", "major", "sites", "GSH", "synthesis", "rats", "GSH", "synthesis", "endothelial", "cells", "process", "required", "activity", "Thus", "study", "increase", "activity", "may", "correspond", "first", "attempt", "overcome", "oxidative", "stress", "producing", "high", "amount", "GSH", "agreement", "result", "increase", "activity", "observed", "liver", "white", "muscle", "Nile", "tilapia", "exposed", "ammonia", "however", "know", "GSH", "decreased", "activity", "increased", "study", "hypothesize", "GSH", "consumed", "scavenging", "process", "oxidation", "induced", "Moreover", "results", "showed", "dietary", "treatment", "alone", "activities", "SOD", "CAT", "GPx", "GR", "hepatopancreas", "intestine", "activity", "intestinal", "GST", "decreased", "significantly", "compared", "control", "group", "Moreover", "significant", "reductions", "SOD", "CAT", "GPx", "GST", "GR", "activity", "also", "observed", "enterocytes", "treated", "certain", "level", "However", "contrast", "reduced", "activities", "enzymes", "mRNA", "levels", "CuZnSOD", "MnSOD", "CAT", "genes", "increased", "increases", "mRNA", "expression", "antioxidant", "enzymes", "may", "related", "adaptive", "mechanism", "stress", "Concurring", "hypothesis", "CAT", "prevents", "injury", "heart", "following", "oxidative", "damage", "induced", "exposure", "anticancer", "agent", "doxorubicin", "Thus", "perhaps", "antioxidant", "enzymes", "largely", "consumed", "defense", "oxidation", "caused", "addition", "Gln", "group", "antioxidant", "enzyme", "activities", "gene", "expressions", "close", "equal", 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1. Introduction {#sec1-foods-09-00918} =============== Global interest for natural food colors is on the rise, especially in Asian countries. Historically, food ingredients and spices like yellow turmeric and red pepper powders are easily prone to food adulteration \[[@B1-foods-09-00918]\]. They play very essential roles in traditional cuisines and are valued for their therapeutic properties irrespective of their color. Current innovation and developmental trend in food processing has led to a paradigm interest in food additives and bioactive compounds with particular focus on their nutritional and organoleptic values. Among the bioactive compounds, betalains and phenolics are the most auspicious with plenty of health benefits, making them easily compatible with food fortification and supplementation. The discovery of betanin, betanidin, and indicaxanthin derivatives following their degradation led to the assumption of their unique nature together with betaxanthin (formerly known as flavonoid) and betacyanin (regarded as nitrogenous anthocyanin) \[[@B2-foods-09-00918]\]. This led to them being grouped as betalains in 1968 \[[@B3-foods-09-00918]\]. Among the acknowledged sources of betalains, i.e., beetroot, swiss chard, dragon fruit, and prickly pear \[[@B3-foods-09-00918],[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918]\], beetroot has gained the most attention since, apart from the whole tuber, even their waste parts such as stalk \[[@B7-foods-09-00918]\], peel \[[@B8-foods-09-00918],[@B9-foods-09-00918]\], and pomace \[[@B10-foods-09-00918],[@B11-foods-09-00918]\] are rich in betalains. The ratio of color contents is decisively influenced by specie source, genetic, cultivar, or the environment of cultivation throughout growing, and the stages of harvesting \[[@B4-foods-09-00918],[@B12-foods-09-00918],[@B13-foods-09-00918]\]. Practically, one kilogram of fresh beetroot contains 300--600 mg of betacyanin and 320--420 mg of betaxanthin \[[@B14-foods-09-00918]\]; compared to the flesh, betalain concentration ranges from 40 to 70% higher in the peel and it also depends on genotypes \[[@B15-foods-09-00918]\]. As reducing agents, phenolic compounds can inhibit oxidation reaction catalyzed by enzymes and can stabilize lipid peroxidases \[[@B16-foods-09-00918]\]. Over 10,000 phenolic compounds exist in several kinds of fruits and vegetables, cereals, tea leaves, coffee beans, and their accumulation in the whole parts of the plant including root, stem, bark, flower, and leaf have been noticed \[[@B17-foods-09-00918]\]. Their extraction from various sources has been boosted by emerging technologies these days, particularly microwave radiation. Some of them are twenty cultivars of tomatoes \[[@B18-foods-09-00918]\], six plant species \[[@B19-foods-09-00918]\], four types of spices \[[@B16-foods-09-00918]\], four types of algae (brown macroalgae species) \[[@B20-foods-09-00918]\], and the rest are discussed in the next section with their proper process optimization. Balasundram and coworkers \[[@B21-foods-09-00918]\] reviewed phenolic compound structures found in by-products of fruit and vegetable processing. Extraction of phenolic compounds from these food products, however, remains a developing field as different techniques are continually being explored. Compared to conventional extraction techniques, non-conventional methods are more pronounced for better quality and quantity of desired products due to advantages such as less use of solvent and lower exposure time to high temperature \[[@B22-foods-09-00918]\]. Specifically, major advantages of microwave treatments are the reduction in operation time and solvent consumption (critically possibility of solvent-free processing), less energy consumption as the rays are delivered directly to the matrix, as well as their ease of handling and processing \[[@B23-foods-09-00918]\]. As reported by Cardoso-Ugarte and coworkers \[[@B24-foods-09-00918]\], the same amount of betalain concentration can be achieved by microwave irradiation ten times faster than the conventional ways with betalain yield scaled up to double. However, with dielectric heating, acceleration in the chemical reaction of target compounds such as epimerization, oxidation, and polarization during microwave processing should be considered \[[@B14-foods-09-00918]\]. The aim of this review was to point out the versatility of microwave irradiation in the recovery of bioactive compounds focusing on phenolics and betalains from various kinds of fruits and vegetables for their improved practical application in food and beverages, pharmaceuticals, cosmetics, and other related sectors. 2. Food Color Compounds {#sec2-foods-09-00918} ======================= Functional additives are compulsory in food processing in order to improve or maintain the existing properties of food during processing \[[@B25-foods-09-00918]\]. Bio-colorants have been explored from plants (flower, root, stalk, seed, fruit, peel, leaf, pomace, rhizome, and stigma), insect (cochineal), algae, bacteria, and fungi \[[@B25-foods-09-00918],[@B26-foods-09-00918],[@B27-foods-09-00918]\] and their existence is essential for pollination and seed disposal as insects and birds are attracted by their hues \[[@B28-foods-09-00918]\]. Carotenoids, phenolics, alkaloids, nitrogen-containing compounds, and organosulfur compounds are major classes of bioactive compounds. Among them, carotenoids (E160, E161, E164), chlorophylls (E140, E141), flavonoids (anthocyanin (E163)), and betalains (E162) are commonly used as natural colorants in food and cosmetic industries ([Table 1](#foods-09-00918-t001){ref-type="table"}) \[[@B3-foods-09-00918],[@B25-foods-09-00918],[@B28-foods-09-00918]\]. Since these compounds have characteristic absorption in the visible spectrum, it is possible to quantify them through UV-visible spectrophotometer within 380--700 nm wavelength range following Beer--Lambert's law. - Carotenoids, 40 carbon atoms possessing terpenoids, are derived from the condensation of geranylgeranyl-PP molecules \[[@B29-foods-09-00918]\]. They are lipid-soluble, basically found in cyanobacteria, algae, plants, some fungi and bacteria, and are produced intracellularly by bioproduction of microorganisms \[[@B29-foods-09-00918],[@B30-foods-09-00918]\]. Based on their chemical structure, they can be classified as hydrocarbon carotenoids and xanthophylls, and their extraction can easily be performed with nonpolar solvents \[[@B29-foods-09-00918]\]. - Flavonoids are water-soluble compounds, consisting of 15 carbon atoms (C6-C3-C6) and belong to the class of phenylpropanoids \[[@B21-foods-09-00918],[@B31-foods-09-00918],[@B32-foods-09-00918]\]. Flavonoids furnish intense color, texture, and taste in fruits and flowers, stretching to a wide range of fruit and vegetable parts mostly leaves, flowers, and skin of the fruits \[[@B17-foods-09-00918],[@B27-foods-09-00918]\]. The color variety and classification differ according to the structural groups such as hydroxyl, methyl, glucosyl, and acyl \[[@B32-foods-09-00918]\]. Anthocyanins (glycosylated and acylated) are groups of flavonoids derived from phenylalanine conferring coloration from pale yellow to blue with respect to pH changes \[[@B32-foods-09-00918]\]. Anthocyanins possess several therapeutic properties as they strongly exhibit free radical scavenging capacity \[[@B17-foods-09-00918]\]. They are abundantly found in berries, blackcurrant, and other purple color giving fruits and vegetables with host taste attributes \[[@B17-foods-09-00918],[@B28-foods-09-00918]\]. - Both anthocyanin and betalain (betacyanin) have UV protectable ability for host plant tissues \[[@B28-foods-09-00918]\]. Betalains have a wider range of pH (3--7) with yellow to red coloration within that range though less stable to temperature and light exposure as compared to anthocyanin \[[@B33-foods-09-00918]\]. Slavov and coworkers \[[@B34-foods-09-00918]\] investigated the color pattern resulting from a mixture of betalain and anthocyanin-rich fruit juices accompanying their functional properties as their coexistence has never been explored. 2.1. Betalains {#sec2dot1-foods-09-00918} -------------- Betalains are the main compounds associated with the displayed red color of flowers, fruits, and other plant tissues. Betalains are derivatives of tyrosine which are normally found in plant groups in the order of Caryophyllales including Amaranthaceae (Beta vulgaris), Cactaceae (Opuntia, Pitaya, or Pitahaya), Nyctaginaceae (Bougainvillea), Phytolaccaceae (Phytolacca americana), and Portulacaceae (Portulaca grandiflora) \[[@B2-foods-09-00918],[@B3-foods-09-00918],[@B27-foods-09-00918],[@B37-foods-09-00918]\]. In Caryophyllaceae and Molluginaceae families, anthocyanin is more pronounced than betalain \[[@B2-foods-09-00918],[@B28-foods-09-00918],[@B32-foods-09-00918]\]. Their existence in high-class fungi, for example, agaric Amanita muscaria, Hygrocybe, and Hygrophorus have been reported as well \[[@B2-foods-09-00918],[@B27-foods-09-00918],[@B33-foods-09-00918],[@B38-foods-09-00918]\]. Betalain compounds are accumulated in plant cell vacuoles, predominantly located in edible parts of plant tissues (epidermal and subepidermal), accompanied by other phytochemical compounds \[[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918],[@B37-foods-09-00918],[@B39-foods-09-00918]\]. Betalains are nitrogen-containing heterocyclic compounds with high water affinity. Their exhibition of antioxidant activity is mainly based on their functional structure, for example, betalamic acid possesses conjugated double bonds that serve as reducing agents via electrons sharing \[[@B4-foods-09-00918]\]. Additionally, their ability to stabilize even after electron donation is responsible for its antioxidant property \[[@B4-foods-09-00918]\]. Depending on the type of groups (imino compounds or amino acid/derivatives) attached to the chromosphere of betalains, which is betalamic acid \[4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridene-2,6-dicarboxylic acid\], two basic color compounds are derived \[[@B2-foods-09-00918],[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918],[@B14-foods-09-00918],[@B28-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918],[@B38-foods-09-00918]\]. Betaxanthin is obtained by conjugating betalamic acid with amines or amino acids, whereas betacyanin is derived from the condensation of cyclo-[l]{.smallcaps}-(3, 4-dihydroxylphenylalanine), known as cyclo-DOPA, or its glucosyl derivatives \[[@B2-foods-09-00918],[@B3-foods-09-00918],[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918],[@B14-foods-09-00918],[@B27-foods-09-00918],[@B28-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918],[@B38-foods-09-00918]\] ([Figure 1](#foods-09-00918-f001){ref-type="fig"}). In the UV visible region, betaxanthin and betacyanin are detectable at the maximum wavelength absorption of 480 and 540 nm, respectively \[[@B27-foods-09-00918]\]. Betacyanin is responsible for the red color but also more tolerant to heat and stable to processing conditions than the yellow color giving betaxanthin \[[@B3-foods-09-00918],[@B9-foods-09-00918],[@B39-foods-09-00918]\]. Betaxanthin sources are limited, which makes for their low market demand despite their availability in the form of essential dietary amino acids, luckily, Celosia species are available as alternative sources of bright yellow color betaxanthin \[[@B40-foods-09-00918]\]. Indicaxanthin with prolin group (abundant in Opuntia species) and vulgaxanthin I ([Figure 1](#foods-09-00918-f001){ref-type="fig"}d) with glutamine substituent, mostly found in Beta vulgaris species, are two prominent yellow color giving betaxanthins \[[@B4-foods-09-00918],[@B27-foods-09-00918],[@B28-foods-09-00918],[@B37-foods-09-00918]\]. Their presence incites the fluorescence property of betaxanthin \[[@B4-foods-09-00918],[@B38-foods-09-00918]\]. From the findings of Mikołajczyk-Bator and Pawlak \[[@B41-foods-09-00918]\], thermal degradation of red-violet pigments was three times lower than yellow color given compounds despite the antioxidant capacity of the latter, which tends to be higher after heat treatments. Betanin, gomphrenin, amaranthin, and bougainvillein are four major types of betacyanin differed by the substituent groups attached to cyclo-DOPA moiety in ortho position ([Figure 1](#foods-09-00918-f001){ref-type="fig"}g) \[[@B3-foods-09-00918],[@B4-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918],[@B38-foods-09-00918]\]. Betanidin is the basic structural unit of most betacyanin derivatives, followed by, betanin (betanidin 5-O-ß-glucoside) derives from glucosylation and acylation of aglycon betanidine (I) \[[@B3-foods-09-00918],[@B27-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918]\]. Betanin is the most stable red color compound, and its antioxidant ability is based on its donation of hydrogen and electron \[[@B4-foods-09-00918]\]. Presence of glucosyl substituent in C-6 hydroxyl group position and amine group in the ring system encourages radical scavenging activity of betacyanin \[[@B33-foods-09-00918],[@B41-foods-09-00918]\], attributes of its anti-oxidative stress-related disorders, anti-cancer, and anti-inflammatory properties in specific plants and vegetables \[[@B33-foods-09-00918]\]. Phyllocathin (betanidin 5-O-ß-malonyl-glucoside) and hylocerenin (betanidin 5-O-ß-\[3″-hydroxyl-3″-methyl-glutaryl\] glucoside) are acylated forms of betacyanin \[[@B37-foods-09-00918]\]. In addition, isobetanin, betanidin, isobetanidin, prebetanin, neobetanin coexist in a small amount apart from betanin \[[@B4-foods-09-00918],[@B34-foods-09-00918]\]. Those neo-derivatives are of interest due to their yellow color appearance derived from thermal degradation of Beta vulgaris and Opuntia species betanin \[[@B37-foods-09-00918]\]. Most degradation processes of betacyanin are isomerization, deglycosylation, dehydrogenation, hydrolysis, and decarboxylation \[[@B5-foods-09-00918],[@B37-foods-09-00918],[@B41-foods-09-00918]\]. Color variety of betalains is influenced by yellow color content to some extent \[[@B25-foods-09-00918]\], and their stabilities are determined by pH, light, heat, water activity, chelating agents, antioxidants, enzymatic reactions \[[@B4-foods-09-00918],[@B37-foods-09-00918],[@B43-foods-09-00918]\]. Peroxidases (POX), polyphenol oxidases (PPO), ß-glucosidase, and betalain oxidase are the most responsible enzymes for betalain degradation during processing \[[@B14-foods-09-00918],[@B37-foods-09-00918]\]. The disturbance of some endogenous enzymes can be overcome by blanching at 70 °C for 2 min \[[@B13-foods-09-00918],[@B27-foods-09-00918]\]. Betalain colorants are available in the form of concentrates \[[@B44-foods-09-00918]\] and dried powder through air, freeze, or spray drying \[[@B43-foods-09-00918],[@B45-foods-09-00918]\], and are normally used in dairy and confectioneries and even in meat products \[[@B27-foods-09-00918],[@B46-foods-09-00918]\], although addition in cold products is preferable because of their thermal sensitiveness. Furthermore, Sivakumar and coworkers \[[@B47-foods-09-00918]\] investigated their application for leather dying through the sonic extraction method. Coating of betalain pigments extracted from skin and pulp of Cactus fruit with acidified mucilage from the pulp of the fruit \[[@B48-foods-09-00918]\] or ionic gelation \[[@B49-foods-09-00918]\] can extend the shelf life as well as the stability of the pigments. Microencapsulation of beetroot pomace extracts and their applications in bakery products have also been explored recently \[[@B50-foods-09-00918]\]. 2.2. Phenolic Compounds {#sec2dot2-foods-09-00918} ----------------------- Phenolic compounds (C~6~H~5~OH), also known as polyphenols, are aromatic metabolites made up of a polar functional (−OH) group bonded directly to an aromatic hydrocarbon ring \[[@B21-foods-09-00918]\]. Their ease of bonding to hydrogen raises their hydrogen bond formation ability for efficient water solubility in aqueous media \[[@B4-foods-09-00918]\]. Phenolic compounds are derivatives of aromatic amino acid phenylalanine ubiquitous in most plant kingdom with different glycosylated forms ranging from a simple phenolic molecule to high complex polymer molecules \[[@B21-foods-09-00918],[@B51-foods-09-00918]\]. They are mostly localized in plant tissues (0.5--5 g in 100 g of dry weight) imparting pigments to their host \[[@B51-foods-09-00918]\]. As aromatic secondary metabolites, phenolics are anti-inflammatory, antimicrobial, and antithrombotic. They are willing to donate their electrons from the outermost shell to fulfil the leakage of free radicals and can extend the shelf-life of food products \[[@B16-foods-09-00918],[@B17-foods-09-00918],[@B21-foods-09-00918],[@B52-foods-09-00918]\]. Consequently, phenoxyl radicals (PhO˙) are developed after transferring the hydrogen atom to free radical, which in turn can conjugate with adjacent hydroxyl or amine groups to enhance their stabilities, making them resistant to further oxidation process \[[@B4-foods-09-00918]\]. Based on carbon atom numbers, phenolics, flavonoids, and tannins upon derivatization possess specific structures \[[@B21-foods-09-00918]\]. Among them, phenolic acids and flavonoids are the most active plant-based phenolic compounds (30% and 60% of total dietary polyphenols content) ([Figure 2](#foods-09-00918-f002){ref-type="fig"}), they can easily be extracted with water, supercritical or subcritical water, and alcoholic solvents \[[@B17-foods-09-00918],[@B53-foods-09-00918],[@B54-foods-09-00918]\]. Benzoic acid derivatives (C~6~--C~1~) and cinnamic acid derivatives (C~6~--C~3~) are the two main classes of phenolic acids \[[@B21-foods-09-00918]\], whereas flavonols, flavones, flavanones, flavanols, isoflavones, flavanonols, and anthocyanidins are unique flavonoid compounds with variation in heterocyclic ring structures \[[@B17-foods-09-00918],[@B21-foods-09-00918],[@B31-foods-09-00918]\]. 3. Generalities of Solid-Liquid Extraction ========================================== Classical extraction of target compounds from different types of matrix can be achieved through several cold or hot extraction methods: distillation; solvent extraction such as percolation, maceration, infusion, enfleurage; and cold compression \[[@B55-foods-09-00918]\]. Solvent selection should be suited with specific properties of compounds of interest. Apart from water, polar solvents such as alcoholic solvents are extensively used to upgrade extraction efficiency, although there are some oppositions of their application in the matter of total solid content increase \[[@B56-foods-09-00918]\]. Between 20%--50% v/v methanol or ethanol are the fundamentally used solvents for complete extraction of betalains. Being a non-toxic green solvent, extractions with ethanol solvent do not need any further purification step, while methanol is preferable for denaturation of enzymes and for overcoming the interference of water-soluble protein \[[@B14-foods-09-00918],[@B27-foods-09-00918],[@B33-foods-09-00918]\]. Biosynthesis reaction of compounds present in the matrix can be handled by acidifying the extraction medium; for example, enzymatic decolorization of betalain can be retarded by ascorbic acid, which interferes with oxidative activity of polyphenol oxidases or ß-gluconolactone and inhibits ß-glucosidase \[[@B33-foods-09-00918]\]. Moreover, ascorbic acid has some preservative effects on Amaranthus betacyanin, improved the stability of red color betalain, and upgraded yield of betanin to the highest as well \[[@B57-foods-09-00918]\]. However, as exogenous antioxidants, care should be taken in the application of ascorbic acids for antioxidant activity determination \[[@B14-foods-09-00918]\]. Chelating agents such as citric acid and EDTA (ethylene-diamine-tetra-acetic acid) are also prominent for acidification extraction of bioactive compounds in order to boost their yield \[[@B4-foods-09-00918]\]. In some cases, inorganic acid like hydrochloric acid is favorable to prevent the activity of endogenous enzymes \[[@B8-foods-09-00918],[@B27-foods-09-00918]\]. 4. Emerging Technology {#sec4-foods-09-00918} ====================== Handling of perishable vegetables is a big challenge for food processing industries and has led to the innovation of processing techniques to assure food quality and at the same time minimize food loss. Minimal processing is the preparation of food with minimal treatment and the smallest changes in food quality through modern processing technology. Attempts have been made to bring practical usage from emerging technologies to minimal food processing in the areas of tempering, vacuum drying, freeze-drying, dehydration, cooking, baking, roasting, pasteurization, sterilization, extraction, blanching, and direct microwave blanching \[[@B58-foods-09-00918]\]. Based on the characteristics of targeted products, thermal treatment methods (ultra-high temperature processing, aseptic or semi-aseptic heat treatment, sous-vid, infrared heating, high frequency or radiofrequency heating, ohmic heating, microwave heating, inductive electrical heating, etc.) and non-thermal treatment methods (low direct current electric fields, ionizing radiation, gamma irradiation, pulsed electric fields (PEF), UV light, pulsed light, laser light heating, ultrasonic wave heating, high-pressure processing, etc.) have been developed \[[@B59-foods-09-00918]\]. Currently used emerging technologies for the extraction of secondary plant metabolites (betalains and phenolic compounds) are microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), high pressure and temperature extraction (HPTE), supercritical fluids extraction (SFE), pressurized liquids extraction (PLE), pulse electric field extraction (PEF), gamma-irradiation-assisted extraction, and extraction by low direct current electrification \[[@B7-foods-09-00918],[@B8-foods-09-00918],[@B16-foods-09-00918],[@B18-foods-09-00918],[@B19-foods-09-00918],[@B20-foods-09-00918],[@B24-foods-09-00918],[@B34-foods-09-00918],[@B42-foods-09-00918],[@B46-foods-09-00918],[@B47-foods-09-00918],[@B52-foods-09-00918],[@B54-foods-09-00918],[@B57-foods-09-00918],[@B60-foods-09-00918],[@B61-foods-09-00918],[@B62-foods-09-00918],[@B63-foods-09-00918],[@B64-foods-09-00918],[@B65-foods-09-00918],[@B66-foods-09-00918],[@B67-foods-09-00918],[@B68-foods-09-00918],[@B69-foods-09-00918],[@B70-foods-09-00918],[@B71-foods-09-00918],[@B72-foods-09-00918],[@B73-foods-09-00918],[@B74-foods-09-00918],[@B75-foods-09-00918],[@B76-foods-09-00918],[@B77-foods-09-00918],[@B78-foods-09-00918],[@B79-foods-09-00918],[@B80-foods-09-00918],[@B81-foods-09-00918],[@B82-foods-09-00918],[@B83-foods-09-00918],[@B84-foods-09-00918],[@B85-foods-09-00918],[@B86-foods-09-00918],[@B87-foods-09-00918],[@B88-foods-09-00918],[@B89-foods-09-00918],[@B90-foods-09-00918]\]. 4.1. Microwave Irradiation {#sec4dot1-foods-09-00918} -------------------------- In green chemistry, MAE is a well known novel technology with comparative advantages of reaction time reduction and lower or no solvent requirement over conventional extraction techniques \[[@B91-foods-09-00918]\]. Electromagnetic waves with frequencies between 300 MHz and 300 GHz are noted as microwave and previously applied for navigation and telecommunication operations \[[@B53-foods-09-00918],[@B92-foods-09-00918]\]. Based on their application purposes, two frequency ranges are classified as 2450 MHz for home usage with adjustable power output \<1000 W and 915 MHz for the industrial one \[[@B23-foods-09-00918],[@B53-foods-09-00918],[@B55-foods-09-00918],[@B91-foods-09-00918]\]. Being a segment of the electromagnetic spectrum, like visible light, the phenomenon of bending, reflection, refraction, and absorption of microwave radiation by the medium through which it passes are unavoidable \[[@B92-foods-09-00918]\]. Likewise, the degree of absorptivity or transmissivity of transmitted waves relies on the electromagnetic properties of the treated object, for instance, dielectric property, polarity, permittivity, and the shape of the object. Another important factor is in situ water content of the matrix as the level of swelling and rupture of the matrix can be varied with it \[[@B93-foods-09-00918]\]. The contradiction of electromagnetic wave is based on its electric and magnetic wave propagation oscillating in a perpendicular direction to each other with wavelength (1 m\~1 mm) \[[@B23-foods-09-00918],[@B53-foods-09-00918],[@B55-foods-09-00918],[@B91-foods-09-00918]\]. Herein, the electric part if only absorbed by natural biological materials \[[@B94-foods-09-00918]\] consequently leads to heating process occurring in the medium with absorption property of microwave \[[@B55-foods-09-00918],[@B91-foods-09-00918]\]. Microwave radiation is non-ionized with photon energy ranging from 3.78 × 10^−6^ eV to 1.01 × 10^−5^ eV, and interaction process occurs by heat conduction mode \[[@B55-foods-09-00918]\]. Moreover, the transformation phenomenon of kinetic to thermal energy is mainly concerned with the polarization potential of the polar molecules and their surroundings \[[@B23-foods-09-00918],[@B92-foods-09-00918]\]. The protocol is that once polar molecules present in a substance are hit by the electromagnetic beams, they become energetic and swing with the alternative movement of electric field, and consequently the alternative action of alignment and realignment of polar molecules creates friction between them, which in turn lead to the heating up of the surroundings \[[@B53-foods-09-00918],[@B92-foods-09-00918]\] ([Figure 3](#foods-09-00918-f003){ref-type="fig"}). Meanwhile, ionic components which are present in the substance migrate according to the electric field \[[@B91-foods-09-00918]\] ([Figure 3](#foods-09-00918-f003){ref-type="fig"}). This intermolecular friction and ionic movements occur several million times (4.9 × 10^9^ times at a frequency of 2450 MHz) per second and raise the internal pressure of cell \[[@B91-foods-09-00918]\]. Consequently, higher internal pressure encountered due to rapid vaporization of in situ water could rupture the cell wall and enclosed substances inside the cell wall could be forced out of the cell at a high rate \[[@B23-foods-09-00918]\]. 4.2. Mathematical Terms {#sec4dot2-foods-09-00918} ----------------------- Electric loss tangent (tan δ) of materials, which is also known as dissipation factor, quantifies the transformation of electric and magnetic energy to thermal energy in the materials; it can be explained in terms of dielectric loss (ε″) and dielectric loss constant (ε′) as given below in (Equation (1)) \[[@B23-foods-09-00918],[@B91-foods-09-00918],[@B92-foods-09-00918],[@B94-foods-09-00918]\]:$$D_{f} = \tan\delta = \frac{\varepsilon^{''}}{\varepsilon^{\prime}}$$ Absorption, transmission, and reflection abilities of different materials, known as electric permittivity, can be tracked in several ways. The absolute permittivity elucidates only how the material interacts with applied electromagnetic waves for nonionizing radiation and nonmagnetic materials \[[@B94-foods-09-00918]\]. Alternatively, it can be said that nonmagnetic materials rely on dielectric permittivity (ε\) to interact with electromagnetic waves and can be expressed by dielectric constant (ε′) and dielectric loss (ε″) shown in (Equation (2)); \[[@B94-foods-09-00918]\]:$$\varepsilon^{}~ = ~\varepsilon_{r}^{\prime} - j\varepsilon_{r}^{''}$$ Real and imaginary parts of permittivity represent dipolar oscillation and damping \[[@B92-foods-09-00918]\], and are valued depending on the motion of dipoles, nature of materials, high or low frequency, elevated or reduced temperature, and also the concentration of aqueous ionic solution \[[@B94-foods-09-00918]\]. Commonly used solvents with their loss tangent values are listed in [Table 2](#foods-09-00918-t002){ref-type="table"}. Relative permittivity (ε) can be estimated from electric displacement field of material (D, C · m^−2^) which is divided by vacuum (D~vacuum~, C · m^−2^) \[[@B92-foods-09-00918]\]. In dielectric material, conversion of electrical energy into thermal energy is defined by its loss factor. Consequently, dipole rotation and electrical conduction are assumed as loss factors in microwave heating, (Equation (3)):$$\varepsilon_{r}^{''}~ = ~\varepsilon_{d}^{''} + \varepsilon_{\sigma}^{''}$$ where ε~d~″ is relative dipole loss and ε~σ~″ is relative ionic loss. If the components are homogeneous, their shape is more responsible for the effective permittivity of the mixtures, especially when the particle size is smaller than the wavelength \[[@B94-foods-09-00918]\]. Literally, ordinary foods possess the penetration depth of electromagnetic waves (10--15 mm) \[[@B94-foods-09-00918]\]. The penetration level of microwave through the substance can be traced by the degree of reflection, transmission, and absorption of the wave by the host substance; for example, pure water has a low degree of reflection and transmission but a high degree of absorption of the wave \[[@B92-foods-09-00918]\]. Dissipated microwave power (P~D~, W) in a material can be estimated from electric field strength (E, V · m^−1^), and frequency (f, Hz) as reported by \[[@B92-foods-09-00918],[@B94-foods-09-00918]\] (Equation (4)):$$P_{D} = k~ \cdot E^{2} \cdot f \cdot \varepsilon^{''}$$ where k is a constant value of 55.61 × 10^−14^ C · m^2^ · V^−1^. Due to direct proportionality of dissipated microwave power (P~D~) to the absorptivity of the material, the attenuation of microwave differs according to the depth of penetration (z) and can be estimated as expressed in Equation (5):$$z = \frac{\mathsf{\lambda}}{2\pi} \cdot \sqrt{\frac{2}{\varepsilon^{\prime}\left( {\sqrt{1 + tan^{2}\delta} - 1} \right)}}$$ Frequency of applied radiation (f, Hz) and speed of the wave (c, m · s^−1^) can be used to evaluate wavelength ($\mathsf{\lambda}$, m) \[[@B92-foods-09-00918]\]. 4.3. Microwave-Assisted Extraction (MAE) of Betalains and Phenolic Compounds {#sec4dot3-foods-09-00918} ---------------------------------------------------------------------------- Microwave irradiation is proposed as an effective alternative way of recovering bioactive compounds from agro-industrial wastes with superior benefits such as high reproducibility within short treatment period, minimal solvent and energy consumption, and simple manipulation as opposed to classical solid--liquid-based extraction methods (soxhlet, rotary, maceration, and heat refluxing), supercritical water extraction, and even ultrasonic extraction \[[@B16-foods-09-00918],[@B18-foods-09-00918],[@B20-foods-09-00918],[@B52-foods-09-00918],[@B54-foods-09-00918],[@B60-foods-09-00918],[@B61-foods-09-00918],[@B62-foods-09-00918],[@B63-foods-09-00918],[@B64-foods-09-00918]\]. In advance, MAE has brought 1.7 times greater yield percent of phenolic \[[@B65-foods-09-00918]\], whilst operation time was two times shorter than UAE \[[@B54-foods-09-00918]\]. Alongside, 5 min of microwave irradiation could bring an equivalent amount of phenolic and flavonoid compounds with an hour of ultrasonic treatment time \[[@B61-foods-09-00918]\]. However, UAE was pointed out as the better choice in the investigations of \[[@B22-foods-09-00918],[@B63-foods-09-00918]\], in this case, the discrepancy in applied frequency and power wattage should be taken into account. Somehow, it is undeniable that both MAE and UAE are better in internal or external mass transfer compared to classical methods. In UAE, the formation of tiny gas bubbles in the solvent medium by mechanical wave encourages several micro-injections into the cell wall, which improves the solvent transport. Herein, sonication of water can propagate highly reactive hydroxyl radicals which may have some effect on the extraction of targeted products \[[@B52-foods-09-00918],[@B66-foods-09-00918]\]. Heating mode, duty cycle, power or heat energy, and temperature in an advanced microwave set up are basic operational parametric modes in MAE of betalains and phenolic compounds. Response surface methodology (RSM) is a suitable tool to approach the efficiency of MAE through the interaction of process variables despite the fact that the predicted values estimated by the model are only within the experimental limit \[[@B9-foods-09-00918],[@B18-foods-09-00918],[@B62-foods-09-00918],[@B63-foods-09-00918],[@B67-foods-09-00918],[@B68-foods-09-00918],[@B69-foods-09-00918],[@B70-foods-09-00918],[@B71-foods-09-00918],[@B72-foods-09-00918],[@B73-foods-09-00918]\]. Central composite design was mostly chosen for the investigation of each or parallel effects of process variables on the corresponding response. Based on the response level whether high or low, the weight of the factor on the response could be judged. Here again, the levels of the factors optimized by RSM support to be in between, i.e., neither at most nor lowest level or else the performed limits are regarded as insufficient for the design to fully predict the optimum condition of the variables within those limits \[[@B26-foods-09-00918]\]. For example, it can be said that the model well predicted the optimum condition of the extraction process if the highest amount of total phenolic compounds (39 mg GAE/gDW) was achieved at ethanol concentration (31.33%), solvent to material ratio (32.21 mL/g), temperature (52.24 °C) among the variables which were ethanol concentration (20--40%), solvent to material ratio (20--40 mL/g), temperature (40--60 °C) \[[@B73-foods-09-00918]\]. [Table 3](#foods-09-00918-t003){ref-type="table"} and [Table 4](#foods-09-00918-t004){ref-type="table"} represent some reported optimum conditions, within experimental variable ranges, for the extraction of betalains and phenolic compounds from fruit and vegetable as well as their wastes. In advance, some example of power output and time setting to accomplish MAE of secondary plant metabolites are demonstrated in [Figure 4](#foods-09-00918-f004){ref-type="fig"} and [Figure 5](#foods-09-00918-f005){ref-type="fig"}. In MAE, both the solvent and the sample undergo transformation by electromagnetic energy of high value based on ionic conduction and dipole rotation (either permanent or induced by the electric field). Ionic conduction, which is due to the electrophoretic migration of dissolved ions, increases solvent penetration into the matrix and facilitates the solvation of targeted compounds \[[@B91-foods-09-00918]\]. Influence of solvent type on microwave extraction of desired phenolic compound quantity has been investigated with pure water, pure and aqueous forms of acetone, ethanol and methanol, and of them, 50% (v/v) ethanol was observed to be most effective \[[@B61-foods-09-00918],[@B64-foods-09-00918],[@B78-foods-09-00918],[@B80-foods-09-00918]\]. Presence of a suitable amount of water in the solvent somehow can improve the swelling of the plant matrix, increasing the contact surface area of the matrix with the solvent \[[@B62-foods-09-00918]\]. However, it is controversial whether inert solvent to microwave radiation is better than the absorbed one or not as acetone performed better in phenolic compounds extraction possibly due to transparent property to microwave radiation subsequently allowing plant cells receive direct contact of the radiation \[[@B19-foods-09-00918]\]. Moreover, it should be noted that microwave can rapidly lose its energy before it reaches the innermost part of the medium if the medium it passed through is in high absorptivity of radiation \[[@B92-foods-09-00918]\]. Applied power output for microwave irradiation influences phenolic compounds \[[@B73-foods-09-00918]\], and it is obvious if irradiation time is short \[[@B59-foods-09-00918]\], which possibly leads to accelerated diffusion of the solvent into the matrix and in turn leaching out of the materials \[[@B23-foods-09-00918]\]. Prolonged heating via elevated temperature treatments can jeopardize pigment retentions; in this case, the acidification method can be considered for decompartmentalization of color compounds after microwave treatment \[[@B24-foods-09-00918]\]. Synergistic effects of processing time and temperature or microwave power, solvent concentration, and matrix-to-solvent ratio on mass transfer rate differ for specific secondary plant compounds, thus process condition adjustment should be focused on targeted compounds in order to distribute radiations uniformly \[[@B17-foods-09-00918],[@B24-foods-09-00918],[@B73-foods-09-00918]\]. Reported SEM (scanning electron microscopy) results showed that cell wall thinning and holes formation were prominent in microwave treatment rather than traditional heating modes \[[@B65-foods-09-00918],[@B80-foods-09-00918],[@B82-foods-09-00918],[@B95-foods-09-00918],[@B96-foods-09-00918]\]. This is because microwave penetrates the matrix and denaturalizes the cell wall in which bioactive compounds are trapped in a short time, subsequently letting them diffuse quickly into the solvent medium. However, the superheating effect can be encountered as a consequence, which is more pronounced with pure water solvent due to its quite low dissipation factor but high dielectric constant, facilitating the absorption of the microwave \[[@B92-foods-09-00918]\]. Since microwave heating is based on the alignment of the polar molecules back and forth, the transformation of kinetic energy to thermal energy is more intensive when their movement cannot catch up the frequency of applied microwave, which is the so-called dumping process \[[@B62-foods-09-00918]\]. That is why intermittent modes of microwave irradiation and cooling in between extraction steps were found to be effective \[[@B21-foods-09-00918],[@B24-foods-09-00918],[@B52-foods-09-00918],[@B61-foods-09-00918],[@B64-foods-09-00918],[@B81-foods-09-00918]\]. 5. Other Applications {#sec5-foods-09-00918} ===================== Apart from betalain and phenolic compounds, MAE has been widely used in the extraction of pectin with some improvements in quantity and quality, degree of esterification, and gel strength \[[@B97-foods-09-00918]\]. Moreover, essential oil extractions from different sources via advanced microwave-assisted techniques have been realized \[[@B93-foods-09-00918],[@B98-foods-09-00918],[@B99-foods-09-00918]\]. The combination of novel extraction methods which are microwave, ultrasonic wave, enzymatic, and also alkali assisted extraction have been complimented as well \[[@B90-foods-09-00918],[@B95-foods-09-00918],[@B100-foods-09-00918],[@B101-foods-09-00918]\] ([Table 5](#foods-09-00918-t005){ref-type="table"}). Other improved MAE methods are in situ extraction of essential oils with microwave oven \[[@B102-foods-09-00918]\], MAE of multi-elements from food materials via perchloric acid and hydrogen peroxide \[[@B103-foods-09-00918]\], MAE and GC-FID quantification of total branched-chain fatty acids in lamb subcutaneous adipose tissue \[[@B104-foods-09-00918]\]. Additionally, some other modified techniques of microwave irradiation such as vacuum microwave-assisted extraction, nitrogen-protected microwave-assisted extraction, dynamic microwave-assisted extraction, pressurized solvent free microwave-assisted extraction, radiation-assisted hydro-distillation, vacuum microwave hydro-distillation, microwave-assisted steam distillation, vacuum microwave-assisted hydro-gravity, microwave dry-diffusion and gravity have been mentioned in the reviews of \[[@B23-foods-09-00918],[@B91-foods-09-00918]\]. Apart from extraction, microwave application has been extended to processing of beetroots which has more effectiveness in oxidative stability of mayonnaise than roasted or boiled ones, thereby preserving betalains and phenolic compounds \[[@B105-foods-09-00918]\]. 6. Conclusions {#sec6-foods-09-00918} ============== As natural bioactive compounds rich in antioxidants, therapeutic properties of betalain and phenolic compounds are numerous. Although they are sensitive to heat, pH, light, etc., not only during processing but also throughout the storage, their presence in food wastes and residues even after processing is noteworthy as well. Currently, their practical usages in food industries, fortification, and supplementation of foods, cosmetics, and pharmaceuticals are thriving. Therefore, further thorough investigations for extraction of these valuable compounds via modern technologies, meantime maintaining their original properties as much as possible, are still needed to be explored. Most of MAE discussed in this review are operated in batch, although semi-continuous or continuous modes are also available. Since microwave irradiation encourages the direct interaction of vast electromagnetic waves and the target material, higher power ensures enough contact of the microwave with the matrix to expedite the isolation of the compounds trapped in the cell wall. Therefore, high temperature or power and short irradiation time is a beneficial combination for MAE of plant materials. Higher extraction time is not necessary once after the equilibrium state between the solvent and the matrix is reached since it can lead to the degradation of most bioactive compounds. All in all, if it is in proper use, microwave can be the best option for bioactive compounds extraction for the sake of simplicity as it can easily be set up or run by the home used microwave oven, high efficiency as the heating is built at the innermost part of the matrix and diffuses to the surrounding, saving energy as the operation can be completed in few minutes, and cleanliness as it is the only way of solvent free or lowest solvent used extraction. We appreciate the Doctoral School of Food Science, Szent Istvan University for the support accomplishing this work as well as the European Union's Horizon 2020 research and innovation programme under the Marie Sklowdowska---Curie grant No: 765860 (AQUAlity). Conceptualization, M.M.Z.; methodology, M.M.Z.; software, M.M.Z., C.B.A.; resources, M.M.Z., C.B.A.; writing---original draft preparation, M.M.Z.; writing---review and editing, M.M.Z., C.B.A.; supervision, S.B. All authors have read and agreed to the published version of the manuscript. This study was financially supported by the European Union and the European Social Fund (grant agreement no.EFOP-3.6.3-VEKOP-16-2017-00005), Tempus Public Foundation, under the Stipendium Hungaricum Scholarship Program. The authors declare no conflict of interest. ![Derivation of betacyanin and betaxanthin color compounds from betalamic acid: (a) Betalamic acid which is core structure of betalain, (b) Amino acid which conjugates with betalamic acid to derive betaxanthin, (c) Yellow color giving betaxanthin, (d) Vulgaxanthin I which is a derivative of betaxanthin, (e) cyclo-DOPA which condenses with betalamic acid to give betacyanin, (f) Red-violet color giving betacyanin, and (g) Betanin which is a derivative of betacyanin \[[@B2-foods-09-00918],[@B5-foods-09-00918],[@B14-foods-09-00918],[@B27-foods-09-00918],[@B28-foods-09-00918],[@B37-foods-09-00918],[@B42-foods-09-00918]\].](foods-09-00918-g001){#foods-09-00918-f001} ![Basic structures of phenolic compounds: (a) Backbone structure of phenolic acid, and (b) Structure of flavonoid moiety \[[@B21-foods-09-00918],[@B31-foods-09-00918]\].](foods-09-00918-g002){#foods-09-00918-f002} ![Treatment of microwave heat radiation for secondary plant metabolites extraction: (a) Domestic microwave oven heating system, (b) Heat diffusion from inside of the matrix to solvent medium and the surroundings, (c) Alignment of dipolar molecules and ions with electromagnetic wave, (d) Friction and rotation of dipolar molecules which heat up the matrix, and (e) Movements of ions with the electromagnetic wave.](foods-09-00918-g003){#foods-09-00918-f003} ![Ranges of power output for extraction of secondary plant metabolites (betalains and phenolic compounds).](foods-09-00918-g004){#foods-09-00918-f004} ![Ranges of microwave irradiation time (min) for extraction of phytochemicals (betalains and phenolic compounds).](foods-09-00918-g005){#foods-09-00918-f005} foods-09-00918-t001_Table 1 ###### Natural colors applied in the food and cosmetic industries, their E numbers, and common sources \[[@B3-foods-09-00918],[@B25-foods-09-00918],[@B28-foods-09-00918],[@B35-foods-09-00918],[@B36-foods-09-00918]\]. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Group Compound/Product Colour Code Common Sources ------------------------------ -------------------------------------------------- --------------------------------------------------------------------------------------------------------------- ------------------- -------------------------------------------------------------------------------------------- \- Curcumin, turmeric oleorisin Yellow-orange E100 Turmeric \- Riboflavin (vitamin B2), riboflavin-5′-phosphate Yellow-orange E101(i), E101(ii) Eggs, green vegetables, milk and other dairy products, meat, mushroom, almond \- Riboflavin-5-sodium phosphate Yellow E106 Eggs, organ meats (kidney and liver), lean meat, milk, green vegetables \- Carminic acid Magenta-red/ crimson E120 Cochineal insect \- Chlorophyll Green E140, E141 Grass, alfalfa leaf, Tagetes erecta (marigold flowers), spinach \- Caramel Brown E150 Modified sugar \- Vegetable carbon Black E153 Vegetables Carotenoid α-carotene,\ Orange-red, red, yellow, amber, brown E160a Carrot, pumpkin, apricot, sweet potato, beans, spinach, kale,\ β-carotene,\ collard greens, papaya, bell peppers, tomatoes, green leafy vegetables γ-carotene Carotenoid Annatto Orange-red E160b Fruit of the achiote tree Carotenoid Paprika oleoresin Red E160c Capsicum annuum or Capsicum frutescens Carotenoid Lycopene Bright to deep red E160d Solanum lycopersicum (tomatoes), watermelon Carotenoid β-Apo-8′-carotenal Orange to orange-red E160e Spinach, citrus fruits Carotenoid Lutein Orange-red to yellow E161b Green leafy vegetables and fruits, yellowish flowers Carotenoid Canthaxanthin Violet E161g Mushroom, crustaceans, fish, eggs Nitrogen containing compound Betalains/ betanin Red violet to yellow (pH \< 3 (more reddish), pH \> 7 (more yellowish)) E162 Mangosteen, beetroot, dragonfruit, red cabbage, swiss chard, Opuntia Phenolic Anthocyanin Dark purple (pH 1 (red), pH 4--5 (colorless), pH \< 7 (purple), pH \< 8 (deep blue), pH \< 12 (yellow/brown)) E163 Black currant, berries, cherry, plum, grape, red cabbage, Opuntia, hibiscus, rose, onion Carotenoid Saffron Yellow-orange-red E164 Crocus sativus ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ foods-09-00918-t002_Table 2 ###### Reported mathematical parametric values associated with commonly used solvents for phytochemical recovery through MAE (Microwave-Assisted Extraction). Solvent Loss Tangent Dielectric Constant (ε′) Dielectric Loss (ε″) Reference ---------- -------------- -------------------------- ---------------------- ----------------------------------------------------------------------- water 0.123 80.4 9.8892 \[[@B23-foods-09-00918],[@B53-foods-09-00918],[@B58-foods-09-00918]\] ethanol 0.941 25.7 24.1837 \[[@B23-foods-09-00918],[@B53-foods-09-00918]\] methanol 0.659 32.7 21.5493 \[[@B23-foods-09-00918],[@B53-foods-09-00918]\] acetone 0.054 20.6 1.1124 \[[@B53-foods-09-00918]\] foods-09-00918-t003_Table 3 ###### Recovery of betalain color compounds through microwave irradiation. Raw Material Solvent Product Process Optimum Conditions Reference ------------------------------- -------------------------------- ------------------------------------ ------------------------------------ ------------------ --------------------- --------------------------- Red beetroot peel acidified water betanin 229.26 mg/L (predicted by RSM) 0.95 min 224.61 W \[[@B8-foods-09-00918]\] ethanol 472.11 mg/L (predicted by RSM) 1.25 min 384.25 W Red beetroot pure water betaxanthin 1.25 mg/g of freeze dried red beet 2.2 min/ 1.7 min 400 W, 100% nominal \[[@B24-foods-09-00918]\] acidified water betacyanin 1.88 mg/g of freeze dried red beet Red beetroot pure water betacyanin 52.2 mg/g of fresh matter 3 min x 4 times 450 W \[[@B34-foods-09-00918]\] betaxanthin 42.8 mg/g of fresh matter Opuntia fruit peel 34.6% methanol betalain 201.6 mg/g of extract 2.5 min 400 W \[[@B63-foods-09-00918]\] Dragon fruit peel pure water betalain 9 mg/L (predicted by RSM) 8 min 100 W \[[@B72-foods-09-00918]\] White-fleshed red pitaya peel pure water betacyanin 1.66 mg/g of dry extract 5 min 600 W \[[@B74-foods-09-00918]\] RSM = response surface methodology. foods-09-00918-t004_Table 4 ###### Total phenolic compound (TPC) recovery by microwave radiation treatment. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Raw Materials Extraction Analytical Method Process Optimum Conditions Reference --------------------------------- ---------------------------- ------------------- ------------------------------------- ----------- ---------------------- --------------------------- Coriandrum sativum (spice) 50% ethanol FC 0.82 mg GAE/g of DW 18 min 200 W, 50% nominal \[[@B16-foods-09-00918]\] Cinnamomum zeylanicum (spice) 16.8 mg GAE/g of DW Cuminum cyminum (spice) 11.6 mg GAE/g of DW Crocus sativus (spice) 29.4 mg GAE/g of DW Lessonia trabeculate\ 70% methanol FC 0.74 mg GAE/g of DW 15 min intermittent \[[@B20-foods-09-00918]\] (Brown algae) Lessonia nigrecens\ 1.07 mg GAE/g of DW (Brown algae) Ascophyllum nodosum\ 1.4 mg GAE/g of DW (Brown algae) Laminaria japonica\ 0.73 mg GAE/g of DW (Brown algae) Rosemary 96% ethanol FC 0.9 mg GAE/g of fresh leaf 7 min 250 W (intermittent) \[[@B52-foods-09-00918]\] pure water 0.1 mg GAE/g of fresh leaf Grape seed methanol FC 67.88 mg GAE/g of DW 60 min 60 W \[[@B60-foods-09-00918]\] Grape skin 7.33 mg GAE/g of DW Eucalyptus leaf 50% ethanol FC 76.6 mg GAE/g of fresh leaf 5 min 600 W (intermittent) \[[@B61-foods-09-00918]\] Olive leaf 50% ethanol FC 88.3 mg TAE/g of DW 15 min intermittent \[[@B64-foods-09-00918]\] Peanut skin 30% ethanol FC 143.6 mg GAE/g of skins\ 0.5 min 950 W, 90% nominal \[[@B67-foods-09-00918]\] (predicted by RSM) Broccoli 72.06% methanol FC 21.39 mg GAE/g of DW 16.94 min 159.33 W \[[@B68-foods-09-00918]\] Wine lee 75% ethanol\ FC 36.4 mg GAE/g of lee extract powder 17 min 200 W \[[@B69-foods-09-00918]\] (acidified) (Ipomoea Batatas)\ 53% ethanol FC 61.26 mg GAE/g DW 2.05 min 302 W \[[@B71-foods-09-00918]\] Sweet potato leaf Melastoma sanguineum Fruit 31.33% ethanol FC 39.02 mg GAE/g of DW 45 min 500 W \[[@B73-foods-09-00918]\] Lycium spp. leaf pure methanol FC 6.65 mg GAE/g of DW 30 min 40 W \[[@B75-foods-09-00918]\] Buckwheat 50% ethanol FC 18.5 mg GAE/g of buckwheat 15 min \- \[[@B76-foods-09-00918]\] Pitaya peel pure water FC 5.8 mg GAE/g of extract 20 min 400 W \[[@B77-foods-09-00918]\] Sour cherry pomace 50% ethanol FC 14.14 mg GAE/g of DW 12 min 700 W \[[@B78-foods-09-00918]\] --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- GAE = gallic acid equivalent, FC = folin-ciocalteu analytical method, TAE = tannic acid equivalent, DW = dry weight. foods-09-00918-t005_Table 5 ###### Some other applications of advanced MAE and their findings. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Sources Methods Advantages References --------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------- Sorghum husks Ultrasonic-microwave assisted extraction of colorant (UMAE) Higher in thermal stabilities and yield percent (3.6 times) with high contents of apigeninidin and luteolinidin than conventional shaking \[[@B90-foods-09-00918]\] Aromatic herbs Enhanced solvent free microwave-assisted extraction (ESFMAE) ESFMAE increased in oxygenated compound content which was more odoriferous than monoterpene hydrocarbons \[[@B93-foods-09-00918]\] Cherry seeds Ultrasonic-microwave-assisted aqueous enzymatic extraction (UMAAEE) Compared to Soxhlet extraction, oil by UMAAEE possessed superior physicochemical properties and higher content of bioactive constituents \[[@B95-foods-09-00918]\] Tunisian cumin (Cuminum cyminum L.) seeds Microwave hydrodiffusion and gravity extraction (MHGE) MHGE successfully improved the EO yield with high amount of oxygenated compounds in shorter extraction time, less electrical consumption, lower carbon dioxide emissions, and smaller volume of waste water \[[@B96-foods-09-00918]\] Rosemary plants Microwave hydro-distillation (MHD) MHD was superior in terms of saving energy and extraction time compared to hydro-distillation \[[@B98-foods-09-00918]\] Foeniculum vulgare Mill. seeds Enhanced solvent free microwave-assisted extraction using double walled reactor (ESFMAE) ESFMAE method was faster, cleaner, less cost and energy usage, and better essential oil composition than hydro-distillation method \[[@B99-foods-09-00918]\] Arabica coffee beans Ultrasonic-microwave assisted extraction of green coffee oil (UMAE) Extraction yields of two diterpenes (cafestol and kahweol) by UMAE were significantly higher than that of solvent method \[[@B100-foods-09-00918]\] Corn brans Ultrasonic-microwave assisted alkali extraction of arabinoxylan (UMAAE) By UMAAE, water-unextractable\ \[[@B101-foods-09-00918]\] arabinoxylan (WUAX) showed good DPPH radical scavenging activity and strong Fe^2^+ chelating activity Schisandra chinensis Baill fruits Ionic liquid-based microwave-assisted extraction (ILMAE) ILMAE method shortened the energy consumption time, improved the extraction efficiency of lignans as to reflux extraction \[[@B106-foods-09-00918]\] Flowers of Ulex europaeus L. Microwave hydrodiffusion and gravity extraction (MHGE) MHGE allowed an efficient water removal from the material, and could be suitable for extraction of antioxidant rich aromatic compounds \[[@B107-foods-09-00918]\] Tomatoes Deep eutectic solvent-based microwave-assisted dispersive liquid--liquid microextraction preconcentration of multiclass pesticide residues in tomato samples (DES-MWA--DLLME) DES-MWA--DLLME represented good repeatability,\ \[[@B108-foods-09-00918]\] high (enrichment factors) EFs, low (limit of detection) LODs ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------	{ 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I. Introduction =============== Aortic aneurysm is a serious condition characterized by the tearing of the inner layer of the aorta. It can lead to sudden death caused by cardiac failure and occasionally, aortic rupture[@B1][@B2]. In addition, hypertension can increase the stress on the weakened aortic wall, increasing its susceptibility to tearing. Aortic aneurysms are divided into Stanford type A and Stanford type B dissections. While type B dissections generally are initially treated medically, with surgery reserved for complications, type A aortic aneurysms, which involve a tear in the aorta ascendens, generally require emergency or quasiemergency surgical treatment[@B2]. Dexmedetomidine (DEX) is an anti-anxiety, sedative, and analgesic medication that can stabilize circulatory dynamics[@B3] and minimize blood pressure fluctuations. We report a case of a 45-year-old morbidly obese man with an aortic aneurysm (Stanford types A and B), in whom invasive oral treatment, namely tooth extraction and cyst enucleation, for the removal of intraoral infectious sources, was successfully performed under local anesthesia with DEX sedation before elective cardiac surgery. II. Case Report =============== A 45-year-old morbidly obese man was transported by ambulance to the Emergency Medical Care Center at Fukuoka University Hospital (Fukuoka, Japan) with suspected acute heart failure after developing a sudden difficulty in breathing with pink frothy sputum. According to his wife, the patient had no significant medical history other than hypertension. Detailed examination revealed an aortic aneurysm (Stanford types A and B), aortic regurgitation, aortic valve ring ectasia, ascending thoracic aorta aneurysm, hypertension, paroxysmal tachycardia, atrial fibrillation, primary lung cancer in the left lower lobe, and morbid obesity (weight, 124 kg; height, 170 cm; body mass index \[BMI\], 42.9 kg/m^2^). The aortic aneurysm required emergency surgery. However, because of the patient\'s morbid obesity, elective vascular graft replacement of the thoracic ascending aorta, aortic valve replacement, pulmonary vein isolation surgery, and segmental left lower lobe resection were planned after gradual weight reduction with dietary therapy. On hospital day 16, the patient underwent a preoperative detailed intraoral examination in our department. At the patient\'s first visit to our department, his weight was 109 kg (BMI, 37.7 kg/m^2^). Intraoral examination revealed six teeth with periapical lesions and two pigeon eggsized radicular cysts that could become potential sources of infection after surgery.([Fig. 1](#F1){ref-type="fig"}) The patient was at increased risk of developing severe complications of aortic aneurysm such as cardiac failure and rupture of the aorta through a sudden increase in blood pressure [@B1][@B2].([Fig. 2](#F2){ref-type="fig"}) The use of intravenous sedation was deemed appropriate to avoid sudden changes in blood pressure and to stabilize the patient\'s hemodynamics. The patient\'s respiratory depression due to morbid obesity also required attention. Accordingly, we planned to use DEX[@B4], which is reported to cause mild respiratory depression, for sedation. The surgery and sedation procedure were explained to the patient, and informed consent was obtained. The surgery involved three oral surgeons, one board-certified dental anesthesiologist, and one nurse. The patient was required to fast for six hours before the procedure. We adopted Fowler\'s position during the surgery, and vital signs (peripheral capillary oxygen saturation, heart rate, blood pressure, and electrocardiogram) were continuously monitored. Oxygen was administered at 3 L/min through a nasal cannula. An initial loading dose of DEX was administered at 4.4 µg/kg/hr for 10 minutes, followed by continuous infusion at 0.075 to 0.3 µg/kg/hr. The procedure took 1 hour 27 minutes to complete, with stable hemodynamics throughout and no marked changes, even during local anesthesia with 2% lidocaine and 1:80,000 epinephrine.([Fig. 3](#F3){ref-type="fig"}) Surgery was safely completed without any complications. For postoperative analgesia, oral celecoxib 400 mg/day was administered until 24 hours after surgery. Thereafter, acetaminophen 3,000 mg/day and loxoprofen sodium hydrate 60 mg were administered as rescue analgesics. Postoperative hemostasis was satisfactory, and no perioperative complications were observed. III. Discussion =============== A dental surgeon should have knowledge of multiple systemic diseases and have the ability to plan and perform appropriate treatment procedures in patients requiring whole body management. Herein, we report successful invasive oral treatment in the form of tooth extractions and cystectomies for the removal of intraoral infectious sources before elective cardiac surgery in a 45-year-old morbidly obese male patient with an aortic aneurysm. Aortic aneurysms are a medical emergency and can quickly lead to death. The prime consideration of the medical management for aortic aneurysms is strict blood pressure control[@B1]. Physical and emotional stress such as anxiety or pain increases blood pressure, and patients undergoing dental treatment are generally anxious. Long surgical duration and intraoperative postural maintenance during oral surgical procedures can cause physical and emotional stress. Moreover, the use of local anesthetics with vasoconstrictors during invasive procedures such as tooth extraction and pain due to an inadequate response to infiltration anesthesia can increase blood pressure. In our patient, the stabilization of circulatory dynamics was very important to prevent the progression and rupture of the aortic aneurysm through a sudden increase in blood pressure. Initially, we considered using 3% mepivacaine without a vasoconstrictor for local anesthesia. However, 2% lidocaine with 1:80,000 epinephrine was selected for the following reasons. First, mepivacaine is believed to have weaker anesthetic effects compared to lidocaine. Second, the effects of epinephrine[@B5] added to the local anesthetic are shortlived, lasting only a few minutes. Finally, an increase in endogenous epinephrine secretion induced by pain can increase blood pressure to a greater extent than exogenous epinephrine. DEX is a popular sedative used in the intensive care unit for patients undergoing mechanical ventilation and has been recently approved for use in Japan in non-intubated patients requiring sedation. Since the patient\'s diaphragmatic movement would be impaired with consequent breathing difficulty in the supine position because of his morbid obesity, the procedures were performed with the patient in Fowler\'s position. Furthermore, the patient had a mild respiratory reserve capacity and was highly sensitive to sedatives, raising the concern of sleep apnea. Therefore, it was considered appropriate to use sedatives that caused mild respiratory depression. Aortic regurgitation is widely known to cause infective endocarditis. Montazem[@B6] reported that infective endocarditis can be observed with high frequency in cases of oral infection. Therefore, the elimination of intraoral infectious sources before cardiac surgery, particularly valve replacement, is important[@B7]. However, severe complications are known to possible during oral surgical procedures in patients with cardiac comorbidities. Care must be taken to prevent the development of life-threatening complications in patients undergoing oral surgical procedures in order to improve the success rate of cardiac surgeries in patients with cardiac comorbidities. Intravenous sedation is extremely useful in stabilizing circulatory dynamics in such patients, and midazolam and propofol are commonly used sedatives. The former induces a dose-related settlement of the tongue root and respiratory depression, while the latter exhibits inhibitory effects on the heart and cardiovascular status. In addition to causing respiratory depression, DEX is an agonist of 2-adrenergic receptors in certain parts of the brain[@B5], causing mild respiratory depression and exhibiting myocardial protective effects and analgesic and sedative effects[@B3]. We believe that DEX was the most suitable for our patient, in whom oral surgical procedures were associated with very high risk and might not have been possible without sedation using DEX. In conclusion, our understanding of risk factors associated with systemic diseases enabled us to plan and safely perform oral surgical procedures with appropriate perioperative whole body management in a patient scheduled to undergo subsequent cardiac surgery. Conflict of Interest: No potential conflict of interest relevant to this article was reported. ![Imaging findings. A. Original panoramic X-ray findings. Periapical lesions are observed in the apical area of the right mandibular third molar, left mandibular first and second molars, and left maxillary central incisor. B. Computed tomography findings (sagittal section image). Cyst-like images are observed in the apical area of the left maxillary lateral incisor and second molar.](jkaoms-42-162-g001){#F1} ![Problems in the invasive treatment of this patient.](jkaoms-42-162-g002){#F2} ![Summary of the therapeutic procedure. 1⃞: Extraction of the left maxillary lateral second molar and cyst enucleation. 2⃞: Extraction of the left maxillary central incisor and lateral incisor and cyst enucleation. 3⃞: Extraction of the left mandibular first and second molars. 4⃞: Extraction of the right mandibular third molar. Observer\'s assessment of alertness/sedation (OAA/S) scale: Score level 5=responds readily to name spoken in normal tone, score level 4=lethargic response to name spoken in normal tone, score level 3=responds only after name is called loudly and/or repeatedly, score level 2=responds only after mild prodding or shaking, score level 1=does not respond to mild prodding or shaking. × to ×: sedative duration, ㉧ to ㉧: operative duration.](jkaoms-42-162-g003){#F3}	{ "pile_set_name": "PubMed Central" }	[ "root", "treatment", "two", "origin", "respond", "impair", "maxillari", "risk", "select", "rais", "mechan", "aortic", "hour", "multipl", "readili", "recent", "sudden", "ventil", "suspect", "gener", "use", "final", "score", "caus", "stanford", "accord", "complet", "imag", "signific", "depart", "becom", "mark", "relev", "tomographi", "right", "also", "montazemb", "molar", "enucl", "emot", "epinephrinefig", "conflict", "perform", "circulatori", "importantb", "graft", "acetaminophen", "dietari", 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INTRODUCTION {#sec1-1} ============ Visual impairment has significant effects on life quality and leads to serious social and economic implications for family and society. It is also a challenging issue for employment and recruitment in many countries. Visual impairment may affect productivity and may lead to injuries. The consequences of visual impairment are an important public health issue with greater impact in the developing countries, where 80% of the world blindness occurs.[@ref1] The World Health Organization (WHO) encourages all countries to monitor the magnitude and causes of visual impairment in order to scrutinize and eliminate avoidable blindness.[@ref2] According to WHO estimates, every 5 seconds one person goes blind in the world. There were 40 to 45 million blind individuals worldwide in 2004 and about three times this figure suffered from visual impairment.[@ref3] It is estimated that the number of blind people worldwide will reach 76 million in 2020.[@ref3],[@ref4] Several studies reporting the causes and prevalence of blindness and visual loss exist in the literature. However, only a limited number of these reports is based on blindness registries, mainly in developed countries. Visual problems have also significant implications in the military in terms of exemption from the military service.[@ref5] As a developing country, Turkey is one of the biggest countries in Europe. The population of Turkey is approximately 77 million in 2013.[@ref6] Comparing to other European countries, Turkey has a considerably young population. According to Turkish Statistical Institute, the number of people between the age of 20-40 years-old is 25 million (32,5%).[@ref6] The Ministry of Health in Turkey does not have a detailed blindness registry. However, because of the compulsory military service in Turkey, each male at the age of 20-years old is examined in Military Recruiting Offices and in the military hospitals. The health status and diseases of every single young man is stored in the database of National Defense Ministry. Those data give valuable information about the prevalence of diseases and causes of disabilities. Detailed results related to musculoskeletal diseases were published previously.[@ref7] This study has been carried out to explore the prevalence and causes of blindness in young Turkish men who have undergone medical examination for compulsory military service between 1 January 2009 and 31 December 2011. METHODS {#sec1-2} ======= The number of young men examined for compulsory military service between 2009 and 2011 was 1.777.500. The number of people used in prevalence calculation in this study was 579.503 for 2009, 583.299 for 2010 and 614.698 for 2011.[@ref7] Military service is compulsory for men in Turkey. Each male at the age of 19-years old is primarily examined in Recruiting Offices where two physicians are available in accordance with Health Ability Regulation document (HARD) of Turkish Armed Forces (TAF) before they start their military service. In case of obligatory reasons, the examinations are performed after the age of 19-year old. During these examinations, disabilities that will prevent from performing military service according to HARD are analyzed. From ophthalmological point of view, proceedings for cases such as enucleation and evisceration are made in Recruiting Offices, whereas cases requiring ophthalmic examination are referred to primary military hospitals, the number of which is 37. If required, the candidates are referred to one of 6 secondary Military Hospitals. The third referral centers are GATA Military Medical School (Ankara) and Haydarpasa GATA Military Medical School (Istanbul). Health reports belonging to the men not suitable for military service are collected in Health Department Directorate of National Defense Ministry (NDM). Descriptions in the Survey {#sec2-1} -------------------------- Decisions regarding health conditions in TAF are made in accordance with the criteria defined in HARD. All diseases have been classified as those suitable for military service, those unfit for military service (UFMS), and those under treatment of an active disease or in the recovery period of a disease. In this report, only those unfit for military service were evaluated. Those fit for military service and those under treatment or having an active disease have been excluded. Ophthalmic examination methods and criteria for UFMS {#sec2-2} ---------------------------------------------------- From ophthalmological point of view, unilateral ophthalmic pathologies causing a visual acuity (VA) under 0.2 (VA≤0,1) and bilateral ophthalmic pathologies causing a total VA (right eye plus left eye) under 1.0 (VA≤0,9) are classified as UFMS. In addition to VA measurements, some high refractive error and anisometropia data and ocular alignment disorders are categorized as UFMS irrespective of VA criteria. These criteria are (1) The sum of high axis of both eyes over 14.00 diopters, (2) anisometropia greater than 4 diopters in hypermetropics and greater than 6 diopters in myopics (by using spherical equivalents), (3) ocular movement disorders more than 'one gaze position in one eye or equal or more than one gaze position in both eyes, (4) visual field loss greater than 2/3 in one eye or greater than 1/2 in both eyes with regard to Humphrey visual field perimeter using central 30/2 SITA Fast program and (5) any night blinding disorder including retinitis pigmentosa, stationary night blinding disorders, etc. The diagnosis of UFMS in candidates with previous evisceration or enucleation due to any cause could be made in the recruitment offices by general practitioners (in case the candidates documented, the surgical report of the disease and evisceration cause). Most of the diagnosis causing UFMS could be made in secondary or tertiary referral military hospitals. Best corrected visual acuity was obtained with Snellen charts. Refraction data was obtained by using auto refractometers 40 minutes after instilling 3 drops of cyclopentolate (Sikloplejin, Abdi Ibrahim, Istanbul, Turkey) with 5 minutes apart. Vertex distance was set as 12.5 mm in the autorefractometers. Globe position and eye movements, pupillary reactions, biomicroscopic and fundoscopic examinations were performed in all cases. The candidates with nyctalopia and/or photophobia were evaluated by full-field electroretinogram (ffERG) (Roland-Consult, Wiesbaden, Germany). The diagnosis of night blinding disorders (retinitis pigmentosa, fundus albipunctatus, congenital stationary night blindness) and cone dystrophies was made by using ffERG. The candidates with macular dystrophy or macular pigmentary changes were evaluated by multifocal electroretinogram (mfERG), pattern electroretinogram (PERG) and ffERG when indicated. The candidates with suspected malingering were evaluated by pattern visual evoked potentials (PVEP) and clinical malingering examination methods that were explained in detail elsewhere.[@ref8] The candidates with optic disc pallor were evaluated by standard ophthalmologic examination, retinal nerve fiber layer thickness analysis, PVEP and orbital and cranial magnetic resonance imaging (MRI). Administrative and Ethical Permissions {#sec2-3} -------------------------------------- The data for this study was acquired from Surgeon General Office of NDM. Ethical permission of the study was obtained from Ethics Committee of Gulhane Military Medical Academy (GMMA). Evaluating the data and statistical analysis {#sec2-4} -------------------------------------------- In order to examine the characteristics of geographic distribution, the provinces where the recruiting offices of the candidates are located were grouped as west, south, central, northern and eastern zoning system as prepared by Turkish Statistical Institute. Since medical examinations before military service began at the ages of 19-20, the ages of the cases have been grouped as "19, 20, 21-24, and 25 years and above" when examining the demographic characteristics. The ophthalmic pathologies responsible for the UFMS were categorized into 4 groups; (1) refractive errors, (2) strabismus and nystagmus, (3) anterior segment causes, and (4) posterior segment causes. Study data was transferred to computer and analyzed with SPSS 15.0 software. RESULTS {#sec1-3} ======= Health data of 1.777.500 military service recruits whose health examinations have been carried out between the years 2009-2011 has been reviewed. It was found that 14.862 cases had ophthalmologic diseases causing unfitness for military service. The prevalence of ophthalmologic diseases causing unfitness for military service was found 0.746% for 2009, 0.871% for 2010 and 0.889% for 2011. The most populous age group was 19 years old (31.8%). The candidates with 25 or more years old comprised 21.8% of the candidates. The most number of the examinations (25.8%) were performed in the central region of Turkey. Detailed age and region distribution of the candidates in years 2009-2011 are presented in [Table-I](#T1){ref-type="table"}. ###### Demographic data of the candidates for military service. 2009 2010 2011 Total -------------- ------ ------- ------ ------- ------- ------- ------- ------- ------ Age groups 19 1546 35.8 1540 30.3 1632 29.9 4718 31.8 20 648 15.0 926 18.2 1037 19.0 2611 17.6 21-24 1208 28.0 1463 28.8 1602 29.3 4273 28.8 25 and above 919 21.2 1149 22.7 1192 21.8 3260 21.8 Region West 739 17,1 901 17,7 920 16,8 2560 17,2 South 426 9,9 568 11,2 552 10,1 1546 10,4 Central 1177 27,2 1328 26,2 1329 24,3 3834 25,8 North 631 14,6 689 13,6 776 14,2 2096 14,1 East 1348 31,2 1592 31,4 1886 34,5 4826 32,5 Total 4321 100.0 5078 100.0 5463 100.0 14862 100.0 Among ophthalmic causes, refractive errors were the most frequent pathology causing UFMS (40.1%). Among refractive errors, 'high axis over 14.00 diopters' was the most common refractive error type (65.58%). Mean prevalence of refractive errors causing UFMS was 3.35 ‰. Detailed refractive error data is presented in [Table-II](#T2){ref-type="table"}. ###### The distribution of ophthalmic causes of unfitness for military service. Diagnoses 2009 2010 2011 Total -------------------------------------------------------------------------------- ------ ------ ------- ------- ------ ------- ------ ------ ------- ------ ------- ------- Refractive Errors 1664 38.5 0.287 2005 39.5 0.344 2292 42.0 0.373 5961 40.1 0.335 High axis over 14 dpt. (OD+OS) (any VA) 1305 30.3 0.225 1607 31.6 0.276 1817 33.3 0.296 4729 31.8 0.266 Anisometropia (VA≤0.1) 359 8.2 0.062 398 7.9 0.068 475 8.7 0.077 1232 8.3 0.069 Ocular Alignment Disorders 596 13.8 0.102 851 16.6 0.146 814 14.9 0.133 2261 15.3 0.128 Strabismus 233 5.4 0.040 344 6.7 0.059 338 6.1 0.055 915 6.2 0.052 a\. Strabismic amblyopia (VA≤0.1) 211 4.9 0.037 300 5.9 0.051 303 5.5 0.049 814 5.5 0.046 b\. EOM paralysis (any VA) 22 0.5 0.003 44 0.8 0.008 35 0.6 0.006 101 0.7 0.006 Nystagmus (VA≤0.1) 130 3.0 0.022 163 3.2 0.028 138 2.7 0.023 431 2.9 0.024 Anterior Segment Pathologies 859 19.8 0.148 984 19.3 0.169 1133 19.8 0.184 2975 20.1 0.167 Corneal and lens pathologies (VA≤0.1) 532 12.3 0.092 595 11.7 0.102 661 12.1 0.107 1788 12.0 0.101 Pseudophakia (VA≤0.1) 117 2.7 0.020 157 3.0 0.027 162 3.1 0.027 436 2.9 0.025 Aphakia (any VA) 187 4.3 0.032 206 4.0 0.035 199 3.6 0.032 592 4.0 0.033 Previous penetrating keratoplasty (VA≤0.1) 13 0.3 0.002 17 0.4 0.003 105 0.9 0.017 134 1.0 0.007 Previous keratorefractive operations (VA≤0.1) 10 0.2 0.002 9 0.2 0.002 6 0.1 0.001 25 0.2 0.001 Posterior Segment Pathologies 1384 31.9 0.294 1540 30.5 0.265 1482 27.1 0.241 4406 29.69 0.249 Previous vitrectomy surgery due to any cause and history of retinal detachment 96 2.2 0.017 116 2.3 0.020 114 2,0 0.018 326 2.19 0.019 Nonsurgical retina, vitreous and optic nerve disease 1194 27.6 0.261 1293 25.6 0.222 1264 23,3 0.206 3751 25,3 0.212 Ocular albinism 71 1.6 0.012 96 1.9 0.017 67 1.2 0.011 234 1.6 0.013 Glaucoma (due to VA ≤0.1 or VF loss) 23 0.5 0.004 35 0.7 0.006 37 0.6 0.006 95 0.6 0.005 Ocular alignment disorders (including strabismus and nystagmus) comprised 9.1% of the ophthalmic causes of UFMS. Strabismic amblyopia was the most common pathology among ocular alignment disorders (60.4%). The prevalence of strabismic amblyopia (blindness, VA≤0, 1 was 0.046%. Nystagmus comprised 2.9% of ophthalmic causes of UFMS. The mean prevalence of nystagmus causing UFMS was 0.024%. Detailed ocular alignment disorders data is seen in [Table-II](#T2){ref-type="table"}. Cornea and lens pathologies were evaluated under the title of anterior segment pathologies. Corneal and lens pathologies other than pseudophakia, aphakia and penetrating keratoplasty comprised of 12% of ophthalmic causes of UFMS. Although detailed data is not available, most of those causes of anterior segment pathologies are corneal scars and cataract. Mean prevalence of these pathologies was 0.101%. Aphakia comprised 4.0% of ophthalmic causes. The mean prevalence of aphakia was 0.033%. Pseudophakia comprised 2.9% of ophthalmic causes and the mean prevalence of pseudophakia was 0.025%. The mean prevalence of previous penetrating keratoplasty was 0.008%. Detailed anterior segment pathologies causing UFMS is presented in [Table-II](#T2){ref-type="table"}. Posterior segment pathologies comprised 31.8% of ophthalmic causes of UFMS. The mean prevalence of blindness from posterior segment pathologies was 0.266%. Posterior segment pathologies causing UFMS is shown in [Table-II](#T2){ref-type="table"}. DISCUSSION {#sec1-4} ========== To our knowledge, this is the first report using official registries in NDM on the causes of severe visual impairment from Turkey. This report includes the results of ophthalmologic examination of all young men coming up to 19-years-old in Turkey. This is because this report presents the real data-not statistics- about the causes of severe visual impairment in Turkey. Currently, in Turkey, the overall prevalence of severe visual impairment in young men is 0.554%. This ratio includes visual field losses (over 2/3 in an eye or over 1/2 in both eyes) related to any cause and excludes uncorrected refractive errors. Nonsurgical retina, vitreous and optic nerve diseases are the most frequent cause of visual impairment (0.212%). The detailed causes are not available in this report. The authors involved in the ophthalmic examination of the candidates for military service (FCG, UY) state that most of those causes are related to optic atrophy and retinitis pigmentosa. Corneal and lens pathologies are the second most frequent cause of blindness (0.101%). Detailed causes are not available in this report. The authors also state that most of the corneal and lens pathologies causing visual impairment are related to corneal leucoma, keratoconus and cataract. The prevalence of visual impairment with pseudophakia and aphakia are 0.025% and 0.033%. The prevalence of visual impairment with previous keratorefractive operations is extremely low (0.001%). To our knowledge, there is no data available on the incidence of visual impairment in Turkey to compare with this study. Refractive errors are the most prevalent cause of visual impairment in young Turkish men. The prevalence of high refractive errors is 0.335%. Visual impairment due to refractive errors is far more than that ratio as that ratio includes only the persons with refractive errors over 14.00 dpt (high axis OD+high axis OS). This report does not include the ratio of uncorrected refractive errors to all refractive errors. Weih et al. reported that 60% of participants with visual impairment due to refractive error and 82% of participants with VA of less than 20/40 to 20/60 were not wearing distance correction in a sample of Australiens.[@ref9] This ratio may somewhat be higher in Turkey due to lower socioeconomic status. The authors also reported that less than 1% of people aged 40-49 years had visual impairment due to uncorrected refractive error. The prevalence of registered blindness was 0.17%-0.22% in Ireland (blindness was defined as BCVA in the better eye of less than 6/60)[@ref10] and 0.26%-0.32% in Israel (using WHO criteria).[@ref11] The incidence of registered blindness was 0.036%-0.029% in Israel (using WHO criteria), 0.02% in Ireland using (US criteria) and 0.012% in Southern Germany (BCVA in the better eye less than 1/50).[@ref11]-[@ref14] The prevalence of blindness in a district of China in male population with an age between 1 to 29-years-old was reported as 0.015% in 2009.[@ref14] The reported blindness increased from 114.7 per 100.000 in 2003 to 145.8 per 100.000 in 2006 to 165.9 per 100.000 in 2009 although the population of the district remained stable. The authors stated that increase in prevalence during 2001-2009 represented increased registration rather than increasing levels of disease. The registries in NDM did not allow us to have the prevalence of blindness in terms of WHO or US criteria. However, from our data, we can state that the prevalence of monocular blindness in Turkey (VA≤0.1) is 0.055%. This prevalence cannot be directly compared to the prevalence of blindness reported from other countries. This article was designed as a further analysis of data set about disability reasons young adult males in Turkey between 2009-2011. The previous paper was published as "The prevalence of disorders causing disability in young adult males in Turkey between 2009-2011; Pak J Med Sci 2013 Vol. 29 No. 5".[@ref4] *Funding:* The authors of this study did not receive any financial support for this study. *Declaration of interest:* None. {#sec1-5} Authors' contribution {#sec2-5} --------------------- FCG: Substantial contributions to conception and design, revising it critically for important intellectual content, final approval of the manuscript. NK: Acquisition of data, drafting and final approval of the manuscript. UY and AI: Substantial contributions to conception and design, drafting and final approval of the manuscript. IA and RA: Acquisition of data, revising it critically for important intellectual content, final approval of the manuscript. 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Body ==== In this issue, one case report, one review article, one brief report, and three original papers were published. Tae-Sung Yeum et al. reported a clinical case of a rare neurologic disorder, anti-N-methyl D-aspartate receptor encephalitis without teratoma, which presented psychotic symptoms. Yeeun Lee et al. wrote a review article focused on the mental health and related environmental factors of ethnic minority youths in the Republic of Korea (ROK). Minha Hong et al. presented a brief report on the differences in the practice of psychiatrists for attention-deficit/hyperactivity disorder (ADHD) among adults in the ROK according to the training background of fellowship for the child and adolescent psychiatry. Minha Hong et al. surveyed to examine the differences and the survey questionnaire used in this study was created by the "Adult ADHD Study Group" organized by the Korean Academy of Child and Adolescent Psychiatry (KACAP) as the first step to establish a practice parameter for ADHD in adults \[[@ref1]\]. Minji Lee and Subin Park suggested that sociodemographic variables, familiarity with the disorder, and personality traits are associated with the lay beliefs about tic disorders and Tourette's syndrome from the findings of an online survey. Geon Ho Bahn et al. determined the cut-off scores of the Korean adult ADHD rating scale (K-AARS). The K-AARS is the first scale developed in ROK to diagnose ADHD in adults. Mi Ae Oh et al. article, The Analysis of Self-Mutilation in Adolescence Based on the Theory of Mentalization: From Sukhvinder in the Novel "Casual Vacancy," is a secondary publication in Englishthatwas first publishedin Koreanandinterpreted the self-cutting behaviors of adolescents in literature. Since Eugene Garfield proposed the idea of impact factor (IF) in the journal "Science" in 1955, the publication of scientific treatises has undergone a tremendous transformation \[[@ref2]\]. Although IF is not a perfect tool to measure the quality of articles, as there is no better technique and it has the advantage of already being in existence, IF is considered to be a good technique for scientific evaluation \[[@ref3]\]. However, we tend to overlook two things about IF. First, according to the 80/20 phenomenon, 20% of the articles may account for 80% of the citations. Second, of the 38 million items cited during the period from 1900 to 2005, only 0.5% were cited more than 200 times and half were not cited at all \[[@ref2]\]. One of the commonalities of frequently cited papers is that they are written in English. Does this suggest that an academic paper has to be written in English in order to be quoted more? The billboard chart is an indicator of the popularity of global music markets. BTS ranked first on the Social 50 in the bill-board chart in the last week of June 2019 \[[@ref4]\]. Interestingly, members of the BTS global official fan club ARMY sing BTS songs, even though their lyrics are not in English. Similar to the BTS ARMY, the frequency of citations of academic papers does not depend on whether the papers are written in English or not, but on how interested the readers were in the paper. The editorial committee of the Journal of the Korean Academy of Child and Adolescent Psychiatry (JKACAP) is looking for ways in which the papers can be cited more and is also trying to prevent the papers published in this journal from becoming orphaned articles. Unless the readers are interested in the articles in JKACAP written in English, the articles will be shunned by readers. As one of the better tactics is for the article to be included in popular bibliographic databases, the editorial committee applied for JKACAP to be indexed in the Emerging Sources Citation Index (ESCI) \[[@ref5]\] on December 18, 2017 and received the acceptance mail on June 17, 2019. ESCI journals are being evaluated on a continual basis for inclusion in the Science Citation Index Expanded (SCIE) and Social Science Citation Index (SSCI). As the editor-in-chief of JKACAP, I am extremely grateful to the members of the editorial committee and the people who support publishing in this journal. With JKACAP being indexed in the ESCI, we will now strive harder to contribute to the development of the mental health of children and adolescents not only in Korea, but throughout Asia. Hansen JR. First man: the life of Neil A. Armstrong. 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Key MessagesAdaptive and adequately resourced health systems are necessary but not sufficient to ensure good health outcomes among adolescent girls in post-conflict settings.Integrating an understanding of the social determinants of health and wellbeing is critical in shaping health service uptake and ultimately outcomes for adolescent girls.Psychosocial outcomes are also central to broader health and wellbeing and need urgent attention in post-conflict developing country settings in particular.Tailored gender- and age-sensitive psychosocial service provisioning is vital in supporting the health and wellbeing of adolescent girls---a cohort often facing complex vulnerabilities in post-conflict settings. Introduction ============ Adaptive and adequately resourced health systems are necessary to ensure good health outcomes in fragile and post-conflict settings. Nevertheless, it is increasingly recognised that domains beyond the health system also significantly impact on broader health and wellbeing. This paper focuses on the importance of psychosocial services for vulnerable populations and specifically adolescent girls in fragile contexts. Adolescence is a pivotal time in the life course when there is considerable opportunity for change. At the same time it is also a period when adolescent girls are especially vulnerable to a range of life-changing experiences including trauma, social isolation, bullying by peers, a lack of supportive adults, and sexual and other forms of violence. The aims of the study are to highlight why current humanitarian and biomedical approaches in the current form are inadequate to address these complexities. It also stresses how critical an understanding of the social determinants of health is to enable adequate and context appropriate responses. This paper argues that it is important to go beyond biomedical approaches and instead investigate the complex social determinants of health critical for achieving broader health and wellbeing, especially amongst the most vulnerable. There is now more evidence that explores the links between gender and adolescence, mental health and development (e.g. [@czx127-B33]; [@czx127-B39]). Yet in many developing countries, health system debates and research have tended to overlook mental health and psychosocial wellbeing generally (e.g. [@czx127-B8]) and particularly the needs of adolescent girls in post-conflict or other fragile settings (for further details of relevant literature see [@czx127-B39]). To contribute to these debates, we draw on qualitative fieldwork from 2014 to 2015 in three countries recently affected by conflict---Gaza, Liberia and Sri Lanka---honing in on the psychosocial vulnerabilities of girls aged 10--19 and the extent to which these are addressed by the existing psychosocial service environment. Situating our study within the wider literature =============================================== Drawing on a large and diverse body of literature, in this section we discuss the conceptual underpinnings of this study, making explicit linkages between the social determinants of health and the mental health and psychosocial wellbeing of adolescent girls. According to the World Health Organization (WHO) 'The social determinants of health (SDH) are the conditions in which people are born, grow, work, live, and age, and the wider set of forces and systems shaping the conditions of daily life. These forces and systems include economic policies and systems, development agendas, social norms, social policies and political systems'[^1^](#czx127-en1){ref-type="fn"}. The ways in which these conditions are experienced by those at risk of psychosocial ill-being are refracted through a number of individual level variables, including age and gender, and family and community level factors, including discriminatory gendered social norms (see e.g. [@czx127-B19] Why the focus on adolescents, particularly girls? ------------------------------------------------- Although definitions vary, the United Nations defines adolescence as the second decade of life between 10 and 19 years ([@czx127-B43]). During this time, girls and boys undergo bodily changes (e.g. the start of menstruation, the development of breasts for girls and deepening voice and hair growth for boys) and begin the transition from childhood to adulthood, when they will assume roles such as parent, spouse, worker and citizen. During adolescence, the constraining role and influence of gendered social norms on girls' lives (in a range of domains, from education and marriage to mobility outside the home) becomes more evident ([@czx127-B20]; [@czx127-B43]). It is also during this period that developmental changes occur and key skills are acquired, such as those that relate to: health, physical and neurological development; social behaviours and attitudes; and education and employment. For girls and young women in many settings, this life stage also heightens deprivation, danger and vulnerability, including an increased risk of sexual and domestic violence. This constrains girls' ability to develop agency and leads to other development deficits, often leading to negative consequences in their adult lives (see e.g. [@czx127-B18] and [@czx127-B14]. Other context-specific factors, such as national laws and local cultural norms and practices also have a major impact on adolescent girls' lives, dictating not only how girls should think and behave but how others expect girls to behave. In this study, the national context (of a fragile or post-conflict state) shapes how girls and their families recover from or continue to face violence, disruption, displacement and loss of livelihoods. These experience are often gendered, as social norms shape how families react to and cope with external shocks and stresses, often with negative outcomes for girls including marrying them off early and pulling them out of school (e.g. [@czx127-B22]). From mental health to psychosocial wellbeing -------------------------------------------- Mental health is now recognised as being crucial to overall health (e.g. [@czx127-B54]) and is defined as including '... subjective well-being, perceived self-efficacy, autonomy, competence, intergenerational dependence, and self-actualization of one's intellectual and emotional potential, among others' ([@czx127-B54], p. 5). Alongside the suffering it causes, mental ill-health can exacerbate household poverty, increase inequality, reduce social capital, and hinder economic growth ([@czx127-B56]). Being exposed to situations common to post-conflict contexts, such as violence, death and difficult living conditions (e.g. lack of food, epidemics, or being forced to flee one's home) is likely to negatively affect the psychosocial wellbeing of individuals and families (see e.g. [@czx127-B4]; [@czx127-B11]). Sexual violence (largely directed against women and girls), a known risk factor for mental ill-health, is also heightened in conflict contexts. Even after a conflict has ended, girls and women often continue to be subject to sexual and other violence. This often reflects a certain 'normalisation' of such violence as well as continuing availability of weapons, lack of punishment for perpetrators, and frustration among men who are no longer able to live up to the role expected of them by traditional norms (see e.g. [@czx127-B2] and [@czx127-B10]). Mental health and psychosocial risks in adolescence --------------------------------------------------- Mental ill-health and psychosocial problems typically start during adolescence; unless young girls or boys receive appropriate treatment, the results of these psychosocial problems will continue to affect them as adults ([@czx127-B55]). One study noted that between 10% and 20% of children and young people have been reported as experiencing diagnosable mental illnesses ([@czx127-B21]). This is leading to rising suicide rates, with young people now the highest-risk group for suicide in around 30% of all countries ([@czx127-B55], [@czx127-B58]); in China, India and the South East Asia region, suicide is the leading cause of death among those aged 15--19 ([@czx127-B32]; [@czx127-B57]). Other negative consequences of mental ill-health and psychosocial problems for young people include poorer school attainment, resorting to substance abuse, greater likelihood of experiencing violence, and increased likelihood of facing reproductive and sexual health-related challenges ([@czx127-B32]; [@czx127-B21]). Despite widespread and increasing awareness of the mental health challenges facing adolescents, their needs in this area are largely unmet, and particularly in developing countries. Reasons for this relative neglect include lack of effective policies targeting adolescents, limited funding and human resources (particularly qualified healthcare professionals), limited training and experience of lower-level health staff, and the ongoing stigma linked to mental ill-health (ibid.). Adolescence is recognised as a troubling time in a person's development, but it brings particular challenges for children and young people in conflict/post-conflict situations. They may experience traumatic events such as the death of (or separation from) their parents; they may be subject to sexual and/or physical violence; they may be abused, abducted, or forced to become involved with fighting forces; and they may miss out on their schooling and on other social and economic opportunities ([@czx127-B7]). All these experiences can bring on serious mental ill-health and emotional trauma, and can have negative and long-term impacts on how adolescents see themselves, interact with each other as well as on their aspirations for the future. These experiences are often gendered---in a study of Sierra Leone's child soldiers [@czx127-B5], for instance, found that girls demonstrated significantly higher scores of depression and anxiety than boys (80% vs 52% and 72% vs 55%, respectively), partly explained by the fact that girls experienced many more cases of rape and sexual abuse than boys (44% vs 5%). Towards a norms-sensitive conceptual framework for understanding psychosocial wellbeing --------------------------------------------------------------------------------------- Humanitarian actors increasingly recognise the impact of conflict and disaster on people's psychosocial wellbeing and in recent years have incorporated mental health and psychosocial support (MHPSS) into their response ([@czx127-B8]; [@czx127-B51]; [@czx127-B52]). The 2004 Indian Ocean tsunami was arguably a turning point, as it required a systematic response to people's mental health as well as physical survival needs, with a focus on building individual and community resilience ([@czx127-B42]; [@czx127-B39]). This has also given rise to new conceptual frameworks that focus on different resource domains that are affected in situations of conflict. These include: human capacity (individuals' mental and physical health, skills and knowledge); social ecology (the relationships that allow people to exist as a community, sometimes called social capital); material and physical environments; and local values and culture ([@czx127-B40]; [@czx127-B15]; [@czx127-B53]). Building on these psychosocial wellbeing frameworks, we apply a social norms lens, positing that social norms affect all domains of adolescents' lives, not just education and marriage but also access to resources (economic, material and human), their health and wellbeing, and their ability to participate in community life (agency and voice) ([@czx127-B35]). Applying a social norms lens helps us to understand and put in context the different paths to wellbeing that are available to adolescents, as well as the challenges faced by girls (and boys), and what works to address or overcome these challenges and barriers. As seen in [Figure 1](#czx127-F1){ref-type="fig"}, this approach puts adolescent girls centre stage, with key areas of wellbeing around them. Girls are situated within their families/households, their wider community and the (fragile) state, characterised by ongoing or recent conflict or other crises related to health, food security or natural disasters. These different areas of girls' lives are in turn influenced by changes in the global context, which include greater learning around adolescents' needs and the rise of MHPSS programming. ![Pathways towards adolescent girls' psychosocial and broader wellbeing](czx127f1){#czx127-F1} The availability of local services also affects girls' psychosocial wellbeing, with tailored services being especially critical to support their evolving needs (e.g. around menstruation, information on family planning, etc.). Such service provisioning needs to be informed by an understanding of the social determinants of health (e.g. gender, ethnicity and fragility) and the importance of integrating a mix of formal, faith-based providers, and informal services. Prevailing gender norms (around education, marriage, sexuality and mobility) also determine girls' vulnerabilities, their access to and uptake of relevant services, and their ability to develop their full capabilities. Services must be carefully targeted and designed to meet girls' needs if they are to maximise girls' psychosocial wellbeing and allow them to develop to their full potential. Methods and case study contexts =============================== The study design combined primary qualitative data collection with a secondary review of literature. This allowed us to situate our research within broader debates on adolescence, fragile states, and mental health and psychosocial services.[^2^](#czx127-en2){ref-type="fn"} The research involved the collection of primary qualitative data, in conjunction with national study partners, in Sri Lanka, Liberia and Gaza in 2014 and 2015. The full research proposal and instruments, which were developed jointly with the national partners, were submitted to and approved by the Overseas Development Institute's ethics committee. The study was also cleared by national research ethics boards in the study countries, i.e. in Liberia the Carter Centre has its own ethics board, in Gaza the study was reviewed by the Helsinki Framework ethics board, and in Sri Lanka the study partners made the necessary arrangements to conduct the study. Phase one of the data collection aimed to map people's perceptions (adults and young people) of the different dimensions of wellbeing (especially those affecting girls) and the factors that affect their wellbeing. The next phase focused on the availability of services, particularly looking at whether MHPSS programmes were sensitive to adolescents' (and especially girls') needs and how services could be made more responsive to girls' needs.[^3^](#czx127-en3){ref-type="fn"}[Table 1](#czx127-T1){ref-type="table"} provides an overview of the study contexts and [Box 1](#czx127-BOX2){ref-type="boxed-text"} provides an overview of the methodology. Table 1.Study sites and relevant country contextsTopicGazaLiberiaSri LankaStudy sitesShajaia neighbourhood Gaza City: high level of violence by Israeli forces leading to high numbers of IDPs: out of a total population on 120 000, 25% are refugees (NECC 2014). High rate of unemployment: \>50% of the residents receive social assistance (Ibid.). Conservative community: median marriage age for women is under 20 years old ([@czx127-B1])New Kru town Montserrado county: densely populated borough of capital city, Monrovia (∼70 000 people) ([@czx127-B35]). One of the communities that suffered most from Ebola virus and the civil war (Montserrado acted as an entry point for rebel armies entering Monrovia) (Ibid.).Diyagama village Polonnaruwa District, North Central Province: Majority of population (total of 7000) belongs Tamil ethnic group ([@czx127-B34]). Until 2006 the area served as a Liberation Tigers of Tamil Eelam stronghold ([@czx127-B12]).Kadalkiraman Batticaloa District, Eastern Province: highest level of poverty in the country (poverty head count index of 20.3%) and highest poverty gap index (5.1 compared with a national of 1.7) (UNDP 2012). Majority of the population (total of 2464 are Tamil ([@czx127-B12]). Located in high-security area with large military camps. Good road: easy access to services (Ibid.).Tubmanburg Bomi county, is a city of 13 114 people in one of the poorest counties in Liberia ([@czx127-B6]). During the civil war Tubmanburg served as rebel army LURD's headquarters. In total Bomi country authorities reported 175 Ebola related deaths since the Liberian outbreak started in 2014 (MoHSW 2015).Population, including youthPopulation: 1.71 million (1.2 are refugees): nearly half the population is under the age of 15 (PCBS 2012a). Ethnicity: 99% are Palestinian Arabs, 1% other (Ibid.).Religion: Predominantly Muslim: 99.3%, Christian 0.7% (Ibid.).Population: 4.19 million with nearly half under 15 and 20.4% between 10 and 17 years old ([@czx127-B59], [@czx127-B60]). Ethnicity: 2.5% Congo people (descendants of former slaves) and 95% indigenous ([@czx127-B35]). Religion: 40% Christian, 20% Muslims and 40% indigenous beliefs (Ibid.).Population: 20.3 million with 23.2% of the population between 15 and 29 years (UNDP 2014). Ethnicity: The majority is Sinhalese and 16.5% are Tamil (UNDP 2012). Religion: majority are Buddhist and 9.3% Muslim (Ibid.).Fragility context---economic situation and conflict legacyFragility: Gaza is marked as a 'hostile entity' by Israel---characterised by blockades (since 2007 and ongoing) and military operations (MoH 2014). Economy: Growth slowed from 6% in 2012 to 2% in 2013 (in Palestine) ([@czx127-B59]a, [@czx127-B60]). Gaza has an HDI of 0.736 (UNDP 2015). Conflict legacy: Conflict-related chronic stress has negatively impacted the public health of the population of Gaza and has, amongst other things, caused high levels of non-communicable diseases including mental health and psychosocial challenges ([@czx127-B1]).Fragility: Civil conflict (1989--2003) due to ethnic tension causing 150.000 deaths and displaced 850.000 Liberians (UN 2015). Ebola outbreak 2014--2015 (WHO 2015). Economy: Growth rate of 7.5% in 2014. HDI of 0.412 in 2013 (UNDP 2014).Fragility: Violent uprisings between 198 and -2009 caused by socioeconomic and ethnic grievances by Tamils. Country was hit by a tsunami in 2004 killing 35 000 people ([@czx127-B12]).Economy: Gained middle income status in 2010 (Ibid.). HDI increased by 28% between 1980 and 2012 (HDI in 2012: 0.715). Human development indicators lagging in several provinces and rural areas (UNDP 2014).Conflict legacy: 48% of the population living in extreme poverty. High unemployment, food insecurity (UN Liberia 2013).Conflict legacy: Conflict caused loss of thousand lives (likely as many as 330.000 deaths) injuries and disability (∼40.000 surgical procedures and 5.000 amputations during the conflict), destruction and displacement (290.000 Sri Lankans displaced from the battle zone) (UN 2011).Gendered effects of conflict including VAWGGendered vulnerabilities: Conflict limits women's already low mobility and restricts their agency and ability to build up social networks ([@czx127-B1]).Many women are displaced and live in temporary mixed housing arrangements that are not constructed gender-sensitively (mixed living room areas and lack of adequate toilet facilities) (Ibid.).Gendered vulnerabilities: Higher rates of illiteracy for women (adult literacy rates: 37% for girls and 63% for boys (UNICEF 2007). With only 58.2% of all women participating in the labour force, most are forced to engage in vulnerable employment (UNDP 2013). Violence: During the civil conflict between 61% and 77% of all women were raped. Normalisation of GBV including FGM/C, early marriage and polygamy, sexual and domestic violence (UN 2013). Over 34% of women reported domestic violence by their partners ([@czx127-B16]).Gendered vulnerabilities: Conflict restricted movement of women and girls impacting negatively on their education and livelihoods ([@czx127-B17]). Conflict changed traditional gender norms: in some cases girls and women became combatants or were responsible for household livelihoods, leading to a form of female empowerment ([@czx127-B37])Violence: Within a context of conflict, brutalisation and militarisation opportunities for sexual violence have been normalised for women (Ibid.).Violence: High level of domestic violence: 51% of married women experienced violence at the hands of her husband ([@czx127-B30]). Girls in temporary shelter arrangements are extra vulnerable for gender based violence and sexual abuse ([@czx127-B1]).Mental health policy and services contextPolicy: National Health Strategy (2012--2016), Public Health Policy for Children (2012) Service providers: Ministry of Health established 54 Primary Health Care centres and 13 hospitals, but only one psychiatric hospital. Other providers include: UNRWA who established 22 centres that provide mental health services for refugees; NGOs that are providing mostly costly secondary and tertiary services in \> 50 clinics; Private-for-profit operators that are mainly focussed on obstetrics and surgical intervention (MoH 2014).Policy: National Mental Health Policy (2009), Basic Package of Health Services (2010).Policy: National Mental Health Policy (2005--2015).Service providers: Policy established by the National Institute for Mental Health: national strategy aims to reduce stigma and discrimination and calls for mental health legislation ([@czx127-B12]). Government provided mental health training for primary health care doctors (WHO 2013). GBV Desks at hospitals intervene in rape and sexual abuses cases and provide counselling to survivors ([@czx127-B12]). Child protection officers raise awareness on the importance of child protection and offer mental health services for children ([@czx127-B12]).Service providers: Only one hospital (private---with only one trained psychiatrist) in Liberia providing mental health services: Grant Memorial Mental Hospital in Monrovia. Primary health personnel lack training. Other mental health services are provided by NGOs or FBOs. Health systems were further damaged by the Ebola outbreak ([@czx127-B35]). Box 1.MethodologyWe used a range of qualitative tools including: in-depth interview (IDI) guides for adolescent girls, boys and parents; key informant interview (KII) guides for service providers and community leaders; focus group discussion (FGD) guides with parents of adolescents and service providers; and guides for carrying out intergenerational trios--where a grandmother/grandfather, mother/father and daughter/son are interviewed individually to obtain information about stasis and change across the generations (for further information see: <http://www.odi.org/sites/odi.org.uk/files/odi-assets/publications-opinion-files/9826.pdf>). Drawing on our conceptual framework, these tools addressed sets of questions around different wellbeing domains and different levels of the social ecology. The aim was not to explore psychometric measures or properties but rather to understand the ways in which different dimensions of adolescent girls' wellbeing are shaped by household, community, national and global factors and contexts.To encourage respondent participation, we also integrated visual techniques into FGDs, including community timelines and mapping exercises. These techniques helped us to gain insights into what services for adolescents exist in the focal communities, changes over time in service provision and their effects. In all countries, a mapping of mental health and psychosocial services was also carried out alongside a validated Health Facility Assessment tool to assess the appropriateness of the mental health and psychosocial services provided at facilities and to identify gaps in human resources, institutional infrastructure, and other resources.A total of 386 respondents participated in the study across the three countries (see table below). The relatively large sample size was determined by a combination of resource parameters, the importance of triangulating findings across diverse informants as well as the principle of research saturation, i.e. reaching a point where no new insights were being garnered by additional interviews.Type of respondent, by tool and country Type of respondentGazaLiberiaSri LankaTotals(1 round of data collection)(2 rounds of data collection)(2 rounds of data collection)Adolescents6 FGDs (44 participants in total---30 girls, 14 boys) 12 IDIs (8 girls, 4 boys)27 IDIs (18 girls, 9 boys) 9 FGDs (5 participants in each)64 IDIs (22 girls, 38 boys)192Adults---parents/caregivers, key informants27 IDIs/KIIs12 IDIs/KIIs 4 FGDS (involving 14 mixed sex adults/community leaders)76 IDIs/KIIs171Service providers1 FGD (6 participants)7 IDIs/KIIs2 FGDs (5 participants in each)23Total respondents89147150386Facility assessments53513In each country local researchers with expertise in mental health and psychosocial wellbeing worked alongside an international researcher. Following an initial 4 day training (which included role plays, discussions on how to deal with sensitive issues, piloting and revision of instruments), the teams spent ∼2 weeks in the field gathering data. The identification and selection of respondents in all countries was guided by local contacts, key stakeholders (e.g. service providers) and NGOs working in the study sites. Following initial interviews, snowball sampling was also used. [Table 1](#czx127-T1){ref-type="table"} provides details of the study sites in each country.After obtaining informed consent from research participants, interviews were recorded, transcribed and translated by in-country research partners. A detailed thematic coding structure was developed jointly by the international and country teams and findings from the interviews were analysed following this set of codes and sub-codes. In terms of quality control, daily debriefing sessions were held to facilitate ongoing analysis and reflection, and a subset of the transcripts (both in local languages and translated) were also read by the in-country study leads to ensure quality and consistency. We also had an external peer reviewer who provided detailed feedback on design and analysis questions at each stage of the study. Finally, in-country consultative meetings were organised with key stakeholders, to share and further examine findings comparing these with existing evidence and tacit knowledge, allowing also for the emergence of alternative interpretations.Limitations of the study included the absence of baseline data prior to conflict episodes and resulting challenges in teasing out conflict-specific drivers shaping the outcomes of the mental health and psychosocial services provided to adolescent girls. Additionally, it was challenging to recruit mental health service users---as originally intended in the study---because of confidentiality requirements and their psychological status. As such, the study teams interviewed both adolescent service users and non-service users and was able to obtain sufficient insights from them, as well as from other respondent (especially parents and service providers), about their perceptions of their own and others' mental health and psychosocial status. Given that we emphasise the importance of context specificity, different research teams gave different emphasis to the study components as appropriate to their settings. For example, in Liberia there was a greater focus on mental health and psychosocial support for sexual violence survivors which is a major part of the post-conflict landscape, whereas in Sri Lanka the team placed greater emphasis on issues of stigma surrounding adolescent experiences and the deeply entrenched gendered social norms associated with pubertal development which permeate both service provider attitudes and uptake of services. Results ======= Understanding adolescent psychosocial vulnerabilities in fragile contexts ------------------------------------------------------------------------- In this section we explore how adolescents in our case study countries perceive the factors underpinning their psychosocial health and the risks to it, linking to a number of key domains mapped in [Figure 1](#czx127-F1){ref-type="fig"}. ### Economic resources Most of the girls in our sample identified access to economic resources (including education and livelihood opportunities) as key to their psychosocial wellbeing. In Sri Lanka, study respondents noted that while adolescent girls and their parents strongly valued education, many girls still dropped out of school due to poverty, romantic relationships or early marriage ('Definitely, they won't come back to school when they elope', 14-year old girl, Diyagama). In Liberia, all our study respondents (girls and their parents/carers) noted that education was vital to young people's future, in spite of substantial challenges around supply (e.g. quality of schooling and educational infrastructure) and demand (e.g. financial constraints):"'. my mother used to advise me... not to go around with boys, that once I had a good education, no man would be able to bluff me' (FGD, adolescent girls, New Kru Town)."In all countries, respondents reported that limited access to economic assets and livelihood opportunities by their parents/carers also affected girls' wellbeing. In Liberia, in particular, girls spoke about this causing both themselves and their parents/carers high levels of stress. It also led them to engage in risky sexual behaviours,[^4^](#czx127-en4){ref-type="fn"} as this quote highlights:"'We engage in sexual activities because at times, the things we wish to have our parents are not able to afford them; ... At times our parents coerce us to get involved in early sex...saying don't you see your friends going out there' (adolescent girl in FGD, New Kru Town)." ### Social connections and close relationships Supportive and caring relationships with family and others played a pivotal role in the psychosocial wellbeing of adolescent girls in our sample. In Sri Lanka, most girls reported one or both parents as their closest relative, providing the most day-to-day support."'I tell everything to amma (mother). I tell amma first and then after the others' (girl, 16, Kadalkiramam)."However, many girls also reported feeling disappointed or frustrated by their parents---for instance, citing 'a father's drunken behaviour' or 'being punished by a parent'. Some girls in our sample were living with an older relative (usually female) either because the family had split or one of her parents had died. While some girls described these relationships as loving and positive, others were treated badly and even traumatised by the actions of these relatives. Girls in Sri Lanka reported that relationships beyond the family---with teachers, neighbours and peers, for example---were also important, especially for obtaining advice, sharing problems and having fun. In Liberia, many girl respondents were not living with their biological families, which was a source of considerable pain and anxiety. Even so, they noted that relationships with family (whether immediate or extended), peers and neighbours were vital to their psychosocial wellbeing."'we also learn from our friends; we learn from other people; how they behave; we learn the good things from our friends not the bad things' (girl, 17, Bomi)."In Gaza, the role of family support was also mixed. While some girl respondents sought advice and support from family members, others were more reluctant, afraid that their parents might not listen or may inadvertently make the problem worse:"'If I share the problem with my mother, the problem could become bigger because I know mom will inform my dad of it' (girl, 14, Gaza)."These problems were also observed by specialists working with adolescents, as the following quote shows:"'Adolescents feel that they are not adequately valued by the family and the community. Families don't understand the needs of adolescents. There are many communication gaps between adolescents and their families: adolescents don't understand parents' concerns and worries about them and also parents don't understand needs, aspirations and desires that adolescents have. It's a mutual misunderstanding' (social protection specialist, Gaza City)." ### Competence and self-worth or self-esteem Our findings were mixed regarding self-worth and self-esteem. Despite strong cultural and economic constraints, a significant number of girls in Liberia and Sri Lanka demonstrated a strong sense of self-esteem, describing ambitious plans for the future:"'I wish to study well and be a teacher, build a house and look after my parents' (girl, 14, Diyagama, Sri Lanka).'I want to own house. I want my own money. I want to be working for myself' (girl, 14, Bomi, Liberia)."Study respondents---girls, their parents, and service providers --cited certain factors that were indicative of good self-esteem, including: having the love and support of parents and other relatives; having space to take part in events at school or in the community; and having hopes for a better future. Obstacles to positive self-esteem included: living in a remote area and lack of opportunities for young people to use their skills and talents; lack of places where girls are allowed to meet and socialise; and negative attitudes towards adolescents and young people. In Gaza, however, girls repeatedly complained that their family and community did not value them equally with boys, particularly when it came to sharing their views and thoughts. One girl aged 15 said: 'My grandfather, when we want to speak, says, "You shut up, what do you think you know? You keep yourself out of things that aren't your business."' ### Physical and mental health A theme common to all three countries (notably Liberia) was the sense of inadequate protection and security, which mapped closely onto the physical and mental health domain. In Liberia, difficult family life and abuse of girls (physical and sexual) were noted as major risks to girls' wellbeing. Nearly all girl respondents in that country reported that parents or guardians regularly beat them as a form of discipline. Moreover, interviews and focus groups revealed that close relatives or teachers often perpetrated the sexual violence girls were subject to. In Tubmanburg, Bomi, one 14-year-old girl explained:"'In school if a girl fails a particular subject and asks the teachers to help her, the teacher will want to sleep with her; she may accept to sleep with the teacher; then the teacher exploits the girl.'"Study respondents in Sri Lanka reported that adolescents' physical and mental wellbeing, and particularly girls' wellbeing, is closely linked to their protection and security. Influences on girls include the experiences of the adults in their lives who have lived through war and conflict. Often, these experiences continue to affect how those adults relate to their children even after the conflict has ended. In Gaza most respondents cited fear of sexual harassment as causing anxiety to girls and young women, especially those living in remote and/or border areas. Other risks cited by respondents in all three countries included experiencing aggression or violence in the home, separation of parents and families, one or both parents migrating to find work, or a parent remarrying. All these circumstances can mean that young girls have to live with their extended family, which typically means taking on more domestic chores, loss of support for them to stay in school, and frequent exposure to harsh discipline and insults. In Gaza, for instance, girls faced the added vulnerability of being displaced from their homes, and reported distress at having to stay in mixed-sex shelters. (These were often schools or community centres temporarily repurposed to support displaced families whose homes were damaged by aerial bombing during the 2014 fighting.) ### Coping responses In all three countries, adolescent girls reported developing strategies to cope with psychosocial vulnerability. Individual-level coping strategies included drawing on 'inner strength' (girls in Sri Lanka); in Gaza, girls would read, draw, paint, write stories, use social media or simply daydream; while girls in all three countries mentioned turning to religion, embracing spirituality or consulting traditional healers."'The temple is the most important place for me. I like the calm environment it has' (girl, 19, Diyagama, Sri Lanka).'I go to church. God saves us from troubles and all the sin we commit. God guards me and protects me from many things (girl, 15, Bomi, Liberia)."Across all three countries, girls cited support from the family (immediate nuclear family as well as extended family) as an important strategy in helping them cope with stresses. Aunts were often attributed with understanding girls more than their mothers did, as the aunts were often closer in age to the adolescent girls."'I'm asking God and my aunt to help me and to take care of my child while I'm in school' (17-year-old adolescent mother, Bomi, Liberia)."However, we found that families can also undermine adolescent girls' psychosocial wellbeing, whether due to violence, neglect and lack of privacy. For example, in Gaza, one adolescent lamented: 'I don't share problems with my mother. For one thing, she tells my father and the problem gets bigger!' Within the community, adolescent girls mentioned friends, teachers and staff working for service providers as being important in helping them cope with life's challenges. Girls in Sri Lanka described how some teachers inspired them, gave them confidence and were willing to listen to their problems. In Sri Lanka and Gaza, girl respondents cited emotional support from a social worker or participation in psychosocial support programmes as helping them cope. Girls in all three countries described socialising and engaging in recreational activities as important coping strategies. Accessing formal service provision ---------------------------------- In Sri Lanka and Gaza, there is a relatively established system of formal MHPSS service provision, whereas in Liberia such services are in their early stages. While the past decade has seen considerable progress in strengthening MHPSS systems in all three countries, our study respondents note that there are still limitations in how services and programmes meet girls' specific age and gender needs. In Gaza, our findings suggest that despite MHPSS services being available, young people often do not access them due to cultural and other barriers. Whether in 'normal' every-day or crisis situations, services rarely target the needs of adolescents, with many programmes focusing on younger children. Similarly, in Sri Lanka, although there are a wide range of MHPSS services, few are designed to deal specifically with adolescents, though the gender-based violence desks in hospitals (which offer psychosocial support to victims and survivors, children and adults) are a notable exception. Respondents in Sri Lanka also suggested the need to focus on social rather than medical interventions as a response to the actions of vulnerable adolescents, such as self-harm or suicide. Liberia also has some health and social support programmes targeting young people, but they vary widely in terms of effectiveness and impact. In terms of gender-sensitive services and programmes, service user perceptions were mixed. Study respondents in Gaza reported that many service providers were enthusiastic and caring and wanted to help those accessing services. However, some (male) providers made judgements about the girls who sought such services and, in some cases, were not willing to treat the girl unless she had a chaperone (usually a family member). Not only did this undermine patient confidentiality, it also fuelled concerns around mental illness being a heritable issue, which could damage girls' marriage prospects. According to one study respondent in Gaza (a caregiver): 'The general physician stopped following up my daughters, especially the older one, and he asked me to stop treating her because she is now a young lady, and continuing receiving mental health services will affect her reputation and she will be stigmatised forever... he said "It is enough. Don't take her to any doctor. This will affect her if people know about her case."' Drawing on respondents' perspectives in all three countries, our findings highlight some critical gaps and disconnects in current MHPSS systems. First, according to study respondents, few activities focus on prevention or identifying groups of adolescents who may be at greater risk of mental ill-health. In Liberia, for instance, despite the existence of counsellors in schools, their role was limited to intervening only at times of crisis. 'The counsellors we have at school only counsel us when we fail---that's when they call us to talk' (16-year-old adolescent girl, Bomi, Liberia). Second, in all three countries, key informants noted that poor-quality and fragmented MHPSS services remain a critical challenge to providing a unified response. Thirdly, respondents noted the need for greater strategic direction for MHPSS services, which are hampered by a short-term approach and limited (or a total lack of) follow-up mechanisms. Fourth, lack of evidence-based practice was mentioned by many respondents. Finally, in all three contexts, lack of essential resources (financial and human) remains an important constraint according to all study respondents. A theme that was brought up by the majority of study respondents was the negative attitudes people displayed towards using mental health services in general and in particular to adolescent girls using such services. In all three countries, people experiencing mental health problems can face substantial stigma and discrimination. In Gaza, for example, most study participants felt that although various efforts had been made to reduce stigma (e.g. through carrying out awareness-raising programmes), they have largely proved ineffective; the result is, according to study respondents, that many people delay seeking help from those services and often do so only after using traditional healers. Adolescent girls' psychosocial wellbeing covers many dimensions and sectors, yet the provision of services remains siloed and constrained by weak coordination. In Gaza, respondents were concerned that there was very limited coordination between psychosocial and mental health service providers and programmes, minimal exchange of information and feedback, and hardly any systematic follow-up of referred cases. In Liberia, respondents also noted that there were few synergies across sectors providing specific MHPSS services or programmes. Moreover, despite mental health professionals having received training in child and adolescent services, they have tended to focus their activities on adults and individuals with serious mental illness. In Sri Lanka, vertical integration between key sectors (health, education and child protection), from community-based services to district- and national-level mechanisms (although there remains a strong focus on adolescent girls' protection and medical needs) was identified in policies and approaches by study respondents. However, on the ground, service providers noted that collaboration across sectors is generally low. The situation is similar for horizontal linkages---while there are many non-government organisations (NGOs) working in different sectors, most, according to study respondents, work independently at local level. Discussion ========== Overall, four broad insights emerged from our study that also contribute to literature on the social determinants of health (e.g. [@czx127-B9], [@czx127-B50]). Firstly, it is vital that social determinants are located within specific political economy understandings to take into account the complex and multi-layered challenges that affect access to all services including mental health and psychosocial support services. In our case study countries the political economy is characterised by fragility in terms of governance, the rule of law and institutional capacities and coordination. This fragility will clearly impact the kind, level, comprehensiveness and quality of service provision as well as the extent to which service are accessible and affordable, and particularly for adolescent girls. While all three case study countries are classified as post-conflict, we found that the political economy dynamics vary significantly, with important implications in terms of psychosocial vulnerabilities and the service environment. In Sri Lanka, poverty emerged as the dominant driver of psychosocial vulnerability among adolescent girls, although the conflict persists as a shadow in their lives. By contrast, in Gaza, conflict (or the risk thereof) due to the Israeli blockade and periodic bombings is ever-present for everyone, but with particular challenges for adolescent girls. In Liberia, despite the fact that the conflict formally ended \>10 years ago, levels of sexual violence and abuse of women remain among the highest in the world.[^5^](#czx127-en5){ref-type="fn"} This reflects what others have termed 'hyper-masculinity', whereby social norms around masculinity normalise and reinforce practices of sexual and gender-based violence (SGBV) rather than seeking to reframe acceptable male attitudes and behaviours as part of the broader peace-building process (see e.g. [@czx127-B3], [@czx127-B10], [@czx127-B13], [@czx127-B61]). Secondly, although attention to social and gendered norms within the social determinants of health literature is growing, it remains too often cursory and analysts fail to disentangle the wide-ranging consequences of social and gendered norms, both on health and wider psychosocial wellbeing. Integrating such a perspective is thus critical and what the current study also strives to do. Thirdly, and linked to the second broad insight, our study highlights the particular psychosocial vulnerabilities adolescent girls face and how they are closely linked to and intertwined with a range of gendered social norms that begin to bear heavily on girls' lives as they enter adolescence. Thus informed by our conceptual framework, which outlines adolescent girls' pathways to psychosocial wellbeing, our study suggests that norms around mobility, marriage, sexuality, education, time use and emotional life underlie and explain much of the behaviour of adolescent girls, including how they perceive and experience mental health and psychosocial distress as well the forms of support, both informal and informal, both available to them and which they are able to access. Yet the role that gendered social norms can play in influencing accessibility and update of services is too often neglected in health systems thinking in many developing country contexts. In Sri Lanka, for instance, barriers to psychosocial service uptake for girls include the stigma associated with admitting to psychosocial problems and the fact that a girl cannot voice questions and interest in adolescence, since it is highly associated with taboos around sex and sexuality which are in turn guided by underlying gendered social norms. Similarly, gendered social norms also continue to hamper girls' ability to take up MHPSS services. Among Muslim communities in Sri Lanka religious institutions and leaders typically mediate in cases that involve sexual abuse and early pregnancy, often 'resolving' the matter by ordering the perpetrator to marry the girl. Hence norms around marriage/early marriage in this case override possibilities of girls accessing MHPSS should they be available. Given this context, services were dedicated to addressing 'risks' and providing 'protection' for girls, framing their activities around regulating girls' 'questionable or immoral behaviour' rather than an overriding concern to improve girls' psychosocial wellbeing. In Gaza, gendered social norms which restrict girls' mobility, largely because of fear that this mobility would damage 'family honour' often result in them facing social isolation which can in turn fuel further mental health and psychosocial distress. Similarly the stigma related to MHPSS service uptake in Gaza also inhibits them seeking support since this will likely harm girls' marriage prospects, and given that marriage is one of the most significant life stages in a person's life, and underlies much of the social order, this becomes more important than seeking on MHPSS services. And in Liberia, aggressive masculinities---a legacy of the conflict---and the normalisation of sexual violence, again a key underlying gendered social norm, remain major threats to girls' physical and psychosocial wellbeing, for which services and support remain highly inadequate. Fourthly, although psychosocial wellbeing is arguably subsumed within the social determinants of health framework, our findings highlight that more explicit attention must be paid to psychosocial wellbeing and mental health in conflict-affected contexts, and that these dimensions should be accorded equal weighting to adolescents' physical health concerns. Finally our findings suggest that unless these insights are well understood, efforts to strengthen the human resources needed to support adolescent girls' health and wellbeing may prove ineffective, and may even inadvertently uphold discriminatory gendered social norms that often underpin local health practices. Conclusions =========== Reflecting on the implications of our findings for future policy and practice, we highlight three broad cross-cutting areas that can also be modified for other contexts. First, our study findings highlight that a range of actions are needed to (1) address the factors that contribute to girls' psychosocial vulnerabilities, (2) boost girls' coping strategies, and (3) mitigate against negative coping responses. Such actions include taking a proactive approach to identifying vulnerable groups (including adolescent girls and those at greatest risk of SGBV) and tailoring services and activities to meet their needs. Low-resource measures for mitigating the risk of psychosocial ill-being in fragile contexts could include: encouraging the use of (peer and other) social networks to support adolescents, providing safe spaces for girls, and providing training (to adolescents and their caregivers) on positive coping strategies, including ways to reduce stress. Building on and working with supportive teachers, as well as harnessing the role of the education sector more broadly to help develop adolescents' self-esteem and self-efficacy, also emerged as a critical area for investment. Finally, the generalised stigma that hinders access to psychosocial services needs to be addressed, with substantial efforts for outreach through the media and community mobilisation. Secondly, our findings also indicate that building the capacities of service providers is critical to stimulate an improved service environment. Areas for capacity-building include: early diagnosis of mental health illnesses, provision of SGBV-related services, and treating substance abuse. The service environment, however, should not be limited to these more medicalised approaches; it should include wide-ranging activities, from promoting girls' participation in social activities, to providing spaces for creative expression, as well as providing safe spaces for girls to play and enjoy leisure time. Finally, there is a need for more investment to make services proactive and integrated. In the education sector, for instance, the quality of school-based psychosocial support, counselling services and protocols need to be improved; similarly, in the justice sector, the police need to receive adequate training on SGBV and how to support adolescent girls who are victims or survivors. A third set of actions concerns the need to enhance policy strategies, and to regulate and coordinate actors providing MHPSS services at different levels (community, sub-national and national). There is also a need to strengthen national institutions and ministries so that they become recognised as legitimate regulators of psychosocial services, and can provide, among other things, improved licensing and accreditation processes. Finally, our findings underscore the need to provide more evidence to inform programming, and particularly the need for more robust and disaggregated data to reflect the realities of adolescents' lives. In sum, our findings highlight the importance of recognising adolescents' psychological needs and their gendered patterning, and working to ensure that their needs are integrated into programme design and implementation. Such efforts need to simultaneously tackle the underlying gendered social norms affecting attitudes and behaviours both around psychosocial and mental health service uptake and service provision. This is particularly pertinent in fragile and post-conflict settings if women and girls are to become active agents in change processes, including those related to peace processes and conflict resolution. Ethical approval ================ Ethical approval was sought and obtained through the ODI ethics review board. In addition, the study was also cleared by national research ethics boards in the study countries, i.e. in Liberia, The University of Liberia, the Pacific Institute for Research and Evaluation (PIRE) approved the research, in Gaza the study was reviewed by the Helsinki Framework ethics board, and in Sri Lanka the study was reviewed by a group of practitioners and researchers with relevant expertise. We would like to acknowledge the support of a number of people. In Liberia, The Liberia Center for Outcomes Research led this work supported by Janice Cooper, with Abayomi Cole and Elton Gbollie as research coordinators. In Sri Lanka the work was led by the Good Practice Group and in particular Kusala Wettesinghe, Sarala Emmanuel and Ananda Galappatti. In Gaza the study was led by Bassam Abu Hamad and Nadja Al Bayoumi from Al Quds University. We would also like to thank all the data collectors in the three countries. We also want to acknowledge support from Maria Stavropoulou for her work on the background literature review and Ingrid Gercama for support on data analysis. The Research in Gender and Ethics (RinGs): Building Stronger Health Systems Partnership (funded by DFID) provided invaluable inputs during the development and revision of this paper. Finally, we would like to thank all respondents for giving us their time and sharing their stories with us. Funding ======= This study was funded by the ReBUILD consortium, a multi-year multi-country UK Department for International Development (DFID) funded research programme, and we would like to thank them for their ongoing support and contribution to this research. The views expressed within this paper are not necessarily those of DFID. Conflict of interest statement. None declared. <http://www.who.int/social_determinants/en/> For details of the literature review please see [@czx127-B39]. In Gaza only one round of data collection took place since it was added to the study at the time when it thought the team would not be able to continue fieldwork in Liberia due to Ebola; in fact the team was able to carry out a shorter second round of data collection in Liberal post the height of the epidemic. Transactional sex is widespread throughout sub-Saharan Africa and there is a large body of literature on this. For a recent overview article please see [@czx127-B36]. Rape of girls and women persists and accounts for over one-third of sexual violence cases. 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"training", "child", "adolescent", "services", "tended", "focus", "activities", "adults", "individuals", "serious", "mental", "illness", "Sri", "Lanka", "vertical", "integration", "key", "sectors", "health", "education", "child", "protection", "services", "mechanisms", "although", "remains", "strong", "focus", "adolescent", "girls", "protection", "medical", "needs", "identified", "policies", "approaches", "study", "respondents", "However", "ground", "service", "providers", "noted", "collaboration", "across", "sectors", "generally", "low", "situation", "similar", "horizontal", "linkages", "many", "organisations", "NGOs", "working", "different", "sectors", "according", "study", "respondents", "work", "independently", "local", "level", "Discussion", "Overall", "four", "broad", "insights", "emerged", "study", "also", "contribute", "literature", "social", "determinants", "health", "Firstly", "vital", "social", "determinants", "located", "within", "specific", "political", "economy", "understandings", "take", "account", "complex", "challenges", "affect", "access", "services", "including", "mental", "health", "psychosocial", "support", "services", "case", "study", "countries", "political", "economy", "characterised", "fragility", "terms", "governance", "rule", "law", "institutional", "capacities", "coordination", "fragility", "clearly", "impact", "kind", "level", "comprehensiveness", "quality", "service", "provision", "well", "extent", "service", "accessible", "affordable", "particularly", "adolescent", "girls", "three", "case", "study", "countries", "classified", "found", "political", "economy", "dynamics", "vary", "significantly", "important", "implications", "terms", "psychosocial", "vulnerabilities", "service", "environment", "Sri", "Lanka", "poverty", "emerged", "dominant", "driver", "psychosocial", "vulnerability", "among", "adolescent", "girls", "although", "conflict", "persists", "shadow", "lives", "contrast", "Gaza", "conflict", "risk", "thereof", "due", "Israeli", "blockade", "periodic", "bombings", "everyone", "particular", "challenges", "adolescent", "girls", "Liberia", "despite", "fact", "conflict", "formally", "ended", "years", "ago", "levels", "sexual", "violence", "abuse", "women", "remain", "among", "highest", "world", "fn", "reflects", "others", "termed", "whereby", "social", "norms", "around", "masculinity", "normalise", "reinforce", "practices", "sexual", "violence", "SGBV", "rather", "seeking", "reframe", "acceptable", "male", "attitudes", "behaviours", "part", "broader", "process", "see", "Secondly", "although", "attention", "social", "gendered", "norms", "within", "social", "determinants", "health", "literature", "growing", "remains", "often", "cursory", "analysts", "fail", "disentangle", "consequences", "social", "gendered", "norms", "health", "wider", "psychosocial", "wellbeing", "Integrating", "perspective", "thus", "critical", "current", "study", "also", "strives", "Thirdly", "linked", "second", "broad", "insight", 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"girls", "psychosocial", "wellbeing", "Gaza", "gendered", "social", "norms", "restrict", "girls", "mobility", "largely", "fear", "mobility", "would", "damage", "honour", "often", "result", "facing", "social", "isolation", "turn", "fuel", "mental", "health", "psychosocial", "distress", "Similarly", "stigma", "related", "MHPSS", "service", "uptake", "Gaza", "also", "inhibits", "seeking", "support", "since", "likely", "harm", "girls", "marriage", "prospects", "given", "marriage", "one", "significant", "life", "stages", "person", "life", "underlies", "much", "social", "order", "becomes", "important", "seeking", "MHPSS", "services", "Liberia", "aggressive", "masculinities", "legacy", "conflict", "normalisation", "sexual", "violence", "key", "underlying", "gendered", "social", "norm", "remain", "major", "threats", "girls", "physical", "psychosocial", "wellbeing", "services", "support", "remain", "highly", "inadequate", "Fourthly", "although", "psychosocial", "wellbeing", "arguably", "subsumed", "within", "social", "determinants", "health", "framework", "findings", "highlight", "explicit", "attention", "must", "paid", "psychosocial", "wellbeing", "mental", "health", "contexts", "dimensions", "accorded", "equal", "weighting", "adolescents", "physical", "health", "concerns", "Finally", "findings", "suggest", "unless", "insights", "well", "understood", "efforts", "strengthen", "human", "resources", "needed", "support", "adolescent", "girls", "health", "wellbeing", "may", "prove", "ineffective", "may", "even", "inadvertently", "uphold", "discriminatory", "gendered", "social", "norms", "often", "underpin", "local", "health", "practices", "Conclusions", "Reflecting", "implications", "findings", "future", "policy", "practice", "highlight", "three", "broad", "areas", "also", "modified", "contexts", "First", "study", "findings", "highlight", "range", "actions", "needed", "address", "factors", "contribute", "girls", "psychosocial", "vulnerabilities", "boost", "girls", 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Introduction {#Sec1} ============ Biotechnological process development, analysis, and control is key to obtain robust processes providing highest product quality attributes as well as a reduced time-to-market latency. Catalyzed by regulatory initiatives for biopharmaceutical products, process analytical technology (PAT) emerged as a major tool that demands for bioprocess analysis and control by frequently measurements ensuring specified final product quality \[[@CR1]\]. Especially in biopharmaceutical production and process development of heterologous protein expression, the physiological state of the cells is highly related to the formation of critical quality attributes \[[@CR2], [@CR3]\]. Therefore, time-resolved knowledge about physiological parameters, such as the specific growth rate or specific substrate uptake rate, is essential in the PAT framework as well as to perform process development, characterization, and validation \[[@CR4]\]. Moreover, those variables frequently serve as targets for control strategies \[[@CR5]--[@CR7]\]. The key to this physiological information is the catalyst concentration---the biomass. However, the required online measurement of biomass is a critical endeavor using hard-type sensors such as dielectric spectroscopy, broth fluorescence or permittivity measurements, each connected to limitations and drawbacks as outlined elsewhere \[[@CR8]\]. Software sensors, or short soft-sensors, provide an elegant, non-invasive way to estimate biomass concentration using different other, easy-accessible measurements \[[@CR9]\]. In this contribution, we want to focus on a dominant biotechnological process mode, the microbial fed-batch fermentation, and on the improvement of one of the most mature soft-sensor implementations using off-gas and substrate-feed measurements. These soft-sensors are established tools for bioprocess control and analysis, which was also frequently shown in practical applications \[[@CR5], [@CR10], [@CR11]\]. Briefly, mass conservation laws are used to calculate turnover rates from online measurements, which might be superimposed with signal errors. In a second step accuracy of turnover rates and constraints, formulated as first-order principles such as mass and energy conservation laws, are used to reconcile the inaccurate turnover rates in order to optimally obey the constraints. Finally, the reconciled turnover rates are used to calculate the biomass formation rate (r ~X~), which leads after simple integration over time to the biomass concentration. The resulting information can be used to calculate specific turnover rates, such as the specific substrate uptake rate (q ~S~), which frequently serves as a control variable \[[@CR12]\]. Therefore, r ~X~ and q ~S~ are regarded as the most prevailing benchmark entities to evaluate biomass estimation---and physiological control---capability. However, the control quality by soft-sensors is limited by measurement errors of raw signals used to derive the measured turnover rates. When it comes to industrial applicability, the ultimate question is: Which measurement accuracy is required in order to obtain a sufficiently accurate estimation of the reconciled rates and the biomass? This question can only be answered if the error sources, their respective impact, and possible counteractions are understood. We note that we use the definition of errors as deviations to the true values, excellently defined elsewhere \[[@CR13]\]. Random errors leading to a lack of signal precision are caused by small changes within the system, e.g., air movement, temperature, and electrostatic fluctuations. A multitude of algorithms exists to smooth signals with random errors ranging from simple median filters to polynomial filters such as Savitzky-Golay filter up to frequency filters such as the Butterworth filter. While random errors can be minimized quite easily, this is not the case for systematic errors caused by miscalibrations, inaccuracy of analytical devices, or a defective feature in the sensor. Those systematic errors can only be detected and possibly reduced by making use of all available information in terms of first-principle constraints and the accuracy of turnover rates in reconciliation procedures as outlined above. First-order principles can be generically formulated for defined processes, whereas the accuracy of turnover rates, which are input to the reconciliation procedure, are not known a priori. They highly depend on the accuracy of the raw signal measurements and dynamically change over time. Previously, this was approximated by propagating the variance of measurement accuracies to the turnover rates \[[@CR19]\]. However, commonly, the expected or maximal error on measurement signals is provided by device manufacturers. Therefore, it is an existing unmet need to establish a methodology that leverages this accuracy information of the raw signals onto the derived turnover rates, which are subsequently used in the reconciliation procedure. Hence, it is the goal of this contribution to develop an error propagation procedure to derive the accuracy of turnover rates from expected measurement errors and demonstrate its benefits in terms of increased physiological accuracy within the soft-sensor framework in microbial fed-batch mode. Moreover, in those previously elaborated methodologies, the impact of measurement error on softsensor accuracies could only be estimated retrospectively, given the process data. Therefore, we want to address the question raised above, and present a novel generic workflow that identifies tolerable measurement errors of combinations of multiple analytical measurements in order to meet the desired accuracy of soft-sensor estimations prior to conducted experiments using mechanistic knowledge. Material and methods {#Sec2} ==================== Aim and relevance of the presented approach {#Sec3} ------------------------------------------- The following study was carried out with in silico generated data. The aim of the in silico data generation was to obtain representative microbial fed-batch fermentation data including an induction phase, the predominant industrial mode for the production of recombinant proteins. The experiments were based on a typical Escherichia coli process with oxidative growth and glucose as substrate. As the batch phase is not part of the discussed soft-sensor, only the fed-batch part was considered here. As commonly used in industry, the modeled fed-batch phase started with an exponential feeding profile. After 8 h, the induction phase started with a linear feed rate. The biomass yield coefficient is dynamic. Due to the metabolic load, typically an especially strong decrease can be observed in the induction phase \[[@CR14]\]. This can be measured by the soft-sensor and was also considered in the data generation process (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1a Simulated feed profile and biomass concentration. b Simulated trajectories of the biomass/substrate yield (Y ~X/S~) and the specific growth rate (μ) The advantages of an in silico study are obvious:It is possible to "run" a bioprocess completely without any errors on the signals and to introduce defined errors into the system. This is not possible with real data, as the exact "real" values without errors on the data cannot be determined.A virtually infinite number of experiments with different combinations of errors can be carried out. This enables a systematic study of errors in a high-dimensional "uncertainty space." Computational environment {#Sec4} ------------------------- All calculations were conducted in a MATLAB environment (2015a, The MathWorks, Inc.). The mechanistic model was created in form of a system of ordinary differential equations. As graphical user interface and bioprocessing toolbox inCyght (2016.02, Exputec GmbH) was used. In silico data generation {#Sec5} ------------------------- ### Main mechanistic assumptions {#Sec6} The main mechanistic assumptions behind data generation and soft-sensor are the same. Substrate, ammonia, and oxygen are converted to biomass and carbon dioxide. In this simple case, the extracellular formation of product or metabolites will be neglected. This assumption is true for many biopharmaceutical processes, as the product formation rate often is several order of magnitudes lower than the biomass formation rate \[[@CR15]\]. For processes were this assumption has to be rejected, the soft-sensor framework has to be extended by online product measurement, e.g., by using spectroscopic techniques \[[@CR16]\].$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_S{\mathrm{CH}}_{\mathrm{pH}}{\mathrm{O}}_{\mathrm{pO}}+{r}_{\mathrm{O}2}{\mathrm{O}}_2+{r}_N{\mathrm{N}\mathrm{H}}_3\ \to {r}_X{\mathrm{CH}}_{zH}{\mathrm{O}}_{zO}{\mathrm{N}}_{zn}+{r}_{\mathrm{CO}2}{\mathrm{CO}}_2 $$\end{document}$$ Two first principle assumptions were made; the carbon balance:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_S + {r}_X + {r}_{C{O}_2}=0 $$\end{document}$$ And the degree of reduction balance:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_S{\gamma}_S + {r}_X{\gamma}_X + {r}_{O_2}{\gamma}_{O_2}=0 $$\end{document}$$ The detailed list of equations for the data generation step is shown in the [Electronic Supplementary Material](#Sec27){ref-type="sec"} (ESM) Section 1. ### Addition of noise and errors on the data {#Sec7} To test the original and new soft-sensors with erroneous data, both systematic as well as random errors were introduced into the model. Based on information of off-gas sensor and mass flow controller manufacturers, as summarized in Table [1](#Tab1){ref-type="table"}, realistic amounts of systematic errors were superimposed to the off-gas data which were used as input for the soft-sensors.Table 1Typical measurement errors of off-gas analyzers and mass flow controllersRelative error to measurement valueMeasurement accuracy (zero deviance)Drift/yearΔF ~a,in~ (mass flow controller)±0.5 % of readout±0.3--1 % of full scale±1 % of full scale$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}} $$\end{document}$ (infrared)n.a.±1 % of full scale±1 % of full scale$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta {y}_{{\mathrm{O}}_2,\mathrm{in}} $$\end{document}$ (paramagnetic)±3 % of readout±0.2 % full scale±2 % value$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta {y}_{{\mathrm{O}}_2,\mathrm{in}} $$\end{document}$ (Galvanic cell)±3 % of readout±0.2 % full scale±2 % value O~2~ and CO~2~ concentrations in the exhaust gas are simply applied on the model output for the off-gas data:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{array}{l}{X}_{{\mathrm{O}}_2,\ \mathrm{measured}}={X}_{{\mathrm{O}}_2,\mathrm{model}}\cdot \left(1+{\upvarepsilon}_{{\mathrm{O}}_2}\right)\kern1em \\ {}{X}_{{\mathrm{CO}}_2,\ \mathrm{measured}}={X}_{{\mathrm{CO}}_2,\mathrm{model}}\cdot \left(1+{\upvarepsilon}_{{\mathrm{CO}}_2}\right)\kern1em \end{array} $$\end{document}$$ As the error on the mass flow controller affects both total oxygen and carbon dioxide input into the system and the resulting final concentrations of O~2~ and CO~2~, the error has to be given as input to the model. The set-points for the MFC are the known values, but the model input and real values are calculated as follows.$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{array}{l}{F}_{{\mathrm{O}}_2,\mathrm{in},\mathrm{model}}=\frac{F_{{\mathrm{O}}_2,\mathrm{in},\mathrm{setpoint}}}{1+{\upvarepsilon}_{MFC}}\hfill \\ {}{F}_{{\mathrm{CO}}_2,\mathrm{in},\mathrm{model}}=\frac{F_{{\mathrm{O}}_2,\mathrm{in},\mathrm{setpoint}}}{1+{\upvarepsilon}_{\mathrm{MFC}}}\hfill \end{array} $$\end{document}$$ For the errors in the feed rate, a relative error on the set-point rate is applied.$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_{S,\mathrm{model}} = \frac{r_{S,\mathrm{setpoint}}}{1+{\upvarepsilon}_{r_S}} $$\end{document}$$ The amounts of systematic errors applied for the different experiments are listed in "[Comparison of soft-sensor estimates to unbiased model data](#Sec13){ref-type="sec"}" section. The model delivered an online value each seven seconds. For the addition of random error, white Gaussian noise was added to the off-gas signals. The noise was generated by using MATLAB's awgn function with a relative standard deviation of 1 % for the off-gas data and 10 % on the feed rate. The noise on the feed rate is typically relatively high, as the signal often is calculated by deriving the scale signal. Quantitative evaluation of bioprocess data and error propagation {#Sec8} ---------------------------------------------------------------- ### Preprocessing {#Sec9} As described in the "[Introduction](#Sec1){ref-type="sec"}" section, random errors can be minimized by using preprocessing methods. We decided to apply a Savitzky-Golay filter with a window size of 30 min and second-degree polygon on the off-gas signals. These parameters in most cases showed a low signal distortion, while on the other hand, the elimination of noise was good. However, it has to be noted that for specific filtering and smoothing problems, better filters and filter parameters may exist. In our experience, most of them are not generically applicable, meaning that if they work very well for a specific problem on a defined signal with specific signal dynamics, they may completely fail on another. ### Data-driven rate calculation {#Sec10} The aim of the next section is to express estimators for those conversion rates derived from measurements. In general, all conversion rates can be formulated using the simple idea, that the conversion rate equals the net accumulation within the reactor minus the inflow into the reactor plus the outflow out of the reactor. For demonstration purpose of the subsequent error propagation, the calculation of the conversion rate for CO~2~ will be shown exemplarily:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_{{\mathrm{CO}}_2}\kern0.5em =\kern0.5em \mathrm{C}\mathrm{E}\mathrm{R}\kern0.5em =\kern0.5em \frac{d\left({\mathrm{CO}}_2\right)}{dt\ }\kern0.5em -\dot{{\mathrm{CO}}_{2,\mathrm{in}}}\kern0.5em +\kern0.5em \dot{{\mathrm{CO}}_{2,\mathrm{out}}} $$\end{document}$$ The term $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \frac{d\left({\mathrm{CO}}_2\right)}{dt\ } $$\end{document}$ can be neglected since it is predominantly a function of pH and temperature, which were kept constant over all in silico simulations. Therefore, the carbon emission rate (CER) formulates to:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{C}\mathrm{E}\mathrm{R}\kern0.5em =\kern0.5em \frac{F_{a,\mathrm{in}}}{V_m}\left({y}_{{\mathrm{CO}}_2,\mathrm{out}}\cdot R{a}_{\mathrm{inert}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}\right) $$\end{document}$$ Where Ra ~inert~ is the inert gas ratio, which connects the inflow to the outflow by:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ R{a}_{\mathrm{inert}}=\frac{F_{a,\mathrm{out}}}{F_{a,\mathrm{in}}} $$\end{document}$$ And is defined as:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ R{a}_{\mathrm{inert}}\kern0.5em =\kern0.5em \frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{y_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}} $$\end{document}$$ Here, y ~wet~ is the oxygen concentration in the off-gas stream without bio-reaction and indirectly relates to water stripping out of the reactor. Well-known procedures can be applied in order to calculate the substrate- and oxygen- uptake rate for a substrate limited E. coli fermentation as described elsewhere \[[@CR15]\]. ### Error propagation {#Sec11} In general, all the input signals for estimating the conversion rates are random variables, associated with a random and systematic error, therefore the estimators itself are random variables, too. As discussed in the "[Introduction](#Sec1){ref-type="sec"}" section, random errors in the raw signals can be minimized using preprocessing methods, whereas systematic errors cannot be removed and propagate directly to the estimated conversion rates. However, via Gaussian error propagation, it is possible to estimate the expected error of the conversion rates. This knowledge will subsequently help us to formulate a much more robust reconciliation procedure and estimation of biomass. The influence of the absolute measurement error (Δy) of the signal y onto a derived signal r can be approximated using a Taylor expansion \[[@CR3]\]:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ r\left(y+\varDelta y\right)=r(y) + \frac{1}{1!}\frac{dr(y)}{dy}\cdotp \varDelta y+\frac{1}{2!}\frac{d{}^2r(y)}{dy{}^2}\cdotp {\left(\varDelta y\right)}^2+\dots $$\end{document}$$ We want to note that the absolute measurement error Δy of the measurement signal can be in most cases calculated from technical device data sheets given by their maximal amplitude (e.g., ±3 % of readout). Therefore, the absolute measurement error Δy can be seen as worst-case error. For an approximate solution, the Taylor expansion can be terminated after the second term and the resulting absolute deviation of the derived signal (Δr) can be written as:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ r\left(y+\varDelta y\right)-r(y) = \varDelta r=\frac{dr(y)}{dy}\cdotp \varDelta y $$\end{document}$$ If the derived signal (here the conversion rate) depends on more than one input variable and the error of the input signal is only known by its boundaries, which is the typical case for biotechnological applications, we can write in analogy:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta r=\left\|\frac{\partial r}{\partial {y}_1}\right\|\cdotp \varDelta {y}_1+\left\|\frac{\partial r}{\partial {y}_2}\right\|\cdotp \varDelta {y}_2+\dots $$\end{document}$$ For the CER, the error propagation formulates to:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{array}{l}\varDelta \mathrm{C}\mathrm{E}\mathrm{R}=\left\|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {F}_{a,\mathrm{in}}}\right\|\cdot \varDelta {F}_{a,\mathrm{in}}+\left\|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {V}_m}\right\|\cdot \varDelta {V}_m+\left\|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {y}_{{\mathrm{CO}}_2,\mathrm{out}}}\right\|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}}+\left\|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial R{a}_{\mathrm{inert}}}\right\|\cdot \varDelta R{a}_{\mathrm{inert}}+\left\|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {y}_{{\mathrm{CO}}_2,\mathrm{in}}}\right\|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{in}}\hfill \\ {}\varDelta \mathrm{C}\mathrm{E}\mathrm{R}=\left\|\frac{1}{V_m}\left({y}_{{\mathrm{CO}}_2,\mathrm{out}}\cdot R{a}_{\mathrm{inert}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}\right)\right\|\cdot \varDelta {F}_{a,\mathrm{in}}+\left\|\frac{F_{a,\mathrm{in}}}{V_m^2}\left({y}_{{\mathrm{CO}}_2,\mathrm{out}}\cdot R{a}_{\mathrm{inert}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}\right)\right\|\cdot \varDelta {V}_m+\left\|\frac{F_{a,\mathrm{in}}}{V_m}\cdot R{a}_{\mathrm{inert}}\right\|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}} + \left\|\frac{F_{a,\mathrm{in}}}{V_m}\cdot {y}_{{\mathrm{CO}}_2,\mathrm{out}}\right\|\cdot \varDelta R{a}_{\mathrm{inert}}+\left\|\frac{F_{a,\mathrm{in}}}{V_m}\right\|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{in}}\hfill \\ {}\varDelta {R}_{a,\mathrm{in}\mathrm{ert}}=\left\|\frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{\left({y}_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}\right){}^2}\right\|\cdot \varDelta {y}_{{\mathrm{O}}_2,\mathrm{out}}+\left\|\frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{{\left({y}_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}\right)}^2}\right\|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}}+\left\|\frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{\frac{1}{y_{{\mathrm{O}}_2,\mathrm{in}}}\cdot \left({y}_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}\right){}^2}\right\|\cdot \varDelta {y}_{\mathrm{wet}}\hfill \end{array} $$\end{document}$$ This procedure can easily be extended to the OUR and the substrate uptake rate (r ~S~). Typical results of error propagation to the off-gas rates are shown in Fig. [2a](#Fig2){ref-type="fig"}.Fig. 2a Time-resolved profiles for OUR and CER are shown together with their respective accuracy as error bars, calculated by error propagation as described in "[Error propagation](#Sec11){ref-type="sec"}" section. b Comparison of biomass (black) and r ~x~ (gray) soft-sensor prediction to the unbiased signals (solid lines). Estimations of traditional soft-sensor implementations, assuming 3 % error of all input rates, are shown dotted; the adapted soft-sensor with error propagation for the input rates is shown in dashed lines. For this particular simulation, raw signals were superimposed by 2 % error for CO~2~ and O~2~ off-gas concentration, respectively, and 1 % error on the r ~S~ and the MFC, respectively For the presented in silico study input signals: F ~a,in~, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{CO}}_2,\mathrm{out}} $$\end{document}$ for CER and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{O}}_2,\mathrm{out}} $$\end{document}$ for the OUR, were regarded as superimposed with considerably relevant systematic measurement error. All other input signals were considered to be perfectly accurate. Typical errors for mass flow controllers and off-gas analytics are given in Table [1](#Tab1){ref-type="table"}. However, the error propagation model could be easily extended to more inputs with systematic error. For the error propagation of r ~S~, the only considerably source of systematic signal error was the concentration of the substrate, which might vary due to evaporation during sterilization procedures. ### Minimum variance rate reconciliation and biomass soft-sensor estimation {#Sec12} In the following section, we want to briefly summarize an established minimum variance reconciliation and biomass estimation procedure in order to reduce systematic error on measured turnover rates (OUR, CER, and r ~S~) using first principles as reported in detail elsewhere \[[@CR15], [@CR17], [@CR18]\]. First principles, such as elemental balances (see "[Main mechanistic assumptions](#Sec6){ref-type="sec"}" section), can be seen as constraints to the bioreactor system. We can formulate many of those constraints and thereby connect components with each other. Commonly, a compact matrix formulation is used to connect conversion rates of components with each other using multiple constraints:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ E\cdot r=0 $$\end{document}$$ E is the elemental composition matrix \[e × n\] with e being the number of elemental balances and n the number of relevant components. r is the vector containing the turnover rates. Under real conditions, the elemental balances do not close due to systematic errors of the rates with a residual vector ε:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ E\cdot r=\varepsilon $$\end{document}$$ For this in silico example, the elemental C balance as well as the degree of reduction (DoR) balance were used as frequently applied elsewhere \[[@CR5]\]. In order to identify gross errors in the system, it is necessary to check if the residual vector (ε) differs significantly from zero. Therefore, Reilly and Carpani introduced a statistical measure (h value) which weights the residuals by their accuracy (covariance matrix). Using this χ ^2^ distributed measure, it is possible to set confidence levels for detecting gross errors \[[@CR18]\]. The presented concept can easily be extended to more elemental balances or energy balances; however, the implementation with C- and DoR-balance is predominantly implemented in industry since additional measurements (e.g., nitrogen or heat transfer) are more complex to realize in practice. The redundancy of the measurable rates (rank of redundancy matrix R \[[@CR15], [@CR17], [@CR18]\]) equals 1, and therefore this redundancy can be used to balance the measured rates in a minimum variance sense and obtain reconciled rates \[[@CR15]\]. After no gross error could be detected with statistical significance and the measured rates are reconciled, those reconciled rates can be finally used to estimate the biomass formation rate, which is the only non-measured rate in this example. For this minimum variance balancing procedure, the covariance matrix of the measured signals is required. A fair assumption is to state that the covariance of the measured rates is diagonal, which assumes non-correlated errors in the measured signals. In current soft-sensor implementations, an empirical approach was chosen and the covariance of all measured rates was assumed to equal 3 % of the readout \[[@CR10], [@CR15]\]. As a unique feature of the presented soft-sensor implementation, we will use at this point the derived error boundaries from above as worst-case estimators for the variances of the signals. Since the herein-derived error boundaries vary dynamically over time, the new approach will be further on called adaptive soft-sensor. Comparison of soft-sensor estimates to unbiased model data {#Sec13} ---------------------------------------------------------- As a methodology to investigate the result of the soft-sensor as a function of the error of the input signals, we investigated 5915 in silico experiments with systematically varied errors on the off-gas measurements, substrate concentration, and mass flow controlled (described in "[Addition of noise and errors on the data](#Sec7){ref-type="sec"}" section). The selected ranges in Table [2](#Tab2){ref-type="table"} were based on technical manufacturer information of MFC and off-gas analyzer (see Table [1](#Tab1){ref-type="table"}).Table 2All errors listed here were combined with each other and applied in 5915 experiments. All those in silico generated data sets were used to test the prediction accuracy of traditional and adaptive soft-sensor approachApplied relative error on in- and outputsStep size$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\varepsilon}_{{\mathrm{CO}}_2,\kern0.75em }{\varepsilon}_{{\mathrm{CO}}_2} $$\end{document}$−3 to +3 %0.5 %$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\varepsilon}_{r_S} $$\end{document}$−3 to +3 %1 %ε ~MFC~−2 to +2 %1 % As a final output of the soft-sensor, the estimated biomass formation rate ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\hat{r}}_X $$\end{document}$) was compared to the true, unbiased biomass formation rate from the in silico model (r ~X,true~), which is known. This comparison was done by calculating the median percentage of difference (MPD) over all data points of the time series according to:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{M}\mathrm{P}\mathrm{D}\kern0.75em =\kern0.5em 100\cdot \mathrm{median}\left(\frac{{\hat{r}}_X-{r}_{X,\mathrm{true}}}{r_{X,\mathrm{true}}}\right) $$\end{document}$$ For each of the 5915 simulations, a MPD value for r ~X~ and q ~S~ was calculated. Those values are displayed as surface plots as shown in Figs. [3](#Fig3){ref-type="fig"} and [4](#Fig4){ref-type="fig"}.Fig. 3Comparison of r ~X~ for traditional (left) and adapted (right) soft-sensor in terms of MPD, showing the deviation between the real biomass formation rate and the soft-sensor values (%) as a function of the errors on the off-gas data. (a) Errors on the off-gas data, but no errors on r~S~ and the MFC. (b) Errors on the off-gas data and 2 % error on r ~S~. (c) Errors on the off-gas data and 2 % error on the MFC Fig. 4Comparison of q ~S~ in terms of MPD (a), the median of the estimated relative standard deviation on the reconciled q ~S~ (b), and the median h values (c) for traditional (left) and adapted (right) soft-sensor, depending on the error level of the off-gas analyzers Results {#Sec14} ======= Comparison of the soft-sensors accuracy {#Sec15} --------------------------------------- ### Biomass formation rate {#Sec16} In the following sections, a comparison between the traditional approach and the adaptive soft-sensor is done. While traditionally, the errors on CER, OUR, and r ~S~ were estimated to be 3 % for all rates and over the whole process, the adapted soft-sensor calculates the accuracy on the rates through error propagation, by making use of the known uncertainty ranges of the raw signals. The herein dynamically resolved accuracy for OUR and CER, assuming 3 % maximal error on the read out of off-gas analytical measurements of O~2~ and CO~2~, are depicted in Fig. [2a](#Fig2){ref-type="fig"}. The accuracy of CER is much higher than the accuracy of OUR. This information is used by the adapted soft-sensor; therefore, we obtain much more accurate biomass and r ~X~ estimates than previous implementations without error propagation, compared to unbiased biomass and r ~X~ signals, as shown in Fig. [2b](#Fig2){ref-type="fig"}. Since Fig. [2b](#Fig2){ref-type="fig"} shows only the results for one particular error combination of errors on O~2~, CO~2~, r ~S~ and the MFC, we have to resolve the predictions with all other error combinations in order to show superiority of the adapted soft-sensor. Figure [3](#Fig3){ref-type="fig"} shows a comparison of the biomass formation rate (r ~X~) between the true rates (model) and the estimated rates (left column: traditional soft-sensor, right column: adaptive soft-sensor) by means of MPD. In each of the subfigures, the MPD is shown as a function of the error on the O~2~ and CO~2~ off-gas concentration, varied between −3 and +3 %. The error on the MFC and on r ~S~ was varied across the rows of the subfigures. When using the traditional approach (subplots on the left side), especially errors on the oxygen signal lead to high errors on the estimated rates as well as much higher MPD values (up to 42 % compared to maximal 19 % for the adaptive soft-sensor) and almost horizontal lines of equivalent MPD lines in Fig. [3](#Fig3){ref-type="fig"}. The adaptive soft-sensor propagates the measurement accuracy to the turnover rates, which makes the OUR less trustworthy than the CER as indicated by the error bars in Fig. [2](#Fig2){ref-type="fig"}. Therefore, also errors on the CO~2~ off-gas measurement have an impact on the MPD values, resulting in a rotation of the equivalent MPD lines to the diagonal direction (subplots on the right of Fig. [3](#Fig3){ref-type="fig"}). This change of influential parameters on the MPD will be observed multiple times throughout this work and is always caused by changing the accuracy of turnover rates to realistic values using the error propagation procedure. In the subplots (a1) and (a2) in Fig. [3](#Fig3){ref-type="fig"}, the MPD values regarding r ~X~ are shown as a function solely of error on O~2~ and CO~2~ off-gas measurement. In the subplots b1 and b2, we added a relative error of 2 % on r ~S~ to the true model values and in subplots c1 and c2, we added an error of 2 % on the MFC set-point. The average MPD values as well as the maximal MPD values (up to 40 vs. 18 %) reached throughout all subplots are much lower for the adaptive soft-sensor compared to the traditional approach. ### Control quality for specific substrate uptake rate {#Sec17} As one of the main applications of the soft-sensor is the process control based on physiological parameters, the two versions of the soft-sensors were also compared in terms of prediction accuracy for the specific substrate uptake rate q ~S~. Here, subplots (a1) and (a2) in Fig. [4](#Fig4){ref-type="fig"} show the MPD for q ~S~ with varying error on O~2~ and CO~2~ measurement and no error on r ~S~ and MFC signal. Again, the adaptive soft-sensor shows on average much lower MPD values as well as much lower maximal MPD values (up to 15 % for the traditional and up to 4 % for the adaptive soft-sensor). b1 and b2 show that the estimated relative standard deviation for both soft-sensors is in the area of 3 %. However, when looking at the results in A1 and A2, only the adaptive soft-sensor delivers the estimated standard deviations, since MPD values are in the range of ±3 %. As shown in Fig. [4](#Fig4){ref-type="fig"}(c1 and c2), the h values of the traditional soft-sensor quickly exceed levels of 3. In this case, the null hypothesis, that there is no gross error in the system, has to be rejected with a confidence level of 95 %. However, the system had no gross error in reality, and as the h values of the adaptive soft-sensor show, the null hypothesis cannot be rejected when using the correctly calculated covariance matrix for the minimum variance reconciliation. Therefore, the h values of the traditional soft-sensor have no statistical significance as the covariance matrix, as explained before, is not correctly estimated. ### Integrated comparison over the entire uncertainty space {#Sec18} The goal of this section is to derive a global parameter which we can use to judge which soft-sensor approach leads to generally more accurate estimations. In general, we face a four-dimensional input space consisting of different errors on the O~2~, CO~2~, r ~S~, and MFC measurement. This space will be subsequently called the uncertainty space. At each point in this uncertainty space, the MPD of the true model value of r ~X~ and q ~S~ is compared to the two soft-sensor approaches. Taking the mean of all those MPD values of the uncertainty space for each soft-sensor approach gives us a clear measure which soft-sensor implementation is generally more accurate. This integrated parameter will be called the global average MPD. Table [3](#Tab3){ref-type="table"} summarizes the results of this kind of analysis and shows for each cell the mean MPD of simulations where the O~2~ and CO~2~ error was varied between −3 and +3 %, analogous to one subplot of Fig. [3](#Fig3){ref-type="fig"}. In the columns of Table [2](#Tab2){ref-type="table"}, the error on the MFC is varied, in the rows the error on r ~S~. The global average MPD regarding r ~X~ of the adaptive soft-sensor is 8.7 % compared to 15.2 % of the traditional approach. This is a reduction of the MPD by 43 %. For the estimation of the specific substrate uptake rate q ~S~, the global average MPD can be even lowered from 7.6 to 2.7 which corresponds to a MPD reduction of 64 %.Table 3Comparison of traditional and adaptive soft-sensor for different error levels. Each of the cells show the mean MPD value of simulations in which the error on O~2~ and CO~2~ was varied between −3 and +3 % Generic workflow to ensure appropriate control quality {#Sec19} ------------------------------------------------------ Besides showing superior behavior of the new soft-sensor implementation over state of the art, we want to present a novel generic workflow to obtain a desired soft-sensor estimate or reconciliation quality by adapting accuracy of measurement devices. This workflow includes the following steps as indicated in Fig. [5](#Fig5){ref-type="fig"}:Fig 5Generic workflow for identification of desired measurement error and noise for robust biomass soft-sensor estimation. Asterisk indicate variables which were superimposed by random noise and systematic error Use a mechanistic process model to generate time-resolved data which will be used to derive rates. These are used as input to the soft-sensor (here O~2~ and CO~2~ concentration of the off-gas stream, substrate concentration and inflowing air controlled by MFC).Obtain biased signals by superimposing them with representative systematic (see manufacturer specifications) and random noise (estimated process noise).Calculate turnover rates including their accuracy as described in "[Data-driven rate calculation](#Sec10){ref-type="sec"}" section. The rates and their accuracy (covariance) are input to the soft-sensor.Use the soft-sensor's first principles to reconcile measured turnover rates unless gross errors are detected. The reconciled rates can be used to estimate the biomass and all related entities (e.g., q ~S~ or biomass).The herein obtained q ~S~ estimate (or biomass estimate) is compared to the true, unbiased model signal. If the estimate does not meet the predefined thresholds (e.g., 5 % global average MPD), more accurate measurement devices and their respective measurement errors are used to continue with step 2 to 5 with reduced systematic error levels. The selection of appropriate measurement devices is driven by technical, manufacturing, and financial constraints, which is not scope of this study.If the estimate meets the predefined thresholds in terms of global average MPD, a robust estimate under industrial relevant process conditions is achieved. As an example, the error ranges of Table [2](#Tab2){ref-type="table"} were taken as a starting position in step 2 of the generic workflow presented in Fig. [5](#Fig5){ref-type="fig"}. It was assumed that the desired control quality could not be reached with the current analytical devices (step 5), therefore, exemplary a higher accuracy of the oxygen sensor and MFC from ±3 to ±0.5 % and ±2 to ±1 %, respectively, was implemented. The results before and after this change are shown in Fig. [6](#Fig6){ref-type="fig"}. After the change, the estimated error surface of q ~S~ is rotated in a favorable direction to enlarge regions of lower error (0 to 2 % error), as depicted in the non-shaded areas of the two subfigures of Fig. [6](#Fig6){ref-type="fig"}. Overall, this results in 10 % reduced global average MPD.Fig. 6Estimation error of q ~S~ before (a ±3 % maximal error on oxygen measurement and ±2 % maximal error on MFC) and after (b ±0.5 % maximal error on oxygen measurement and ±1 % maximal error on MFC) the increase of the input signal accuracies. Not only values outside the uncertainty range can be excluded but also the mean MPD inside the uncertainty space is more accurate Discussion {#Sec20} ========== Superior accuracy for the estimated rates of the adaptive soft-sensor {#Sec21} --------------------------------------------------------------------- As perceived in Figs. [2](#Fig2){ref-type="fig"}, [3](#Fig3){ref-type="fig"}, and [4](#Fig4){ref-type="fig"}(a1 and a2) as well as summarized in Table [1](#Tab1){ref-type="table"}, the adaptive soft-sensor delivers more accurate estimates of r ~X~, which integrates to biomass, and q ~S~. Moreover, maximal MPD values for r ~X~ and q ~S~ are much lower for the adaptive soft-sensor which implies that the biomass estimate as well as the control of q ~S~ can be performed much more robust under real process conditions since large deviations to the true values of r ~X~ and q ~S~ can be avoided. This is due to the fact that the covariance matrix for the minimum variance reconciliation procedure is arbitrary chosen for the traditional soft-sensor, which assumes a too low uncertainty range for the OUR, as shown exemplarily in Fig. [2](#Fig2){ref-type="fig"}. The adaptive soft-sensor on the other hand dynamically uses all available information (uncertainty ranges of off-gas analysis) to calculate a realistic covariance matrix. This leads to a much more robust estimate of r ~X~ and q ~S~. For each subfigure, there are some "sweet spots" for certain error combinations, where the classical soft-sensor shows a better accuracy in the prediction of r ~X~. Since the exact combination of the present errors on the input signals is not known a priori, this is no advantage under real process conditions. In a previous approach, the uncertainty of turnover rates was approximated by propagation of variance \[[@CR19]\]. However, under industrial applications, the maximal expected error is provided or known as an empirical parameter. Moreover, in the previous work, simulations were not used to systematically investigate the true error obtained by softsensor estimations. Therefore, our approach including the generic workflow offers the possibility to pre-access the expected dynamics of the process and influence of measurement errors on soft-sensor predictions using mechanistic modeling. Statistical meaningfulness of standard deviation and h value {#Sec22} -------------------------------------------------------------- The covariance matrix is a critical input for the minimum variance reconciliation procedure. As for the traditional soft-sensor, the covariance matrix consists of arbitrary values which do not represent the true and dynamically changing uncertainties; all calculated statistical measures, i.e., standard deviation and h value, lose their significance. This is different for the adaptive soft-sensor. As shown in Fig. [4](#Fig4){ref-type="fig"}(b1 and b2), the relative standard deviations of the estimated rates are in the range of 3 % and almost identical for traditional and adaptive soft-sensor. However, when comparing these standard deviations to the actually measured errors in terms of MPD in Fig. [4](#Fig4){ref-type="fig"}(a1 and a2), it becomes clear that the calculated standard deviations do not fit to these errors for the traditional soft-sensor; the measured MPDs go up to 15 % in the considered area. For the adaptive soft-sensor, the standard deviations are meaningful and on the same magnitude as the actually measured MPDs. Under real process conditions, the calculated standard deviation of an estimated rate is the only available measure to evaluate their prediction accuracy and expected uncertainty and is therefore of critical importance. As shown in Fig. [4](#Fig4){ref-type="fig"}(c1 and c2), the h values of the traditional soft-sensor quickly exceed levels of 3. As already explained in "[Control quality for specific substrate uptake rate](#Sec17){ref-type="sec"}" section, the calculated h values are statistically not meaningful. This means that they cannot be used to detect a gross error in the system with a defined level of significance. They only can be used to relatively compare similar processes or detect gross errors when the h values are magnitudes higher than expected. This is not true for the adaptive soft-sensor, as over the whole uncertainty space the h values are below 3, and no false positive detection of gross errors occurred with 95 % confidence. Applicability of the generic workflow to set measurement accuracies and ensure desired accuracy of soft-sensor estimations {#Sec23} -------------------------------------------------------------------------------------------------------------------------- The question about the required measurement accuracy of raw signals to meet the desired accuracy of derived variables, such as the soft-sensor estimation for bioprocess control, is equally urged by device manufacturer as by process engineers. This is due to the fact that measurement accuracy is often correlated to higher asset costs of advanced devices or more frequent maintenance intervals of existing devices. In "[Generic workflow to ensure appropriate control quality](#Sec19){ref-type="sec"}" section, we present a generic workflow to answer this question. Since the measurement accuracy of the derived soft-sensor estimate is not only a function of the accuracy of the input signals (step 2 of the workflow) but also of the dynamics of the process, a mechanistic model has to provide this information (step 1 of the workflow). If one has multiple possibilities of exchanging devices or maintenance intervals to increase accuracy of input signals, this can be solved iteratively in the workflow by testing different of those combinations and evaluating if the resulting accuracy of the soft-sensor is sufficient. Moreover, as shown in Fig. [6](#Fig6){ref-type="fig"}, it is thereby possible to not only get rid of areas with high levels of MPD but rather additionally increase the accuracy in the reduced uncertainty space due to the introduction of supplementary knowledge about the accuracy of input signals. Extrapolations of the adaptive soft-sensor and the generic workflow to other application areas {#Sec24} ---------------------------------------------------------------------------------------------- The presented error propagation approach as well as the presented workflow are generically applicable to include additional sources of information. For example, the consideration of energy balances based on the metabolic heat production during a process \[[@CR20]\] or the already mentioned combination of the soft-sensor with spectroscopic techniques \[[@CR16]\] could be included. This would result in an even more robust and diverse applicable package. Conclusion {#Sec25} ========== In this contribution, we aim to present an error propagation procedure increasing the accuracy and robustness of the soft-sensor estimates. Traditionally, the uncertainties for conversion rates (CER, OUR, r ~s~) were arbitrarily assumed and static over the whole process. Here, we established a novel procedure to obtain meaningful uncertainties, dynamically changing over time, which are used as representative knowledge source together with first principles in the soft-sensor framework. In this in silico case study, the new approach using the adaptive soft-sensor, the error on the estimates could be reduced by 43 % for the estimated biomass growth rate (r ~X~) compared to traditional soft-sensor implementations. For the estimation of the specific substrate uptake rate q ~S~, the error on the estimate could even be lowered by 64 %. When using the traditional soft-sensor approach, the resulting h values could not be used to statistically reject the null hypothesis of detecting gross errors, since estimations of covariance of the turnover rates were arbitrarily chosen and static over time. The new approach delivers both statistically meaningful h values for the detection of gross errors and informative standard deviations on the estimated rates. Latter ones are essential under real process conditions to judge soft-sensor estimation quality, as obviously there exist no possibility to evaluate the control quality by comparing the estimates to unbiased model values. Additionally, we presented a new generic approach to ensure a predefined control quality of the soft-sensor estimate by iteratively evaluating the effect of the different errors on the raw signal measurements. It has been demonstrated that by following this generic workflow, it is possible to additionally significantly increase the adaptive soft-sensor accuracy. The presented approach can be generically applied taking also additional error sources into account. The new methodology is practically applicable to industrial conditions, where maximal errors of measurement devices are used to obtain dynamically changing accuracies of derived turnover rates as shown in Fig. [2a](#Fig2){ref-type="fig"}. Electronic supplementary material ================================= {#Sec27} Below is the link to the electronic supplementary material.ESM 1(PDF 651 kb) CER : Carbon dioxide evolution rate (mol h^−1^) E : Elemental composition matrix F~a,in~ : Air flow in (L min^−1^) F~a,out~ : Air flow out (L min^−1^) MFC : Mass flow controller MPD : Median percentage of difference OUR : Oxygen uptake rate (mol h^−1^) q~S~ : Specific substrate uptake rate (mol mol^−1^ h^−1^) Ra~inert~ : Inert gas ratio (−) r~i~ : Consumption/formation rate for species i (mol h^−1^) r~X~ : Biomass formation rate (mol h^−1^) S : Substrate, C-normalized (mol) V~m~ : Molar volume (L mol^−1^) X : Biomass, C-normalized (mol) µ : Specific growth rate (h^−1^) ε : Residual vector for non-closing balances y~wet~ : Oxygen fraction in the off-gas without microbial activity (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{O}}_2,\mathrm{out}} $$\end{document}$ : Oxygen fraction in the off-gas stream (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{O}}_2,\mathrm{in}} $$\end{document}$ : Oxygen fraction in the inlet air (−) γ~i~ : Degree of reduction for species i (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{CO}}_2,\mathrm{out}} $$\end{document}$ : Carbon dioxide fraction in the off-gas stream (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{CO}}_2,\mathrm{in}} $$\end{document}$ : Carbon dioxide fraction in the inlet air (−) Y~X/S~ : Biomass/substrate yield coefficient ε~i~ : Applied relative error on species i (−) Δy : Absolute measurement error of signal y Valentin Steinwandter and Thomas Zahel contributed equally to this work. Open access funding provided by TU Wien (TUW). Authors' contributions {#d29e4286} ====================== Thomas Zahel developed and implemented the error propagation procedure within the softsensor framework as well as the design of the generic workflow to identify tolerable measurement accuracy to deliver predefined softsensor accuracy. Valentin Steinwandter developed the in silico simulation environment, consisting of the representative mechanistic model as well as the test environment to systematically investigate the uncertainty space and implemented the generic workflow. Both authors contributed equally to the writing of the presented work. Funding {#FPar1} ======= Financial support was provided by the Austrian research funding association (FFG) under the scope of the COMET program within the research project "Industrial Methods for Process Analytical Chemistry---From Measurement Technologies to Information Systems (imPACts)" (contract \# 843546). Conflict of interest {#FPar2} ==================== The authors declare that they have no conflict of interest.	{ "pile_set_name": "PubMed Central" }	[ "amount", "two", "paramagnetic±", "almost", "control", "given", "work", "origin", "divers", "induct", "program", "eg", "nitrogen", "decreas", "ie", "purpos", "inaccuraci", "exist", "subfigur", "savitzkygolay", "identifi", "written", "kind", "rais", "select", "fund", "take", "valu", "maxim", "convers", "multipl", "signal", "may", "toolbox", "taylor", "softsensor", "therebi", "graphic", "q", "use", "gener", "amplitud", "hatrx", "judg", "noisecalcul", "constraint", "final", "support", "fedbatch", "caus", "complet", "yfracfracdrydycdotp", "multitud", "prior", "accord", "focu", "signific", "establish", "ecdot", "usepackagewasysym", "gross", "full", "carbon", "temperatur", "becom", "emiss", "comet", "relev", "sizedocumentclassptminim", "process", "excel", "right", "also", "growth", "benchmark", "molar", "fracrsmathrmsetpointupvarepsilonr", 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Introduction {#s1} ============ Inhibitory control, the ability to suppress planned or ongoing cognitive or motor processes is necessary to ensure flexible and adapted goal-directed behavior in ever-changing environments (Aron, [@B3]; Dillon and Pizzagalli, [@B23]). Converging functional neuroimaging, transcranial magnetic stimulation and lesion data indicate that inhibitory control relies on a cortico-subcortical network involving the right inferior frontal gyrus (rIFG), the pre-supplementary motor area (SMA) and the basal ganglia (Garavan et al., [@B31]; Aron et al., [@B6]; Chambers et al., [@B19]; Majid et al., [@B51]), and manifesting at latencies of 150--400 ms after the onset of the stimuli associated with the inhibition goals (Kaiser et al., [@B39]; Smith and Douglas, [@B72]). Although inhibitory control performance mostly depends on how the stimuli triggering the inhibition are processed, mounting evidence indicates that the cognitive- and brain-states preceding the presentation of these stimuli also contribute to the success of the inhibition. In Eriksen flanker tasks, contrasts between event-related potentials (ERPs) time-locked to the motor responses to trial preceding error vs. accurate trials revealed a specific ERP component peaking 50--100 ms post-response onset over frontal electrode sites \[The "error related positivity" (EPP) Ridderinkhof et al., [@B69]; Allain et al., [@B1]; Hajcak et al., [@B34]\]. Britz and Michel ([@B13]) extended these results by showing that dorsolateral prefrontal cortices were engaged differentially during the 100 ms preceding the stimulus onset in error vs. correct trials during a classical color stroop task. These collective results suggest that errors are foreshadowed by a disruption of prefrontal task monitoring systems before the actual need for inhibitory control (see also Eichele et al., [@B26]; Masaki et al., [@B53]; Steinhauser et al., [@B76]). Additional support for the critical role of pre-stimulus brain states in inhibitory control performance comes from studies in which the occurrence of the inhibition stimuli was predictable. In such cases, proactive inhibitory mechanisms are engaged before the stimulus presentation and interact with stimulus-driven reactive inhibitory mechanisms to eventually enhance inhibitory control (Claffey et al., [@B20]; Jahfari et al., [@B38]; Aron, [@B4]; Cai et al., [@B18]; Duque et al., [@B24]; Majid et al., [@B51]). By manipulating the degree of predictability of inhibition trials, these studies showed that when response inhibition can be prepared, effector-selective proactive inhibition mechanisms, mediated by the dorsolateral prefrontal cortex, are engaged to support fronto-basal reactive inhibition mechanisms (Aron, [@B4] for review). Criaud et al. ([@B21]) further demonstrated that proactive mechanisms persist once established at the beginning of each trial, suggesting that proactive inhibition has not only a transient effect but also modifies the general response mode of the participants. Taken together, these findings suggest that inhibitory control performance does not solely depend on how participants manage the conflict induced by the stimulus but also on the state of the task-monitoring and control systems before the trial. However, the studies conducted so far on the state-dependency of inhibitory control were based on response- or stimulus-evoked ERPs and thus could not reveal whether spontaneous, not-time-locked fluctuations of ongoing brain activity preceding unpredictable inhibition-stimuli impact on inhibitory control performance. The averaging of the EEG signal in ERP studies indeed canceled out the activity not time-locked to the event of interest and thus dismissed this substantial fraction of the variability of the raw electrical brain activity (e.g., Arieli et al., [@B2]). Based on the current evidence for the state-dependency of behavioral and brain responses during various types of perceptual tasks (e.g., Lehmann et al., [@B45]; Ress et al., [@B68]; Fox et al., [@B30]; Fox and Raichle, [@B29]; Britz et al., [@B12], [@B14]), we hypothesize that spontaneous brain states immediately preceding the presentation of inhibition-triggering stimuli might also influence the success or failure to inhibit responses during inhibitory control tasks. To address this question, we used well-established methods of single-trial topographic analyses of EEG to determine whether specific voltage topographies present at the moment of the onset of unpredictable were associated with correct rejection (CR) vs. false alarms (FAs) to NoGo stimuli during a Go/NoGo task (Lehmann et al., [@B45]; Kondakor et al., [@B43], [@B42]; Koenig et al., [@B40]; Muller et al., [@B58]; Britz et al., [@B12]; Brodbeck et al., [@B15]). The current investigation was based on a reanalysis of the data from Manuel et al. ([@B52]) in which EEG was recorded in eleven healthy participants during an auditory spatial Go/NoGo task. Material and methods ==================== Participants ------------ Eleven healthy volunteers participated in the study, all male and right-handed (Oldfield, [@B61]), aged 22--39 years (mean ± SD, 29.4 ± 1.6 years). Each participant provided written, informed consent to participate in the study. No participant had a history of neurological or psychiatric illness, and all reported normal hearing. All procedures were approved by the Ethics Committee of the Faculty of Biology and Medicine of the Vaudois University Hospital Center and University of Lausanne. Stimuli ------- Auditory stimuli were 150-ms noise bursts (200--500 Hz bandpass filtered; 5 ms rise/fall), lateralized by means of a right- or left-ear leading interaural time difference of 770 μ s resulting in a perceived lateralization of \~80° from the central midline (Blauert, [@B9]). The sounds were presented via ER-4P Etymotic earphones. Procedure and task ------------------ The current study is based on a reanalysis of the data from Manuel et al. ([@B52]), in which the procedure and task are already detailed; we thus present only the main task parameters here. Participants were seated in an electrically shielded and sound-attenuated booth in front of a 19-in screen. Stimulus delivery and response recording were controlled using E-prime 2.0. The paradigm comprised an auditory spatial Go/NoGo task in which participants had to respond as quickly as possible via a button to left-lateralized sounds (Go stimuli, hereafter termed LG) and to withhold responses to right-lateralized sounds (NoGo stimuli, termed RNG). Each trial started with the presentation of a centrally presented gray cross on a black background for a randomly determined duration ranging from 1000 to 1900 ms. At the same time that the cross disappeared, the LG and RNG sounds were presented and response collection window was opened. In the Go conditions, a feedback on accuracy and response speed was provided immediately after the response. LG and RNG trials were presented with an equal probability of 0.5. The experiment was divided into three sessions. Each session started with a calibration block of 16 randomly presented trials (eight LG and eight RNG), followed by two test blocks of 80 randomly presented trials each (40 LG and 40 RNG). The calibration blocks were used to individually adjust the task difficulty and to maintain time pressure across the whole experiment. During each calibration phase, the mean response time (RT) to LG trials was calculated online and used to determine the individual participant\'s RT threshold (RTt), which was set at 80% of the mean RT from the calibration block. During the test block, a Go response RT was considered as correct if it was below the 80% RTt of the immediately preceding calibration phase. Otherwise, a feedback screen indicating "too late!" was displayed immediately after the Go response (slow hit). On each trial, the mean percentage of correct trials, including fast hit and CR, was displayed. Participants were not informed about this thresholding procedure. Except for the global accuracy, no visual feedback was displayed after fast hits or FAs (see Vocat et al., [@B79] for a similar procedure). The whole Go/NoGo training session included a total of 528 stimuli (160 stimuli in the test block + 16 stimuli in the calibration block × 3 sessions = 528) and lasted for a total of \~ 35 min. After the completion of each session, a rest period of 10 min was provided to participants. EEG acquisition and preprocessing --------------------------------- Continuous EEG was acquired at 1024 Hz through a 128-channel Biosemi ActiveTwo system referenced to the common mode sense/driven right leg ground. All the EEG analyses were conducted with the Cartool software (Brunet et al., [@B16]). Before the single trial analyses, data at artifact electrodes from each participant were interpolated (Perrin et al., [@B65]). EEG epochs of the 50 ms preceding the stimulus onset were extracted from the raw EEG data, for each participant, for RNG CR and for the RNG FAs conditions. Because there was less FA than CR (see the behavioral results), we balanced the number of epochs included in each condition before the analyses to ensure that any potential differences between the two conditions did not follow from difference in statistical power. First, the same number of CR as FA epochs was randomly extracted for each participant separately. On the resulting epochs, a ±80 μV artifact rejection criterion was applied to exclude trials with eye blinks or other artifacts. When necessary, the number of epochs was then again balanced across conditions to eventually result in the inclusion of 25.4 ± 16.8 (mean ± SD) epochs in each condition for the single-trial analyses. Behavioral data analyses ------------------------ Behavioral data were analyzed to determine whether FA commission occurred randomly within the sequence of NoGo stimuli (see the Discussion section). We analyzed the pattern of FA occurrence by calculating the temporal auto-correlation function of the FA response type. If there was a relationship between FA occurrence at one trial with FA occurrence at previous or subsequent trials, as could for instance occur during periods of decrease in attention inducing series of FA, it should manifest as an increase in the autocorrelation coefficient (Britz et al., [@B12], [@B14]; Bernasconi et al., [@B8]). We first performed a binary classification on the FAs and the Hits and Misses (respectively responded and missed Go trials), as well as CR of NoGo trials. To determine if the patterns of FA of the participants were different from a random distribution, we permuted 1000 times the sequence of each participant and then compared the autocorrelation coefficients of the sequence of the participant to the distribution of the autocorrelation coefficients of the randomized sequence of the participant. The autocorrelation coefficients of the FA occurrence were computed for each trial n with that in trial n + m for m = 1 − 20. Single-trial topographic analyses --------------------------------- The time-locked averaging of the EEG signal across multiple repetitions of an event (e.g., the presentation of a stimulus or a behavioral response) to build ERPs cancels out all the fluctuations of brain activity not time-locked to the event of interest, because by definition, the phase of these fluctuations varies across trials. To circumvent this problem and to investigate whether non-stimulus-locked variations in the prestimulus activity impact on inhibitory control proficiency, we utilized previously published methods of single-trial EEG analysis (Lehmann et al., [@B45]; Kondakor et al., [@B43], [@B42]; Koenig et al., [@B41]; Mohr et al., [@B57]; Britz et al., [@B12]; see for review Britz and Michel, [@B13]; Eichele et al., [@B26]; Steinhauser et al., [@B76]). This approach is based on evidence that evoked and induced ongoing EEG signal is not random, but rather organized in a succession of quasi-stable topographies of the electric potentials. Since the configuration of the electric potentials at the scalp reflects the sum of all active sources in the brain at each moment in time (Lehmann et al., [@B46]), the observation of stable topographies or "maps" suggest that during these time intervals, the functional state of the brain is stable (e.g., Michel et al., [@B55]). The short periods of functional stability have been referred to as "functional microstate" and typically comprise 80--120 long segments of stable configuration of the scalp-recorded voltage topography of the electric potential (Lehmann and Skrandies, [@B47]; Koenig et al., [@B41]). While EEG topography remains stable during a microstate, the strength of the electric potentials, as indexed, for example, by the Global Field Power (GFP), increases and then decreases. The GFP represents a single-number index of the strength of electric potentials; it is calculated as the spatial standard deviation of the electric potentials: the square root of the sum of all squared potentials divided by the number of electrodes (Lehmann and Skrandies, [@B47]; Murray et al., [@B59]). Compelling relationships have been found between spontaneously occurring microstates preceding the presentation of a stimulus and the behavioral and brain response to this stimulus (Lehmann et al., [@B45]; Kondakor et al., [@B43], [@B42]; Mohr et al., [@B57]; Muller et al., [@B58]; Britz et al., [@B12]), suggesting that microstate-based single-trial EEG topographic analyses enable reliable investigations of the effects of variations in the raw ongoing EEG signal. The single-trial topographic analysis comprises the following processing steps, which mainly consist in reducing the raw EEG data into a limited number of common stable microstates within and then across participants (for similar procedures see also Koenig et al., [@B41]; Mohr et al., [@B57]; Britz et al., [@B12], [@B14]). The first step involved determining the topographies differentiating the FAs and CR conditions for each participant: (1) We extracted from each EEG epoch, for each participant and each condition the topography manifesting at the single time frame when the GFP was maximal (i.e., when the signal-to noise ratio of the microstate was the highest) within the 50 ms pre-stimulus onset. We selected this pre-stimulus time frame because it corresponds to the half of the duration of an average microstate and thus this procedure enables to extract topography at the time frame best representing the microstate present at the moment when the stimulus is presented (the total length of a microstate ranges between 80 to 120 ms; Britz et al., [@B14]); (2) For each participant separately, we applied a spatial k-means cluster analysis (e.g., Pascual-Marqui et al., [@B63]) on these topographies to identify the most dominant topographies among all topographies extracted in step (1). (3) The optimal number of clusters, or "template topographies," was determined based on a modified Krzanowski-Lai criterion (Krzanowski and Lai, [@B44]); (4) A fitting procedure, in which each original topography of step (1) was relabeled with the template map of step (2) with which it best correlated (Pegna et al., [@B64]), enabled us to identify the two template topographies that best differentiated the FA and CR conditions: the template map with the highest frequency of occurrence in the FA and in the CR conditions were retained. The second step comprised determining the topographies differentiating the FA and CR across participants: (5) We applied a second k-mean cluster analysis on the groups of FA and CR map identified during the first step for each participant and back-fitted the resulting template topographies on the original data using the same procedure as steps (1), (2), and (3). The statistical comparison between the frequency of occurrence of the maps in the FA and CR conditions revealed the topographies differentiating the two conditions at the group level. Electrical source estimations ----------------------------- Electrical sources underlying the template map(s) of interest were estimated using a distributed linear inverse solution based on a local autoregressive average (LAURA) regularization approach (Grave De Peralta Menendez et al., [@B33]; Grave-De Peralta et al., [@B32]; see also Michel et al., [@B56] for a comparison of inverse solution methods) implemented in the Cartool software (Brunet et al., [@B16]). LAURA selects the source configuration that mimics the biophysical behavior of electric potential (i.e., activity at one point depends on the activity at neighboring points). The solution space is based on a realistic head model and included 3005 solution points homogeneously distributed within the gray matter of the average brain of the Montreal Neurological Institute (courtesy of R. Grave de Peralta Menendez and S. Gonzalez Andino, University Hospital of Geneva, Geneva, Switzerland). Results {#s2} ======= Behavior -------- Reaction time to Go stimuli was 251 ± 21 ms (mean ± SEM). The percentage of FAs was 10.9 ± 0.95% (Manuel et al., [@B52]). The autocorrelation coefficients of the sequence of FA of the participants were all between z = −0.37 and 0.64 SD from the mean of the coefficients from the randomized sequences, indicating no significant (−1.96 \< z \< 1.96; p \> 0.05) differences between the distributions of FA in the random vs. actual sequences for lags 1--20 (Figure [1](#F1){ref-type="fig"}). ![Analysis of the sequence of false alarms. For lags 1--20, the maximal (blue), mean (green), and minimal (red) autocorrelation coefficients are represented, for the sequences of the participants (plain lines) and for randomized sequences (dotted lines). The sequences of FA from the participant did not differ statistically from the random sequences (see the Results section), supporting that random fluctuations in the pre-stimulus period may account for FA occurrence.](fnhum-07-00238-g0001){#F1} Single-trial topographic analyses --------------------------------- Consistent with previous applications of the current single-trial topographic analysis (Britz et al., [@B12], [@B14]), the k-means cluster analysis applied during the first step of the analysis (see the Method section) identified on average 5.9 ± 1.6 maps for each participant as optimally explaining the data. These template maps accounted for 72.7 ± 7.3% of the global explained variance. The maps were then grouped for each conditions across participants and a second cluster analysis was applied. The best clustering during the second step explained 93% of the variance with 11 template maps. The resulting template maps were then back-fitted to the original individual subject data for each conditions to determine their frequency of occurrence. Two-tailed pairwise t-tests were conducted to compare the frequency of occurrence of each template map between the CR and FA conditions. A single topographic map was found to differ in its frequency of occurrence between the FA and CR conditions (Figure [2](#F2){ref-type="fig"}, violet square): This topography occurred significantly more often in the CR than FA condition \[t~(10)~ = 2.83; p = 0.018; d~z~ = 0.9; Figure [2](#F2){ref-type="fig"}\]. To test for the probability of type 1 errors, we generated 1000 randomized permutations of the raw data maps and computed, for each of the permutation, the same t-tests as those we applied to the actual data. The results revealed a probability of 1.7% to have the same pattern as in the real data (i.e., one p-values \< 0.019 among the 11 tests) in the 1000 permutations, i.e., when there was no structure in the data. ![Topographies of the prestimulus microstates as revealed by the cluster analysis. The map that significantly differentiated the correct rejections is framed in violet. The red lines indicate the t-test p \< 0.05 significance threshold and an asterisk is placed where significance has been reached.](fnhum-07-00238-g0002){#F2} Electrical source estimations ----------------------------- The estimation of the electric sources at the origin of the template topography differentiating the CR from the FA condition showed that it resulted from the activation of a right hemispheric parieto-frontal network extending from the inferior parietal lobule to the frontal gyri (Figure [3](#F3){ref-type="fig"}). The largest hub of activation was centered around the supramarginal gyrus/primary auditory cortex. Importantly, this result is only a visualization of the sources underlying the EEG topography and not the result of a statistical contrast; it should thus be interpreted with caution. ![LAURA distributed source estimations corresponding to the topography identified as best characterizing the pre-stimulus period before correct rejections identified a right hemispheric fronto-temporal prestimulus activation.](fnhum-07-00238-g0003){#F3} Discussion {#s3} ========== The present study suggests that performance in a classical Go/NoGo task is influenced by the momentary state of the ongoing brain activity immediately preceding the onset of unpredictable NoGo stimuli. A specific EEG voltage topography manifested more frequently before successful response-inhibition to NoGo stimuli than before FAs. There was no evidence for topographies specifically preceding FA trials. Electrical source estimations localized the source of the topography preceding successful inhibition within a right fronto-temporal network. Our results contribute to current knowledge on the influence of pre-stimulus brain state on inhibitory control by indicating that inhibition performance is modulated by the momentary brain state present when the stimuli are presented. Fluctuations in the ongoing brain activity have been shown to represent almost 90% of the variability in the EEG signal (Eichele et al., [@B26]). This source of variability was mostly dismissed in previous ERPs studies on the effect of pre-stimulus activity because time-locked averaging of the EEG signal were applied to extract ERPs (e.g., Hajcak et al., [@B34]; Masaki et al., [@B53]); in such procedures, the signal not time-locked to the event is cancelled out and usually considered as background physiological noise. In line with our findings, mounting evidence indicate that ongoing spontaneous EEG fluctuations are actually functionally relevant and account for a substantial fraction of the variability in the behavioral and brain responses to stimuli in various experimental contexts (Picton et al., [@B66]; O\'Connell et al., [@B60] for the role of pre-stimulus endogenous modulation in oscillatory activity in task performance; Britz and Michel, [@B13] for discussion). However, the dependency of sensory-cognitive processes to single trial variability in brain activity was so far demonstrated in perceptual but not executive tasks. For instance, Mohr et al. ([@B57]) showed that a specific topographic map with a left anterior-right posterior dipole orientation spontaneously manifesting before word presentation predicted an enhanced discrimination of emotional word when the word was presented in the left visual field but only for men during a bilateral lexical decision task. Lehmann et al. ([@B45]) and Kondakor et al. ([@B43], [@B42]) further demonstrated that the brain responses to identical tones depended on the spontaneously occurring topography preceding their presentation. Corroborating and extending these findings, our results suggest that spontaneous fluctuations of ongoing brain activity not only modulate perceptual processes but also high-order, top-down executive mechanisms as those supporting inhibitory control in conflict tasks. The sources of the scalp topography preceding more frequently CR than FAs were maximal within a right fronto-parietal network. The right rIFG has been repeatedly involved in inhibitory control by functional neuroimaging (Garavan et al., [@B31]; Rubia et al., [@B71]; Aron and Poldrack, [@B7]; Aron et al., [@B5]) and lesion studies (Decary and Richer, [@B22]; Aron et al., [@B6]; Rieger et al., [@B70]; Floden and Stuss, [@B28]; Picton et al., [@B67]). The rIFG is thought to trigger motor inhibition via its connections to the subthalamic nucleus (STN; Inase et al., [@B37]; Aron et al., [@B5]; Aron, [@B4]). Most of these studies, however, involved the rIFG in inhibitory control by contrasting the post-stimulus brain responses to stimuli associated with vs. without response inhibition. In the current study, we showed that random fluctuations in the activity of rIFG before the demand for inhibition also play a critical role in inhibitory control proficiency. In conditions when the onset of the NoGo stimuli cannot be predicted, if the NoGo stimuli are presented when the activity of the rIFG is high, the probability of a correct inhibition increases. Although speculative, an account for this effect could be that a pre-activation of the rIFG increases the speed of inhibition process because less time is needed to reach the elicitation threshold of the inhibitory command from frontal to subcortical structures. Right inferior parietal areas have been previously involved in response inhibition (Garavan et al., [@B31]; Liddle et al., [@B49]; Menon et al., [@B54]; Rubia et al., [@B71]) or response conflict resolution (Braver et al., [@B11]; Van Veen et al., [@B78]). These studies suggest that inferior parietal cortices mediate attention to the task and might thus, in turn, modulate performance (see also Hampshire et al., [@B35]). Increase in the activity of the precentral gyrus in condition of successful inhibition has also been reported in stop-signal task (though more superior as in the current study; Li et al., [@B48]), and have been interpreted as "negative motor areas" (Ikeda et al., [@B36]; Yazawa et al., [@B80]), whose direct stimulation elicits response inhibition (Luders et al., [@B50]). Alternatively, since participants had to respond with their right hand, an activation of ipsilateral (right) motor areas might have inhibited contralateral, homotopic motor areas via interhemispheric inhibition mechanisms and in turn facilitated the rejection of NoGo stimuli (e.g., Ferbert et al., [@B27]). Hand and arm motor representations are, however, higher along the central sulcus as the area found in the current study. The precise role of the right temporal structures in inhibitory control is more difficult to delineate. These areas have been shown to interact with higher level prefrontal regions during inhibitory control tasks (Egner and Hirsch, [@B25]) and to be modulated in inhibition-related disorders (Tamm et al., [@B77]; Solanto et al., [@B73]), but their precise role remains unclear. An alternative account for the role of right temporal areas in FA commission would be their involvement in the processing of the auditory spatial features distinguishing Go from NoGo stimuli in the current task. Go and NoGo goals activation indeed first require discriminating whether the stimulus is presented on the left or right auditory hemifield. Right temporal areas have been repeatedly involved in the early stage of auditory spatial processing (Spierer et al., [@B75], [@B74]) and their pre-activation could have facilitated the detection of NoGo stimuli and in turn response inhibition. In line with this hypothesis, Manuel et al. ([@B52]) suggested an important role of early auditory analyses of the lateralization of the stimuli in inhibitory control proficiency during the current auditory spatial Go/NoGo task. Importantly, however, we would like to emphasize that our source estimations were only visualizations, rather than a statistical analysis, of the network likely underlying the topography preceding FA commission. These results should thus be interpreted with caution. Although our pattern of result is highly consistent with current evidence for a crucial role of the rIGF in stimulus-induced inhibitory control processes, it contrasts with previous data on the brain regions whose pre-stimulus activity modulates inhibition proficiency. Electrophysiological studies on trials preceding error during an Eriksen flankers task identified a specific response-locked ERP component at 100 ms (EPP; Ridderinkhof et al., [@B69]; Allain et al., [@B1]; Hajcak et al., [@B34]). The EPP was specific to trials preceding an error and its amplitude was larger on frontal, central and parietal midline electrodes, compatible with a generator located within the anterior cingulate cortex (ACC). Using a stroop task, Britz and Michel ([@B13]) showed a specific pattern of stimulus-locked fronto-parietal activity before erroneous vs. correct trials. The modulation of prefrontal activity before error commission found in the studies reviewed above was interpreted as reflecting a transient disruption of action monitoring and executive control systems. Our results complement these finding by demonstrating that in addition to the impact of maintaining a minimal level of action monitoring during the task found by response- or stimulus-locked ERP approaches, spontaneous ongoing activity within the networks mediating stimulus-induced response inhibition also influences inhibitory control performance. In line with this assumption, growing evidence indicate that as soon as the need for response inhibition can be predicted, proactive inhibitory control mechanisms are engaged to facilitate the inhibitory control of task-irrelevant responses (Claffey et al., [@B20]; Jahfari et al., [@B38]; see Aron, [@B4] for review; Cai et al., [@B18]; Criaud et al., [@B21]). "Active breaking" mechanisms would be engaged before the stimulus onset to decrease the activity of motor area and in turn facilitate response inhibition (Cai et al., [@B18]; Duque et al., [@B24]). Anticipatory mechanisms have been shown to rely on a fronto-striatal network partly overlapping with the estimated sources of the topography prominently associated with successful inhibition in the current study. We cannot rule out that the frequency of occurrence of the topography preceding successful inhibition was influenced by proactive preparatory mechanisms in the current study. According to this hypothesis, the anticipation of NoGo stimuli would have increased the frequency of occurrence of the "facilitatory" topography or, in other word, pre-activated inhibitory control mechanisms. Speaking against this hypothesis, there was no strategic advantage of intentionally maintaining inhibitory control networks active in our task because such decrease in the response elicitation threshold would have resulted in a general decrease in response speed to Go stimuli. In our paradigm, emphasis was put on response speed: slow Hits (correctly responded Go stimuli but above the RTt determined in calibration blocks) were considered as error and reported explicitly as such by the visual feedback provided after each trial. Although there was a probability of 0.5 for NoGo stimuli occurrence, response prepotency was maintained very high by the recurrent feedback on responses speed. The threshold for considering a response to Go trials to slow was indeed individually adjusted to 80% of the mean response speed calculated during the calibration blocks intervening between experimental blocks (see the Method section). Hence, we interpret our results as indicating that when the fronto-temporal brain network was already (spontaneously) activated before a NoGo stimulus was presented, inhibition performance was improved. Compatible with this hypothesis, the analyses of the distribution of FA occurrence across the experimental session showed that participants committed FA following the same pattern as when FA were randomly distributed within the sequence. The adoption of any strategy by the participant would have most likely resulted in non-random sequences consisting of series of FA or CR because a specific task set could unlikely be maintained constantly during the whole experiment (Palva and Palva, [@B62]). Our results cannot disentangle the relationship between proactive inhibition and the occurrence of specific microstates. To our knowledge, no study has so far investigated whether top-down, strategic implementation of task sets can impact on the frequency of occurrence-specific microstate and thus whether proactive mechanism could have influenced our pattern of result. Further studies focusing on longer epochs and manipulating the engagement of proactive inhibitory mechanisms are necessary to elucidate this question. Of note, we found a specific EEG topography preceding successful trials, but no specific topography preceding errors. This pattern of result suggests that while the engagement of specific cognitive set may enhance motor responses inhibition in conditions of high response prepotency, many different deviations from this optimal brain-state could result in inhibition failure, in turn explaining that we did not identify a specific topographic map preceding errors. In this regard, a limitation of the current study is that by defining our FA and CR conditions only based on RT (and lack thereof), we possibly included in the CR condition some trials in which erroneous motor activation was actually engaged but inhibited before reaching activation threshold. Referred to as "covert" errors, these partial-error trials can only be detected with the recording of electromyography activations and have been suggested to depend on distinct mechanisms as overt errors (Burle et al., [@B17]; Allain et al., [@B1]; Boulinguez et al., [@B10]). In any case, the inclusion of covert error trials in the CR condition would have increased noise in the data and thus most likely increased the probability of type 2 but not type 1 errors. Second, because FAs were quite rare in our data, our single trial topographic analyses included only a limited number of trials. While the capacity to deal with small numbers of EEG epochs is an advantage of this approach and we reach reliable statistical results, how the number of epochs impacts the statistical outcome of our approach remains to be determined. Collectively, our results point out that spontaneous fluctuations within inhibitory control networks at the moment when NoGo stimuli are presented influence inhibition performance. The question remains open, however, as to whether and how prestimulus modulations impact on the processing of the inhibition-related stimuli. It would be notably interesting to link directly the current results with previous literature on pre-target predictor of inhibition performance and to examine potential differences in the early sensory processing of the NoGo stimuli between FA and CR trials. Further studies comparing the pre-stimulus with the post-stimulus activity would be necessary to elucidate this question. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by a grant from the Swiss National Science Foundation to Lucas Spierer (\#320030_143348). The Cartool software has been programmed by Denis Brunet (Functional Brain Mapping Laboratory, Geneva, Switzerland) and supported by the Center for Biomedical Imaging of Geneva and Lausanne. We thank David Magezi, David Souto and the reviewers for their valuable comments. [^1]: Edited by: John J. Foxe, Albert Einstein College of Medicine, USA [^2]: Reviewed by: Redmond O\'Connell, Trinity College Dublin, Ireland; Kevin Whittingstall, Université de Sherbrooke, Canada	{ "pile_set_name": "PubMed Central" }	[ "root", "sma", "rest", "probabl", "two", "almost", "control", "refer", "anticipatori", "work", "origin", "remain", "program", "eg", "decreas", "compris", "hz", "ie", "compat", "respond", "pariet", "frontotempor", "interhemispher", "soundattenu", "μv", "collect", "thank", "masaki", "sum", "ikeda", "frontal", "ress", "anticip", "put", "identifi", "basal", "network", "length", "written", "block", "cai", "classif", "retain", "select", "grant", "execut", "mechan", "commit", "maxim", "task", "locat", "multipl", "signal", "may", "squar", "acquir", "separ", "flanker", "gener", "use", "autocorrel", "amplitud", "hajcak", "ms", "ongo", "support", "interven", "majid", "focus", "stimulusdriven", "poldrack", "complet", "imag", "accord", "paradigm", "signific", "gyri", "establish", "build", 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"et", "Liddle", "et", "Menon", "et", "Rubia", "et", "response", "conflict", "resolution", "Braver", "et", "Van", "Veen", "et", "studies", "suggest", "inferior", "parietal", "cortices", "mediate", "attention", "task", "might", "thus", "turn", "modulate", "performance", "see", "also", "Hampshire", "et", "Increase", "activity", "precentral", "gyrus", "condition", "successful", "inhibition", "also", "reported", "task", "though", "superior", "current", "study", "Li", "et", "interpreted", "negative", "motor", "areas", "Ikeda", "et", "Yazawa", "et", "whose", "direct", "stimulation", "elicits", "response", "inhibition", "Luders", "et", "Alternatively", "since", "participants", "respond", "right", "hand", "activation", "ipsilateral", "right", "motor", "areas", "might", "inhibited", "contralateral", "homotopic", "motor", "areas", "via", "interhemispheric", "inhibition", "mechanisms", "turn", "facilitated", "rejection", "NoGo", "stimuli", "Ferbert", "et", "Hand", "arm", "motor", "representations", "however", "higher", "along", "central", "sulcus", "area", "found", "current", "study", "precise", "role", "right", "temporal", "structures", "inhibitory", "control", "difficult", "delineate", "areas", "shown", "interact", "higher", "level", "prefrontal", "regions", "inhibitory", "control", "tasks", "Egner", "Hirsch", "modulated", "disorders", "Tamm", "et", "Solanto", "et", "precise", "role", "remains", "unclear", "alternative", "account", "role", "right", "temporal", "areas", "FA", "commission", "would", "involvement", "processing", "auditory", "spatial", "features", "distinguishing", "Go", "NoGo", "stimuli", "current", "task", "Go", "NoGo", "goals", "activation", "indeed", "first", "require", "discriminating", "whether", "stimulus", "presented", "left", "right", "auditory", "hemifield", "Right", "temporal", "areas", "repeatedly", "involved", "early", "stage", "auditory", "spatial", "processing", "Spierer", "et", "could", "facilitated", "detection", "NoGo", "stimuli", "turn", "response", "inhibition", "line", "hypothesis", "Manuel", "et", "al", "suggested", "important", "role", "early", "auditory", "analyses", "lateralization", "stimuli", "inhibitory", "control", "proficiency", "current", "auditory", "spatial", "task", "Importantly", "however", "would", "like", "emphasize", "source", "estimations", "visualizations", "rather", "statistical", "analysis", "network", "likely", "underlying", "topography", "preceding", "FA", "commission", "results", "thus", "interpreted", "caution", "Although", "pattern", "result", "highly", "consistent", "current", "evidence", "crucial", "role", "rIGF", "inhibitory", "control", "processes", "contrasts", "previous", "data", "brain", "regions", "whose", "activity", "modulates", "inhibition", "proficiency", "Electrophysiological", "studies", "trials", "preceding", "error", "Eriksen", "flankers", "task", "identified", "specific", "ERP", "component", "ms", "EPP", "Ridderinkhof", "et", "Allain", "et", "Hajcak", "et", "EPP", "specific", "trials", "preceding", "error", "amplitude", "larger", "frontal", "central", "parietal", "midline", "electrodes", "compatible", "generator", "located", "within", "anterior", "cingulate", "cortex", "ACC", "Using", "stroop", "task", "Britz", "Michel", "showed", "specific", "pattern", "activity", "erroneous", "correct", "trials", "modulation", "prefrontal", "activity", "error", "commission", "found", "studies", "reviewed", "interpreted", "reflecting", "transient", "disruption", "action", "monitoring", "executive", "control", "systems", "results", "complement", "finding", "demonstrating", "addition", "impact", "maintaining", "minimal", "level", "action", "monitoring", "task", "found", "ERP", "approaches", "spontaneous", "ongoing", "activity", "within", "networks", "mediating", "response", "inhibition", "also", "influences", "inhibitory", "control", "performance", "line", "assumption", "growing", "evidence", "indicate", "soon", "need", "response", "inhibition", "predicted", "proactive", "inhibitory", "control", "mechanisms", "engaged", "facilitate", "inhibitory", "control", "responses", "Claffey", "et", "Jahfari", "et", "see", "Aron", "review", "Cai", "et", "Criaud", "et", "Active", "breaking", "mechanisms", "would", "engaged", "stimulus", "onset", "decrease", "activity", "motor", "area", "turn", "facilitate", "response", "inhibition", "Cai", "et", "Duque", "et", "Anticipatory", "mechanisms", "shown", "rely", "network", "partly", "overlapping", "estimated", "sources", "topography", "prominently", "associated", "successful", "inhibition", "current", "study", "rule", "frequency", "occurrence", "topography", "preceding", "successful", "inhibition", "influenced", "proactive", "preparatory", "mechanisms", "current", "study", "According", "hypothesis", "anticipation", "NoGo", "stimuli", "would", "increased", "frequency", "occurrence", "facilitatory", "topography", "word", "inhibitory", "control", "mechanisms", "Speaking", "hypothesis", "strategic", 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"necessary", "elucidate", "question", "note", "found", "specific", "EEG", "topography", "preceding", "successful", "trials", "specific", "topography", "preceding", "errors", "pattern", "result", "suggests", "engagement", "specific", "cognitive", "set", "may", "enhance", "motor", "responses", "inhibition", "conditions", "high", "response", "prepotency", "many", "different", "deviations", "optimal", "could", "result", "inhibition", "failure", "turn", "explaining", "identify", "specific", "topographic", "map", "preceding", "errors", "regard", "limitation", "current", "study", "defining", "FA", "CR", "conditions", "based", "RT", "lack", "thereof", "possibly", "included", "CR", "condition", "trials", "erroneous", "motor", "activation", "actually", "engaged", "inhibited", "reaching", "activation", "threshold", "Referred", "covert", "errors", "trials", "detected", "recording", "electromyography", "activations", "suggested", "depend", "distinct", "mechanisms", "overt", "errors", "Burle", "et", "Allain", "et", "Boulinguez", "et", "case", "inclusion", "covert", "error", "trials", "CR", "condition", "would", "increased", "noise", "data", "thus", "likely", "increased", "probability", "type", "type", "errors", "Second", "FAs", "quite", "rare", "data", "single", "trial", "topographic", "analyses", "included", "limited", "number", "trials", "capacity", "deal", "small", "numbers", "EEG", "epochs", "advantage", "approach", "reach", "reliable", "statistical", "results", "number", "epochs", "impacts", "statistical", "outcome", "approach", "remains", "determined", "Collectively", "results", "point", "spontaneous", "fluctuations", "within", "inhibitory", "control", "networks", "moment", "NoGo", "stimuli", "presented", "influence", "inhibition", "performance", "question", "remains", "open", "however", "whether", "prestimulus", "modulations", "impact", "processing", "stimuli", "would", "notably", "interesting", "link", "directly", "current", "results", "previous", "literature", 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"de", "Sherbrooke", "Canada" ]
Introduction {#section1-2324709613492503} ============ "Pseudo--Ludwig's angina" is a descriptor that has been used for sublingual swelling due to noninfectious causes, as opposed to the true condition, Ludwig's angina, which is a manifestation of regional suppuration in the neck. Multiple case reports have described sublingual hematoma secondary to hemorrhage in patients anticoagulated with warfarin.^[@bibr1-2324709613492503][@bibr2-2324709613492503][@bibr3-2324709613492503][@bibr4-2324709613492503][@bibr5-2324709613492503][@bibr6-2324709613492503][@bibr7-2324709613492503][@bibr8-2324709613492503]-[@bibr9-2324709613492503]^ Sublingual hematoma with airway compromise secondary to brodifacoum, a common long-acting anticoagulant rodenticide, has only been reported in the veterinary literature.^[@bibr10-2324709613492503]^ A large sublingual hematoma can mimic Ludwig's angina; either etiology can compromise the airway. Initial treatment includes either reversal of the bleeding diathesis for the former or appropriate antibiotic coverage for the latter. Angioedema is yet another potential cause of upper airway obstruction due to swelling, but this etiology is not typically invoked as underlying "pseudo--Ludwig's angina." We report a case of massive tongue swelling and impending airway compromise in the context of an intentional ingestion of the long-acting anticoagulant brodifacoum. This presentation was initially suspected to be due to local hemorrhage, consistent with coagulopathy and the previously reported pattern of "pseudo-Ludwig's angina" associated with warfarin misadventure. Ultimately, however, imaging excluded the presence of hematoma and the clinical course was more consistent with angioedema, temporally implicated in response to the brodifacoum ingestion. Case Report {#section2-2324709613492503} =========== A 32-year-old African American male-to-female transgender patient presented to the emergency department, complaining of inability to swallow and difficulty speaking for the previous 2 days. She denied odynophagia, fever, trauma, seizure, or significant respiratory symptoms. Past medical history included depression, chronic pain, and a remote suicide attempt. On physical exam, she was afebrile and had a normal respiratory rate and oxygenation by pulse oximetry. The oral examination was remarkable for a massively swollen tongue, elevation of the sublingual tissue, and fullness of the anterior neck. She could not vocalize. The remainder of the physical examination was unremarkable. The patient was given empiric intravenous clindamycin to cover for possible infection and dexamethasone to reduce swelling. Anesthesia and otolaryngology services were consulted for emergent airway management and the patient was taken directly to the operating room for fiber-optic nasal intubation. While in the operating room, prior to intubation, she communicated by writing that she had swallowed a rat poison 4 weeks prior to presentation in an intentional suicide attempt. Her initial international normalization rate (INR), which returned a short time later, was greater than 13.7. During intubation, she was noted to have tongue, nasopharyngeal, oropharyngeal, left arytenoid, and epiglottic swelling. After intubation, she was admitted to the intensive care unit for management of her airway. To treat her coagulopathy, she was administered high-dose vitamin K and fresh frozen plasma. Dexamethasone and clindamycin were continued. A computed tomography scan of the head was obtained on hospital day 2. This showed diffuse tongue, floor of mouth, submental, and submandibular space edema but no abnormal increased density that would be expected with a discrete hematoma or diffuse interstitial bleeding ([Figure 1](#fig1-2324709613492503){ref-type="fig"}). Her airway swelling improved slowly and she was successfully extubated on hospital day seven. The patient was able to converse with minimal difficulty, being able to describe the brand packaging and price of the rat poison she ingested, which confirmed that it was a brodifacoum-containing product. She denied any co-ingestion or that she was taking any other daily medications at that time. The patient's coagulopathy initially required vitamin K doses of 100 mg every 6 hours per feeding tube as well as intermittent fresh frozen plasma to maintain an INR less than 3. On hospital day 13, she was transferred out of the intensive care unit. Her corticosteroid therapy was tapered without recurrence of swelling. She was ultimately transitioned to vitamin K by mouth, which was safely reduced to a total daily dose of 25 mg daily. A psychiatric consultation determined that she was no longer a danger to herself and she was discharged on hospital day 17 with the plan to further taper her vitamin K by 2.5 mg every week and monitor weekly INR values. A brodifacoum level was not quantified. ![Axial contrast-enhanced computed tomography scan through the oropharynx demonstrates diffuse edema throughout the swollen tongue (\), which fills the entire oral cavity resulting in severe narrowing of the pharyngeal airway with patency maintained by endotracheal tube and nasogastric tube (arrow)](10.1177_2324709613492503-fig1){#fig1-2324709613492503} Discussion {#section3-2324709613492503} ========== This is the first case report of airway obstruction associated with over-anticoagulation yet without hematoma. Hematoma leading to such obstruction, referred to as "Pseudo--Ludwig's angina," is a well-established warfarin-associated phenomenon (including a case report from our own institution).^[@bibr1-2324709613492503][@bibr2-2324709613492503][@bibr3-2324709613492503][@bibr4-2324709613492503][@bibr5-2324709613492503][@bibr6-2324709613492503][@bibr7-2324709613492503][@bibr8-2324709613492503]-[@bibr9-2324709613492503]^ It has only been documented previously in association with brodifacoum in the veterinary literature.^[@bibr10-2324709613492503]^ The absence of hematoma, coupled with the clinical course, leads us to conclude that the patient's presentation is best explained by angioedema and that this angioedema can be attributed to brodifacoum. Although angioedema is a well-recognized complication of many pharmaceuticals, it has neither been reported with anticoagulants nor is it listed in the package insert for Coumadin.^[@bibr11-2324709613492503]^ Nonetheless, this potential complication of long-acting anticoagulants, even if unusual, is important because such ingestions are common. In 2010, there were 10 488 human exposures to similar long-acting anticoagulant rodenticides reported to US Poison Control centers, of which 2774 were treated in health care facilities.^[@bibr12-2324709613492503]^ Similar to warfarin, brodifacoum inhibits the hepatic synthesis of vitamin K--dependent clotting factors. The addition of the brominated polycyclic hydrocarbon side chain to the basic warfarin structure has several properties that serve to promote anticoagulation: It allows brodifacoum to have a higher affinity for hepatic tissue, it interrupts the vitamin K(1)-epoxide cycle at more than one point, it has greater lipid solubility, and it manifests enterohepatic recirculation.^[@bibr13-2324709613492503]^ Several of these attributes lead to a markedly longer half-life for brodifacoum as compared with warfarin.^[@bibr14-2324709613492503]^ Management of hemorrhage secondary to overdose has been discussed in several studies.^[@bibr15-2324709613492503][@bibr16-2324709613492503][@bibr17-2324709613492503][@bibr18-2324709613492503][@bibr19-2324709613492503][@bibr20-2324709613492503]-[@bibr21-2324709613492503]^ Bromine-containing drugs as a class have certain unusual features. Exposure to bromine and brominated products has been reported to cause a plaque forming dermatitis, termed "bromoderma." The exact mechanism is unknown, but it is postulated to be either an inflammatory or delayed hypersensitivity reaction.^[@bibr22-2324709613492503],[@bibr23-2324709613492503]^ This, however, is not a classic angioedema pattern. Nonetheless, it does underscore that bromine is associated with delayed responses and could signal a different risk profile than that of warfarin. Warfarin is not bromine containing and has been long and widely used without reported angioedema. Indeed, warfarin has been suggested as a potential treatment to prevent angioedema.^[@bibr24-2324709613492503][@bibr25-2324709613492503][@bibr26-2324709613492503]-[@bibr27-2324709613492503]^ Based on our review of the literature, we are unable to establish a clear mechanism of action for angioedema caused by brodifacoum. Angioedema is a well-recognized risk of angiotensin-converting enzyme (ACE) inhibitors.^[@bibr28-2324709613492503]^ The chemical structure of brodifacoum, however, does not appear to be similar to that class of medications and there is no evidence to suggest that it functionally interacts with ACE receptors^[@bibr29-2324709613492503]^ and it is not otherwise known to inhibit the metabolism of ACE. We did not rechallenge the patient to brodifacoum or another bromine-containing substance to support a causal association, nor can we absolutely exclude that another unreported exposure may have been involved or perhaps interacted with the brodifacoum. Conclusion {#section4-2324709613492503} ========== Airway compromise secondary to over anticoagulation, although rare, previously has been well documented from local hematoma, rather than a result of the angioedema we report here. In addition to hemorrhage, airway angioedema should remain in the differential for the etiology of airway compromise, even when coagulopathy from an anticoagulant is present. Declaration of Conflicting Interests:* The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. 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Introduction {#s1} ============ Thyrotropin-releasing hormone (TRH) was originally isolated as the first hypothalamic hormone [@pone.0040437-Boler1], [@pone.0040437-Burgus1] and a major stimulator of the secretion of thyrotropin (TSH) from the anterior pituitary gland [@pone.0040437-Jackson1]. Subsequently, TRH was also found to promote production of TSH in part by stimulating transcription of the TSHβ and α genes. TRH binds to its receptor in the anterior pituitary and activates phospholipase C, leading to calcium mobilization and protein kinase C activation [@pone.0040437-Carr1]--[@pone.0040437-Kiley1] and also stimulation of the MAPK pathway [@pone.0040437-Kanda1], [@pone.0040437-Ohmichi1]. The actions of these intracellular signaling pathways ultimately lead to an increase in transcription of the TSHβ and α genes [@pone.0040437-Shupnik1], [@pone.0040437-Sun1]. However, precisely how TRH mediates transcription of the TSHβ gene in vivo still remains unclear. A pituitary-specific transcription factor, Pit1, was first postulated as a candidate protein that influences TRH-induced stimulation of the TSHβ gene. Pit1 which contains two transactivation domains termed the POU-specific domain and POU homeo domain is expressed in somatotrophs, lactotrophs and thyrotrophs, and is critical for the development of pituitary thyrotrophs [@pone.0040437-Andersen1]. In fact, a patient with a mutation of the Pit1 gene exhibited TSH, PRL- and GH- deficiency [@pone.0040437-Tatsumi1], [@pone.0040437-Cohen1]. Pit1 has also been reported to be important for regulation of the TSHβ gene by TRH [@pone.0040437-Andersen1]. TRH-dependent phosphorylation of Pit1 has been suggested to increase Pit1-binding to low-affinity TSHβ promoter-binding sites, and overexpression of a mutant Pit1 containing the DNA-binding domain but lacking the major transactivation domain substantially blocked the TRH-induction of the TSHβ promoter activity in GH3 cells [@pone.0040437-Steinfelder1]. Therefore, TRH may exert its function by changing the state of the Pit1 protein. The second candidate for a protein involved with TRH-induced stimulation of the TSHβ gene is GATA2 [@pone.0040437-Gordon1]. GATA2 belongs to a subtype of transcription factors, the GATA family, that binds through its Zn finger domain with the GATA-responsive element (GATA-RE), which has high homology among all GATA family members [@pone.0040437-Ko1]. GATA2 is expressed in thyrotrophs and gonadotrophs in the pituitary [@pone.0040437-Dasen1]. It has been reported that TRH enhanced GATA2- dependent activation of the TSHβ promoter and that this stimulation was abolished by an amino-acid substitution of the GATA2-Zn finger domain or a mutation of the GATA-responsive element of the TSHβ gene. In addition, an recent EMSA study by Oba et al revealed that TRH increased the DNA-binding capacity of GATA2 on the gene [@pone.0040437-Ohba1]. We generated TRH-deficient mice using homologous recombination in embryonic stem cells [@pone.0040437-Yamada1]. These mice show characteristic phenotypes, including tertiary hypothyroidism and mild hyperglycemia. The basal serum TSH level was unexpectedly elevated, and the result of the TRH test suggested that the secreted TSH had reduced biological activity. An ontogeny based analysis of these mice demonstrated that there was no requirement for TRH in the development of embryonic thyrotrophs in the pituitary, but TRH was required for the maintenance of the normal function of pituitary thyrotrophs [@pone.0040437-Shibusawa1]. NR4A1 (also known as Nur77, NGFI-B or TR3) belongs to a superfamily of orphan nuclear receptors and was originally isolated as an immediate-early response gene induced by a nerve growth factor in a pheochromocytoma cell line, PC12 [@pone.0040437-Milbrandt1]. NR4A1 is also regulated by many physiological stimuli including growth factors, inflammatory signals and hormones, and implicated in a wide range of important biological processes including apoptosis, brain development, glucose metabolism, and vascular remodeling [@pone.0040437-MartnezGonzlez1]--[@pone.0040437-Liu1]. Expression of NR4A1 has also been identified in several endocrine organs including the anterior pituitary, ovary, adrenal gland, and testis [@pone.0040437-Stocco1]--[@pone.0040437-Smith1]. Several investigators have demonstrated that NR4A1 plays an important role in the function of the hypothalamo-pituitary-adrenal (HPA) axis and hypothalamo-pituitary-gonadal (HPG) axis [@pone.0040437-Philips1], [@pone.0040437-Philips2], [@pone.0040437-Inaoka1], [@pone.0040437-Fernandez1]. However, there is no report regarding the involvement of NR4A1 in the hypothalamo-pituitary-thyroid axis, since there has been no appropriate animal model to use. Pit1 is necessary but not sufficient for basal transcription of the TSHβ gene, in fact, the basal activity of the TSHβ promoter was minimally stimulated when co-transfected with a mouse Pit1 in the thyrotroph cell lines αTSH and TtT-97 [@pone.0040437-Gordon2]. In addition, several studies indicated that TRH-induced activation of the TSHß gene requires both PKC and MAPK pathways [@pone.0040437-Carr1]--[@pone.0040437-Ohmichi1], [@pone.0040437-Sun1]. TRH-induced stimulation of GATA2-activation of the TSHβ promoter depends on PKC but not MAPK in GH3 cells [@pone.0040437-Ohba1]. Therefore, there may be additional factors involved with TRH-induced stimulation of the TSHβ gene in vivo. In the present study, to elucidate the mechanisms underlying TRH-induced stimulation of the TSHβ gene in vivo, we first examined the role of TRH in the secretion and synthesis of TSH by measuring serum TSH and pituitary TSH mRNA levels in different thyroid status in TRH knockout mice. We then attempted to determine factors related to action of TRH on the TSHβ gene through expression profiling of pituitary mRNAs using wild-type and TRH-deficient pituitary supplemented with thyroid hormone. We discovered that an orphan nuclear receptor, NR4A1, played a critical role in TRH-mediated stimulation of the pituitary TSHβ gene in vivo. Results {#s2} ======= TRH Regulates the Basal Activity and Responsiveness to Thyroid Hormone of the TSHβ Gene In vivo {#s2a} ----------------------------------------------------------------------------------------------- As previously reported, the serum TSH levels in homozygotes of TRH knockout mice (TRHKO) (143.9±7.9 ng/ml, n = 7) were slightly but significantly higher that those in the wild-type (71.3±4.1 ng/ml, n = 7, p\<0.01) and heterozygous mice (81.2±2.5, n = 6, p\<0.01). Previous studies suggested that the elevated serum TSH levels in TRHKO reduced the biological activity [@pone.0040437-Shibusawa1], [@pone.0040437-Nikrodhanond1]. In order to examine the feedback system of the hypothalamo-pituitary-thyroid axis in TRHKO, we first measured serum TSH levels in euthyroid TRHKO given daily injections of T4. The decreased serum thyroid hormone level normalized (1.92±0.05 ng/ml, n = 6) following the daily subcutaneous injection of thyroid hormone (T4, 1.5 μg/100 g body weight) for 14 days. In these euthyroid TRHKO, the elevated serum TSH levels reverted to normal (75.8±3.7 ng/ml, n = 6). These results suggested the serum TSH level to be normally regulated by thyroid hormone in the absence of TRH ([Fig. 1A](#pone-0040437-g001){ref-type="fig"}). To determine the mechanism of the normalization of the serum TSH level in euthyroid TRHKO, we next measured TSHβ and α mRNA levels in the pituitary by real-time PCR with TaqMan probes. Although serum TSH levels in the intact TRHKO were significantly elevated, pituitary TSHβ and α mRNA levels were paradoxically decreased to 47.4±16.2% and 71.8±5.0% of those in the control wild-type mice, respectively. In contrast to the change of serum TSH level in the euthyroid TRHKO, both TSHβ and α mRNA levels were further reduced to 10.1±1.1% and 30.1±3.0% of the control values ([Fig.1B and C](#pone-0040437-g001){ref-type="fig"}). Furthermore, a predominant reduction was observed in the TSHβ mRNA level as compared to the TSHα mRNA level. These results suggested that the normalization of the serum TSH level in TRHKO by thyroid hormone may be, at least in part, due to a decrease in the synthesis of TSH in the pituitary. Since there were no reports of thyroidectomy in mice, we first established a technique using dissection microscopy [@pone.0040437-Mori1]. The thyroidectomy for mice was basically the same as that for rats, except for the use of microscopy. The success rate of this procedure was approximately 70%, and the rest of the mice died due to respiratory failure with damage to the phrenic nerve. In the wild-type mice, thyroidectomy caused a significant decrease in the serum thyroid hormone levels from 1.14±0.55 ng/ml (n = 6) to 0.50±0.11 ng/ml (n = 6) within two weeks. Although the serum T4 level in intact TRHKO was approximately 60% of that in the wild-type mice (0.7±0.05 ng/ml, n = 7), thyroidectomy further decreased these levels to 0.28±0.02 ng/ml (n = 5)([Fig. 1D](#pone-0040437-g001){ref-type="fig"}). Reflecting the hypothyroid status, the serum TSH level of the wild-type mice (64.2±4.4 ng/ml) was significantly increased approximately 25-fold of the control (2349.5±255.5 ng/ml) after two weeks. In contrast, although the serum TSH level was higher in the intact TRHKO than wild-type mice, the increase was insufficient (611±240 ng/ml, n = 6) compared to that in the wild-type control ([Fig 1E](#pone-0040437-g001){ref-type="fig"}). ![The hypothalamic-pituitary-thyroid axis in TRH deficient mice.\ A) Serum TSH levels were measured in wild-type mice (Wt), TRH knockout mice (TRHKO) heterozygotes (Hetero) and homozygotes (TRH−/−) and TRHKO supplemented with thyroid hormone (TRH−/− + T4). TRHKO (n = 7) were supplemented with thyroid hormone by the daily injection of T4 (1.5 μg/100 g body weight) for 14 days to be euthyroid (n = 6). The serum TSH level in TRH−/−+T4 mice was similar to that in the wild-type (n = 7) and heterozygous mice (n = 6). Values are represented as the mean ± SEM. \\, p\<0.01; \, N.S., not significant. B) TSHβ mRNA levels were measured in pituitaries of the wild-type mice, TRHKO, and TRHKO supplemented with thyroid hormone (TRH−/−+T4). The TSHβ mRNA level in TRH−/− was decreased to about 50% of that in the wild-type, and that of TRH−/−+T4 mice was further decreased to about 10% (n = 3, p\<0.01). \\, p\<0.01. C) TSHα mRNA levels showed a similar profile to the TSHβ mRNA level shown in [Fig 1B](#pone-0040437-g001){ref-type="fig"}. However, the change in TSHβ mRNA was more profound than that in TSHα mRNA. \\, p\<0.01. D) Two weeks after thyroidectomy, serum free T4 levels were significantly reduced in both the wild-type mice (n = 6) and TRHKO mice (TRH−/−) (n = 5). The serum T4 levels were significantly lower in TRH−/− mice than wild-type mice. \\, p\<0.01. E) Reflecting hypothyroidism induced by thyroidectomy (TX), the serum TSH level in the wild-type mice showed an approximately 25-fold increase (Wt+TX, n = 6), but TRHKO mice (TRH−/−) showed only about a 6-fold increase (TRH−/−+TX, n = 6). \\, p\<0.01. F) The TSHβ mRNA levels in thyroidectomized wild-type mice (Wt+TX) were significantly increased after two weeks to approximately 6-fold the control value (Wt) (n = 3). However, the TRHKO mice (TRH−/−) showed a lesser increase (TRH−/−+TX)(n = 3). \\, p\<0.01; G) A similar profile was observed in TSHα mRNA levels in the pituitary. But a milder effect was observed compared to those of TSHβ mRNA levels. \\, p\<0.01; n = 3. H) Correlation between serum thyroid hormone levels and the corresponding pituitary TSHβ mRNA levels. Circles represent values for the wild-types and the squares, those for TRH knockout mice (TRH−/−). When comparing the slopes of the curve, it was demonstrated that TRH altered the basal activity and responsiveness of the TSHβ gene to thyroid hormone. The arrow indicates the effect of TRH, taking approximately 80% responsibility for the value of the TSHβ mRNA level in the wild-type pituitary. I) Correlation between serum thyroid hormone levels and the corresponding serum TSH levels. In mild hypothyroidism, the serum TSH levels in TRHKO mice (TRH−/−) were similar to those in the wild-type mice. However a lack of TRH induced a marked impairment of the serum TSH level in severe hypothyroid status.](pone.0040437.g001){#pone-0040437-g001} To evaluate the mechanism of the insufficient increase in the serum TSH levels in TRHKO in response to hypothyroidism induced by thyroidectomy, we measured TSHβ and α mRNA levels in the TRHKO pituitary and compared them to those in the wild-type. As expected thyroidectomy caused a significant increase in the TSHβ mRNA level, to approximately 6-fold (584.9±13.4%) that in the wild-type pituitary. Similarly, the TSHα mRNA level was increased to 199.0±2.4%. However, the increase of the TSHβ mRNA level in TRHKO was only approximately 3 fold (296±7.8%), and similarly, the TSHα mRNA level was increased to 105±1.3% of the control ([Fig 1F, and G](#pone-0040437-g001){ref-type="fig"}). The experimental hypothyroidism induced by MMI treatment with regular chow that contained iodine in a previous study induced a milder hypothyroidism than that due to thyroidectomy [@pone.0040437-Shibusawa1]. The serum thyroid hormone level in the MMI-induced hypothyroid wild-type mice was similar to that in the simple TRHKO. The treatment with MMI increased the TSHβ mRNA level to 300.0±15.5%. To analyze the correlation between the serum thyroid hormone levels and the corresponding TSHβ mRNA levels and serum TSH levels, all the data was plotted in. As shown in [Fig 1H](#pone-0040437-g001){ref-type="fig"}, it is clear that each pituitary TSHβ mRNA level corresponding to the serum thyroid hormone level in the TRHKO pituitary was significantly lower than those of the wild-type controls. Furthermore, when compared to the slope of the correlation curve, the slope for the TRHKO was almost parallel to that in the wild-type mice, suggesting that TRH affected the basal activity and the responsiveness of the TSH gene to thyroid hormone. When the TSHβ mRNA level at the middle of this curve was calculated, approximately 80% (83,7% at serum T4, 0.7 ng/ml) depended on the presence of TRH. It is important to note that although the serum TSH levels in TRHKO with mild hypothyroidism were similar to those in the wild-type mice, the lack of TRH impaired the raising of serum TSH levels, particularly in mice with severe hypothyroidism, as shown in [Fig 1I](#pone-0040437-g001){ref-type="fig"}. cDNA Microarray Analysis of the TRH-deficient Pituitary with and without Thyroid Hormone Replacement {#s2b} ---------------------------------------------------------------------------------------------------- In order to identify the factors involved with regulation of the pituitary TSH gene by TRH, we examined the gene expression in TRH-deficient pituitary with or without thyroid hormone replacement. Total RNAs were extracted from the pituitary of wild-type (WT signal), TRHKO (Homo Signal), and euthyroid TRHKO with thyroid hormone replacement (T4 signal) (TRHKO +T4∶1.5 μg T4/100 g body weight, subcutaneously for 14 days) and subjected to a cDNA microarray analysis using the Affymetrix Mouse Gene Chip 320 2.0 Array. After exclusion of absent signals (less than 100 signals), 10445 probes were expressed out of 45102 probes and subjected to a cluster analysis using a K-means algorithm. As shown in [Fig 2A](#pone-0040437-g002){ref-type="fig"}, it was classified into ten clusters according to profiles of changes among three groups and the expression level of the gene. Most genes belonging to clusters other than cluster 5 showed an increase in expression with thyroid hormone replacement. In contrast, cluster 5 consists of genes showing a significant decrease with both a deficiency of TRH and thyroid hormone replacement. It is important to note that among 10445 probes, the sole gene in the entire genome showing the largest and most consistent decrease in the absence of TRH and by supplement with thyroid hormone was the TSHβ gene showing 63% expression of the wild-type pituitary in the absence of TRH, and a further decrease to 6% of the wild-type ([Fig 2B, C, and D](#pone-0040437-g002){ref-type="fig"}). These findings demonstrated that TRH is a highly specific regulator of the TSHβ gene in the entire genome. Indeed, genes for other anterior-pituitary hormones such as POMC, FSH, LH, GH, and prolactin, and genes reported to regulate expression of the TSHβ gene including Pit1 and GATA2 were found to show no significant change ([Fig. 2C and D](#pone-0040437-g002){ref-type="fig"}). ![Microarray and K-means cluster analyses of genes regulated by TRH and by thyroid hormone in TRH-deficient pituitary.\ RNA samples from pituitaries of wild-type, TRHKO (TRH−/−), and euthyroid TRHKO mice with thryroid hormone replacement (TRH−/−+T4) were subjected to a whole-genome microarray using Affymetrix Mouse Gene chip 320 2.0 Array. After exclusion of absent signals (less than 100 signals), 10445 out of 45102 genes expressed in the TRHKO pituitary were subjected to the cluster analysis using a K-means algorithm. Hierarchical cluster analysis was carried out using a computer program, Genowiz™ 3.2. A) Genes were divided into ten hierarchical clusters according to changes and profiles among three groups, wild-type (WT signal), TRH−/− (Homo signal), and TRH−/− + T4 (T4 signal). B) In addition to the TSHβ genes, cluster 5 contained the Egr-1, NR4A1, Glt2, and Tmsb10 genes. C) mRNA levels of other anterior pituitary hormones including POMC, GH, LHβ, FSHβ, and prolactin (PRL) were not altered among the three groups, suggesting the expression to be TRH-independent. D) Profiles of the NR4A1, NR4A2, Egr-1, Pit1 and GATA2 genes are depicted. Expression of NR4A1, NR4A2 and Egr-1 might be TRH-dependent. E) Confirmation of results of the microarray analysis of NR4A1 mRNA levels in the TRH-deficient pituitary by real-time PCR. TRHKO mice (TRH−/−, n = 3) were supplemented with thyroid hormone by the daily injection of T4 (TRH−/−+T4, n = 3) and with TRH by 1 mg/kg body weight/day with an Alzet pomp for 7 days (TRH−/−+TRH n = 3). Total RNA obtained from these pituitaries was subjected to real-time PCR. The NR4A1 mRNA level in the TRHKO pituitary was decreased to about 25% of that of the wild-type. The decreased NR4A1mRNA level was not altered by thyroid hormone replacement, but completely reversed to the normal level by TRH administration, suggesting expression of NR4A1 mRNA in the pituitary to be TRH-, but not thyroid hormone-dependent.](pone.0040437.g002){#pone-0040437-g002} Other genes classified into cluster 5 were the NR4A1, Egr-1, Gtl2 and Tmsb10 genes as shown in [Fig. 2B](#pone-0040437-g002){ref-type="fig"}. The Gtl2 gene is an imprinted gene which is maternally expressed and appears to function as an RNA molecule; multiple splice variants are observed in the available sequence data, and a pituitary transcript variant has been reported to inhibit cell proliferation [@pone.0040437-Zhao1]. The tymosin-beta 10 gene (Tmsb10) is related to cell growth through proliferation, and its expression is associated with brain development in rats and humans. In addition, previous reports indicated enhanced Tmsb10 expression in a variety of human tumors such as renal and thyroid medullary carcinomas and melanomas [@pone.0040437-Santelli1], [@pone.0040437-Takano1]. Although these two genes were found in the same cluster, cluster 5 to which the TSHβ gene belonged, the signals slightly increased in the absence of TRH, suggesting that these two genes were expressed in a TRH-independent manner. The other two genes, NR4A1 and Egr-1, showed a profile of change similar to that of the TSHβ gene. The NR4A1 mRNA signal in the TRH-deficient pituitary decreased to 70% of that of wild-type mice, and further decreased to 52% of the wild-type level in euthyroid TRH-deficient pituitary (TRHKO+T4). Similarly, the Egr-1 signal in the TRH-deficient pituitary was decreased to 39% of the wild-type value, and reduced to 40% of the wild-type value in the euthyroid TRH-deficient pituitary (TRHKO +T4). Therefore, the reduction of these two genes in TRHKO mice was not recovered by thyroid hormone replacement, indicating that the expression of these two genes was TRH-dependent and probably up-regulated by TRH. The NR4A1 and Egr-1 genes are known as immediate-early response genes. Egr1- knockout mice have been reported to have LHβ deficiency [@pone.0040437-Lee1] and impaired liver regeneration [@pone.0040437-Liao1] but normal thyroid function and intact thyrotrophs in the pituitary, therefore we excluded Egr-1 from the study [@pone.0040437-Topilko1]. In contrast, NR4A1 is known to be an important signal for both the hypothalamo-pituitary-adrenal axis, particularly the pituitary POMC gene and the adrenal Cyp11β2 and HSD3β genes, and the hypothalamo-pituitary-gonadal axis including the 20α--HSD gene in the ovary and the Cyp17 and HSD3β genes in the testis [@pone.0040437-Stocco1]--[@pone.0040437-Smith1], however, its role in the hypothalamo-pituitary-thyroid axis (HPT axis) remains unclear. We therefore examined the importance of NR4A1 in the HPA axis in subsequent experiments. Furthermore, the previous analysis of NR4A1 knockout mice demonstrated normal functioning of the adrenal grand, suggesting other homologous factors such as NR4A2 and NR4A3 to compensate for the function of NR4A1 [@pone.0040437-Crawford1]. However, in the present microarray analysis, NR4A2 mRNA expression was not significantly changed and NR4A3 mRNA was not found in the pituitary ([Fig. 2D](#pone-0040437-g002){ref-type="fig"}), suggesting that no such compensation in the TRH-deficient mice. Furthermore, there was no report regarding the H-P-T axis and thyroid function in either NR4A1 knockout mice or NR4A1/NR4A3 double knockout mice [@pone.0040437-Crawford1]--[@pone.0040437-Mullican1]. NR4A1 mRNA Level was Regulated by TRH In vivo {#s2c} --------------------------------------------- To confirm the results of the microarray analysis, we measured NR4A1 mRNA levels in the pituitary gland by real-time PCR with the TaqMan probe of NR4A1 ([Fig 2E](#pone-0040437-g002){ref-type="fig"}). We first confirmed that the NR4A1 mRNA level in the TRH deficient-pituitary was decreased to about 25% of the wild-type level (1.2±0.1 vs. 3.8±0.2, NR4A1 mRNA/GAPDH mRNA, n = 3, p\<0.01). When the TRHKO were treated with thyroid hormone to be euthyroid (1.5 μg T4/100 g body weight, injected subcutaneously for 14 days), the level of NR4A1 mRNA was not significantly changed (1.5±0.1, n = 3). However, when TRH was given to TRHKO mice by Alzet pump subcutaneously for 7 days, the NR4A1mRNA level completely reverted to normal (2.8±0.4, n = 3) ([Fig. 2E](#pone-0040437-g002){ref-type="fig"}), suggesting the expression of NR4A1 mRNA to be TRH-dependent but thyroid hormone-independent. Furthermore, the NR4A2 mRNA level did not differ among the wild-type (0.9±0.1, n = 3), TRHKO (1.0±0.4, n = 3), and TRHKO +T4 (0.8±0.1, n = 3), and NR4A3 mRNA was not detected in the pituitary showing the Ct value of the real-time PCR as 29.5±0.9, while those of NR4A1 and NR4A2mRNA were 24.4±1.65, 27.3±1.0, respectively, suggesting again that compensation of the role of NR4A1 by NR4A2 and 3 might not occur in the anterior pituitary. Expression of NR4A1 in the Thyrotrophs and Regulation by TRH In vivo {#s2d} -------------------------------------------------------------------- We next investigated in vivo* expression and regulation by TRH of NR4A1 in the pituitary thyrotrophs using double-fluorescent immunohistochemistry. As shown in the left panel of [Fig 3A](#pone-0040437-g003){ref-type="fig"}, in the wild-type pituitary, NR4A1 shown with green signals was observed in the nucleus of most cells in the anterior, intermediate and posterior lobes. We found in numbers of cells, the coexistence of NR4A1 with TSHβ immunoreactivity shown by red signals in the cytoplasm of the anterior lobe. In contrast, in TRHKO pituitary shown in the right panel, a significant decrease in the staining of TSHβ was observed. However, careful observation indicated that in the remaining weakly-stained cells, NR4A1 expression was absent as shown by a black hole, suggesting the suppressed expression of NR4A1 in the nucleus of the thyrotrophs to be due to the absence of TRH. ![Expression and regulation of NR4A1 by TRH in pituitary thyrotrophs in vivo.\ The expression of NR4A1 in pituitary thyrotrophs was confirmed by double fluorescent immunohistochemistry. In numbers of cells in the anterior pituitary NR4A1 were expressed and stained as green signals in the nucleus. A) Numbers of cells expressing NR4A1 were also stained with antibody against TSHβ (red signals) in the wild-type pituitary (left panel). As shown in the right panel, the intensity and number of the TSHβ-immunopositive cells were remarkably decreased in the TRH-deficient pituitary, and at X400, most of the NR4A1 expression was reduced or lost in these cells as seen as black dots (indicated by white arrows). B) As observed in [Fig 4B and C](#pone-0040437-g004){ref-type="fig"}, the expression of ACTH (B) and FSH (C) was also observed as red signals in the cytoplasm in the anterior lobe. As expected, these ACTH- and FSH- positive cells expressed NR4A1, but the staining intensity was not altered in the TRH-deficient pituitary (right panel).](pone.0040437.g003){#pone-0040437-g003} ![Stimulation of the promoter activity of the TSHβ gene by NR4A1 in vitro.\ A) The human TSHβ promoter region between bp --1192 and +37 linked to a luciferase reporter gene (pA3TSHβ(−1192∼+37)-Luc) was transfected into CV-1 cells. Expression of Pit1 and GATA2 was necessary for the basal promoter activity of the TSHβ gene. When an expression vector for Pit1 was co-transfected, the promoter activity of the TSHβ was stimulated by 10-fold, but co-transfection with GATA2 did not significantly alter it. However, synergistic stimulation of the promoter activity was observed when Pit1 and GATA2 were simultaneously expressed. B) Under the above conditions expressing Pit1 and GATA2, the TSHβ promoter activity was stimulated by NR4A1 in a dose-dependent manner. When 1 μg of expression vector of NR4A1/3 wells was co-trasfected, the promoter activity was stimulated about 2-fold, and when 3 μg, it reached a plateau at about 6-fold. Relative luciferase activity against the value without expression of NR4A1 is shown. C) Stimulation of the promoter activity by NR4A1 was specific for the TSHβ gene (TSHB). The promoter activities of the thyrotropin-releasing hormone (TRH), TSHα, and prolactin (PRL) genes was not stimulated by overexpression of NR4A1. Relative luciferase activity against the value without expression of NR4A1 in each promoter is shown. Values are represented as the mean ± SEM from at least three experiments. \\, p\<0.01; N.S., not significant.](pone.0040437.g004){#pone-0040437-g004} We also identified ACTH- and FSH- producing cells using specific antibodies in the anterior pituitary. As shown in [Fig. 3B and C](#pone-0040437-g003){ref-type="fig"}, many ACTH- and FSH- producing cells expressed NR4A1 in the anterior pituitary. However, the expression of NR4A1 in the ACTH- and FSH- producing cells was not changed by the absence of TRH as shown in the right panel of [Fig. 3B and C](#pone-0040437-g003){ref-type="fig"}, suggesting that NR4A1 in the ACTH- or FSH- producing cells was not regulated by TRH in vivo. Activation of the TSHβ Promoter by NR4A1 {#s2e} ---------------------------------------- We next examined whether NR4A1 stimulates the activity of the TSHβ promoter in vitro. As previously reported by us and others, for the full expression of the TSHβ promoter Pit1 and GATA2 were required in CV-1 cells ([Fig. 4A](#pone-0040437-g004){ref-type="fig"}) [@pone.0040437-Gordon1], [@pone.0040437-Nakajima1]. As shown in [Fig 4B](#pone-0040437-g004){ref-type="fig"}, in the presence of Pit1 and GATA2, overexpression of NR4A1 significantly increased the TSHβ promoter activity in a dose-dependent manner and when 4.0 μg of expression vector per plate was used, the activity of the promoter was stimulated by 6-fold the basal level. The effect of NR4A1 could be observed only on the TSHβ promoter, not the TRH promoter or prolactin promoter, suggesting that the effect was specific for the TSHβ promoter ([Fig. 4C](#pone-0040437-g004){ref-type="fig"}). Furthermore, the activity of the TSHα promoter was also not stimulated by overexpression of NR4A1. It may be due to the small effect of the absence of TRH on the TSHα mRNA level as compared to the TSHβ mRNA level as shown in [Fig. 1C](#pone-0040437-g001){ref-type="fig"}. In order to determine the region responsible for the effect of NR4A1 on the TSHβ promoter, we first searched for an NR4A1 response element (NurRE)(AAAT(G/A)(C/T)CA) in the TSHβ promoter and found a complete consensus sequence of NurRE (5′-aaatatca-3′) in the region between bp -1091 and -1083 [@pone.0040437-Philips2], [@pone.0040437-Maira1]. However, as shown in [Fig 5A](#pone-0040437-g005){ref-type="fig"}, transient transfection analysis showed that the deletion of this region of the TSHβ promoter (-1078 ∼ +37 construct) caused a similar response to NR4A1, indicating that this element did not act as a NurRE in the TSHβ gene. Furthermore, a series of deletion mutants including the construct containing the region between -138 and +37 showed a significant response to NR4A1, suggesting that the region between -138 and +37 included the area responsible for the NR4A1-induced stimulation of the TSHβ gene. It has been reported by several investigators that Pit1 and GATA2 bound to this region of the TSHβ gene, and this region is also responsible for the TRH-induced stimulation ([Fig. 5A](#pone-0040437-g005){ref-type="fig"}) [@pone.0040437-Gordon1], [@pone.0040437-Gordon3], [@pone.0040437-Kashiwabara1]. ![Characterization of NR4A1-induced stimulation of the promoter activity of the TSHβ\ gene. A) To determine the region responsible for the NR4A1-induced stimulation of the TSHβ promoter activity, a series of deletion constructs of the human TSHβ promoter were established. The consensus sequence of the NR4A1 response element (NurRE) was identified in the region between -1091 and -1083 from the transcription start site. The right figure shows the fold-stimulation by NR4A in each deletion mutant of the TSHβ gene. Putative Pit1 and GATA2 binding sites, and the negative thyroid hormone response element (NRE) were indicated as pit1, GATA2 and NRE. All the deletion constructs including pA3TSHβ (−138 ∼ +37) -Luc showed similar stimulation by NR4A1, suggesting the region responsible to be within this area. Fold-increase in activity against that without TRH is shown. B) Chromatin-immunoprecipitation (ChIP) assays were performed with anti-NR4A1 antibody (NR4A1) and normal mouse IgG as a negative control (IgG). GH4C1 cells were transfected with pA3TSHβ (-1192 ∼ +37)-Luc in the presence or absence of 100 n M TRH. Amplified PCR products were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX. Chip assays demonstrated that NR4A1 was recruited to the region between -138 and +13 of the TSHβ promoter (-138/+13), but not the region containing NurRE (NuRE). Addition of TRH did not alter recruitment of NR4A1 on the gene (data not shown). All ChIP assays were repeated at least three times. C) The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE and a fragment containing the human TSHβ bp -123∼-87. There was no binding of NR4A1 to the TSHβ gene in the absence (−) or presence (+) of 100 nM TRH, while the POMC promoter fragment bound to NR4A1. Arrows indicate NR4A1 bound to the POMC promoter as monomers and dimers. NR4A1 was synthesized by the TNT-coupled reticulocyte lysate system (NR4A1). Lysate indicates un-programmed lysate. D) Effect of knockdown of NR4A1 on the TRH-induced stimulation of the promoter activity of the TSHβ gene. D-1. The NR4A1 mRNA levels were measured after transfection with siCONTROL or siNR4A1 in GH4C1 cells. Transfection of siNR4A1 led to an approximately 80% reduction of the mRNA level after 48 hr. Data are presented as the mean ± SEM from three experiments. \\, p\<0.01 D-2. GH4C1 cells were transfected with siNR4A1 or siCONTROL, and then TSHβ (-1192 ∼ +37)-Luc reporter, Pit1, and GATA2 expression vectors were cotransfected. After incubation with 100 nM TRH for 24 hr, the promoter activity was measured. In the vehicle transfected with siCONTROL, incubation with TRH stimulated promoter activity of the TSHβ gene showing 242±33% of that without TRH stimulation. Knockdown of NR4A1 resulted in significant reduction of TRH-induced stimulation (156% of that without TRH stimulation). Data are presented as the mean ± SEM from three experiments. \, p\<0.05 E) Negative regulation of the TSHβ gene by thyroid hormone was examined in the presence or absence of NR4A1 in CV-1 cells. Expression vector for thyroid hormone receptor β 1 (TRβ1) was also co-transfected. The responsiveness of the TSHβ promoter activity to thyroid hormone (T3) without expression of NR4A1 was significantly reduced as compared to that with expression of NR4A1, suggesting NR4A1 increased the basal promoter activity and responsiveness to T3 of the TSHβ promoter. F) Effect of NR4A1 and Egr-1 on the promoter activity of the TSHβ gene. Egr-1 affected neither the basal promoter activity of the TSHβ gene nor the NR4A1-induced stimulation of the gene. The amount of vector/3 wells transfected is indicated below the graph. \\, p\<0.01; \, p\<0.05; N.S. not significant.](pone.0040437.g005){#pone-0040437-g005} NR4A1 was Recruited to the Region between −138 and +13 {#s2f} ------------------------------------------------------ There was no consensus sequence for NurRE in the region between bp -138 and +37 of the TSHβ gene, and the EMSA study showed no binding of NR4A1 in the region −123∼−87 of the TSHβ gene, which has been reported to bind to Pit1 and GATA2 and be responsible for TRH-induced stimulation, while the POMC promoter fragment bound to NR4A1 as both dimers and monomers ([Fig. 5C](#pone-0040437-g005){ref-type="fig"}). Therefore, we expected indirect recruitment, and examined whether NR4A1 was recruited to this region by a chromatin-immunoprecipitation (ChIP) assay and a specific antibody against NR4A1. The ChIP assay demonstrated that NR4A1 appeared to be recruited both in the presence or absence of TRH, while the region containing a NurRE sequence (between -1091 and -1083) did not recruit NR4A1 ([Fig. 5B](#pone-0040437-g005){ref-type="fig"}). Knockdown of NR4A1 by siRNA Reduced TRH-induced Stimulation of the TSHβ Gene {#s2g} ---------------------------------------------------------------------------- In order to examine the importance of NR4A1 for activation of the TSHβ promoter by TRH, we next knocked-down NR4A1 by siRNA and examined the effect of NR4A1 on the gene. Treatment with siRNA (siNR4A1) induced a reduction of NR4A1 mRNA to 20% of the control siRNA (siControl) in a rat pituitary cell line, GH4C1 cells ([Fig. 5D](#pone-0040437-g005){ref-type="fig"}-1). The level of TSHβ promoter activity induced by TRH was 2.5-fold high in the control cells transfected with siControl, while activation by TRH was significantly suppressed to about 40% of that of siControl when NR4A1 expression was knocked down ([Fig. 5D](#pone-0040437-g005){ref-type="fig"}-2), suggesting the importance of NR4A1 for TRH-induced stimulation of TSHβ promoter activity. Expression of NR4A1 Shifted the Dose-response Curve of the TSHβ Gene Against Thyroid Hormone {#s2h} -------------------------------------------------------------------------------------------- When compared to the curve of dose-response repression of the TSHβ gene by thyroid hormone in the presence or absence of overexpression of NR4A1 in CV-1 cells, the curve was shifted to the right in the presence of NR4A1, indicating that NR4A1 increased the responsiveness of the TSHβ gene to thyroid hormone in vitro ([Fig 5E](#pone-0040437-g005){ref-type="fig"}). This phenomenon was similar to that of the in vivo experiment comparing TSHβ mRNA levels between the wild-type mice and TRHKO differing thyroid hormone status shown in [Fig. 1D](#pone-0040437-g001){ref-type="fig"}. Furthermore, another typical early response gene, Egr-1, did not alter the promoter activity of the TSHβ gene in the presence or absence of NR4A1 ([Fig 5F](#pone-0040437-g005){ref-type="fig"}). Immediate-early Response of NR4A1 mRNA via TRH through the PKC and MAPK Pathways {#s2i} -------------------------------------------------------------------------------- We next examined how TRH stimulates the expression of endogenous NR4A1mRNA in GH4C1 cells. Incubation with 100 nM TRH induced a marked and rapid increase in NR4A1 mRNA levels with a peak at 60 mins that was 51-fold the basal level, and then a rapid reduction to 19% of the basal level within 120 mins ([Fig 6A](#pone-0040437-g006){ref-type="fig"}). Furthermore, treatment with TRH increased NR4A1 mRNA levels in a dose-dependent manner with a minimum dose of 0.1 nM and a plateau at 10 nM ([Fig. 6B](#pone-0040437-g006){ref-type="fig"}), suggesting the NR4A1 mRNA to act as an immediate-early response gene for TRH-stimulation. Furthermore, Western blot analysis of GH4C1 cells demonstrated that incubation with 100 nM TRH increased NR4A1 expression at the protein level within 2 h with a peak at 2 h, and then a decrease in 4 h ([Fig 6C](#pone-0040437-g006){ref-type="fig"}). In addition, TRH did not alter the pit1 mRNA level, and GATA2 and POMC mRNAs were not detected in GH4C1 cells (data not shown). ![Immediate-early response of NR4A1 mRNA and protein levels to TRH in GH4C1 cells.\ A) Incubation with 1μM TRH led to a remarkable increase of the endogenous NR4A1 mRNA level with a peak within 1 hr (51-fold) and then an immediate reduction in 120 mins in GH4C1 cells. B) The TRH-induced increase of the NR4A1 mRNA level was observed in a dose-dependent manner. The minimum effect was observed with 0.1 nM TRH and then reached a plateau at 10 nM. C) Western blot analysis in GH4C1 cells demonstrated that incubation with 100 nM TRH led to a significant increase of NR4A1 protein (NR4A1) within 2 h, and NR4A1 was then degradated in 4 h, while the level of GAPDH was not altered.](pone.0040437.g006){#pone-0040437-g006} To explore which signal transduction pathway was involved with TRH-induced NR4A1 mRNA expression, we used several specific inhibitors. As shown in [Fig. 7A](#pone-0040437-g007){ref-type="fig"}, treatment with 100 nM TRH significantly stimulated the expression of endogenous NR4A1 mRNA in 60 mins, however, an prior incubation with a inhibitor for PKC (1 nM), staurosporine significantly inhibited this stimulation by 60%, and 10 nM staurosporine showed about 90% inhibition. Similarly, an inhibitor for MAPK, PD98059 (1--1000 nM), showed significant inhibition of TRH-induced stimulation of NR4A1 mRNA (90% of the control)([Fig. 7B](#pone-0040437-g007){ref-type="fig"}). In contrast, an inhibitor for Ca2+ channels, nimodipine, or for PKA, KT5720, did not affect it ([Fig. 7C and D](#pone-0040437-g007){ref-type="fig"}). These findings suggested that TRH-induced NR4A1 mRNA expression was mediated through both PKC and MAPK pathways. ![Regulation of NR4A1 mRNA by TRH through the PKC and MAPK pathways.\ The signal transduction pathways for the TRH-induced stimulation of NR4A1 mRNA expression were determined using several inhibitors in GH4C1 cells. After overnight incubation in DMEM without serum, GH4C1 cells were treated for 1 hr with a PKC inhibitor, Staurosporine (A), a MAPK inhibitor, PD98059 (B), a Ca channel inhibitor, nimozipine (C), and a PKA inhibitor, KT5720 (D). Significant inhibition of 100 nM TRH-induced stimulation of endogenous NR4A1 mRNA expression was observed with 1.0∼ nM Staurosporine and 1.0 ∼ nM PD98059, but not 0.5 nM nimozipine or 1 μM KT5720. The value relative to that without TRH is shown. Data are presented as the mean ± SEM. from three experiments. \\, p\<0.01.](pone.0040437.g007){#pone-0040437-g007} Stimulation of the NR4A1 Promoter Activity by TRH through TRH Receptors {#s2j} ----------------------------------------------------------------------- To examine whether TRH stimulates transcription of the NR4A1 gene, we performed a transient transfection assay with a NR4A1 promoter in GH4C1 cells expressing endogenous TRH receptors and CV-1 cells without the expression. In GH4C1 cells, incubation with 100 nM TRH induced a significant stimulation of the NR4A1 promoter activity (257±22% of the control. n = 3, p\<0.05). In contrast, no significant stimulation was observed in CV-1 cells ([Fig. 8A](#pone-0040437-g008){ref-type="fig"}). To determine whether TRH-induced NR4A1 mRNA expression is mediated through TRH receptors, we performed a similar experiment in CV-1 cells with and without expression of TRH receptors. As shown in [Fig 8B](#pone-0040437-g008){ref-type="fig"}, incubation with 100 nM TRH significantly stimulated the activity of the NR4A1 promoter with expression of TRH receptors, but, no change was observed without, suggesting the effect of TRH to be mediated through its receptors. Conversely, the POMC promoter activity was significantly stimulated by overexpression of NR4A1 to about 6-fold the basal level in At-T20 cells expressing endogenously POMC gene ([Fig. 8C](#pone-0040437-g008){ref-type="fig"}). However, incubation with TRH did not alter endogenous the POMC mRNA levels in At-T20 cells with and without TRH receptors ([Fig. 8D](#pone-0040437-g008){ref-type="fig"}). In addition, the POMC promoter was not affected by incubation with TRH in the presence or absence of TRH receptors ([Fig. 8E](#pone-0040437-g008){ref-type="fig"}). These findings suggested that the effect of TRH on NR4A1 mRNA and subsequent stimulation of the TSHβ promoter occurred in a receptor- and promoter-specific manner. ![TRH stimulates the promoter activity of the NR4A1 gene through TRH receptors.\ A) A fragment spanning bp −1295 and +152 of the promoter of the human NR4A1 gene fused to a luciferase reporter gene (pA3NR4A1(−1295∼+152)-Luc) was transfected into CV-1 cells (CV-1) that express no endogenous TRH receptors (TRHRs) and GH4C1 cells (GH4C1) expressing endogenous TRHRs. In CV-1 cells, 100 nM TRH did not stimulate the promoter activity of the NR4A1 gene, in contrast, it led to a significant increase in GH4C1 cells (255±33% of the control without TRH, n = 3, p\<0.05). B) When TRH receptors were expressed in CV-1 cells (CV-1+TRHR), 10^−7^ M TRH induced a significant increase of NR4A1 promoter activity, suggesting that TRH exhibited stimulation of the NR4A1 gene through TRH receptors. Fold-increase on incubation with 100 nM TRH is represented. The value in GH4C1 cells is also shown as a control. C) The rat POMC promoter containing the region from bp −706 to +64 fused to a luciferase reporter (POMC-Luc) was transfected into At-T20 cells expressing the endogenous POMC gene. The promoter activity was significantly increased about 6-fold by overexpression of NR4A1. D) The level of endogenous POMC mRNA was not altered either by incubation with expression of TR and T3 or by incubation with expression of TRH receptors (TRHRs) and 100 nM TRH in At-T20 cells. E) The POMC promoter activity was not stimulated by incubation with 100 nM TRH even with expression of TRH receptors (TRHRs), suggesting that the effect of TRH occurred in a promoter-specific manner. Data are presented as the mean ± SEM. from three experiments. \, p\<0.05; N.S., not significant.](pone.0040437.g008){#pone-0040437-g008} Discussion {#s3} ========== In the present study, we first demonstrated that TRH altered the basal expression of the TSHβ gene and the responsiveness of the gene to thyroid hormone in vivo* by using TRHKO differing in thyroid hormone status. When TRH-deficient mice were supplemented with thyroid hormone, the elevated serum TSH level became normal. In contrast, the decreased TSH mRNA levels in the TRH-deficient pituitary were further decreased, and this reduction was found predominantly in the TSHβ subunit. Although thyroidectomy caused severe hypothyroidism in TRH-deficient mice, the increases in serum TSH levels and pituitary TSHβ mRNA levels were insufficient. Analysis of the correlation between the serum thyroid hormone level and the corresponding serum TSH and TSHβ mRNA levels clearly demonstrated that TRH altered the basal activity and responsiveness to thyroid hormone of the gene. In addition, the serum TSH level was more profoundly affected in animals with severe hypothyroidism in the absence of TRH. These findings demonstrate that TRH is a major up-regulator of TSH synthesis, altering the basal activity and the responsiveness to thyroid hormone of the gene, and TRH also affected TSH production by post-transcriptional mechanisms in vivo. In hypothyroid status TRH and thyroid hormone receptors (TRs) both may be required for hypothalamic-pituitary-thyroid axis for stimulation of the production and secretion of TSH in vivo [@pone.0040437-Chiamolera1]. Unliganded thyroid hormone receptors exert a silencing effect in genes regulated positively by thyroid hormone but, we and others reported that unliganded TRs have a constitutively stimulating effect on the genes regulated negatively by thyroid hormone in vitro, such as the TSH gene [@pone.0040437-Satoh1]--[@pone.0040437-Lazar1]. Therefore, one may expect that unliganded TRs function as an up-regulator in hypothyroid animals. However, when animals who possessed no TRs (TRα1−/−β−/−) were rendered to hypothyroidism, their serum TSH and TSHβ mRNA levels in the pituitary were equivalent to those in the wild-type mice, demonstrating that TRs are not required and not involved with the up-regulation of the TSH gene in the hypothyroid status [@pone.0040437-Gthe1]. Furthermore, we have recently reported the predominant action of TRH compared to TRs in the hypothalamic-pituitary-thyroid axis using TRβ/TRH double knockout mice (KO) [@pone.0040437-Nikrodhanond1]. TRβKO mice had an impairment of negative feedback regulation by thyroid hormone, resulting in a significant rise in serum T4 levels and showed resistance to thyroid hormone with elevated serum TSH levels compared with wild-type mice [@pone.0040437-Nikrodhanond1], [@pone.0040437-Shibusawa2]--[@pone.0040437-Forrest1]. Double KO showed a reduced serum thyroid hormone level compared with simple TRβKO and showed only a slight increase in TSH levels compared with wild-type mice. In addition, when TRβKO and double KO were rendered to severe hypothyroid by treatment with PTU, double KO failed to show a significant rise in serum TSH levels as compared to TRβKO. Therefore, TRH is essential for the stimulation of the TSHβ gene under both euthyroid and hypothyroid conditions. How does TRH up-regulate TSHβ gene expression in vivo? In the present study, the cDNA microarray and K-means cluster analysis with TRH-deficient pituitary revealed that among whole genes a gene in which most dramatic effect of TRH was observed is the TSHβ gene, and no changes were observed in other pituitary hormones including GH, POMC, PRL, FSH, or LH. In addition, NR4A1 was a gene showing TRH-dependent expression and showed a similar profile to the TSHβ gene. However, expression of Pit1 and GATA2, other candidates for a factor related to TRH-induced stimulation of the TSHβ gene, was not altered by TRH or thyroid hormone replacement in vivo. Therefore, we focused on the effect of NR4A1 on the transcription of the TSHβ gene in vitro. Overexpression of NR4A1 clearly stimulated the promoter activity of the TSHβ gene in a time- and dose-dependent manner. In addition, we found that incubation with TRH markedly stimulated expression of NR4A1 mRNA by over 50-fold in an early time within 60 minutes. Furthermore, immunocytochemistry studies demonstrated co-expression of NR4A1 with TSHβ in the anterior pituitary, and in addition the expression was significantly decreased in the TRH-knockout mice. To our knowledge, this study is the first demonstration of the presence of NR4A1 even in ACTH- and FSH- producing cells by immunohistochemistry [@pone.0040437-Lavoie1]. Taken together, we demonstrated that NR4A1 is an up-regulator working down-stream of TRH in the anterior pituitary in vivo. The analysis with a series of deletions of the promoter region of the TSHβ gene demonstrated that the responsible region of TRH-NR4A1 induced-stimulation of the TSHβ gene was between bp -138 bp and +37 from the transcription start site (TSS). Furthermore, although no consensus sequence for binding site for NR4A1 was found in this region, Chip assay with a specific antibody against NR4A1 revealed that NR4A1 was recruited to this region. Steinfelder et al, also demonstrated with a transient transfection study and deletion analysis of the human TSHβ gene that the region between -128 and +8 in relation to TSS is responsible for TRH-induced stimulation of the gene [@pone.0040437-Steinfelder1]. This region was close to the TSS and contained binding sites for Pit1 and GATA2, and the thyroid hormone inhibitory element (nTRE). Steinfelder et al. reported that the response element of the TSHβ gene to TRH was located in two discrete regions (-128 to -92 base pairs and -28 and +8 base pairs). The upstream site contains a DNA sequence with homology to the DNA-binding site for a pituitary specific transcription factor, Pit1, and the downstream site overlaps with the thyroid hormone inhibitory element [@pone.0040437-Steinfelder1], [@pone.0040437-Gordon1]. Furthermore, a recent study of pituitary-specific GATA2 Knockout mice demonstrated reduced pituitary TSH content and serum TSH levels, and pituitary TSHβ mRNA showed a defective response to hypothyroidism [@pone.0040437-Charles1]. Two regions of the mouse TSHβ gene have a sequence homologous to a consensus motif of the GATA binding site, AGATGC, at the region between -110 and -105 to the TSS, and AGATAA between -98 and -93 [@pone.0040437-Gordon1], and these sequences are perfectly conserved in rodents and humans [@pone.0040437-Ohba1]. Although both Pit1 and GATA2 have been reported to be essential for the expression of the TSHβ gene in thyrotrophs, the present study demonstrated that knockdown of NR4A1 with siRNA significantly reduced TRH-induced stimulation of the promoter activity of the TSHβ gene. Therefore, as shown in [Figure 9](#pone-0040437-g009){ref-type="fig"} as a proposed model of action of TRH on the TSHβ gene, NR4A1 may also be critical and cooperatively work with Pit1 and GATA2 as an up-regulator of TRH-induced stimulation of the TSHβ gene through this region -128 and +8 close to the TSS. Although interactions of NR4A1 with several transcription factors and coregulators, including silencing mediator for retinoid and thyroid hormones (SMRT), steroid receptor coactivator (SRC-1), TRAP220, and SIN3B/KDM5A and Z3 (ZMYND8-containing) complex have been reported, those with Pit1 and GATA2 remain to be studied [@pone.0040437-Gordon3], [@pone.0040437-Kelly1]--[@pone.0040437-Malovannaya1]. ![Proposed models of the TRH-NR4A1-TSHβ pathway.\ TRH stimulates the transcription of the NR4A1 gene through TRH receptors, and PKC and MAPK pathways within 1 hr. The increased amount of NR4A1 stimulates TSHβ promoter activity, which may act cooperatively with Pit1 and GATA2 on the gene.](pone.0040437.g009){#pone-0040437-g009} NR4A1 was originally isolated as a gene induced by nerve growth factor in PC12 cells [@pone.0040437-Milbrandt1]. Subsequently, it has been reported to act as a stimulus-induced transcriptional activator in many tissues and organs, and recently been implicated in apoptosis, cell cycle regulation, inflammation, carcinogenesis and metabolism [@pone.0040437-MartnezGonzlez1]--[@pone.0040437-Liu1]. In addition, NR4A1 plays an important role in the hypothalamo-pituitary-adrenal (HPA) axis [@pone.0040437-Philips1], [@pone.0040437-Philips2], [@pone.0040437-Inaoka1], [@pone.0040437-Fernandez1], [@pone.0040437-Maira1]. Philips et al. reported that the regulation of transcription of the POMC gene by CRH was mediated through activation of NR4A [@pone.0040437-Philips2]. However, NR4A1-deficient mice showed normal adrenocortical function, but subsequent studies demonstrated that other members of a subfamily of nuclear receptors such as NR4A2 (Nurr1) and NR4A3 (Nor-1) might compensate for a loss of NR4A1 and maintain sufficiently normal steroidogenesis in vivo [@pone.0040437-Fernandez1], [@pone.0040437-Crawford1]. We also examined expression of NR4A2 and NR4A3 mRNA in GH4C1 cells, however, neither significant expression nor effect of TRH was observed on these mRNA levels. In addition, as mentioned above knockdown of NR4A1 in GH4C1 cells significantly reduced TRH-induced stimulation of the TSHβ gene. Therefore, NR4A1 appeared to be essential for thyrotrophs in the pituitary gland. Furthermore, the pituitary--thyroid--axis in NR4A1-deficient mice has not been characterized, and so will be interesting to study in the future [@pone.0040437-Crawford1], [@pone.0040437-Lee2]. NR4A1 has also been well characterized as a regulator in the hypothalamo-pituitary-gonadal (HPG) axis. GnRH stimulated NR4A1 expression in pituitary gonadotropes and stimulated transcription of the LH gene [@pone.0040437-Kakar1], [@pone.0040437-Wurmbach1]. In addition, in the testis and ovary, expression of the steroidogenic acute regulatory (StAR) gene and steroidogenesis are regulated by the LH-NR4A1 pathway [@pone.0040437-Manna1]--[@pone.0040437-Martin1]. Thus, NR4A1 would be a common transducer of transcriptional signals of hypothalamic hormones for the regulation of targeted pituitary hormone genes in the anterior pituitary gland. Methods {#s4} ======= Reagents and Solutions {#s4a} ---------------------- Inhibitors (Staurosporine, PD98059, nimodipine and KT5720) were purchased from Sigma--Aldrich (St. Louis, MO). Staurosporine, PD98059 and KT5720 were dissolved in DMSO, and nimodipine in methanol. All vehicles were incubated with the same concentration of dissolved solvent. TRH was kindly provided by Tanabe-Mitsubishi Pharmaceutical Company, Japan. Animals {#s4b} ------- Procedures for animal care and use in this study were approved by the Review Committee on Animal Use at Gunma University, Maebashi, Japan. Animals were maintained on a 12-hour light and 12-hour dark schedule (lights on at 6.00 h) and fed laboratory chow and tap water ad libitum. All experiments were performed between 9.00 and 11.00 h. The mice with thyroxin (T~4~) replacement received 1.5 μg T4/100 g body weight, subcutaneously for 14 days before the experiment. TRH replacement was performed with 1 mg TRH/kg body weight/day by Alzet pump (micro-osmotic pomp model 1002, Alzet, U.S.A.) subcutaneously for 7 days before the experiment. Experimental hypothyroid mice were generated by thyroidectomy under microscopy. Thyroidectomized mice were given with 1% CaCl2 in drinking water to prevent hypocalcemia due to the simultaneous removal of the parathyroid gland. A mild hypothyroidism was induced by 0.1% MMI in drinking water for 7 days with regular chow that contained iodine. Radioimmunoassay (RIA) {#s4c} ---------------------- Serum TSH levels were measured by a specific mouse TSH RIA with mouse TSH/LH reference (AFP51718MP), mouse TSH antiserum (AFP98991) and rat TSH antigen (NIDDK-rTSH-I-9), all of which were obtained from Dr. A.F. Parlow (Harbor-UCLA Medical Center, Torrance, USA). Serum free T~4~ levels were determined by RIA (GammaCoat Free T4, DiaSorin Inc. USA). All assays were performed in duplicate and had a detection limit of 0.16 ng/dl. RNA Extraction and Real-time PCR {#s4d} -------------------------------- Total RNA was prepared from mouse pituitary, GH4C1 cells and At-T20 cells using ISOGEN (Nippongene, Toyama, Japan) according to the manufacturer's instructions. Then cDNA was reverse-transcribed from 300 ng of total RNA (TaqMan Reverse Transcription Reagents, Applied Biosystems, Tokyo, Japan), and 0.5 μl was subjected to real-time PCR. All reactions were performed in triplicate using TaqMan probes and an Applied Biosystems 7500 sequence detection system. TaqMan probes for NR4A1 (Mm01300401, Rn01533237), NR4A2 (Mm00443060, Rn00570936), NR4A3 (Mm01354011, Rn00569312), Pit1 (Mm00476852, Rn00564562), GATA2 (Mm00492300, Rn00583735), POMC (Mm00435874, Rn00595020) and GAPDH (Mm99999915, Rn99999916) were purchased from Applied Biosystems. The expression level of each mRNA relative to that of GAPDH was calculated using a standard curve, and the relative quantification method was performed as described in ABI User Bulletin \#2. All experiments were repeated at least twice. cDNA Microarray Analysis and K-means Cluster Analysis {#s4e} ----------------------------------------------------- Total RNA was prepared from each mouse pituitary using ISOGEN (Nippongene, Toyama, Japan) according to the manufacturer's instructions. cRNA was then prepared and hybridized to an Affymetrix Mouse Gene chip 320 2.0 Array. Only genes with raw data greater than 100 signals for all conditions were included in the analysis. Genes after exclusion of absent signals were subjected to a cluster analysis using a K-means algorithm. The hierarchical cluster analysis was carried out using the computer program Genowiz™ (ver. 3.2). Double-fluorescent Immunohistochemistry {#s4f} --------------------------------------- A rabbit polyclonal antibody for NR4A1 (LS-B114) was purchased from LifeSpan BioSciene (Seattle, WA, USA). Antibodies for mouse TSHβ (M-16: sc-7815) and was purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA). An ACTH antibody was purchased from DAKO Corp. (Glostrup, Denmark), and a follicle-stimulating hormone (FSH) antibody (MON5012) was obtained from MONOSAN (MONOSAN, Uden, Netherlands). Fluorescently labeled secondary antibodies used were TSHβ (red), Alexa Fluor 594 donkey anti-goat IgG (Invitrogen, California, USA), NR4A1(green), FITC: polyclonal goat rabbit immunoglobulins (HRP) (DAKO Glostrup Denmark); ACTH (red), Alexa Fluor 594 rabbit anti-mouse IgG (Invitrogen, California, USA), NR4A1(green), FITC: polyclonal goat rabbit immunoglobulins (HRP) (DAKO Glostrup, Denmark); FSH (red, Alexa Fluor 594 donkey anti-mouse-IgG(H+L) (Invitrogen, California, USA), NR4A1 (green), and Alexa Fluor 488 goat anti-rabbit-IgG (H+L) (Invitrogen, California, USA). Pituitaries were excised and fixed with 4% paraformaldehyde at 4°C overnight. Tissues were embedded in paraffin wax and sectioned sagittally in 4 μm-thick slices. The sections were pretreated with 0.3% hydrogen peroxide for 30 min for the quenching of endogenous peroxidase activity and then autoclaved for 10 min at 120°C. They were rinsed in TBS (Tris-buffer saline) and incubated with skim milk for 30 min at room temperature to block non-specific antibody binding, and treated with the above antibodies (TSHβ, ACTH, or FSH) at 4°C overnight. After another three washes in TBS, the sections were incubated with the secondary antibodies, then washed in TBS, and treated with the NR4A1 antibody at 4°C overnight. After three washes in TBS, the sections were incubated with the secondary antibodies, washed in TBS, and treated with Vectashield mounting medium (VECTOR Laboratories, CA, USA). We observed the slides with a BX52 biological microscope and DP70 digital camera (OLYMPUS, Tokyo, Japan). Mammalian Cell Culture {#s4g} ---------------------- CV-1 (a monkey kidney cell line) [@pone.0040437-JENSEN1], GH4C1 (a rat pituitary cell line) [@pone.0040437-Tashjian1] and At-T20 (a mouse pituitary adenoma cell line) [@pone.0040437-Gumbiner1] cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), as described previously [@pone.0040437-Lin1]. In some experiments, GH4C1 cells were starved in DMEM without serum overnight, then pre-treated with several inhibitors for signal transduction at the indicated concentrations 30 min before the treatment with TRH. Plasmid Constructions {#s4h} --------------------- Expression vectors for the mouse NR4A1, Egr-1, GATA2, Pit1 and TRH receptor (TRHR) were prepared by PCR and verified by sequencing of the DNA. The mouse NR4A1, Egr-1, GATA2 and Pit1 cDNAs were subcloned into the vector pcDNA^TM^3.1 D/V5-His-TOPO, and TRHR was subcloned into the vector pCDM8 for in vitro transcription/translation and transient expression analyses. The human TRβ~1~ cDNAs were subcloned into the vector pKCR~2.~ The human NR4A1 promoter containing 1,295 bp of the 5′ flanking sequence and 152 bp of exon 1(pA3NR4A1(-1295∼+152)-Luc), the rat POMC promoter containing 706 bp of the 5′ flanking sequence and 64 bp of exon 1, the human TSHβ-Luc containing 1,192 bp of the 5′ flanking sequence and 37 bp of exon 1 (pA3TSHβ(−1192∼+37)-Luc), the human prolactin gene containing 332 bp of the 5′ flanking sequence and 65 bp of exon 1 (pA3PRL-Luc), and the human TSHα-Luc containing 486 bp of the 5′ flanking sequence and 44 bp of exon 1 (pA3TSHα-Luc) [@pone.0040437-Hashimoto1] were fused to a firefly luciferase reporter plasmid (pA3Luc). Cell Transfection and Luciferase Assay {#s4i} -------------------------------------- Twenty-four hours before transfection, cells were split into 6-well plates at subconfluency. The transient transfection was performed using a calcium phosphate precipitation method, as described previously [@pone.0040437-Lin1]. The total amount of transfected plasmid was adjusted by adding an empty expression vector in all experiments. Sixteen hours after transfection, the medium was changed to DMEM supplemented with 10% FBS treated with AG1-X8 resin (Bio-Rad) and activated charcoal (Sigma) to remove thyroid hormones. Cells were further incubated in the presence or absence of TRH at the indicated concentration. To determine luciferase activity, cell monolayers were rinsed twice with PBS, then lysed with 300 μl of 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO~4,~ 4 mM EGTA, 1 mM dithiothreitol, and 1% v/v Triton X-100. Cells were scraped from the dishes and centrifuged at 12.000× g for 5 min at 4°C. Assays for Luc activity were performed using 150 μl aliquots of cell lysate and 210 μl of 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO~4,~ 4 mM EGTA, 3.3 mM KPO~4,~ 1 mM dithiothreitol, and 0.45 mM ATP. The reaction was initiated by addition of 200 μl of 0.2 mM d-luciferin and light emission was measured for 10 seconds using a luminometer. Luciferase activity was expressed as arbitrary light units per microgram of cellular protein. All the transfection experiments were repeated at least twice with triplicate determinants. Western Blot Analysis {#s4j} --------------------- GH~4~C~1~ cell lysates were prepared in RIPA buffer and 100 μg/ml PMSF, 30 μl/ml Aprotinin, and 1 μmol/ml sodium orthovanadate, and then passed through a 23-gauge needle on ice. For cell and tissue extracts, the samples were incubated on ice for 30 min in RIPA buffer. Insoluble cell debris was removed by centrifugation at 10.000 X g for 10 min. Aliquots of protein-containing supernatant were stored at −80°C. Protein concentrations were determined by the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc. Tokyo, Japan). The lysates (40 μg) were resolved by SDS-PAGE gel (10%) and transferred to a polyvinylidene fluoride membrane (Hybond-P, Amersham Biosciences, Tokyo, Japan) with a semidry system (BIO CRAFT, Tokyo, Japan) for detection of NR4A1. The blots were blocked for 1 h with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and probed for 16 h with a primary antibody against NR4A1 (LS-B114, LifeSpan BioScienes, Seattle, WA, USA). After three washes with Tris-buffered saline with 0.1% Tween 20, antigen-antibody complexes were detected using a peroxidase-conjugated secondary rabbit antibody and an enhanced fluoro-chemiluminent system (ECL-plus; Amersham Biosciences). Small Interfering RNA (siRNA) Experiments {#s4k} ----------------------------------------- Pooled siRNA oligonucleotides targeting NR4A1 were designed, synthesized and annealed at Dharmacon Research, Lafayette, CO (siGENOME SMART pool NR4A1, L-100466-01)(siNR4A1). Pooled unrelated siRNA (siCONTROL non-targeting siRNA pool, D-001810-0X)(siControl) was used as a control. These siRNAs were transfected into GH4C1 cells by the lipofection method (Lipofectamine RNAiMAX™, Invitrogen, California, USA). Briefly, in the 6-well format, 100 pmol of siRNA per well was transfected into the GH4C1 cells. After 6 h, the medium was changed to DMEM containing 10% FBS. Twenty-four hours after the first transfection, the transient transfection of TSHβ-Luc, Pit1 and GATA2 was performed using a calcium phosphate precipitation method. Sixteen hours after the second transfection, the medium was changed to DMEM supplemented with 10% FBS treated with AG1-X8 resin and activated charcoal. Cells were further incubated in the presence or absence of TRH. After twenty-four hours incubation, a luciferase assay and RNA extraction were performed. Electrophoretic Mobility Shift Assay (EMSA) {#s4l} ------------------------------------------- The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE (5′-gatcctagtgatatttacctccaaatgccagga-3′) and a fragment containing the human TSHβ bp −123∼−87 (5′-cagtatgaattttcaatagatgcttttcagataagaaa-3′), which has been reported to be responsible for TRH-induced stimulation [@pone.0040437-Ohba1], [@pone.0040437-Philips1]. Double-stranded oligonucleotides were labeled with \[α^32^P\]dCTP by a fill-in reaction using a Klenow fragment of DNA polymerase I. NR4A1 was synthesized by in vitro transcription/translation from pcDNA3.1-NR4A1 using T~7~ RNA polymerase and the TNT-coupled reticulocyte lysate system (Promega Corporation). The binding reaction, gel electrophoresis, and autoradiography were performed under conditions described previously [@pone.0040437-Nakajima1]. Chromatin-immunoprecipitation (ChIP) Assay {#s4m} ------------------------------------------ ChIP assays were performed as we previously reported, using a kit from Upstate Biotechnology (Massachusetts, USA) [@pone.0040437-Hashimoto2]--[@pone.0040437-Ishii1]. GH4C1 cells were transfected with pA3TSHβ(−1192∼+37-Luc and incubated in medium containing 10% fetal bovine serum. After incubation overnight, 37% of formaldehyde was directly added to the culture at a final concentration of 1%, and the cells were incubated for 15 min at room temperature to crosslink protein to DNA. The whole cell extracts were pelleted and resuspended in 500 μl of lyses buffer (1% SDS/50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1 mM PMSF and 1 mg/ml aprotinin) for 10 min at 4°C. The lysate was sonicated 3 times with 10-sec pulses using a sonicator set at 70% of maximum power to reduce DNA length to between 200 and 1000 bp. The chromatin solution (500 μl) was used for each ChIP assay with 0.6 mg of a rabbit anti-NR4A1 antibody (LS-B114, LifeSpan BioScienes, Seattle, WA, USA). As a negative control, normal mouse IgG (sc-2025, Santa Cruz Biotechnology, CA, USA) was used. The primers used for the region between bp --138 and +13 were as follows, forward primer, 5′-GGTAAAGATATTGTGAGCTTGTTTGTCTAA-3′, and reverse primer, 5′- GCTGTGGTGACCCAAACTAAAAGC-3′, and the length of the predicted PCR product was 151 bp. The primers used for the region between bp -1192 and -1049 containing putative NurRE sequences (AAATATCA: -1091 bp and -1083 bp) were 5′-GGATCCCTTCCCTACACCATATAGAAAAAT-3′, and 5′-GTCATGAAATCTCTA CCCGGTCCTACATCT-3′, and the predicted size was 143 bp. Conventional PCR was performed in 50 μl with platinum high-fidelity (Invitrogen) for 30 cycles (annealing temperature of 60°C), and signals were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX (Bio-Rad). All ChIP assays were repeated at least three times. Statistical Analysis {#s4n} -------------------- Statistical analyses were performed with ANOVA and Student's t test or the Wilcoxon/Kruskal-Wallis test using JMP (SAS Institute Inc., Cary NC). Competing Interests:The authors have declared that no competing interests exist. Funding:This study was supported, in part, by grant from Health and Labor Sciences Research Grants, Research on Measures for Intractable Disease Hyptahalamo-Pituitary Dysfunction Research Group (MM), and Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (MY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: YN MY NS. Performed the experiments: YN MY RT NS. Analyzed the data: YN MY RT NS AO TT KH TS TT SO TS MM. 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Background ========== The traditional method for harvesting this vein involves the use of a long incision from ankle to groin. A frequent observation after coronary artery bypass graft surgery is that many patients complain more about their leg wound than they do about their median sternotomy. The long incision is associated with significant intra-operative and postoperative complications \[[@B1]\]. Impaired leg wound healing has been reported to occur in up to 25% of patients \[[@B2]\]. Wound complications including infections, hematoma formation, cellulitis and saphenous neuropathy prolong recovery \[[@B3]\]. The use of minimal invasive saphenous vein harvesting has been advocated in an effort to minimize such wound related problems \[[@B4]-[@B6]\]. There is already some evidence that these methods may reduce leg wound complications \[[@B6]\], although they have not yet gained widespread use. Several techniques are available for minimal invasive vein harvesting, but all necessitate traction on the vein to maximize surgical visibility and enable side branch ligation. Excessive surgical manipulation of saphenous vein impairs endothelial cell function and reduces the bioavailability of nitric oxide \[[@B7]-[@B9]\]. This endothelial injury promotes platelet and leukocyte adhesion that in turn can result in smooth muscle cell proliferation that exacerbates the intimal hyperplasia that is a common cause of vein graft occlusion \[[@B10]-[@B12]\]. Thus, functional integrity of the harvested and prepared saphenous vein has important implications for immediate and long-term graft patency Materials and methods ===================== The patients ------------ From May 2002 to May 2003, 80 patients scheduled for elective first time coronary artery bypass grafting were recruited into the study. Study participants had isolated coronary artery disease that required at least part of their revascularization to be done using the LSV. Exclusion criteria included patients undergoing emergency coronary artery bypass grafting and those with severe varicose veins. The study was approved by the Local Research and Ethics Board at Glasgow Royal Infirmary. Before enrollment and randomization each participant provided written informed consent. Surgical techniques ------------------- Two consultant cardiac surgeons (UN and AM) carried out all of the operative procedures reported in this study. ### Long incision (open) saphenous vein harvesting (Group A) The incision was commenced just above the medial malleolus. The vein was identified and cleared of all adventitia and connective tissue using sharp and blunt dissection. The skin was incised over the whole length of the vein to the required length and careful dissection was used to isolate the vein in situ, with attention given to avoid unnecessary trauma to the vein or its tributaries. Side branches were ligated with 4/0 ethibond ligatures on the vein side and metal clips on the patient side. The leg wound was closed in layers and a full length pressure dressing was applied. ### Minimal invasive technique for long saphenous vein harvest (Group B) A 2 cm longitudinal incision was made above the medial malleolus. The long saphenous vein was identified and cleared of all adventitial and connective tissue using sharp dissection. The distal end of vein was tied. The proximal part was also tied and about 6--8 cms of thread was left with that end. The vein was then divided between the two tied ends. The end with a thread was passed through the ring in the Mayo vein stripper and forward pressure was applied to the vein stripper in the direction of usual vein anatomy while applying traction on the vein through the length of thread. Whenever resistance was felt on the vein stripper, a small incision (2 to 3 cm) at the area where resistance was felt was made. With a combination of sharp and blunt dissection any branch of the saphenous vein at that site was isolated and ligated using a 4/0 ethibond ligature on the vein side and a metal clip on the patient side. The same process was repeated with multiple short incisions until the required length of vein was obtained. The skin incisions were closed with skin staples and full-length pressure dressing applied. In common to both techniques of harvest, the vein was inflated with heparinised blood to check for any unidentified side branches or tears in the vein. Any that were identified were either ligated with a 4/0 ethibond suture or closed with a 6-0 prolene suture. Randomization ------------- Patients were randomized immediately prior to surgery by minimization using a computer program \[[@B13]\]. Minimization has the advantage that differences in important patient variables that might otherwise occur by chance can be avoided. The following patient characteristics were employed for minimization: diabetes, peripheral vascular disease, age and gender. Wound complications ------------------- Wound complications were assessed using the ASEPSIS \[[@B14]\] scoring method (Table [1](#T1){ref-type="table"}). All incisions were carefully assessed by a single observer on each day of the in-patient stay. The ASEPSIS score measures erythema, exudates and wound separation. A numerical score is calculated according to the proportion of the wound affected by each of these characteristics (table [5](#T7){ref-type="table"}). All discharging wound were swabbed and any bacterial growth identified by standard methods. Since some patients were discharged by the 5th postoperative day we calculated the sum of the first five daily scores for each patient. Pain measurement ---------------- A visual analogue score was used to assess postoperative leg pain \[[@B15],[@B16]\]. This consisted of a 10 cm long straight line with extreme limits marked with perpendicular lines and appropriate labels, but with no words or numbers between the endpoints. The patient was asked to mark the line with a cross at a point that represented their current level of pain. The sensitivity of this type of pain evaluation has been previously validated \[[@B15]\]. Pain assessment was carried out daily until hospital discharge. Vasomotor studies ----------------- Of the 79 patients in the study 29 were evaluated for vasomotor studies, 14 from Group A and 15 from Group B. A saphenous vein segment was removed prior to distension and immediately rinsed without pressure, immersed in iced oxygenated Krebs-Henseleit buffer \[[@B17]\] and transported to the laboratory. These vein segments were divided into three or four rings approximately 4 mm long that were mounted in a 25 ml organ bath. The tension was recorded directly onto a computer. Optimal resting tension was determined in baseline studies. Rings were then pre-contracted with phenylephrine (30 mmol/l). Endothelium-dependent relaxation was evaluated by cumulative addition of calcium ionophore A23187 (0.1 to 10 mmol/l) and endothelium-independent relaxation was evaluated by sodium nitroprusside (0.001 to 0.1 mmol/l) \[[@B18]\]. Relaxation to the NAD (P) H oxidase inhibitor apocynin (10 to 1000 mmol/l) was studied to assess the contribution of super oxide to vasoconstriction. Statistics ---------- Statistical analysis were performed using Student\'s t-test to compare parametrically distributed variables, the Mann Whitney test to compare non-parametrically distributed variables and the Chi-squared to compare discrete variables. Results ======= Of eighty patients undergoing coronary artery surgery in the study period, one patient was unavoidably excluded from the study before vein harvest commenced. Two patients who died within the first 48 hours were excluded from further analysis, leaving 38 patients in group A and 39 in group B. There were no differences in the demography of the patients between the two groups (Table [2](#T4){ref-type="table"}). No patient from group B was converted to the traditional open technique. ###### The ASEPSIS wound score Criterion Description Points ---------------------------------------------- -------------------------------------------- -------- AAdditional treatment Antibiotics 10 Drainage of pus under local anesthetics 5 Debridement of wound (General anesthetics) 10 SSerous discharge Daily 0--5 EErythema Daily 0--5 PPurulent exudates Daily 0--10 SSeparation of deep tissues Daily 0--10 IIsolation of bacteria 10 SStay in hospital prolonged over 14 days 5 ###### Points scale for the daily wound inspection Wound characteristic Proportion of wound affected (%) ---------------------------- ---------------------------------- --- --- --- --- ---- Serous exudates 0 1 2 3 4 5 Erythema 0 1 2 3 4 5 Purulent exudates 0 2 4 6 8 10 Separation of deep tissues 0 2 4 6 8 10 ###### The total score with the category of infection Total score Category of infection ------------- -------------------------- 0--10 Satisfactory healing 11--20 Disturbance of healing 21--30 Minor wound infection 31--40 Moderate wound infection \>40 Severe wound infection ###### Preoperative demographics Group A Group B ----------------------------- ----------- ----------- Age (years: mean ± 1sd) 63 (8) 65 (8) Male sex 29 29 NYHA class (I/II/III/IV) 0/8/14/15 0/8/15/16 Smoker 14 14 Diabetic Type 1 3 2 Type 2 6 8 Obese (BMI\>30) 10 8 Peripheral vascular disease 9 9 Venous stasis disease 5 9 ###### Intraoperative data Group A Group B ------------------------------------------- ----------- ----------- ------------ CPB time (mins: mean ± 1sd) 85 (23) 78 (15) ns Theatre time (mins: mean ± 1sd) 200 (32) 192 (24) ns Vein harvest site: Calf only 35 35 Calf and thigh 3 4 Total no of grafts (mean ± 1sd) 3.3 (0.7) 3.2 (0.5) ns No of vein grafts (mean ± 1sd) 1.9 (0.7) 1.6 (0.5) ns Length of vein harvested (cm: mean ± 1sd) 31 (13) 25 (9) p = 0.018 Length of wound (cm: mean ± 1sd) 35 (13) 16 (6) p \< 0.001 Number of incisions (mean ± 1sd) 1.2 (0.4) 6.9 (2.7) p \< 0.001 Vein harvest time (mins: median + IQR) Stage 1 16 (10) 11 (14) p = 0.01 Stage 2 9 (6) 2 (1) p \< 0.001 Stage 3 2 (2) 2 (4) p = 0.5 Total 26 (16) 15 (22) p = 0.002 Intra operative findings ------------------------ The total operative time taken to harvest vein was divided into 3 parts; stage 1: start of skin incision to removal of vein, stage 2: incision closure and stage 3: vein preparation. Adding the 3 gives the total vein operation time (Table [3](#T5){ref-type="table"}). Technique B was significantly quicker whilst requiring no increase in the time required to prepare the vein. Time was saved both during removal of the vein and during skin closure. There was no statistical difference in length of vein harvested but total wound length and number of incisions was less in Group B (Table [3](#T5){ref-type="table"}). Postoperative pain ------------------ Although the analogue scale pain scores were less in Group B than in Group A, the differences did not reach statistical significance either for maximum pain score or total pain score over the 5 first postoperative days. (Table [4](#T6){ref-type="table"}) ###### Pain scores in first 5 post operative days Group A Group B Student\' s t-test --------------------------------- ------------ ----------- -------------------- Maximum pain score (mean ± 1sd) 4.4 (2.0) 3.8 (2.1) P = 0.17 Total pain score (mean ± 1sd) 10.2 (5.5) 9.1 (5.8) P = 0.43 ###### Relaxation of long saphenous vein rings in response to calcium ionophore, sodium nitroprusside and apocynin. Agonist Group A Group B ------------------------------------- ------------- ------------- --------- Apocynin (mean ± 1 sem) 22.3 (8.7) 20.6 (8.6) p = 0.7 Calcium Ionophore (mean ± 1 sem) 18.4 (7.0) 17.6 (7.5) p = 0.4 Sodium Nitroprusside (mean ± 1 sem) 37.6 (12.1) 40.1 (13.1) p = 0.6 Infection and healing --------------------- The accumulated ASEPSIS scores for the first five postoperative days are shown in Figure [1](#F1){ref-type="fig"}. When the scores are categorized according to the grades given in Table [1c](#T3){ref-type="table"}, there were 8 patients in Group A with a minor wound infection or worse, compared to only 2 in Group B. This reaches statistical significance using a Chi-squared test (p \< 0.001). ![](1749-8090-1-15-1){#F1} Vasomotor studies ----------------- Long saphenous vein rings harvested from Group A and Group B showed similar relaxation to calcium ionophore, sodium nitroprusside and apocynin. (Table [5](#T7){ref-type="table"}) Discussion ========== Modern management of CABG surgery patients emphasizes an early return to normal activities. In this regard early mobilization after surgery plays an important part in the process of recovery. In turn, any reduction in morbidity from the saphenous vein harvest procedure will promote early mobilization and speed rehabilitation. In this randomized study we found that compared to the traditional open method of harvesting long saphenous vein, a less invasive technique employing a Mayo vein stripper was quicker and provided significantly improved wound healing. There are other instruments available but the Mayo stripper has the advantage of being reusable and is therefore less expensive than disposable devices. Although we only examined endothelial function in a sample of the harvested veins, we found no evidence that the manipulation of the vein, consequent upon using the Mayo stripper, affected endothelial function any more than the traditional open harvest method. In order to eliminate the effect of surgical expertise, we used 2 very experienced surgeons who were equally comfortable with either technique to carry out all of the vein harvesting. However, we see no reason why the less invasive technique cannot be taught to surgical trainees and surgical assistants, or why they should not be able to carry it out to an equally high standard. We failed to demonstrate any significant difference in the perception of pain from the leg wounds. In view of the fact that there is a significant difference in the overall length of the skin incisions between the two patient groups this is a little surprising, and does not reflect our subjective observations. In conclusion, this study supports the notion that harvesting vein through multiple incisions using the Mayo vein stripper is quicker, results in better wound healing and has no deleterious effect on endothelial function compared to the open technique.	{ "pile_set_name": "PubMed Central" }	[ "treatment", "rest", "two", "intim", "given", "program", "ctreftypet", "theatr", "impair", "longterm", "sum", "employ", "identifi", "length", "discharg", "mayo", "written", "student", "maxim", "hour", "commenc", "multipl", "may", "revascular", "branch", "separ", "use", "gener", "endotheliumdepend", "medial", "moder", "forward", "score", "support", "bath", "stasi", "caus", "accord", "prior", "signific", "full", "bacteri", "mark", "traine", "process", "also", "growth", "directli", "criterion", "cell", "tension", "perform", "graft", "numer", "longitudin", "criteria", "thigh", "anatomi", "sutur", "normal", "straight", "long", "distribut", "expertis", "first", "metal", "remov", "whole", "organ", 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Background {#Sec1} ========== Cancer of the stomach (GC) and the distal esophagus and esophagogastric junction (AEG) is a substantial global health problem with around 1 million new cases and 750,000 deaths per year, accounting for estimated 10% of all cancer-related deaths \[[@CR1], [@CR2]\]. In Europe and North America, the overall 5-year survival for GC is approximately 25% \[[@CR3]\], while superior outcomes with 5-year survival rates of approximately 60% are reported in East Asia \[[@CR4]\]. The optimal medical treatment for advanced GC and AEG is still a source of debate, but after the publication of the randomized "MAGIC Trial" and "ACCORD Trial", neoadjuvant chemotherapy has become first choice for the treatment of locally advanced GC and AEG, and reported improved 5-year survival rates of 36 and 38% respectively \[[@CR5], [@CR6]\]. This situation is similar for esophageal and cardia cancers where the recently published randomized "CROSS Trial", using neoadjuvant chemoradiation, reported 3-year survival rates of 59% \[[@CR7]\]. Objective assessment of the treatment effect after neoadjuvant treatment is only possible by histopathology in the resected specimens. In patients with AEG and GC, the presence of \< 10% of vital residual tumor cells is considered as a major pathologic response, and is associated with a significant survival benefit \[[@CR8]--[@CR10]\]. The difficulty is to identify patients who do not respond or progress under neoadjuvant treatment. Those patients may not profit from neoadjuvant treatment, still suffer from adverse events, and finally risk tumor progression. In the setting of esophageal cancer, measurement of early changes in tumor glucose uptake by use of 18-fluorodeoxyglucose-PET (PET) and later PET-CT yielded promising results for predicting response following neoadjuvant chemotherapy \[[@CR11]\]. Metabolic tumor activity can be quantified by the standardized uptake value (SUV), and it was shown that a drop of ≥35% measured after 2 weeks of induction chemotherapy was an accurate cut-off value to predict response \[[@CR12]\]. This cut-off at ≥35% was prospectively studied in the MUNICON-I trial including 119 patients with AEG I and II \[[@CR13]\]. Metabolic response evaluation by PET-CT accurately identified all non-responding tumors within two weeks of treatment. In addition, there was a significant survival difference between metabolic responders and non-responders. No data are currently available for early metabolic response evaluation by PET-CT in patients with AEG III and GC, and the potential benefit is therefore unclear. We studied patients with AEG II/III and GC in a cohort of patients using the criteria for early metabolic response from the MUNICON-I trial to evaluate the accuracy and feasibility of metabolic response evaluation by early PET-CT following neoadjuvant CTX. Methods {#Sec2} ======= A retrospective cohort of 72 consecutive patients with biopsy proven GC or AEG Siewert type II-III \[[@CR14]\] was included in this study. All patients underwent routine staging, including, laboratory tests, upper GI-endoscopy with endoscopic ultrasound (EUS) and ^18^FDG PET-CT as reported previously \[[@CR15]\]. Patients with stage cT2N+ or cT3--4, Nx by EUS and PET-CT (UICC TNM Classification, 7th edition, \[[@CR16]\] underwent diagnostic laparoscopy to exclude occult peritoneal carcinomatosis prior to neoCTX. All patients were discussed in a multidisciplinary specialized tumor board prior to treatment initiation. The study was approved by the local ethics committee (PB 2016--00769). Imaging by ^18^FDG positron emission tomography-CT {#Sec3} -------------------------------------------------- A baseline PET-CT was performed as part of the staging procedure and an early response PET-CT, 14 days after the first cycle of neoadjuvant CTX. Imaging was performed on an in-line system (Discovery RX or Discovery VCT; GE Healthcare). These systems integrate a state-of-the art full ring PET scanner with a multi-slice helical CT (LightSpeed 16 or VCT 64 slice; GE Healthcare) allowing for acquisition of co-registered CT and PET images in one session. Patients fasted for at least 4 h before scanning, which started 50--60 min after the injection of a standard dose of 340--370 MBq 18F-FDG. A low dose CT (80 mA, 140 kV, 0.5-s tube rotation, 4.25-mm section thickness, 867-mm scan length, and 22.5-s data acquisition time) was performed first. Immediately after CT, the PET emission scan was acquired, with 2 min emission time per cradle position (total PET-CT acquisition time 12--16 min). PET images were reconstructed using a standard 3-dimensional iterative algorithm (ordered-subset expectation maximization). Image reading was done on screen using a commercially available software package (Advantage workstation, version 4.4; GE Healthcare). For quantitative measurement, a circular region of interest was placed over the tumor in the slice with maximum \[18F\]-FDG uptake in the baseline scan. A tumor was defined as negative or non-avid when there was no measurable activity over background \[18F\]-FDG uptake in the tumor area defined by endoscopy and/or demonstrated as mass in the integrated multi-slice CT. In the second PET scan, the region of interest was placed according to the baseline study using the surrounding anatomical landmarks. Patients with a decrease of ≥35% SUV were classified as metabolic responders (12,13). Neoadjuvant chemotherapy {#Sec4} ------------------------ Two neoadjuvant chemotherapy regimens were applied, either 3 cycles ECF according the "MAGIC" regimen \[[@CR5]\], or 3 cycles of FLOT, consisting of biweekly oxaliplatin 85 mg/m2, day1; docetaxel 50 mg/m2, day2; and continuous infusion 5-FU 2600 mg/m2 days 1--2 \[[@CR17], [@CR18]\]. Adverse events were reported according to the National Cancer Institute Criteria, version 3.0. Surgery {#Sec5} ------- Standardized resections were performed including subtotal (80%) gastrectomy for distal GC, gastrectomy for middle or proximal third GC and transhiatal extended gastrectomy for AEG Siewert type II-III tumors \[[@CR19]\]. Systematic D2-lymphadenectomy (LAD) was routinely performed \[[@CR20]\], and additionally LAD of the lower mediastinum for AEG types II/III. In few selected patients, para-aortic lymph node dissection (D3-LAD, \[[@CR20]\]) was performed. Complications were recorded using the Clavien-Dindo classification \[[@CR21]\]. Pathology {#Sec6} --------- Pathologic tumor regression was evaluated using a published validated scoring system \[[@CR8]\]. Patients with less than 10% residual tumor cells were classified as responders. All other patients were classified as non-responders. All specimens were reviewed by an experienced gastrointestinal pathologist (AW). Follow-up {#Sec7} --------- Patients were followed clinically and by contrast CT (local tumor recurrence, lymph node metastases, systemic metastases including peritoneal carcinomatosis) at 4-month intervals during the first year after surgery and at 6-month intervals thereafter, and endoscopy at 6 months and yearly thereafter. Survival was calculated from the day of study inclusion. Statistical analysis {#Sec8} -------------------- Differences in proportions were analyzed using Fisher's exact test. Inter-individual comparisons of quantitative data were done by use of a Wilcoxon signed rank test. Survival was estimated according to Kaplan-Meier. Statistical comparisons between different groups of patients were done with a log-rank test. All tests were two-sided and done at the 5% level of significance with the use of SPSS for Windows, version 11.50 (SPSS Inc., Chicago, IL, USA). Results {#Sec9} ======= Study population {#Sec10} ---------------- From October 1, 2008 to October 31, 2013, 72 consecutive patients with resectable, locally-advanced GC or AEG II/III were included. Among them, 28 patients were finally not eligible for neoadjuvant chemotherapy due to severe comorbidities, patient's decision, or peritoneal carcinomatosis at diagnostic laparoscopy. In 44 patients planned for neoCTX, 14 (32%) did not show FDG uptake and could therefore not be further evaluated. The remaining 30 patients (68%) underwent neoCTX and were restaged by PET-CT 14 days after beginning neoCTX. Patient characteristics are shown in Table [1](#Tab1){ref-type="table"}.Table 1Patient characteristicsParameterMedian (range)n = 30%Age (years)57.4 (36.9--78.9)Gender male2273 emale827Body Mass Index (kg/m^2^)23.2 (16.7--30.8)Charlson-comorbidity-Index 22377 3--5413 \> 6310ECOG score 02273 1827Localization AEG Siewert Type II1240 AEG Siewert Type III1034 Gastric cancer826Grading G21447 G31653Laurén's classification Intestinal2480 Mixed310 Diffuse310uT-category uT2413 uT32274 uT4413cN-category cN0310 cN+2790cM-category cM02480 cM1620Clinical TNM staging is based on EUS (uT) and/or CT or PET-CT (cN) Chemotherapy {#Sec11} ------------ Nine patients (30%) received neoCTX with ECF \[[@CR5]\], and 21 (70%) received FLOT \[[@CR17], [@CR18]\]. 8/9 patients pretreated with ECF received the planned 3 cycles and one patient refused the third cycle because of side effects. 20/21 (95%) patients with FLOT received 3 full cycles and 1 patient received 2 cycles due to severe bone marrow depression. Surgery and perioperative complications {#Sec12} --------------------------------------- All but one patient proceeded to surgical resection within 3 weeks, while it had to be postponed to week 5 due to bone marrow depression. Radical lymphadenectomy was performed in all patients with a high median number of resected lymph nodes of 43 (range 23--113). Perioperative morbidity according to the Clavien-Dindo classification was observed in 50% of patients, including major complications grade IIIb in 4 (13%) and grade IV in 1 (4%) patient. There was no in-hospital or 90-day mortality. Surgical details are summarized in Table [2](#Tab2){ref-type="table"}.Table 2Surgical characteristicsParametern = 30%Type of resection Subtotal gastrectomy27 Gastrectomy27 Extended gastrectomy27 Transhiatal extended gastrectomy^a^2272 Esophagectomy27Type of lymphadenectomy D2-lymphadenectomy413 D2+ lower mediastinum2013 D3-paraaortic ± lower mediastinum467 2-field (abdominal and extended mediastinal)27R-category R02583 R1/R2517Postoperative complications (Clavien-Dindo) none1550 Grade I310 Grade II723 Grade IIIa00 Grade IIIb413 Grade IVa14 Grade IVb00 Grade V00^a^Splenectomy: n = 1 Pathology {#Sec13} --------- Most patients had locally-advanced ypT3--4 tumors (83%), and positive lymph nodes (67%). Advanced N-categories ypN2--3 were detected in 50% of resected specimens. Major pathologic regression occurred in 11/30 (36.7%) tumors with only 2/30 (7%) showing a pathologic complete remission. Major pathologic response in R0-resected patients was significantly associated with improved median survival rates (not reached) compared to minor response (28.2 months; 95% CI 16.7--39.7 months) by log-rank test (p = 0.04). Results are summarized in Table [3](#Tab3){ref-type="table"}.Table 3Histopathology after neoadjuvant chemotherapyParameterMedian (range)n = 30%ypT-category ypT027 ypT127 ypT213 ypT32170 ypT4a310 ypT4b13Number of removed lymph nodes43 (21--113)ypN-category ypN01033 ypN1517 ypN2723 ypN3827 ypN3 (AEG)310 ypN3a (GC)414 ypN3b (GC)13Tumor regression grading Complete regression (Ia)27 \< 10% residual tumor (Ib)930 ≥ 10% and \< 50% residual tumor (II)620 ≥ 50% residual tumor (III)827 no regression (IV)516Modified tumor regression grading Minor (grade II - IV, ≥ 10% residual tumor)1963 Major (grade Ia - Ib, \< 10% residual tumor)1137 Metabolic response {#Sec14} ------------------ In 30 patients with PET positive primary tumors, median SUV significantly decreased from 10.4 (range 4.0--29.8) to 5.0 (range 0--25.2) 14 days after the first cycle neoCTX (p \< 0.0001). Metabolic response was observed in 20 (66.7%), and no response in 10 (33.3%) patients. Prediction of pathologic response by metabolic response on PET-CT resulted in a sensitivity of 90.9% (95%-CI: 57.1--99.5%), specificity 47.3% (95%-CI: 25.2--70.5%), positive predictive value (PPV) 50% (95%-CI: 27.8--72.1%), negative predictive value (NPV) 90% (95%-CI: 54.1--99.4%) and overall accuracy of 63.3% (95% CI: 38.5--78.6%). Although the overall accuracy is low, the NPV is high with a correct identification in 9/10 true non-responding tumors. Metabolic response and prognosis {#Sec15} -------------------------------- Median follow-up was 22.4 months (range 3.2--61.8) for surviving patients. Median overall survival was significantly better for metabolic responders than for non-responders (median survival not reached and 28.2 months, 95%-CI: 7.2--10.7 months, respectively, log-rank p = 0.04). Survival curves are shown in Fig. [1](#Fig1){ref-type="fig"}.Fig. 1Overall survival according response PET-CT Overall survival estimates of n = 30 patients according to Kaplan-Meier curves based on their response PET-CT after two cycles of neoCTX. Numbers at risk are shown in 12 month intervals Discussion {#Sec16} ========== This cohort study in patients with locally advanced AEG II/III and GC shows that metabolic response two weeks after starting neoadjuvant therapy evaluation by FDG-PET-CT, using the validated threshold for metabolic responders from the MUNICON-I intervention trial (13) of ≥35% reduction of SUV does not accurately predict overall pathologic response. It does however identify a subgroup of patients that does not respond to neoCTX with a specificity of 90%. In this cohort, this subgroup compromised 14% (10/72) of the study population with an inferior prognosis compared to PET responders. The results of our study compare well to the results of the MUNICON-1 trial in patients with esophageal cancer, where 110 patients with AEG I/II were evaluated by PET-CT \[[@CR13]\]. The authors claim that responders can be identified by early metabolic imaging, however, 42% of the 50 PET responders showed a minor regression like the 50% observed in our study. The NPV for the metabolic response was 100% in the MUNICON trial, which is comparable to the 90% in our study and the 10% difference is likely due to the low patient numbers and different tumor entities. We therefore conclude that metabolic response evaluation by PET-CT does not accurately predict overall response but identifies non-responders in both trials. We also confirmed that PET-responders have a better prognosis than non-responders (p = 0.04) with remarkably close survival rates (median survival was not reached in PET-responders in both trials and 26 months and 28.2 months respectively for PET non-responders). In the MUNICON-I intervention trial, chemotherapy was discontinued in metabolic non-responders, thereby saving time, and reducing side-effects and costs without compromising the outcome. Less data is available for patients with gastric cancer. Vallböhmer et al. \[[@CR22]\] found no predictive value for the FDG uptake in 40 gastric cancer patients. Ott and colleagues \[[@CR23]\] prospectively studied 49 GC patients including Siewert type III tumors with a metabolic response (SUV reduction ≥35%) by PET. Overall, 23/49 (47%) patients had non-intestinal type cancers and 38/49 (78%) were in the proximal third. Metabolic response correctly predicted histopathologic regression in 11/16 responding and 27/33 non-responding tumors. This resulted in a sensitivity and specificity of 69 and 82%. PPV and NPV were 65 and 84% and overall accuracy was 78%. Median survival of metabolic responders was not reached, and significantly (p = 0.037) better than for non-responders (24.1 months). Again, remarkably similar results were obtained in our study. The lower NPV is likely attributable to the higher proportion of non-intestinal tumors (46.9%) compared to our study (20%) and the significantly lower baseline SUV obtained in non-intestinal-type tumors. In contrast to patients receiving chemotherapy alone, early metabolic response evaluation by PET-CT was not successful in patients receiving neoadjuvant chemoradiation for esophageal and esophagogastric junction cancers. \[[@CR24]--[@CR26]\]. Technically, a higher cut-off value might be better to predict histopathological response. Indeed, a previous study from the Munich group \[[@CR12]\] showed that a 45% or more decrease in SUV would result in higher specificity for histological response (86% versus 75%) but most important, in a lower NPV. This was also true in our study with a cut-off at 50% (data not shown). The FDG uptake is not uniform among the subgroups. Esophageal tumors, AEG I, show about 100% FDG uptake, much higher than AEG III or GC, or diffuse type cancer \[[@CR15]\]. Therefore, the use of PET-CT is not uniformly recommended, particularly in non-intestinal gastric cancer including signet ring cell cancer \[[@CR23], [@CR27]--[@CR29]\]. Distal gastric cancer with low differentiation grades, including diffuse types are less likely to achieve major tumor regression after chemotherapy \[[@CR30]\]. A risk score was evaluated in 410 patients receiving neoCTX for GC. Well-differentiated tumor grading, intestinal tumor type histology and tumor localization in the middle third of the stomach were identified as the significant positive predictive factors for histopathologic response and prognosis. A prognostic index could be created based on tumor localization, grading and type according to Lauren classification that identified 3 risk groups (low, intermediate and high) with significantly different clinical and histopathological response rates and overall survival times. \[[@CR31]\]. Several molecular markers have been investigated in view of characterizing tumor entities and predicting tumor response and prognosis following neoadjuvant treatment \[[@CR32], [@CR33]\], since a variety of novel targeted therapeutic approaches are introduced in cancer treatment \[[@CR34]\]. In HER2-positive advanced GC and AEG, the international phase III trastuzumab for GC (ToGA) study showed a significant improvement in the median overall survival of patients upon the addition of trastuzumab to cisplatin and fluoropyrimidine backbone therapy \[[@CR35]\]. In the MUNICON-II trial, salvage neoadjuvant radiochemotherapy in metabolic non-responders lead to local remissions in a considerable number of patients but was not able to change the clinical course \[[@CR36]\]. Altogether, metabolic non-responders may profit from a therapeutic switch to these novel approaches. Our study clearly has its limitations, mainly attributable to the small study population which does not allow more sophisticated statistical analysis including multivariate testing, and all test results must therefore be interpreted with caution. Despite these limitations, our results are remarkably close to the results of two published comparable studies, the MUNICON PET-CT trial in Siewert type I/II tumors \[[@CR13]\] and the PET-study in gastric cancer including Siewert type III tumors \[[@CR23]\] using early metabolic response evaluation. Furher research could also include assessing survival outcomes in patients classified as non-responders by PET-CT with subsequent discontinuation of neoadjuvant chemotherapy and comparing them with those remaining on treatment. Conclusions {#Sec17} =========== In conclusion, our study in patients with AEG and GC adds further evidence that response PET-CT reliably detects metabolic non-responders and can therefore identify patients who should either immediately proceed to resection or receive a modified multimodality therapy. PET-CT-guided neoadjuvant chemotherapy appears feasible in patients with AEG and GC but important issues remain to be addressed in future trials especially standardization for metabolic imaging as planned by the EORTC GI Group and NEOPEC Trial Group \[[@CR37], [@CR38]\]. AEG : Adenocarcinoma of the esophagogastric junction CT : Computed tomography EUS : Endoscopic ultrasound GC : Gastric cancer MDCT : Multidetector spiral computed tomography neoCTX : neoadjuvant chemotherapy PET : 18-fluorodeoxyglucose (^18^FDG) positron emission tomography PET-CT : Combined positron emission tomography and computed tomography We thank Miss Monika Stentström, Clinical Nurse for assistance with patients follow-up. Availability of data and materials {#FPar1} ================================== The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. PMS: consultant surgeon in all patients, analyzed and interpreted the patient data, manuscript preparation DE: retrieved, analyzed and interpreted the patient data, manuscript editing TR: chemotherapy and oncological follow-up of patients, manuscript editing DV: interpreted the patient data, manuscript editing PV: radiologic response evaluation, interpreted the patient data, manuscript editing AW: histological examination, interpreted the patient data, manuscript editing PB: endoscopic classification of AEG, endoscopic ultrasound, endoscopic follow-up of patients, interpreted the patient data, manuscript editing PS: chemotherapy and oncological follow-up of patients, manuscript editing KL: retrieved, analyzed and interpreted the patient data and was a major contributor in the writing the manuscript. Ethics approval and consent to participate {#FPar2} ========================================== The study was approved by the local ethics committee, Kantonale Ethikkommission Zürich, Stampfenbachstrasse 121, 8090 Zürich, Tel + 41 (0)43259 79 70 and registered under registration number PB 2016--00769. The need for written informed consent form from each patient has been waived by ethics committee due to retrospective manner of the study. Competing interests {#FPar3} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar4} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	{ "pile_set_name": "PubMed Central" }	[ "treatment", "ypna", "sophist", "choic", "two", "induct", "remain", "tumor", "cours", "decreas", "respond", "ethikkommiss", "laparoscopi", "thank", "biopsi", "identifi", "healthcar", "length", "risk", "classif", "written", "select", "mdct", "valu", "week", "maxim", "monika", "may", "cisplatin", "therebi", "emalebodi", "recent", "pb", "acquir", "cancergrad", "use", "gastrectomi", "final", "score", "ppv", "imag", "complet", "accord", "prior", "signific", "multidetector", "full", "ypta", "kaplanmei", "becom", "emiss", "ps", "tomographi", "helic", "also", "waiv", "intervent", "ctn", "multivari", "cmclinic", "iiia", "iva", "cell", "landmark", "dv", "perform", "metastas", "commerci", "popul", "screen", "proven", "remiss", "retrospect", "vct", "criteria", "accuraci", "neoctx", "fisher", 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Introduction {#s1} ============ Glioblastoma (GBM) is one of the most common and malignant subtype of brain tumors in adults ([@B1]). Although the incident rate is relatively low, GBM is very invasive that leads to rapid neurological destruction and a disproportionately high mortality. Despite the improvement of new surgical and radiation techniques, the median survival rate of patients with GBM is rather low ([@B2], [@B3]). A blood-brain-barrier permeable DNA alkylating agent temozolomide ([@B4]) is currently the first line chemotherapy for treating GBM. In clinics, combination of radiotherapy with temozolomide significantly prolongs the survival rate for GBM patients ([@B5], [@B6]). Unfortunately, tumor often regrows after radio- and chemotherapies ([@B7], [@B8]) resulting in poor prognosis ([@B9], [@B10]). The recurrence is caused at least in part, by the resistance of GBM to conventional chemo- and radio-therapies ([@B11]--[@B14]), highlighting an urgent necessity of designing new strategies for the treatment of GBM. Sonic hedgehog (Shh) is a member of the hedgehog (Hh) family which functions as a chemical signal in transmitting information to the embryonic cells required for normal development. Shh plays a critical role in the regulation of vertebrate organogenesis and the development of brain and spinal cord including midbrain and ventral forebrain neuronal differentiation and proliferation, and many other parts of the body ([@B15]--[@B18]). Because of its role in embryonic development, aberrant or dysregulation of Shh signaling has been implicated in the initiation and/or maintenance of different types of tumor ([@B19]--[@B21]) including GBM ([@B22]). Consistent with these reports, Shh antagonists have been shown to possess anti-tumor activity in patients with basal cell carcinoma and medulloblastoma ([@B21], [@B23], [@B24]). Cancer stem cells (CSCs) are cancer cells that possess the ability to replenish tumors through the self-renewal and differentiation into multiple cell types ([@B25], [@B26]). CSCs have been identified in the breast, colon, brain and other areas and can differentiate into all the cell phenotypes of the parental tumor ([@B27], [@B28]). CSCs are hypothesized to be associated with chemo- and radio-resistance that lead to recurrence of tumor formation. In this theory, conventional radio- and chemotherapies kill differentiated or differentiating cells which form the bulk of the tumor, while sparing CSCs. Therefore, targeting CSCs offers a promising approach to improve cancer treatment or even cure cancer ([@B29]). In the present study, we isolated glioma stem-like cells (GSCs) from human GBM cell line U87MG (U87) using CD133 as a marker. We found that Shh expression is higher in the CD133^+^ cells than in the CD133^−^ cells. LDE225, a smoothened antagonist ([@B30], [@B31]), delayed GBM growth in vivo and significantly reduced the number of tumor spheroids derived from CD133^+^ cells. Furthermore, tumor growth was much slower in Shh knockdown mice suggesting that glioma growth may be dependent on a small population of CD133^+^ cells that are regulated by the Shh pathway. Materials and Methods {#s2} ===================== Animals ------- The BALB/cAnN.Cg-Foxn1^nu^/CrlNarl mice were purchased from the National Laboratory Animal Center (NLAC). Five mice were housed in a cage with controlled temperature (22 ±2°C) and humidity (55 ± 5%), kept on a 12 h light/dark cycle, and were given free access to water and food. Care and use of laboratory animals were in accordance with National Institutes of Health (NIH) guidelines. All the procedures were approved by the Institutional Animal Care and Use Committee of the College of Medicine, NCKU, with project approval number (\#104064 and \#107106). Cell Culture ------------ The human glioblastoma (GBM) cell lines U87MG (U87) was provided by Dr. Michael Hsiao (Genomics Research Center, Academia Sinica, Taiwan). GBM patient-derived cell line P\#5 was developed by Dr. Jian Ying Chuang. Both cells were cultured in Dulbecco\'s Modified Eagle medium-high glucose (DMEM-high glucose, Caisson) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Caisson). All cells were maintained in a humidified incubator with 5% CO~2~ at 37°C. Isolation and Characterization of Cancer Stem Cells From Glioblastoma Cell Line ------------------------------------------------------------------------------- For magnetic-activated cell sorting (MACS) purification, fresh GBM and spheroids were dissociated, washed, and incubated either with PE conjugated CD133/2 or IgG2b (Miltenyi Biotech, 1:11) at a concentration of 10^8^ nucleated cells per ml at room temperature for 15 min. EasySep^?^ PE selection cocktail at 100 μl/ml cells was added and mixed down for more than 5 min. Magnetic cell separation was performed using manual FalconTM polystyrene round-bottom tubes and an EasySep^?^ Magnet machine. Tube was removed from magnet and cells resuspended in an appropriate amount of desired medium. The CD133^+^ cells were incubated in neural stem cells selection medium (NeuroCult^TM^ NS-A Basal Medium, NeuroCult^TM^ NS-A Proliferaction Supplement, bFGF 10 ng/mL, EGF 10 ng/mL, 20 μg/mL Heparin; STEMCELL, Canada) and gave rise to non-adherent spheres on Ultra Low Attachment Multiple Well Plates (CORNING). CD133^+^ cells were initially allowed to form tumor spheroids in suspension culture, dissociated using Accutase (BD Biosciences) at 37°C for 30 min, and then split 1:3 to 1:5. The number of tumor spheroids formed by CD133^+^ cells treated with or without LDE225 (25 μM) for 7 days in culture were determined with an Olympus DP72 image analysis system and Inverted fluorescence microscope Olympus IX71. Cell Proliferation Assay ------------------------ Cell proliferation was measured using WST1 \[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium\] assay (Clontech Laboratories, California, USA). The CD133^+^ cells were seeded at a concentration of 8,000 cells/well in 200 μl culture medium containing various concentrations of LDE225 or cyclopamine (e.g., final concentration of 1--100 μM) into 96-well plates. The cells were incubated for 48 h at 37°C and 5% CO~2~. WST-1 reagent (10 μl/well) was added, and cells were incubated for 1 h at 37°C and 5% CO~2~. The absorbance of the product was measured at 440 nm with a microplate (ELISA) reader. The cell counts were determined by the percentage of the absorption relative to the vehicle-treated control culture. Western Blotting Assay ---------------------- Cell pellets were collected, centrifuged at 4,000 rpm and stored at −80°C. Drugs- or vehicle-treated cell pellets were lysed in a RIPA lysis buffer containing 50 mM Tris--HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor (Roche) and phosphatase inhibitor (Roche). Lysates were shaken at 40 rpm on ice for 1 h and then centrifuged at 12,000 rpm for 30 min at 4°C. The mouse brain tissues were homogenized in a lysis buffer (50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA and protease/phosphatase inhibitor cocktail (Roche, Nutley, USA). Supernatants were collected and then protein concentration was measured by Bradford assay. The protein was resuspended in 5X sample buffer (12.5 mM Tris, 25% glycerol, 4% SDS, 1.54% DTT, and 0.02% Bromophenol blue). The total protein was denatured at 95°C for 5 min. Protein electrophoresis on 15 or 8% SDS-polyacrylamide gel under 120 volt, and the separated protein was transferred to a PVDF membrane (Immunobilon transfer membranes, Millipore) by semi-dry transfer system (BIO-RAD) under 350 mA, 20 volt for 1.5 h. Membranes were incubated with blocking buffer (3% bovine serum albumin in PBS) for 1 h. Membranes were incubated with the following primary antibodies: rabbit anti-Shh (1:3,000, Millipore, Darmstadt, Germany), rabbit anti-P62 (1:2,000, Cell Signaling Technology, Danvers, USA), mouse anti-LC3 (1:2,000, MBL, Japan), mouse anti-CD133 (1:5,000, Millipore, Darmstadt, Germany), rabbit anti-Ptch1 (Protintech, Illinois, USA), rat anti-Gli1 (Sigma, Darmstadt, Germany) antibody overnight at 4°C, followed by treatment with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Lab., West Grove, USA) for 1 h at room temperature. After three rinses with 0.3% Triton X-100 in PBS for 10 min each, the ECL-plus chemical reagents (PerkinElmer) were added to the membrane. Films (Fuji, Japan) were exposed at different time points to ensure the optimum density. The band intensities were analyzed for densitometry by using Windows Image-J version 1.29. The protein levels in all groups were expressed as a percentage of those in controls. Immunofluorescent Staining -------------------------- For immunostaining of tumor spheroids, CD133^+^ cells were initially allowed to form spheroids in suspension culture for 7 days. The cells were then fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature. The cells were then incubated for 1 h in blocking solution (1% bovine serum albumin), and then incubated in primary antibodies at 4°C overnight. The following primary antibodies were used: mouse anti-CD133 (1:100, Millipore, Darmstadt, Germany), rabbit anti-SOX-2 (1:1,000, Abcam, Cambridge, UK) and rabbit anti-Shh (1:200, Millipore, Darmstadt, Germany), rabbit anti-ALDH1A1 (Protintech, Illinois, USA), rat anti-ABCG2 (Santa Cruz, Texas, US), rabbit anti-LC3 (Novus, Colorado, US). After three rinses with PBS containing 0.1% Triton X-100 for 10 min each, the cells were incubated in secondary antibodies at room temperature for 1 h. The following secondary antibodies were used: Alexa Fluor®594-conjugated Goat-anti-rabbit IgG (Jackson ImmunoResearch Lab., West Grove, USA) and Alexa Fluor®488-conjugated Sheep-anti-mouse IgG and Alexa Fluor®488-conjugated Goat-anti-rat IgG (Jackson ImmunoResearch Lab., West Grove, USA). DAPI (4\',6-diamidino-2-phenylindole) (1:1,000, Sigma-Aldrich, St. Louis, USA) dye was used as nuclear counterstain. The cells are washed in PBS containing 0.1% Triton X-100 three times for 10 min. The sections were cover-slipped with Prolong^?^ gold anti-fade reagent (Life Technologies, Carlsbad, USA). Fluorescence signals were detected with a Leica DM 2500 microscope and MetaMorph image software used for counting the cells number. JC-1 Mitochondrial Membrane Potential Assay ------------------------------------------- Early apoptosis was measured by using JC-1 Mitochondrial Membrane Potential assay (Cayman Chemical, Michigan, USA). The CD133^+^ cells were seeded at a concentration of 5,000 cells/well in 100 μl culture medium containing 25 μM LDE225 or 0.1% DMSO as vehicle control into black 96-well plates. The cells were incubated for 48 h at 37°C and 5 % CO~2~. JC-1 Staining solution (10 μl/well) was added, and cells were incubated for 30 min at 37°C and 5% CO~2~. Plates were centrifuged at 300 × g at room temperature. The supernatant was removed, and JC-1 buffer was used to suspend JC-1 stained cells. The fluorescence intensity of J-aggregate and JC-1 monomer was measured with excitation and emission at 535/595 nm and 485/535 nm with a microplate (ELISA) reader. The ratio of J-aggregate and JC-1 was determined as early apoptosis. Caspase-Glo® 3/7 Assay ---------------------- The activity of caspase 3 and 7 was measured by using Caspase-Glo® 3/7 Assay (Promega, Wisconsin, US). The CD133^+^ cells were seeded at a concentration of 1,000 cells/well in 100 μl culture medium containing 25 μM LDE225 or 0.1% DMSO as vehicle control into white 96-well plates. The cells were incubated for 48 h at 37°C and 5% CO~2~. Caspase-Glo®3/7 Reagent (10 μl/well) was added, and cells were incubated for 1 h at 37°C and 5% CO~2~. The luminescence was recorded with a microplate (ELISA) reader. The activity of caspase 3 and 7 was determined by the percentage of the luminescence signal relative to the control culture. In vivo Intracranial Xenograft Animal Model and Bioluminescence Imaging ------------------------------------------------------------------------- U87 GBM cells were transduced with lentiviral vector expressing GFP and firefly luciferase. GFP/Luc expressing cells were sorted out for further passages (FACS-Aria, BD Biosciences). For tumorigenesis, luciferase-expressing GBM cells were inoculated intracranially into the 8- to 10-week-old male nude mice (BALB/cAnN-Foxnlnu/CrlNarl mice, National Laboratory Animal Center). Nude mice were anesthetized with chloral hydrate and placed on a stereotaxic device. Subsequently, a hamilton syringe with 30-gauge needle was mounted on a stereotaxic device, and luciferase-expressing GBM cells were injected into the left side of the brains, 1.5 mm caudal and lateral to the bregma, and at a depth of 3.5 to 4 mm. LDE225 (Cayman) was injected intraperitoneally injected at a dose of 20 mg/kg twice weekly. Tumor growth was monitored by IVIS spectrum Live Imaging System (IVIS-200, Xenogen) twice weekly. Before monitoring, mice were injected with 150 mg/kg D-luciferin (PerkinElmer), and simultaneously anesthetized with isoflurane. The results of luciferase radiance were quantitated by Live Imaging Software (Xenogen) and the results were analyzed by using GraphPad Prism software. Shh shRNA Lentivirus Production --------------------------------- Production of lentivirus was initiated by triple transfection of HEK293T cells by a Lipofectamine® LTX Reagent (Life Technologies, Carlsbad, USA) method using small hairpin interfering RNA (shRNA) together with pCMV-dR8.91 and pMD2.G. The Shh-shRNA or scrambled shRNA conjugated on the vector of pLKO.1 with puromycin-resistant region was provided by National RNAi Core Facility (Institute of Molecular Biology, Academia Sinica, Taiwan). Cells were harvested 48 h later, medium containing lentiviruses filtered with 0.45 μm filters and viral particles were concentrated from the supernatant by Lenti-X™ Concentrator (Clontech Laboratories, Mountain View, USA) and purified to yield 1 × 10^8^ transducing units/ml storing at −80°C until use. The target sequence of Shh-shRNA is described as follows: Shh-shRNA: 5′-GCGGAAGGTATGAAGGGAAGA-3′. Shh-Over-Expression --------------------- To create lentiviruses over-expressing Shh, full-length Shh open reading frames (ORFs) (NM_000193; GenScript, New Jersey, USA) was amplified by PCR and was inserted into pLVX-IRES-ZsGreen1 expression vector (Clontech Laboratories, California, USA). The pLVX-NES1-IRES-ZsGreens1 vector encoding Shh (or empty vector) and the two packaging plasmids (pCMV-dR8.91 and pMD2.G) were co-transfected into HEK293T cells by lipofectamine® LTX Reagent (Life Technologies, Carlsbad, USA). Lentiviruses were harvested at 48 h after transfection, filter lentivirus supernatant through a 0.45 μm PVDF membrane filters, concentrated by Lenti-X™ Concentrator (Clontech Laboratories, Mountain View, USA), purified to yield 1 × 10^8^ transducing units/ml and stored at −80°Cuntil use. Statistical Analysis -------------------- Experiments were performed at least in triplicate. All results were presented as mean ± standard error of the mean (SEM). Independent experiments were analyzed by unpaired t-test. Two-way ANOVA was used to analyze the differences in tumor spheroids numbers in vitro and intracranial tumor growth in vivo at different times of treatment. Levels of p \< 0.05 were considered to be of statistical significance. Results {#s3} ======= CD133 has been reported as a marker of human neural and brain tumor stem cells ([@B32], [@B33]). We previously have isolated cancer stem-like cells with CD133 from glioblastoma (GBM) cell lines using magnetic bead cell sorting ([@B34]). Then CD133^+^ and CD133^−^ cell populations were collected and cultured separately. Shh Expression Is Higher in CD133^+^ Cells ------------------------------------------ We examined Shh expression in CD133^+^ and CD133^−^ cells. Western blotting analysis showed that Shh expression was higher in the CD133^+^ cells than in the CD133^−^ cells ([Figure 1A](#F1){ref-type="fig"}). Immunofluorescence staining revealed that most of CD133^+^ cells were positive and co-localized with Shh ([Figure 1B](#F1){ref-type="fig"}). SRY (sex determining region Y)-box 2 (SOX2) is a transcription factor that plays an important role in maintaining embryonic and neural stem cells ([@B35], [@B36]). [Figure 1C](#F1){ref-type="fig"} showed that SOX2 expression was higher in the CD133^+^ cells than in the CD133^−^ cells.These Shh-positive cells were also co-immunolabeled with SOX2 ([Figure 1D](#F1){ref-type="fig"}). ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily. The expression level of ABCG2 has been implicated in multidrug resistance (MDR) in cancer chemotherapy and the ability of self-renewal which correlates with CD133 ([@B37], [@B38]). AlDH1A1 is a member of the highly conserved ALDH family that is observed in several cancer stem cells, and is often used to isolate and functionally characterize cancer stem cells ([@B39]). We also examined the expression of ABCG2 and AlDH1A1 by immunofluorescence in parental and CD133^+^ cells. The expression of ABCG2 and ALDH1A1 is higher in CD133^+^ cells than in parental cells ([Supplemental Figure 1](#SM1){ref-type="supplementary-material"}). ![Tumor spheroids derived from CD133^+^ cells exhibit higher level of Shh expression. (A) Expression of Shh in CD133^+^ and CD133^−^ cells determined by Western blotting analysis. The CD133^+^ cells exhibited higher level of Shh expression. (B) Immunofluorescence staining of CD133 and Shh in tumor spheroids derived from CD133^+^ cells. CD133 and Shh were co-localized at tumor spheroids. (C) Expression of SOX2 in CD133^+^ and CD133^−^ cells determined by Western blotting analysis. The CD133^+^ cells exhibited higher level of SOX2 expression. (D) Immunofluorescence staining of CD133, Shh, and SOX2 in tumor spheroids derived from CD133^+^ cells. CD133 and SOX2, Shh, and SOX2 were co-localized at tumor spheroids.](fonc-10-01233-g0001){#F1} We used a potent and selective smoothened antagonist LDE225 ([@B30]) to determine the requirement of Shh signaling in the proliferation and tumor growth of GBM cell lines. We first confirmed the effect of LDE225 on tumor growth by using intracranial injection model. Luc-expressing parental U87 cells were injected intracranially into athymic mice and tumor growth was monitored using IVIS-200 imaging system. At day 7, LDE225 or DMSO as vehicle was injected intraperitoneally twice per week (20 mg/kg) into the mice and tumor growth was observed for 22 more days. Comparing with vehicle control, [Figure 2A](#F2){ref-type="fig"} showed that LDE225 was able to delay tumor growth. A two-way ANOVA revealed a main effect of group (LDE225 vs. control) \[F~(1,\ 42)~ =6.126, p \< 0.05\], interaction \[F~(6,\ 42)~ = 2.297, p = 0.0524\] and days after application \[F~(6,\ 42)~ = 5.849, p \< 0.001\] ([Figure 2B](#F2){ref-type="fig"}). Kaplan--Meier analysis of the survival data of vehicle control and LDE225-treated mice displayed in [Figure 2C](#F2){ref-type="fig"}. We also confirmed whether LDE225 inhibits Shh signal pathway. CD133^+^ cells were treated with LDE225 (25 μM) or vehicle for 48 h and the expression of Patch1 and Gli1 were determined by Western blotting analysis. As shown in [Figure 3A](#F3){ref-type="fig"}, LDE225 downregulated the expression of PATCH1 and GLI1. Next, CD133^+^ and CD133^−^ cells were treated with LDE225 (25 μM) for 48 h and cell viability was assessed using WST-1 assay. Concentration dependent relationship estimated the IC~50~ of about 20 μM for LDE225 to reduce CD133^+^ cell viability ([Figure 3B](#F3){ref-type="fig"}), while IC~50~ of LDE225 was about 50 μM for parental cells ([Supplemental Figure 2A](#SM2){ref-type="supplementary-material"}). As shown in [Figure 3C](#F3){ref-type="fig"}, CD133^+^ cells were more sensitive to LDE225 inhibition than that of CD133^−^ cells. At the concentration of 25 μM, LDE225 inhibited the proliferation of CD133^+^ and CD133^−^ cells by 57.57 ± 3.17% (n = 3) and 26.18 ± 6.22% (n = 4), respectively \[t~(5)~ = 4.013, p \< 0.05\]. We also determined the effect of LDE225 on patient-derived cell line P\#5 which displays various characteristics of GBM ([@B40], [@B41]). As illustrated in [Supplemental Figure 2B](#SM2){ref-type="supplementary-material"}, LDE225 inhibited cell viability with IC~50~ \~423 μM. Similar inhibition of CD133^+^ was observed by another Shh inhibitor cyclopamine, which binds to and inactivates smoothened protein ([@B42]) (IC~50~ ≈ 100 μM, [Supplemental Figure 2C](#SM2){ref-type="supplementary-material"}). We determined the effect of Shh inhibition on self-renew capacity of CD133^+^ cells ([Figure 3D](#F3){ref-type="fig"}). CD133^+^ cells were treated with LDE225 (25 μM) or vehicle and CD133^+^-derived tumor spheroids were counted at 1, 3, 7, and 14 days. Treatment with LDE225 significantly reduced the number of tumor spheroids. A two-way ANOVA revealed a main effect of drug (LDE225 vs. vehicle) \[F~(1,\ 16)~ = 100.9, p \< 0.001\], interaction \[F~(3,\ 16)~ = 44.27, p \< 0.001\] and days after application \[F~(3,\ 16)~ = 44.25, p \< 0.001\] ([Figure 3E](#F3){ref-type="fig"}). ![LDE225 delays tumor growth in an intracranial tumor model. (A) Luc-expressing parental U87 cells (3 × 10^5^ cells) were injected intracranially into athymic mice and tumor growth was monitored using the IVIS-200 imaging system. (B) Tumor growth was slower in LDE225 treated mice than in vehicle control mice. \\p \< 0.001 vs. vehicle control. (C) Kaplan--Meier analysis of LDE225 treatment on survival.](fonc-10-01233-g0002){#F2} ![Effects of Shh inhibitors LDE225 on the cell viability of CD133^+^ and CD133^−^ cells. (A) CD133^+^ cells were treated with LDE-225 (25 μM) for 48 h and the expression of Patched1 and Gli1 was determined by the Western blotting analysis. LDE225 downregulated Patched1 and Gli1 in CD133^+^ cells. (B) Concentration-dependent effect of LDE225 on the cell viability of CD133^+^ cells. \\p \< 0.01, \\\*p \< 0.001 vs. vehicle. (C) CD133^+^ and CD133^−^ cells were treated with LDE225 (25 μM) for 48 h and cell viability was assessed using WST-1 assay. CD133^+^ cells were more sensitive to LDE225 inhibition than that of CD133^−^ cells. \*p \< 0.05 vs. CD133^−^ cells. (D,E) Effects of LDE225 (25 μM) on the number of tumor spheroids derived from CD133^+^ cells. Primary tumor spheroids derived from CD133^+^ were dissociated and cultured. They were then treated with LDE225 (25 μM) for the times as indicated in (E). The medium was replaced every 2 days in the presence of LDE225. \*p \< 0.05, \\p \< 0.01 vs. Vehicle.](fonc-10-01233-g0003){#F3} Inhibition of Shh Induces Autophagy in CD133^+^ Cells ----------------------------------------------------- In LDE225-treated cells, the observation by transmission electronic microscope showed the appearance of large membranous vacuoles in the cytoplasm which is a characteristic feature of cells undergoing autophagy ([Figure 4A](#F4){ref-type="fig"}). In addition, LC3 is distributed in the autophagosome membrane ([@B43]). The conversion of LC3-I to LC3-II is a common biomarker for autophagy activation ([@B44], [@B45]). We determined the conversion of LC3-I to LC3-II with anti-LC3 antibody. Immunoblotting using lysates from LDE225 (25 μM)-treated CD133^+^ cells revealed a significant increase in processed LC3-II \[t~(5)~ = 3.39, p \< 0.05\] ([Figure 4B](#F4){ref-type="fig"}). This was accompanied by the reduced expression of Mushashi-1, a RNA--binding protein selectively expressed in neural progenitor cells ([@B46]). The intensity of LC3 fluorescence punctate also increased after LDE225 treatment ([Figure 4C](#F4){ref-type="fig"}). Furthermore, LDE225-induced cell death was rescued by autophagy inhibitor 3-methyladenine (3-MA) ([@B47]) ([Figure 4D](#F4){ref-type="fig"}) suggesting that autophagy plays a critical role in LDE225-induced cytotoxicity in CD133^+^ cells. ![LDE225 induces autophagy in the CD133^+^ cells. (A) The graphs of transmission electron microscopy represented CD133^+^ cells treated with LDE225 (25 μM) or vehicle for 48 h. Arrows indicate autophagosomes. (B) Western blot analysis of LC3-I/LC3-II and Mushashi-1 expression in the CD133^+^ cells treated with LDE225 (25 μM) for 48 h. LDE225 induced conversion of LC3-I to LC3-II which was accompanied by the downregulation of Mushashi-1. (C) LC3 expression in CD133^+^ cells treated with LDE225 (25 μM) or vehicle for 48 h. The fluorescence intensity of LC3 punctate increased after LDE225 treatment. (D) Block of LDE225-induced cell death by autophagy inhibitor 3-MA. CD133^+^ cells were pretreated with 3-MA (3 μM) 1 h before the exposure to LDE225 (25 μM). Pretreatment with 3-MA attenuated LDE225-induced cell death. \*p \< 0.05.](fonc-10-01233-g0004){#F4} We performed different apoptosis assays to evaluate whether LDE-induced cell death involves apoptosis. We used JC-1 mitochondrial membrane potential assay which measures the mitochondrial membrane potential as the indicator of cell health. Change in fluorescent property of JC-1 dye can be utilized to evaluate early apoptosis. CD133^+^ cells were treated with LDE225 (25 μM) or 0.1% DMSO as vehicle control for 48 h. [Supplemental Figure 3A](#SM3){ref-type="supplementary-material"} shows that vehicle led to mitochondrial depolarization and decreased the ratio of J-aggregate/JC-1 monomer while that was not different between vehicle and LDE225 (25 μM) treatment. Caspase-Glo assay also revealed that Caspase 3/7 activity was not different between LDE225 and vehicle ([Supplemental Figure 3B](#SM3){ref-type="supplementary-material"}). These results suggest that LDE225-induced cell death likely was not mediated primarily through apoptosis. Knockdown of Shh Slows Tumor Growth in an Intracranial Tumor Model ------------------------------------------------------------------ We clarified whether Shh plays a role in tumor growth under in vivo conditions using an orthotopic GBM model. To monitor intracranial tumor growth, we infected Luc-expressing CD133^+^ cells with lentiviruses carrying the expression vector containing Shh shRNA. Transduced CD133^+^ cells (8 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using IVIS-200 imaging system. [Figure 5A](#F5){ref-type="fig"} shows that tumor growth was much slower in Shh shRNA-knockdown mice than in control RNA-transfected mice. A two-way ANOVA revealed a main effect of group (Shh shRNA vs. control) \[F~(1,\ 11)~ = 4.980, p \< 0.05\], interaction \[F~(7,\ 77)~ = 3.110, p \< 0.01\] and days after application \[F~(7,\ 77)~ = 4.853, p \< 0.001\] ([Figure 5B](#F5){ref-type="fig"}). Kaplan--Meier analysis of the survival data demonstrated a statistically significant difference (p \< 0.05) in the median survival between vector control and Shh shRNA-treated mice ([Figure 5C](#F5){ref-type="fig"}). Brain tumor growth was analyzed in coronal brain slices by H&E staining. As shown in [Figure 5D](#F5){ref-type="fig"}, in comparison with vector control, shRNA-Shh-transfection generated significantly smaller intracranial tumor. ![Knockdown of Shh attenuates tumor growth in an intracranial tumor model. (A) Luc-expressing CD133^+^ cells were infected with lentiviruses carrying the expression vector containing Shh shRNA. Transduced CD133^+^ cells (8 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using the IVIS-200 imaging system. (B) Tumor growth was much slower in Shh shRNA- transfected mice than in control RNA-transfected mice. \*p \< 0.05, \\\*p \< 0.001 vs. control RNA-transfected. (C) Kaplan--Meier analysis of Shh shRNA transfection on survival. (D) Brains of mice harboring tumors generated from control RNA-transfected mice and Shh shRNA-transfected mice and stained by H&E. Note that tumor in Shh shRNA-transfected mice was significantly larger than control RNA-transfected tumor.](fonc-10-01233-g0005){#F5} To examine whether knockdown of Shh affected autophagy and stemness in vivo, we examined the expression of LC3-II in Shh shRNA-treated mice. [Supplemental Figure 4](#SM4){ref-type="supplementary-material"} shows that the levels of Shh, CD133, mushashi-1 and SOX2 were lower whereas the conversion of LC3-I to LC3-II was higher in mice of Shh knockdown CD133^+^ cells compared to CD133^+^ cells of control mice. These results suggest that knockdown of Shh reduces stemness and GBM tumor growth. Over-Expression of Shh Promotes Tumor Growth -------------------------------------------- To over-express Shh, we cloned the mouse Shh gene into parental U87 cells with lentiviruses carrying the expression vector containing Shh gene. The empty vector (LV-Vehicle) served as control. The LV-Shh or LV-vehicle transfected parental U87 GBM cells (8 × 10^4^ cells) was injected intracranially into athymic mice. As shown in [Figures 6A,B](#F6){ref-type="fig"}, tumor growth was much faster in LV-Shh-transfected mice than in LV-vehicle-transfected mice. A two-way ANOVA revealed a main effect of group (LV-Shh vs. control) \[F~(1,\ 6)~ = 7.665, p \< 0.05\], interaction \[F~(9,\ 54)~ = 4.022, p \< 0.001\] and days after application \[F~(9,\ 54)~ = 9.691, p \< 0.001\]. Kaplan--Meier analysis of the survival data demonstrated a statistically significant difference (p \< 0.05) in median survival between control and LV-Shh-treated mice ([Figure 6C](#F6){ref-type="fig"}). Brain tumor size was analyzed in coronal brain slices. As shown in [Figure 6D](#F6){ref-type="fig"}, in comparison with vector control, LV-Shh-transfection generated significantly larger intracranial tumor. ![Over-expression of Shh promotes tumor growth. (A) Shh overexpression in parental U87 cells was transduced by lentiviruses carrying the expression vector containing mouse Shh gene (LV-Shh). The empty vector (LV-Vehicle) served as control. The LV-Shh or LV-Vehicle infected U87 (8 × 10^4^ cells) was injected intracranially into athymic mice. Tumor growth was monitored by IVIS-200 imaging system. (B) Tumor growth was much faster in LV-Shh-transfected mice than in LV-vehicle-transfected mice. \*p \< 0.05, \\p \< 0.01, \\\*p \< 0.001 vs. vector control. (C) Kaplan--Meier analysis of over-expression of Shh transfection on survival. (D) Brains of mice harboring tumors generated from LV- Vehicle and LV-Shh-transfection and stained by H&E. Note that LV-Shh-transfection tumor was significantly larger than vector control tumor.](fonc-10-01233-g0006){#F6} To examine whether over-expression of Shh affected autophagy and stemness in vivo, we examined the expression of LC3-II in LV-Shh-treated mice. [Supplemental Figure 5](#SM5){ref-type="supplementary-material"} shows that the levels of Shh, CD133, mushashi-1 and SOX2 were higher whereas the conversion of LC3-I to LC3-II was lower in Shh over-expression GBM compared to that of control. These results suggest that over-expression of Shh promotes cancer stemness and GBM tumor growth. Additive Effect of Rapamycin and LDE225 on The Viability of CD133^+^ Cells -------------------------------------------------------------------------- Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced by the mTOR inhibitor rapamycin ([@B48]--[@B50]). To confirm our hypothesis, we tested the effect of rapamycin on CD133^+^ cells. Single treatment of CD133^+^ cells with LDE225 (25 μM) or rapamycin (100 nM) inhibited the cell viability by 32.0 ± 7.9% (n = 6) and 24.6 ± 9.9% (n = 6), respectively. As shown in [Figure 7A](#F7){ref-type="fig"}, combination of LDE225 and rapamycin resulted in 54.6 ± 3.3% (n = 6) inhibition (p \< 0.05 vs. single treatment). Interestingly, combined treatment resulted in additive conversion of LC3-I to LC3-II. The conversion of LC3-I to LC3-II after LDE225 or rapamycin treatment was 159.8 ± 14.7% (n = 7) and 138.8 ± 14.5% (n = 7) of control, respectively. Combined treatment resulted in 217.7 ± 19.7% (n = 7) of control (p \< 0.05 vs. LDE225, p \< 0.01 vs. rapamycin) ([Figure 7B](#F7){ref-type="fig"}). ![LDE225 and rapamycin in combination additively reduces cell viability and induces conversion of LC3-I to LC3-II in CD133^+^ cells. (A) CD133^+^ cells were treated with LDE225 (25 μM) and rapamycin (100 nM) alone or in combination for 48 h and cell viability was assessed using WST-1 assay. (B) CD133^+^ cells were treated with LDE225 (25 μM) and rapamycin (100 nM) alone or in combination for 48 h and the levels of LC3-I and LC3-II were measured by Western blotting analysis. (C) CD133^+^ cells were treated with LDE225 (25 μM) and rapamycin (100 nM) alone or in combination for 48 h and the levels of p-mTOR and mTOR were measured by Western blotting analysis. Combined treatment with LDE225 and rapamycin reduced p-mTOR to the level not different from that treated with rapamycin alone (p = 0.2797). \*p \< 0.05, \\p \< 0.01.](fonc-10-01233-g0007){#F7} Autophagy can also be induced through mTOR-independent pathway ([@B50]). We determined the effect of LDE225 (25 μM) on mTOR phosphorylation. LDE225 (25 μM) did not significantly influence the phosphorylated level of mTOR (p-mTOR) whereas rapamycin (100 nM) reduced it. Combined treatment with LDE225 and rapamycin reduced p-mTOR to the level not significantly different from treatment with rapamycin alone (p = 0.2797) ([Figure 7C](#F7){ref-type="fig"}). Thus, it is likely that LDE225 induced autophagy through mTOR-independent pathway. Amiodarone Reduces Stem-Like Cell Viability and Inhibits Tumor Formation ------------------------------------------------------------------------ Amiodarone, a clinically used anti-arrhythmic drug, could induce autophagy via mTOR-independent signaling ([@B51]). We determined whether amiodarone reduced cancer stem-like cell viability in vitro and exhibited anti-tumor activity in vivo. CD133^+^ cells were treated with amiodarone for 48 h and cell viability was assessed using WST-1 assay. As shown in [Figure 8A](#F8){ref-type="fig"}, amiodarone dose-dependently reduced cell viability \[F~(7,\ 24)~ = 16.71, p \< 0.001\]. Immunoblotting using lysates from amiodarone (5 μM)-treated CD133^+^ cells revealed a significant increase in processed LC3-II \[t~(4)~ = 4.3976, p \< 0.05\] ([Figure 8B](#F8){ref-type="fig"}). Furthermore, amiodarone-induced cell death was reversed by autophagy inhibitor 3-methyladenine (3-MA) ([Figure 8C](#F8){ref-type="fig"}) suggesting that autophagy plays a critical role in amiodarone-induced cell death of CD133^+^ cells. CD133^+^ cells were treated with amiodarone (5 μM) or vehicle and CD133^+^-derived tumor spheroids were counted at 1, 3, 7, and 14 days. Treatment with amiodarone significantly reduced the number of tumor spheroids \[t~(4)~ = 10.09, p \< 0.001, at 14 days culture\] ([Figure 8D](#F8){ref-type="fig"}). ![Effects of amiodarone on the cell viability and tumor spheroid formation of CD133^+^ cells. (A) Concentration-dependent effect of amiodarone on the cell viability of CD133^+^ cells. \\p \< 0.01, \\\*p \< 0.001 vs. vehicle. (B) Western blot analysis of LC3-I and LC3-II expression in the CD133^+^ cells treated with amiodarone (5 μM) for 48 h. (C) Block of amiodarone-induced cell death by autophagy inhibitor 3-MA. CD133^+^ cells were pretreated with 3-MA (3 μM) 1 h before the exposure to amiodarone (5 μM). Pretreatment with 3-MA attenuated amiodarone-induced cell death. \\p \< 0.01. (D) Effects of amiodarone (5 μM) on the number of tumor spheroids derived from CD133^+^ cells. Primary tumor spheroids derived from CD133^+^ were dissociated and cultured. They were then treated with amiodarone (5 μM) for the times as indicated. The medium was replaced every 2 days in the presence of amiodarone. \*p \< 0.05, \\p \< 0.01, \\\*p \< 0.001 vs. Vehicle.](fonc-10-01233-g0008){#F8} We determined whether amiodarone inhibited tumor formation. Transduced CD133^+^ cells (6 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using IVIS-200 imaging system. At day 11, amiodarone was injected intraperitoneally once per day for 8 days (80 mg/kg) into the mice and tumor growth was observed for 17 more days. [Figure 9A](#F9){ref-type="fig"} shows that tumor growth at day 35 was much slower in amiodarone-treated mice than in control vehicle-treated mice \[t~(8)~ = 2.758, p \< 0.05\]. Brain tumor size was analyzed in coronal brain slices. As shown in [Figure 9B](#F9){ref-type="fig"}, in comparison with vehicle control, amiodarone-treated mice generated significantly smaller intracranial tumor. ![Amiodarone slows tumor growth in an intracranial tumor model. (A) Transduced CD133^+^ cells (6 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using the IVIS-200 imaging system. At day 11, amiodarone was injected intraperitoneally once per day for 8 days (80 mg/kg) into the mice and tumor growth was observed for 17 more days. \*p \< 0.05 vs. vehicle. (B) Amiodarone-treated mice generated significantly smaller intracranial tumor by H&E staining.](fonc-10-01233-g0009){#F9} Discussion {#s4} ========== Roles of Shh Pathway in CD133^+^ Cells-Derived Tumor Spheroid Formation and Tumorigenesis ----------------------------------------------------------------------------------------- We have isolated CD133-positive cells from human U87MG (U87) GBM cells using magnetic bead cell sorting ([@B34]). CD133^+^-derived cell clones were able to grow in vitro in tumor spheroids and generate a tumor in vivo by intracranial cell injection in immunocompromised mice. The developmental signal transduction pathway mediated by Shh plays an important role during embryogenesis by regulating body patterning, cell proliferation and differentiation ([@B52], [@B53]). Activation of Shh has been causatively correlated with initiation and/or maintenance of cancer ([@B54]). In the present study, using Western blotting analysis and immunofluorescent staining, we showed that CD133^+^ cells exhibited higher Shh expression compared to CD133^−^ cells. Treatment with smoothened antagonist LDE225 significantly reduced the number of CD133^+^ cells-derived tumor spheroids suggesting Shh signaling likely contributes to tumor spheroids formation in vitro. Silencing Shh gene with small hairpin interfering RNA inhibited tumor growth in an intracranial mouse model. Conversely, over-expression of Shh gene facilitated tumor growth. Taken together, these results suggest that Shh signaling is involved in in vitro tumor spheroid formation and in vivo tumor growth. Shh Inhibitor Reduces the Number of CD133^+^-Derived Tumor Spheroids by Inducing Autophagy ------------------------------------------------------------------------------------------ The reports about the relationship between Shh and autophagy pathway in GBM are limited. One study in SHSY5Y cells showed that cyclopamine reduced serum starvation-induced LC3 positive cells but increased the levels of cleaved caspase 3 ([@B54]). They suggested that Shh inhibitor induced cell death by reducing autophagic processes concomitant with facilitating apoptotic cell death. In the present study, we demonstrated that LDE225 induced cell death in CD133^+^ cells in a concentration-dependent manner with IC~50~ of \~20 μM. Similar inhibition of cell survival was observed after application of another Shh inhibitor cyclopamine. Electron microscopy examination revealed that, after exposure to LDE225, CD133^+^ cells exhibited large membranous vacuoles in the cytoplasm that is a characteristic feature of cells undergoing autophagy. Further experiments of the conversion of LC3-I to LC3-II and attenuation of cell death by autophagy inhibitor 3-MA confirmed that LDE225-induced autophagy in the CD133^+^ cells. This study, in contrast to previous results, suggests that Shh inhibitor produces cytotoxicity in CD133^+^ cells by inducing autophagic cell death. To further test this hypothesis, we used rapamycin which is a mTOR inhibitor and is capable of inducing autophagy ([@B49], [@B51]). We found that rapamycin reduced CD133^+^ cell viability. Interestingly, the effects of LDE225 and rapamycin were additive. Furthermore, LDE225 at the concentration that induced autophagy and reduced cell viability of CD133^+^ cells did not affect the phosphorylated level of mTOR. Combined LDE225 and rapamycin failed to show further reduction of p-mTOR compared with rapamycin alone. These results suggest that LDE225 induces autophagy through mTOR-independent pathway. These findings also provide additional support that Shh regulation of autophagy plays an important role in the survival of GSCs. Thus, Shh pathway inhibitor could act as a sensitizer to increase efficiency of conventional chemotherapeutic agents in GBM by inducing cancer stem cell autophagic death. Anti-tumorigenesis of Amiodarone -------------------------------- Amiodarone, a frequently prescribed anti-arrhythmic drugs in clinics, has been identified as a mTOR-independent autophagy enhancer ([@B55], [@B56]). If LDE225-induced cell death was mediated by the mTOR-independent autophagy, then amiodarone should produce similar effects as LDE225. Indeed, this was the case. Amiodarone reduced CD133^+^ cell viability and tumor spheroid formation in vitro and exhibited anti-tumor efficacy in vivo. In summary, there was a small percentage of human U87 GBM cells which were CD133-positive, exhibited cancer stem cell markers and were capable of forming tumor spheroids in vitro and tumor in vivo. These CD133^+^ cells showed higher Shh expression and inhibition of Shh pathway with LDE225 reduced their survival and self-renew property. In LDE225-treated CD133^+^ cells, there appeared large membranous vacuoles in the cytoplasm and the conversion of LC3-I to LC3-II. LDE225-induced cell death was reversed by autophagy inhibitor 3-MA, suggesting that LDE225-induced autophagic cell death in CD133^+^ cells. Further, LDE225 did not affect the level of p-mTOR and combined LDE225 and rapamycin failed to show further reduction of p-mTOR when compared with rapamycin alone. Taken together, these results suggest that targeting Shh signal pathway may overcome chemoresistance and provide a therapeutic strategy for the treatment of malignant gliomas. Data Availability Statement {#s5} =========================== All datasets generated for this study are included in the article/[Supplementary Material](#s9){ref-type="sec"}. Ethics Statement {#s6} ================ The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the College of Medicine, NCKU. Author Contributions {#s7} ==================== J-YC, C-LS, and P-WG contributed to the conception and design of the experiments. H-CH and C-CL performed the experiments and statistical analysis. C-LS and P-WG wrote the paper. All authors contributed to the article and approved the submitted version. Conflict of Interest {#s8} ==================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Dr. Yang-Kao Wang for providing lentiviral package plasmids, Dr. Shih-Chieh Lin for providing GFP/Luciferase plasmid, Dr. Min-Der Lai for correcting the grammar and scientific editing of the manuscript, and the Laboratory Animal Center, College ofMedicine, National Cheng Kung University and Taiwan Animal Consortium for the technical support in IVIS. Funding. This study was supported by grants MOST 104-2320-B-006-007-MY3 and MOST-108-2320-B-006-008 from the Ministry of Sciences and Technology of Taiwan. Supplementary Material {#s9} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fonc.2020.01233/full#supplementary-material> ###### The differential expression of CD133 and cancer stem cell markers in parental GBM cells and tumor spheroids derived from CD133^+^ cells. (A) Immunofluorescence staining of CD133, SOX2, ABCG2, and AlDH1A1 in parental U87 cells. (B) Immunofluorescence staining of ABCG2 and AlDH1A1 in tumor spheroids derived from CD133^+^ cells. ###### Click here for additional data file. ###### The inhibitory effects of shh inhibition on cell proliferation. (A) Concentration-dependent effect of LDE225 on the cell viability of parental U87 cells. ^\^p* \< 0.05, ^\\\^p* \< 0.001 vs. vehicle. (B) Concentration-dependent effect of LDE225 on the cell viability of patient-derived P\#5 cell line. ^\^p* \< 0.05, ^\\\^p* \< 0.001 vs. vehicle. (C) Concentration-dependent effect of cyclopamine on the cell viability of CD133^+^ cells. ^\^p* \< 0.05, ^\\\^p* \< 0.001 vs. vehicle. ###### Click here for additional data file. ###### LDE225-induced cell death is not mediated primarily through apoptosis. CD133^+^ cells were treated with LDE225 (25 μM) or vehicle for 48 h. (A) JC-1 assay detect the changes of mitochondria potential which is able to measure early apoptosis. Vehicle (0.1% DMSO) led to mitochondrial depolarization with the decreased ratio of J-aggregate/JC-1 monomer. There was not different from LDE225 (25 μM) treatment. (B) Caspase/Glo assay revealed the activity of Caspase 3/7. There was not different between LDE225 and vehicle treatment. ###### Click here for additional data file. ###### The conversion of LC3-I to LC3-II was enhanced in Shh shRNA transfection CD133^+^-bearing mice. Tumor tissues were collected from Shh shRNA or vector-control transfection CD133^+^-bearing mice. (A) The efficiency of Shh shRNA-mediated knockdown of Shh was confirmed by western blot analysis. (B--D) The levels of CD133 (B), mushashi-1 (C), and SOX2 (D) were lower in Shh shRNA transfection CD133^+^-bearing mice. (E) The conversion of LC3-I to LC3-II was enhanced by Shh shRNA transfection. ^\^p* \< 0.05 vs. control. ###### Click here for additional data file. ###### The conversion of LC3-I to LC3-II was lower in Shh over-expression GBM-bearing mice. Tumor tissues were collected from LV-Shh or vector-control transfection GBM -bearing mice. (A) The efficiency of LV-Shh-mediated over-expression of Shh was confirmed by western blot analysis. (B--D) The levels of CD133 (B), mushashi-1 (C), and SOX2 (D) were higher in LV-Shh transfection GBM-bearing mice. (E) The conversion of LC3-I to LC3-II was reduced by LV-Shh transfection. ^\^p* \< 0.05, ^\\^p \< 0.01 vs. control. ###### Click here for additional data file. [^1]: Edited by: Liam Chen, Johns Hopkins University, United States [^2]: Reviewed by: Han Shen, Westmead Institute for Medical Research, Australia; Sujatha Venkataraman, University of Colorado Denver, United States [^3]: This article was submitted to Neuro-Oncology and Neurosurgical Oncology, a section of the journal Frontiers in Oncology	{ "pile_set_name": "PubMed Central" }	[ "cord", "amount", "treatment", "roch", "graphpad", "athym", "two", "control", "shrnatreat", "viabil", "ofmedicin", "given", "dosedepend", "starvationinduc", "tumor", "eg", "decreas", "machin", "pwg", "overnight", "caspas", "punctat", "disproportion", "hch", "collect", "sucros", "thank", "umg", "gene", "abcam", "identifi", "basal", "spheroidsfoncgf", "mtorindepend", "autophag", "spectrum", "smoothen", "sort", "block", "grant", "select", "fund", "forebrain", "sheepantimous", "week", "convers", "fix", "lvshhtreat", "lysi", "grammar", "multipl", "pmdg", "signal", "may", "split", "shrnashhtransfect", "theori", 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Introduction ============ Microtubule-targeting agents (MTAs) have been used for a long time against a wide range of malignancies. It is generally believed that MTAs kill cancer cells by causing cell cycle arrest in M-phase, followed by activation of apoptotic pathways and cell death.[@b1-ott-10-5633]--[@b4-ott-10-5633] Many studies have used this rationale to explain the potent radiosensitization effects exerted by MTAs,[@b5-ott-10-5633],[@b6-ott-10-5633] ie, by inducing mitotic arrest, MTAs increase the proportion of tumor cells in a phase of the cell cycle that is very susceptible to DNA damage.[@b7-ott-10-5633],[@b8-ott-10-5633] Currently, chemoradiotherapy regimens including MTAs have been proven effective for the treatment of breast cancer, esophageal cancer and a variety of other neoplasms.[@b9-ott-10-5633],[@b10-ott-10-5633] Support for a mitosis-based mechanism for the therapeutic effects of MTAs has been derived from the characteristic side effects of these drugs, which include hair loss, neutropenia and gastrointestinal upset. These deleterious effects demonstrate the profound sensitivity of rapidly dividing tissues to MTAs. However, the action of MTAs on mitotic cells fails to explain their clinical efficacy against many slow-growing solid tumors with exceptionally low mitotic indices.[@b11-ott-10-5633] Most human tumors have a doubling time of 30--60 days or longer, making it unlikely that mitotic arrest serves as a critical mechanism of MTA-induced therapeutic benefit.[@b12-ott-10-5633] A prime example of this "proliferation rate paradox" is the significant activity of MTAs against adenocarcinoma of the prostate, a highly indolent cancer.[@b13-ott-10-5633],[@b14-ott-10-5633] Thus, a number of interphase-based mechanisms for the efficacy of MTAs in cancer therapy have been proposed, although not without controversy.[@b15-ott-10-5633],[@b16-ott-10-5633] A recent study has shown that the MTAs, vincristine (VCR) and paclitaxel, can delay DNA damage repair.[@b17-ott-10-5633] These MTAs were also shown to interfere with the trafficking of DNA damage response (DDR) proteins, including ATM, ATR and p53, from the cytosol to the nucleus, strongly suggesting that MTAs can sensitize cells to radiation by blocking microtubule-based transport of DDR proteins into the nucleus during interphase. It is challenging to physically separate mitotic from interphase cells in the presence of an MTA, as this results in a steady accumulation of new mitotic cells as long as the MTA is present. Thus, the question remains as to what extent the role of MTAs in radiosensitization is caused by interference with microtubule-facilitated nuclear import. To address this question, we took advantage of the fact that ionizing radiation (IR) treatment induces G2--M cell cycle arrest, thereby transiently eliminating the mitotic cell population and strongly enriching for interphase cells. Using glioblastoma cells and the MTA, mebendazole (MBZ), as a model system, we show that the effect of MBZ in interphase is responsible for the majority of the radiosensitization effect of this MTA. Materials and methods ===================== Cell lines and reagents ----------------------- GL261 (glioma) cells were obtained from the National Cancer Institute (Frederick, MD, USA). Cells were cultured in macrophage serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 1% penicillin/streptomycin and 1 mM L-glutamine. GBM14 cells have been described previously,[@b18-ott-10-5633] and they were obtained by Dr J Sarkaria at Mayo Clinic (Rochester, MN, USA) and cultured in StemPro media (Thermo Fisher Scientific), as directed by the manufacturer. Drug treatment -------------- For each experiment, GL261 and GBM14 cells were cultured without drug or treated with MBZ (Sigma-Aldrich Co., St Louis, MO, USA). MBZ stocks were prepared by dissolving the drug in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co.). About 24 hours after plating, glioma cells were treated with MBZ, while control cells were treated with 0.01% DMSO. Irradiation procedure --------------------- For all experiments requiring radiation, cells were irradiated using a biological irradiator (RS2000; Rad Source Technologies, Buford, GA, USA). Antibodies ---------- Phospho-MPM2 was obtained from EMD Millipore (Billerica, MA, USA), and γH2AX, H2AX, Chk2, Nbs1 and GAPDH were purchased from Cell Signaling (Danvers, MA, USA). Immunofluorescence ------------------ GL261 cells were seeded at a density of 30,000 cells/well in 24-well plates containing cover slips coated with laminin (Sigma-Aldrich Co.). The following day, cells were exposed to 6 Gy of IR. At designated time points post IR, cells were washed in PBS and fixed in 4% formaldehyde in PBS at room temperature. Once the cells were fixed, immunofluorescence was performed using an MPM2 antibody (EMD Millipore) and counterstained with 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI). For each condition, a total of 10 fluorescence micrograph images were taken with a Zeiss Axiovert 200M inverted microscope (Thornwood, NY, USA), running on Axiovision software. For each image field, the total number of cells and the number of MPM2-positive cells were quantified. The mitotic index was calculated at each time point using the number of MPM2-positive cells as a percentage of the total number of cells counted in all 10 fields. Cell viability assays --------------------- GL261 cells were seeded in 96-well plates at a density of 1,000 cells/well. GBM14 cells were seeded at a density of 10,000 cells/well. About 24 hours after seeding, cells were treated with 25--150 nM MBZ and irradiated with 3, 6 or 9 Gy. Drug treatment was applied for a 6-hour time window beginning either 6 hours pre IR or 3 hours post IR. Following the 6-hour window, the drug was washed out and replaced with fresh medium. Control cells were either left nonirradiated or irradiated with 3, 6 or 9 Gy of IR. Cell viability was examined 72 hours post IR using a WST-1 cell viability assay (Abcam, Cambridge, MA, USA) at an absorbance of 450 nm. For any given radiation dose--response, data were normalized by the fraction of viable cells treated with a given dose of drug in the absence of radiation. The radiosensitizing effect of MBZ and VCR was quantified using the dose enhancement factor (DEF) at the point of 50% (DEF~50~) cell viability. The DEF was calculated for each MBZ concentration using the following formula: (surviving fraction with radiation alone)/(surviving fraction with radiation + MBZ). Assessment of the DDR --------------------- GL261 cells were seeded at a density of 800,000 cells/well in 6-cm dishes. About 24 hours after seeding, cells were irradiated with 6 Gy of IR, followed by treatment with 150 nM MBZ during 3--9 hours post IR. Control cells were either left nonirradiated or irradiated with 6 Gy. All cells were harvested in lysis buffer at 0.5, 3, 6 and 9 hours post IR. The lysis buffer was composed of 50 mM Tris--HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% Nonidet P-40, 1× protease inhibitor (Thermo Fisher Scientific), phosphatase inhibitor (Hoffman-La Roche Ltd., Basel, Switzerland) and 0.4 U/mL Benzonase (EMD Millipore). For each condition, 60 μg of cell lysate was diluted in 1× NuPAGE Sample Reducing Agent (Thermo Fisher Scientific) and 1× NuPAGE LDS Sample Buffer (Thermo Fisher Scientific). All samples were loaded onto a NuPAGE, 4%--12% Bis-Tris Protein Gel (Thermo Fisher Scientific) for Western blot analysis. Histone H2AX and γH2AX were detected by Western blot using anti-histone H2AX or anti-γ-H2AX (Ser139) monoclonal antibodies (Cell Signaling). Nuclear and cytoplasmic fractionation ------------------------------------- GL261 cells were seeded at a density of 500,000 cells in 6-cm dishes. The following day, cells were treated with 3 Gy. MBZ treatment (25--250 nM) was performed for a 6-hour time window beginning 3 hours post IR. Irradiated control cells were treated with 3 Gy followed by 0.01% DMSO in medium during the same time period. Nonirradiated control cells were treated with 0.01% DMSO in medium for a total of 6 hours. After the 6-hour period of MBZ treatment, cells were harvested for protein analysis in cytoplasmic (C) and nuclear (N) fractions. The C and N fractions were collected according to the protocol of the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific). For each sample, 20% of the final volume of the C or N fractions was diluted in 1× NuPAGE Sample Reducing Agent and 1× NuPAGE LDS Sample Buffer. All samples were loaded onto a NuPAGE, 4%--12% Bis-Tris Protein Gel for Western blot analysis. Blots were incubated in antibodies: Nbs1, Chk2, GAPDH and H2AX monoclonal antibodies (all from Cell signaling). Cytoplasmic and nuclear fractions were normalized by GAPDH and H2AX levels, respectively. The percentage of cytoplasmic retention of DDR proteins was calculated by the following formula: \[C/(C + N)\] × 100%. Statistical analysis -------------------- All analyses were performed using Graphpad Prism 7 software. Radiosensitization experiments were analyzed using the two-way ANOVA method to compare the mean cell viability between treatment groups. The interaction between the MBZ treatment group and radiation dose was also examined. One-way ANOVA was used to compare the concentration of a drug that gives half-maximal response (EC~50~) values for each effect as outlined in [Table 1](#t1-ott-10-5633){ref-type="table"}. If a significant difference between means was found by ANOVA, then multiple comparisons between treatment groups were conducted. The Tukey--Kramer method was used to adjust for multiple comparisons. Studies examining the effect of MBZ on intracellular transport of DDR proteins were analyzed with unpaired two-tailed Student's t-test for direct comparisons of means. The same method was used to compare means in the analysis of H2AX phosphorylation following exposure to IR. For all studies, results were considered statistically significant for values of P\<0.05. Results ======= IR transiently eliminates the mitotic cell population ----------------------------------------------------- To select for a population composed entirely of interphase cells, we took advantage of the fact that IR treatment induces G2--M cell cycle arrest and transiently eliminates the mitotic cell population. Glioma cells were treated with 6 Gy of IR. To monitor the mitotic index, we quantified the proportion of MPM2-positive cells by immunofluorescence at several time points post IR. MPM2 is an antibody that recognizes a phosphorylated serine/threonine epitope found in proteins that are phosphorylated at the onset of mitosis.[@b19-ott-10-5633] MPM2 is recognized as a reliable mitotic marker in the literature and has been used to assess the mitotic index in cells exposed to radiation and a number of chemotherapeutic agents.[@b20-ott-10-5633]--[@b22-ott-10-5633] The baseline mitotic index of the glioma cell population was 3.5% and was strongly inhibited during a period of 3--10 hours after exposure to 6 Gy of IR. Recovery of the mitotic cell population begins after 10 hours post IR and completes by 24 hours post IR. To confirm that the mitotic index remained strongly inhibited even in the presence of MBZ, we applied MBZ during 3--9 hours post IR. In the presence of MBZ, the mitotic index remained suppressed at 4 hours (mitotic index =0.58%), 6 hours (mitotic index =0.12%) and 9 hours (mitotic index =0.28%) post IR. Thus, the treatment of GL261 glioma cells with 6 Gy of IR eliminated the mitotic cell population for a time period of \~6 hours ([Figure 1](#f1-ott-10-5633){ref-type="fig"}). MTAs sensitize interphase cells to IR ------------------------------------- In the following experiments, we compared the radiosensitizing effect of MTAs when applied during different time periods with respect to IR, using both murine GL261 cells and primary patient-derived GBM14 cells. A simplified schematic of all three treatment conditions is shown in [Figure 2](#f2-ott-10-5633){ref-type="fig"}. To examine whether MTAs can sensitize glioma cells to IR, we treated GL261 cells with MBZ ([Figures 3](#f3-ott-10-5633){ref-type="fig"} and [4](#f4-ott-10-5633){ref-type="fig"}) or VCR ([Figure 5](#f5-ott-10-5633){ref-type="fig"}). In order to test, whether MTAs can sensitize interphase cells to IR, we treated glioma cells with 25--150 nM MBZ during 3--9 hours post IR ([Figures 3A](#f3-ott-10-5633){ref-type="fig"} and [4A](#f4-ott-10-5633){ref-type="fig"}). The use of MBZ after exposure to IR made it possible to study the impact of MBZ independent of its effect on mitotic cells. Cells were exposed to MBZ after different doses of IR. After this 6-hour time frame, the drug was washed out and replaced with a drug-free medium. The radiosensitizing effect of MBZ on interphase cells was compared with the effect of MBZ on a cell population composed of both interphase and mitotic cells. Thus, cells were treated with MBZ for a 6-hour time window immediately prior to irradiation, and subsequently the drug was either washed out and replaced with drug-free medium ([Figures 3B](#f3-ott-10-5633){ref-type="fig"} and [4B](#f4-ott-10-5633){ref-type="fig"}) or left in the original medium until the end of the assay, 72 hours post IR ([Figures 3C](#f3-ott-10-5633){ref-type="fig"} and [4C](#f4-ott-10-5633){ref-type="fig"}). Treatment with MBZ post IR sensitized GL261 cells to IR with a maximal DEF at 50% viability (DEF~50~) of 1.34 ([Figure 3D](#f3-ott-10-5633){ref-type="fig"}). Treatment for a 6-hour time window pre IR, which increased the mitotic index to 9.2% from a baseline value of 2.8%,[@b23-ott-10-5633] sensitized glioma cells to IR with a maximal DEF~50~ of 1.2 in GL261 cells ([Figure 3E](#f3-ott-10-5633){ref-type="fig"}) and 1.33 in GBM14 cells ([Figure 4E](#f4-ott-10-5633){ref-type="fig"}). In the GL261 cell line, maximal radiosensitization was observed when the drug was applied pre IR and left in the medium for 72 hours, DEF~50~ =1.41 ([Figure 3F](#f3-ott-10-5633){ref-type="fig"}). In the GBM14 cell line, the application of the drug pre IR followed by 72-hour incubation sensitized cells to IR with a DEF~50~ =1.60 ([Figure 4F](#f4-ott-10-5633){ref-type="fig"}). Maximal radiosensitization in the GBM14 cell line was achieved when MBZ was applied post IR, DEF~50~ =1.69 ([Figure 4D](#f4-ott-10-5633){ref-type="fig"}). These observations show a maximal or near-maximal radiosensitization effect of MBZ when applied to cells during interphase. To investigate whether these findings can be generalized to other MTAs, we performed an identical set of experiments in GL261 cells using VCR, an established MTA that is frequently used in clinical practice. The radiosensitizing effect of VCR was greatest when applied post IR, DEF~50~ =1.53 ([Figure 5A and D](#f5-ott-10-5633){ref-type="fig"}). The application of VCR for a 6-hour period pre IR radiosensitized GL261 cells with a DEF~50~ of 1.34 ([Figure 5B and E](#f5-ott-10-5633){ref-type="fig"}). When VCR was applied pre IR and left in the medium for the remaining 72 hours, the magnitude of the radiosensitizing effect was quite similar, DEF~50~ =1.30 ([Figure 5C and F](#f5-ott-10-5633){ref-type="fig"}). Thus, it appears that the radiosensitizing effect of both MBZ and VCR is largely determined by the impact of these agents on interphase cells. MBZ treatment prolongs DDR after irradiation -------------------------------------------- To confirm that MTAs sensitize interphase cells to IR by interfering with the DDR, we examined the level of IR-induced phosphorylated H2AX (γH2AX), which is a highly sensitive marker of DNA damage.[@b24-ott-10-5633],[@b25-ott-10-5633] We exposed glioma cells to 6 Gy of IR, followed by treatment with 150 nM MBZ during 3--9 hours post IR. Cells were harvested at different time points post IR, and γH2AX was quantified by Western blot analysis. As shown in [Figure 6](#f6-ott-10-5633){ref-type="fig"}, treatment with MBZ led to more sustained γH2AX levels in response to IR, indicating a delay in the DDR. This finding demonstrated that MTAs sensitize interphase cells to IR by interfering with the DDR. MBZ interferes with the trafficking of DDR proteins --------------------------------------------------- To investigate whether MTAs synergize with IR by disrupting intracellular transport of DDR proteins, we performed cytoplasmic and nuclear (C/N) fractionation studies with glioma cells that had been exposed to IR followed by treatment with MBZ. Similar to the cell viability studies, MBZ was applied during 3--9 hours post IR (only when the mitotic cell population was absent). Glioma cells were treated with different concentrations of MBZ followed by C/N fractionation. Each fraction was analyzed by Western blot. We selected two DDR proteins, Chk2 and Nbs1, to serve as potential targets of MTA-mediated toxicity. Chk2 is a key protein kinase involved in the DDR that is responsible for cell cycle checkpoint activation and DNA repair following DNA damage induced by IR.[@b26-ott-10-5633] Nbs1 is a crucial component of an enzymatic complex that repairs double strand breaks (DSBs) following irradiation or heat shock.[@b27-ott-10-5633] In control cells, which had not been treated with MBZ, Chk2 and Nbs1 were localized entirely in the nucleus ([Figure 7A](#f7-ott-10-5633){ref-type="fig"}). Radiation treatment did not significantly alter the intracellular distribution of DDR proteins ([Figure 7A](#f7-ott-10-5633){ref-type="fig"}). Treatment of glioma cells with MBZ sequestered Chk2 and Nbs1 in the cytoplasm in a dose-dependent manner ([Figure 7B and C](#f7-ott-10-5633){ref-type="fig"}). These results demonstrate that MBZ, like other MTAs,[@b17-ott-10-5633] inhibits the trafficking of DDR proteins from the cytoplasm to the nucleus. MBZ sensitizes glioma cells to IR by interfering with the trafficking of DDR proteins ------------------------------------------------------------------------------------- To more closely examine the relationship between the effects of MBZ on intracellular trafficking of DDR proteins and IR sensitization, we compared the EC~50~ values for radiosensitization (DEF), cytoplasmic sequestration of DDR proteins and induction of mitotic arrest by MBZ ([Table 1](#t1-ott-10-5633){ref-type="table"}) in GL261 cells. The EC~50~ was defined as the concentration of MBZ required to achieve a half-maximal effect. The EC~50~ of cytoplasmic sequestration for Chk2 (31 nM) and Nbs1 (25 nM) was significantly lower than the EC~50~ of mitotic arrest (192 nM). Most notably, the EC~50~ of radiosensitization (35 nM) was very similar to the EC~50~ for cytoplasmic sequestration of DDR proteins, but significantly lower than the EC~50~ of mitotic arrest (P\<0.01). Similarly, the EC~50~ for radiosensitization by VCR (\<0.5 nM) is significantly lower than the EC~50~ for mitotic arrest in GL261 cells (2.5 nM).[@b23-ott-10-5633] These findings support the hypothesis that MBZ indeed radiosensitizes GL261 cells in large part by interfering with intracellular trafficking of DDR proteins during interphase. Discussion ========== Studying the mechanisms of action of MTAs in cell toxicity, and in particular whether MTAs target cells in mitotic phase or interphase, has been hampered by the difficulty in separating these two cell populations. In this study, we transiently eliminated the mitotic cell population by irradiating glioblastoma cells, which allowed us to examine the extent to which the MTA, MBZ, can radiosensitize these cells in a cell population that is essentially made up of interphase cells only. Our results show that in this system, the effect of MBZ in interphase is responsible for the majority of the radiosensitization effect caused by this MTA. For a long time, MTAs have been believed to inhibit tumor growth primarily by targeting mitotic cells, but this hypothesis has come under considerable scrutiny.[@b11-ott-10-5633],[@b12-ott-10-5633],[@b15-ott-10-5633],[@b16-ott-10-5633] We examined the role of interphase microtubules by determining the effect of MBZ as a radiosensitizer, when present during a time window in which mitosis is prevented by G2--M cell cycle arrest. We showed that this regimen is better at radiosensitization of tumor cells than when MBZ is present before IR administration, leading to an increase in the number of mitotic cells, and is very similar to that caused by chronic treatment with MBZ, strongly indicating that interphase microtubules are indeed the targets of MBZ. In addition, the EC~50~ of MBZ for radiosensitization is much lower than that for inducing mitotic arrest, further supporting the notion that the radiosensitizing effect of MBZ is independent of its effect on mitosis. We found essentially the same results for VCR. Thus, most likely this conclusion holds for a wide range of MTAs. We also observed that MBZ, even when applied for only 6 hours, leads to a strong delay in the DDR. Thus, our results strongly support the hypothesis that MTAs prolong DNA damage repair by interfering with the trafficking of DDR proteins from the cytosol to the nucleus.[@b17-ott-10-5633] Interestingly, the EC~50~ for radiosensitization by MBZ (35 nM) is very similar to that of cytoplasmic sequestration of DDR proteins by MBZ (25 nM), which is much lower than the EC~50~ of MBZ for the induction of mitotic arrest (184 nM), further supporting the notion that MTAs radiosensitize by blocking trafficking of DDR proteins to the nucleus. The low EC~50~ of MBZ for the inhibition of DDR protein trafficking is surprising, because it is also much lower than the EC~50~ for microtubule depolymerization (132 nM) that we determined recently.[@b23-ott-10-5633] This EC~50~ largely reflects that of the depolymerization of interphase microtubules, as most of the cells are in interphase. Although the mechanistic basis for the relatively low EC~50~ of MBZ for the inhibition of DDR protein trafficking remains to be determined, it will be of great interest to examine whether our findings with MBZ extend to other MTAs. Our observation that radiosensitization can be accomplished at a concentration of MBZ that is significantly lower than the concentration needed for cell killing on its own also has important clinical implications, as it suggests the possibility to utilize a dose of MBZ that minimizes toxicity. Importantly, our results also have implications for the optimal timing of administration of MTAs when used as radiosensitizers. Indeed, we show that optimal radiosensitization is obtained when inhibition of microtubule formation is achieved during the post-IR DNA repair period. This study also underlines the critical role of microtubule-based transport in the response to DNA damage. Thus, further elucidation of these mechanisms may lead to the identification of novel therapeutic targets for radiosensitization. Conclusion ========== We have shown that MBZ sensitizes cancer cells to IR in a manner that is largely independent of the induction of mitotic arrest by this microtubule inhibitor. We also provide evidence that MBZ-induced radiosensitization is mediated by inhibiting DDR protein accumulation into the nucleus. Thus, this study strongly supports a critical role for DDR protein trafficking in the response to radiation, suggesting that elements of the protein trafficking machinery can be mined for additional radiosensitization targets. GBM14 cells were kindly provided by Dr J Sarkaria, Mayo Clinic, Rochester, MN, USA. All data generated and analyzed during this study are included in this publication. This study was supported by grants to MS from the Swim Across America Foundation and the Project To Cure Foundation and to RR from the Zankel Foundation. Author contributions All authors contributed toward data analysis, drafting and critically revising the paper and agree to be accountable for all aspects of the work. Disclosure The authors report no conflicts of interest in this work. ![IR temporarily eliminates the mitotic cell population.\ Notes: GL261 cells were exposed to 6 Gy of IR, and the proportion of MPM2-positive cells was quantified by immunofluorescence as described in the "Immunofluorescence" section. MPM2 was utilized as a marker of the mitotic index. Data are expressed as the average ± SEM of three independent experiments.\ Abbreviations: IR, ionizing radiation; SEM, standard error of the mean.](ott-10-5633Fig1){#f1-ott-10-5633} ![Timing of MBZ application with respect to IR.\ Notes: (A) MBZ was applied for a 6-hour time frame beginning 6 hours pre IR followed by washout of the drug. During this time period, both mitotic and interphase cells were present. (B) MBZ was applied for a 6-hour time frame beginning 3 hours post IR followed by washout of the drug. During this time period, the mitotic cell population was absent (composed entirely of interphase cells). (C) MBZ was applied beginning 6 hour pre IR and left in the medium for the duration of the experiment.\ Abbreviations: IR, ionizing radiation; MBZ, mebendazole.](ott-10-5633Fig2){#f2-ott-10-5633} ![MBZ sensitizes GL261 cells to IR.\ Notes: GL261 cells were exposed to 25--150 nM of MBZ in conjunction with 3--9 Gy of IR. MBZ treatment was applied at different time points with respect to IR, and cell viability was determined by the WST assay as described in the "Cell viability assays" section. All data are expressed as the average ± SEM of four independent experiments. For each radiation dose--response curve, data were normalized by the fraction of viable cells treated with a given dose of MBZ in the absence of IR. DEFs were determined at the point of 50% cell viability. (A) Cells were treated with MBZ during 3--9 hours post IR. (B) Cells were treated with MBZ for a 6-hour time window immediately prior to irradiation. (C) Cells were treated with MBZ starting 6 hours prior to irradiation and until 72 hours post IR. P-values were \<0.05 for all treatments points when doses of MBZ ≥50 nM. (D--F) The respective DEFs were calculated as described in the "Cell viability assays" section. \*P\<0.05, \\P\<0.01, \\\*P\<0.001.\ Abbreviations: DEF, dose enhancement factor; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean.](ott-10-5633Fig3){#f3-ott-10-5633} ![MBZ sensitizes GBM14 cells to IR.\ Notes: GBM14 cells were exposed to 50 or 150 nM of MBZ in conjunction with 3--9 Gy of IR. MBZ treatment was applied at different time points with respect to IR, and cell viability was determined by the WST assay as described in the "Cell viability assays" section. All data are expressed as the average ± SEM of three independent experiments. For each radiation dose--response curve, data were normalized by the fraction of viable cells treated with a given dose of MBZ in the absence of IR. DEFs were determined at the point of 50% cell viability. (A) Cells were treated with MBZ during 3--9 hours post IR. (B) Cells were treated with MBZ for a 6-hour time window immediately prior to irradiation. (C) Cells were treated with MBZ starting 6 hours prior to irradiation until 72 hours post IR. (D--F) The respective DEFs were calculated as described in the "Cell viability assays" section. \\P\<0.01, \\\*P\<0.001.\ Abbreviations: DEF, dose enhancement factor; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean.](ott-10-5633Fig4){#f4-ott-10-5633} ![VCR sensitizes GL261 cells to IR.\ Notes: GL261 cells were exposed to 0.5--2 nM of VCR in conjunction with 3--9 Gy of IR. Experiments with VCR were performed in an identical fashion to those with MBZ. All data are expressed as the average ± SEM of three independent experiments. For each radiation dose--response curve, data were normalized by the fraction of viable cells treated with a given dose of VCR in the absence of IR. DEFs were determined at the point of 50% cell viability. (A) Cells were treated with VCR during 3--9 hours post IR. (B) Cells were treated with VCR for a 6-hour time window immediately prior to irradiation. (C) Cells were treated with VCR starting 6 hours prior to irradiation until 72 hours post IR. (D--F) The respective DEFs were calculated as described in the "Cell viability assays" section. \*P\<0.05, \\P\<0.01, \\\*P\<0.001.\ Abbreviations: DEF, dose enhancement factor; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean; VCR, vincristine.](ott-10-5633Fig5){#f5-ott-10-5633} ![MBZ prolongs the DDR after exposure to IR.\ Notes: (A) Western blot analysis. (B) Quantification of the Western blot bands. GL261 cells were treated with 6 Gy of IR followed by treatment with 150 nM of MBZ during 3--9 hours post IR. Control cells were treated only with IR. Following treatment, cell lysates were harvested at different time points (0.5--9 hours) post IR as described in the "Assessment of the DNA-damage response" section. Western blot analysis of γH2AX and H2AX levels was conducted at each time point post IR, and γH2AX levels were used as a measure of DNA damage. Data for each time point are expressed as the average ± SEM of three independent experiments.\ Abbreviations: DDR, DNA damage response; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean.](ott-10-5633Fig6){#f6-ott-10-5633} ![MBZ interferes with the trafficking of DDR proteins from the cytoplasm to the nucleus.\ Notes: (A) GL261 cell were exposed to IR for 9 hours or left untreated. Cell lysates were collected using cytoplasmic and nuclear fractionation as described in the "Nuclear and cytoplasmic fractionation" section. In untreated GL261 cells, DDR proteins are localized to the nucleus. Exposure to IR did not alter the intracellular distribution of DDR proteins. (B) GL261 cells were exposed to IR followed by application of 25--250 nM of MBZ during 3--9 hours post IR. Western blot analysis of the cytoplasmic and nuclear fractions was conducted, and the levels of DDR proteins, Chk2 and Nbs1, were quantified for each fraction. Exposure to MBZ post IR resulted in the dose-dependent sequestration of DDR proteins in the cytoplasm. (C) The histogram shows quantification of Chk2 and Nbs1 levels from three independent experiments ± SE. \*P\<0.001.\ Abbreviations: C, cytoplasmic; DDR, DNA damage response; IR, ionizing radiation; MBZ, mebendazole; N, nuclear; SE, standard error.](ott-10-5633Fig7){#f7-ott-10-5633} ###### DEF~50~ for MBZ-mediated radiosensitization, cytoplasmic sequestration of DDR proteins and induction of mitotic arrest Treatment EC~50~(nM) 95% CI ------------------------------ ------------------------------------------------- ---------- Radiosensitization (DEF~50~) 35 9--50 Nuclear trafficking (Chk2) 31 17--45 Nuclear trafficking (Nbs1) 25 18--32 Mitotic arrest 192[\](#tfn2-ott-10-5633){ref-type="table-fn"} 127--257 Notes:* Each EC~50~ value represents the average of three independent experiments with the representative 95% CI. DEFs were determined from WST assays conducted in GL261 cells with MBZ treatment during the period of 3--9 hours post IR. The EC~50~ of radiosensitization is defined by the half-maximal DEF at 50% cell viability (DEF~50~). Data used to determine the EC~50~ for induction of mitotic arrest by MBZ were obtained previously, also using GL261 cells.[@b23-ott-10-5633] P\<0.01. Abbreviations: CI, confidence interval; DDR, DNA damage response; DEF, dose enhancement factor; EC~50~, concentration of a drug that gives half-maximal response; IR, ionizing radiation; MBZ, mebendazole.	{ "pile_set_name": "PubMed Central" }	[ "atm", "treatment", "roch", "mitosisbott", "graphpad", "two", "sulfoxid", "control", "mtamedi", "viabil", "given", "dosedepend", "origin", "work", "remain", "induct", "tumor", "administr", "ie", "postir", "collect", "abcam", "mayo", "block", "tottreftypet", "grant", "select", "dnadamag", "mechan", "phosphompm", "student", "nmbott", "mbzmediat", "valu", "hour", "fix", "maxim", "lysi", "neper", "multipl", "signal", "may", "therebi", "vincristineottfigfott", "recent", "pb", "dish", "toward", "separ", "use", "gener", "mine", "ms", "support", "room", "final", "caus", "imag", "ny", "accord", "complet", "prior", "rad", "signific", "buford", "establish", "cover", "temperatur", "nb", "exposur", "slip", "suggest", "paradox", "bovin", "hair", "nucleu", 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INTRODUCTION {#sec1-1} ============ Vitamin D is a fat-soluble secosteroid required for intestinal absorption of calcium, magnesium, and phosphate. It has direct as well as indirect effects on bone. It is also essential for optimal functioning of various organs and tissues. It exerts its effect by binding to Vitamin D receptors (VDRs) on target cells. Vitamin D is derived from food and ultraviolet B photons. It undergoes hydroxylation in liver to form 25(OH)D3 and further in kidneys by enzyme 1α hydroxylase (1αOHase) to form 1, 25,(OH)2D3 (calcitriol), an active form. This active form is also produced at extrarenal sites. Enzyme 1αOHase (CYP27B1) required for this conversion is expressed by several target cells. In cases of infection, the pathogen-associated molecular patterns such as lipopolysaccharides, interferon gamma, and nitric oxide get attracted to toll-like receptors of macrophages. This regulates cytochrome P27B1 (CYP27B1) genes of the target cells. The expressed enzyme locally hydroxylates the inactive form (25\[OH\] D3) resulting in the production of calcitriol (1,25\[OH\] 2D3).\[[@ref1]\] This calcitriol exerts its effect on target cells which express VDR. Calcitriol enters the target cell through caveolae, containing VDR or by endocytic uptake of 25(OH)D3-D-binding protein, with the help of glycoproteins such as cubilin and megalin.\[[@ref2]\] After entering the cell, 1,25(OH)2D3 ligates with VDR in nucleus and generates vital biological responses. The 1,25(OH)2D3-VDR complex heterodimerizes with retinoid X receptor and shows DNA binding at Vitamin D response element, leading to RNA transcription and biological actions which include modulation of inflammatory response via promoting monocyte differentiation to macrophages; preventing release of inflammatory cytokines; preventing cell surface expression of major histocompatibility complex-II;\[[@ref3]\] downregulating T-cells and monocytes; downregulating the expression and production of several pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-8; and decreasing the nuclear factor-κB activity which is a transcription factor having a key role in immunomodulation.\[[@ref4]\] Vitamin D also regulates the expression of specific endogenous antimicrobial peptides.\[[@ref5]\] Since the discovery of VDR, three decades ago, more than fifty target cells have been identified, involving the receptor-mediated Vitamin D functions.\[[@ref6]\] To the best of our knowledge, very few studies have mentioned the presence of VDR in periodontal ligament (PDL) tissue. Investigations by Andrukhov et al.\[[@ref7]\] Tang and Meng,\[[@ref8]\] McMahon et al.,\[[@ref9]\] and Liu et al.\[[@ref10]\] have shown VDR-mediated activities in PDL tissue cells. These studies were however investigated on cell cultures and according to Bhalla et al.\[[@ref11]\] VDRs actually may not be present in tissues but may be acquired in cultures. They also stated that receptors may appear in vivo when cells are exposed to vascular or inflammatory cell products. Study by Onishi et al. in 2003\[[@ref12]\] and Chen in 2012\[[@ref13]\] elucidated immunolocalization of VDR during and at completion of root formation. VDR was detected in periodontium. However, these investigations do not confirm the presence of VDR in human PDL (hPDL) tissue of completely erupted teeth in adults. The present study was intended to evaluate the presence of VDRs in PDL tissue and study its response to serum 25(OH)D3 levels. Detection of VDR in hPDL tissue would implicate the involvement of Vitamin D-mediated response in periodontal tissue. MATERIALS AND METHODS {#sec1-2} ===================== Study design {#sec2-1} ------------ A total of 19 chronic periodontitis patients of both sexes with age ranging from 35 to 65 years, visiting the outpatient department of periodontology (from February 2015 to March 2017), were recruited for the present study. Their medical history was taken followed by a detailed periodontal examination. Individuals with a history of any systemic disease, medications (such as bisphosphonates, barbiturates, cardiac glycosides, cholestyramine, glucocorticoids, and laxatives), hypersensitivity reactions, Vitamin D or calcium supplements, smokers, pregnant women, and those with a history of antibiotic therapy within the last 3 months were excluded from the study. Periodontal examination {#sec2-2} ----------------------- The selected patients were examined clinically. Participants diagnosed with moderate-to-severe chronic periodontitis requiring extractions of at least two teeth (e.g., teeth with compound or complex pockets having 70%--75% bone loss on the involved surfaces and 50%--60% bone loss on the remaining surfaces; teeth with Grade III furcation involvement with severe vertical bone loss in interradicular area with 50%--60% bone loss on mesial and distal surfaces; teeth with severe recession on one side and 50%--60% bone loss on other sides along with mobility; patients with partially edentulous arches with 50%--60% bone loss on few remaining teeth and indicated for extraction for prosthodontic purposes) were considered for the study. PDL tissue was scraped from the remaining surfaces for evaluation of VDRs in the study. The study was approved by the Institutional Ethics Committee (reference number: Perio-II-1/2015--2016). Detailed informed written consent was obtained from all the selected participants, indicating voluntary participation in the study. Venous blood sample of approximately 2--3 ml was withdrawn in a vacutainer (with clot activator and gel for serum separation) from all the 19 chronic periodontitis patients for assessment of serum 25(OH)D3 levels. ADVIA Centaur Vitamin D Total (Vitamin D) assay (antibody competitive immunoassay) was used for the determination of total 25(OH)D3 in human serum. The reference range\[[@ref14]\] used for the study was as follows: deficiency (\<20 ng/mL), insufficiency (20--30 ng/mL), sufficiency (30--100 ng/mL), and toxicity (above 100 ng/mL). The sensitivity range of ADVIA Centaur Vitamin D assay is 4.2--150 ng/mL. Periodontal Phase I therapy (nonsupportive periodontal therapy) and extraction of tooth were carried out. The participants were then referred to physician for supplementation of Vitamin D3. The dose of cholecalciferol (Torrent Pharmaceuticals Ltd, Ahmedabad, India) was 60,000 IU once a week for 6 weeks. After supplementation, the participants were tested for serum 25(OH)D3 levels. This was followed by extraction of the second tooth. PDL tissue of the extracted tooth was used for the evaluation of VDR concentration. First, the extracted tooth was collected in a vial containing solution of 0.9%W/V sodium chloride, 50 U/mL of penicillin, and 50 μg/mL of streptomycin. The tooth was then washed with the same solution to remove blood completely. PDL tissue was scraped from the root surfaces with sterile curettes and placed in Eppendorf tube containing 1 ml of phosphate-buffered saline. The tubes were centrifuged at 4°C at 5000 gravity for 10 min. The supernatant was removed. Pellet was resuspended in 0.3ml of phosphate buffered saline. The tube was then subjected to three freeze-thaw cycles to facilitate cell lysis. Enzyme-linked immunosorbent assay procedure {#sec2-3} ------------------------------------------- PDL tissue samples were analyzed by using Human VDR ELISA Kit (Elabscience Catalog no E-EL-H2043) for in vitro quantitative determination of human VDR concentrations. The minimum detectable dose of human VDR by this kit was 0.38 ng/ml. The detection range was 0.63--40 ng/ml. The enzyme-linked immunosorbent assay (ELISA) procedure was performed as per the manufacturer\'s instructions. After preparing standard stock solution of 40 ng/ml and its serial dilutions, optical density (OD) values were obtained. Standard concentrations and OD values were plotted to get a standard curve. Actual ELISA for samples was then assayed using stock solution of 40 ng/ml as control. 100 μl of PDL tissue samples was added in respective wells of ELISA microplate in duplicate. They were incubated for 90 min at 37°C. 100 μl of diluted biotinylated detection antibody was added to all the wells. After incubation for 60 min at 37°C, the wells were washed with wash buffer for 3 times. 100 μl of horseradish peroxide conjugate was added and incubated for 30 min at 37°C. The wash process was repeated 5 times followed by addition of 90 μl of substrate reagent and incubation for 15 min at 37°C. 50 μl of stop solution was then added to it. ELISA plate was read on microplate reader at 450 nm. The OD readings of the samples were plotted on standard graph to get its VDR concentration. Statistical analysis {#sec2-4} -------------------- Data were statistically analyzed using SPSS version 16.0, IBM Corporation, Armonk, NY, USA. The intragroup comparison was done by paired t- test. To study the predictive reliability (goodness of fit) of the regression models, percentage R^2^ as a coefficient of determination was used. Correlation analysis was done using Pearson\'s method (Pearson\'s r value). P \<0.05 indicated statistically significant results (P \< 0.05, \\P \< 0.001, \\\*P \< 0.001), and NS indicated statistically nonsignificant results. Power of the study was 80%. RESULTS {#sec1-3} ======= A total of 19 chronic periodontitis patients were evaluated for 25(OH)D3 levels in serum and VDR concentration in human PDL tissue in the present study. The mean serum level before supplementation was 13.96 ng/mL with standard deviation of 3.36. The participants then underwent Vitamin D3 supplementation as prescribed by the physician. The mean serum 25(OH)D3 levels 6 weeks after supplementation of 60,000 IU Vitamin D3 increased to 35.12 ng/mL with a standard deviation of 5.27. The results are presented in [Table 1](#T1){ref-type="table"} and [Figure 1](#F1){ref-type="fig"} which depict significant increment in serum 25(OH)D3 levels after supplementation of Vitamin D3 (P \< 0.001). ###### Comparison of mean serum 25(OH)D3 levels before and after supplementation of Vitamin D3 Before supplementation (n=19) After supplementation (n=19) P ------------------------ --------------------------------- -------------------------------- ------- ------ ------------- Serum 25(OH)D3 (ng/ml) 13.96 3.36 35.12 5.27 0.001\\\* \Is a standard notation used to determine the level of significance.P* value by independent sample "t-" test. P\<0.05 is considered to be statistically significant. \*P\<0.05, \\P\<0.01, \\\*P\<0.001. SD -- Standard deviation; n -- Sample size; NS -- Statistically nonsignificant ![Comparison of mean serum 25(OH)D3 levels before and after supplementation](JISP-23-100-g001){#F1} The present study also involved the investigation of VDR in PDL tissue isolated from 19 participants of the above study group at both time intervals. VDR analysis was done by using Human Vitamin D Receptor ELISA kit. The mean VDR concentration in PDL tissue was calculated from OD values obtained from ELISA readings. The mean VDR concentration in periodontal ligament tissue of Vitamin D deficient, chronic periodontitis subjects was -1.443/ml with standard deviation of 0.604. The values increased to 2.38 /ml with standard deviation of 2.64 after supplementation, indicating highly significant increment (P-value \< 0.001) \[[Table 2](#T2){ref-type="table"} and [Figure 2](#F2){ref-type="fig"}\]. ###### Comparison of mean Vitamin D receptor concentrations before and after supplementation of Vitamin D3 Before supplementation (n=19) After supplementation (n=19) P --------------------------- --------------------------------- -------------------------------- ------ ------ ------------- VDR concentration (ng/ml) 3.31 1.73 8.78 3.24 0.001\\\* \Is a standard notation used to determine the level of significance. P* value by independent sample t-test. P\<0.05 is considered to be statistically significant. \*P\<0.05, \\P\<0.01, \\\*P\<0.001. VDR -- Vitamin D receptor; SD -- Standard deviation; n -- Sample size; NS -- Statistically nonsignificant ![Comparison of mean Vitamin D receptor concentration before and after supplementation](JISP-23-100-g002){#F2} The study also points out the correlation between VDR concentration and serum 25(OH)D3. When correlation was studied between VDR concentration and serum 25(OH)D3. The correlation coefficient "r" value before supplementation was 0.433. The p value obtained was 0.063 which indicated no significant correlation before supplementation. After supplementation the correlation coefficient "r" value was 0.480. The P value obtained was 0.048 which indicated significant positive correlation \[[Table 3](#T3){ref-type="table"}\]. The Linear relationship, before as well as after supplementation is presented in Figure [3a](#F3){ref-type="fig"} and [3b](#F3){ref-type="fig"}. ###### Correlation analysis of 25(OH)D3 levels and Vitamin D receptor concentrations before and after supplementation Before supplementation (n=19) After supplementation (n=19) --------------------------------- --------------------------------- -------------------------------- ------- --------- 25(OH)D3 with VDR concentration 0.476 0.039\* 0.480 0.038\* \\Is a standard notation used to determine the level of significance. Pearson's r value, P\<0.05 indicated statistically significant correlation. \*P\<0.05, \\P\<0.01, \\\*P\<0.001. There was a statistically significant positive correlation between 25(OH)D3 levels and VDR concentrations before supplementation (P\<0.05) as well as after supplementation (P\<0.05). NS -- Statistically nonsignificant; VDR -- Vitamin D receptors; n -- Sample size ![(a) Linear relationship between serum 25(OH)D3 and VDR concentration in PDL tissue in chronic periodontitis subjects during deficiency state. On pearson\'s correlation analysis the "r" value before supplementation was 0.433 and the P value obtained was 0.063 indicating no significant positive correlation (P \> 0.05) between 25(OH)D3 and VDR concentration in PDL tissue when subjects were deficient; (b) Linear relationship between serum 25(OH)D3 and VDR concentration in periodontitis cases after attaining sufficient range. The correlation coefficient "r" value on supplementation was 0.480 with P value of 0.048. A significant positive correlation was noted (P\< 0.05) between serum 25(OH)D3 levels and VDR concentration as subjects became sufficient in D3](JISP-23-100-g003){#F3} The results of present study clearly demonstrated that VDR concentration in periodontal ligament tissue is fully dependent on serum 25(OH)D3 levels. Lower serum concentration of 25(OH)D3 was associated with decreased expression of VDRs and increase in 25(OH)D3 levels in serum was associated with increased expression of VDRs in periodontal ligament tissue. DISCUSSION {#sec1-4} ========== The present study evaluated serum concentrations of 25(OH)D3 and VDR concentration in PDL tissue in chronic periodontitis patients, before as well as after supplementation of Vitamin D3. The study also attempted to correlate serum 25(OH)D3 levels with VDR concentration in PDL tissue. The results of the study revealed that all the chronic periodontic patients were found to be deficient in Vitamin D3. According to Ritu,\[[@ref15]\] Vitamin D3 deficiency is common in the Indian population due to dark skin (increased melanin pigmentation), social and religious norms to cover maximum part of the body, tendency to protect self from sun exposure, vegetarian diet (which lacks Vitamin D), less Vitamin D fortification of food products, and because people live in highly dense areas. Marwaha,\[[@ref16]\] Sahu,\[[@ref17]\] and Khadilkar\[[@ref18]\] have reported high prevalence of Vitamin D deficiency in various groups of the Indian population including healthy School children, adolescents, rural girls, pregnant women, and health-care professionals. The study by Patil\[[@ref19]\] also confirmed Vitamin D deficiency in Indian population. They carried out a study on 48 chronic periodontitis and 37 periodontally healthy controls. In their study, 97.9% of chronic periodonttis patients demonstrated 25(OH)D3 deficiency or insufficiency, whereas only 2.1% of the participants had sufficient levels of 25(OH)D3. Studies by Boggess et al.,\[[@ref20]\] Chang et al.,\[[@ref21]\] Antonoglou et al.,\[[@ref22]\] and Abreu et al.\[[@ref23]\] have shown association of low Vitamin D3 levels and periodontitis. Thus, Vitamin D deficiency may be considered a risk factor for periodontitis and therefore its evaluation needs to be considered in periodontic patients. Comparison of 25(OH)D3 levels in the study group determined significant increase after supplementation of Vitamin D3 in all the study participants \[[Table 1](#T1){ref-type="table"} and [Figure 1](#F1){ref-type="fig"}\]. The results corroborated with earlier findings. Harinarayan\[[@ref24]\] showed that Vitamin D of 9572 IU/day for 8 weeks significantly raised the serum levels of 25(OH)D and that supplementation up to upper tolerable intake levels is safe and sufficient. The present study suggests that loading dose of cholecalciferol (60,000 IU weekly)\[[@ref25]\] for 6 weeks helps in increasing the levels of Vitamin D3 to sufficient range (≥30 ng/ml). In the present study, Vitamin D3 supplementation was given instead of Vitamin D2. Evidence suggests that cholecalciferol (D3) is superior to ergocalciferol (D2) in terms of potency, elevating and sustaining 25(OH)D concentrations, and maintaining the storage form of Vitamin D.\[[@ref26][@ref27]\] Studies by Trang et al.,\[[@ref28]\] Armas et al.,\[[@ref29]\] and Heaney\[[@ref30]\] also found that the D3 supplement was more effective in increasing serum 25(OH)D3 than D2. The dosage used in our study was 60,000 IU/week for 6 weeks as recommended by Vieth et al.\[[@ref31]\] and Hathcock et al.\[[@ref32]\] Similarly, Gupta et al.\[[@ref33]\] and Goswami et al.\[[@ref25]\] also used comparable dose and found serum 25(OH)D3 levels to increase. ELISA kit with D6 antibody was used to determine detectable levels of VDR in the PDL tissue. VDR concentration was found to be very less. Lower VDR concentration has also been demonstrated earlier in infectious condition. Marshall in 2008\[[@ref34]\] determined that sulphono-lipid capnine substance produced by some gliding bacteria disabled the VDR. Capnine binds to and inactivates the VDR leading to exaggerated inflammatory responses. Waterhouse et al.\[[@ref35]\] found that intracellular bacteria in chronic infectious condition dysregulated Vitamin D metabolism by causing VDR dysfunction within phagocytes. These data suggest the mechanism for reduced VDR levels in chronic infectious condition. In inflammatory conditions, the active macrophages through CYP27B1 vigorously convert 25(OH)D3 to its active form (1,25\[OH\] 2D3), further increasing the expression of VDR.\[[@ref36]\] Conversion of 25(OH)D3 into 1,25(OH)2D3 up to toxic levels has been demonstrated by Marshall in 2008\[[@ref34]\] and Waterhouse et al.\[[@ref36]\] In contrast to the above findings, the VDR concentration in the present study was found to be very low. The lower concentration of VDR was probably because of severe deficiency of Vitamin D in chronic periodontitis patients; hence, less 25(OH)D3 was available for conversion to active form, further leading to less expression of VDRs. Moreover, as Phase I therapy was initiated prior to the supplementation of Vitamin D3, there was considerable reduction of plaque-induced inflammation. As inflammation reduced, the response to inflammation was low. The results of the present study thus indicate that in cases of Vitamin D deficiency and infection or inflammation, the concentration of VDR decreases as seen in chronic periodontitis. The decreased levels of VDR would in turn be responsible for reduced effect of Vitamin D-mediated generation of adequate immune and inflammatory response. The VDR concentration in PDL tissue in chronic periodontic patients in the present study showed direct correlation with serum 25(OH)D3 levels after supplementation with Vitamin D3. After supplementation, D3 ligand binds to VDR and brings about conformational changes in VDR, making it less vulnerable to proteolytic attack. The following studies determined the increased stability of VDR with Vitamin D3. Li et al.\[[@ref37]\] found that VDR is degraded by ubiquitin/proteosome pathway. However, after supplementation, D3 bound to VDR and inhibited ubiquitination and retarded degradation, resulting in elevated VDR levels. Kongsbak et al.,\[[@ref38]\] Costa et al.,\[[@ref39]\] Zineb et al.,\[[@ref40]\] and Wiese et al.\[[@ref41]\] also found that 1,25(OH)2D3 stabilized and upregulated VDR protein expression by approximately 2--2.3 folds, by protecting it from proteasomal degradation, thus increasing half-life and mean lifetime of the VDR protein. In our study also, supplementation of Vitamin D3 after periodontal Phase I therapy may have helped in decreased degradation/increased stability of VDR and increased availability of 25(OH)D3 for conversion to 1,25(OH)2D3, increased binding of 1,25(OH)2D3 to VDR, thus leading to increased expression of VDRs. The results of the present study showed that 25(OH)D3 levels in serum and VDR concentration in periodontal ligament tissue was very less in chronic periodontitis patients before supplementation, which increased significantly after supplementation of Vitamin D3. In addition, a significant positive correlation was seen between serum 25(OH)D3 and VDR concentration in Periodontal Ligament tissue, after supplementation of Vitamin D3. Limitations {#sec2-5} ----------- The limitation of the study was patient compliance regarding supplementation of Vitamin D3. CONCLUSION {#sec1-5} ========== The present article determined the presence of VDRs in PDL tissue which responded to levels of Vitamin D3 in serum. The VDR levels were very low in subjects patients with Vitamin D3 deficiency, which increased significantly after supplementation of Vitamin D3 for 6 weeks. Vitamin D3 through VDR-mediated effect plays a crucial role in generating immune and anti-inflammatory responses (decreased production of pro-inflammatory cytokines such as interferon-γ, IL-17, IL-21, decreased levels of IL-6, IL-1β, and TNF-α, and increased apoptotic mechanisms in pro-inflammatory cells). Low levels of VDR are indicative of low immune response in these patients. Vitamin D supplementation could enhance VDR levels which in turn may contribute to enhanced immune response. The innovative finding of expression of VDRs in PDL tissue in fully erupted teeth has opened a new gate for further research. Mechanisms to understand its effect on periodontal regeneration (PDL fibroblast uniqueness and bone formation) need to be studied. Financial support and sponsorship {#sec2-6} --------------------------------- Nil. Conflicts of interest {#sec2-7} --------------------- There are no conflicts of interest.	{ "pile_set_name": "PubMed Central" }	[ "root", "nonsupport", "probabl", "two", "halflif", "control", "refer", "given", "remain", "tendenc", "tumor", "eg", "decreas", "respond", "ligand", "purpos", "lifetim", "collect", "vdr", "gene", "centaur", "identifi", "pearson", "moderatetosever", "healthcar", "written", "risk", "complexiiref", "select", "rais", "iu", "mechan", "indirect", "convers", "week", "valu", "lysi", "enter", "may", "periodontti", "ohddbind", "attack", "pb", "live", "proteolyt", "acquir", "separ", "gener", "use", "graph", "eppendorf", "sahuref", "februari", "plaqueinduc", "supplement", "support", "complet", "caus", "accord", "ny", "prior", "intak", "retard", "μgml", "signific", "depart", "kidney", "modul", "cover", "li", "exposur", "suggest", "elisa", "process", "norm", "nucleu", "also", "state", "attempt", "immunomodulationref", "cell", "conflict", "women", "perform", "factorα", 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"Vitamin", "Receptor", "ELISA", "kit", "mean", "VDR", "concentration", "PDL", "tissue", "calculated", "OD", "values", "obtained", "ELISA", "readings", "mean", "VDR", "concentration", "periodontal", "ligament", "tissue", "Vitamin", "deficient", "chronic", "periodontitis", "subjects", "standard", "deviation", "values", "increased", "standard", "deviation", "supplementation", "indicating", "highly", "significant", "increment", "P", "Table", "table", "Figure", "fig", "Comparison", "mean", "Vitamin", "receptor", "concentrations", "supplementation", "Vitamin", "supplementation", "n", "supplementation", "n", "P", "VDR", "concentration", "standard", "notation", "used", "determine", "level", "significance", "P", "value", "independent", "sample", "P", "considered", "statistically", "significant", "P", "P", "P", "VDR", "Vitamin", "receptor", "SD", "Standard", "deviation", "n", "Sample", "size", "NS", "Statistically", "nonsignificant", "Comparison", "mean", "Vitamin", "receptor", "concentration", 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"VDR", "concentrations", "supplementation", "P", "well", "supplementation", "P", "NS", "Statistically", "nonsignificant", "VDR", "Vitamin", "receptors", "n", "Sample", "size", "Linear", "relationship", "serum", "OH", "VDR", "concentration", "PDL", "tissue", "chronic", "periodontitis", "subjects", "deficiency", "state", "correlation", "analysis", "r", "value", "supplementation", "P", "value", "obtained", "indicating", "significant", "positive", "correlation", "P", "OH", "VDR", "concentration", "PDL", "tissue", "subjects", "deficient", "b", "Linear", "relationship", "serum", "OH", "VDR", "concentration", "periodontitis", "cases", "attaining", "sufficient", "range", "correlation", "coefficient", "r", "value", "supplementation", "P", "value", "significant", "positive", "correlation", "noted", "P", "serum", "OH", "levels", "VDR", "concentration", "subjects", "became", "sufficient", "results", "present", "study", "clearly", "demonstrated", "VDR", "concentration", "periodontal", "ligament", "tissue", "fully", "dependent", "serum", "OH", "levels", "Lower", "serum", "concentration", "OH", "associated", "decreased", "expression", "VDRs", "increase", "OH", "levels", "serum", "associated", "increased", "expression", "VDRs", "periodontal", "ligament", "tissue", "DISCUSSION", "present", "study", "evaluated", "serum", "concentrations", "OH", "VDR", "concentration", "PDL", "tissue", "chronic", "periodontitis", "patients", "well", "supplementation", "Vitamin", "study", "also", "attempted", "correlate", "serum", "OH", "levels", "VDR", "concentration", "PDL", "tissue", "results", "study", "revealed", "chronic", "periodontic", "patients", "found", "deficient", "Vitamin", "According", "Ritu", "Vitamin", "deficiency", "common", "Indian", "population", "due", "dark", "skin", "increased", "melanin", "pigmentation", "social", "religious", "norms", "cover", "maximum", "part", "body", "tendency", "protect", "self", "sun", "exposure", "vegetarian", "diet", "lacks", "Vitamin", "less", 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Among Latinas living in the United States, breast cancer is the most common form of cancer and the leading cause of cancer death \[[@CR1]\]. Compared to non-Latina White women, Latinas are more likely to be diagnosed with regional or distant cancer \[[@CR1]\]. They are also more likely to undergo mastectomy (versus breast-conserving surgery) and are more likely to die of breast cancer than non-Latina White women diagnosed at a similar age and stage of cancer \[[@CR1]--[@CR4]\]. Given the higher rates of more advanced cancer and rigorous treatments reported in Latinas, the psychological and physical burden of breast cancer may be particularly grave for this ethnic group. In the large body of research on cancer-related quality of life (QOL), however, relatively few studies have focused on the breast cancer experience of ethnic minority women, with the possible exception of Black women \[[@CR5]\]. A diagnosis of breast cancer might be especially disruptive for Latinas in light of culturally specific causal attributions about cancer, including the notion that cancer represents punishment for one's sins, as well as fatalistic beliefs about cancer \[[@CR6]--[@CR8]\]. The importance of attending to QOL in Latinas is underscored by the observations that the Latino population is currently the largest and fastest growing ethnic minority population in the United States \[[@CR9]\], and Latinos are overrepresented among low-income and underserved groups \[[@CR10]\]. Quality of life is a multidimensional construct that encompasses various areas of functioning, including emotional, physical, sexual, and social domains \[[@CR11]\]. The physical domain pertains to a subjective evaluation of health status and bodily functioning (e.g., pain, fatigue, incontinence, lymphedema), whereas the emotional component encompasses psychological functioning, including positive and negative indicators of mood (e.g., anxiety, depressive symptoms, distress, affect) and perceived limitations due to emotional problems. Sexual quality of life generally refers to perceived sexual functioning, changes in sexual desire, and body image distress. The social domain often includes the impact of the disease on an individual's social role and the perceived utility of social support. These domains are not orthogonal, but rather are inter-related \[[@CR11], [@CR12]\]. For example, the experience of pain or fatigue may limit social and familial role performance. Additional indicators of quality of life include perceived cognitive functioning (e.g., memory) and spiritual well-being. The primary aim of this report is to present a systematic review of the literature to compare the QOL of Latinas diagnosed with breast cancer to QOL in women of other racial/ethnic groups. We consider evidence on socio-demographic factors, treatment-related variables, and other characteristics, such as level of acculturation, that may account for differences in QOL. Finally, we discuss directions for research and application. Method {#Sec1} ====== Defining quality of life {#Sec2} ------------------------ Our operational definition of quality of life was guided by findings from previous empirical studies and reviews that sought to illuminate the QOL of patients with breast cancer \[[@CR5], [@CR12], [@CR13]\]. The current review suggests four primary domains of QOL: mental, physical, social, and sexual. Low quality of life was operationalized as disruption in one of the aforementioned domains, presence of negative mood, or lack of positive mood. To illustrate, assessing the frequency of sexual activity in the absence of collecting information regarding its impact on functioning was not considered an assessment of QOL. Indices of QOL could be general or cancer-specific. Inclusion and exclusion criteria {#Sec3} -------------------------------- The current review included research on samples of women of self-identified Latina/Hispanic ethnic status with any stage of breast cancer. The U.S. Census Bureau \[[@CR14]\] defines Latino as individuals who identify themselves or their heritage as Mexican, Mexican American, Chicano, Puerto Rican, Cuban, or those whose origins are from Spain, the Spanish-speaking countries of Central or South America, and the Dominican Republic. To be included in the review, studies were required to provide a comparison of QOL between Latinas and at least one other ethnic/racial group. Studies were excluded if they did not report the QOL of Latinas as a separate group within the study. Dissertations, book chapters, and reviews were excluded. A systematic review of the literature from 1985 to 2009 was conducted using PubMed and PsycInfo. Studies had to be available online by December of 2009. In light of the advances in medical treatment for breast cancer over the past two decades, studies before 1985 were not considered. Manuscripts were retrieved using primary keywords (i.e., QOL, Latina/Hispanic, breast cancer) in combination with other words (e.g., fatigue, SF-36, depression), amounting to 30 key phrases[1](#Fn1){ref-type="fn"}. Another search was conducted to identify additional publications of all the authors whose manuscripts met entry criteria. References of selected studies were also reviewed to identify additional eligible studies. In total, 375 studies were reviewed. Of these studies, 22 met criteria.[2](#Fn2){ref-type="fn"} Studies were initially coded by the first author on 18 predetermined categories[3](#Fn3){ref-type="fn"} previously employed in a review on quality of life in African American cancer survivors \[[@CR5]\], and 25% of those were coded by the second author. Inter-rater reliability regarding agreement on the coding was 90%. Discrepancies were resolved through discussion among the authors. Results {#Sec4} ======= See Table [1](#Tab1){ref-type="table"} for socio-demographic and cancer-related descriptive data on each study's sample and Table [2](#Tab2){ref-type="table"} for information on research design, measures, and a summary of findings[4](#Fn4){ref-type="fn"}. With regard to research design, two qualitative studies, two longitudinal quantitative reports, and 18 cross-sectional quantitative reports met criteria for inclusion. Of the 20 quantitative reports, eight contained identical or overlapping samples. Most studies were conducted in large urban areas (Miami, Los Angeles, New York). Sample sizes for Latinas in quantitative studies ranged from 16 to 492 women (with the exception of Clauser et al. \[[@CR15]\]). Most women had early-stage disease. Breast cancer diagnosis duration was variable, with some relatively recently diagnosed samples (\<1 year) and some samples of longer-term survivors (\>2 years). Of the few studies that reported statistical tests comparing race/ethnicity by demographic and cancer-related variables, results showed that Latinas reported lower income and were more likely to be younger than other groups of women. Latinas were also more likely to be diagnosed at later stages compared to non-Latina Whites. Table 1Demographic data for studies included in the reviewAuthorsStudy SampleAgeCancer StageTime Since Diagnosis or Surgery^1^Income or EducationAshing-Giwa et al., 2004n = 26 LatinaLatina M = 56 years, range = 40--730 Latinas stage 0Latina M = 4 yearsNot providedn = 18 non-Latina White5 Latinas stage 1n = 24 Black7 Latinas stage 2n = 34 Asian American5 Latinas stage 30 Latinas stage 49 Latinas stage unknownAshing-Giwa et al., 2007^5^n = 183 LatinaLatina M = 5018 Latinas stage 0Latina M = 2.9 years87 Latinas \<25 Kn = 179 non-Latina White59 Latinas stage 136 Latinas 25--45 Kn = 135 Blacks75 Latinas stage 227 Latinas 45--75 Kn = 206 Asian American28 Latinas stage 323 Latinas \>75 KLatinas reported significantly lower income than other groups of womenAshing-Giwa et al., 2009n = 183 Latina\M* = 55.03 years, range 29--62\332 women stage 1\M = 2.98 years, SD = 1.67\205 women \<25 Kn* = 179 non-Latina White267 women stage 2312 women 25--75 Kn = 135 Blacks95 women stage 3186 women \>75 Kn = 206 Asian AmericansLatinas significantly younger at diagnosis than other ethnic groupsLim et al., 2009n = 183 LatinaLatinas M = 53.45 years, SD = 11.518 Latinas stage 0Latinas M = 2.93 years, SD = 1.690 Latinas \<25 Kn = 206 Asian59 Latinas stage 139 Latinas 25 K -- 45 K75 Latinas stage 229 Latinas \>45 K--75 KNo significant differences in age between Latinas and Asian women28 Latinas stage 3No significant differences between ethnic groups25 Latinas \>75 KLatinas significantly more likely to be diagnosed at a later stage than AsiansLatinas reported significantly lower income than AsiansBowen et al., 2007n = 95 Latina\M* = 55.55 years, SD = 10.4\179 women in situ\6.1, 24, and 35 months\54 women \< = 10 Kn* = 486 non-Latina White453 women localized86 women \>10 K--20 Kn = 199 Black172 women regional (unstaged)93 women \>20 K--30 Kn = 24 Other168 women \>30 K--50 K211 women \>50 K--70 K113 women \>70 KCarver et al., 2003^6^n = 60 Latina\M^2^ = 53.59 years, SD* = 12.41\10 women stage 0\≤1 year post surgery\*M = 14.25 years, SD = 2.87Study 1n = 26 Black135 women stage 1\\<8 weeksn* = 149 non-Latina White90 women stage 2Study 2n = 16 Latina\M^2^ = 50.37 years, SD* = 9.24\9 women stage 0\Years of education for full sample 15.71 years, SD = 5.33n = 5 Black47 women stage 1n = 72 non-Latina White41 women stage 2n = 4 OtherPetronis et al., 2003n = 62 Latina\*M = 53.61 years, SD = 12.42\11 women stage 0\≤1 year post surgery\Years of education at diagnosis for full sample 14.25 years, SD* = 2.89n = 25 Black135 women stage 1n = 150 non-Latina White90 women stage 2Spencer et al., 1999n = 48 Latina\*M = 53.75 years, SD = 12.62\10 women stage 0\≤1 year post surgery\Years of education at diagnosis for full sample 14.39 years, SD* = 2.80n = 24 Black128 women stage 1n = 151 non-Latina White85 women stage 2Carver et al., 2005^7^n = 32 Latina\* M = 54.18 years Age at diagnosis, SD = 10.61\5 women stage 0\5--13 years post surgery\Years of education at diagnosis for full sample 14.18 years, SD* = 3.44n = 114 non-Latina White101 women stage 1n = 17 Black57 women stage 2Carver et al., 2006n = 32 Latina\* M = 54.18 years Age at diagnosis, SD = 10.61\5 women stage 0\5--13 years after diagnosis\* Years of education at diagnosis for full sample 14.18 years, SD = 3.44n = 114 non-Latina White101 women stage 1n = 17 Black57 women stage 2Clauser et al., 2008\n* = over 5,500Not provided for women with breast cancerNot providedNot providedNot providedCulver et al., 2002Sample 1Sample 1Sample 1\2 days prior to surgerySample 1n* = 4 Latina\M* = 53.72 years, SD = 11.14\* 55 women stage 17 days post surgery\M* = 14.63 years, SD = 2.56n = 1 Black20 women stage 23, 6, 12 months post surgeryn = 70 non-Latina WhiteSample 2Sample 2Sample 2Sample 2n = 49 Latina\M* = 56.29 years, SD = 10.27\5 women stage 0\M = 9.04 years, SD = 4.24^4^n = 7 Black21 women stage 130 women stage 2Eversley et al., 2005n = 29 LatinaLatina M = 47 years, range 29--687 Latinas stage 1\All women diagnosed within the past 2 yearsLatina M* monthly income \$1,119, range \$0--\$3,500n = 35 Black13 Latinas stage 2n = 35 non-Latina White7 Latinas stage 3Non-Latina Whites reported significantly greater income than all other groups of women.n = 17 Other2 Latinas stage 4Whites significantly more likely to be diagnosed with stage 1 than women from all other groups.Fatone et al., 2007n = 12 Latina\*M = 54.10 years, SD = 10.54\9 women stage 1--2\13 women diagnosed between 1998--2001^3^\7 women some high school or lessn* = 8 Black3 women stage 3--47 women diagnosed in 1997 or earlier13 women high school graduate or some college8 unknown stageFobair et al., 2006n = 45 Latina\n* = 107 Age \<40 years\* 88 women in situ\253 women = 2--4 months\102 women 12 years or less of schooln = 389 non-Latina Whiten = 442 Age ≥40 years248 women localized275 women = 5--7 months\446 women \>12 years of schooln* = 29 Black213 women regional (unstaged)20 women = 8 months or moren = 86 AsianFriedman et al., 2006.n = 22 Latina\*M = 52 years, SD = 10.2Not provided\*M = 26 months, SD = 33.5\14 less than 8 yearsn* = 45 Black14 some high schooln = 14 non-Latina White24 high school/GED22 some college7 college graduateFu et al., 2009n = 63 Latinan = 22 Latinas \<452 Latinas stage 042 Latinas \<2 years29 Latinas \<10 Kn = 58 non-Latina Whiten = 16 Latinas 45--5417 Latinas stage 118 Latinas 2--5 years11 Latinas 10 K--50 Kn = 18 Blackn = 13 Latinas 55--6427 Latinas stage 23 Latinas \>5 years8 Latinas 50 K--100 Kn = 9 Latinas 65+8 Latinas stage 33 Latinas \>100 K\*M = 52.5, range = 26--909 Latinas stage not reported12 Latinas income not reportedGiedzinska et al., 2004n = 78 LatinaM = 51.4, range 30--74 Latinas\* Women were stage 0, 1, or 2Latina M = 3.07 years, SD = 1.213 Latinas \<15 Kn = 233 Black18 Latinas 15 K--30 Kn = 77 Asian American25 Latinas 30 K--45 Kn = 233 non-Latina WhiteLatinas significantly younger than non-Latina Whites and Blacks8 Latinas 45 K--60 KNo significant differences between ethnic groups11 Latinas 60 K--75 K4 Latinas 75 K--100 K5 Latinas \>100 KNon-Latina Whites and Asians reported significantly higher income than Latinas.Janz et al., 2009n = 344 LatinaLatinas high acculturationLatina high acculturation\Women diagnosed between 2005--2007Latinas high acculturationn* = 386 Blackn = 34 age \<50 years16 stage 022 \< High School diploman = 726 non-Latina Whiten = 50 age 50--70 years41 stage 117 High School diploman = 160 high acculturation Latinan = 16 \> 70 years29 stage 261 \> High School diploman = 184 low acculturation LatinaLatina low acculturation14 stage 3Latina low acculturationn = 30 age \<50 yearsLatina low acculturation70 \< High School diploman = 58 age 50--70 years21 stage 020 High School diploman = 13 \> 70 years35 stage 110 \> High School diplomaLatinas more likely to be \<50 years than non-Latina Whites33 stage 212 stage 3Maly et al., 2010n = 492 Latina\* M = 50.8 years, SD = 9.5Not provided\M* = 180 days, SD = 20.8\* 377 women \< High Schooln = 54 Black534 women ≥ High Schooln = 292 non-Latina Whiten = 68 Asian/Pacific Islandern = 15 OtherMaly et al., 2008n = 99 Latina\* M = 68.7 years, SD = 8.5\Only 8% of sample had greater than stage 2\ M = 8.20 months, SD = 2.751.5% of Latinas \< 20 Kn = 66 Black10.9% of Whites \< 20 Kn = 92 non-Latina WhiteNo significant differences between ethnic groupsNo significant differences between racial/ethnic groupsLatinas reported significantly lower income than non-Latina WhitesYoon et al., 2008n = 103 Latina English speaker\n* = 416 Age 50--59\173 women in situ\ M = 223 days after diagnosis\288 women \< 20 Kn =* 104 Latina Spanish speakern = 198 Age 60--64487 women stage 1244 women 20 K--39 Kn = 112 Blackn = 349 Age 65--74368 women stage 2687 women 40 K+n = 834 non-Latina Whiten = 256 Age 75--9950 women stage 3n = 66 Other19 women stage 4122 women unknown stage\Data only for breast cancer patients belonging to all ethnic groups^1^When time since diagnosis was not available time since surgery was provided^2^Data for the study comes from two recruitment samples^3^Study published in 2007 and no other information on time since diagnosis provided^4^Mean values on education were reported for each recruitment sample^5^Ashing Giwa et al., 2007, and Ashing Giwa et al., 2009 are the same data set and Lim et al., 2009 is from an overlapping dataset^6^Spencer et al., 1999, Carver et al, 2003 and Petronis et al., 2003 are overlapping data sets^7^Carver et al. (2005) and Carver et al. (2006) used the same data setTable 2Methodological data for studies included in the reviewAuthorAnalyses of DataMeasures or Interview TopicsResults SummaryAshing-Giwa et al., 2004QualitativeFear and anxiety, body image, intimate relationships.Latinas and Asian Americans discussed how their feelings about their body affected their QOL. Latinas discussed feeling embarrassed, sad, frustrated, ugly, and incomplete. Regarding the major theme of overall stress/effect of breast cancer, Blacks, Asians, and Latinas reported decrements in QOL whereas non-Latina Whites emphasized positive aspects. Latinas emphasized deleterious treatment side effects.Ashing-Giwa et al., 2007QuantitativeFACT-G (all subscales)Latinas were significantly lower on physical, social, emotional, and functional quality of life on FACT-G than non-Latina Whites, Blacks, and Asian Americans. After controlling for covariates, analysis on the FACT-G summary score indicated that ethnic differences were no longer significant. Latinas were significantly lower on SF-36 role limitation and emotional well-being compared to all other groups of women.Cross-sectionalRand/SF-36 (Role Limitation/Emotional Well-being)Ashing Giwa et al., 2009QuantitativeSF-36 (Physical and Mental)Latina ethnicity was significantly related to poorer Mental Health on the SF-36; this disparity was no longer significant when job type and stress were entered into the model. No significant relationship between Latina ethnicity and physical health QOL.Cross-sectionalLim et al., 2008QuantitativeFACT-G (Emotional and Physical)Emotional and Physical Well-being scores for Asian Americans were significantly higher than Latinas' scores.Cross-sectionalBowen et al., 2007QuantitativeSF-36 (Physical and Mental)Adjusting for covariates, Black women reported significantly lower impact of cancer on cognitive/mood and incontinence subscales assessing hormone-related symptoms than Latinas and Latinas reported more fear of recurrence than Black women. In a similar set of analyses examining differences in SF-36 Mental and Physical Health summary scores, also adjusting for covariates, Black women evidenced significantly lower physical functioning scores, compared with Latinas, but higher mental health scores. Two additional models were conducted with non-Latina White entered as the referent group. After controlling for covariates, no significant differences between Latinas and non-Latina Whites emerged on physical health, but Latinas evidenced significantly poorer mental health on the SF-36. Latinas did not significantly differ from non-Latina Whites on the scale to assess the social/emotional impact of breast cancer.Cross-sectionalFear of Recurrence ScaleHormone-related symptomsImpact of breast cancer(Social/Emotional)Carver et al., 2003QuantitativeSIP (Social Activities, Recreation and Pastime Activities subscales)Findings were similar to those of Spencer et al. (1999). Latinas reported more POMS fatigue than non-Latina Whites. Study 2 yielded no racial/ethnic differences in QOL on social disruption, fatigue, and distress.Cross-sectionalPOMS (Fatigue subscale)\Emotional distressPetronis et al., 2003QuantitativeEmotional distress (CES-D, 11 items selected from Andrews and Withey SQOL 10 items selected from Carver et al., 1993)In models controlling for covariates, Latinas indicated significantly greater distress and more social disruption than non-Latinas, but groups did not differ on the PAIS.Cross-sectionalSIP (Social Activities, Recreation and Pastime Activities subscales)PAIS (Sexual subscale)PCBCSpencer et al., 1999QuantitativeEmotional disruption (CES-D, POMS, 11 items selected from Andrews and Withey SQOL )After controlling for covariates, Latinas reported poorer adjustment than non-Latina White and Black women in all domains of QOL.Cross-sectionalSIP (Social Activities and Recreational Pastimes subscales)PAIS (Sexual relations subscale)Carver et al., 2005QuantitativeCES-DLatinas had higher scores on the CES-D and SIP than non-Latinas (i.e., non-Latina Whites and Blacks). No significant differences between ethnic groups on distress (ABS/POMS) or self-rated QOL. Initial values on the two other dependent variables (SIP, CES-D) were entered simultaneously with demographic /medical factors. Latina ethnic status was not a significant predictor of change in depressive symptoms and social disruption.LongitudinalABS/POMSSIP (Social Activities, Recreation and Pastime Activities subscales)10 questions from Andrews and Withey SQOL (1976)Carver et al., 2006QuantitativeQLACSLatinas reported significant elevations in negative feelings, social avoidance, distress about family's future, and distress about recurrence compared to non-Latinas, even when covariates were controlled. Groups did not differ on cognitive impairment and fatigue subscales.Cross-sectionalClauser et al., 2008QuantitativeSF-36 (Mental and Physical)Asians evidenced the highest physical QOL, followed by non-Latina Whites, Latinas, and Blacks. Asians evidenced the highest mental QOL, followed by non-Latina Whites, Blacks, and Hispanics.Cross-sectionalCulver et al., 2002QuantitativeCES-DAfter controlling for covariates, Latinas did not significantly differ on distress from non-Latinas across time points. However, between 6 and 12 months after surgery, Latinas evidenced a significant increase in distress compared to non-Latina Whites. With covariates controlled, Black women reported significantly lower depressive symptoms than Latinas.Longitudinal\DistressEversley et al., 2005QuantitativeCES-DLatinas had significantly higher depressive symptoms on the CES-D and fatigue on the Piper Fatigue Scale (PFS) when compared to Black and non-Latina White women. Latinas also evidenced higher rates of pain on the Brief Pain Inventory (BPI) and lymphedema-related swelling (measured by a single item) when compared to non-Latina White women. Latina ethnic status remained a correlate of total number of post-treatment symptoms even when covariates were controlled.Cross-sectionalPFSBPILymphedema-related SwellingFatone et al., 2007QualitativePhysical, Social/Functional, Psychological, Sexual, Cognitive, ExistentialThe primary domain of concern for the Latinas was psychological, whereas the primary domain of concern for Black women was physical. Main psychological themes for Latinas were sadness, crying, anxiety, and irritability. In descending order of importance, the remaining areas of concern for Latinas were physical, social/functional, spiritual/existential, cognitive, and sexual.Fobair et al., 2005QuantitativeMOS (Sexual Problems)After controlling for covariates, multiple regression analysis indicated significantly fewer sexual problems among Latinas compared to non-Latina White women.Cross-sectionalFriedman et al., 2006QuantitativeFACT-G (Emotional, Functional, Social/Family subscales)No significant differences for race/ethnicity on QOL.Cross-sectionalIES (Intrusions)Latinas, non-Latina Whites, and Blacks did not differ significantly on any of the measures.POMS-SF (all 6 subscales)Fu et al., 2009QuantitativeMSAS-SF (Depression, Hormone-related Symptoms, Pain-related Symptoms, Chemotherapy-related Symptoms)Latinas had more chemotherapy- and pain-related symptoms on the Memorial Symptoms Assessment Scale Short Form than non-Latina Whites when controlling for covariates. No significant ethnic differences emerged on the other areas of QOL.Cross-sectionalGiedzinska et al., 2004QuantitativeSF-36 (all subscales), CARES (Sexual Dysfunction, Sexual Interest, Body Image subscales)Black women reported significantly higher SF-36 Emotional Well-Being than Latinas, even after covariates were controlled for. No significant differences on the other SF-36 scales were reported. Mean differences in sexual dysfunction were not significant; however, when covariates were controlled, Black women evidenced less sexual dysfunction than Latinas. Black women reported higher Mental Health on the SF-36 than Latinas, even after controlling for covariates. Latinas scored higher than any other ethnic group on symptoms related to breast cancer treatment (BCPT summary score). After controlling for covariates, Latinas did not significantly differ on the BCPT from non-Latina Whites but remained higher than Black and Asian American women. No significant differences for Latinas emerged on the CES-D and Revised Dyadic Adjustment Scale.Cross-sectionalBCPTCES-DDASJanz et al., 2009QuantitativeFACT-B (Physical, Emotional, Functional, Social/Family, Breast Concerns)Low-acculturated and high-acculturated Latinas reported significantly lower physical well-being and more breast-specific concerns than non-Latina White women. Low-acculturated Latinas also reported significantly lower scores on functional well-being, emotional well-being, and social well-being compared to non-Latina Whites, who did not differ from high-acculturated Latinas. In the final model which included all covariates, Latinas low in acculturation reported significantly lower functional well-being and emotional well-being than non-Latina Whites, and more breast-specific concerns than non-Latina Whites, but did not significantly differ on physical and social well-being. Low-acculturated Latinas reported lower levels of functional and emotional well-being than Latinas high in acculturation and Black women.Cross-sectionalMaly et al., 2010Quantitative\Self-reported symptoms and self-reported symptom resolution (nausea/vomiting, pain, depressive symptoms)In analyses controlling for covariates, both less-acculturated and more-acculturated Latinas were significantly more likely to report having experienced nausea than non-Latina Whites. Less acculturated Latinas were significantly less likely to report having experienced pain than non-Latina White women. Latinas did not significantly differ from non-Latina Whites on depressive symptoms. Less-acculturated Latinas were significantly less likely to perceive depressive symptom resolution than non-Latina White women in the unadjusted and adjusted models. Latinas were less likely that non-Latina Whites to perceive pain resolution; however, no differences emerged on the resolution of nausea or pain in the adjusted models.Cross-sectionalMaly et al., 2008QuantitativeSF-36 (all subscales)Latinas reported poorer QOL compared to non-Latina Whites. With control variables entered in the structural equation model, non-Latina ethnic status was directly associated with better QOL.Cross-sectionalCES-DSTAIBreast Cancer Specific AnxietyYoon et al., 2008Quantitative\* Physical symptoms (nausea/vomiting, difficulty sleeping, arm problems, vaginal dryness, hot flashes)After controlling for covariates, Spanish-speaking Latinas were approximately 60% less likely to report having any severe cancer-related symptoms than non-Latina White women, who did not differ from English-speaking Latinas. English-speaking Latinas were significantly less likely to report hot flashes and that Spanish-speaking Latinas were significantly less likely to report difficulty sleeping than non-Latina Whites.Cross-sectionalAshing Giwa et al., 2007, and Ashing Giwa et al., 2009 are the same data set and Lim et al., 2009 is an overlapping dataset. Carver et al. (2005) and Carver et al. (2006) used the same data set. Spencer et al., 1999, Carver et al, 2003 and Petronis et al., 2003 are overlapping data sets. Scales with reported alphas ranged from .70 to .96\Author constructed scale. No other information on scale provided^+^QOL only measured at 1 time pointCES-D* Center for Epidemiologic Studies Depression; SF-36 Rand Short-Form 36; CARES Cancer Rehabilitation Evaluation System; BCPT Breast Cancer Prevention Trial Symptom Checklist; POMS Profile of Mood States; MSAS-SF Memorial Symptoms Assessment Scale -- Short Form; QLACS Quality of Life in Adult Cancer Survivors; FACT Functional Assessment of Cancer Therapy. FACT-G (general cancer), and FACT-B (breast cancer specific) are the same measure except FACT-B contains an additional measure of breast cancer specific QOL; IES Impact of Event Scale; STAI-S State-Trait Anxiety Inventory; ABS Affects Balance Scale; SIP Sickness Impact Profile; SQOL Self-Rated Quality of Life; PCBC Profile of Concerns about Breast Cancer; QOL Quality of Life; PAIS Psychological Adjustment to Illness Scale; PFS Piper Fatigue Scale; BCPC Breast Cancer Problems Checklist; BPI Brief Pain Inventory; DAS Dyadic Adjustment Scale Results are organized by QOL domain. We reviewed findings by each domain and most-commonly employed measures within domains. In the event that two separate measures were used to assess a QOL domain, we report findings for each measure. We also provide findings by measure subscale if reported in the study's results section. Not all studies in the review disentangled domains of QOL but rather reported total scores on measures of QOL that included some combination of the four domains assessed in this review (e.g., mental, physical, social, sexual). In these cases, the relationships between racial/ethnic status and distinct domains of one of the four QOL domains could not be determined. Therefore, for purposes of this review, an additional section labeled Combined QOL Domains was included. Finally, only QOL findings that pertain to Latinas are reported. In other words, QOL comparisons between African American, Asian American, and non-Latina White women are not reported. Findings related to mental health {#Sec5} --------------------------------- Findings related to mental health are divided into two sections: 1) general emotional functioning which includes mental health QOL, anxiety, distress, fear of recurrence, or a combination of these variables, and 2) depressive symptoms. Despite the variations in measurement, a consistent finding among quantitative studies is that Latinas generally report poorer emotional functioning than non-Latinas. The most commonly used measures of general emotional functioning were the Rand Short-Form 36 (SF-36) and Functional Assessment of Cancer Therapy (FACT). In 12 studies, Latinas demonstrated significantly poorer QOL than non-Latina Whites, Blacks, and/or Asian Americans on at least one measure of emotional functioning compared to non-Latina Whites, Blacks, and/or Asian Americans \[[@CR15]--[@CR26]\]. In general, Latinas reported poorer mental health on the SF-36 and the FACT. They also reported greater fear of recurrence and distress. Eight of the 12 studies assessing mental health controlled for relevant covariates and continued to report significantly poorer QOL among Latinas compared to other women. Four studies that assessed emotional functioning reported no significant differences between groups on at least one measure of general emotional functioning among Latinas, non-Latina Whites, and/or Blacks \[[@CR16], [@CR22], [@CR25], [@CR27]\]. Two of these four studies present both positive and negative findings for Latinas. Janz et al. \[[@CR22]\] reported that both low-acculturated and high-acculturated Latinas evidenced poorer QOL compared to non-Latina Whites. Their findings also revealed that high-acculturated Latinas did not significantly differ from non-Latina Whites on some QOL measures and that disparities in QOL were stronger among low-acculturated Latinas than high-acculturated Latinas \[[@CR22]\]. Although Culver et al. \[[@CR16]\] did not find significant ethnic group differences in distress, they did report a significant increase in distress over time in Latinas compared to non-Latina Whites. Finally, no study reported that Latinas evidenced better emotional functioning than women of other ethnic groups. The most commonly used measure of depressive symptoms was the Center for Epidemiologic Studies Depression scale (CES-D). Four quantitative studies \[[@CR16], [@CR28]--[@CR30]\] reported significantly greater levels of depressive symptoms or less depressive symptom resolution among Latinas than non-Latina Whites and/or Blacks on at least one measure. Three of these studies continued to report significant differences in depressive symptomatology despite controlling for covariates. Three studies reported no significant differences between groups on depressive symptoms compared to non-Latina Whites and/or Blacks \[[@CR17], [@CR30], [@CR31]\] on at least one measure. Of note, Maly et al. \[[@CR30]\] reported that Latinas did not significantly differ from non-Latina Whites on depressive symptoms, but less-acculturated Latinas were less likely to report depressive symptom resolution compared to non-Latina Whites. No study reported that Latinas evidenced significantly lower depressive symptomatology than other groups of women. Taken together, these findings suggest that Latina breast cancer patients report greater depressive symptoms and poorer mental health than non-Latinas. Findings related to physical health {#Sec6} ----------------------------------- Although less consistent than the findings pertaining to mental health, the majority of studies suggested that Latinas evidenced worse QOL related to physical health. The most commonly employed measures of physical health were the SF-36 and FACT. Other measures included treatment-related symptoms such as fatigue pain, nausea, and lymphedema. Ten studies reported that Latinas evidenced poorer QOL compared to non-Latina Whites, Blacks, and/or Asian Americans on at least one measure of physical health \[[@CR15], [@CR17], [@CR18], [@CR20], [@CR22], [@CR23], [@CR25], [@CR29]--[@CR31]\]. In general, Latinas reported poorer general physical QOL on the SF-36 and FACT and more cancer treatment-related symptoms such as fatigue, pain, and lymphedema swelling. Of these 10 studies, six reported significantly poorer QOL among Latinas than other groups of women even after controlling for relevant covariates. Five studies reported no significant differences between Latinas and non-Latina Whites, Blacks, and/or Asian Americans on at least one measure of physical health \[[@CR17], [@CR19], [@CR21], [@CR25], [@CR31]\], and three studies reported that Latinas evidenced superior QOL on at least one measure of physical health compared to non-Latina Whites and Blacks \[[@CR20], [@CR30], [@CR32]\]. Among the studies reporting superior physical QOL, findings indicated that Latinas evidenced better physical functioning on the SF-36, less pain, fewer hot flashes, and less difficulty sleeping. Findings related to social functioning {#Sec7} -------------------------------------- Seven studies assessed social functioning. Commonly used measures of social functioning included the SF-36, FACT, and Sickness Impact Profile. Six studies reported that Latinas demonstrated poorer social functioning than non-Latina Whites, Blacks, and/or Asian Americans \[[@CR18], [@CR21], [@CR22], [@CR24], [@CR26], [@CR28]\]. Of these six studies, three reported significantly poorer QOL among Latinas after controlling for relevant covariates. Five reported no significant differences between Latinas and non-Latina Whites and Black women \[[@CR17], [@CR20], [@CR22], [@CR25], [@CR27]\]. Of note, Janz et al. \[[@CR22]\] reported that low-acculturated Latinas but not high-acculturated Latinas reported significantly lower scores on social well-being. No study reported better social functioning among Latinas compared to other groups of women. The pattern of findings suggests that Latinas are more likely to report poor social functioning compared to non-Latinas. Findings related to sexual health {#Sec8} --------------------------------- Four studies reported data pertaining to sexual health. Two of the four studies reported greater sexual dysfunction among Latinas compared to non-Latina Whites, Blacks, and Asian Americans \[[@CR17], [@CR24]\]. Both of these studies reported significant differences after controlling for relevant covariates. One study reported fewer sexual problems among Latinas \[[@CR33]\] compared to non-Latina Whites, and one study reported no differences in sexual disruption between Latina and non-Latina Whites and Black women \[[@CR26]\]. Given the dearth of studies assessing sexual health and lack of consistent findings, it is difficult draw to any conclusion for this QOL domain. Findings related to combined QOL domains {#Sec9} ---------------------------------------- One study combined several domains of QOL. Using a multiethnic sample of non-Latina White, Latina, and Black breast cancer patients, Maly et al. \[[@CR34]\] employed a structural equation model to examine predictors of QOL. Quality of life was a latent variable consisting of the Medical Outcomes Study 36-Item Short Form (SF-36), the Center for Epidemiologic Studies Depression Scale (CES-D), the State-Trait Anxiety Inventory-State (STAI-S), and a scale on breast cancer emotional health. Findings indicate that Latinas reported poorer QOL compared to non-Latina Whites. In a structural equation model with control variables included, non-Latina ethnic status was directly associated with better QOL \[[@CR34]\]. Maly et al. \[[@CR34]\] primarily assessed mental health, and the study's results are consistent with the Findings Related to Mental Health section, in which the majority of studies reported significantly poorer mental health among Latinas compared to other ethnic groups. Qualitative studies {#Sec10} ------------------- Fatone et al. \[[@CR35]\] conducted semi-structured interviews with a sample of Latina and Black women. The primary domain of concern for the Latinas was psychological, whereas the primary domain of concern for Black women was physical. Main psychological themes for Latinas were sadness, crying, anxiety, and irritability. In descending order of importance, the remaining areas of concern for Latinas were physical, social/functional, spiritual/existential, cognitive, and sexual. Another qualitative study sought to understand the breast cancer experiences of Black, Asian American, non-Latina White, and Latina breast cancer survivors \[[@CR36]\]. Participants were interviewed in a focus group format. Two themes related to QOL were feelings about body and overall stress/effect of breast cancer. Latinas and Asian Americans discussed how the impact of their feelings about their body affected their QOL. Latinas discussed feeling embarrassed, sad, frustrated, ugly, and incomplete. Regarding the major theme of overall stress/effect of breast cancer, Blacks, Asians, and Latinas reported decrements in QOL whereas non-Latina Whites emphasized positive aspects. Latinas emphasized deleterious treatment side effects. Discussion {#Sec11} ========== Summary of findings {#Sec12} ------------------- Twenty-two studies met criteria for this review of QOL in Latinas with breast cancer compared to other racial/ethnic groups, of which eight used identical or overlapping samples \[[@CR18], [@CR19], [@CR21], [@CR23]--[@CR26], [@CR28]\]. Synthesizing the findings is complicated by marked variability in the available studies' sample sizes, operationalizations of QOL, approach to statistical control of demographic and other variables, and cultural diversity within the Latina population. Overall, findings suggest several important conclusions: 1) Latinas appear to be at risk for poor QOL following breast cancer diagnosis relative to non-Latinas; 2) relatively little is known about the mechanisms that explain these health-related discrepancies; and 3) research is needed to determine whether and how psychosocial interventions will improve QOL for this group of women. We begin by first providing a summary in which the results are organized according to study findings: studies that reported no significant racial/ethnic differences pertaining to Latinas on all QOL measures, studies in which Latinas consistently evidence higher QOL, studies in which Latinas consistently evidence lower QOL, and studies that yielded mixed findings on the relationship between race/ethnicity and QOL. The remainder of the discussion focuses on interpretations of study findings, explanatory variables, and future directions. Only one study reported no significant differences between Latinas and other women on all measures of QOL \[[@CR27]\]. Of note, this study had a very small sample of non-Latina Whites. One study reported that Latinas evidenced higher QOL than other groups of women on all measures of QOL \[[@CR33]\]. Six studies found that Latinas reported poorer QOL than other groups of women on all measures \[[@CR15], [@CR18], [@CR23], [@CR24], [@CR29], [@CR34]\]. However, after controlling for relevant covariates Ashing-Giwa et al. reported that some ethnic differences between Latinas and other ethnic groups lost statistical significance \[[@CR18]\]. The majority of studies (12) yielded mixed findings \[[@CR16], [@CR17], [@CR19]--[@CR22], [@CR25], [@CR26], [@CR28], [@CR30]--[@CR32]\] in which Latinas generally demonstrated significantly worse QOL on most, but not all, measures. Carver et al. \[[@CR25]\] reported findings from two separate studies within one manuscript. Findings from Study 1 revealed racial/ethnic disparities in QOL whereas findings from Study 2 revealed no significant differences in QOL by race/ethnicity. Of significance, the sample in Study 2 contained only 16 Latinas. The pattern of results indicates that Latinas diagnosed with breast cancer may be at risk for poorer QOL than non-Latinas. Specifically, Latinas are more likely to report poor mental, physical health, and social disruption than do non-Latinas. Measurement of sexual health was too limited to draw any conclusions. Findings from two qualitative studies emphasized the psychological impact of breast cancer (e.g., sadness, embarrassment, frustration), treatment-related symptoms, decrements in physical functioning, and the impact of cancer on social well-being, which are consistent with findings from quantitative studies. Qualitative studies add to the literature by providing a nuanced understanding of the breast cancer experience. For example, Ashing-Giwa et al. \[[@CR36]\] underscored the importance of attending to the impact of Latinas' embarrassment regarding changes to their bodies and the subsequent impact on relationships, which was not fully captured by quantitative studies. Explanatory mechanisms {#Sec13} ---------------------- Stress-based theories of health posit that minority ethnic status may confer risk for poor health outcomes as a result of fewer economic and social resources, race-based discrimination, and additional sources of stress exposure caused in part by social disadvantage \[[@CR37]--[@CR39]\]. Numerous factors can account for the obtained racial/ethnic differences in QOL outcomes. Socio-demographic factors such as income and education can affect QOL and vary by racial/ethnic status, with Latinas usually of lower SES than their White counterparts \[[@CR27], [@CR34]\]. Treatment-related factors have also been associated with QOL outcomes. Women diagnosed with more advanced stages of cancer have reported lower QOL than women diagnosed at earlier stages \[[@CR40]\]. Stage of diagnosis may be especially relevant for Latinas as they are likely to be diagnosed with more advanced stages of cancer \[[@CR1]\]. Studies included in the review that examined differences in socio-demographic and treatment-related factors indicated that Latinas generally reported lower SES and were diagnosed at a later stage than other groups of women. Even when socio-demographic and/or treatment-related factors were controlled statistically, however, the majority of studies still demonstrated lower QOL among Latinas on at least one indicator, and in several cases the findings did not change \[[@CR24], [@CR25], [@CR29]\]. Based on findings from the reviewed studies, we suggest that socio-demographic and treatment-related variables may account for some of the disparities in QOL, but they are not likely responsible for all of the observed racial/ethnic differences and therefore additional explanatory factors must be investigated. After a breast cancer diagnosis, Latinas may face additional stressors that are culturally-relevant. Only four studies examined language use or level of acculturation and QOL among Latinas. Findings from these four studies suggest that low-acculturated Latinas may be at risk for poor QOL, specifically mental health, relative to high-acculturated Latinas and non-Latina Whites \[[@CR22], [@CR23], [@CR30], [@CR32]\]. Janz et al. \[[@CR22]\] compared low-acculturated Latinas to high-acculturated Latinas and non-Latina Whites and found that that low-acculturated Latinas evidenced the poorest QOL. Low-acculturated Latinas reported poorer emotional, physical, and functional well-being on the FACT and more breast-specific concerns relative to non-Latina Whites. Low-acculturated Latinas also reported lower levels of functional and emotional well-being relative to high-acculturated Latinas. Maly et al. \[[@CR30]\] compared low-acculturated Latinas to non-Latina Whites and reported mixed findings regarding level of acculturation and QOL. Findings indicated that less-acculturated Latinas were more likely to experience nausea than non-Latina Whites and less likely to experience pain compared to non-Latina Whites. Less-acculturated Latinas were also less likely to perceive depressive symptom resolution and pain resolution compared to non-Latina Whites \[[@CR30]\]. Of the studies assessing level of acculturation, one indicated that Spanish-speaking Latinas were consistently less likely to report the presence of cancer-related symptoms compared to their White counterparts \[[@CR32]\]. Although not directly tested, Lim et al. revealed that greater acculturation was indirectly related to better mental health among Latinas diagnosed with breast cancer \[[@CR23]\]. The finding that low-acculturated Latinas generally reported poorer mental health contrasts with research in the general Latino population which suggests that longer residence or birth in the United States, versus foreign nativity, are associated with poorer mental health \[[@CR41], [@CR42]\]. In the context of breast cancer, however, low acculturation might be accompanied by lower levels of breast cancer knowledge, greater misconceptions about breast cancer, fatalistic beliefs about cancer, and less information on health resources \[[@CR6], [@CR7], [@CR43]--[@CR45]\]. Due to a potential lack of familiarity with the U.S. medical system and language barriers, Latinas, especially recently immigrated Latinas, may have difficulty navigating the medical system and interacting with health care providers. Studies in the review emphasize the importance of patient-physician relationships among Latinas. A better quality patient-physician relationship, for example, was associated with better QOL among Latinas \[[@CR18], [@CR23]\]. Thus, the racial/ethnic disparities in QOL seen in the current review may be attributed in part to sociodemographic and cancer-related factors, but also likely influenced by barriers related to language and differences in culturally-relevant beliefs and expectations which may shape various aspects of the cancer experience, from coping with the news of diagnosis to interacting with physicians and adjusting to survivorship. More research is needed to shed light on the association between acculturation and QOL among Latina cancer patients and to establish moderating factors in this relationship. Future directions {#Sec14} ----------------- ### Areas for research growth {#Sec15} Studies included in this review were limited in several ways. Few studies assessed sexual health outcomes. Only a limited number of studies included women with metastatic disease, and findings cannot be generalized to this understudied group. Eight of the 20 quantitative studies reported on identical or overlapping samples, which may have inflated patterns of consistent findings across studies. As studies were primarily conducted in large urban areas, generalization to non-urban Latina populations and to specific sub-populations of Latinas (e.g., Cuban American, Mexican American) also requires study. There is limited, existing evidence to suggest that women living in rural areas are at increased risk for poorer QOL relative to women in urban areas \[[@CR46]\]. More research is needed to examine the QOL among Latinas living in rural areas, especially in light of projections that Latinos will become the largest ethnic minority rural group in the United States \[[@CR47]\]. No study assessed major depressive disorder. As future studies continue to assess the QOL of Latina patients, it will be important to distinguish between symptoms and psychological disorders. Latinos in the general population report more depressive symptoms compared to non-Latino Whites \[[@CR48]\]. However, more research is needed to determine whether Latina breast cancer patients are more likely to meet diagnostic criteria compared to non-Latina Whites. The mechanisms through which ethnicity might influence adjustment have not been comprehensively explored. Ethnicity is an atheoretical construct that in itself provides little insight into psychological and physical phenomena, but rather may best be understood in the context of socio-cultural factors that can inform interventions targeted at improving QOL for Latinas \[[@CR49]\]. Latinas' reports of sadness regarding treatment-related changes to their bodies and feelings of incompleteness may constitute one pathway for their compromised QOL \[[@CR36]\]. Therefore, Latinas might be more likely to benefit from reconstructive surgery or from psychosocial interventions to enhance body integrity than non-Latina whites, but more studies are needed to elaborate Latinas' preferences for reconstructive treatment. As another example, a construct of interest in the psycho-oncology literature of particular relevance to the Latino population is spirituality \[[@CR50]\]. Latinas diagnosed with breast cancer report more religious coping than non-Latina White women \[[@CR16], [@CR51]\] and religious and spiritual beliefs have been associated with higher QOL among Latinas \[[@CR52]\]. ### Research design {#Sec16} More longitudinal studies are required to assess change in QOL over time, which would inform healthcare providers of periods during which Latinas might benefit from interventions. Although some studies in the current review were longitudinal, only two studies in this review assessed QOL across time by race/ethnicity, one of which revealed an increase in distress among Latinas, when compared to non-Latina White and Black women, from 6 to12 months after surgery \[[@CR16]\]. Longitudinal studies also can provide valuable information on the relevance of different indicators of QOL throughout the cancer trajectory. For example, distress may be especially prevalent within the first year after diagnosis for Latinas, whereas fear of cancer recurrence may be more pertinent later in cancer survivorship. Not all studies in the review reported average time since diagnosis, and those that did focused on women within the first few years of diagnosis. With the exception of two studies \[[@CR16], [@CR28]\], no study investigated group differences in change in QOL across time, and few studies assessed QOL within the first months following a diagnosis. Additional research on the trajectories of QOL following cancer diagnosis and treatment may provide valuable information as to when Latinas are at greatest risk for poor QOL and may benefit from interventions. ### Measurement {#Sec17} It is important to consider the appropriateness of the measurement tools used in the assessment of QOL among minorities. Measurement tools that have not been validated among Latino populations may produce findings that are difficult to interpret. For example, Yoon et al. \[[@CR32]\] used author-constructed questions to assess QOL and reported that only Spanish-speaking Latinas (versus English-speaking Latinas) were less likely to report any severe physical symptoms compared to non-Latina Whites \[[@CR32]\]. However, 20% of Spanish-speaking respondents answered that they did not know or declined to answer the question for at least one of the five symptoms compared to 5% of Whites and 6% of Blacks, suggesting that Spanish-speaking Latinas may be less familiar with labeling symptoms or may be more uncomfortable endorsing certain symptoms when being interviewed \[[@CR32]\]. More attention to matching the best approach for measuring QOL among Latinas of varying level of acculturation, education, and language proficiency is required. The most commonly employed measures used in the 20 quantitative reports have been validated in Latino populations (e.g., CES-D, SF-36, FACT). Although the CES-D, FACT, and SF-36 have similar psychometric properties for non-Latino Whites and Latinos \[[@CR53]--[@CR55]\], not all research suggests that established measures of QOL are valid in Latino populations, as differences in the factor structure of the CES-D between Latinos and Whites have been observed \[[@CR56]\]. More research is needed to attend to cross-cultural validity of measures. Latino stigma surrounding mental health and additional life stressors experienced by ethnic minorities, for example, may lead to greater reporting of depressive symptomatology through somatic expression than through affective symptomatology \[[@CR57], [@CR58]\]. Among Latina cancer patients, greater somatic complaints may be mistakenly viewed as sequelae of the cancer treatment (e.g., radiation-induced fatigue) rather than as a reflection of depression. Research is needed to elucidate the presentation of depressive symptoms among Latinas with breast cancer. ### Effect sizes {#Sec18} Although most studies noted that Latinas report worse QOL than non-Latinas, these findings do not provide an indication of the magnitude of the effects. Eleven of the 20 quantitative studies provided an effect size (i.e., correlation coefficient, standardized beta, adjusted or unadjusted odds ratio \[OR\]) which varied across quantitative studies. We discuss effect sizes for one study which provided odds ratios comparing Latinas to non-Latina Whites. Maly et al. \[[@CR30]\] reported that, relative to non-Latina Whites, both less and more acculturated Latinas were significantly more likely to report nausea (adjusted OR = 1.79 and 2.09), that less acculturated Latinas were less likely to report pain (adjusted OR = .62), and that less acculturated Latinas were less likely to perceive depressive symptom resolution (adjusted OR = .35). As the literature expands, quantitative approaches such as meta-analysis will be warranted to determine effect sizes across domains of QOL. ### Clinical implications {#Sec19} A general conclusion we draw from this review is that Latinas are likely to benefit from interventions that seek to improve QOL, and studies are needed to determine the efficacy of culturally-tailored interventions which seek to reduce distress and improve QOL for Latinas. Moreover, there is evidence to indicate that Latinas, specifically Spanish-speaking Latinas, would like to receive cancer survivorship-related information about impact on mental health, relationships, and sexual functioning \[[@CR59]\]. To date, stress management interventions for women with breast cancer have proven to be efficacious among samples of predominantly non-Latina White women \[[@CR60], [@CR61]\]. Additional research is needed to determine whether existing interventions for breast cancer patients are equally efficacious among Latinas or whether modifications to existing interventions would benefit Latinas as there are some findings in the literature to suggest that Spanish-speaking Latinas prefer to work with Spanish-speaking Latino health-care providers and would benefit from community-based interventions that consider Latino traditions and beliefs \[[@CR62], [@CR63]\]. In addition, findings from quantitative and qualitative studies suggest that low-acculturated Latinas may benefit from interventions related to enhancing patient-physician communication \[[@CR18], [@CR23]\] and support groups which provide women the opportunity to process the effects of treatment on body image and relationships \[[@CR36]\]. Research is warranted to develop efficacious, culturally-tailored psychosocial interventions for Latina breast cancer patients to reduce cancer-related distress and enhance well-being. Conclusions {#Sec20} ----------- To the best of our knowledge, this is the first systematic review comparing QOL between Latina and non-Latinas with breast cancer. Mounting evidence suggests that Latinas might be at risk for poor QOL outcomes following a breast cancer diagnosis, especially when compared to non-Latina White women. Enhancing the QOL of Latina breast cancer patients requires a multifaceted approach including efforts aimed at improving cancer screening rates to reduce disparities in stage of diagnosis and in treatment, as well as interventions aimed at reducing life disruption and improving well-being. Continuing research in this area will be crucial to this rapidly growing population and the health-care providers with whom they interact. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. A list of key phrases is available from the authors. One study by Tomich and Helgeson (2004) reported racial/ethnic differences in QOL. However, given the small subsample of Latinas, approximately 1% (n = 4), this study was not included. List of variables: 1) study purpose, 2) number of participants, 3) location of study, 4) participants' heritage, 5) age, 6) stage of cancer, 7) cancer treatment, 8) comorbidities, 9) first or recurrent diagnosis, 10) time since diagnosis, 11) health insurance, 12) language spoken, 13) SES, 14) study design, 15) recruitment setting, 16) measures of QOL and corresponding estimates of internal consistencies, 17) data analytic plan, 18) results. 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Introduction ============ Community-acquired sepsis at an early stage is common, but haemodynamic alterations remain unclear. The aim of the study was to characterize cardiovascular alterations in patients of our ProFS (monocentric observational) study, which was to characterize patients with sepsis in the emergency department. Methods ======= Systemic vascular resistance (SVR) and cardiac output (CO) were measured non-invasively using a TaskForce monitor (CNSystems, Graz, Austria) after admission, 24 hours and 72 hours. Indexed values were calculated (SVRI (dyn·second/cm^5^/m^2^), CI (l/minute/m^2^)). Procalcitonin (PCT, ng/ml) was measured in serum. Results ======= A sample of 64 patients of 208 included patients received haemodynamic examination. Mean age was 61.8 ± 18.0 years, 62.7% were male. Patients were divided by PCT \<2 and ≥2. Age, gender and previous medical history were comparable in both groups. The heart rate was 99.8 ± 21.6 vs 104.6 ± 23.0/minute (P= NS) and the mean artery pressure was 89.5 ± 15.6 vs 81.6 ± 21.5 mmHg (P\< 0.01). Mean SVRI in patients with PCT \<2 was 2,934 ± 1,045 vs 2,376 ± 842, P\< 0.05 at the time of admission. No difference was found after 24 hours (2,959 ± 1,002 vs 2,924 ± 1,324, P= NS) and 72 hours (3,123 ± 931 vs 3,556 ± 1,524, P= NS). On the contrary, for patients with PCT ≥2 the increase after 72 hours was significant (P\< 0.05). Differences after admission could not be observed for CI between patients with PCT \<2 vs ≥2. Mean values after admission were 2.7 ± 1.0 vs 2.8 ± 0.8, after 24 hours 2.5 ± 0.8 vs 2.5 ± 0.5, and after 72 hours 2.3 ± 0.7 vs 2.3 ± 0.6 (all: P= NS). See Figure [1](#F1){ref-type="fig"} ![Systemic vascular resistance index and cardiac index in sepsis in the emergency department.](cc8613-1){#F1} Conclusions =========== Patients with community-acquired sepsis in the emergency department had an elevated SVRI. At the time of admission patients with high PCT had a significantly lower SVRI than patients with low PCT. Cardiac index at the time of admission was at a lower limit of normal range in all patients. These findings are in strong contrast to the classic pattern of sepsis on the ICU, where SVRI is keenly reduced and CI elevated. They implicate that patients with sepsis in the emergency department may benefit more from application of fluid and positive inotrope substances than from vasopressor.	{ "pile_set_name": "PubMed Central" }	[ "vs", "find", "posit", "figur", "remain", "serum", "applic", "monocentr", "method", "aim", "vascular", "includ", "classic", "valu", "hour", "calcul", "contrari", "receiv", "lminutem", "minut", "may", "pattern", "contrast", "ngml", "system", "lower", "year", "implic", "use", "graz", "conclus", "resist", "signific", "depart", "p", "result", "mean", "alter", "introduct", "≥", "haemodynam", "svri", "rate", "patient", "measur", "index", "noninvas", "vasopressor", "departmentccf", "admiss", "fluid", "cnsystem", "unclear", "male", "keenli", "normal", "pct", "medic", "observ", "mmhg", "see", "procalcitonin", "rang", "co", "low", "dyn·secondcmm", "sepsi", "emerg", "monitor", "high", "freftypefig", "icu", "limit", "taskforc", "pressur", "ci", "examin", "elev", "age", "divid", "output", "common", "reduc", "strong", "compar", "benefit", "svr", "austria", "sampl", "time", "could", "stage", "ns", "substanc", "cardiovascular", "communityacquir", "significantli", "heart", "prof", "found", "group", "differ", "studi", "±", "gender", "increas", "character", "arteri", "inotrop", "previou", "earli", "histori", "cardiac" ]	22,225	[ "Introduction", "sepsis", "early", "stage", "common", "haemodynamic", "alterations", "remain", "unclear", "aim", "study", "characterize", "cardiovascular", "alterations", "patients", "ProFS", "monocentric", "observational", "study", "characterize", "patients", "sepsis", "emergency", "department", "Methods", "Systemic", "vascular", "resistance", "SVR", "cardiac", "output", "CO", "measured", "using", "TaskForce", "monitor", "CNSystems", "Graz", "Austria", "admission", "hours", "hours", "Indexed", "values", "calculated", "SVRI", "CI", "Procalcitonin", "PCT", "measured", "serum", "Results", "sample", "patients", "included", "patients", "received", "haemodynamic", "examination", "Mean", "age", "years", "male", "Patients", "divided", "PCT", "Age", "gender", "previous", "medical", "history", "comparable", "groups", "heart", "rate", "vs", "P", "NS", "mean", "artery", "pressure", "vs", "mmHg", "P", "Mean", "SVRI", "patients", "PCT", "vs", "P", "time", "admission", "difference", "found", "hours", "vs", "P", "NS", "hours", "vs", "P", "NS", "contrary", "patients", "PCT", "increase", "hours", "significant", "P", "Differences", "admission", "could", "observed", "CI", "patients", "PCT", "vs", "Mean", "values", "admission", "vs", "hours", "vs", "hours", "vs", "P", "NS", "See", "Figure", "fig", "Systemic", "vascular", "resistance", "index", "cardiac", "index", "sepsis", "emergency", "department", "Conclusions", "Patients", "sepsis", "emergency", "department", "elevated", "SVRI", "time", "admission", "patients", "high", "PCT", "significantly", "lower", "SVRI", "patients", "low", "PCT", "Cardiac", "index", "time", "admission", "lower", "limit", "normal", "range", "patients", "findings", "strong", "contrast", "classic", "pattern", "sepsis", "ICU", "SVRI", "keenly", "reduced", "CI", "elevated", "implicate", "patients", "sepsis", "emergency", "department", "may", "benefit", "application", "fluid", "positive", "inotrope", "substances", "vasopressor" ]
Introduction ============ Mitochondria are key eukaryotic organelles involved not only in the well-known synthesis of ATP through oxidative phosphorylation (OXPHOS; [@evw187-B109]; [@evw187-B172]), but also in many other biological functions, such as intracellular signaling, cell differentiation, programmed cellular death, fertilization, and aging ([@evw187-B145]; [@evw187-B162]; [@evw187-B170]; [@evw187-B90]; [@evw187-B98]; [@evw187-B157]; [@evw187-B27]). Currently, it is widely accepted that mitochondria originated from a single endosymbiotic event that took place over a billion of years ago ([@evw187-B66], [@evw187-B67]; [@evw187-B148]; [@evw187-B68]). It has been proposed that the putative ancestor of all mitochondria was an alpha-proteobacterium ([@evw187-B114]; [@evw187-B67]; [@evw187-B54]; [@evw187-B176]; [@evw187-B9]; [@evw187-B3]; [@evw187-B165]; [@evw187-B40]). The following evolution of mitochondria is still unclear and a matter of debate. In animals, mitochondrial DNA (mtDNA) is a small (∼16 kb) and compact circular molecule that typically contains 13 OXPHOS-related genes, 2 rRNAs encoding for the two subunits of mitochondrial ribosomes, and an array of tRNAs used for translation within the organelle ([@evw187-B16]; [@evw187-B21]). Most genes of the original endosymbiont were therefore lost or have been transferred to the nucleus during a process called Genome Reductive Evolution (GRE) ([@evw187-B8]; [@evw187-B83]; [@evw187-B58]; [@evw187-B81]). As claimed by [@evw187-B103], in mammals no less than 1,500 proteins are needed to keep mitochondria alive and working. Why then only a small cluster of protein coding genes (PCGs) was spared by GRE---the typical 13 in metazoans, and even down to 3 in apicomplexans ([@evw187-B52]; [@evw187-B134])? Many tentative answers were proposed to this question. It is well known, for example, that mitochondria employ a genetic code slightly different from that of the nucleus, which would possibly lead to erroneous translations of some genes if transferred to nucleus ([@evw187-B4]); however, this is true for animals, but not for plants ([@evw187-B79]), which also underwent mitochondrial GRE. The high hydrophobicity of some of these proteins would hamper the import from the cytosol as well ([@evw187-B171]; [@evw187-B129]; [@evw187-B29]; [@evw187-B124], [@evw187-B123]; [@evw187-B38]; [@evw187-B55]; [@evw187-B4]). [@evw187-B101] also claimed that certain gene products are toxic in the cytoplasm. Finally, it has been proposed that the genes still encoded by mtDNAs must be efficiently and directly regulated by mitochondrial redox conditions, so they need mandatory colocation for redox regulation (CoRR) ([@evw187-B133]; [@evw187-B5], [@evw187-B6]; [@evw187-B91]). The sequencing and characterization of several complete mitochondrial genomes is a key to address GRE of mitochondria. Pioneering comparative works of large mtDNA datasets could recently, e.g., shed light on the reduction and ultimate loss in animals' mitochondria of ribosomal protein genes ([@evw187-B100]) and establish that the mitochondrion-encoded genes of almost all eukaryotes are different subsets of the mitochondrial gene complement of jakobids, a group of free-living, heterotrophic flagellates ([@evw187-B81]). At the same time, mitochondrial genomes of single metazoan groups often exhibit peculiar evolution that deserves a careful examination. In this regard, bivalves (Mollusca, Bivalvia) are among the most notable animal taxa, because of several interesting features. Most strikingly, some bivalves follow a non-canonical way of mitochondrial inheritance, the Doubly Uniparental Inheritance (DUI; [@evw187-B153], [@evw187-B154]; [@evw187-B187], [@evw187-B188]). In these species, two separate sex-linked mitochondrial lineages (and their respective mtDNAs) are present, namely, the F and the M ([@evw187-B20]; [@evw187-B121]; [@evw187-B189]; [@evw187-B21]). F mitochondria are passed from the mother to the complete offspring, and they are typically found in the soma and in the female germline (eggs); the M mitochondria are passed from the father to the male offspring, and they are typically found in the male germline (sperms). However, these general DUI rules may hold imperfectly, thus resulting in a leakage of the M mitochondrial lineage in somatic cells of either sex ([@evw187-B26]; [@evw187-B11]; [@evw187-B89]; [@evw187-B59]; [@evw187-B118]). DUI represents a case of a triple genomic conflict (nucleus vs. F mtDNA; nucleus vs. M mtDNA; F vs. M mtDNAs) ([@evw187-B121]; [@evw187-B21]). There are more outstanding features of bivalves mtDNA that are generally shared across the entire class. In fact, bivalve mitochondrial genomes are often very large, up to the 46,985 bp of Scapharca broughtonii ([@evw187-B96]); they present many putative unassigned regions (URs) ([@evw187-B58]); they may encode for supernumerary open reading frames (ORFans) ([@evw187-B18]; [@evw187-B105]); they show an unpaired degree of gene rearrangement ([@evw187-B169]; [@evw187-B150]; [@evw187-B127]); they exhibit strong differences in strand usage (see Additional file 6 in [@evw187-B127]). All this considered, the interest of bivalves in the wider picture of the evolution of animal mitochondria is self-evident. In this work, we present the first meta-analysis of 100 bivalve mtDNAs, corresponding to all the species whose mtDNA was available in GenBank in August, 2014. The polyphyly of bivalves in molecular phylogenetic reconstructions is a well-known artifact ([@evw187-B61]; [@evw187-B60]; [@evw187-B1]; [@evw187-B2]; [@evw187-B147]; [@evw187-B127]; [@evw187-B160]; [@evw187-B14]) and it is probably related to the fact that a small group of bivalves (the protobranchs) separated very early and, regarding mitochondrial markers, before the burst of genomic novelties described above ([@evw187-B127]). In the present work, we do not address the issue of bivalve monophyly: therefore, the only protobranch bivalve whose complete mitochondrial genome is available in GenBank in August, 2014 is used as outgroup for phylogenetic analyses. The comparative mitogenomics of bivalves is critically explored to (i) achieve a deeper knowledge about factors that shaped mitochondrial evolution in these metazoans, and (ii) focus on the specific features of bivalve mitochondrial PCGs. Materials and Methods ===================== The Database ------------ Hundred bivalve complete mitochondrial genomes ([supplementary file S1](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online) were downloaded from GenBank in August, 2014 using CLC Sequence Viewer 7.5 (Qiagen A/S, Aarhus). Annotations and sequences were imported and managed in Microsoft Excel^®^ 2007 through custom functions and VBA macros that were also used to output files in the correct format for downstream analyses. In August, 2014 the annotation of Tegillarca granosa ([@evw187-B161]) was already published, but the sequence was not available yet; the sequence of Nucula nucleus (GenBank accession number EF211991) is still incomplete and unpublished: as a consequence, these species were included in the overall analysis, but not in alignments presented in this paper. Alignment Algorithms -------------------- The first step of most analyses was a structural alignment and data masking: to this purpose, a custom tool was written in bash and R ([@evw187-B132]) environments, loading the package seqinr ([@evw187-B28]). The atp8 gene and tRNAs were excluded from alignments beacuse of many uncertainties in annotations. This tool was called masking_package and is available with a brief tutorial from the GitHub repository <https://github.com/mozoo/masking_package.git>. It performs: structural alignment using T-Coffee ([@evw187-B117]) and nested packages PSI-BLAST ([@evw187-B7]), Muscle ([@evw187-B49]), ProbconsRNA ([@evw187-B41]), RNAplfold ([@evw187-B99]), and MAFFT ([@evw187-B82]), using the option series PSI-Coffee \> Expresso \> accurate for Protein Coding Gene (PCG) amino acids and the MR-Coffee mode for rRNA nucleotides;alignment masking in order to eliminate phylogenetic noise using the four softwares Aliscore 2.0 ([@evw187-B108]), BMGE 1.1 ([@evw187-B35]), Gblocks 0.91b ([@evw187-B25]), and Noisy ([@evw187-B44]), setting adequate options for distantly related sequences;comparison of outputs from different masking strategies in terms of number of sites and percentage of removed sites and output of a final concatenated alignment where only sites kept by at least k softwares are present (where k is up to the user and was set to 2, 3, or 4);computation of the number of sites selected by all the possible combinations of the four softwares. All the masking_package scripts adapted for amino acid (PCGs) and nucleotide (rRNAs) alignments of the present study are available as [supplementary files S2 and S3](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online, respectively. Split rrnL genes of ostreids were concatenated prior to alignment. Moreover, all Crassostrea species (with the exception of C. virginica) have a duplicated rrnS gene. In these cases, the two copies were aligned with MR-Coffee, a consensus sequence was computed using seqinr and the result was inserted into the final alignment. Genomic Features ---------------- Five large-scale features of bivalve mtDNAs were extracted: length, percentage of URs, number of genes (including newly annotated ones; see below), and A-T and G-C skew following [@evw187-B138]. Instead of a typical ANOVA, because of non-normality and non-homoscedasticity of data, differences between subclasses had to be tested using a non-parametric alternative: we used the Kruskal--Wallis test ([@evw187-B88]), while relative pairwise comparisons were carried out using the Dunn\'s test ([@evw187-B48]). Given the poor sample size of Anomalodesmata (N = 1) and Opponobranchia (N = 2), these subclasses were excluded from these preliminary analyses. Afterwards, single alignments were used as input for several custom bash and R scripts that were used to: subsample the original dataset, producing subsets corresponding to largest families (Mytilidae, Ostreidae, Pectinidae, Unionidae, Veneridae) and subclasses (Heterodonta, Palaeoheterodonta, Pteriomoprhia) to be aligned and masked;compute the percentage of cases in which each site of each gene was kept after the masking phase, using the agreement of at least 3 out of 4 masking softwares (across the five families, the three subclasses, and in the complete-dataset analysis);comparing masking results using a one-sided t-test;back-translate the original, unmasked amino acid alignment into nucleotides;use EMBOSS ([@evw187-B139]) to compute different metrics of pairwise and overall distances, both on total gene length (using distmat) and over a sliding window (using plotcon);compare nucleotide and amino acid distances through the Kolmogorov--Smirnov test;test alignments for saturation by plotting uncorrected p-distance over the Jin--Nei distance (for nucleotides; [@evw187-B77]) or the Kimura distance (for amino acids; [@evw187-B84]);perform a Principal Component Analysis (PCA) on concatenated distance matrices, using the FactoMineR ([@evw187-B94]) package for computations and ggplot2 ([@evw187-B175]) for graphics. The ratio of non-synonymous vs. synonymous substitutions (d~N~/d~S~) was computed for back-translated alignments using PAML 4.8a ([@evw187-B183], [@evw187-B184]); d~N~/d~S~ was computed both for each pair of sequences and along the phylogenetic tree (see below). In the latter case, we used the Likelihood Ratio Test (LRT) to compare the fitting of single d~N~/d~S~ for the complete tree with the use of 12 different d~N~/d~S~, allowing different d~N~/d~S~ ratios for different clades. All LRTs were carried out in the R environment; to be conservative, we used a chi-square distribution with 11 degrees of freedom ([@evw187-B177]). Phylogenetic Analyses --------------------- Using the original annotations available in GenBank, phylogenetic analysis was carried out on PCGs and rRNAs; given many uncertainties in annotation and orthology, the atp8 gene and tRNAs were excluded from the analysis. The best-fitting partitioning scheme and molecular evolution models were estimated using PartitionFinderProtein and PartitionFinder 1.1.0 ([@evw187-B92]) under the Bayesian Information Criterion and a greedy approach. Gaps were coded following the simple indel method of [@evw187-B152] as implemented in GapCoder ([@evw187-B185]). As a result, the final alignment spanned over 14 genes and three types of data: amino acids for PCGs, nucleotides for rRNAs, and binary data for coded gaps. The software RAxML 8.2.0 ([@evw187-B159]) was used to infer Maximum Likelihood (ML) phylogeny. The strand usage is variable across different bivalve mtDNAs: while in most cases all genes locate to the same strand, genes are evenly distributed on both strand in N. nucleus, S. velum, and Unionidae. As a strand usage bias can be associated with a compositional bias, which may in turn lead to phylogenetic artifacts, preliminary analyses with 500 bootstrap replicates were separately conducted on those genes mapping on the "+" and on the "−" strand of unionids. If a significant compositional bias is working, we expect to detect possible phylogenetic artifacts by recovering different topologies from the two analyses. The final tree was computed as follows; explicit RAxML commands are listed in [supplementary file S4](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. five randomized maximum parsimony (MP) starting trees were generated;a maximum likelihood (ML) tree was inferred from each MP starting tree with a fixed rearrangement radius of 10 and with an automatically determined one, choosing the setting yielding the best results;a ML tree was inferred from each MP starting tree with the selected initial rearrangement option under different number of rate categories (10, 25, 40, or 55) for the CAT model accounting for evolutionary rate heterogeneity ([@evw187-B158], choosing the setting yielding the best results;the best-known likelihood tree was inferred from the original alignment under the selected rearrangement and rate categories options calling 10 runs from 10 randomized MP starting trees;500 bootstrap replicates were run; andconfidence values were computed from bootstrap replicates and annotated on the best-known likelihood tree. The software MrBayes 3.2.1 ([@evw187-B140]) was used to carry out Bayesian Inference (BI) using 2 separate runs, 4 chains, and 10,000,000 generations of MC^3^, sampling every 100 trees. Convergence between runs and burn-in were estimated looking to standard deviation of average split frequencies sampled every 1,000 generation and to Potential Scale Reduction Factor (PSRF) ([@evw187-B57]). The best-known likelihood tree computed by RAxML was used to obtain a time-scaled chronogram using r8s 1.70 ([@evw187-B144]) and the r8s bootstrap kit by Torsten Eriksson (downloaded from <https://github.com/TorstenEriksson/r8s-bootstrap> in February, 2015). First appearance data for six calibration point were downloaded from the Paleobiology Database (<http://fossilworks.org>) in February, 2015: the root of all bivalves, Mytilidae, Ostreidae, Pectinidae, Unionidae, and Veneridae ([@evw187-B102]; [@evw187-B166]; [@evw187-B36]; [@evw187-B47]; [@evw187-B115]; [@evw187-B17]; [@evw187-B10]; [@evw187-B65]; [@evw187-B23]; [@evw187-B104]; [@evw187-B87]; [@evw187-B116]). We used the Penalized Likelihood method and the truncated Newton algorithm; the cross-validation approach allowed us to estimate the best smoothing parameter up to the sixth decimal digit and five restarts and five guesses were used each round. Following the r8s bootstrap kit procedure, 163 bootstrap replicates of the original alignment were generated and the original tree was optimized on each of them; node ages were estimated under the same r8s setting, estimating the best smoothing parameter up to the second decimal digit. The 99% confidence intervals of each node age were computed using custom R script loading the package ape ([@evw187-B120]) and following methodological recommendations of [@evw187-B76], i.e., type = 8. Trees were graphically edited using the software PhyloWidget ([@evw187-B78]) and Dendroscope 3.3.2 ([@evw187-B75]). Phylogenetic Informativeness (PI) was investigated on the amino acid dataset using the PhyDesign portal ([@evw187-B97]) and Rate4Site ([@evw187-B130]) to estimate site-specific evolutionary rates. Best-fitting amino acid evolutionary models were selected using ProtTest 3.4 ([@evw187-B39]) and PhyML ([@evw187-B70]). PI was computed over five different epochs, whose boundaries were taken from the International Stratigraphic Chart v2015/01 ([@evw187-B30]): Quaternary, "Cenozoic" (Paleogene + Neogene), Mesozoic, "Paleozoic" (from Ordovician to Permian), and "Cambrian" (from 520 to 485.4 Mya). Following [@evw187-B167] and [@evw187-B151], the phylogenetic signal and noise analysis was carried out in a state space of five. Finally, we investigated the correlation between gene rearrangements and substitution rates, which has been demonstrated for insects by [@evw187-B146] and hypothesized for mollusks by [@evw187-B160]. However, the RGR test of [@evw187-B43] and its modified version of [@evw187-B182] cannot be useful in the case of bivalves to describe gene arrangement variability because it is a relative rate test that involves a comparison with the ancestral gene arrangement. As detailed in [@evw187-B127], this is most likely that of the chiton Katharina tunicata, but, with the notable exception of Solemya velum and Nucula nucleus ([@evw187-B127]), most bivalve gene orders are not comparable with each other and seem to be equally very different from that. Therefore, while there is no remarkable difference in terms of distance from the ancestral state, higher rates of mitochondrial gene rearrangement are straightforward in some clusters with respect to others, but this would not be detected by RGR: for this reason, we quantified rates of mitochondrial rearrangement in the clades obtained in the best-known likelihood tree using the architecture rate (AR) as introduced by [@evw187-B62]. This is given by $$AR = \frac{N_{GA} - 1}{N_{OTU} - 1}$$ where N~GA~ is the number of different gene arrangements found in a clade and N~OTU~ is its number of OTUs. In fact, this index conservatively estimates the number of gene rearrangement events along a clade\'s evolutionary history, as it must be at least as large as the number of different gene arrangements found in extant taxa, and normalizes it with the number of species, so that clades of different sizes are comparable. AR rate ranges from 0 (the whole clade shares the same gene arrangement) to 1 (each OTU shows a different gene arrangement). The sum of branch lengths for a given clade was computed with the software Phylocom 4.2 ([@evw187-B174]), thus estimating the total number of expected substitution per site in a given cluster; this was divided by the root age in millions of years (see above). The correlation of AR rate and substitution rate was assessed with the Spearman\'s rho and Kendall\'s tau tests using R. Annotation of the atp8 Gene and of ORFans ----------------------------------------- The EMBOSS suite was used to find all the possible Open Reading Frames (ORFs) in all the complete mitochondrial genomes, using all the six possible frames. A Hidden Markov Model (HMM) was constructed for each ORF using HHblits 2.0 ([@evw187-B135]) and the latest PDB70 release. All the HMMs were merged in a custom database, which was called Biv_mtDNA_ORFs. All the annotated atp8 genes were aligned using the T-Coffee (again following the PSI-Coffee \> Expresso \> accurate option series). A Hidden Markov Model (HMM) was constructed using HHblits 2.0 and the latest Uniprot release and a HMM-HMM alignment was run against the Biv_mtDNA_ORFs database to check for homology with atp8. Positive results were manually screened and original annotations were consequently updated. Bivalve supernumerary mitochondrial ORFs do not have known homologs and are therefore considered as ORFans ([@evw187-B53]). However, it is possible to find homologies at least within Bivalvia. All the published supernumerary ORFs ([@evw187-B18]; [@evw187-B105]; [@evw187-B127]; [@evw187-B13]; GenBank accession numbers HM856636, HQ283344, KC848655, KF030963, NC_015310, NC_015476, NC_015477, NC_015479, NC_015481, NC_015483, NC_018763, NC_022803, NC_023250, NC_023942) were annotated; a HHblits database of all the known ORFans was created as above using Biv_mtDNA_ORFs and was called Biv_mtDNA_ORFans; finally, HHblits was used for each ORFan against this custom database to investigate putative relationships between known ORFans. Summarizing Data ---------------- A PCA was carried out as above using many different parameters ([supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online): nucleotide composition; AT content; A-T and G-C skews; use of between-genes overlapping nucleotides; length; percentage of Unassigned Regions (URs); total number of genes; the strand usage skew (SU skew); number of truncated T--/TA- stop codons in the genome; the Amount of Mitochondrial Identical Gene Arrangements (AMIGA). SU skew and AMIGA were defined as follows. $$SUskew = \frac{H - L}{H + L}$$ where H is the number of genes on the H strand and L is the number of genes on the L strand. If the strand usage is perfectly balanced (i.e., H = L), the SU skew is equal to 0; a negative SU skew indicates a bias towards the L strand, while a positive SU skew indicates a bias towards the H strand. $$AMIGA = \frac{N_{IGA} - 1}{N - 1}$$ where N~IGA~ is the number of taxa in the analyzed sample that share an Identical Gene Arrangement and N is the total number of taxa. As a consequence, unique gene arrangements will have an AMIGA score equal to 0. Conversely, when all the taxa in a given sample share the same gene arrangement, each AMIGA score will be equal to 1. An intermediate value expresses an intermediate value of conservation. Given many uncertainties in annotations, rRNAs and tRNAs were excluded from the analysis, therefore the AMIGA index relies solely on PCGs. The HERMES Index ---------------- A widespread method of quantifying molecular evolution of mitochondrial genomes in different species and clusters is still lacking for mitogenomic analyses. We developed a new index in this regard, which relies on maximum likelihood factor analysis to summarize different measures that are typically found to be linked with evolutionary rates; it is intended to be computed a posteriori, i.e. after the phylogenetic and genomic analysis. As different empirical measures are merged together in a single score, this is a "hyper-empirical" index. Moreover, a taxon retaining most genomic plesiomorphies of the group (at least following state-of-art knowledge) is selected as a benchmark: thus, it is a relative measure, and that taxon was in our case N. nucleus. All this considered, the index was called Hyper-Empirical Relative Mitochondrial Evolutionary Speed (HERMES) index. We explored the use of different subsets of the following variables to compute the HERMES index: the AT content;the genome length;the number of (annotated) genes;the percentage of URs;the absolute value of SU skew;AMIGA;the root-to-tip distance computed on the best-known likelihood tree using Phylocom 4.2;the ML distance from N. nucleus computed with RAxML specifying the model as above. The factor analysis was carried out using the psych ([@evw187-B137]) package of R; the plot was prepared using the ggplot2 package. Normalization and varimax rotation were used, factor scores were found using correlation preserving, and correlations were found using the Pearson method; given the possible presence of a missing value (as T. granosa was not included in the phylogeny and we were therefore unable to compute either the root-to-tip and the ML distance), missing data were set to be imputed using the median. All the variables were pooled together for each species into the value of a single loading: we define this score as the HERMES score of a given species. The best-performing variable set and the goodness-of-fit of the analysis was assessed following the recommendations of [@evw187-B74]: Tucker--Lewis Index (TLI) ([@evw187-B168]) \> 0.95; root mean square of the residuals (SRMR) \< 0.08; root mean squared error of approximation (RMSEA) \< 0.06; moreover, the Kaiser--Meyer--Olkin index (KMO) ([@evw187-B80]) was taken into account on this regard. A Python script was written to compute the HERMES scores of a given assemblage of species, providing the GenBank annotation, gene alignments and a phylogenetic tree; this software can be downloaded from the GitHub repository at the URL <https://github.com/mozoo/HERMES.git>, along with sample data and a tutorial. Results ======= Overall Genomic Features ------------------------ The 100 mitochondrial genomes (mtDNAs) of the present study range from 14,622 (Lanternula elliptica) to 46,985 (Scapharca broughtonii) bp in length. Excluding the abnormally large genomes of Arcidae, the longest mtDNA is that of Placopecten magellanicus (32,115), followed by the female genome of Venerupis philippinarum (22,676 bp). Conversely, the proportion of putatively Unstranslated Regions (URs) span from 1.44% (L. elliptica) and 2.13% (S. velum) to the high scores of Pectinidae (29.86--51.04%) and Arcidae (52.30--70.76%). The number of annotated genes on the molecule is much more stable: exception made for Scapharca kagoshimensis and S. broughtonii (55 and 54, respectively), this number span from 30 (Mizuhopecten yessoensis) to 46 (P. magellanicus), with a mean value of 38.11 ± 3.20. As summarized in [figure 1](#evw187-F1){ref-type="fig"}, mtDNA length and %UR increases in the order Palaeoheterodonta \< Pteriomorphia \< Heterodonta. Palaeoheterodonta are in both cases significantly different from either Pteriomorphia or Heterodonta (P \< 0.001\\\; [supplementary file S6](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). The Kruskal--Wallis test detected a significant location difference also for the number of annotated genes, which is due to the Pteriomorphia/Heterodonta comparison only (P* \< 0.05\; [supplementary file S6](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Finally, each subclass was significantly different from each other when considering A-T and G-C skews---again, comparisons involving Palaeoheterodonta showed the highest significance values (P* \< 0.001\\\; [supplementary file S6](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Single-species data used in this study are extensively listed in [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. F[ig]{.smallcaps}. 1.---Main features of mitochondrial genomes. Five large-scale mitochondrial features are compared across four bivalvian subclasses (the fifth subclass, Anomalodesmata, was excluded because only one mitogenome was available): Op, Opponobranchia (purple); Pa, Palaeoheterodonta (blue); Pt, Pteriomorphia (red); He, Heterodonta (green). Length, median length of the genome; %UR, median percentage of Untranslated Regions; Annotated genes, mean number of annotated genes; A-T skew, median A-T skew; G-C skew, median G-C skew. Differences between subclasses were tested using the Krukal--Wallis and the Dunn\'s test; Anomaldesmata and Opponobranchia were excluded because of low sample size (N* = 1 and N = 2, respectively). Asterisks above columns refer to levels of significance in both pairwise comparisons or in a single one (bracketed); \*P \< 0.05; \\P \< 0.01, \\\*P \< 0.001. Notwithstanding the large variability, especially in length and %UR, the genome content is quite stable: if we do not take tRNAs into account, all the mtDNAs share the same gene content and few duplication events are present. The most evident duplication is that of the rrnS gene in all species of the genus Crassostrea (with the exception of C. virginica), whose genes show a divergence between 0.11% (C. iredalei) and 5.05% (C. ariakensis), as estimated using uncorrected p-distance (mean = 2.92%). Moreover, as already signaled ([@evw187-B106]; [@evw187-B136]), in all Ostreidae species the rrnL gene is split in two separate fragments. Finally, a duplication of cox2 was already signaled in Musculista senhousia, male type ([@evw187-B122]), and V. philippinarum, female type (GenBank Accession Number AB065375). Alignments ---------- Protein Coding Genes (PCGs) alignment lengths range from 186 amino acids of nad3 to 1,313 amino acids of cox2 (before masking). The amount and percentage of sites selected by at least 2, 3, or 4 softwares is detailed in [supplementary file S7](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. The use of three masking softwares out of four is a good compromise between the elimination of noise and the elimination of apparently noisy useful sites, therefore this was taken as our preferred setting. All alignments are available from FP upon request. The masking step affected different genes differently: cox1 and cytb are the genes that were least affected during masking phase (68.63% and 60.13% of their sites were kept, respectively), while cox2, nad4L, and nad6 alignments were heavily reduced after this phase (to the 16.45%, 18.32%, and 21.73% of the original size, respectively). As expected, rRNA alignments are much longer (4,210 and 2,891 bp for rrnL and rrnS) and much more shortened after the masking phase (to the 14.06% and 11.45% of the original size, respectively). The amount of shortening was not reduced when smaller, more-related subsets were analyzed ([supplementary file S8](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Differences in the percentages of kept sites among different subsets were never significantly higher when moving towards a smaller, nested taxon, but rather in some cases they were significantly lower (Bivalvia \> Pteriomorphia, P \< 0.001\\\; Palaeoheterodonta \> Unionidae, P* \< 0.05\; Pteriomorphia \> Pectinidae, P* \< 0.01\\). Actually, in all the cases, phylogenetically-informative sites are almost the same, being the dataset a single family, a subclass, or the whole class ([fig. 2](#evw187-F2){ref-type="fig"}). The longest conserved domains are those of cox1 and cytb, even if also nad genes (with the exception of nad4L and nad6) show extended well-aligned regions. Interestingly, nad4 is the only case of a conserved domain that appears with reduced and less variable alignments: about 120 amino acids at the N-terminus of the protein are not conserved when the complete dataset is considered, but are increasingly more conserved at the subclass and family level ([fig. 2](#evw187-F2){ref-type="fig"}). The similarity measure of plotcon over a sliding window show highest values for the most conserved regions, as expected ([supplementary files S9 and S10](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). F[ig]{.smallcaps}. 2.---Conserved sites of protein coding genes. Single genes were aligned, masked using four different approaches, and finally only sites selected as phylogenetically informative by at least three softwares were kept. Whenever possible in terms of sample size, the same procedure was carried out for reduced datasets, corresponding to single subclasses (3 datasets) and single families (5 families). Each chart shows the proportion of datasets keeping single sites: this is 0 (discarded site) or 100 (kept site) at the class level, and ranges from 0 to 100 at the subclass and family level, because more datasets are available. Uncorrected (p-) distances are slightly, but significantly (P = 0), higher for amino acids than for nucleotides; however, when a more complex model is used to account for multiple substitution events at a single site, the situation is the opposite, and nucleotide distances are significantly (P = 0) higher than amino acid ones ([fig. 3](#evw187-F3){ref-type="fig"}). Moreover, with the exception of the low-scoring cox1, all uncorrected distances are comparable among genes, while using the Jin--Nei/Kimura method (for nucleotides/amino acids, respectively), some genes (namely, atp6, nad2, nad4L, and nad6) have higher distance values than others ([fig. 3](#evw187-F3){ref-type="fig"}). A similar scenario is retrieved through saturation plots. The uncorrected distance was plotted on Jin--Nei/Kimura distance: while in many cases uncorrected distances tend to increase with the other model, the same four genes show a plateau, indicating that p-distance is not able to uncover multiple hits and that a significant degree of saturation is present. As an example, cytb and nad6 plots are shown in [figure 4](#evw187-F4){ref-type="fig"}, while all genes are detailed in [supplementary file S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. Expectedly, aminocid (aa) plot is farther from the plateau than nucleotide (nt) plot in all cases. F[ig]{.smallcaps}. 3.---Nucleotide and amino acid genetic distances. The mean genetic distance across the complete dataset ± one standard deviation is shown for each PCG; brown, nucleotides; green, amino acids. Differences in distance distributions were tested using the Kolmogorov--Smirnov test; \\\*P \< 0.001. F[ig]{.smallcaps}. 4.---Example of four saturation plots. Pairwise p-distance is plotted over Jin--Nei/Kimura distance for nucleotides (above)/amino acids (below), respectively; nt, nucleotides; aa, amino acids. All saturation plots are available as [supplementary file S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. A PCA was carried out using pairwise nucleotide and amino acids distances as variables for each gene; the result is shown in [figure 5](#evw187-F5){ref-type="fig"}. Taken together, the first two principal components explain the 85.64% of the variance, but it has to be noted that the first component alone explains the 77.62%. The PCA (and specifically the first component) clearly separates atp6, nad2, nad4L, and nad6 from other genes. F[ig]{.smallcaps}. 5.---Distance PCA. Single-gene pairwise distance matrices were used as input data in order to investigate differences between PCGs. Finally, the d~N~/d~S~ ratios for each gene are given in [table 1](#evw187-T1){ref-type="table"}. In all cases, the null hypothesis that a single d~N~/d~S~ applies to all the tree branches was not rejected by the LRT (P = 1); the d~N~/d~S~ value computed along the entire tree is one or two orders of magnitude higher than the median of pairwise comparisons ([table 1](#evw187-T1){ref-type="table"}). The highest values are shown by nad6 (d~N~/d~S~ = 0.344; median of pairwise comparisons = 0.045), while the lowest are shown by cox1 (d~N~/d~S~ = 0.080; median of pairwise comparisons = 0.004). However, namely in atp6, nad4L, and nad6, a quite large number of high (i.e., greater than 10) pairwise d~N~/d~S~ values were computed ([table 1](#evw187-T1){ref-type="table"}). In the vast majority of cases, these values expectedly come from pairs of distantly related species. Table 1Values of d~N~/d~S~Genesd~N~/d~S~Pairwise mediand~N~/d~S~ \> 10atp60.2550.023 ± 6.03041cox10.0800.004 ± 1.4361cox20.2220.008 ± 1.4361cox30.1550.009 ± 0.0270cytb0.1640.008 ± 0.0170nad10.1620.009 ± 0.0260nad20.3100.019 ± 2.9499nad30.2160.009 ± 1.4612nad40.2300.010 ± 0.0220nad4L0.3120.032 ± 8.44167nad50.2520.008 ± 0.0200nad60.3440.045 ± 11.223103[^2] Phylogenetic Analysis --------------------- The two subsets of markers (i.e., genes that are on the "+" strand and genes that are on the "−" strand of Unionoidea) yielded the same topology in the preliminary analyses, therefore the final tree was computed on the complete set of genes. The best partitioning scheme selected by PartitionFinder separated atp6+nad genes, cytochrome subunit genes, and rRNAs (which were taken together; see [supplementary file S12](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online, for details). The best-known likelihood tree annotated with support values is shown in [figure 6](#evw187-F6){ref-type="fig"} over a geological timescale, while original phylograms are shown in [supplementary files S13](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online (best-known likelihood tree with bootstrap proportions) and 14 (Bayesian consensus tree). Both Bootstrap Proportions (BPs) and Bayesian Posterior Probabilities (PPs) are generally high: as BP is typically lower than PP, we conservatively show the consensus tree after collapsing nodes with BP \> 70. After the separation of the outgroup S. velum, the tree is divided into two major branches: Palaeoheterodonta (BP = 100, PP = 1.00) and Amarsipobranchia sensu [@evw187-B125] (BP = 97, PP = 1.00). Within Palaeoheterodonta, M and F genomes (if non-DUI species genomes are considered as F ones) cluster separately, both with BP = 100/PP = 1.00; the branching order is also the same. Within Amarsipobranchia, relationships between L. elliptica (Anomalodesmata), Pteriomoprhia (BP = 91, PP = 1.00), and Heterodonta (BP = 73, PP = 1.00) are not resolved. Within Pteriomorphia, Mytilidae (BP = 100, PP = 1.00) are the sister group of all remaining pteriomorphians (BP = 79, PP = 1.00). With the exception of M. californianus M, DUI genomes of Mytilus spp. cluster by sex, as in Palaeoheterodonta, while M. senhousia genomes cluster by species. Within Heterodonta, Lucinindae (Loripes lacteus+Lucinella divaricata, BP = 100, PP = 1.00) are the sister group of all remaining heterodonts (BP = 100, PP = 1.00). All heterodont DUI genomes (i.e., M. lamarckii and V. philippinarum) cluster by species. F[ig]{.smallcaps}. 6.---The mitochondrial phylogeny of bivalves. Ultrametric tree of 98 bivalve species computed using r8s starting from the best-known likelihood tree computed with RAxML and the consensus Bayesian tree. RAxML bootstrap values/MrBayes posterior probabilities are printed at each node (an asterisk means a bootstrap of 100 and a posterior probability of 1.00). Below the tree the phylogenetic informativeness for each PCG is shown. The geological timescale in Mya follows [@evw187-B30]. The main split of the class (Palaeoheterodonta, one side, and Amarsipobranchia, the other side) took place about 500 million years ago (Mya), with a confidence interval (CI) of about 4.5 million years (My). The origin of pteriomorphians is dated at 484 mya (CI = 6 My), while the origin of heterodonts is dated slightly later, 454 mya (CI = 16 My). Oldest families are those of mytilids (421 Mya), pectinids (384 Mya), and venerids (336 Mya). All details about time calibration, including node names, mean estimates across bootstrap replicates, and CIs are shown in [supplementary files S15 and S16](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. Different genes have different Phylogenetic Informativeness (PI) scores for different periods ([fig. 6](#evw187-F6){ref-type="fig"}). Most nad genes and the cytb gene reach their peak in the Cenozoic, while cox genes and atp6 reach it in the Cretaceous. Notably, the peak of cox1 informativeness is shifted back to the Jurassic-Cretaceous limit and decreases slowly towards the deeper past. In noise vs. signal analysis ([supplementary file S17](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online) cox1, cytb, nad1, and nad5 typically outperformed all the other markers, although the complete dataset always shows much higher values than single genes. The AR and substitution rates ([supplementary file S18](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online) showed a good correlation when six clades were used, corresponding to the major clades of the tree (Palaeoheterodonta F, Palaeoheterodonta M, Mytilidae, other Pteriomorphia, Lucinidae, other Heterodonta). The value of Spearman\'s rho was 0.90 (P \< 0.01\\) and that of Kendall\'s tau was 0.83 (P \< 0.05\). Annotation of the atp8 Gene and of ORFans ----------------------------------------- Twenty atp8* genes were annotated using the HHblits approach; they are listed in [table 2](#evw187-T2){ref-type="table"}. All the newly annotated atp8 had a homology probability \> 95%, E-value \< 0.01, and P-value \< 1 × 10^−6^. The only exceptions to these are the atp8s in Paphia euglypta (Prob. = 92.7%, E-value = 0.071, P-value = 4.8 × 10^−6^), Acanthocardia tuberculata (Prob. = 86.6%, E-value = 0.46, P-value = 3.1 × 10^−5^), and Musculista senhousia M (Prob. = 80.6%, E-value = 0.74, P-value = 5.0 × 10^−5^): as the atp8 gene was not annotated in these species, it is highly probable that the homology is correct, but they anyway should be regarded as only tentatively annotated; the complete list of HHblits scores is available as [supplementary file S19](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. The annotation of atp8 led us to make small changes to the original GenBank annotations; these, along with other corrections of minor flaws that we detected during the analyses, are listed in [supplementary file S20](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online, where the complete, updated annotations of all the present mtDNAs are given. Table 2Newly annotated atp8 genesSpeciesStartStopLengthaaMytilus galloprovincialis M8,5308,871345115Mytilus edulis M9,78910,133345115Mytilus californianus F8,7359,037303101Mytilus galloprovincialis F8,8029,06226488Mytilus edulis F10,39610,65626488Mytilus californianus M8,2628,570312104Ruditapes philippinarum M4,6304,75212642Arctica islandica10,34310,49315150Semele scabra11,96912,09712943Solecurtus divaricata11,32111,45213545Nuttallia olivacea12,93013,05813244Soletellina diphos11,21411,34213244Mimachlamys nobilis7,9378,08615351Moerella iredescens11,62511,75313244Meretrix petechialis8,5328,66914147Meretrix meretrix8,5328,66914147Ruditapes philippinarum F5,9686,08412040Paphia euglypta12,99413,10711739Acanthocardia tuberculata12,54612,64810334Musculista senhousia M7,4037,59119264[^3] Conversely, ORFans seem to be poorly connectible across the entire class: the complete list of HHblits clusters of homologs shows that any given ORFan shows sharp similarities only with ORFans of strictly related species ([supplementary file S21](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Indeed, ORFans from Unionidae are phylogenetically clumped in the same clusters that appear in the tree ([fig. 6](#evw187-F6){ref-type="fig"}) and, notably, F and M ORFans are never intermingled. The same apply for F ORFans of mytilids, but not for M ORFans, whose homology scores are always unsignificant for all entries of the database. The only exception to this is given by Musculista senhousia. Three ORFans were described on the F mtDNA of this species ([@evw187-B69]): while no significant hits were retrieved for one of these, the other two and the single M ORFan are clearly homolog ([supplementary file S21](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online; see also [@evw187-B69]). Finally, S. velum ORFans did not show significant homologies with other ORFans. Summarizing Data: PCA and HERMES Score -------------------------------------- All bivalve mitogenomic features are listed in [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. The resulting PCA is shown in [figure 7](#evw187-F7){ref-type="fig"}; the first two principal components account for the 66.61% of the dataset variability. In the PCA plot, larger taxonomic assemblages are easy identified: Palaeoheterodonta on one side and the wide cluster of Amarsipobranchia on the other side. Most families create also a cluster, like Pectinidae, Ostreidae, and Margaritiferidae. Contrastingly, some points are sharply separated from others: N. nucleus, S. velum, Scapharca spp., P. magellanicus, and L. elliptica. F[ig]{.smallcaps}. 7.---Bivalve mitogenomics PCA. Input data are shown in [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online (see text for details). Different subclasses are indicated with different shades: shades of blue, Palaeoheterodonta; shades of violet, Opponobranchia; shades of red, Pteriomorphia; shades of green, Heterodonta + Anomalodesmata; species of the same family are indicated with the same color. The best-performing variable set to compute the HERMES score was the following: percentage of URs;absolute value of SU skew;AMIGA;root-to-tip distance; andML distance from N. nucleus. For example, when inserting also the AT content, the goodness-of-fit parameters become only slightly better, but AT content was given a communality of 0.25%; therefore, we may conclude that this variable is not highly linked with the other five and thus it not significant in quantifying molecular evolution of mitochondrial genomes. The HERMES factor analysis index ([fig. 8](#evw187-F8){ref-type="fig"}) shows good levels of correlation between the selected variables: TLI = 0.965, SRMR = 0.061, RMSEA 95% CI = 0.025-0.233, KMO = 0.764, which are all within boundaries suggested by [@evw187-B74]. The lowest communality was scored by %UR (13.20%), while the highest was scored by the ML distance from N. nucleus (98.66%); the mean communality of the model was 60.62%, meaning that the HERMES index accounts for the 60.62% of the total variability of the source matrix. F[ig]{.smallcaps}. 8.---The HERMES index. To highlight HERMES differences between subclasses, species are horizontally listed by subclass and then in alphabetical order. Discussion ========== To the best of our knowledge, the present study is the first overall and detailed appraisal to the mitogenomics of bivalve mollusks. Many similar studies have been published in the past on similar topics, but generally they focused on a single family, like Mytilidae ([@evw187-B19]), Unionidae ([@evw187-B18]), Pectinidae ([@evw187-B181]), or on the DUI phenomenon ([@evw187-B42]). As stated in the introduction, however, large comparative studies are essential to understand the main pathways followed by the evolution of mitochondrial genomes. At the class level, we decided to especially concentrate on PCGs, as annotations are more trustworthy, homology is certain, and the same set of genes is present in all genomes under study. Actually, we find only two duplication events (cox2 in M. senhousia M and V. philippinarum F) and we were able to detect the atp8 gene in 20 species where this gene was originally described as missing ([table 2](#evw187-T2){ref-type="table"}): some of these atp8 genes were already signaled ([@evw187-B160]), namely those of A. tuberculata, H. arctica, and V. philippinarum ([@evw187-B45]); Mytilus spp. ([@evw187-B22]; [@evw187-B155]); Unionidae ([@evw187-B42]). After our analysis, 71 species out of 100 have an annotated atp8. The nucleotide/amino acid sequence of this gene is scarcely conserved, and this probably led to annotation flaws, as already stated ([@evw187-B127]; [@evw187-B189]; [@evw187-B13]; [@evw187-B56]). Structural analyses are needed to recover some homology with known atp8, and we may suggest the importance of such analyses in other groups where atp8 is reported as missing, like nematodes ([@evw187-B119]), platyhelminths ([@evw187-B95]), and chaetognaths ([@evw187-B15]). Contrastingly, the GenBank annotation of rRNAs is still commonly obtained by the boundaries of the upstream and downstream genes, despite accurate ongoing work of re-annotation ([@evw187-B12]; [@evw187-B160]). The use of these genes is therefore problematic for analyses like nucleotide composition, skews, or biases. However, phylogenetic methods should be robust enough to overcome this issue (but a masking phase is required): for this reason, we decided to insert rRNAs in the phylogenetic analysis anyway. Finally, tRNAs are extremely prone to gene rearrangement ([@evw187-B16]; [@evw187-B169]; [@evw187-B182]; [@evw187-B62]) and recruitment ([@evw187-B93]; [@evw187-B178]; Wu, Li, Li, Yu, et al. [@evw187-B179]), and therefore they have to be assessed at a lower taxonomical level ([@evw187-B180]). PCGs in Bivalve Mitochondrial Genomes ------------------------------------- The distance analyses reveal a high degree of divergence for single genes. p-Distance, which is an underestimation of the true distance in that it does not account for multiple substitution events, is generally comprised between 40% and 60% ([fig. 3](#evw187-F3){ref-type="fig"}). However, when the correct model of molecular evolution is used, it is clear that most of this variability is found in synonymous mutations ([fig. 3](#evw187-F3){ref-type="fig"}), which is clearly demonstrated by the low values of d~N~/d~S~ ([table 1](#evw187-T1){ref-type="table"}). Pairwise d~N~/d~S~ have very low medians, but a very high standard deviations, probably due to high d~N~/d~S~ computed for very distantly related species; therefore, we consider as the best estimation of d~N~/d~S~ in our dataset the value computed along the phylogenetic tree, which is comprised between 0.080 (cox1) and 0.344 (nad6; [table 1](#evw187-T1){ref-type="table"}). Such a pattern of overall negative selection was already detected for the species of genus Mytilus ([@evw187-B186]; [@evw187-B56]). However, conservation and variability should not be assessed at the general gene level, because our results clearly indicate a sharp contrast between strongly conserved domains and highly variable regions. Those domains are shown in [figure 2](#evw187-F2){ref-type="fig"} (see also [supplementary files S9 and S10](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Concluding, some domains of the mitochondrial PCGs are currently under severe purifying selection in bivalves, while, in most cases, variability is due to indel events typical of some species (see, e.g., the cox2 gene). If the general pattern is the conservation of specific domains in each PCG, some genes seem to follow different evolutionary pathways. In most analyses, atp6, nad2, nad4L, and nad6 behave differently from other PCGs: they are more heavily affected by masking phase ([supplementary file S7](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), show higher d~N~/d~S~ values ([table 1](#evw187-T1){ref-type="table"}), are heavily saturated ([fig. 4](#evw187-F4){ref-type="fig"} and [supplementary file S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), and are much more variable ([fig. 3](#evw187-F3){ref-type="fig"}). In a nutshell, these genes are driven by different evolutionary constraints with respect to the others ([fig. 5](#evw187-F5){ref-type="fig"}); although the second principal component accounts for only the 8.02% of the variability, this may indicate that, furthermore, each of these genes follows its own evolutionary pathway. History of Bivalve Mitochondria ------------------------------- The present phylogeny ([fig. 6](#evw187-F6){ref-type="fig"}) corroborates a view of bivalve evolution where most extant families were essentially already present in the Lower Ordovician ([@evw187-B111]). Expectedly, the mitogenomic differentiation of the main clades of extant bivalves predates the paleontological evidences of the well-known Ordovician bivalve radiation ([@evw187-B33]; [@evw187-B51]; [@evw187-B143]; [@evw187-B50]; [@evw187-B128]; [@evw187-B111]). Conversely, the root of most families is placed after the Ordovician, probably because of limited taxon sampling: the same would hold for Palaeoheterodonta, but they appear to be much more recent (Early Triassic) since only members of the superfamily Unionoidea were actually inserted in this phylogenetic analysis. The topology of our phylogenetic tree is in perfect agreement with our previous results ([@evw187-B126]; [@evw187-B125]). It is particularly noteworthy the presence of the Amarsipobranchia clade, i.e., Pteriomorphia + Heterodonta. Our previous analyses were based on four mitochondrial genes (cox1, cytb, rrnL, and rrnS), and the use of the complete mitochondrial gene array led to the same result, even with a larger sample. Furthermore, the signal vs. noise analysis indicates that the phylogenetic signal of all genes is suitable ([supplementary file S17](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), and it becomes even stronger for the complete, concatenated dataset. Indeed, the same Amarsipobranchia clade was also retrieved by other studies using mitochondrial markers ([@evw187-B60]; [@evw187-B42]; [@evw187-B160]). On the other side, the use of morphological characters leads generally to the Heteroconchia sensu [@evw187-B173] clade (i.e., Palaeoheterodonta + Heterodonta; [@evw187-B60]; [@evw187-B14])---however, some morphological analyses retrieved instead the Amarsipobranchia clade ([@evw187-B33]). Recent analyses based on nuclear genes and transcriptomes ([@evw187-B85]; [@evw187-B156]; [@evw187-B147]; [@evw187-B63]) also recovered the Heteroconchia clade. Thus, the discrepancy between phylogenies based on morphology/nuclear genes vs. mitochondrial genes still holds. The amount of data in mitochondrial genomes is clearly restricted with respect to nuclear genomes. It is also possible that specific mitochondrial features may lead to a wrong relationship between heterodonts and pteriomorphians, thus disrupting Heteroconchia. Nucleotide composition may be one of these features: Amarsipobranchia have all negative A-T and positive G-C skews, while the situation is reversed for Palaeoheterodonta ([supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Another feature which is correlated with nucleotide composition is the substitution rate ([@evw187-B86]; [@evw187-B149]; [@evw187-B64]; [@evw187-B72]), which we show is linked with AR rate ([supplementary file S18](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), as already hypothesized by [@evw187-B160]. However, our HERMES factor analysis demonstrates that the AT content practically does not explain other phylogenetic parameters like root-to-tip distance and ML distance from N. nucleus, and the protobranch Solemya velum show the same situation of Palaeoheterodonta, corroborating the idea that this is the plesiomorphic condition of bivalves. The same can be seen from the general PCA ([fig. 7](#evw187-F7){ref-type="fig"}): Amarsipobranchia and Palaeoheterodonta are separated, many features are considered (not only nucleotide composition), and Opponobranchia are shifted towards Palaeoheterodonta. Conclusions =========== The HERMES Index: Tempo and Mode of Mitochondrial Evolution ----------------------------------------------------------- The use of a single score to quantify mitochondrial evolution is a complex task, as many different parameters (that, furthermore, are linked together to different extents) can be considered and have to be summarized into a single number. High goodness-of-fit test results (given the complexity of the dataset) show that HERMES ([fig. 8](#evw187-F8){ref-type="fig"}) is indeed a suitable measure of this evolution. We are planning to apply the HERMES index in other taxa, and this will lead us to the identification of taxon-specific mitochondrial features that are suitable to measure molecular evolution; a Python script was written and made publicly available for this purpose (available at <https://github.com/mozoo/HERMES.git>). Concerning bivalves, there is consistence between all the aforementioned data and the HERMES measure: S. velum is the slowest-evolving mitogenome, followed by those of Palaeoheterodonta. Amarsipobranchia have similar HERMES scores, typically higher than Palaeoheterodonta; finally, the position of L. elliptica is intriguing and deserves further investigation. Within Palaeoheterodonta, F genomes have HERMES scores that are sharply lower than the M counterparts. The very early onset of DUI in Palaeoheterodonta led to the highest inter-sex distance values for DUI species ([@evw187-B13]) and to the well-known gender-joining pattern in phylogenetic trees ([@evw187-B37]; [@evw187-B42]; [@evw187-B189]; [fig. 6](#evw187-F6){ref-type="fig"}). The divergence of the two lineages started at least from the origin of Unionidae, ∼250 Mya ([@evw187-B166]; [@evw187-B36]; [@evw187-B47]; [@evw187-B116]; [fig. 6](#evw187-F6){ref-type="fig"}; [supplementary file S16](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online): F and M genomes are separately evolving since then. Recall that the organization of the ancestral bivalve mitochondrial genome should have resembled that of Solemya and Nucula ([@evw187-B127]), we can conclude that, while somehow the F mtDNAs of unionids retain much of this original condition, the M mtDNAs seem to have diverged more quickly on their own. DUI in other bivalve lineages appears to be a much more recent phenomenon ([fig. 6](#evw187-F6){ref-type="fig"}), or masked by masculinization role reversals ([@evw187-B73]; [@evw187-B131]; [@evw187-B189]), therefore the gender-joining pattern of unionids remains essentially unique across the phylogeny of the entire class. It is tempting to explore links between the molecular evolution of bivalve mitochondrial genomes as depicted by the HERMES score (and the mitogenomic feature-based PCA as well; [fig. 7](#evw187-F7){ref-type="fig"}) and the fossil evidences of the class. First known bivalves originated in the Early-Middle Cambrian and slowly faded away during the Late Cambrian; no Cambrian species are known from the Ordovician ([@evw187-B51]; [@evw187-B143]; [@evw187-B50]; [@evw187-B32]; [@evw187-B128]; [@evw187-B110]). However, some of them gave rise to extant bivalve clades in the Ordovician period ([@evw187-B34]; [@evw187-B143]; [@evw187-B128]). Protobranch forms were the first branching clade ([@evw187-B113]; [@evw187-B31]; [@evw187-B85]; [@evw187-B125]; [@evw187-B156]; [@evw187-B50]; [@evw187-B147]; [@evw187-B14]; [@evw187-B63]; and reference therein) before the evolution of the true feeding gill of all remaining autobranchs. The strict similarity of the mitochondrial genome of Solemya (and Nucula as well) with some gastropods ([@evw187-B127]) strengthens this hypothesis and allows to set a direction in the evolution of bivalve mtDNAs after the original appearance of extant bivalves; the HERMES index detects two main phases of this evolution ([fig. 8](#evw187-F8){ref-type="fig"}). According to the HERMES pattern, a first phase was the split of palaeoheterodonts from Amarsipobranchia. Again, fossil data may shed light on this issue. The most ancient families of Ordovician bivalves (dating to the Lower Ordovician, upper Tremadoc, ∼480 Mya) are Ucumariidae, Modiolopsidae (Goniophorinidae), and Lipanellidae ([@evw187-B142]; [@evw187-B50]): following current revised bivalve systematics, they should be classified within Heterodonta, Pteriomorphia, and Heterodonta, respectively ([@evw187-B24]). However, the state-of-art phylogenetic reconstruction is Lipanellidae + (Modiolopsidae + Ucumariidae) ([@evw187-B142], p. 117; [@evw187-B50], p. 12), and Heterodonta should be therefore considered as polyphyletic at their root. Moreover, Ucumariidae are interpreted as connected to extant anamalodesmatans ([@evw187-B142]; [@evw187-B50]), which are currently considered as nested within heterodonts ([@evw187-B61]; [@evw187-B46]; [@evw187-B60]; [@evw187-B71]; [@evw187-B164], [@evw187-B163]; [@evw187-B147]; [@evw187-B14]; [@evw187-B63]). Indeed, our phylogenetic reconstruction and the Amarsipobranchia hypothesis retrieve the same clade; namely, in the present phylogenetic tree it exhibits a basal tritomy (Heterodonta + Pteriomorphia + Anomalodesmata; [fig. 6](#evw187-F6){ref-type="fig"}). Furthermore, it is worth recalling the fossil record of the family Thoraliidae, which is also known from the Lower Ordovician ([@evw187-B112]; [@evw187-B141]; [@evw187-B32]) and it is currently classified within Palaeoheterodonta ([@evw187-B24]). Thus, in the Lower Ordovician a monophyletic Amarsipobranchia-like clade is hypothesized, while the first palaeoheterodonts have been found from the same epoch: concluding, phylogenetic paleontological reconstructions are not discordant with our molecular phylogentic tree or with the HERMES pattern. Eventually, the second phase of bivalve mitochondrial evolution as depicted by the HERMES score is the diversification of Amarsipobranchia, which definitely lost most plesiomorphic mitogenomic features ([fig. 7](#evw187-F7){ref-type="fig"}): all genes, with rarest exceptions, migrated on the same coding strand; an increase in length was coupled to an increase of the genomic regions not assigned to canonical genes; there was the inversion in A-T and G-C skews ([fig. 1](#evw187-F1){ref-type="fig"}; [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online); most strikingly, the gene rearrangement rate was given an unprecedented boost ([supplementary file S18](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Supplementary Material ====================== [Supplementary file S1--S21](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) are available at Genome Biology and Evolution online (<http://www.gbe.oxfordjournals.org/>). ###### Supplementary Data We would like to thank Fabrizio Ghiselli, Liliana Milani, Stefano Bettinazzi, and Mariangela Iannello for suggestions and stimulating discussion. Thanks are also due to Davide Guerra for his unvaluable guide to the taming of R and to Liliana Silva for having introduced us to the basics of factor analysis. The original manuscript was greatly improved by comments and suggestions by two anonymous reviewers. This work was financed by the "Canziani Bequest" fund (University of Bologna, grant number A.31.CANZELSEW). [^1]: Associate editor: Liliana Milani [^2]: d~N~/d~S~, value of d~N~/d~S~ computed by PAML using the best-known likelihood tree (see text for details) as the given phylogeny; pairwise median, median of all pairwise comparisons ± standard deviation; d~N~/d~S~ \> 10, number of pairwise d~N~/d~S~ ratios greater than 10. [^3]: Start and stop refer to the complete mitochondrial genome as referenced in [supplementary file S1](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online; length, nucleotide length without stop codon; aa, amino acid length. All newly annotated atp8 are on the "+" strand; ORFs are listed in order of hit, as in [supplementary file S19](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online.	{ "pile_set_name": "PubMed Central" }	[ "ab", "nterminu", "eukaryot", "ie", "purpos", "pearson", "basal", "factomin", "round", "grant", "august", "heterodont", "signal", "back", "split", "graphic", "squar", "fifth", "bivalvian", "nonhomoscedast", "cat", "constraint", "plesiomorphi", "sharpli", "discrep", "resultsa", "dui", "process", "benchmark", "paphia", "state", "shift", "trustworthi", "conflict", "paleontolog", "liliana", "hidden", "seqinr", "ostreida", "higher", "tcoffe", "even", "remov", "run", "organ", "phylowidget", "h", "hundr", "pairwis", "intersex", "crossvalid", "singlespeci", "reannot", "quickli", "venerid", "farther", "supernumerari", "deserv", "gre", "taxonom", "strongli", "thu", 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"genomes", "Mytilus", "spp", "cluster", "sex", "Palaeoheterodonta", "senhousia", "genomes", "cluster", "species", "Within", "Heterodonta", "Lucinindae", "Loripes", "lacteus", "Lucinella", "divaricata", "BP", "PP", "sister", "group", "remaining", "heterodonts", "BP", "PP", "heterodont", "DUI", "genomes", "lamarckii", "philippinarum", "cluster", "species", "F", "ig", "mitochondrial", "phylogeny", "bivalves", "Ultrametric", "tree", "bivalve", "species", "computed", "using", "starting", "likelihood", "tree", "computed", "RAxML", "consensus", "Bayesian", "tree", "RAxML", "bootstrap", "posterior", "probabilities", "printed", "node", "asterisk", "means", "bootstrap", "posterior", "probability", "tree", "phylogenetic", "informativeness", "PCG", "shown", "geological", "timescale", "Mya", "follows", "main", "split", "class", "Palaeoheterodonta", "one", "side", "Amarsipobranchia", "side", "took", "place", "million", "years", "ago", "Mya", "confidence", "interval", "CI", "million", "years", 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"Material", "http", "online", "resulting", "PCA", "shown", "figure", "fig", "first", "two", "principal", "components", "account", "dataset", "variability", "PCA", "plot", "larger", "taxonomic", "assemblages", "easy", "identified", "Palaeoheterodonta", "one", "side", "wide", "cluster", "Amarsipobranchia", "side", "families", "create", "also", "cluster", "like", "Pectinidae", "Ostreidae", "Margaritiferidae", "Contrastingly", "points", "sharply", "separated", "others", "nucleus", "velum", "Scapharca", "magellanicus", "elliptica", "F", "ig", "mitogenomics", "PCA", "Input", "data", "shown", "supplementary", "file", "http", "Supplementary", "Material", "http", "online", "see", "text", "details", "Different", "subclasses", "indicated", "different", "shades", "shades", "blue", "Palaeoheterodonta", "shades", "violet", "Opponobranchia", "shades", "red", "Pteriomorphia", "shades", "green", "Heterodonta", "Anomalodesmata", "species", "family", "indicated", "color", "variable", "set", "compute", 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Introduction {#sec1-1} ============ Nigeria's health sector is a part of its economy, which require critical discourse, especially concerning issues surrounding infrastructure, government funding, health workers motivation and satisfaction, among others. Considering the strategic role that health workers play within this sector, it is imperative that researchers and practitioners give the required attention to exploring and interrogating issues that can enhance their performance, to ensure that they function optimally \[[@ref1]\], \[[@ref2]\]. It is not untrue that where the workers in the Nigerian health sector are unfairly treated, the ailing patients will be at the receiving end of such injustice. Consequently, existing studies have attempted to show the relationships between healthcare workers satisfaction and the motivation towards work \[[@ref3]\]. There have also been studies to demonstrate the factors that influence health workers satisfaction with their work \[[@ref4]\], \[[@ref5]\]. Not withstanding, the case with Nigeria requires more than simply understanding the basic elements that explain job satisfaction of health workers. As \[[@ref6]\] opined, there are insufficient empirical details in the literature to explain health workers' motives for linking their level of job satisfaction with their motives to remain committed, or otherwise, to their organisation. Literature has asserted that employees' perception about treatments of diversity at the workplace is critical to enhancing, or otherwise, their behaviours and commitment to the job \[[@ref7]\]. According to \[[@ref8]\], \[[@ref9]\] it is not sufficient to measure employees' levels of satisfaction, but workplace diversity can influence the type of commitment they show to the organisation. In the Nigerian health sector, employees' perceptions about treatments of diversity are crucial not only to their satisfaction with the job but equally to their commitment to the organisation \[[@ref10]\], \[[@ref11]\]. The importance of this view of the public health sector, is that it could induce political behaviours, result in conflict situations and hence promote tensed work environments, if not properly managed. Despite these facts, there is again, a dearth of existing literature that shows an element of empirical analysis to demonstrate the moderating influence of workforce diversity on job satisfaction and organisational commitment of public healthcare employees in Nigeria. Therefore, this study is focused on investigating the mediating effect of employees' perceived treatments of diversity in the workplace on the relationship between job satisfaction and their commitment to the organisation. Material and Methods {#sec1-2} ==================== The method of survey was used to collect data. This research involved 133 public health workers from the Ministry of State Health of Lagos in Nigeria. Public health workers, especially in the Ministry of Health of Nigeria, are important for this research because of the increased awareness of the diverse workforce of the Ministry and the need to manage this diversity in a way that does not hurt employee satisfaction and commitment to the organisation \[[@ref14]\]. Furthermore, due to the importance of the Ministry of Health for the population and the overall well-being of each country, this research is considered essential to maintaining employee interest and motivation in the provision of quality services \[[@ref15]\]. Sample Size & Sampling Procedure {#sec2-1} -------------------------------- The sample size was determined using a sample size determination formula with a 5 per cent sampling error. Apply the Yamane sample size procedure \[[@ref16]\]. The formula N = N / (1 + N(e)^2^) where n = sampling size e = sampling error (0.05) N= the population (that is 200 employees in the healthcare location) n = 200 / (1+200 (0.05)^2^) n = 200 / 1+ 200 (0.0025) n = 200 / 1+ 0.5 n = 200 / 1.5 n = 133.33 (approximately 133) n = 133 Therefore, the sample size is 133 For this research, the technique adopted is the simple random sampling technique. Simple random sampling which is a subdivision of the probability sampling is used and is applicable as a result of the small number and similarity of the population and all subdivision of the population have an equal chance of being selected. This technique allows every member of the population to be a respondent by selecting the respondents without any form of partiality. Execution of Field Research {#sec2-2} --------------------------- The research used questionnaires as the main tools to perform this research. The questionnaire was used in this study for the following reasons; it allowed the respondents to express themselves more freely and clearly and to gather the answers in a standardized manner; It saved time by allowing information to be collected as soon as possible and also by facilitating the collection of possible information from a large sample of respondents. The research questionnaire consisted of a 5-point Likert scale in which the respondent was expected to agree strongly, agree, disagree and strongly disagree with carefully constructed questions, ranging from very positive to very negative, to measure the influence of diversity in the workforce on the commitment and satisfaction of employees. The questionnaire has been tested by the pilot using the Cronbach reliability test. The investigation questionnaire was divided into two parts. Section A was designed to provide information on the background of respondents, while section B was designed to provide information on the nature of workforce diversity, employee satisfaction and level of commitment (affective, regulatory and continuing) in the Ajeromi General Hospital. Measures {#sec2-3} -------- This research took advantage of the ideas from existing research studies. Workforce diversity issues were developed based on \[[@ref17]\], \[[@ref18]\], work satisfaction items were developed based on \[[@ref19]\], while organisational commitment items were adapted from \[[@ref20]\], \[[@ref21]\]. The data collected was encoded and entered into version 22 of SPSS. The analysis of data was carried out using descriptive statistics and structural equation modelling (SEM). Ethical Consideration {#sec2-4} --------------------- The management of the questionnaire was based on the readiness of respondents to respond to the research instrument. The participants in the study were also assured of confidentiality and anonymity, as their names were not reflected in the questionnaire. Validity and Reliability of Research Instrument {#sec2-5} ----------------------------------------------- The alpha coefficient (Cronbach's alpha) has been used in this research to test the reliability of the measurement scale. The Cronbach alpha is a method to determine the internal consistency of a measuring instrument by identifying all possible ways to split the items in the instrument and then investigate the correlation degree. Values of the coefficient from 0 to 1. A highly reliable instrument has a coefficient value near 1, where a score very close to 0 indicates a very low or no reliability of the instrument. It is widely accepted that the score above 0.7 shows the instrument's reliability. The SPSS (Social Science Statistical Package) was used to test the reliability of the research instrument. The statistics on the reliability of the tools used in this study were 0.747. The validity of the instrument was determined by the validity of the content. Statistical Analysis {#sec2-6} -------------------- The statistical analysis was carried out using SPSS (version 22) and AMOS (version 23) software for this study. Descriptive analysis showed the total number of respondents according to the analysed categories as well as the percentages of each category. The inferential statistics which established the multivariate relationships between public healthcare workers' diversity issues and commitment to their organisation was analysed using structural equation modelling (SEM) at a significant level of p \< 0.000. Results {#sec1-3} ======= Sixty-four (48.1%) were male, and the remaining 69 (51.9%) were female. This research involves more female participants than male respondents. The age distribution of the respondents revealed that 68 respondents were aged between 18 and 34 years; 45 respondents were aged between 35 and 44 years. The range of 13 respondents is 45 to 54 and 7 falls within the range of 55 years. This means that the age group was between 18 and 34 years old, with the majority of respondents and had 68 participants in this research. Based on years of working experience (40.6%) of respondents worked for less than 5 years, the table shows that (36.8%) of respondents worked for 5-10 years and (12.0%) for 11-15 years. The table shows that there were some respondents 16 years ago (10.5%). [Table 4](#T1 T2){ref-type="table"} above shows the background of the students surveyed 24 (18.0%) were O.N.D., 9 (6.8%) were N.C.E., 43 (32.3%) were H.N.D., 35 (26.3%) were Bachelor, and 22 (16.5%) were Masters. This means that the majority of the education background was (32.3%), meaning H.N.D. There were 43 respondents. ###### Socio-demographic Distribution of Respondent Demographic Variable Frequency Percentage --------------------------- ------------------- ------------ ------ Gender Male 64 48.1 Female 69 51.9 Age 18-34 68 51.1 35-44 45 33.8 45-54 13 9.8 55 and above 7 5.3 Years of Work experience less than 5 years 54 40.6 5-10 years 49 36.8 11-15 years 16 12.0 16 years above 14 10.5 Educational Qualification O.N.D 24 18.0 N.C.E 9 6.8 H.N.D 43 32.3 Bachelor's degree 35 26.3 Master's degree 22 16.5 ###### Structural Regression Weights for Job Satisfaction, Workforce Diversity and Employee Commitment Estimate S.E. C.R. P Label ----------- --------- ----------- ---------- ------- -------- -------- -------------------- Education \<\-\-- JobSatisf 0.215 0.098 2.184 \\\* Relationship Exist Gender \<\-\-- JobSatisf 0.418 0.067 6.223 \\\* Relationship Exist Religion \<\-\-- JobSatisf -0.179 0.078 -2.290 \\\* Relationship Exist Ethnicity \<\-\-- JobSatisf 0.301 0.057 5.312 \\\* Relationship Exist Position \<\-\-- JobSatisf 0.118 0.069 1.723 \\\* Relationship Exist AffectCom \<\-\-- Education 0.092 0.048 1.901 \\\* Relationship Exist NormCom \<\-\-- Education 0.110 0.049 2.239 \\\* Relationship Exist ContCom \<\-\-- Education 0.121 0.059 2.040 \\\* Relationship Exist AffectCom \<\-\-- Gender 0.245 0.067 3.642 \\\* Relationship Exist NormCom \<\-\-- Gender 0.341 0.069 4.954 \\\* Relationship Exist ContCom \<\-\-- Gender 0.129 0.083 1.553 0.120 No Relationship AffectCom \<\-\-- Religion 0.029 0.059 0.486 0.627 No Relationship NormCom \<\-\-- Religion 0.025 0.060 0.417 0.677 No Relationship ContCom \<\-\-- Religion 0.121 0.072 1.670 \\\* Relationship Exist AffectCom \<\-\-- Ethnicity 0.078 0.079 0.983 0.326 No Relationship NormCom \<\-\-- Ethnicity -0.025 0.081 -0.312 0.755 No Relationship ContCom \<\-\-- Ethnicity -0.099 0.098 -1.011 0.312 No Relationship AffectCom \<\-\-- Position 0.005 0.066 0.074 0.941 No Relationship NormCom \<\-\-- Position -0.041 0.068 -0.612 0.540 No Relationship ContCom \<\-\-- Position 0.074 0.081 0.909 0.363 No Relationship ![Regression Path of Job Satisfaction, Workforce Diversity & Employee Commitment](OAMJMS-7-2031-g001){#F1} [Figure 1](#F1){ref-type="fig"} shows the model for analysing the multifaceted relationships between workforce diversity, job satisfaction and commitment of employees. [Table 2](#T2){ref-type="table"} also shows structural regression weights of multi-variable analysis. Chi-square / Degree of Freedom (Cmin / df) = 3.876, Goodness of Fit Index (GFI) = 0.949, Normal Fit Index (NFI) = 0.854, Comparative Fit Index (CFI) = 0.872, Root Mean Square Error Approximation (RMSEA) = 0.148 are used to ensure the model fit of the analysis. The values are important based on the arguments presented in the study \[[@ref28]\], \[[@ref29]\], \[[@ref30]\]. The data supports a relationship between diversity of workforce and job satisfaction, diversity of workforce and organizational commitment and influence on the organizational commitment to work. The results of these data support existing research results \[[@ref21]\], \[[@ref22]\], \[[@ref23]\], \[[@ref24]\]. Specifically, the results from the statistical analysis indicate that job satisfaction significantly relates with all dimensions of workforce diversity in the following ways: education (r = 0.19), gender (r = 0.48), religion (r = -0.20), ethnicity (r = 0.42) and position (r = 0.15). The mediating effects of workforce diversity on the relationship between job satisfaction and employee commitment is also evident from the statistical analysis, especially with respect to education (affective commitment = 0.16, normative commitment = 0.18, continuance commitment = 0.18); gender (affective commitment = 0.32, normative commitment = 0.42); and religion (continuance commitment = 0.14). Discussion {#sec1-4} ========== The study focused on investigating the mediating effects of diversity in the workforce on job satisfaction and the organisational commitment of public health workers in Nigeria. For the analysis of data collected from respondents, the structural equation model was used. This study fills a research gap in the existing literature by empirically demonstrating the moderating influence of diversity in the workforce on the satisfaction of public health workers in Nigeria and their organisational activities. Through the understanding of such relationships, managers and governments in the healthcare sector are ably posed to curbing incidences that could arise as a result of adverse politicking and conflicts. Moreover, the findings of this study has been corroborated by existing literature that argued that diversity at the workplace can be harnessed not only to ensure sustainable satisfaction of employees, but to motivate them into creativity and commitment in the organization \[[@ref13]\], \[[@ref14]\], \[[@ref15]\], \[[@ref16]\], \[[@ref17]\], \[[@ref18]\], \[[@ref19]\], \[[@ref20]\], \[[@ref21]\], \[[@ref22]\], \[[@ref23]\], \[[@ref24]\], \[[@ref25]\]. This study constitutes a significant departure from conventional studies on workforce diversity by adopting a mediating perspective of the concept. In other words, the strength of workplace diversity is projected as a means of achieving higher levels of satisfaction from healthcare workers. The perspective adopted in this study is significant to enhancing passion and emotional affinity of healthcare workers toward caring for patients, hence sustaining a healthy community \[[@ref10]\], \[[@ref11]\], \[[@ref12]\]. Based on the findings from the study, educational diversity at the workplace is a very strategic linkage between job satisfaction and employees' commitment (including affective, normative and continuance commitment). Indeed, the need to appreciate the roles of educational diversity reflects in the capacity of each to function across different cadres, yet achieving the common goal of the organisation \[[@ref26]\], \[[@ref27]\]. In the same way, diversities based on gender and religion has been evidence from the statistical point of view of this study to be credible leverages for stimulating employees' interest and commitment to the organisation. This study concludes that not only is job satisfaction significant to ensuring the commitment of healthcare workers to their organisation, but more critical is the role of workforce diversity as viable leverage for transiting the interest of employees from the level of job satisfaction to organisational commitment. Specifically, three levels of workforce diversity, namely; education, gender and religion, were found to be strategic to enhancing healthcare workers perception about diversity treatments in the healthcare sector. Consequently, this study recommends that policymakers and managers in the Nigerian health care sector must pay adequate attention to creating a balance in the educational, gender and religious diversity of healthcare workers in Nigeria to sustain their satisfaction and stimulate their commitment to the organisation. Authors of this research work express sincere appreciation to the Management of Covenant University for giving full sponsorship to the publication of the research work in this journal. Funding: This research was financially supported by the Management of Covenant University, Nigeria Competing Interests: The authors have declared that no competing interests exist	{ "pile_set_name": "PubMed Central" }	[ "cfi", "treatment", "root", "probabl", "gather", "two", "freedom", "work", "divers", "remain", "respond", "collect", "exist", "identifi", "healthcar", "select", "fund", "commit", "execut", "readi", "student", "valu", "locat", "enter", "split", "squar", "toward", "use", "gener", "workforc", "moder", "score", "support", "focus", "accord", "abli", "signific", "establish", "constitut", "full", "journal", "unfairli", "norm", "commitmentoamjmsgf", "also", "wellb", "discours", "state", "multivari", "attempt", "equat", "emot", "leverag", "conflict", "item", "perform", "popul", "sustain", "qualif", "normal", "inferenti", "dimens", "convent", "transit", "old", "distribut", "higher", "anonym", "organ", "tool", "cronbach", "contcom", "practition", "involv", "hurt", "likert", 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1. Introduction =============== Safe and reliable venous access is an essential procedure in modern oncology,^\[[@R1],[@R2]\]^ as long-term venous infusion is occasionally required in cancer patients for chemotherapy, fluid resuscitation, and nutritional treatment.^\[[@R3]\]^ Implantable central venous devices can play an important role in minimizing the discomfort of frequent vein puncture and cannulation.^\[[@R4],[@R5]\]^ Recently, the internal jugular vein (IJV) approach using real-time ultrasonography (USG) has gained popularity and has been performed widely; however, this approach might increase patients' discomfort and might cause inconvenience in maintaining an aseptic occlusive dressing.^\[[@R6]\]^ In this study, we aimed to introduce our surgical technique of cephalic venous access and to identify its safety, feasibility, and long-term functional outcomes compared to those of conventional methods. In addition, we investigated the possible risk factors that might affect the dysfunction of the implanted venous port. 2. Materials and methods ======================== 2.1. Study design ----------------- This study was approved by the institutional review board of Bucheon St. Mary\'s Hospital, College of Medicine, The Catholic University of Korea (HC19RESI0002). We retrospectively reviewed the medical records of patients who underwent implantable central venous access performed by surgeons between January 2012 and December 2014 at our hospital. The cases that were conducted by interventional radiologists were excluded. All patients gave written consent to the procedure and data collection for our database. The data, including patient demographics, cause of catheter insertion, venous approach methods, operation time, length of functional availability, and cause of device removal, were collected and analyzed. We classified our patients into 4 groups according to the venous access routes: IJV approach with real-time USG; external jugular vein (EJV) access with a cut-down method; subclavian vein access with the Seldinger landmark technique; and cephalic vein access. The operation methods were decided randomly according to the surgeons' competence and preference. We compared surgical and clinical outcomes between each group and performed additional analyses to identify risk factors for long-term functional impairment of the implanted venous devices. 2.2. Procedure for the cephalic vein approach --------------------------------------------- The patient was placed in a supine position, and his/her arm on the side of the procedure was extended to fully expose the outline of the deltoid and pectoralis muscles. After local anesthesia using diluted lidocaine was applied, a skin incision was made in the upper anterior chest wall along the deltopectoral groove (Fig. [1](#F1){ref-type="fig"}). Sharp dissection was carried out using electrocautery and curved mosquito forceps through the plane between the deltoid and pectoralis major muscles until the cephalic vein was identified (Fig. [2](#F2){ref-type="fig"}). The cephalic vein was then ligated proximally, and transverse venotomy was performed (Fig. [3](#F3){ref-type="fig"}), through which the venous catheter was inserted (Fig. [4](#F4){ref-type="fig"}). After radiologic examination for accurate placement of the catheter tip between the superior vena cava and right atrium, the reservoir was implanted with a suture on the pectoralis fascia (Fig. [5](#F5){ref-type="fig"}). The final catheter position was checked with a postoperative chest X-ray. ![Skin incision in the upper anterior chest wall along the deltopectoral groove.](medi-98-e18007-g001){#F1} ![Identifying the cephalic vein.](medi-98-e18007-g002){#F2} ![Ligating the cephalic vein at the proximal and peripheral sides.](medi-98-e18007-g003){#F3} ![Catheter insertion after transverse venotomy.](medi-98-e18007-g004){#F4} ![Reservoir implantation.](medi-98-e18007-g005){#F5} 2.3. Statistical analysis ------------------------- Summary statistics are presented as numbers and percentages for categorical variables and as the mean ± standard deviation for continuous variables. The Chi-square test or Fisher exact test was used to compare categorical variables, and the independent t test was used for continuous variables. Univariate and multivariate logistic regression analyses were applied to analyze the correlation of the possible risk factors with delayed malfunction of the implanted venous devices. We considered a 2-sided P-value \< .05 to be statistically significant. All statistical analyses were performed using the software package SAS 9.4 (SAS Institute, Inc, Cary, NC). 3. Results ========== A total of 508 patients were included in this study: 230 patients were included in the subclavian group; 134 in the IJV group; 25 in the EJV group; and 119 in the cephalic vein group. Regarding demographic characteristics (Table [1](#T1){ref-type="table"}), the cephalic group had a large portion of the colorectal malignancies compared to that of the IJV and subclavian groups (53.7% vs 34.3%, P \< .01; 53.7% vs 35.2%, respectively, P \< .01), and the IJV group included more female patients than the cephalic group (70% vs 57%, P = .03). The body mass index (BMI), patient age, and side of the access were similar between the cephalic and other groups. The mean operation time of the entire cohort was 46.9 minutes. The cephalic group had a longer operation time than the subclavian group (51.5 minutes vs 38.1 minutes, P \< .01); however, there was no difference in the operation time between the cephalic and IJV groups (51.5 minutes vs 53.7 minutes, P = .59). Comparatively, the operation time was longer in the EJV group than in the cephalic group (69.1 minutes vs 51.5 minutes, P \< .01). The length of catheter use and cycles of chemotherapy were comparable between the cephalic and other groups. In addition, the incidence of unscheduled catheter removal caused by late complications was similar between the cephalic and other groups as well. Infection and malfunction of the catheter were found to be late complications causing impairment in cyclic chemotherapy. We found 6 cases (1.2%) of immediate complications after the operation: the 4 cases in the subclavian group were all pneumothorax, and the 2 cases in the cephalic group were pneumothorax and postoperative bleeding. The statistical analysis was difficult for a few complicated cases. ###### Demographic characteristics and clinical outcomes according to the venous access. ![](medi-98-e18007-g006) Univariate analysis for the risk factors of late complications was performed according to the venous access routes, BMI, operation time, and age; however, the results were nonsignificant: odds ratios were 0.959 for the noncephalic approach (P = .93), 1.031 for BMI (P = .56), 1.004 for operation time (P = .64), and 0.979 for age (P = .21). In the multivariate logistic regression analysis (Table [2](#T2){ref-type="table"}), no factor was demonstrated to increase the incidence of late complications, including infection and catheter malfunction. ###### Multivariate risk factors analysis for the late complications. ![](medi-98-e18007-g007) 4. Discussion ============= This study revealed that the cephalic vein approach can be safely performed and has acceptable surgical, clinical, and functional outcomes compared to conventional percutaneous techniques. Central venous access devices in cancer patients were first introduced in 1982^\[[@R7]\]^ to reduce complications associated with frequent venous puncture during chemotherapy. There are several approaches for implantable central venous access. While the IJV approach with real-time USG has been widely performed because it has a good success rate without increasing the procedure duration,^\[[@R8],[@R9]\]^ we noticed that the surgical and clinical outcomes of the existing central venous approaches seem to be comparable.^\[[@R10]--[@R12]\]^ Despite its safety and feasibility, the conventional IJV approach requires specialized and expensive medical resources, including percutaneous vascular surgical kits, tunneling instruments, vascular USG, and/or fluoroscopic techniques; therefore, this approach might preclude wide clinical applications in small-volume regional hospitals or private clinics. Comparatively, the subclavian vein approach using the Seldinger landmark technique without a USG can be considered alternatively because it is easily performed with no special equipment; however, this approach might cause life-threatening complications such as hemothorax, pneumothorax, mediastinal hematoma, vascular perforation, and myocardial injury.^\[[@R13]\]^ Consequently, we employed the cephalic vein cut-down approach to reduce the risk of immediate complications^\[[@R14]\]^ while achieving cost-effectiveness and patient comfort. Several studies have reported that the cephalic vein approach can avoid possible immediate fatal complications, including pneumothorax and arterial puncture, although this approach might have a low success rate.^\[[@R15]--[@R19]\]^ In our study, the general surgeons performed central venous catheter insertion, and the failure rate of cephalic vein access was found to be 12.5%, which was superior to that of previous reports, ranging from 12% to 20%.^\[[@R11],[@R17]--[@R21]\]^ This result is similar to that of IJV and subclavian access.^\[[@R9],[@R19]\]^ Of the 136 patients who received the cephalic vein approach, the diameter of the cephalic vein was recorded in 62 cases. The mean diameters were 3.1 mm in the success group and 2.2 mm in the failure group, which were statistically significant (P \< .01); therefore, we carefully suggest that identifying the diameter of the cephalic vein would be helpful in successful procedure attempts. Two cases of immediate complications were found in the cephalic vein group (1.7%), and late complications occurred in 7 cephalic vein cases (5.9%). These results were similar to those of cephalic vein access in previous studies.^\[[@R16]--[@R23]\]^ Additionally, the complication rate of cephalic vein access in this study was lower than that of IJV access and subclavian access in other studies.^\[[@R9],[@R18],[@R19]\]^ Accordingly, we suggest that the cephalic vein cut-down approach can be safely performed with a comparable success rate and without increasing acute and late catheter-related complications compared to those of conventional percutaneous approaches. In addition, previous studies have shown that age, underlying cancer type, prior anticoagulant therapy, catheter tip position, and surgeon are independent prognostic factors for catheter complications.^\[[@R9],[@R24]--[@R26]\]^ However, in our study, patient BMI, age, and operation time did not increase the risk of long-term catheter dysfunction in univariate and multivariate logistic regression analyses; therefore, it seems reasonable that a central venous port can be considered in elderly or obese patients. Since our procedures were performed by several surgeons, there might have been interoperator variations as well as other confounding biases associated with the retrospective study design. Moreover, there might have been a reporting bias in analyzing the cephalic vein diameter between success and failure groups because the small size of the cephalic vein tended to be emphasized when the procedure failed. Nevertheless, our study was performed with a sufficiently large sample size, and the surgical records were documented by the specialized medical recorder immediately after the surgery. All surgeries were performed according to identical protocols. We suggest that these features might enhance the level of evidence of our study. 5. Conclusions ============== The present study demonstrates that cut-down central venous catheter insertion through the cephalic vein can be performed safely by nonvascular surgeons with no differences in surgical and clinical outcomes compared to those of conventional percutaneous approaches. Moreover, this approach requires no specialized equipment, including percutaneous vascular kits, tunneling instruments, and intraoperative USG. Therefore, the cephalic vein cut-down technique might incur less medical expenses than conventional approaches and would be helpful for both patients and surgeons. Author contributions ==================== Conceptualization: Jinbeom Cho, Kang Woong Jun, Byung Joo Song, Kiyoung Sung. Data curation: Jiyoung Rhu. Writing -- original draft: Jiyoung Rhu. Writing -- review and editing: Jinbeom Cho. Jinbeom Cho orcid: 0000-0002-6329-016X. Abbreviations: BMI = body mass index, EJV = external jugular vein, IJV = internal jugular vein, USG = ultrasonography. How to cite this article: Rhu J, Jun KW, Song BJ, Sung K, Cho J. Cephalic vein approach for the implantable central venous access. Medicine. 2019;98:46(e18007). Statistical consultation was supported by the Department of Biostatistics of the Catholic Research Coordinating Center, College of Medicine, The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, 06591, Seoul, Republic of Korea (ID. 1810-1439-01). Statistical consultation was supported by the Department of Biostatistics of the Catholic Research Coordinating Center, College of Medicine, The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, 06591, Seoul, Republic of Korea (ID. 1810-1439-01). The authors have no conflicts of interest to disclose.	{ "pile_set_name": "PubMed Central" }	[ "deltoid", "two", "origin", "impair", "collect", "longterm", "exist", "interoper", "korea", "identifi", "employ", "length", "risk", "written", "perfor", "id", "republ", "recent", "use", "gener", "chest", "univari", "final", "support", "ijv", "caus", "accord", "prior", "signific", "depart", "cathet", "suggest", "hcresi", "cari", "right", "intervent", "transvers", "multivari", "difficult", "attempt", "landmark", "conflict", "perform", "deltopector", "jugular", "seoul", "retrospect", "kang", "inconveni", "cannulationrr", "featur", "sutur", "underli", "fisher", "comparablerr", "medic", "convent", "longer", "first", "remov", "safe", "popular", "nc", "odd", "mari", "wide", "major", "selding", "achiev", "author", "andor", "biostatist", "orcid", "tend", "reduc", "regard", "subclavian", "compar", "cathol", "immedi", "bleed", "center", "infect", "complicationsrrr", "carri", "appli", "time", "type", 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Background {#Sec1} ========== Globally, cancers are the second most common cause of death with about one in every six deaths attributable to cancer \[[@CR1]\]. In 2012, there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide \[[@CR1]\]. Over the last few years, despite advances in early diagnosis and treatment, and in the survival rate, there has been an increase in the overall incidence of cancers \[[@CR2]\]. The cost of cancer treatment has also increased with advancements in early diagnosis and treatment \[[@CR2]\]. Among Head and Neck Cancers (HNC), the most commonly occurring are Oro-Pharyngeal Cancers (OPC), which include the lip, oral cavity, and oropharynx. Globally, OPC are the sixth most common malignancy \[[@CR3]\]. Despite the technological advancement, cancer awareness and improvement in survival rates for some cancers \[[@CR2]\], no significant improvement has been reported in the five-year survival rates for OPC \[[@CR4], [@CR5]\]. OPC is one of the few oral diseases encountered by the oral health team that has significant morbidity and premature mortality Tobacco products, alcohol consumption and sun exposure are the most recognized risk factors for OPC \[[@CR6]\]. But the incidence of Human Papilloma Virus (HPV) related OPC has also been on the rise, particularly in younger adults who have never smoked or used other tobacco products \[[@CR7], [@CR8]\]. In Australia, HNC are the seventh most common cancers with an incidence rate of 17 cases per 100,000 persons \[[@CR9]\]. In 2017, estimated deaths due to HNC is well over 1000 individuals (with three fourths of the reported deaths in males), that amounts to roughly 3.7% (n = 4956) of all new cancers diagnosed \[[@CR9]\]. A recent effectiveness review on oral cancer screening, demonstrated conventional oral examination to be a feasible and satisfactory option for opportunistic screening in dental settings with sensitivity and specificity similar to breast and cervical cancer screening programs \[[@CR4], [@CR10]--[@CR12]\]. Early diagnosis of OPC can greatly increase the five-year survival rates from 50% to more than 80% \[[@CR6], [@CR13]\]. OPC are known to be amenable to early detection as they primarily occur at sites that are accessible and visible during a non-invasive examination \[[@CR4]\]. They are often preceded by a visible precancerous lesion enabling early detection and treatment \[[@CR14]\]. The tumors' proliferative factor and stage at the time of diagnosis largely determines the prognosis of cancers \[[@CR15]\]. However, only 30% of OPC are identified at an early stage \[[@CR16]\] with the majority (50%) being diagnosed at an advanced stage of metastasis (stage III or IV). This is largely due to late presentation, delayed diagnosis and lack of a clear referral pathway between doctors and dentists \[[@CR17]--[@CR21]\]. This forms the strongest argument for early diagnosis of OPC and initiating early treatment. It is essential that OHPs such as dentists, dental hygienists (DHs), dental therapists (DTs), and oral health therapists (OHTs), understand the importance of conducting a thorough oral screening examination for malignant and potentially-malignant lesions as part of their routine clinical assessments, even in younger populations originally considered at lower risk for oral cancer \[[@CR22], [@CR23]\]. The World Dental Federation and Dental Associations, including the Australian Dental Association, proactively encourage Oral Health Professionals (OHP) to incorporate oral mucosal examinations as part of routine assessment \[[@CR6], [@CR24]--[@CR27]\]. Several studies have assessed dentists' knowledge, attitudes and practices regarding oral cancer \[[@CR28]--[@CR33]\]. However, only a few studies include DH, DT, and OHT. Thus, clinical screening practices regarding oral cancer among the complete range of oral health professionals remain largely unknown. Screening programs should be designed to detect precancerous lesions or malignancies in their asymptomatic phases for all patients, not only in the older population groups, traditionally known to be at higher risk, but also in younger patient groups amongst which there has been a rising incidence of OPC \[[@CR4], [@CR7], [@CR8], [@CR34]\]. Nonetheless, studies among oral health professionals in Australia identified lack of training, lack of confidence and time constraints as the three most important barriers for oral cancer screening \[[@CR4], [@CR21]\]. Expansions of early diagnosis and treatment of oral cancers should be a priority from a public health viewpoint. The purpose of this study was to investigate oral cancer screening practices of OHPs in Victoria, Australia as well as evaluate their oral cancer-related opinions and attitudes; and to identify factors associated with the likelihood of an OHP performing an oral cancer screening examination. This information can be used to identify gaps in oral cancer knowledge among OHPs, and subsequently to inform the development of continuing education programs specifically focused on oral cancer prevention, identification and management of malignant and potentially-malignant lesions of the oral cavity and oropharynx at the state level. Methods {#Sec2} ======= The study was a cross-sectional survey of OHP in Victoria, Australia. As of September 2014, there were 4781 registered OHPs in Victoria, which amounts to 23% of the total Australian dental work force. Of them, 3715 are dentists and another 585 are registered DH/DT/OHTs in Victoria \[[@CR35]\]. With the approval of the Human Research Ethics Committee at the University of Melbourne, a request was submitted to the professional associations representing Victorian OHPs, including the Australian Dental Association, the Dental Hygienists Association of Australia (Victorian Branch) and the Victorian Dental and Oral Health Therapists Association, to distribute the survey to their members. OHPs were initially contacted by mail in September 2014. Two weeks and four weeks after the first mailing, postcards were sent to thank those who had returned the questionnaire and to remind the others of the importance of the study, and encourage them to complete the survey. In an attempt to maximize the response rate, participants were offered a choice between completing either a paper-and-pencil survey or a web-based survey (i.e. invitation-letter with URL of online survey, questionnaire and reply paid envelope). Additionally, after the initial postal contact, the Australian Dental Association indicated a preference that e-mails be sent with the link to the online survey, as a reminder. Two reminders were sent in March and April 2015. According to the literature \[[@CR36]--[@CR38]\] the expected response rate among OHPs is around 30%. However, an Australian study involving medical doctors, reported a mean response rate of 19.7% when using a similar simultaneous mixed-mode survey (a paper questionnaire and login details sent together) \[[@CR39]\]. Therefore, we expected a final sample size of around 611 dentists and 89 dental hygienists/dental therapists/oral health therapists. The data collection period was from September 2014 to March 2015. This sample size would be large enough to conduct the required statistical analyses as the minimum sample size necessary to study the relationship between a dependent variable (e.g., knowledge of risk factors for oral cancers) and a set of independent variables based on the case of 10 independent variables accounting for 5% of the variance (a conservative estimate) in the dependent variable, indicates that a sample size of 335 will yield a power of 0.80, that is, an 80% chance of explaining that proportion of the variance at a p-value of 0.05 \[[@CR40]\]. Survey data was derived from a questionnaire consisting of four parts: socio-demographic and work characteristics (6 items); knowledge of risk factors for oral cancers (14 items); level of confidence in discussing health behaviors with patients (5 items); and oral cancer screening practices (11 items). The socio-demographic information included sex, age-range and location of practice. By occupational sub-stratification, participants were classified into three professional groups: 'Dentists'; 'Dental Hygienists (DH)'; and 'Oral Health Therapists and Dental Therapists (OHT/DT)'. Dentists were further sub-divided into General Dental Practitioners (GDP) and 'Dental Specialists'. Professional experience was classified into six categories: '5 years or less'; '6 to 10 years'; '11 to 15 years'; '16 to 20 years'; '21 to 25 years'; and 'More than 25 years'. Employment history information included the postal code(s) of their main geographical location of practice. Using the Australia Post's local delivery service guidelines, work locations were classified as being 'Urban', or 'Rural' \[[@CR41]\]. Participants were asked to self-assess their level of knowledge about risk for oral cancer, utilizing a 10-point numerical scale (0 = 'Very poor' to 10 = 'Excellent'). Knowledge regarding risk factors for oral cancer included 11 factors (smoking, periodontal disease, caffeine consumption, family history of oral cancer, chewing betel nut, human papillomavirus \[HPV\] infections, hepatitis C infection, chewing tobacco, level of alcohol consumption, herpes simplex virus infections and history of oral cancer). Participants were asked to select which of the 11 factors were risk factors for oral cancers utilising a three-option response of 'Yes, 'No' or 'Unsure'. To quantify the degree of knowledge of oral cancer risk factors among OHPs, a score system was developed, which involved adding the weighted answers for each of the 11 questions on risk factors. Scores ranged from 0 to 9. OHPs were also asked two questions regarding their perceived need for more training and education on oral cancers with response categories of 'Yes'; 'No'; or 'Unsure'. Questions related to oral cancer screening attitudes and practices asked: a) whether OHP should routinely screen for oral cancer; b) self-reported frequency of screenings (response options were: 'Very rarely'; 'With less than 50% of patients'; 'With 50% or more of patients'; and 'With every patient'); c) factors that prompt OHPs to initiate discussion of risk factors with patients; d) age groups routinely screened for oral cancer; e) whether OHPs inform the patient about being screened; f) if patients are informed what the screening involves; g) if they have ever used additional diagnostic tools to detect malignant lesions; and h) their course of action when an abnormal lesion was detected. Participants were also asked whether they had ever referred a patient to an oral medicine specialist. Items focusing on OHPs' level of confidence in discussing health behaviors with patients, utilized a numerical 10-point scale (0 = not at all confident to 10 = completely confident). These behaviors included; tobacco use, alcohol consumption, sexual behaviors, oral hygiene practices and diet and nutrition. Data analysis {#Sec3} ------------- The analysis provides descriptive information on the participants' work and various socio-demographics. Bivariate associations were evaluated with Chi-squared analysis for nominal or ordinal variables. For variables on an interval scale, results were analyzed using one-way analysis of variance (ANOVA). A significant ANOVA was followed by post-hoc comparisons using Tukey's Honestly Significant Differences tests. To better understand the association between the combination of socio-demographic, work and psychosocial variables and the probability of conducting a screening exam, a stepwise logistic regression analysis (LRA) was performed. All p-values \<0.05 were considered significant. Data manipulation and analysis were conducted using IBM SPSS Statistics (Version 21.0, IBM Corporation, Endicott, NY, USA). Results {#Sec4} ======= A total of 380 OHPs responded, achieving an overall response rate of 9.4% (ranging from 9.3% among dentists to 22.9% among DH/DT/OHTs). Fourteen respondents indicated that they do not practice dentistry anymore while another 31 mentioned that they do not routinely treat adult patients. These 45 OHPs were excluded from further analysis. Thus, a total of 335 were included in the final analysis. Most this group were dentists (72%; n = 241), either GDP (63.6%) or Dental Specialists (8.4%). Another 13.7% (n = 46) were DHs; 12.2% (n = 41) were OHTs, and the remaining 2.1% (n = 7) were DTs. By gender, the majority were female (58.2%). Among dentist participants, a marginally higher percentage were male (55.8%), while more than 90% of DH/DT/OHTs were female. By age, more than half the total participants (55.5%) were 45 years or younger; however, the largest age group was the '46 to 55 years old' group (24.8%), with 10.7% of the participants in the '25 years of age or younger' group. Differences in age and gender were statistically significant by oral health profession (p \< 0.001). Those working exclusively as OHT were younger than those working as dentists or DHs (See Table [1](#Tab1){ref-type="table"}).Table 1Demographic, work characteristics of oral health professionals in VictoriaDentistsDHsOHT/DTsTotal(n = 242)(n = 45)(n = 48)(n = 335)Age of group\* 25 or less7.42.135.510.7 26--3519.011.150.022.4 36--4523.235.66.222.4 46--5525.242.36.224.8 \> 5525.28.92.119.7Gender\* Male55.82.28.341.8 Female44.297.891.758.2Duration of practicing\* 5 or less15.711.180.523.0 6--1012.411.117.112.8 11--1511.613.32.411.0 16--209.915.609.9 21--259.920.00.09.9 \> 2540.528.90.033.4Location of workplace Urban76.677.374.576.4 Rural23.422.725.523.6\Chi- squared test; p-value: 0.001 When participants were asked about the location of their workplace, the majority (76.4%) indicated an 'Urban' location, with no statistically significant difference by professional background. Regarding the length of time practicing as an OHP, 33.4% indicated more than 25 years of practice; 30.8% between 11 and 25 years; 23.0% reported five years or less of practice; and the remaining 12.8% reported between 6 and 10 years of practice. As expected, differences by duration of practice between groups were statistically significant (P* \< 0.001). Oral cancer screening examination. Almost all OHPs (95.2%) indicated the importance of and need for routine oral cancer screening in their clinical practice. However, only half of those surveyed (51.3%) reported conducting a comprehensive oral cancer screening for all of their patients (Table [2](#Tab2){ref-type="table"}). Another 21.8% reported conducting a comprehensive oral cancer screening for '50% or more' of their patients. OHPs who reported performing oral cancer screening in 'less than 50% of patients' or 'Very Rarely' comprised 14.0% and 12.8% of respondents, respectively. Differences by professional background for completing oral health screening were not statistically significant. The vast majority of respondents (92.2%) routinely screened patients 40 years of age or older for oral cancer. This proportion was 68.4% for 20--39 years old patients, and 38.5% for patients younger than 20 years of age.Table 2Oral screening behaviors of oral health professionals in VictoriaDentistsDHsOHT/DTsTotal(n = 242)(n = 45)(n = 48)(n = 335)How frequently do you complete a comprehensive oral cancer screening? With every patient51.746.754.251.4 With 50% or more of patients21.922.220.821.8 With less than 50% of patients13.217.814.614.0 Very rarely13.213.310.412.8How often do you discuss risk factors for oral cancer with your patients? With every patient5.88.98.46.6 With 50% or more of patients22.740.033.326.6 With less than 50% of patients47.935.541.745.3 Very rarely23.615.616.621.5What factors influence your decision to perform an oral cancer screening examination?\ (Multiple answers) Patient complains of a problem32.231.141.433.6 Age of the patient31.425.243.832.4 Medical history26.931.143.829.9 Other16.526.625.019.1 Among those OHPs who reported not conducting a cancer screening for all their patients (n = 193), the decision to screen was influenced by the patient complaining of an oral health problem (65.6%); the patient's age (63.8%); or the patient's medical history (53.3%). Another 35.0% of respondents indicated other factors influencing their decision to conduct an oral cancer screening examination, such as exposure to risk factors (i.e. alcohol consumption and smoking). Of note, only the 'age of the patient' as an influencing variable reached statistical significance (p \< 0.05). When excluding those who 'Very rarely' conducted an oral cancer screening examination on their patients, just over half of respondents (51.9%) indicated that they 'Sometimes' informed the patient that they were screening for oral cancer lesions. Another 19.9% 'Always' informed patients that they were screening for potentially malignant or malignant lesions. More importantly, almost one third (28.2%) of the OHPs, 'Never' informed patients that such screening assessments were taking place. No statistically significant differences were found by professional background. After excluding those who reported 'Very rarely' conducting oral cancer screenings, for almost all OHPs (97.6%) the examination involved a visual inspection of the patient's oral cavity and 81.1% also included an extra-oral visual inspection (Table [3](#Tab3){ref-type="table"}). Meanwhile, a screening examination that "Always" included either a visual inspection of the oropharynx or conducting neck palpation was reported at lower rates (52.3% and 28.1, respectively).Table 3Oral cancer screening examination behaviors of oral health professionals who performed screening more frequently than "Very rarely"DentistsDHsOHT/DTsTotal(n = 210)(n = 39)(n = 43)(n = 292)Screening examination involved:\ Extra-oral visual inspection of the oral cavity Always80.482.183.781.1 Sometimes2.95.10.02.7 Never16.712.816.316.2A visual inspection of the oral cavity Always98.193.010097.6 Sometimes1.47.00.02.1 Never0.50.00.00.3A visual inspection of the oropharynx Always50.564.150.052.3 Sometimes40.335.942.940.0 Never9.20.07.17.7Neck palpationAlways25.523.744.2\\ 28.1Sometimes56.952.632.552.6Never17.633.721.319.3\Chi- squared test; p-value: 0.05 Apart from neck palpation, which was significantly more frequently conducted among OHTs than other professional groups (p \< 0.05), none of the other screening techniques reached statistical significance levels by OHP background. Thirty-three respondents (11.3%), most of whom were dentists, indicated using additional diagnostic aids/tools to identify potentially malignant lesions. The most commonly utilized additional diagnostic tools included biopsies and radiographs. When asked if respondents had ever referred a patient to an oral medicine specialist to have a lesion investigated, which subsequently was diagnosed as a malignancy, 54.2% of respondents had either never encountered such a clinical scenario, or were unsure. Of those who had referred a patient (n = 136), the most common intraoral site of the malignant lesion (n = 41) was cancer of the tongue, followed by cancer of the floor of the mouth (n = 22); hard palate (n = 18); and the lip (n = 14). There were also eleven cases of bone cancer. OHP respondents were asked about their clinical decision pathway once an abnormal lesion was detected and the practitioner could not reach a diagnosis. The most commonly reported courses of action included: 'Referral to an oral medicine specialist' (85.7%); 'Follow-up at a later appointment' (53.7%); and 'Consultation with another colleague' (45.4%). Least mentioned courses of action included: 'Ask the patient to monitor the lesion' (38.2%); and 'Referral to the patient's general medical practitioner' (14.3%). Differences by professional background were statistically significant for: 'Consultation with another colleague' (p \< 0.001), where DHs and OHT/DTs were more likely to pursue this course of action; and 'Asking the patient to monitor the lesion', whereby OHT/DTs (55.8%) were more likely to ask the patient to monitor the lesion compared with 35.9% and 35.9% for dentists and DHs, respectively (p \< 0.05). Regarding knowledge of oral cancer risk factors, 99.4% of participants identified smoking as a risk factor for oral cancer. The second most frequently identified risk factor was chewing betel nut and chewing tobacco products (98.2%). A history of oral cancer was the third most frequently identified risk factor (97.0%), followed by family history of oral cancer (96.1%), and alcohol consumption (94.6%). The oral cancer risk knowledge score ranged from 0 to 88 with a mean of 68.5 (s.d. 10.1). When OHPs were asked how often they discussed risk factors for oral cancer with their patients, the largest group (45.3%) indicated discussing risk factors for oral cancer with less than 50% of their patients (Table [2](#Tab2){ref-type="table"}). Another 21.5% reported they 'Very rarely' discussed risks factors with their patients. Discussion of risk factors in 'More than 50% of patients' or 'With every patient' was reported by 26.6% and 6.6% of OHP respondents, respectively. No significant differences were demonstrated by professional background. Concerning participants' level of confidence in discussing behavioral issues that may affect oral health with their patients, participants generally felt confident discussing oral hygiene practices (Mean 8.9; s.d. 1.6), diet and nutrition (Mean 8.3; s.d. 1.7), tobacco use (Mean 8.2; s.d. 1.8), and to some extent alcohol consumption (Mean 7.3; s.d. 2.2). In contrast, the mean level of confidence in discussing sexual behaviors (i.e. oral sex practices) was comparatively low at 3.6 (s.d. 1.8). The probability of conducting an oral health screening examination ("Very rarely": 0 vs. Other categories \[i.e., 'With every patient'; 'With 50% or more of patients'; and 'With less than 50% of patients'\]: 1) was explored utilizing LRA with age, sex, profession, rurality and number of years of work experience, risk factor knowledge index, self-assessed oral cancer knowledge, and self-confidence items as independent variables (Table [4](#Tab4){ref-type="table"}). After controlling for the other independent variables included in the model, four variables remained statistically significant \[χ^2^(4) = 40.689; p \< 0.0001\]. Results indicated that dental specialists, were less likely to perform an oral cancer screening examination (OR = 0.18; 95% CI 0.07 to 0.52). As the level of self-assessed knowledge of oral cancer increased, so too did the likelihood of performing an oral cancer screening examination (OR = 1.35; 95% CI 1.09 to 1.67). Additionally, when the OHP's level of confidence in discussing oral hygiene with the patient increased, the likelihood of conducting an oral cancer screening examination also increased (OR = 1.25; 95% CI 1.03 to 1.52). On the other hand, when the 'Patient complained of a problem' the likelihood of performing an oral cancer screening examination decreased (OR = 0.21; 95% CI 0.10 to 0.44). The variance for oral cancer screening examination, using the full model, was 22.5% (Nagelkerke ^2^r = 0.225).Table 4Regression coefficient, odds ratios and 95% confidence interval for odds ratios for the factors associated with the probability of conducting an oral health screening examination among oral health professionals in Victoria, Australiaβ coefficientOdds ratio95% Confidence intervalDental specialist−1.700.180.07 to 0.52Self-assessed knowledge0.301.351.09 to 1.67Level of confidence (oral hygiene)0.221.251.03 to 1.52Patient complained of a problem−1.560.210.10 to 0.44Constant−1.04Nagelkerke r^2^ = 0.225 Discussion {#Sec5} ========== The present study represents an attempt to explore oral cancer-related practices and attitudes among different OHPs working in the Australian state of Victoria. This study's results show little over 50% of OHPs perform comprehensive oral cancer screening with all patients. Although these results are not directly comparable due to wording of questions and response categories, they would contrast with earlier studies done in Australia where OHPs routinely performed oral mucosal screening on 85--95% of their recall and new patients \[[@CR4], [@CR21]\]. In the same manner, studies done elsewhere, reported that 85--89% of dentists and 66--78% of dental hygienists performed oral cancer examination for their patients, although they did not indicate frequency of performing these examinations \[[@CR29], [@CR30]\]. Apart from this, the present study is largely in line with one other previous study done among DH/DT and OHTs in Australia on the importance and need for routine oral cancer screening \[[@CR33]\]. A high proportion indicated that they did so in almost all patients older than 40 years. However, only about two-thirds of respondents routinely screened patients 20--39 years of age. The study also indicated that OHPs, who screened all patients, were less likely to always include neck palpation, and to a lesser extent, visual inspection of the oropharynx in their oral cancer screening practice. This may be due to lack of confidence in undertaking a head and neck examination as evidenced in other study done in Australia \[[@CR21], [@CR33]\]. These findings are cause for some concern as many oral cancers may not present with visibly detectable signs or symptoms during the pre-malignant or localized stages, which represents a clinical window in which these lesions are most amenable to treatment \[[@CR29]\]. Such early stage lesions could otherwise be detected by a comprehensive and thorough visual examination of high risk sites \[[@CR22], [@CR42]\] and facilitating patient education about oral cancer. Furthermore, a comprehensive oral cancer screening examination would seem essential, particularly given the growing incidence of HPV positive oropharyngeal cancers \[[@CR4], [@CR22], [@CR42], [@CR43]\]. Consequently, it is important for OHPs to maintain and develop competence and confidence in neck palpation techniques and visual examination of the oropharynx as part of their oral cancer screening practices \[[@CR22]\]. Where necessary, appropriate education programs should be developed to address the knowledge and skill gaps in this area \[[@CR4], [@CR21]\]. The present study showed that OHTs performed oral cancer screening somewhat more frequently than other OHP groups. As the incidence of oral cancer continues to rise \[[@CR9]\], the role of oral health professionals in the prevention, early identification and management of oral malignancies will become increasingly relevant to public health. In order to reduce the morbidity and mortality of oral cancer, it is necessary to implement programs aimed at prevention, early identification and diagnosis. Early identification would involve facilitating patient recognition of signs and symptoms and providing education about when to consult a health professional. Patient delays in seeking care could be reduced by self-examination for early clinical features and by population level educational interventions, particularly targeting those in the higher risk groups for oral cancer. In recent years, awareness of cancer risk factors has increased due to wider media coverage of the issue \[[@CR44]\]. However, studies indicate that in contrast to other types of cancers, the majority of the population are ignorant about oral cancer \[[@CR45], [@CR46]\]. The public may have a tendency to interpret oral cancer symptoms and signs as relatively minor, as problems that are likely to resolve on their own. Given the lack of general public understanding of oral cancers and inability to recognise clinical symptoms and signs \[[@CR22]\], it can be inferred that patient presentation to an OHP regarding an oral lesion may be either absent or too late, which would adversely impact on early detection and treatment outcomes. In addition to these issues, some cancers may be asymptomatic and the absence of notable symptoms may further contribute to late diagnosis. For these reasons, opportunistic oral cancer screening examinations conducted by OHPs remain an important mode for early identification and diagnosis. Results from the multivariate analysis indicate that after controlling for other variables, dental specialists tend to perform less oral screening compared with other OHPs. It is possible that dental specialists rely on GDPs to conduct comprehensive dental examinations while they focus solely on the condition for which the patient was referred them; however, this cannot be stated categorically as the relevant information was not collected in the survey. The odds of conducting an oral screening examination also decreased when the patient complained of oral pain. It could be inferred, although once again respondents were not asked about this in the survey, that OHPs addressed the presenting complaint on these occasions (e.g. pain) and did not conduct a more comprehensive oral examination. Unsurprisingly, the data indicated that OHPs with a higher self-evaluated confidence level in oral cancer-related knowledge had greater confidence advising patients and were more likely to perform oral cancer screening examinations \[[@CR21]\]. The ultimate goal would be to have the whole oral health team collaborate with an integrated approach to oral health care, particularly in the context of oral cancer knowledge, screening practices and patient and community education. When considering these results, certain limitations must be acknowledged. The low response rate may have introduced non-response bias, and combined with the self-reported nature of the data means it is not possible to generalize findings to all OHPs working in Victoria, Australia. Furthermore, response rates to online surveys have been demonstrated to be less than for surveys administered using a paper-based approach \[[@CR47], [@CR48]\]. However, the response rate is also related to the size of the population under study with larger populations requiring smaller response rates \[[@CR48]\]. Thus, although the number of responses received was not as high as was hoped, the achieved overall response rate was within the expected range for online surveys among oral health professionals (2.5% to 26%) \[[@CR49], [@CR50]\]. Despite some minor differences, this study group is largely representative of the Victorian OHP population in terms of age, profession, gender and distribution. There was an overrepresentation of DH/DT/OHTs in the study sample, who make up 28% of total respondents but only 17% of all registered dental practitioners in Victoria \[[@CR35]\]. When considering the socio-demographic profile, there is no available data to determine the age range of each dental practitioner group in the wider profession in the state of Victoria. However, the age profile of survey respondents (55.5% aged 45 years or younger) is very similar that for all dental practitioners in Australia at the time of the survey (56.7% aged less than 45 years old) \[[@CR35]\]. The proportion of female dentists in the study also mirrored figures reported for the state of Victoria (57%) in 2015. Most dentists worked in urban areas (80.0%) \[[@CR51]\]. Conclusions {#Sec6} =========== Together with risk behavior change, oral cancer screening has been shown to contribute to reductions in oral cancer mortality rates among high-risk individuals \[[@CR22], [@CR52]\]. It is highly probable that OHPs will see patients with oral cancer and many more with potentially malignant lesions in their professional lives \[[@CR4], [@CR21]\]. Present results suggest that continued efforts to enhance the quality and consistency of oral cancer screening practices are required, which should include education and training of OHPs in up-to-date and evidence-based screening methods. The recommendation to see an oral health professional at least once a year would only be beneficial if patients are routinely screening for oral cancer. The results from this study may assist in the development of oral cancer-related education and training programs for OHPs. Increased emphasis on regular training in oral cancer screening protocols and in patient education and counselling practices are imperative to improve oral cancer prevention and rates of early detection, to reduce the mortality-burden resulting from oral cancers. DHs : Dental Hygienists DT : Dental Therapists GDP : General Dental Practitioners HNC : Head and Neck Cancer HPV : Human Papilloma Virus OHP : Oral Health Professional OHTs : Oral Health Therapists OPC : Oro-Pharyngeal Cancers Not applicable Funding {#FPar1} ======= This study was partially funded by a grant received from DENTSPLY Research & Innovation Grants. Availability of data and materials {#FPar2} ================================== Ethics approvals were granted on the basis that only researchers involved in the study could access the de-identified data. Raw data have been stored securely at the Melbourne Dental School, The University of Melbourne. The minimum retention period is 5 years from publication or public release of the results. Supporting documents are available upon request to the corresponding author. RM1 contributed with the conception of the study, design, draft of the manuscript, read and approved the final manuscript. SH carried out the fieldwork, contributed with the data analysis and draft of the manuscript, and read and approved the final manuscript. RM2 contributed with the conception of the study, and the design and draft of the manuscript, and read and approved the final manuscript. DB contributed with the conception of the study, and the design and draft of the manuscript, and read and approved the final manuscript. MM1 contributed with the conception of the study, interpretation of the data and the design and draft of the manuscript. RM3 contributed with interpretation of the data and final drafting of the manuscript. BK carried out part of the fieldwork, interpretation of the data and final drafting of the manuscript. AC contributed with interpretation of the data and drafting of the manuscript. All the authors revised and approved the manuscript. MM2 contributed with the conception of the study, and the design and draft of the manuscript, and read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== This study was approved by The Human Research Ethics Committee of The University of Melbourne, Australia (\#1442325.1). All participants conseted to participate in this study. It was explained to potential participants that reception of completed surveys, was assumed as consent for participation in the study. Consent for publication {#FPar4} ======================= Not Applicable Competing interests {#FPar5} =================== The authors declare that they have no competing interest. RM is a Section Editor for BMC Oral Health. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	{ "pile_set_name": "PubMed Central" }	[ "amount", "treatment", "cervic", "probabl", "choic", "two", "almost", "control", "refer", "given", "work", "origin", "hope", "program", "remain", "tumor", "eg", "cours", "compris", "decreas", "nomin", "tendenc", "ie", "respond", "ohtdt", "purpos", "collect", "thank", "biopsi", "employ", "identifi", "length", "risk", "grant", "select", "fund", "neck", "take", "week", "maxim", "locat", "occas", "multipl", "may", "team", "caffein", "recent", "branch", "squar", "septemb", "live", "use", "gener", "constraint", "strongest", "final", "score", "collabor", "ever", "world", "focus", "support", "caus", "complet", "accord", "ny", "overrepresent", "signific", "focu", "db", "full", "guidelin", "becom", "exposur", "thirtythre", "suggest", "relev", "excel", "endicott", "proactiv", "also", "paperbas", 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1. INTRODUCTION {#sec1} =============== Calcifying Epithelial Odontogenic Tumor (CEOT) is an uncommon jaw lesion with a benign and slow-growing pattern but locally aggressive course \[[@r1]\]. The lesions are most prevalent among patients aged 30 to 50 years, and no sex predilection has been observed \[[@r1], [@r2]\]. Clinically, the CEOT appears as a slowly asymptomatic expansion with radiolucent honeycomb appearance in the posterior areas of the mandible \[[@r2]\]. The CEOT is predominantly an intraosseous tumor in approximately 94% of cases \[[@r3]\]. The peripheral variant is a rare tumor (6%) with less aggressive behavior that was described by Pindborg in 1966 \[[@r4]\]. The classical histopathological aspects include sheets and islands of eosinophilic polyhedral epithelial cells in association with homogeneous pink amyloid-like deposits and areas of calcification \[[@r5]-[@r22]\]. One unusual histological finding is the presence of clear cells, as reported in 14 cases of peripheral CEOT in the English literature \[[@r2], [@r5]-[@r19], [@r22], [@r23]\]. CEOT is an odontogenic tumor; the consensus regarding the best therapeutic approach is surgical excision with safe margins recommended for the intraosseous variant \[[@r3], [@r4]\]. However, the peripheral lesions are commonly treated through conservative surgery with no review of this approach \[[@r3]\]. The present study reports a case of a 21-year-old Caucasian female patient with a mandibular peripheral CEOT rich in clear cells. An additional critical literature review focuses on the treatment protocols for the peripheral variant of the tumor. 2. CASE DESCRIPTION AND RESULTS {#sec2} =============================== A 21-years-old Caucasian woman presented to a private dental clinic with a chief complaint of asymptomatic swelling in the gingiva observed four years prior. A gradual increase in size and no history of previous treatment were also reported during the anamnesis. The patient signed the informed consent, which represents the ethical approval of the faculty committee. Her medical and socio-economic histories were not contributory. The extra-oral evaluation did not reveal changes. The intraoral examination revealed a sessile nodule with a color similar to that of the mucosa and a focal erythematous area with a fibro-elastic consistency measuring 1.5 cm in the largest diameter extending from the inferior right lateral incisor to the inferior right first premolar. The lesion involved the vestibular and lingual gingiva, causing displacement of the inferior right canine (Fig. [1](#F1){ref-type="fig"}). Panoramic reconstruction and parasagittal slices of the Cone Beam Computed Tomography (CBCT) showed a slightly superficial hypodense area between the inferior right lateral incisor and inferior right canine with reabsorption of the alveolar crest (Fig. [2](#F2){ref-type="fig"}). Based on the clinical and immunological aspects, the main diagnosis hypotheses included peripheral ossifying fibroma, peripheral giant cell lesion, and ancient pyogenic granuloma. The peripheral odontogenic tumors were also included as a differential diagnosis. An excisional biopsy was performed and a clear separation was noted between the lesion and mandible bone during the trans-surgical approach. The histopathological analysis revealed a well-circumscribed proliferation comprising numerous islands and strands of epithelial polyhedral cells with well-defined borders and marked round nucleus in the connective tissue under the mucosal epithelium. Numerous nests, cords, and small islands of polyhedral cells with clear and vacuolated abundant cytoplasm were observed interspersed with the amorphous eosinophilic deposits (Fig. [3](#F3){ref-type="fig"}). Immunohistochemistry was performed, which yielded positive results for CK-19 in the epithelial cells, except for the clear cells. Congo red staining showed the presence of amyloid-like deposits with apple-green birefringence under polarized light (Fig. [4](#F4){ref-type="fig"}). A final diagnosis of a peripheral CEOT rich in clear cells was reached. No complications were observed in the postoperative appointment and a follow-up schedule was established. The patient has had no recurrence after 22 months (Fig. [5](#F5){ref-type="fig"}). 3. PERIPHERAL CEOT CASES {#sec3} ======================== Long-term follow-up of the central variant of CEOT has shown the best results, with no recurrence following block resection with safe margins; however, well-documented cases are scarce \[[@r1], [@r24]\]. Additionally, there is discussion regarding aggressive behavior involving clear cells in the CEOT \[[@r1], [@r21], [@r24]\]. Other odontogenic lesions with clear cells including ameloblastoma and clear cell odontogenic tumors have been reclassified as malignant, in which a more aggressive course can be expected \[[@r24], [@r25]\]. Nonetheless, this premise has not been confirmed for CEOT because the latest review of all central variants showed no conclusive data regarding the worse course for this type of lesion \[[@r1], [@r24], [@r25]\]. Peripheral CEOT is considered a harmless lesion, but the recommended approach for peripheral cases in the presence or lack of clear cells is not emphasized in previous reports \[[@r2], [@r4]-[@r6], [@r8]-[@r20], [@r22]\]. In this context, a search of the English literature was performed in the PubMed database using the keywords "calcifying epithelial odontogenic tumor" with "Pindborg," "peripheral," "clear cell," and "treatment." All manuscripts on peripheral CEOT published until May 2018 were considered. Cross-references were included. Studies involving mixed odontogenic tumors in association with CEOT, CEOT associated with other conditions, no exclusive extraosseous tumors, absence of treatment modality, and no full-text database were excluded. The anatomic sites, duration, clinical and imaging aspects, types of treatment, recurrence, and follow-up are summarized in Table [1](#T1){ref-type="table"}. 4. DISCUSSION {#sec4} ============= The peripheral variant is a rare presentation of CEOT, with a differential diagnosis including gingival reactive lesions such as ancient pyogenic granuloma, peripheral giant cell lesion, and peripheral ossifying fibroma in addition to other peripheral odontogenic tumors. The histopathological aspects of the extraosseous are similar to those of the intraosseous CEOT counterparts and Congo red staining confirms the presence of amyloid-like material such as immunohistochemistry keratin markers confirm the odontogenic epithelial origin \[[@r21], [@r22]\]. In addition, clear cells have been observed in some odontogenic lesions and, although previous authors have speculated their relationship with more aggressive CEOT, no role has yet been shown for the relationship between the behavior and this histopathological presentation \[[@r21], [@r22]\]. Extraosseous CEOT is considered a less aggressive tumor and conservative surgery is performed in most cases. However, no previous articles have evaluated the best approach for peripheral CEOT based on recurrence and follow-up data. The current review identified no clinical significance in relation to the aggressive aspects. This finding is supported by the observation that approximately 80% of cases were treated with conservative management, with only one recurrent case without a clear cell component \[[@r2], [@r4]-[@r6], [@r8]-[@r18], [@r22]\]. A detailed evaluation of all articles on peripheral CEOT found no reported recurrence during follow-up in 96% of cases \[[@r2], [@r5], [@r6], [@r8]-[@r20], [@r22], [@r23], [@r26]\]. A conservative soft tissue excision was the main approach in the literature and was also sufficient for the complete resolution of the current case. These findings suggest an indolent, local, non-infiltrative course of peripheral lesions when compared to intraosseous CEOT. The discrete cupping or erosion of superficial bone may be caused by compression rather than by invasive behavior, supporting the non-aggressive behavior of the lesion. Other peripheral odontogenic tumors, including ameloblastoma, ameloblastic fibroma, calcifying cystic odontogenic tumor, and adenomatoid odontogenic tumor, present similar characteristics \[[@r27], [@r28]\]. An isolated case with recurrence was observed with posterior intraosseous involvement; however, it was a unique case with bilateral peripheral lesions \[[@r18]\]. Clear cells were also not observed and there was no recurrence after the second conservative surgery \[[@r18]\]. CONCLUSION ========== Our review confirms that conservative enucleation is the most appropriate management for peripheral CEOT. Moreover, we identified no association between clear cells and clinical aggressiveness in peripheral lesions as was previously suggested for the central counterparty. Declared none. CONSENT FOR PUBLICATION ======================= Informed consent was obtained from all the patients prior to being enrolled in the study. CONFLICT OF INTEREST ==================== The authors declare no conflict of interest, financial or otherwise. ![](TODENTJ-12-856_F1){#F1} ![](TODENTJ-12-856_F2){#F2} ![](TODENTJ-12-856_F3){#F3} ![](TODENTJ-12-856_F4){#F4} ![](TODENTJ-12-856_F5){#F5} ###### Previous articles about peripheral calcifying epithelial odontogenic tumor including clear cell variant with emphasis in the treatment. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Authors Anatomic Site Duration Clinical Presentation Imaging\ Treatment Recurrence Folow-up Presentation ----------------------------------- ------------------- -------------- -------------------------------- --------------------- ------------------------------------------------- ---------------- -------------- Pindborg, 1966^5^ Max.\ 5 years Painless firm\ NCI Simple excision No NCI gingiva mass Abrams and Howell, 1967^10^\\ Mand. gingiva NA Painless firm\ Crest\ Simple excision No 3 years mass resorption Decker and Laffitte, 1967^11^ Mand.\ 5years Painless firm\ NCI Simple excision No NCI gingiva mass Patterson et al, 1969^12^ Mand. gingiva 1 year Painless firm\ NCI Simple excision No NCI mass Krolls and Pindborg, 1974^13^ Mand. gingiva NA Painless firm\ NCI Simple excision No NCI mass Wherteimer et al., 1977^14^\\ Max. gingiva NA Painless firm\ No Simple excision No NA mass Ai-ru et al., 1982^15^\\ Mand. gingiva 10 years Painless firm\ NCI Ressection No 2 years mass Ai-ru et al., 1982^15^\\ Mand. gingiva 2 years Painless firm\ No Partial\ No 10 years mass ressection Takeda et al., 1983^16^ Max\ NA Painless firm\ NCI Excision underlying bone No NCI gingiva mass KH Ng et al., 1996^17^ Max.\ 1 year Painless firm\ Erosion Excision No NA gingiva swelling Houston & Fowler, 1997^18^\\ Max.\ 5 months Ulcerated\ No Simple excision No 4 years gingiva mass Houston & Fowler, 1997^18^\\ Mand.\ 5 months Ulcerated\ Erosion\* Simple excision No 4 years gingiva mass Orsini et al., 2000^9^\\ Max. gingiva 6 months Painless red\ Not\ Simple\ No 4 years mass performed excision Mesquita et al., 2003^6^\\ Max. gingiva 10 months Painless firm\ No Excision No 2.5 years nodule de Oliveira et al., 2009^2^\\ Max. gingiva NA Painless exophitic mass No Excision No 1 year de Oliveira et al., 2009^2^\\ Mand. gingiva NA Painless exophitic mass Superficial cupping Excision No 1 year Abrahão et al., 2009^19b^ Mand\ 3 months Painfull erithematous swelling No^a^ Simple excision^c^ Yes^d^ 3.5 years gingiva Habibi et al., 2009^20^\\ Max.\ 11 years Ulcerated\ NCI Excisional biopsy with 5-mm safety margins No NA gingiva mass Marino et al., 2013^21^ Max. gingiva NA Painless\ Bone\ Conservative surgery including teeth extraction No 2 years swelling resorption Afrogheh\ Mand. gingiva 6 months Painless\ Erosion Complete excision No 1.5 years et al., 2014^7^\\ swelling Shetty et al., 2016^22^\\ Mand. gingiva 8 months Painless swelling No Excisional biopsy No 6 months de Carvalho et al., 2016^26^ Mand.\ 1 month Painless swelling No Excisional\ No 1 year gingiva biopsy Bajpai M,\ Max.\ 9 months Pink-coloured\ Loss of lamina dura Complete\ No 6\ 2018^23^\\ gingiva swelling excision months Flores et al., 2018\\¡ Mand. gingiva 4 years Painless\ Superficial\ Conservative surgery No 22 months swelling hypodense\ cupping --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Max.= maxillary Mand. = mandible NA = Not available NCI = Not clearly identified a = osseous resorption and calcifications (recurrent lesions) b = bilateral c = soft tissue excision followed bone curettage d = after 1 year \Observed only during the surgical procedure \\*Clear cell variant ¡ Current case	{ "pile_set_name": "PubMed Central" }	[ "cord", 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Introduction {#sec1_1} ============ Adrenal myelolipomas are benign tumors which are usually asymptomatic and diagnosed incidentally on imaging or during autopsy. They were first described by Gierke in 1905 and were termed as myelolipomas by Oberling in 1929 \[[@B1], [@B2]\]. They are uncommon benign tumors of the adrenal cortex constituting approximately for 3--5% of all primary adrenal tumors and are increasingly being diagnosed due to improved imaging techniques \[[@B3], [@B4]\]. Adrenal myelolipomas are most frequently detected after the fifth decade and are seen almost equally in both genders. They are commonly found as unilateral masses, but occasional bilateral masses have been documented \[[@B5]\]. A right-sided predominance in unilateral tumors has also been reported \[[@B6]\]. They are usually asymptomatic but myelolipomas may attain large sizes and present as abdominal masses \[[@B7], [@B8]\]. Patients may also present with symptoms of hematuria and hypertension \[[@B9]\]. Their size may also cause mass effect by compressing other intra-abdominal organs \[[@B10]\]. Malignant transformation has not been documented in these tumors. Diagnosis of giant adrenal myelolipomas in a timely manner may prevent complications due to hemorrhage and can be lifesaving. Case Description {#sec1_2} ================ A 52-year-old man with a past medical history significant for atrial fibrillation, diabetes type 2 and hypertension for 30 years was admitted with early satiety for the past 3 months and progressive dyspnea on exertion for 3 weeks. He also complained of gradual unintentional weight loss of approximately 100 pounds over the last 10 years. He had been a non-smoker and a moderate alcohol user for the last 30 years. On initial presentation to the hospital, he denied shortness of breath at rest or chest discomfort and was in no acute distress. His blood pressure was 204/93 mm Hg on the left arm and 194/88 mm Hg on the right arm with an irregular heart rate of 54 beats/min and oxygen saturation of 96% on room air. The lungs were clear to auscultation, jugular venous distension was noted at 13 cm above the sternal angle and 3+ bilateral leg edema with scrotal edema. Cardiac auscultation was pertinent for 3/6 systolic ejection murmur over the left sternal border. His abdomen felt firm and lacking in tympanicity, but it was non-tender in all four quadrants. Bowel sounds were audible but faint. Laboratory values were notable for a brain natriuretic peptide of 980 ng/l, creatinine of 1.2 mg/dl, BUN of 20 mg/dl, HCO~3~ of 31 mEq/l, INR of 1.23, Hb of 12.5 g/dl, Na of 140 mEq/l, K of 3.7 mEq/l and negative cardiac troponins. He was monitored in the Intensive Care Unit for decompensated heart failure with uncontrolled hypertension. To promote diuresis and reduce afterload, he was administered intravenous furosemide 80 mg, oral metolazone 2.5 mg, oral hydralazine 75 mg every 6 h, oral clonidine 0.3 mg, lisinopril 40 mg, isosorbide mononitrate 120 mg and losartan 100 mg. Overnight, he had an episode of moderate self-limited epistaxis with minimal improvement in blood pressure control. He remained in slow atrial fibrillation with a lowest recorded heart rate of 38 beats/min. However, he denied dizziness at any time despite a negative fluid balance of 1.8 liters. Renal function worsened with an increase of creatinine to 1.6 mg/dl. An echocardiogram showed a left ventricular ejection fraction of 30% with four-chamber dilatation, moderately reduced right ventricular systolic function, severe mitral regurgitation and moderate pulmonary hypertension. He underwent right heart catheterization showing a right atrial pressure of 12 mm Hg, right ventricular pressure (systolic/diastolic) of 60/12 mm Hg, pulmonary artery pressure of 60/23 mm Hg and pulmonary capillary wedge pressure of 20 mm Hg and a cardiac index of 2.3 liters/min. A left heart catheterization revealed clean coronaries, and alcohol was thought to be the likely etiology of non-ischemic cardiomyopathy in this patient. Urine catecholamine and vanillylmandelic acid were measured and a magnetic resonance angiography of the renal arteries was performed to evaluate for secondary causes of hypertension. This showed non-obstructed renal arteries bilaterally. A minimally enhancing 22 × 16 × 23 cm mass was visualized in the left upper and mid-abdomen displacing the spleen and stomach posterosuperiorly, the pancreas anteriorly and the left kidney inferiorly (fig. [1b](#F1){ref-type="fig"}). The left adrenal gland was not visible. A contrast-enhanced CT scan of the abdomen to further characterize the mass showed no infiltration of the surrounding vasculature or organs, and the left adrenal gland was visualized although it could not be confirmed if the mass was arising from the adrenals (fig. [1a](#F1){ref-type="fig"}). The suggested radiological differential diagnoses were: liposarcoma, teratoma and myelolipoma. A needle biopsy of this mass showed adipose tissue with foci of extramedullary hematopoiesis consistent with myelolipoma without any evidence of malignancy, confirming this mass to be a benign myelolipoma (fig. [2a, b](#F2){ref-type="fig"}). Urinary epinephrine, metanephrine and vanillylmandelic acid were within normal limits. A renal perfusion scan showed a small left kidney with reduced perfusion contributing 22% to the overall tubular function without obstructive uropathy. The lower extremity edema was partly ascribed to be due to inferior vena cava compression, and diuretics were decreased due to worsening renal function. Given the bradycardia and severe congestive heart failure, an electrophysiologist was consulted for evaluation and need for pacemaker implantation. Bradycardia was ascribed to be partly due to the Cushing reflex secondary to elevated blood pressure, and as the patient had no symptoms secondary to a low heart rate, pacemaker implantation was not advised. Despite maximum doses of clonidine, isosorbide, hydralazine, losartan and lisinopril, at best a moderate blood pressure control was achieved preoperatively. The patient was taken for surgical removal of the mass. During surgery, the mass was found to be soft, highly vascular but well encapsulated and attached only to the left adrenal gland via a small portion that was resected along with the mass (fig. [3a, b](#F3){ref-type="fig"}). There was minimal blood loss during the surgery, and the patient had an uneventful recovery in the postoperative period. A renal perfusion scan done 8 days after surgical removal showed 36% contribution to the overall tubular function. Blood pressure control improved after surgery and the patient was gradually titrated off clonidine, hydralazine and lisinopril. Bradycardia resolved with few episodes of atrial fibrillation with rapid ventricular response that were controlled with β-blockers and digoxin. The antihypertensive medications that the patient was discharged on were carvedilol 40 mg, valsartan 320 mg and isosorbide mononitrate 240 mg. Discussion {#sec1_3} ========== Myelolipomas are composed of fat and hematopoietic tissue and are usually biochemically inactive. They are thought to arise from the reticuloendothelial cells of the adrenal gland in response to repeated stimulation by stress, inflammation and ACTH \[[@B11], [@B12], [@B13]\]. Both the adipose and the hematopoietic cells have been noted to have the same pattern of X-chromosome inactivation and have been postulated to arise by clonal proliferation from a common mesenchymal stem cell \[[@B14]\]. Moreover, the chromosomal translocation (3;21)(q25;p11) has been detected in myelolipomas in patients with hematological malignancies, which has led to the speculation of bone marrow origin of these tumors \[[@B15]\]. Extra-adrenal myelolipomas have been documented in the mediastinum, lung, thoracic spine, liver mesentery, spleen, kidney and pelvis \[[@B16], [@B17], [@B18], [@B19], [@B20], [@B21]\]. They have been noted to be more common in women \[[@B22]\]. Extra-adrenal myelolipomas may mimic extramedullary hematopoietic tumors especially since bone spicules have been reported \[[@B23]\]. Extramedullary hematopoietic tumors generally have a higher content of erythroid cells and less fat tissue compared to extra-adrenal myelolipomas which have a relatively higher number of lymphoid cells \[[@B24]\]. Adrenal myelolipomas are best imaged by CT due to the high negative attenuation value. However, calcification is seen in approximately 27% of the cases and the presence of a higher proportion of hematopoietic tissues may mimic an adrenal adenoma \[[@B4], [@B25], [@B26]\]. Ultrasonography is not a very useful diagnostic modality, especially if the tumors are smaller in size (\<2 cm). An MRI has a similar diagnostic utility as a CT scan in adrenal myelolipomas with a high fat content \[[@B27]\]. The gold standard for diagnosis is tissue biopsy which typically shows hematopoietic cells admixed with mature adipose cells \[[@B6]\]. Adrenal myelolipomas can be associated with hyperaldosteronism, congenital adrenal hyperplasia and Cushing\'s syndrome \[[@B28]\]. They are known to be biochemically inactive but a case of adrenal myelolipoma secreting cortisol has been reported \[[@B8]\]. Adrenal myelolipomas \<5 cm are conservatively managed with sequential imaging, but larger masses are preferably resected due to the risk of hemorrhage \[[@B29]\]. Laparoscopic resection has been successfully used to reduce hospital stay and improve perioperative outcomes, but it is not preferable for tumors \>10 cm or in tumors with infiltration or extensive adhesions \[[@B30], [@B31]\]. Our patient was fortunate not to have any hemorrhagic complications. His clinical presentation was compatible with symptoms related to mass effect of the giant myelolipoma, which resolved after surgical resection. Conclusion {#sec1_4} ========== Adrenal myelolipomas are benign tumors with favorable outcomes when diagnosed in a timely manner. Their insidious clinical course becomes apparent only when mass effects are manifested in the form of an abdominal mass, flank pain, hypertension and pedal edema. It is important to differentiate them from other malignancies, such as liposarcoma, and to ensure that the myelolipoma is not biochemically active. Adequate follow-up of small masses and surgical resection of large masses are the standard of care which provide for lesser morbidity and mortality. Disclosure Statement {#sec1_5} ==================== The authors declare that there was no funding for this study. ![a CT scan of the abdominal mass seen in transverse view. b MRI scan of the abdominal mass seen in coronal view.](cro-0007-0182-g01){#F1} ![a Low-power magnification of the biopsy showing capsule (red arrowhead), adrenal tissue (black arrowhead) and an admixture of adipose and hematopoietic cells. b High-power magnification of the biopsy showing adipocytes (black arrowhead), erythroid precursors (cyan arrowhead), lymphoid precursors (blue arrowhead) and megakaryocytes (green arrowhead). Bony spicules and sinusoids are conspicuously absent from the specimen confirming it is a myelolipoma.](cro-0007-0182-g02){#F2} ![a Resected specimen showing a well-encapsulated tumor. b Resected specimen cut surface revealing adipose tissue with interspersed areas of hemorrhage.](cro-0007-0182-g03){#F3}	{ "pile_set_name": "PubMed Central" }	[ "auscult", "lowpow", "rest", "interspers", "almost", "control", "given", "origin", "remain", "tumor", "cours", "decreas", "overnight", "compat", "bowel", "biopsi", "creatinin", "discharg", "risk", "spicul", "fund", "week", "valu", "urinari", "mri", "hematopoiet", "may", "fifth", "mononitr", "clonidin", "cush", "gener", "use", "chest", "etiolog", "moder", "room", "posterosuperiorli", "imag", "caus", "intraabdomin", "signific", "constitut", "kidney", "becom", "suggest", "deni", "right", "also", "boni", "gierk", "transvers", "cell", "women", "uncontrol", "perform", "jugular", "metolazon", "qp", "dizzi", "normal", "afterload", "attain", "syndrom", 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Core tip: Recognizing patterns of subcutaneous sarcoidosis is important for hand surgeons and other surgical specialties that do not commonly see this patient population in order to rapidly identify and diagnose a disease that has extra-cutaneous manifestations and can lead to greater morbidity and mortality when not diagnosed or treated early. INTRODUCTION ============ Sarcoidosis is a chronic systemic granulomatous disease of unknown etiology characte-rized by the presence of non-caseating granulomas in affected organs\[[@B1]\]. he non-caseating granulomas of sarcoidosis can be found anywhere on the body and involve many different organs. In the head and neck, sarcoidosis typically impacts the cervical lymph nodes, globe, parotid, and larynx with up to 5% of patients demonstrating facial nerve involvement\[[@B2]\]. Ocular involvement is also seen, presenting as uveitis, scleritis, and chorioretinitis\[[@B2]\]. Typically, the lungs are the primary site of disease with cutaneous manifestations being the second most common site. Cutaneous manifestations of Sarcoidosis are seen in up to 9%-37% of patients\[[@B3],[@B4]\]. One particular manifestation of cutaneous involvement, subcutaneous sarcoid nodules, is a rare finding. Previous estimates of the frequency of subcutaneous sarcoidosis ranged from 1.4%-6%, with more recent studies suggesting an occurrence rate of 11.8%-16% among patients presenting with cutaneous sarcoid involvement\[[@B3],[@B5]-[@B7]\]. This variant of Sarcoidosis is defined clinically by asymptomatic, non-tender, flesh colored nodules usually ranging between 0.5-2.0 cm\[[@B6],[@B8]\]. Histologically, subcutaneous sarcoidosis is defined by the presence of non-caseating granulomas present in the subcutaneous tissue\[[@B6]\]. On ultrasound imaging these lesions present as an irregularly defined mass with hyper and hypoechoic areas\[[@B9]\]. Fludeoxyglucose (FDG) positron emission tomography (PET) / computed tomography (CT) has also been used to identify sarcoid lesions and presents as increased uptake in subcutaneous areas\[[@B10]\]. While helpful, FDG PET/CT may not be able to differentiate between connective tissue diseases as Sjrogen's Syndrome also presents as increased uptake\[[@B11]\]. Additionally, many soft tissue diseases can look similar on magnetic resonance imaging (MRI) making defining imaging characteristics of each disease important to diagnosis. On MRI imaging Sarcoid lesions involving the face and neck will appear with high signal intensity on T2-weighted images and enhancement on contrast-enhanced images\[[@B2]\]. Given that Wegener's Granulomatosis can mimic Sarcoidosis in the head and neck, MRI imaging helps differentiate these two soft tissue diseases as Wegener's Granulomatosis will appear as hypodense on T1 and T2-weighted images with variable degrees of enhancement with contrast\[[@B2]\]. Similarly, both sarcoidosis and Scleroma can impact the cervical lymph nodes making diagnosis difficult. MR imaging is again a useful tool in differentiating these two diseases in the lymph nodes as Scleroma will appear as low signal intensity on T1 and high signal intensity on T2 imaging with homogeneous pattern of contrast enhancement, and Sarcoidosis will have a foamy appearance on T1 weighted imaging\[[@B2],[@B12]\]. Lupus Both Sarcoidosis and another soft tissue disease like scleroderma can impact the lymph nodes in the neck Recent literature has suggested a strong correlation between subcutaneous sarcoidosis and evidence of systemic sarcoid involvement, and that sarcoid lesions may be an early finding indicative of systemic disease\[[@B7],[@B13],[@B14]\]. Given its correlation with systemic illness, the ability to correctly identify subcutaneous sarcoid lesions is an important diagnostic tool for physicians in the early stages of sarcoidosis. We describe here a case of subcutaneous sarcoidosis and review the literature to determine if there are any commonalities in the presentation of this disease among patients, and to better assist clinicians with diagnosing this rare disorder. A literature search was performed of the MEDLINE and PubMed database using keywords as "Subcutaneous sarcoidosis" and "Subcutaneous Sarcoidosis", combined with "hand", "hand surgery", "Upper extremity surgery", yielding 202 results. The search includes all articles published since 2000. The search was limited to studies published in English and performed on humans. Cases without either a serum angiotensin-converting enzyme (ACE) level or a chest imaging study for all patients reported were excluded. Ultimately 29 articles were selected using the diagnostic criteria of subcutaneous sarcoidosis first proposed by Vainsencher et al\[[@B6]\]. These 29 articles represent 82 cases of subcutaneous sarcoidosis dating back to 1966. CASE PRESENTATION ================= Chief complaints ---------------- A 38-year-old Caucasian female was referred to Plastic Surgery from Dermatology after presenting with an 8-mo history of 4 firm, asymptomatic, skin-colored nodules on her left and right upper extremities and neck. History of past illness ----------------------- A past medical history of arthritis and recurrent nephrolithiasis. Personal and family history --------------------------- She denied any family history of soft tissue masses or autoimmune disorders. Physical examination upon admission ----------------------------------- The mass on the posterior aspect of her neck measured 0.5 cm x 5 cm. The nodule on her left forearm measured 2 cm x 3 cm (Figure [1A and B](#F1){ref-type="fig"}). On the extensor surface of her right forearm were two masses measuring 1 cm x 1.4 cm and 3.5 cm x 4.5 cm respectively (Figure [1C and D](#F1){ref-type="fig"}). The overlying skin was normal. At the time of the plastic surgery consult, the patient was compliant with her medication regimen of dextroamphetamine-amphetamine 30 mg by mouth once daily, and acetaminophen 500 mg Tab as needed for arthritic pain. ![Subcutaneous nodule with normal overlying skin on the left forearm (A and B), subcutaneous nodules with normal overlying skin on the right forearm (C and D).](WJCC-7-2505-g001){#F1} Laboratory examinations ----------------------- The complete blood count with differential was normal. Comprehensive metabolic panel was normal. Erythrocyte sedimentation rate, C-Reactive Protein, Anti-SCL, Rheumatoid Factor, and antinuclear antibody were all within normal limits. Her Serum ACE level was also within normal limits with a value of 53 U/L (normal 9-67 U/L). While chest radiograph was normal, chest computed tomography revealed mild mediastinal lymphadenopathy. FINAL DIAGNOSIS =============== Based on the histopathological findings of the cutaneous nodules and the computed tomography finding of mediastinal lymphadenopathy, a diagnosis of subcutaneous sarcoidosis was made. TREATMENT ========= Patient was advised on various treatment options and chose to have the lesions surgically excised by a plastic surgeon. OUTCOME AND FOLLOW-UP ===================== Needle biopsy and post-excisional pathology report both revealed the presence of well-formed, dense, non-caseating granulomas located in the subcutaneous tissue. There was no evidence of organisms with Acid-Fast and Grocott's Methamine Silver staining. Among the articles selected for this literature review, 4 were hospital based retrospective chart reviews. DISCUSSION ========== The largest study was conducted by Ahmed et al\[[@B14]\] in 2006 out of the Mayo Clinic\[[@B14]\]. The authors reviewed all cases filed under the diagnosis of sarcoidosis, nonspecific granulomas and granulomatous panniculitis between 1966-2001. Ultimately 21 cases of subcutaneous sarcoidosis were reviewed. Among the 21 patients diagnosed with subcutaneous sarcoidosis, 15 were female and 6 were male. The mean age was 46.3. In 20/21 patients, lesions were located on more than one anatomical site. The most common anatomical site was the upper extremity with all 21 patients presenting with a lesion in this area. Lower extremity lesions were also common as they were found in 16/21 patients. In 15/21 patients, other types of cutaneous lesions of sarcoidosis co-existed with the subcutaneous lesion with plaques presenting in 6 patients, papules in 4 patient, erythema nodosum in 4 patients, and scar sarcoidosis in 1 patient. Out of the 20 patients who were evaluated for systemic involvement, 16 had pulmonic involvement evidenced by an abnormal chest radiograph. 15/16 patients exhibited bilateral hilar lymphadenopathy, with 6 of these cases exhibiting an additional finding of paratracheal and pulmonary infiltrates. The most common systemic involvements, other than the lung, included arthritis, peripheral neuropathy and renal dysfunction. Out of the 11 patients tested for Serum ACE, 3 patients had elevated levels. In 2016, Ando et al\[[@B15]\] reviewed the charts of 130 patients diagnosed with systemic sarcoidosis between 2000-2012 out of Oita University of Japan medical center. 37/130 patients presented with cutaneous sarcoid lesions with 9/37 presenting with subcutaneous sarcoidosis. Among their cohort were 8 female patients and 1 male patient with an average age of 52.5 years. Six of the patients only had lesions on their lower extremities. The other 3 patients had lesions on their upper extremity and trunk, upper and lower extremities, and hip respectively. Two patients presented with sarcoid plaques and scars in addition to their subcutaneous nodules. All 9 patients were found to have lung involvement with 4 of these patients presenting with an additional involvement of their eyes, and 3 patients with an involvement of their muscles. On chest radiograph 3 patients had lymphadenopathy, and 6 patients had lymphadenopathy with pulmonary infiltrates. In 2005, Marcoval et al\[[@B7]\] conducted a retrospective chart review analyzing 480 patients admitted with systemic sarcoidosis from 1974-2002 at the University Hospital of Bellvitge in Barcelona, Spain. A total of 85/480 patients demonstrated sarcoid cutaneous involvement with 10/85 demonstrating subcutaneous sarcoidosis. 9/10 of the patients were female, and the average age of presentation was 52.6 years. All of the patients presented with nodules on their upper extremities with 5 patients presenting with additional nodules on their lower extremities. In addition to subcutaneous nodules, 4 patients presented with erythema nodosum, and 1 patient presented with sarcoid plaques and papules. 8 patients presented with lymphade-nopathy on chest radiograph, and 1 patient presented with lymphadenopathy and pulmonary infiltrate. In 2011 the same lead author, Marcoval et al\[[@B3]\], conducted a similar retrospective chart review analyzing 86 patients with systemic sarcoidosis who presented with cutaneous involvement to the Sarcoid Clinic of Bellvitge University Hospital in Barcelona, Spain. A total of 14/86 patients presented with subcutaneous nodules. Among the 14 patients, 11 were female and 3 were male. All 14 patients had nodules limited to the upper and lower extremities with 6 patients presenting with lesions on their arms, 1 patient presenting with lesions on their legs, and 7 patients presenting with lesions on both their upper and lower extremities. 13/14 patients were found to have abnormal chest radiograph findings with 11 patients exhibiting hilar lymphadenopathy, and 2 exhibiting hilar lymphadenopathy and lung infiltrate. Among the 14 patients, 12 had systemic involvement with arthritis being the most common presentation in 6 of the patients (Table [1](#T1){ref-type="table"}). ###### Subcutaneous sarcoidosis retrospective chart reviews Authors Sex Average age Serum angiotensin-converting enzyme Chest X-ray Most common site of lesion Most common site of systemic involvement other than the lungs ------------------------------- --------- ----------------- ----------------------------------------- ------------------------------------------------------------------------- -------------------------------- ------------------------------------------------------------------- Ahmed et al\[[@B14]\], 2006 15F/6M 46.3 3/11 Elevated 9/16 Lymphadenopathy, 6/16 Pulmonary infiltration Upper extremities Joints-arthritis Ando et al\[[@B15]\], 2016 8F/1M 52.5 7/9 Elevated 3/9 Lymphadenopathy, 6/9 Lymphadenopathy with pulmonary infiltration Upper extremities Eyes Marcoval et al\[[@B7]\], 2005 9F/1M 52.6 NA 8/10 Lymphadenopathy, 1/10 Lymphadenopathy with pulmonary infiltration Upper extremities NA Marcoval et al\[[@B3]\], 2011 11F/3M N/A NA 11/14 Lymphadenopathy, 2/14 Lymphadenopathy with pulmonary infiltration Upper and lower extremities Joints-arthritis In our literature review we found 25 case reports representing 28 unique cases of subcutaneous sarcoidosis between 2000-2019. Notable features of these cases are listed in Table [2](#T2){ref-type="table"}. The average age of patients among all reports was 53 years old and 21/28 of the patients were female. ###### Features of 25 case reports from 2000-2019 Authors Sex Average age Serum angiotensin-converting enzyme Chest computed tomography Chest X-ray Site of lesion Extracutaneous involvement other than the lungs ----------------------------------------- --------- ----------------- ----------------------------------------- ---------------------------------------------------------------------------------- --------------------- ----------------------- ----------------------------------------------------- Barnadas et al\[[@B16]\], 2000 F 38 Normal Normal Normal Upper and lower limbs Malaise, joint pains Girão et al\[[@B17]\], 2000 M 37 Elevated Lymphadenopathy Lower limb Hands and feet arthralgia Dalle Vedove et al\[[@B18]\], 2011 1F/1M 75 2/2 Elevated 1/2 Mediastinal Lymphadenopathy, 1/2 Lymphadenopathy with pulmonary infiltration 2/2 Normal Upper and lower limbs 1/2 Uveitis Kim et al\[[@B19]\], 2014 M 61 Elevated Normal Trunk Renal Fichtel et al\[[@B20]\], 2006 F 42 Elevated Normal Upper and lower limbs None Bosnic et al\[[@B21]\], 2010 F 51 Elevated Normal Face None Kim et al\[[@B22]\], 2017 M 33 Lymphadenopathy with pulmonary infiltration Face, toe None Won et al\[[@B23]\], 2016 F 54 Normal Lower limb None Marcoval et al\[[@B24]\], 2008 F 49 Elevated Lymphadenopathy Upper and lower limbs None Dulgueroy et al\[[@B25]\], 2015 F 34 Elevated Lymphadenopathy with pulmonary infiltration Face None Ruangchaijatuporn et al\[[@B26]\], 2016 M 56 Normal Lower limb None Watanbe et al\[[@B27]\], 2007 F 70 Elevated Lymphadenopathy Lower limb Polyneuropathy of limbs Janegova et al\[[@B28]\], 2016 F 59 Lymphadenopathy with pulmonary infiltration Foot None Yamaguchi et al\[[@B29]\], 2013 F 85 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Upper and lower limbs Joints arthralgia Mori et al\[[@B30]\], 2018 F 72 Elevated Lower limb Renal and cardiac dysfunction Kwan et al\[[@B31]\], 2015 F 53 Elevated Lymphadenopathy Upper and lower limbs None Miida et al\[[@B32]\], 2009 F 62 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Upper limbs Uveitis, renal dysfunction, splenic nodules Bianchini et al\[[@B33]\], 2010 F 38 Elevated Normal Normal Face None Kerner et al\[[@B34]\], 2008 F 53 Lymphadenopathy Lymphadenopathy Upper and lower limbs Facial nerve palsy, arthralgia Kim et al\[[@B35]\], 2013 F 52 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Upper and lower limbs None Guccione et al\[[@B36]\], 2017 M 40 Lymphadenopathy Upper limbs, trunk None Meyer-Gonzalez et al\[[@B37]\], 2011 3F 52.6 1/3 Normal, 2/3 Elevated 1/3 Lymphadenopathy, 2/3 Lymphadenopathy with pulmonary infiltration 3/3 Lymphadenopathy Upper and lower limbs Dactylitis, lower limb weakness Moscatelli et al\[[@B38]\], 2011 M 41 Lymphadenopathy with pulmonary infiltration Normal Hand None Shigemitsu et al\[[@B39]\], 2008 F 65 Lymphadenopathy Upper extremity None Celik et al\[[@B40]\], 2010 F 53 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Foot None Out of the 20 reported cases that checked for serum ACE, elevated levels were found in 18 patients. Abnormal Chest Computed Tomography results were found in 16/21 patients. Abnormal Chest Radiograph results were found in 12/19 patients. The most common site of lesion seemed to be the upper and lower limbs with 17/25 patients presenting with subcutaneous sarcoid nodules in one or both of these locations. Most reports did not comment on extracutaneous involvement other than the lung, but those did reported a range of systemic findings including arthritis, renal dysfunction, uveitis, dactylitis, and limb weakness. Sarcoidosis is a chronic systemic granulomatous disease of unknown etiology\[[@B1]\]. Although the lungs are typically the primary site of disease, cutaneous manifestations of the Sarcoidosis can be seen in up to 9-37% of patients\[[@B3],[@B4]\]. Subcutaneous sarcoid nodules, is a rare cutaneous sarcoidosis finding that typically presents as asymptomatic, non-tender, flesh colored nodules ranging in size between 0.5-2.0 cm\[[@B6],[@B8]\]. Histological examination of a subcutaneous sarcoid nodule reveals the presence of non-caseating granulomas present in the subcutaneous tissue\[[@B6]\]. Our patient, a 38 year old female, presented with an 8 month history of 4 subcu-taneous nodules ranging in size from 0.5cm-5cm. Her past medical history was significant for arthritis and recurrent nephrolithiasis. Upon testing we found that she had a normal chest radiograph and her serum ACE levels were within normal limits. On chest computed tomography we found evidence of mild mediastinal lymphadenopathy, pathognomonic for sarcoidosis. In analyzing the 29 publications from 2000-2019 along with our own case, we reviewed 83 cases of subcutaneous sarcoidosis. Among the 83 patients, 65 (78.3%) were female and the average age of presentation was 51.1 years old. The upper and lower extremities were the most common site of subcutaneous sarcoidosis development with 76/83 (91.6%) patients presenting with at least one lesion in these anatomical areas. In our analysis we learned that findings of lymphadenopathy or lymphadenopathy with pulmonary infiltrate was a very common chest radiograph finding among patients presenting with subcutaneous lesions. In total, 58/69 (84.1%) patients had abnormal chest radiograph findings. Among the 22 patients that received a chest computed tomography scan, abnormal findings of lymphadenopathy or pulmonary infiltrate were found in 17/22 (77.2%) patients. Elevated levels of serum ACE is also a common finding, although not as prevalent as lung involvement. In total, among the cases that measured serum ACE, 28/41 (68.3%) patients presented with elevated levels. Instances of sarcoidosis organ involvement other than the lung seems to be a rarer finding presenting in only 29/49 (59.1%) patients. Assessment of the number of patients with systemic involvement other than the lung, however, was difficult as some articles did not include this information within their study. In conclusion, our literature review shows that subcutaneous sarcoidosis primarily impacts middle-aged women, is most frequently found on the upper or lower limbs, and commonly presents with abnormal findings of lymphadenopathy or pulmonary infiltration on chest imaging as well as elevated levels of serum ACE. These patterns and findings are important for hand surgeons and other surgical specialties that do not commonly see this patient population to be able to rapidly identify and diagnose a disease that has extra-cutaneous manifestations and can lead to greater morbidity and mortality when not diagnosed and treated early. Informed consent statement: Informed consent was obtained from the patient prior to the writing of this case report. Conflict-of-interest statement: The authors declare there is no conflict of interest CARE Checklist (2016) statement: This paper is in accordance with guidelines from CARE Checklist 2016. Manuscript source: Invited Manuscript Peer-review started: March 28, 2019 First decision: June 17, 2019 Article in press: July 27, 2019 Specialty type: Medicine, research and experimental Country of origin: United States Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: El-Razek AA S-Editor: Cui LJ L-Editor: A E-Editor: Xing YX [^1]: Author contributions: All authors added work and value to the manuscript. Corresponding author: Raman Mehrzad, MD, Academic Fellow, Doctor, Staff Physician, Department of Plastic and Reconstructive Surgery, Rhode Island Hospital, the Warren Alpert Medical School of Brown University, 235 Plain St, Providence, RI 02903, United States. <[email protected]> Telephone: +1-774-2400060	{ "pile_set_name": "PubMed Central" }	[ "cervic", "treatment", "conflictofinterest", "two", "noncas", "refer", "given", "work", "origin", "eeditor", "biopsi", "identifi", "feet", "mayo", "classif", "select", "neck", "mri", "valu", "locat", "tweight", "signal", "may", "back", "hip", "recent", "toe", "larynx", "use", "chest", "etiolog", "guccion", "invit", "final", "paratrach", "imag", "complet", "accord", "prior", "mr", "signific", "depart", "dextroamphetamineamphetamin", "hypodens", "guidelin", "emiss", "suggest", "deni", "excel", "tomographi", "right", "also", "nodul", "jointsarthr", "state", "difficult", "chief", "janegova", "nerv", "dwjccgf", "conflict", "women", "perform", 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Introduction {#s1} ============ According to the 'efficient-coding hypothesis' [@pcbi.1000053-Barlow1], the sensory neurons are adapted to the statistical properties of the signals to which they are exposed. Because not all signals are equally likely, sensory systems should best encode those signals that occur most frequently. This idea was first tested by Laughlin [@pcbi.1000053-Laughlin1] in a pioneering study of first order interneurons in the insect compound eye, the large monopolar cells, which code for contrast fluctuations. He showed that the response function of these graded potential cells, measured by intracellular recording, approximates the cumulative probability distribution function of contrast levels measured in the natural fly\'s habitat with a photodiode. The efficient coding hypothesis has been much studied in the visual system [@pcbi.1000053-Laughlin1]--[@pcbi.1000053-Laughlin2]; reviewed in [@pcbi.1000053-Simoncelli1] and to a lesser extent in the auditory system [@pcbi.1000053-Lewicki1],[@pcbi.1000053-Smith1]. However, it has been rarely discussed in the context of olfactory sensory neurons [@pcbi.1000053-Kostal1],[@pcbi.1000053-Rospars1]. With a nonlinear stimulus-response function, the neuron encodes differently an equal change in stimulus intensity depending on the actual concentration ([Figure 1A](#pcbi-1000053-g001){ref-type="fig"}). The key question is, how should a neuron weigh its input so as to transfer as much information as possible? Information theory [@pcbi.1000053-Cover1],[@pcbi.1000053-Dayan1] provides the solution. In the simplest scenario (with no other constraints on the response range), the inputs should be encoded so that all responses are used with the same frequency [@pcbi.1000053-Laughlin1]. The optimal stimulus statistics is given by the stimulus probability distribution ([Figure 1B](#pcbi-1000053-g001){ref-type="fig"}), which is obtained directly from the stimulus-response curve. This simple solution, however, does not hold in the case of olfaction because of the large differences in reaction time at different stimulus concentrations. This is a major difference with respect to Laughlin\'s approach, in which all response states were assumed to be equiprobable. ![Amount of information transferred by a neuron in the case where all response states are equiprobable.\ (A) Stimulus-response function. The amount of transferred information is limited by the finite range of possible response states. Due to the non-linearity of the stimulus-response function, each response state encodes different relative changes in stimulus intensity. (B) Corresponding probability density function (pdf). Maximum information is transferred if all response states are used equally, i.e., if the area under the stimulus pdf is equal for each response state, as shown. In the limit of vanishingly small response states, the optimal stimulus CDF corresponds to the (normalized) stimulus-response function (adapted from [@pcbi.1000053-Laughlin1]).](pcbi.1000053.g001){#pcbi-1000053-g001} In this paper, we paralleled Laughlin\'s approach [@pcbi.1000053-Laughlin1], adapting his method to suit the specificity of olfaction. We chose a well studied olfactory receptor neuron, the pheromone receptor neuron of male moths, to investigate its adaptation to the natural signal it processes, the sexual pheromone emitted by conspecific females. To our knowledge this neuron and its stimulus provide the only example in olfaction for which enough data are available on the odorant plume and the neuron transduction mechanisms to make a quantitative comparison possible between the predicted optimum signal and the natural signal. Flying male moths rely on the detection of pheromone molecules released by immobile conspecific females for mating. The atmospheric turbulence causes strong mixing of the air and creates a wide spectrum of spatio-temporal variations in the pheromonal signal ([Figure 2](#pcbi-1000053-g002){ref-type="fig"}). The largest eddies are hundreds of metres in size and may take minutes to pass a fixed point, while the smallest spatial variations are less than a millimetre in size and last for milliseconds only [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis1]. Due to inhomogeneous mixing, a very high concentration of pheromone can be found in a wide range of distances from the source, though their frequency decreases with distance [@pcbi.1000053-Jones1]. Because of its complicated and inhomogeneous structure, the description of the plume must rely on statistical methods, notably the histogram of the fluctuations in pheromone concentration [@pcbi.1000053-Jones1]--[@pcbi.1000053-Mylne2]. These fluctuations are essential for the insect to locate the source of the stimulus. Experiments in wind tunnels showed that moths would not fly upwind in a uniform cloud of pheromone [@pcbi.1000053-Kennedy1]--[@pcbi.1000053-Willis1]. Characteristics like the frequency and intensity of the intermittent stimulation play a key role in maintaining the proper direction of flight [@pcbi.1000053-Vickers1]. ![Visualization of a pheromone plume.\ The figure is extracted and adapted from a digitized image of a smoke plume filmed in a wind tunnel 1 m across and 2 m long with source on the left side [@pcbi.1000053-Belanger1]. Though the average pheromone concentration in the air decreases with distance, high pheromone concentrations can be found relatively far from the source due to the imperfect mixing of odorant with air. The signal detected by both moving and stationary detectors is therefore always intermittent, consisting of pulses of relatively undiluted pheromone.](pcbi.1000053.g002){#pcbi-1000053-g002} The goal of this paper is to present arguments specifying in which sense the perireception and reception processes occuring in pheromone olfactory receptor neurons (ORNs) can be considered as optimally adapted to their natural stimulus. Although, in the light of previous studies on similar sensory neurons, the ORN may be considered a priori as adapted to the pheromone plume, the exact nature of this adaptation and its proof are more challenging questions. Despite widespread agreement that environmental statistics must influence neural processing [@pcbi.1000053-Lungarella1], precise quantification of the link proved difficult to obtain [@pcbi.1000053-Simoncelli1]. So, the main aim of this paper was to identify the specific characteristics to which the pheromone ORN is adapted and to provide quantitative evidence for their adaptation. We proceeded in two steps. First, using the statistical theory of information, we predicted the characteristics of the optimal pheromonal signal that the ORN is best capable of encoding based on the properties of the initial steps of signal transduction. Second, we compared these theoretically-derived properties with statistical characteristics most often determined in experimental measurements, i.e., the probability distribution function of the fluctuations in pheromone concentration, the spectral density function of the stimulation course and the intermittency of the odorant signal. Results {#s2} ======= Model of Pheromone Reception {#s2a} ---------------------------- Pheromone components are detected by specialized ORNs located in the male antenna. We considered a specific ORN type of the moth Antheraea polyphemus detecting (E,Z)-6,11-hexadecadienyl acetate, the major component of the sexual pheromone in this species, for which a wealth of precise information is available (reviewed in [@pcbi.1000053-Kaissling1]). The pheromone molecules are adsorbed on the cuticle, diffuse inside the sensory hair to the neuron membrane and are thought to be enzymatically deactivated [@pcbi.1000053-Kaissling1] then degraded. The initial cell response is triggered by the binding of the pheromone molecules to the receptor molecules borne by the dendritic membrane and the ensuing receptor activation. A cascade of events follows, amplifying this initial response and finally leading to the generation of a train of action potentials conveyed to the brain. The pheromone concentration at each instant determines the ORN response. However the extreme temporal variability of pheromone concentration in plumes prevents a full description of stimulus-response relationships by direct electrophysiological measurements. For this reason we based our study on a model of perireception and reception processes describing how any stimulus (concentration of pheromone in the air) is transformed into the receptor response (concentration of activated receptors). This model, based on extensive biochemical, radiochemical and electrophysiological experiments, was developed by Kaissling and coworkers [@pcbi.1000053-Kaissling1],[@pcbi.1000053-Kaissling2]. It involves the following system of chemical reactions: The network includes (1) the translocation of the ligand from the air (input pheromone signal L~air~) to the hair lumen (L); (2) the reversible binding of L to receptor R and the reversible change of the complex R~L~ to an activated state R^\^ (output signal); (3) the reversible binding of L to a deactivating enzyme N and its deactivation to product P which is no longer able to interact with the receptor. The concentrations of individual components in the network 1--3 are denoted by square brackets and the concentration values are functions of time. For simplicity we omit here the explicit dependence on the time variable t* and adopt the following notation for the individual concentrations: L ~air~ = \[L~air~\](t), = \[L\](t), R = \[R\](t), R ~L~ = \[R~L~\](t), R ^\^ = \[R^\^\](t), N = \[N\](t), P = \[P\](t) and N ~L~ = \[N~L~\](t). The evolution of the system 1--3 in time given the external signal L ~air~ is fully described by five first order ordinary differential Equations 4--8 and two conservation Equations 9 and 10: Equations 9 and 10 follow from the fact that the total concentration of the receptor molecules, R ~tot~ = R+R ~L~+R ^\^, as well as the total concentration of the deactivating enzyme, N* ~tot~ = N+N ~L~, do not change over time. We assume that at t = 0 the concentrations L, R ~L~, R ^\^, N* ~L~ and P are zero. The parameter values, derived from extensive experimental investigations, are given in [Table 1](#pcbi-1000053-t001){ref-type="table"}. 10.1371/journal.pcbi.1000053.t001 ###### Parameters of the perireceptor and receptor model. ![](pcbi.1000053.t001){#pcbi-1000053-t001-1} Parameter Value Unit Parameter Value Unit -------------- ------- ------------- -------------- -------- ------- k ~3~ = 0.209 s^−1^µM^−1^ k ~−3~ = 7.9 s^−1^ k ~4~ = 16.8 s^−1^ k ~−4~ = 98 s^−1^ k ~5~ = 4 s^−1^µM^−1^ k ~−5~ = 98.9 s^−1^ k ~6~ = 29.7 s^−1^ k~I~ = 29,000 s^−1^ R ~tot~ = 1.64 µM N ~tot~ = 1 µM From [@pcbi.1000053-Kaissling1],[@pcbi.1000053-Kaissling2]. Basic Stimulus-Response Properties {#s2b} ---------------------------------- The efficiency of information transfer in the system 1--3 depends critically on its stimulus-response relationship under single and repeated stimulus pulses. For transferring as much information as possible the response states must be optimally utilized. The actual amount of information transferred is limited by biological constraints. In the system studied, information transfer from L ~air~ (stimulus) to R ^\^ (response) presents three main limitations. First, it is limited by the finite number of receptor molecules per neuron which places an upper bound on the range of responses. Whatever the pheromone concentration (height of the step) the concentration of activated receptors cannot exceed at any time [@pcbi.1000053-Kaissling2]. Second, temporal details in the stimulus course shorter than a certain lower limit Δt* cannot be analyzed by the system. The smallest period of stimulation of the model studied here is 0.4 s [@pcbi.1000053-Kaissling2],[@pcbi.1000053-Rospars2], in agreement with experimental measurements [@pcbi.1000053-Kodadov1],[@pcbi.1000053-Rumbo1]. With smaller periods, at higher frequencies, the amplitude of the oscillations of R ^\^ becomes too small to be effective. Therefore we set Δt* = 0.4 s. Two successive pheromone pulses separated by a time shorter than Δt cannot be distinguished. Third, information transfer in time is also limited by the response duration, which depends on the deactivation rate of the activated receptors. The time course of R ^\^ in response to stimulations of different heights L* ~air~ and limited duration (0.4 s) is shown in the inset of [Figure 3A](#pcbi-1000053-g003){ref-type="fig"}. The concentration of activated receptors rises at first, reaches R ~Δ~ ^\^ at the end of the stimulus pulse, i.e., R* ~Δ~ ^\^ = R* ^\^(t* = Δt), and finally decreases. We consider R ~Δ~ ^\^ as the "response" of the system and for the sake of simplicity in the following, we omit index Δ. The duration of the falling phase (receptor deactivation) gets progressively longer for higher pheromone concentrations. This deactivation takes typically much longer than the time resolution parameter Δt. The falling phase is often described by the half-fall time, τ(R* ^\^), which is the time required for R* ^\^(t) to decrease from R* ^\^ to R* ^\^/2. The relationship between R* ^\^ and τ(R* ^\^) is shown in [Figure 3A](#pcbi-1000053-g003){ref-type="fig"}. A unique value of R* ^\^ corresponds to each value L~air~, which defines the stimulus-response curve ([Figure 3B](#pcbi-1000053-g003){ref-type="fig"}). The fact that the deactivation of activated receptors is relatively slow suggests that the reception system cannot encode a long sequence of pheromone pulses in arbitrarily quick succession. This observation plays an important role in the definition of the optimal stimulus course. ![Response properties of the olfactory reception model.\ (A) Temporal properties. Inset: concentration of activated receptors, R* ^\^(t), as a function of time for single pulses of pheromone of fixed duration (0.4 s) and different intensities L* ~air~ (1, 5, 10 and 20 nM). The maximum of R ^\^(t) is reached slightly after the end of the stimulation. The prolongation of the falling time with increasing intensities is quantified by the half-fall time, τ, as a function of R* ^\^ at the end of stimulation. (B) Stimulus-response function R* ^\^(L* ~air~) for single pulses of the same duration as in (A). This curve depends on the temporal resolution and the choice of the response intensity. (C) Optimal cumulative distribution function of the responses, F~R~(R ^\^), determined by maximizing the information transfer per average half-time (see [Methods](#s4){ref-type="sec"}). The functions R* ^\^(L* ~air~) and F~R~(R ^\^) were used for calculating the optimal stimulus probability distribution (shown in [Figure 5B](#pcbi-1000053-g005){ref-type="fig"}).](pcbi.1000053.g003){#pcbi-1000053-g003} Optimal Stimulus Course {#s2c} ----------------------- In the simplest scenario (with no other constraints on the response range and stimulus-independent additive noise), the inputs should be encoded so that all responses are used with the same frequency [@pcbi.1000053-Laughlin1],[@pcbi.1000053-Brunel1]. The optimal stimulus is thus described by its probability distribution function, which is obtained directly from the stimulus-response curve. Due to the large differences in reaction times at different stimulus concentrations, all response values R* ^\^ from 0 to 0.24 µM cannot be considered as equally "usable" (the long falling phases decrease the efficacy of the information transfer). Therefore, the longer the half-fall time of a given response R* ^\^ (i.e. the greater concentration R* ^\^ is) the less frequent it must be. The particular form of the optimal response cumulative probability distribution function (CDF), F~R~(R* ^\^), which was determined by maximizing the information transferred and minimizing the average half-fall time (see [Methods](#s4){ref-type="sec"}), is shown in [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}. Then, based on the three factors mentioned (stimulus-response curve, [Figure 3B](#pcbi-1000053-g003){ref-type="fig"}; time resolution Δt* = 0.4 s; and optimal response probability distribution, [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}), an optimum stimulus course in time can be predicted as explained in the [Methods](#s4){ref-type="sec"} section. Examples of predicted temporal fluctuations in pheromone concentration are shown in [Figure 4](#pcbi-1000053-g004){ref-type="fig"} at various time scales and compared to experimental observations. Even though the time resolution of the system studied here is only 0.4 s, it seems sufficient to capture the main bursts of pheromone (see the 10 s sample in [Figure 4A](#pcbi-1000053-g004){ref-type="fig"}). The comparison can be made more precise by describing statistically the heights and occurences in time of the pulses. ![Qualitative comparison of reconstructed optimal pheromone stimulations L ~air~ with experimentally-measured fluctuations in concentration of tracers (in arbitrary units), at various time scales.\ 10 s (A), 50 s (B) and 350 s (C). Temporal positions of pulses in experiments and simulations do not need to coincide. Quantitative comparisons are done in [Figures 5](#pcbi-1000053-g005){ref-type="fig"} and [6](#pcbi-1000053-g006){ref-type="fig"} and in [Table 2](#pcbi-1000053-t002){ref-type="table"}. (A) Ion signal measured using Langmuir probe in the field, 2.5 m from the source (top, from [@pcbi.1000053-Murlis2]); theoretical prediction (bottom) shows reasonable correspondence: the temporal resolution Δt = 0.4 s is sufficient to capture the main bursts of pheromone. (B) Ion signal, averaged over 330 ms, distance up to 30 m from the source (top, from [@pcbi.1000053-Murlis3]); the predicted signal (bottom) captures the overall character of the natural stimulation. (C) Propylene source, 67 m from the source (top, from [@pcbi.1000053-Mylne1]); the longest pauses (over 1 minute) are caused by the global meandering of the plume: they are absent in the prediction (bottom) because moths are assumed to stay within the pheromone plume.](pcbi.1000053.g004){#pcbi-1000053-g004} 10.1371/journal.pcbi.1000053.t002 ###### Comparison of statistical characteristics of optimal and actual plumes. ![](pcbi.1000053.t002){#pcbi-1000053-t002-2} Characteristics[a](#nt102){ref-type="table-fn"} Predicted Values[b](#nt103){ref-type="table-fn"} Experimental Values -------------------------------------------------------------------- -------------------------------------------------- ---------------------------------------------------------------------------------- Concentration CDF ([Figure 5](#pcbi-1000053-g005){ref-type="fig"}) Exponential Exponential [@pcbi.1000053-Mylne1],[@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna1] Spectra ([Figure 5](#pcbi-1000053-g005){ref-type="fig"}) Approx. flat to 0.2 Hz, Approx. flat to 0.1 Hz or 0.5 Hz Close to −2/3 slope after −2/3 slope to 1 Hz [@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2]) Intermittency 20% 10--40% [@pcbi.1000053-Murlis2],[@pcbi.1000053-Murlis3] 10--20% [@pcbi.1000053-Jones1] Total mean L ~air~ 1.0×10^−4^ µM -- Total std. dev. of L ~air~ 3.0×10^−4^ µM -- Peak value of L ~air~ 3.8×10^−3^ µM -- Peak/mean ratio 37 \>20 [@pcbi.1000053-Murlis2],[@pcbi.1000053-Murlis3] 30--150 [@pcbi.1000053-Jones1] Peak/std.dev. ratio 13 \>3 [@pcbi.1000053-Mylne1] The mean concentration, standard deviation and their ratios are calculated from the complete stimulus course, including parts of zero concentration (see [Methods](#s4){ref-type="sec"}). Based on a simulated sample 4000 s long. Predicted Temporal Pattern of Pulses {#s2d} ------------------------------------ Concerning temporal aspects, the bursts of non-zero signal do not occur at periodic intervals but appear randomly. An important descriptor of the temporal structure is the intermittency [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis1], which is the fraction of total time when the signal is present. The intermittency of the predicted optimal stimulus is 20%, which is in relatively good agreement with experimental data. It has been shown using various types of ion detectors [@pcbi.1000053-Murlis2],[@pcbi.1000053-Mylne2] as well as electroantennogram responses [@pcbi.1000053-Murlis2],[@pcbi.1000053-Baker1], that the natural signal is always present less than 50% of the total time, and usually smaller values are found. The average intermittency values reported are 10--20% [@pcbi.1000053-Jones1] and 10--40% [@pcbi.1000053-Murlis1],[@pcbi.1000053-Murlis2], depending on the experimental conditions, such as the detector size or the global meandering of the plume (see [Discussion](#s3){ref-type="sec"}). Predicted Concentrations of Pheromone Pulses {#s2e} -------------------------------------------- Concerning pulse height, the overall character of the predicted stimulus course is that pulses of high concentration are much rarer than those of low concentration. This feature of the predicted stimulus can be best quantified by the CDF, P(L ~air~), of the stimulus. The shape of the CDF is one of the most important properties for comparing theoretical predictions to experimental measurements because it describes the relative distribution of odorant concentrations throughout the plume. In fact, because measuring pheromone concentration in the field is not presently feasible [@pcbi.1000053-Murlis2], pheromone molecules must be replaced by measurable tracers. Relative quantities are valid for both pheromones and tracers (see [Discussion](#s3){ref-type="sec"}). They are the only quantities known experimentally for pheromone plumes. So, although our model predicts them, we cannot compare values of L ~air~ to actual measurements. Given the definition of the optimal stimulus, function P(L ~air~) can be directly computed (see [Methods](#s4){ref-type="sec"}). [Figure 5](#pcbi-1000053-g005){ref-type="fig"} shows a comparison between experimentally measured (A) and predicted (B) concentration CDF. The optimal pheromone concentration CDF ([Figure 5B](#pcbi-1000053-g005){ref-type="fig"}, solid line) is not known in analytical form but it can be well approximated by an exponential CDF ([Figure 5C](#pcbi-1000053-g005){ref-type="fig"}, dashed line). The differences between the predicted and true exponential shape can be considered as non-significant, namely, very high values of L ~air~ are predicted to be less frequent than in the exponential model. The exponential CDF is in agreement with experimental CDF ([Figure 5A](#pcbi-1000053-g005){ref-type="fig"}), [@pcbi.1000053-Mylne1],[@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna1],[@pcbi.1000053-Hanna2] and holds well especially for observations closer to the source (less than 100 m). Although the precise form of the CDF varies with distance from the plume centerline [@pcbi.1000053-Mylne2] and may be affected by the measurement technique, the shape is always highly skewed. ![Experimental and theoretical cumulative probability distribution functions (CDF) P(C) of dimensionless odorant concentration C/\<C\> (concentration divided by the total mean concentration).\ (A) Experimental CDF (solid) as measured at 75 m from a propylene (passive tracer) source and its best exponential fit (dashed) plotted on a logarithmic scale (taken from [@pcbi.1000053-Mylne2]). The intermittency is included in the plots in the non-zero value of P(C) for zero concentrations (see [Methods](#s4){ref-type="sec"}). The experimental data clearly follow the exponential CDF, except close to C = 0, which is caused by technical issues in the measurement process [@pcbi.1000053-Mylne2]. The relatively high value of measured intermittency (close to 47%) is caused mainly by initial data processing [@pcbi.1000053-Mylne2]. (B) CDF predicted by the pheromone reception model together with its best exponential fit, the scales correspond to panel (A) After correcting (see [Methods](#s4){ref-type="sec"}) for the fact that the intermittency predicted by the pheromone reception model (20%) is lower than that measured in [@pcbi.1000053-Mylne2] (as explained in the [Discussion](#s3){ref-type="sec"}), the predictions correspond well to the measured data in (A), except at very high values of L ~air~ where they are less frequent than expected. Since this deviation is apparent only for events occurring with probability P\<0.01, it can be considered as non-significant.](pcbi.1000053.g005){#pcbi-1000053-g005} Other predicted relative quantities (peak-to-mean ratios, dimensionless concentrations L ~air~/〈L ~air~〉) were compared with their experimental counterparts. The results, summarized in [Table 2](#pcbi-1000053-t002){ref-type="table"}, show that the predicted statistical properties of the stimulus are not contradicted by the experimental observations. Spectral Density Functions of the Stimulus Course {#s2f} ------------------------------------------------- Spectral density functions of the concentration time course, which analyze the contribution of various frequencies to the overall stimulus course, characterize other properties of the plume which are independent on the nature of the odorant (pheromone or ion source) [@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2]. Furthermore, spectral density function represents a point of view different from the concentration probability distribution. Several spectral density functions, shown in [Figure 6](#pcbi-1000053-g006){ref-type="fig"}, were calculated from the predicted optimal pheromone stimulation (see [Methods](#s4){ref-type="sec"}). The spectral shapes seem to be almost flat from 0.02 Hz to 0.2 Hz with a decreasing slope close to −2/3 above 0.2 Hz. The same slope −2/3, which is theoretically predicted by the inertial subrange theory [@pcbi.1000053-Mylne2], was reported in the spectral densities obtained from measurements close to the source (less than 100 m), in the range 0.1 Hz (or 0.5 Hz, depending on records) to 1 Hz [@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2], although the precise range may depend on the technique of measurement. ![Spectral density functions of the predicted fluctuations in time of pheromone concentration.\ Several spectral density functions were calculated from the predicted optimal pheromone stimulations (such as shown in [Figure 4](#pcbi-1000053-g004){ref-type="fig"}, bottom panels), for different initial random seeds. Calculated spectral shapes are usually almost flat from 0.02 Hz to 0.2 Hz, although exceptions are sometimes observed at lower frequencies, which are also found in experimental data [@pcbi.1000053-Mylne2]. Above 0.2 Hz there is a decreasing slope close to −2/3. Flat spectrum up to 0.2 Hz and true −2/3 slope beyond are shown for comparison (thick line). Spectra from experimental measurements (not shown) on propylene plume obtained close to the source are reported to exhibit a similar flat region followed by −2/3 slope [@pcbi.1000053-Mylne2].](pcbi.1000053.g006){#pcbi-1000053-g006} Discussion {#s3} ========== The goals of this study were to determine to which extent early olfactory transduction in olfactory receptor neurons can be considered adapted (in the evolutionary sense) to odorant plumes and to specify the plume characteristics to which it is adapted. The formulation and resolution of this problem benefited from successful studies of efficient sensory coding undertaken in the field of vision and audition. However, transposition from these sensory modalities to olfaction is not straigthforward, which may explain in part why it has not been attempted earlier. Specificities of olfaction concern both the odorant plume and the sensory system. Odor Plumes {#s3a} ----------- In theory and in practice, the quantitative description of odor plumes and their spatiotemporal distribution is less straightforward than that of visual or auditory scenes. Contrary to light and sound, for which the physical description is essentially complete, the turbulent phenomena which underlie the plume characteristics are still an incompletely mastered domain of physics [@pcbi.1000053-Ruelle1]. In Laughlin\'s classical experiment in vision a single time-independent variable, the contrast level, was measured [@pcbi.1000053-Laughlin1] and directly compared with experimental data. In olfaction, however, the odorant concentration (an analogue to the contrast level) is essentially time dependent which results in a complex optimal stimulus course ([Figure 4](#pcbi-1000053-g004){ref-type="fig"}). Complexity and time dependence make a meaningful direct comparison between predictions and experimental records, but also between different experimental records, impossible. Instead, the comparison must rely on global, statistical descriptors [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis2],[@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2]. We identified 5 such descriptors of odor plumes, actually measured and usable in the present context (see [Table 2](#pcbi-1000053-t002){ref-type="table"}), which summarize the present knowledge on odor plumes. Moreover, there are no easy-to-use instruments to measure odor plumes in the field, comparable to luxmeters and microphones. For example, the absolute pheromone concentration cannot be easily known in field experiments [@pcbi.1000053-Murlis2]. This explains why no experimental values were given for this descriptor in [Table 2](#pcbi-1000053-t002){ref-type="table"}. In practice, only ratios of concentrations are presented because they are independent of the dispersed molecules. The pheromone is often substituted by an ion or a passive tracer (polypropylene for example) whose concentration can be measured [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis2],[@pcbi.1000053-Mylne2]. Because both pheromone and tracer compounds in the air are governed by the same physical laws, the relative (dimensionless) values are conserved, as confirmed by independent experiments with different sources [@pcbi.1000053-Jones1]--[@pcbi.1000053-Murlis2],[@pcbi.1000053-Hanna2]. More generally, this limitation explains why we compared only relative quantities (i.e. shape of probability distributions, spectral density functions, peak-to-mean ratios, dimensionless concentrations L ~air~/〈L ~air~〉 and intermittency values). Other limitations of plume measurements are discussed below. Model of Early Transduction {#s3b} --------------------------- The essentially multidimensional and stochastic nature of the odor stimulus has a profound influence on the analysis of olfactory transduction system in its natural context, as undertaken here. Indeed to investigate the problems at hand, the kinetic responses of the system to a very large number of stimuli, varying in intensity, duration and temporal sequence must be known in order to simulate the diversity of stimuli encountered in a natural plume. This task is difficult, if not impossible, to manage in a purely experimental approach. However, this difficulty can be overcome with an exact dynamic model of the system because its response to the diverse conditions mentioned can be computed, provided it includes all initial steps from molecules in the air to the early neural response. This is the case of the perireception and reception stages of the moth pheromonal ORN and the reason why it was chosen in the present study. This choice brings about two questions, one about the validity of the model, the other on its position within a larger context. The computational model employed has been thoroughly researched and improved over the last three decades [@pcbi.1000053-Kaissling1], [@pcbi.1000053-Kaissling3]--[@pcbi.1000053-Minor1]. It describes perireceptor and receptor events in the ORN cell type sensitive to the main pheromone component of the saturniid moth Antheraea polyphemus. At the time of writing it represents the most completely researched computational models of its kind, agreeing with extensive experimental data from various authors and a wide range of experimental techniques. This model is the best description presently available for early events in any ORN and it summarizes in a nutshell a wealth of dispersed knowledge. This model is based on ordinary differential equations 4--8, following the law of mass action for chemical reactions, and is therefore purely deterministic. This approximation is acceptable when the concentrations of reactants are high enough above single-molecular levels, so that the stochastic fluctuations can be neglected. In this paper, the concentration of R ^\^ is always well above that corresponding to one activated receptor molecule per neuron (approximately 10^−6.2^ µM) because we do not investigate the effect of extremely small pheromone doses. Then, the response of the system can be considered as deterministic, in accordance with the efficient coding hypothesis [@pcbi.1000053-Simoncelli1]. The system studied here constitutes only a small part of the whole pheromonal system, although its role is absolutely essential and all other parts depend on it. First, in ORNs, post-receptor mechanisms modify the receptor signal, primarily by a large amplification factor and by sensory adaptation. Second, the ORN population includes cell types with different properties, e.g. the ORN type sensitive to the minor pheromone components can follow periodic pulses up to 10 Hz [@pcbi.1000053-Rumbo1], a performance not yet accounted for in present models [@pcbi.1000053-Rospars2]. Third, in the brain antennal lobe, convergence of a large number of ORNs on a few projection neurons (PNs) provides another amplification and supports the ability of some PNs to follow periodic signals at 10 Hz or greater [@pcbi.1000053-Christensen1]. Evolutionary adaptation of an integrated ORN response is difficult to study at the present time because no complete model of the ORN from receptors to the generation of the receptor potential and the ensuing spike train, is yet available, at least with the required degree of precision. The same argument holds a fortiori for higher order processes. Notwithstanding, the study of the early sensory events is not as restrictive as it may seem because any incoming odor signal must be first transduced in the population of membrane receptors. No information can be extracted by the post-receptor transduction system which has not been encoded by the receptors in the first place. For this reason it is essential to investigate the nature of the adaptation of the initial events (pheromone interaction with receptors) to the pheromone signal. Determination of the Optimal Stimulus {#s3c} ------------------------------------- Different response states of the pheromone reception system have different efficacies from the coding point of view: the "high" states, with large concentrations of activated receptors, take much more time to deactivate than the "low" states, so that for some time after its exposition to a large concentration of pheromone the system is "dazzled". It means that in the optimal stimulus the low pheromone concentrations must be more frequent than the high ones. This is a difference with respect to the classical problem where the efficacy of all response states at transferring information is considered the same, as in the vision of contrasts for example. The problem to solve is to find the right balance between two conflicting demands: to use all response states (including the high ones) and to react rapidly (the short transient responses must be as frequent as possible), i.e. to maximimize the information transferred per time unit. The solution to this optimization problem is provided by information theory as detailed in the [Methods](#s4){ref-type="sec"} section. The optimal balance derives from Equation 19 which relates the average half-fall time and the maximum response entropy distribution. The key factor to consider in the optimization is the average half-fall time, which characterizes globally the "swiftness" of the system -- smaller average half-fall time means faster stimulation rate. In other words, the average half-fall time characterizes the bias towards "low" response states. Simultaneously, the condition of maximum response entropy guarantees that the temporal dynamics of the system is as varied as possible and that during the course of stimulation every possible response state is used (with appropriate frequency). By taking into account only the average half-fall time, and not the precise sequence of its individual values, we therefore do not neglect or limit the temporal dynamics of receptor molecules activation. It is important to note, that the average half-fall time is not a free parameter of the problem; it is not set a priori: its optimal value follows from the optimization procedure (Equation 20). The resulting optimal response CDF is highly biased towards low response states, as expected (see [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}). Nature of the System Adaptation {#s3d} ------------------------------- The main achievement of the present investigation was to predict the characteristics of the stimulus optimally processed by the receptor system based on its biochemical characteristics and an information theoretic approach. The predicted optimal plume was shown to be close to the actual plumes for a series of characteristics, namely intermittency, peak/mean ratio and peak/standard deviation ratio of pheromone pulses, probability distribution of dimensionless pheromone concentration and spectral density function of pheromone concentration ([Table 2](#pcbi-1000053-t002){ref-type="table"}, [Figures 4](#pcbi-1000053-g004){ref-type="fig"}--[](#pcbi-1000053-g005){ref-type="fig"} [6](#pcbi-1000053-g006){ref-type="fig"}). The correspondence between the predictions and measurements is very good for the last two characteristics (probability distributions) and fair for the first three (numerical values). These differences in precision of the predictions may be interpreted by taking into account technical factors. Increasing the noise rejection threshold leads to a decrease of the measured intermittency [@pcbi.1000053-Jones1],[@pcbi.1000053-Mylne2], while increasing the detector size or averaging the signal over longer time windows has the opposite effect [@pcbi.1000053-Murlis3]. So, for example, the small size of olfactory sensilla with respect to detectors may explain in part why in [Figure 4B](#pcbi-1000053-g004){ref-type="fig"}, the predicted intermittency seems lower than that in the corresponding experimental record sample, and also why the peak-to-mean ratio and peak-to-standard deviation ratio are relatively higher. The immobility of the measurement devices, in contrast with the active movements of the moths, is another significant factor. For example, long pauses (of the order of minutes) of zero signal are missing in the prediction but visible in the longest available field record (350 s, [Figure 4C](#pcbi-1000053-g004){ref-type="fig"}). They are caused simply by the plume being blown away from the immobile field detector. First, this loss of signal is clearly an extraneous effect, which cannot be included in our optimal signal predictions and therefore cannot be seen in our results. Second, the moth is not subjected to this extraneous effect, or at least not to the same extent, because, in case of signal loss, it actively seeks the pheromone plume, whereas the fixed detector must passively wait for its return. This difference of mobility may substantially affect the intermittency values, but does not affect the shape of probability distributions (see [Methods](#s4){ref-type="sec"}), hence the better quality of the fits in the latter case. In conclusion, the results obtained suggest that the perireceptor and receptor system investigated here is evolutionary adapted to the pheromone plumes. Even if one considers that the pheromone olfactory system must be a priori* adapted to the average characteristics of the pheromone plumes, it does not logically follow that the system studied is itself necessarily well adapted. Indeed, it is conceivable that the global adaptation results mainly, not from perireception and reception processes but from other downhill intra- and intercellular processes involved in higher signal processing. The respective importance of the former and latter processes in global adaptation cannot be decided a priori. Therefore, the relatively close correspondence between predicted and observed plume characteristics presented here is not trivial. It suggests that the adaptation at the level of receptors is already substantial, and consequently that the global adaptation is not predominantly the result of post-receptor mechanisms involving amplification, sensory adaptation, convergence of different ORN types in the antennal lobes etc. The role of these mechanisms in the global adaptation of the animal remains to be established, as well as the relative importance of the various components of the olfactory system (receptor population, ORN as a whole, population of pheromonal ORNs in the antenna, projection neurons in the antennal lobes, etc.). The response characteristics of these other subsystems, e.g. their various temporal resolutions, will have also to be interpreted, maybe in relation with changing plume characteristics with distance to the source and other factors yet to be identified. Methods {#s4} ======= Optimal Response Probability Distribution Function {#s4a} -------------------------------------------------- As mentioned in the Results section, information transfer in the pheromone reception system is limited by the finite response range, (), and by the deactivation rate of the activated receptors for each concentration value R ^\^. This deactivation rate is described by the half-fall time τ(R* ^\^). The optimal performance of the system is thus reached by a trade-off between two conflicting demands: to employ full response range (maximum information) vs. to employ only the "fastest" responses (minimum average half-fall time). In other words we need to maximize the information transferred per average half-fall time. In the following we provide the mathematical framework that enabled us to find the probability distribution function over the response states R* ^\^ that realizes this trade-off. ### Information transferred {#s4a1} The information transferred by the pheromone reception system in a selected time window (t,t+Δt) is described by the relation between all possible stimulus values, L* ~air~, and the corresponding response values, R ^\^. This relation is explicitly quantified by the mutual information, I(L* ~air~; R ^\^) (see [@pcbi.1000053-Cover1] for details)where H(R* ^\^) is the entropy of the response probability distribution function and the conditional entropy H(R* ^\^\\|L* ~air~) measures the uncertainty in the output given the input, or equivalently, the amount of noise in the information transduction [@pcbi.1000053-Cover1],[@pcbi.1000053-Dayan1]. The model of pheromone reception employed here is deterministic and therefore H(R ^\^\\|L* ~air~) = 0. Thus maximizing the mutual information corresponds to maximizing the response entropy H(R ^\^). (Note that in the usual setting of signal independent and additive noise the term H(R* ^\^\\|L* ~air~) is constant and then maximization of I(L ~air~;R ^\^) again corresponds to maximization of H(R* ^\^).) The available response range, (), is naturally discrete, since it is comprised of individual receptor molecules. The expression of H(R* ^\^) is ([@pcbi.1000053-Cover1], p.14)where p(R* ^\^) is the probability of having R* ^\^ (expressed as a number of molecules). (In the following we use the base of logarithm 2 only to express all information-related quantities in the usual units of "bit"). The value of R* ^\^ corresponding to one activated receptor molecule per neuron is approximately ΔR* ^\^ = 10^−6.2^ µM [@pcbi.1000053-Kaissling2], which gives a total of different response states. Since N* is so large, the impractical Equation 12 can be replaced by a continuous approximation based on differential entropy, h(R ^\^), defined as ([@pcbi.1000053-Cover1], p.243)where f(R* ^\^) is the response probability density function. An approximative relation between H(R* ^\^) and h(R* ^\^) is given in ([@pcbi.1000053-Cover1] p.248)In the present case the approximation is excellent because the discretization step ΔR* ^\^ is very small compared to the whole response range (). From relation 14 the mutual information 11 can be expressed in terms of differential entropyMaximizing the information transferred is thus achieved by maximizing the differential entropy h(R* ^\^). The advantage of employing differential entropy is that it lends itself to an elegant approach for entropy maximization in terms of integrals. ### Information optimization {#s4a2} We adopt the standard procedure for maximizing the differential entropy of a continuous probability distribution constrained by a known function τ(R* ^\^). "Constraining" means that the average value 〈τ〉 of τ(R* ^\^) is under our control (see [@pcbi.1000053-Cover1], p.409)The task is to find a probability density function, f~R~(R* ^\^), which (i) maximizes the value of h(R* ^\^) (Equation 13) and (ii) is such that the average 〈τ〉 (Equation 19) taken over f~R~(R* ^\^) is equal to the value we set. The well known solution to this problem (see [@pcbi.1000053-Cover1], p.410 or [@pcbi.1000053-Jaynes1] for its derivation) iswhereIt depends on new parameter, λ, called Lagrange multiplier. In the standard setting of maximum-entropy problems ([@pcbi.1000053-Cover1] p.409 or [@pcbi.1000053-Simoncelli1],[@pcbi.1000053-Jaynes1]) the mean value of the constraint function, 〈τ〉, is known a priori. The value of λ* is then determined by substituting f(R ^\^) = f* ~R~(R ^\^) in Equation 16, so that the following equation between 〈τ〉 and λ* holdsIn the case of pheromone reception, however, the value of 〈τ〉 and consequently of λ is unknown. The value of λ must be determined by finding a compromise between maximum information transferred (Equation 15) and minimum average half-fall time (Equation 19). This compromise is made explicit by a simple requirementIn other words we maximize the information transfer per half-fall time. ### Application to pheromone reception {#s4a3} In order to simplify practical calculations we substitute f(R ^\^) = f~R~(R* ^\^) into the definition of differential entropy 13 so that Equation 15 reduces toNow we have all the necessary information to calculate (i) the mutual information I(L* ~air~;R ^\^) from Equation 21 (shown in [Figure 7A](#pcbi-1000053-g007){ref-type="fig"}), (ii) the mean half-fall time from Equation 19 ([Figure 7B](#pcbi-1000053-g007){ref-type="fig"}) and (iii) their ratio from Equation 20 ([Figure 7C](#pcbi-1000053-g007){ref-type="fig"}) in dependence on the Lagrange multiplier λ. [Figure 7A](#pcbi-1000053-g007){ref-type="fig"} shows that the mutual information is maximized (18.5 bits) for λ* = 0 which corresponds to the uniform probability distribution function over the whole response range. Generally, since τ(R ^\^) is a monotonously increasing function of R* ^\^, the optimal probability density function f~R~(R* ^\^) (Equation 17) is either monotonously increasing (λ\<0), monotonously decreasing (λ\>0), or constant (λ* = 0). The multiplier λ thus decides whether f~R~(R ^\^) puts more weight on the "slow" response states (λ\<0) or on the "fast" response states (λ\>0). These observations are confirmed in [Figure 7B](#pcbi-1000053-g007){ref-type="fig"} where the mean half-time monotonically decreases with increasing λ. [Figure 7C](#pcbi-1000053-g007){ref-type="fig"} shows the information transferred per average half-time, i.e., it shows the compromise between the "slowness" or "reactivity" of the system and the transferred information. Clearly, there cannot be a maximum for λ\<0 where the system is both "slow" and below its information capacity (note the sharp decrease of mutual information in [Figure 7A](#pcbi-1000053-g007){ref-type="fig"} for λ\<0). The optimal balance between reactivity and information transfer is reached for λ≈6 at 8 bits/s. By substituting λ* = 6 into formula 17 we obtain the desired optimal response probability density function, f~R~(R ^\^), which maximizes the information transfer per average half-time. The corresponding CDF F~R~(R* ^\^), shown in [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}, is given by ![Determination of the optimal Lagrange multiplier λ* giving the optimal response probability distribution.\ (A) Mutual information for the pheromone reception model in dependence on λ (Equation 21). (B) Mean half-fall time in dependence on λ (Equation 19). (C) Information transferred per average half-time representing the balance between the reactivity of the system and information transferred (Equation 20). Maximum occurs for λ≈6.](pcbi.1000053.g007){#pcbi-1000053-g007} The maximum of information transferred per average half-time ([Figure 7C](#pcbi-1000053-g007){ref-type="fig"}) is not sharply defined, namely, the transfer of 7--8 bits/s persists with values of λ greater than the optimal value. At the same time, both mutual information ([Figure 7A](#pcbi-1000053-g007){ref-type="fig"}) and average half-time ([Figure 7B](#pcbi-1000053-g007){ref-type="fig"}) decrease slowly in the corresponding region, indicating that the shape of the optimal response probability distribution changes slowly with respect to λ. Indeed, as we verified numerically, varying λ within reasonable limits (so that information transferred stays close to 8 bits/s) has no impact on the results presented in this work. Optimal Stimulus Course {#s4b} ----------------------- The optimal stimulus course in time was calculated as follows. First, at time t ~0~ = 0 a random value p ~0~ is drawn randomly from a uniform probability distribution function over the range \[0,1\]. The concentration corresponding to probability p ~0~ is obtained by solving the equationwhere F~R~(R ^\^) is the optimal CDF given by formula 22 ([Figure 3C](#pcbi-1000053-g003){ref-type="fig"}). The predicted optimal concentration L* ~air,0~ for a pheromone pulse of duration Δt = 0.4 s which corresponds to is obtained by solving the equationwhere R ^\^(L* ~air~) is the stimulus-response function ([Figure 3B](#pcbi-1000053-g003){ref-type="fig"}). The value L ~air,0~ is plotted at t ~0~ ([Figure 4](#pcbi-1000053-g004){ref-type="fig"}). Second, the concentration L ~air,1~ and time of appearance t ~1~ of the next pulse are determined. Time t ~1~ follows from the falling phase of activated receptors: optimality requires that no pheromone pulse appears before R ^\^ returns to its resting level. In practice it is considered that the resting level is reached when R* ^\^ falls below 0.01 µM (less than 5% of the coding range). The concentration L* ~air,1~ of the pulse at t ~1~ is determined in the same way as for the pulse at t ~0~ by drawing a new random number p ~1~ from the uniform probability distribution function over \[0,1\]. The same process can be repeated as many times as needed to create an optimal pheromone pulse train of arbitrary length. Optimal Stimulus Probability Distribution Function {#s4c} -------------------------------------------------- It is common in the literature on the statistical analysis of plumes [@pcbi.1000053-Jones1],[@pcbi.1000053-Mylne1],[@pcbi.1000053-Mylne2] to define two types of mean concentrations. The total mean concentration, 〈L ~air~〉, describes the "true" mean concentration obtained from the whole record of concentration fluctuations in time, i.e., including the parts where no signal was available. On the other hand, the conditional mean concentration, 〈L ~air~〉~cond~, describes the mean concentration inside the plume, i.e., with zero concentrations excluded. The intermittency, γ, relates the two means as [@pcbi.1000053-Mylne2] (Analogously, the total variances and total standard deviations are calculated by taking into account also the parts where no signal is available [@pcbi.1000053-Mylne2].) By combining Equations 23 and 24 we may symbolically express the optimal CDF of the stimulus, P(L ~air~), asThough P(L ~air~) cannot be expressed in a closed form, it can be well approximated by the exponential CDFwhere ξ = (5.24±0.01)×10^−4^ µM is the estimated value of 〈L ~air~〉~cond~ by least-squares fitting of F ~exp~(L ~air~) to P(L ~air~). In order to compare concentration probability distribution functions from different measurements meaningfully, authors [@pcbi.1000053-Mylne2] plot the CDF for a dimensionless concentration L ~air~/〈L ~air~〉. (In the [Figure 5A](#pcbi-1000053-g005){ref-type="fig"} C/〈C〉 is used, since the data plotted were obtained using a propylene source, not pheromone), see [Figure 5A](#pcbi-1000053-g005){ref-type="fig"}. The scale of such plots is affected by intermittency due to the presence of the total mean in the ratio. Furthermore, information about intermittency is included explicitly in the plots by letting the probability P(L ~air~ = 0) of zero concentration beConsequently the CDF P(L ~air~) must be renormalized [@pcbi.1000053-Mylne2]. Intermittency affects only the dimensionless scale, L ~air~/〈L ~air~〉, and the value of P(L ~air~ = 0) but not the overall shape of CDF [@pcbi.1000053-Mylne2]. Therefore we can use formulas 25 and 28 to compare our predictions with experimentally measured data by correcting for different intermittency values. Spectral Density Function of the Stimulus Course {#s4d} ------------------------------------------------ The optimal stimulus course is represented by pulses of different pheromone concentrations, L ~air~, occurring in time intervals 0.4 s long. In order to calculate the spectral density function of such stimulation course we sample the time axis with step Δt = 0.4 s. Thus we obtain a series of pheromone concentrations at these time points, {L ~air,j~}, j = 1...n, where n should be even. The discrete Fourier transform, φ~k~, of {L ~air,j~} is defined for k = 1,...,n values as [@pcbi.1000053-Champeney1] where i is the complex unit. The zero-frequency term is thus at position k = 1. The spectral density, S(f), of the complete time course of the stimulus can be calculated for a total of n/2+1 values of frequency f (given in Hz) [@pcbi.1000053-Oppenheim1] where m = 0, 1, 2,..., n/2−1, n/2 and f = m/(nΔt) are the frequency values. The function Π(f) is the Fourier transform of a pulse of unit height, 0.4 s long and starting at t = 0 [@pcbi.1000053-Champeney1],where a = 2.5 and δ = −0.5. The function Π(f) appears in formula 30 because the whole stimulus course (such as shown in [Figure 4](#pcbi-1000053-g004){ref-type="fig"}, bottom panels) can be reconstructed by convolving the discrete series {L ~air,j~} with such a pulse of unit height in the time domain [@pcbi.1000053-Champeney1]. The authors are grateful to Christine Young for linguistic corrections. The authors have declared that no competing interests exist. This work was supported by Marie-Curie fellowship HPMT-CT-2001-00244 to LK, by ECO-NET 12644PF from the French Ministere des Affaires Etrangeres, by Research project AV0Z50110509, Centre for Neuroscience LC554, and by Academy of Sciences of the Czech Republic grants 1ET400110401 and KJB100110701. [^1]: Contributed reagents/materials/analysis tools: PL. Wrote the paper: LK JR. Performed the research: LK. 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Introduction {#s1} ============ The kisspeptins are a family of peptides essential for the onset of puberty and the regulation of reproductive function. The absence of a functional kisspeptin receptor (KiSS1r) or Kiss-1 gene results in low gonadotrophin levels and failure to undergo pubertal development in both mice and humans [@pone.0097611-deRoux1]--[@pone.0097611-dAnglemontdeTassigny1]. Administration of exogenous kisspeptin potently stimulates gonadotropin secretion in rodents and man [@pone.0097611-dAnglemontdeTassigny1]--[@pone.0097611-Dhillo1]. Kisspeptin is the endogenous ligand of the G-protein coupled receptor Kiss1r [@pone.0097611-Muir1]--[@pone.0097611-Ohtaki1]. The stimulatory effects of kisspeptin on the reproductive axis appear to be mediated by a direct activation of KiSS1r-expressing gonadotrophin releasing hormone (GnRH) neurons [@pone.0097611-Irwig1]--[@pone.0097611-Kirilov1]. In humans, KISS1 encodes a 145 amino acid precursor peptide which is cleaved to form a 54-amino-acid peptide, known as kisspeptin-54 or metastin [@pone.0097611-Ohtaki1], and shorter fragments 14, 13 and 10 amino acids long [@pone.0097611-Kotani1]. All kisspeptin share a common 10 amino acid C-terminal section, which is required for kisspeptin receptor activation. Kisspeptin-54, -14, -13 and -10 all bind to Kiss1 with equal affinity and efficacy in vitro [@pone.0097611-Kotani1]. The largest proteolytic product in rodents is a 52 amino-acid peptide. Although this peptide shares only relatively low overall homology (52%) with human kisspeptin-54, the C-terminal 10 amino-acid signalling sequences is highly conserved between mouse and human KiSS1 proteins, varying by only one amino acid \[Tyr 10 (Y) in rodents to Phe 10 (F) in humans\] [@pone.0097611-Stafford1]. Expression of Kiss1 mRNA has been observed in tissues including the placenta, pancreas, small intestine, and brain in humans [@pone.0097611-Muir1]--[@pone.0097611-Ohtaki1]. Within the human CNS, kisspeptin immunoreactive cell bodies are predominantly located in the infundibular nucleus of the hypothalamus [@pone.0097611-Hrabovszky1]. In rodents, kisspeptin perikarya are located in two major populations in the hypothalamus, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) [@pone.0097611-Gottsch1], [@pone.0097611-Mikkelsen1]. Kisspeptin mRNA expression has been quantified using in situ hybridisation (ISH) and quantitative polymerase chain reaction (qPCR). This provides valuable information on gene transcription, but it is widely recognised that changes in mRNA are not always mirrored by protein levels [@pone.0097611-Schwanhausser1], [@pone.0097611-Vogel1]. In humans, circulating kisspeptin levels have been quantified by radioimmunoassay, and immunocytochemical (ICC) methods have been used to localise kisspeptin expression in humans and rodents [@pone.0097611-Dhillo1], [@pone.0097611-Franceschini1]. However, to date, kisspeptin peptide levels in the rodent have not been measured. Western blotting can be used to measure protein levels, but it can be challenging to detect small changes in kisspeptin--IR levels using this technique. In the current study we have developed a sensitive and specific radioimmunoassay (RIA) which enables the quantification of kisspeptin levels within rodent tissues. Materials and Methods {#s2} ===================== Materials {#s2a} --------- Synthetic kisspeptin-10 was obtained from the Advanced Biotechnology Centre, Imperial College, London, UK. Kisspeptin-52 was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). The kisspeptin polyclonal antibody was purchased from Millipore (Watford, UK). Rat Tissue {#s2b} ---------- Female Wistar rats (220--250 g) obtained from Charles River (Margate, UK) were housed in single cages (specific pathogen free, Imperial College London, UK) and maintained in a controlled environment (temperature 21--23°C, 12-h light--dark cycle, lights on at 07∶00) with ad libitum access to food (RM1 diet; SDS UK, Witham, Essex, UK) and water. All animal procedures were approved by the British Home Office Animals (Scientific Procedures) Act 1986. Estrus cyclicty was monitored daily by vaginal lavage, slides were examined under a light microscope to determine the dominant cell type. On the morning of the diestrus phase of the cycle, animals were killed by decapitation, and either whole brain or hypothalamus dissected and snap frozen then stored at −80°C. Pregnant female rats were obtained from Charles River. At pregnancy day 20 rats were killed and the placenta removed, snap frozen and stored at −20°C. An additional cohort of female Wistar rats were bilaterally ovarectomized (OVX) and implanted with an estradiol (E~2~) filled (150 ug E~2~/ml) silicone capsule. This model has previously been shown to mediate changes in kisspeptin expression [@pone.0097611-Roa1]. Two weeks later animals were killed by decapitation and whole brain dissected, snap frozen and stored at −80°C`.` Radioimmunoassay {#s2c} ---------------- An RIA directed against rodent kisspeptin was developed. The rabbit kisspeptin-10 polyclonal antibody used has been demonstrated to detect markedly diminished immunoreactivity in Kiss1 ^−/−^ mice compared to wild type mice when used for immunocytochemistry [@pone.0097611-Lapatto1], and to have minimal cross-reactivity with other RF-amide peptides [@pone.0097611-Desroziers1]. ^125^I-kisspeptin-10 label was prepared using the chloramine-T method and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column (Waters, Milford, MA) over a 32--38% 90-min gradient of acetonitrile (AcN)/water/0.1% trifluoroacetic acid (TFA). The specific activity of kisspeptin label was 56 Bq/fmol. The assay was set up as previously described [@pone.0097611-Bloom1]. The final antibody dilution used was 1∶87 000. Kisspeptin-10 label was used at 20 Bq/tube. The assay was performed in 0·06 M phosphate buffer (pH 7·2) containing 0·3% (v/v) bovine serum albumin (BSA) and 0·02% (v/v) Tween 20. After 3 days of incubation at 4°C, antibody-bound and free fractions were separated by charcoal (2.4 g charcoal, 0.24 dextran in 100 ml phosphate buffer) adsorption of the free fraction. Unless stated, all tissues were homogenised using a Kimble Kontes pellet pestle (Sigma-Aldrich, Dorset, UK) in 450 µl of acid ethanol extraction buffer (0.15% hydrochloric acid in 25% ethanol) [@pone.0097611-Murphy1]. To assess parallelism of the assay, standard curves of 1, 2, 3, 5, 10, 15, 20, 30, 50 and 100 fmol/tube of kisspeptin-10 and kisspeptin-52 were set up in duplicate in conjunction with dilution curves of hypothalamus tissue extracts (5, 10, 20 and 30 µl of extract, also in duplicate) (n = 5). To assess the recovery efficiency of the assay concentrations from 50 to 200 fmol/tube kisspeptin-10 were added to 450 µl rat cerebellum tissue extract and assayed. For the quantification of kisspeptin-IR in tissues, a standard curve consisting of 1, 2, 3, 5, 10, 15, 20, 30, 50 and 100 fmol/tube kisspeptin-10 in duplicate was used. Chromatography {#s2d} -------------- Synthetic kisspeptin-52 (2 nmol in 150 ul water) was fractionated using RP-HPLC Phenomenex Jupiter 4 µm Proteo 90 Å column and eluted with a 30--35% gradient of ACN plus 0·05% (v/v) water/0·1% (v/v) 50 min at a flow rate of 1 ml/min per fraction. Fractions were collected every min. An additional set of hypothalamic tissue samples (n = 4) was extracted using the protocol described above, centrifuged at 15 000 g for 3 min, and the supernatants filtered through 0·2 µm hydrophilic membranes (Sartorius, Göttingen, Germany). Samples of 150 ul from each extract were then loaded separately onto the HPLC column and eluted under the same conditions described above. Fractions from all runs were freeze-dried, reconstituted in assay buffer and the kisspeptin content determined by RIA. Quantification of AVPV, ARC and Placenta Kisspeptin {#s2e} --------------------------------------------------- Several groups have confirmed kisspeptin expression in the placenta and the hypothalamic AVPV and ARC [@pone.0097611-Mark1], [@pone.0097611-Smith1]. Brain sections (300µm) were cut on a cryostat, and bilateral punches (1mm diameter) of the AVPV were taken from Bregma +0.2 to −0.4mm, single midline punch (1mm diameter) that included both ARC was taken from bregma −1.7 to −3.9 according to the rat brain atlas [@pone.0097611-Paxinos1], following the micropunch method of Palkovits [@pone.0097611-Palkovits1]. Hypothalamic punches, cerebellum and placental tissue (350 mg) were homogenised and extracted in acid ethanol buffer as described above, and kisspeptin content measured by radioimmunoassay. A separate set of tissues were also extracted by boiling for 15min in 0·5 M acetic acid [@pone.0097611-Murphy1]. Statistical Analysis {#s2f} -------------------- All data are presented as mean ± standard error of the mean (SEM). Levels of kisspeptin expression were analysed using an unpaired t-test (GraphPad Prism version 5 for Windows; GraphPad Software, San Diego, CA). Results {#s3} ======= Radioimmunoassay {#s3a} ---------------- The assay demonstrated 100% cross-reactivity to both rodent kisspeptin-10 and rodent kisspeptin-52. The assay had a sensitivity of 0.81±0·12 fmol/tube (mean ± SEM), n = 3 with 95% confidence. The least detectable tissue concentration was \<1.21 fmol/tube, and the midrange was 18.14 fmol/tube. Inter- and intra-assay variation were established to be 8·2%±0·7 and 6·8%±1·7 respectively, n = 5. Recovery of kisspeptin from tissue homogenate was between 78% and 96%. The dilution curve for hypothalamic extract was almost parallel to that of kisspeptin-10 and kisspeptin-52 standard, n = 5 ([Figure 1](#pone-0097611-g001){ref-type="fig"}). ![HPLC profile of kisspeptin-immunoreactivity from hypothalamic extracts.\ Solid lines- Kisspeptin concentration; broken lines-% acetonitrile (ACN). ↓KP-52 represents the elution position of Kisspeptin-52 standard. The recovery of Kisspeptin-IR in the tissue extract from each column run was above 80% (means S.E.M.; n = 4).](pone.0097611.g001){#pone-0097611-g001} Chromatography {#s3b} -------------- HPLC of hypothalamic extracts resulted in a distinct kisspeptin-IR peak at the same point at which synthetic kisspeptin-52 peptide eluted ([Figure 2](#pone-0097611-g002){ref-type="fig"}). The recovery of kisspeptin-IR for each column run of the tissue extracts was \>80%. ![Parallelism of standard and tissue extract for novel rodent Kisspeptin RIA.\ Kisspeptin −10 (KP-10) and hypothalamus (Hypo) (n = 5).](pone.0097611.g002){#pone-0097611-g002} Quantification of AVPV and ARC Punches and Placenta {#s3c} --------------------------------------------------- Kisspeptin-IR in the acid ethanol extracted AVPV and ARC of female rat was determined. Significantly more kisspeptin-IR was detected in the ARC than in the AVPV (47.1±6.0 vs. 7.6±1.3 fmol/punch, P\<0.001, n = 15, [Figure 3](#pone-0097611-g003){ref-type="fig"}). As anticipated, a significant increase in AVPV kisspeptin-IR was observed in OVX+E~2~ compared with the OVX rats (16±0.9 vs. 12.5±1 fmol/punch, mean ± SEM P\<0.05, n = 6--8, [Figure 4A](#pone-0097611-g004){ref-type="fig"}), and a significant decrease observed in ARC kisspeptin-IR in OVX+E~2~ compared with OVX rats (43.3±2.9 vs. 66.2±2.1 fmol/punch, mean ± SEM, P\<0.001, n = 6--8, [Figure 4B](#pone-0097611-g004){ref-type="fig"}). We detected 1.26±0.15 fmol/mg (n = 5) kisspeptin-IR in acid ethanol extracted placental tissue. No kisspeptin-IR was detected in the cerebellum tissue. No measurable kisspeptin-IR levels were detected in tissues extracted by boiling for 15min in 0·5 M acetic acid (data not shown). ![Quantification of kisspeptin-IR in the rat brain (A) Diagram of a rat brain coronal section showing the position of the punch microdissections for the Hypothalamic anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC).\ AVPV at bregma −0.26mm and the ARC at bregma −3.30mm according to the rat brain atlas of Paxinos and Watson. Black circles denote punch positions. Ac, anterior commissure; 3v, third cerebral ventricle. (B) Quantification of kisspeptin-IR in the AVPV and ARC of female rats by RIA. Significantly more kisspeptin-IR was detected in the ARC than in the AVPV. (Mean S.E.M, n = 15--16, \\\P\<0.001, Student's t-test).](pone.0097611.g003){#pone-0097611-g003} ![The effect of E~2~ on kisspeptin-IR in the hypothalamus of OVX female rats.\ (A) E~2~ significantly increased kisspeptin-IR in the AVPV. (B) E~2~ significantly decreased kisspeptin-IR in the ARC. Quantification of kisspeptin-IR by RIA. (Mean S.E.M, n = 6--8, \P\<0.05, \\\P\<0.001, Student's t-test).](pone.0097611.g004){#pone-0097611-g004} Discussion {#s4} ========== In the present study, we have characterised the development of a novel kisspeptin RIA and demonstrated its utility in quantifying kisspeptin-IR within the AVPV and ARC of female rats. We have demonstrated that this assay can detect changes in kisspeptin expression in response to E~2~* feedback. In accord with studies using other methods, we observed suppression of kisspeptin-IR in the ARC and increased kisspeptin-IR in the AVPV in response to E~2~ in ovariectomised female rats [@pone.0097611-Smith2]. Numerous studies have demonstrated a wide distribution of kisspeptin expression throughout the rodent brain [@pone.0097611-Brailoiu1], [@pone.0097611-Clarkson1]. Initial studies examining the distribution of kisspeptin-IR rodents were hampered by the limited specificity of available antibodies [@pone.0097611-Brailoiu1]. However, the development of reliable antibodies which recognise rodent, sheep [@pone.0097611-Franceschini1] and human [@pone.0097611-Dhillo1] kisspeptin has enabled the distribution of kisspeptin-IR to be fully mapped. Furthermore, we observe a single IR peak following HPLC of hypothalamic extracts, at the point we would expect kisspeptin-52 to elute, thus supporting previous findings regarding antibody specificity [@pone.0097611-Franceschini1]. In addition to mapping kisspeptin distribution, ICC methodologies have been utilised to quantify changes in expression under different experimental and physiological conditions [@pone.0097611-Clarkson1], [@pone.0097611-Clarkson2]. Kisspeptin-IR has been successfully quantified in the AVPV, and also in the ARC, though the ARC has presented difficulties due to the dense plexus of kisspeptin fibres present [@pone.0097611-Clarkson1], [@pone.0097611-Castellano1], [@pone.0097611-MittelmanSmith1]. The limitations of ICC preclude the quantification of the absolute number of kisspeptin molecules or its concentration in a sample. Further, though semi-quantitative methodologies provide information about relative quantities of kisspeptin-IR within each study, it is difficult to compare the levels observed between different studies and models. Kisspeptin expression can be quantified using ISH [@pone.0097611-Gottsch1] or qPCR [@pone.0097611-Yamada1]. However, it is becoming increasingly evident that examination of mRNA levels may be an unreliable proxy for protein concentrations [@pone.0097611-deSousaAbreu1], [@pone.0097611-Maier1], with only ∼40% protein levels being explained by mRNA abundances in mammalian cell lines [@pone.0097611-Schwanhausser1], [@pone.0097611-Vogel1]. Differences in the distribution of kisspeptin mRNA and immunoreactivity have previously been reported. For example, kisspeptin-IR cell bodies and fibres were described in the dorsomedial nucleus of mice [@pone.0097611-Clarkson1], though ISH failed to identify kisspeptin mRNA within this region [@pone.0097611-Smith1]. A subsequent study suggested that the IR detected within the dorsomedial nucleus may be the result of cross-reactivity of the antibody used with neuropeptide FF [@pone.0097611-Iijima1], though it is interesting to note that a recent study detected kisspeptin-immunoreactivity in some DMN cells in the mouse using a different antibody (Franceschini et al 2013). In the current investigation, the higher level of kisspeptin-IR in the ARC than the AVPV is in accord with the relative levels predicted by assessment of mRNA levels. Kisspeptin-IR was also present in the placenta, albeit at relatively low levels. Previous studies have suggested that the expression of kisspeptin mRNA in the placenta varies during pregnancy, and it may be that higher levels would have been detected at another time point [@pone.0097611-Mark1]. Our data also suggest that the majority of hypothalamic kisspeptin-IR is in the form of kisspeptin-52, though it is possible that our extraction methods favour this particular form, and that other kisspeptin forms are present at lower levels. Further studies are thus necessary to conclusively demonstrate the relative quantities of the different kisspeptins in the hypothalamus. In summary, we have developed a novel radioimmunoassay for the measurement of kisspeptin-IR within rat tissue. When combined with the Palkovits punch micro-dissection methodology, this assay enables quantification of kisspeptin-IR within specific brain nuclei. [^1]: Competing Interests:The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: KGM SRB. Performed the experiments: JSKJ KEB JC KTO XFL. Analyzed the data: JSKJ KGM. Wrote the paper: JSKJ KGM.	{ "pile_set_name": "PubMed Central" }	[ "graphpad", "immunocytochem", "two", "almost", "control", "poneyamada", "watson", "lavag", "decreas", "avpv", "administr", "ligand", "immunocytochemistri", "fmolpunch", "abund", "collect", "ovariectomis", "midrang", "favour", "exist", "anticip", "silicon", "gene", "identifi", "charcoal", "∼", "icc", "witham", "fmoltub", "student", "week", "locat", "signal", "may", "ponelapatto", "recent", "proteolyt", "bqtube", "separ", "mrna", "use", "ponemikkelsen", "germani", "final", "support", "accord", "decapit", "signific", "antibodybound", "establish", "temperatur", "becom", "suggest", "bovin", "nucleu", "also", "state", "difficult", "cell", "qpcr", "perform", "popul", "numer", "tyr", "paxino", "sartoriu", "radioimmunoassay", "plexu", "though", "long", "assay", "immunoreact", "solid", "reconstitut", "particular", "distribut", "higher", "ponedhillo", "mous", "remov", "run", "whole", "mirror", "acetonitril", "μm", "h", "light", 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INTRODUCTION ============ Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent chronic liver disease. NAFLD encompasses a wide spectrum of liver pathologies ranging from simple liver steatosis to nonalcoholic steatohepatitis (NASH), the latter of which is characterized by steatosis with features of cellular injury, including inflammation, and hepatocyte apoptosis.^[@R1]^ Simple steatosis was originally thought to be a benign condition; however, increasing evidence indicates that fatty livers are more vulnerable to factors that promote inflammation and fibrosis.^[@R2]^ In a long-term follow-up study in the United States, up to 5.4% of NAFLD cases ultimately progress to cirrhosis, liver failure, and hepatocellular carcinoma.^[@R3]^ Alarmingly, Chinese patients with NAFLD frequently exhibit significant hepatic necroinflammation and progressive liver fibrosis.^[@R4],[@R5]^ NAFLD is now believed to be a major cause of liver-related morbidity and mortality; therefore, early treatment for NAFLD should be widely considered. Insulin resistance is a key pathogenic factor in both the initiation and development of NAFLD. Several insulin sensitizers, including thiazolidinediones and metformin, have been tested in pilot studies and have been shown to significantly reduce liver inflammation and steatosis in NAFLD patients^[@R6],[@R7]^; however, these compounds are associated with weight gain and raise concerns about cardiovascular safety.^[@R8]^ Thus, the side effects of insulin sensitizers are underestimated, particularly for simple cases of NAFLD in the absence of other complications. Dietary and lifestyle changes are recommended as the primary treatment for NAFLD.^[@R9]^ Increased intakes of fruits and vegetables are likely to have important health benefits for NAFLD patients.^[@R10]^ In addition to vitamins, minerals, and fiber, fruits and vegetables contain high concentrations of bioactive compounds, such as carotenoids, tocopherols, and polyphenols. Among them, anthocyanin polyphenols are of particular interest since several epidemiological studies demonstrated their potential to reduce the risk of insulin resistance-related diseases, such as type 2 diabetes mellitus (T2DM) and cardiovascular disease.^[@R11]--[@R13]^ In a cross-sectional investigation of female adults in England, higher anthocyanin intake was associated with improvements in insulin resistance and inflammatory status.^[@R14]^ The beneficial effects of anthocyanins (either anthocyanin extracts from different plants or pure anthocyanin) have been demonstrated in several animal studies, with respect to oxidative stress, insulin resistance, hepatic steatosis, and inflammation, which are key features of NAFLD.^[@R15]--[@R17]^ Recent clinical trials have investigated the impact of consuming anthocyanins-rich foods on the development and progression of NAFLD. Consumption of purple sweet potato beverages was associated with favorable effects in lowering serum γ-glutamyl transferase and alanine aminotransferase (ALT) levels in healthy men with borderline hepatitis.^[@R18]^ Our previous study demonstrated that bayberry juice can protect against NAFLD in young adults by improving plasma antioxidant status as well as by inhibiting the inflammatory and apoptotic responses involved in this disease,^[@R19]^ but the effect of purified anthocyanin on NAFLD in humans has not been reported. Given the potentially protective effects of anthocyanin on insulin resistance and liver injury and the need for effective therapies for NAFLD, the present study was designed to assess the effects of anthocyanin supplementation in NAFLD patients in a double-blinded placebo-controlled trial. Our objective was to evaluate the effect of anthocyanin purified from berries on selected biomarkers of liver injury. We also examined the effects of anthocyanin on body mass, blood pressure, lipid profile, and glucose homeostasis. SUBJECTS AND METHODS ==================== Subjects -------- The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Ethics Committee of Sun Yat-Sen University prior to initiation of recruitment. Participants diagnosed with NAFLD ranging in age from 25 to 65 were recruited at the Shaoguan Railway Hospital, Shaoguan, China. A diagnosis of fatty liver was indicated based on ultrasound (Voluson E8, GE healthcare, Waukesha, WI) if 2 of the following criteria were met: increased hepatic echogenicity compared with cortical of the right kidney, blurring of liver vasculature, and deep attenuation of the ultrasonographic signal^[@R20]^; and plasma ALT \>30 U/L in men and 19 U/L in women. Criteria were added after recruitment began, as a large fraction of recruited NAFLD patients had normal or even very low ALT levels that would reduce the likelihood of observing significant changes. Hepatic ultrasonography scanning was performed independently by 2 trained radiologists who were blinded to the participants' details and to each other\'s diagnosis. Subjects were included if they were consistently classified as hepatic steatosis by both radiologists. Subjects with excessive alcohol consumption (ethanol \>140 g/wk for men and \>70 g/wk for women), cirrhosis, viral hepatitis, cardiovascular disease, cancer, and any consumption of nonsteroidal anti-inflammatory drugs, corticosteroids, or prescriptive medicines that affect liver function, lipid, or glucose metabolism were excluded. Recruitment began on June 30, 2013, with the first participant identified on July 5, and ended with the last visit on October 30, 2013. A total of 116 subjects were screened, and 74 subjects (39 men and 35 women) were enrolled in the study. Written informed consent was obtained from each patient included in the study. All participants were compensated for their participation in the study. This trial was registered at clinicaltrials.gov as NCT01940263. Study Design ------------ This pilot study was designed according to the CONSORT 2010 statement.^[@R21]^ Participants were assigned in a 1:1 ratio into anthocyanin or placebo groups in a random and double-blinded manner. Randomization was performed using a computer-generated list of random numbers. Randomization was stratified by gender to ensure equal numbers of males and females in each group. Study nurses or clinical physicians enrolled participants. Scheduled participants were consecutively assigned by a medical technologist, who was unaware of enrolment status, to treatment codes that corresponded to labels on otherwise identical concealed containers. Participants, investigators, and outcome assessors were blinded to the treatment for the duration of the study. Treatment assignments were not revealed prior to data collection and analysis. Anthocyanin and placebo capsules (Biolink AS, Sandnes, Norway) were identical in appearance and packaging. Anthocyanin capsules contained 80 mg of anthocyanins that were extracted from bilberry (blueberry) (Vaccinium myrtillus) and black currant (Ribesnigrum), as detailed previously.^[@R22]^ Placebo capsules consisted of maltodextrin and blue color. Two capsules were self-administered each morning and evening, yielding a daily intake of 320 mg anthocyanin. Our previous studies indicated beneficial effects of anthocyanin intervention on lipid profile and inflammatory status in dyslipidemic subjects when treated for 12--24 weeks^[@R22],[@R23]^; consequently we carried out a 24-week intervention in this study. However, during the follow-up period, considering the high dropout rate and the coming Chinese New Year, it was difficult for subjects to remain on the prescribed diet during the Spring Festival, and the trial was terminated at the interim. The patients attended the clinic at weeks 4 and 8 for anthropometric assessments, and returned the remaining capsules and received the capsules for the next interval. Patient compliance was evaluated by counting the remaining capsules. Use of pharmacological agents, for example, metformin, thiazolidinediones, statins, and probiotics, or any medicines that may affect body weight, insulin resistance, lipid profile, or fatty liver was not allowed during the trial. Subjects were asked to maintain their normal lifestyle and not to use any anthocyanin-rich nutritional supplements, red wine, grapes, blueberry, etc, during this period. Adverse events were defined as injuries related to or caused by the study treatment. At each visit, patients were specifically asked about adverse events, and the physician checked for any association between treatment and adverse events. Clinical and Laboratory Evaluation ---------------------------------- Before and after the 12-week intervention, height, weight, waist, and hip circumference, and blood pressure were measured. Body mass index (BMI) was calculated as follows: body weight (kg)/\[body height (m)\]^2^. Waist circumference was measured at a level midway between the lower rib margin and iliac crest with the measuring tape encircling the body in a horizontal position. In addition, a short form (International Physical Activity Questionnaire \[IPAQ\]) and a 3-day dietary record were used to evaluate physical exercise and dietary intake. Plasma glucose, lipid profile, and liver enzymes were tested using a fully automated biochemical analyzer (AU680, Beckman Coulter, Krefeld, Germany). Additional blood variables (hemoglobin, platelets, and white blood cells) were evaluated using an automated hematology analyzer (Sysmex 9000, Toa Medical Electronics, Kobe, Japan). Plasma insulin was determined by enzyme-linked immunosorbent assay (Human insulin ELISA kit, EMD Millipore Corporation, Billerica, MA). Insulin resistance was evaluated by homeostasis model assessment (HOMA-IR) and calculated as (fasting insulin \[mU/L\] × fasting glucose \[mmol/L\])/22.5.^[@R24]^ Plasma cytokeratin-18 fragment M30 (CK-18 M30) was measured using the M30 Apoptosense ELISA kit (PEVIVA, Bromma, Sweden) according to the manufacturer\'s protocols. Plasma myeloperoxidase (MPO) concentrations were measured using a commercially available ELISA kit (Hycult Biotec, Uden, The Netherlands). All plasma samples were analyzed in duplicate in the same run. According to the willingness of participants, a 2-hour oral glucose tolerance tests (OGTT) was performed before and after intervention. Briefly, patients fasted for 10 hours, after which the first blood samples were drawn at 8:00 [am]{.smallcaps} to test fasting glucose. Patients were then given 75 g glucose dissolved in 300 mL water, and the second blood samples were drawn 2 hours after their first drink. The participants were instructed to avoid strenuous exercise, eat any food, or drink any beverage except water for 2 hours. Impaired glucose tolerance (IGT) was defined as 2-hour glucose \>7.8 mmol/L, but \<11.1 mmol/L, according to the Chinese Type 2 Diabetes Prevention Guide 2010.^[@R25]^ A noninvasive scoring system was used to assess the severity of liver injury in NAFLD patients. The score for each test was calculated according to the following formulas derived from the original studies: NAFLD fibrosis score (NFS) = −1.675 + 0.037 × age (years) + 0.094 × BMI (kg/m^2^) + 1.13 × impaired fasting glucose/diabetes (yes = 1, no = 0) + 0.99 × aspartate aminotransferase (AST)/ALT ratio − 0.013 × platelet (×10^9^/L) −0.66 × albumin (g/dL).^[@R26]^ Study Outcomes -------------- The primary outcome measure was the difference in plasma ALT concentration. Secondary outcome measures included anthropometric measurements, fasting plasma levels of insulin, glucose, lipids, MPO, CK-18 M30, and NAFLD fibrosis score. Statistical Analysis -------------------- Sample size calculations were performed using the nQuery Advisor 7.0 software (Statistical Solutions Ltd., Cork, Ireland) and were based on data from a preliminary study in Japan showing decreased ALT levels (from 51.3 to 42.8 U/L) in subjects consuming an anthocyanin-rich purple sweet potato beverage for 12 weeks.^[@R18]^ Assuming that a 15% decrease of ALT in the intervention group and a 5% increase of ALT in the control group would be detected, a sample size of 30 patients per group was necessary to detect a difference with 83% power at a 5% significance level using the 2-sided test. To account for dropouts, 74 participants were included in our study. Data were analyzed according to the intention-to-treat principle. Patients missing laboratory measurements of the primary and secondary outcome measures were imputed using estimating-equation methods.^[@R27]^ SPSS 19.0 software (IBM-SPSS Inc, Chicago, IL) was used for statistical analysis of resulting data. The Kolmogorov--Smirnov test was performed to test normal distributions. Variables that were not normally distributed were logarithmically transformed to achieve normal distribution. Variables followed normal distributions and were described as means ± standard deviation. Data not exhibiting a normal distribution after transformation were expressed as medians with interquartile ranges. The percent change was calculated as follows: (value at 12 weeks -- value at baseline)/value at baseline × 100. Percent changes expressed as medians and interquartile range for nonnormal distributions or the means and 95% confidence interval for normal distributions. For primary or transformed data with a normal distribution, an independent Student t test was used to compare the differences at baseline between groups, and a paired Student t test was used to compare the differences before and after the intervention within groups. Otherwise, a Wilcoxon matched pairs signed rank test and a Mann--Whitney U test were used to compare characteristics in each group before and after intervention and differences between the 2 groups, respectively. Pearson correlation coefficients (r) were used to determine the association between the changes in the plasma CK-18 M30 concentrations and the changes in ALT in the 12-week study. RESULTS ======= Patient Characteristics ----------------------- A total of 74 volunteers were recruited. During the follow-up periods, 8 subjects dropped out by declining to return and 3 subjects were withdrawn by their physicians. In total, 63 participants (34 in the anthocyanin group and 29 in the control group) finished the whole procedure (Figure [1](#F1){ref-type="fig"}); however, all randomized patients were included in the intention-to-treat analysis. Among these subjects, an OGTT was administered to 33 patients (20 in the anthocyanin group, 13 in the control group). Based on the count of the recalled capsules at every visit, the level of compliance was very good, with capsule intake rates of 91.3% and 89.5% in the anthocyanin and placebo groups, respectively. None of the subjects experienced any serious adverse events resulting from the consumption of either the anthocyanin or placebo capsules throughout the trial period. ![Consolidated standards of reporting trials flow diagram of the study participants.](medi-94-e758-g001){#F1} At baseline assessment, no significant differences in anthropometric characteristics were observed between the 2 groups. The 12-week intervention with purified anthocyanin had no impact on BMI, waist and hip circumference, or blood pressure in anthocyanin group compared to the placebo control group (Table [1](#T1){ref-type="table"}). ###### Anthropometric Characteristics Across Groups at Baseline and 12 wk ![](medi-94-e758-g002) Based on the 3-day dietary record and the IPAQ, there were no significant differences in daily nutrient intake or physical activity between the 2 groups at baseline and after 12 weeks' intervention (Supplementary Table 1, <http://links.lww.com/MD/A272>). Primary Outcome --------------- At baseline, there were no significant differences in plasma liver enzymes between the 2 groups (Table [2](#T2){ref-type="table"}). After intervention, although no difference was observed between 2 groups after male-to-male or female-to-female comparisons, the total mean reduction of ALT levels in the anthocyanin group was greater than that of the placebo group (−19.1% vs −3.1%, P = 0.02). Compared to baseline, ALT levels were decreased significantly in both genders in the anthocyanin group; however, only females in the placebo group exhibited a significant drop in ALT levels (Figure [2](#F2){ref-type="fig"}). In addition, the mean value of both genders did not change significantly in the placebo group, which reflected a favorable effect of anthocyanin on chronic liver injury in the study participants. ###### Effects of Anthocyanin Supplementation on Biomarkers of Liver Injury ![](medi-94-e758-g003) ![Effect of the 12-week anthocyanin intervention on ALT levels in NAFLD patients. The columns and error bars indicate medians with quartiles. After intervention, no differences were observed between the 2 groups across intragender comparisons; however, the total mean reduction of ALT levels in the anthocyanin group was greater than that in the placebo group (a), P compared difference between 2 groups by Mann--Whitney U test. Compared with baseline, there is a remarkable decline in ALT after a 12-week intervention in males, females, and total patients in the anthocyanin group (b, c), but only in females in the placebo group (b, d). P compared with the basal values in each group by Wilcoxon matched pairs signed rank test.](medi-94-e758-g004){#F2} Secondary Outcomes ------------------ ### Lipid Profile and Insulin Resistance As shown in Table [3](#T3){ref-type="table"}, ingestion of anthocyanin for 12 weeks significantly decreased plasma triglyceride (TG) levels; however, the mean percentage changes were not significantly different from placebo controls. No effects on total plasma cholesterol or low-density lipoprotein-cholesterol were observed. Plasma levels of high-density lipoprotein-cholesterol increased moderately in both the groups, but this trend did not reach statistical significance across the 2 groups. ###### Effects of Anthocyanin Supplementation on Fasting Plasma Lipid Profile and Glucose and Insulin Concentrations ![](medi-94-e758-g005) Fasting blood glucose and HOMA-IR decreased significantly from baseline in the anthocyanin group, but did not differ from the placebo values (Table [3](#T3){ref-type="table"}). Insulin levels after 12 weeks was comparable to baseline in both anthocyanin and placebo groups. To examine insulin sensitivity and β-cell function, an oral glucose tolerance test was performed in 33 subjects, including 20 in anthocyanin group and 13 in the control group. Among these patients, 10 (50.0%) in the anthocyanin group and 6 (46.2%) in the control exhibited IGT. The anthropometric characteristics, dietary intake, physical activities, fasting blood glucose and insulin, and HOMA-IR were comparable between the 2 groups at baseline (Supplementary Table 2, <http://links.lww.com/MD/A272>). Anthocyanin supplementation significantly decreased the 2-hour OGTT glucose levels compared to the placebo control (−18.7% vs −3.8%, P = 0.02; Figure [3](#F3){ref-type="fig"}). ![Changes in glucose concentration measured 2 h after a 75-g oral glucose load in the 2 treatments during the study period. P compared difference between 2 groups by independent-sample t test.](medi-94-e758-g006){#F3} ### Other Liver Injury Biomarkers Anthocyanin supplementation did not affect the release of AST, another important enzyme in the liver (Table [2](#T2){ref-type="table"}). Plasma levels of CK-18 M30, a predictor of steatohepatitis,^[@R28]^ decreased by 8.8% after anthocyanin treatment and increased by 5.6% in the placebo group (P = 0.04). Inflammation and oxidative stress are involved in NAFLD progression, and MPO is linked to both inflammation and oxidative stress based on its location in leukocytes and role in catalyzing the formation of oxidizing agents.^[@R29]^ We next evaluated plasma levels of MPO and observed a significant difference in the effects of the 2 capsules on circulating MPO levels (−75.0% vs −44.8%, P \< 0.01). Compared with baseline, the NAFLD fibrosis score increased by 22.3% in the placebo group (P \< 0.05) but only 8.4% in the anthocyanin group (not significant), respectively. Furthermore, a positive correlation (r = 0.49, P \< 0.01) between the plasma ALT and CK-18 M30 levels was observed in our study subjects (Figure [4](#F4){ref-type="fig"}). ![Correlation between changes in plasma ALT activities and the levels of CK-18 M30; n = 37 in each group. The data were evaluated by using Pearson correlation coefficients (r).](medi-94-e758-g007){#F4} DISCUSSION ========== The results of this pilot study suggest that purified anthocyanin supplements can have favorable effects on insulin resistance and several plasma biomarkers of liver injury in patients with NAFLD. Although the patients in our study had relatively mild disease, they represent the spectrum of patients who are commonly seen in a primary care setting. The participants were able to maintain a constant body weight throughout the study (Supplementary Table 3, <http://links.lww.com/MD/A272>), excluding weight changes as a confounding factor that contributed to the improved features of NAFLD. Furthermore, participants were instructed not to alter their physical activity during the study. Therefore, in light of these findings, we suggest that an increase of dietary anthocyanin intake may help slow or reverse the clinical evolution of fatty liver disease, especially in early-stage NAFLD patients. Previous studies of foods rich in anthocyanin have yielded positive findings on whole-body inflammatory status and insulin sensitivity in obese subjects.^[@R30],[@R31]^ In the current study, a significant improvement or normalization of ALT, along with a marked improvement of glucose homeostasis, occurred in NAFLD patients after 12 weeks of anthocyanin treatment. Increasing evidence provided by cohort studies suggests that fatty liver may be a predictor of T2DM and cardiovascular disease,^[@R3]^ and our results are consistent with the observed association between higher anthocyanin intake and lower incidence of T2DM or cardiovascular disease.^[@R11],[@R13]^ However, in contrast to the results in certain trials,^[@R22],[@R31],[@R32]^ we did not observe any effect of anthocyanin on plasma lipid profile. It is possible that the efficacy of anthocyanins on lipid profile responses depends, to some extent, on the population studied and the duration of the intervention. Another important finding of this study is the marked decrease in plasma levels of caspase-cleaved CK-18 fragments in participants receiving anthocyanin treatment. CK-18 is the predominant keratin expressed in the liver and is a well-known substrate of caspases during apoptotic hepatocyte cell death.^[@R33]^ Other investigators have shown that CK-18 M30 has high overall accuracy in differentiating NAFLD from control subjects and moderate accuracy in differentiating NASH from simple steatosis.^[@R34]^ Our previous in vitro study showed that anthocyanin cyanidin 3-O-β-glucoside improved hepatocyte function and cell survival in response to high glucose stress.^[@R35]^ In this study, we observed a positive correlation between changes in plasma ALT activities and the levels of CK-18 M30 in anthocyanin-treated patients. These results suggest that CK-18 M30 has potential as a marker for predicting NAFLD progression. Oxidative stress and inflammation are important mechanisms in the pathogenesis of NAFLD. MPO is a heme-containing peroxidase abundantly expressed in neutrophils and to a lesser extent in monocytes.^[@R29]^ Upon cell activation, MPO is released and MPO-derived oxidants have been associated with tissue damage in many diseases, particularly those characterized by acute or chronic inflammation.^[@R36]^ Increased numbers of MPO-expressing cells and accumulation of HOCl-modified and nitrated proteins in steatotic livers enhance progression of simple steatosis to steatohepatitis. In contrast, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in mice on a high fat diet.^[@R37]^ A recent study demonstrated that plasma MPO was positively associated with insulin resistance and inflammation parameters in overweight subjects.^[@R38]^ In NASH patients, both hepatic MPO expression and plasma MPO levels were elevated compared with patients with similar BMI but without NASH.^[@R39]^ Our results suggest that anthocyanin supplementation reduced plasma MPO and showed a trend toward improvement in NAFLD fibrosis score. It is tempting to speculate that MPO may be a factor that directly links the hepatoprotective effects of anthocyanin to its well-documented anti-inflammatory and anti-oxidative properties.^[@R40]^ The majority of studies investigating anthocyanin intervention in humans have used foods containing multiple types of polyphenols. To the best of our knowledge, this is the first study comparing the effects of purified anthocyanin on blood-based biomarkers of NAFLD using a randomized double-blind design with placebo controls. Nevertheless, this study has some intrinsic limitations that must be acknowledged. First, as liver biopsies were not performed, NAFLD features such as steatosis and necroinflammation could not be evaluated. Second, our study was mildly underpowered by small sample size and a relatively short intervention period. We experienced a significant number of screen failures in the enrolment, and patient dropout rates were higher than anticipated, resulting in a lower number of evaluable patients than is estimated to be necessary to detect differences between groups, which may have led to a type II error. Finally, our patients had relatively low BMIs compared to those reported in Western studies; however, Asians generally develop visceral obesity and NAFLD at a lower BMI.^[@R5]^ In summary, our findings above linking the dietary anthocyanin supplement to improved characteristics of NAFLD are particularly important in light of the increasing incidence of NAFLD worldwide. Furthermore, this supplementation is an inexpensive, nontoxic therapy that results in overall health improvement, reducing the risk not only of chronic liver disease, but also T2DM and cardiovascular disease. Future large, prospective, well-designed, multicenter studies are needed to explore the durability of the benefits of anthocyanin. The authors would like to express our gratitude to Dr Yichun Zhong (Shaoguan Railway Hospital) for his kind support of this work. Abbreviations: ALT = alanine aminotransferase, BMI = body mass index, CK-18 M30 = cytokeratin-18 fragment M30, HOMA-IR = homeostasis model assessment of insulin resistance, MPO = myeloperoxidase, NAFLD = nonalcoholic fatty liver disease, NASH = nonalcoholic steatohepatitis, OGTT = oral glucose tolerance test, T2DM = type 2 diabetes mellitus. This work was funded by grants from the National Basic Research Program (973 Program, 2012CB517506), National Natural Science Foundation (81372994, 81172655), and Guangdong Industry-University Research Foundation (2013B090600138). All authors have read and approved the submitted version of the manuscript. Study concept: H-HG; data acquisition: P-WZ; data analysis and interpretation: F-XC, DL; drafting of the manuscript: P-WZ; critical revision of the manuscript for important intellectual content: W-HL; study supervision: H-HG. The authors have no conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal\'s Website ([www.md-journal.com](www.md-journal.com)).	{ "pile_set_name": "PubMed Central" }	[ "miner", "treatment", "hoclmodifi", "cholesterol", "two", "control", "hepatoprotect", "given", "work", "origin", "conceal", "remain", "program", "decreas", "bloodbas", "caspas", "impair", "quartil", "collect", "testmediegf", "longterm", "biopsi", "anticip", "identifi", "healthcar", "currant", "pearson", "basal", "placebo", "anthropometr", "spectrum", "risk", "written", "pathogenesi", "kind", "rais", "select", "fund", "grant", "mechan", "student", "week", "hour", "valu", "locat", "weeksr", "multipl", "may", "hip", "recent", "logarithm", "toward", "signalr", "use", "nonalcohol", "dropout", "beckman", "gener", "moder", "cb", "germani", "interim", "score", "final", "supplement", "support", "keratin", "steatot", "caus", "accord", "prior", "intak", "signific", "purpl", 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"predictor", "cardiovascular", "disease", "results", "consistent", "observed", "association", "higher", "anthocyanin", "intake", "lower", "incidence", "cardiovascular", "However", "contrast", "results", "certain", "trials", "observe", "effect", "anthocyanin", "plasma", "lipid", "profile", "possible", "efficacy", "anthocyanins", "lipid", "profile", "responses", "depends", "extent", "population", "studied", "duration", "intervention", "Another", "important", "finding", "study", "marked", "decrease", "plasma", "levels", "fragments", "participants", "receiving", "anthocyanin", "treatment", "predominant", "keratin", "expressed", "liver", "substrate", "caspases", "apoptotic", "hepatocyte", "cell", "investigators", "shown", "high", "overall", "accuracy", "differentiating", "NAFLD", "control", "subjects", "moderate", "accuracy", "differentiating", "NASH", "simple", "previous", "vitro", "study", "showed", "anthocyanin", "cyanidin", "improved", "hepatocyte", "function", "cell", "survival", "response", "high", "glucose", "study", "observed", "positive", "correlation", "changes", "plasma", "ALT", "activities", "levels", "patients", "results", "suggest", "potential", "marker", "predicting", "NAFLD", "progression", "Oxidative", "stress", "inflammation", "important", "mechanisms", "pathogenesis", "NAFLD", "MPO", "peroxidase", "abundantly", "expressed", "neutrophils", "lesser", "extent", "Upon", "cell", "activation", "MPO", "released", "oxidants", "associated", "tissue", "damage", "many", "diseases", "particularly", "characterized", "acute", "chronic", "Increased", "numbers", "cells", "accumulation", "nitrated", "proteins", "steatotic", "livers", "enhance", "progression", "simple", "steatosis", "steatohepatitis", "contrast", "MPO", "deficiency", "attenuates", "development", "NASH", "diminishes", "adipose", "tissue", "inflammation", "mice", "high", "fat", "recent", "study", "demonstrated", "plasma", "MPO", "positively", "associated", "insulin", "resistance", "inflammation", "parameters", "overweight", "NASH", "patients", "hepatic", "MPO", "expression", "plasma", "MPO", "levels", "elevated", "compared", "patients", "similar", "BMI", "without", "results", "suggest", "anthocyanin", "supplementation", "reduced", "plasma", "MPO", "showed", "trend", "toward", "improvement", "NAFLD", "fibrosis", "score", "tempting", "speculate", "MPO", "may", "factor", "directly", "links", "hepatoprotective", "effects", "anthocyanin", "majority", "studies", "investigating", "anthocyanin", "intervention", "humans", "used", "foods", "containing", "multiple", "types", "polyphenols", "best", "knowledge", "first", "study", "comparing", "effects", "purified", "anthocyanin", "biomarkers", "NAFLD", "using", "randomized", "design", "placebo", "controls", "Nevertheless", "study", "intrinsic", "limitations", "must", "acknowledged", "First", "liver", "biopsies", "performed", "NAFLD", "features", "steatosis", "necroinflammation", "could", "evaluated", "Second", "study", "mildly", "underpowered", "small", "sample", "size", "relatively", "short", "intervention", "period", "experienced", "significant", "number", "screen", "failures", "enrolment", "patient", "dropout", "rates", "higher", "anticipated", "resulting", "lower", "number", "evaluable", "patients", "estimated", "necessary", "detect", "differences", "groups", "may", "led", "type", "II", "error", "Finally", "patients", "relatively", "low", "BMIs", "compared", "reported", "Western", "studies", "however", "Asians", "generally", "develop", "visceral", "obesity", "NAFLD", "lower", "summary", "findings", "linking", "dietary", "anthocyanin", "supplement", "improved", "characteristics", "NAFLD", "particularly", "important", "light", "increasing", "incidence", "NAFLD", "worldwide", "Furthermore", "supplementation", "inexpensive", "nontoxic", "therapy", "results", "overall", "health", "improvement", "reducing", "risk", "chronic", "liver", "disease", "also", "cardiovascular", "disease", "Future", "large", "prospective", "multicenter", "studies", "needed", "explore", "durability", "benefits", "anthocyanin", "authors", "would", "like", "express", "gratitude", "Dr", "Yichun", "Zhong", "Shaoguan", "Railway", "Hospital", "kind", "support", "work", "Abbreviations", "ALT", "alanine", "aminotransferase", "BMI", "body", "mass", "index", "fragment", "homeostasis", "model", "assessment", "insulin", "resistance", "MPO", "myeloperoxidase", "NAFLD", "nonalcoholic", "fatty", "liver", "disease", "NASH", "nonalcoholic", "steatohepatitis", "OGTT", "oral", "glucose", "tolerance", "test", "type", "diabetes", "mellitus", "work", "funded", "grants", "National", "Basic", "Research", "Program", "Program", "National", "Natural", "Science", "Foundation", "Guangdong", "Research", "Foundation", "authors", "read", "approved", "submitted", "version", "manuscript", "Study", "concept", "data", "acquisition", "data", "analysis", "interpretation", "DL", "drafting", "manuscript", "critical", "revision", "manuscript", "important", "intellectual", "content", "study", "supervision", "authors", "conflicts", "interest", "disclose", "Supplemental", "digital", "content", "available", "article", "Direct", "URL", "citations", "appear", "printed", "text", "provided", "HTML", "PDF", "versions", "article", "Website" ]
Suggested citation: EFSA GMO Panel (EFSA Panel on genetically modified organisms) , Naegeli H, Bresson J‐L, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogué F, Rostoks N, Sánchez Serrano JJ, Savoini G, Veromann E, Veronesi F, Álvarez F, Ardizzone M, Papadopoulou N, Paraskevopoulos K, 2019 Scientific Opinion on the assessment of genetically modified soybean A2704‐12 for renewal of authorisation under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐RX‐009). EFSA Journal 2019;17(1):5523, 12 pp. 10.2903/j.efsa.2019.5523 Requestor: European Commission (DG SANTE) Question number: EFSA‐Q‐2017‐00721 Panel members: Hanspeter Naegeli, Jean‐Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michele Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Francisco Javier Moreno, Ewen Mullins, Fabien Nogué, Nils Rostoks, Jose Juan Sánchez Serrano, Giovanni Savoini, Eve Veromann and Fabio Veronesi. Acknowledgements: The Panel wishes to thank the members of its standing Working Groups on Molecular Characterisation, Food/Feed and Environmental Risk Assessment for the preparatory work on this scientific opinion, and the EFSA staff members Andrea Gennaro, Sylvie Mestdagh, Claudia Paoletti and Irene Muñoz Guajardo for the support provided to this scientific opinion. Adopted: 29 November 2018 Summary {#efs25523-sec-0001} ======= Following the submission of application EFSA‐GMO‐RX‐009 under Regulation (EC) No 1829/2003 from Bayer CropScience N.V., the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel) was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the herbicide‐tolerant genetically modified soybean A2704‐12. The scope of renewal application EFSA‐GMO‐RX‐009 is for placing on the market of products containing, consisting of, or produced from soybean A2704‐12, excluding cultivation within the European Union. In delivering its scientific opinion, the GMO Panel took into account application EFSA‐GMO‐RX‐009, additional information provided by the applicant, scientific comments submitted by the Member States and relevant scientific publications. The data received in the context of the renewal application EFSA‐GMO‐RX‐009 contained: post‐market environmental monitoring reports, an evaluation of the literature retrieved by a systematic search, updated bioinformatics analyses, and additional studies performed by or on behalf of the applicant. In addition, the applicant provided sequence data on the soybean A2704‐12 event using material from a commercial variety currently on the market and intended to be marketed in the coming years. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. The GMO Panel concludes that there is no evidence in renewal application EFSA‐GMO‐RX‐009 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean A2704‐12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). 1. Introduction {#efs25523-sec-0003} =============== 1.1. Background {#efs25523-sec-0004} --------------- On 3 November 2017, the European Food Safety Authority (EFSA) received from the European Commission (DG SANTE) application EFSA‐GMO‐RX‐009 by Bayer CropScience N.V. for the renewal of authorisation of genetically modified (GM) soybean A2704‐12 (Unique Identifier ACS‐GMØØ5--3) for the placing on the market of products containing, consisting of, or produced from this GM soybean for import and processing submitted within the framework of Regulation (EC) No 1829/2003[1](#efs25523-note-1006){ref-type="fn"}. Before sending the application to EFSA, the European Commission confirmed whether the data submitted in the context of this renewal application were in line with the legal requirements laid down in Articles 11 and 23 of Regulation (EC) No 1829/2003. After receiving application EFSA‐GMO‐RX‐009, and in accordance with Articles 5(2)(b) and 17(2)(b) of Regulation (EC) No 1829/2003, EFSA informed Member States and made the summary of the application available to the public on the EFSA website.[2](#efs25523-note-1007){ref-type="fn"} On 9 March 2018, EFSA declared the application valid in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003. EFSA made the valid application available to Member States and the European Commission, and consulted nominated risk assessment bodies of Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC following the requirements of Articles 6(4) and 18(4) of Regulation (EC) No 1829/2003, to request their scientific opinion. Member States had three months after the opening of the Member State commenting period (until 9 June 2018) to make their opinion known. Following the submission of application EFSA‐GMO‐NL‐2005‐18 and the publication of the EFSA scientific opinion (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}), the placing on the market of soybean A2704‐12 for products containing, consisting of, or produced from this GM soybean, excluding cultivation in the European Union, was authorised by Commission Decision 2008/837/EC[3](#efs25523-note-1008){ref-type="fn"}. A copy of this authorisation was provided by the applicant.[4](#efs25523-note-1009){ref-type="fn"} EFSA requested additional information on 26 March 2018 and 8 May 2018. The applicant submitted their replies on 27 April 2018 and 19 June 2018, respectively. In giving its scientific opinion to the European Commission, the Member States and the applicant, and in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003, EFSA has endeavoured to respect a time limit of six months from the acknowledgement of the valid application. As additional information was requested by the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel), the time limit of 6 months was extended accordingly, in line with Articles 6(1), 6(2), 18(1), and 18(2) of Regulation (EC) No 1829/2003. According to Regulation (EC) No 1829/2003, this scientific opinion is to be seen as the report requested under Articles 6(6) and 18(6) of that Regulation and thus will be part of the EFSA overall opinion in accordance with Articles 6(5) and 18(5). 1.2. Terms of Reference as provided by the requestor {#efs25523-sec-0005} ---------------------------------------------------- The GMO Panel was requested to carry out a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the placing on the market of products containing, consisting of, or produced from GM soybean A2704‐12, in accordance with Articles 11 and 23 of Regulation (EC) No 1829/2003. Where applicable, any conditions or restrictions which should be imposed on the placing on the market and/or specific conditions or restrictions for use and handling, including post‐market monitoring requirements based on the outcome of the risk assessment and, in the case of GMOs or food and feed containing or consisting of GMOs, conditions for the protection of particular ecosystems/environment and/or geographical areas should be indicated in accordance with Articles 6(5)(e) and 18(5)(e) of Regulation (EC) No 1829/2003. The GMO Panel was not requested to give an opinion on information required under Annex II to the Cartagena Protocol. Furthermore, the GMO Panel did not consider proposals for labelling and methods of detection (including sampling and the identification of the specific transformation event in the food and feed and/or food and feed produced from it), which are matters related to risk management. 2. Data and methodologies {#efs25523-sec-0006} ========================= 2.1. Data {#efs25523-sec-0007} --------- The data for application EFSA‐GMO‐RX‐009 provided by the applicant at the time of submission, or in reply to requests for additional information, are specified below. The applicant submitted sequence data on soybean A2704‐12 single event[5](#efs25523-note-1010){ref-type="fn"} derived from material of an early generation (T4) of soybean A2704‐12 (fourth generation after the original transformant) and from material of a commercially available soybean A2704‐12 variety currently on the market and indicated that these sequences are identical. The applicant clarified that the soybean A2704‐12 commercial variety has been available on the market since 2016 and is intended to be marketed in the coming years. The applicant also clarified that the A2704‐12 event sequence reported in this renewal application is the sequence submitted in the original application EFSA‐GMO‐NL‐2005‐18 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}), corrected for eight nucleotides, two of which affect newly created open reading frames (ORFs) within the insert and spanning the junction sites (EFSA, [2018](#efs25523-bib-0006){ref-type="ref"}). ### 2.1.1. Post‐market monitoring reports[6](#efs25523-note-1011){ref-type="fn"} {#efs25523-sec-0008} Based on the outcome of the initial food and feed risk assessment, a post‐market monitoring plan for monitoring of GM food and feed was not required by the authorisation decision. The implementation of a post‐market environmental monitoring (PMEM) plan, consisting of a general surveillance plan to check for any adverse effects on the environment arising from soybean A2704‐12, was a condition for the authorisation. As no potential adverse environmental effects were identified in the environmental risk assessment of soybean A2704‐12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}), case‐specific monitoring was not considered necessary by the GMO Panel. The applicant provided eight annual PMEM reports covering a reporting period from September 2008 to June 2016. The annual PMEM plans submitted by the applicant included (1) the description of a centralised system established by EuropaBio for the collection of information recorded by various operators (federations involved in soybean seeds import and processing) on any observed adverse effect(s) on human health and the environment arising from handling of soybean possibly containing soybean A2704012; (2) the reports of the surveillance activities conducted by such operators; and (3) the review of relevant scientific peer‐reviewed studies retrieved from literature searches. ### 2.1.2. Systematic search and evaluation of literature[7](#efs25523-note-1012){ref-type="fn"} {#efs25523-sec-0009} In addition to the eight separate literature searches provided as part of the annual PMEM reports, the applicant performed a systematic literature search for soybean A2704012 and the newly expressed phosphinothricin N‐acetyltransferase (PAT) protein covering the period from 1 September 2007 till 10 January 2018, in accordance with the recommendations on literature searching outlined in EFSA ([2010](#efs25523-bib-0002){ref-type="ref"}, [2017a](#efs25523-bib-0004){ref-type="ref"}). Searches against electronic bibliographic databases and internet searches to specialist databases were performed to identify relevant publications. Altogether, 564 publications were retrieved. After applying the eligibility/inclusion criteria defined a priori by the applicant, six publications were identified as relevant for food and feed safety assessment, molecular characterisation and environmental safety assessment. The list of relevant publications is provided in Appendix [A](#efs25523-sec-1001){ref-type="sec"}. ### 2.1.3. Updated bioinformatic data[8](#efs25523-note-1013){ref-type="fn"} {#efs25523-sec-0010} At the time of submission of the renewal dossier, the applicant provided a complete bioinformatic dataset for the soybean A2704‐12 event (using the corrected sequence) including an analysis of the insert and flanking sequences, an analysis of the potential similarity to allergens and toxins of the newly expressed protein and of all possible ORFs within the insert and spanning the junction sites, and an analysis of possible horizontal gene transfer (EFSA, [2017b](#efs25523-bib-0005){ref-type="ref"}). The outcome of the updated bioinformatic analyses is presented in Section [3.3](#efs25523-sec-0018){ref-type="sec"}. On 8 May 2018, EFSA requested supplementary information on the partial deletion of the endogenous gene previously reported in the frame of the original application EFSA‐GMO‐NL‐2005‐18. On 19 June 2018, the applicant provided the supplementary information which included information on the function of the interrupted endogenous gene and a risk assessment of its interruption with respect to the agronomic--phenotypic characteristics and composition of soybean A2704‐12. ### 2.1.4. Additional documents or studies provided by the applicant[9](#efs25523-note-1014){ref-type="fn"} {#efs25523-sec-0011} The applicant provided an overview on the worldwide approvals of soybean A2704‐12 and a list containing the summaries of all studies performed by or on behalf of the applicant over the course of the authorisation period and not previously submitted to the EU (Appendix [B](#efs25523-sec-1002){ref-type="sec"}). The relevance of the listed studies for molecular characterisation, human and animal safety and the environment was assessed by the applicant. On 26 March 2018, the GMO Panel requested the applicant to provide the full study reports of five of these studies considered potentially relevant for safety assessment. The applicant submitted the requested information on 27 April 2018. ### 2.1.5. Overall assessment as provided by the applicant[10](#efs25523-note-1015){ref-type="fn"} {#efs25523-sec-0012} The applicant provided an overall assessment concluding that information provided in the application for renewal of authorisation of soybean A2704‐12 for food and feed use and processing in the EU does not change the outcome of the original risk assessment (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). ### 2.1.6. Monitoring plan and proposal for improving the conditions of the original authorisation[11](#efs25523-note-1016){ref-type="fn"} {#efs25523-sec-0013} The applicant indicated in the dossier that the environmental monitoring plan is appropriate and does not need any changes. 2.2. Methodologies {#efs25523-sec-0014} ------------------ The GMO Panel assessed the application for renewal of the authorisation of soybean A2704‐12 for food and feed uses, import and processing in accordance with Articles 11 and 23 of Regulation (EC) No 1829/2003. The GMO Panel took into account the requirements described in its guideline for the risk assessment of renewal applications of GM food and feed authorised under Regulation (EC) No 1829/2003 (EFSA GMO Panel, [2015](#efs25523-bib-0003){ref-type="ref"}). The comments raised by Member States are addressed in Annex G of EFSA\'s overall opinion[12](#efs25523-note-1017){ref-type="fn"} and were taken into consideration during the scientific risk assessment. 3. Assessment {#efs25523-sec-0015} ============= 3.1. Evaluation of the post‐market monitoring reports {#efs25523-sec-0016} ----------------------------------------------------- During the general surveillance activities covering the authorisation period of soybean A2704‐12, no adverse effects were reported by the applicant. 3.2. Evaluation of the systematic search and evaluation of literature {#efs25523-sec-0017} --------------------------------------------------------------------- The GMO Panel assessed the applicant\'s literature searches on soybean A2704‐12. Although the overall quality of the performed literature searches is acceptable, the GMO Panel considers that future searches could be improved. The GMO Panel therefore recommends the applicant for future searches to: ensure that enough search term variation is used (covering possible synonyms, related terms, acronyms, spelling variants, old and new terminology, brand and generic names, lay and scientific terminology, common typos, translation issues);include trait terms;include controlled vocabulary (subject indexing) in the searches when available (in addition to/combination with text words);use wider proximity operators (5W). The GMO Panel acknowledges that no publications raising a safety concern for human and animal health and the environment which would change the original risk assessment conclusions on soybean A2704‐12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}) have been identified by the applicant. 3.3. Evaluation of the updated bioinformatic data {#efs25523-sec-0018} ------------------------------------------------- The results of the updated bioinformatic analyses of soybean A2704‐12 using the corrected event sequence (EFSA, [2018](#efs25523-bib-0006){ref-type="ref"}) confirm the previous conclusions on the partial deletion of an endogenous gene. Based on the information from updated databases submitted in the frame of this renewal application, this gene is identified as SEOe, a member of the sieve element occlusion (SEO) multigene family encoding for Glycine max SEO proteins. The SEOe gene is one of four genes in subgroup 5 of the SEO gene family, thereby indicating that functional redundancy for this gene in the soybean genome is likely. In addition, the original agronomic, phenotypic and compositional analyses of soybean A2704‐12 (including endpoints that may be relevant to the reported biological role of SEO proteins in photoassimilate translocation via the phloem network such as plant height, yield and fiber content) did not indicate relevant differences as compared to the non‐GM comparator. Analyses of the amino acid sequence of the newly expressed PAT protein reveal no significant similarities to toxins or allergens. In addition, bioinformatic analyses of the newly created ORFs (using the corrected event sequence) within the insert or spanning the junctions with genomic DNA reveal no significant similarities to toxins and allergens. Updated bioinformatic analyses of the recombinant DNA present in soybean A2704‐12 reveal sufficient sequence identity at both ends of the inserted cassette by the disrupted bla gene. This could facilitate double homologous recombination with bla genes as they can occur in environmental bacteria. The recombination event, however, would result in the disruption of such genes in the recipients. In addition, the insertion of a pat gene codon‐optimised for plants regulated by a plant virus promoter would probably lack PAT activity in potential recipient bacteria. Therefore, bacteria in which the described double homologous recombination would occur would lose their antibiotic resistance and acquire a plant codon‐optimised pat gene for which a selective advantage, compared to natural variants of pat genes as they occur in environmental bacteria, cannot be expected. The results of updated bioinformatic analyses confirm the previous conclusions that the unlikely, but theoretically possible, horizontal transfer of recombinant genes from soybean A2704‐12 to bacteria does not raise any environmental safety concern (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). 3.4. Evaluation of the additional documents or studies provided by the applicant {#efs25523-sec-0019} -------------------------------------------------------------------------------- The GMO Panel evaluated the summary and/or full study reports of the additional studies provided and (Appendix [B](#efs25523-sec-1002){ref-type="sec"}). This new information does not raise any concern for human and animal health and the environment, which would change the original risk assessment conclusions on soybean A2704‐12. 3.5. Evaluation of the overall assessment as provided by the applicant {#efs25523-sec-0020} ---------------------------------------------------------------------- The GMO Panel evaluated the overall assessment provided by the applicant and confirms that there is no evidence in renewal application EFSA‐GMO‐RX‐009 indicating new hazards, relevant changes in exposure or scientific uncertainties that would change previous conclusions on soybean A2704‐12. 3.6. Evaluation of the monitoring plan and proposal for improving the conditions of the original authorisation {#efs25523-sec-0021} -------------------------------------------------------------------------------------------------------------- The PMEM plan covers general surveillance of imported GM plant material, including soybean A2704‐12. This general surveillance is coordinated by EuropaBio and implemented by selected operators (federations involved in soybean seeds import and processing). In addition, the applicant reviews relevant scientific publications retrieved from literature searches on an annual basis. The GMO Panel is of the opinion that the scope of the plan provided by the applicant is consistent with the scope of soybean A2704‐12, but reminds that monitoring is related to risk management, and thus, the final adoption and implementation of the PMEM plan falls outside the mandate of EFSA. 4. Conclusions {#efs25523-sec-0022} ============== Based on the data provided, the GMO Panel concludes that there is no evidence in the renewal application EFSA‐GMO‐RX‐009 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean A2704--12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). Documentation provided to EFSA {#efs25523-sec-0023} ============================== Letter from the European Commission to EFSA received on 3 November 2017 for the continued marketing of genetically modified soybean A2704‐12 in accordance with articles 11 and 23 of Regulation (EC) No 1829/2003 by Bayer CropScience N.V. (EFSA‐GMO‐RX‐009).Acknowledgement letter, dated 7 November 2017, from EFSA to the European Commission.Letter from EFSA to applicant dated 15 December 2017 requesting additional information under completeness check.Letter from applicant to EFSA received on 16 February 2018 providing additional information under completeness check.Letter from EFSA to applicant dated 9 March 2018 delivering the 'Statement of Validity' for application EFSA‐GMO‐RX‐009.Letter from EFSA to applicant dated 26 March 2018 requesting additional information and stopping the clock.Letter from applicant to EFSA received on 27 April 2018 providing additional information.Letter from EFSA to applicant dated 30 April 2018 re‐starting the clock from 27 April 2018.Letter from EFSA to applicant dated 8 May 2018 requesting additional information and stopping the clock.Letter from applicant to EFSA received on 19 June 2018 providing additional information.Letter from EFSA to applicant dated 19 June 2018 re‐starting the clock from 19 June 2018. Abbreviations {#efs25523-sec-0024} ============= GMgenetically modifiedGMOgenetically modified organismsGMOPanel EFSA Panel on Genetically Modified OrganismsORFsopen reading framesPATphosphinothricin N‐acetyltransferasePMEMpost‐market environmental monitoringSEOsieve element occlusion Appendix A -- List of relevant publications identified by the applicant through the systematic literature search (1/9/2007--10/1/2018) {#efs25523-sec-1001} ====================================================================================================================================== {#efs25523-sec-0025} ReferenceFard NA et al., 2013. In silico allergenicity assessment of novel proteins derived from GMHR crops. Ortuno F and Rojas I \[Eds\]. Proceedings IWBBIO.Fard NA, Minuchehr Z and Mousavi A, 2013. Allergenicity study of genetically modified herbicide resistant crops (bioinformatics assessment). Bulletin of Environment, Pharmacology and Life Sciences, 2, 24--32.Nagy A, Pauk J, Takacs K and Gelencser E, 2008. Nutritional evaluation of the proteins of broad range herbicide resistant spring wheat (Triticum aestivum L.) lines. II. Resistance to digestion of marker proteins in rat model. Acta Alimentaria, 37, 159--166.Oh J, Ko M and Lee H, 2009. Evaluation for allergenicity for genetically modified organic foods. Journal of Allergy and Clinical Immunology, 123, S244.Schafer BW, Embrey SK and Herman RA, 2016. Rapid simulated gastric fluid digestion of in‐seed/grain proteins expressed in genetically engineered crops. Regulatory Toxicology and Pharmacology, 81, 106--112.Verma AK, Misra A, Subash S, Das M and Dwivedi PD, 2011. Computational allergenicity prediction of transgenic proteins expressed in genetically modified crops. Immunopharmacology and Immunotoxicology, 33, 410--422. Appendix B -- List of additional studies performed by or on behalf of the applicant over the course of the authorisation period and not previously submitted to the EU with regard to the evaluation of the safety of the food and feed for humans, animal or the environment from soybean A2704‐12 {#efs25523-sec-1002} =================================================================================================================================================================================================================================================================================================== {#efs25523-sec-0026} Study identificationTitleM--413199‐01‐1Agronomic characteristics of glufosinate‐tolerant soybean A2704‐12, USA, 2006M‐534302‐01‐1Structural stability analysis of early and recent generations of Glycine max A2704‐12M‐540738‐01‐1A2704‐12 soybean ‐ Inheritance of the insert over two generationsM‐131221‐04‐1[a](#efs25523-note-1018){ref-type="fn"}PAT/pat protein: Heat stability studyM‐214021‐03‐1PAT/pat protein: in vitro digestibility study in human simulated intestinal fluidM‐214025‐05‐1[a](#efs25523-note-1018){ref-type="fn"}PAT/pat protein ‐ In vitro digestibility study in human simulated gastric fluidM‐344158‐01‐1[a](#efs25523-note-1018){ref-type="fn"}PAT protein (encoded the pat gene) ‐ Acute toxicity (48 hours) to rainbow trout (Oncorhynchus mykiss) by intraperitoneal injectionM‐395296‐01‐1Measurement of PAT protein in soybean oil samples using the QualiPlate Kit for PAT from EnviroLogixM‐475440‐01‐1[a](#efs25523-note-1018){ref-type="fn"}PAT/pat protein ‐ Acute toxicity by oral gavage in miceM‐500889‐01‐1[a](#efs25523-note-1018){ref-type="fn"}The effect of temperature on microbially‐produced PAT/pat as assessed by ELISA[^1] Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, 18.10.2003, p. 1--23. Available online: <http://registerofquestions.efsa.europa.eu/roqFrontend/questionDocumentsLoader?question=EFSA-Q-2017-00721> COMMISSION DECISION of 8 September 2008 authorising the placing on the market of products containing, consisting of, or produced from genetically modified soybean A2704‐12 (ACS‐GMØØ5‐3) pursuant to Regulation (EC) No 1829/2003 of the European Parliament and of the Council. Official Journal of the European Union L 247/50, 16.9.2008. Dossier: Soybean A2704‐12 renewal -- Annex 1. Dossier: Soybean A2704‐12 renewal -- Section 3. Dossier: Soybean A2704‐12 renewal -- Section 2. Dossier Soybean A2704‐12 renewal -- Section 3a; additional information: 27/4/2018. Dossier: Soybean A2704‐12 renewal -- Section 3b; additional information: 19/6/2018. Dossier: Soybean A2704‐12 renewal -- Section 3c; additional information: 27/4/2018. Dossier: Soybean A2704‐12 renewal -- Section 4. Dossier: Soybean A2704‐12 renewal -- Section 5. Available online: <http://registerofquestions.efsa.europa.eu/roqFrontend/questionLoader?question=EFSA-Q-2018-00992> [^1]: Studies for which the full report was requested by the GMO Panel.	{ "pile_set_name": "PubMed Central" }	[ "referencefard", "toxicolog", "wheat", "probabl", "two", "control", "refer", "work", "origin", "cours", "nomin", "sylvi", "ian", "veronesi", "dossier", "ewen", "collect", "thank", "agronomicphenotyp", "gene", "codon‐optimis", "identifi", "network", "risk", "hazard", "crawford", "rais", "select", "endeavour", "websiteefsnotereftypefn", "hour", "may", "micem‐‐‐aefsnotereftypefnth", "therebi", "sschafer", "recent", "a‐m‐‐‐a‐", "septemb", "acquir", "envirologixm‐‐‐aefsnotereftypefnpatpat", "separ", "use", "gener", "support", "dewhurst", "final", "februari", "pauk", "complet", "accord", "signific", "nagi", "establish", "cover", "full", "journal", "guidelin", "temperatur", "exposur", "suggest", "relev", "elisa", "process", "gelencs", "market", "also", "state", 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"glufosinate‐toler", "document", "intraperiton", "addit", "w", "subash", "activ", "befsbibreftyperef", "limit", "opinionefsnotereftypefn", "statement", "rat", "feed", "concern", "approv", "agronom", "framespatphosphinothricin", "juan", "qualiti", "eligibilityinclus", "offici", "disrupt", "paoletti", "environment", "took", "savoini", "previous", "stand", "multigen", "height", "lay", "correct", "amino", "misra", "sampl", "plant", "databas", "annex", "frame", "immunotoxicolog", "identif", "f", "case", "materi", "deriv", "organismsorfsopen", "inherit", "álvarez", "jan", "httpregisterofquestionsefsaeuropaeuroqfrontendquestiondocumentsloaderquestionefsaq", "allergen", "council", "specialist", "molecular", "studi", "line", "methodolog", "delet", "open", "ident", "three", "bioinformat", "simul", "claudia", "toxin", "usa", "fluidm‐‐‐aefsnotereftypefnpat", "product", "earli", "ko", "n‐acetyltransferasepmempost‐market", "spring", "moreno", "bibliograph", "papadopoul", "recipi", "valid", "mykiss", 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It is widely accepted that magnetic reconnection plays an important role in plasmas, particularly in solar eruptive events, such as flares[@b1][@b2][@b3][@b4][@b5], filament eruptions[@b6], coronal mass ejections[@b7][@b8] and jets[@b9]. In the models of magnetic reconnection, magnetic field lines with an oppositely directed component approach each other in a current sheet or at a magnetic null point, break up and reconnect to form new magnetic lines. Magnetic energy is thereby released into thermal and kinetic energy of the plasma, potentially leading to large-scale phenomena. Evidence of reconnection on the Sun has mostly been limited to single aspects and has been indirect, showing changes of magnetic connections[@b10], reconnection inflows[@b11][@b12][@b13] and outflows[@b14][@b15][@b16][@b17], hot cusp-shaped structures at their interface[@b11][@b13][@b14], supra-arcade downflows[@b15][@b18], loop shrinkage[@b19][@b20], sudden brightenings[@b21][@b22], current sheets[@b15][@b20][@b23], plasmoid ejections[@b24][@b25], loop-top hard X-ray sources[@b20][@b26], pulsating radio emissions[@b27], and coronal heating in the interface between emerging and ambient magnetic flux[@b28]. In recent years, direct and more comprehensive evidence of reconnection has been discovered in a couple of energetic events on the Sun. Two very clear cases of reconnection, including inflowing cool loops, outflowing hot loops and plasma heated to \>10 MK, were observed in solar flares through extreme ultraviolet (EUV) and X-ray data[@b12][@b13]. The reconnection inflow and outflow velocities could be inferred in these flares to lie in the ranges of 10--90 and 90--460 km s^−1^, respectively, constraining the reconnection rate to the range of 0.05--0.5. Inflows, outflows and the formation of new loops could also be studied in a well-observed case of reconnection between two sets of small-scale chromospheric loops, imaged at high resolution in H-alpha[@b22]. This study showed a transition from slow to fast reconnection. The three-dimensional (3D) topology of reconnecting loops and their heating has been inferred using the combined perspectives and multiple EUV channels of two spacecraft[@b29]. Here we present comprehensive observational evidence of reconnection between a set of chromospheric fibrils and the threads of an erupting filament, which gave rise to a small flare. We observe the in- and outflows and hot cusp-shaped structures at the ends of a small-scale reconnecting current sheet, as well as newly formed loops that demonstrate the change of magnetic connectivity. We estimate that the reconnection is fast. The intriguing rotational motion of the erupted filament is found to show the untwisting of a flux rope, enabled by the reconnection with the chromospheric fibrils, which extend to essentially current-free magnetic flux in the corona. Our detailed study is made possible using H-alpha images from the New Vacuum Solar Telescope (NVST)[@b30] and EUV images from the Solar Dynamics Observatory (SDO)[@b31] (see the Methods section for details). Hot coronal plasma is also imaged using the X-Ray Telescope (XRT) on board Hinode[@b32] and the Soft X-ray Imager (SXI) on board the Geostationary Operational Environmental Satellite (GOES)[@b33]. Photospheric vector magnetograms taken by the Helioseismic and Magnetic Imager (HMI)[@b34] on board SDO allow us to obtain the 3D field structure of the reconnection region, independently demonstrating the change of topology. The dynamics of this source region model are studied in a data-constrained magnetohydrodynamic (MHD) simulation, which reproduces the observed features of the eruption and confirms that the untwisting of the erupted flux is enabled by reconnection with ambient current-free flux. Results ======= Confined partial eruption of the filament ----------------------------------------- On 3 October 2014, a filament is observed in active region (AR) 12178 (as designated by the National Oceanic and Atmospheric Administration \[NOAA\]) near the disk centre. The filament is composed of a western and an eastern section, which join smoothly at a bend point near solar (x,y)=(−70,−185). Many thin threads extending along the filament spine make up its fine structure (see the red arrows in [Fig. 1a](#f1){ref-type="fig"}). The threads in the western section show indications of twist of approximately one turn, consistent with the widely but not universally adopted assumption that the magnetic structure of filaments is that of a weakly twisted magnetic flux rope[@b35][@b36][@b37]. [Figure 1b](#f1){ref-type="fig"} shows the position of the filament in the magnetogram. Positive photospheric polarity is given at the western footpoint and southern side of the filament, and negative polarity is given at the eastern footpoint and northern side, so the filament has sinistral chirality[@b38]. This corresponds to positive (right handed) magnetic helicity, because the axial current of the filament must also point eastward for a force-free equilibrium to exist in the given ambient flux distribution. [Figure 1c--h](#f1){ref-type="fig"} and the related [Supplementary Movie 1](#S1){ref-type="supplementary-material"} show the eruption of the filament in H-alpha images taken by the NVST. The eruption commences near the bend point at ∼07:26 UT, as a motion of the dark threads with a southwestward projected direction, which later turns more southward. Associated brightenings, indicating heating due to reconnection, appear soon but only after the onset of the motion. The eruption comprises the whole western filament section and part of the eastern section. Some of the threads in the western section follow the main eruption with a small delay. The threads in the eastern section\'s part adjacent to the bend point (−100≲x≲−70) connect smoothly to the threads in the western section and move jointly with them (see the blue-dotted lines in [Fig. 1c,d](#f1){ref-type="fig"}). Subsequently, other threads become visible in their place; these, as well as all other threads in the eastern filament section, remain stable (see the red arrows in [Fig. 1f--h](#f1){ref-type="fig"}). We conclude that part of the magnetic flux in the filament experiences an instability, which causes the motion and subsequently triggers reconnection. The unstable flux comprises the whole western section and part of the eastern section, where unstable flux lies on top of the stable flux. The unstable flux extends into the eastern section at least up to approximately x=−100 (the apparent end region of the erupting threads), but possibly up to approximately x=−120 (the extension of the triggered brightenings); the stable flux extends from the bend point to the edge of the active region. Because the indicated twist of approximately one turn lies below the threshold of the helical kink instability[@b39][@b40] and the erupted filament does not build up a clear helical shape as a whole (see the blue-dotted line in [Fig. 1c--h](#f1){ref-type="fig"}), the torus instability[@b41] is the primary candidate mechanism for the onset of eruption. All southward motions end by 07:50 UT, and all material is subsequently seen to slide down towards the western end point of the filament in this confined eruption, which does not produce a coronal mass ejection. Obviously, the southward end region of the erupted filament threads is the highest part of the visible erupted structure. The data do not definitely reveal whether the erupted flux is still magnetically rooted in the eastern filament section or if it now connects to other negative flux in the photosphere. However, the erupted threads point approximately towards the neighbouring AR 12179 in the southeast, away from the direction of the eastern filament section, but similar to pre-existing interconnecting loops between the two ARs. This is suggestive of reconnection between the erupting filament and these loops. After ∼09:30 UT, a new filament forms along the original western filament section, initially separate from the surviving filament in the eastern section, but 2 h later beginning to join it. The eruption gives rise to two signatures of magnetic reconnection. The two prominent brightenings that develop at the sides of the eastern filament section ([Fig. 1c--h](#f1){ref-type="fig"}) represent an indirect signature, which agrees perfectly with the reconnection assumed to release the energy in the so-called standard flare model[@b1][@b2][@b3][@b4][@b5]. Ambient flux passing over the filament is lifted; subsequently, its legs approach each other and reconnect in a vertical current sheet that is known to form under the erupting flux, but not imaged in the present event. The released energy is channelled downward along the field lines, producing the chromospheric brightenings where the reconnecting ambient flux is rooted. This can likewise be seen under the western filament section ([Fig. 1d,e](#f1){ref-type="fig"}), but it is much weaker there, in agreement with the much smaller amount of ambient flux in this area ([Fig. 1b](#f1){ref-type="fig"}). After ∼07:38 UT, a second, direct signature of reconnection is revealed. Chromospheric fibrils south of the bend point reconnect with the trailing threads of the erupting western filament section on the north side of the bend point. The observations of the reconnecting current sheet are analysed in detail in the following section, where we find that it also forms under the erupting flux. However, here it is the erupting flux that reconnects with ambient flux, a process not envisioned in the standard flare model. In addition, the erupted filament displays an intriguing motion that is highly suggestive of a rotation about its main direction ([Supplementary Movie 1](#S1){ref-type="supplementary-material"}). The motion is also seen in the Atmospheric Imaging Assembly (AIA)[@b42] data ([Supplementary Movie 2](#S1){ref-type="supplementary-material"}). At least one full turn is indicated. The motion of the better visible threads on the upper side is also shown in a time-slice plot (white-dotted lines in [Fig. 1i](#f1){ref-type="fig"}). When looking along the filament toward the western footpoint, the rotation is clockwise. Erupted flux is generally assumed to have the structure of a flux rope[@b5][@b6][@b7][@b8]. The positive helicity inferred above implies that the twist of the rope is right handed. The clockwise rotation thus represents an untwisting that is equivalent to the relaxation of magnetic tension and supports the conjecture of a flux rope structure for the erupted filament. Erupting flux ropes show an apparent untwisting simply as the result of their expansion. While the total number of field line turns is preserved (in the absence of reconnection), the twist per unit length decreases. The stretching can appear as a propagation of twist if only a part of the rope displays a twist pattern. Although the threads of the erupting filament do not display a pronounced twist pattern in images ([Fig. 1c--h](#f1){ref-type="fig"}), the animation in [Supplementary Movie 1](#S1){ref-type="supplementary-material"} is consistent with this effect in the southward expanding end region of the threads. The stretching can also mimic a rotation in a time-slice plot, as in [Figure 1i](#f1){ref-type="fig"}. However, it cannot explain two effects visible in the present event: many threads shift nearly completely to the other side of the rope (cyan lines in [Fig. 1g,h](#f1){ref-type="fig"}) and their southern end points appear to rotate about the rope axis. True untwisting results from the conversion of twist into writhe of the flux rope axis and from reconnection with less twisted flux. The first effect is negligible here because a clear helical shape does not develop ([Fig. 1c--h](#f1){ref-type="fig"}). Following reconnection with less twisted flux, the twist tends to equilibrate along the new structure. Again, the total number of turns present after the reconnection is preserved, but the equilibration occurs as a result of a true propagation of twist from the more twisted to the less twisted part (as a torsional Alfvén wave packet) and involves a true rotation about the axis of the new structure. Hence, the observed rotational motion indicates that the erupted filament flux reconnects with ambient flux, which usually has no twist. The H-alpha observations reveal only a small part of that flux (the chromospheric fibrils south of the bend point of the filament channel), but a consistent whole picture is provided below by our models for the coronal field of the AR and by the evolution of one of these models in an MHD simulation. Imaging small-scale magnetic reconnection ----------------------------------------- The reconnection process and formation of the associated current sheet are shown in [Fig. 2](#f2){ref-type="fig"}. Their full evolution can be better seen in [Supplementary Movie 1](#S1){ref-type="supplementary-material"}. [Figure 2a](#f2){ref-type="fig"} shows the positions of the original structures in an H-alpha image overlaid by a line-of-sight magnetogram. The reconnection occurs between two sets of magnetic loops with opposite directions (see the arrows in [Fig. 2a](#f2){ref-type="fig"}). One set of magnetic loops, that is, the filament threads whose eruption is delayed, is indicated by the red arrow before the reconnection. The other set, that is, the chromospheric fibrils, is indicated by the white arrow. The reconnection occurs in a small region (marked by the black rectangle in [Fig. 2b](#f2){ref-type="fig"}). In [Fig. 2c](#f2){ref-type="fig"}, two typical loops (red- and white-dotted lines with red and white arrows, respectively) are indicated just before their reconnection. They further approach each other, and their interaction results in a reconnection at ∼ 07:38 UT. During the reconnection process, two cusp-shaped structures are formed (blue-dotted lines in [Fig. 2d](#f2){ref-type="fig"}). Simultaneously, new loops appear on the other side of the cusp structure northeast of the reconnection region (the black arrows in [Fig. 2d,e](#f2){ref-type="fig"}). Subsequently, these loops move away from the cusp. With the two sets of outer loops continuing to move towards each other, the reconnection continues from ∼07:38 to ∼07:50 UT. After the reconnection ceases, the newly formed loops can be seen more obviously (marked by black arrows in [Fig. 2d--h](#f2){ref-type="fig"}). They have accumulated in the northeast reconnection outflow region. The new loops, which form early in the southwest of the reconnection region (at the right cusp), follow the filament eruption ([Supplementary Movie 1](#S1){ref-type="supplementary-material"}), while those that form there later do not rise further and can be clearly seen near the end of the event (indicated by the yellow arrow in [Fig. 2h](#f2){ref-type="fig"}). During the eruptive process the reconnection region is stretched, apparently to form a current sheet, which is visible in H-alpha as a bright linear structure extending between the tips of the two cusps ([Fig. 2f](#f2){ref-type="fig"}). The average length and width of the current sheet are estimated to be 4.3 × 10^3^ and 1.06 × 10^3^ km, respectively. At the same time, we find that the plasma at the tip of the northeast cusp structure is significantly heated, causing the brightening ([Fig. 2g](#f2){ref-type="fig"}). The cusp and current sheet can be seen in multiple channels ([Fig. 3a--d](#f3){ref-type="fig"}; [Supplementary Movie 2](#S1){ref-type="supplementary-material"}). The northeastern bright cusp appears very clearly in all EUV channels of AIA (T≈0.02−10 MK). The southwestern cusp structure is weaker, as is to be expected from lower plasma densities at the upper end of a current sheet that is formed and stretched out upward by the eruption. This also shows up only intermittently owing to absorption by the moving threads of the erupted filament, which demonstrates that the current sheet forms under the erupting flux. The current sheet appears as a bright linear structure in all AIA channels, most clearly at the highest temperatures (131 Å; T=11 MK). The hot cusp structure can also be clearly seen in the XRT and SXI images during the reconnection process ([Fig. 3e,f](#f3){ref-type="fig"}). The unusual simultaneous visibility of the current sheet and cusp in H-alpha and EUV emissions ([Figs 2f](#f2){ref-type="fig"} and [3a--d](#f3){ref-type="fig"}) is probably due to the fact that cool and dense threads and fibrils are embedded in the reconnection inflow. The major brightening of the current sheet and cusp in H-alpha immediately follows the inflow and fading of a major filament thread (prominent in the current sheet in [Fig. 2c--e](#f2){ref-type="fig"}), suggesting heating of the thread by the reconnection. The emission measure (EM) maps at the different temperature bins ([Fig. 3g--i](#f3){ref-type="fig"}) show that the northeastern, newly formed loops are dominated by EM at temperatures of 4--8 MK, several times higher than the unperturbed corona. On the other hand, the tips of the two cusp structures (circles A and B) and the current sheet between them are most prominent at even higher temperatures, that is, T\>10 MK ([Supplementary Movie 3](#S1){ref-type="supplementary-material"}). This is similar to earlier observations of a pair of hard X-ray sources located at the two ends of the conjectured current sheet in flares[@b20]. The EUV images at high temperatures ([Fig. 3d](#f3){ref-type="fig"}) and the EM maps ([Fig. 3i](#f3){ref-type="fig"}) also show a diffuse area of enhanced temperature in the northwest inflow region after ∼07:35 UT, which begins to cool down ∼10 min later and then transiently brightens in H-alpha ([Fig. 1h](#f1){ref-type="fig"}). The magnetic field inhibits heat conduction from the current sheet into the inflow region. Guided by the MHD simulation, which shows the formation of additional weak current layers north of the erupting filament, we interpret these enhancements as a signature of additional energy release in the complex ambient coronal field when it is perturbed by the eruption, followed by downward heat conduction. To quantitatively investigate the motions of the plasma in the reconnection region, we construct the three time-slice plots in [Fig. 4a--c](#f4){ref-type="fig"}, which yield projected velocities along the lines marked in [Fig. 2](#f2){ref-type="fig"}. [Figure 4a](#f4){ref-type="fig"} shows the inward motions of loops, that is, the reconnection inflow. The apparent velocities of these loops are in the range of 3.7--25.0 km s^−1^ for the various filament threads on the northwest side and 6.3--16.8 km s^−1^ for the various fibrils on the southeast side. [Figure 4b](#f4){ref-type="fig"} displays outward motions of bright (hot) plasma, that is, the reconnection outflow, seen immediately downstream of the northeastern cusp. The outflow velocities lie in the range of 41.7--43.7 km s^−1^, with an average value of 42.7 km s^−1^. At the same time, two newly formed loops containing cooled plasma, dark in H-alpha (the white-dotted lines), can be seen in [Fig. 4b](#f4){ref-type="fig"}. These shrink slowly, with an average velocity of 2.6 km s^−1^. In addition, a mixture of bright and dark plasma moves down the legs of the cusp structure, that is, in the interface between the reconnection inflow and outflow ([Fig. 4c](#f4){ref-type="fig"}). This apparent mix of inflowing and outflowing plasma, as well as the intermittently irregular structure of the cusp legs ([Supplementary Movie 1](#S1){ref-type="supplementary-material"}), whose observation is made possible by the high resolution of the NVST, provide support for the recent numerical finding of turbulence in reconnection developing at the separatrices[@b43]. The downflows at the cusps show velocities in the range of 17.6--80.2 km s^−1^ with a decreasing trend. The brightenings at the end of the current sheet were observed in multiple channels. To investigate their evolution, [Fig. 4d](#f4){ref-type="fig"} displays the intensities in the H-alpha, 304, 171 and 335 Å channels, integrated over the area of the circle in [Fig. 2g](#f2){ref-type="fig"}. The early brightenings are caused by the eruption of the filament. The onset of reconnection, as marked by the rise of the EUV emissions at 07:38 UT, coincides with the arrival of the first inflow trace at the forming current sheet ([Fig. 4a](#f4){ref-type="fig"}) and with the onset of outflows from the current sheet ([Fig. 4b,c](#f4){ref-type="fig"}). The bright structures and the outflows show some degree of intermittency, which is a characteristic of plasmoid-mediated reconnection in very long current sheets, but here is probably caused mainly by the inhomogeneity of the inflowing plasma. All tracers of the reconnection process decay after ∼07:45 UT, by which time the length of the current sheet has shortened considerably because of the accumulation of new loops in the downward (northeast) outflow region and the approximate stationarity of the upper tip of the sheet under the erupted but no longer rising filament. 3D NLFFF configuration of the reconnection region ------------------------------------------------- To investigate the 3D magnetic field structure of the reconnection region, we carry out extrapolations of the HMI vector magnetograms at 07:00 and 08:24 UT based on the nonlinear force-free field (NLFFF) assumption for the coronal field ([Fig. 5](#f5){ref-type="fig"}). In the eastern section of the filament, an incoherent flux rope structure is obtained, similar to several recent extrapolation results for active regions before an eruption[@b44][@b45]. In the western section, the strong axial flux expected in a filament is found, but the poloidal (twist) field component is largely missing. We expect that the latter results from the low signal-to-noise ratio in the extended area of the weak field around the western filament section ([Fig. 1b](#f1){ref-type="fig"}). The overall shape of the filament is nevertheless well matched. The extrapolation also correctly indicates that the field in the area of the western filament section has a similar structure sometime after the eruption, providing the prerequisite for the observed formation of a new filament in the western section. These findings yield confidence in the large-scale structure of the extrapolated NLFFF. The extrapolations demonstrate the change of magnetic connections in agreement with the observed change. They further show that the ambient field rooted in the southeast reconnection inflow region (grey field lines in [Fig. 5a](#f5){ref-type="fig"}) first follows the low-lying reconnecting fibrils seen in H-alpha in the westward direction, but then bends strongly upward, forming high-reaching connections to the neighbouring AR 12179 (see [Fig. 6](#f6){ref-type="fig"}). These field lines exchange footpoints, that is, reconnect, with a set of lower field lines running in the northwest inflow region and the western section of the filament channel (also coloured grey). The resulting low-lying loop ([Fig. 5b](#f5){ref-type="fig"}) corresponds very well to the newly formed H-alpha loops downstream of the observed cusp ([Fig. 2d--g](#f2){ref-type="fig"}). The other resulting set of field lines indicates that flux in the erupting western part of the filament reconnects with the high-reaching field lines that extend to the neighbouring active region. This corresponds very well to the southward bending of the erupting flux (blue-dotted lines in [Fig. 1c--h](#f1){ref-type="fig"}) and supports our conjecture above that the strong untwisting motion is realized by a torsional Alfvén wave packet propagating to ambient, essentially untwisted flux. MHD simulation -------------- To further substantiate the occurrence of reconnection triggered by the erupting filament, we model the event in an MHD simulation whose initial and boundary conditions are constrained by the HMI data. Because the extrapolation largely fails to reproduce the twist in the western part of the filament, we construct a new NLFFF model of the AR from the pre-eruption magnetogram using the Flux Rope Insertion method, as detailed in the Methods section. On the basis of the observation that the western filament section erupts fully and the eastern section only partly, two flux ropes are inserted along the path of the filament. A low-lying rope is inserted along the eastern section. A higher-lying rope models the western section and extends into the eastern section as far as the moving threads at the onset of the event ([Fig. 6a](#f6){ref-type="fig"}). Numerical relaxation partly merges them into a smooth configuration, which is essentially a flux rope split into two branches in the eastern part. From models for a range of values of the inserted fluxes, we select the one that yields an unstable western section, with approximately one field line turn, and a stable eastern section to serve as the initial condition of the MHD simulation ([Figs 6b](#f6){ref-type="fig"} and [7a--d](#f7){ref-type="fig"}). The model also includes the high-reaching field lines anchored in positive flux southeast of the filament (grey in [Fig. 5a](#f5){ref-type="fig"} and left set of open field lines in [Fig. 7c](#f7){ref-type="fig"}), the bottom part of which represents the reconnecting chromospheric fibrils (white arrows in [Fig. 2a,c,e](#f2){ref-type="fig"}). [Figure 6b](#f6){ref-type="fig"} shows that these field lines connect to the neighbouring AR 12179. Similarly, the low-lying field lines north of the filament bend point (grey in [Fig. 5a](#f5){ref-type="fig"}) are here also seen to be part of the flux in the western filament section (right set of weakly twisted field lines in [Fig. 7c](#f7){ref-type="fig"}). This flux models the trailing threads of the erupting western filament section (red arrows in [Fig. 2a,c,e](#f2){ref-type="fig"}), which reconnect with the chromospheric fibrils. A wide range of agreement with the observations is obtained. The simulation shows a confined eruption in the southern direction, comprising the full western and part of the eastern filament section ([Fig. 7](#f7){ref-type="fig"}; [Supplementary Movie 4](#S1){ref-type="supplementary-material"}). Magnetic reconnection is triggered very similarly to that shown by the NVST and SDO data. A current sheet is dragged upward into the corona between the rising and remaining flux along the filament section eastward of the original bend point ([Fig. 7](#f7){ref-type="fig"}, fourth column). The ambient flux on the southeast side of the filament moves towards the sheet to reconnect at the observed position with approaching flux from the western section of the flux rope ([Fig. 7](#f7){ref-type="fig"}, third column). As shown above, while being in agreement with the NVST observations, this differs partly from the standard view of reconnection in solar eruptions, which supposes that only ambient flux reconnects under a rising flux rope[@b5][@b6][@b7][@b8][@b46]. The reconnection forms small loops that accumulate under the current sheet at the position of the newly formed H-alpha loops ([Figs 2d--g](#f2){ref-type="fig"}, [5b](#f5){ref-type="fig"} and [7o](#f7){ref-type="fig"}). The other parts of the reconnected field lines yield new, high-reaching magnetic connections from the western footpoint of the erupted flux to AR 12179 ([Fig. 7](#f7){ref-type="fig"}, left two columns; compare with the evolution of the blue-dotted line in [Fig. 1c--h](#f1){ref-type="fig"}). The untwisting of the erupted flux via propagation of twist along these new connections is very clear. These results are similar to the indications from the dynamics of the erupted filament in the H-alpha data (the draining of filament material in the westward direction along the whole length of the erupted filament and the indication of a rotational motion). They are also in agreement with the new connections in the extrapolated field ([Fig. 5b](#f5){ref-type="fig"}). The accumulation of reconnected flux causes a gradual rise of the cusp that forms at the bottom tip of the current sheet ([Fig. 7h,l,p](#f7){ref-type="fig"}). As the rise of the erupted flux is stalled, but the bottom cusp continues to rise as a result of the reconnection, the current sheet weakens and eventually decays. The simulation shows further reconnection in a current sheet forming in the western filament section in the interface between rising and overlying flux. This rebuilds connections from the positive footpoint area of the flux rope to the strong negative photospheric flux patches adjacent to the eastern filament section ([Fig. 7i,m](#f7){ref-type="fig"}). The current sheet is not imaged by NVST and SDO because it is not aligned with the line of sight; however, the resulting connections match the filament that reforms in the western section after 09:30 UT. Discussion ========== In this paper, we study in detail a small-scale magnetic reconnection event triggered by a filament eruption and obtain a comprehensive set of solid observational evidence for the occurrence of reconnection which is unprecedented. This includes the reconnection inflows at both sides of the current sheet at projected velocities of 3.7--25.0 km s^−1^; the reconnection outflow at one side of the current sheet, moving downward at projected velocities of 41.7--43.7 km s^−1^; the formation of a long, thin structure suggestive of a current sheet and of two cusp-shaped structures at its end points, both occurring simultaneously with the onset of reconnection; newly formed loops in both outflow regions, demonstrating the change of magnetic connectivity, those on the downward side shrinking with an average projected velocity of 2.6 km s^−1^; and fast downward motion at projected velocities of 17.6--80.2 km s^−1^ of cool and hot plasma at the cusp-shaped boundary between inflow and outflow. Moreover, the newly formed loops, current sheet and cusps show enhanced emission measure in a broad temperature range, with the loops being dominated by plasma at 4--8 MK and the current sheet and cusps by plasma at T\>10 MK. The length, L, and width, d, of the current sheet are estimated to be 4.3 × 10^3^ and 1.06 × 10^3^ km, respectively. The reconnection rate is an important physical parameter in the theory of reconnection and can be estimated from our observations. It can be expressed as the Alfvénic Mach number, M~A~, of the inflow velocity because the reconnection outflow generally approaches the Alfvén velocity, V~A~ (ref. [@b47]). Assuming that the observed outflow velocity is of the order of V~A~ and neglecting projection effects, M~A~≈V~in~/V~out~, which lies in the range of ∼0.08−0.60 for our measurements. In a steady-state reconnection, the reconnection rate also equals the inverse aspect ratio of the current sheet[@b47], which is obtained as d/L≈0.25, consistent with the estimated range for M~A~. Both indicate that the reconnection is fast. Our estimate tends to be relatively high, but is consistent with the range of previous estimates for solar events, for example, 0.001--0.03 (ref. [@b11]), 0.01--0.23 (ref. [@b48]), 0.055--0.20 (ref. [@b12]), 0.16 (ref. [@b17]) and 0.05--0.5 (ref. [@b13]). Because the measured reconnection outflow in our event hits the accumulating newly formed loops after only a short distance ([Fig. 2](#f2){ref-type="fig"}), it may not have reached the Alfvén velocity. Furthermore, the current sheet width obtained from the images, although resolved by the NVST, is considered an upper limit for the actual width of the field reversal, which tends to occur at microscopic scales[@b47]. Therefore, the estimated reconnection rate is also considered an upper limit. Finally, the confinement of the eruption may be due to a strong restraining force of the overlying flux or due to the cancelation of the upward Lorentz force in the filament by its reconnection with ambient flux. The former effect is equivalent to saturation of the torus instability in an ambient field that decreases too slowly with height, which can be quantified by the decay index, n(z)=−d log* B~p,\ hor~(z)/d log z, where B~p~,~ hor~(z) is the horizontal component of the potential field at the x--y* position of interest[@b41]. The decay index profile at the observed position of the current sheet indicates instability (n\>3/2) in a small interval around z=0.03R~⊙~, where the upper flux rope is located in our NLFFF model, and at z\>0.13R~⊙~, with the intermediate height range being stable. In this stable range, the flux rope reconnects in our MHD simulation. Thus, both potential reasons for confinement may be relevant in the event. Methods ======= Differential emission measure calculation ----------------------------------------- We calculate the differential emission measure (DEM) using the almost simultaneous observations of six AIA EUV lines (131, 94, 335, 211, 193 and 171 Å) formed at coronal temperatures. The DEM is determined by where I~i~ is the observed intensity of the waveband i, R~i~(T) represents the temperature response function of waveband i, and DEM(T) is the DEM of coronal plasma, which is computed using the routine xrt_dem_iterative2.pro in the Solar Software package. This code was first written for the Hinode/X-ray telescope data[@b49][@b50], and then modified for the SDO/AIA data[@b51]. In this work, log T is set in the range of 5.5--7.5, where the DEM is generally well constrained[@b52][@b53]. To obtain the emission measure in the temperature range (T~min~,T~max~), we evaluate NLFFF extrapolation ------------------- The 3D NLFFF structures of the reconnection region and associated filament ([Fig. 5](#f5){ref-type="fig"}) are reconstructed using an optimization algorithm[@b54][@b55]. A preprocessing procedure is first used to deal with the bottom vector data to remove most of the net force and torque that usually results in an inconsistency between the photospheric magnetic field and the force-free assumption in the NLFFF models[@b56]. The visualization of 3D magnetic field is realized by the software Paraview. The field lines shown are traced from nearly identical points in the photosphere. Flux rope insertion method and MHD simulation --------------------------------------------- The magnetic field model used as the initial condition of the MHD simulation is constructed through Flux Rope Insertion. This method involves computing the potential field in the volume of interest by extrapolation, inserting a magnetic flux rope along the observed path of the filament and numerically relaxing the configuration to an NLFFF[@b36][@b57][@b58]. The active region of interest ([Fig. 6a](#f6){ref-type="fig"}) is modelled with high spatial resolution (0.002R~⊙~), and the more distant regions are modelled with a lower-resolution (1°) global potential field. The high-resolution region is derived from the line-of-sight photospheric magnetogram taken by SDO/HMI on 3 October 2014 at 07:26 UT, and the global field is constructed based on the HMI synoptic map. The high-resolution computational domain extends about 25° in longitude, 15° in latitude and up to 1.75 R~⊙~ from Sun centre. Two flux ropes are inserted along the path of the filament (blue lines) and anchored in the photosphere (at the blue circles), with path 1 chosen to run higher than path 2. The axial and poloidal fluxes along paths 1 and 2 are 4 × 10^20^ Mx and 10^11^ Mx cm^−1^, and 10^20^ Mx and 10^10^ Mx cm^−1^, respectively, giving the western section higher free magnetic energy and twist. The numerical relaxation partly merges the flux ropes into a smooth configuration and changes the end positions slightly ([Fig. 6b](#f6){ref-type="fig"}). All flux connecting to photospheric sources outside AR 12178 corresponds well to the large-scale coronal loops imaged in the AIA 171-Å channel (which are not shown here, but can be accessed at the public source <http://helioviewer.org/>). The MHD simulation code[@b59] is used with numerical settings analogous to an earlier data-constrained simulation of a solar eruption[@b60]. In particular, the velocity in the bottom plane is set to zero, to model the inertia of the photosphere. The vertical component of the HMI magnetogram thus remains invariant. Software availability --------------------- The Solar Software package is available at <http://www.lmsal.com/solarsoft/>. Data availability ----------------- The NVST is a ground-based telescope with a 986-mm clear aperture in the Fuxian Solar Observatory of the Yunnan Observatories, Chinese Academy of Sciences. It is designed to observe the fine structures on the Sun and their activities in multiple layers (photosphere and chromosphere) with high spatial resolution (0.165 arcsec per pixel) and high temporal resolution (∼12 s). We use data from the H-alpha line-centre channel at 6,562.8 Å, corresponding to chromospheric temperatures. These data can be accessed at <http://fso.ynao.ac.cn/dataarchive_ql.aspx>. EUV and far-ultraviolet images obtained by the AIA on board SDO, including the 304, 171, 193, 335, 211, 94, 131 and 1,600 Å channels, display magnetic reconnection and the associated current sheet at higher temperature, that is, primarily in the corona. Photospheric magnetograms are provided by the HMI Instrument on SDO. The SDO data are publicly available at <http://jsoc.stanford.edu/ajax/lookdata.html> and at [http://helioviewer.org/](http://helioviewer.org/.%20Soft%20X-ray%20images%20of%20the%20Sun%20by%20Hinode/XRT%20and%20GOES/SXI). Soft X-ray images of the Sun by Hinode/XRT and GOES/SXI are available at <http://darts.isas.jaxa.jp/solar/hinode/query.php?A01=Go%20to%20Search/> and <http://sxi.ngdc.noaa.gov/sxi/servlet/sxisearch/>, respectively. Additional information ====================== How to cite this article: Xue, Z. et al. Observing the release of twist by magnetic reconnection in a solar filament eruption. Nat. Commun. 7:11837 doi: 10.1038/ncomms11837 (2016). Supplementary Material {#S1} ====================== ###### Supplementary Movie 1 The NVST H-alpha movie. (a) The general appearances of the filament and its eruption in which the red rectangle indicates the field of view (FOV) of panel b. (b) The process of magnetic reconnection. ###### Supplementary Movie 2 The EUV channel movie observed by SDO/AIA. (a-h) 304, 1600, 171, 193, 211, 335, 94, and 131 Å channel movies respectively. ###### Supplementary Movie 3 The EM map movie. In each panel, the EM maps at the different temperatures are shown. ###### Supplementary Movie 4 MHD simulation of the filament eruption. Field lines of the two flux ropes representing the unstable (rainbow-colored) and stable (green) filament sections are shown in vertical and perspective views (snapshots of which are shown in the left two columns in Fig. 6. The HMI line-of-sight magnetogram is included in the bottom plane. We thank the NVST, SDO, GOES/SXI, Hinode and BBSO teams for providing the data. SDO is a mission of NASA\'s Living With a Star Program. Hinode is a Japanese mission developed and launched by ISAS/JAXA, with NAOJ as a domestic partner, and NASA and STFC (UK) as international partners. It is operated by these agencies in co-operation with ESA and NSC (Norway). This work is sponsored by the National Science Foundation of China under the grant numbers 11503080, 11373066, 11573012, 11373065, 11303016, 11533008 and 11473071, Key Laboratory of Solar Activity of Chinese Academy of Sciences (CAS) under numbers KLSA201412, KLSA201407, Yunnan Science Foundation of China under number 2013FB086, CAS 'Light of West China\' Program, Youth Innovation Promotion Association CAS (no. 2011056), and the national basic research program of China (973 program, 2011CB811400). B.K. is supported by the CAS under grant no. 2012T1J0017 and by the DFG. Y.N.S is supported by the One Hundred Talent Program of Chinese Academy of Sciences. Author contributions Z.K.X. developed the ideas, performed analysis of the data, discussed the results and wrote the first manuscript. X.L.Y. developed the ideas and lead the discussion of the manuscript. X.C. and K.Y. carried out the NLFFF extrapolation, DEM analysis and discussion. L.H.Y. carried out the analysis of the XRT and GOES data, and contributed to the discussion. Y.N.S. performed NLFFF modelling of the AR to provide the initial condition for the MHD simulations carried out by B.K.; Y.N.S. and B.K. also contributed to the discussion. B.K. and X.L.Y made major revisions of the manuscript. J.Z. developed ideas and contributed to the discussion. Z.L., Y.B., Y.Y.X and L.Z. contributed to the original observational data. All authors reviewed the manuscript. ![Structure and eruption of the filament.\ (a) NVST H-alpha image showing the general appearance of the filament (indicated by the red arrows) at the onset of the eruption. (b) HMI line-of-sight magnetogram of AR 12178, with positive (negative) photospheric flux shown in white (black) and the red line representing the position of the filament. (c--h) The process of filament eruption in H-alpha images. The original position is marked by the red-dotted line. The erupting part is indicated by the blue-dotted lines and blue arrows. Cyan-dotted lines in f--h indicate some of the filament threads that rise with a delay. The stable eastern filament section is marked by the red arrows in f--h. (i) Time-slice plot acquired at the position A--B, with the white-dotted lines marking the filament threads. The motion of these threads indicates the untwisting of the filament during its eruption.](ncomms11837-f1){#f1} ![Evolution of reconnection.\ (a,h) NVST H-alpha images in the early and late phases of the reconnection event, overlaid by red (blue) contours representing positive (negative) photospheric polarity from near-simultaneous HMI data. (b--g) NVST H-alpha images of the reconnection process in which the red- and white-dotted lines indicate the magnetic loops before the reconnection (with red arrows marking the filament threads and white arrows marking the chromospheric fibrils), the blue-dotted lines mark the cusp-shaped structures, and the black (yellow), cyan and pink arrows point to newly formed loops, current sheet and brightening, respectively. The three time-slice plots shown in [Fig. 4](#f4){ref-type="fig"} are made along the white solid line C--D, the blue solid line E--F and the black solid line G--H, respectively.](ncomms11837-f2){#f2} ![EUV/X-ray images and EM maps.\ (a--d) AIA EUV images at 304, 171, 335 and 131 Å channels, in which the cusp-shaped structures are marked by the dotted lines and the current sheet is indicated by the white arrows. (e,f) Cusp structure seen in X-ray images observed by Hinode/XRT and GOES/SXI, respectively. (g--i) EM maps in three temperature ranges showing the dominant temperatures of the inflow region, current sheet, cusps (encircled and marked 'A\' and 'B\' in i), and reconnected loops.](ncomms11837-f3){#f3} ![Time-slice plots and EUV brightening.\ (a--c) H-alpha time-slice plots made by stacking the slits along the slices C--D, E--F and G--H in [Fig. 2](#f2){ref-type="fig"}, respectively. The red-, blue-, white- and pink-dotted lines represent reconnection inflows, outflows, shrinking loops, and downward motions, respectively. (d) Temporal evolution of the brightenings in the region marked by the black circle in [Fig. 2g](#f2){ref-type="fig"}, using H-alpha, 304, 171 and 335 Å images, where each light curve is normalized by its maximum. The vertical black-dotted line in each panel indicates the onset of the observed fast reconnection.](ncomms11837-f4){#f4} ![3D configuration of the reconnection region from NLFFF extrapolation.\ (a,b) 3D NLFFF configuration before (07:00 UT on 3 October 2014) and after (08:24 UT on 3 October 2014) the reconnection. The background images indicate the radial component of the vector magnetic field in the photosphere. The grey lines represent the magnetic loops involved in the reconnection. The pink lines show the magnetic structures of the filament.](ncomms11837-f5){#f5} ![Magnetic field model constructed using the Flux Rope Insertion method.\ (a) Line-of-sight magnetogram observed by SDO/HMI at 07:26 UT on 3 October 2014. The two blue curves with a circle at each end show the paths of the inserted flux ropes. (b) H-alpha image taken by Big Bear Solar Observatory at 23:00 UT on 2 October 2014, overlaid with red (positive) and green (negative) contours representing the magnetic field shown in a. The colour lines show selected field lines from the magnetic field model, and the horizontal size of the MHD computation box (height of 0.75R~⊙~ and fully within the high-resolution region) is indicated by the black square.](ncomms11837-f6){#f6} ![MHD simulation of the confined filament eruption and magnetic reconnection.\ The left two columns show field lines of the fully erupting western flux rope section (rainbow colours) and the largely stable eastern flux rope section (green) and the line-of-sight magnetogram in a cube of 0.25R~⊙~ per side. The inset at t=0 indicates the area shown in the third column, and the yellow line shows the position of the vertical cut plane in the fourth column. The third column shows selected field lines in the area of the observed reconnection in a cube of 0.06R~⊙~ per side. Field lines rooted in positive photospheric flux (white) initially mostly extend to AR 12179 eastward of AR 12178 ([Fig. 6b](#f6){ref-type="fig"}), while field lines rooted in negative flux (black) initially mostly join the unstable western flux rope section. The fourth column displays the current density and in-plane velocity vectors in a vertical cut through the reconnecting current sheet (0.06R~⊙~ per side). At t=0, the inserted, unstable flux rope is seen, with velocities set to zero. 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INTRODUCTION ============ LPA (lysophosphatidic acid) is a potentially bioactive phospholipid that mediates a number of physiological processes, including cell adhesion, proliferation, differentiation, survival and migration. GPCRs (G-protein-coupled receptors) in the EDG (endothelial differentiation gene) and P2Y families are specific receptors for LPA in the plasma membrane, and intracellular signalling via these GPCRs has been well characterized \[[@B1]\]. However, less is known about the physiological regulation of LPA production or how LPA-specific receptors in the plasma membrane are stimulated in vivo. At least two plausible enzymatic pathways for the production of LPA have been described. One of these is the deacylation of PA (phosphatidic acid) by activated PLA (phospholipase A) \[[@B2]\]. In support of this idea, a PA-selective PLA~1~ (phospholipase A~1~) called mPA-PLA~1~α/LIPH (membrane-associated phosphatidic acid-selective phospholipase A1α/lipase member H) has been identified that hydrolyses acyl residues at the sn−1 position of PA and enhances release of LPA from the plasma membrane \[[@B3],[@B4]\]. The other pathway for LPA production is direct production of equimolar amounts of LPA and choline from LPC (lysophosphatidylcholine), as catalysed by lysoPLD (lysophospholipase D) \[[@B5],[@B6]\]. The physiological importance of LPA production in serum by lysoPLD was suggested by Tokumura et al. \[[@B7]\] approximately 25 years ago. In circulating blood, LPA modulates blood pressure \[[@B8],[@B9]\], maintenance of pregnancy \[[@B10]\] and maturation of embryonic vessel systems \[[@B11]\]. Previously, a secreted protein with lysoPLD activity was purified from plasma and serum, and was found to be identical with a molecule known as autotaxin \[[@B12],[@B13]\]. Autotaxin \[ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2)\] was originally cloned as a tumour cell motility-stimulating factor and was recognized as a nucleotide pyrophosphatase on the basis of its primary structure and in vitro enzymatic characteristics \[[@B14]\]. LPC, which can bind albumin and lipoproteins, is released into circulation from the liver and may be generated by lecithin/cholesterol acyltransferase. Autotaxin might hydrolyse these LPCs as substrates, and LPA accumulated to levels greater than 50 μM in plasma, presumably due to very low activity of LPA-degrading enzymes in that context \[[@B7]\]. When cells are stimulated by hormones and proliferation factors, equimolar amounts of free fatty acid and LPC are produced from PC (phosphatidylcholine) by the activated PLA~2~ (phospholipase A~2~) \[[@B15]\]. The free fatty acids that are released, including arachidonic acid, which is present at position sn−2 of PC prior to cleavage, are converted into eicosanoids. Whereas the metabolism of arachidonic acid has been well characterized, the metabolism of LPC is less well understood. Because the accumulation of LPC in cells induces cell lysis \[[@B16]\], it has been thought that LPC was easily hydrolysed by lysophospholipases we cloned in a previous study \[[@B17]\], followed by conversion into a non-bioactive saturated free fatty acid and GPC (glycerophosphorylcholine). However, LPC itself was recognized recently as a bioactive phospholipid that can induce chemotaxis \[[@B18]\], signal-responsive kinase activities \[[@B19]\] and atherosclerosis \[[@B20]\]. The first enzyme with lysoPLD activity was isolated from rat brain microsomes by Wykle and Schremmer \[[@B5]\]. The characteristics of this intracellular enzyme, such as substrate specificity, cation requirement and optimal pH, are different from those of autotaxin. We purified a novel lysoPLD from the rat brain that is a Mg^2+^-dependent enzyme and utilizes lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) as a substrate \[[@B21]\], similar to the enzyme reported by Wykle and Schremmer \[[@B5]\]. This enzyme might be involved in the production of LPA from LPC, which is produced from PC hydrolysed by PLA~2~ in cells. Previously, it has been reported that LPA receptors are expressed in perinuclear areas \[[@B22],[@B23]\] and, furthermore, that the transcription factor PPARγ (peroxisome proliferation-activator receptor γ) is bound to LPA \[[@B24]\]. Based on these observations, it seems reasonable to propose that intracellular lysoPLD is important for cellular responses. In the present study, we have identified a purified protein with lysoPLD activity from the rat brain as the heterotrimeric G protein subunits Gα~q~ and Gβ~1~. Mutations predicted to affect the binding of nucleotide phosphates or Mg^2+^ to the Gα~q~ subunit resulted in a dramatic reduction of lysoPLD activity. Moreover, several types of Gα subunits, including Gα~q~ and Gα~11~, exhibit lysoPLD activity when expressed and purified as tagged proteins. EXPERIMENTAL ============ Materials --------- LPC (1-palmitoyl, 16:0; stearoyl, 18:0; and oleoyl, 18:1) was obtained from Sigma--Aldrich. LysoPAF was from Alexis Biochemicals. 1-\[^14^C\]Palmitoyl-2-lyso-phosphatidylcholine was purchased from GE Healthcare. HPPA \[3-(4-hydroxyphenyl) propionic acid\], peroxidase and choline oxidase were purchased from Wako Chemicals. Silica Gel 60 plates and tag-purified G(β~1~ and γ~2~) complex overexpressed in Spodoptera frugiperda insect cells were purchased from Merck, and CHAPS was purchased from Dojindo. Antibodies against Gα~q/11~, Gβ~1--4~, and Gγ~5~ were obtained from Santa Cruz Biotechnology, and anti-FLAG antibody, anti-FLAG affinity gels, FLAG peptides, anti-actin antibody and GTP were purchased from Sigma. The monoclonal anti-autotaxin antibody was obtained from Dr Junken Aoki (Graduate School of Pharmaceutical Sciences, Tohoku University, Japan). Identification of purified lysoPLD ---------------------------------- Purification of lysoPLD from rat brain tissue was performed exactly as described previously \[[@B21]\]. All steps in enzyme purification were carried out at 4°C. Briefly, rat brains were minced and homogenized with a Teflon homogenizer in 9 volumes of buffer A \[0.3 M sucrose in 50 mM Tris/HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol and 0.1 mM PMSF). Nuclear fractions were obtained by centrifugation at 600 *g* for 10 min and resuspended in buffer A. To dissolve lysoPLD in the nuclear pellets, 10 mM CHAPS was added and the suspension was sonicated with a Microson Ultrasonic cell disruptor (Misonix). The suspensions were then centrifuged at 10000 *g* for 10 min and the supernatants were used as a source of enzyme. The supernatants were applied to a DEAE Cellulofine A-500 column equilibrated with buffer B \[20 mM Tris/HCl (pH 7.5), 0.1 mM PMSF and 5 mM CHAPS), and the enzyme was eluted with a NaCl gradient. Active fractions were sequentially applied to three different types of columns. The enzyme fractions were concentrated using Microcon centrifugal filter devices (Millipore) and then applied to a Superdex 200 10/300 GL column. As a final step, the eluted active fractions were applied to a HiTrap DEAE FF column using a Waters 650 (Millipore). The final eluted fraction was then subjected to SDS/PAGE (10% gel) and the gel was stained with EZ Stain Silver (Atto). The major protein bands were excised for in-gel digestion with MS grade trypsin (Wako Chemicals). The mass spectra of extracted peptides were analysed by MALDI--TOF MS (matrix-assisted laser desorption ionization--time-of-flight MS) (Voyager Elite/STR Perspective, Life Technologies), and the proteins were subsequently identified using MS-Fit (<http://prospector.ucsf.edu>). SDS/PAGE and immunoblotting --------------------------- Proteins from rat brains and the FLAG tag affinity gel-purified fractions (10 μl) were separated on 10% acrylamide gels, and stained with a silver staining kit (Wako Chemicals) or transferred on to PVDF membranes using a semi-dry transfer apparatus (Nippon Eido) and blocked with 5% skimmed milk in TBS-T buffer \[150 mM NaCl, 20 mM Tris/HCl (pH 8.0) and 0.05% Tween 20\]. The membranes were incubated overnight at 4°C with primary antibody diluted 1:1000 (anti-Gα~q/11~, -Gβ~1--4~, -Gγ~5~, -actin or -autotaxin) or 1:5000 (anti-FLAG) in TBS-T and then for 1 h at room temperature (20°C) with a peroxide-conjugated secondary antibody diluted 1:5000 in TBS-T. Reactive bands were detected by chemiluminescence using the Bio-Rad Laboratories ChemiDoc XRS+ system. Plasmid construction -------------------- N-terminally FLAG-tagged Gα~q~ (GenBank® accession number NM_031036.1), Gα~11~ (GenBank® accession number NM_031033.1) or Gβ~1~ (GenBank® accession number NM_030987.2) cDNAs were amplified from cDNAs from a rat brain cDNA library using a forward primer containing the FLAG tag sequence, then subcloned into the pcDNA3.1 V5/His TOPO TA vector (Invitrogen). PCR was performed with the following pairs of primers: Gα~q~, 5′-GGAAGAATGGACTACAAGGACGACGATGACAAGACTCTGGAGTCCATCATGG-3′ and 5′-TCACACCAGATTGTACTCCTTCAGG-3′; Gα~11~, 5′-GCGACGATGGACTACAAGGACGACGATGACAAGACTCTGGAGTCCATGATG-3′ and 5′-TCACACCAGATTGTACTCCTTCAGG-3′; and Gβ~1~, 5′-GTGAAGATGGACTACAAGGACGACGATGACAAGAGTGAACTTGACCAGCTGC-3′ and 5′-GTTCCAGATCTTGAGGAAG-3′. The Gα~i~ (GenBank® accession number NM_010305.1) and Gα~s~ (GenBank® accession number AK168996.1) cDNAs were amplified from a mouse brain cDNA library with the primer pairs 5′-AAAGAATTCGCCACCATGGGCTGCAC-3′ and 5′-AAACTCGAGTTCGAAGAGACCACAGTCTTT-3′, and 5′-AAAGAATTCGCCGCCATGGGCTGCCTC-3′ and 5′-AAACTCGAGTTAGAGCAGCTCGTATTGGCG-3′ respectively. The amplicons were cloned into pcDNA3.1 His/V5 after digestion with EcoRI and XhoI. The FLAG tag was added via a site-directed mutagenesis approach using the following primers paired with their complimentary sequences: Gα~i~, 5′-GAATTCGCCACCATGGACTACAAGGACGACGATGACAAGGGCTGCACATTGAG-3′; and Gα~s~, 5′-GAATTCGCCGCCATGGACTACAAGGACGACGATGACAAGGGCTGCCTCGGC-3′. Mutations were introduced by site-directed mutagenesis using the following primers paired with complimentary sequences: Gα~q~ (G48V), 5′-CTGCTGCTGGGGACAGTCGAGAGTGGCAAG-3′; Gα~q~ (G48A), 5′-CTGCTGCTGGGGACAGCGGAGAGTGGCAAG-3′; Gα~q~ (K52A), 5′-GACAGGGGAGAGTGGCGCGAGTACCTTCATAAG-3′; Gα~q~ (T186A), 5′-GTTCGAGTCCCCGCCACAGGGATCATTG-3′; Gα~q~ (D205A), 5′-TCTTCAGAATGGTCGCTGTAGGAGGCCAAAGG-3′; Gα~q~ (Q209L), 5′-TGTAGGAGGCCTAAGGTCAGAGA-3′; and Gβ~1~ (H311A), 5′-GTCCTAGCTGGAGCTGACAACCGAGTCAGC-3′. The sequences of the constructs were verified by direct DNA sequencing (ABI PRISM 377-XL, Applied Biosystems). LysoPLD assay ------------- The isotopic lysoPLD activity assay was performed as described previously \[[@B21]\]. Briefly, 30 μl of the source of enzyme was incubated at 37°C for 6 h in a reaction mixture containing 0.15 mM 1-\[^14^C\]palmitoyl-GPC (6000 d.p.m./nmol), 20 mM Tris/HCl (pH 7.0), 1 mM Na~3~VO~4~, and 50 mM MgCl~2~ with or without 1 mM GTP in a final volume of 75 μl. The reactions were terminated by the addition of 15 μl of 2 M HCl and 187.5 μl of chloroform/methanol/HCl \[100:200:1 (v/v)\]. The lipids were extracted by the addition of 93.75 μl each of chloroform and 2 M KCl, followed by centrifugation at 500 *g* for 10 min at 20°C. The extracted lipids were subjected to two-dimensional TLC \[chloroform/methanol/28% ammonia at 65:35:5 (v/v) for the first dimension and chloroform/acetone/methanol/acetic acid/water at 45:20:10:13:5 (v/v) for the second dimension\]. The LPA spots were visualized and quantified using the Fuji BAS2000 system (Fujifilm). For the colorimetric assay to detect lysoPLD activity, the amount of choline released from choline lysophospholipids was used as a measure of enzyme activity. Purified proteins (40 μl) were incubated at 37°C with 0.15 mM (0.0375--0.6 mM) choline lysophospholipids in the presence of 20 mM Tris/HCl (pH 7.0) and 1 mM Na~3~VO~4~ with or without 50 mM MgCl~2~ and/or 100 mM EDTA in a total reaction volume of 200 μl. After incubation for the given lengths of time, the reactions were terminated by boiling. To determine the amount of choline released, a second reaction was performed at 37°C for 15 min in a 500 μl reaction mixture containing the first assay mixture plus 50 mM Tris/HCl (pH 8.5), 0.5 mM HPPA, 0.033 unit/ml horseradish peroxidase and 1 unit/ml choline oxidase. The fluorescence intensity of each mixture was determined by excitation at 320 nm and collection at 404 nm using an RF5300 instrument (Shimadzu). Purification of FLAG-tagged proteins ------------------------------------ Mouse hepatocytoma cells (Hepa-1 cells), COS-7 cells or Neuro2A cells were seeded into 100 mm dishes in DMEM (Dulbecco\'s modified Eagle\'s medium; Wako Chemicals) with 10% fetal bovine serum (Gibco) 1 day prior to transfection. A total of 24 μg of empty vector or FLAG-tagged protein expression vector were transfected into cells using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer\'s instructions. At 1 day (24 h) post-transfection, the cells in one or five dishes were collected in 800 μl/dish of buffer C \[0.25 M sucrose in 50 mM Tris/HCl (pH 7.4), 1 mM EDTA and 1 mM PMSF\] and disrupted by sonication (three to eight pulses of 10 s each) with a Microson Ultrasonic cell disruptor. The homogenates were then centrifuged at 105000 *g* for 1 h at 4°C and the supernatants were removed or used for FLAG-tagged protein purification as indicated below. The pellets were resuspended in 400 μl/dish of buffer C with 10 mM CHAPS, and homogenized with an Ultrasonic cell disruptor. The suspensions were diluted to 1600 μl/dish of buffer C, then centrifuged at 21500 *g* for 10 min at 4°C and the supernatants were mixed with 250 μl of anti-FLAG M2 affinity gel (Sigma--Aldrich) suspended in 1.6 ml/dish of TBS buffer \[50 mM Tris/HCl (pH 7.4), 150 mM NaCl and 1mM PMSF\]. After incubation with gentle agitation at 4°C for several hours, the gel was washed with 5 ml of wash buffer (TBS with 0.1% CHAPS), and then tagged proteins were eluted with 250 μl of 100 μg/ml FLAG peptide in wash buffer four times. The viability of cells transfected with control or FLAG-tagged protein expression vectors did not change over 24 h and nearly equal amounts of protein were obtained in each sample. Effect of mouse Gα~q~ and Gα~11~ siRNAs (small interfering RNAs) on lysoPLD activity in Hepa-1 cells ---------------------------------------------------------------------------------------------------- Stealth siRNA, a 25-base pair duplex oligoribonucleotide against mouse Gα~q~ or Gα~11~, was purchased from Invitrogen. The target sense sequences were: Gα~q~, 5′-CCCUUUGACUUACAAAGUGUCAUUU-3′; and Gα~11~, 5′-CCAAGUUGGUGUACCAGAACAUCUU-3′. Hepa-1 cells (1×10^5^) were cultured in 60 mm dishes for 24 h, and then transfected with both siRNAs by using Lipofectamine™ 2000 (Invitrogen) according to manufacturer\'s instructions. After 48 h, the cells were collected in 400 μl/dish of buffer C and disrupted by sonication (three to eight pulses of 10 s each) with a Microson Ultrasonic cell disruptor. The homogenates were then centrifuged at 105000 *g* for 1 h at 4°C and the supernatants were removed. The pellets were resuspended in 300 μl/dish of buffer C with 10 mM CHAPS, and homogenized with an Ultrasonic cell disruptor. The suspensions were centrifuged at 21500 *g* for 10 min at 4°C, and the supernatants were used as microsomal fractions for lysoPLD assay with 10 μg of the protein for a 30 min incubation. Statistical analysis -------------------- All values are expressed as the means±S.D. (n≥3). Group means were compared using the Student\'s t test or ANOVA to determine the significance of the differences among the individual means. Statistical significance was assumed at P\<0.05. Each experiment was repeated at least twice with similar results. RESULTS ======= A protein with lysoPLD activity is highly associated with heterotrimeric G protein ---------------------------------------------------------------------------------- In a previous study, we purified a novel protein with lysoPLD activity from rat brain tissue and analysed its enzymatic characteristics, which were different from those of the secreted lysoPLD autotaxin. Initially, a 35 kDa protein was the main band in purified lysoPLD from rat brain and did not react with an anti-autotaxin antibody \[[@B21]\]. However, as shown in [Figure 1](#F1){ref-type="fig"}(A), two bands (faint 42 kDa and 35 kDa bands, indicated by arrows on the right-hand side) were observed following repeated purifications as reported previously \[[@B21]\], separation by SDS/PAGE and silver staining. The 42 kDa protein was present at lower levels and was not recognized as potentially important at first. To identify their molecular species, the bands were analysed by PMF (peptide mass fingerprinting). The 42 kDa and 35 kDa proteins were identified as Gα~q~ ([Figure 1](#F1){ref-type="fig"}D) and Gβ~1~ ([Figure 1](#F1){ref-type="fig"}E) respectively. ![Identification of G protein subunits Gα~q~ and Gβ~1~ in purified lysoPLD fractions from the rat brain\ (A and B) Fractions eluted after HiTrap DEAE FF purification, the final step in a previously reported protocol for purification of a lysoPLD activity from rat brain tissue, were subjected to SDS/PAGE and detected by silver staining (A) and immunoblotting (B). The right-hand side arrows in (A) indicate protein bands at 42 kDa (upper) and 35 kDa (lower). (C) LysoPLD activities of the same purified samples obtained from rat brain tissue as determined using a colorimetric assay. (D and E) Identification of the purified 42 kDa and 35 kDa proteins from rat brain tissues by PMF. The 42 kDa (D) and 35 kDa (E) proteins were identified as Gα~q~ and Gβ~1~ respectively. Amino acid sequences identified by PMF analysis are indicated with underlining. Fr., fraction.](bic536i001){#F1} To confirm this result, the fractions obtained from the final step of enzyme purification with HiTrap DEAE FF ([Figure 1](#F1){ref-type="fig"}C) were subjected to immunoblot analysis with antibodies specific against Gα~q/11~ and Gβ~1--4~. As shown in [Figure 1](#F1){ref-type="fig"}(B), both Gα~q~ and Gβ~1~ proteins were detected in those fractions and the levels of those proteins correlated well with lysoPLD activities in each fraction. On the basis of these results, we speculated that the heterotrimeric G protein complex is highly associated with lysoPLD activity. Tag-purified G protein exhibits lysoPLD activity ------------------------------------------------ FLAG-tagged Gα~q~ or Gβ~1~ proteins were overexpressed in the mouse hepatocytoma cell line Hepa-1 and then purified via immune-affinity chromatography with an anti-FLAG antibody. When overexpressed, FLAG--Gα~q~ was purified with FLAG peptides, endogenous Gβ co-purified and, similarly, endogenous Gα~q/11~ co-purified with FLAG-tagged Gβ~1~ ([Figure 2](#F2){ref-type="fig"}A). Gγ~5~ also co-purified with FLAG--Gα~q~ ([Figure 2](#F2){ref-type="fig"}A). These results suggest that heterotrimeric G protein complexes can form with overexpressed FLAG--Gα~q~. The density of the immunoreactive bands with anti-Gα~q/11~ or anti-Gβ~1--4~ antibody and lysoPLD activities coincided well ([Figures 2](#F2){ref-type="fig"}A and [2](#F2){ref-type="fig"}B); thus we used fraction 4 for subsequent analyses. The significant increase in lysoPLD activity was not observed in fractions prepared from cells transfected with empty vector. The lysoPLD activity was confirmed by choline production from lysoPAF ([Figure 2](#F2){ref-type="fig"}C) and LPA production from \[^14^C\]palmitoyl-labelled LPC, ([Figures 2](#F2){ref-type="fig"}D and [2](#F2){ref-type="fig"}E). Significant increases in both choline and/or LPA production via lysoPLD activity were detected in the eluates prepared from cells overexpressing FLAG--Gα~q~ or FLAG--Gβ~1~. Autotaxin was not detected in the tag-purified fraction by immunoblot analysis ([Figure 2](#F2){ref-type="fig"}A, indicated by the arrow), and lower bands were non-specific bands. LysoPLD activity of autotaxin is not dependent on Mg^2+^ supplementation \[[@B25]\]. These results suggest that the lysoPLD activities were attributable to Gα~q~ or Gβ~1~ themselves, or to a protein(s) in a complex with these proteins. ![LysoPLD activity of tag-purified G protein subunits Gα~q~ and Gβ~1~\ Empty vector (Cont) or a vector encoding FLAG-tagged Gα~q~ or Gβ~1~ was transfected into Hepa-1 cells and FLAG-tagged proteins (F-Gα~q~ or F-Gβ~1~) were purified from one dish. (A) Immunoblot analysis of tag-purified G protein subunits Gα~q~ and Gβ~1~. A total of 10 μl of each sample prior to purification (lane 1), eluted with a wash buffer (lane 2) or eluted with elution buffers containing FLAG-peptides (lanes 3--6) was separated by SDS/PAGE (10% gel) and transferred on to PVDF membranes. The immunoreacted Gα~q~, Gβ~1~, Gγ~5~, or autotoxin proteins (ATX) were detected with anti-Gα~q/11~, -Gβ, -Gγ~5~ or -autotoxin antibodies. As expected, the molecular masses of tagged proteins were approximately 2 kDa larger than endogenous proteins. The immunoreacted Gγ~5~ in lane 1 and other lanes were determined in separated lanes, but on the same membrane. The experiments were repeated several times and similar results were obtained each time. (B) A total of 40 μl of each fraction (lanes 2--6 in A) from Hepa-1 cells overexpressing empty vector (open square) or FLAG-tagged Gα~q~ (closed diamond) was incubated with 0.15 mM lysoPAF and then lysoPLD activities were calculated using a colorimetric assay. (C) A total of 40 μl of the second fractions eluted with FLAG peptides (lane 4 in A) obtained from Hepa-1 cells overexpressing empty vector (white bar), F-Gα~q~ (black bar) or F-Gβ~1~ (grey bar) was incubated with 0.15 mM lysoPAF for 6 h and then lysoPLD activities were calculated using a colorimetric assay. Results are means±S.D. (n=6). \*P\<0.001 compared with the vector control. (D and E) LPA production from \[^14^C\]-labelled LPC by incubation with the purified enzyme from Hepa-1 cells overexpressing empty vector (Cont) (white bar), FLAG-tagged Gα~q~ (F-Gα~q~) (black bar) or FLAG-tagged Gβ~1~ (F-Gβ~1~) (grey bar) for 6 h was visualized after lipids were extracted and subjected to TLC analysis. Results are means±S.D (n=3). \*P\<0.012 compared with the vector control. Arrows (E) indicate the positions of LPA. The experiments were repeated more than three times with similar results.](bic536i002){#F2} Time course, substrate specificity and Mg^2+^ cation requirement of lysoPLD activity of Gα~q~ --------------------------------------------------------------------------------------------- To examine the time course of choline production from lysoPAF hydrolysed by purified FLAG-tagged Gα~q~ subunit, the enzyme was incubated with 0.15 mM lysoPAF for 0--6 h and activities were tested using a colorimetric assay. As shown in [Figure 3](#F3){ref-type="fig"}(A), the levels of the hydrolysed product, choline, significantly increased in the presence of purified FLAG-tagged Gα~q~ for up to 6 h of incubation. ![Time dependence, substrate specificity and requirement of Mg^2+^ cations of the lysoPLD activity of tag-purified Gα~q~\ (A) A total of 40 μl of the tag-purified fractions obtained from Hepa-1 cells overexpressing empty vector (open squares, n=6) or FLAG-tagged Gα~q~ (F-Gα~q~) (closed diamonds, n=6) was incubated with 0.15 mM lysoPAF for the indicated times and then subjected to a colorimetric lysoPLD assay. \*P\<0.01 compared with the vector control. (B) The purified F-Gα~q~ (40 μl) was incubated with 0.15 mM of various choline lysophospholipids \[16:0 (black bar, n=7), 18:0 (grey bar, n=3), 18:1 (horizontally striped bar, n=6) or lysoPAF (white bar, n=5)\] for 6 h. Relative choline production as compared with lysoPC (16:0) was analysed using a colorimetric assay. \*P\<0.01 compared with 16:0; \\P\<0.001 compared with the other substrates. (C) The lysoPLD activities of the fractions (40 μl) obtained from Hepa-1 cells overexpressing empty vector (white bar) or F-Gα~q~ (black bar) were incubated with or without Mg^2+^ and/or EDTA (n=3). Relative choline production as compared with F-Gα~q~ and Mg^2+^ was analysed using a colorimetric assay. \*P\<0.001 compared with F-Gα~q~ and Mg^2+^~.~ (D) The supernatants of homogenates from Hepa-1 cells overexpressing FLAG--Gα~q~ after centrifugation at 105000 *g* were applied on to immune-affinity chromatography with anti-FLAG antibody, and immunoblot analysis of tag-purified Gα~q~ (F-Gα~q~) was performed (top panel). The enzymatic activity in the purified fraction was assayed (bottom panel). Results are means±S.D. The experiments were repeated several times with similar results. Cont, control.](bic536i003){#F3} To identify a preferred substrate, purified FLAG-tagged Gα~q~ was incubated for 6 h with several different choline lysophospholipid substrates ([Figure 3](#F3){ref-type="fig"}B). The preferred substrate identified in this assay is lysoPAF (P\<0.01). The enzyme was more efficient at hydrolysis of 1-palmitoyl-2-lyso-GPC (16:0) than hydrolysis of 1-oleoyl-2-lyso-GPC (18:1) (P\<0.01). The substrate specificity of Gα~q~ for lysophospholipids is very similar to what was observed for the enzyme activity previously purified from the rat brain \[[@B21]\]. Choline production from lysoPAF by the purified FLAG--Gα~q~ was dependent on incubation with Mg^2+^ ([Figure 3](#F3){ref-type="fig"}C), similar to what was observed for the enzyme purified from rat brain tissue \[[@B21]\]. Although after centrifugation at 105000 *g* high levels of FLAG--Gα~q~ could be purified from the supernatants of homogenates from cells overexpressing the protein, enzymatic activity was not detected ([Figure 3](#F3){ref-type="fig"}D). The lysoPLD activity of Gα~q~ and Gβ~1~ mutant proteins ------------------------------------------------------- As FLAG--Gα~q~ purified from Hepa-1 cells overexpressing the protein co-purified with endogenous Gβ and purified FLAG-Gβ~1~ co-purified with endogenous Gα~q/11~ ([Figure 2](#F2){ref-type="fig"}A), we next tried to clarify which subunit is responsible for the lysoPLD activity we observed. Autotaxin had been identified previously as a member of the ecto-nucleotide PDE (pyrophosphatase/phosphodiesterase) family due to similarity in its primary structure \[[@B14],[@B26]\]. Later, it became clear that the ester bond between phosphate and choline in LPC was also hydrolysed by autotaxin \[[@B12],[@B13]\]. Therefore we speculated that Gα~q~ subunit might exhibit lysoPLD activity because it hydrolyses GTP to GDP via its intrinsic GTPase activity. The Gβ~1~ subunit would be the candidate for the protein with lysoPLD activity, as a histidine acid phosphatase consensus motif is present in its C-terminus (V^305^GILSGHDNAVSCLGV^320^, analysed by GENETYX). To test this, we introduced several mutations at amino acid residues thought to be important for Gα~q~ or Gβ~1~ enzyme activities. The mutant proteins were then purified and their lysoPLD activities were assayed. Tag-purified Gβ~1~--Gγ~2~ complex overexpressed in S. frugiperda insect cells was also obtained, and its lysoPLD activity was determined. As shown in [Figure 4](#F4){ref-type="fig"}(A), the K52A, T186A, D205A, G48A and Q209L mutant forms of Gα~q~ showed a significant reduction of lysoPLD activity, whereas G48V did not. The decrease in lysoPLD activity of G48A and Q209L was low. Based on amino acid sequence similarity to p21^ras^, the amino acid sequence motif G^46^TGESGKS^53^ is predicted to form a loop in which main-chain amide hydrogens of several amino acids and the ϵ-amino group of Lys^52^ might form bonds with the β- and γ-phosphate of GTP \[[@B27]\]. This site also corresponds to the GTP-binding site. Therefore we speculated that the side chain of Lys^52^ might be important for the binding of phosphate at the sn−3 position of LPC, whereas the side chain of Gly^48^ is not likely to be important for this binding. This suggests that the GTP- and LPC-binding sites or interacting amino acids of Gα~q~ seem to be not completely the same. Moreover, the side chain of Thr^186^ may point away from the bound nucleotide in the GDP-bound form, but flip towards the nucleotide in the GTP-bound form, where it might directly interact with a Mg^2+^ ion coordinated with oxygen molecules in the β- and γ-phosphates of GTP. On the basis of crystal structure analysis, it has been suggested that the side chain of Asp^205^ in Gα~q~ might bind catalytic Mg^2+^ via an intervening water molecule \[[@B27],[@B28]\]. The well-known dominant negative Gα~q~ (Q209L) construct was prepared and its activity was assayed. Only slight reduction of lysoPLD activity of Q209L was detected. The Gβ~1~ mutant H311A, which disrupts a conserved amino acid residue in the histidine acid phosphatase consensus, did not show a significant change in lysoPLD activity as compared with the wild-type Gβ~1~ subunit ([Figure 4](#F4){ref-type="fig"}B). Tag-purified Gβ~1~--Gγ~2~ complex overexpressed in insect cells did not exhibit lysoPLD activity. These results strongly suggest that Gα~q~ exhibits lysoPLD activity and, furthermore, that the predicted nucleotide phosphate and Mg^2+^-binding sites of the protein are important for lysoPLD enzymatic activity. When we purified lysoPLD from rat brain or culture cells, Gα~q~ and Gβ were always co-purified. It is possible that lysoPLD activity of Gα~q~ is dependent on the presence of Gβ protein. ![LysoPLD activity of mutant forms of the G protein subunits Gα~q~ and Gβ~1~, and tag-purified Gβ~1~--Gγ~2~ complex\ Empty vector (Cont), FLAG-tagged wild-type Gα~q~ (A, F-Gα~q~) or Gβ~1~ (B, F-Gβ~1~), or mutant forms (A, T186A, K52A, D205A, G48V, G48A, Q209L mutations of Gα~q~, and B, F-Gβ~1~m for Gβ~1~) were expressed in Hepa-1 cells and purified from one dish. Top panels: the levels of unmodified or mutant FLAG-tagged Gα~q~ and Gβ~1~ subunits in 10 μl of the purified fractions, and tag-purified Gβ~1~--Gγ~2~ complex (3 ng). The immunoreacted wild-type Gα~q~ and mutant forms in (A) were not determined in the same experiments, but all experiments were repeated several times and almost equivalent levels of proteins compared with F-Gα~q~ were obtained each time. (A) Bottom panel: relative choline production by tag-purified enzymes (40 μl) from cells overexpressing empty vector (Cont; white bar, n=6), T186A (vertically striped bar, n=5), K52A (horizontally striped bar, n=3), D205A (left oblique striped bar, n=3), G48V (grey bar, n=4), G48A (right oblique striped bar, n=3) or Q209L (crosswise striped bar, n=3) as compared with unmodified FLAG-tagged Gα~q~ (black bar, n=8) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 6 h. \*P\<0.001 compared with FLAG-tagged Gα~q~. (B) Bottom panel: relative choline production by tag-purified enzymes from cells overexpressing empty vector (Cont; white bar, n=6), F-Gβ~1~m (horizontally striped bar; n=6) or tag-purified Gβ~1~--Gγ~2~ complex (12 ng) \[G(β~1~ and γ~2~); black bar, n=3\] as compared with F-Gβ~1~ (grey bar; n=6). \*P\<0.001 compared with F-Gβ~1~. Results are means±S.D. The experiments were repeated more than three times with similar results.](bic536i004){#F4} LysoPLD activity of Gα~q~ protein and a mutant form, T186A, highly purified from COS-7 cells -------------------------------------------------------------------------------------------- To eliminate the possibility that the detection of lysoPLD activity from purified tagged Gα~q~ protein is cell-line dependent, we performed the same experiment with COS-7 cells, which do not exhibit autotaxin activity and express relatively low endogenous levels of G proteins. As shown in [Figure 5](#F5){ref-type="fig"}(A), highly purified wild-type FLAG--Gα~q~ protein and a mutant form, T186A, could be detected following ectopic expression in COS-7 cells. The levels of the hydrolysed product, choline, significantly increased over time in the presence of purified FLAG-tagged Gα~q~ ([Figure 5](#F5){ref-type="fig"}B). The activity was dependent on incubation with Mg^2+^ cations ([Figure 5](#F5){ref-type="fig"}C). The presence of lysoPLD activity was confirmed by the significant production of choline from lysoPAF ([Figures 5B--5](#F5){ref-type="fig"}D) and LPA from LPC ([Figures 5](#F5){ref-type="fig"}E and [5](#F5){ref-type="fig"}F). The T186A mutant form of Gα~q~ showed significantly less lysoPLD activity than the wild-type FLAG--Gα~q~ protein. These results clearly show that Gα~q~ has lysoPLD activity. As shown in [Figure 5](#F5){ref-type="fig"}(A) (bottom panel), a small amount of endogenous Gβ in COS-7 cells was also co-purified with intact or mutated FLAG-tagged Gα~q~. ![LysoPLD activity of highly purified Gα~q~ protein and a mutant form, T186A, from COS-7 cells\ Empty vector (Cont), FLAG-tagged wild-type Gα~q~ (F-Gα~q~) or a FLAG-tagged mutant form (T186A) was expressed in COS-7 cells and purified from five dishes. (A) Silver staining (top panel) or immunoblot analysis (bottom panel) with anti-Gα~q/11~ and Gβ of vector control (Cont), tag-purified wild-type Gα~q~ (F-Gα~q~) or T186A in the purified fractions. (B) A total of 40 μl of the tag-purified fractions obtained from COS-7 cells overexpressing empty vector (Cont) (open squares, n=3) or FLAG-tagged Gα~q~ (F-Gα~q~) (closed diamonds, n=3) was incubated with 0.15 mM lysoPAF for the indicated times and then subjected to a colorimetric lysoPLD assay. \*P\<0.001 compared with the vector control. (C) The lysoPLD activities of the purified fractions (40 μl) obtained from COS-7 cells overexpressing empty vector (open bars) or F-Gα~q~ (closed bars) were incubated with or without Mg^2+^ and/or EDTA (n=3). Relative choline production as compared with the vector control was analysed using a colorimetric assay. \*P\<0.05 compared with the vector control and F-Gα~q~ with or without Mg^2+^ and EDTA. (D) Choline production by tag-purified enzymes (40 μl) from cells overexpressing unmodified FLAG-tagged Gα~q~ (F-Gα~q~) (black bar, n=3) or T186A (vertically striped bar, n=3) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 6 h. \\P\<0.001 compared with F-Gα~q~. (E and F) LPA production by tag-purified enzymes (30 μl) from cells overexpressing unmodified FLAG-tagged Gα~q~ (F-Gα~q~) (black bar, n=3) or T186A (vertically striped bar, n=3) was analysed with an isotopic assay after incubation with 0.15 mM \[^14^C\]-labelled LPC for 6 h. LPA production was visualized after lipids were extracted and subjected to TLC analysis. (E) \*P\<0.03 compared with F-Gα~q~. (F) Arrows indicate the positions of LPA. Results are means±S.D. The experiments were repeated at least twice with similar results.](bic536i005){#F5} LysoPLD activity of Gβ~1~ protein highly purified from COS-7 cells ------------------------------------------------------------------ To examine the possibility that purified FLAG--Gβ~1~ obtained from COS-7 cells also has lysoPLD activity, we repeated the experiment described above with these cells. A small amount of endogenous Gα~q/11~ in COS-7 cells was co-purified with FLAG--Gβ~1~ ([Figure 6](#F6){ref-type="fig"}A). Almost the same lysoPLD activity as for purified FLAG--Gα~q~ protein was observed, in FLAG--Gβ~1~ with the production of choline from lysoPAF ([Figure 6](#F6){ref-type="fig"}B) and LPA from LPC ([Figure 6](#F6){ref-type="fig"}C). When purified FLAG--Gα~q~ and FLAG--Gβ~1~ proteins were incubated together in vitro, an additive effect of lysoPLD activity was observed, as specific activity did not change ([Figure 6](#F6){ref-type="fig"}B). ![LysoPLD activity of highly purified Gβ~1~ protein from COS-7 cells\ FLAG-tagged wild-type Gα~q~ (F-Gα~q~) or a FLAG-tagged wild-type Gβ~1~ (F-Gβ~1~) was expressed in COS-7 cells and purified from five dishes. (A) Immunoblot analysis with anti-Gα~q/11~ (top panel) and anti-Gβ (middle panel), or silver staining (bottom panel) of tag-purified wild-type Gα~q~ (F-Gα~q~) or Gβ~1~ (F-Gβ~1~) in the purified fractions. (B) A total of 40 μl of the tag-purified fractions obtained from COS-7 cells overexpressing FLAG-tagged Gα~q~ (F-Gα~q~) (black bar, n=3), FLAG-tagged Gβ~1~ (F-Gβ~1~) (grey bar, n=3) or equal amounts of both F-Gα~q~ and F-Gβ~1~ (white bar, n=3) was incubated with 0.15 mM lysoPAF for 6 h and then subjected to a colorimetric lysoPLD assay. (C) The lysoPLD activity of the purified fraction (30 μl) obtained from COS-7 cells overexpressing F-Gα~q~ (black bar, n=3) or F-Gβ~1~ (grey bar, n=3) was analysed with an isotopic assay after incubation with 0.15 mM \[^14^C\]-labelled LPC for 6 h. LPA production was visualized after lipids were extracted and subjected to TLC analysis. Results are means±S.D. The experiments were repeated at least twice with similar results.](bic536i006){#F6} Effect of substrate concentration and GTP on the activity of FLAG-purified lysoPLD from Neuro2A cells overexpressing FLAG--Gα~q~ -------------------------------------------------------------------------------------------------------------------------------- Because the lysoPLD enzyme that we have purified previously was from the brain, we also tested overexpression and tag-affinity purification of FLAG--Gα~q~ using Neuro2A cells. As shown in [Figures 7](#F7){ref-type="fig"}(A) and [7](#F7){ref-type="fig"}(B), the level of lysoPLD activity of purified FLAG-Gα~q~ was dependent on cell type (COS-7\<Hepa-1\<Neuro2A cells). Notably, the enzyme activity of purified FLAG-tagged Gα~q~ expressed in COS-7 cells was approximately 4 nmol/min per mg, whereas the activity from Neuro2A cells was much higher, i.e. 137.4 nmol/min per mg. The calculated K~m~ and V~max~ values for lysoPAF using Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively ([Figures 7](#F7){ref-type="fig"}B and [7](#F7){ref-type="fig"}C). The K~m~ and V~max~ values of lysoPLD obtained from rat brain for lysoPAF were 26.7 μM and 0.29 μmol/min per mg respectively. These results using Neuro2A cells and rat brain were almost equivalent with each other. The slightly lower V~max~ value in Neuro2A cells is probably due to the purity of the enzyme or the disturbance of enzyme activity by N-terminal FLAG peptides. ![Effect of substrate concentration and GTP on the activity of FLAG-purified lysoPLD obtained from Neuro2A cells overexpressing FLAG--Gα~q~\ Empty vector (Cont) or FLAG-tagged wild-type Gα~q~ (F-Gα~q~) was expressed in Hepa-1, COS-7 or Neuro2A (N2A) cells and purified from five dishes. (A) Immunoblot analysis with anti-Gα~q/11~ antibody (upper panel) of tag-purified Gα~q~ (F-Gα~q~) obtained from Hepa-1, COS-7 or Neuro2A cells overexpressing FLAG-Gα~q~. Choline production (lower panel) by tag-purified enzyme (40 μl) obtained from cells overexpressing unmodified FLAG-tagged Gα~q~ (F-Gα~q~) (n=3) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 6 h. Results are means±S.D. The experiments were repeated at least two times with similar results. Tag-purified enzyme from Neuro2A cells overexpressing unmodified FLAG-tagged Gα~q~ was incubated with various concentrations from 0.0375 to 0.6 mM of lysoPAF for 6 h, and choline production was analysed with a colorimetric assay. An untransformed plot (B) and Lineweaver--Burk (double-reciprocal) plot (C) are shown. (D) The lysoPLD activity of the purified fraction (30 μl) obtained from Neuro2A cells overexpressing empty vector or F-Gα~q~ was analysed with an isotopic assay after incubation with 0.15 mM \[^14^C\]-labelled LPC for 6 h with (black bar, n=3) or without (white bar, n=3) 1 mM GTP. Relative LPA production was determined as compared with FLAG-tagged Gα~q~ without GTP. LPA production was visualized after lipids were extracted and subjected to TLC analysis. Results are means±S.D. The experiments were repeated at least two times with similar results.](bic536i007){#F7} As shown in [Figure 7](#F7){ref-type="fig"}(D), 1 mM GTP did not disturb the lysoPLD activity (0.15 mM LPC) of FLAG--Gα~q~ obtained from Neuro2A cells. As the mutation at the GTP-binding region (Gly^48^) did not decrease lysoPLD activity of Gα~q~ much ([Figure 4](#F4){ref-type="fig"}A), the GTP- and LPC-binding sites seem to not be completely the same. Moreover, the exchange of GDP to GTP and activation of GTPase activity of Gα~q~ possibly rarely occurred in this experiment. As for most of the GTPases, the rate of exchange of GDP to GTP and GTPase activity of Gα~q~ are quite low without ligand-stimulated receptor and GAPs (GTPase-activating proteins) \[[@B29]--[@B31]\]. LysoPLD activity of autotaxin may be inhibited by incubation with GTP because autotaxin catalyses the hydrolysis of GTP as well as ATP. LysoPLD activity of other Gα subfamily members and the effect of Gα~q~ and Gα~11~ siRNAs ---------------------------------------------------------------------------------------- In the human genome, 16 different Gα subunits have been identified and these can be divided into four subfamilies based on their amino acid similarities and the machinery for their signal transduction. Thus we were interested in the possibility that other Gα protein family members might also exhibit lysoPLD activity. To test this, we first constructed plasmids encoding FLAG-tagged Gα~11~, Gα~i~ and Gα~s~. The plasmids were introduced into Hepa-1 cells and the activities of the purified overexpressed proteins were analysed. As shown in [Figure 8](#F8){ref-type="fig"}(A), purified FLAG--Gα~11,~ which has 89% sequence similarity with Gα~q~ and belongs to the same subfamily, exhibited a high lysoPLD activity (P\<0.01), whereas FLAG--Gα~i~ and FLAG--Gα~s~ did not. However, a slight increase in lysoPLD activity of FLAG--Gα~s~ was observed. ![Testing LysoPLD activity of other Gα subfamily members and effect of Gα~q~ and Gα~11~ siRNAs\ (A) FLAG-tagged Gα subtypes F-Gα~q~ (black bar, n=18), F-Gα~11~ (grey bar, n=6), F-Gα~i~ (horizontally striped bar, n=6), F-Gα~s~ (vertically striped bar, n=6) or empty vector (Cont; white bar, n=12) were overexpressed in Hepa-1 cells and tagged proteins were purified from one dish. Levels of tag-purified Gα subfamily proteins (top panel) and co-purified Gβ proteins (middle panel) detected using anti-FLAG and anti-Gβ~1--4~ antibodies respectively~.~ Note that Gα~s~ migrates at a higher molecular mass than the others. The bands of the same size present in the control and experimental samples are non-specific. Bottom panel: relative choline production by 40 μl of purified enzyme incubated with lysoPAF as compared with FLAG-tagged Gα~q~, detected with a colorimetric assay. \*P\<0.001 compared with F-Gα~q~. Results are means±S.D. The experiment was repeated several times with similar results. (B) Hepa-1 cells were pretreated with control siRNA or mouse Gα~q~ and Gα~11~ siRNAs for 48 h. Levels of endogenous Gα~q~ and Gα~11~ (top panel) and actin (middle panel) detected using anti-Gα~q/11~ and anti-actin antibodies respectively~.~ Bottom panel: lysoPLD activity in a crude microsomal fraction (10 μg) from Hepa-1 cells pretreated with control siRNA (white bar, n=3) or Gα~q~ and Gα~11~ siRNAs (black bar, n=3) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 30 min.](bic536i008){#F8} As shown in [Figure 8](#F8){ref-type="fig"}(B), pretreatment of Gα~q~ and Gα~11~ siRNAs clearly decreased the endogenous Gα~q~ and Gα~11~ in Hepa-1 cells. However, lysoPLD activity in the crude microsomal fraction did not changed compared with the control. In the crude microsome, we speculated that other undetermined G proteins or contaminated autotaxin may be responsible for the high endogenous lysoPLD activity and that the lysoPLD activity of the interested protein may be masked. DISCUSSION ========== We were interested to molecularly identify a lysoPLD activity that had been reported previously. Two proteins from the rat brain exhibiting lysoPLD activity (faint 42 kDa and 35 kDa bands by gel electrophoresis) can be observed following a method reported previously for purification \[[@B21]\]. As shown in [Figure 1](#F1){ref-type="fig"}(A), the 42 kDa protein was present at lower levels and, at first, was not recognized as potentially important. The enzymatic properties of the fractions containing both proteins, including optimum pH, substrate specificity and cation requirement, were different from those of a secreted protein with lysoPLD activity, autotaxin. In the present study, we analysed the 42 kDa and 35 kDa proteins by MS and identified them as the heterotrimeric G protein subunits Gα~q~ and Gβ~1~ respectively. When FLAG-tagged Gα~q~ or Gβ~1~ was overexpressed in Hepa-1 cells and purified, both purified subunits exhibited lysoPLD activity ([Figure 2](#F2){ref-type="fig"}). Mutations in conserved amino acid residues of Gα~q~ known to be important for GTPase activity and interaction with nucleotide phosphates or Mg^2+^ significantly reduced lysoPLD activity ([Figure 4](#F4){ref-type="fig"}). Furthermore, highly purified FLAG--Gα~q~ protein ectopically expressed in COS-7 cells clearly hydrolysed LPC to LPA, whereas a mutant form, T186A, showed significantly less lysoPLD activity ([Figure 5](#F5){ref-type="fig"}). The calculated K~m~ and V~max~ values for lysoPAF of the FLAG-purified enzyme obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively ([Figures 7](#F7){ref-type="fig"}B and [7](#F7){ref-type="fig"}C). These values were similar to the enzyme purified from the rat brain (K~m~=26.7 μM and V~max~=0.29 μmol/min per mg) (21). Although Gβ~1~ detected by silver staining was the main protein of purified lysoPLD from rat brain, Gβ~1~ is not likely to be the main component for lysoPLD activity because of following four reasons: (i) FLAG--Gβ~1~ mutant (H311A) exhibited significant lysoPLD activity ([Figure 4](#F4){ref-type="fig"}B); (ii) FLAG--Gα~11~ co-purified with lower amounts of Gβ~1~ exhibited lysoPLD activity comparable with Gα~q~ ([Figure 8](#F8){ref-type="fig"}A); (iii) FLAG--Gα~i~ and FLAG--Gα~s~ did not exhibit as high a lysoPLD activity as FLAG--Gα~q~ when they were co-purified with Gβ ([Figure 8](#F8){ref-type="fig"}A); and (iv) tag-purified Gβ~1~ and Gγ~2~ overexpressed in insect cells did not exhibit lysoPLD activity ([Figure 4](#F4){ref-type="fig"}A). However, the potential importance of Gβ~1~ for lysoPLD activity of Gα~q~ may be possible because they were always co-purified with respect to lysoPLD activity. These results reveal a new function for Gα~q~ and Gβ~1~, which are well known as multifunctional signal transduction proteins, as an enzyme with lysoPLD activity. Although FLAG-purified Gα~q~ obtained from the microsomal fraction exhibited significant lysoPLD activity, the purified enzyme from supernatants after 105000 *g* centrifugation did not have lysoPLD activity ([Figure 3](#F3){ref-type="fig"}D). The level of lysoPLD activity of purified FLAG--Gα~q~ from the microsomal fraction was dependent on cell type (COS-7\<Hepa-1\<Neuro2A cells) ([Figure 7](#F7){ref-type="fig"}A). These results suggest that post-translational modification(s), or some kind of activator(s) present in the microsome, is important for lysoPLD activity of Gα~q~. However, it is also possible that Gα~q~ proteins in supernatants were misfolded after translation. Agonist-activated GPCRs enhance GDP/GTP exchange on the G protein α subunit, thereby generating an active GTPbound Gα subunit, followed by dissociation from the receptor and Gβγ subunit. The onset and termination of G protein signalling is determined by the length of time spent in the active GTP-bound state. GTP is hydrolysed to GDP by the intrinsic GTPase activity of the Gα subunit, resulting in re-association of the Gα subunit with the Gβγ subunit and termination of signalling \[[@B29]\]. As for most of GTPases, the rates of exchange of GDP to GTP and GTPase activity of Gα~q~ are quite low. However, the GTPase activity of Gα~q~ can be dramatically increased by in vitro incubation with ligand-stimulated cholinergic receptor and GAPs, such as the RGS (regulator of G protein signalling) proteins \[[@B30]\] or phospholipase C \[[@B31]\]. In the present study, we found evidence that the lysoPLD activity of FLAG-tagged Gα~q~ is exhibited in the presence of Gβ and Gγ~5~, as endogenous Gβ and Gγ~5~ co-purified with FLAG--Gα~q~ ([Figure 2](#F2){ref-type="fig"}). We speculate that the heterotrimeric form of G protein subunits may exhibit lysoPLD activity. Previously, GTPγS was found to be bound to the purified activators \[[@B32]\] and, additionally, that purified PI-PLC (phosphoinositide phospholipase C) from the bovine brain could be activated by purified Gα~q~ in the presence of GTPγS (0.1--1 mM) \[[@B33]\]. On the basis of our results, we propose that a possible role of the novel lysoPLD identified in the present study for Gα~q~ and Gβ~1~ is the production of LPA inside cells in response to ligand stimulation, which is then followed by sequential activation of PI-PLC (Ca^2+^ influx) and PLA~2~s that hydrolyse PC to free fatty acid and LPC \[[@B15]\]. It was reported previously that LPA receptors can be detected on the nuclear membrane \[[@B22],[@B23]\] and that the transcription factor PPARγ acts as a LPA receptor \[[@B24]\]. It seems reasonable to speculate that the Gα~q~ and Gβ~1~ subunits mediate the signal to these intracellular receptors via lysoPLD activity. After testing several family members as shown in [Figure 8](#F8){ref-type="fig"}(A), Gα~q~ and Gα~11~ exhibit a high lysoPLD activity, but Gα~i~ and Gα~s~ do not. Therefore we conclude that the lysoPLD activity of the Gα subunit is strictly dependent on its subfamily. However, treatment of Hepa-1 cells with Gα~q~ and Gα~11~ siRNAs did not affect lysoPLD activity in the microsomal fraction ([Figure 8](#F8){ref-type="fig"}B). It may be possible that other undetermined G proteins and/or different kinds of lysoPLD, such as autotaxin, are responsible for the high endogenous lysoPLD activity and that they masked the reduced activity. Furthermore, our preliminary experiments showed that the stimulation of radio-labelled cells by carbachol that activates Gα~q/11~ through the muscarinic receptor did not affect the basal amount of LPA (results not shown). We speculate that the degradation of LPA in cells was rapid or that the stimulation system was inadequate. Further investigation is required to help elucidate the importance of LPA production by Gα~q~ in signal transduction. AUTHOR CONTRIBUTION =================== The project strategy was devised by, and most of the experiments were performed by, Chieko Aoyama and Hiroyuki Sugimoto. Purification of proteins was helped by Hiromi Ando, Satoko Yamashita and Sayaka Sugimoto. siRNA experiments and PMF analysis were performed by Yasuhiro Horibata and Motoyasu Satou respectively. ACKNOWLEDGEMENTS ================ We thank Dr Takashi Namatame of the Medical Research Center for help with DNA sequencing and the Research Support Center for allowing us to use the facilities at Dokkyo Medical University School of Medicine. We also thank Dr Junken Aoki (Graduate School of Pharmaceutical Science, Tohoku University, Japan) for providing the monoclonal anti-autotaxin antibody. FUNDING ======= This work was supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 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1. Background {#sec61617} ============= Laparoscopic cholecystectomy (LC) is an established, universally accepted procedure for cholelithiasis treatment, to decrease patient discomfort. This procedure is well tolerated by patients, however, an increased incidence of postoperative nausea and vomiting (PONV) predisposes patients to psychological distress increase and delayed recovery and discharge times ([@A16710R1], [@A16710R2]). Although the PONV has multifactorial etiology, one its main predisposing factors is postoperative pain ([@A16710R1]). Opioids are routinely used for pain relief and control, but they increase the incidence of PONV by opioid receptor stimulation in the trigger zone ([@A16710R3]). It seems that some surgical and anesthetic interventions can decrease postoperative pain and subsequently the opioid use, leading to a reduced level of PONV. Pain following LC can be divided into three major groups: 1. Parietal pain caused by surgical incision; 2. Visceral pain originating from gallbladder bed and caused by visceral manipulation during the operation and finally 3. Shoulder pain secondary to distension of diaphragm by neuropraxia of phrenic nerve ([@A16710R4], [@A16710R5]). Due to the different pathophysiological mechanisms of these pain components, different local anesthetic interventions have been developed to reduce postoperative pain, like peritoneal infiltration, instillation into the peritoneal cavity or subdiaphragmatic area, intraperitoneal spraying above the gallbladder and combined peritoneal-peritrocal ropivacaine ([@A16710R6]-[@A16710R12]). However, there is controversy about the characteristics of the pain components and also about the pain reducing the effects of intraperitoneal or peritrocal local anesthetics. 2. Objectives {#sec61618} ============= The present study aims to evaluate the efficacy of intraperitoneal local anesthetic bupivacaine on PONV, after managing the visceral and shoulder pains. To avoid any confusion in different components of pain, we decided to eliminate parietal pain by injection bupivacaine into the abdominal wall, in both groups. So that we could fully concentrate on visceral and shoulder pain and to see if IP bupivacaine injection could affect the opioid demand by influencing visceral and shoulder pain. Intra-abdominal dull pain that cannot exactly be located is considered as visceral pain, while the sharp pain felt in the abdominal wall is deemed as the parietal pain. 3. Patients and Methods {#sec61619} ======================= This was a double-blind, randomized clinical trial conducted on 66 patients of both sexes (aged 20 to 60 years), September 2010 to April 2011. All patients were registered for LC operation, according to the American Society of Anesthesiologists physical status I and II. All studies were performed in accordance with the ethical guidelines, set by the ethical committee of Faculty of Medical Sciences, Kermanshah University of Medical Sciences (Iran). The study was registered in Iranian Registry of Clinical Trials (IRCT13880112946N1). All patients were well informed of the study and all signed a written consent. The exclusion criteria were obesity (body mass index higher than 30 kg/m^2^), history of opioids abuse, antiemetic and steroids administrations within 24 hours prior to the surgery, history of motion sickness or PONV and patients with migraine whose surgery changed to open cholecystectomy. Patients were randomly assigned to two groups using a computer based randomization method. All patients received the same anesthetic agent and protocol. General anesthesia was induced by 0.2 µg/kg sufentanil, followed by 3-5 mg/kg thiopental and 0.6 mg/kg atracurium to facilitate tracheal intubation. Anesthesia was maintained with propofol infusion (200 µg/kg/minute) and injection of 0.1 µg/kg sufentanil, every 15 minutes. Patients in bupivacaine group received 20 mL of bupivacaine in the gallbladder bed, after abdominal CO~2~ insufflation, as well as 20 mL of bupivacaine 0.25% in the gallbladder bed, after resection of gallbladder, whereas control group (n = 33) did not get such injections. During operation, patients were placed in the reverse Trendelenburg position. Pneumoperitoneum was created with a closed Veress needle technique and LC was performed using four trocars, placed in the standard position. The gallbladder was retracted via a supraumbilical trocar port. During laparoscopy, intra-abdominal pressure was maintained at 12 mmHg. CO~2~ was carefully evacuated at the end of the operation by manual compression of the abdomen with open trocars. For elimination of the parietal pain and concentration on the visceral and shoulder pains, both groups received local administration of 5 mL bupivacaine 0.25% at incision of each trocar and different layers of abdomen. For reversal of muscle relaxation, 40 µg/kg neostigmine and 20 µg/kg atropine were administered and patients were transferred to the post anesthesia care unit (PACU) after tracheal extubation. The incidence and severity of nausea and postoperative pain intensity at rest, when coughing and changing positions from supine to sitting were measured, using the visual analog scale (VAS) at one, two, three and four hours after the operation. The incidence of vomiting was evaluated by a "yes" or "no" question at the same time. At the first, second, third and fourth hours after surgery, when examining the intensity of pain and nausea, the patients were asked if they had vomiting. If the answer was positive, the frequency was recorded. All assessments were recorded by trained nurses who were blinded to the study and group assignments. Patients could request for rescue analgesia and antiemetic at any time after operation. Fifty milligram tramadol and 10 mg metoclopramide was intravenously administered as a rescue analgesic and antiemetic, respectively (rescue antiemetic treatment in VAS \> 3). The statistical analysis was performed using SPSS package (SPSS, Chicago, IL, USA, version 16). For statistical analysis of the demographic data and for comparison of the two groups, Chi square, Mann-Whitney U-test and the student t-test analyses were performed. 4. Results {#sec61620} ========== A total of 73 patients were assessed for eligibility, of which 66 patients were enrolled into the study. There were no differences among the groups with respect to patients\' characteristics and operative data. Bupivacaine and control groups did not differ significantly in mean age (39.7 ± 11.3 vs. 42.39 ± 12 years; P = 0.368) and intraoperative opioid usage (32.61 ± 7.92 vs. 30.88 ± 7.20 microgram sufentanil; P = 0.381). Female patients comprised 97.1% of the bupivacaine group and 85.5% of the control group, indicating no significant difference in this regard (P = 0.166). The means values for the pain VAS during resting, coughing and changing positions for four hours following operation are presented in [Figure 1](#fig12236){ref-type="fig"}. ![Comparison of Pain at Rest in the Two Groups](aapm-04-03-16710-i001){#fig12236} The highest pain levels were experienced during the first postoperative hour and these levels were gradually diminished with time. No significant difference was found between the two groups in terms of incidence of nausea ([Figure 2](#fig12237){ref-type="fig"}) and vomiting ([Figure 3](#fig12238){ref-type="fig"}). Vomiting occurred for six patients in the bupivacaine group and seven in the control group, who were then treated with 10 mg metoclopramide. Furthermore, both groups were similar with respect to opioid consumption, during the first postoperative four hours. ![Comparison of Postoperative Nausea in Bupivacaine and Control Groups](aapm-04-03-16710-i002){#fig12237} ![Comparison of Postoperative Vomiting in Bupivacaine and Control Groups](aapm-04-03-16710-i003){#fig12238} There was no significant difference in parietal pain between the two groups. Visceral and shoulder pains showed significant differences between the control and bupivacaine groups only in 4th post-operative hours. Although the viseral and shoulder pain intensity at the other time was lower in Bupivacaine group. That was not statistically significant ([Table 1](#tbl15741){ref-type="table"}). ###### Pain Component Data in the Two Groups Post-Operative Pain Control Group, (n = 33) Bupivacaine Group, (n = 33) P Value ---------------------- ------------------------- ----------------------------- --------- Shoulder pain, h 1 11 8 0.587 2 14 8 0.191 3 10 8 0.783 4 18 10 0.046 Visceral Pain 1 22 23 0.792 2 24 19 0.301 3 23 23 0.999 4 22 14 0.048 Parietal Pain 1 2 0 0.492 2 1 0 0.999 3 2 0 0.492 4 1 0 0.999 Variables related to analgesic consumption in the bupivacaine and control group is shown in [Table 2](#tbl15742){ref-type="table"}. ###### Comparison of Variables Related to Analgesic Consumption in the Bupivacaine and Control Group ^[a](#fn12277){ref-type="table-fn"}^ variables Control Group Bupivacaine Group P Value ---------------------------------------------------------------- --------------- ------------------- --------- First analgesic request, h 1.68 ± 0.98 1.27 ± 0.90 0.126 Pain intensity based on VAS in first analgesic request 7.40 ± 3.49 7.04 ± 3.24 0.711 Mean dose of received analgesic after surgery, mg tramadol 57.14 ± 29.55 64.1 ± 45.69 0.435 ^a^ Data are presented as Mean ± SD. 5. Discussion {#sec61621} ============= This randomized, double-blind, placebo-controlled trial aimed to investigate the effects of IP administration of bupivacaine on visceral and shoulder pain, as well as its effects on PONV after LC. Our results showed that IP administration of 20 mL bupivacaine 0.25% in the gallbladder bed after abdominal CO~2~ insufflation and also after resection of gallbladder did not reduce the patient's demand for extra opioids and had no effect on PONV. The origin of pain and PONV, following LC is not entirely cleared, but it is probably multifactorial. Insufflation of CO~2~, resulting in stretching of the peritoneum, residual pneumoperitoneum after CO~2~ insufflation, peritoneum distension, diaphragm irritation and visceral organ irritation and manipulation have been reported to influence the severity of pain and PONV ([@A16710R5], [@A16710R13]). Although, in several studies using local anesthetics in peritoneal cavity reduced postoperative pain and PONV, the use of different pain control techniques, including the application of intraperitoneal and/or peritrocar local anesthetics has still remained controversial ([@A16710R6], [@A16710R12], [@A16710R14]). Visceral and shoulder pain were significantly different at the 4th post-operative hour between the two groups. Some studies have shown that parietal pain can be blocked by infiltration of local anesthetic agents at incisions and visceral and shoulder pains by IP administration of local anesthetics ([@A16710R5]-[@A16710R9]). A meta-analysis performed by Boddy et al. showed the efficacy of IP local anesthetics in relieving post-laparoscopic cholecystectomy pain, without side effect of analgesic toxicity ([@A16710R15]). Chou YJ et al. demonstrated that IP bupivacaine administration, is effective on the visceral pain reduction, both after trocar incision and at the end of the surgery whereas, with no efficient reduction in abdominal and right shoulder pain and opioid demand ([@A16710R16]). In this study, parietal pain in both groups could be diminished through trocar bupivacaine administration to assess the effects of visceral and shoulder pains on PONV following LC. Our findings showed that IP administration of bupivacaine reduced both visceral and shoulder pains but had no effect on reduction of rescue analgesic. On the other hand, IP bupivacaine had not any impact on reduction of PONV and the incidence of nausea and vomiting were similar in both groups. Based on the findings of this study, it can be concluded that use of opioids has more effects than pain itself to increase PONV. It has been shown that opioid use increases the incidence of PONV by stimulation of opioid receptor in chemoreceptor trigger zone (CTZ) ([@A16710R3]). Due to the presence of the blood-brain barrier at the CTZ, neurons at this region are stimulated by opioids present in the systemic circulation ([@A16710R17]). Furthermore, opioid receptors are involved in reduction of bowel motility. This effect results in bowel distension, increased GI emptying time and constipation, leading to visceral chemoreceptors and mechanoreceptors stimulation. This effect is often responsible for nausea and vomiting in patients receiving opioid drugs ([@A16710R17]-[@A16710R20]). Findings of this study demonstrated that 20 mL intraperitoneal bupivacaine 0.25% administration at the beginning and also at the end of the laparoscopic cholecystectomy process, reduced only visceral and shoulder pains at the 4th postoperative hour, but had no effect on reducing neither PONV, nor opioid requirement, during the first four postoperative hours. However, further studies are needed on larger populations with different doses and concentrations of bupivacaine and also combination of bupivacaine and other analgesic drugs, to gain the maximum benefits of intraperitoneal analgesic in postoperative pain treatment. We would like to sincerely thank Shokoufe Khodakarami, Saeedeh Pourchamani, Shiva Azami and Parastu Yari kia for their help and kind cooperation. Authors' Contributions:Mitra Yari took part in study concept and design and participated in manuscript preparation. Parisa Golfam prepared the manuscript and helped with study design and submission of the manuscript. Bahman Roshani helped in manuscript preparationand Nahid Nazari collected the data. Funding/Support:Kermanshah University of Medical Sciences, Kermanshah, Iran	{ "pile_set_name": "PubMed Central" }	[ "treatment", "rest", "probabl", "two", "control", "origin", "intub", "remain", "decreas", "compris", "administr", "pariet", "iran", "bahman", "laparoscopi", "collect", "thank", "bowel", "chemoreceptor", "discharg", "written", "block", "kind", "mechan", "controversi", "student", "valu", "hour", "locat", "squar", "septemb", "pourchamani", "bloodbrain", "use", "peritroc", "gener", "etiolog", "final", "μgkg", "caus", "intraabdomin", "accord", "prior", "signific", "establish", "guidelin", "process", "fundingsupportkermanshah", "khodakarami", "bupivacain", "also", "right", "intervent", "mgkg", "nerv", "perform", "nazari", "popul", "ip", "criteria", "steroid", "medic", "higher", "first", "organ", "h", "involv", "thiopent", "distress", "reliev", "particip", "major", "chi", "ttest", "pressur", "assess", "diminish", "gradual", "base", "author", "andor", "peritonealperitroc", 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Background ========== Type 2 diabetes (T2D) is a chronic disorder of carbohydrate, fat and protein metabolism. The prevalence of diabetes is increasing worldwide. According to the recent World Health Organization (WHO) estimates, more than 180 million people worldwide have diabetes. This number is likely to be more than double by 2030. There will be 300 million people with diabetes by the year 2025 \[[@B1]\]. It is estimated that the developing countries will bear the brunt of diabetes epidemics in the 21st century \[[@B1],[@B2]\]. Mounting evidence indicates that obesity along with increased rates of other chronic inflammatory diseases, such as hypertension (HT) and cardiovascular diseases (CVD), are highly associated with T2D \[[@B3]\]. How all theses complications arise and manifest together is yet to be solved. Thus, understanding the underlying molecular mechanisms of T2D is essential for developing more targeted and effective therapies and preventive approaches against diabetes-related complications. The integration of computational biology and \"omics\" science facilitates an understanding of the mechanisms of diseases in an integrative way, including elucidation of complex networks of genes and proteins along with their regulatory networks \[[@B4]-[@B7]\]. Protein-protein interactions (PPI) regulate nearly every living process. Availability of high-throughput genomic data has enabled unprecedented views of gene and protein co-expression, co-regulations and interactions in cellular systems. The approach of this present study was to build an integrated network model system comprised of high-throughput genomic data along with associated computational analysis that would enable predictions of systems level view of normal and aberrant genes, their connections and their functions- to elucidate the underlying molecular mechanisms. Herein we mined genes differentially regulated through T2D and their \"wiring\" which will provide a more complete understanding of the overall gene network topology and their roles in disease progression. By elucidating these networks, exploring how they have been linked and characterizing how they have evolved- novel disease markers can be identified. In this present study, by analyzing the genes related to T2D, HT and OBS, a highly regulated gene-disease integrated network model was developed. PPI sub-networks containing the hub genes involved in T2D-associated signaling pathways revealed that the oxidative stress mediated regulation cascade, is the link, associated with the mechanism of pathogenesis of type 2 diabetes, hypertension and other inflammatory diseases such as obesity. Methods ======= The present work has developed gene interaction network models, comprised of high-throughput genomic and proteomic data for type 2 diabetes (T2D). The overall strategy of the integrated network model generation has been illustrated in Figure [1](#F1){ref-type="fig"}. The network model has been built in three successive stages. The first stage consisted of the \"parts list\" generation using biomedical literature mining. The second stage was the topological model generation by combining all the pooled genes and their interactions from all available interaction databases. The co-regulated and co-expression data fetched helped to identify the involved genes, proteins and their interactions. In third and final stage the fetched interaction data were filtered against experimentally validated interaction (e.g., yeast 2 hybrid, Chip- chip interaction, immuno-coprecipitation, arrays co-expression) data sets and the functional annotations were analyzed using computational approaches. By identifying the genes, the pathways they are involved in and the interactions they are part of, using a systems approach; the integrated networks of T2D have been generated considering all experimentally verified PPI data of the regulating genes. Finally from the integrated network systems the \'hubs\' or key genes that play central roles in these diseases have been identified. Interestingly along the way of building up the network for T2D, gene networks of other related diseases like obesity and hypertension were also generated. The aim of this study was to identify the key switches of regulation and from that interpreting how these chronic diseases correlate and co-occur. ![Concept and overall strategy for generating the integrated network of T2D.](1755-8794-3-45-1){#F1} Stage 1: Initial disease related gene pool generation through extensive text mining ----------------------------------------------------------------------------------- The most important collection of scientific publications is PubMed <http://www.ncbi.nlm.nih.gov/pubmed>, which currently contains more than 19 million citations of biomedical articles from MEDLINE and Life Science journals. Citations include links to full-text articles from PubMed Central (PMC) <http://www.ncbi.nlm.nih.gov/pmc/> or publisher web sites. Currently the number of publications is growing by more than 500K documents per year. PMC is the U.S. National Institutes of Health\'s (NIH) free digital archive of biomedical and Life Sciences journal literature serving the critical role of providing access to published literature toward the first step in the synthesis and translation of genomic research in an easy and comprehensive way. PubMed basically supports keyword based searching and information Retrieval (IR). There have been a significant number of web-based text mining tools available to biologists to discover hidden relationships among biological entities. Some of them are quite effective in generating annotated relationships among graphs in the form of networks. AliBaba <http://alibaba.informatik.hu-berlin.de> is a tool that is capable of automatically and interactively extracting the most valuable information and graphically summarizing search results from PubMed \[[@B8]\]. It uses a dictionary-based approach for recognizing biomedical objects. Dictionaries consist of regular expressions depicting terms and spelling variations. The dictionaries are collected from different sources. To find associations among entities, AliBaba uses two different techniques in parallel: pattern matching and co-occurrence filtering. It mines relationships among cells, diseases, drugs, proteins, species and tissues based on a user query involving text key terms. Thus, AliBaba can be used to turn unstructured text into structured data records \[[@B8]\]. Specifically, for increasing the accuracy and efficiency for discovering relationships between important biological entities, e.g., protein-to-disease associations- the present study combines manually locating and querying information about disease genes entity from the biomedical literature together with automated computational tools to identify the genes and gene products by computationally capturing the related knowledge embedded in textual data. Stage 2: Using the pooled disease genes protein-protein interaction (PPI) maps construction ------------------------------------------------------------------------------------------- PPI maps have considerable impact on the discovery and synthesis of molecular networks. Thus, generating human protein interaction maps has become an important tool in biomedical research for the elucidation of molecular mechanisms and the identification of new modulators of disease processes. There have been a number of web-based tools available to biologists. The Unified Human Interactome database (UniHI, <http://www.unihi.org>) provides a comprehensive platform to query and access human protein-protein interaction (PPI) data \[[@B9]\]. The latest update of UniHI includes over 250,000 interactions between \~22,300 unique proteins collected from 14 major PPI sources \[[@B9]\]. However, this amount of data has its challenges. Even searches with a small number of query proteins can lead to large, highly connected and often unstructured networks (frequently referred to as \'hairballs\'). UniHi has integrated separate PPI resources to provide a comprehensive platform for querying the human interactome (Figure [2](#F2){ref-type="fig"}). UniHI is not intended to replace single databases, but to offer a convenient single portal access to human protein interaction data for the biomedical research community. Additionally, it allows the identification of network topologies which would not be detectable if PPI resources were examined separately. ![Steps of network model generation.](1755-8794-3-45-2){#F2} Pathway information can provide highly useful clues about the functions and dynamics of interactions. Especially for the elucidation of local network structures, knowledge of interrelated pathways can be of crucial importance. UniHI provides the possibility to examine the intersection of canonical pathways from Kyoto Encyclopaedia of Genes and Genomes (KEGG) with the extracted networks \[[@B10]\]. UniHI Scanner does not only show the proteins included in the pathway but also the KEGG annotation of the interactions (e.g. phosphorylation, activation or inhibition) between nodes \[[@B11]\]. In this way, it enables researchers to detect possible modifiers of pathways as well as proteins involved in the cross-talk between pathways and users can switch between the graphical display of the complete network and the intersection with selected pathways \[[@B11]\]. Stage 3: Filtering and validation of the PPI data ------------------------------------------------- Advances in recent genome-wide interactome projects have generated a wealth of PPI data. In order to understand the complexity, it is essential to extract meaningful information in the context of physiological systems. This necessitates identification of not only the function of individual proteins, but also to validate the physical interactions and biological processes in which they participate. The emergence of large scale protein-protein interaction maps has opened up new possibilities in systematically surveying and studying the underlying biological system. UniHI (the Unified Human Interactome database, <http://www.unihi.org>) integrates protein interaction data with pathway data from the Human Gene Expression Atlas \[[@B11]\]. In UniHI, PPI maps have been assembled using eight publicly available large-scale interaction maps: three literature-based, three orthology-based and two Y2H-based. Interactions in different maps are compared. For normalization of the intersections however, only the number of interactions between common proteins is used. Thus, the interaction overlapis defined as the average percentage of shared interactions between common proteins \[[@B12]\]. In UniHI, maps are subsequently clustered based on the interaction overlap. For all comparisons, it is notably larger than zero, which is the expected value for comparison of random maps. The observed concurrence of interaction maps does not occur merely by chance as it is validated by two permutation tests for pair-wise comparisons of graphs, where the observed overlap of interactions generated is highly significant for all comparisons (P\< 0.01) \[[@B12]\]. To measure the conservation of connectivity between pairs of networks, UniHI correlates the number of interactions of proteins in the two networks using Spearman correlation for the set of common proteins \[[@B12]\]. A high correlation between two maps signifies that the interaction-rich (interaction-poor) proteins in one map are also interaction-rich (interaction-poor) in the other map. Finally, for the functional coherency of maps, UniHI employs the gene annotations available in GO \[[@B12]\]. Using UniHI, users can filter interacting proteins by requiring a minimum expression threshold, and the PPI network retrieved from UniHI can be reduced to include only highly expressed proteins or extended to include lowly expressed proteins. Additionally, the PPI resource to be queried can be specified. In this work, the fetched interactions data have been filtered against experimentally validated interaction (yeast 2 hybrid, Chip-chip interaction, immuno-precipitation, arrays co-expression) datasets. Integrated network visualization and identifying the \'hubs\' ------------------------------------------------------------- Flexible visualization is a crucial prerequisite for the display and evaluation of network structures. UniHI \[[@B9]\] provides users to switch between the graphical display of the complete network and the intersection with selected pathways. *Cytoscape<http://www.cytoscape.org/> is another open source bioinformatics software platform for visualizing molecular interaction networks and biological pathways and integrating these networks with annotations, gene expression profiles and other state data \[[@B13]\]. Although Cytoscape was originally designed for biological research, now it is a general platform for complex network analysis and visualization. Cytoscape coredistribution provides a basic set of features for data integration and visualization. A variety of layout algorithms are available, including cyclic, tree, force-directed, edge-weight, and yFiles organic layouts. Additional features are available as pluginswhich are available for network and molecular profiling analyses, new layouts, additional file format support, scripting, and connection with databases \[[@B13]\]. Cytoscape supports a lot of standard network and annotation file formats including: SIF, GML, XGMML, BioPAX, PSI-MI, SBML, OBO, and Gene Association. In this work along with UniHI \[[@B11]\], the interactions among the differentially expressed genes were visualized by using Cytoscape 2.5.1 software \[[@B13]\]. Candidate genes, which were common within various diseases, were identified. Network-neighbours around the fetched genes of T2D, HT, OBS and ROS been studied extensively to identify the \'hubs\'. Genes with thirty or more gene degree in the PPI network were considered as hubs. Results ======= Gene pool generation through extensive text mining -------------------------------------------------- Candidate genes, responsible for the diseases (T2D, HT, OBS, ROS) were extracted and sorted out both manually (through literature review) and by using text-mining soft wares (Figure [3](#F3){ref-type="fig"}). At the beginning, the present work only focused on to understand the correlation between type 2 diabetes and hypertension (HT). But later on, ROS (Reactive Oxygen Species) and obesity (OBS) were also considered as they were found to be highly related to first two diseases. So, at the end candidate genes, responsible for all the diseases were pooled through text-mining. A number of genes for T2D, HT, OBS and ROS were identified. Among them the candidate genes were selected based on the following criteria\'s: i) Genes experimentally proved to be associated with any one of the above mentioned diseases, ii) Genes experimentally proved to be associated with two or more concerned diseases, iii) Genes predicted computationally to be associated with any individual disease, iv) Genes predicted computationally to be associated with two or more concerned diseases and v) Genes found in KEGG pathways database to be associated with any individual disease. So basically, a gene-disease network is generated by defining two genes as \'connected\' if they have been studied for association with the same disease(s). The identified genes were further verified through literature review to get insight about their functions and also with KEGG for annotation in different biological pathways. These genes were also confirmed with Phenopedia (HuGEpedia: an integrated, searchable knowledge base of genetic associations and human genome epidemiology) \[[@B14]\] which provides the total number of reported genes that have genetic association with T2D, HT and other related diseases (Additional file [1](#S1){ref-type="supplementary-material"}, [2](#S2){ref-type="supplementary-material"}). After this first stage of text mining using literature databases, the number of genes identified and pooled for: Type 2 Diabetes (T2D) \~ 257, Hypertension \~100, Obesity \~239 and ROS \~55. ![Text mined data and the initial gene pool list.](1755-8794-3-45-3){#F3} Finding Protein-Protein Interaction partners (PIPs) --------------------------------------------------- Genes do not act as individual units; they collaborate in overlapping pathways, the co-regulation of which is a hallmark for the disease pathogenesis. A simple enrichment analysis has been applied in order to characterize the T2D set on the network level including pathways and protein-protein interactions. The computational approach used in this study was based on functional links derived from co-expression and co-regulation profiles. The co-expressed and co-regulated genes generally have a higher likelihood of having a direct physical interaction. The gene interaction data used to build the network was based on direct physical interactions that are either experimentally derived (e.g., Y2H or co-affinity purification) or computationally predicted. In order to integrate pathway information and to derive cellular network information on the selected genes, functional annotation from pathway databases such as KEGG, Reactome, BioCyc \[[@B15]-[@B17]\], protein-protein interaction databases such as BIND, DIP, REACTOME, COCIT, HPRD BIN, CCSB, MDC and IntAct was added \[[@B18]\]. To facilitate the findings, UniHi (United Human Interactome) \[[@B9]\], a web based PIP finding tool was used. The present work tried to find significantly interconnected sub-networks to interrogate whether the specific pattern formed by a pre-specified list of genes is significant (Figure [4](#F4){ref-type="fig"}). At this stage the number of significant candidate genes of inflammation related diseases were T2D \~1231, HT \~1000, OBS \~1845 and ROS \~417. ![Mined PPI data and graphical visualization using UniHI.](1755-8794-3-45-4){#F4} Compiling of data and network visualization ------------------------------------------- Network subgraphs can be network modules, motif clusters, or other network neighborhoods. A network module can be defined as a subgraph consisting of highly interconnected nodes that may fulfill a particular biological function \[[@B19]-[@B21]\]. At this stage of network building, the pooled important common genes for various diseases were integrated into network models and visualized using UniHI \[[@B18]\], Cytoscape \[[@B13]\]. The models have been generated through several stages of screening where in subsequent steps the pooled data have been filtered, verified for authentication and most importantly, validated with experimentally derived data to make the output graph generated for different diseases meaningful and interpretable. This work constructed four disease networks (Additional file [2](#S2){ref-type="supplementary-material"}), each containing a set of proteins that are associated with the diseases. The hub nodes form the backbone of a network, they are considered to be an important measurement for the similarity between protein interaction networks. Moreover, backbone network has also been shown to be highly conserved in maintaining biological function of the cell \[[@B22]\]. Within the networks the \"hubs\" are defined as the protein nodes with degrees ≥ 15 in a disease network \[[@B22]\]. The generated model of T2D has predicted the highest number of hub proteins among the four diseases we studied so far, probably due to the fact that it has the highest number of genes that were used to construct the disease network (Additional file [3](#S3){ref-type="supplementary-material"}). Molecular linkage among T2D, HT, OBS and Inflammation ----------------------------------------------------- To investigate the molecular cross-talk within these interrelated disease mechanisms, the individual disease networks were looked thoroughly at their \'hubs\' to understand the underlying connections between genes and the disease mechanism. This was done in three phases. First, we developed the gene-disease network using the text miner Ali-Baba \[[@B8]\] (Figure [5](#F5){ref-type="fig"}). Thorough analyses of the gene patterns and their relatedness within different diseases, the way they were connected, the cross talk - a gene list was generated containing the connections in all four diseases. In the second phase, the PPI patterns around the selected hub genes were studied extensively using UniHI \[[@B9]\], validated the connections using Gene Ontology (GO) \[[@B23]\] and Genopedia and Phenopedia \[[@B14]\] databases for annotation (Figure [6](#F6){ref-type="fig"} and Additional file [4](#S4){ref-type="supplementary-material"}). And finally in the third phase, the selected genes were cross-validated and clustered using the pathway database KEGG \[[@B10]\] (Figure [7](#F7){ref-type="fig"}). During the whole process a list of 42 co-regulated genes were analysed and these candidate genes were significantly clustered in 19 pathways. The candidate genes were looked for common interaction partners and the highly integrated gene regulatory network was generated (Figure [8](#F8){ref-type="fig"}). ![Cross-linkage between various diseases.](1755-8794-3-45-5){#F5} ![Main \'hub\' genes and connections.](1755-8794-3-45-6){#F6} ![Important signaling pathways integration with gene network.](1755-8794-3-45-7){#F7} ![Gene regulatory network around the highly inter-connected key hubs.](1755-8794-3-45-8){#F8} Discussion ========== In this study, a text mining approach was used to identify the disease-related genes of T2D and HT. Along the way of querying, genes associated with oxidative stress (reactive oxygen species, ROS) and OBS was also included based on- the two diseases are \'connected\' if they have been studied for association with the same gene(s) hypothesis. These data were further analysed using various novel bioinformatics tools and resources to identify the key candidate hub genes associated with various diseases. Literature-based annotation analyses showed that all these diseases T2D, HT, OBS are somehow correlated with oxidative stress and inflammation (Figure [5](#F5){ref-type="fig"}). Further PPI studies, generated the disease-gene network within T2D, HT, OBS, ROS and Inflammation. In this work, the genes for inflammation were never pooled separately but from the cross-talk between the networks of T2D, HT and OBS, the strong co-regulated inflammation genes were identified. Although at this point it was not completely understandable how ROS or inflammation plays role in disease mechanism. Further pathway-based investigation, using both Genopedia \[[@B14]\] and KEGG \[[@B15]\] provided more insights about the roles of these genes in association with different disease phenotypes. This work predicted the involvement of about 20 enriched pathways, some of which are very commonly involved in diabetes and/or obesity like the insulin signaling, PPAR signaling, Adipocytokine signaling, MAPK signaling, Jak-STAT signaling pathways. But along them, the involvement of Wnt signaling, p53 signaling, ErbB signalling, TGF-beta signalling, Focal adhesion, toll-like receptor signaling pathways were predicted, whose associations are more widely linked with cancer metastasis (Figure [8](#F8){ref-type="fig"} and Additional file [4](#S4){ref-type="supplementary-material"}). From pathway analysis (Figure [7](#F7){ref-type="fig"}), the involvement of p53 or Wnt signaling pathway shed new light to focus at the PPI networks. After further filtering of the data (through network and/or PubMed interaction partners, IPs) along with cross validation through literature annotation, the most interesting 46 candidate \'hub\' genes were identified. Careful investigation around these \'hubs\' provide more meaningful insights about the cross- talk within gene-disease networks in terms of disease phenotype association with ROS and inflammation (Figure [6](#F6){ref-type="fig"}). From network analysis, common IPs revealed that all the diseases T2D, HT, OBS are linked via two common gene regulatory cascades: (i) EP300/TP53/MYC/CDKN1A/STAT3/CXCR4 and (ii) PPARG/PPARA/IGF1R/AKT1/LEP/LEPR. Basically, these regulatory routes connect insulin signaling pathway through oxidative stress and inflammation. From investigating the \'hub\' gene network connections and broadly studying the interconnection, one hypothesis could be, the components of the second routes can only be regulated through the first or vise-versa (Figure [8](#F8){ref-type="fig"}). To further investigate the hypothesis, and to address how the connections regulated within the network that could be associated with genesis of inter-connections between various diseases, the \'gene-links\' associating two or more hubs were identified. In course of the analysis, regulations around EP300, PPARG, PPARA, STAT3, IGF1R, CXCR4, MYC, TP53, CDNK1A, CDKN2A, PCAF, HIF1A, CREBBP, CEBPB, RELA, HSP90AA1, MAPK3, MAPK8, JAK2, JAK3, PTPN11, TFAP2A, LEP, TCF7L2, CTNNB1, FOXO1A, FOXO3A and GSK3B seem to play crucial roles in the regulation of T2D and other related disorders (Figure [8](#F8){ref-type="fig"}). Interestingly, the genes like GSK3B, PCAF, HIF1A, CREBBP, CEBPB, RELA, TCF7L2, CTNNB1, MYC, TP53, CDNK1A are known as important role players in Wnt and p53 signaling pathways, which would provide a putative link between T2D and certain types of cancer. The molecular events leading to β-cell failure in the diabetic environment, in particular high levels of glucose and free fatty acids, exert toxic effects on the β-cell \[[@B24]\]. A number of signaling pathways have been implicated in β-cell failure, including insulin signaling \[[@B25]\] and oxidative stress \[[@B26]\]. Ectopic overexpression of the Wnt target gene c-myc in mice has been shown to cause β-cell apoptosis and diabetes \[[@B27]\]. Also extensive recent investigations have revealed the existence of molecular crosstalk between insulin and Wnt signaling pathway \[[@B28]-[@B32]\]. A number of genetic studies have confirmed that TCF7L2, a direct downstream target of β-catenin of the Wnt pathway, to be involved in β-cell dysfunction and the etiology of T2D \[[@B24],[@B33]\]. There is clear evidence that TCF7L2 regulates insulin secretion rather than insulin action. A central feature of the Wnt/β-catenin pathway is the regulation of cytosolic β-catenin protein levels via a destruction complex containing glycogen synthase kinase-3b (GSK-3b). GSK-3B has long been known as an important mediator for impaired insulin action on peripheral tissue and in the development of insulin resistance \[[@B34]\]. However, only recently, studies have shed light on the growth regulatory properties of GSK-3B in β-cells. Two elegant studies from the same group reported recently that GSK-3B overexpression in mice induces β-cell mass restriction and the development of diabetes \[[@B35]\] and that the genetic disruption of GSK-3B in β-cells results in increased β-cell mass in those transgenic mice \[[@B36]\]. Importantly, β-catenin is able to interact with the FoxO family of transcription factors, which defend against oxidative stress by stimulating the transcription of oxidant scavenging enzymes such as superoxide dismutase and catalase \[[@B37]\]. Since FOXO and TCF proteins compete for a limited pool of β-cat, enhanced FOXO activity during ageing and oxidative stress may attenuate Wnt-mediated activities. The decrease of β-catenin not only attenuates Wnt signaling but also unleashes the expression of PPARγ, which is normally suppressed by β-catenin \[[@B38]-[@B40]\]. Oxidative stress activates the FoxO family of transcription factors, which in turn attenuate β-catenin/TCF-mediated transcription, leading to derepression of PPARγ transcription. The increase in PPARγ levels serves as an additional β-catenin sink by sequestering it and activating its proteasomal degradation \[[@B41]\]. Adipogenesis is also affected by oxygenation, low oxygen tensions activate HIF (hypoxia inducible factor)-1α, inhibiting differentiation by repressing PPARγ (peroxisome proliferator-activated receptor γ) \[[@B42]\] whereas higher oxygenation tends to induce differentiation \[[@B43]-[@B45]\]. HIF-1α has been shown to compete with TCF4/TCF7L for direct binding to β-catenin \[[@B46]\]. A recent work showed that Wnt signaling (which is up-regulated in high oxygen) has been associated with increased β-cell proliferation \[[@B47]\]. Thus, the increase in Wnt activity associated with higher oxygen availability might have the twofold effect of increasing (a) endocrine cell mass by self replication and (b) exocrine cell mass by progenitor cell expansion \[[@B48]\]. Another study showed the involvement of p53 expression in adipose tissue is crucial in the development of insulin resistance. The work showed in animal model with T2D-like disease, excessive calorie intake led to the accumulation of oxidative stress in the adipose tissue and promoted increased activity of senescence-associated β-galactosidase, increased expression of p53 and increased production of pro-inflammatory cytokines. Conversely, up-regulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance \[[@B49]\]. It has also been reported that production of reactive oxygen species (ROS) is selectively increased in the adipose tissue of obese mice and that increased oxidative stress in fat is a key mechanism underlying the occurrence of insulin resistance related to obesity \[[@B50]\]. The ROS-induced p53 activation causes NF-κB-dependent induction of inflammatory cytokines and thus accelerates the development of diabetes. Once oxidative stress is initiated it affects multiple systems. By reaction of ROS with NO, oxidative stress is increased while NO is diminished, thus promoting inflammation and endothelial dysfunction \[[@B51]\]. Specifically, ROS were implicated in mitogen-activated protein kinase (MAPK) pathways, which induce activation of various nuclear transcription factors, such as nuclear factor (NF)-κB, activator protein (AP)-1, hypoxia-inducible factor (HIF)-1a, sterol regulatory element binding proteins (SREBPs) and GATA-4 \[[@B52]-[@B54]\]. NF-κB participates in obesity and the metabolic syndrome; it induces inflammatory and atherosclerotic consequences. Obesity increases the formation of reactive oxygen species in fat cells, and ultimately results in activation of the p53 tumor suppressor, inflammation and the promotion of insulin resistance \[[@B55]\]. Also, insulin resistance is affected by oxidative stress and, when combined with up regulated NF-κB activity, may promote type 2 diabetes. Conversely, hyperglycaemia was also shown to trigger increased formation of ROS via glucose auto-oxidation. Accordingly, consumption of a high free-glucose diet promoted the development of oxidative stress \[[@B56]-[@B58]\]. HIF-1a, is also associated co morbidities such as hypertension \[[@B59],[@B60]\] and hyperlipidaemia \[[@B61]\]. High glucose concentration up-regulates SREBP-1c and insulin resistance \[[@B62]\]. However, none of these mechanisms are yet fully elucidated \[[@B63]\]. Proposed hypothetical model of mechanism ---------------------------------------- Based on an elaborate study of the key \'hubs\' regulations, their association, along with detailed literature reviewing - in this work we put the puzzle pieces together and proposed a hypothetical mechanism for co-regulation of various inflammatory diseases like T2D, HT and OBS (Figure [9](#F9){ref-type="fig"}). From the generated regulatory networks around key \'hub\' genes it is very much visible that via EP300 the main pathways like insulin signaling, PPAR signaling, calcium signaling, adipocytokine signaling, Jak-STAT signaling pathway, MAPK signaling pathways that are well recognised in association with disease like T2D, OBS and HT. Interestingly, the analyses of the regulatory cascades showed that the genes involved in main disease pathways are all connected and regulated via the genes involved in Wnt-signaling and p53 signaling pathways. ![Proposed hypothetical model of T2D mechanism.](1755-8794-3-45-9){#F9} In detailed study of the gene-disease network and the regulation around the \'hubs\', this work suggests EP300/TP53/CDKN1A/STAT3/TCF7L2/MYC/CXCR4 is the main regulatory cascade that generates the highly connected, co-regulated network of insulin signaling which in turn, could be responsible for the genesis of molecular cross-talk within various diseases. It has been suggested that insulin signaling crosstalk with Wnt signaling, due to the existence of common downstream target genes and the shared negative mediator GSK-3 \[[@B33]\]. However, mechanisms underlying this potential crosstalk still remain unclear \[[@B33]\]. β-catenin, an intracellular signaling molecule is essential for Wnt activation. Without Wnt signal, β-catenin, is constantly phosphorylated and thereby inactivated by GSK3B, is eventually degraded to prevent its accumulation. Inactivation of GSK3B, in turn can no longer phosphorylate β-catenin. This leads to nuclear translocation of β-catenin and subsequently, which coactivates TCF/LEF transcription factors \[[@B64]\]. Our regulatory gene- network could fit all those observations and explain a bit more these underlying molecular mechanisms. Wnts induce glucose-stimulated insulin secretion and β-cell proliferation \[[@B65]\]. This signaling system might senses insulin sensitivity via the regulator gene GDK3B inactivation/activation (phosphorylation/dephosphorylation) by increasing insulin effects through AKT1/IGF1R/STAT3/MYC/CDKN1A/TP53/EP300 cascade via activating Akt/PKB and inhibiting the MAPK pathways (Figures [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"} and [9](#F9){ref-type="fig"}). In the absence of insulin, FOXOs upregulate the expression of a set of target genes via AKT1/IGF1R/CXCR4/STAT3/EP300/PPARG cascade, thereby promoting cell cycle arrest, stress resistance and apoptosis \[[@B65]\]. In the presence of insulin, FOXOs are phosphorylated by AKT/PKB protein kinase and stay in the cell cytosol \[[@B66]\]. In contrast to the effect on insulin signaling, oxidative stress (ROS) induces the activation of FOXO signaling \[[@B67]\]. This might be due to the activation via the Jak/STAT signaling pathway \[[@B68]\]. T2D, age, obesity- all increases ROS that in turn increases lipid oxidation, via ROS/FOXO/PPARG/PPARA/β-catenin regulation leads to PPARG activation and repression of Wnt-signaling. So, when PPARG activates it attenuates Wnt-signaling, which induces lipid oxidation that in turn increases ROS. Increased oxidative stress diminished Wnt signaling that may leads to β-cell destruction via FOXO apoptosis \[[@B69]\]. Regulatory gene network (Figure [8](#F8){ref-type="fig"}) suggests that one possibility could be via CREBBP regulation on MYC/TP53/EP300/TCF7L2 cascade, the system might sense signal to FOXO. Since FOXO and TCF compete for limited pool of β-cat, enhanced FOXO activity during oxidative stress may attenuate WNT- mediated activation \[[@B70]\]. Thus, oxidative stress lead to FOXO mediated gene transcription and reduced TCF mediated gene transcription. Conversely, activation of Wnt pathway through inactivation of GSK3B, stabilise β-catenin, that regulates β-cell proliferation via TCF7L2, a down stream effector of this cascade \[[@B69]\]. This in turn coactivates PPARG mediated transcription on the glucokinase gene promoter. Glucokinase is the key regulator of glucose-sensing in pancreatic beta-cells, thereby offering a model for the adipocyte-induced hyper secretion of insulin \[[@B71]\]. Although TCF7L2 expression was positively correlated with the expression of INS, which encodes insulin, it was inversely correlated with glucose-stimulated insulin release \[[@B72]\]. There is clear evidence that TCF7L2 regulates insulin secretion rather than insulin action \[[@B65]\]. Thus, β-catenin interaction with TCF7L2 with nuclear co-activators, such as EP300, results in the stimulation of Wnt or β-cat/TCF downstream target gene transcription \[[@B73]-[@B75]\]. From the gene regulation network, it can be seen that insulin may also stimulates the transcription via MYC, a known downstream target of Wnt signaling, via a PI3K-dependent but Akt/GSK3B-independent mechanism, via IGF1R/MYC/TP53/EP300 regulatory cascade. Indeed, several other studies have also reported the stimulatory effect of insulin or IGF1 on β-cat nuclear localization and cat/TCF mediated gene transcription \[[@B29],[@B33],[@B76],[@B77]\]. Thus, fitting all these evidences in the proposed hypothetical model (Figure [9](#F9){ref-type="fig"}), it can be suggested that insulin signaling may crosstalk with Wnt signaling, due to the existence of common downstream target genes, the shared negative mediator GSK3B, and insulin resistance are capable of explaining the association of T2D co-occurrences with other inflammatory disease like HT, and OBS. T2D and/or OBS can develop independently, due to genetic, behavioral or lifestyle-related variables but both T2D and OBS lead to oxidative stress generation \[[@B78]\]. The pathogenic mechanisms by which diabetes and oxidative stress induce inflammation are not certain at the present time \[[@B79]\]. But the predicted model, at least provided much more logical explanation to fit what so far been known about the inflammation mediated consequences. Excessive calorie intake led to the accumulation of oxidative stress with T2D that can promote increased activity of senescence-associated β-galactosidase, increased expression of p53 and increased production of proinflammatory cytokines. Conversely, upregulation of p53 in adipose tissue can cause an inflammatory response that led to insulin resistance \[[@B79]\]. Also oxidative stress through TCF7L2/NF-kB/MAPK/HIF1A/SREBP regulatory cascade via insulin, MAPK and Calcium signaling pathways, may activate inflammatory responses than in turn causes insulin resistance. Hyperglycemia could lead to increase lipid oxidation and endothelial dysfunction regulated through PPARG/STAT3/CXCR4/ICAM/VCAM cascade via Chemokine, Adipocytokine and Jak-STAT signaling pathways that could cause increase expression ICAM1, VCAM1 levels which in turn increases inflammation and obviously, increases oxidative stress \[[@B80],[@B81]\]. Once oxidative stress is initiated it affects multiple systems-via AKT1/NOS3 regulation. Increased oxidative stress can diminish NO production, thus promoting inflammation and endothelial dysfunction \[[@B51]\]. Inflammatory pathway activation will lead increased expression of adhesion molecules, cytokines and associated with increased risk of HT. Obesity, insulin resistance and hypertension commonly cluster with other risk factors for CDV or chronic kidney disease to form the metabolic syndrome \[[@B65]\]. Conclusion ========== It can be hypothesised from the generated model (Figure [9](#F9){ref-type="fig"}) that oxidative stress is a direct cause for the Wnt pathway activation via the high glucose-induced β-catenin activation through a highly inter-connected gene regulatory cascade, where EP300 plays a role as a key regulator in T2D. Even though these predictions need further experimental validation, the hypothetical model presented in this study could be a starting point to visualize in a systems approach understanding the molecular cross- talk between diseases. The findings provide a novel integrated approach for understanding the pathogenesis of various co-associated diseases by combining the power of pathway analysis with gene regulatory network evaluation. The integrated network model developed by this work provides useful functional linkages among groups of genes and thus addressing how different inflammatory diseases such as obesity, diabetes, and hypertension are connected and propagated at genetic level. It is anticipated that derived models will be of great benefit to a wide research audience, including those involved in disease biomarker identification and drug development. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= Jesmin, MSMR and HJ coordinated the text mining and information extraction, Jesmin, MSMR mined and computed the interaction data, Jesmin, HJ, JB-R, and RH analysed and interpreted the result, drafted and revised the manuscript. All authors have read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1755-8794/3/45/prepub> Supplementary Material ====================== ###### Additional file 1 Statistics of the reported genes associated with T2D, HT, OBS and ROS. ###### Click here for file ###### Additional file 2 Complete \'text-mined\' data set. ###### Click here for file ###### Additional file 3 Predicted integrated network model of the four diseases (using Cytoscape). ###### Click here for file ###### Additional file 4 Gene annotation using GO, Genopedia and Phenopedia*. ###### Click here for file Acknowledgements ================ We thank Dr Zaheed Husain (Division of Interdisciplinary Medicine & Biotechnology, BIDMC, Department of Medicine, Harvard Medical School, Boston) for valuable comments and suggestions that helped improve the manuscript.	{ "pile_set_name": "PubMed Central" }	[ "unifi", "miner", "amount", "glucoseinduc", 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"High", "glucose", "concentration", "insulin", "resistance", "However", "none", "mechanisms", "yet", "fully", "elucidated", "Proposed", "hypothetical", "model", "mechanism", "Based", "elaborate", "study", "key", "regulations", "association", "along", "detailed", "literature", "reviewing", "work", "put", "puzzle", "pieces", "together", "proposed", "hypothetical", "mechanism", "various", "inflammatory", "diseases", "like", "HT", "OBS", "Figure", "fig", "generated", "regulatory", "networks", "around", "key", "genes", "much", "visible", "via", "main", "pathways", "like", "insulin", "signaling", "PPAR", "signaling", "calcium", "signaling", "adipocytokine", "signaling", "signaling", "pathway", "MAPK", "signaling", "pathways", "well", "recognised", "association", "disease", "like", "OBS", "HT", "Interestingly", "analyses", "regulatory", "cascades", "showed", "genes", "involved", "main", "disease", "pathways", "connected", "regulated", "via", "genes", "involved", "signaling", "pathways", 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"Division", "Interdisciplinary", "Medicine", "Biotechnology", "BIDMC", "Department", "Medicine", "Harvard", "Medical", "School", "Boston", "valuable", "comments", "suggestions", "helped", "improve", "manuscript" ]
Organ formation and modifications in tissue architecture occurring during development involve deconstructing both cell adhesion and the extracellular matrix[@b1][@b2][@b3], thereby converting cells into a migratory state with a new tissue identity; this is known as epithelial--mesenchymal transition[@b4][@b5]. Although epithelial cells and mesenchymal cells have contrasting properties, the molecular mechanisms behind these properties are not entirely mutually exclusive. Some epithelial cells acquire the ability to migrate while retaining cell--cell adhesion, allowing these cells to migrate as a group[@b6][@b7][@b8][@b9]. This is possible due to the polarized nature of the epithelia; the apical side retains the cell--cell interface, while migratory protrusions form on the basal cell surface. On reaching their destination, these migratory epithelia are integrated into the target tissues. However, how migratory epithelia find their target tissues, establish new cell--cell interfaces and integrate with the target tissue are poorly understood. Studies of the formation of adhesive interfaces in cultured cells showed that contact with actin-enriched filopodia initiates the assembly of the adherens junction[@b10][@b11] and suggested that microtubules are involved in this process[@b12]. As the formation of cell junctions involves coordinating the assembly of the cell adhesion complex and the generation of tension in each side, the cells forming the junction must be observed simultaneously to understand how mechanical coupling is controlled at the tissue level. Here, to examine how cell adhesion and cell contractility are coupled to establish a new cell interface, we examined the anastomosis formation in the Drosophila tracheal system. In this process, a pair of tip cell (hereafter called fusion cell, FC) forms an adherens junction de novo ([Fig. 1a,b](#f1){ref-type="fig"}) and deposit extracellular matrix materials into newly forming lumen. Each FC is converted into a torus shape by plasma membrane fusion via the inner leaflet[@b16][@b17][@b18], under the control of endosome and microtubule-associated protein Arl3 (refs [@b8], [@b19], [@b20]). Interestingly, plasma membrane fusion always occurs simultaneously in each FC, suggesting cell shape change and membrane trafficking in each FC pair is highly coordinated. However, how cell adhesion, cell-shape change and membrane trafficking are regulated coordinately in pairs of FCs is poorly understood. We show that FCs form a de novo adherens junction at the contact site through stabilization by a mechanism requiring actomyosin and microtubules; this mechanism applies a balanced pulling force to flatten the FCs. In addition to this cell-intrinsic contractile force, the preferential deposition of molecules via the apical secretion pathway promotes maturation of the matrix in the lumen that forms in the FC contact interface and helps to fuse the plasma membranes. Results ======= F-actin and microtubule dynamics in migrating FCs ------------------------------------------------- FCs expressing the F-actin markers GFP-moesin ([Fig. 1c](#f1){ref-type="fig"}; see also [Supplementary Movie 1](#S1){ref-type="supplementary-material"}) or lifeact-GFP ([Supplementary Fig. 1a](#S1){ref-type="supplementary-material"}), each driven by an FC-specific enhancer (esg_FC enhancer; see Methods), were closely associated with the basal surface of the dorsal epidermis and showed numerous F-actin-enriched filopodia extending towards the dorsal midline[@b18][@b21]. We also observed invasive filopodia that extended vertically from these FCs into the epidermal layer ([Fig. 1c](#f1){ref-type="fig"} open triangles, [Fig. 1c′](#f1){ref-type="fig"} and see also [Supplementary Fig. 1a](#S1){ref-type="supplementary-material"}); their location did not correspond to epidermal cell junctions, suggesting that the filopodia penetrated the epidermal cells. While migrating, the FCs appeared flat with numerous forward-extending filopodia ([Fig. 1c](#f1){ref-type="fig"}, time 0:00 h), but on reaching their target, their shape became more compact, with the lumen extending from the side of the stalk ([Fig. 1c](#f1){ref-type="fig"}, time 1:54 h). Microtubule labelling with GFP-tau revealed extensive microtubule arrays emanating from the microtubule-organizing centre (MTOC) at the proximal side of the cell ([Fig. 1d](#f1){ref-type="fig"}, time 0:00 h; see also [Supplementary Fig. 1b,c](#S1){ref-type="supplementary-material"} and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}) and F-actin labelling revealed many, F-actin-rich protrusions extending forward to reach the target cell ([Fig. 1e](#f1){ref-type="fig"} and see also [Supplementary Movie 3](#S1){ref-type="supplementary-material"}). On FC contact with its partner FC, the MTOC moved to the contact site ([Fig. 1d](#f1){ref-type="fig"}, time 1:25 h). The vertical protrusions revealed by F-actin marking were not observable with microtubule labelling (compare [Fig. 1c′](#f1){ref-type="fig"} and [Supplementary Fig. 1b](#S1){ref-type="supplementary-material"} for another microtubule marker GFP-Jupiter). A polarized flux of E-Cadherin to the new cell junction ------------------------------------------------------- When two FCs came within a proximity of 15.2 μm (on average), fluorescent markers for E-cadherin ([Fig. 2a](#f2){ref-type="fig"})[@b22] and its associated protein p120ctn ([Supplementary Fig. 3a](#S1){ref-type="supplementary-material"})[@b23], accumulated at the contact site. Immunostaining revealed endogenous E-cadherin localized to the extending edge of forward-reaching filopodia on the tip cells ([Fig. 2b](#f2){ref-type="fig"} top). This punctate E-cadherin localization was not observed on the filopodia of terminal cells or stalk cells ([Fig. 2b](#f2){ref-type="fig"} bottom). To monitor the dynamics of E-cadherin transport, we imaged E-cadherin-GFP fluorescence during the fusion process. We found that the E-cadherin-GFP level remained fairly constant at the junction between the FC and stalk cell ([Fig. 2a](#f2){ref-type="fig"}, position and plot marked with 'L\' and 'R\'), but increased steadily at the FC interface, surpassing the intensity at the FC--stalk cell interface within 20 min after contact ([Fig. 2a](#f2){ref-type="fig"}). We next monitored the E-cadherin-GFP turnover rate at each cell interface by fluorescence recovery after photobleaching (FRAP) analysis. All three E-cadherin-GFP foci (L, R and C) were photobleached simultaneously and their rate of fluorescence recovery was measured ([Fig. 2c](#f2){ref-type="fig"} arrowheads). The fluorescence recovery rate and the mobile E-cadherin-GFP fraction were significantly higher at the FC interface than at the L and R foci ([Fig. 2d,e](#f2){ref-type="fig"}). Imaging with high spatiotemporal resolution revealed that E-cadherin-GFP appeared on the free surface of the plasma membrane, with occasional streams from the cytoplasm to the membrane surface, and then accumulated at the FC contact site ([Fig. 2e](#f2){ref-type="fig"} and [Supplementary Movie 4](#S1){ref-type="supplementary-material"}). In addition, Golgi apparatus in FCs became progressively enriched near the contact site ([Fig. 2g](#f2){ref-type="fig"}). The low E-cadherin-GFP turnover at the FC--stalk cell interface suggests that the exchange rate of the E-cadherin complex at this junction, which was already present at the time of dorsal branch (DB) migration, is low. To analyse E-cadherin\'s contribution, we reduced its synthesis by double-stranded RNA-mediated gene knockdown ([Supplementary Fig. 3b](#S1){ref-type="supplementary-material"}). Expressing E-cadherin double-stranded RNA in tracheal cells with the trachea-specific btl-Gal4 driver reduced the level of E-cadherin in DB stalk cell junction by ∼27% (Methods), but did not compromise the overall tracheal morphology or the migration speed of the DB before the FCs came into contact with each other ([Fig. 2h,i](#f2){ref-type="fig"}), suggesting that the amount of E-cadherin gene product in this experimental condition was sufficient to sustain the tracheal tissue architecture. However, the FC contraction was significantly delayed after contact and the interface between the FCs failed to mature ([Fig. 2h,i](#f2){ref-type="fig"}). These results suggest that newly synthesized E-cadherin is preferentially partitioned to the distal side, to form the new cell interface. FC contraction by a myosin-dependent pulling force -------------------------------------------------- We next examined the role of myosin II in the contractile force within the FC. When expressed in FCs, a green fluorescent protein (GFP) fusion protein of the myosin heavy chain Zipper formed longitudinal bundles at the time of FC--FC contact, to connect the adherens junctions on each FC ([Fig. 3a](#f3){ref-type="fig"} and [Supplementary Movie 5](#S1){ref-type="supplementary-material"}). These myosin bundles became shorter as the FCs contracted and became more compact ([Fig. 3a](#f3){ref-type="fig"}). Similar myosin bundles were observed in the dorsal-trunk FCs ([Supplementary Fig. 4a](#S1){ref-type="supplementary-material"}). To determine whether FC contraction is driven by a cell-autonomous force, we assessed the contractile state of laser-perturbed FCs. A microwave-length infrared laser (1,440 nm) effectively penetrated the epidermal layer and produced sufficient heat at the focal point to induce a heat-shock response[@b24][@b25]. Infrared laser illumination targeted to one of the paired FCs caused a cell-specific relaxation of cell contractility ([Fig. 3b](#f3){ref-type="fig"}), with a simultaneous excessive contraction in the intact FC. The progression of the fusion process, assessed by shortening of the L--R length, was also arrested ([Fig. 3c](#f3){ref-type="fig"} and [Supplementary Movie 6](#S1){ref-type="supplementary-material"}, N=3). This result supports the idea that FC pairs normally exert nearly equal pull on each other to maintain their symmetric appearance, and that this balanced pulling force is required to pull FCs closer together. Furthermore, expressing a dominant-negative form of the myosin heavy chain interfered with FC contraction ([Fig. 3d](#f3){ref-type="fig"} and [Supplementary Movie 7](#S1){ref-type="supplementary-material"}, 100% N=9, 3 embryos) and fusion ([Supplementary Fig. 4e,f](#S1){ref-type="supplementary-material"}). Polarized microtubule organization in the FC -------------------------------------------- We next studied the detailed organization of microtubules. The microtubule minus-end marker Nod-LacZ (ref. [@b26]) and the centriole marker GFP-PACT (ref. [@b27]) were concentrated near the FC--stalk cell interface ([Fig. 4a,b](#f4){ref-type="fig"}). From this proximal MOTC, microtubules extended in a fan-shaped pattern to the migrating FC\'s leading edge ([Fig. 1d](#f1){ref-type="fig"} and [Supplementary Fig. 1c](#S1){ref-type="supplementary-material"}). We also imaged the microtubule plus-end marker EB1-GFP in FCs ([Fig. 4c](#f4){ref-type="fig"} and [Supplementary Movie 8](#S1){ref-type="supplementary-material"}); particle image velocimetry analysis revealed rapid, comet-like EB1-GFP signals with a mode velocity of 5 μm min^−1^ moving towards the FC\'s leading edge ([Fig. 4c](#f4){ref-type="fig"}) and an EB1-GFP population moving at even higher velocities (\>10 μm min^−1^) in the periphery ([Fig. 4c](#f4){ref-type="fig"}). Dual-colour imaging of EB1-GFP and the cell membrane revealed that the microtubule plus ends projected to the cortical region of the leading edge and extended repeatedly into the filopodial protrusions ([Fig. 4d](#f4){ref-type="fig"} and [Supplementary Movie 9](#S1){ref-type="supplementary-material"}). The microtubule-filled protrusions were found most frequently in the leading edge, which made contact with the partner FC at the midline and accumulated E-cadherin-GFP ([Fig. 4e](#f4){ref-type="fig"} and [Supplementary Movie 10](#S1){ref-type="supplementary-material"}). Requirement of microtubules for tracheal fusion ----------------------------------------------- To reveal the role of polarized microtubule arrays in tracheal-tube fusion, we perturbed the FC microtubule organization by expressing Katanin p60 (Kat60), a catalytic subunit of the microtubule-severing factor Kat60/p80 (refs [@b28], [@b29], [@b30]). Microtubules in control FCs, visualized by the marker GFP-tau, formed dense central bundles directed towards the leading edge, with diffuse fractions filling the rest of the cell ([Fig. 4f](#f4){ref-type="fig"} top and [Supplementary Movie 11](#S1){ref-type="supplementary-material"}). Analysis of the signal variance in a 200-s interval (20 frames) revealed stable central microtubule bundles and an unstable diffuse fraction ([Fig. 4f](#f4){ref-type="fig"} bottom). In FCs expressing Kat60, the microtubule bundle was misdirected and became unstable (high signal variance, [Fig. 4f](#f4){ref-type="fig"}), indicating that the ectopic Kat60 could destabilize the microtubules. In this condition, endogenous Zip cables connecting the adherence junctions of FCs was reduced ([Supplementary Fig. 2](#S1){ref-type="supplementary-material"}). DBs expressing Kat60 migrated towards the dorsal midline and maintained normal-shaped stalk cells and the FCs contacted each other using filopodia. However, the fusion process was arrested, because the filopodia contact site failed to accumulate E-cadherin or mature into a tight cell contact[@b18] ([Fig. 4g,h](#f4){ref-type="fig"}). In control trachea, signals from the adherens junction marker p120ctn-mRFP increased steadily in the contact site. However, in cells expressing Kat60, the p120ctn-mRFP accumulation was unstable, showing a 'blinking\' pattern ([Fig. 4I](#f4){ref-type="fig"} and see also [Supplementary Movie 12](#S1){ref-type="supplementary-material"}). These results indicate that microtubule destabilization inhibited the accumulation of the E-cadherin complex during fusion[@b18]. To further elucidate the role of microtubules in forming the adherens junctions, we expressed Spastin (Spas), another microtubule-severing factor[@b31], in FCs. Spas was previously demonstrated to inhibit tracheal branch migration and fusion[@b15][@b18]. We found Spas to be less disruptive to the fusion process than Kat60 (Methods), making it suitable for quantifying the E-cadherin-GFP accumulation at the contact site over time. The rate of E-cadherin-GFP accumulation was estimated by the slope of a fitted line ([Fig. 4j,k](#f4){ref-type="fig"}) and the coefficient of determination (R^2^-value) was used as a measure of fluctuation ([Fig. 4l](#f4){ref-type="fig"}). Spas reduced the E-cadherin-GFP accumulation rate and increased the fluctuation (lower R^2^-value; [Fig. 4l](#f4){ref-type="fig"}). Despite these defects, FCs expressing Spas eventually accumulated E-cadherin-GFP and completed branch fusion. Taken together, these results indicate that proper microtubule organization is required for stable, persistent E-cadherin accumulation to the new cell-adhesion sites in tracheal-branch fusion. Microtubules coordinate deformation of paired FCs ------------------------------------------------- We next analysed the dynamics of cell-shape change during the fusion process by monitoring the distance between the FC contact site (C) and the FC--stalk cell boundaries at the left (L) and right (R) side branch. In control embryos, FC pairs maintained their symmetry, with nearly equal C--L and C--R lengths ([Fig. 5a--c](#f5){ref-type="fig"} and [Supplementary Movie 13](#S1){ref-type="supplementary-material"}). However, FCs that expressed Spas deformed more slowly, as measured by the speed at which the L--R length decreased ([Fig. 5d](#f5){ref-type="fig"}), and failed to maintain a balanced bilateral cell contraction ([Fig. 5a](#f5){ref-type="fig"}) as measured by the L/R balance; the L/R balance was calculated as (a−b)/(a+b), where a=C--L and b=C--R ([Fig. 5b](#f5){ref-type="fig"}). The degree of fluctuation, based on the s.d. of the L/R balance, remained low in control cells. However, imbalances in cell length began appearing in esg_FC\>Spas cells 5 min after the first contact between FC cells ([Fig. 5c](#f5){ref-type="fig"}). These findings indicated that proper microtubule organization is required for balanced cell contraction. We further analysed the mechanism of the balanced cell contraction. We hypothesized that asymmetry in the length of paired FCs is caused by an imbalance in contractile forces between the cells, creating a back-and-forth tug-of-war (ToW) situation. To demonstrate this concept, we calculated the contraction speed of each FC at every time interval after contact. We used the contraction speed of viscoelastic cell materials, which is proportional to the contractile force[@b32], as a surrogate of cellular force. Negative and positive values indicate contractile and relaxed state of each FC, respectively. If both FCs contracted or relaxed simultaneously at a given time point after contact, the contractile force was considered balanced. If one cell contracted while the other relaxed, the event was considered imbalanced and in the ToW state. We pooled a number of such cell-shape change events and calculated the percentage of ToW imbalances ([Fig. 5e](#f5){ref-type="fig"}; ToW ratio, solid vertical bar). The ToW ratio was higher in the esg_FC\>Spas-expressing cells (62.2%, 7 time series) than in control cells (46.8%, 4 time series). To determine whether this deviation might be caused by random drift, we ran a simulation to calculate the ToW ratio; this simulation assumed that FCs contracted independently with the deformation rate in normal distribution ([Fig. 5e](#f5){ref-type="fig"}; mean displacement, −0.22±0.65 μm min^−1^ for control and −0.17±0.79 μm min^−1^ for esg_FC\>Spas cells; see Methods). The calculated probability distributions of control and esg_FC\>Spas conditions were similar to the control value ([Fig. 5e](#f5){ref-type="fig"}, thin curves, 10^4^ trials each), but deviated greatly from the measured esg_FC\>Spas value. Therefore, the ToW ratio deviation observed in esg_FC\>Spas cell pairs was due to an imbalance of forces in the paired FCs, leading to ToW-type contractions. Directed apical Serp secretion to the FC interface -------------------------------------------------- Based on our observation that the Golgi apparatus moved towards the site of contact between FCs ([Fig. 2g](#f2){ref-type="fig"}), we reasoned that the localization of secretory machineries is probably influenced by microtubule polarity. Rab9 is required for the endosome-to-Golgi trafficking of a key luminal component, the chitin deacetylase Serpentine (Serp)[@b33][@b34][@b35]. RFP-Rab9\'s localization in FCs was similar to that of the Golgi apparatus and of Arl3, another small GTPase that is essential for tracheal-branch fusion ([Fig. 6a,b](#f6){ref-type="fig"})[@b19][@b20]. We therefore examined the secretion of luminal components into the new lumen formed between FCs. Fusion points of dorsal trunk were observed for this study, because detailed analysis of lumen formation is possible with their large tube diameter. In arl3 mutants, isolated chitin-filled lumen was found at the contact site of dorsal-trunk FCs ([Fig. 6c](#f6){ref-type="fig"}) and the lumen contained secreted luminal proteins: the chitin deacetylase Verm[@b33][@b34], the ZP-domain protein Pio[@b36] and the chitin-binding protein Gasp[@b37] ([Fig. 6d--f](#f6){ref-type="fig"}). However, Serp was specifically missing in the isolated lumen ([Fig. 6c](#f6){ref-type="fig"}). To determine whether FCs can secrete Serp, we expressed Serp-GFP by the FC driver and found that Serp-GFP was secreted and filled the entire tracheal lumen ([Fig. 6g](#f6){ref-type="fig"}). In contrast, in an arl3-mutant background, Serp-GFP was absent at the FC contact site but filled the rest of the tracheal lumen ([Fig. 6h](#f6){ref-type="fig"}). These results indicate that Arl3 is specifically required for the directed secretion of Serp into the lumen in the interface between paired FCs, but not for its secretion into FC--stalk cell interfaces. Rab9 overexpression overrides the requirement for Arl3 ------------------------------------------------------ The aforementioned results suggested that Rab9-dependent Serp trafficking might be an important function of Arl3. To test this idea, we examined whether Rab9 overexpression could rescue the arl3-mutant phenotype. We found that GFP-Rab9 expressed by btl-Gal4 or esg_FC-Gal4 rescued the fusion defect of arl3 mutants ([Fig. 7a--d,g](#f7){ref-type="fig"}). However, GFP-fusion proteins of other small GTPases, Rab11 and Rab6, or of Serp failed to rescue the arl3 phenotype ([Figs. 7e--g](#f7){ref-type="fig"} and [6h](#f6){ref-type="fig"}). These results indicate that Rab9-dependent recycling and apical secretion is a crucial component of the Arl3-dependent conversion of paired FCs into a torus shape. Discussion ========== Previous studies showed that F-actin-enriched cell protrusions form in the tip of migrating tracheal branches[@b16][@b17][@b18][@b38]. Here we showed that tracheal FCs form polarized microtubule bundles oriented towards the leading edge of the migrating cells. The function of these microtubules is twofold: to concentrate E-cadherin to the newly contacted cell interface and to initiate the formation of new adherens junctions. We found that the E-cadherin that accumulated at the new cell interface is not recycled from the cell surface, but is instead drawn from a newly synthesized pool and recruited preferentially to the FC contact site, and not to existing adherens junctions between FCs and stalk cells. We speculate that the forward reorientation of the MTOC and the polarization of the microtubule plus ends towards the leading edge underlie the preferential deposition of E-cadherin at the FC contact site[@b39]. We considered one possible mechanism of preferential deposition, in which microtubules transport endosomes containing E-cadherin towards the contact site and we attempted to test this model by imaging vesicular trafficking of the complex containing E-cadherin-GFP and other adherens junction components. We did not find definitive evidence for this model. However, we observed that the Golgi apparatus shifted forward, towards the FC contact site, and that an E-cadherin-GFP signal increased in the plasma membrane before becoming concentrated at the contact site. Based on these observations, we favour a model in which the relocalization of the Golgi apparatus near the leading edge of the FC provides a source for E-cadherin that is deposited locally in the plasma membrane and the trans association of E-cadherin between the FCs nucleates a further concentration of E-cadherin via cis clustering[@b40]. Brodu and colleagues[@b15][@b41] have shown that MTOC components are located apically in stalk cells and the microtubule function is required for the apical assembly of adherens junction proteins Par-3 and E-cadherin through regulation of recycling endosomes. This mechanism appears different from FCs, as assembly of new adherens junction occurs in the cell interface enriched with microtubule plus ends opposite to the centriole located in the proximal side. A second microtubule function was discovered in this study, which was to equalize the contraction in FC pairs after contact. The coordinated contraction in FC pairs pulls two FC--stalk cell junctions simultaneously towards the FC contact site. The contractile force comes from a myosin-driven process; the microtubules may serve as a 'ratchet\' to fix the length of the FCs after each round of contraction. When microtubules were inhibited, the FCs relaxed to their original length after contracting, which delayed the overall fusion process. When microtubules were destabilized, branch fusion proceeded, albeit with delays and imbalances, and fusion was eventually completed. Even in this condition, the conversion of the FC cells into a torus shape occurred simultaneously, suggesting that there is a mechanism to coordinate the fusion event in FC pairs. The proposed ratchet-like function of microtubule must in some way be linked to the contractile activity of myosin. One good candidate molecule for coupling the actomyosin contraction to the microtubule function is Short-stop (Shot), which belongs to the conserved spectraplakin family of cytoskeletal proteins[@b42], and was shown to be required for tracheal branch fusion[@b43]. The involvement of microtubules and Shot in the ratchet-like mechanisms observed in several contraction-dependent morphogenetic events[@b44][@b45][@b46] should be tested in the future. It is interesting to note that balancing of the force applied to the E-cadherin conjugated cell interface via microtubule plus ends is similar in configuration to the mitotic spindle, where microtubule plus ends attached to the kinetochore of each of paired sister chromosomes applies pulling force to each spindle pole[@b47]. The equal number of cadherin-catenin complex in each side of the FC interface associated with trans-conjugated E-cadherin pairs in the FC interface may provide a platform for microtubule plus end attachment for generating balanced contractile force. When FCs are fully contracted, the plasma membrane of the two adherens junctions in each FC are connected in a single burst and cell pairs are converted simultaneously into a torus shape. This process requires Arl3 GTPase, which associates with the microtubules and intracellular vesicles concentrated at the FC contact site[@b19][@b20]. Here we showed that Arl3 is required for directed Serp trafficking, and that GFP-Rab9 overexpression overrides the requirement for Arl3. We propose that the microtubule-dependent transport of the Golgi apparatus and endosomes facilitates the concentration of Rab9 and Arl3 at the FC contact site, where they act together to increase the concentration of Serp in the lumen. The deacetylation of chitin converts it to the more hydrophilic form chitosan. The increase in water absorption by chitosan would cause the luminal matrix gel to swell, simultaneously pushing the plasma membranes of the FC interface closer to the plasma membrane of the FC--stalk cell interface so that the membrane-fusion machinery triggers the conversion of the paired FCs into a torus shape. A number of issues remain to be explained. Lumen formation in FCs was clearly detected arl3 mutants, but not in the normal context. This is probably because very small lumen is sufficient to trigger fusion of wild-type FCs. Although Arl3 is absolutely required for fusion, Rab9 and Serp are not, suggesting that the proposed luminal-matrix swelling due to chitin deacetylation is not the sole mechanism of plasma membrane fusion, and additional Arl3-regulated process of fusion control must exist. Moreover, additional Rab9 cargo that acts together with Serp to rescue the arl3 mutants is predicted. To uncover the entire fusion process, it will be necessary to search for additional Arl3 and Rab9 targets, and to analyse FC-specific membrane trafficking and secretion. Methods ======= Drosophila strains -------------------- The esg_FC-Gal4 strains used here were constructed by injecting a plasmid containing a genomic fragment upstream of the esg gene into the pGaTB vector[@b48] and by isolating insertions in the second and third chromosomes. The fusion-cell enhancer was identified during a study of esg regulatory elements[@b49][@b50]. The esg_FC-Gal4 strain were constructed by inserting the 7,259 bp of genomic DNA (position 15313739--15320998 of NT_033779) upstream of esg into the P{CaSpeR-hs} vector (FBtp0000164), whereas YY, MT-M and SH were associated with the Genetic Resource Center, National Institute of Genetics in Mishima, Japan. This construct was described as pAs-Gal4 expressed in the peripheral amnioserosa[@b51]. UAS-p120ctn-tagRFP was constructed with a p120ctn sequence amplified from the complementary DNA library[@b52] and tagRFP (Evrogen) was constructed as described elsewhere[@b23]. UAS-3xtagRFP was constructed by cloning three copies of tagRFP as a tandem fusion. UAS-tagRFP-tau was constructed by fusing tau\'s microtubule-binding domain (amino acids 165--327) to the carboxy terminus of tagRFP. UAS-TagRFP-Jupiter was constructed by fusing Jupiter open reading frame spanning codon 2 to terminator sequence to the C-terminal end of Tag-RFP. UAS-EB1-GFP was constructed by Masako Kaido and Leo Tsuda. All of the fluorescent-protein constructs were cloned into pUAST vector and transformed into germ lines[@b48][@b53]. Kat60\'s microtubule-severing activity was originally discovered in a screen of Gene Search strains[@b54] for effectors interfering with tracheal morphogenesis (Kenzi Oshima, personal communication; P{w^+mC^=GSV6}GS10360 insertion). The following fly strains were used: btl-Gal4 (ref. [@b55]), UAS-GFP-moe[@b56], UAS-Spas[@b57], UAS-GFP-PACT[@b27], UAS-DE-cad-GFP[@b58], UAS-GFP-Zip and UAS-GFP-DN-Zip[@b59]; the UAS-IR strain for DE-cad RNA interference (RNAi) (NIG Stock Center, 3327R-2), UAS-tau-GFP[@b60], UAS-Nod-lacZ[@b26], UAS-GFPS65T inserted in 2L (FBti0013280), UAS-Grasp65-GFP.2 (FBti0040816) and UAS-Serp-GFP[@b34]; rab9^56^, rab9^199^ and UAS-TagRFP-Rab9 (ref. [@b35]); and arl3^1^ and UAS-mCherry-CAAX[@b19]. To compare the effect of Spas and Kat60 overexpression on the tracheal branch fusion, we imaged DB migration under condition of each effector expressed by btl-Gal4. DB migration was delayed by Kat60 and fusion failed frequently (31% n=26), whereas success rate was higher in Spas (85%, n=26) or control (100%, n=11). Staining -------- The following antibodies and dilution were used for staining: rabbit anti-GFP (Medical & Biological Laboratories Co., Ltd, \#598, 500 × ), anti-beta-galactosidase (MP Biomedicals, \#0855976, 1000 × ), rat anti-DE-cad (DCAD2, 20 × )[@b61], mouse anti-Gasp (2A12, DSHB, 2 × ), anti-Serp (300 × ) and Verm (300 × )[@b34], anti-Pio (50 × )[@b36], anti-Arl3 (500 × )[@b19] and CBP-Alexa 488 (50 × )[@b35]. Embryos were dechorionated with 1/2 diluted commercial bleach and were fixed by mixing vigorously in the mixture of 2 ml 4% paraformaldehyde in PBS and 2 ml heptane in liquid scintillation vial for 30 min at room temperature. After removal of heptane, embryos were devitelinized by addition of 2 ml methanol and vigorous mixing. Devitellinized embryos were washed two times with methanol and rehydrated with PBS. Embryos were stained as described previously[@b56]. Blocking, antibody incubation and washing were performed with 1% BSA and 0.2% Triton X-100 in PBS. The anti-Katanin 60 antibody (100 × ) was generously donated by Naoyuki Fuse and Fumio Matsuzaki. Microscopy ---------- Time-lapse imaging of living embryos was performed with Olympus FV1000 with GaAsP detectors[@b19]. FRAP analyses were performed on tracheal cells expressing DE-cadherin-GFP using a confocal microscope equipped with a second scanner and a × 60 oil-immersion lens (FV-1000, Olympus). The sampling rate was every 10 s and specific locations were bleached with a 50-mW, 405-nm diode laser. EB1-GFP was imaged using a TCS SP5 II (Leica) equipped with a × 63 oil-immersion lens (numerical aperture=1.4). Optical sections (0.46 μm × 13) were scanned at 200 frames every 0.098 s. Infrared laser illumination was applied as a 1-s pulse at 60 mW and the embryos were imaged in 30-s intervals[@b25]. Measurement and data processing ------------------------------- All imaging data were processed and measured with applications developed in-house; these data were written in C and Objective-C and compiled as figures with ImageJ (Rasband, <http://imagej.nih.gov/ij/>). Measurements were processed with Ruby (<http://www.ruby-lang.org/>) and R (R Development Core Team, <http://www.R-project.org/>). The spatial coordinates (x--y--z--t) of areas with high signal intensity (E-cadherin-GFP, p120ctn-RFP or GFP-PACT) were tracked using software that searches for the local maximum within a three-dimensional volume manually specified by operators. Because of its concentrated localization, the E-cadherin fluorescence intensity at FC contact sites was estimated by taking the mean of the pixel intensities within a circle centred at the peak coordinate with a radius of 10 pixels (0.65 μm). The mobile fractions for FRAP analyses were estimated by fitting the fluorescence intensities to the following exponential equation: FC deformation was assessed by the shortening of the distance between the two FC--stalk cell interfaces (L--R) or between the FC contact site and the stalk-cell contact site (C--L and C--R). The particle image velocimetry analysis of EB1-GFP was performed on maximal z-projections of a confocal stack. In this analysis, 0.52-μm square windows were placed on the image for every 0.38 μm of x--y coordinates and the shift of the EB1-GFP signal in each window was estimated by searching within the next time point for the adjacent pixels by shifting original window up to 8 pixels along both x and y axes. The shift value showing the highest correlation of image pattern to the original window was recorded as vector. Tracks computed along 200 frames were mapped on a 25 × 25 grid of 0.38-μm squares and their direction and velocity were represented as bars. Numerical calculation of tug-of-war frequency --------------------------------------------- To compare the frequency of tug-of-war occurrence between wild-type and MT perturbed conditions, we measured the distance from FC contact site to FC/SC contact site (left and right) in every frames and calculated their change per time frame (displacement). Pairs of displacement value at each time point are then multiplied and the time points of which resulted in negative products were defined as ToW situation. Frequency of ToW situation was 72/154 (46.8%, 6 time series) and 112/180 (62.2%, 7 time series) for FC pairs in control and microtubule destabilized conditions, respectively. This result showed the frequencies of ToWs were significantly increased in MT-destabilized condition (Fisher\'s exact test, P=0.006). To simulate ToWs in silico, 300 pairs of random numbers were generated from Gaussian distribution with sample mean and s.d. of experimentally measured FC deformations (−0.22±0.65 μm min^−1^ for control and −0.17±0.79 for spas). Probability distributions of simulated ToW frequencies mimicking both conditions were estimated by repeating this trial for 10,000 times. Quantifying the effect of E-cadherin RNAi in the trachea -------------------------------------------------------- UAS-shg RNAi constructs were expressed with btl-Gal4 and embryos were fixed, stained with anti-E-cadherin and confocal image stacks were taken from stage-15 embryos. E-cadherin signal intensity in the epidermis and trachea (stalk cells of DB) was measured by setting region of interest with line tool (ten cell boundaries for each tissue per embryo), background subtracted and the intensity ratio of trachea and epidermis was calculated. Signal intensity ratio was 1.62±0.33 for control (N=6) and 1.18±0.14 for E-cad RNAi (N=4). This is ∼27% reduction of E-cadherin protein level (P=0.0397, Student\'s t-test, two tailed). Additional information ====================== How to cite this article: Kato, K. et al. Microtubule-dependent balanced cell contraction and luminal-matrix modification accelerate epithelial tube fusion. Nat. Commun. 7:11141 doi: 10.1038/ncomms11141 (2016). Supplementary Material {#S1} ====================== ###### Supplementary Figures Supplementary Figures 1-4 ###### Supplementary Movie 1 Time course of dorsal branch migration and fusion and their relationship to the epidermis. GFP-moe was expressed in tracheal FCs and pAs by esg_FC-Gal4, and in the sqh-GFP-moe. Lower panel corresponds to a stacked x-z section in the boxed area of dorsal view in top panel. Scale bar in lower right is 10 μm and time stamp is shown in top-right. Original movie used to prepare Fig. 1c. ###### Supplementary Movie 2 Microtubule dynamics in tracheal FC and pAs revealed by tau-GFP marker expressed by the esg_FCGal4 driver. Original movie used to prepare Fig. 1d. ###### Supplementary Movie 3 Double labeling of tau-RFP (top, red) and GFP-moe (middle, green) driven by btl-Gal4. Related to Fig. 1e. ###### Supplementary Movie 4 High-speed imaging of E-cadherin trafficking during the first 3 minutes after contact of FCs. Related to Fig. 2f. ###### Supplementary Movie 5 Dynamics of myosin localization in DB fusion site revealed by esg_FC \> GFP-Zip. Related to Fig. 3a. ###### Supplementary Movie 6 Laser perturbation of fusion cell contraction. Embryos carrying btl-Gal4, UAS-GFP-moe and UASp120ctn-RFP was illuminated with 1440 nm laser (60mW) for 1 second at one of fusion cells (left) of DB5 (t=0). The embryo was imaged with PlanApo 60x NA1.40 oil IR lens with 4x zoom. Then 512 x 512 pixel images of 1-mm-thick sections were captured every 30 sec for 20 minutes. Original movie used to prepare Fig. 3b. ###### Supplementary Movie 7 Effect of myosin inhibition on fusion of DB. GFP-DN-Zip was co-expressed with p120ctn-RFP by btlGal4. Top panel; x-y view, bottom panel: y-z view. Most of FCs reached the midline and accumulated p120ctn-RFP at the interface. But p120ctn-RFP was not concentrated into a spot and FC failed to contract. Original movie used to prepare Fig. 3d. ###### Supplementary Movie 8 Dynamics of microtubule plus end. FC and TC of btl \> EB1-GFP embryo were imaged. The branch was directed toward 10 o\'clock direction. Left: original movie. Right: overlay of PIV tracks. Original movie used to prepare Fig. 4c. ###### Supplementary Movie 9 Double labeling of EB1-GFP (green) and membrane marker (red) imaged in a btl \> EB1-GFP, mRFPCAAX embryo. Dorsal branch was directed toward 2 o\'clock direction. EB1 comet repeatedly entered into filopodia. Original movie used to prepare Fig. 4d. Scale bar: 10μm. ###### Supplementary Movie 10 Double labeling of tau-RFP (top, megenta) and E-cadherin-GFP (middle, green). Horizontal view of the focal plane covering FC. Related to Fig. 4e. ###### Supplementary Movie 11 Effect of Kat60 on microtubule stability visualized by tau-RFP imaging. Left: control, right: Kat60 expressed by btl-Gal4. Related to Fig. 4F. ###### Supplementary Movie 12 Effect of Kat60 on adherens junction formation. p120ctn-RFP expression in FCs was imaged in control (top) and Kat60 (bottom) expressing trachea. Central signal correspond to FC contact site. Frame rate 18 sec. Original movie used to prepare Fig. 4i. ###### Supplementary Movie 13 Microtubule inhibition caused unbalanced FC contraction. Control (top) and Spas expressing FCs are labeled with E-cadherin-GFP and tagRFP. esg_FC-Gal4 driver used here additionally labeled pAs. Each panel consists of x-y and x-z stacks of dorsal views of fusion event in one metamere. Vertical lines mark fusion cell adhesion site (L, C, R). Original movie used to prepare Fig. 5a. We thank the Kyoto and Bloomington Drosophila Stock Centers and the Developmental Studies Hybridoma Bank, and Andrea Dega, Hiroki Oda, Yukiko Yamashita, Leo Tsuda, Masako Kaido, Naoyuki Fuse and Fumio Matsuzaki for fly stocks and antibodies. We thank Kenzi Oshima for suggesting the use of Katanin 60 to perturb microtubules and Stefan Luschnig for communication of unpublished work. We also thank the members of the Hayashi laboratory for helpful discussions. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (22111007 to S.H.) from The Ministry of Education, Culture, Sports, Science and Technology, Japan, by the RIKEN Special Postdoctoral Researcher Program (K.K.) and by the RIKEN Foreign Postdoctoral Researcher Program (B.D.). Author contributions K.K. performed the microtubule and cadherin experiments and analysed data with help from H.W. B.D. analysed the functions of myosin, Arl3 and Serp. Y.Y. identified the FC enhancer and M.T.-M. constructed the driver strain. K.K., B.D. and S.H. designed experiments, analysed the data and wrote the manuscript. ![Dorsal-branch migration and fusion of the Drosophila trachea.\ (a) A stack of confocal GFP images shows the tracheal system of a stage-15 embryo carrying btl\>GFP-moe. (b) Spatial relationship of the migrating tracheal branch and the epidermis; FCs, terminal cells and stalk cells of the dorsal branch and peripheral amnioserosa (pAs) are indicated. (c) Time course of dorsal-branch migration and fusion, shown by F-actin labelling with esg_FC-Gal4 and UAS-GFP-moe (FC and pAs), and with sqh-GFP-moe (epidermis). Each panel shows dorsal (top half) and transverse views (bottom half) of images at the indicated time points (top left of the image). Filled triangles indicate F-actin-rich denticles of the apical surface of the epidermis; open triangles indicate FC filopodia penetrating vertically into the epidermis and nearly reaching the apical surface. Red dotted line indicates the basal epidermal surface. (c\') Enlarged view of the vertical filopodia (1:30). (d) Microtubule distribution during tracheal-branch fusion, revealed by expressing the tau-GFP marker in FCs and pAs by the esg_FC-Gal4 driver. Magenta triangles indicate MTOCs. (d\') Enlargement of d, 0:30 time point. (e) Double labelling of microtubules (GFP-tau) and F-actin (GFP-moe). Scale bar, 10 μm.](ncomms11141-f1){#f1} ![E-cadherin accumulation and trafficking.\ (a) E-cadherin accumulation during formation of the cell junction (C) between FCs is shown by time-lapse movie (left) and by quantification of the accumulated E-cadherin-GFP (right). L and R, left and right FC--stalk boundaries. (b) E-cadherin accumulated at two sites in FC filopodia: in FCs making contact (arrow) and at the tip of forward-extending filopodia (white arrowheads). Red arrowhead, FC--stalk boundary; S, stalk cell; T, terminal cell. FRAP analysis (c) and quantification (d) of E-cadherin-GFP accumulation. Error bar indicate s.d. (e) A scatter plot of C versus L/R signals (Fc) after recovery showing the preferential addition of E-cadherin-GFP to the newly forming junction between FCs. (f) Intracellular E-cadherin-GFP trafficking: the GFP signal accumulated progressively in the FC contact site (C, white arrow) over a period of ∼3 min. Yellow arrows in the central panel show cytoplasmic E-cadherin-GFP streaming towards the plasma membrane. (g) Relocalization of the Golgi apparatus to the front end of FCs during new junction formation, visualized by imaging Grasp65-GFP driven by esg_FC_Gal4. (h) RNAi inhibition of de novo E-cadherin synthesis interfered with branch fusion. (i) Velocity measurements demonstrated that branch migration before branch contact was normal, but the cell deformation after contact was significantly delayed in the E-cadherin RNAi embryo (\Student\'s t-test, P\<0.05). Scale bar, 10 μm.](ncomms11141-f2){#f2} ![FC contraction.\ (a) GFP-myosin heavy chain formed a longitudinal track connecting two adherens junctions in an FC. (b) Laser perturbation of FC contraction: the left-side cell (asterisk) in a new FC pair was illuminated by infrared laser at the time of contact. (c) The location of each cell-adhesion site (L, C and R) was tracked and the distance between sites was plotted. Laser perturbation caused significant deviations in L/R balance values (N=3, a representative case is shown). (d) Fusion was inhibited by a dominant-negative form of myosin. White arrow indicates the FC contact site. Scale bar, 10 μm.](ncomms11141-f3){#f3} ![Microtubule dynamics in FCs.\ (a) The polarization of FC microtubules during migration. The FC is outlined with a dotted line. The microtubule minus-end marker Nod-LacZ was concentrated at the FC--stalk cell boundary, which was marked with E-cadherin and lumen (chitin) staining. (b) The centriole marker GFP-PACT localized near the FC--stalk cell boundary; the lower panel shows the relative GFP-PACT localization along the axis connecting the FC--FC and FC--stalk cell boundaries during fusion. (c) Localization of the microtubule plus-end marker EB1-GFP and the flux and velocity distribution deduced by particle image velocimetry (PIV) analysis. (d) Multiple EB1-GFP comets entered a single filopodia. (e) E-cadherin-GFP accumulated on microtubule tracks at the FC contact site. (f) Kat60 destabilized the microtubule lattice in FCs. The distribution of tau-RFP (top, averaged for 20 frames of 10-s intervals) and its pixel-wise coefficient of variation in a 100-s period (bottom, averaged as in the top panel) are shown. (g) Kat60 inhibited E-cadherin accumulation at the FC contact site. (h) Kat60 inhibited tracheal-branch fusion. FCs contacted each other using filopodia, but did not form a stable adhesion interface. (i) Time course of p120ctn-RFP accumulation at the FC contact site. The region of interest (ROI) was set at the FC contact site (yellow box in inset); its intensity profile over a 15-min period is shown. (j) Time course of E-cadherin-GFP accumulation (arbitrary unit) in control and esg_FC-Gal4\>Spas* FCs. (k) The rate of E-cadherin-GFP accumulation. (l) Fluctuation of E-cadherin-GFP accumulation (R^2^ value). Error bar indicates s.d. Scale bar, 10 μm.](ncomms11141-f4){#f4} ![The balanced contraction of paired FCs depends on microtubules.\ (a) Microtubule inhibition disrupted the balanced contraction: x--z views show FC pairs in control and Spas-expressing embryos (esg_FC-Gal4). Green, E-cadherin-GFP; magenta, tagRFP. Arrowheads and lines indicate cell-adhesion sites. (b) Plot of the L/R balance values. Spas expression caused greater deviations from a balanced condition, which was represented by a value of 0. The onset of E-cadherin-GFP accumulation at the FC--FC interface was set as 0 min in the time series of each embryo. (c) Fluctuations in contraction: the s.d. of the L/R balance value was plotted against time. (d) A contraction rate plot based on the L--R length decrease (\Student\'s t-test, P\<0.05). (e) Evaluation of ToW conditions: vertical bars indicate the frequency of contraction/relaxation in FC pairs for each time interval measured in the two genotypes. Curves indicate the simulated distribution of numerically calculated frequencies of paired contraction/retraction events, assuming that contraction and retraction occur randomly in each FC (10,000 trials). The value calculated for control FCs was slightly smaller than that for Spas-expressing FCs due to the difference in contraction rate (d). The ToW value for Spas-expressing FCs (62.2%) was far greater than the value expected from the random hypothesis, suggesting that uncoordinated contraction/retraction events occurred more frequently in the FCs expressing Spas. Scale bar, 10 μm.](ncomms11141-f5){#f5} ![RFP-Rab9 vesicle trafficking in dorsal-branch FCs.\ (a) In dorsal-branch FCs (indicated by arrowheads and dotted outlines), RFP-Rab9 vesicle clusters (asterisks) were localized near the FC contact site. Anterior, left. (a\') Clustering of RFP-Rab9 vesicles was inhibited when microtubule was disrupted by Kat60. (b) Arl3 was also concentrated adjacent to the apical cell membrane (labelled with Uif) and co-localized with GFP-Rab9 at the FC contact site of the dorsal trunk (yellow arrowheads). (c--f) A specific secretion defect in arl3-mutant embryos: fusion points of stage-16 dorsal trunk were stained for CBP and Serp (c), Verm (d), Pio (e) or Gasp (f). Chitin, Verm, Pio and 2A12 were present in the lumen isolated from the FC contact site (blue arrowheads), whereas Serp (yellow arrowheads) was missing. (g,h) Test of directed Serp trafficking: Serp-GFP was expressed in FCs (blue arrowheads) using the esg_FC-Gal4* driver in control (arl3^1^/+, g) and arl3^1^ mutant (h) embryos, respectively. Secreted Serp-GFP was present throughout the lumen in control embryos (yellow arrowheads in g) but absent from the FC contact site in arl3^1^ embryos (blue arrowheads in h). (g\', h\') Enlargement of the boxed regions in g and h, respectively. Scale bar, 10 μm.](ncomms11141-f6){#f6} ![GFP-Rab9 overexpression rescued the arl3-mutant phenotype.\ (a,b) Control and arl3-mutant embryos were stained with CBP. Yellow arrowheads indicate a failure of branch fusion in the dorsal trunk of arl3-mutants. (c,d) GFP-Rab9 expressed specifically in the whole tracheal system by the btl-GAL4 driver or in FCs by the esg_FC-Gal4 driver rescued the branch-fusion defect in arl3 mutants. Yellow arrowheads indicate fusion-failure points remaining in the rescued embryos. GFP-Rab11 (e) and Serp-GFP (f) expression failed to rescue the arl3 phenotype. (g) Quantification of the dorsal trunk fusion rate in different genotypes; 100% corresponds to nine successful fusion events in the ten tracheal metameres on one side of the embryo. Rescue activities of GFP-Rab9, GFP-Rab11, GFP-Rab6 and Serp-GFP were tested. Error bars indicate s.d. \\The fusion rate was significantly higher in arl3 mutants overexpressing GFP-Rab9 than in arl3 mutants without GFP-Rab9 overexpression, by Student\'s t-test (P\<0.01). Scale bar, 50 μm (a--f).](ncomms11141-f7){#f7} [^1]: Present address: Department of Imaging Science, Center for Novel Science Initiatives, National Institutes of Natural Sciences, Myodaiji-Cho, Okazaki, Aichi 444-8787, Japan [^2]: Present address: College of Marine Life Sciences, Ocean University of China, No. 5 Yushan Road, Qingdao 266003, China	{ "pile_set_name": "PubMed Central" }	[ "amount", "destabil", "rest", "probabl", "discov", "evrogen", "two", "ab", "control", "gfpmyosin", "deform", "given", "origin", "work", "remain", "program", "asreftypesupplementarymateri", "decreas", "cours", "ocean", "penetr", "punctat", "trachealtub", "rubi", "uasgfpmo", "myodaijicho", "intervalsb", "httpwwwrubylangorg", "carboxi", "previouslyb", "ten", "thank", "esgfcgal", "exist", "favour", "tow", "gene", "identifi", "basal", "pctn", "length", "tracheaspecif", "∼", "round", "block", "written", "retain", "bank", "mechan", "cf", "take", "student", "valu", "convers", "fix", "maxim", "katp", "locat", 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"inhibited", "microtubule", "disrupted", "b", "also", "concentrated", "adjacent", "apical", "cell", "membrane", "labelled", "Uif", "FC", "contact", "site", "dorsal", "trunk", "yellow", "arrowheads", "c", "f", "specific", "secretion", "defect", "embryos", "fusion", "points", "dorsal", "trunk", "stained", "CBP", "Serp", "c", "Verm", "Pio", "e", "Gasp", "f", "Chitin", "Verm", "Pio", "present", "lumen", "isolated", "FC", "contact", "site", "blue", "arrowheads", "whereas", "Serp", "yellow", "arrowheads", "missing", "g", "h", "Test", "directed", "Serp", "trafficking", "expressed", "FCs", "blue", "arrowheads", "using", "driver", "control", "g", "mutant", "h", "embryos", "respectively", "Secreted", "present", "throughout", "lumen", "control", "embryos", "yellow", "arrowheads", "g", "absent", "FC", "contact", "site", "embryos", "blue", "arrowheads", "h", "Enlargement", "boxed", "regions", "g", "h", "respectively", "Scale", "bar", "μm", "overexpression", "rescued", "b", "Control", "embryos", "stained", "CBP", "Yellow", "arrowheads", "indicate", "failure", "branch", "fusion", "dorsal", "trunk", "c", "expressed", "specifically", "whole", "tracheal", "system", "btl", "driver", "FCs", "driver", "rescued", "defect", "mutants", "Yellow", "arrowheads", "indicate", "points", "remaining", "rescued", "embryos", "e", "f", "expression", "failed", "rescue", "phenotype", "g", "Quantification", "dorsal", "trunk", "fusion", "rate", "different", "genotypes", "corresponds", "nine", "successful", "fusion", "events", "ten", "tracheal", "metameres", "one", "side", "embryo", "Rescue", "activities", "tested", "Error", "bars", "indicate", "fusion", "rate", "significantly", "higher", "mutants", "overexpressing", "mutants", "without", "overexpression", "P", "Scale", "bar", "μm", "f", "Present", "address", "Department", "Imaging", "Science", "Center", "Novel", "Science", "Initiatives", "National", "Institutes", "Natural", "Sciences", "Okazaki", "Aichi", "Japan", "Present", "address", "College", "Marine", "Life", "Sciences", "Ocean", "University", "China", "Yushan", "Road", "Qingdao", "China" ]
Chemical context {#sec1} ================== Naphthalene and its derivatives are known for their wide range of applications in the field of pharmaceuticals. They are also used in the manufacturing of colorants, surface-active agents, resins, disinfectants and insecticides. These derivatives play a vital role in the control of microbial infection (Rokade & Sayyed, 2009[@bb12]) and in the chemical defence against biological enemies (Wright et al., 2000[@bb18]). Compounds with a naphthalene moiety have been shown to exhibit significant anti-TB activity (Upadhayaya et al., 2010[@bb17]). Structural commentary {#sec2} ======================= The molecular structure of the title compound is illustrated in Fig. 1[▸](#fig1){ref-type="fig"}. The benzene ring (C9--C14) of the naphthalene moiety is substituted by a dimethylphenyl ring (C2--C4/C6--C8) and a methoxyphenyl ring (C19--C24) para to each other. The naphthalene ring system is slightly bent with the two aryl rings being inclined to each other by 3.06 (15)°. Its mean plane makes dihedral angles of 65.24 (12)° with the dimethylphenyl ring (C2--C4/C6--C8) and 55.82 (12)° with methoxyphenyl ring (C19--C24). The latter two rings are inclined to each other by 59.28 (14)°. The methoxy group (C22/O1/C25) lies out of the plane of the benzene ring (C19--C24) to which it is attached by 11.3 (3)°. The bond lengths and bond angles are similar to those reported for 1,4-diphenylnaphthalene, which crystallized with two independent molecules in the asymmetric unit (Lima et al., 2012[@bb7]). Supramolecular features {#sec3} ========================== In the crystal, there is only one significant intermolecular interaction present, viz. a C---H⋯π interaction linking adjacent molecules to form chains propagating along the a-axis direction (Table 1[▸](#table1){ref-type="table"} and Fig. 2[▸](#fig2){ref-type="fig"}). Database survey {#sec4} ================= A search of the Cambridge Structural Database (Version 5.39, last update February 2018; Groom et al., 2016[@bb5]) for the aromatic skeleton of the title compound yielded ten hits. They include 1,4-diphenylnaphthalene itself, which crystallized with two independent molecules in the asymmetric unit (CSD refcode ZAXJEP: Lima et al., 2012[@bb7]). There are also a number of copper(II) complexes (LAYQOU: Chen et al., 2017[@bb3]; BOSHIC: Cai et al., 2014[@bb2]; PUBSOV: Lin et al., 2009[@bb8]) of the tetracarboxylic acid derivative, 5,5′-(naphthalene-1,4-diyl)diisophthalic acid, all of which are metal--organic frameworks. Analysis of the Hirshfeld surfaces, interaction energies and energy frameworks {#sec5} ================================================================================= The Hirshfeld surfaces and two-dimensional fingerprint plots were generated in order to explore and quantify the weak intermolecular interactions using the program CrystalExplorer 17.5 (Turner et al., 2017[@bb16]). The electrostatic potentials were calculated using TONTO, integrated in the program CrystalExplorer (Spackman et al., 2008[@bb15]; Jayatilaka et al., 2005[@bb6]). The Hirshfeld surfaces of the title compound were mapped over d ~norm~, electrostatic potential, curvedness and shape index (Fig. 3[▸](#fig3){ref-type="fig"} a--3d); depending upon the closeness to the adjacent molecules, the colour patches are mapped differently on the Hirshfeld surface (Fig. 3[▸](#fig3){ref-type="fig"} e). Two-dimensional fingerprint plots showing the result of all intermolecular contacts (McKinnon et al., 2007[@bb11]) are presented in Fig. 4[▸](#fig4){ref-type="fig"} a; d ~i~ (x axis) and d ~e~ (y axis) are the closest internal and external distance from a given point on the Hirshfeld surface. The fingerprint plot of H⋯H contacts, which represent the largest contribution to the Hirshfeld surface (64.6%), are shown as a distinct pattern with a minimum value of d ~e~ = d ~i~ ≃ 1.2 Å (Fig. 4[▸](#fig4){ref-type="fig"} b). The C⋯H/H⋯C interactions appear as the next largest region of the fingerprint plot, highly concentrated at the edges, having almost the same d ~e~ + d ~i~ ≃ 2.7 Å (Fig. 4[▸](#fig4){ref-type="fig"} c), with on overall contribution of 27.1%. The O⋯H/H⋯O interactions on the fingerprint plot, which contribute 5.2% of the total Hirshfeld surfaces, with d ~e~ + d ~i~ ≃ 2.8 Å (Fig. 4[▸](#fig4){ref-type="fig"} d) are shown as two symmetrical wings. The C⋯C contacts, which are the measure of π--π stacking interactions, occupy 3.1% of the Hirshfeld surfaces and appear as a unique triangle at about d ~e~ = d ~i~ ≃ 1.8 Å (Fig. 4[▸](#fig4){ref-type="fig"} e). These weak interactions mostly contribute to the packing of the title compound. The interaction energies between the molecules are obtained using monomer wavefunctions at the B3LYP/6-31G(p,d) level. The total interaction energy, which is the sum of scaled components, was calculated for a 3.8 Å radius cluster of molecules around the selected molecule (Fig. 5[▸](#fig5){ref-type="fig"} a). The scale factors used in the CE-B3LYP benchmarked energy model (Mackenzie et al., 2017[@bb9]) are given in Table 2[▸](#table2){ref-type="table"}. The energies calculated by the energy model reveals that the dispersion energy contributes significantly to the interactions in the crystal (Table 3[▸](#table3){ref-type="table"}). The energy framework calculations were performed for a cluster of molecules present in 2 × 2 × 2 unit cells using the CE-B3LYP energy model. Energies between molecular pairs are represented as cylinders joining the centroids of pairs of molecules with the cylinder radius proportional to the magnitude of the interaction energy. Energy frameworks were constructed for E ~elec~ as red cylinders, E ~dis~ as green and E ~tot~ as blue (Fig. 5[▸](#fig5){ref-type="fig"} b--5d) and these cylinders represent the relative strength of molecular packing in different directions. Thus the supramolecular architecture of the crystal structure is visualized uniquely by energy frameworks. Synthesis and crystallization {#sec6} =============================== A reaction scheme for the synthesis of the title compound is illustrated in Fig. 6[▸](#fig6){ref-type="fig"}. To a solution of m-xylyl-p-anisyl tethered benzo\[c\]furan (0.16 g, 0.49 mmol) in dry xylenes (15 ml) was added tetrathiafulvalene (TTF) (0.10 g, 0.49 mmol) and the mixture was refluxed until the consumption of benzo\[c\]furan was complete; monitored by the disappearance of the fluorescent colour after 6 h. After the removal of xylenes in vacuo, the crude adduct was dissolved in dry CH~2~Cl~2~ (15 ml) and then kept at 273 K. To this, triflic acid (0.02 g, 0.13 mmol) was added and the mixture stirred at room temperature for 10 min. After completion of the reaction (monitored by TLC), it was poured into ice--water (20 ml) and then extracted with CH~2~Cl~2~ (2 × 10 ml). The organic layers were combined and washed with aq. NaHCO~3~ (2 × 10 ml) and then dried (Na~2~SO~4~). Removal of the solvent followed by column chromatographic purification (silica gel, 5% ethyl acetate in hexane) afforded the title compound as a yellow solid (0.20 g, 79%). Yellow block-like crystals of the title compound, suitable for X-ray diffraction analysis, were obtained by slow evaporation of a solution in CHCl~3~ (m.p. 351--353 K). Refinement {#sec7} ============ Crystal data collection and structure refinement details are summarized in Table 4[▸](#table4){ref-type="table"}. All H atoms were positioned geometrically and refined using a riding model: C---H = 0.93--0.96 Å with U ~iso~(H) = 1.5 U ~eq~(C-methyl) and 1.2U ~eq~(C) for other H atoms. Supplementary Material ====================== Crystal structure: contains datablock(s) I, Global. DOI: [10.1107/S2056989018008332/su5445sup1.cif](https://doi.org/10.1107/S2056989018008332/su5445sup1.cif) Structure factors: contains datablock(s) I. DOI: [10.1107/S2056989018008332/su5445Isup2.hkl](https://doi.org/10.1107/S2056989018008332/su5445Isup2.hkl) ###### Click here for additional data file. Supporting information file. DOI: [10.1107/S2056989018008332/su5445Isup3.cml](https://doi.org/10.1107/S2056989018008332/su5445Isup3.cml) CCDC reference: [1841351](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1841351) Additional supporting information: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?su5445&file=su5445sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?su5445sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?su5445&checkcif=yes) The authors thank the Central Instrumentation Facility (DST--FIST), Queen Mary's College (A), Chennai-4, for the computing facilities and the SAIF, IIT, Madras, for the X-ray data collection facility. Crystal data {#d1e138} ============ ------------------------ ---------------------------------------- C~25~H~22~O Z = 2 M~r~ = 338.42 F(000) = 360 Triclinic, P1 D~x~ = 1.223 Mg m^−3^ a = 9.1670 (9) Å Mo Kα radiation, λ = 0.71073 Å b = 10.4566 (10) Å Cell parameters from 19154 reflections c = 11.2499 (11) Å θ = 2.3--22.7° α = 64.707 (4)° µ = 0.07 mm^−1^ β = 71.312 (4)° T = 296 K γ = 77.032 (4)° Block, yellow V = 918.75 (16) Å^3^ 0.15 × 0.10 × 0.10 mm ------------------------ ---------------------------------------- Data collection {#d1e264} =============== ---------------------------------------------------------- -------------------------------------- Bruker Kappa APEXII CCD diffractometer 1777 reflections with I \> 2σ(I) Radiation source: fine-focus sealed tube R~int~ = 0.060 ω and φ scan θ~max~ = 26.6°, θ~min~ = 2.1° Absorption correction: multi-scan (SADABS; Bruker, 2012) h = −11→11 T~min~ = 0.900, T~max~ = 0.945 k = −13→13 19120 measured reflections l = −14→14 3828 independent reflections ---------------------------------------------------------- -------------------------------------- Refinement {#d1e377} ========== ------------------------------------- ------------------------------------------------------------------------------------------------- Refinement on F^2^ Primary atom site location: structure-invariant direct methods Least-squares matrix: full Secondary atom site location: difference Fourier map R\[F^2^ \> 2σ(F^2^)\] = 0.057 Hydrogen site location: inferred from neighbouring sites wR(F^2^) = 0.193 H-atom parameters constrained S = 1.00 w = 1/\[σ^2^(F~o~^2^) + (0.0922P)^2^ + 0.0768P\] where P = (F~o~^2^ + 2F~c~^2^)/3 3828 reflections (Δ/σ)~max~ \< 0.001 238 parameters Δρ~max~ = 0.27 e Å^−3^ 0 restraints Δρ~min~ = −0.19 e Å^−3^ ------------------------------------- ------------------------------------------------------------------------------------------------- Special details {#d1e533} =============== ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#d1e552} ================================================================================================== ------ ------------- ------------ ------------ -------------------- -- x y z U~iso~\/U~eq~ C1 0.7907 (4) 0.8345 (4) 0.2984 (3) 0.0771 (10) H1A 0.7017 0.8047 0.2937 0.116\ H1B 0.7750 0.9350 0.2771 0.116\* H1C 0.8053 0.7849 0.3885 0.116\* C2 0.9307 (3) 0.8015 (3) 0.1987 (3) 0.0509 (7) C3 1.0610 (3) 0.8748 (3) 0.1542 (3) 0.0578 (8) H3 1.0583 0.9411 0.1898 0.069\* C4 1.1927 (3) 0.8530 (3) 0.0602 (3) 0.0573 (8) C5 1.3309 (4) 0.9324 (4) 0.0196 (4) 0.0863 (11) H5A 1.4237 0.8679 0.0169 0.129\* H5B 1.3205 0.9724 0.0848 0.129\* H5C 1.3365 1.0071 −0.0686 0.129\* C6 1.1935 (3) 0.7571 (3) 0.0065 (3) 0.0598 (8) H6 1.2803 0.7415 −0.0585 0.072\* C7 1.0668 (3) 0.6835 (3) 0.0480 (3) 0.0541 (7) H7 1.0699 0.6198 0.0093 0.065\* C8 0.9353 (3) 0.7015 (3) 0.1453 (3) 0.0446 (7) C9 0.8049 (3) 0.6158 (3) 0.1869 (3) 0.0437 (6) C10 0.7284 (3) 0.6328 (3) 0.0942 (3) 0.0537 (7) H10 0.7534 0.7037 0.0079 0.064\* C11 0.6138 (3) 0.5472 (3) 0.1247 (3) 0.0527 (7) H11 0.5648 0.5633 0.0581 0.063\* C12 0.5716 (3) 0.4408 (3) 0.2488 (3) 0.0426 (6) C13 0.6437 (3) 0.4220 (3) 0.3514 (2) 0.0393 (6) C14 0.7621 (3) 0.5075 (3) 0.3195 (2) 0.0403 (6) C15 0.8360 (3) 0.4807 (3) 0.4219 (3) 0.0507 (7) H15 0.9173 0.5323 0.4017 0.061\* C16 0.7907 (3) 0.3810 (3) 0.5493 (3) 0.0602 (8) H16 0.8414 0.3649 0.6147 0.072\* C17 0.6690 (3) 0.3029 (3) 0.5823 (3) 0.0612 (8) H17 0.6361 0.2374 0.6706 0.073\* C18 0.5983 (3) 0.3221 (3) 0.4860 (3) 0.0518 (7) H18 0.5180 0.2682 0.5092 0.062\* C19 0.4540 (3) 0.3483 (3) 0.2743 (2) 0.0432 (6) C20 0.3108 (3) 0.4064 (3) 0.2491 (3) 0.0504 (7) H20 0.2877 0.5046 0.2182 0.060\* C21 0.2007 (3) 0.3235 (3) 0.2682 (3) 0.0525 (7) H21 0.1051 0.3658 0.2507 0.063\* C22 0.2331 (3) 0.1787 (3) 0.3130 (3) 0.0510 (7) C23 0.3750 (3) 0.1177 (3) 0.3379 (3) 0.0601 (8) H23 0.3977 0.0195 0.3676 0.072\* C24 0.4841 (3) 0.2011 (3) 0.3193 (3) 0.0543 (7) H24 0.5794 0.1581 0.3371 0.065\* C25 −0.0236 (4) 0.1430 (4) 0.3343 (4) 0.0834 (11) H25A −0.0854 0.0667 0.3644 0.125\* H25B −0.0284 0.2070 0.2437 0.125\* H25C −0.0624 0.1932 0.3942 0.125\* O1 0.1324 (2) 0.0867 (2) 0.3352 (2) 0.0742 (7) ------ ------------- ------------ ------------ -------------------- -- Atomic displacement parameters (Å^2^) {#d1e1180} ===================================== ----- ------------- ------------- ------------- -------------- -------------- -------------- U^11^ U^22^ U^33^ U^12^ U^13^ U^23^ C1 0.076 (2) 0.079 (2) 0.081 (2) −0.0173 (19) −0.0011 (18) −0.044 (2) C2 0.0503 (17) 0.0521 (17) 0.0505 (17) −0.0106 (14) −0.0132 (14) −0.0171 (14) C3 0.068 (2) 0.0485 (17) 0.0622 (19) −0.0158 (15) −0.0235 (16) −0.0169 (15) C4 0.0534 (18) 0.0526 (18) 0.0532 (18) −0.0149 (15) −0.0173 (15) −0.0013 (15) C5 0.069 (2) 0.091 (3) 0.088 (3) −0.040 (2) −0.0228 (19) −0.007 (2) C6 0.0523 (18) 0.0585 (19) 0.0533 (18) −0.0127 (15) −0.0040 (14) −0.0106 (16) C7 0.0547 (17) 0.0528 (18) 0.0498 (17) −0.0128 (14) −0.0061 (14) −0.0169 (14) C8 0.0491 (16) 0.0419 (15) 0.0427 (15) −0.0124 (13) −0.0133 (13) −0.0113 (13) C9 0.0458 (15) 0.0475 (16) 0.0415 (15) −0.0104 (13) −0.0101 (12) −0.0186 (13) C10 0.0621 (18) 0.0550 (18) 0.0421 (16) −0.0192 (15) −0.0150 (14) −0.0093 (14) C11 0.0593 (18) 0.0571 (18) 0.0424 (17) −0.0159 (15) −0.0180 (14) −0.0113 (14) C12 0.0412 (15) 0.0454 (15) 0.0438 (16) −0.0070 (12) −0.0088 (12) −0.0197 (13) C13 0.0393 (14) 0.0416 (15) 0.0365 (15) −0.0044 (12) −0.0067 (11) −0.0165 (12) C14 0.0404 (14) 0.0416 (15) 0.0408 (15) −0.0055 (12) −0.0091 (12) −0.0178 (13) C15 0.0515 (16) 0.0574 (18) 0.0482 (17) −0.0131 (14) −0.0149 (14) −0.0197 (15) C16 0.069 (2) 0.070 (2) 0.0467 (18) −0.0158 (17) −0.0230 (15) −0.0174 (16) C17 0.073 (2) 0.067 (2) 0.0392 (17) −0.0215 (17) −0.0110 (15) −0.0115 (15) C18 0.0526 (17) 0.0563 (18) 0.0454 (17) −0.0149 (14) −0.0063 (13) −0.0185 (15) C19 0.0435 (15) 0.0456 (16) 0.0436 (15) −0.0065 (13) −0.0088 (12) −0.0206 (13) C20 0.0522 (17) 0.0465 (16) 0.0551 (17) −0.0068 (14) −0.0183 (13) −0.0179 (14) C21 0.0458 (16) 0.0576 (19) 0.0610 (18) −0.0057 (14) −0.0174 (13) −0.0261 (15) C22 0.0521 (18) 0.0548 (19) 0.0544 (18) −0.0148 (15) −0.0075 (14) −0.0285 (15) C23 0.060 (2) 0.0463 (17) 0.076 (2) −0.0056 (16) −0.0136 (16) −0.0280 (16) C24 0.0451 (16) 0.0518 (18) 0.0666 (19) −0.0019 (14) −0.0130 (14) −0.0256 (15) C25 0.060 (2) 0.102 (3) 0.108 (3) −0.029 (2) −0.0172 (19) −0.051 (2) O1 0.0641 (14) 0.0687 (14) 0.1023 (18) −0.0233 (12) −0.0140 (12) −0.0415 (13) ----- ------------- ------------- ------------- -------------- -------------- -------------- Geometric parameters (Å, º) {#d1e1774} =========================== ----------------------- ------------ ----------------------- ------------ C1---C2 1.495 (4) C13---C18 1.413 (3) C1---H1A 0.9600 C13---C14 1.421 (3) C1---H1B 0.9600 C14---C15 1.416 (3) C1---H1C 0.9600 C15---C16 1.360 (4) C2---C8 1.399 (4) C15---H15 0.9300 C2---C3 1.401 (3) C16---C17 1.390 (3) C3---C4 1.375 (4) C16---H16 0.9300 C3---H3 0.9300 C17---C18 1.357 (4) C4---C6 1.373 (4) C17---H17 0.9300 C4---C5 1.510 (4) C18---H18 0.9300 C5---H5A 0.9600 C19---C20 1.380 (4) C5---H5B 0.9600 C19---C24 1.391 (4) C5---H5C 0.9600 C20---C21 1.383 (3) C6---C7 1.381 (3) C20---H20 0.9300 C6---H6 0.9300 C21---C22 1.370 (4) C7---C8 1.385 (3) C21---H21 0.9300 C7---H7 0.9300 C22---O1 1.373 (3) C8---C9 1.488 (3) C22---C23 1.373 (4) C9---C10 1.366 (3) C23---C24 1.381 (3) C9---C14 1.430 (3) C23---H23 0.9300 C10---C11 1.397 (3) C24---H24 0.9300 C10---H10 0.9300 C25---O1 1.420 (4) C11---C12 1.363 (4) C25---H25A 0.9600 C11---H11 0.9300 C25---H25B 0.9600 C12---C13 1.430 (3) C25---H25C 0.9600 C12---C19 1.488 (3) C2---C1---H1A 109.5 C18---C13---C12 121.9 (2) C2---C1---H1B 109.5 C14---C13---C12 119.8 (2) H1A---C1---H1B 109.5 C15---C14---C13 118.0 (2) C2---C1---H1C 109.5 C15---C14---C9 121.8 (2) H1A---C1---H1C 109.5 C13---C14---C9 120.2 (2) H1B---C1---H1C 109.5 C16---C15---C14 121.4 (3) C8---C2---C3 118.4 (2) C16---C15---H15 119.3 C8---C2---C1 122.1 (2) C14---C15---H15 119.3 C3---C2---C1 119.5 (3) C15---C16---C17 120.4 (3) C4---C3---C2 122.9 (3) C15---C16---H16 119.8 C4---C3---H3 118.5 C17---C16---H16 119.8 C2---C3---H3 118.5 C18---C17---C16 120.1 (3) C6---C4---C3 117.8 (3) C18---C17---H17 120.0 C6---C4---C5 121.9 (3) C16---C17---H17 120.0 C3---C4---C5 120.3 (3) C17---C18---C13 121.6 (3) C4---C5---H5A 109.5 C17---C18---H18 119.2 C4---C5---H5B 109.5 C13---C18---H18 119.2 H5A---C5---H5B 109.5 C20---C19---C24 117.0 (2) C4---C5---H5C 109.5 C20---C19---C12 120.9 (2) H5A---C5---H5C 109.5 C24---C19---C12 122.1 (2) H5B---C5---H5C 109.5 C19---C20---C21 122.2 (3) C4---C6---C7 120.7 (3) C19---C20---H20 118.9 C4---C6---H6 119.7 C21---C20---H20 118.9 C7---C6---H6 119.7 C22---C21---C20 119.7 (3) C6---C7---C8 122.1 (3) C22---C21---H21 120.2 C6---C7---H7 119.0 C20---C21---H21 120.2 C8---C7---H7 119.0 C21---C22---O1 124.4 (3) C7---C8---C2 118.1 (2) C21---C22---C23 119.5 (3) C7---C8---C9 118.8 (2) O1---C22---C23 116.0 (3) C2---C8---C9 123.1 (2) C22---C23---C24 120.5 (3) C10---C9---C14 117.6 (2) C22---C23---H23 119.8 C10---C9---C8 120.0 (2) C24---C23---H23 119.8 C14---C9---C8 122.3 (2) C23---C24---C19 121.2 (3) C9---C10---C11 122.3 (3) C23---C24---H24 119.4 C9---C10---H10 118.9 C19---C24---H24 119.4 C11---C10---H10 118.9 O1---C25---H25A 109.5 C12---C11---C10 122.1 (3) O1---C25---H25B 109.5 C12---C11---H11 119.0 H25A---C25---H25B 109.5 C10---C11---H11 119.0 O1---C25---H25C 109.5 C11---C12---C13 118.0 (2) H25A---C25---H25C 109.5 C11---C12---C19 120.0 (2) H25B---C25---H25C 109.5 C13---C12---C19 122.0 (2) C22---O1---C25 117.3 (2) C18---C13---C14 118.4 (2) C8---C2---C3---C4 0.2 (4) C12---C13---C14---C9 2.4 (3) C1---C2---C3---C4 −178.4 (3) C10---C9---C14---C15 179.2 (2) C2---C3---C4---C6 1.4 (4) C8---C9---C14---C15 3.5 (4) C2---C3---C4---C5 −178.2 (3) C10---C9---C14---C13 0.0 (3) C3---C4---C6---C7 −1.2 (4) C8---C9---C14---C13 −175.7 (2) C5---C4---C6---C7 178.4 (3) C13---C14---C15---C16 −3.2 (4) C4---C6---C7---C8 −0.7 (4) C9---C14---C15---C16 177.6 (2) C6---C7---C8---C2 2.3 (4) C14---C15---C16---C17 −0.3 (4) C6---C7---C8---C9 −178.6 (2) C15---C16---C17---C18 2.4 (4) C3---C2---C8---C7 −2.0 (4) C16---C17---C18---C13 −0.9 (4) C1---C2---C8---C7 176.6 (3) C14---C13---C18---C17 −2.6 (4) C3---C2---C8---C9 178.9 (2) C12---C13---C18---C17 178.8 (3) C1---C2---C8---C9 −2.5 (4) C11---C12---C19---C20 −53.8 (3) C7---C8---C9---C10 −63.5 (3) C13---C12---C19---C20 126.4 (3) C2---C8---C9---C10 115.5 (3) C11---C12---C19---C24 123.9 (3) C7---C8---C9---C14 112.1 (3) C13---C12---C19---C24 −56.0 (3) C2---C8---C9---C14 −68.9 (3) C24---C19---C20---C21 0.5 (4) C14---C9---C10---C11 −1.1 (4) C12---C19---C20---C21 178.2 (2) C8---C9---C10---C11 174.7 (2) C19---C20---C21---C22 −0.3 (4) C9---C10---C11---C12 −0.3 (4) C20---C21---C22---O1 −179.9 (2) C10---C11---C12---C13 2.7 (4) C20---C21---C22---C23 −0.2 (4) C10---C11---C12---C19 −177.2 (2) C21---C22---C23---C24 0.6 (4) C11---C12---C13---C18 174.9 (2) O1---C22---C23---C24 −179.7 (2) C19---C12---C13---C18 −5.2 (3) C22---C23---C24---C19 −0.4 (4) C11---C12---C13---C14 −3.7 (3) C20---C19---C24---C23 −0.1 (4) C19---C12---C13---C14 176.2 (2) C12---C19---C24---C23 −177.8 (2) C18---C13---C14---C15 4.5 (3) C21---C22---O1---C25 −11.4 (4) C12---C13---C14---C15 −176.8 (2) C23---C22---O1---C25 168.9 (3) C18---C13---C14---C9 −176.2 (2) ----------------------- ------------ ----------------------- ------------ Hydrogen-bond geometry (Å, º) {#d1e2748} ============================= Cg is the centroid of the C13--C18 ring. ------------------------ --------- --------- ----------- --------------- D---H···A D---H H···A D···A D---H···A C25---H25C···Cg^i^ 0.96 2.78 3.597 (5) 144 ------------------------ --------- --------- ----------- --------------- Symmetry code: (i) x−1, y, z. ![The molecular structure of the title compound, with the atom labelling. Displacement ellipsoids are drawn at 50% probability level.](e-74-00939-fig1){#fig1} ![The crystal packing of the title compound, viewed along the b axis. The C---H⋯π interactions (see Table 1[▸](#table1){ref-type="table"}) are shown as dashed lines, and only the H atom H25C (grey ball) has been included.](e-74-00939-fig2){#fig2} ![Hirshfeld surfaces mapped over (a) d ~norm~, (b) electrostatic potential, (c) shape index and (d) curvedness and (e) fragment patches.](e-74-00939-fig3){#fig3} ![Two-dimensional fingerprint plot for the title compound showing the contributions of individual types of interactions: (a) all intermolecular contacts, (b) H⋯H contacts, (c) C⋯H/H⋯C contacts, (d) H⋯O/O⋯H contacts, (e) C⋯C contacts. The outline of the full fingerprint is shown in grey. Surfaces to the right highlight the relevant surface patches associated with the specific contacts with d ~norm~ mapped.](e-74-00939-fig4){#fig4} ![(a) Interaction between the selected molecule and the molecules present in a 3.8 Å cluster around it, (b) Coulombic energy, (c) dispersion energy and (d) total energy.](e-74-00939-fig5){#fig5} ![Reaction scheme.](e-74-00939-fig6){#fig6} ###### Hydrogen-bond geometry (Å, °) Cg is the centroid of the C13--C18 ring. D---H⋯A D---H H⋯A D⋯A D---H⋯A ----------------------- --------- ------- ----------- ------------- C25---H25C⋯Cg ^i^ 0.96 2.78 3.597 (5) 144 Symmetry code: (i) . ###### Scale factors for the benchmarked energy model Energy model k ~elec~ k ~pol~ k ~energy-dispersive~ k ~rep~ ----------------------------------------------- ------------ ----------- ------------------------- ----------- CE-B3LYP⋯B3LYP/6--31G(d,p) electron densities 1.057 0.740 0.871 0.618 ###### Interaction energies (kJ mol^−1^) R is the distance between molecular centroids (mean atomic position) in Å and N is the number of molecules at that distance. Colour N symop R E ~elec~ E ~pol~ E ~energy-dispersive~ E ~rep~ E ~total~ ------------- ----- ------------------ ------- ------------ ----------- ------------------------- ----------- ------------- Red 2 x, y, z 15.38 −2.2 −0.6 −11.2 6.2 −8.6 Orange 1 −x, −y, −z 15.99 −4.3 −0.8 −11.5 4.2 −12.5 Yellow 1 −x, −y, −z 7.45 −6.2 −1.3 −39.2 19.3 −29.7 Lime 2 x, y, z 9.17 −10.0 −1.8 −44.0 26.8 −33.6 Green 2 x, y, z 10.46 −0.1 −0.1 −6.6 1.5 −5.0 Aquamarine 1 −x, −y, −z 6.86 −6.8 −0.9 −39.9 18.7 −31.0 Cyan 1 −x, −y, −z 10.11 −0.3 −0.4 −19.8 6.7 −13.7 Blue 1 −x, −y, −z 5.37 −3.3 −1.9 −69.2 32.2 −45.2 Violet 1 −x, −y, −z 9.31 −6.5 −0.8 −36.0 20.7 −26.0 Orchid 2 x, y, z 14.01 0.1 0.0 −2.0 0.0 −1.7 Magenta 1 −x, −y, −z 11.61 −3.3 −1.0 −41.6 20.4 −27.9 ###### Experimental details -------------------------------------------------------------------------- ------------------------------------------- Crystal data Chemical formula C~25~H~22~O M ~r~ 338.42 Crystal system, space group Triclinic, P Temperature (K) 296 a, b, c (Å) 9.1670 (9), 10.4566 (10), 11.2499 (11) α, β, γ (°) 64.707 (4), 71.312 (4), 77.032 (4) V (Å^3^) 918.75 (16) Z 2 Radiation type Mo Kα μ (mm^−1^) 0.07 Crystal size (mm) 0.15 × 0.10 × 0.10 Data collection Diffractometer Bruker Kappa APEXII CCD Absorption correction Multi-scan (SADABS; Bruker, 2012[@bb1]) T ~min~, T ~max~ 0.900, 0.945 No. of measured, independent and observed \[I \> 2σ(I)\] reflections 19120, 3828, 1777 R ~int~ 0.060 (sin θ/λ)~max~ (Å^−1^) 0.631 Refinement R\[F ^2^ \> 2σ(F ^2^)\], wR(F ^2^), S 0.057, 0.193, 1.00 No. of reflections 3828 No. of parameters 238 H-atom treatment H-atom parameters constrained Δρ~max~, Δρ~min~ (e Å^−3^) 0.27, −0.19 -------------------------------------------------------------------------- ------------------------------------------- Computer programs: APEX2, SAINT and XPREP (Bruker, 2012[@bb1]), SHELXT2014 (Sheldrick, 2015a [@bb13]), SHELXL2014 (Sheldrick, 2015b [@bb14]), ORTEP-3 for Windows (Farrugia, 2012[@bb4]) 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"et", "al" ]
In the past decades, surface plasmons (SPs) have been extensively investigated in the optical frequencies[@b1]. Due to their extremely strong and confined optical fields, SPs have shown various potential applications in the photonic circuits[@b2][@b3], near-field microscopy[@b4][@b5], biological sensors[@b6][@b7][@b8], photovoltaics[@b9][@b10], etc. Since the spoof (or designer) surface plasmon polaritons (SPPs) allow a route to imitate the optical SPPs[@b11][@b12], a variety of advanced plasmonic researches have been put forward and experimentally realized at low frequencies ranging from the microwave to infrared spectra, such as conformal surface plasmons waveguide[@b13], negative-index waveguide[@b14], and terahertz SPPs[@b15][@b16][@b17][@b18]. Recently, an important discovery showed that periodically textured perfectly electrical conducting (PEC) particles either in two or three-dimensions is able to mimic the natural localized surface plasmons (LSPs) at lower frequencies, which is called as spoof LSPs[@b19]. More recently, an ultrathin plasmonic spoof LSP resonator has been proposed, which is easy for fabrication and integration[@b20]. Based on the design of these plasmonic metamaterial particles, the conventional LSPs at optical frequencies can be directly scaled down to lower frequencies[@b21]. Apart from the features of conventional LSPs shown in the optical frequencies, many phenomena have been predicted and experimentally verified for spoof LSPs, such as the vertical transportation[@b22], unusual electric and magnetic LSPs resonances[@b23] and high-order electric LSPs resonances[@b24][@b25]. However, all earlier investigations and experiments on spoof LSPs are limited to the microwave frequency. With the rapid development of terahertz technology in recent years, the implementation of terahertz spoof LSPs resonators may become possible, which will facilitate the device miniaturization and system integration in the terahertz band. In this article, we propose the LSP particle in the terahertz frequency using ultrathin spirally corrugated metallic disk and generate the LSP resonances. We demonstrate that both spoof electric and magnetic LSPs are supported by the proposed ultrathin structure in the terahertz band, which is in deep-subwavelength scale and highly relies on the incident angle of terahertz waves. By slightly tuning the geometrical parameters of the proposed structure, we could change the resonant frequencies and further increase the subwavelength level. Besides the geometrical parameters, arrangements and separations have also significant effects on the resonance frequency[@b26][@b27]. These approaches extends the capabilities of spoof LSPs, and can be applied to the terahertz integrated circuits. Using the proposed magnetic LSPs, we further construct an electromagnetically induced transparency (EIT) metamaterial in the terahertz spectrum[@b28][@b29]. Numerical simulations, experimental measurements are conducted to evaluate and validate the performance of the proposed spoof LSPs, promising in a number of potential terahertz applications such slow lights, filters, and sensing. Results ======= The structure of the designed LSPs particle is schematically illustrated in [Fig. 1(a)](#f1){ref-type="fig"}, which is composed of four gold spiral arms and one internal gold disk with ultrathin thickness 0.2 μm. The radius r of the inner disk and length R of the outer spiral arms are 6.5 μm and 33 μm, respectively. The four spiral arms have the same linewidth w = 5 μm, and are separated by a gap g = 5 μm. The designed LSPs particle is fabricated on a polyimide substrate with 20 μm thickness (ε = 3, tan = 0.03). We first consider the case when the particle is normally illuminated with an x-polarized plane wave. The extinction cross sections (ECSs) are calculated using commercial software, the CST Microwave Studio 2013, as shown in [Fig. 1b](#f1){ref-type="fig"}. Here, the ECSs are sums of absorption cross sections and scattering cross sections. In [Fig. 1b](#f1){ref-type="fig"}, a peak can be clearly observed at 0.73 THz. To gain more insights into the resonance, we give the simulated electric field distributions (E~z~) in the x-y plane 5 μm above the metallic structure at 0.73 THz, as shown in [Fig. 1c](#f1){ref-type="fig"}. The electric fields distribution matches well with the previously reported spoof LSPs in the microwave frequency[@b23]. Meanwhile, we plot in [Fig. 1d](#f1){ref-type="fig"} the magnitude of the electric fields and electric field lines of this mode in a cross section y = 0. We can see that the arrows go from left of the structure to right. Clearly, this mode profiles correspond to the electric-dipole mode, indicating that the proposed structure supports spoof LSPs resonance at terahertz frequency. In [Fig. 2(a)](#f2){ref-type="fig"}, we show the calculated ECS spectra as a function of the outer radius R. When the radius R of the particle increases from 29 to 37 μm, the resonance frequency experiences an obvious redshift from 0.94 to 0.577 THz. To reveal the reason for the redshift in the proposed spoof LSP resonator, we first investigate the nature of spoof SPPs, since the spoof LSP resonator arises from the spoof SPP strip and the work frequency of the spoof SPPs has many influences on the resonance frequency of the relevant spoof LSPs. Textured surfaces can support spoof SPs due to the cavity-waveguide mode inside the groove, which is analogous to the penetration of fields into the metal. Either in straight or spiral groove, the frequency of existing SPs depends mainly on the depth of grooves. Here, although grooves in the proposed structure are spiral, it has been proved that the spiral grooves behave like the straight grooves approximatively[@b23]. The mentioned spoof SPP structure is illustrated in the inset of [Fig. 2(b)](#f2){ref-type="fig"}, whose parameters are set as s = 6.5 μm, p = 10 μm, and a = 5 μm. The depths of the straight grooves h are equal to the lengths of spiral curves in spoof LSP resonators, which are 45.7 μm, 53.8 μm, 62.5 μm, 71.8 μm, and 81.7 μm according to R = 29 μm, 31 μm, 33 μm, 35 μm, and 37 μm. The thicknesses of the metal and substrate are 0.2 μm and 20 μm, respectively. The calculated dispersion relations of spoof SPPs are presented in [Fig. 2(b)](#f2){ref-type="fig"}, from which we obtain that the deeper grooves correspond to the lower asymptotic frequencies. Actually the spoof LSP is a standing wave on the spoof LSP loop. Meanwhile because deeper grooves have lower frequencies for the same wavenumber, as shown in [Fig. 2(b)](#f2){ref-type="fig"}, hence the spoof LSP resonators composed of deeper grooves have lower resonance frequencies. Here, the deeper groove means longer spiral curve in the particle, which represents that the particle has bigger radius. Therefore, the resonance frequencies get smaller as the radiuses become bigger, as plotted in [Fig. 2(a)](#f2){ref-type="fig"}. In addition, we note that the radius R is much less than the resonant wavelength in the present design, making it a deep subwavelength structure. To gain an intuitional insight into their relationship, [Fig. 3](#f3){ref-type="fig"} further gives the resonance wavelengths λ~res~ as a function of the outer radius R, in which we see that the resonance wavelengths increase from about 319 to 520 μm as R increases from 29 to 37 μm. It can be calculated that a 27% change in size (from R = 29 μm to R = 37 μm) results in a 201 μm (or 63%) increase in resonance wavelength, demonstrating that λ~res~ can be tuned in large range by simply varying the radius R. Most importantly, by calculating the equivalent electrical length of the case when R equals 29 and 37 μm, we found that the bigger the radius R, the more the deep subwavelength effect is. This can be attributed to the large electrical length formed by the spiral grooves of particle, which effectively reduce the resonant frequency. Moreover, small change in radius R will result in great variation in the effective length of spiral grooves. The resonance frequency is not only highly dependent on the size of the meta-atom, but also depends on arrangement due to the electromagnetic interactions between neighbor particles. To evaluate these interactions, we simulate the transmission spectra using periodic boundaries. [Figure 4(a)](#f4){ref-type="fig"} shows the results for square arrays of meta-atoms with lattice constant p = 80--180 μm. As array spacing decreases, the resonance frequency first blue shifts for p larger than 80 μm and then redshifts for smaller distances. To analyze the unit coupling effects, we introduce a simple semi-analytical model which can help understand this trend qualitatively[@b26][@b27]. In this model, the retarded dipole sum S can reveal the variation of the spectra with arranged period. It was demonstrated that the real parts of S imply the resonant frequency shifts. The positive value corresponds to red shift of λ~max~, while the negative value represents blue shift. This sum S is given by where d~ij~ is the distance between the ith and jth particles, θ~ij~ is the angle between the vector from units i to j and the polarization vector. From [Fig. 4(a)](#f4){ref-type="fig"}, we find that the resonant frequencies are approximately 0.75 THz (400 μm). Then we calculate the values of S as the period p changes from 180 to 100 μm for λ = 400 μm. The results are presented in [Table 1](#t1){ref-type="table"}. [Table 1](#t1){ref-type="table"} demonstrates that the real part of S is negative when p changes from 180 to 100 μm, while is positive for p = 80 μm. The real part becomes less negative as the period p decreases and becomes positive until p = 80 μm. In other words, blue shifts occur for p = 180 to 100 μm and red shifts for p = 80 μm, which have good agreement with the trend in [Fig. 4(a)](#f4){ref-type="fig"}. Actually, the expression for S can be divided into two parts, one of which has 1/d component, representing the radiative dipole interaction, and the other of which has 1/d^3^ component, showing the static dipole coupling. Hence we find that for big lattice constant the radiative dipole interaction is dominant, whereas the static dipole coupling plays a more important role as the period p decreases. And the resonance blue shifts (negative value in real parts of S) are the result of the radiative dipole interaction for bigger separations, whereas red shifts due to the static dipole coupling for smaller lattice spacing. To experimentally verify the performance of the proposed spoof LSPs arrays, we fabricate samples, whose unit cells are distributed on a two-dimensional surface with lattice constants p from 80 to 180 μm. Each sample occupies an area of 15 × 15 mm, which is wide enough to cover the focused THz beam in experiments. [Figure 4(c)](#f4){ref-type="fig"} illustrate the sample with p = 180 μm and his zoomed image taken by an optical microscope (VHX-5000, Keyence Company). The measured transmission spectra are plotted in [Fig. 4(b)](#f4){ref-type="fig"}. In the measured spectra, the resonance wavelength first blue shifts and then red shifts, the switch in behavior occurring at 80 μm, which match with the trend in [Fig. 4(a)](#f4){ref-type="fig"}. Both calculation and experiment demonstrate that meta-atom arrangement and separation play an important role in resonance frequency. So far, magnetic resonance is not observed in the proposed structure under the normal incidence. Then we proceed to calculate the extinction cross sections (ECSs) of a single meta-atom under grazing incidence, aiming to excite magnetic resonance, shown in [Fig. 5(a)](#f5){ref-type="fig"}. The ECSs spectrum shows two distinct peaks, and this phenomenon closely resembles that discovered at microwave frequencies in previous published works[@b23][@b25]. The lower frequency at 0.73 THz corresponds to the electric resonance, which is same to the frequency of the resonance peak under the normal incidence. Then we plot the magnetic field distribution (H~z~) of the higher peak at 1.06 THz in [Fig. 5(b)](#f5){ref-type="fig"}. The magnetic field distribution is similar to a magnetic dipole. To confirm this mode, the magnitude of the magnetic fields and magnetic field lines in transverse plane y = 0 are simulated, shown in [Fig. 5(c)](#f5){ref-type="fig"}. The H-field lines is circulating around the disk, and thus the mode was termed as magnetic LSPs. We have demonstrated that for spoof LSPs resonance, normal incidence only excite the electric LSPs mode directly while the magnetic LSPs mode needs to be generated by grazing incidence. By this feature, magnetic LSPs mode is able to serve as EIT dark element. Generally, EIT spectral response is attributed to the destructive interference between bright and dark mode resonances, which corresponds to broad resonance and sharp resonance, respectively. Therefore, a metallic wire with length 90 μm and linewidth 5 μm is used to generate bright mode, as displayed in the left inset of [Fig. 6(a)](#f6){ref-type="fig"}. These wires are distributed on a polyimide substrate with lattice constant p = 180 μm. The simulated transmittance of the wires array under y-polarized (along the wire) normally incident terahertz wave are given in [Fig. 6(a)](#f6){ref-type="fig"} (black curve). The wires array show a low quality factor and broad resonance. To visualize this resonance mode, [Fig. 6(c)](#f6){ref-type="fig"} plots the simulated E~z~ near-field distribution at 1.07 THz, which shows a typical dipolar resonance. In this case, the metallic wires are strongly coupled to the normal incident wave, which enable them to be bright mode. Next, we place the straight wires close to the spoof LSPs resonators with distance of 5 μm, as shown in the right inset of [Fig. 6(a)](#f6){ref-type="fig"}. In this case, the normal terahertz waves can drive the wires directly while the spoof magnetic LSPs resonators have no response. However, the near field coupling between them excites the magnetic resonance in the spoof LSPs resonator. The destructive interference between the dipole and magnetic LSPs resonances results in a transparency window at 1.07 THz in the simulated transmission spectra, as illustrated in [Fig. 6(a)](#f6){ref-type="fig"} (the red line). To visually verify the strong EIT effect arising from destructive interference, the simulated E~z~ near-field distribution of the unit cell is shown in [Fig. 6(d)](#f6){ref-type="fig"}, in which strong spoof magnetic LSPs resonance can be clearly observed[@b23][@b25]. Comparing with [Fig. 6(c)](#f6){ref-type="fig"}, the dipole resonance in [Fig. 6(d)](#f6){ref-type="fig"} is very weak due the destructive interference effect. In order to experimentally verify the EIT behavior, a sample combining both wires and LSPs resonators is fabricated, as shown in the inset of [Fig. 6(b)](#f6){ref-type="fig"}. The experimental results of transmissions presented in [Fig. 6(b)](#f6){ref-type="fig"}, which are in good agreement with the simulation results. However, one may notice that the quality factor of the measured spectra is lower than simulation, which can be also observed in [Fig. 4(b)](#f4){ref-type="fig"}. This discrepancy is most likely due to surface roughness and the higher loss of metallic structure in the fabricated sample. Conclusion ========== In summary, we have numerically and experimentally demonstrated that spoof electric and magnetic LSPs can be realized at terahertz by metamaterial. The dimension of the meta-atoms is much smaller than resonance wavelength, making the proposed structure to be deep subwavelength scale. Both dimensions and arrangements can affect the resonance frequency. It is found that a sharp transparent transmission window can be achieved by placing the spoof LSPs resonator close to a metallic cut wire, between which destructive interference occurs. This work not only offers a way to obtain spoof LSPs resonance at terahertz frequency, but also make use of it to exhibit a EIT effect. Such plasmonic metamaterials may find interesting applications in plasmonic devices at terahertz frequency. Methods ======= Manufacturing method -------------------- The detailed fabrication processes are described as follows. First, a 20 μm thick polyimide is coated to a silicon wafer as the substrate. Then, it was cured on hotplate at 80, 120, 180 and 250 °C for 5, 5, 5 and 20 minutes, respectively. A standard photolithography was then conducted before depositing a Ti/Au layer (10/180 nm) with electron beam evaporation, after which a lift-off process helped form the final metallic pattern. Finally, we peeled off the sample from the silicon wafer by immersing it in HF solvent for 30 minutes. Simulation method ----------------- All numerical simulations are performed by the commercial software, CST Microwave Studio 2013. The relevant substrate is polyimide, whose permittivity and loss tangent are 3 and 0.03, respectively. The metal is chosen as gold, which is selected in the material library in CST. For ECS calculation, a plane-wave excitation and a broadband far-field monitor are applied to record absorption cross sections and scattering cross sections. The transmissions are simulated by the CST transient solver, in which the periodic boundary conditions are used. The calculation of dispersion relation is based on the CST eigenmode solver, in which the eigen frequencies of a unite cell is analyzed. Experimental setup and measurement ---------------------------------- The experimental setup is a commercial Terahertz Spectroscopic Systems (TAS7400SP). The samples are placed on a platform, as shown in [Fig. 7](#f7){ref-type="fig"}. All the experiments were measured in nitrogen environment (humidity smaller than 1%) under 25 °C. All spectra are normalized to the transmission without sample. Additional Information ====================== How to cite this article: Liao, Z. et al. Electromagnetically induced transparency metamaterial based on spoof localized surface plasmons at terahertz frequencies. Sci. Rep. 6, 27596; doi: 10.1038/srep27596 (2016). This work was supported by the National Science Foundation of China (61302018, 61401089, 61571117, 61501112, 61501117, and 61138001), the 111 Project (111-2-05), the National Instrumentation Program (2013YQ200647), the Natural Science Foundation of Jiangsu Province (BK20130854), the Scientific Research Foundation of Graduate School at Southeast University (YBJJ1436), the Program for Postgraduate Research Innovation in University of Jiangsu Province (3204004910), and the Natural Science Foundation of Jiangsu Province under BK20150020. Author Contributions Z.L. and T.J.C. designed the samples and performed the simulations. Z.L., S.L., H.F.M., C.L., B.J. and T.J.C. conducted the experiment. Z.L., H.F.M. and T.J.C. wrote the manuscript. ![(a) Schematic diagram of the ultrathin metallic spiral structure. (b) The calculated ECS spectra at normal incidence. (c) The simulated E~z~ near-field pattern at the resonance frequency. (d) The magnitude of the electric fields at the resonance frequency, in which the arrows show the electric field lines.](srep27596-f1){#f1} ![(a) The simulated ECS spectra with respect to different outer radius. (b) The dispersion relations of spoof SPPs with different depth. The inset shows the schematic picture of spoof SPPs strip.](srep27596-f2){#f2} ![The resonant wavelengths of metamaterial particle as a function of the radius R.](srep27596-f3){#f3} ![(a) The simulated amplitude transmission spectra for the two-dimensional array with lattice constants p from 80 to 180 μm. (b) The measured amplitude transmission spectra for the two-dimensional array with lattice constants p from 80 to 180 μm. (c) The photo of sample and microscopic image of the fabricated structure.](srep27596-f4){#f4} ![(a) The calculated ECSs spectra at grazing incidence. (b) The simulation result of H~z~ near-magnetic-field distribution corresponding to the magnetic resonance. (c) The magnitude of the magnetic fields at the resonance frequency. The arrows show the magnetic field lines.](srep27596-f5){#f5} ![(a) The simulated amplitude transmission spectra of the sole-wire (the black line), and the EIT metamaterial (the red line). The insets present the structure of the simulated unit cell. (b) The measured amplitude transmission spectra of the sole-wire (the black line), and the EIT metamaterial sample (the red line). The insets present the microscopy images of the fabricated sample. The simulated electric field (E~z~) distributions of (c) the sole-wire and (d) the EIT metamaterial at 1.07 THz.](srep27596-f6){#f6} ![Photograph of the experimental setup used to measure the transmission of samples.](srep27596-f7){#f7} ###### The dipole sum S and its two components for λ = 400 μm. p(μm) S S(1/d^3^) S(1/d) ------- ---------------- --------------- --------------- 80 0.52 + 6.04i 3 + 3.61i −2.48 + 2.43i 100 −2.71 + 7.8i 2.21 + 4.29i −4.92 + 3.54i 120 −4.49 + 6.86i 1.23 + 4.8i −5.72 + 2.06i 140 −8.27 + 7.19i 0.15 + 5.14i −8.43 + 2.05i 160 −10.58 + 4.85i −1.04 + 5.25i −9.54-0.4i 180 −14.06 + 2.91i −2.3 + 5.1i −11.76-2.2i	{ "pile_set_name": "PubMed Central" }	[ "electricdipol", "sppsbb", "discov", "keyenc", "two", "extinct", "phenomenon", "terahertz", "given", "work", "program", "nitrogen", "decreas", "hz", "penetr", "tiau", "sppsbbbb", "sum", "nearfield", "exist", "silicon", "put", "wafer", "length", "asymptot", "spectrum", "bigger", "select", "valu", "nearmagneticfield", 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Glioblastomas (GBMs) are one of the most aggressive and deadly forms of human cancer. GBM treatment usually consists of surgical resection followed by radiotherapy combined with the alkylating agent temozolomide (TMZ)[@b1]. Although this therapeutic approach slightly improves the survival rate of GBM patients, a large fraction of these patients suffer from tumour recurrence[@b1]. Accumulating evidence suggests that tumour relapse may be driven by a component of heterogeneous tumour cells that retain stem cell-like properties, called "cancer stem cells" (CSCs). The potent tumourigenic capacity of glioma CSCs (GSCs), coupled with evidence of radio- and chemo-resistance, suggests that a stem cell-orientated therapy may represent an innovative strategy to reduce tumour recurrence and improve GBM prognosis[@b2]. Two main strategies are currently exploited to eradicate the heterogeneous population of GBM and GSCs: (a) chemotherapeutic regimens that specifically drive GSCs into cell death, and (b) driving GSCs into differentiation, thereby depleting the tumour reservoir. The latter strategy appears the most promising, considering that differentiated cells are in general more sensitive to chemotherapeutic agents with respect to CSCs[@b3]. Studies on human GBM samples have uncovered that the deregulation of signal transduction pathways is one of the most prominent[@b4][@b5]. The disruption of signal transduction in GBM occurs through over-expression or a gain-of-function mutation of tyrosine-kinase receptors[@b6][@b7], thus leading, among other events, to constitutive activation of Ras/extracellular signal-regulated kinase (ERK), AKT/mammalian target of rapamycin (mTOR). As a result, AKT is elevated in the majority of examined GBMs[@b8][@b9] with the subsequent amplification of pro-survival signals and blockage of oncosuppressor controls. The inactivation of the oncosuppressor protein p53 is certainly one of the main phenomena that allow GBM cells to escape cell cycle checkpoints. In particular, the intracellular levels of p53 are maintained low due to an excessive stimulation (mediated by AKT constitutive activation[@b10]) of the ubiquitin-ligase murine double minute 2 homologue (MDM2), the predominant natural endogenous inhibitor of the protein p53[@b11][@b12]. In addition to accelerating p53 degradation, MDM2 prevents p53 binding to DNA, blocking its transcriptional activity. As GBM cells typically express p53 with a wild-type amino acid sequence, the re-activation of p53 functionality can be restored through the inhibition of the oncogenic block exerted by the AKT/mTOR pathway, which causes an excessive stimulation of MDM2. In this respect, while agents inhibiting either the AKT/mTOR pathway[@b13][@b14][@b15] or the MDM2/p53 interaction[@b16][@b17][@b18] have provided some survival benefit in GBM, the effects of a co-therapy have not been deeply investigated to date, either in GBMs or in their stem cells. In acute myeloid leukaemia, the PI3K/mTOR inhibitor PI-103 acts synergistically with the MDM2 inhibitor nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion[@b19], supporting the aforementioned mechanistic rationale. In our previous work, a series of 2-oxindole derivatives (OXIDs) have been described[@b20] and demonstrated to act as inhibitors of the AKT/mTOR pathway. Herein, we identified FC85 as a new ligand, useful in establishing the preclinical proof of concept for the AKT/mTOR pathway, and whose activity could be amplified by co-treatment with an MDM2 inhibitor. The mechanism of action of FC85 was examined alone or in combination with an already characterized inhibitor of MDM2, ISA27[@b18], both in GBM cells and in their derived GSCs. In parallel experiments, the oral mTOR inhibitor everolimus[@b21][@b22] and the MDM2 inhibitor nutlin-3[@b17][@b18]were also used as reference compounds. Globally, our findings demonstrated that AKT/mTOR inhibitors actively enhance downstream p53 signalling and that a combination strategy aimed at inhibiting the PI3K/AKT/mTOR pathway and activating p53 signalling is potentially effective in GBMs and in GSCs ([Fig. 1a](#f1){ref-type="fig"}). Results ======= Design and Synthesis -------------------- Over recent years, new compounds with an indole/oxindole core have been widely investigated as agents able to target the activity of the serine/threonine kinases PDK1 and/or AKT[@b23]. Recently, we synthesized new OXIDs compounds by the combination of a tetrahydroisoquinoline nucleus with the 2-oxindole nucleus throughout a methylenamido moiety, and anchoring the 3-position of oxindole core to different heterocycles ([Fig. 1b and c](#f1){ref-type="fig"}). The new OXIDs[@b24] induced cell cycle arrest and inhibited AKT phosphorylation in non-small cell lung cancer cells (which overexpress the PI3K/AKT/mTOR pathway and show resistance to EGFR inhibitors), suggesting that the OXID nucleus can be used as central core to develop inhibitors of the PI3K/AKT/mTOR pathway. Specifically, we afforded the replacement of the amido moiety of OXIDs ([Fig. 1b and c](#f1){ref-type="fig"}) with its bioisosteric amidosulfonyl group. Sulfonamide is a well-known pharmacofore notorious as key element to confer anticancer properties, among others[@b25][@b26]. FC85 was obtained as depicted in [Figure 1b](#f1){ref-type="fig"}. Briefly, the 5-amino-2-oxindole 1 reacted with p-toluenesulfonyl chloride to give 4-methyl-N-(2-oxoindolin-5-yl)benzenesulfonamide 2. The subsequent Knoevenagel condensation of 2 with the 1H-imidazole-5-carboxaldehyde afforded the target compound as a Z-isomer. In co-treatment experiments, we used the small-molecule MDM2 inhibitor with a spirooxoindolepyrrolidine core structure, ISA27, which was synthesized as previously described[@b27]. Effects of FC85 on the AKT/mTOR signalling pathway in U87MG cells ----------------------------------------------------------------- As a representative GBM cell line, we used U87MG cells, which is an appropriate model to study the interaction between the AKT/mTOR and MDM2-p53 pathways because of the following characteristics: i) U87MG cells maintain a wild type status of p53, and ii) U87MG cells are deficient for the tumour suppressor phosphatase and tensin homologue (PTEN), a negative regulator of the PI3K/AKT pathway; moreover, PTEN deficiency leads to MDM2 nuclear accumulation, thus inhibiting p53 functions[@b28]. To assess whether FC85 could effectively inhibit the mTOR/AKT pathway in U87MG cells, the effects of the new compound on AKT and mTOR activity were examined. FC85, tested at 100 nM, 1 µM and 10 µM, significantly inhibited AKT phosphorylation at Ser473 ([Suppl. Fig. 1a](#s1){ref-type="supplementary-material"}), as reported for the already published OXIDs[@b24]. The new compound reduced also mTOR constitutive phosphorylation at Ser2448 ([Suppl. Fig. 1b](#s1){ref-type="supplementary-material"}). These findings demonstrated that FC85 was able to target the AKT/mTOR signalling pathway in U87MG cells. Effects of FC85 on U87MG cell viability --------------------------------------- To examine the effects of FC85 on GBM cell growth/survival, U87MG cells were incubated with different concentrations of the compound for 24 h this new compound showed a dose-dependent inhibitory effect on the growth/survival of U87MG cells, with an IC~50~ value of 468.9±48.9 nM ([Fig. 2a](#f2){ref-type="fig"}). Trypan blue exclusion experiments confirmed a concentration-dependent decrease of live cells and an increase of dead cells ([Fig. 2b](#f2){ref-type="fig"}). To assess whether U87MG cells could resume proliferation, cells were treated for 96 h with the compound at 10 µM. At the end of the treatment period, the culture medium was replaced with fresh medium not containing the drug. As depicted in [Figure 2c](#f2){ref-type="fig"}, the number of viable cells did not significantly increase at either 1 or 3 days of wash-out with respect to control samples, suggesting the inability of cells to recover normal growth. To determine compound toxicity in non-tumour cells, we examined the effects of FC85 on the viability of normal Human Umbilical Vein Endothelial Cells (HUVECs). As shown in [Figure 2d](#f2){ref-type="fig"}, the viability of HUVECs was significantly decreased after a 48 h incubation with FC85 used at 1 µM and 10 µM. However, the effects on cell viability were not strictly concentration dependent, and the percentages of cell viability reduction were significantly lower with respect to those observed in U87MG cells, suggesting that the anti-proliferative effect elicited by FC85 was directed preferentially towards tumour cells. Effect of a combined therapy with an inhibitor of MDM2/p53 complex on U87MG cell viability ------------------------------------------------------------------------------------------ Because the inhibition of PI3K/AKT/mTOR signalling can augment p53-mediated apoptosis[@b29], AKT/mTOR may synergize with inhibitors of the MDM2/p53 complex to promote anti-tumoural activity in GBM cells that retain p53 function. To this end, we investigated the effects on U87MG cell viability of combining doses of FC85 with the already characterized MDM2 inhibitor, ISA27[@b18]. The theoretical additive IC~50,add~ values for the two drugs were calculated for two fixed ratios (1:2, 1:5), and the experimental IC~50,mix~values were determined for the same fixed-ratio combinations in the viability assay ([Fig. 3a](#f3){ref-type="fig"}). Statistical analysis of the data from isobolographic analysis revealed synergistic interactions between FC85 and ISA27 for the two examined fixed-ratio combinations ([Suppl. Table S1](#s1){ref-type="supplementary-material"}; [Fig. 3a](#f3){ref-type="fig"}). Similar experiments were performed using the oral mTOR inhibitor everolimus, currently in clinical use for several type of solid tumours[@b21][@b22], including GBMs (NCT00823459, NCT00831324, NCT00613132, NCT00553150, NCT00387400, NCT00085566, NCT00805961) and the MDM2 inhibitor nutlin-3, whose oral formulation has completed the first early phase clinical trials for both solid cancers and haematological malignancies[@b30][@b31]. Everolimus and nutlin-3 alone inhibited U87MG cell proliferation, yielding an IC~50~ of 50.0±2.8 nM and 10.3±1.1 µM, respectively ([Suppl. Fig. 2a](#s1){ref-type="supplementary-material"}). When combined, the two reference agents showed a synergic reduction of U87MG cell viability for both the examined fixed-ratio combinations ([Suppl. Table S2](#s1){ref-type="supplementary-material"}; [Suppl. Fig. 2b](#s1){ref-type="supplementary-material"}). Globally, these results confirmed that a combined therapy with an inhibitor of MDM2/p53 complex and an inhibitor of the AKT/mTOR pathway could be a useful anti-proliferative strategy in GBM cells. We then examined the effects of cell treatment with FC85, alone or in combination with ISA27, on the reactivation of p53 pathway, assessing accumulation of p53 protein and transcription induction of p53 target genes. The incubation of GBM cells with FC85 alone for 6 h led to a slight but significant increase in p53 protein levels ([Fig. 3b and c](#f3){ref-type="fig"}). As expected, challenging cells with ISA27 caused a higher accumulation of p53. When cells were treated with FC85 and ISA27, a significant enhancement of p53 protein accumulation with respect to cells treated with FC85 or ISA27 alone was observed ([Fig. 3b and c](#f3){ref-type="fig"}). In line with these data, short-term (6 h) treatment of U87MG cells with FC85 or ISA27 led to a significant increase in the mRNA levels of certain p53 target genes ([Fig. 3d](#f3){ref-type="fig"}): p21, a cell cycle inhibitor; PUMA, a gene product required for p53-controlled intrinsic apoptosis pathway; and MDM2, physiological inhibitor of p53, and its main transcriptional target. These results suggested that p53 stabilization in FC85-treated GBM cells led to an increase in MDM2, PUMA and p21 mRNA levels in a manner that was consistent with the activation of the p53 pathway. Moreover, a significant enhancement of the transcription of p53 target genes was observed in FC85-ISA27 co-treated cells with respect to single agent-treated cells ([Fig. 3d](#f3){ref-type="fig"}). In parallel experiments, neither nutlin-3 (10 µM) nor everolimus (50 nM) alone induced a significant accumulation of p53 protein in short-term (6 h) treatment ([Suppl. Fig. 3a and b](#s1){ref-type="supplementary-material"}). These data are consistent with those previously reported for nutlin-3[@b18]. When combined, the reference agents produced a significant p53 accumulation after 6 h of treatment ([Suppl. Fig. 3a and b](#s1){ref-type="supplementary-material"}). Real time RT-PCR experiments showed that nutlin-3 did not induce any significant increase in p53 target gene products after 6 h of treatment ([Suppl. Fig. 3c](#s1){ref-type="supplementary-material"}), according to published data[@b18]. Similar results were obtained with everolimus ([Suppl. Fig. 3c](#s1){ref-type="supplementary-material"}). When the two drugs were used together, a significant increase in MDM2 mRNA was noticed ([Suppl. Fig. 3c](#s1){ref-type="supplementary-material"}). These data are consistent with those obtained in western blotting analysis and suggest that the combined therapy could reactivate p53 function with increased efficacy compared with single therapy. To examine whether the p53 function was effectively reactivated after a prolonged exposure time, U87MG cells were incubated with everolimus and/or nutlin-3 for 24 h. Both agents caused a significant induction of MDM2 and p21 mRNA levels ([Suppl. Fig. 3d](#s1){ref-type="supplementary-material"}). Moreover, a synergistic and significant induction of PUMA mRNA level was noticed when the two drugs were combined ([Suppl. Fig. 3d](#s1){ref-type="supplementary-material"}). Globally, these results demonstrate that nutlin-3 and everolimus caused a slower re-activation kinetics of p53 function with respect to FC85 and ISA27. Effect of FC85, ISA27 and of their combined treatment on the induction of apoptosis and cell cycle block -------------------------------------------------------------------------------------------------------- Challenging U87MG cells with FC85 (500 nM) induced a significant phosphatidylserine externalization, both in the absence (early apoptosis) or presence of 7-amino-actinomysin binding to DNA (late apoptosis/death; [Fig. 4a and b](#f4){ref-type="fig"}). These data are consistent with the observed increase of PUMA mRNA level ([Fig. 3d](#f3){ref-type="fig"}). Similar results were observed with the MDM2 inhibitor ISA27 ([Fig. 4a and b](#f4){ref-type="fig"}). When the two drugs were combined together, a significant enhancement in the percentage of cells in late apoptosis/death was detected, with respect to cells treated with ISA27 or FC85 alone ([Fig. 4a and b](#f4){ref-type="fig"}). These findings suggest that the combined therapy may accelerate apoptosis induction in U87MG cells. In contrast, everolimus or nutlin-3 did not induced a significant level of apoptosis in U87MG cells within 24 h of treatment ([Suppl. Fig. 4](#s1){ref-type="supplementary-material"}), consistent with previously published data[@b17][@b18][@b32][@b33]. However, the co-treatment induced a slight but significant induction of apoptosis ([Suppl. Fig. 4](#s1){ref-type="supplementary-material"}), suggesting that everolimus could accelerate the induction of apoptosis elicited by nutlin-3 in more prolonged treatment[@b17][@b18]. These data are consistent with those observed in real-time RT-PCR experiments: after 24 h of incubation, the levels of PUMA mRNA, a p53-target gene product mainly involved in cellular apoptosis, were indeed significantly higher only when nutlin-3 and everolimus were used in combination. Cell cycle analysis demonstrated that challenging U87MG cells with FC85, as well as with ISA27, for 24 h arrested cell-cycle progression in the G0/G1-phase ([Fig. 4c and d](#f4){ref-type="fig"}). U87MG incubation with both FC85 and ISA27 was able to induce a significantly higher blockage in the G0-G1 phase compared with single-compound treated cells ([Fig. 4c and d](#f4){ref-type="fig"}). These results suggest that cell cycle arrest may have been a contributing factor in the observed increased sensitivity of GBM cells to the combined therapy. Effects of FC85, ISA27 and of their combined treatment on GSC viability ----------------------------------------------------------------------- A hyper activation of the AKT/mTOR pathway has been reported to play a pivotal role in GBM proliferation as well as in the self-renewal and propagation of GSCs. On this basis, we evaluated the effects of FC85 on GSCs, isolated from U87MG cells as previously reported[@b34]. As depicted in [Figure 5a](#f5){ref-type="fig"}, when GSCs were incubated with this new compound (1 µM or 10 µM), a significant inhibition of cell proliferation occurred starting from four days of treatment. Similar results were obtained using ISA27 ([Fig. 5b](#f5){ref-type="fig"}). A dose-response curve of FC85 and ISA27 was then performed after seven days of treatment: the two compounds were able to induce a concentration-dependent inhibition of GSC proliferation, with IC~50~ values of 104±12 nM and 540±68 nM, respectively ([Fig. 5c](#f5){ref-type="fig"}). In parallel experiments, nutlin-3 reduced GSC proliferation starting from four days of incubation, yielding an IC~50~ value of 18.5±2.1 µM after seven days of incubation ([Suppl. Fig. 5b and c](#s1){ref-type="supplementary-material"}). Everolimus exhibited a high ability in reducing GCS proliferation starting from 24 h of cell treatment and showing an IC~50~ value of 15.0±1.2 nM ([Suppl. Fig. 5a and c](#s1){ref-type="supplementary-material"}). The effects of a combined administration of FC85 and ISA27 were then investigated in GSCs. Isobolographic analysis revealed a marked synergic effect on the reduction of GSC proliferation, when the two compounds were used together ([Suppl. Table S3](#s1){ref-type="supplementary-material"}; [Fig. 5d](#f5){ref-type="fig"}), suggesting that a combined therapy with an inhibitor of the MDM2/p53 complex and an inhibitor of the AKT/mTOR pathway can be useful in GSC-orientated therapy. Similar synergistic interactions were confirmed also using everolimus and nutlin-3 ([Suppl. Fig. 5d](#s1){ref-type="supplementary-material"}; [Suppl. Table S4](#s1){ref-type="supplementary-material"}). Next, we assessed whether FC85 and/or ISA27-treated cells could resume proliferation after drug removal. After GSC challenging with FC85 or ISA27, and a wash-out period of seven and fourteen days, the percentages of proliferating/viable cells did not significantly increase, suggesting an overall inability to recover normal growth ([Fig. 5e](#f5){ref-type="fig"}). These effects were particularly evident when the two compounds were used simultaneously ([Fig. 5e](#f5){ref-type="fig"}). Effects of FC85, ISA27 and of their combined treatment on the induction of GSC apoptosis ---------------------------------------------------------------------------------------- We then investigated whether the reduction of GSC proliferation elicited by the two compounds was associated with apoptosis. After seven days of GSC treatment, both FC85 and ISA27 induced a significant phosphatidylserine externalization, both in the absence (early apoptosis) or in the presence of 7-amino-actinomysin 11 binding to DNA (late apoptosis/death; [Fig. 6a and b](#f6){ref-type="fig"}). The simultaneous incubation of GSCs with the two compounds induced both early and late apoptosis; the percentage of late apoptosis-death was significantly higher with respect to those observed with the two compounds alone ([Fig. 6a and b](#f6){ref-type="fig"}). Of note, whereas nutlin-3 alone did not show any significant effects on GSC apoptosis, everolimus induced slight but significant early apoptosis after seven days of treatment ([Suppl. Fig. 6](#s1){ref-type="supplementary-material"}). When used together, the two reference drugs caused a strong induction of early apoptosis, with significantly higher effects with respect to those obtained in single agent-treated GSCs ([Suppl. Fig. 6](#s1){ref-type="supplementary-material"}). These results confirmed that the chosen combined therapy could accelerate the induction of GSC apoptosis. Effects of FC85, ISA27 and of their combined treatment on GSC morphology and differentiation -------------------------------------------------------------------------------------------- The effects of FC85 and ISA27 on GSC morphology were evaluated by quantifying the area occupied by the cells in culture plates as well as the outgrowth of cellular processes. When GSCs were incubated with FC85 for seven days, an almost complete reduction in the area occupied by the floating spheres was noticed, and the cells showed prominent outgrowth of processes ([Fig. 7a-c](#f7){ref-type="fig"}), as previously reported as a response to other AKT/mTOR inhibitors[@b35][@b36]. Similar results were obtained with the MDM2/p53 inhibitor ([Fig. 7a-c](#f7){ref-type="fig"}), suggesting that the reactivation of the p53 pathway could also induce GSC differentiation. In both cases, the morphology of differentiated cells appeared to be heterogeneous, suggesting that FC85 and ISA27 could promote cell differentiation towards more than one phenotype. When FC85 and ISA27 were combined, a marked reduction in the size of neurospheres was evidenced, with few cells presenting small cellular processes ([Fig. 7a-c](#f7){ref-type="fig"}). To investigate the mechanisms through which FC85 and ISA27 affect GSC morphology, we then assessed the levels of stem and differentiation markers upon stimulation with the two compounds. In FC85-treated GSCs, a significant decrease in the stemness markers, CD133 and Nestin, was noticed ([Fig. 6c](#f6){ref-type="fig"}) accompanied by a significant increase of MAP and GFAP content, demonstrating that FC85 was able to promote GSC differentiation towards a neuronal and a glial phenotype. Similar results were obtained with ISA27 ([Fig. 6c](#f6){ref-type="fig"}). Surprisingly, despite the fact that no significant cellular processes had been noticed in the morphological analysis, when cells were treated with the two drugs, a significant decrease in the stemness markers was evidenced, together with a marked increase in the MAP, GFAP and Olig2 content ([Fig. 6c](#f6){ref-type="fig"}). Moreover, the enhancement of the neuronal and of the astrocyte markers were significantly higher in FC85+ISA27-treated cells with respect to that observed in single agent-treated cells ([Fig. 6c](#f6){ref-type="fig"}). Comparing the morphological/apoptosis analysis and RT-PCR data indicated that combined ISA27 and FC85 not only induced the apoptosis of GSCs but also promoted the differentiation of these cells. Effects of FC85, ISA27 and of their combined treatment on the kinetics of AKT and ERK1/2 phosphorylation -------------------------------------------------------------------------------------------------------- The putative intracellular signalling pathways underlying the effects elicited by the MDM2/p53 and the AKT/mTOR inhibitors were then investigated. Different signalling pathways have been demonstrated to play a pivotal role in GSC proliferation and differentiation[@b37]; among these, several evidence suggested that a crosstalk between the PI3K/AKT/mTOR and MEK/ERK pathways in the maintenance of self-renewal and tumourigenicity of GSCs[@b38]. To study in depth the mechanism underlying the observed effects, the kinetics of AKT and ERK1/2 inhibition were investigated. GSC incubation with FC85 and/or ISA27 did not alter total ERK or AKT levels ([Suppl. Fig. 7](#s1){ref-type="supplementary-material"}). Both FC85 and ISA27 induced a fast and significant inhibition of AKT phosphorylation, which persisted up to 60 min ([Fig. 8a](#f8){ref-type="fig"}). Challenging cells with both compounds prolonged p-AKT inhibition up to 120 min, with significantly higher percentages of inhibition with respect to single agent-treated cells ([Fig. 8a](#f8){ref-type="fig"}). Both ligands also affected p-ERK1/2, although showing a transient blockage (up to 30 min of GSC incubation, [Fig. 8b](#f8){ref-type="fig"}). In contrast, FC85+ISA27 produced a significant and sustained inhibition of ERK1/2 activity (up to 120 min); as in the case of p-AKT, the percentages of inhibition were significantly higher with respect to single agent-treated cells at 60 and 120 min of GSC treatment ([Fig. 8b](#f8){ref-type="fig"}). These results are in line with the additive/synergic effect on GSC differentiation elicited by the co-treatment FC85+ISA27, and support the previous demonstration that the dual and sustained inhibition of PI3K/AKT/mTOR and MEK/ERK signalling induced differentiation and inhibited the tumourigenicity of GSCs[@b38]. Discussion ========== In this study, we report the interactions of PI3K/AKT/mTOR and MDM2/p53 pathways in GBM cells, following the simultaneous inhibition of AKT/mTOR signalling and re-activation of the p53 pathway. First, we showed that a combined therapy utilizing the novel mTOR/AKT inhibitor FC85 and the MDM2/p53 inhibitor ISA27 produced a synergistic effect on the inhibition of GBM cell viability as well as on the reactivation of p53 pathway. Most importantly, similar synergistic effects were shown in GBM-derived CSCs, where the simultaneous use of the two compounds induced a strong differentiation of GSCs, as well as apoptosis. A synergistic inhibition of cell viability of both GBM and its derived CSCs was evidenced also using the mTOR inhibitor everolimus and the MDM2 inhibitor nutlin-3 as reference compounds. These findings suggest that a combination strategy aimed at inhibiting AKT/mTOR signalling and re-activating p53 signalling may be potentially effective in GBMs and GSCs. The multi-targeted strategy has assumed great importance in GBM therapy, based on the evidence that single-agent therapy is often not sufficient to control this tumour[@b39][@b40][@b41]. Because the hyper-activation of the PI3K/AKT/mTOR pathway and the inactivation of wild-type p53 by MDM2 over-expression are frequent molecular events in highly proliferative tumours, and the aforementioned pathways are directly related[@b29], a combined therapy with an inhibitor of AKT/mTOR pathway and a compound able to inhibit the MDM2/p53 interaction may represent a valuable approach in GBM. In this respect, a cell-based screen has demonstrated the efficacy of such combined therapy in several cancer cells[@b42], thus providing a framework for the rational design of new clinical trials. We found that FC85 inhibited mTOR and AKT constitutive activity in U87MG cells; as a result, the new compound decreased cell viability, induced cell cycle block, and triggered apoptosis. Moreover, the inhibition of cell viability was long-lasting and directed preferentially towards tumour cells, as demonstrated by the significantly lower effects observed in HUVECs. Real time RT-PCR and western blotting analyses demonstrated that FC85 alone led to the accumulation of p53 protein levels and to an increase of the mRNA levels of p53-target genes, demonstrating a reactivation of the p53 pathway. Challenging cells with FC85 and the MDM2/p53 inhibitor ISA27 caused an amplification response on the reactivation of the p53 signalling pathway. As a result, such combined therapy produced synergistic effects on the inhibition of cell viability and enhanced the induction of p53-mediated apoptosis and cell cycle block with respect to those elicited by the use of a single agent. A synergistic inhibition of GBM cell viability was confirmed using the mTOR inhibitor everolimus and the MDM2 inhibitor nutlin-3. However, slight differences were noted. When used alone, everolimus and nutlin-3 caused slower reactivation kinetics of p53 function compared with FC85 and ISA27, displaying a significant induction of p53 target genes only after 24 h of cell treatment, accordingly to data previously reported for the MDM2 inhibitor[@b18]. Moreover, neither everolimus nor nutlin-3 alone induced apoptosis of U87MG cells within 24 h of treatment, in line with literature findings, reporting a non-apoptotic mechanism for everolimus[@b32][@b33] and a tardive induction of apoptosis for nutlin-3[@b17][@b18][@b41]. Interestingly, the co-treatment induced a slight but significant induction of apoptosis, suggesting that everolimus could accelerate the induction of apoptosis elicited by nutlin-3 in prolonged treatment[@b17][@b18]. The different reactivation kinetics of p53 function and induction of apoptosis elicited by the two MDM2 inhibitors could be attributed to the more rapid accumulation of p53 protein levels caused by ISA27 with respect to nutlin-3[@b18]. With regard to everolimus and FC85, it is important to mention the intricacy of the AKT/mTOR pathway. mTOR protein kinase interacts with multiple proteins via its two distinct multiprotein complexes, mTORC1 and mTORC2[@b43][@b44], which have been shown to have distinct signalling in GBM[@b45]. Rapamycin and rapalogs, including everolimus, suppress mTOR activity via an allosteric mechanism and have been found to be incomplete inhibitors of mTORC1[@b46][@b47]. On this basis, and considering the lack of information on the detailed mechanism of AKT/mTOR inhibition elicited by FC85, further investigations are needed to obtain insight into the signalling pathways that are the basis of such differences. We next characterized our compounds, alone or in combination, in GSCs isolated from U87MG cells. First, we demonstrated that both FC85 and ISA27 induced a time- and concentration-dependent inhibition of GSC proliferation and triggered apoptosis. In addition, in GSCs, the combined therapy produced synergisticc effects, accelerating the induction of apoptosis and enhancing the inhibition of GSC viability. Such synergistic interactions were shown also using the reference compounds nutlin-3 and everolimus, thus confirming that a combined therapy with an inhibitor of MDM2/p53 complex and an inhibitor of the AKT/mTOR pathway is useful in anti-proliferative strategy in GSCs. To investigate the mechanism underlying these effects, the morphology and stemness/differentiation of GSCs upon treatment with the compounds were evaluated. Morphological and RT-PCR analyses demonstrated that FC85 and ISA27 alone significantly decreased the stemness of GBM-derived cells, and promoted their differentiation toward a neuronal-glial phenotype. Globally, these effects elicited by FC85 and ISA27 are in line with the differentiating/pro-apoptotic properties previously reported for AKT/mTOR inhibitors in GSCs[@b36][@b48], as well as with the abilities of p53 to induce differentiation and to suppress self-renewal in human embryonic stem cells[@b49]and in breast CSCs[@b50], respectively. When cells were concomitantly treated with the two drugs, a significant decrease in the stemness markers was evidenced, together with a marked increase in neuronal and glial markers, demonstrating that combining ISA27 and FC85 not only induced apoptosis of GSCs but also induced the differentiation of these cells. Then, the signalling pathways most likely implicated in the aforementioned effects were investigated. It has been demonstrated that the AKT/mTOR signaling pathway is critical for the maintenance of the properties of glioma CSCs[@b36][@b48]; on the other hand, in the same cells the MEK-ERK signalling is required for MDM2 expression, which prevents p53 activation and subsequent suppression of O(6)-methyl-guanine DNA methyltransferase expression, thus contributing to TMZ resistance[@b51]. In this study, we showed that FC85 and ISA27 alone inhibited AKT and ERK1/2 phosphorylation up to 30 or 60 min, respectively. Challenging cells with both compounds prolonged such inhibitions up to 120 min, with significant higher percentages of inhibition with respect to single agent-treated cells. These results are in line with the additive/synergic effect on GSC differentiation elicited by the co-treatment FC85+ISA27, and support the previous demonstrations that the dual inhibition of PI3K/AKT/mTOR and MEK/ERK signalling induced the differentiation of and inhibited the tumourigenic potential of GSCs[@b38]. Globally, our findings suggest that FC85 actively enhances downstream p53 signalling and that a combination strategy aimed at inhibiting PI3K/AKT/mTOR signalling and activating p53 signalling is potentially effective in GBM and in its CSC subpopulation, where TP53 mutations are rare, and the downstream p53 signalling is intact. The promising anti-tumour effects, confirmed by the combined use of everolimus and nutlin-3, sustain the strength of the present pharmacological approach, and encourage the extension of preclinical investigations that, above all, focus on molecular mechanisms at the basis of such cellular responses. Methods ======= Chemical synthesis ------------------ Melting points were determined on a Kofler apparatus and are uncorrected. Chemical shift (δ) is reported as part per million downfield from tetramethylsilane and referenced from solvent references.^1^H NMR and ^13^C NMR spectra of all compounds were obtained with a Varian Gemini 200 spectrometer (Labexchange, Burladingen, Germany) operating at 200 MHz, in a \~2% solution of DMSO-d~6~. The \>95% purity of tested compounds was confirmed by combustion analysis. Analytical TLC was performed on Merck 0.2-mm precoated silica gel aluminium (60 F~254~) sheets t hat were visualized under a UV lamp. Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was used as the drying agent. Commercially available chemicals were purchased from Sigma-Aldrich. The 5-amino-1,3-dihydro-2H-indol-2-one (1) was prepared according to reported procedures[@b20]. 4-methyl-N-(2-oxoindolin-5-yl)benzenesulfonamide (2) ------------------------------------------------------ To a stirred solution of 5-amino-2-oxindole 1 (148 mg, 1 mmol) in water (10 mL was added TsCl (228.8 mg, 1.2 mmol) at room temperature, and stirring was continued until the reaction was complete. Then, the solution was evaporated to dryness. The crude residue was diluted with MeOH, and the solid product was filtered off from the mixture. The collected solid was washed with diethyl ether and air-dried. (109 mg, 0.36 mmol, 36% yield): mp 210--212°C.[@b1]H NMR: δ 2.37 (s, 3H, Me); 3.44 (s, 2H, CH~2~); 6.65 (d, 1H, J = 8.2 Hz, Ar); 6.84 (d, 1H, J = 8.2 Hz, Ar); 6.95 (s, 1H, Ar); 7.32 (d, 1H, J = 8.1 Hz, Ar); 7.58 (d, 1H, J = 8.1 Hz, Ar); 9.89 (br s, 1H); 10.30 (br s, 1H) ppm. ^13^C NMR: δ 170.06, 141.81, 137.46, 135.73, 133.61, 128.85, 128.11, 127.25, 121.99, 117.19, 110.54, 36.42, 21.87 ppm. Anal. Calcd. for C~15~H~14~N~2~O~3~S: C, 59.59; H, 4.67; N, 9.27. Found: C, 59.38; H, 4.46; N, 9.03. (3Z)-N-(3-((1H-imidazol-5yl)-methylene)-2-oxoindolin-5-yl)-4-methylbenzenesulfonamide (FC85) ---------------------------------------------------------------------------------------------- To a solution of 4-methyl-N-(2-oxoindolin-5-yl)benzenesulfonamide (2) (118 mg, 0.39 mmol) in ethanol (7 mL) was added the 1H-imidazole-5-carboxaldehyde (41.32 mg, 0.43 mmol) and a catalytic amount of piperidine. The resulting solution was stirred and refluxed for 12 h; then, the solution was evaporated to dryness. The crude residue was purified by crystallization from EtOH, affording FC85 as Z-isomer (79 mg, 0.21 mmol, 53% yield): mp 219--217°C. ^1^H NMR: δ 2.31 (s, 3H, Me); 6.70--6.82 (m, 2H, Ar); 7.32 (d, 1H, J = 8.1 Hz, Ar); 7.40 (s, 1H, Ar); 7.59 (d, 1H, J = 8.1 Hz, Ar); 7.73 (s, 1H, Ar); 7.77 (s, 1H, Ar); 8.02 (s, 1H, H-vinyl); 9.94 (br s, 1H); 10.95 (br s, 1H) ppm. ^13^C NMR: δ 168.90, 139.05, 136.74, 135.45, 134.95, 131.88, 131.40, 130.72, 128.46, 127.59, 127.44, 126.68, 121.33, 117.95, 111.01, 110.12, 21.30 ppm. Anal. Calcd. for C~19~H~16~N~4~O~3~S: C, 59.99; H, 4.24; N, 14.73. Found: C, 60.12; H, 4.46; N, 14.87. GBM cell culture and GSC isolation ---------------------------------- The U87MG cell line was obtained from the National Institute for Cancer Research of Genoa (Italy) and cultured as described[@b18]. To isolate GSCs, approximately 2.0 × 10^6^ cells were suspended in 1 mL of a defined serum-free Neural Stem Cell (NSC) medium[@b34]. After 3--4 days of culture, the neurospheres were collected, suspended in NSC medium, dissociated into single cells, and plated for the assays. For the long-term treatment of cells, NSC or complete medium containing drugs was replaced every two to three days. Cell proliferation/viability assays of GBM cells and CSCs --------------------------------------------------------- The human U87MG cells or GSCs were seeded at a density of 3 × 10^3^ cells/well. After 24 h, the cells were treated for one to 7 days with fresh growth medium containing different concentrations of FC85, ISA27, everolimus or nutlin-3. Following the treatment period, cell viability was determined using the MTS assay according to manufacturer\'s instruction. The absorbance of formazan at 490 nM was measured in a colorimetric assay with an automated plate reader (Victor Wallac 2, Perkin Elmer). For wash-out experiments, U87MG cells or GSCs were treated with FC85 and/or ISA27 for 96 h (U87MG cells) or seven days (GSCs). At the end of treatments, medium containing drugs was replaced by fresh medium, and cells were allowed to grow for the indicated days (one or three days in the case of U87MG, seven or fourteen days in the case of GSCs). At the end of treatments, cell viability was measured by the MTS assay. The results were calculated by subtracting the mean background from the values obtained from each evaluation and were expressed as the percentage of the control (untreated cells). Sigmoid dose-response curve was generated, from which the IC~50~ values were derived. The effects of compound treatment on U87MG cell viability were also evaluated using the trypan blue exclusion assay. Cells were treated with different concentrations (100 nM, 1 µM or 10 µM) of FC85. Following the treatment period, cells were collected and centrifuged at 300 × g for 5 minutes. The harvested cells were mixed with an equal volume of 0.4% trypan blue dye, and the blue (dead cells) and white (living cells) cells in each well were manually counted. The number of live cells for each condition was reported as the percentage of living cells relative to that in the control sample. Isobolar analysis ----------------- A graphical assessment of synergy with regard to growth inhibition was performed using isobolographic analysis[@b52]. In the present study, the dose of ISA27 or nutlin-3 required to give a 50% effect[@b18] was plotted on the abscissa, and the iso-effective dose of FC85 or everolimus was plotted on the ordinate. The theoretical additive effect of the two drugs is represented by the straight line connecting the two points. If the experimentally determined data points and their confidence interval fall on this line, the drug effects are additive (no interaction). If the points lie below this line, there is superadditivity (synergy), and if the points lie above this line, there is subadditivity (antagonism). To determine whether the interaction between the two drugs was synergistic, additive or antagonistic, the theoretical additive IC~50,add~was estimated from the dose-response curves of each drug administered individually. The interaction index, denoted by γ, is an assessment of the degree of synergism or antagonism. The index is defined by the isobolar relationship as follows[@b53][@b54]: γ = a/A + b/B where A and B are the doses of drug A (alone) and B (alone) that give the specified effect, and (a,b) are the combination doses that produce the same effect. p53 stabilization analysis in U87MG cells ----------------------------------------- The western blot analysis for the evaluation of p53 protein levels was performed as previously described[@b18]. In brief, U87MG cells were treated with DMSO (control) or with 500 nM FC85 and 2.5 µM ISA27, alone or in combination. Alternatively, cells were incubated with 50 nM everolimus and/or 10 µM nutlin-3, alone or in combination. Cells were then lysed for 60 min at 4°C, and equal amount of the cell extracts (40 μg of proteins) were diluted in Laemmli solution and resolved by SDS-PAGE[@b41]. RNA extraction and Real Time PCR analysis in U87MG cells and in CSCs -------------------------------------------------------------------- U87MG cells or GSCs were treated with DMSO (control), 500 nM FC85 and/or 2.5 µM ISA27 for 6 h or seven days, respectively. In some experiments, U87MG cells were incubated with 50 nM everolimus and/or 10 µM nutlin-3 for 6 h or 24 h. At the end of treatment, the cells were collected, and total RNA was extracted using RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\'s instructions. cDNA synthesis was performed with 500 ng of RNA using the i-Script cDNA synthesis kit (BioRad, Hercules, USA) following the manufacturer\'s instructions. RT-PCR reactions consisted of 25 µL of Fluocycle® II SYBR® (Euroclone, Milan, Italy), 1.5 µL of both 10 µM forward and reverse primers, 3 µL of cDNA, and 19 µL of H~2~O. All reactions were performed for 40 cycles using the following temperature profiles: 98 °C for 30 seconds (initial denaturation); T °C (see [Suppl. Table S5](#s1){ref-type="supplementary-material"}) for 30 seconds (annealing); and 72°C for 3 seconds (extension)[@b34]. Cell cycle analysis in U87MG cells ---------------------------------- The measurement of the percentage of cells in the different cell phases was performed using the Muse™ Cell Analyser, Merck KGaA, Darmstadt, Germany). Briefly, U87MG cells were treated for 24 h with DMSO or 500 nM FC85 and 2.5 µM ISA27, alone or in combination. Adherent cells were collected and centrifuged at 300 × g for 5 minutes. The pellet was washed with PBS and suspended in 100 µl of PBS; finally, the cells were slowly added to 1 ml of ice cold 70% ethanol and maintained o/n at −20°C. Then, a cell suspension aliquot (containing at least 2 × 10^5^ cells) was centrifuged at 300 × g for 5 minutes, washed once with PBS and suspended in the fluorescent reagent (Muse™ Cell Cycle reagent)[@b41]. Annexin V and 7-AAD staining in U87MG cells and in GSCs ------------------------------------------------------- Dual staining with Annexin V conjugated to fluorescein-isothiocyanate (FITC) and 7-amino-actinomysin (7-AAD) was performed using the commercially available kit (Muse Annexin V and Dead Cell Kit; Merck KGaA, Darmstadt, Germany). U87MG cells or GSCs were treated with DMSO (control), FC85 and/or ISA27 for 6 h or seven days, respectively. In some experiments, U87MG cells were incubated with 50 nM everolimus and/or 10 µM nutlin-3 for 24 h. At the end of the treatment periods, the percentages of living, apoptotic and dead cells were acquired and analysed by Muse™ Cell Analyser, as previously described[@b41]. Quantification of the occupied area and the cellular processes of neurospheres ------------------------------------------------------------------------------ GSCs were plated in complete growth medium (day 0) and treated for seven or fourteen days with 500 nM FC85 and/or 2.5 µM ISA27. At the end of the treatment period, the drug-containing media were replaced with fresh NSC medium, and the GSCs were allowed to grow for another 7 or 14 days. Images of the neurospheres were captured at days 0, 7, 14 and 21. Three different wells were analysed for each condition, and 15 images of each well were captured[@b34][@b55]. The response of the cultures to the various treatments was quantified by measuring the area occupied by neurospheres that had formed, using the ImageJ program (version 1.41; Bethesda, MD, USA). The cellular processes extending from the six to eight differentiating neurospheres per condition in three independent experiments were evaluated. ERK and AKT phosphorylation assays ---------------------------------- U87MG cells were cultured in 96-well microplates (5.000 cells/well) and treated for 2 hours with different FC85 concentrations (100 nM, 1 µM and 10 µM). In kinetic experiments, GSCs were treated for different times with 500 nM FC85 and/or 2.5 µM ISA27. At the end of the treatment period, the GSCs were centrifuged at 500 × g for 3 minutes; cells were washed twice using fresh saline and rapidly fixed with 4% (for adherent U87MG cells) or 8% (for suspension GSCs) formaldehyde to preserve the activation of specific protein modification. The levels of total and phosphorylated AKT and ERK1/2 were determined using specific primary antibodies. The subsequent incubation with a secondary HRP-conjugated antibody and the developing solution allowed for the colorimetric quantification of the levels of total and phosphorylated proteins. The relative number of cells in each well was then determined using the crystal violet assay. The results were calculated by subtracting the mean background value from the values obtained under each test condition: values were normalized to the number of cells in each well and are expressed as the percentages of the control (untreated cells) values. Statistical analyses -------------------- The nonlinear multipurpose curve-fitting program Graph-Pad Prism (GraphPad Software Inc., San Diego, CA) was used for data analysis and graphic presentations. All data are presented as the mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni\'s corrected t-test for post-hoc pair-wise comparisons. P\<0.05 was considered statistically significant. Author Contributions ==================== S.D., E.Z. and E.D.P. performed most of the biological work. S.S., G.N. and P.C. synthesized the compounds. B.C. and S.R. conceived the idea and conducted the design. C.M. coordinated the project. S.D., B.C. and S.R. wrote the main manuscript text. L.M. and E.N. provided important help in the significance of the results and in writing article discussion section. All authors reviewed the manuscript. Supplementary Material {#s1} ====================== ###### Supplementary Information Supplementary Information This work was supported by the FIRB, Bando Futuro in Ricerca 2010 (Grant RBFR10ZJQT). ![(a) Diagram of AKT/mTOR and MDM2/p53 signalling crosstalk in GBM and in GSCs.\ AKT/mTOR deactivation decreases MDM2 and p53 phosphorylation and increases stable p53, which triggers its downstream targets. Simultaneously, p53 may increase PTEN to suppress AKT activation further. FC85 inhibited both AKT (Ser473) and mTOR (Ser2448) phosphorylation. ISA27 dissociated the MDM2-p53 complex, thus re-activating p53. The combined therapy with FC85+ISA27 more efficiently re-activated the p53 pathway, producing a synergic effect on the inhibition of GBM cell viability; most importantly, the simultaneous inhibition of AKT/mTOR and of the MDM2-p53 complex led to a synergic effect in triggering cellular differentiation/apoptosis of GSC subpopulation. (b) The design of FC85 starting from the general structure of OXIDs. (c) The synthetic procedure for the preparation of FC85.](srep09956-f1){#f1} ![Effect of FC85 on U87MG and HUVEC cell viability.\ (a) U87MG cells were treated in complete medium with the indicated FC85 concentrations for 24 h. At the end of treatment, cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. (b) U87MG cells were treated as in (a); live and dead cells were then estimated using the trypan blue exclusion test. The data are expressed as the cell number per well and are the mean values ± SEM of two independent experiments, each performed in triplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \\\* p\<0.001 vs. control live cells; \#\#p\<0.01, \#\#\# p\<0.001 vs. control dead cells. (c) U87MG cells were treated with 10 µM FC85 for 24 h or 96 h, and then, the medium was replaced with drug-free fresh medium for another 24 h or 72 h. At the end of the treatment period, cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \\\* p\<0.001 vs. control. (d) U87MG (black bars) or HUVEC (white bars) cells were treated with different FC85 concentrations for 48 h. Cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \\\* p\<0.001 vs. respective control; \#p\<0.05, \#\#p\<0.01 vs. U87MG cells.](srep09956-f2){#f2} ![Synergistic effect of FC85 and ISA27 on the survival/growth of U87MG cells and on the reactivation of p53 pathway.\ (a) Isobologram 2-D showing the interactions between FC85 and ISA27 in MTS viability tests performed in U87MG cells treated for 24 h with FC85 and/or ISA27. The IC~50~values for FC85 and ISA27 are shown on the X- and Y-axis, respectively. The open points (○) on the additivity line depict the theoretical IC~50,add~values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. The solid points (●) depict the experimental IC~50,mix~values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. (b) U87MG cells were treated for 6 h with DMSO (control), 500 nM FC85 or 2.5 µM ISA27, alone or in combination. Lysates were subjected to western blot analysis using an antibody to p53. One representative Western blot is presented (b) for each cell treatment. β-actin was used as the loading control. The bar graph (c) shows the quantitative analysis of the Western blots, performed using ImageJ. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, set to 100%, and are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \p0.05, \\\p\<0.001 vs. control; \#\# p\<0.01 vs. FC85 alone; §§ p\<0.01 vs. ISA27 alone. Full-length blots are presented in the [Supplementary Information](#s1){ref-type="supplementary-material"} section titled ''Full-length blots relative to the cropped images showed in the main Figures". (d) U87MG cells were treated as in b. The relative mRNA quantification of p53 target genes (PUMA, p21 and MDM2) was performed by real-time RT-PCR as describe in the Methods section. The data are the mean values ± SEM of three different experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \\\* p\<0.001 vs. control; \#\# p\<0.01, \#\#\# p\<0.001 vs. FC85 alone; §§ p\<0.01, §§§ p\<0.001 vs. ISA27 alone.](srep09956-f3){#f3} ![The effect of FC85 and/or ISA27 on U87MG cell apoptosis and cell cycle.\ (a, b) U87MG cells were treated for 24 h with DMSO (control), or 500 nM FC85 or 2.5 µM ISA27, alone or in combination. At the end of the treatment period, the cells were collected, and the level of phosphatidylserine externalisation was evaluated using the Annexin V-staining protocol, as described in the Methods section. b) The data are expressed as the percentage of apoptotic cells (the data for the early-stage apoptotic cells shown in white, and the data for the late-stage apoptotic/necrotic cells shown in grey) versus the total number of cells. The data shown represent the mean ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \\\* p\<0.001 vs. control; \#\#\# p\<0.001 vs. FC85 alone; §§§ p\<0.001 vs. ISA27 alone. (c, d) U87MG cells were treated as in a. At the end of the treatment periods, the cell cycle was analysed as described in the Methods section. Representative cell cycle histograms of untreated and treated cells are shown (c). The data are expressed as the percentage of cells in the different phases (G0/G1, G2 or S) versus the total cell number and are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \\ p\<0.01 vs. control in the respective cellular phase; \# p\<0.05, \#\# p\<0.01 vs. FC85 alone; § p\<0.05, §§ p\<0.01 vs. ISA27 alone.](srep09956-f4){#f4} ![Effect of FC85 and/or ISA27 on GSC proliferation/viability.\ U87MG-derived GSCs were incubated with the indicated concentrations of FC85 (a) or ISA27 (b) for 24 h, four days or seven days. At the end of the treatment periods, cell viability was measured using the MTS assay. (c) U87MG-derived GSCs were incubated for 7 days with increasing concentrations of FC85 or ISA27, and cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \\ p\<0.01, \\\* p\<0.001 vs. control. (d) Isobologram 2-D showing the interactions between FC85 and ISA27 in MTS viability tests performed in U87MG-derived CSCs treated for seven days with FC85 and/or ISA27. The IC~50~ values for FC85 and ISA27 are shown on the X- and Y-axis, respectively. The open points (○) on the additivity line depict the theoretical IC~50,add~values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. The solid points (●) depict the experimental IC~50,mix~ values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. (e) U87MG-derived GSCs were incubated with FC85 and/or ISA27 for seven days; then, the medium was replaced with drug-free fresh medium for other seven or fourteen days. At the end of the treatment period, cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \\p\<0.01, \\\* p\<0.001 vs. control.](srep09956-f5){#f5} ![The effect of FC85 and/or ISA27 on GSC apoptosis/differentiation.\ (a, b) GSCs were treated for 7 days with NSC medium containing DMSO (control), or 500 nM FC85 or 2.5 µM ISA27, alone or in combination. At the end of treatments, the cells were collected and the degree of phosphatidylserine externalisation was evaluated using the Annexin V protocol, as described in the Method section. b) The data were expressed as the percentage of apoptotic cells (early-apoptotic in white, late-apoptotic/necrotic in grey) relative to the total number of cells. The data are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \\\* p\<0.001 vs. control; \#\#\# p\<0.001 vs. FC85 alone; §§§ p\<0.001 vs. ISA27 alone. (c) GSCs were treated as in a; total RNA was extracted, and the relative mRNA quantification of the stem cell markers CD133 and nestin, the neuronal marker MAP, the astrocyte marker GFAP, and of the oligodendrocyte marker Olig2 was performed by RT-PCR. The data are expressed as the fold change vs. the levels of the control and are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \\ p \<0.01, \\\* p\<0.001 vs. control; \#\#\# p\<0.001 vs. FC85 alone; §§ p\<0.01, §§§ p\<0.001 vs. ISA27 alone.](srep09956-f6){#f6} ![Effect of FC85 and/or ISA27 on sphere-derived cell morphology.\ GSCs were treated for seven or fourteen days with complete NSC medium containing DMSO (control), or 500 nM FC85, or 2.5 µM ISA27, alone or in combination. (a) Representative cell micrographs after seven and fourteen days of treatment are shown. The area of the culture plates occupied by the spheres (b) and the length of cellular processes (c) were scored after seven and fourteen days of treatment. The counts represent the mean values ± SEM of two independent experiments. The significance of differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \\\* p\<0.001 vs. control; \# p\<0.05 vs. FC85 alone; § p\<0.05 vs. ISA27 alone.](srep09956-f7){#f7} ![Kinetics of FC85 and/or ISA27 inhibition of AKT and ERK1/2 phosphorylation.\ U87MG-derived CSCs were treated for the indicated times (0--120 min) with NSC medium containing DMSO (control), or 500 nM FC85, or 2.5 µM ISA27, alone or in combination. At the end of the treatment periods, the levels of AKT (a) and ERK 1/2 (b) phosphorylation were evaluated using ELISA kits, as described in the Methods section. The data are expressed as the percentage of phosphorylated AKT or ERK1/2 relative to those of untreated cells (control), which were set at 100%, and are the mean values ± SEM of three independent experiments performed in triplicate. The significance of differences was performed using one-way ANOVA with Bonferroni post-test: \* p\< 0.05, \\ p\< 0.01, \\\* p\<0.001 vs. control; \# p\<0.05, \#\#p\< 0.01 vs. FC85 alone; § p\<0.05, §§ p\<0.01, §§§ p\<0.001 vs. ISA27 alone.](srep09956-f8){#f8}	{ "pile_set_name": "PubMed Central" }	[ "amount", "bc", "treatment", "graphpad", "two", "ab", "almost", "control", "viabil", "refer", "work", "dosedepend", "induct", "program", "asreftypesupplementarymateri", "decreas", "hz", "administr", "ligand", "reflux", "referencesh", "collect", "latestag", "longterm", "umg", "gene", "put", "identifi", "uv", "length", "aktmtor", "methyltransferas", "block", "pi", "mini", "retain", "anticanc", "grant", "analysisb", "mechan", "valu", "mmol", "fix", "downfield", "hour", "reportedb", "multipl", "leukaemia", "signal", "may", "burladingen", "yaxi", "therebi", "merck", "graphic", "milan", "recent", "perkin", "γ", "pb", "fctreat", "live", "acquir", "toward", "embryon", "gener", "use", "mrna", 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Introduction {#sec1-1} ============ Placebo seems indispensable in modern clinical investigations. A placebo is a sham medical intervention that can produce a placebo effect. Common placebos are usually inert tablets or sham surgery based on false information.\[[@ref1]\] Since the publication of Henry K. Beecher\'s in 1955, the phenomenon has been considered to be clinically important.\[[@ref2]\] Placebo effects were shown to be genuine psychobiological events attributed to the overall therapeutic context.\[[@ref3][@ref4]\] From a psychological point of view, many mechanisms might contribute to placebo effects, including expectations, conditioning, learning, motivation, memory, reward and anxiety reduction, etc.\[[@ref5][@ref6]\] Owing to the placebo effect, it is sometimes difficult to evaluate new treatments. Apparent benefits of a new drug may derive from the placebo effect but not from the drug per se. Therefore, modern clinical trials control for this effect by using a placebo, in which the subjects are blinded as to whether they receive a drug treatment or a placebo. In this way, placebo-controlled trials might provide information about the real effectiveness of a drug. The view of placebo effect was notably challenged when a systematic review of clinical trials in 2001 concluded that there was no evidence of clinically important effects, except perhaps in the treatment of pain and other continuous subjective outcomes.\[[@ref7]\] Since evidence-based medicine (EBM) is increasingly emphasized, placebo is still widely used in clinical trials other than in the treatment of pain and continuous subjective outcomes. Currently, international journals as well as clinical researchers are inclined to avoid or even ignore the ethical aspects of placebo. However, accumulating ethical concern has arisen from the worldwide use of placebo in randomized control trials (RCTs), because the investigators in a certain trial may render its participants without early and optimal treatment. Where for compelling and scientifically sound methodological reasons the use of placebo is necessary to determine the efficacy or safety of an intervention and the patients who receive placebo or no treatment will not be subject to any risk of serious or irreversible harm. Extreme care must be taken to avoid abuse of this option. (Extracted from the Declaration of Helsinki)\[[@ref8]\] A recent study, the European Cooperative Acute Stroke Study III (ECASS III) reported by Bluhmki et al and by Hacke et al supported the use of alteplase three hours after the onset of stroke symptoms, while together with other studies, ECASS III raised some concerns about thrombolytic studies.\[[@ref9][@ref10]\] Despite repeated emphases of early treatment on acute ischemia, the control group in ECASS III failed to receive any treatment except the placebo. This is contradictory to the common sense "Time is brain".\[[@ref11]\] The lack of evidence-based therapies should not justify the exclusion of empirical use of anti-platelet agents, or others. In this regard, it is almost always the case in China, since traditional Chinese medicine has been a routine for patients suffering from cerebrovascular ischemia.\[[@ref12]\] Another example of clinical trial that may raise ethical concerns is the one conducted by Kappos, et al, about oral fingolimod in treating relapsing multiple sclerosis (MS). This placebo-controlled trial revealed that fingolimod improved the relapse rate, the risk of disability progression, and end points on MRI.\[[@ref13]\] However, the longer the trial lasts, the longer the patients in the placebo group must go without treatment. In this context, the well-acknowledged and widely used therapeutic approaches should be considered first and foremost as controls, when RCTs are designed. Although placebos may be helpful in treating MS, the better-acknowledged ways such as interferon-beta and glatiramer acetate can potently alleviate the sufferings of patients with MS\[[@ref14]\] and can readily serve as positive controls in that study. As a clinician, I feel suffocated when confronted with such ethically problematic studies. Where is the Hippocratic Oath? And where is Declaration of Helsinki. I hate the unreasonable use of placebos but cannot do anything. The sanctification of placebo is partly due to the repeated emphasis of RCTs as the gold standard of clinical trials. Thus for the time being, the ending of this sanctification seems incoercible. Dichotomy seems necessary in evaluating the role of placebo in scientific research; the need for placebo-controlled design cannot be denied at certain stage of drug development, whereas for larger phase IV studies, better alternatives should be considered. I thus would like to propose some solutions to such an ethical conundrum. Ethically, the clinicians are always expected to provide their patients with the best choices of treatment other than placebo, and the best available treatment as a positive control other than placebos is preferable to the patients. In neurological field, for example, aspirin is widely accepted as a control in trials with regard to prevention or treatment of cerebral ischemic disorders,\[[@ref15]\] because its prophylactic as well as therapeutic effects on cardiovascular events are well established.\[[@ref16][@ref17]\] Although the control group is a must in RCTs, an alternative way such as a delayed-start approach,\[[@ref18]\] can be utilized to avoid ethical issues that may arise. Delayed-start studies are designed primarily to test the disease-modifying effect of a drug, whereas they can reduce the ethical concerns that patients in the placebo group must go without treatment until the end of the study. The control subjects enrolled in the aforementioned thrombolytic study should at least start to accept conventional medication therapies immediately after the time-window for thrombolytic studies. As an old Chinese saying goes, doctors ought to be parentally considerate. Similarly, clinicians should always bear in mind that no treatment equals killing for their patients. Clinicians/researchers need to understand the implications of offering no treatment in certain situations before enrolling experimental subjects. More importantly, a thorough explanation about the aforementioned possible implications should be performed by the clinicians. The placebo effect is produced on the premise that the subjects believe the effectiveness of placebo. This is different from the practice in RCTs in which the subjects are informed of the possibility of accepting only a sham medical intervention. Informed consent for a study is usually required to be considered ethical, including the disclosure that some patients are to receive placebos only. In this case, the subjects do not know whether they might be getting a real treatment or a sham one, and thus might suspect the efficacy of the medical interventions even before the trials.\[[@ref19]\] As a result, placebos do not work as strongly in clinical trials, as in basic research. This may explain the insignificant clinically important effects as summarized by the said review.\[[@ref7]\] What\'s worse, the deception involved in the use of placebos creates conflicts between the Hippocratic Oath and the honesty of the doctor--patient relationship.\[[@ref20][@ref21]\] The purpose of the clinical trials should be explained in detail in the process of obtaining informed consent from the participants, while it is not always the case, especially in low-income countries. For patients in low-income countries, the participation in clinical trials is intriguing simply because poor patients have the chance of getting free treatment, or they can get rewards for attending the research. This may lead to selection bias as well as ethical debates because the poor participants have no better choice than attending the trials. The honest relationship between doctors and patients is further worsened because of the notion of the patients that they are utilized. In summary, although the ethics of placebos have been debated frequently in history,\[[@ref21]\] even in the revision process of the Declaration of Helsinki, placebos have given rise to ethical debates much more often than ever before. As the pilgrimage of placebo is still on the way, refinement of controls in RCTs is worth paying close attention to. This study was supported by the China Scholarship Council. 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"investig", "safeti", "pain", "brainref", "especi", "acut", "sham", "lowincom", "improv", "revis", "report", "aspect", "offer", "point", "issu", "emphasi", "summari", "progress", "contradictori", "nil", "seriou", "attribut", "therefor", "inert", "clinician" ]	22,245	[ "Introduction", "Placebo", "seems", "indispensable", "modern", "clinical", "investigations", "placebo", "sham", "medical", "intervention", "produce", "placebo", "effect", "Common", "placebos", "usually", "inert", "tablets", "sham", "surgery", "based", "false", "Since", "publication", "Henry", "phenomenon", "considered", "clinically", "Placebo", "effects", "shown", "genuine", "psychobiological", "events", "attributed", "overall", "therapeutic", "psychological", "point", "view", "many", "mechanisms", "might", "contribute", "placebo", "effects", "including", "expectations", "conditioning", "learning", "motivation", "memory", "reward", "anxiety", "reduction", "Owing", "placebo", "effect", "sometimes", "difficult", "evaluate", 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"neurological", "field", "example", "aspirin", "widely", "accepted", "control", "trials", "regard", "prevention", "treatment", "cerebral", "ischemic", "disorders", "prophylactic", "well", "therapeutic", "effects", "cardiovascular", "events", "well", "Although", "control", "group", "must", "RCTs", "alternative", "way", "approach", "utilized", "avoid", "ethical", "issues", "may", "arise", "studies", "designed", "primarily", "test", "effect", "drug", "whereas", "reduce", "ethical", "concerns", "patients", "placebo", "group", "must", "go", "without", "treatment", "end", "study", "control", "subjects", "enrolled", "aforementioned", "thrombolytic", "study", "least", "start", "accept", "conventional", "medication", "therapies", "immediately", "thrombolytic", "studies", "old", "Chinese", "saying", "goes", "doctors", "ought", "parentally", "considerate", "Similarly", "clinicians", "always", "bear", "mind", "treatment", "equals", "killing", "patients", "need", "understand", "implications", 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1. Introduction {#sec1} =============== 1.1. Anaerobic Methane-Oxidizing Archaea (ANME) {#sec1.1} ----------------------------------------------- Anaerobic methane-oxidizing archaea (ANME) perform anaerobic oxidation of methane (AOM) via reversal of the methanogenic pathway. ANME were first discovered in marine sediments where AOM was coupled to sulfate reduction (SR) ([Table 1](#tab1){ref-type="table"}, reaction (1)). Here, ANME formed metabolically interdependent consortia with sulfate-reducing bacteria (SRB) that belong to the Deltaproteobacteria \[[@B1]--[@B3]\]. Three distinct methanotrophic groups were identified: ANME-1 (subclusters a and b), ANME-2 (subclusters a, b, and c), and ANME-3. The ANME-1 cluster is related to Methanomicrobiales and Methanosarcinales but forms a separate cluster \[[@B2]\], ANME-2 are related to cultivated members of the Methanosarcinales \[[@B4]\], and ANME-3 are more related toMethanococcoides spp. \[[@B5]\] ([Figure 1](#fig1){ref-type="fig"}). The ANME clades are not monophyletic with each other and the phylogenetic distance between the subgroups is large, with 16S rRNA gene sequence similarity of only 75--92% \[[@B6]\]. Subclusters ANME-2a and ANME-2b form a coherent clade that is distinguished from ANME-2c \[[@B7]\] and are therefore often grouped together as ANME-2a/b ([Figure 1](#fig1){ref-type="fig"}). The wide phylogenetic distribution is reflected in the ecological niche adaptation of the different ANME clades. ANME clades involved in sulfate-dependent AOM (S-AOM) co-occur in many different marine environments, except for ANME-3 that was mainly found in mud volcanoes and in some seep sediments \[[@B6], [@B8], [@B9]\]. In marine sediments, a distinct zonation occurs where ANME-2a/b dominate upper layers and ANME-2c and/or ANME-1 abundance increases in deeper zones, indicating ecological niche separation \[[@B10]--[@B15]\]. ANME also form a versatile partnership with non-SRB such as beta-proteobacteria \[[@B16]\] and Verrucomicrobia \[[@B17]\]. ANME, and especially ANME-1, have also been observed without a (closely associated) bacterial partner \[[@B5], [@B12], [@B13], [@B18]--[@B22]\]. It was therefore suggested that ANME could perform AOM alone, using electron acceptors such as metal oxides, or perform other processes such as methanogenesis \[[@B23], [@B24]\]. Indications exist that AOM can be coupled to the reduction of different metal (oxides) ([Table 1](#tab1){ref-type="table"}, reactions (3)--(5)), but limited experimental evidence exists to date that ANME are responsible for this reaction (discussed in [Section 3.3](#sec3.3){ref-type="sec"}). Besides marine environments, ANME involved in S-AOM can be found in terrestrial \[[@B25], [@B26]\] and freshwater ecosystems \[[@B27]\]. A member of a fourth subcluster, "Candidatus (Ca.) Methanoperedens nitroreducens," was recently discovered to perform nitrate-dependent AOM (N-AOM) \[[@B28]\] ([Table 1](#tab1){ref-type="table"}, reaction (2)). This cluster was named "ANME-2d" \[[@B29]\] but later renamed to "GOM Arc I" \[[@B30]\] and "AOM-associated archaea (AAA)" \[[@B6]\]. Phylogenetic analysis shows that the ANME-2d cluster is monophyletic with "Ca. M. nitroreducens" and other AAA/GoM Arc I sequences, but distinct from other ANME-2 subclusters ([Figure 1](#fig1){ref-type="fig"}). ANME-2d were initially enriched in bioreactors inoculated with freshwater samples \[[@B28], [@B31]--[@B33]\]. As ANME-2d archaea were only recently recognized, their environmental preferences remain insufficiently studied. So far they have been found in freshwater canals \[[@B31]\], soils and rice paddy fields \[[@B34]--[@B36]\], lakes and rivers \[[@B35]\], and wastewater treatment plants \[[@B33]\]. In situ AOM activity of ANME-2d was determined recently for the first time \[[@B36]\]. More ANME phylotypes in different environments and possibly new archaeal clades involved in AOM may yet have to be discovered. For example, methyl coenzyme M reductase A genes (mcrA) from Bathyarchaeota (formerly known as Miscellaneous Crenarchaeota Group) and from the new archaeal phylum Verstraetearchaeota were recently found, indicating their involvement in methane metabolism \[[@B37], [@B38]\]. This review focusses on archaea performing AOM through the reversal of the methanogenesis pathway. We describe the reversibility of the central methanogenic pathway, including the key enzyme in methanogenesis and anaerobic methanotrophy (i.e., methyl coenzyme M reductase, Mcr). The possibility of methanogens to perform methane oxidation and of ANME to perform methanogenesis is also addressed. Lastly, the physiological adaptations of ANME to perform respiration using different electron acceptors during AOM are discussed. 1.2. ANME versus Methanogens: Domain Based (Meta)Genome Comparison {#sec1.2} ------------------------------------------------------------------ In order to find additional differences between archaeal methanotrophs and related methanogens that could validate and complement findings in the literature, we performed domain based (meta)genome comparison between selected metagenomes of ANME and genomes of methanogens, as done previously for bacterial genomes \[[@B39]\]. For archaeal methanotrophs, we used the metagenomes of ANME-1 \[[@B40], [@B41]\], ANME-2a \[[@B42]\], and ANME-2d \[[@B28], [@B43]\]. For methanogens, we used genomes of closely and distantly related species able to perform acetoclastic methanogenesis (A:Methanosaeta concilii GP6), methylotrophic methanogenesis (M-1:Methanococcoides burtonii DSM6242, M-2:Methanolobus tindarius DSM2278, and M-3:Methanohalophilus mahiiDSM5219), hydrogenotrophic methanogenesis (H-1:Methanospirillum hungatei JF-1, H-2:Methanobacterium formicicum DSM3637, H-3:Methanococcus maripaludis C5, and H-4:Methanoregula formicica SMSP), and both acetoclastic and methylotrophic methanogenesis (AM:Methanosarcina acetivoransC2A). The genome of a sulfate-reducing archaeon that contained most enzymes for methanogenesis except for Mcr (S:Archaeoglobus fulgidus DSM 4304) was also included in the comparison. For each dataset the protein domains were obtained through InterProScan 5.17-56.0 using the TIGRFAM, ProDom, SMART, PROSITE, PfamA, PRINTS, SUPERFAMILY, COILS, and Gene3D domain databases. Results of the analysis are given in [Table 2](#tab2){ref-type="table"} and Table S1 of the Supplementary Material available online at <https://doi.org/10.1155/2017/1654237>. Since the ANME-1 metagenome assembled by Stokke et al. 2012 \[[@B40]\] contained many bacterial genes, we did not refer to this data for the domain based (meta)genome comparison but only used the ANME-1 metagenome described by Meyerdierks et al., 2010 \[[@B41]\]. We included both ANME-1 metagenomes to analyze the organization of genes for the formaldehyde-activating enzyme (Table S2) and the iron-only hydrogenase (Table S3). 2. Reversal of the Methanogenesis Pathway {#sec2} ========================================= 2.1. The Central Methanogenesis Pathway {#sec2.1} --------------------------------------- ANME are described to perform "reverse methanogenesis" \[[@B44]\] which implies the complete reversal of methanogenesis from H~2~ and CO~2~, that is, hydrogenotrophic methanogenesis (for kinetic and thermodynamic considerations, the reader is referred to \[[@B45]\]). During "forward" hydrogenotrophic methanogenesis, CO~2~ is reduced to CH~4~ with reducing equivalents derived from H~2~ ([Figure 2](#fig2){ref-type="fig"}). During methylotrophic methanogenesis, this pathway is already partly reversed. Methylotrophic methanogens utilize one-carbon compounds such as methylamines, methanol or methylated sulfur compounds (methanethiol, dimethyl sulfide) that are activated to methyl coenzyme M. About 75% of the methyl coenzyme M (CH~3~-CoM) is reduced to produce CH~4~ and about 25% of CH~3~-CoM is oxidized to CO~2~ using the methanogenesis pathway in reverse during methylotrophic growth. The oxidative part provides reducing equivalents that are needed for the generation of the proton motive force in the methanogenic respiratory chain and the reduction of CH~3~-CoM by methyl coenzyme M reductase (Mcr) \[[@B46]\] ([Figure 3](#fig3){ref-type="fig"}). In all methanogens, the Mcr reaction operates in the forward reaction and yields methane and the heterodisulfide of coenzyme B and coenzyme M (CoB-S-S-CoM):$$\begin{matrix} \left. {}{CH}_{3}\text{-}CoM + CoB\text{-}SH\longrightarrow{CH}_{4} + CoB\text{-}S\text{-}S\text{-}CoM; \right. \\ {\mspace{21210mu}\Delta G^{0} = - 30\, kJ\,{mol}^{- 1}\left\lbrack 52 \right\rbrack} \\ \end{matrix}$$The heterodisulfide is a central intermediate and acts as terminal electron acceptor in all methanogens. In hydrogenotrophic methanogens without cytochromes, it is the electron acceptor of the cytoplasmic electron-bifurcating CoB-S-S-CoM reductase (HdrABC) and F~420~-nonreducing hydrogenase (MvhADG) complex \[[@B47], [@B48]\] that is needed to provide reduced ferredoxin for the first step in methanogenesis; the reduction of CO~2~ to a formyl group. Within the methanogens with cytochromes, only a few members of the genusMethanosarcinaare able to grow on H~2~/CO~2~. They use a ferredoxin-dependent hydrogenase (Ech) for ferredoxin reduction and an additional membrane bound methanophenazine-dependent hydrogenase (Vho) for H~2~ oxidation coupled to the reduction of the heterodisulfide by the membrane bound CoB-S-S-CoM reductase (HdrDE). F~420~ cycling can be accomplished using the F~420~-dependent hydrogenase (Frh) and F~420~H~2~: phenazine oxidoreductase (Fpo) complex \[[@B48]\] ([Figure 2](#fig2){ref-type="fig"}). For methanogens it is of crucial importance that Mcr operates in the forward reaction to yield methane and the heterodisulfide. If all reactions of the methanogenic pathway are reversed such as during AOM, the pathway requires the input of energy and produces electron donors (Figures [4](#fig4){ref-type="fig"} [](#fig5){ref-type="fig"}--[6](#fig6){ref-type="fig"}). Therefore, during AOM, an external electron acceptor is needed which makes the reaction thermodynamically favourable ([Table 1](#tab1){ref-type="table"}). The reverse reaction of Mcr is therefore an essential step in AOM and is discussed in [Section 2.2](#sec2.2){ref-type="sec"}. The respiratory chain that is needed for using different terminal electron acceptors will be discussed in [Section 3](#sec3){ref-type="sec"}. Evidence that ANME use the reverse methanogenesis pathway during AOM is derived from metagenomic and metatranscriptomic analyses. This showed that all genes for the (reverse) methanogenic pathway were present and expressed in ANME-2a \[[@B42]\] ([Figure 4](#fig4){ref-type="fig"}) and ANME-2d \[[@B28]\] ([Figure 5](#fig5){ref-type="fig"}). ANME-1 were consistently lacking the gene encoding N^5^,N^10^-methylene- tetrahydromethanopterin (H~4~MPT) reductase (Mer), which is an enzyme needed to oxidize methyl-H~4~MPT during methane oxidation \[[@B40], [@B41], [@B44], [@B49]\] ([Figure 6](#fig6){ref-type="fig"}, [Table 2](#tab2){ref-type="table"}). Possible explanations could be that (1) themer gene is present but not yet detected, (2) ANME-1 used a bypass of this step, and (3) Mer was replaced by a structural analogue. The first possibility is highly unlikely. Although no closed genome of ANME-1 is publicly available to date, all ANME-1 metagenomes consistently only lack Mer and no other methanogenic genes. The second possibility was proposed previously where ANME-1 uses a bypass of Mer via the formation of methanol or methylamine \[[@B41]\], as was detected in deletion mutants ofMethanosarcina \[[@B50], [@B51]\]. Here, CH~3~-CoM was presumably converted to methanol by a methyltransferase and subsequently to formaldehyde by a methanol dehydrogenase (Mdh). Then, formaldehyde would be converted to N^5^,N^10^ methylene-H~4~MPT using a fusion protein of the formaldehyde-activating enzyme (Fae) and a hexulose-6-phosphate synthase (Hps) \[[@B51]\] ([Figure 6](#fig6){ref-type="fig"}). Both Fae and Hps were found in the ANME-1 metagenome \[[@B41]\] and metaproteome \[[@B40], [@B49]\]. However, no genes coding for enzymes involved in methanol metabolism were detected in these ANME-1 datasets \[[@B40], [@B41], [@B49]\] ([Table 2](#tab2){ref-type="table"}), indicating that this alternative pathway probably does not occur. The presence of the Fae/Hps fusion protein in ANME-1 during AOM could also be explained by its involvement in ribose phosphate synthesis and not in AOM \[[@B52]\]. Indeed, the Fae gene domains of ANME-1 were located in between ribulose-phosphate binding barrel and ribosomal protein S5 domains (Table S2). The third possibility of a structural analogue is most likely since an analogue of N^5^,N^10^-methylene tetrahydrofolate (H~4~HF) reductase (MetF) was expressed by ANME-1 during AOM which could replace Mer \[[@B40]\] ([Figure 6](#fig6){ref-type="fig"}). 2.2. Methyl Coenzyme M Reductase (Mcr) {#sec2.2} -------------------------------------- The enzymatic reaction of a purified Mcr from ANME has not been measured to date. The key question is whether a methanogenic Mcr can explain the observed in situ AOM rates or if the Mcr of ANME is structurally altered. There are three main factors to be considered: the kinetic limitations as defined by enzyme properties (i.e., half-maximal activity at a specific K ~M~ value), the thermodynamic constraints of the enzymatic reaction, and the maximal or ambient rate of the enzymatic reaction. For the Mcr fromMethanothermobacter marburgensis, kinetic parameters have been determined to illustrate the reversibility of reaction ([1](#EEq1){ref-type="disp-formula"}). In the methanogenic reaction, the purified Mcr isoform I \[[@B53]\] catalyzes the production of methane with a V ~max~ of 30 U mg^−1^ and a K ~M~ of 5 mM for CH~3~-CoM. The same (methanogenic) enzyme was able to oxidize methane to CH~3~-CoM with a rate of 0.0114 U mg^−1^ and a K ~M~ for methane of \~10 mM \[[@B54]\]. To answer if the observed AOM rates are in accordance with the measured methane oxidation rate for the purified Mcr enzyme fromM. marburgensis, the Mcr activity during AOM is needed. Estimates for AOM rates in terms of activity (per cell dry mass) range between \<1 and 20 mmol day^−1^ and g cell dry mass^−1^ \[[@B54]--[@B64]\]. This equals an activity of 0.7 to \~14 nmol min and mg cell dry mass^−1^. About half of the cell dry mass is protein, so the activity for the ANME archaea would approximate 1.4 to 28 nmol min and mg protein^−1^. To estimate the activity per mg of Mcr, the proportion of Mcr to cellular protein is needed. It was reported that 7% of the protein of ANME microbial mats from the Black Sea \[[@B65], [@B66]\] and 10.4% of peptides from Hydrate Ridge mesocosms is Mcr \[[@B49]\]. As these were no pure cultures, the actual percentages of Mcr in ANME cells may be higher. Transcriptome data for ANME-2d \[[@B43]\] showed that about 19% of the total transcripts were derived from themcr genes indicating (though not demonstrating) a high Mcr content in ANME-2d. Estimating that 10% of the cellular protein would be Mcr, the specific activity of the enzyme would be between 14 and 280 nmol min and mg Mcr protein^−1^, which is up to 25 times higher than the measured reverse reaction rate of theM. marburgensis enzyme (\~12 nmol min and mg Mcr^−1^ \[[@B54]\]). However, the reverse reaction rate of theM. marburgensis Mcr was determined under nonsaturating substrate conditions and was therefore not possibly representing the true maximum rate. Nevertheless, both reverse reaction rates are in the same order of magnitude, other than the forward reaction of 30,000--100,000 nmol min and mg Mcr^−1^. Thus, it seems that the Mcr in ANME may have similar catalytic properties as the methanogenic enzyme and that the high amount of Mcr per mg total cell biomass in ANME may in part compensates for the apparently relatively slow catalysis. Considering the thermodynamic constraints, the Gibbs free energy change of the Mcr forward reaction under standard conditions is around −30 kJ mol^−1^ \[[@B54]\]. Therefore, the reverse reaction is endergonic under standard conditions and will not proceed. However, high methane concentrations (10^5^ according to reaction ([1](#EEq1){ref-type="disp-formula"}) \[[@B67], [@B68]\]) may lead to a favourable change in the Gibbs free energy in the direction of AOM. High methane partial pressure prevails at many habitats where AOM has been detected. The solubility of methane at atmospheric pressure is only 1.3 mM \[[@B69]\]. Consequently, increased AOM rates were reported upon pressurizing samples of different geographical origin \[[@B57], [@B58], [@B70], [@B71]\]. The K ~M~ of Mcr ofM. marburgensis for methane was determined at or above 10 mM and reported K ~M~ values of S-AOM varied from (at least) 1.1 mM \[[@B72]\], a few mM \[[@B55]\], to even 37 mM (equivalent to 3 MPa CH~4~) \[[@B56]\]. Thus, high pressure and therefore high concentrations of methane in the natural habitat accelerate the oxidation rate of methane by Mcr. Future research to accurately determine K ~M~ values and rates for Mcr at different methane partial pressures is however needed. This may seem difficult, but microbial activity measurements at in situ methane partial pressure were shown to be successful in the laboratory \[[@B73]\]. It was suggested that the Mcr reaction is the rate limiting step in reverse methanogenesis \[[@B54]\] which is in line with the above described challenges. Supporting these findings, there does not seem to be a major change in the amino acid structure that determines whether the backwards or the forward reaction of Mcr is favoured. Amino acid alignments \[[@B65]\] and the crystal structure of ANME-1 Mcr \[[@B74]\] indicated high overall similarity of the methanogenic and methanotrophic enzyme and unambiguously demonstrated that CoM-SH and CoB-SH are substrates of the methanotrophic enzyme. However, several posttranslational modifications of amino acids were different between methanogens and ANME archaea, and the cofactor F~430~ (the prosthetic group of Mcr) is modified in ANME-1, but not in ANME-2 or ANME-3 archaea \[[@B49], [@B61], [@B65], [@B66], [@B74], [@B75]\]. Furthermore, ANME-1 seems to lack the noncatalytic protein D domain of themcrgene that is present in all other methanogens and methanotrophs but of which the function is unknown (IPR003901, [Table 2](#tab2){ref-type="table"}) \[[@B49]\]. A metabolically engineeredMethanosarcina acetivoranswas able to convert methane and CO~2~ to acetate with a plasmid containing Mcr derived from ANME-1 \[[@B76]\]. It is thus unclear if only thermodynamic constraints and the abundance of Mcr ensure AOM activity, or if also specific modifications can have an effect on the reverse activity of Mcr. 2.3. Methane Oxidation by Methanogens {#sec2.3} ------------------------------------- Pure cultures of methanogens were not able to oxidize methane under high methane and low hydrogen concentrations (reviewed in \[[@B77], [@B78]\]). Methanogens are only able to oxidize methane during net methane production \[[@B79]\]. Labeled methane addition (^13^C or ^14^C) to pure cultures of methanogens showed production of labeled CO~2~ during net methane production. This characteristic was confirmed with several pure cultures of methanogens \[[@B80]--[@B82]\]. The process was called "trace methane oxidation" (TMO), since the CO~2~ was formed in trace amounts relative to the methane produced \[[@B81]\]. It is not clear if TMO is due to the reported reversibility of individual enzymes \[[@B64]\], or if it is an active microbial process from which energy can be conserved. TMO was speculated to be an active metabolic process for three reasons: (1) the amount of methane oxidized varied between different species of methanogens grown on the same methanogenic substrate; (2) the amount of methane oxidized varied between different methanogenic substrates; and (3) TMO products varied between different methanogenic substrates \[[@B81], [@B82]\]. For instance, when grown on acetate,Methanosarcina acetivorans produced labeled acetate from labeled methane. When grown on carbon monoxide, it produced both labeled acetate and methyl sulfides from labeled methane \[[@B82]\]. During hydrogenotrophic and methylotrophic methanogenesis, TMO mainly produced CO~2~ from labeled methane \[[@B81]\]. However, in contrast with AOM, TMO rates never exceeded methanogenesis rates, even during long-term incubation with methane and sulfate \[[@B83]\]. It seems that methanogens are not able to conserve energy from TMO, even under thermodynamically favourable conditions. TMO occurs both in absence and presence of an external electron acceptor and only during net methanogenesis. It is therefore most likely caused by the reported back flux of individual enzymes of the methanogenic pathway \[[@B64]\]. TMO also occurred in granular sludge and in freshwater and terrestrial samples. These mixed communities showed higher TMO rates than pure cultures, reaching up to 90% of the methane produced \[[@B27], [@B83]--[@B85]\]. TMO should therefore be carefully considered in the experimental setup and interpretation of results when studying AOM in environmental samples, especially since TMO rates were, like AOM, stimulated by a high methane partial pressure \[[@B70], [@B84], [@B87]\]. Sulfate reduction was also stimulated by higher methane partial pressures \[[@B83]\]. Thus, a high methane partial pressure can have a stimulating effect on methane oxidation (either through AOM or TMO) and SR, which could be unrelated to S-AOM. Moreover, addition of iron sulfate (FeSO~4~) or manganese oxide (MnO~2~) also increased TMO rates \[[@B84]\]. Therefore, methane-dependent SR and sulfate- or metal-dependent methane oxidation are not necessarily indications for AOM in mixed cultures. In conclusion, when studying complex "black-box" communities, only net methane oxidation is proof for AOM activity. 2.4. Methane Production by ANME {#sec2.4} ------------------------------- The process of S-AOM is at the energetic limit for sustaining life, with estimates of Gibbs free energy yields between −18 and −35 kJ mol^−1^ \[[@B45], [@B77], [@B88]--[@B90]\] and doubling times between 1.1 and 7.5 months \[[@B63], [@B70], [@B71], [@B91], [@B92]\]. Since S-AOM operates close to its thermodynamic equilibrium, the reversibility of individual enzymes leads to measurable back flux, producing methane (3--7% of AOM) and sulfate (5.5--13% of SR) during S-AOM \[[@B64]\]. This "trace methane production" was observed in situ \[[@B11]\] and in sediment slurries, with methanogenesis around 10% \[[@B60], [@B93]\] or even as high as 50% of AOM \[[@B34]\]. When sulfate becomes depleted, Gibbs free energy yields become even lower (less negative) and the enzymatic back flux becomes even more apparent, up to 78% of net AOM \[[@B94]\]. Previous measurements of ^13^C depletion below the sulfate-methane transition zone (SMTZ) of marine sediments that were thought to be indicative for methanogenesis could therefore instead be attributed to the back flux of AOM \[[@B94]\]. The occurrence of ANME-1 without a bacterial partner in sediment layers where sulfate was depleted was previously interpreted as evidence that ANME-1 perform methanogenesis \[[@B24]\], but in light of the above it could also indicate AOM. There are indeed reports of AOM activity below the SMTZ in the methanogenic zone \[[@B95]--[@B98]\]. In contrast, AOM with other electron acceptors than sulfate operates far from the thermodynamic equilibrium with Gibbs free energy changes between −517.2 and −841.4 kJ mol methane^−1^ ([Table 1](#tab1){ref-type="table"}). Here, the reported anaerobic back flux \[[@B64]\] is expected to be less apparent. In laboratory incubations, researchers were not able to stimulate net methanogenesis through addition of methanogenic substrates to AOM performing sediments \[[@B60], [@B93]\]. In two cases, researchers were successful \[[@B23], [@B99]\]. In one of these cases, sediment-free long-term AOM enrichments that were dominated by ANME/SRB were incubated with methanogenic substrates. The resulting methanogenic activity most likely came from the enrichment of a minor population of methanogens (up to 7‰ of total archaeal gene tag sequences) that was present in the inoculum \[[@B99]\]. In the second study, methanogenic substrates were added to ANME-1 and ANME-2 dominated microbial mat samples and methanogenesis also occurred \[[@B23]\]. However, no information was provided for the total archaeal community composition, which makes it impossible to exclude methanogens as the responsible organisms. Genomic information of ANME also gives indication on potential methanogenic routes. Considering methylotrophic methanogenesis, no gene homologues catalyzing methyl transfer from methylated substrates to coenzyme M were detected in ANME ([Table 2](#tab2){ref-type="table"}) \[[@B40]--[@B43]\]. Acetoclastic methanogenesis needs either the AMP- and ADP-forming acetyl-coenzyme A synthetase (Acs and Acd, resp.) or proceeds via acetate kinase and phosphotransacetylase. In ANME-1, only the alpha subunit of a homologue of Acd was expressed during AOM \[[@B41]\], but in an ANME-1 proteome of active AOM biomass, no Acd was detected \[[@B40]\]. The Acd gene was detected in the single-aggregate genome and transcriptome of ANME-2a \[[@B42]\] and in ANME-2d \[[@B43]\]. However, gene domains for Acd are also present in methanogens unable to use acetate as substrate ([Table 2](#tab2){ref-type="table"}) and are probably used for lipid metabolism. In hydrogenotrophic methanogenesis, hydrogenases are used to replenish reduced coenzyme B and to recycle oxidized F~420~ (discussed in [Section 2.1](#sec2.1){ref-type="sec"}). Both the cytoplasmic Mvh complex and the membrane bound Vho were not present in ANME-2d \[[@B43]\] and not expressed in ANME-2a (which also lacked Ech and F~420~-dependent hydrogenase (Frh)) \[[@B42]\], making hydrogenotrophic methanogenesis unlikely. In ANME-1, both the cytoplasmic HdrABC and MvhD are present, as well as homologues of Frh and Ech, but these were lacking catalytic subunits \[[@B40], [@B41]\]. An iron hydrogenase was found in both ANME-1 metagenomes but not in any other methanotroph or methanogen \[[@B41]\] ([Table 2](#tab2){ref-type="table"}). This iron hydrogenase domain is part of a gene that is 70% identical to a \[FeFe\]-hydrogenase ofDehalococcoides mccartyi. \[FeFe\]-hydrogenases catalyze reversible H~2~ production and uptake, but these were presumed to have no key function in AOM \[[@B41]\]. However, the gene is part of a gene cluster of three genes containing a 51 kDa NADH: ubiquinone oxidoreductase subunit (Table S3), which could potentially form a complex that generates a proton motive force during hydrogen oxidation. Therefore, hydrogenotrophic methanogenesis by ANME-1 cannot be excluded yet. 3. Respiration during Anaerobic Oxidation of Methane {#sec3} ==================================================== For net AOM to occur, an external electron acceptor is needed which results in a favourable Gibbs free energy change ([Table 1](#tab1){ref-type="table"}). A variety of terminal electron acceptors have been discovered for AOM which will be discussed in Sections 3.1--3.3. 3.1. Sulfate-Dependent AOM {#sec3.1} -------------------------- During sulfate-dependent AOM, electrons are transferred from ANME to the sulfate-reducing bacterial partner. Previous work tried to uncover how electrons were transferred and most compounds that could act as interspecies electron carrier (IEC) were excluded to be involved in AOM, such as methanol, hydrogen, methanethiol, acetate, and carbon monoxide \[[@B55], [@B59], [@B60], [@B93], [@B100]\]. Indications that polysulfide could act as IEC were found, and ANME-2a archaea were thought to perform both AOM and sulfate reduction (SR) \[[@B101]\]. However, in marine seeps, hydrothermal vents, and other nondiffusion based sediments, AOM rates are high and ANME form close associations with SRB in dense aggregates \[[@B1], [@B5], [@B102], [@B103]\]. In these aggregates, the high AOM rates could not be explained by diffusion of an IEC, which made direct interspecies electron transfer (DIET) a more plausible mechanism \[[@B88], [@B104], [@B105]\]. Cellular activities were independent of aggregate type and distance between the syntrophic partners within the aggregate, which is best explained by DIET \[[@B106]\]. DIET is normally achieved using multiheme cytochrome c proteins (MHCs) and conductive pili (i.e., nanowires) which are mainly found in bacteria that donate electrons extracellularly, such asGeobacter andShewanella species \[[@B107]--[@B113]\]. Indeed, ANME-2a from seep-sediment samples seem to transfer electrons directly using large MHCs \[[@B106]\], which were found in the metagenome of ANME-2a \[[@B106], [@B114]\]. ANME-1 and the associated bacterial partner also overexpressed genes for extracellular MHCs during AOM \[[@B115]\], which complements previous findings of transcription \[[@B41]\] and translation \[[@B40]\] of ANME-1 related MHC genes. Domain based (meta)genome analysis shows the high abundance of MHC domains in different ANME as compared to methanogens ([Table 2](#tab2){ref-type="table"}). A recently isolated bacterial partner of ANME-1 ("HotSeep-1") also produced cell-to-cell connections using pili-derived nanowires \[[@B115]\], which explain previously detectedDeltaproteobacteria-related pili genes in an AOM sample dominated by ANME-1 \[[@B40]\]. How ANME use MHCs to donate electrons to the bacterial partner is not yet clear. For ANME-2a, the electrons probably flow from methanophenazine to membrane integrated di-heme cytochromes (cytochrome b/b ~6~) that transfer the electrons through the S-layer via MHC/S-layer fusion proteins to extracellular MHCs ([Figure 4](#fig4){ref-type="fig"}) (Figure 4 in \[[@B106]\]). Exosortases and archaea-specific archaeosortases are involved in export of cell surface proteins, such as the archaeal S-layer proteins. These transpeptidases recognize specific signal peptides for protein- sorting; that is, archaeosortase A recognizes the protein-sorting signal PGF-CTERM and archaeosortase C recognizes the PEF-CTERM signal \[[@B116]\]. Both ANME-2a and 2d show presence of di-heme cytochromes, archaeosortase A (IPR014522), archaeosortase C (IPR022504), and other exosortase gene domains ([Table 2](#tab2){ref-type="table"}). Moreover, some genes of both ANME-2a and 2d contained both MHC and PGF or PEF-CTERM domains. Lastly, some genes of both ANME-2a and 2d contained both MHC and S-layer domains \[[@B106]\], indicating that these could form the above-stated MHC/S-layer fusion proteins. ANME-1 do not seem to have di-heme cytochromes ([Table 2](#tab2){ref-type="table"}) \[[@B114]\]. PGF related domains (IPR026453 and IPR026371) were present in all ANME but PEF-CTERM (IPR017474) related domains were absent in ANME-1 ([Table 2](#tab2){ref-type="table"}). Moreover, ANME-1 lacked archaeosortase A (IPR014522) and archaeosortase C gene domains (IPR022504), as well as some other exosortases ([Table 2](#tab2){ref-type="table"}). A search in NCBI\'s conserved domains database (CCD, \[[@B117]\]) and the EMBL InterPro database \[[@B118]\] of amino acids sequences of all genes from ANME metagenomes that contain MHC domains showed that ANME-1 did not have any protein-sorting signal or S-layer domains within these genes. In fact, S-layer domains were completely absent in ANME-1 ([Table 2](#tab2){ref-type="table"}). These results imply that ANME-1 do not use di-heme cytochromes for electron transfer to MHCs and do not produce an S-layer ([Figure 6](#fig6){ref-type="fig"}). This implies that ANME-1 use a different mechanism for DIET and could explain the need for less MHCs by ANME-1 ([Table 2](#tab2){ref-type="table"}) and the observed pili-derived nanowires produced by the bacterial partner \[[@B115]\]. The genome of the bacterial partner of ANME-1 ("HotSeep-1") encoded 24c-type cytochromes of which 10 were similar to secreted MHCs ofGeobacter sulfurreducens\[[@B119]\] which also uses pili for DIET \[[@B120]\]. In the case of ANME-2a, it is not clear if pili (i.e., nanowires) were formed during AOM. It was previously thought that electrically conductive pili seemed to be a prerequisite for current production and DIET \[[@B121], [@B122]\], even when syntrophs were closely associated \[[@B123]\] such as within ANME-2/SRB aggregates. However, conductive materials such as granular activated carbon were shown to be able to substitute pili in DIET \[[@B123]\]. Although in previous work conductive materials such as phenazines or humic acids did not seem to stimulate AOM rates \[[@B59]\], in a recent study AOM was decoupled from SR using artificial electron acceptors \[[@B124]\]. This indicates that conductive materials can indeed replace pili and that ANME-2a/b could possibly couple AOM to metal oxide reduction or any other suitable electron acceptor (discussed in [Section 3.3](#sec3.3){ref-type="sec"}). However, it needs to be proven if in ANME-2/SRB aggregates no pili are formed and if the mechanism of DIET is fundamentally different from ANME-1. As for the polysulfide shuttling theory \[[@B101]\], canonical genes for dissimilatory sulfate reduction such as adenylyltransferase (Sat), APS reductase (Apr), and dissimilatory sulfite reductase (Dsr) which are all present in the sulfate-reducing archeonA. fulgidus (Table S2) were not found in metagenomes of ANME-1 \[[@B41]\] and ANME-2a (Table S1). The enzymes Sat and Dsr were also not detected in ANME cells using fluorescent immunolabelling \[[@B125]\]. ANME-1 were previously found to encode most proteins for assimilatory sulfate reduction \[[@B41]\]. ANME-2d only harbor gene domains that encode Sat and assimilatory ATP sulfurylase, APS kinase, and sodium/sulfate symporters, which were not present in ANME-2a (Table S1). It is therefore clear that at least ANME-2a cannot donate electrons to sulfate but need to donate electrons to a sulfate-reducing partner. 3.2. Nitrate-Dependent AOM {#sec3.2} -------------------------- Unlike during S-AOM, ANME-2d that perform N-AOM do not need a bacterial partner but transfer electrons directly to a membrane bound nitrate reductase (Nar) \[[@B28], [@B43]\] ([Figure 5](#fig5){ref-type="fig"}). The ANME-2d genomes contain most MHCs found so far in archaea \[[@B106], [@B114]\] ([Table 2](#tab2){ref-type="table"}). Of the 87 proteins that contained a CxxCH binding motif, of which 43 seemed to be truec-type cytochromes \[[@B114]\], 23 CxxCH motif-encoding transcripts were expressed during N-AOM \[[@B43]\]. The function of most of these MHCs is not known, but they are likely involved in nitrate reduction sincec-type cytochromes are capable of operating in the wide range of redox potentials that couple nitrate reduction (E ^0′^ (NO~3~ ^−^/NO~2~ ^−^) = +433 mV) and methane oxidation (E ^0′^ (CoM-S-S-CoB/CoM-SH+CoB-SH) = −143 mV) \[[@B43]\]. Nitrate as terminal electron acceptor in anaerobic respiration has been found in halophilic and thermophilic archaea \[[@B126]\]. Thenar gene cluster of "Ca. M. nitroreducens" comprises several genes including the catalytic alpha (NarG, molybdopterin) and beta (NarH, iron sulfur cluster) subunit of nitrate reductase \[[@B28], [@B43]\]. The (halo)archaeal nitrate reductase complex was reported to be located at the extracellular side of the cytoplasmic membrane \[[@B127]\] and in most archaea associated with the cytoplasmic membrane via NarM \[[@B128]\]. The "Ca.M. nitroreducens" genome does not encode NarM but encodes a TAT signal peptide at the N-terminus of NarG for translocation across the cytoplasmic membrane \[[@B28], [@B43]\]. Interestingly, the NarG and NarH seem to have been acquired from the Proteobacteria via lateral gene transfer \[[@B28]\]. It is not yet clear at which point in the metabolism "Ca. M. nitroreducens" conserves energy. During reverse methanogenesis, N^5^-methyl-H~4~MPT:CoM methyltransferase (Mtr) dissipates the sodium ion potential across the cytoplasmic membrane so subsequent steps in N-AOM have to be coupled to the build-up of a proton or sodium motive force to make the overall process energetically favourable. The analysis of an environmental genome \[[@B43]\] indicated presence of several protein complexes involved in electron transport and energy conservation. Electrons that enter the respiratory chain could be transported by membrane-integral electron carriers (i.e., menaquinones) to a Rieske-cytochromeb complex that may use cytochromec as electron acceptor. This in turn may be the electron donor for the unusual nitrate reductase complex. Energy conservation is thermodynamically and mechanistically feasible at the F~420~H~2~ dehydrogenase and the Rieske-cytochromebcomplex ([Figure 5](#fig5){ref-type="fig"}) (Figure 2 in \[[@B43]\]). Further investigations are needed to determine whether nitrate reductase is also involved in energy conservation, but this working hypothesis is strengthened by the presence of cupredoxin, multicopper oxidase domains, and copper centers related to the periplasmic domain of cytochromec oxidase subunit II (HCO II) in ANME-2d (Table S1). Both ANME-2d genomes discussed here are derived from bioreactors where "Ca. M. nitroreducens" formed syntrophic cultures with nitrite scavenging bacteria, either with"Ca.Kuenenia stuttgartiensis" (anammox bacteria) \[[@B28]\] or"Ca.Methylomirabilis oxyfera" (NC10 bacteria) \[[@B31], [@B43]\]. This indicated that ANME-2d could be dependent on those bacteria for nitrite removal. However, in addition to nitrite, "Ca. M. nitroreducens" may also produce ammonium during AOM by a pentahemec-type nitrite reductase (NrfAH) encoded in the genome \[[@B43]\] ([Figure 5](#fig5){ref-type="fig"}). In fact, both ANME-2d genomes contain domains for NrfA (IPR003321) and NrfH (IPR017571) (Table S1) implying that both ANME-2d species are not necessarily dependent on a nitrite scavenger during AOM. 3.3. Metal-Dependent AOM {#sec3.3} ------------------------ Evidence for metal-dependent AOM was found in marine sediments \[[@B129]--[@B131]\]. In nonmarine environments, AOM was also hypothesized to be coupled to iron and/or manganese oxide reduction \[[@B26], [@B132]--[@B135]\] or even coupled to the reduction of humic acids \[[@B135], [@B136]\]. However, organisms responsible for metal-dependent AOM were not identified in these studies. It was speculated that JS1 bacteria, methanogenic archaea, andMethanohalobium/ANME-3 could be responsible for iron-dependent AOM \[[@B137]\]. Other researchers speculated that either ANME-1 orMethanococcoides/ANME-3 together with a bacterial partner were responsible for manganese-dependent AOM \[[@B138]\]. In another study where AOM was decoupled from SR, ANME were not detected, which leaves open the possibility that other archaeal clades besides ANME could perform metal-dependent AOM \[[@B130]\]. It was recently observed that cultures containing ANME-2a and ANME-2c could decouple AOM from SR in the presence of artificial electron acceptors, humic acids, and soluble iron \[[@B124]\], which confirmed previous findings of AOM not connected to SR in ANME dominated samples \[[@B139]\]. This suggests that ANME-2 could also use insoluble metal oxide minerals as electron acceptor during AOM. The MHCs of ANME-2a/b and ANME-2d are larger than those inShewanella andGeobacter species \[[@B106]\], which are known to be capable of extracellular electron conduction. It was speculated that both ANME-2d andFerroglobus placidus, of which the latter can perform solid iron reduction, can fold CxxCH motifs into extracellular conductive structures or pili \[[@B114]\]. Many of the MHCs of ANME-2d were not expressed when grown with nitrate (discussed in section 3.2), implying that these are not needed for nitrate reduction \[[@B43]\]. This strengthens the hypothesis that also ANME-2d could couple AOM to reduction of other extracellular electron acceptors than nitrate and even to insoluble metal oxides. Indeed, recent work showed that ANME-2d could be involved in AOM coupled to chromium(VI) reduction \[[@B140]\] ([Table 1](#tab1){ref-type="table"}, reaction (5)) and in AOM coupled to the reduction of soluble iron and insoluble ferrihydrite and birnessite minerals \[[@B141]\] ([Table 1](#tab1){ref-type="table"}, reactions (3) and (4)). ANME-2d could even possibly donate electrons to a bacterial partner: besides nitrate-rich environments, ANME-2d archaea have been found in wells of an aquifer where sulfate and methane concentrations overlap \[[@B142]\]. Moreover, ANME-2d was the only clade detected in sediments of a freshwater lake where S-AOM occurred \[[@B143]\]. Sulfate concentrations in these studies were low, but above 1 mM and thus higher than the lowest reported concentrations for S-AOM to occur \[[@B144], [@B145]\]. Lastly, sequences of ANME-2d were relatively more abundant in freshwater sediments fed with methane and sulfate than in sediments fed with only methane or only sulfate and no N-AOM activity was detected when fed with nitrate and methane \[[@B27]\]. These indications hold promise that direct experimental evidence for sulfate-dependent AOM by ANME-2d could be found in the future. The ANME-1 genome contains fewer MHC gene domains as compared to ANME-2a and ANME-2d and some other archaea, such as some methylotrophic methanogens ([Table 2](#tab2){ref-type="table"}) and some members of the Archaeoglobales \[[@B114]\]. The MHCs of ANME-1 also have a smaller heme count as compared to other ANME and some other archaea, with the largest being an octaheme cytochrome \[[@B106]\]. Each heme within a MHC has its own redox potential and therefore structurally different MHCs represent a large range of redox potentials that can be used for bioenergetic electron transfer (reviewed in \[[@B146]\]). For instance, metal oxide reduction inShewanella oneidensis MR-1 is catalyzed by a chain of a tetraheme (CymA), two decaheme (MtrA and MtrB), and eventually extracellular decaheme cytochromes OmcA/MtrC that reduce the iron minerals \[[@B120], [@B147], [@B148]\]. InGeobacter sulfurreducens, iron mineral reduction seems to be catalyzed by the tetraheme cytochrome OmcE and hexaheme cytochrome OmcS transferring electrons from the outer membrane to type IV pili that transmit the electrons to iron minerals \[[@B120], [@B149], [@B150]\]. Since ANME-1 lack MHCs of the correct size and lack gene domains for pili production \[[@B115]\], they seem unable to reduce minerals via both mechanisms present inShewanellaandGeobacter. It can therefore be speculated that ANME-1 are less versatile in electron acceptor use and are not able to reduce solid metal oxides. However, DIET mechanisms are still not well understood and the true differences between MHCs of different ANME types need to be investigated using biochemical methods. This would allow uncovering the true capabilities concerning DIET. 3.4. Menaquinones and Methanophenazines {#sec3.4} --------------------------------------- ANME-2a encoded a protein with a domain specific for PhzF, which is an enzyme involved in phenazine biosynthesis inPseudomonas fluorescens \[[@B151], [@B152]\]. The respective gene is not present in all methanophenazine containing methanogens (in our genome comparison only inM. acetivorans andM. formicica, [Table 2](#tab2){ref-type="table"}) so it is unclear whether this enzyme is really involved in methanophenazine biosynthesis. It is however likely that ANME-2a use methanophenazines in their respiratory chain since gene domains for menaquinone biosynthesis were absent ([Table 2](#tab2){ref-type="table"}). "Ca. M. nitroreducens" probably uses menaquinones as membrane-integral electron carrier since environmental genomes \[[@B28], [@B43]\] encoded the futalosine (mqn) biosynthesis pathway as reported forArchaeoglobus fulgidus\[[@B153]\] ([Table 2](#tab2){ref-type="table"}). Moreover, "Ca. M. nitroreducens" enrichment cultures showed absence of methanophenazines \[[@B43]\]. ANME-1 also contained gene domains for menaquinone biosynthesis via the futalosine (mqn) biosynthesis pathway ([Table 2](#tab2){ref-type="table"}). Indications for a quinone biosynthesis pathway in ANME-1 were previously found to be weak since only some of the Ubi homologues of the oxic ubiquinone biosynthesis pathway were detected \[[@B41]\], but the futalosine (mqn) biosynthesis pathway was overlooked in that particular analysis. Additionally, ANME-1 have Fqo homologues similar toArchaeoglobus fulgidus\[[@B44]\] and expressed the catalytic subunit FqoF \[[@B41]\]. However, since the phenazine biosynthesis domain PhzF was also present in the ANME-1 genomes ([Table 2](#tab2){ref-type="table"}) and Fpo and Fqo are homologues, we cannot conclude on genomic information alone which redox shuttle is used by ANME-1 during AOM. If ANME-1 would use menaquinones, this would have implications for subsequent electron transfer to MHCs since methanophenazine (E ^0^ = −165 mV) \[[@B154]\] and menaquinone (E ^0^ = −80 mV) \[[@B155]\] have different redox potentials. 3.5. Cell Adhesion {#sec3.5} ------------------ Some gene domains involved in cellular adhesion were more abundant in ANME than in methanogens ([Table 2](#tab2){ref-type="table"}), especially in ANME-2a and ANME-1 that are known to form syntrophic interactions for electron transfer during respiration. These domains include HYR (IPR003410) and CARDB domains (IPR011635) that both have a direct role in cellular adhesion \[[@B156]\] ([Table 2](#tab2){ref-type="table"}). Interestingly, also domains related to the cellulosome ofClostridium species, termed dockerin and cohesin, were highly abundant in both ANME-1 and ANME-2a as compared to ANME-2d and methanogens, together with many carbohydrate binding domains ([Table 2](#tab2){ref-type="table"}). InClostridium species, dockerin and cohesin form an anchor to the bacterial cell wall that contains a scaffold with cellulose-degrading enzymes and a carbohydrate binding module that binds cellulose, altogether forming the cellulosome \[[@B157]\]. These domains have been found in all domains of life irrespective of cellulose utilization, but the function of such proteins outside of the cellulosome is not known \[[@B158]\]. Therefore, dockerin, cohesin, and carbohydrate binding domains in ANME could hypothetically form a construct that binds to carbohydrates and could possibly have a function in cell-to-cell contact or MHC adhesion, but this needs further investigation. 4. Future Challenges {#sec4} ==================== Advances in (meta)genomics, transcriptomics, and proteomics have produced the valuable metabolic blueprints of different ANME with hypotheses on how central metabolism, electron transport, and energy conservation may function. Future experiments are needed to biochemically demonstrate that these hypotheses are correct. The bottleneck for biochemical studies of ANME is the lack of pure cultures due to the slow and syntrophic growth. However, the recent milestone discovery of direct electron transfer provides opportunities to grow ANME, as stated previously, on electron accepting electrodes (i.e., anodes) \[[@B159]\]. In this way, pure or highly enriched cultures of different ANME without their respective syntrophic partner could be obtained and MHCs responsible for electric conduction could be biochemically characterized. It seems that ANME-1 are limited by the size and abundance of MHCs which could relate to differences in behavior on an anode. The partner bacteria of ANME-1 and ANME-2 could be investigated on an electron donating electrode (i.e., cathode), with specific focus on the ability to produce pili (i.e., nanowires). Also worth investigating is if besides ANME-2d, ANME-2a/b could also use insoluble electron acceptors and if ANME-2d could donate electrons to a bacterial partner. Another future challenge is to isolate and characterize Mcr from different ANME clades. It needs to be investigated if Mcr abundance in ANME cells compensates for the slow reverse activity or if modifications in the Mcr of ANME-1 also contribute to a better reverse activity of Mcr. Moreover, the effect of methane partial pressure on reaction rates and enzyme kinetics needs to be determined in situ. ANME-1 are potentially able to perform hydrogenotrophic methanogenesis due to the presence of a hydrogenase in the genome. Genetic indications of menaquinones as electron carrier in ANME-1 and the various cellulosome and cell adhesion related gene domains in all ANME are also topics that could be explored further. Methanogenic archaea are likely not able to perform AOM, but additional studies on TMO and more genetic modifications to stimulate AOM in methanogens could help in understanding the parameters needed for AOM to occur. Ultimately, physiological understanding of ANME will help to explain the observed ecological niche separation of different ANME clades and the occurrence of ANME without a bacterial partner. This would greatly enhance our knowledge of the methane cycle in anoxic environments. Supplementary Material {#supplementary-material-sec} ====================== ###### This supplementary material contains data on domain based (meta)genome comparison of selected metagenomes of methanotrophs and genomes of selected archaea that belongs to the article "Reverse methanogenesis, and respiration in methanotrophic archaea" by Peer H.A. Timmers, Cornelia U. Welte, jasper J. Koehorst, Caroline M. Plugge, Mike S. M. Jetten and Alfons J.M. Stams. The authors thank Stefanie Berger (RU, Nijmegen) for critical reading of the manuscript. This research is supported by the Soehngen Institute of Anaerobic Microbiology (SIAM) Gravitation grant (024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Organisation for Scientific Research (NWO). Mike S. M. Jetten was further supported by ERC AG 339880 Eco-MoM and Alfons J. M. Stams was supported by ERC AG 323009 Novel Anaerobes. Competing Interests =================== The authors declare that there is no conflict of interests regarding the publication of this paper. ![Phylogenetic tree of full length archaeal 16S rRNA sequences showing all methanotrophic clades so far described (grey) and other archaeal clades used in our domain based (meta)genome comparison (black). The tree was constructed with the ARB software package (version arb-6.0.1.rev12565) \[[@B160]\] using 2800 sequences from the SILVA SSURef NR 99 database (release 119.1) \[[@B161]\]. Trees were calculated by maximum likelihood analysis (RAxML, PHYML) and the ARB neighbor-joining method with terminal filtering and the Jukes-Cantor correction. Resulting trees were compared manually and a consensus tree was constructed. Sulfolobales as outgroup was removed after tree calculations. The scale bar represents the percentage of changes per nucleotide position.](ARCHAEA2017-1654237.001){#fig1} ![Hydrogenotrophic methanogenesis in cytochrome containingMethanosarcina barkeri. Black lines represent presence of conversions. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.002){#fig2} ![Methylotrophic methanogenesis in cytochrome containingMethanosarcina barkeri. Black lines represent presence of conversions and red lines indicate reversal of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.003){#fig3} ![Methanotrophic pathway during S-AOM by ANME-2a \[[@B42]\]. Red lines indicate reversal of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.004){#fig4} ![Methanotrophic pathway during N-AOM by "Ca.M. nitroreducens" MPEBLZ (ANME-2d) \[[@B43]\]. Red lines indicate reversal of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.005){#fig5} ![Methanotrophic pathway during S-AOM by ANME-1 \[[@B40], [@B41]\]. Red lines indicate reversal of the hydrogenotrophic methanogenic pathway, grey lines represent absence of conversions, and blue lines indicate a bypass of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.006){#fig6} ###### Gibbs free energy changes under standard conditions (ΔG ^0^) for anaerobic methane oxidation coupled to different electron acceptors (possibly) performed by ANME. Reaction Gibbs free energy (ΔG ^0^, kJ mol^−1^) -------------------------------------------------------------------------------- ------------------------------------------ \(1\) CH~4~ + SO~4~ ^2−^ → HCO~3~ ^−^ + HS^−^+ H~2~O −16.3 \(2\) CH~4~ + 4 NO~3~ ^−^ → HCO~3~ ^−^ + 4 NO~2~ ^−^ + H~2~O + H^+^ −517.2 \(3\) CH~4~ + 8 Fe(OH)~3~ + 16 H^+^ → CO~2~ + 8 Fe^2+^ + 22 H~2~O −571.2 \(4\) CH~4~ + 4 MnO~2~ + 8 H^+^ → CO~2~ + 4 Mn^2+^ + 6 H~2~O −763.2 \(5\) CH~4~ + 4/3 Cr~2~O~7~ ^2−^ + 32/3 H^+^ → 8/3 Cr^3+^ + CO~2~ + 22/3 H~2~O −841.4 ###### Domain based (meta)genome comparison of selected metagenomes of methanotrophs and selected genomes of other archaea. Domain abundance in every (meta)genome is indicated by numbers. S-AOM performing ANME include ANME-1-s \[[@B40]\], ANME-1-m \[[@B41]\], and ANME-2a \[[@B42]\]. N-AOM performing ANME include ANME-2d-h \[[@B28]\] and ANME-2d-a \[[@B43]\]. The acetoclastic (A) and methylotrophic (M) methanogens include Methanosarcina acetivoransC2A (AM), Methanosaeta concilii GP6 (A), Methanococcoides burtonii DSM6242 (M-1), Methanolobus tindarius DSM2278 (M-2), and Methanohalophilus mahiiDSM5219 (M-3). Hydrogenotrophic methanogens (H) include Methanospirillum hungatei JF-1 (H-1), Methanobacterium formicicum DSM3637 (H-2), Methanococcus maripaludis C5 (H-3), and Methanoregula formicica SMSP (H-4). The sulfate-reducing archaeon (S) is Archaeoglobus fulgidus DSM 4304. InterPro ID ANME-1 S ANME-1 M ANME-2a ANME-2d H ANME-2d A AM A M-1 M-2 M-3 H-1 H-2 H-3 H-4 S ---------------------------------------------------------------------- ------------- ---------- ---------- --------- ----------- ----------- ---- ---- ----- ----- ----- ----- ----- ----- ----- ---- Central pathway Mcr, alpha subunit, N-terminal IPR003183 4 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, alpha subunit, N-terminal subdomain 1 IPR015811 2 3 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, alpha subunit, N-terminal subdomain 2 IPR015823 2 2 2 2 2 2 2 2 2 2 2 4 2 4 0 Mcr, alpha subunit, C-terminal IPR009047 3 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, alpha/beta subunit, C-terminal IPR008924 6 4 2 2 2 2 2 2 2 2 2 4 2 4 0 Mcr, alpha/beta subunit, domain 2, C-terminal IPR022681 9 6 3 3 3 3 3 3 3 3 3 6 3 6 0 Mcr, beta subunit IPR003179 1 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, beta subunit, C-terminal IPR022679 3 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, beta subunit, N-terminal IPR022680 4 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, gamma subunit IPR003178 14 8 4 4 4 4 4 4 4 4 4 8 4 8 0 Mcr, protein C IPR007687 2 1 1 1 1 1 1 1 1 1 1 1 1 1 0 Mcr, protein C-like IPR026327 5 2 2 2 2 2 2 1 2 2 2 2 2 1 0 Mcr, protein D IPR003901 0 0 3 6 6 3 3 3 3 3 3 9 3 6 0 Mcr, ferredoxin-like fold IPR009024 12 6 3 3 3 3 3 3 3 3 3 6 3 6 0 5,10-methylenetetrahydromethanopterin reductase IPR019946 0 0 2 1 1 1 1 1 1 1 1 1 1 1 1 Acetoclastic methanogenesis CO dehydrogenase/acetyl-CoA synthase complex alpha subunit IPR004460 0 1 1 1 1 3 1 1 1 1 1 1 1 1 2 CO dehydrogenase/acetyl-CoA synthase complex beta subunit IPR004461 13 4 2 2 2 4 4 2 2 2 2 2 2 4 2 CO dehydrogenase/acetyl-CoA synthase delta subunit IPR004486 5 1 1 1 1 2 1 1 1 1 1 1 1 1 1 CO dehydrogenase/acetyl-CoA synthase delta subunit, TIM barrel IPR016041 11 2 5 3 2 4 2 2 3 3 3 2 2 5 2 CO dehydrogenase b subunit/acetyl-CoA synthase epsilon subunit IPR003704 3 2 2 2 2 4 2 2 2 2 2 2 2 2 4 Methylotrophic methanogenesis Methyltransferase MtaA/CmuA IPR006360 0 0 0 0 0 7 0 4 5 6 0 0 1 0 0 Methanol-cobalamin methyltransferase, B subunit IPR021079 0 0 0 0 0 3 0 2 2 2 0 0 0 0 0 Monomethylamine methyltransferase MtmB IPR008031 3 0 0 0 0 12 0 12 21 15 0 0 0 0 0 Trimethylamine methyltransferase IPR010426 11 0 0 0 0 4 0 5 2 2 0 0 0 0 0 Dimethylamine methyltransferase MtbB IPR012653 0 0 0 0 0 9 0 6 6 6 0 0 0 0 0 Trimethylamine methyltransferase, Methanosarcina IPR012740 0 0 0 0 0 2 0 1 1 1 0 0 0 0 0 Methylamine methyltransferase corrinoid protein reductive activase IPR026339 0 0 0 0 0 4 0 2 2 2 0 0 0 0 0 C-type cytochromes Di-haem cytochrome, transmembrane, nitrate reduction IPR016174 2 0 4 3 7 4 0 0 4 0 1 1 0 0 1 Doubled CXXCH motif IPR010177 33 0 3 2 3 0 0 0 0 0 0 0 0 0 0 Cytochrome c-like domain IPR009056 2 0 6 0 17 15 0 4 8 6 0 0 0 0 4 Class III cytochrome C (tetraheme cytochrome) IPR020942 2 0 0 3 3 0 0 0 0 0 0 0 0 0 0 Tetraheme cytochrome domain, flavocytochrome c3 (Shewanella) IPR012286 4 0 4 5 4 2 0 0 1 0 1 0 0 0 2 Octaheme c-type cytochrome IPR024673 1 0 2 0 0 0 0 0 2 2 0 0 0 0 1 Methanogenesis multiheme c-type cytochrome IPR027594 0 0 1 0 0 1 0 1 1 1 0 0 0 0 0 Multiheme cytochrome IPR011031 78 15 52 80 73 3 0 6 8 14 0 0 0 1 7 S-layer domains S-layer family duplication domain IPR006457 13 0 29 16 26 34 19 16 44 17 0 0 0 0 0 Sarcinarray family protein IPR026476 0 0 6 0 4 1 0 0 2 1 0 0 0 0 0 S-layer homology domain IPR001119 1 0 2 0 0 0 0 0 0 0 0 0 0 0 0 Cell export and cell adhesion HYR domain IPR003410 1 5 2 0 0 0 0 0 0 0 0 0 0 0 0 CARDB domain IPR011635 64 14 15 6 6 30 4 1 4 0 9 0 0 3 8 Collagen-binding surface protein Cna-like, B-type domain IPR008454 4 1 2 1 8 0 6 0 2 2 0 0 0 0 0 von Willebrand factor, type A IPR002035 55 28 17 32 14 37 23 17 42 9 28 8 0 27 7 VWA N-terminal IPR013608 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 Adhesion lipoprotein IPR006128 8 4 4 5 0 0 5 3 0 4 4 5 0 0 0 Adhesin B IPR006129 0 3 4 5 0 0 3 0 0 4 0 3 0 0 0 Invasin/intimin cell-adhesion fragments IPR008964 17 0 3 2 6 7 0 2 7 0 1 2 0 2 2 Putative cell wall binding repeat 2 IPR007253 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 Archaeosortase A IPR014522 2 0 1 2 2 1 1 1 1 1 1 0 0 2 1 Archaeosortase B IPR026430 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 Archaeosortase C IPR022504 0 0 1 2 1 0 0 1 1 0 0 0 0 0 0 Exosortase/archaeosortase domain IPR026392 5 0 2 5 3 1 1 2 2 2 1 0 1 2 1 Exosortase, EpsH IPR013426 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 Exosortase EpsH-related IPR019127 4 2 2 3 2 1 1 2 1 1 1 0 1 2 1 Archaeosortase family protein ArtE IPR026485 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 Cell wall hydrolase/autolysin, catalytic IPR002508 2 2 1 0 0 0 0 0 0 0 0 0 0 1 0 PEF-CTERM protein sorting domain IPR017474 0 0 3 4 1 0 0 10 19 0 0 0 0 0 0 PGF-pre-PGF domain IPR026453 1 1 18 9 8 24 0 7 21 11 0 0 0 1 1 PGF-CTERM archaeal protein-sorting signal IPR026371 9 4 6 2 1 2 0 2 1 2 1 0 0 1 3 LPXTG cell wall anchor domain IPR019931 3 0 1 0 0 0 1 1 0 0 0 0 0 0 0 VPXXXP-CTERM protein sorting domain IPR026428 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 Cellulosome-related/Dockerin Cellulosome anchoring protein, cohesin domain IPR002102 80 77 52 4 12 2 0 4 15 3 1 1 0 0 4 Dockerin domain IPR016134 149 112 44 4 6 5 17 2 4 0 9 0 0 0 2 Dockerin type I repeat IPR002105 1 0 5 0 0 0 0 0 0 0 0 0 0 0 1 Carbohydrate-binding domains Carbohydrate-binding domain IPR008965 50 42 39 2 6 2 0 3 13 2 3 0 1 0 2 Carbohydrate-binding-like fold IPR013784 14 5 3 10 19 0 0 0 0 1 3 0 0 6 1 Carbohydrate-binding, CenC-like IPR003305 1 0 7 0 17 0 0 0 0 0 0 0 0 0 0 Carbohydrate-binding/sugar hydrolysis domain IPR006633 27 22 5 2 2 27 3 7 8 8 0 5 2 1 8 Carbohydrate-binding domain, family 9 IPR010502 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 Bacteroidetes-associated carbohydrate-binding often N-terminal IPR024361 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Carbohydrate binding module, xylan-binding domain IPR031768 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 Galactose-binding domain-like IPR008979 12 1 17 6 39 4 4 2 1 3 0 0 0 0 0 Menaquinones 3-Demethylubiquinone-9 3-methyltransferase IPR028973 0 0 0 3 3 0 0 0 0 0 0 1 0 0 0 Succinate dehydrogenase/fumarate reductase, flavoprotein subunit IPR014006 0 0 1 2 1 0 0 0 0 1 0 0 0 0 1 UbiE/COQ5 methyltransferase IPR004033 3 0 0 2 2 1 0 1 0 0 0 0 0 0 0 UbiE/COQ5 methyltransferase, conserved site IPR023576 3 0 0 2 2 0 0 0 1 0 0 0 0 0 0 NrfD family IPR005614 3 0 0 4 3 0 0 0 0 0 0 0 0 0 3 Futalosine hydrolase IPR019963 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 Cyclic dehypoxanthine futalosine synthase IPR022431 1 1 0 1 1 0 0 0 0 0 0 0 0 0 1 Aminodeoxyfutalosine synthase IPR022432 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 Menaquinone synthesis (chorismate dehydratase & naphthoate synthase) IPR003773 8 6 0 2 2 0 0 0 0 0 0 0 0 0 2 FO synthase, subunit 2 IPR020050 3 3 1 3 3 2 1 2 1 2 2 2 2 2 3 Phenazines Phenazine biosynthesis PhzF protein IPR003719 1 1 4 0 0 2 0 0 0 0 0 0 0 2 0 ###### Nomenclature. -------------------------------- --------------------------------------------------------------------------------- Central methanogenic pathway Fmd Formylmethanofuran (CHO-MFR) dehydrogenase Ftr Formylmethanofuran-tetrahydromethanopterin (H~4~MPT) formyltransferase Mch N^5^,N^10^-methenyl-H~4~MPT cyclohydrolase Mtd F~420~H~2~-dependent methylene -H~4~MPT dehydrogenase Mer N^5^,N^10^-methylene-H~4~MPT reductase Mtr N^5^-methyl-H~4~MPT:coenzyme M (CoM) methyltransferase Mcr Methyl coenzyme M (CH~3~-CoM) reductase Mdh Methanol dehydrogenase Fae/Hps Fusion protein of formaldehyde activating enzyme/ hexulose-6-phosphate synthase MetF N^5^,N^10^-methylene tetrahydrofolate (H~4~HF) reductase analogue Electron transport Mvh F~420~-nonreducing hydrogenase Vho Methanophenazine-dependent hydrogenase Fpo F~420~H~2~:phenazine oxidoreductase Fqo F~420~H~2~:quinone oxidoreductase Hdr Coenzyme B-coenzyme M heterodisulfide (CoB-S-S-CoM) reductase Frh F~420~-dependent hydrogenase Ech Ferredoxin-dependent hydrogenase MePh/MePhH~2~ Methanophenazine MQ/MQH~2~ Menaquinone Cyt b Cytochrome b Cyt c Cytochrome c MHC Multiheme c-type cytochrome Rieske Rieske cytochrome b complex Orf7 Pseudoperiplasmic b-type cytochrome Nar Nitrate reductase Nap Periplasmic nitrate reductase Nrf Nitrite reductase -------------------------------- --------------------------------------------------------------------------------- [^1]: Academic Editor: Michael W. 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"domain", "flavocytochrome", "Shewanella", "Octaheme", "cytochrome", "Methanogenesis", "multiheme", "cytochrome", "Multiheme", "cytochrome", "domains", "family", "duplication", "domain", "Sarcinarray", "family", "protein", "homology", "domain", "Cell", "export", "cell", "adhesion", "HYR", "domain", "CARDB", "domain", "surface", "protein", "domain", "von", "Willebrand", "factor", "type", "VWA", "Adhesion", "lipoprotein", "Adhesin", "B", "fragments", "Putative", "cell", "wall", "binding", "repeat", "Archaeosortase", "Archaeosortase", "B", "Archaeosortase", "C", "domain", "Exosortase", "EpsH", "Exosortase", "Archaeosortase", "family", "protein", "ArtE", "Cell", "wall", "catalytic", "protein", "sorting", "domain", "domain", "archaeal", "signal", "LPXTG", "cell", "wall", "anchor", "domain", "protein", "sorting", "domain", "Cellulosome", "anchoring", "protein", "cohesin", "domain", "Dockerin", "domain", "Dockerin", "type", "repeat", "domains", "domain", "fold", "hydrolysis", "domain", 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Background {#Sec1} ========== It is well recognized that patients with end stage renal disease (ESRD) requiring intermittent hemodialysis (HD) suffer excess cardiac morbidity and mortality \[[@CR1]\]. There are disproportinaltely higher rates of heart failure and cardiac arrhythmia \[[@CR2]\] in this population and cardiovascular disease remains the leading cause of mortaliy. In addition to the traditional cardiovascular risk factors, intra-dialytic hypotension (IDH) may be a key contributor to myocardial injury, and repeated injury during routine dialysis further precipitate progression of cardiovascular deterioration. For instance, previous investigations have found evidence of reduction in myocardial blood flow during dialysis, resulting in ischemia and myocardial stunning. Over time, such repeated injury can lead to ventricular remodeling that may further contribute to hemodynamic instability and dysregulation \[[@CR3], [@CR4]\]. Intra-dialytic hypotension is one of the most common complication of HD treatment, occurring in approximately 20--30% of HD sessions \[[@CR5]\]. Clinically, IDH often leads to premature cessation of dialysis sessions, or decreased fluid removal, both of which not only decrease dialysis efficiency and effect, but also leads to decreased quality of life. Furthermore, IDH has been associated with other medical complications such as vascular access thrombosis, and mesenteric ischemia \[[@CR6]\]. Current treatment strategies for IDH are aimed to address the rate of fluid shift through altering the ultrafiltration rate and dialysate electrolyte concentrations. Vasoconstrictive medications such as midodrine are commonly used with some success, however, effect tends to diminish over time and their safety has been called into question \[[@CR7]\]. Novel strategies are needed to further address this issue. One such approach to the management of IDH is mechanical augmentation via the application of lower limb compression stockings during dialysis \[[@CR8]--[@CR10]\]. These devices increase venous return and contributes to additional intravascular volume, preload, and in turn, cardiac output. In particular, the non-pneumatic anti-shock compression garments have been used since the early 1900s to manage shock \[[@CR11]\]. Benefit has been demonstrated in patients with hypovolemic shock, such as traumatic injury, abdominal aortic aneurysm rupture, and postpartum hemorrhage; and in distributive shock, such as in the setting of anaphylaxis \[[@CR11]\]. In addition to improving cardiac pre-load, when applied to the abdomen, the anti-shock garments may exert hormonal effects via sympathetic stimulation, and further improve blood pressure \[[@CR12]\]. In the dialysis population, the benefit of compression stockings is conflicting. Two studies have examined the benefit of pneumatic compression stockings: a randomized crossover study by Tai et al. did not find significant benefit \[[@CR13]\], while Alvares et al. \[[@CR14]\], also using a randomized crossover design, did find a reduction in IDH with the use of pneumatic compression in the first hour of dialysis. However, the use of non-pneumatic devices remains un-explored. In contrast to pneumatic compression stockings which provide thigh high intermittent compression, non-pneumatic anti-shock compression garments apply continuous pressure to not only the lower limbs but also upper thigh and abdomen. The ZOEX Non-Inflatable Anti-Shock Garment (NASG) is a specific type of anti-shock garment originally developed from NASA anti-G-force technology which received FDA device approval for management of haemorrhagic shock in 1991. They are a lightweight neoprene garment that is made up of six segments that close tightly with Velcro. This garment is worn on the lower extremities (legs) and applies 20--40 mmHg pressure to the lower body and abdomen. The utility of NASG to raise systemic blood pressure is already well established through work done on obstetrical haemorrhages in remote locations. Two independent groups working in Pakistan report a marked elevation in systolic blood pressure within minutes of application of NASG to patient suffering from obstetrical haemorrhage \[[@CR15], [@CR16]\]. Later prospective work by Miller et al. demonstrated a decrease in both blood loss, morbidity and mortality in patients with postpartum bleeding using NASG \[[@CR17]--[@CR19]\]. Furthermore, this work demonstrated that the use of NASG over extended periods is safe. Over a 36-h period of continuous use, no adverse events were reported in the study group \[[@CR17]--[@CR19]\]. The immediacy in which the Zoex NASG modulates hemodynamic variables combined with the lack of adverse effects even over extended use makes this device promising for use in the dialysis setting. The proven benefit in other settings, the relatively low cost, and the ease of application, make these devices a worthwhile option for further exploration. We therefore conducted this pilot study to examine the effects of non-pneumatic compression garments on individuals with varying degree of renal dysfunction. Through this small and focused trial. We aim 1) to assess the tolerability of these garments, 2) to examine the hemodynamic effects of these compression garments through continuous hemodynamic monitoring using the Finapres technology, and 3) to assess the feasibility for potential larger scale clinical trials. Methods {#Sec2} ======= Study design {#Sec3} ------------ In this prospective, interventional, pilot study, non-pneumatic anti-shock compression garments were applied to individuals with varying degrees of renal disease. Three groups of participants were analyzed: 1) Healthy participants with no known kidney disease which served as a control group, 2) participants with stage 4 or 5 chronic kidney disease (CKD) not receiving dialysis, and 3) individuals with end stage renal disease currently receiving intermittent hemodialysis. Participants {#Sec4} ------------ Five patients were recruited for each arm of the study. Healthy participants were recruited via poster board advertisement at London Health Science Centre (LHSC), Victoria Campus, London, Ontario, while CDK patients were recruited from the LHSC CKD clinic based at a community dialysis site. Dialysis patients were recruited from the Victoria Hospital dialysis unit population, also at LHSC. Inclusion and exclusion criteria {#Sec5} -------------------------------- All participants were required to be over the age of 18 and were of both sexes. Healthy participants had no clinical diagnosis of CKD. Non-dialysis CKD patients were required to have diagnosed stage 4 or 5 CKD but were not receiving and had never had any form of dialysis. Dialysis patients were required to be undergoing active intermittent hemodialysis at the time of the study protocol. Participants were excluded if they had New York Heart Association Class IV heart failure, absence of lower limbs or lower limb injury, or individuals with established peripheral vascular disease or clear symptoms of claudication. Additionally, patients who declined participation, were pregnant, or did not meet inclusion criteria were excluded. Compression garments {#Sec6} -------------------- ZOEX Non-Inflatable Anti-Shock Garment (NASG), supplied by ZOEX NIASC, Coloma, CA, USA were used for all participants of this study. They are a compression garment consisting of 6 segments that close tightly with Velcro that applies a continuous pressure of 20--40 mmHg to the lower limbs and abdomen. Finometer {#Sec7} --------- The use of the Finometer (Finapres Medical Systems, Arnhem, Netherlands) in dialysis patients has been described in detail elsewhere \[[@CR20]\] and was used to monitor a variety of hemodynamic parameters, including cardiac output and mean arterial pressure, during the study protocol. Study protocol {#Sec8} -------------- On the day of the study visit, participants were subjected to a basic history review and a baseline set of routine vitals were taken to calibrate the Finometer. Patients were weighed, and their current weight was compared against their ideal weight as an estimate of fluid balance (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Participants were laid supine on an examination table with the Finometer attached and without compression garments on. Baseline data for each participant was collected for a total of 15 min. Five minutes into the session a baseline echocardiogram was performed. Following this first interval, participants donned the compression garments and were again laid supine on an examination table. Data was again collected for 15 continuous minutes with an echocardiogram being performed 5 min after application of the compression garments. For dialysis patients, the study visit occurred on a day they would normally receive dialysis, but before their dialysis session. Regarding statistical analysis {#Sec9} ------------------------------ This pilot study was designed to characterize the hemodynamic of patients with varying degrees of renal dysfunction in the presence and absence of lower body compression. This study was not sufficiently powered to show an effect on cardiac output or mean arterial pressure with the application compression garments and as such a statistical analysis to that end was not performed. Descriptive and summary statistics were computed with Microsoft Excel 2018. Results {#Sec10} ======= Patients {#Sec11} -------- Baseline characteristics for study participants are presented in Table [1](#Tab1){ref-type="table"}. Significant heterogeneity was observed between individuals within groups, particularly with regards to their medications and comorbidities. Healthy participants were significantly younger than both CKD and dialysis participants and represent a cardiovascularly healthy optimal control. Table 1Baseline characteristics for study participants, each arm contained five patientsGroupDialysis (n = 5)CKD (n = 5)Healthy (n = 5)Average Age66.374.446.8Causes for CKD (n) Diabetes Mellitus41N/ABaseline SBP (mmHg)136.5155.8135.8Baseline DBP (mmHg)83.581.488.4Baseline HR (bmp)94.57372.6Medications (n) Beta-Blocker120 Calcium Channel Blocker130 Diuretic020 ACE inhibitor/ARB010 MRA010Cardiovascular Comorbidities (n) CAD100 Hypertension440 Atrial Fibrillation400Note: CKD Chronic kidney disease, SBP Systolic blood pressure, DBP Diastolic blood pressure, HR Heart rate, ACE Angiotensin converting enzyme, ARB Angiotensin receptor blocker, MRA Mineralocorticoid receptor antagonist, CAD Coronary artery disease Hemodynamic response {#Sec12} -------------------- Figure [1](#Fig1){ref-type="fig"} illustrates the continuous cardiac output of a representative individual from each group over the course of the study procedure. Mild fluctuations in cardiac output are seen throughout in each patient, however a stable baseline is readily apparent. No obvious or reproducible changes in cardiac output were observed over the 15-min interval in which the compression garments were worn, indicating that any hemodynamic effect caused by application of the garments was immediate and sustained. Similar scatter plots were generated for each study participant for several hemodynamic parameters. Individual responses to the compression garments were quite variable (Fig. [2](#Fig2){ref-type="fig"}, Table [2](#Tab2){ref-type="table"}) and no clear or significant trend was observed in any group with the exception of CKD participants who uniformly experienced an increase in mean arterial pressure when wearing the compression garments. Fig. 1Representative data series of cardiac output from individual patients of each cohort. Baseline characteristics in approximately 15-min intervals displayed in orange. Application of non-pneumatic compression stocks occurred subsequently, and hemodynamic response is shown in blue Fig. 2Hemodynamic response of patients with kidney disease to non-pneumatic compression stockings. The relative change in cardiac output, mean arterial pressure and global longitudinal strain after the application of non-pneumatic compression stockings in heathy individuals, CKD patients not on dialysis, and dialysis patients are displayed. Cardiac output and mean arterial pressure data were collected via Finometer while global longitudinal strain was obtained via echocardiography Table 2Hemodynamic response to non-pneumatic compression stockings. Hemodynamic parameters from healthy individuals (H), CKD patients (C) and participants receiving hemodialysis (D) are displayed. Data is related as a percent change from baseline (without compression garments)GroupHeart RateStroke VolumeCardiac OutputPeripheral ResistanceSystolic PressureMean Arterial PressureDiastolic PressureH1−0.9%−1.7%−2.7%5.8%1.0%1.3%2.7%H2−3.3%20.2%16.8%−13.1%9.6%6.1%2.7%H31.1%−1.5%2.5%4.7%1.5%0.4%0.5%H4−8.4%−3.2%−4.1%14.5%3.2%4.3%6.1%H54.2%−13.0%−9.4%21.8%2.8%5.3%8.1%Mean−1.5%0.1%0.6%6.7%3.6%3.5%4.0%St. Dev.4.8%12.0%10.0%13.0%3.0%3.0%3.0%C18.2%−10.6%−8.8%17.8%6.2%5.6%8.6%C2−0.5%−3.2%−3.0%11.6%3.5%5.4%4.9%C3−1.0%−19.8%−20.6%47.1%−2.3%5.5%11.4%C414.5%−16.5%−12.4%43.8%8.9%15.9%16.1%C54.4%16.0%21.3%−11.1%15.1%10.1%5.5%Mean5.1%−6.8%−4.7%21.8%6.3%8.5%9.3%St. Dev.6.5%14.2%15.9%24.1%6.4%4.6%4.6%D1−3.0%11.4%8.0%8.6%21.0%15.7%14.1%D2−3.4%3.0%1.7%10.4%11.5%13.1%9.0%D3−3.0%−7.8%−12.0%0.1%−16.9%−12.3%−9.9%D4−3.8%17.7%19.2%−19.2%4.6%3.4%2.7%D58.4%−15.7%−9.1%−12.5%−22.4%− 17.8%−15.9%Mean−0.9%1.7%1.5%−2.5%−0.4%0.4%0.0%St. Dev.5.3%13.6%12.7%13.0%18.6%15.0%12.6% Discussion {#Sec13} ========== In this pilot study we assessed the tolerability and hemodynamic response of a small group of patients with varying degrees of kidney disease to the application of non-pneumatic anti-shock compression garments. In general, the compression garments were well tolerated by all study participants, however, individuals with pre-existing peripheral vascular disease were noted to most likely decline participation in the trial. We performed continuous hemodynamic monitoring via the Finapres system and obtained multiple cardiac parameters. Interestingly, despite complete and robust data collection from each patient, no consistent trend was observed within or between groups of varying renal function. However, there seem to be greater degree of variability in the CKD and HD groups, as compared to the healthy group. The cause of the variability in response to compression garments is likely multifactorial. In this small scale study, there is significant heterogeneity in participant's characteristics. Underlying medical comorbidity, cardiovascular health, and hemodynamic modifying mediations are among some of the factors that have likely contributed to the response of each individual in our study. For instance, in the HD group, the experiments were conducted prior to dialysis sessions for ease of participant scheduling. As a result, these participants may be relatively hypervolemic, and augmentation of preload in this state may have diminished benefit, or even detrimental effect. Therefore, the lack of consistent response may simply reflect the variable fluid status in these individuals. Patient fluid balance as estimated by change from the patient's ideal weight (Additional file [1](#MOESM1){ref-type="media"}: Table S1) did not appear to be related to the hemodynamic response observed. Furthermore, the higher degree of variability between individuals was not unexpected as the increase in pre-load may be substantial, and different responses may reflect underlying cardiovascular fitness and right heart function. In comparison between groups, our qualitative observation of higher degrees of hemodynamic variability in the two groups with renal dysfunction may be at least partly explained by impaired autonomic regulation, and inability to respond to the increased pre-load in these populations. Another source of variability is the concurrent use of beta blockers or renin-aldosterone system blockade agents which may further impair this regulation system \[[@CR21]\], leading to additional sources of variability. In light of our data, large scale studies such as the study by Tai et al. may not observe a significant benefit statistically as the underlying variability due to patient heterogeneity may dilute the true effect. As such, study of generalized application of these compression stockings may not be the best approach. Our study highlights the need for further characterization of factors that would predict beneficial response. For instance, compression garments may be more suitable to individuals with certain known echocardiographic features, or application of these garments may be reserved on an as-needed basis, rather than universal application. However, in clinical setting, this may be difficult, as clinical variables such as intravascular volume status is difficult to determine accurately, especially since this variability naturally exist between individuals and within each individual across time. Therefore, such tailored but restricted approach may not be pragmatic in a clinical setting. Our study is limited in its small size, and descriptive in nature. However, our protocol reflected the pragmatic approach that would be applied in a large scaled study, and the smaller sample size allowed for mechanistic assessment of the intervention. Despite excellent tolerability, we found significant variation in hemodynamic response. This inconsistency calls into questions the likely utility of universal application of our compression garments to patients with renal dysfunction and informs that large scale study based on similar a protocol will likely result in statistically negative trial. Our results provide mechanistic insights into the use of these garments and poses questions for future research. Conclusions {#Sec14} =========== Non-pneumatic anti-shock compression garments inexpensive and readily available devices that are tolerable and appear feasible to utilize in the CKD and dialysis setting from a practical perspective. We have also highlighted the significant heterogeneity of hemodynamic responses to lower body compression in patients with renal dysfunction, which may explain previous studies' failure to show benefit with generalized application of these garments. Further study is warranted to refine patient selection and identify candidate who would be most likely to receive benefit from nonpneumatic compression garments and we remain optimistic for novel application methods of these devices. Supplementary information ========================= {#Sec15} Additional file 1: Table S1. Study participants fluid balance as estimated by change from ideal weight. CKD participants (C) and dialysis participants (D) were weighted on the day of study. This weight was compared to their ideal weight, obtained from their recent nephrology clinic notes. The difference between study weight and ideal weight is intended to provide an estimate to the fluid balance of the study participant on the day of study. ACE : Angiotensin converting enzyme ARB : Angiotensin receptor blocker CAD : Coronary artery disease CKD : Chronic kidney disease DBP : Diastolic blood pressure ESRD : End stage renal disease HD : Hemodialysis HR : Heart rate IDH : Intra-dialytic hypotension LHSC : London Health Science Centre MRA : Mineralocorticoid receptor antagonist NASG : Non-inflatable anti-shock garment SBP : Systolic blood pressure Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. R. Marinovich and Z. Li are co-first authors. Supplementary information ========================= Supplementary information accompanies this paper at 10.1186/s12882-019-1680-8. Not applicable. RM and ZL with the literature review, organized the study, recruited participants, were responsible of executing the study protocol as well as data interpretation, analysis, manuscript preparation and submission. TT was involved with experimental design, execution of the study protocol and data collection and interpretation. KQ, and SW were involved with the literature review, experimental design and bringing the study through the approval process. CM is credited for the original research question and acted as supervising principal investigator for this study. He was involved the literature review, experimental design, data analysis and manuscript preparation. All authors have read and approved the final manuscript. Funding requirements were minimal for this study, as most materials used were already available to our research group. Purchase of the ZOEX NASG was supported by departmental funds. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. This trial was reviewed and approved by the University of Western Ontario Health Sciences Research Ethics Board prior to initiation of any study related procedures. Written, informed consent was obtained from all participants in this trial. All participants in this study have provided written, informed consent that their data will be made available for publication. 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Introduction {#s1} ============ Prior to the emergence of the highly pathogenic severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in 2003 [@ppat.1004166-Drosten1]--[@ppat.1004166-Peiris1] only two circulating human coronaviruses (HCoVs), HCoV-229E [@ppat.1004166-Hamre1] and HCoV-OC43 [@ppat.1004166-McIntosh1] causing relatively mild common cold-like respiratory tract infections, were known, and coronaviruses have not been regarded as significant threat for human health. Now, more than ten years later, the emergence of another highly pathogenic coronavirus of zoonotic origin, the Middle East respiratory syndrome coronavirus (MERS-CoV) [@ppat.1004166-Bermingham1]--[@ppat.1004166-Zaki1], boosted community awareness towards the pending need to develop effective therapeutic options to combat coronavirus infections. Coronaviruses are enveloped viruses and their positive strand RNA genome, the largest of all RNA viruses, encodes for as many as 16 non-structural proteins (nsps), 4 major structural proteins, and up to 8 accessory proteins (reviewed in [@ppat.1004166-Perlman1]). Many of these proteins provide essential, frequently enzymatic, functions during the viral life cycle and are therefore attractive targets for antiviral intervention. Antiviral strategies are mainly proposed for targeting coronavirus entry and essential enzymatic functions, such as coronavirus protease or RNA-dependent RNA polymerase (RdRp) activities. For example, the spike (S) protein mediates binding of different HCoVs to their specific cellular receptors [@ppat.1004166-Yeager1]--[@ppat.1004166-Raj1], an event associated with preferential virus tropism for either ciliated or non-ciliated cells of the airway epithelium [@ppat.1004166-Dijkman1]. The S protein also mediates fusion between lipids of the viral envelope and the host cell plasma membrane or membranes of endocytic vesicles to promote delivery of viral genomic RNA into the cytoplasm. Virus binding and cell entry events can be inhibited by antibodies directed against the S protein, antibodies or small molecules interfering with the virus receptors, or synthetic peptides derived from the fusion-triggering heptad repeat regions of the S protein (reviewed in [@ppat.1004166-Cinatl1]). Following virus entry, the coronavirus genome, a positive sense, capped and polyadenylated RNA strand, is directly translated resulting in the synthesis of coronavirus replicase gene-encoded nsps. Coronavirus nsps are translated as two large polyproteins harboring proteolytic enzymes, namely papain-like and chymotrypsin-like proteinases that extensively process coronavirus polyproteins to liberate up to 16 nsps (nsp 1--16) [@ppat.1004166-Perlman1], [@ppat.1004166-Gorbalenya1]--[@ppat.1004166-Ziebuhr2]. These proteolytic functions are considered essential for coronavirus replication and, consequently, a number of candidate drugs were reported to inhibit coronavirus polyprotein processing [@ppat.1004166-Anand1]--[@ppat.1004166-Anand2]. Likewise, the coronavirus RdRp activities, which reside in nsp8 [@ppat.1004166-Imbert1] and nsp12 [@ppat.1004166-Gorbalenya2], are considered essential for coronavirus replication and attractive targets for antiviral intervention. In addition to these classical drug targets, coronaviruses encode an array of RNA-processing enzymes representing additional candidate targets. These include a helicase activity linked to an NTPase activity in nsp13, a 3′-5′-exonuclease activity linked to a N7-methyltransferase activity in nsp14, an endonuclease activity in nsp15, and a 2′-O-methyltransferase activity in nsp16 (reviewed in [@ppat.1004166-Gorbalenya2]). Like all positive strand RNA viruses, coronaviruses synthesize viral RNA at organelle-like structures in order to compartmentalize this critical step of the viral life cycle to a specialized environment that is enriched in replicative viral and host-cell factors, and at the same time protected from antiviral host defense mechanisms [@ppat.1004166-Ahlquist1]--[@ppat.1004166-Overby1]. There is now a growing body of knowledge concerning the involvement, rearrangement and requirement of cellular membranes for RNA synthesis of a number of positive-strand RNA viruses, including coronaviruses [@ppat.1004166-denBoon1], [@ppat.1004166-Knoops1]--[@ppat.1004166-Welsch1]. Three coronaviral nsps, i.e., nsp3, nsp4, and nsp6 [@ppat.1004166-Perlman1], [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1] are thought to participate in formation of these sites for viral RNA synthesis. In particular, these proteins contain multiple trans-membrane domains that are thought to anchor the coronavirus replication complex through recruitment of intracellular membranes to form a reticulovesicular network (RVN) of modified, frequently paired, membranes that includes convoluted membranes [@ppat.1004166-Knoops1] and double membrane vesicles (DVM) [@ppat.1004166-Gosert1] interconnected via the outer membrane with the rough ER [@ppat.1004166-Knoops1]. Indeed, Angelini and colleagues [@ppat.1004166-Angelini1] have recently shown that co-expression of all three transmembrane domain-containing SARS-CoV nsps (nsp3, nsp4, and nsp6) is required to induce DMVs that are similar to those observed in SARS-CoV-infected cells. Such organelle-like compartments harboring membrane-bound replication complexes show remarkable parallels amongst a broad range of positive-strand RNA virus families, and are potentially evolutionary linked to similar mechanisms in the life cycle of double-strand (ds)RNA, reverse-transcribing, and cytoplasmic replicating DNA viruses [@ppat.1004166-Ahlquist1]. Coronavirus ER-derived DMVs are induced early after virus entry into the host cell cytoplasm [@ppat.1004166-Perlman1], [@ppat.1004166-Knoops1], [@ppat.1004166-Ulasli1], [@ppat.1004166-Gosert1]--[@ppat.1004166-Hagemeijer1], and display striking similarities to DMVs induced by hepatitis C virus [@ppat.1004166-RomeroBrey1]. The evolutionary conservation of engaging host cell-derived organelle-like membranous structures for virus RNA synthesis and genetic evidence that impairment of coronavirus DMV integrity is associated with severe reduction of virus replication [@ppat.1004166-Clementz1], [@ppat.1004166-Gadlage1] suggest that antiviral intervention by targeting membranes involved in virus replication represents an attractive, however yet largely unexplored approach. In this work, we describe a novel inhibitor of coronavirus replication that specifically interferes with membrane-bound coronaviral RNA synthesis. This novel mode-of-action is characterized by severe impairment of DMV formation that results in near-complete inhibition of RNA synthesis. Notably, the inhibitor displayed antiviral activity against a broad range of animal and human coronaviruses, including the recently emerging MERS-CoV. Results {#s2} ======= Identification of anti-HCoV-229E hit compound K22 {#s2a} ------------------------------------------------- To identify novel inhibitors of coronavirus infectivity we screened the ChemBioNet collection of 16671 compounds for antiviral activity against HCoV-229E. To this end, MRC-5 cells growing on 384-well plates were supplemented with a specific library compound (20 µM) and then inoculated with HCoV-229E. Compounds that reduced or abolished viral cytopathic effect were re-tested in 24-well plate format for more precise evaluation of their antiviral potential. This two-step screening procedure resulted in several hits including two structurally similar compounds referred to as K22 ([Figure 1A](#ppat-1004166-g001){ref-type="fig"}) and J15 ([Figure S1A](#ppat.1004166.s001){ref-type="supplementary-material"}). The former compound, K22, whose structural name is (Z)-N-(3-(4-(4-bromophenyl)-4-hydroxypiperidin-1-yl)-3-oxo-1-phenylprop-1-en-2-yl)benzamide was examined in detail. The compound was completely soluble in medium up to 50 µM. The concentration of K22 that inhibited the number of HCoV-229E plaques by 50% (IC~50~) was 0.7 µM ([Figure 1B](#ppat-1004166-g001){ref-type="fig"}). K22 did not reduce viability of MRC-5 cells by \>50% (CC~50~) at a concentration range of 0.032--500 µM ([Figure 1C](#ppat-1004166-g001){ref-type="fig"}). However this compound decreased proliferation of MRC-5 cells with a CC~50~ value of 110 µM ([Figure 1C](#ppat-1004166-g001){ref-type="fig"}). Hence, using the CC~50~ value determined in cell proliferation assay, the selective index for K22, i.e. the CC~50~/IC~50~ quotient, was 157. Compound J15, although showing anti-HCoV-229E activity similar to that of K22 exhibited a somewhat less favorable cytotoxicity profile in the cell viability assay ([Figure S1B](#ppat.1004166.s001){ref-type="supplementary-material"}). ![K22 structure, antiviral activity, and cytotoxicity.\ (A) K22 structure. (B) Anti-HCoV-229E activity of K22 in MRC-5 cells. K22 and HCoV-229E were added to MRC-5 cells, and the number of viral plaques developed after 48 h were assessed. Data shown are means (±SD) of duplicate determinations from three independent experiments. PFU, plaque forming unit. (C) Viability and proliferation of MRC-5 cells in the presence of K22. MRC-5 cells were incubated with K22 or DMSO solvent for 48 h at 37°C and the cell viability determined using tetrazolium-based reagent while cell proliferation was assayed by counting of cells. Data shown are means (±SD) of duplicate determinations from two independent experiments. (D) K22 affects the post-entry phase of viral life cycle. K22 (4 µM) or DMSO solvent were incubated with cells for a period of 2 h either before (−2 h), during (0 h) or after a 2 h period of cell inoculation with HCoV-229E, and the number of viral plaques developed after 48 h were assessed. Data shown are means of duplicate determinations from three independent experiments.\*P\<0.05; n = 3. \\\*P\<0.005; n = 3. (E-F) K22 exhibits potent antiviral activity when added up to 6 h after infection of cells. MRC-5 cells were inoculated with HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) added at specific time points relative to the end of inoculation period. The culture medium and cells were harvested after 24 h of incubation at 37°C, and the viral RNA (E) and infectivity (F) determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. EX, extracellular medium; CA, cell-associated sample.](ppat.1004166.g001){#ppat-1004166-g001} K22 inhibits HCoV-229E during the early, post entry phase of the viral life cycle {#s2b} --------------------------------------------------------------------------------- To assess which step of the HCoV-229E life cycle is affected by K22, a time-of-addition/removal experiment was performed. K22 (4 µM) was incubated with cells for a period of only two hours either prior to, during, or after infection with HCoV-229E. As shown in [Figure 1D, K](#ppat-1004166-g001){ref-type="fig"}22 treatment prior to infection resulted in only marginal reduction of virus infectivity thus excluding blockade of cellular receptor(s) for HCoV-229E as its mode-of-action. Simultaneous addition of K22 and virus resulted in ∼50% reduction of virus infectivity suggesting that the compound may interact with viral particles thus inactivating their binding or cell-entry activity. To clarify this possibility, the virus was incubated with ∼70 IC~50~ doses of K22 or DMSO solvent for 15 min at 37°C, followed by virus dilution and its titration at non-inhibitory compound concentrations. Similar titers were observed for the virus treated with K22 (7.2×10^5^/ml±8.9%) and DMSO (7.5×10^5^/ml±4.7%) (n = 2; two experiments), indicating that K22 exhibited no virus particle-inactivating activity. Thus, the ∼50% reduction in plaque number ([Figure 1D](#ppat-1004166-g001){ref-type="fig"}) observed by simultaneous addition of K22 and virus is likely due to cellular uptake of K22 and inhibitory activity of probably not yet metabolically processed compound during a very early step of virus replication rather than the drug binding to viral particles and interference with their penetration into cells. This idea is further corroborated by the most pronounced inhibition of HCoV-229E replication when K22 was added after infection ([Figure 1D](#ppat-1004166-g001){ref-type="fig"}). To more precisely determine the time window of efficient K22-mediated inhibition of HCoV-229E, K22 (10 µM) was added to infected cells at different time points post infection (p.i.), and intra- and extracellular viral RNA, and infectious particles were quantified at 24 hours p.i.. As shown in [Figures 1E-F, K](#ppat-1004166-g001){ref-type="fig"}22 addition within the first 6 hours p.i. resulted in near complete inhibition of viral RNA synthesis and ∼1000-fold reduction of produced infectious virus, suggesting that K22 inhibits most potently post virus entry during the early phase of the HCoV-229E life cycle. K22 resistant mutants contain substitutions in nsp6 {#s2c} --------------------------------------------------- To obtain further insight concerning the target of K22 inhibition we aimed to generate K22-resistant mutants and therefore subjected plaque purified HCoV-229E to 10--13 consecutive passages on MRC-5 cells in presence of increasing concentrations of K22 (2--16 µM). In two independent experiments we isolated and plaque purified several variants displaying moderate (∼2-fold) to strong (∼12-fold) K22 resistance (IC~50~ of 1.6--8.5 µM; [Table 1](#ppat-1004166-t001){ref-type="table"}). Whole genome sequencing analysis of wild type (wt) HCoV-229E, mock passaged virus, and K22 passaged virus revealed two amino acid substitutions within nsp 6 (H121L; M159V) that were associated with strong K22 resistance ([Table 1](#ppat-1004166-t001){ref-type="table"}). Sequence alignment and prediction of potential transmembrane regions of nsp6 homologs of HCoV-229E and other coronaviruses used in this study, revealed presence of 7 potential membrane-spanning domains ([Figure 2](#ppat-1004166-g002){ref-type="fig"}) 6 of which are proposed to be used as membrane anchors in other coronaviruses [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1], and that mutations conferring resistance to K22 are located in or near these regions ([Figure 3A](#ppat-1004166-g003){ref-type="fig"}). Subsequent generation of recombinant mutants, designated HCoV-229E^H121L^, HCoV-229E^M159V^, and HCoV-229E^H121L/M159V^, carrying the nsp6 mutations individually or combined by reverse genetics confirmed that these mutations confer resistance to K22 inhibition as revealed by plaque inhibition ([Table 1](#ppat-1004166-t001){ref-type="table"}) and the time-of-addition ([Figures 3B-C](#ppat-1004166-g003){ref-type="fig"}) assays. Thus, as expected from the previous experiment ([Figure 1E](#ppat-1004166-g001){ref-type="fig"}), K22 addition within the first 6 hours p.i. with the wt HCoV-229E resulted in near complete inhibition of viral RNA synthesis ([Figure 3C](#ppat-1004166-g003){ref-type="fig"}), an effect completely abrogated in the drug-resistant recombinant mutant viruses ([Figure 3B](#ppat-1004166-g003){ref-type="fig"}). Notably, although the amount of intracellular ([Figure 3D](#ppat-1004166-g003){ref-type="fig"}) and extracellular ([Figure 3E](#ppat-1004166-g003){ref-type="fig"}) viral RNA was comparable between K22-resistant mutants and parental wt HCoV-229E, production of infectious particles during infection with K22-resistant mutant viruses was greatly reduced (up to 34 fold at 48h p.i.) ([Figure 3F](#ppat-1004166-g003){ref-type="fig"}). This difference cannot be attributed to the presence of free viral RNA in preparations of extracellular virus, since the treatment of K22-resistant HCoV-229E^M159V^ mutant virus with ribonuclease A did not reduce the quantity of viral RNA ([Figure S2](#ppat.1004166.s002){ref-type="supplementary-material"}). This observation suggests that K22 resistance-conferring mutations in nsp6 are associated with a fitness cost (reduced specific infectivity). ![Alignment of coronavirus nsp6 sequences.\ Alignment of nsp6 sequences derived from coronaviruses used in this study was performed with Geneious Software (Biomatters Ltd, New Zealand). Coronavirus species and corresponding GenBank accession numbers are indicated. Membrane domains predicted by TMHMM Server v. 2.0 (<http://www.cbs.dtu.dk/services/TMHMM/>) are indicated by cyan shading while conserved amino acid residues are highlighted by black/grey shading. K22 resistance-conferring mutations in HCoV-229E nsp6, identified in this study, are depicted.](ppat.1004166.g002){#ppat-1004166-g002} ![Analysis of recombinant HCoV-229E nsp6 mutants.\ (A) Predicted topological structure of HCoV-229E nsp6 indicating the location of K22 resistance mutations. Concerning transmembrane domains VI and VII two proposed topologies are shown. (B-C) Recombinant nsp6 mutant viruses are resistant to K22. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L^, HCoV-229E^M159V^, HCoV-229E^H121L/M159V^ or wild-type HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) was added at specific time points relative to the end of inoculation period. The infectious cell culture medium and cells were harvested after 24 h of incubation at 37°C, and copy numbers of cell-associated (CA) or extracellular (EX) viral RNA was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. (D-F) Replication kinetics of recombinant nsp6 mutant viruses. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L^, HCoV-229E^M159V^, HCoV-229E^H121L/M159V^ or wild-type HCoV-229E at an moi of 0.05 for 1 h at 4°C. The infectious cell culture medium and cells were harvested at specific time points relative to the end of inoculation period, and copy numbers of cell-associated (CA; D) or extracellular (EX; E) viral RNA and infectivity (F) was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments.](ppat.1004166.g003){#ppat-1004166-g003} 10.1371/journal.ppat.1004166.t001 ###### Alterations detected in the K22 resistant variants of HCoV-229E. ![](ppat.1004166.t001){#ppat-1004166-t001-1} Alteration[a](#nt101){ref-type="table-fn"} ------------------------------------------------------- -------------------------------------------- ---------------------- ------------------------------------------ ---------- Initial[b](#nt102){ref-type="table-fn"} None None 0.7[c](#nt103){ref-type="table-fn"} KF293664 K22 passage 10 a10455t H121L (Nsp6) 9.8 (14)[d](#nt104){ref-type="table-fn"} KF293666 c19463t T281I (Nsp15) c26667t P328S (Nucleocapsid) A a10455t H121L (Nsp6) 8.2 (12) KF285470 B a10455t H121L (Nsp6) 8.2 (12) KF285471 D a10455t H121L (Nsp6) 7.6 (11) KF285472 G a10455t H121L (Nsp6) 6.9 (10) KF285473 K c19463t T281I (Nsp15) 1.6 (2) KF285481 c26667t P328S (Nucleocapsid) KF293662 L c19463t T281I (Nsp15) 2.2 (3) KF285482 c26667t P328S (Nucleocapsid) KF293663 K22 passage 13 - M[e](#nt105){ref-type="table-fn"} a10568g M159V (Nsp6) 6.7 (10) KF285474 a23130c N854T (Spike) KF285480 N a10568g M159V (Nsp6) 7.1 (10) KF285475 O a10568g M159V (Nsp6) 7.7 (11) KF285476 P a10568g M159V (Nsp6) 8.5 (12) KF285477 Q a10568g M159V (Nsp6) 7.7 (11) KF285478 R a10568g M159V (Nsp6) 6.8 (10) KF285479 HCoV-229E[f](#nt106){ref-type="table-fn"} 0.6 [g](#nt107){ref-type="table-fn"}HCoV-229E^H121L^ a10455t H121L (Nsp6) 7.2 (12) [g](#nt107){ref-type="table-fn"}HCoV-229E^M159V^ a10568g M159V (Nsp6) 6.3 (11) [g](#nt107){ref-type="table-fn"}HCoV229E^H121L/M159V^ a10455t H121L (Nsp6) 8.2 (14) a10568g M159V (Nsp6) Detected by comparison of the nucleotide sequences of HCoV-229E subjected to 10--13 passages in the presence of K22 including its plaque purified variants A-R with those of initial virus or mock-passaged virus (accession number KF293665). Plaque purified HCoV-229E that served as initial material for the virus passages. IC50 (µM). Fold resistance to K22 as related to initial virus is shown in parentheses. Virus preparation and its plaque purified variants M-R obtained in separate K22 selection experiment. The virus used for preparation of recombinant nsp6 mutants. K22 resistant recombinant viruses. K22 treatment results in loss of DMVs {#s2d} ------------------------------------- The observation that amino acid substitutions in nsp6 confer K22 resistance strongly suggests a mode-of-action based on interference with host cell membranes required for coronavirus replication. Nsp6 is expressed as a membrane-spanning integral component of the viral replication complex, and is, together with nsp3 and nsp4, implicated in anchoring the coronavirus replicase complex to DMVs or related membrane structures [@ppat.1004166-Perlman1], [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1], [@ppat.1004166-Angelini1], [@ppat.1004166-Hagemeijer1]. Indeed, there is genetic and experimental evidence concerning nsp4-mediated alterations of coronavirus DMVs [@ppat.1004166-Clementz1], [@ppat.1004166-Gadlage1], and that ectopic expression of nsp6 results in the formation of ER-derived vesicles [@ppat.1004166-Cottam1]. We therefore assessed if K22 may impact the formation of coronavirus-induced DMV by electron microscopy ([Figure 4](#ppat-1004166-g004){ref-type="fig"}). As expected, perinuclear DMV clusters as well as viral particles were readily detectable in wt HCoV-229E-infected cells ([Figure 4A](#ppat-1004166-g004){ref-type="fig"}). In sharp contrast, no DMV clusters or viral particles were detectable in wt HCoV-229E-infected and K22-treated (4 µM) cells ([Figure 4A](#ppat-1004166-g004){ref-type="fig"}). Since double-stranded (ds) RNA is indicative of coronavirus replication and has been shown to reside predominantly within the inner lumen of coronavirus-induced DMVs [@ppat.1004166-Knoops1] we also performed immunofluorescence analysis and stained HCoV-229E-infected cells for viral replicase complex (nsp8) and dsRNA. Strikingly, the characteristic perinuclear immunofluorescence staining pattern for viral replicase complexes and dsRNA visible in wt HCoV-229E-infected cells was completely absent under K22 treatment ([Figure 5](#ppat-1004166-g005){ref-type="fig"}), confirming the remarkable efficacy of K22-mediated inhibition of viral replication and supporting the notion that K22 blocks the formation of DMVs. In contrast to parental wt HCoV-229E and irrespectively whether K22 was applied, recombinant K22 escape mutants were still capable of inducing the formation of DMVs ([Figure 4B](#ppat-1004166-g004){ref-type="fig"}) and displayed the characteristic staining pattern for replicase complexes and dsRNA ([Figure 5](#ppat-1004166-g005){ref-type="fig"}). Likewise, compound J15 efficiently blocked replication ([Figure S1B](#ppat.1004166.s001){ref-type="supplementary-material"}) and DMV formation of wt HCoV-229E but not K22 resistant nsp6 recombinant HCoV-229E^M159V^ ([Figure S3](#ppat.1004166.s003){ref-type="supplementary-material"}) suggesting that J15 may have the same target and mode-of-action. Notably, in cells infected with K22 escape mutants the overall number of DMVs per cell was reduced (30.3±29.7 in HCoV-229E^M159V^ versus 65±50.1 in wt HCoV-229E infected cells; P\<0.05; n = 20), similar as previously described for mouse hepatitis virus (MHV) nsp4 mutants [@ppat.1004166-Clementz1], [@ppat.1004166-Gadlage1], while the number of intracellular viral particles that were often packed in tubular vesicle-like structures ([Figures 4A-B](#ppat-1004166-g004){ref-type="fig"}) was comparable to that of wt virus (471.8±212.6 in HCoV-229E^M159V^ versus 438.3±96.8 in wt virus infected cells; n = 10). We could also frequently detect DMVs displaying partially collapsed inner membranes in cells infected with K22 escape mutants (irrespectively whether or not K22 was applied; [Figure 4B](#ppat-1004166-g004){ref-type="fig"}), again similarly as reported for MHV nsp4 mutants [@ppat.1004166-Gadlage1], suggesting that nsp6, like nsp4, has a pivotal role in coronavirus DMV formation. Overall, these findings demonstrate that the antiviral activity of K22 (and that of the structurally similar compound J15) results in complete loss of DMVs. This efficient block in replication can be overcome by resistance mutations in nsp6, and DMVs induced by nsp6 mutant viruses are reduced in numbers and structurally impaired -- both findings concurring with the established function of nsp6 in DMV formation. ![K22 affects formation of double membrane vesicles (DMVs).\ MRC-5 cells growing on Melinex polyester film were infected with wild type HCoV-229E (WT) or with K22-resistant recombinant nsp6 mutant HCoV-229E^M159V^ (M159V) and incubated for 18 h at 37°C with or without K22. The cells were then fixed with glutaraldehyde and processed for electron microscopy without their scrapping or pelleting. (A) Electron micrographs of cells infected with WT virus show presence of perinuclear clusters of DMVs (arrow) and viral particles (arrowhead), and the lack of their production upon K22 treatment (4 µM). (B) Note presence of DMVs and viral particles in cells infected with K22-resistant nsp6 recombinant HCoV-229E^M159V^ (M159V) irrespective of the addition of K22. Each image shown was selected from a pool of over 30 images captured in three separate experiments.](ppat.1004166.g004){#ppat-1004166-g004} ![K22 affects formation of coronavirus replication complex in cells.\ MRC-5 cells were infected with wild type HCoV-229E (WT) and K22-resistant recombinants HCoV-229E^H121L^ (H121L), HCoV-229E^M159V^ (M159V), and HCoV-229E^H121L/M159V^ (H121L/M159V) and incubated for 18 h with or without the presence of K22. The cells were then fixed with 4% paraformaldehyde and immunostained for immunofluorescence analysis. Note the lack of detection of dsRNA and nsp8 upon K22 treatment (4 µM) of cells infected with WT but not recombinant viruses. Scale bar is 10 µM.](ppat.1004166.g005){#ppat-1004166-g005} K22 does not impact cellular autophagy {#s2e} -------------------------------------- Our data show that K22 targets a very early step in the HCoV-229E life cycle, and the appearance of resistance-conferring mutations in nsp6 suggests that K22 impairs DMV formation. We therefore assessed if K22 treatment may, independent of virus infection, impact autophagy, a cellular process displaying similarities to coronaviral DMV formation. To this end we first transfected Huh7 cells with a plasmid encoding LC3B-GFP in order to trace rapamycin-induced autophagsomes by life imaging. This analysis revealed that three to six hours after adding rapamycin to the culture medium green fluorescent autophagocytic vesicles become apparent, irrespectively if K22 (20 µM) was added or not (data not shown). We corroborated this result by immunofluorescence analysis of Huh7 cells that were stained for endogenous LC3B at six hours post addition of rapamycin. As shown in supplementary [Figure S4](#ppat.1004166.s004){ref-type="supplementary-material"} rapamycin-incuced autophagocytic vesicles were again readily detectable in the presence of K22 (20 µM), suggesting that K22 does not impact cellular autophagy. K22 inhibits a number of diverse coronaviruses {#s2f} ---------------------------------------------- Since K22 inhibits a crucial step in the HCoV-229E life cycle, we assessed the antiviral activity of K22 against a panel of diverse coronaviruses representing the major phylogenetic lineages of α-, β- and ???-coronaviruses. As shown in [Figure 6A-D](#ppat-1004166-g006){ref-type="fig"} and supplementary [Figure S5](#ppat.1004166.s005){ref-type="supplementary-material"}, K22 indeed displayed antiviral activity against recombinant MHV (strain A59 [@ppat.1004166-Coley1]) expressing Gaussia luciferase as marker for virus replication, recombinant type-I feline coronavirus (FCoV; strain Black [@ppat.1004166-Tekes1]) expressing Renilla luciferase as marker for virus replication, avian infectious bronchitis virus (IBV; strain Beaudette [@ppat.1004166-Casais1]), and SARS- CoV (strain Frankfurt-1 [@ppat.1004166-Thiel1]), suggesting that K22 targets a broad range of coronaviruses. Furthermore, there was no cytotoxicity detectable in cells of feline (FCWF cells), murine (L929 cells), and primate (Vero cells) origin in the K22 concentration range assessed, and analysis of K22 cytostatic activities in the cell proliferation assay revealed CC~50~ values ≥40 µM ([Table S1](#ppat.1004166.s007){ref-type="supplementary-material"}), i.e., the highest drug concentration used in antiviral assays. Notably, the efficacy of K22-mediated inhibition varied amongst different coronaviruses, however whether this is related, as in HCoV-229E, to nsp6 function would require generation and analysis of K22 resistant variants for all coronaviruses tested. In contrast, K22 exhibited little or no effect on replication of poliovirus ([Figure S6](#ppat.1004166.s006){ref-type="supplementary-material"}), a pathogen that like coronaviruses induces rearrangement of cellular membranes to assist RNA replication. ![K22 affects replication of diverse coronaviruses including MERS-CoV.\ (A-D) The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 during MHV-Gluc (A), FCoV-RL (B), SARS-CoV (C) and IBV (D) infection on representative continuous cell lines of murine (L-929 cells; A), feline (FCWF cells; B), or primate (Vero cells; C-D) origin. Data are shown as mean (±SD) of a representative experiment, from two independent experiments performed in triplicate. Toxicity values for Vero cells in panels C and D are derived from the same experiments. (E-F). The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 in HCoV-229E-ren (E) and MERS-CoV (F) infected differentiated human airway epithelial (HAE) cultures. Data are shown as mean (±SD) of three independent experiments performed in triplicate (log reduction), or mean (±SD) of a representative experiment, from two independent experiments performed in triplicate (cell viability). (G-H) Immunofluorescence analysis of HAE cultures infected with MERS-CoV in presence or absence of K22 in a representative overview (G, 20x; H, 40x) confocal Z-stack image. Stainings were performed using antibodies directed against (G) dsRNA (green), and DAPI (cell nucleus; blue), and (H) dsRNA, DAPI, β-tubulin (ciliated cells; white), and ZO1 (tight junctions, red). Scale bars are 50 (G) or 20 (H) µm.](ppat.1004166.g006){#ppat-1004166-g006} Inhibition of HCoV-229E and MERS-CoV in primary human airway epithelia cultures {#s2g} ------------------------------------------------------------------------------- Finally, we assessed the efficacy of K22 inhibition in the primary target cells of respiratory virus infection, the human airway epithelium. Fully differentiated primary human airway epithelia (HAE) cultures [@ppat.1004166-Dijkman1], [@ppat.1004166-Kindler1] derived from three different donors and grown under air-liquid interphase conditions were infected with a recombinant HCoV-229E expressing Renilla luciferase as marker for virus replication [@ppat.1004166-vandenWorm1], and with MERS-CoV [@ppat.1004166-Zaki1], [@ppat.1004166-Kindler1]. MERS-CoV was first described in 2012 and was isolated from a 60-year old man with acute pneumonia, renal failure and fatal outcome in Saudi Arabia [@ppat.1004166-Zaki1]. The virus is most likely of zoonotic origin [@ppat.1004166-vanBoheemen1], [@ppat.1004166-Reusken1] and by February 2014 the number of laboratory-confirmed cases of MERS-CoV infection reported to the World Health Organization exceeded 182, including more than 79 cases with fatal outcome. We have previously shown that MERS-CoV can readily replicate on primary HAE cells [@ppat.1004166-Kindler1] by infecting non-ciliated cells expressing the cellular receptor dipeptidyl peptidase 4 [@ppat.1004166-Raj1]. As shown in [Figure 6](#ppat-1004166-g006){ref-type="fig"}, HCoV-229E and MERS-CoV infections were inhibited by K22 treatment with remarkable efficacy, illustrated by reduction of viral replication by several orders of magnitude ([Figure 6E-F](#ppat-1004166-g006){ref-type="fig"}) and substantial reduction of dsRNA in MERS-CoV-infected primary HAE cultures ([Figure 6G-H](#ppat-1004166-g006){ref-type="fig"}). This result demonstrates that the broad anti-coronaviral activity of K22 makes this compound particularly promising for the development of efficacious treatment options for emerging coronaviruses, such as MERS-CoV. Discussion {#s3} ========== Here we describe the discovery of a novel class of inhibitor and propose a mode-of-action that targets membrane-bound viral replication. Like all positive strand RNA viruses, coronaviruses employ host cell membranes to assemble the viral replicase complex. This evolutionary conserved strategy provides a compartment for viral RNA synthesis that is enriched in replicative viral and host cell-derived proteins and believed to protect from antiviral host cell defense mechanisms. The remarkable efficacy of K22-mediated inhibition of coronavirus replication confirms that the employment of host cell membranes for viral RNA synthesis is a crucial step in the coronavirus life cycle, and importantly, demonstrates that this step is extremely vulnerable and also druggable for antiviral intervention. The observation that K22 resistance is mediated through mutations in nsp6 defines transmembrane domain-containing nsps implicated in anchoring viral replicase complexes to host cell-derived membranes, as novel targets for anti-coronaviral intervention. Moreover, we expect this mode-of-action to serve as a paradigm for the development of similar antiviral drugs to combat infections caused by many other positive strand RNA viruses. Notably, resistance conferring mutations in nsp6 emerged only after 10--13 consecutive passages of HCoV-229E under K22 selection, and we were so far not successful in obtaining K22-resistant MHV-A59 mutants (data not shown). This suggests that escape mutations in membrane domain-containing coronavirus nsps compatible with maintaining efficient RNA synthesis are limited. In addition, the nsp6 escape mutants we have obtained for HCoV-229E display a remarkable reduction of specific infectivity. Thus, although RNA synthesis appears to be unaffected and viral RNA detected in preparations of extracellular virus was ribonuclease insensitive implying its adequate package in viral particles, mutations in nsp6 seem to reduce virus fitness. Thus, it is conceivable that the nsp6 mutants may be functionally impaired during an early step in the viral life cycle. Since dsRNA is localized in DMVs and nsp6 escape mutants induced decreased number of DMVs that are structurally impaired, it is possible that the reduced specific infectivity of these viruses could be related to dsRNA-triggered innate immune responses. SARS-CoV nsp6 was recently found to contribute to the establishment of the virus-induced RVN by promoting vesicle formation in transfected cells [@ppat.1004166-Angelini1], and our observation that K22 resistant mutants generated decreased number of DMVs implies that specific alterations may adversely affect the vesicle-forming capability of nsp6. Nsp6 of HCoV-229E (this report), MHV, and SARS-CoV [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1] is predicted as a hexaspaning protein comprising a conserved C-terminal cytoplasmic tail. The latter domain may serve as a wedge-like amphipathic helix which upon insertion into the lipid membrane can trigger its bending due to induction of positive membrane curvature (reviewed in [@ppat.1004166-McMahon1]). The vesicle formation would also require a putative ion channel activity that depolarizes curved membranes thus facilitating their fusion and vesicle scission. The question as to whether nsp6 or other components of the coronavirus replicase complex exhibit such activities would require further investigation. Although our data reveal that the K22 escape mutations occur in nsp6, further binding experiments are required to clarify whether K22 targets nsp6 directly. We observed that K22 is most active in inhibiting replication of the tested α-coronaviruses (HCoV-229E, FCoV) and the γ-coronavirus IBV, whereas amongst β-coronaviruses K22 was highly active in inhibiting MERS-CoV, but only moderately against MHV or SARS-CoV ([Figure 6](#ppat-1004166-g006){ref-type="fig"}). It is conceivable that K22 may strong inhibit α-coronaviruses, since K22 has been identified by screening for anti-HCoV-229E activity. However, the limited nsp6 sequence similarity between coronaviruses ([Figure 2](#ppat-1004166-g002){ref-type="fig"}) does not allow predicting the strength of K22-mediated inhibition of replication based on nsp6 homology. We also like to address in future studies a question of how the moderately resistant virus variant L (containing mutations in nsp15 and nucleocapsid) can escape K22-mediated inhibition of replication. This variant, in contrast to these containing resistance mutations in nsp6, exhibited only moderate resistance to K22 (∼2-3-fold) and was not consistently selected in separate selection experiments. Although nsp15 and nucleocapsid protein have not yet been described as being directly involved in DMV formation, these proteins are components of the replicase complex that may somehow affect/modulate nsp6 functions, and compensatory mutations in these proteins may partially relieve K22 blockade of nsp6. An alternative possibility is that the actual K22 target may be a cellular protein or a process of recruitment of a cellular protein that participates in coronavirus-induced membrane rearrangements by interacting with nsp6. While we could not observe any detectable impact of K22 on the formation of autophagosomes, further studies are required to address if K22 may target similar vesicles, such as EDEMosomes [@ppat.1004166-Reggiori1]. Both possibilities are compatible with the observed phenotype of DMV impairment and the detection of resistance mutations at regions of HCoV-229E nsp6 that are structurally conserved while displaying only limited sequence similarity. It is thus conceivable that membrane domain-containing nsp3 and nsp4 may represent additional drug targets. Similar as described for the related arteriviruses, where co-expression of membrane-spanning nsp2 and nsp3 results in membrane alterations and DMV formation similar to those observed during arterivirus infection [@ppat.1004166-Snijder2], [@ppat.1004166-Posthuma1], co-expression of coronavirus nsp3, nsp4 and nsp6 is required to produce coronavirus-like membrane rearrangements including DMVs [@ppat.1004166-Angelini1]. Expression of nsp3, nsp4 or nsp6 alone or in combinations of two induces aberrant membrane rearrangements that only partially mimic membrane structures known from coronavirus infection [@ppat.1004166-Angelini1]. Thus, there is growing evidence that nsp3, nsp4, nsp6, and possibly ER membrane-resident host cell proteins [@ppat.1004166-Reggiori1], [@ppat.1004166-Bernasconi1], orchestrate critical events that lead to the development of suitable membrane structures facilitating coronavirus RNA synthesis. Since K22 apparently interferes with these processes, inhibitors like K22 and corresponding escape mutants will likely become valuable tools to further our understanding on the induction of membrane alterations and DMV formation that take place during the early phase of the coronavirus life cycle. For example, co-expression of nsp3, nsp4 and native or mutated nsp6 in the absence of virus replication, similar as described by Angelini and colleagues [@ppat.1004166-Angelini1], may help to clarify whether presence of K22 would affect formation of DMV by directly targeting nsp6 or cellular protein(s) required and recruited for DMV formation. We emphasize that the identification of K22 and its proposed mode-of-action is only the very first step towards an approved drug for therapeutic use in animals or humans. Specifically, we are currently focusing on the structure-activity relationship analysis of K22 analogs, with the aim to identify compounds with improved antiviral and cytotoxic profiles prior to their assessment in vivo. However, one important lesson of the past SARS-CoV and recent MERS-CoV outbreaks is that zoonotic transmission of coronaviruses into the human population can pose considerable threat to human health and that it is warranted to eventually invest significant efforts to developing efficacious and approved drugs to increase preparedness and combat coronavirus infections. The antiviral activity against a number of diverse coronaviruses makes K22 an ideal candidate for further development towards an efficacious "pan-coronavirus inhibitor". Broad anti-coronaviral activity has been proposed for inhibitors targeting highly conserved enzymatic functions, such as coronavirus proteinase activities [@ppat.1004166-Anand2], [@ppat.1004166-Ren1], or more recently, for compounds targeting host cell factors required for efficient replication, such as cyclophilins [@ppat.1004166-Pfefferle1], [@ppat.1004166-deWilde1]. The concept of targeting multiple key functions of viral replication led to the development of efficacious treatment regimens against HIV and hepatitis C virus by combining multiple antiviral drugs [@ppat.1004166-Deeks1], [@ppat.1004166-Delang1] and it is tempting to speculate that this concept will be applicable to combat coronavirus infections in the future. Moreover, with the identification of K22, we demonstrate that there are yet additional critical steps in the life cycle of positive strand RNA viruses to explore as targets for antiviral intervention. Materials and Methods {#s4} ===================== Ethics statement {#s4a} ---------------- Human bronchial epithelial cells were isolated from patients (\>18 years old) who underwent bronchoscopy and/or surgical lung resection in their diagnostic pathway for any pulmonary disease and that gave written informed consent. This was done in accordance with local regulation of the Kanton St. Gallen, Switzerland, as part of the St. Gallen Lung Biopsy Biobank (SGLBB) of the Kantonal Hospital, St. Gallen, which received approval by the ethics committee of the Kanton St. Gallen (EKSG 11/044, EKSG 11/103). Cells and viruses {#s4b} ----------------- Human embryonic lung diploid fibroblasts (MRC-5), African green monkey kidney cells (Vero), baby hamster kidney cells (BHK-21), felis catus whole fetus 4 cells (FCWF-4), were purchased from the American Type Culture Collection (ATCC), murine fibroblast cells (L929), African green monkey kidney cells (CV-1) were purchased from the European Collection of Cell Cultures. D980R cells were a kind gift from G. L. Smith, Imperial College, London, United Kingdom. African green monkey kidney (GMK AH1) cells were obtained from the Swedish Institute for Infectious Disease Control, Stockholm. Cells were grown in Eagle\'s minimum essential medium (EMEM) (MRC-5, CV-1, D980R, L929, BHK-21, GMK AH1 cells) or in Dulbecco\'s modified EMEM (DMEM) (FCWF-4, Vero cells), supplemented with 5--10% heat-inactivated fetal calf serum, (HI-FCS), 1% L-glutamine, penicillin (60 µg/ml) and streptomycin (100 µg/ml) (PEST). Isolation and cultivation of primary human bronchial epithelial cells to form pseudostratified/differentiated human airway epithelial (HAE) cultures was performed as described previously [@ppat.1004166-Dijkman1], [@ppat.1004166-Dijkman2]. Human CoV strain 229E [@ppat.1004166-Hamre1] (HCoV-229E) was obtained from ATCC (VR-740). HCoV-229E stocks were prepared from virus passages 6--8 in MRC-5 cells growing in EMEM supplemented with 2% HI-FCS, 1% L-glutamine, HEPES (10 mM) and PEST (EMEM-FP). In some experiments, the virus was concentrated by centrifugation of infectious culture fluid of MRC-5 cells over a 1.5 ml cushion of 20% sucrose for 2 h at 22000 rpm (SW28.1 rotor, Beckman). The pellet was covered with PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO), left overnight at 4°C, and then gently suspended by pipetting. The following viruses and their propagation were described previously: recombinant HCoV- 229E [@ppat.1004166-Thiel2], recombinant HCoV-229E-Ren expressing Renilla luciferase [@ppat.1004166-vandenWorm1], recombinant feline coronavirus (strain Black) expressing Renilla luciferase (recFCoV-RL) [@ppat.1004166-Tekes1], SARS-CoV strain Frankfurt-1 [@ppat.1004166-Thiel1], recombinant avian infectious bronchitis virus (IBV, strain Beaudette) [@ppat.1004166-Casais1], MERS-CoV [@ppat.1004166-Zaki1], [@ppat.1004166-Kindler1]. Recombinant MHV strain A59 expressing Gaussia luciferase (MHV-Gluc) was generated based on the previously described reverse genetics system [@ppat.1004166-Coley1], [@ppat.1004166-Eriksson1]. Briefly, the MHV-A59 accessory gene 4 was replaced by the gene encoding the codon-optimized Gaussia luciferase [@ppat.1004166-Tannous1] (hGLuc) using vaccinia-virus-mediated homologous recombination essentially as described for the generation of MHV-GP33-GFP [@ppat.1004166-Zust1]. The plasmid DNA used for recombination contained MHV-A59 nucleotides (nts) 27500--27967, the hGLuc Gaussia luciferase gene, and MHV-A59 nts 28265--28700. Recombinant HCoV-229E containing mutations conferring K22 resistance in nsp6 were generated based on the previously described reverse genetics system [@ppat.1004166-Thiel2], [@ppat.1004166-Eriksson1]. Briefly, vaccinia virus HCoV-inf1 (containing the full-length HCoV-229E cDNA) [@ppat.1004166-Thiel2] was used to recombine with a plasmid based on pGPT1 [@ppat.1004166-Hertzig1] where the Escherichia coli guanine phosphoribosyltransferase (GPT) gene was flanked by HCoV-229E nts 9398--10098 and 10930--11580. The resulting GPT-positive vaccinia virus was then used to recombine with plasmids containing the HCoV-229E nts 9398--11580 with modification of nucleotide 10455 (A to T; HCoV-229E^H121L^), or nt 10568 (A to G; HCoV-229E^M159V^), or both nts 10455 and 10568 (HCoV-229E^H121L/M159V^). The resulting vaccinia viruses were then used to rescue HCoV-229E^H121L^, HCoV-229E^M159V^, and HCoV-229E^H121L/M159V^ as described previously [@ppat.1004166-Thiel2], [@ppat.1004166-Eriksson1]. The identity of plasmid DNA and recombinant vaccinia viruses and recombinant coronaviruses was confirmed by sequencing. In some experiments poliovirus 1 strain Sabin (obtained from the Swedish Institute for Infectious Disease Control, Stockholm) was used. Reagents {#s4c} -------- The ChemBioNet diversity library of 16671 compounds was obtained from the Leibniz Institute for Molecular Pharmacology (Berlin, Germany). Library was provided in a 384 well plate format, each well containing 5 µl of a compound solubilized in DMSO to a final concentration of 10 mM. Hit compound K22 was purchased from ChemDiv (San Diego, CA; catalog number 4295--0370). The correct structure and purity of K22 (\>95%) was confirmed in our laboratory by NMR and LCMS analyses. Immunofluorescence analysis {#s4d} --------------------------- MRC-5 cells were infected at a multiplicity of infection (moi) of 0.05 with wtHCoV-229E and K22-resistant recombinants HCoV-229E^H121L^, HCoV-229E^M159V^, and HCoV-229E^H121L/M159V^ with or without the presence of K22 (4 µM). The cells were fixed at 18 h p.i. with 4% paraformaldehyde (PFA) and immunostained [@ppat.1004166-Dijkman3] using the mouse monoclonal anti-dsRNA (J2, English & Scientific Consulting Bt.) and rabbit anti-HCoV-229E nsp8 [@ppat.1004166-Ziebuhr3] (kindly provided by John Ziebuhr, University of Giessen, Germany) as primary antibodies for detection of double-stranded (ds) RNA and viral replication complexes. Donkey derived, Dylight 488 labeled, anti-mouse IgG (H+L) and Dylight 647 labeled, anti-rabbit IgG (H+L) (Jackson Immunoresearch) were applied as secondary antibodies. Cells were counterstained with DAPI (4\',6-diamidino-2-phenylindole; Invitrogen) to visualize nuclei. HAE cell cultures were inoculated with 40000 plaque forming units (PFU), with or without the presence of K22 (50 µM) and fixed with 4% PFA 48 h p.i. Staining was performed with the mouse monoclonal antibody directed against dsRNA (J2) and goat polyclonal anti-ZO1 (tight junctions; ab99462, Abcam) as primary antibodies. Dylight 488-labeled donkey anti-mouse IgG (H+L), Dylight 546-labeled donkey anti-goat IgG (H+L) (Jackson Immunoresearch) were applied as secondary antibodies, followed by two separate incubation steps with Alexa Fluor647-conjugated rabbit monoclonal anti-beta-Tubulin antibody (ciliated cells; 9F3, Cell Signal) and DAPI (Invitrogen). Images were acquired using EC-plan Neofluar 20x/50 M27 or EC Plan-Neofluar 40x/1.30 Oil DIC M27 objectives on a Zeiss LSM 710 confocal microscope. Image capture, analysis and processing were performed using the ZEN 2010 (Zeiss) and Imaris (Bitplane Scientific Software) software packages. Anti-coronavirus compound screening assay {#s4e} ----------------------------------------- The screening assay was performed as described previously for respiratory syncytial virus [@ppat.1004166-Lundin1]. Briefly, MRC-5 cells were seeded in 384 well plates (CLS-3701; Costar-Corning, NY, USA) to become ∼70--90% confluent after one day of culture. The growth medium was removed, and the cells supplemented consecutively with 25 µl of EMEM-FP medium, 1 µl volumes of library compounds at 1 mM concentration, and ∼350 PFU of HCoV-229E in 25 µl of EMEM-FP. The last two columns of the 384 well plate received either virus or EMEM-FP medium to serve as controls. The cells were observed under the microscope for their protection from the virus-induced cytopathic effect after 3 and 6 days of incubation at 37°C. Antiviral assays {#s4f} ---------------- Plaque reduction assay to determine the antiviral effect of K22 on HCoV-229E was done as follows. MRC-5 cells were seeded in 12-well plates to become nearly confluent after one day of culture. Serial fivefold dilution of K22 (0--100 µM) and 100 PFU of HCoV-229E virus in 0.5 ml of EMEM-FP medium were added to and incubated with cells for 3 h at 37°C, 5% CO2. Subsequently, the virus-compound mixtures were removed from cells, and 1.5 ml volumes of 1% methylcellulose (MC) solution in EMEM-FP medium supplemented with the same concentration of K22 were added. The plates with cells were further incubated at 37°C, 5% CO2 for 2--3 days, and then stained with 0.2% solution of crystal violet to visualize the viral plaques. Viral yield reduction assays were done to determine the antiviral effect of K22 on HCoV-229E-Ren, recFCoV-RL, MHV-Gluc, SARS-CoV, IBV, MERS-CoV, and poliovirus replication. Briefly, K22 or its DMSO solvent in medium was added at the indicated concentrations to nearly confluent monolayers of corresponding cell lines or to HAE cultures at the basolateral side and incubated for 4 h at 37°C, 5% CO2. The cells were then inoculated with recFCoV-RL (moi = 0.1 on FCWF-4 cells), MHV-Gluc (moi = 0.001 on L929 cells), SARS-CoV (moi = 0.001 on Vero cells), IBV (moi = 1 on Vero cells), HCoV-229E-Ren (4×10^3^ PFU on HAE cultures apically), MERS-CoV (4×10^3^ PFU on HAE cultures apically) or poliovirus (moi = 0.001 on GMK AH1 cells). After 2 h the viral inoculum was removed, cells were rinsed three times with PBS, and fresh medium containing the same concentrations of K22 or DMSO was added. Coronavirus replication was assessed from cell culture supernatant by determining titer as TCID50 (tissue culture infectious dose that will produce pathological change in 50% of cell cultures inoculated) for IBV or poliovirus at 48 h p.i., by determining the amount of viral genome RNA produced by qRT-PCR specific for SARS-CoV and MERS-CoV at 48 h p.i. as described previously [@ppat.1004166-Kindler1], or by determining the level of Renilla expression at 48 h p.i. (HCoV-229E-Ren) or 72 h p.i. (recFCoV-RL) using Renilla Luciferase Assay System (Promega, E2820), or Gaussia luciferase expression (MHV-Gluc) at 24 h p.i. using the BioLux Gaussia Luciferase Assay Kit (NEB,E3300), respectively. For the virucidal assay, 200 µl of HCoV-229E suspension (∼3×10^4^ PFU) in EMEM-FP medium was mixed with 50 µM K22 and incubated for 15 min at 37°C. In the control sample, virus was incubated with the DMSO solvent at a final concentration corresponding to that present in the test compound. Then, both mixtures were diluted serially tenfold in EMEM-FP medium and the residual virus infectivity determined by the viral plaque assay. Cell toxicity and proliferation assays {#s4g} -------------------------------------- The toxicity of K22 or its solvent (DMSO) for MRC-5 cells was evaluated using the tetrazolium-based CellTiter 96 AQueous One Solution cytotoxicity assay (Promega; G3580). The effect of K22 or its solvent on proliferation of MRC-5 cells was studied as follows. The cells were seeded in 48 well plates to become ∼50% confluent after one day of culture. The growth medium was removed, and cells incubated with specific concentrations of K22 or its solvent in EMEM-FP medium for 72 h at 37°C. The cells were then dissociated with trypsin/EDTA solution and counted. The effect of K22 or DMSO on viability of Vero, L929, and FCFW-4 cells was assessed using the CytoTox-Glo Cytotoxicity Assay kit (Promega, G9291) while the toxicity of test compound for differentiated HAE cultures was evaluated with CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, G7571). Time-of-addition assay {#s4h} ---------------------- MRC-5 cells growing in 12 well plates were precooled for 15 min at room temperature and for another 15 min at 4°C. The cells were rinsed once with 500 µl of cold EMEM-FP and inoculated with HCoV-229E at moi of 0.05. Following virus adsorption to cells for 45 min at 4°C, the cells were rinsed twice with 500 µl of cold EMEM-FP, and 990 µl of warm EMEM-FP medium was added. Subsequently 10 µl of 1 mM K22 was added at specific time points relative to the end of the virus adsorption period, and the infectious cell culture medium and cells harvested at the time point 24 h. The cell culture supernatant medium was clarified by centrifugation at 1000×g for 5 min while the pelleted cells were suspended in RNase-free water and stored at −80°C until quantification in RT-PCR assay. To study the effect of K22 on early virus-cell interaction the "time-of-addition" assay was modified as follows. MRC-5 cells were rinsed once with 1 ml of EMEM-FP and 500 µl of EMEM-FP supplemented with 4 µM K22 was added. The compound was incubated with cells for 2 h at 37°C either prior to, during or after a 2 h period of infection of cells with ∼100 PFU of 229E virus in 500 µl of EMEM-FP. The cells were washed once with 1 ml of EMEM-FP after each 2 h period of their incubation with compound and/or virus. Finally, the cells were overlaid with the MC solution, and after incubation for 2 days at 37°C stained with crystal violet to visualize the viral plaques. RT-PCR {#s4i} ------ The RT TaqMan PCR was carried out as described by Brittain-Long et al. [@ppat.1004166-BrittainLong1]. Briefly, the extraction of RNA was conducted in the Magnapure LC robot using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Mannheim, Germany), and amplification was performed using a TaqMan 7300 Real Time PCR system (Applied Biosystems, Foster City, CA), with a pair of forward 5′-CAGTCAAATGGGCTGATGCA-3′ and reverse 5′-AAAGGGCTATAAAGAGAATAAGGTATTCT-3′ primers as well as a probe 3′CCCTGACGACCACGTTGTGGTTCA 5′ specific for HCoV-229E genome fragment coding for nucleocapsid protein [@ppat.1004166-Gunson1]. The number of HCoV-229E RNA copies was determined by relating the detected cycle threshold values to a standard curve prepared based on five tenfold dilutions of the specific plasmid (pUC57) comprising a 94 bp insert from the nucleocapsid sequence of HCoV-229E. qRT-PCR assays to quantify SARS-CoV and MERS-CoV genomic RNA have been described previously [@ppat.1004166-Kindler1]. Preparation of drug-resistant variants of HCoV-229E and sequencing analysis {#s4j} --------------------------------------------------------------------------- A procedure described previously for respiratory syncytial virus [@ppat.1004166-Lundin1] was used. Briefly, plaque purified HCoV-229E was subjected to 10--13 consecutive passages in MRC-5 cells in the presence of increasing concentrations (2--16 µM) of K22. For control purposes, the same virus was also passaged in MRC-5 cells in the absence of inhibitor. The virus was then subjected to two rounds of plaque purification in the presence of inhibitor, and its relative drug-resistance tested using the viral plaque reduction assay. Genomic RNA of original, mock-passaged, and the K22-resistant virus from passage 10--13 was extracted from extracellular fluid of the 229E-infected MRC-5 cells using the QIAamp viral RNA purification kit (Qiagen). Overlapping DNA fragments covering the entire coding sequence were produced by reverse transcription PCR and subjected to nucleotide sequencing using the ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems). Nucleotide sequence analysis was performed using Sequencher 4.9 software (Gene Codes Corporation). HCoV-229E replication kinetics {#s4k} ------------------------------ MRC-5 cells growing in 12 well plates were precooled for 15 min at room temperature and for another 15 min at 4°C. The cells were rinsed once with 500 µl of cold EMEM-FP and inoculated with concentrated preparation (see the Cells and Viruses section) of HCoV-229E (moi = 0.05). Following virus adsorption to cells for 1 h at 4°C, the cells were rinsed thrice with 500 µl of cold EMEM-FP, and 500 µl of warm EMEM-FP medium was added. The supernatant fluid and infected cells were harvested at specific time points relative to the end of the virus adsorption period, and processed for determination of viral RNA and infectivity as described under the "time-of-addition" assay. Ribonuclease treatment of HCoV-229E {#s4l} ----------------------------------- The infectious culture medium comprising HCoV-229E or recombinant nsp6 mutant HCoV-229E M159V were clarified by centrifugation at 1000×g for 5 min, and then 100 µl volumes of the supernatant were supplemented with 2 µl (20 µg) of ribonuclease A (Thermo Fisher Scientific; EN0531) or its solvent. All samples were spiked with ∼7 µg of RNA purified from human respiratory syncytial virus (RSV) to serve as an internal control of ribonuclease activity. Following incubation of the virus-enzyme mixture for 30 min at 37°C, the coronaviral and RSV RNA were quantified by RT TaqMan PCR as described by Brittain-Long et al. [@ppat.1004166-BrittainLong1] while coronavirus infectivity was determined by plaque titration. Autophagy {#s4m} --------- To assess the time-frame where autophagy vesicle formation occurs we seeded Huh-7 cells (100.000 cells) on glass bottom 12-well cluster plates (MatTek). Forty-eight hours prior to stimulation cells were transfected with LC3B-GFP plasmid [@ppat.1004166-Kabeya1] using lipofectamine2000 (Invitrogen), according to manufactures protocol. Hereafter cells were exposed to 100 nM of rapamycin (Invivogen) alone or in presence of either 20 µM of K22 or an equal volume of DMSO for the duration of 18 hours at 37°C. Fluorescent and differential interference contrast (DIC) images were acquired with 30 minute interval using EC Plan Neo-fluar 40x/1.30 Oil DIC M27 objective on a Zeiss LSM 710 confocal microscope. Image capture, analysis and processing were performed using the ZEN 2010 (Zeiss). To determine whether K22 inhibits endogenous autophagy vesicle formation we stimulated Huh-7 cells (40.000 cells) with 100 nM of rapamycin alone or in presence of either 20 µM of K22 or an equal volume of DMSO for duration of six hours at 37°C. Unstimulated cells were used as mock control. Cells were fixed and immunostained as previously described [@ppat.1004166-Dijkman3]. Rabbit polyclonal anit-LC3B (L7543, Sigma Aldrich) was applied as primary antibody for the detection of autophagy vesicles. Goat derived, Cy3 labeled, anti-rabbit IgG (H+L; Jackson ImmunoResearch) was applied as secondary antibody. Thereafter cells were counterstained with DAPI (Invitrogen). Fluorescent images were acquired using a PLAPON 60xO/1.42 objective on an Olympus FV-1000 confocal microscope. Image capture, analysis and processing were performed using the Olympus Fluoview software. Electron microscopy {#s4n} ------------------- MRC-5 cells growing on a Melinex polyester film (Agar Scientific Ltd., Stansted, U.K.) in 24 well cluster plates were infected with HCoV-229E (moi = 0.04) in the presence of 10 µM of K22. After 18 h of infection at 37°C, the culture medium was removed, the cells rinsed twice with Eagle\'s medium, and a fresh Eagle\'s medium supplemented with 2.5% glutaraldehyde was added and incubated for 45 min at 37°C. The cells were washed twice with 0.05 M Tris-HCl buffer (pH 7.4) supplemented with 2 mM CaCl2, and further processed for electron microscopy as described [@ppat.1004166-Widehn1]. Experiments with recombinant nsp6 mutant viruses and original virus were carried out in a similar manner except that the cells were inoculated at a moi of ∼0.25 and incubated with or without the presence of 4 µM K22. Supporting Information {#s5} ====================== ###### J15 structure, antiviral activity, and cytotoxicity. (A) J15 structure. (B) Anti-HCoV-229E activity and cytotoxicity of J15 in MRC-5 cells. J15 and wild type (WT) HCoV-229E or nsp6 recombinant HCoV-229E^M159V^ (M159V) were added to MRC-5 cells, and the number of viral plaques developed after 48 h were assessed. For cytotoxicity assessment, MRC-5 cells were incubated with J15 for 48 h at 37°C and the cell viability determined using tetrazolium-based reagent. Data shown are means (±SD) of duplicate determinations from two independent experiments. PFU, plaque forming unit. (TIF) ###### Click here for additional data file. ###### Ribonuclease treatment of HCoV-229E. Infectious culture medium comprising wild type HCoV-229E or mutant nsp6 recombinant HCoV-229E^M159V^ (M159V) was spiked with RNA purified from human respiratory syncytial virus (RSV) and then incubated for 30 min at 37°C in the presence of ribonuclease A (RNase) or without this enzyme (mock). The number of copies of coronaviral RNA (A) or control RSV RNA (B) was determined by qPCR while titer of infectious coronavirus (C) by viral plaque assay. Data shown are means (±SD) of four determinations obtained in four independent experiments (qPCR) or duplicate determinations from two independent experiments (infectivity). PFU, plaque forming unit; n.d., not detectable; n.s., not significant. (TIF) ###### Click here for additional data file. ###### J15 affects formation of double membrane vesicles (DMVs). MRC-5 cells growing on Melinex polyester film were infected with wild type HCoV-229E (WT) or with K22-resistant recombinant nsp6 mutant HCoV-229E^M159V^ (M159V) and incubated for 18 h at 37°C with or without J15. The cells were then fixed with glutaraldehyde and processed for electron microscopy without their scrapping or pelleting. (A) Electron micrographs of cells infected with WT virus show presence of clusters of DMVs (arrow) and viral particles (arrowhead), and the lack of their production upon J15 treatment (4 µM). (B) Electron micrographs of MRC-5 cells infected with K22-resistant recombinant nsp6 mutant M159V showing presence of DMVs and viral particles irrespective of the addition of J15. (TIF) ###### Click here for additional data file. ###### K22 does not inhibit autophagy vesicle formation. To determine whether K22 inhibits autophagy vesicle formation Huh-7 cells were stimulated with rapamycin alone or in presence of either 20 µM of K22 or an equal volume of DMSO solvent for 6 h at 37°C. Unstimulated cells were used as mock control. Fixed cells were stained with Anti-LC3B (red) and DAPI (blue) to annotate autophagy vesicles and cell nucleus, respectively. (TIF) ###### Click here for additional data file. ###### K22 affects replication of diverse coronaviruses including MERS-CoV. (A-D) The antiviral activity (bars) and cell toxicity (data points above bars) of K22 (black bars) or DMSO solvent (white bars) during MHV-Gluc (A), FCoV-RL (B), SARS-CoV (C) and IBV (D) infection on representative continuous cell lines of murine (L-929 cells; A), feline (FCWF cells; B), or primate (Vero cells; C-D) origin. Data are shown as mean (±SD) of a representative experiment, from two independent experiments performed in triplicate. (E-F). The antiviral activity (bars) and cell toxicity (data points above bars) of K22 (black bars) or DMSO solvent (white bars) in HCoV-229E-ren (E) and MERS-CoV (F) infected differentiated human airway epithelial (HAE) cultures. Data are shown as mean (±SD) of three independent experiments performed in triplicate (viral yield), or mean (±SD) of a representative experiment, from two independent experiments performed in triplicate (cell viability). Ns, not significant (P\>0.05); \* P\<0.05; \\ P\<0.01 (paired t-test). (TIF) ###### Click here for additional data file. ###### K22 exhibits little or no activity against poliovirus 1. GMK AH1 cells were pretreated with K22 (black bars) or DMSO solvent (white bars) for 4 h at 37°C and then infected with poliovirus 1 Sabin strain at a moi of 0.001. Following incubation of infected cells in the presence of K22 or DMSO for 48 h at 37°C, the titer of extracellular infectious virus in culture medium was determined. The results shown are means of duplicate determinations from two separate experiments. TCID~50~, tissue culture infectious dose. (TIF) ###### Click here for additional data file. ###### Effect of K22 on proliferation and viability of cultured cells. (DOCX) ###### Click here for additional data file. We are grateful to Dr. Regulo Rodriguez and Dr. Christoph Zeisel, Kantonal Hospital St.Gallen, Switzerland, for their support to obtain human lung tissue, to Sibylle Widehn, Department of Pathology, University of Gothenburg, Sweden, for help with electron microscopy, and to Dr. Alexandra Trkola, Dr. Silke Stertz and Dr. Jovan Pavlovic, Institute of Medical Virology, University of Zürich, Switzerland, for their generous help and availability of the BSL3+ facility. [^1]: JK is employed by a commercial company, Merck Animal Health. This does not alter our adherence to all PLOS Pathogens policies on sharing data and materials. [^2]: Conceived and designed the experiments: AL TB CH BA VT ET. Performed the experiments: AL RD EK HRJ JK DM. Analyzed the data: AL BA CH NK ET VT RD EK HRJ DM MAM CD MF. Contributed reagents/materials/analysis tools: NK. Wrote the paper: VT ET RD AL. 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1. Introduction {#s0005} =============== The leishmaniases, caused by protozoan parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae), are worldwide vector-borne diseases transmitted by phlebotomine sand flies. Leishmania spp. infect a wide range of hosts including sylvatic and domestic animals. In humans, the leishmaniases are an enormous public health problem with a global prevalence of 12 million cases and a yearly incidence of 2 million cases ([@b0120]). Infection with Leishmania can cause a broad spectrum of clinical presentations ranging from asymptomatic to simple cutaneous or destructive mucocutaneous lesions, or severe visceral leishmaniasis (VL) that is fatal without effective chemotherapy. Historically, the population structure of Leishmania has been considered to be fundamentally clonal ([@b0110; @b0105]) but with some limited historical evidence of recombination, for example, between Leishmania braziliensis and Leishmania panamensis/guyanensis, from multilocus enzyme electrophoresis (MLEE) and random amplification of polymorphic DNA ([@b0015; @b0010]). Diversity observed within natural populations was thus generally attributed to the accumulation of mutations over time, with perhaps rare instances of genetic exchange. Nevertheless, evidence of genetic exchange between different Leishmania spp. in natural populations has been reported on several occasions ([@b0015; @b0040; @b0010; @b0080; @b0065; @b0100; @b0075]). However, at the intra-species level, the identification of hybrids among natural populations has been more difficult and more equivocal due to the limited discriminatory power of methods such as MLEE. A few recent studies characterising natural Leishmania populations using highly polymorphic microsatellite markers have suggested the presence of genetic exchange ([@b0020; @b0030]). In addition, [@b0090] described evidence of inbreeding in natural populations of both Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis based on linkage disequilibrium (LD), yet with a deficit of heterozygosity. The proposed reproductive strategy is therefore an alternating model of clonality in both the vertebrate and invertebrate hosts with non-obligatory sexual recombination in the sand fly. Whole genome sequencing of vector-isolated Leishmania infantum from southeastern Turkey supported the occurrence of hybridization and subsequent selfing; one of the parental genotypes was not identified but was likely to be from the Leishmania donovani complex ([@b0085]). The extant capacity of Leishmania spp. to undergo genetic exchange in the sand fly has been proven by generating experimental Leishmania major hybrids following co-passage of transgenic strains ([@b0005; @b0035]). Hybrids isolated from the sand fly gut had genotypes consistent with meiosis. Furthermore, experimental crosses of fluorescent transgenic L. donovani in the sand fly produced dual expression in single cells, consistent with intra-species genetic exchange ([@b0095]). Genetic exchange has potential implications for heterosis (hybrid vigour), the emergence and spread of virulent strains, resistance to chemotherapeutics, exploitation of different hosts and vectors, and adaptation to new ecological niches that may provide a selective advantage. For example, a startling observation by [@b0115] provided clear evidence that L. infantum/major hybrids possess enhanced transmission potential. Leishmania infantum is not normally able to infect the broad ranging Phlebotomus papatasi but, remarkably, the resultant hybrids were able to do so with potentially profound epidemiological consequences. Leishmania braziliensis/Leishmania peruviana hybrids have also been implicated as agents of destructive forms of mucocutaneous leishmaniasis ([@b0065]). The work described here was designed to accomplish three main objectives applied to a panel of 11 natural isolates of north Ethiopian origin: (i) to characterise four putative hybrids using a range of high resolution markers; (ii) to identify likely parental genotypes among seven north Ethiopian L. donovani representatives that did not have hybrid-like microsatellite genotypes; and (iii) to discount the possibility that hybrid-like microsatellite genotypes were the result of mixed infections. We applied a novel combination of multilocus sequence typing (MLST) targets and highly resolutive multilocus microsatellite typing (MLMT) markers ([@b0045; @b0030]) in conjunction with mitochondrial (maxicircle) sequencing and DNA content analysis. We demonstrate that four natural isolates are bona fide hybrids and not the result of mixed infection. The likely parental origins and epidemiological implications are discussed. 2. Materials and methods {#s0010} ======================== 2.1. Parasite strains and DNA isolation {#s0015} --------------------------------------- A panel of 11 north Ethiopian L. donovani isolates was available, originating from a population of Ethiopian isolates containing some putatively characterised as hybrids by [@b0030], participants were enroled according to written informed consent procedures and approved by the Institutional Review Boards of the Faculty of Medicine, Addis Ababa University, Addis Ababa. Full World Health Organization (WHO) isolate labels are shown in [Table 1](#t0005){ref-type="table"} and isolate characteristics are shown in [Supplementary Table S1](#s0075){ref-type="sec"}. Isolates that were available comprised four putative hybrids, DM19, DM62, DM295 and DM299. Two of the hybrids were isolated from an HIV-infected patient; DM62 and DM299 were isolated from the same HIV/VL co-infected patient during different episodes of the disease. Parent-like representatives were DM20, DM297 (designated hypothetical parent group A), and DM256, DM257, DM259, DM481, DM559 (designated hypothetical parent group B, [Fig. 1](#f0005){ref-type="fig"}, [Table 1](#t0005){ref-type="table"}). A total of 90 biological clones were generated, a maximum of 10 per isolate (DM19, eight clones; DM62, 10 clones; DM256, nine clones; DM257, nine clones; DM259, nine clones; DM295, nine clones; DM 297, nine clones; DM299, eight clones; DM481, nine clones; DM599, 10 clones). Leishmania were cultured in supplemented RPMI liquid medium at 28 °C, as previously described ([@b0055]). Isolates were then cloned on solid media as described by [@b0125]. Once single colonies became visible they were removed and inoculated into liquid culture medium. DNA was isolated from each clonal culture using DNA isolation kits (Promega, UK) following the manufacturer's instructions. 2.2. MLMT {#s0020} --------- Five microsatellite markers were selected ([Table 2](#t0010){ref-type="table"}) based on their ability to distinguish putative hybrids from parents ([@b0030]). Fluorescent-conjugated forward and non-conjugated reverse primers (Eurofins, Germany) were used for amplification. The amplification conditions and reaction cycles were performed as described by [@b0070] with slight modifications. After an initial denaturation step at 95 °C for 10 min, samples were processed through 35 cycles consisting of 95 °C for 30 s, annealing for 30 s at the temperature (T~A~) indicated in [Table 2](#t0010){ref-type="table"} and 72 °C for 1 min, followed by a terminal elongation step at 72 °C for 10 min. Each reaction was performed in a final volume of 20 μl consisting of 1× ThermoPol reaction buffer (New England Biolabs (NEB), UK), 1.5 mM MgCl~2~, 0.2 mM dNTPs, 10--25 pmol of each primer, 1 unit of Taq polymerase (NEB, UK) and 20--30 ng of genomic DNA. Allele sizes were estimated using an automated capillary sequencer (ABI3730, Applied Biosystems, UK) and were checked manually. 2.3. MLST {#s0025} --------- MLST markers were amplified from genomic DNA extracted from the isolates and biological clones. [Table 3](#t0015){ref-type="table"} summarises PCR amplification primers and annealing temperatures. Genetic markers consisted of four coding nuclear markers (glucose-6-phosphate isomerase, GPI; isocitrate dehydrogenase, ICD ; Ch28; Ch36-1190) and one non-coding nuclear marker (Ch36-0350). Targets were chosen following screening of a panel of markers known to exhibit sequence diversity within the L. donovani complex on the basis of GenBank sequences. Amplifications were performed with an initial denaturation step at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, with annealing and elongation steps for each marker as described herein, and a final elongation step at 72 °C for 10 min. The annealing temperatures for GPI and ICD were 57 °C and 61 °C, respectively, for 90 s and for CH28-0190, CH36-1130 and CH36-0350 were 53 °C, 59 °C and 61 °C, respectively, for 60 s. Each reaction was performed in a 20 μl total volume containing: 20 ng of genomic DNA, 20 pmol of each primer, 2 mM dNTPs, 1.5 mM MgCl~2,~ and 5 U Taq (BIO-21086, Bioline, UK). PCR products were visualised on 1.5% agarose gels and the appropriate bands excised and purified using a QIAGEN Gel Extraction Kits (Qiagen, UK) or SureClean (Bioline). Bi-directional sequencing was performed, using internal primers where required ([Table 3](#t0015){ref-type="table"}), with Big Dye Terminator Cycle Sequencing V3.1 (Applied Biosystems) and an ABI PRISM 377 DNA Sequencer (Applied Biosystems) according to the manufacturer's protocols. Sequence data were assembled manually in BioEdit v7.0.9.0 (Ibis Biosciences, USA) and ambiguous peripheral regions of aligned sequences were discarded to produce unambiguous partial gene sequences for each isolate or clone. Chromatograms were examined visually in both directions for the presence of heterozygous bi-allelic single nucleotide polymorphisms (SNPs) at a single locus ([Fig. 2](#f0010){ref-type="fig"}). Re-sequencing was undertaken if results were ambiguous. SNP differences between isolates and clones were recorded and tabulated together with their associated positions ([Fig. 2](#f0010){ref-type="fig"}). 2.4. Mitochondrial maxicircle gene sequencing (mMLST) {#s0030} ----------------------------------------------------- The cytochrome b gene (CYTb) was amplified and sequenced using primer pairs LCBF: GGTGTAGGTTTTAGTTTAGG and LCBR2: CTACAATAAACAAATCATAATATACAATT ([@b0050]). Cycle conditions consisted of an initial denaturation at 94 °C for 30 s followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 68 °C for 5 min and a final extension step at 72 °C for 7 min. The amplified fragments were sequenced in both directions using the same set of primers and following the sequencing methods described above. 2.5. DNA content analysis {#s0035} ------------------------- Approximately 1 × 10^7^ cloned L. donovani parasites were harvested from mid-log phase liquid cultures. Parasites were pelleted by centrifugation at 3,000g (20 min), washed twice in ice-cold PBS and fixed with 100% methanol for 48 h at 4 °C. Two subsequent washes with ice-cold PBS were performed, after which cells were resuspended in PBS to a final density of 1 × 10^6^ cells/ml. Propidium iodide and RNAse A were added to a final concentration of 10 μg/ml and the mixture incubated for 45 min at 37 °C in the dark. Fluorescence was detected using a FACSCalibur flow cytometer, on channel FL2. For each sample a minimum of 10,000 events was counted. For each strain/clone, two independent assays were performed. Data analysis used FlowJo software (Tree Star Inc., Oregon, USA). Gates were created for G1-0 (2n) peaks and for G2-M (4n) peaks. Mean G1-0 values were taken to infer relative DNA content. Relative DNA content values were calculated as a ratio in comparison with an internal standard (L. major, Friedlin strain). For putative hybrids, the ratios relative to each putative parent (A or B) were also recorded. 3. Results {#s0040} ========== 3.1. MLMT hybrids, parental genotypes and exclusion of mixed infections {#s0045} ----------------------------------------------------------------------- Uncloned stocks from Ethiopia had identified a subset of putative hybrids possessing heterozygous profiles, with MLMT genotypes composed of alleles that were otherwise restricted to two distinct L. donovani populations in northern Ethiopia ([@b0030]). Heterozygous genotypes could have been the result of amplification of multiple alleles from multiclonal sources. We therefore prepared a maximum set of 10 biological clones for each of the 11 selected strains and conducted MLMT analyses. MLMT profiles across five loci are summarised in [Table 1](#t0005){ref-type="table"}. Overall in the four putative hybrids, MLMT analyses of clones showed high levels of heterozygosity that were not a result of multiclonality, in stark contrast with the high levels of homozygosity in putative parents (group A and group B). In more detail, the two non-hybrid (group A) strains and all of their derived clones were consistently homozygous across the five microsatellite markers. Similarly, the five non-hybrid (group B) strains and derived clones were generally homozygous across the five loci, with the exception of the heterozygous genotypes DM259 at locus Li41-56 and DM559 at loci Li41-56 and Li71-7. Three of the four hybrid strains (and their biological clones) were heterozygous across all five loci. The exception was DM19, which was heterozygous in three loci (Li46-67, Li22-35 and Li71-33) and homozygous at Li41-56 and Li71-7. Thus the heterozygous alleles in the five hybrid microsatellite loci matched with corresponding homozygous alleles among the parent-like isolates. However there were four examples where the origins of donor alleles in the putative hybrids could not be determined. As shown in [Table 1](#t0005){ref-type="table"}, these were: (i) the 103 bp allele at the heterozygous Li71-33 site in the hybrids was absent from both putative parental groups; (ii) for the heterozygous loci at Li41-56 (89/91), Li46-67 (70/72) and Li71-7 (90/92) in the hybrids one allele, 89, 72 or 92, respectively, could be found among both parental groups; and (iii) at locus (Li71-7), one isolate among parental group B was heterozygous (90/92). An exception was also seen at a single site for the MLST target ICD (see Section [3.2](#s0050){ref-type="sec"}). Taken alone, microsatellite data were less robust than MLST data (Section [3.2](#s0050){ref-type="sec"}) in identifying likely parental donors. 3.2. MLST comparisons reveal hybrids and putative parents {#s0050} --------------------------------------------------------- Although MLMT data detected consistent hybrid profiles, allele sharing across hypothetical non-hybrid groups (A and B) meant that inferring likely parental multilocus genotype donors was not possible. We therefore sought to analyse nucleotide sequences from several nuclear loci, reasoning that patterns of SNP marker inheritance would be more discriminatory. Gene identities (IDs) were confirmed by BLAST searches and submitted to TriTypDB 4.2 (<http://TriTrypDB.org>) to determine chromosomal locations. Locations for each of the nuclear gene fragments are shown in [Table 3](#t0015){ref-type="table"}. Two gene fragments (Ch36-0350 and Ch36-1190) were located on chromosome (Ch)36 (P:95153--94109 and P:429635--431277, respectively). The remaining genes were on independent chromosomes 10, 12 and 28. Nucleotide sequence data reported in this paper are available in the GenBank™ database under the Accession Nos. KJ907394- KJ907448. We characterised a total of 90 biological clones originating from both the putative hybrids (four isolates) and parent-like strains (seven isolates). The GPI locus was monomorphic and homozygous across all 11 strains and for the 90 biological clones; GPI was therefore not included in subsequent analysis. A summary of the distribution of SNPs and their relative positions in the remaining four loci is shown in [Fig. 1](#f0005){ref-type="fig"}. A total of 34 unambiguous SNP positions were detected across all loci. Locus Ch28 was the most diverse fragment with 18 SNPs, followed by Ch36-1190 with eight, ICD with five and Ch36-0350 with three. Heterozygous sequence profiles were found only in the hybrids; 16 SNPs displayed bi-allelic heterozygous profiles in at least one isolate: locus Ch28 with one such heterozygous site, Ch36-1190 with seven, ICD with five and Ch36-0350 with three. In contrast the putative parent-like isolates (A and B) were homozygous across all SNP positions. The high number of heterozygous sites seen in the hybrids, in comparison with the relative rarity of heterozygosity in the putative parents, is consistent with the patterns of heterozygosity and homozygosity seen in the MLMT data. Each of the four hybrid strains had a unique multilocus genetic profile. Interestingly, even hybrid isolates DM62 and DM299, which were isolated sequentially from the same patient, had distinct Ch36-0350 and ICD sequences, with one and four SNP differences, respectively, suggesting either multiclonality in this patient or clonal diversification in vitro. Variable sites were then examined with respect to their presence/absence in strains and clones from the hybrid group ([Fig. 1](#f0005){ref-type="fig"}). Three classes of marker distribution were observed. In some cases SNP distribution in hybrid group strains was consistent with acquisition of a group A and a group B allele, for example, DM62, DM299 and DM295 had heterozygous sequences compatible with inheritance of an A-like and a B-like Ch36 sequence. In other cases the hybrid group strains were homozygous for a group A-like or a group B-like sequence, for example, DM19 for the Ch28 and Ch36 loci and DM299 for the ICD locus. There were also several instances where the sequences in hybrid group strains were predominantly homozygous for an A-like or B-like sequence but with clear heterozygosity at the extreme 5′ and/or 3′ regions, e.g. DM62 and DM299 had Ch28 sequences that were homozygous for A-like SNPs at all informative positions except the most 5′ site (position 273) for which it had both an A-like and B-like SNP. Finally, both heterozygous sites for ICD in DM62, DM295 and DM299 were not present in non-hybrid groups A and B and were not informative with respect to inheritance. Thus, although it is clear the MLST data overall support the hypothesis that the hybrid group strains are derived from a hybridization event between group A and group B, the distribution of specific markers suggests additional complexity that may be a result of several post-hybridization events including loss of heterozygosity due to gene conversion or subsequent rounds of hybridization (inbreeding), or that the diversity within groups A and B may not have been sampled adequately. We next examined the hybrid and parental strains in order to identify instances of strain-specific marker inheritance. There was only one intra-group SNP in non-hybrid group A (Ch36-1190, position 545, G/C); the 'G' allele was uniquely present in group A strain DM20 and three of the hybrid group strains, DM19, DM62 and DM299 ([Fig. 1](#f0005){ref-type="fig"}). This indicates that DM20 is a more similar candidate A parent of these hybrids than DM297. There was a greater degree of intra-group variation in non-hybrid group B. The group B-like Ch28 and ICD alleles present in hybrid group strain DM295 were clearly most closely related to the group B strain DM481. Where informative markers were present for hybrid group strains DM62, DM299 and DM19, the sequences indicated higher identity with group B strains DM256, DM257 and DM259 than DM481 or DM559. The high number of SNP differences separating the different likely B-like allele donors for DM295 and DM62, DM299 and DM19 suggest two independent hybridization events, although a single event followed by significant subsequent diversification cannot be ruled out. It is also possible that DM19 stems from a third independent event, particularly given its distinct MLMT profile. In summary the MLST data showed a pattern of marked but not fixed heterozygosity in the hybrids in comparison with the rarity of heterozygosity in non-hybrid group strains, similar to that observed for the MLMT data. Taken alone, microsatellite data are less robust than MLST data in identifying likely parental contributors. But taken together, both the MLMT and MLST data indicate overwhelmingly that strains DM19, DM62, DM295 and DM299 are genetic hybrids. The distribution of SNP markers allowed some tentative parent-hybrid relationships to be identified and indicated the hybrid group strains have either three independent origins or, less likely, a single origin with extensive subsequent divergence. 3.3. Kinetoplast DNA (kDNA) maxicircle-encoded CYTb sequences support uniparental mitochondrial inheritance {#s0055} ----------------------------------------------------------------------------------------------------------- SNP markers were identified in a region of the CYTb gene to determine inheritance patterns of the mitochondrial maxicircles. Six SNPs were identified spanning a 748 bp fragment of the CYTb locus. Relative positions were p19 (G, T), p350 (A, T), p499 (G, A), p566 (A, C), p583 (A, G) and p649 (G, A). Two maxicircle genotypes were present among the isolates and clones studied here, with either TTACGA or GAGAAG SNP profiles across the six SNP sites. All hybrids and derived hybrid clones (a minimum of four clones per strain) had the type GAGAAG SNP profile, consistent with uniparental inheritance of the maxicircle DNA, in contrast to the biparental inheritance of the nuclear markers. There were no other combinations of maxicircle profiles and there was no heterozygosity. The GAGAAG maxicircle profiles found in the hybrid group strains were identical in both non-hybrid A strains (DM20, DM297) and in group B strains (DM559, DM481). Within group B, both mitochondrial genotypes were present. 3.4. Flow cytometry measurement of cellular DNA content {#s0060} ------------------------------------------------------- An example of the DNA content determinations on fixed parasites of hybrid and parents, stained with the fluorescent DNA binding dye propidium iodide, is shown in [Fig. 2](#f0010){ref-type="fig"}. A reference diploid (2n) strain was included in each run and duplicate runs were performed. All isolates produced clear G1-0 (2n) and G2-M (4n) fluorescence peaks. There was no significant difference in relative DNA contents between any of the hybrid or non-hybrid strains. The results are a strong indication of diploidy in all clones that were analysed. 4. Discussion {#s0065} ============= Here we provide evidence for bona fide hybrid strains and ancestral-like genotypes selected from a natural population of L. donovani causing VL in northern Ethiopia. Our evidence is based on high resolution MLMT and novel MLST targets applied to the nuclear genomes of 11 north Ethiopian L. donovani isolates and 90 clones derived from those. Importantly, the genotypes of all of the clones corresponded with the genotypes of the original isolates, and these results were therefore not confounded by artefactual analysis of isolate mixtures. By comparing genetic profiles of the north Ethiopian isolates and their derivative clones, four of the isolates were shown to be hybrids. The hybrids were heterozygous at both MLST and MLMT nuclear loci, with significant corresponding homozygosity present among putative parental strains of groups A and B. Breakpoints within individual alleles were not detected when applied to recombination algorithms (RDP V4), suggesting the inheritance of a discrete allele from each of the parents. Strikingly, the heterozygosity in the MLST data from hybrids almost precisely matched the homozygous SNPs at the same sites in the deduced pairs of parents. Results were mirrored in the MLMT analysis, although the technique is less robust and not suitable to fully resolve parents and donors using the five loci. However, together MLST analysis and MLMT spanning nine loci proved to be powerful and complementary techniques for identifying hybrids of L. donovani. As such, the novel combination of markers may be useful for screening whole populations for hybrids when sequencing is not feasible. Not all of the four L. donovani hybrid isolates were identical and there were varying degrees of SNP differences between them. Two hybrids (DM62 and DM299) had very similar genetic profiles, possibly originating from the same event. Remaining hybrids (DM295 and DM19) were more distinct. Our data show that DM62 and DM299 appear to possess a different parent (B) allele than DM295 and we consider this suggests more than one recombination event. Hybrid DM19 was an unusual outlier exhibiting reduced heterozygosity, relative to the other hybrids, yet still retaining a hybrid signal in one MLST locus and two MLMT loci. One interpretation of these data is another recombination event, although long-term divergence from hybrids (DM62 and DM299) or divergence via gene conversion cannot be entirely excluded. Recent whole genome sequencing of 11 L. infantum isolates from a focus of cutaneous leishmaniasis (CL) in southeastern Turkey ([@b0085]) suggested that they derived from a single cross of two diverse strains followed by recombination within the population. Although one of the parental types was not present, it was likely to be from the L. donovani complex. They concluded that recombination in L. infantum is probably rare in the field as judged by the length of parental sequence blocks, and the pattern of linkage between SNPs. Concerning our data, the allelic contributions from both parental types were present and appear relatively conserved, also suggesting recent genetic exchange events. However, further sampling would be required at the population level to resolve these processes and to assess more accurately the presence and frequency of endogamy which, at high frequencies, would be characterised by a trend towards homozygosity within a population ([@b0090]). Not surprisingly, the genotypes considered here to be parental did not correspond precisely with those of predicted parents; for example one of the SNPs at the heterozygous ICD locus is missing from the putative parents. There are a number of explanations for differences between the hybrids and putative parent-like isolates. Firstly, it is most likely that multiple other genotypes circulating naturally were not sampled and some of these may be even more similar to the predicted parents. Secondly, some gene conversion or mutation is inevitable with time since hybridization, restoring homozygosity or generating divergent genotypes at some loci. Differences between the hybrids could be also accounted for by repeated selfing, although the largely corresponding heterozygous SNPs and corresponding homozygous "donors" in MLST and MLMT outputs make this unlikely. We also investigated the L. donovani maxicircle kDNA inheritance patterns by sequencing the CYTb locus. Several SNPs in CYTb were identified. Identified SNPs formed two distinct mitochondrial marker combinations among the non-hybrid L. donovani isolates and clones. As expected, for haploid mitochondrial markers no heterozygosity was observed. We found that all four hybrid isolates and their derivative clones only inherited a single maxicircle DNA genotype. Furthermore, this was the mitochondrial type genotype of DM20, one of the most likely parent-like genotypes of all four hybrids. Thus, we find uniparental inheritance of mitochondrial DNA. This accords with mitochondrial inheritance of the experimental hybrids of L. major, in which 12 of 18 hybrids inherited the mitochondrial genotype of one parent and six inherited the genotype of the other parent ([@b0005]). We determined the DNA contents of the hybrids relative to the putative parents by fluorescence activated cell sorting (FACS analysis). The FACS analysis demonstrated that hybrids, non-hybrids and resultant clones were all diploid. In contrast, stable hybrid aneuploids have been described in L. major following extensive serial passage in vitro ([@b0005; @b0035]). Reversion to diploidy over longer time periods cannot be ruled out and has been observed with aneuploid genomes that are a product of parasexual processes. One hybrid tetraploid clone of L. major, stable in vitro and passaged through mice, resulted in the recovery of diploids ([@b0035]). Although there is striking synteny at the gene content level between different Leishmania genomes, there is extensive variation in some at the level of chromosomes ([@b0025]) and the characteristics of recombination in L. donovani may not be identical to that seen in L. major. On balance, the hybrids observed here among the north Ethiopian population of L. donovani appear compatible with products of meiosis in that: there are high levels of heterozygosity relative to the parents, with clearly identifiable donor SNPs; one homologue is present from each parent and recombinant genotypes are observed across multiple loci, consisting of nuclear and microsatellite markers; there is uniparental maxicircle inheritance, and a majority 2n ploidy. The routine generation of experimental hybrids would be needed to fully address the nature of recombination in L. donovani and whether hybridization follows the pattern of orthodox meiosis. It is interesting to note that two of the reported isolates (Dm62 and Dm299) were from a single HIV co-infected patient during different episodes of the disease in northern Ethiopia. Although the two hybrids were similar, SNP differences were apparent. The presence of multiple genotypes in a single severely immunocompromised patient may increase the likelihood of recombination between genotypes within the vertebrate host ([@b0080]). Alternatively, immunocompromised individuals may be more susceptible to infection with a wider range of genotypes from a sand fly vector in which genetic exchange has occurred, as is supported by the experimental proof of capacity for genetic exchange in the sand fly. Additionally, an immunocompromised status may enable less fit hybrids to persist when they would otherwise be cleared from immune competent individuals ([@b0060]). The full consequences of genetic exchange in L. donovani are as yet uncertain. For example, do the hybrids described here also differ in their ability to exploit different hosts and vectors or cause differential pathology in humans? The L. donovani hybrid and parental isolates characterised provide an excellent opportunity for follow up analysis and for further experiments on hybridization, the mechanisms of genetic exchange and the associated generation of phenotypic diversity. Appendix A. Supplementary data {#s0075} ============================== Supplementary data 1 We wish to thank Bethlehem Getachew (Leishmaniasis Research and Diagnostic Laboratory, School of Medicine, Addis Ababa University, Ethiopia) for her help with parasite cultivation. We also thank the Leishmaniasis Research and Treatment Centres in Gondar University and Arba Minch Hospital, Ethiopia. We specifically thank Dr Zewdu Hurissa and Dr Workagegnehu Hailu (Gondar University) and Dr. Teklu Weldegebreal (Arba Minch Hospital) for their help during field studies. This work was supported by the Wellcome Trust. ![Summary of single nucleotide polymorphism distributions across four multilocus sequence typing loci (Chromosome (Ch)28, Ch36-0350, Ch36-1190 and isocitrate dehydrogenase (ICD)) in Leishmania clones. Data from the glucose-6-phosphate isomerase (GPI) locus is not shown (all sequences were identical). Isolate names are abbreviated with World Health Organization strain codes shown in [Table 1](#t0005){ref-type="table"}. DM19, DM62, DM295 and DM299 hybrids possess heterozygous single nucleotide polymorphisms. DM20 and DM297 (in colour) represent parent-like A donors and DM256, DM257, DM259, DM481 and DM559 (in colour) represent possible parent-like B donors. Full IUPAC codes for heterozygote single nucleotide polymorphisms are as follows: K(G,T); M(A,C); R(G,A), S(G,C); Y(T,C). Numbers indicate the nucleotide position within the loci. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)](gr1){#f0005} ![Flow cytometric analysis and sequencing electropherograms. (A) Flow cytometric analysis of relative DNA content in Leishmania clones. Data is plotted as a FL2 area histogram with gates were created for G1-0 (2n) peaks and for G2-M (4n) peaks and overlaid DNA histograms illustrating comparable (2n) DNA content in hybrid and parent-like isolates. (B) Electropherograms showing two variable single nucleotide polymorphisms present in homozygous non-hybrids (DM20, DM256) and the equivalent bi-allelic locus present in hybrid DM62 at the same locus.](gr2){#f0010} ###### Microsatellite profiles at five loci of hybrids and their corresponding putative parents for Leishmania isolates. Strain WHO code Marker ----------------------------------- ---------------------------------------------------- -------- ------- --------- --------- ------- Hypothetical parent A (n = 2) MHOM/ET/2007/DM20 89/89 72/72 78/78 99/99 92/92 MHOM/ET/2008/DM297 83/83 70/70 78/78 113/113 92/92    Hybrid isolates (n = 4) MHOM/ET/2007/DM19[a](#tblfn1){ref-type="table-fn"} 91/91 70/72 78/98 99/103 92/92 MHOM/ET/2007/DM62 89/91 70/72 78/98 99/103 90/92 MHOM/ET/2008/DM295 89/91 70/72 78/98 99/103 90/92 MHOM/ET/2008/DM299 89/91 70/72 78/98 99/103 90/92    Hypothetical parental B (n = 5) MHOM/ET/2008/DM256 91/91 72/72 98/98 117/117 92/92 MHOM/ET/2008/DM257 91/91 72/72 98/98 117/117 92/92 MHOM/ET/2008/DM259 83/89 72/72 98/98 117/117 92/92 MHOM/ET/2009/DM481 89/89 72/72 98/98 113/113 92/92 MHOM/ET/2009/DM559 83/89 72/72 98/98 113/113 90/92 WHO, World Health Organization. Hybrid DM19 is heterozygous at three loci unlike DM62, DM295 and DM299, which are heterozygous across all five loci, as is congruent with multilocus sequence typing (MLST) data ([Fig. 2](#f0010){ref-type="fig"}). ###### Properties of microsatellite markers used in this study. Marker T~A~ (°C) Chromosome Forward (5′--3′) Reverse (5′--3) --------- ------------- ------------ ------------------------ ----------------------- Li22-35 52 1 CTTGATGTTCGGGTTAGCAAG ATGCACACCAAAAATCATGTG Li41-56 50 36 TTGCTTCATGATAACAACTTGG CCTGTTGGTGTGAGTTCGTG Li46-67 50 31 TCTTCTTTCGTTAGCTGAGTGC CTGTATCACCCATGAGGGGC Li71-7 50 30 GCTGCAGCAGATGAGAAGG GTGAGAAGGCAGGGATTCAA Li71-33 50 31 CTCCTTTCACACCCGCCTCT GAGAGAAGACGAGCCGAAGT T~A~, temperature of annealing. ###### Multilocus sequence typing targets for Leishmania donovani. ------------------------------------------------------------------------------------------------------------------------------------------------------------------- Locus Chromosome number No. of polymorphic sites/SNPs Primer sequences (5′--3′) Annealing temp (T~A~ °C) bp -------------------------------------------- ------------------- ------------------------------- ------------------------------ ---------------------------- ------ GPI[a](#tblfn2){ref-type="table-fn"} 12 0 F:GACCGAGGCACTTGAAG\ 57 1150 R6: TGAATGAGCTGGTAGAATG ICD[a](#tblfn2){ref-type="table-fn"} 10 5 F:ATGTTCCGCCATGTTTCGG\ 61 1267 R:TTACGCGCTCATCGCCTT Ch28[a](#tblfn2){ref-type="table-fn"} 28 18 INTF:GTCGCAGTCCAACTCCCATA\ 53 1530 INTR:CGCATAGCAAAAGCCAAA Ch36-1190[a](#tblfn2){ref-type="table-fn"} 36 8 F:GCTTCTCGCTATTGCTCGTC\ 59 1640 R:ACTGGCAGGCACACATCAG\ INTFA: GCGGCTACCTCGCCCTCAGT\ INTFB:GTGAAGGACCAAGCTGCCTGG\ INTRA;TGTGAAGCACCAGCAGGACGG Ch36-0350[b](#tblfn3){ref-type="table-fn"} 36 3 F:ACTTGGTCTTGGTACGG\ 61 850 R:TGGAGGACGGAGAGACTTTG\ INTF:GTGAATGGAGGGCAGACG\ INTR:GTCGTGAAAAGCGAGAAGGT ------------------------------------------------------------------------------------------------------------------------------------------------------------------- SNP, single nucleotide polymorphism; T~A~, temperature of annealing; GPI, glucose-6-phosphate isomerase; ICD, isocitrate dehydrogenase; Ch, chromosome. 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"found", "hybrid", "group", "strains", "identical", "strains", "group", "B", "strains", "Within", "group", "B", "mitochondrial", "genotypes", "present", "Flow", "cytometry", "measurement", "cellular", "DNA", "content", "example", "DNA", "content", "determinations", "fixed", "parasites", "hybrid", "parents", "stained", "fluorescent", "DNA", "binding", "dye", "propidium", "iodide", "shown", "Fig", "fig", "reference", "diploid", "strain", "included", "run", "duplicate", "runs", "performed", "isolates", "produced", "clear", "fluorescence", "peaks", "significant", "difference", "relative", "DNA", "contents", "hybrid", "strains", "results", "strong", "indication", "diploidy", "clones", "analysed", "Discussion", "provide", "evidence", "bona", "fide", "hybrid", "strains", "genotypes", "selected", "natural", "population", "donovani", "causing", "VL", "northern", "Ethiopia", "evidence", "based", "high", "resolution", "MLMT", "novel", "MLST", "targets", "applied", "nuclear", "genomes", "north", 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"parents", "donors", "using", "five", "loci", "However", "together", "MLST", "analysis", "MLMT", "spanning", "nine", "loci", "proved", "powerful", "complementary", "techniques", "identifying", "hybrids", "donovani", "novel", "combination", "markers", "may", "useful", "screening", "whole", "populations", "hybrids", "sequencing", "feasible", "four", "donovani", "hybrid", "isolates", "identical", "varying", "degrees", "SNP", "differences", "Two", "hybrids", "similar", "genetic", "profiles", "possibly", "originating", "event", "Remaining", "hybrids", "distinct", "data", "show", "appear", "possess", "different", "parent", "B", "allele", "consider", "suggests", "one", "recombination", "event", "Hybrid", "unusual", "outlier", "exhibiting", "reduced", "heterozygosity", "relative", "hybrids", "yet", "still", "retaining", "hybrid", "signal", "one", "MLST", "locus", "two", "MLMT", "loci", "One", "interpretation", "data", "another", "recombination", "event", "although", "divergence", "hybrids", "divergence", "via", "gene", "conversion", "entirely", "excluded", "Recent", "whole", "genome", "sequencing", "infantum", "isolates", "focus", "cutaneous", "leishmaniasis", "CL", "southeastern", "Turkey", "suggested", "derived", "single", "cross", "two", "diverse", "strains", "followed", "recombination", "within", "population", "Although", "one", "parental", "types", "present", "likely", "donovani", "complex", "concluded", "recombination", "infantum", "probably", "rare", "field", "judged", "length", "parental", "sequence", "blocks", "pattern", "linkage", "SNPs", "Concerning", "data", "allelic", "contributions", "parental", "types", "present", "appear", "relatively", "conserved", "also", "suggesting", "recent", "genetic", "exchange", "events", "However", "sampling", "would", "required", "population", "level", "resolve", "processes", "assess", "accurately", "presence", "frequency", "endogamy", "high", "frequencies", "would", "characterised", "trend", "towards", "homozygosity", "within", 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"figure", "legend", "reader", "referred", "web", "version", "article", "Flow", "cytometric", "analysis", "sequencing", "electropherograms", "Flow", "cytometric", "analysis", "relative", "DNA", "content", "Leishmania", "clones", "Data", "plotted", "area", "histogram", "gates", "created", "n", "peaks", "n", "peaks", "overlaid", "DNA", "histograms", "illustrating", "comparable", "n", "DNA", "content", "hybrid", "isolates", "B", "Electropherograms", "showing", "two", "variable", "single", "nucleotide", "polymorphisms", "present", "homozygous", "equivalent", "locus", "present", "hybrid", "locus", "Microsatellite", "profiles", "five", "loci", "hybrids", "corresponding", "putative", "parents", "Leishmania", "isolates", "Strain", "code", "Marker", "Hypothetical", "parent", "n", "Hybrid", "isolates", "n", "Hypothetical", "parental", "B", "n", "World", "Health", "Organization", "Hybrid", "heterozygous", "three", "loci", "unlike", "heterozygous", "across", "five", "loci", "congruent", "multilocus", "sequence", "typing", "MLST", "data", "Fig", "fig", "Properties", "microsatellite", "markers", "used", "study", "Marker", "Chromosome", "Forward", "Reverse", "CTTGATGTTCGGGTTAGCAAG", "ATGCACACCAAAAATCATGTG", "TTGCTTCATGATAACAACTTGG", "CCTGTTGGTGTGAGTTCGTG", "TCTTCTTTCGTTAGCTGAGTGC", "CTGTATCACCCATGAGGGGC", "GCTGCAGCAGATGAGAAGG", "GTGAGAAGGCAGGGATTCAA", "CTCCTTTCACACCCGCCTCT", "GAGAGAAGACGAGCCGAAGT", "temperature", "annealing", "Multilocus", "sequence", "typing", "targets", "Leishmania", "donovani", "Locus", "Chromosome", "number", "polymorphic", "Primer", "sequences", "Annealing", "temp", "bp", "GPI", "F", "TGAATGAGCTGGTAGAATG", "ICD", "F", "R", "TTACGCGCTCATCGCCTT", "INTF", "INTR", "CGCATAGCAAAAGCCAAA", "F", "R", "INTFA", "INTFB", "INTRA", "TGTGAAGCACCAGCAGGACGG", "b", "F", "R", "INTF", "INTR", "GTCGTGAAAAGCGAGAAGGT", "SNP", "single", "nucleotide", "polymorphism", "temperature", "annealing", "GPI", "isomerase", "ICD", "isocitrate", "dehydrogenase", "Ch", "chromosome", "Coding", "nuclear", "marker", "nuclear", "marker" ]
T[he Biobanking and]{.smallcaps} Cohort Network (BCNet) was established by the International Agency for Research on Cancer (IARC) in 2013, as an opportunity for low- and middle-income countries (LMICs) to work together in a coordinated manner to jointly address many of the challenges in biobanking infrastructure, the acquisition and maintenance of high-quality samples and data, and governance and regulatory frameworks to guide in the sharing and reuse of resources for research. The BCNet currently has 34 LMIC member organizations from across the world and 13 partner organizations that have biobanking in their core activities. The BCNet initiative arose from the realization that despite improvements in developed countries, population cohorts and biobanking facilities are either underdeveloped or nonexistent in LMICs.^[@B1]^ In this context and in line with IARC\'s mission to contribute to worldwide cancer research, BCNet was set up as an opportunity for LMICs to work together in a coordinated and effective manner and jointly address the many challenges in biobanking infrastructure, including ethical, legal, and social issues.^[@B2],[@B3]^ In addition, BCNet facilitates the sharing of resources (e.g., expertise and protocols) and the development of joint projects, strengthening the competitiveness of LMIC biobanks in applying for international funding. IARC\'s Dr. Maimuna Mendy has expertly coordinated the BCNet with the help of a steering committee team focused on education and training, ethics and legal issues, and informatics (<http://bcnet.iarc.fr>). The BCNet steering committee has organized several training initiatives and resources; it has focused on the informatics necessary to support LMIC biobanking, in partnership with B3Africa; and has examined ethics and legal issues within and across LMIC biobanking, including sample and data sharing.^[@B4]^ The International Society for Biological and Environmental Repositories (ISBER) is one of the founding partner organizations of the BCNet and has taken a key role in the education and training portion of its outreach activities. ISBER entered into a partnership agreement with IARC-BCNet in 2015 to allow very low-cost access to the ISBER educational platform and the ISBER online biobanking training modules, Introduction to Biobanking, developed by the Canadian Tissue Resource Network. The purpose of the course is to give the viewer a general overview of the key issues in establishing, maintaining, and accessing a biobank. The education program includes nine online modules designed to provide "how-to" knowledge for researchers and biobankers. This course is currently available in the English and French languages. Now ISBER, in line with its mission to strengthen international collaboration^[@B5]^ and based on further recent evidence,^[@B6]^ has entered into a stronger partnership agreement with IARC BCNet. ISBER has made a firm commitment over the past few years to lower costs for resource-constrained biobankers and organizations, in addition to the partnership with IARC-BCNet. The new agreement between the two organizations welcomes all registered LMIC organizations that are members of BCNet into ISBER in a complimentary membership capacity. This new status provides BCNet members with full access to ISBER tools, including the ISBER forum discussion board, participation in working groups and committees, the ISBER best practices, science policy resources, and recorded conference sessions and webinars. In addition, BCNet members will be able to communicate with the ISBER membership through the provision of complimentary news features and the creation of a joint working group or task force for developing further educational material that would be LMICs specific and freely available to the members of both organizations. ISBER looks forward to this newly strengthened relationship to support our strategic commitment to education and harmonization of best practices in biobanking across the world, in all resource settings. 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An outbreak of severe acute respiratory syndrome (SARS) began in Guangdong, China, on November 16, 2002. The first three SARS cases in Singapore were confirmed on March 6, 2003. By May 5, a total of 204 cases, including 27 deaths, had been confirmed. The last case was isolated on May 11, and by July 30, the end of the outbreak, 205 patients had recovered and 33 had died ([@R1]). Since SARS infection may come from ordinary contact with acquaintances, colleagues, or strangers, outbreaks can trigger anxiety and influence public perception of susceptibility, causing serious economic and social disruption. The need for health information and for crisis management by public health authorities is also high. We examine four areas of public reaction to the SARS outbreak in Singapore: preventive practices, perception of self-health, knowledge of SARS, and appraisal of SARS crisis management. Materials and Methods ===================== Sample ------ We interviewed a representative stratified random sample of 1,202 adults (≥21 years of age). To minimize personal contact during the outbreak, participants were interviewed by telephone instead of face-to-face. The residential telephone sampling covered 90% of households in Singapore. The response rate was 62.3%, and the sampling error ±3.5% ([Table 1](#T1){ref-type="table"}). We used Random Digit Dialing+1, a system commonly used in public health studies, to capture unlisted telephone numbers ([@R3]). ###### Demographic characteristics of study and total population Characteristics Study population Total population^a^ ------------------------------------------ --------------------------------------------- ------------------------------------------------------------- Ethnicity Chinese Malay Indian Other 900 (75.0) 172 (14.0) 82 (7.0) 47 (4.0) 2,505,400 (76.8) 453,600 (13.9) 257,800 (7.9) 46,400 (1.4) Age 21--29^b^ 30--39 40--49 50 and older 233 (19.0) 313 (26.0) 312 (26.0) 343 (28.0) 480,191 (20.4) 613,944 (26.1) 575,674 (24.5) 681,282 (29.0) Sex Male Female 599 (49.9) 602 (50.1) 1,630,293 (49.9) 1,632,916 (50.1) ^a^Ref. [@R2]. ^b^Total population figures refer to ages 20--29 years. Data Collection --------------- We modified and expanded a structured questionnaire provided by researchers from the Department of Community Medicine, University of Hong Kong (A.J. Hedley, T.H. Tan, G.M. Leung, B.H.Y. Chan, S.Y. Ho, L.M. Ho, unpub. data). The modified questionnaire ([Appendix](#SD1){ref-type="local-data"}) was translated into Mandarin, Malay, and English; interviews were conducted from May 5 to May 10, 2003. Factor analysis and logistic regression (SPSS for Windows \[Version 11.5\]) examined trends among four factors (SARS prevention, perception of self-health, knowledge of SARS, and perception of health authorities' crisis management). We also assessed how prevention measures correlated with other factors, including respondents' demographic characteristics. Preventive Measures ------------------- Eight questions focused on respondents' prevention practices in the 3 days before the interview. We constructed a composite index indicating the total number (from 0 to 8) of preventive measures taken. A dichotomous indicator of preventive behavior was calculated based on the mean number of precautions taken (4.68): "low" (≤5) versus "high" (≥6). Self-Health Perception ---------------------- Three sets of questions addressed respondents' perception of their own health. The first set covered nine physical health complaints. We created a composite index of symptoms by adding all instances of health complaints over the previous 2 weeks. This index was 0 to 7 in our study since no one reported having more than seven of the nine symptoms. The second set was a "frame of mind" index fashioned after B.A. Thyer's Clinical Anxiety Scale ([@R4]). Scores for positive items were 1 (not at all) to 4 (very much); negative item scores were reversed, so lower total scores indicated higher anxiety. The scale had an Alpha reliability coefficient of 0.8244. The third set addressed respondents' perceived susceptibility to SARS. Scores were 4 (very likely) to 0 (don't know). On the basis of the average score (1.5; standard deviation \[SD\] 1.01), we created a dichotomous variable to contrast respondents who believed they were susceptible to contracting SARS (scores 3 and 4) with those who did not (scores 0--2). Knowledge of SARS ----------------- Three questions tested SARS knowledge. Responses were scored 0 (incorrect) or 1 (correct); a composite index indicated the number of correct answers, from none correct (0) to all three correct ([@R3]). Appraisal of Crisis Management ------------------------------ Four sets of questions addressed respondents' appraisal of crisis management, but we discuss the three most relevant. The first set of five questions (Alpha reliability 0.8136) assessed opinions on information distribution. Scores were 1 (very negative) to 6 (very positive). On the basis of the mean score (4.83; SD 0.617), we calculated a dichotomous index: negative appraisal (scores ≤4.7) versus positive appraisal (scores ≥4.8). The second set of questions addressed openness of communication. Scores were 1 (very negative) to 6 (very positive). By using the sample's mean score (4.31; SD 1.25), this variable was dichotomized into "disagreement" (scores 1--3) and "agreement" (scores 4--6). The third set referred to the public's acceptance of quarantine regulations. The scores were dichotomized into "agreement" ([@R1]) versus "no agreement" and "don't know" ([@R2]). Results ======= Responses to the survey questions are summarized in [Table 2](#T2){ref-type="table"}. Variables were examined by using odds ratios (ORs) at 95% confidence intervals (CI). The statistically significant ORs are reported in [Table 3](#T3){ref-type="table"} with their respective level of significance from the Mantel-Haenszel common odds ratio estimates. ###### Variables used in analysis of public reaction and perspective of SARS crisis Variable \% Mean SD ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------- -------- -------- Symptoms (0--8) over past 2 weeks None One or more Main SARS-related symptoms Persistent high fever ≥38°C Cough Rapid breathing 77.6 22.4 1.0 9.0 1.0 0.3639 0.8286 Anxiety level High (1.0--2.2) Moderate (2.3--3.2) Low (3.3--4.0) 2.9 42.4 54.7 3.2307 .4819 Perceived likelihood of contracting SARS Very likely (4) Likely (3) Not very likely (2) Not likely at all (1) Don't know (0) 4.0 10.0 39.0 29.0 18.0 1.5304 1.014 Knowledge of SARS No knowledge (0 of 3 answers correct) (0) 1 of 3 answers correct (1) 2 of 3 answers correct (2) 3 of 3 answers correct (3) 11.7 25.0 42.5 20.7 1.7227 .9222 Appraisal of crisis management "Strongly agree" and "Agree" that information by health authorities is: Accurate Clear Sufficient Timely Trustworthy Population has chance to express personal views and concerns to the authorities, "strongly agree" or "agree." Agreeable to 10-day quarantine after nonclose contact with SARS-infected person and no symptoms Agree Don't agree Don't know 82.2 86.3 84.5 84.4 87.8 66.3 71.6 22.4 6.0 Years of formal education ≤10 years ≥11 years 57.1 42.9 10.07 3.9642 Practice of preventive measures Practicing each of eight measures "always" or "most of the time" during the past 3 days: Covered mouth with tissue when sneezing or coughing Covered mouth with bare hand when sneezing or coughing Washed hands after sneezing, coughing, or clearing nose Used soap or liquid hand-wash when washing hands Wore a mask Used serving utensils for shared food Took preventive measures when touching objects Washed hands after touching objects Preventive measures taken over past 3 days (score 0--8) Five or fewer of the eight measures Six or more of the eight measures 62.0 47.0 72.0 81.0 4.0 28.0 15.0 48.0 69.3 30.7 4.686 1.5286 ###### Practice of SARS preventive measures, 3 days before interviews^a^ Variable No. OR 95% CI ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------- ------------------------------------- ------------------------------------------------------------------------------------------- Personal health evaluation Symptoms in past 2 weeks None One or more Anxiety^b^ Moderate or high (score ≤3.25) Low anxiety (score \>3.25) Perceived likelihood of SARS Not likely Likely 932 269 544 657 1,034 167 1.012 0.960 0.861 1.140 1.031 0.833 0.947 to 1.082 0.766 to 1.203 0.757 to 0.978 1.031 to 1.283 0.979 to 1.085 0.621 to 1.118 Knowledge of SARS Two or fewer correct answers Three correct answers 952 249 1.012 0.954 0.950 to 1.079 0.753 to 1.079 Appraisal of crisis management Quality of official information Below average (negative) Above average (positive) Have chance to express opinion^c^ Disagree Agree Agreeable to quarantine when non-close contact with SARS-infected person and no symptoms Agree Do not agree or don't know 290 911 271 930 860 341 1.164 0.955 1.434 0.909 0.969 1.084 0.928 to 1.460 0.893 to 1.020 1.115 to 1.846 0.855 to 0.966 0.899 to 1.045 0.888 to 1.323 Demographic characteristics Years of formal education^d^ ≤10 \>10 Sex^c^ Male Female Age^c^ (y) \<35 ≥35 686 515 599 602 391 809 0.909 1.143 1.339 0.770 1.365 .872 0.821 to 1.006 0.985 to 1.325 1.166 to 1.539 0.689 to 0.861 1.123 to 1.658 0.806 to 0.943 ^a^SARS, severe acute respiratory syndrome; OR, odds ratio; CI, 95% confidence interval. ^b^Asymptotic significance (2-sided) ≤0.05. ^c^Asymptotic significance (2-sided) ≤0.001. ^d^Asymptotic significance (2-sided) ≤0.10. Recommended preventive measures were not practiced uniformly. The most practiced measures 3 days before the interview were using soap when washing hands (81%) and washing hands after sneezing, coughing, or clearing the nose (72%). The least practiced measure was wearing a mask over the mouth. A total of 4% wore masks, and most did so only when visiting a clinic or hospital or when the mask was part of a uniform (as in healthcare workers). The index of preventive measures indicates that most people (69.3%) took some preventive measures. Respondents' perception of their health was generally positive. A relatively low proportion (22.4%) of respondents reported having any of our nine physical health complaints over the previous 2 weeks, and fewer than 1% reported the three classic symptoms of SARS (fever ≥38°C, cough, rapid breathing). The mean number of health complaints reported in our sample was 0.369 (SD 0.828). The survey also showed low anxiety; only 2.9% of respondents reported high anxiety. The mean anxiety score was 3.23 (SD 0.48). Most respondents (68%) thought they were not very likely or not likely at all to contract SARS, and 18% were not sure of their likelihood. Those who thought they were likely to get the disease reported slightly more anxiety. Of the three aspects of health perception, only anxiety was associated with taking precautions (OR 0.861; 95% CI 0.757--0.978). In the high-anxiety group, 34% followed six or more of the eight preventive measures, in contrast to 28% of respondents who had low anxiety. Regarding knowledge of SARS, the sample correctly answered an average of 1.722 (SD 0.922) of 3 questions on SARS transmission. Approximately 63% answered two or more questions correctly; 11.7% did not answer any questions correctly. Respondents had a generally high opinion of authorities' crisis management. More than 80% thought official information was accurate, clear, sufficient, timely, and trustworthy, and 72% were prepared to accept a 10-day quarantine, even in the absence of SARS symptoms or close contact with a SARS patient. Of the three crisis management aspects, one had significant influence on preventive action: respondents' opinion of authorities' openness to communication. People who thought that authorities were open to communication were more inclined to practice six or more of the eight SARS preventive measures (OR 0.909; 95% CI 0.855 to 0.966) than those who thought they had no chance to express their concerns to the authorities (OR 1.434; 95% CI 1.115 to 1.846). Three demographic characteristics were associated with taking preventive measures against SARS: sex, age, and estimated years of formal education. Women were more inclined (OR 0.770; 95% CI 0.689 to 0.861) than men (OR 1.339; 95% CI 1.166 to 1.539) to take preventive measures; this finding is consistent with other studies on health behavior in Singapore ([@R5],[@R6]). People ≥35 years of age were more inclined to take preventive measures (OR 0.872; 95% CI 0.806 to 0.943) than their younger counterparts (OR 1.365; 95% CI 1.123 to 1.658). The association with education disappeared when controlled for sex. Discussion ========== Information regarding the SARS outbreak was widely distributed by the media and government; while this information was essential to keep the public informed of the risks for infection and preventive measures, it also could increase anxiety. However, we found low levels of anxiety in Singapore, and few reported health complaints. Reporting health complaints was not associated with taking precautions against SARS, possibly because the nine symptoms of SARS covered in our questionnaire are associated with other common diseases in Singapore (e.g., dengue fever, the incidence of which was 86.2 per 100,000 in May 2003) and are not usually deemed serious. In fact, familiarity with symptoms was a key initial obstacle in preventing SARS spread in hospitals ([@R7]) and remains an impediment to raising community alertness. In our sample, anxiety appeared to motivate preventive behavior; those in the highest anxiety group took more precautions. However, anxiety was not associated with the perceived likelihood of contracting SARS. The low percentage of respondents who viewed SARS as a personal risk (14%, compared to 22% found in a similar survey in Toronto \[[@R8]\]) could be explained by the fact that healthcare workers were among the first SARS patients. By the time the interviews began, two physicians had died, and two hospitals had clusters of cases. Lay respondents (those with no contact with hospitals or healthcare workers) may have perceived SARS an occupational hazard. Distribution of SARS information and prevention advice in Singapore increased rapidly over the 2 months preceding the interviews. All types of media were used, including a public television channel, the "SARS Channel," established to give current and comprehensive information on world infection trends and Singapore's situation. The Ministry of Health provided SARS information on its Web site ([@R9]), taking advantage of the fact that, as of December 2001, Singapore had 1.9 million Internet subscribers (out of 3.3 million population) ([@R10]). Of respondents, 20.7% were able to correctly answer all three SARS questions, and these did not differ in the practice of preventive measures from those who had less SARS knowledge. The absence of a correlation between knowledge and behavior confirms that knowledge of a disease is not sufficient to trigger preventive action ([@R5],[@R6],[@R11]--[@R13]). Since SARS appeared unexpectedly, healthcare experts were uncertain how to control the epidemic. Consequently, assessing public opinion of authorities' crisis management in our survey was relevant to Singapore. Of the aspects we examined, only public opinion of authorities' openness to communication was correlated with taking preventive measures. The other two aspects (information dissemination and acceptance of quarantine regulations) did not affect preventive action, probably because of their very positive rating. The public's highly positive assessment of Singapore authorities' crisis management is distinctive. History shows that epidemics are politically perilous to governments as, among other things, they challenge their resolve, efficiency, and state of readiness ([@R14]). Political leaders of other SARS-affected Asian countries witnessed this principle directly. The SARS outbreak in Singapore appears to have worked in an opposite way: it corroborated the usefulness of public health and environmental regulations. In addition, this study's findings parallel the population's response to quarantine and other restrictive measures, confirming previous observations of a relatively high level of social discipline in the population ([@R15],[@R16]). Conclusion ========== Singapore was taken out of the official list of SARS-infected countries by the World Health Organization on May 30, 2003. The epidemic has left the crisis phase and entered a new phase, normalization and vigilance. As a new disease, SARS demands continuous scrutiny on all fronts, from the laboratory to the homes of the people. Supplementary Material ====================== ###### Appendix Questionnaire given to SARS survey respondents Suggested citation for this article: Quah SR, Lee H-P. Crisis prevention and management during SARS outbreak, Singapore. Emerg Infect Dis \[serial online\] Feb 2004 \[date cited\]. Available from: URL: <http://www.cdc.gov/ncidod/EID/vol10no2/03-0418.htm> We express our appreciation to the persons who consented to be interviewed for this study; John Wong, Director of the National University of Singapore--Office of Life Sciences, who facilitated the grant application; Calvin Fones, David Koh, and Paulin Straughan for their comments and suggestions on the questionnaire; two anonymous reviewers for their constructive feedback; and A.J. Hedley for sending us the questionnaire his research team applied in Hong Kong (April 2003) and suggesting that we apply it in Singapore. This study was supported by National University of Singapore research grant R-111-000-045-712. Dr. Quah is a professor of sociology at the National University of Singapore. Among her areas of interest are medical sociology and social policy. Dr. Lee is professor of public health at the National University of Singapore. 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"SARS", "knowledge", "answers", "correct", "answers", "correct", "answers", "correct", "answers", "correct", "Appraisal", "crisis", "management", "Strongly", "agree", "Agree", "information", "health", "authorities", "Accurate", "Clear", "Sufficient", "Timely", "Trustworthy", "Population", "chance", "express", "personal", "views", "concerns", "authorities", "strongly", "agree", "agree", "Agreeable", "quarantine", "nonclose", "contact", "person", "symptoms", "Agree", "agree", "know", "Years", "formal", "education", "years", "years", "Practice", "preventive", "measures", "Practicing", "eight", "measures", "always", "time", "past", "days", "Covered", "mouth", "tissue", "sneezing", "coughing", "Covered", "mouth", "bare", "hand", "sneezing", "coughing", "Washed", "hands", "sneezing", "coughing", "clearing", "nose", "Used", "soap", "liquid", "washing", "hands", "Wore", "mask", "Used", "serving", "utensils", "shared", "food", "Took", "preventive", "measures", "touching", "objects", "Washed", "hands", "touching", "objects", "Preventive", "measures", "taken", "past", "days", "score", "Five", "fewer", "eight", "measures", "Six", "eight", "measures", "Practice", "SARS", "preventive", "measures", "days", "Variable", "CI", "Personal", "health", "evaluation", "Symptoms", "past", "weeks", "None", "One", "Moderate", "high", "score", "Low", "anxiety", "score", "Perceived", "likelihood", "SARS", "likely", "Likely", "Knowledge", "SARS", "Two", "fewer", "correct", "answers", "Three", "correct", "answers", "Appraisal", "crisis", "management", "Quality", "official", "information", "average", "negative", "average", "positive", "chance", "express", "Disagree", "Agree", "Agreeable", "quarantine", "contact", "person", "symptoms", "Agree", "agree", "know", "Demographic", "characteristics", "Years", "formal", "Male", "Female", "severe", "acute", "respiratory", "syndrome", "odds", "ratio", "CI", "confidence", "interval", "significance", "significance", "significance", "Recommended", "preventive", "measures", "practiced", "uniformly", "practiced", "measures", "days", "interview", "using", "soap", "washing", "hands", "washing", "hands", "sneezing", "coughing", "clearing", "nose", "least", "practiced", "measure", "wearing", "mask", "mouth", "total", "wore", "masks", "visiting", "clinic", "hospital", "mask", "part", "uniform", "healthcare", "workers", "index", "preventive", "measures", "indicates", "people", "took", "preventive", "measures", "Respondents", "perception", "health", "generally", "positive", "relatively", "low", "proportion", "respondents", "reported", "nine", "physical", "health", "complaints", "previous", "weeks", "fewer", "reported", "three", "classic", "symptoms", "SARS", "fever", "cough", "rapid", "breathing", "mean", "number", "health", "complaints", "reported", "sample", "SD", "survey", "also", "showed", "low", "anxiety", "respondents", "reported", "high", "anxiety", "mean", "anxiety", "score", "SD", "respondents", "thought", "likely", "likely", "contract", "SARS", "sure", "likelihood", "thought", "likely", "get", "disease", "reported", "slightly", "anxiety", "three", "aspects", "health", "perception", "anxiety", "associated", "taking", "precautions", "CI", "group", "followed", "six", "eight", "preventive", "measures", "contrast", "respondents", "low", "anxiety", "Regarding", "knowledge", "SARS", "sample", "correctly", "answered", "average", "SD", "questions", "SARS", "transmission", "Approximately", "answered", "two", "questions", "correctly", "answer", "questions", "correctly", "Respondents", "generally", "high", "opinion", "authorities", "crisis", "management", "thought", "official", "information", "accurate", "clear", "sufficient", "timely", "trustworthy", "prepared", "accept", "quarantine", "even", "absence", "SARS", "symptoms", "close", "contact", "SARS", "patient", "three", "crisis", "management", "aspects", "one", "significant", "influence", "preventive", "action", "respondents", "opinion", "authorities", "openness", "communication", "People", "thought", "authorities", "open", "communication", "inclined", "practice", "six", "eight", "SARS", "preventive", "measures", "CI", "thought", "chance", "express", "concerns", "authorities", "CI", "Three", "demographic", "characteristics", "associated", "taking", "preventive", "measures", "SARS", "sex", "age", "estimated", "years", "formal", "education", "Women", "inclined", "CI", "men", "CI", "take", "preventive", "measures", "finding", "consistent", "studies", "health", "behavior", "Singapore", "People", "years", "age", "inclined", "take", "preventive", "measures", "CI", "younger", "counterparts", "CI", "association", "education", "disappeared", "controlled", "sex", "Discussion", "Information", "regarding", "SARS", "outbreak", "widely", "distributed", "media", "government", "information", "essential", "keep", "public", "informed", "risks", "infection", "preventive", "measures", "also", "could", "increase", "anxiety", "However", "found", "low", "levels", "anxiety", "Singapore", "reported", "health", "complaints", "Reporting", "health", "complaints", "associated", "taking", "precautions", "SARS", "possibly", "nine", "symptoms", "SARS", "covered", "questionnaire", "associated", "common", "diseases", "Singapore", "dengue", "fever", "incidence", "per", "May", "usually", "deemed", "serious", "fact", "familiarity", "symptoms", "key", "initial", "obstacle", "preventing", "SARS", "spread", "hospitals", "remains", "impediment", "raising", "community", "alertness", "sample", "anxiety", "appeared", "motivate", "preventive", "behavior", "highest", "anxiety", "group", "took", "precautions", "However", "anxiety", "associated", "perceived", "likelihood", "contracting", "SARS", "low", "percentage", "respondents", "viewed", "SARS", "personal", "risk", "compared", "found", "similar", "survey", "Toronto", "could", "explained", "fact", "healthcare", "workers", "among", "first", "SARS", "patients", "time", "interviews", "began", "two", "physicians", "died", "two", "hospitals", "clusters", "cases", "Lay", "respondents", "contact", "hospitals", "healthcare", "workers", "may", "perceived", "SARS", "occupational", "hazard", "Distribution", "SARS", "information", "prevention", "advice", "Singapore", "increased", "rapidly", "months", "preceding", "interviews", "types", "media", "used", "including", "public", "television", "channel", "SARS", "Channel", "established", "give", "current", "comprehensive", "information", "world", "infection", "trends", "Singapore", "situation", "Ministry", "Health", "provided", "SARS", "information", "Web", "site", "taking", "advantage", "fact", "December", "Singapore", "million", "Internet", "subscribers", "million", "population", "respondents", "able", "correctly", "answer", "three", "SARS", "questions", "differ", "practice", "preventive", "measures", "less", "SARS", "knowledge", "absence", "correlation", "knowledge", "behavior", "confirms", "knowledge", "disease", "sufficient", "trigger", "preventive", "action", "Since", "SARS", "appeared", "unexpectedly", "healthcare", "experts", "uncertain", "control", "epidemic", "Consequently", "assessing", "public", "opinion", "authorities", "crisis", "management", "survey", "relevant", "Singapore", "aspects", "examined", "public", "opinion", "authorities", "openness", "communication", "correlated", "taking", "preventive", "measures", "two", "aspects", "information", "dissemination", "acceptance", "quarantine", "regulations", "affect", "preventive", "action", "probably", "positive", "rating", "public", "highly", "positive", "assessment", "Singapore", "authorities", "crisis", "management", "distinctive", "History", "shows", "epidemics", "politically", "perilous", "governments", "among", "things", "challenge", "resolve", "efficiency", "state", "readiness", "Political", "leaders", "Asian", "countries", "witnessed", "principle", "directly", "SARS", "outbreak", "Singapore", "appears", "worked", "opposite", "way", "corroborated", "usefulness", 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"Director", "National", "University", "Singapore", "Office", "Life", "Sciences", "facilitated", "grant", "application", "Calvin", "Fones", "David", "Koh", "Paulin", "Straughan", "comments", "suggestions", "questionnaire", "two", "anonymous", "reviewers", "constructive", "feedback", "Hedley", "sending", "us", "questionnaire", "research", "team", "applied", "Hong", "Kong", "April", "suggesting", "apply", "Singapore", "study", "supported", "National", "University", "Singapore", "research", "grant", "Quah", "professor", "sociology", "National", "University", "Singapore", "Among", "areas", "interest", "medical", "sociology", "social", "policy", "Lee", "professor", "public", "health", "National", "University", "Singapore", "Among", "areas", "interest", "cancer", "epidemiology", "community", "medicine" ]
1. Introduction {#sec1-ijms-18-00554} =============== Resveratrol is a stilbene-based, polyphenolic compound occurring naturally in nuts, berries, and the skin of grapes, and is present in many food items, particularly red wine \[[@B1-ijms-18-00554]\]. In recent years, the clinical effects of resveratrol have been studied intensively owing to a plethora of promising effects in cells and animals; among these are improvement in terms of the various consequences of obesity, e.g., impaired insulin sensitivity and low-grade inflammation \[[@B2-ijms-18-00554],[@B3-ijms-18-00554],[@B4-ijms-18-00554]\]. A landmark study demonstrated a shortened life span in diet-induced obese mice and a resveratrol-mediated improvement in longevity despite obesity \[[@B5-ijms-18-00554]\]. Owing to its purported salutary effects, resveratrol is already being sold as an over-the-counter dietary supplement. Unfortunately, translating the effects of resveratrol to a human setting has proven difficult. Some clinical studies have confirmed the pre-clinical results of resveratrol-mediated improved insulin sensitivity, diminished low-grade inflammation, reduced blood pressure, and reduced amounts of liver fat accumulation \[[@B6-ijms-18-00554],[@B7-ijms-18-00554]\]. In contrast, other studies fail to detect any positive physiological effects \[[@B8-ijms-18-00554],[@B9-ijms-18-00554]\]. These conflicting results may in part be attributable to differences in study designs, populations, and resveratrol formulations. Some studies have been performed in healthy individuals, and the ability of resveratrol to improve glucose handling in situations with normal glucose homoeostasis has been questioned \[[@B8-ijms-18-00554]\]. In contrast, it is feasible that the effect of a food ingredient is too weak to demonstrate any measurable consequences in full-blown type 2 diabetes. Thus, subjects suffering from modest degrees of insulin resistance are most likely optimal for the purpose of investigating the possible effects of resveratrol on insulin sensitivity, and probably most comparable to the original mice studies using diet-induced obese mice. Study length may also be of importance. Most studies include intervention periods of only a few weeks \[[@B6-ijms-18-00554],[@B9-ijms-18-00554]\]. Obviously, longer studies provide a better determination of the possible effects of resveratrol on chronic conditions such as insulin resistance and low-grade inflammation. Finally, the methods currently used in most studies on resveratrol are targeted towards whole-body effects. Insulin sensitivity, for instance, has been assessed via measurements of specific targets such as plasma insulin, glucose, and HbA1c \[[@B6-ijms-18-00554],[@B8-ijms-18-00554],[@B9-ijms-18-00554]\]. These techniques have their strengths, such as being very specific and applicable in daily clinics; however, the drawback is that findings will be limited to the pre-specified objectives. Likewise, when confining assessment to plasma samples, intracellular effects may be missed. Recently, the so-called metabolomics approach has been developed. Metabolomic analysis profiles small-molecule metabolites using either a targeted or non-targeted approach \[[@B10-ijms-18-00554]\]. Through systematic identification and quantification of metabolites in biological systems, e.g. tissues, organs, or fluids at a certain time point, metabolomics directly sample metabolic modulations. Targeted metabolomics allows for very precise measurement of a specific target or an isolated group of related metabolites, whereas non-targeted metabolomics assesses as wide a range of different metabolites as possible. This technique provides a simultaneous screening of multiple metabolic pathways and captures an instantaneous integrated snapshot of the entire physiology of an organism. Metabolomics has the advantage over other "omics" that it integrates changes in gene expression, protein levels, enzymatic activity, and post-translational changes. Previously, metabolomics made it possible to determine changes in the human plasma metabolome caused by various conditions and diseases like sleep restriction \[[@B11-ijms-18-00554],[@B12-ijms-18-00554]\], diabetes \[[@B13-ijms-18-00554]\], and bariatric surgery \[[@B14-ijms-18-00554]\]. Studies have demonstrated a weak correlation between plasma and muscle metabolite levels, indicating that plasma measurements are poor indicators of skeletal muscle metabolism \[[@B15-ijms-18-00554]\]. Consequently, metabolomic analysis requires tissue biopsies to allow a detailed examination of changes in the intracellular pathways within this specific tissue. Such metabolic profiling offers the possibility to identify biochemical signatures of cellular metabolism involved in different pathways like glucose, lipid, and protein handling. Massive research interest has gone into resveratrol as a potential protective compound in the management of obesity-associated complications, e.g., metabolic syndrome and low-grade inflammation. However, the particular pathways involved in potential salutary effects are by and large undetermined. Non-targeted metabolomic profiling may provide new insight into these matters. We have recently conducted a four-month randomized, placebo-controlled clinical trial describing the effects of resveratrol treatment in middle-aged males with metabolic syndrome and reported the findings on bone \[[@B16-ijms-18-00554]\], circulating steroids \[[@B17-ijms-18-00554]\], and glucose metabolism and inflammation \[[@B18-ijms-18-00554]\]. The aim of the present study is to provide a comprehensive metabolomic analysis of the changes caused by four months of high-dose resveratrol treatment in these middle-aged men with metabolic syndrome. As the effects of resveratrol in humans are incompletely characterized, we used a non-targeted metabolomics approach based on four different matrices: plasma, urine, adipose tissue, and skeletal muscle tissue from each study subject. In our study on circulating steroids and prostate size we established a dose-dependent decrease in plasma androgen precursors by resveratrol \[[@B17-ijms-18-00554]\]. In order to maximize the chances of detecting even subtle resveratrol-induced pathway changes, we therefore decided to employ the metabolomics analysis in the high-dose resveratrol (hRSV) (500 mg twice daily) group compared to the placebo group. 2. Results {#sec2-ijms-18-00554} ========== 2.1. Clinical Features {#sec2dot1-ijms-18-00554} ---------------------- The basic characteristics of the participants are outlined in [Table 1](#ijms-18-00554-t001){ref-type="table"}. Apart from a slight difference in age, the two groups were comparable at baseline and post-treatment. 2.2. Global Metabolic Profiling {#sec2dot2-ijms-18-00554} ------------------------------- Based on a combination of two Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) platforms and one Gas Chromatography--Mass Spectroscopy (GS-MS) platform, 405 metabolites in plasma, 282 metabolites in adipose tissue, 446 metabolites in skeletal muscle, and 604 metabolites in urine were identified. General platform methods, data analysis, and metabolite detection identification are described in detail in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"} and [Supplementary Tables S1--S4](#app1-ijms-18-00554){ref-type="app"}). A total of 88 identified metabolites were present in all four matrices, as illustrated in [Figure 1](#ijms-18-00554-f001){ref-type="fig"}. The relationship between significantly hRSV-changed metabolites in the four matrices is depicted in [Figure 2](#ijms-18-00554-f002){ref-type="fig"}. Adipose tissue and urine had the greatest overlap in terms of significantly changed metabolites. One metabolite, dehydroisoandrosterone sulfate (DHEA-S), was significantly changed in all four matrices. Random forest analysis (RF analysis) of adipose tissue, muscle tissue, urine, and plasma metabolic profiles resulted in 75%, 87%, 82%, and 89% accuracy in differentiating the hRSV and placebo groups, respectively, indicating that the differences in biochemical profiles between the two groups were quite pronounced. Random Forest classification results, methods, and data analysis are described in detail in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"} and [Supplementary Table S7](#app1-ijms-18-00554){ref-type="app"}). Among the 30 top-ranking metabolites resulting from the RF analysis, biochemicals involved in amino acid metabolism and lipid metabolism were particularly important with regards to the separation of the two groups. Many of the metabolites embraced in the RF-analysis are also included in the hRSV-altered metabolic pathways. Steroid hormones, especially, were well represented in the RF importance plots, in addition to being significantly altered by hRSV intervention. 2.3. Metabolic Profiling in Adipose Tissue {#sec2dot3-ijms-18-00554} ------------------------------------------ Of the 282 biochemicals identified in adipose, tissue 45 displayed a significant change (p ≤ 0.05) in response to hRSV treatment: 31 of these were elevated and 14 were reduced. Furthermore, for 28 compounds there was a trend towards a change (p \< 0.10), of which 24 compounds were increased and four compounds were reduced by hRSV treatment. The pathways that differed significantly between the hRSV group and controls in adipose tissue (p \< 0.05) are shown in [Figure 3](#ijms-18-00554-f003){ref-type="fig"}. Exploring the respective significantly changed pathways in detail reveals the specific hRSV-affected compounds. As illustrated in [Table 2](#ijms-18-00554-t002){ref-type="table"}, four out of 15 identified lipids are significantly elevated in the long-chain fatty acid pathway (myristate (14:0), myristoleate (14:1n5), palmitate (16:0), and palmitoleate (16:1n7)). These lipids depict the significant fold change between the hRSV group and the placebo group. In addition, examining the polyunsaturated fatty acid pathway demonstrated that six out of 13 identified lipids are significantly elevated in the hRSV group (stearidonate (18:4n3), docosapentaenoate (n3 DPA; 22:5n3), docosahexaenoate (DHA; 22:6n3) linoleate (18:2n6) adrenate (22:4n6), and mead acid (20:3n9)). Also of interest, the steroid hormone pathway reveals significant reductions in both of the identified steroids, dehydroisoandrosterone sulfate (DHEA-S) and 4-androsten-3β, 17β-diol disulfate. The most interesting trending changes (p \< 0.10) in adipose tissue pathways were displayed in the glycolysis, gluconeogenesis, and pyruvate metabolism pathway and the pentose phosphate pathway. As illustrated in [Table 2](#ijms-18-00554-t002){ref-type="table"}, trending elevation in three out of nine identified glycolytic pathway intermediates (glucose-6-phosphate (G6P), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, phosphoenolpyruvate (PEP)), and one in the pentose phosphate pathway intermediate was found (sedoheptulose-7-phosphate). 2.4. Metabolic Profiling in Plasma {#sec2dot4-ijms-18-00554} ---------------------------------- Of the 405 named compounds in plasma, 38 were statistically different between the two groups (p ≤ 0.05); 13 of these were elevated and 25 were reduced. In addition, 11 compounds showed a trend towards a change by hRSV (p \< 0.10), of which eight were increased and three were reduced. The pathways that are significantly different (p \< 0.05) between the hRSV group and controls are shown in [Figure 4](#ijms-18-00554-f004){ref-type="fig"}. Particularly interesting changes in response to hRSV treatment were found in the steroid hormone pathway with consistent significant reductions in circulating levels of cholesterol-derived steroid hormones and sulfated steroid hormones. As illustrated in [Table 3](#ijms-18-00554-t003){ref-type="table"}, 13 out of 19 detected steroid hormones were significantly reduced. Also noteworthy, significant elevations in histidine and the related biochemical marker of muscle protein turnover 3-methylhistidine along with its acetylated derivative N-acetyl-3-methylhistidine were observed in plasma in response to hRSV treatment. In addition, subtle but consistent reductions in the dicarboxylic acids dodecanedioate, tetradecanedioate, sebacate, hexadecanedioate, and octadecanedioate were observed in the hRSV group. 2.5. Metabolic Profiling in Skeletal Muscle Tissue {#sec2dot5-ijms-18-00554} -------------------------------------------------- Of the 446 named biochemicals in skeletal muscle tissue, 24 changed significantly (p ≤ 0.05); 12 of these were elevated and 12 reduced by hRSV treatment. Additionally, 19 biochemicals showed a trend towards a change by hRSV (p \< 0.10), with 12 biochemicals increased and seven reduced. The pathways that are significantly different (p \< 0.05) between the hRSV group and controls are shown in [Figure 5](#ijms-18-00554-f005){ref-type="fig"}. The most striking change in muscle tissue was the effect of hRSV on the steroid hormone pathway, with four out of six compounds being significantly reduced, as illustrated in [Table 4](#ijms-18-00554-t004){ref-type="table"}. All four compounds comprised sulfated steroid metabolites (dehydroisoandrosterone sulfate (DHEA-S), epiandrosterone sulfate, androsterone sulfate, 4-androsten-3β, and 17β-diol disulfate 1). Regarding skeletal muscle lipid metabolism, our analysis revealed significant hRSV alterations in the short-chain fatty acids, medium-chain fatty acid, and long-chain fatty acid pathways. Generally, the hRSV group had lower levels of intracellular lipids. In addition, two out of 12 biochemicals in the polyunsaturated fatty acid pathway were increased (stearidonate (18:4n3) and linolenate \[α or γ; (18:3n3 or 6)\]). 2.6. Metabolic Profiling in Urine {#sec2dot6-ijms-18-00554} --------------------------------- Urine data were analyzed in both non-normalized and osmolality-normalized formats, and because normalization to osmolality values did not substantially affect results, non-normalized data were utilized for data interpretation. Of the 604 named biochemicals in urine, 43 changed significantly (p ≤ 0.05); 31 of these were elevated and 12 were reduced by hRSV treatment. Additionally, 27 biochemicals showed a trend towards a change by hRSV (p \< 0.10), with 22 biochemicals increased and five reduced. Pathways that demonstrated significantly modifications (p \< 0.05) in response to hRSV are shown in [Figure 6](#ijms-18-00554-f006){ref-type="fig"}. The most striking hRSV-induced changes in urine were observed in the steroid hormone pathway. Ten out of 28 named steroids were significantly changed, with the majority being elevated, as illustrated in [Table 5](#ijms-18-00554-t005){ref-type="table"}. Furthermore, several metabolites derived from aromatic amino acids that have a contribution from or are exclusively produced by the gut microbiota were altered in the urine. The tyrosine-derived metabolites tyramine, phenol sulfate and homovanillate (HVA), were changed in the hRSV group. Similarly, urinary levels of derivatives from the tryptophan metabolism (tryptamine and indolelactate), the phenyalanine metabolism (2-hydroxyphenylacetate), and the histidine metabolism (imidazole propionate) were significantly altered by hRSV treatment, as depicted in [Table 5](#ijms-18-00554-t005){ref-type="table"}. 3. Discussion {#sec3-ijms-18-00554} ============= The effects of resveratrol in human trials have proven to be very inconsistent. Most data represent standard biochemical analyses of selected parameters. The focus has been on inflammation \[[@B8-ijms-18-00554],[@B19-ijms-18-00554]\], glucose metabolism \[[@B9-ijms-18-00554],[@B20-ijms-18-00554]\], lipids \[[@B21-ijms-18-00554]\], liver function \[[@B22-ijms-18-00554]\], and steroids \[[@B17-ijms-18-00554]\], assessed by measurement in blood samples, thus representing a picture of whole body metabolism. The present paper describes an exhaustive metabolomic profile of blood, adipose tissue, skeletal muscle tissue, and urine from a comprehensive clinical trial of high-dose resveratrol-treated, middle-aged males with metabolic syndrome. This approach allows for a more elaborate investigation of subtle resveratrol effects on distinct intracellular pathways than what is possible by measurements confined to blood. In addition, this approach of unbiased measurements in various body compartments may help us to determine the cause of some of the effects we have previously detected. Recently, we reported that the androgen precursors in blood were significantly reduced after resveratrol treatment for four months \[[@B17-ijms-18-00554]\]. The presented metabolomic analysis was performed in the same study cohort, and by using this advanced technique we are able to verify the decrease in sulfated androgen precursors in the blood \[[@B17-ijms-18-00554]\]. In addition, we find significantly lower intracellular amounts of sulfated androgen precursors in skeletal muscle tissue as well as adipose tissue. Sulfation is a common modification to control levels of active steroid hormones at target sites by increasing solubility and renal excretion. The metabolomic profiling of urine revealed increased urinary excretion of the majority of the measured sulfated steroid hormones. In the original paper on reduced androgen precursors, we discussed whether the reduction was caused by decreased formation or increased excretion. The present results propose that at least part of the decrease in sulfated androgen precursors in blood and tissues is caused by an increase in urinary excretion of the sulfated steroids. The metabolomic analysis of lipids points to a striking accumulation of long-chain saturated, monounsaturated, and polyunsaturated (n3 and n6) free fatty acids in adipose tissue. However, in skeletal muscle two long-chain lipids were significantly changed; one was elevated (10-heptadecenoate (17:1n7)) whereas the other was significantly reduced (arachidate (20:0)). Deducing how resveratrol induces the increase in many of the long-chain polyunsaturated fatty acids (n3 and n6) in adipose tissue is complex. Usually these long-chain polyunsaturated fatty acids derive from increased intake of seafood or special plant oils. However, the two essential fatty acids alpha-linolenic acid (ALA; 18:3n3) and linoleic acid (LA; 18:2n6) can be metabolized into longer-chain polyunsaturated fatty acids through a series of desaturation and elongation processes \[[@B23-ijms-18-00554]\]. The literature describes a gender difference in the conversion of ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), with women having a much higher conversion rate than men \[[@B24-ijms-18-00554],[@B25-ijms-18-00554]\]. This difference depends upon sex steroids, and testosterone supplementation to female transsexuals reduced DHA concentration by 22% \[[@B26-ijms-18-00554]\]. Clearly, this indicates that the enzymes responsible for the desaturation and elongation conversion of ALA to DHA are negatively regulated by androgens. Based on this knowledge, we speculate that resveratrol might stimulate the conversion process of ALA and LA, resulting in an increase in long-chain polyunsaturated fatty acids through the robust reduction in androgen precursors. The health impacts of changes in levels of long-chain polyunsaturated fatty acids are still not fully determined. Long-chain polyunsaturated fatty acids have been associated with a positive effect on insulin signaling via the insulin receptor \[[@B27-ijms-18-00554]\], and even a minor increase of ALA content in adipose tissue of 1% has been associated with a 5 mmHg decrease in blood pressure \[[@B28-ijms-18-00554]\]. However, intervention studies providing ALA have yielded conflicting results on blood pressure, cholesterol levels, and triglyceride levels, as reviewed by Baker et al. \[[@B23-ijms-18-00554]\]. Also, despite the increase in these fatty acids in adipose tissue after resveratrol treatment, no clinically relevant improvements in blood pressure or insulin sensitivity were observed in our study population \[[@B18-ijms-18-00554]\]. In the adipose tissue we also detected a significant increase in intracellular glycerol and free fatty acids in the resveratrol-treated subjects. In support of this notion, previously published cell culture studies revealed a resveratrol-mediated enhancement of adrenergic stimulated lipolysis in fat cells \[[@B29-ijms-18-00554],[@B30-ijms-18-00554]\]. Moreover, resveratrol treatment caused consistent reductions in several dicarboxylic fatty acids in plasma. These lipids can be produced from peroxisomal-mediated degradation of very long chain fatty acids (\>22 carbons) and subsequently be metabolized via beta-oxidation. Collectively, a decrease in the dicarboxylic fatty acids in plasma could be a result of increased beta-oxidation in adipose tissue. Regarding glucose metabolism, we found trends toward elevation in glycolytic intermediates and the pentose phosphate pathway (PPP) intermediates (p \< 0.10) in adipose tissue in response to resveratrol. Stimulation of the PPP pathway is associated with increased insulin resistance, increased NADPH production, and thus, increased production of free fatty acids \[[@B31-ijms-18-00554]\]. Hence, our metabolomics findings may indicate increased glucose availability and utilization through glycolysis and the pentose phosphate pathway, owing to decreased insulin sensitivity in the males receiving resveratrol. Clinical parameters in the same study subjects support this assumption, as resveratrol increased fructosamine levels after four months of treatment, indicating poorer insulin sensitivity \[[@B18-ijms-18-00554]\]. In the resveratrol-treated group we demonstrated elevation in metabolites related to metabolism of the amino acid histidine. In particular, histidine was significantly increased in the blood and a trending elevation in the related biochemical marker of the muscle protein turnover 3-methylhistidine along with the acetylated derivative N-acetyl-3-methylhistidine was also observed. Increase in 3-methylhistidine, which is a post-translationally modified amino acid derived from the muscle contractile proteins actin and myosin, and N-acetyl-3-methylhistidine indicates increased muscle turnover in men treated with resveratrol. This finding ties in well with the study by Olesen et al. \[[@B32-ijms-18-00554]\], in which resveratrol blunted the positive effects of exercise training. In contrast, Scribbans et al. found that resveratrol in addition to exercise had positive effects on muscle metabolism with an increase in mitochondrial capacity \[[@B33-ijms-18-00554]\], while others found no effect in humans \[[@B34-ijms-18-00554]\]. Most rodent studies have found positive effects of resveratrol on muscle function \[[@B5-ijms-18-00554],[@B35-ijms-18-00554],[@B36-ijms-18-00554]\]. Clearly, more clinical studies are needed to determine the exact effects of resveratrol on skeletal muscle function. Finally, we found urinary changes in several tyrosine-derived, tryptophan-derived, phenylalanine-derived, and histidine-derived metabolites. Tryptophan, histidine, and phenylalanine are classified as essential amino acids, which cannot be synthesized de novo by the body. However, gut bacteria may contribute to their production and degradation \[[@B37-ijms-18-00554]\]. Fermentation of tyrosine and tryptophan by colonic bacteria is found to produce phenols and indoles, which are excreted in the urine \[[@B38-ijms-18-00554]\]. Also, imidazole propionate is known to be excreted in the urine and direct degradation of urocanate to imidazole propionate by the intestinal flora was demonstrated in an animal study \[[@B39-ijms-18-00554]\]. Changes in microbiota-related urinary metabolites are proposed to be of relevance to human health. For instance hippurate, phenylacetylglutamine, 4-cresylsulfate, and 4-hydroxyphenylpropionate have been related to body weight, blood pressure, and metabolic syndrome \[[@B40-ijms-18-00554]\], while urinary imiadazole propionate, which we found to be elevated by resveratrol, has been related to intestinal dysfunction \[[@B39-ijms-18-00554]\]. Therefore, the changes in urinary amino acids and derivatives revealed by the metabolomic analysis may imply a resveratrol-induced modulation of the gut flora in men with metabolic syndrome. This is in accordance with studies in rodents where resveratrol has been shown to modulate the gut microbiota directly \[[@B41-ijms-18-00554],[@B42-ijms-18-00554],[@B43-ijms-18-00554]\]. The strengths of the present clinical trial are a carefully selected, strong study design and the advanced metabolomics approach. A high dosage of resveratrol in a relatively long-term study was chosen. Participants were selected because they had metabolic syndrome, a condition considered a prime target for resveratrol. Furthermore, the advanced metabolomics technique was applied in an unbiased approach to four different matrices. However, a limitation of our study is the lack of liver tissue samples. The liver is a highly metabolic organ and a number of experimental studies have provided evidence of the hepatic benefits of resveratrol treatment \[[@B44-ijms-18-00554]\]. Metabolomic profiling of liver tissue from the same study subjects would help complete the metabolic puzzle of resveratrol-induced biochemical effects. Also, metabolomic analysis on feces would have provided insight into possible beneficial effects on the gut microbiota. Finally, it must be kept in mind that the present study used a very high dose of resveratrol (1000 mg), which is impossible to reach from natural food items. Resveratrol has a very low bioavailability owing to a rapid metabolism to glucuronide and sulfate conjugates in the liver. However, it is possible that ingestion of food items rich in resveratrol and other polyphenols might inhibit the metabolism or increase the absorption of resveratrol, resulting in a better bioavailability from food items than from purified resveratrol products \[[@B45-ijms-18-00554],[@B46-ijms-18-00554]\]. 4. Materials and Methods {#sec4-ijms-18-00554} ======================== 4.1. Study Design {#sec4dot1-ijms-18-00554} ----------------- Data in the present study comprise a subgroup from our original study describing the effects of resveratrol on bone \[[@B16-ijms-18-00554]\]. As we only obtained tissue samples from 24 subjects in the placebo group and 21 in the hRSV group, the sample size is lower compared to the original paper. The protocol was approved by the Regional Committee on Health Research Ethics (M-20110111) (24 May 2011) and the Danish Data Protection Agency, and the study was conducted in agreement with the Declaration of Helsinki II. Participants were given oral and written information about the purpose and nature of all procedures before informed consent was obtained. The protocol was registered at clinicaltrials.gov (NCT01412645). The study was a randomized, placebo-controlled, double-blinded, single-center study. Male test subjects with metabolic syndrome (MetS) were randomized to treatment for four months with tablets containing a placebo, low-dose resveratrol (75 mg twice daily), or high-dose resveratrol (500 mg twice daily). The study comprised 66 test subjects. Inclusion criteria were: Male gender, age between 30 and 60 years, and MetS. MetS was defined according to the International Diabetes Federation \[[@B47-ijms-18-00554]\] as central obesity (Waist circumference ≥94 cm and/or BMI \> 30 kg/m^2^) plus any two of the following: raised triglycerides (≥1.7 mmol/L), reduced high-density lipoprotein (HDL) (≤1.03 mmol/L), raised blood pressure (systolic ≥130 mmHg or diastolic ≥85 mmHg), raised fasting plasma glucose (≥5.6 mmol/L), or drug treatment for the individual features. The trial was performed under conditions of weight stability, unchanged diet, unchanged dietary supplements, and strict compliance with the study drug. Metabolomics results from the high-dose resveratrol (1000 mg daily) and the placebo groups after four months' treatment are reported in the present study. 4.2. Samples {#sec4dot2-ijms-18-00554} ------------ Blood and urine samples were collected between 7:30 and 11:00 a.m. after an overnight fast. Biopsy of the abdominal subcutaneous adipose tissue depot was obtained using a liposuction cannula and skeletal muscle biopsy was harvested from the musculus vastus lateralis using a Bergström cannula. All procedures were performed under sterile conditions. Before both biopsies were performed, the area was anesthetized using 5 to 10 mL lidocaine. 4.3. Metabolomic Analysis {#sec4dot3-ijms-18-00554} ------------------------- Urine, blood, adipose tissue, and skeletal muscle tissue samples were shipped to Metabolon, Inc.^®^, (Durham, NC, USA) on dry ice. Following receipt, samples were stored at −80 °C until analyzed. A recovery standard was added prior to the first step in preparation for quality control (QC). To remove protein, dissociate small molecules bound to protein or trapped in the protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for two minutes followed by centrifugation. The resulting extracts were divided into five fractions: one for analysis by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) with positive ion mode electrospray ionization, one for analysis by UPLC-MS/MS with negative ion mode electrospray ionization, one for LC polar platform, one for analysis by Gas Chromatography-Mass Spectroscopy (GC-MS), and one sample was reserved for backup. Raw data were extracted, peak-identified, and processed using Metabolon's hardware and software. More than 3300 commercially available purified standard compounds were used for the identification of compounds in the samples. General platform methods and metabolite detection identification are described in detail in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"}, [Tables S8 and S9](#app1-ijms-18-00554){ref-type="app"}, [Figures S1 and S2](#app1-ijms-18-00554){ref-type="app"}, and [Supplementary Tables S1--S4](#app1-ijms-18-00554){ref-type="app"}). 4.4. Statistical Analysis {#sec4dot4-ijms-18-00554} ------------------------- Baseline comparisons of the study population were evaluated by unpaired Student's t-test after being checked for normality and equal variance using SPSS Statistics version 20 software. Results are expressed as mean ± SEM. The level of significance was 0.05. The pathway enrichment value (PEV) used in [Figure 3](#ijms-18-00554-f003){ref-type="fig"}, [Figure 4](#ijms-18-00554-f004){ref-type="fig"}, [Figure 5](#ijms-18-00554-f005){ref-type="fig"} and [Figure 6](#ijms-18-00554-f006){ref-type="fig"} is calculated based on the following equation: A PEV greater than one indicates that the pathway holds more hRSV-regulated compounds relative to the study overall, suggesting that the pathway may be a target of interest in the intervention effects. In the metabolomic analysis Welch's two-sample t-test was used to identify biochemicals that differed significantly between treatment groups, following log transformation and, if necessary, imputation of missing values with the minimum observed value for each compound. p-Values ≤ 0.05 were considered statistically significant and values approaching significance (p \< 0.10) were reported as well. An estimate of the false discovery rate (q-value method) was calculated to take into account the multiple comparisons that normally occur in metabolomics-based studies. Metabolomics statistical methods are described in details in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"} and [Table S10](#app1-ijms-18-00554){ref-type="app"}). 5. Conclusions {#sec5-ijms-18-00554} ============== Our comprehensive metabolomic analysis revealed small but robust changes in response to high-dose resveratrol treatment. The main finding is that resveratrol lowered sulfated androgen precursors in blood, adipose tissue, and muscle tissue and concurrently increased the content in urine, indicating increased urinary excretion of these sulfated steroids. Furthermore, the content of long-chain polyunsaturated fatty acids in adipose tissue was increased, probably by increased conversion of ALA and LA mediated through reduced androgen precursors. Several metabolites derived from aromatic amino acids associated with the gut microbiota changed, suggesting that resveratrol may affect either the composition or the metabolism of the gut flora. Considering the increasingly recognized role of the gut microbiota in human health and disease, this effect on gut flora activity may represent a potential and import mode of action of resveratrol. Lastly, our results indicate that non-targeted metabolomics is able to broaden our understanding of the many intra-cellular processes regulated by resveratrol treatment. Yet, our study also demonstrates that it is difficult to conclude solely from a single matric, since only a minor overlap between the four matrices we examined was found. Based on this, we suggest that metabolomics are performed in the tissue of interest. Future clinical studies on resveratrol should focus on the steroid metabolism pathway and the direct impact on human gut microbiota, preferably including liver tissue and feces samples. We wish to thank Lenette Pedersen and Pia Hornbek for their laboratory assistance. The study was supported by the Rasmus Riisfort Foundation, the Ejnar Danielsens Foundation, and the AP Møller Maersk Foundation. The study is part of the research program LIRMOI Research Center ([www.LIRMOI.com](www.LIRMOI.com)), which is supported by the Danish Council for Strategic Research (Grant 10-093499). Supplementary materials can be found at [www.mdpi.com/1422-0067/18/3/554/s1](www.mdpi.com/1422-0067/18/3/554/s1). ###### Click here for additional data file. Anne Sofie Korsholm took part in formulating the study design, analyzed the data, and wrote the manuscript with feedback from the co-authors. Marie Juul Ornstrup and Thomas Nordstrøm Kjær performed the experiment and took part in formulating the study design, drafting the manuscript, and revising it critically. Steen Bønløkke Pedersen conceived and designed the experiment, took part in analyzing the data, drafting the manuscript, and revising it critically, and contributed to important intellectual content of the manuscript. The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. hRSV High-dose resveratrol RF Random forest analysis PEV Pathway enrichment value DHA Docosahexaenoate ALA α-linolenic acid LA Linoleic acid MetS Metabolic syndrome ![Venn diagram of all detected metabolites in the four different matrices (for metabolite details see [Supplementary Table S5](#app1-ijms-18-00554){ref-type="app"}).](ijms-18-00554-g001){#ijms-18-00554-f001} ![Venn diagram of significantly hRSV-altered metabolites detected in the four different matrices (for metabolite details see [Supplementary Table S6](#app1-ijms-18-00554){ref-type="app"}).](ijms-18-00554-g002){#ijms-18-00554-f002} ![Resveratrol-mediated changes in adipose tissue, expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites); hRSV: high-dose resveratrol; Ctrl: Placebo group.](ijms-18-00554-g003){#ijms-18-00554-f003} ![Resveratrol-mediated changes in plasma expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites).](ijms-18-00554-g004){#ijms-18-00554-f004} ![Resveratrol-mediated changes in skeletal muscle tissue expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites); hRSV: high-dose resveratrol; Ctrl: Placebo group.](ijms-18-00554-g005){#ijms-18-00554-f005} ![Resveratrol-mediated changes in urine expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites); hRSV: high-dose resveratrol; Ctrl: Placebo group.](ijms-18-00554-g006){#ijms-18-00554-f006} ijms-18-00554-t001_Table 1 ###### Clinical features at baseline and post-treatment. Characteristic Placebo (n = 24) hRSV (n = 21) p-Value -------------------------------- -------------------- ----------------- ------------ ------------ ------------- Age, years 47.8 ± 1.3 51.9 ± 1.3 p \< 0.05 Body mass index, kg/m^2^ 34.1 ± 0.8 33.4 ± 0.2 33.8 ± 0.7 33.7 ± 0.2 NS HOMA-IR 4.36 ± 0.5 4.19 ± 0.3 3.87 ± 0.5 4.50 ± 0.3 NS Systolic blood pressure, mmHg 150 ± 3 142 ± 3 146 ± 2 140 ± 3 NS Diastolic blood pressure, mmHg 91.3 ± 2.1 86.0 ± 1.3 89.3 ± 1.7 87.8 ± 1.4 NS ^1^ Data are expressed as mean ± SEM. NS = no significant difference. Comparisons between groups were evaluated by unpaired Student's t-test. The level of significance was 0.05. ijms-18-00554-t002_Table 2 ###### Summary of resveratrol-induced changes in adipose tissue. Identified metabolites in the long-chain fatty acid, polyunsaturated fatty acid (n3 and n6), steroids, glycolysis, gluconeogenesis, and pyruvate metabolism, and pentose phosphate pathways in adipose tissue. Red: Significant elevation (p ≤ 0.05). Orange: Trending elevation (p \< 0.10); Green: Significant reduction (p ≤ 0.05). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul} Ctrl p-Value q-Value ------------------------------------------------------------------------------------------------ -------------------------------------------------------------- ------------------------------ ------------------ ----------- ----------- Lipid Long-Chain Fatty Acid Myristate (14:0) 1.10 0.003 0.111 Myristoleate (14:1n5) 1.67 0.017 0.182 Pentadecanoate (15:0) 1.01 0.977 0.816 Palmitate (16:0) 1.10 0.033 0.240 Palmitoleate (16:1n7) 1.41 0.003 0.109 Margarate (17:0) 0.98 0.500 0.672 10-heptadecenoate (17:1n7) 1.12 0.086 0.309 Stearate (18:0) 1.02 0.610 0.731 Oleate (18:1n9) 1.17 0.109 0.328 cis-vaccenate (18:1n7) 1.17 0.304 0.538 Nonadecanoate (19:0) 1.00 0.866 0.794 10-nonadecenoate (19:1n9) 1.08 0.569 0.703 Arachidate (20:0) 0.94 0.472 0.660 Eicosenoate (20:1n9 or 11) 1.12 0.335 0.566 Erucate (22:1n9) 0.96 0.657 0.734 Polyunsaturated Fatty Acid (n3 and n6) Stearidonate (18:4n3) 1.50 0.045 0.240 Eicosapentaenoate (EPA; 20:5n3) 1.25 0.093 0.311 Docosapentaenoate (n3 DPA; 22:5n3) 1.31 0.012 0.177 Docosahexaenoate (DHA; 22:6n3) 1.38 0.042 0.240 Linoleate (18:2n6) 1.24 0.021 0.203 Linolenate \[α or γ; (18:3n3 or 6)\] 1.21 0.060 0.253 Dihomo-linolenate (20:3n3 or n6) 1.19 0.065 0.261 Arachidonate (20:4n6) 1.17 0.107 0.328 Adrenate (22:4n6) 1.69 0.010 0.177 Docosapentaenoate (n6 DPA; 22:5n6) 1.63 0.056 0.253 Docosadienoate (22:2n6) 1.10 0.275 0.498 Dihomo-linoleate (20:2n6) 1.11 0.216 0.443 Mead acid (20:3n9) 1.33 0.045 0.240 Steroid Dehydroisoandrosterone sulfate (DHEA-S) 0.40 0.013 0.177 4-androsten-3β,17β-diol disulfate (1) 0.37 0.009 0.177 Carbohydrate Glycolysis, Gluconeogenesis, and pyruvate metabolism pathway 1,5-anhydroglucitol (1,5-AG) 0.86 0.235 0.464 Glucose 0.98 0.594 0.722 Glucose-6-phosphate (G6P) 1.23 0.059 0.253 Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate, myo-inositol 1,4 or 1,3-Diphosphate 1.16 0.159 0.394 Dihydroxyacetone phosphate (DHAP) 1.30 0.080 0.303 3-phosphoglycerate 1.75 0.057 0.253 Phosphoenolpyruvate (PEP) 1.39 0.128 0.357 Lactate 1.04 0.396 0.599 Glycerate 1.08 0.771 0.777 Pentose phosphate pathway Sedoheptulose-7-phosphate 1.27 0.051 0.253 ijms-18-00554-t003_Table 3 ###### Summary of resveratrol-induced changes in plasma. Identified metabolites in the steroid, fatty acid dicarboxylate, and histidine metabolism pathways in plasma. Red: Significant elevation (p ≤ 0.05). Orange: Trending elevation (p \< 0.10); Green: Significant reduction (p ≤ 0.05). Light green: Trending reduction (p \< 0.10). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul} Ctrl p-Value q-Value ------------------------------------------------------ ---------------------- ---------------------- ------------------ ----------- ----------- Lipid Steroid Pregnenolone sulfate 0.63 0.001 0.018 21-hydroxypregnenolone disulfate 0.41 \<0.001 \<0.001 5α-pregnan-3β,20α-diol disulfate 0.42 \<0.001 \<0.001 5α-pregnan-3α,20β-diol disulfate 1 0.93 0.140 0.762 Pregnen-diol disulfate 0.53 0.001 0.015 Pregn steroid monosulfate 0.72 0.046 0.515 Pregnanediol-3-glucuronide 0.92 0.644 0.927 Cortisol 0.92 0.745 0.941 Cortisone 0.93 0.285 0.824 Dehydroisoandrosterone sulfate (DHEA-S) 0.57 \<0.001 0.013 Epiandrosterone sulfate 0.42 \<0.001 0.004 Androsterone sulfate 0.40 \<0.001 0.004 4-androsten-3β,17β-diol disulfate 1 0.51 \<0.001 0.016 4-androsten-3β,17β-diol disulfate 2 0.49 \<0.001 \<0.001 5α-androstan-3β,17α-diol disulfate 0.37 \<0.001 0.001 5α-androstan-3α,17β-diol disulfate 0.71 0.071 0.659 5α-androstan-3β,17β-diol disulfate 0.37 \<0.001 0.002 Andro steroid monosulfate 2 0.31 \<0.001 \<0.001 Estrone 3-sulfate 0.87 0.132 0.762 Fatty Acid, Dicarboxylate 2-hydroxyglutarate 0.98 0.660 0.934 Sebacate (decanedioate) 0.73 0.101 0.762 Dodecanedioate 0.61 \<0.001 0.001 Tetradecanedioate 0.75 0.021 0.278 Hexadecanedioate 0.85 0.206 0.762 Octadecanedioate 0.88 0.255 0.811 3-carboxy-4-methyl-5-propyl-2-furanpropanoate (CMPF) 0.95 0.955 0.964 Amino Acid Histidine Metabolism Histidine 1.16 0.008 0.147 3-methylhistidine 1.93 0.056 0.570 N-acetyl-3-methylhistidine 1.23 0.084 0.721 trans-urocanate 1.38 0.128 0.762 ijms-18-00554-t004_Table 4 ###### Summary of resveratrol-induced changes in skeletal muscle tissue. Identified metabolites in the steroid, short-chain fatty acid, medium-chain fatty acid, long-chain fatty acid, and polyunsaturated fatty acid (n3 and n6) pathways in skeletal muscle. Red: Significant elevation (p ≤ 0.05). Orange: Trending elevation (p \< 0.10); Green: Significant reduction (p ≤ 0.05). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul} Ctrl p-Value q-Value ----------------------------------------- ----------------------- ------------------ ------------------ ----------- ----------- Lipid Steroid Cortisol 1.40 0.211 0.962 Cortisone 1.14 0.778 0.987 Dehydroisoandrosterone sulfate (DHEA-S) 0.48 \<0.001 0.025 Epiandrosterone sulfate 0.29 \<0.001 0.001 Androsterone sulfate 0.24 \<0.001 0.001 4-androsten-3β,17β-diol disulfate (1) 0.46 0.002 0.125 Short-Chain Fatty Acid Valerate 0.86 \<0.001 0.026 Medium-Chain Fatty Acid Caproate (6:0) 1.09 0.442 0.962 Heptanoate (7:0) 0.82 \<0.001 0.032 Caprylate (8:0) 1.08 0.619 0.962 Undecanoate (11:0) 0.89 0.002 0.125 Long-Chain Fatty Acid Myristoleate (14:1n5) 1.78 0.252 0.962 Palmitate (16:0) 0.98 0.449 0.962 Margarate (17:0) 0.95 0.268 0.962 10-heptadecenoate (17:1n7) 1.09 0.029 0.655 Stearate (18:0) 0.97 0.209 0.962 Oleate (18:1n9) 1.01 0.994 0.996 cis-vaccenate (18:1n7) 1.02 0.904 0.995 10-nonadecenoate (19:1n9) 0.97 0.882 0.995 Arachidate (20:0) 0.84 0.003 0.168 Eicosenoate (20:1n9 or 11) 1.01 0.958 0.996 Erucate (22:1n9) 0.88 0.193 0.962 Polyunsaturated Fatty Acid (n3 and n6) Stearidonate (18:4n3) 1.45 0.015 0.474 Eicosapentaenoate (EPA; 20:5n3) 0.98 0.554 0.962 Docosapentaenoate (n3 DPA; 22:5n3) 1.04 0.511 0.962 Docosahexaenoate (DHA; 22:6n3) 1.07 0.344 0.962 Linoleate (18:2n6) 1.02 0.714 0.980 Linolenate \[α or γ; (18:3n3 or 6)\] 1.16 0.065 0.962 Dihomo-linolenate (20:3n3 or n6) 1.02 0.873 0.995 Arachidonate (20:4n6) 1.00 0.990 0.996 Docosapentaenoate (n6 DPA; 22:5n6) 0.99 0.994 0.996 Docosadienoate (22:2n6) 0.93 0.680 0.962 Dihomo-linoleate (20:2n6) 0.83 0.287 0.962 Mead acid (20:3n9) 0.93 0.619 0.962 ijms-18-00554-t005_Table 5 ###### Summary of resveratrol-induced changes in urine. Identified metabolites in the steroid, tryptophan metabolism, phenylalanine and tyrosine metabolism, and histidine metabolism pathways in urine. Red: Significant elevation (p ≤ 0.05). Orange: Trending elevation (p \< 0.10); Green: Significant reduction (p ≤ 0.05). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul}Ctrl p-Value q-Value ------------------------------------------------------ ----------------------- ---------------------------------- ----------------- ----------- ----------- Lipid Steroid 21-hydroxypregnenolone disulfate 0.50 0.012 0.313 Pregnen-diol disulfate 0.79 0.421 0.861 Pregn steroid monosulfate 2.10 0.008 0.281 Pregnanediol-3-glucuronide 0.87 0.910 0.938 Cortisol 1.29 0.734 0.915 Cortisol glucuronide 1.36 0.600 0.893 Cortisone 1.13 0.757 0.915 Tetrahydrocortisone 1.54 0.270 0.762 Dehydroisoandrosterone sulfate (DHEA-S) 3.95 \<0.001 0.001 16a-hydroxy DHEA 3-sulfate 1.04 0.201 0.735 Epiandrosterone 0.87 0.967 0.938 Epiandrosterone sulfate 1.43 0.036 0.575 Androsterone sulfate 1.22 0.171 0.724 4-androsten-3β,17β-diol monosulfate (1) 3.75 \<0.001 0.001 4-androsten-3β,17β-diol monosulfate (2) 3.50 0.003 0.149 4-androsten-3α,17α-diol monosulfate (2) 6.78 \<0.001 0.001 4-androsten-3β,17β-diol disulfate (1) 0.72 0.804 0.920 4-androsten-3β,17β-diol disulfate (2) 0.89 0.736 0.915 5α-androstan-3β,17α-diol disulfate 0.20 0.002 0.127 5α-androstan-3β,17β-diol disulfate 0.68 0.414 0.861 Andro steroid monosulfate (1) 0.45 0.004 0.170 Testosterone sulfate 3.74 0.009 0.281 11-ketoetiocholanolone glucuronide 0.98 0.793 0.920 Etiocholanolone glucuronide 0.85 0.766 0.915 17α-hydroxypregnanolone glucuronide 1.10 0.216 0.747 5β-pregnan-3α,21-diol-11,20-dione 21-Glucosiduronate 1.51 0.604 0.893 11-ketoetiocholanolone sulfate 1.23 0.957 0.938 Dehydroepiandrosterone glucuronide 0.83 0.585 0.881 Amino Acid Tryptophan Metabolism Tryptophan 0.99 0.908 0.938 N-acetyltryptophan 1.37 0.194 0.726 Tryptamine 0.37 \<0.001 0.010 Indolelactate 1.56 0.050 0.613 Indoleacetate 0.58 0.201 0.735 3-indoxyl sulfate 1.39 0.124 0.724 Kynurenine 1.29 0.637 0.905 Kynurenate 1.17 0.325 0.793 Anthranilate 1.16 0.446 0.861 3-hydroxykynurenine 1.43 0.480 0.874 3-hydroxyanthranilate 1.59 0.057 0.638 Xanthurenate 1.23 0.231 0.748 Picolinate 1.16 0.180 0.724 5-hydroxytryptophan 1.11 0.383 0.848 5-hydroxyindoleacetate 1.02 0.563 0.881 Serotonin (5HT) 1.03 0.782 0.920 Indoleacetylglutamine 0.64 0.824 0.920 Tryptophan betaine 1.14 0.976 0.940 Indole-3-carboxylic acid 0.93 0.696 0.914 C-glycosyltryptophan 1.03 0.693 0.914 N-acetylkynurenine (2) 2.14 0.047 0.613 Phenylalanine and Tyrosine Metabolism Phenylalanine 1.11 0.588 0.881 N-acetylphenylalanine 1.31 0.185 0.726 Phenyllactate (PLA) 1.86 0.148 0.724 4-hydroxyphenylacetate 1.02 0.915 0.938 3-hydroxyphenylacetate 1.52 0.096 0.724 Phenylacetylglycine 1.13 0.801 0.920 Phenylacetylglutamine 0.97 0.720 0.914 Tyrosine 1.06 0.693 0.914 N-acetyltyrosine 1.47 0.051 0.613 4-hydroxycinnamate 1.83 0.172 0.724 Tyramine 0.60 0.001 0.075 m-tyramine 0.99 0.923 0.938 4-hydroxyphenylpyruvate 2.23 0.012 0.313 3-(4-hydroxyphenyl)lactate 1.33 0.280 0.762 Phenol sulfate 1.74 0.016 0.365 p-cresol sulfate 0.87 0.465 0.874 o-cresol sulfate 1.41 0.301 0.782 Dopamine 0.37 0.998 0.947 Vanillylmandelate (VMA) 1.08 0.392 0.858 3-methoxytyrosine 1.08 0.967 0.938 3-methoxytyramine 1.07 0.226 0.748 3-methoxytyramine sulfate 0.96 0.301 0.782 3,4-dihydroxyphenylacetate 1.32 0.107 0.724 Homovanillate (HVA) 1.33 0.040 0.583 Homovanillate sulfate 0.98 0.565 0.881 Gentisate 1.37 0.125 0.724 Phenylpropionylglycine 0.95 0.946 0.938 3-\[3-(sulfooxy)phenyl\]propanoic acid 0.66 0.602 0.893 3-(3-hydroxyphenyl)propionate 1.01 0.903 0.938 5-hydroxymethyl-2-furoic acid 0.77 0.442 0.861 2-hydroxyphenylacetate 1.31 0.075 0.721 Phenylacetylcarnitine 0.82 0.542 0.881 Dopamine sulfate (1) 0.48 0.963 0.938 Dopamine sulfate (2) 0.46 0.893 0.938 p-cresol-glucuronide 0.95 0.704 0.914 Tyramine O-sulfate 0.69 0.202 0.735 Vanillic alcohol sulfate 0.91 0.271 0.762 4-hydroxycinnamate sulfate 0.47 0.240 0.748 3,4-dihydroxyphenylacetate sulfate 0.69 0.548 0.881 Histidine Metabolism Histidine 1.10 0.402 0.861 N-acetylhistidine 1.12 0.152 0.724 1-methylhistidine 1.09 0.712 0.914 3-methylhistidine 2.02 0.080 0.724 N-acetyl-3-methylhistidine 2.10 0.037 0.576 N-acetyl-1-methylhistidine 1.07 0.482 0.874 Hydantoin-5-propionic acid 1.01 0.954 0.938 trans-urocanate 1.14 0.280 0.762 cis-urocanate 1.11 0.632 0.905 Formiminoglutamate 1.03 0.984 0.943 Imidazole propionate 1.67 0.010 0.290 Imidazole lactate 1.31 0.280 0.762 1-methylimidazoleacetate 1.13 0.424 0.861 4-imidazoleacetate 1.16 0.703 0.914 N-acetylhistamine 0.80 0.554 0.881	{ "pile_set_name": "PubMed Central" }	[ "amount", "treatment", "testosteron", "probabl", "cholesterol", "two", "control", "given", "dosedepend", "origin", "divers", "program", "eg", "dihomolinolen", "decreas", "compris", "overnight", "mead", "impair", "purpos", "appendixappijmsreftypeapp", "pregnanediolglucuronid", "ten", "collect", "fullblown", "lowgrad", "highdos", "resveratrolmedi", "biopsi", "longterm", "thank", "gene", "employ", "identifi", "gluconeogenesi", "length", "hydroxyanthranil", "gentis", "placebo", "classif", "written", "lastli", "select", "rais", 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Introduction ============ Systemic anticancer therapy (SACT) ---------------------------------- There are many different treatment options for lung cancer, and these are influenced by type, extent and progression of disease. These treatments can be categorised into surgery, radiotherapy and systemic anticancer therapy (SACT), combinations of which are also possible. While surgery and radiotherapy tend to be of use in local, early-stage disease, SACT is largely used as first-line treatment in advanced stage IIIb-IV non-small-cell lung cancer (NSCLC), with extensive nodal involvement and metastases, to 'improve survival, disease control and quality of life' and is the preferred treatment for both limited and extensive small-cell lung cancer (SCLC) \[[@ref1]\]. It is planned in cycles, between which the patient is reviewed for response to therapy. SACT can be divided into three categories, based on mechanism of action in treating cancer. The most extensive group, chemotherapy, involves the use of cytotoxic drugs to directly destroy tumour cells. It has multiple applications in NSCLC, due to the various intentions of use across most stages of lung cancer. As an adjunct or in combination with radiotherapy, it can be considered pre-operatively in early-disease (stage I--II) patients suitable for surgery; adjuvant chemotherapy aiming to kill cancer cells following radiotherapy is an option in stage II--III disease \[[@ref2]\]. There are multiple classes of chemotherapy drug available, with a variety of intracellular targets such as DNA and microtubules. Biological therapies, the second group, are anticancer agents that prevent the spread of cancer by 'interfering with specific molecules involved in tumour growth and progression' \[[@ref3]\], as opposed to killing tumour cells directly. These targeted therapies have further developed as a result of advancements in tumour analysis for protein mutations that cause uncontrolled proliferation, such as those in epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) \[[@ref4]\]. The third mechanism of SACT is immunotherapy, where monoclonal antibodies recruit the immune system to recognise and attack malignant cells \[[@ref5]\]. [Table 1](#table1){ref-type="table"} details the SACT agents recommended for use in lung cancer. Application of SACT involves the use of combination therapies. The most recent statistics of the commonest regimens for lung cancer are shown in [Figure 1](#figure1){ref-type="fig"}. Supportive care medicines are often prescribed as part of the SACT regimen to prevent side effects from occurring. These include various antiemetics such as aprepitant, domperidone, and ondansetron, to combat nausea and vomiting, a common adverse effect of most anticancer agents \[[@ref6]\]. Notably, some have higher potential for nausea than others, particularly cisplatin, and different regimens thus require varying doses of antiemetic \[[@ref17]\]. Corticosteroids, such as dexamethasone, also counter nausea but additionally reduce the incidence and severity of skin rash, another frequent adverse reaction \[[@ref6]\]. Other medicines include folic acid and vitamin B12 for the antifolate drug pemetrexed \[[@ref18], [@ref19]\] and hydration guidelines for the nephrotoxic platinum-based compounds \[[@ref20]\], encompassing combinations of oral furosemide, intravenous saline, and magnesium or potassium salt solutions \[[@ref21], [@ref22]\]. Drug--drug interactions ======================= As discussed, cancer patients for whom SACT is indicated take a multitude of medicines, either within a complex regimen or due to a range of supportive therapies \[[@ref23]\]. This increases the likelihood of drug--drug interactions (DDIs), where concurrent administration of two drugs allows one to influence the activity of another \[[@ref24]\]. Moreover, interlinking these factors, older patients are the typical demographic of cancer and thus are more likely to be taking multiple regular medicines due to increased comorbidities \[[@ref25]--[@ref27]\]. DDIs, including those involving SACT medicines, can be beneficial and lead to a synergistic (augmented) effect of both drugs, a concept utilised in combination anticancer therapy \[[@ref28]\]. However, there is potential for negative outcomes; due to the large number and difference in mechanisms of action between anticancer agents, there are a multitude of adverse DDIs involving chemotherapy drugs \[[@ref28], [@ref29]\]. Importantly, several SACT agents, including cisplatin, crizotinib, and EGFR tyrosine kinase inhibitors (TKIs), are CYP450 substrates, meaning they can influence hepatic metabolism of many drugs \[[@ref29], [@ref30]\]. Other possible DDIs include platinum-based compounds with nephrotoxic agents such as NSAIDs, a synergistic interaction causing impaired renal function \[[@ref31]\], and EGFR-TKIs with antacids, which raise stomach pH and thus prevent absorption of the anticancer agent \[[@ref32]\]. The impact of DDIs directly on healthcare is poorly characterised. However, the broader group of adverse drug reactions (ADRs) place a significant strain on patients and hospitals, accounting for 6.5% of all hospital admissions, with a total cost per year of over £500,000 to the NHS \[[@ref33], [@ref34]\]. The proportion of this attributed to DDIs is debatable; some suggest an increased risk of readmission related to DDIs \[[@ref35]\], particularly in the elderly \[[@ref36]\], while other reviews propose the opposite \[[@ref37], [@ref38]\]. Nevertheless, as the potential for DDIs in SACT patients is high, methods to increase awareness are crucial to minimise risk of adverse events with which they are associated \[[@ref39], [@ref40]\]. Medicines reconciliation ------------------------ With regard to safe medicine prescribing, it is important that healthcare professionals (HCPs) are conscientious in maintaining the efficacy of medicines, in order for patients to get the best out of their treatment \[[@ref27], [@ref41]\]. This concept is known as medicines optimisation and comprises four guiding principles to improve patient outcome, as outlined by the Royal Pharmaceutical Society \[[@ref42]\]: aiming to understand the patient's experience; evidence-based choice of medicines; ensuring medicines use is as safe as possible; and making medicines optimisation part of routine practice. A major aspect of medicines optimisation that contributes to these aims is medicines reconciliation (Med-Rec), the process of ensuring the medicines a patient is taking are correctly documented \[[@ref43]\]. This involves 'creating and maintaining the most accurate list possible' of the patient's medicines, and then 'comparing... with the current list in use, recognising any discrepancies, and documenting any changes, thereby resulting in a complete list of medicines, accurately communicated' \[[@ref27], [@ref44]\], as defined by the Institute of Healthcare Improvement (IHI). These tasks can be summarised into three elements to form 'reliable' reconciliation: verification of the list of current medicines; validation (a review of the current medicines by a trained and competent HCP, noting whether to continue or alter any doses); and clarification, where the current list is compared with the prescribed 'medication order' \[[@ref41], [@ref44]\]. Previous studies highlight the potential for problems in healthcare without a formal Med-Rec procedure; unintentional discrepancies were found in 70% of medicines prescribed on admission (covering 60% of patients) in a large systematic review by Garfield \[[@ref41], [@ref45]\]. Actively implementing the process is also found to be beneficial; it decreases the rate of 'medication errors' by 70% and ADRs by 15% \[[@ref44], [@ref46], [@ref47]\] in one hospital setting, while another trial found that it reduced potential ADRs by 80% \[[@ref48]\]. This suggests Med-Rec is an important part of preventing harm to patients. Rationale for study ------------------- As clarified, there is a considerable risk of DDIs occurring between anticancer agents, plus various supportive care medicines as part of SACT regimens, and other medicines being taken by patients. The complexity of regimens in lung cancer specifically, utilising drugs with various mechanisms, adds to the potential risk of harm. It was therefore of interest to characterise the severity of these potential DDIs (PDDIs), and review whether established processes of Med-Rec, or simply documentation of PDDIs, could have a role in preventing harm. Numerous studies report aspects of these separately: DDIs involving general chemotherapy have been identified retrospectively in several studies \[[@ref49]--[@ref55]\]; while outcomes of pharmacy-led intervention with Med-Rec \[[@ref39], [@ref56]--[@ref62]\] have also been analysed. However, combining severity of PDDIs and improvement of patient safety has not been carried out previously. Aims, objectives and standards ============================== Aims ---- The aim is to evaluate the potential for DDIs between medicines in SACT regimens and other medicines taken by lung cancer patients treated at Chelsea and Westminster Hospital (CWH). Objectives ---------- 1. To identify and characterise PDDIs present between SACT medicines (comprising anticancer agents and supportive care medicines) and other medicines taken by the patients. 2. To assess the process of Med-Rec and evaluate documentation of PDDIs by prescribing HCPs (clinical audit). Standards for clinical audit ---------------------------- 1. 100% of consultations with a prescribing HCP since the patient started their current SACT regimen include a documented Med-Rec. 2. 100% of PDDIs between SACT medicines and other medicines had been acknowledged and documented by a prescribing HCP. Methodology =========== Study design, inclusion, and exclusion criteria ----------------------------------------------- This was a single-centre, retrospective case series study. Patients were selected for inclusion if diagnosed with lung cancer and undergoing a SACT regimen as of 31st March 2016 under the care of the Oncology team (led by Professor Mark Bower and Dr Tom Newsom-Davis) at CWH. Data collection --------------- For each patient, every instance of documented patient contact at CWH during their SACT regimen was compiled from the patient records. The chosen start point was the last consultation with a prescribing HCP before starting the current SACT regimen in which a full medicines history was taken. For patients on a maintenance SACT regimen, data were recorded from the last consultation in which a full medicines history was elicited before starting the SACT regimen. The endpoint was the cut-off date of 31st March 2016. Data recorded for each consultation include documentation of medicines being taken and changes to the medicine profile as a result of the consultation. Identifying PDDIs ----------------- A 'drug chart' overviewing how the medicine profile changed for each patient during their SACT regimen was then created from the data collection spreadsheet ([Appendix 1](#app1){ref-type="app"}). PDDIs were identified using three primary sources: the British National Formulary (BNF); Summary of Product Characteristics (SPC) at <www.medicines.org.uk>; and the London Cancer Alliance (LCA) protocols for each SACT regimen. Only PDDIs involving a SACT medicine---either the anticancer agent, or any prescribed supportive care medicine---were noted, and not between any two non-SACT medicines taken concurrently. Component drugs within a preparation were studied individually. Assessment of Med-Rec and documentation of PDDIs ------------------------------------------------ The same patient consultation records were analysed for documentation both of Med-Rec being performed and of any DDIs by prescribing HCPs. Definitions ----------- 'PDDI' (potential drug--drug interaction) is defined as a possible DDI between two medicines (as reported in the BNF, SPC, or LCA protocols) that may have occurred when the patient was taking both concurrently. 'Anticipated DDI' is defined as a possible DDI that was identified but did not occur due to intervention before both medicines were being taken simultaneously. There is therefore no possibility of an adverse event due to this DDI occurring. A DDI is 'identified' if noted by the author during retrospective analysis of the collected data; 'acknowledged' or 'documented' DDIs are those identified and written down during a consultation by a prescribing HCP. Results ======= Twenty-three patients met the criteria for inclusion in this study. SACT regimens being followed, listed by route of administration, are shown in [Table 2](#table2){ref-type="table"}. Identified PDDIs ---------------- A total of 88 instances of PDDIs involving SACT medicines across 21 patients were identified. This total includes anticipated DDIs (n = 13). [Figure 2](#figure2){ref-type="fig"} presents the SACT medicines with at least one identified PDDI. In order to present these data qualitatively, PDDIs were grouped based on effect of interaction and mechanism of the interacting medicine. These 30 distinct DDIs are summarised in [Table 3](#table3){ref-type="table"}. The most common identified DDI was that of dexamethasone + antihypertensives (12.5%), followed by: platinum-based compounds + nephrotoxic drugs (8.0%); crizotinib + UGT substrates, dexamethasone + antidiabetics; pemetrexed + nephrotoxic drugs (each 6.8%). The remaining PDDIs had five or fewer instances. In terms of severity, there were six 'potentially serious' DDIs, equating to 8.0% of the total number. Medicines reconciliation and documentation of PDDIs --------------------------------------------------- Standard 1: 100% of consultations with a prescribing HCP since the patient started their current SACT regimen include a documented Med-Rec. Outcome 1 (standard not met): 3.0% of consultations with a prescribing HCP since the patient started their current SACT regimen include a documented Med-Rec. A total of 480 instances of documented patient contact were recorded across all 23 patients. Forty-eight of these were excluded from further analysis: eight were phone calls, three were radiology scan appointments, and 37 were consultations with non-prescribing HCPs. Of the remaining 432 instances, 13 (3.0%, across 7 patients) included a documented Med-Rec. Additionally, 95.7% of patients had at least one full medicines history elicited by a prescribing HCP. This translates to a full medicines history being taken in 11.8% of consultations. Standard 2: 100% of identified PDDIs between SACT medicines and other medicines had been acknowledged and documented by a prescribing HCP. Outcome 2 (standard not met): 15.9% of identified PDDIs between SACT medicines and other medicines had been acknowledged and documented by a prescribing HCP. Of the 88 instances of PDDIs, 14 (15.9%, across 8 patients) were acknowledged and documented. These are summarised in [Table 4](#table4){ref-type="table"}. Comparison of severity and probability of documentation was performed using Fisher's exact test. Instances of PDDI with an unknown severity (n = 30 across both documented and non-documented DDIs) were omitted. Using a standard alpha-level of 0.05, the null hypothesis can be rejected (d.f.=1, two-tailed p = 0.019), indicating that potentially serious DDIs were significantly more likely to be documented. Documented PDDIs were also analysed in relation to Med-Rec processes. None of the 14 PDDIs were acknowledged by a prescribing HCP in the same consultation as a documented Med-Rec. Two (14.3%) were elicited after a full medicines history was taken in the same consultation. Thirteen of the 14 documented PDDIs can be considered anticipated DDIs, where intervention took place before the interaction could occur. The remaining one instance, gefitinib + warfarin, was acknowledged and documented after both medicines were taken concurrently. Discussion ========== Identified PDDIs ---------------- The wide-ranging nature of this study means there are several different aspects suitable for analysis. Firstly, the variety of SACT regimens---10 in total---confirm the multitude of treatment options for late-stage lung cancer patients and compare reasonably well with national statistics \[[@ref16]\]. Regarding the interactions, nearly all patients (91.3%) had at least one identified PDDI, highlighting the importance of characterising them well. Of particular significance were the PDDIs involving supportive care medicines, which accounted for 55% of the total. Most previous studies looking at DDIs involving chemotherapy only consider the anticancer agent \[[@ref50], [@ref51], [@ref54]\], although van Leeuwen did report supportive care medicines to be involved in 86% of all identified PDDIs for the cohort studied \[[@ref55]\], further supporting their clinical relevance in DDIs. The major SACT medicine of note here was dexamethasone, which comprised 39% of the total number. Moreover, two further PDDIs were identified involving dexamethasone prescribed separately from the SACT regimen. DDIs with corticosteroids have been reported in the literature---van Leeuwen's retrospective study found that dexamethasone was the major supportive care medicine involved \[[@ref55]\], and Lam considered it a 'cancer-related drug' with a variety of mechanisms of interaction \[[@ref49]\]. Additionally, a quarter of PDDIs involving dexamethasone were classified as potentially serious. This raises the question of its safety, particularly as it was a prescribed component of all intravenous combination SACT regimens for these patients ([Appendix 2](#app2){ref-type="app"}). However, regimens are carefully designed in terms of dose, timing, and frequency to minimise harm, with low-dose dexamethasone being taken for only three days for every cycle in these LCA-approved regimens. Nevertheless, caution should be exercised considering the wide range of drugs with which it potentially interacts, especially antihypertensives, which was the single-most common PDDI. Indeed, the high frequency reflects the high prevalence of hypertension in the population, particularly the elderly \[[@ref63]\] and therefore suggests it is an important comorbidity to consider. The next most commonly involved SACT medicine were the platinum-based compounds. This meant that cisplatin and carboplatin were the anticancer agents with the most PDDIs, a finding corroborated by Mouzon in a retrospective study \[[@ref50]\] and a review by Scripture \[[@ref29]\]. All PDDIs for these were linked with nephrotoxicity, which was also the potential outcome in the interactions involving pemetrexed, another nephrotoxic anticancer agent. Indeed, combining the PDDIs for all three makes kidney damage the most common potential outcome (20.4%). This is particularly dangerous because patients tend to be older and are more likely to have existing renal impairment \[[@ref64]\], putting them at greater risk. Other notable interactions are the remaining potentially serious PDDIs. Although only six of the 30 were classified in this way, each with low frequencies and thus constituting only 8.0% of the total PDDIs, possible outcomes such as increased risk of bleeding and adrenal suppression mean they should be actively avoided. It is also important to note that 10 PDDIs (totalling 27 instances) did not have a classified severity because they were identified from sources other than the BNF, and so any number could also be potentially serious DDIs, limiting the validity of the results. Medicines reconciliation and documentation of PDDIs --------------------------------------------------- The extent of identified interactions, with each patient on average having four PDDIs involving SACT medicines, highlights the necessity of safe prescribing and maintaining patient safety. Clinical audit was an effective method to measure this, identifying results as below expected. For the first standard, a remarkably low value of 3.0% of consultations including a documented Med-Rec implies there is almost a complete lack of the process occurring. Indeed, this was the case: the 13 documented Med-Recs were only performed when a patient was discharged from CWH, and never in clinic. UK and US guidelines focus primarily on Med-Rec for inpatients, with guidance stating it should be performed for any 'transfer of care', either on admission to hospital, during transfer between wards or on discharge \[[@ref27]\]. There is no formally established procedure for patients in ambulatory care at CWH, including this cohort of lung cancer patients attending regular Oncology clinics. This implies there is scope for implementation of Med-Rec processes for outpatients. IHI suggest collecting a full medicines history and then reviewing if there have been any changes during the consultation, after which a list can be kept 'on file' and verified for each subsequent appointment \[[@ref65]\]. Several intervention studies have assessed the advantages of reminders for patients to bring in their regular medicines to clinic followed by correction of the medicines list by patients themselves \[[@ref66]--[@ref68]\], all with encouraging results. A further justification to perform Med-Rec for these patients is that attending clinic could be seen as a 'temporary' transfer of care, during which the processes of Med-Rec should be carried out. These shortcomings are also reflected in the second standard regarding PDDI documentation. This again fell well below 100%, suggesting prescribing HCPs do not regularly review the safety of medicines being taken by the patient. Moreover, some were acknowledged simultaneously, meaning the 14 documented PDDIs were across eight consultations only. This below-standard practice could be due to the assumption made by HCPs that reviewing medicines comes under the remit of Pharmacy; involving pharmacists in a multidisciplinary team to ensure accurate medicines lists could improve the documentation of PDDIs and thus prevention of interactions, as seen in Lopez-Martin's single-centre study in Oncology outpatients \[[@ref61]\]. While increasing documentation of PDDIs is clearly key, there were some interesting patterns in the acknowledgement of certain categories. The results revealed the potentially serious interactions were significantly more likely to be documented than non-serious PDDIs. There are also three classes of interacting medicine that constitute the majority (71.4%) of the documented interactions: anticoagulants, PPIs, and antiretrovirals. This suggests that there may be awareness about certain types of DDI involving specific drugs, without the need for research. Finally, combining the aspects of the study, it appears that performing Med-Rec does not determine whether PDDIs are documented, which was reasonably assessed by the occurrence of both processes in the same consultation. However, this does not take into account the fact that Med-Rec may not always be carried out in a consultation, even though face-to-face Med-Rec is recommended in local guidelines \[[@ref41]\]; it could feasibly be done by comparing to the current medicines list without patient input. This means that there is possible underreporting of Med-Rec, potentially skewing results. Despite this, even just 'verification' correlates poorly (14.3%) with documentation of PDDIs, suggesting that the majority of acknowledged PDDIs were independent of the taking of a full medicines history in that consultation. Thus, it is beneficial to look at some of the many techniques already established to improve identification of potential interactions, such as the pharmacist-led PINCER \[[@ref69]\], the screening tools for elderly patients STOPP/START \[[@ref70]\], and My Medicines Passport, a patient-oriented booklet for recording medicines launched in CWH \[[@ref71]\]. Conclusion ========== In summary, this study provides a broad review of the common and potentially serious DDIs between SACT medicines and other medicines taken in a cohort of lung cancer patients. It also confirms the lack of established procedure for Med-Rec in these patients and highlights the scope for improvement in identifying more potential interactions, particularly as cancer patients are at risk of polypharmacy. However, the multidisciplinary care required in oncology, including general practice, must be taken into account; thus, implementing these processes in a hospital setting would not eliminate all risk of drug-related harm, but rather, contribute to the bigger picture of ensuring safe medicine prescribing across the whole of the patient journey. Ethical considerations ====================== Ethics approval is not required for this work as it is part of a service evaluation and improvement project. An ethics waiver was given by Chelsea and Westminster Hospital NHS Foundation Trust Research and Development lead and NRES for CLAHRC NWL Medicines Optimisation projects. ![](can-11-764fig3){#figure3} ---------------------------------------------------------------------------------------------------------------------------- SACT regimen Route of regimen Supportive care medicines used Route of supportive care medicine -------------------------------- ---------------------- -------------------------------- ----------------------------------- Pemetrexed\ Intravenous Folic acid Oral (maintenance) Ondansetron Intravenous and oral Vitamin B12 (hydroxocobalamin) Intramuscular Pemetrexed + cisplatin Intravenous Aprepitant Oral Dexamethasone Domperidone Folic acid Magnesium sulphate Intravenous Ondansetron Intravenous and oral Vitamin B12 (hydroxocobalamin) Intramuscular Gemcitabine + carboplatin Intravenous Domperidone Oral Dexamethasone Intravenous and oral Ondansetron Nivolumab Intravenous None N/A Etoposide +\ Intravenous Dexamethasone Intravenous carboplatin Ondansetron Pemetrexed + carboplatin Intravenous Aprepitant Oral Dexamethasone Domperidone Folic acid Magnesium sulphate Intravenous Ondansetron Intravenous and oral Vitamin B12 (hydroxocobalamin) Intramuscular Gefitinib Oral None N/A Erlotinib Oral None N/A Crizotinib Oral None N/A Carboplatin + vinorelbine Intravenous and oral Aprepitant Oral Dexamethasone Intravenous and oral Ondansetron Magnesium sulphate Intravenous Potassium chloride Docetaxel +\ Intravenous and oral Domperidone Oral nintedanib Dexamethasone Intravenous and oral Ondansetron ---------------------------------------------------------------------------------------------------------------------------- ![Most common SACT regimens for lung cancer between January and December 2014. Key: GEMCARBO=gemcitabine + carboplatin. Taken from \[[@ref16]\].](can-11-764fig1){#figure1} ![Frequency of PDDIs (total n = 88) for each of the 13 SACT medicines that had at least one identified PDDI.](can-11-764fig2){#figure2} ###### Summary of anticancer agents used in lung cancer with indications, class and mechanism of action. Taken and adapted with kind permission from \[[@ref6]\], contributions from \[[@ref1], [@ref2], [@ref4], [@ref7]--[@ref15]\]. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Anticancer agent Indication(s) in lung cancer Class of anticancer agent Main mechanism -------------------------------------------- ------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------- Cyclophosphamide Extensive SCLC Nitrogen mustard Intrastrand cross linking of DNA Carboplatin, cisplatin Stage I--II NSCLC (adjuvant) Platinum-based compound Stage IIIb--IV NSCLC (palliative) All-stage SCLC (palliative) Pemetrexed Stage IIIb--IV non-squamous NSCLC\ Folate antagonist Blocking the synthesis of DNA and/or RNA (first line) Gemcitabine Stage IIIb--IV squamous NSCLC (first-line) Pyrimidine pathway antimetabolite Doxorubicin Extensive SCLC Anthracycline Multiple effects on DNA/RNA synthesis and topoisomerase action Docetaxel, paclitaxel Stage IIIb--IV NSCLC (first line) Taxane Microtubule assembly; prevents spindle formation Vincristine Extensive SCLC Vinca alkaloid Vinorelbine Stage IIIb--IV squamous NSCLC (first line) Topotecan Relapsed SCLC Campothecin Inhibition of topoisomerase Etoposide All-stage SCLC (palliative) Other plant derivative Afatinib, erlotinib, gefitinib Stage IV--NSCLC + EGFR mutation\ Epidermal growth factor receptor\ Inhibition of kinases involved in growth factor receptor transduction (first line) (EGFR)-tyrosine kinase inhibitor (TKI) Stage IV NSCLC (second line if refractory) Crizotinib Stage IIIb--IV NSCLC + ALK translocation (second line) Anaplastic lymphoma kinase (ALK) inhibitor Nintedanib Stage IIIb--IV non-squamous NSCLC (second line, if progressive disease after first line) Vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-3) and platelet-derived growth factor receptor (PDGFRα,β) inhibitor Nivolumab Stage IV squamous and non-squamous NSCLC Anti-programmed cell death-1\ Recruitment of T cells (PD-1) monoclonal antibody -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ###### SACT regimens of all patients (n = 23). SACT regimen Frequency (%) ------------------------------------- --------------- Intravenous 14 (60.9) -- Pemetrexed (maintenance) -- 7 (30.4) -- Post-pemetrexed + cisplatin -- 6 (26.1) -- Post-gemcitabine + carboplatin -- 1 (4.3) -- Nivolumab -- 3 (13.0) -- Gemcitabine + carboplatin -- 2 (8.7) -- Etoposide + carboplatin -- 1 (4.3) -- Pemetrexed + carboplatin -- 1 (4.3) Oral 7 (30.4) -- Gefitinib -- 4 (17.4) -- Erlotinib -- 2 (8.7) -- Crizotinib -- 1 (4.3) Combined (intravenous and oral) 2 (8.7) -- Carboplatin + vinorelbine -- 1 (4.3) -- Docetaxel + nintedanib -- 1 (4.3) ###### Summary of the identified PDDIs (n=30). Key: UGT=UDP-glucuronosyltransferase; ACEIs=angiotensin-converting enzyme (ACE) inhibitors; CCBs=calcium channel blockers; ARBs=angiotensin-II receptor blockers; PPIs=proton pump inhibitors. SACT medicine Interacting (class of) drug Possible outcome (effect of interaction) Severity of interaction n ---------------------------------------------------- -------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------- ------------------------- ---- Aprepitant Ritonavir Aprepitant toxicity (increased exposure) Non-serious 1 Warfarin Reduced anticoagulation (reduced exposure to warfarin) Non-serious 1 Crizotinib UGT substrates (amoxicillin, colecalciferol, diazepam, levomepromazine, metoclo-pramide, mirtazapine) Various (increased exposure to UGT substrates) Unknown 6 Dexamethasone Poor crizotinib efficacy (reduced exposure) Unknown 1 Dexamethasone Antihypertensives (ACEIs, CCBs, ARBs, beta-blockers, nitrates) Raised blood pressure (antagonised effect of antihypertensives) Non-serious 11 Antidiabetics (metformin, gliclazide, linagliptin) Raised blood glucose (antagonised effect of antidiabetics) Non-serious 6 Diuretics (furosemide, bendroflumethiazide) Hypokalaemia and associated signs and symptoms Non-serious 5 Aspirin Gastrointestinal bleeding and ulceration (reduced exposure to salicylate) Non-serious 5 Calcium carbonate Hypocalcaemia and associated signs and symptoms (reduced exposure to calcium salts) Non-serious 4 Coumarins Enhanced (high-dose corticosteroids) or reduced anticoagulation (increased or reduced exposure to coumarins) Potentially serious 1 Phenindione Enhanced or reduced anticoagulation (increased or reduced exposure to phenindione) Non-serious 1 Ritonavir Adrenal suppression (increased exposure to corticosteroids) Potentially serious 1 Docetaxel CYP3A inhibitors (paracetamol, PPIs) Docetaxel toxicity (increased exposure) Unknown 2 Clarithromycin Myelosuppression; docetaxel toxicity (increased exposure) Potentially serious 1 Domperidone Opioid analgesics (codeine, morphine) Gastroparesis (antagonised gastrointestinal effects of domperidone) Non-serious 5 Clarithromycin Domperidone toxicity (increased exposure to domperidone); ventricular arrhythmias Potentially serious 2 Tiotropium Gastroparesis (antagonised gastrointestinal effects of domperidone) Non-serious 1 Erlotinib PPIs/H2 antagonists/antacids (lansoprazole, ranitidine, sodium bicarbonate) Poor efficacy (reduced exposure to erlotinib) Unknown 3 Statins Myopathy Unknown 1 Folic acid Sodium bicarbonate Folate deficiency and associated signs and symptoms (reduced exposure to folic acid) Non-serious 2 Gefitinib CYP3A4 inhibitors (diclofenac, clindamycin) Gefitinib toxicity (increased exposure) Unknown 2 PPIs (lansoprazole, omeprazole) Poor gefitinib efficacy (reduced exposure) Unknown 2 CYP3A4 inducers (nevirapine, flucloxacillin) Poor gefitinib efficacy (reduced exposure) Unknown 2 Warfarin Enhanced anticoagulation (increased exposure to warfarin) Potentially serious 1 Nivolumab Dexamethasone Systemic immunosuppression Unknown 1 Ondansetron Sertraline Enhanced serotonergic effects Non-serious 1 Pemetrexed Nephrotoxic drugs (ibuprofen, ACEIs, sulphamethoxazole) Nephrotoxicity; pemetrexed toxicity (increased exposure) Non-serious 6 Platinum-based compounds (cisplatin, carboplatin) Nephrotoxic drugs (aspirin, ibuprofen, ACEIs, sulphamethoxazole) Nephrotoxicity Unknown 7 Diuretics (bendroflumethiazide, furosemide) Nephrotoxicity; ototoxicity Non-serious 5 Potassium chloride Irbesartan Hyperkalaemia and associated signs and symptoms Potentially serious 1 ###### Summary of acknowledged and documented instances of PDDIs (n = 14). ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Patient \# SACT medicine Interacting medicine Effect of interaction Severity of interaction --------------- --------------- -------------------------------------------------------------------------------------- ---------------------------------------------- ------------------------- 1 Aprepitant Ritonavir Increased exposure to aprepitant Non-serious Dexamethasone Ritonavir Increased exposure to corticosteroids, causing increased risk of adrenal suppression Potentially serious 2 Nivolumab Dexamethasone Increased risk of systemic immunosuppression Unknown 3 Aprepitant Warfarin Reduced anticoagulant effect of warfarin Non-serious Dexamethasone Coumarins Enhanced (high-dose corticosteroids) or\ Potentially serious reduced anticoagulant effect of coumarins Dexamethasone Phenindione Enhanced or reduced anticoagulant effect of phenindione Non-serious 4 Gefitinib Warfarin Enhanced anticoagulant effect of warfarin Potentially serious 5 Gefitinib Lansoprazole Reduced exposure to gefitinib Unknown 6 Gefitinib Flucloxacillin Reduced exposure to gefitinib Unknown 7 Erlotinib Lansoprazole Reduced exposure to erlotinib Unknown Erlotinib Statin Increased risk of myopathy Unknown 8 Gefitinib Nevirapine Reduced exposure to gefitinib Unknown Gefitinib Omeprazole Reduced exposure to gefitinib Unknown Gefitinib Diclofenac Increased exposure to gefitinib Unknown -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------	{ "pile_set_name": "PubMed Central" }	[ "treatment", "probabl", "postgemcitabin", "choic", "two", "nevirapin", "almost", "control", "given", "work", "remain", "block", "nitrogen", "decreas", "compris", "administr", "nonsquam", "impair", "nre", "postpemetrex", "collect", "latestag", "highdos", "exist", "anticip", "put", "identifi", "healthcar", "remit", "discharg", "risk", "written", "newsomdavi", "regularli", "anticanc", "rais", "select", "bigger", "kind", "mechan", "lca", "take", "vegfr", "valu", "task", "multipl", "cisplatin", "may", "team", "potassium", 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All relevant data are within the paper and its Supporting Information files. Introduction {#sec005} ============ Buruli ulcer (BU) is a Neglected Tropical Disease for which the World Health Organization (WHO) stressed the need to improve treatment \[[@pntd.0004076.ref001]\]. BU remains endemic in areas in West Africa, especially in Benin and Ghana \[[@pntd.0004076.ref002],[@pntd.0004076.ref003]\]. In Benin, prevalence rates between 5.4 and 60.7 per 10.000 inhabitants have been reported each year \[[@pntd.0004076.ref004]\] while in Ghana, prevalence rates have fluctuated between 2.0 and 15.0 per 10.000 inhabitants \[[@pntd.0004076.ref005]\]. BU destroys skin, subcutaneous fat, and sometimes bone \[[@pntd.0004076.ref006]\]. Patients typically present with non-ulcerated lesions; papules, nodules, plaques or edema, or undermined ulcers \[[@pntd.0004076.ref006],[@pntd.0004076.ref007]\]. Treatment entails antibiotics complemented with surgery if needed, together with dressing changes of which the frequency depends on the wound, and physiotherapy \[[@pntd.0004076.ref008]\]. Specialized treatment centers deliver the necessary care while antimicrobial treatment and dressings may also be delegated to local health centers \[[@pntd.0004076.ref009]\]. Irrespective of therapy (antibiotics or surgery), 47% of patients are left with functional limitations after healing \[[@pntd.0004076.ref010]\]. Although BU is described as relatively painless \[[@pntd.0004076.ref011]--[@pntd.0004076.ref013]\] clinical observations reveal that at least some patients experience considerable pain during their treatment---especially during wound care and physiotherapy. This is supported by two studies, i.e., one in Japanese patients \[[@pntd.0004076.ref014]\], and one in Ghanaian patients \[[@pntd.0004076.ref015]\]. Ghanaian BU patients sometimes complained of pain at the lesion site just before and during ulceration. This suggests a recovered sensation---perhaps due to a decrease in mycolactone concentrations \[[@pntd.0004076.ref016]\] in tissues at advanced disease stages, however this suggestion warrants verification by clinical studies. Clinical studies on pain and pain treatment among BU patients are lacking. The recognition, management and treatment of pain is a challenge worldwide, but limited resources in low- and middle-income countries further increase the risk of under treatment of pain \[[@pntd.0004076.ref017],[@pntd.0004076.ref018]\] Health care personnel (HCP) treating BU in Ghana and Benin stressed the need to include pain management \[[@pntd.0004076.ref019]\]. The WHO recommends integrating pain treatment as part of general treatment of patients globally \[[@pntd.0004076.ref018]\]. Pain management should follow a practice guideline, which is currently not available for BU treatment, including at least an analgesic ladder, i.e. the 'pain ladder', initially developed by the WHO originally for cancer pain control \[[@pntd.0004076.ref020],[@pntd.0004076.ref021]\], but now used for pain relief among patients with wound pain \[[@pntd.0004076.ref022]\]. The ladder consists of three steps of drugs with increasing analgesic effects. Previous studies showed that perceptions and beliefs of health professionals are important in pain treatment \[[@pntd.0004076.ref023]\]. In addition, barriers related to the availability of- and access to analgesics, a lack of education and information among HCP, and legal barriers such as regulatory restrictions on opioids exists, preventing effective pediatric pain treatment in sub-Saharan Africa \[[@pntd.0004076.ref024]\]. Furthermore, language barriers, fear of addiction to opioids, and cultural differences between patients and health professionals, influenced pain management in Central Africa \[[@pntd.0004076.ref025]\]. Firstly, this study investigates current pain practices in BU in Ghana and Benin, including the prescription of pain medication, and the use of the WHO pain ladder by the HCP. Secondly, this study aims to explore factors that might influence the success of a possible future guideline implementation. Methods {#sec006} ======= Design and participants {#sec007} ----------------------- A mixed methods approach was used, consisting of information on prescribed pain medication, retrieved from medical records from Ghana and Benin, and semi-structured interviews. Data collection was performed in the same period in both countries. Two out of the four BU treatment centers in Benin, and two out of the four BU treatment centers in Ghana were selected for the study. Information from the medical records of patients treated between 2008 and 2012 was collected between November 2012 and August 2013. In the two treatment centers in Ghana, medical records of all PCR confirmed BU patients admitted and treated in the selected time period, were included (n = 69). In the two treatment centers in Benin, a larger number of records of PCR confirmed BU cases was found in the selected time period, thus a systematic selection (every 8 ^th^ medical record) was performed in both centers to arrive at 40 records per center. Thus, in Ghana 69 medical records of patients were included, and in Benin 80 medical records of patients leading to a total of 149 patients. Eligible HCP were selected by purposive sampling based on their involvement in BU wound care, their profession---we selected medical doctors, nurses, physiotherapists and local health workers, to ensure heterogeneity of the interviewees-, and the ability to speak English. In total 13 HCP were eligible and approached. Interviews were conducted between August 2012 and May 2013. Data collection {#sec008} --------------- The interviews were held in private settings in the hospital or health center in Ghana, and were conducted by one of the authors (MA, JDZ or SL). Interviews were tape recorded and lasted between 60 and 90 min. Materials {#sec009} --------- Data on general characteristics and diagnostic tests were collected from the records. The type of pain medication and its indication were retrieved as well. An interview topic guide was developed, covering HCPs perceptions on current pain assessment and treatment. Specific topics included were; current practice, current prescription of pain medication, HCPs preferences for prescribing pain medication, satisfaction with current practice, wishes for improvements, perceptions on pain, pain assessment, and the acceptability of showing pain. Questions were derived from previously published work \[[@pntd.0004076.ref024]\]. Probes were used where necessary. Face and content validity of the interview guide were assessed by the study team, ensuring that questions adequately covered the objectives and were relevant. Data analysis {#sec010} ------------- Data on prescribing behavior and patient characteristics from the records were analyzed using Statistical Package for Social Science version 20. Descriptive analysis was performed on age and sex of patients whose records were included, including the type of lesion, indication for pain medication, types of pain medication prescribed during hospitalization and the types of pain medication prescribed in line with the WHO pain ladder. The total number of times that pain medication was prescribed for each step of the WHO pain ladder was counted. Interviews were transcribed verbatim by three different researchers (MA, JDZ and SL). A qualitative content analysis was performed using open coding and axial coding. To ensure reliability, interviews were analyzed consecutively by two researchers (MA and JDZ). These two researchers developed an initial codebook independently using one interview, and taking into account the interview questions. Both researchers participated in weekly meetings to extensively discuss the open coding analysis until consensus was reached, after which codes were merged into one codebook. The codebook was adapted throughout the analysis, based on new codes emerging from the data. A third and fourth researcher (AVR, YS) were consulted in case of disagreement, and ensured an acceptable coding of the data, ordering of the codes, and selection of themes. Qualitative and quantitative data were combined according to parallel data analysis \[[@pntd.0004076.ref026]\]. Ethics {#sec011} ------ Ethical clearance was obtained from the Medical Ethical Review Committee of the Kwame Nkrumah University of Science and Technology; School of Medical sciences, Komfo Anokye teaching hospital in Ghana (ref: CHRPE/AP/230/12). Data of medical records was handled in line with Good Clinical Practice. Written informed consent was obtained from all HCP. Before the interview started, voluntary participation, confidentiality, the aim, the topic and type of questions, the rights to withhold certain information, and to withdraw during the interview, were explained. Written informed consent was obtained from all HCP. In order to ensure anonymity and confidentiality, a number was assigned to each interview. Results {#sec012} ======= Prescribed pain medication {#sec013} -------------------------- ### Patient characteristics from medical records {#sec014} In total, 149 medical records were reviewed (69 from Ghana, 80 from Benin), of which 14 were incomplete, resulting in 135 records included in the analysis. In 113 (84%) of the 135 included records, pain medication was prescribed at least once during hospitalization. The median weeks of hospitalization was 17 (IQR: 10--23). The median age of the patients receiving pain medication was 14 years (IQR: 8--28), and 65 (58%) was male ([Table 1](#pntd.0004076.t001){ref-type="table"}). In 64% of the medical records, ulcers were reported, 15% plaque, 10% edema and 3% nodules (8% missing). 10.1371/journal.pntd.0004076.t001 ###### Patient characteristics (n = 113). ![](pntd.0004076.t001){#pntd.0004076.t001g} Total ---------------------------------------------------- -------------- ------------- Sex (male) 65 (58%) Age (Median, IQR) 14 (8--28) Type of ulcer Ulcer 64% Plaque 15% Edema 10% Nodule 3% Not reported 8% Duration of hospitalization in weeks (Median, IQR) 17 (10--23) ### Type of pain medication (different generics) prescribed during hospitalization {#sec015} In most records (43.4%; n = 49), two types of pain medication were prescribed, while in 35.4% of records (n = 40), one type of pain medication was prescribed. In 13.3% of the records (n = 15), three types were prescribed and in 7.9% of the records (n = 9), four or five types of medication were prescribed. ### Pain medication according to WHO ladder {#sec016} The total number of times pain medication was prescribed was 482. Step 1 medication (i.e. simple analgesics) was mostly prescribed (n = 430), for several indications. Pain medication is mostly prescribed after a surgical intervention (n = 165; 34%) ([Table 2](#pntd.0004076.t002){ref-type="table"}), however, often, no indication was noted in the records (n = 233; 48%). Step 2 and 3 medications (weak and strong opioids, respectively) were rarely prescribed (n = 52) ([Table 2](#pntd.0004076.t002){ref-type="table"}). Predominantly, pain medication is prescribed after an intervention, and less often for background pain. 10.1371/journal.pntd.0004076.t002 ###### Pain medication prescribed along different indications according to WHO ladder (n = 482). ![](pntd.0004076.t002){#pntd.0004076.t002g} Indication Step 1[\](#t002fn001){ref-type="table-fn"} Step 2[\\](#t002fn002){ref-type="table-fn"} Step 3[\\\](#t002fn003){ref-type="table-fn"} ------------------------------------------------------- --------------------------------------------- ----------------------------------------------- ------------------------------------------------- Post-surgical pain 151 12 2 (before) wound dressing 5 5 0 Other interventions (e.g. removal of necrotic tissue) 14 3 0 Infected wound/wound related pain 12 2 0 Generalized body pain 18 1 0 Not related to pain (e.g. fever, diarrhea) 22 2 0 Missing indication 208‡ 24 1 Total 430 49 3 \Paracetamol/acetaminophen, ibuprofen, acetylsalicylic, diclofenac and diclofenac sodium. \\* paracetamol/codeine, tramadol and dihydrocodeine. \\\* pethidin ‡ of the missing indications 136 times paracetamol/acetaminophen was prescribed Current pain assessment and treatment-results from the semi-structured interviews- {#sec017} ---------------------------------------------------------------------------------- 11/13 eligible HCP agreed to participate in the semi structured interview; nurses (4), medical doctors (2), BU coordinators (2), physiotherapist (1), health worker (1) and pharmacy technologist (1). Two participants refused because of a lack of time. Three factors in current pain assessment and treatment were important, i.e., perceptions on pain, pain assessment and pain treatment, including the use of the WHO pain ladder. Perceptions on pain in BU {#sec018} ------------------------- Pain is described as 'an uncomfortable feeling' or 'an alarm'. In early stages of the disease, BU is painless, however, in later stages patients start to feel pain: 'So when they start to heal, they start to feel pains'. Perceived causes of pain were; wound pus, ulceration of the lesion, inflammation, nerve exposure, secondary infection, a graft at the donor site, proximity to bone or joint. An increase in pain is mentioned after wound treatment. > 'Yes, normally, patients with BU, actually don't have pain. Unless there is a secondary bacterial infection. There is involvement of the nerves and involvement of the bone tissue'. Pain threshold is described by the HCP as 'The extent to which a person can handle pain', or as 'How patients react to pain'. According to the participating HCP, patients differ in their pain threshold and pain tolerance. Factors related to differences in pain tolerance are; previous experiences, gender differences, age of the patient, and size of the ulcer. Expression of pain and its assessment {#sec019} ------------------------------------- Nurses and a physiotherapist reported not to be trained in assessing mild pain, neither do they ask patients about mild pain. They attributed this to their culture; patients are able to handle mild pain. > 'We \[HCP\] are not trained to recognize the mild ones quickly, it is a cultural problem, we know people take the mild ones, so I don't ask, suggest pain to you \[patient\].' Instead of focusing on whether patients report pain, professionals pay attention to facial expression, body language, wound characteristics, individual characteristics, medical background, and patients' behavior. > 'But you can see from the face whether it is painful or not. So we look for signs, because otherwise, we are going to get wrong feedback.' > 'Assuming you have a lesion, and I touch it, and you don't complain, that means you are ok, there is no pain. But if I touch it \[the lesion\] and you pull your hand, I mean the little thing I do, hurts to your hand, and you are preventing me. The moment you start and the patient is starting crying and screaming and all these things, you can easily know that now, the patient has a severe pain.' Suppressing pain expression is ascribed to cultural factors; which is especially common among adult males, patients from the northern part of the country, patients from rural areas, and patients with a lower educational level. This cultural tendency hampers a proper pain assessment. > 'Location wise, you see people from the northern part of the country, they are very good adapted at taking pain. I would rather classify them as the affluent compared to the less affluent or the region poor or urban and the village. If it is in the city, well-educated parents know that they shouldn't be treated this way, so they get better treatment when you have more enlightened parents. People try to contain pain. It is a country wide a cultural thing, we are brought up to be able to contain an amount of pain, so you are a cry baby if you express too much pain.' On the other hand, the majority of the HCP mentioned that patients exaggerate pain. This paradox could imply that---since HCP are part of the Ghanaian culture in which suppression of pain expression is commonly seen---they believe that patients who express pain overtly, are exaggerating. > If it is true pain, or fake pain, we don't know, you measure, you look at the patient.' > 'You rate it for the patient, but not what the patient tells you. Because psychologically, the patient would tell you 'oh, it is very painful'. Meanwhile, it is not like that.' Pain treatment {#sec020} -------------- Important factors in current pain treatment include the financial constrains that patients often have to pay for pain medication. Moreover, HCP report a fear of side effects of pain medication, a fluctuation in availability of pain medication, and a shortage of time among medical doctors. > 'We shouldn't over rush in giving pain killers, because there are side-effects attached to the drugs'. Furthermore, HCP report a discrepancy between their own as compared to their patients' expectations regarding pain relief. While professionals expect patients to endure pain to some extent, patients expect to be relieved from pain during hospital admission, according to the HCP. > 'They \[patients\] believe as soon as they're in the hospital, everything must be stopped. As soon as they're operated they should have no pain at all, even not the wound pain. I always tell them that operation does not mean that everything is healed, that pain stopped, the wound is there, so it pains'. Besides, non-pharmacological factors in the current pain treatment include the different coping strategies used by the HCP to help the patient to handle pain. Examples are: counseling (providing information, reassurance, showing empathy) and giving advice. > 'Explaining to patient, oh no, this is just a little thing that I would just only have to give you, but it will minimize the pain that you are going through, because you fell from the bike. So your pain will minimize.' Furthermore, while the HCP were not explicitly familiar with the WHO ladder, they reported to use the basic principles of the tool. > 'It is about, if someone is in pain then you start with paracetamol and then if someone has more severe pain you can give paracetamol and another pain killer which is more effective and then in the end you can give something like pethidin.' Discussion {#sec021} ========== This study aimed to explore current pain practice in BU in Ghana and Benin. For most BU patients, pain medication was prescribed, and pain management mainly focused on severe pain. Professionals perceived later stages of BU as painful, and reported an increase in pain after wound treatment, and after a skin graft at the donor site. HCP reported a suppressed pain expression as well as exaggeration in patients, and differences in expectations between professionals and patients on what is an acceptable pain level without medication. Mainly WHO step 1 pain ladder medication was prescribed, while strong opioids were hardly prescribed. Explanations provided by the HCP on the mild prescriptions were; fear of side effects of strong opioids, fluctuation of availability of pain medication, and the shortage of time among medical doctors. These findings are in line with literature mentioning resistance among HCP to use morphine \[[@pntd.0004076.ref027]\]. Alternative explanations are that HCP did not ask whether patients experienced pain, or a lack of attention for mild pain. At the same time, the prescribed pain medication is typically for mild pain, according to the WHO pain ladder indicating that severe pain is treated with mild pain medication. It appeared that the indication for pain medication was often not documented in the medical records. Effective pain relief ultimately depends on accurate pain assessment, and the nature, severity, location and duration of pain should be assessed and documented to understand the possible etiology and to effectively treat pain \[[@pntd.0004076.ref028]\]. Both the finding that HCP believed that patients exaggerated their pain, and the expectation that patients should endure pain, might be influenced by sociocultural factors, since pain expression varies across cultures worldwide \[[@pntd.0004076.ref028]\]. In different African ethnic groups, stoicism is a valued response to pain \[[@pntd.0004076.ref029],[@pntd.0004076.ref029],[@pntd.0004076.ref030]\]. Despite the cultural factors, differences in pain expression can be due to individual differences \[[@pntd.0004076.ref031]\]. An important implication of our finding is that it complicates pain assessment for professionals. Patients and professionals differed in their expectation on acceptable pain levels without medication. HCP noticed that patients expected to be pain free during hospitalization, a statement that should be confirmed in studies using a patients' perspective. If these results can be confirmed, patients' expectations could be addressed during the preoperative pain assessment by collaboratively setting goals for pain control \[[@pntd.0004076.ref032]\], or during the intake for hospitalization. Furthermore, if documentation of pain will be integrated in daily practice, pain can be monitored, which is essential for adequate treatment. This study had several limitations. For the part on the medical records, an information bias might have occurred due to the retrospective design of the study. In a small proportion of medical records, no information on pain medication was found, and in case pain medication was prescribed, the indication was often missing. In addition, by using medical records, only the prescribing behavior of HCP was studied, while the actual intake remained unknown. A limitation of the interviews was the possible bias introduced by the role of non-native interviewers. Although the interviewers spent time in the different BU settings, cultural differences remained. This might have influenced the interviews. To conclude, these findings, together with a study on wound care-related pain in BU \[[@pntd.0004076.ref015]\], suggest that there is room for improvement to arrive at adequate pain treatment. Several factors could be taken into account when developing a pain guideline, such as the current practice on prescribing pain medication, the discrepancy between HCP and patients about pain relief, the views on pain expression and suppression, recognition and treatment of mild pain, and the lack of recorded indications. Furthermore, the findings that HCP tried to help the patient to cope with pain by providing information, reassurance, showing empathy, giving advice, as well as the finding that HCP were aware of the basic principles of the WHO ladder are useful in the development phase of such a protocol. We would like to thank the health care personnel from Benin and Ghana who participated in the interviews, as well as the personnel who assisted us with collecting the medical records of former BU patients. In addition, we would like to thank the board members of the participating hospitals for facilitating the study. [^1]: The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: JdZ MA YTB GS ROP TSvdW AVR YS. Performed the experiments: JdZ MA SL. Analyzed the data: JdZ MA YTB BM MP AVR YS. 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Introduction {#section1-1524838017697312} ============ Timely access to supportive and therapeutic resources for child sexual abuse (CSA) survivors can mitigate risk to the health and mental health well-being of children, youth, and adults. Identifying and understanding factors that promote or inhibit CSA disclosures have the potential to facilitate earlier disclosures, assist survivors to receive services without delay, and potentially prevent further sexual victimization. Increased knowledge on both the factors and the processes involved in CSA disclosures is timely when research continues to show high rates of delayed disclosures ([@bibr8-1524838017697312]; [@bibr9-1524838017697312]; [@bibr11-1524838017697312]; [@bibr17-1524838017697312]; [@bibr20-1524838017697312]; [@bibr25-1524838017697312]; [@bibr31-1524838017697312]; [@bibr42-1524838017697312]). Incidence studies in the United States and Canada report decreasing CSA rates ([@bibr13-1524838017697312]; [@bibr14-1524838017697312]; [@bibr49-1524838017697312], [@bibr50-1524838017697312]), while at the same time global trends from systematic reviews and meta-analyses have found concerning rates of CSA, with averages of 18--20% for females and of 8--10% for males ([@bibr37-1524838017697312]). The highest rates found for girls is in Australia (21.5%) and for boys in Africa (19.3%), with the lowest rates for both girls (11.3%) and boys (4.1%) reported in Asia ([@bibr46-1524838017697312]). These findings point to the incongruence between the low number of official reports of CSA to authorities and the high rates reported in prevalence studies. For example, a meta-analysis conducted by [@bibr46-1524838017697312]) combining estimations of CSA in 217 studies published between 1980 and 2008 revealed rates of CSA to be more than 30 times greater in studies relying on self-reports (127 in 1,000) than in official report inquiries, such as those based on data from child protection services and the police (4 in 1,000) ([@bibr23-1524838017697312]; [@bibr45-1524838017697312]). In other words, while 1 out of 8 people retrospectively report having experienced CSA, official incidence estimates indicate only 1 per 250 children. In a survey of Swiss child services, [@bibr35-1524838017697312]) further found 2.68 cases per 1,000 of CSA disclosures, while in a recent comprehensive review [@bibr31-1524838017697312]) details the high prevalence of delayed, partial, and nondisclosures in childhood indicating a persistent trend toward withholding CSA disclosure. It is our view that incidence statistics are likely an underestimation of CSA disclosures, and this drives the rationale for the current review. Given the persistence of delayed disclosures with research showing a large number of survivors only disclosing in adulthood ([@bibr8-1524838017697312]; [@bibr11-1524838017697312]; [@bibr21-1524838017697312]; [@bibr31-1524838017697312]; [@bibr42-1524838017697312]), these issues should be a concern for practitioners, policy makers, and the general public ([@bibr31-1524838017697312]). The longer disclosures are delayed, the longer individuals potentially live with serious negative effects and mental health problems such as depression, anxiety, trauma disorders, and addictions, without receiving necessary treatment. This also increases the likelihood of more victims falling prey to undetected offenders. Learning more about CSA disclosure factors and processes to help advance our knowledge base may help professionals to facilitate earlier disclosures. Previous literature reviews examining factors influencing CSA disclosure have served the field well but are no longer current. Important contributions on CSA disclosures include [@bibr36-1524838017697312] original review covering the literature largely from the premillennium era, followed by [@bibr30-1524838017697312] subsequent review, which may not have captured publications affected by "lag to print" delays so common in peer-reviewed journals. These reviews are now dated and therefore do not take into account the plethora of research that has been accumulated over the past 15 years. Other recent reviews exist but with distinct contributions on the dialogical relational processes of disclosure ([@bibr39-1524838017697312]), CSA disclosures in adulthood ([@bibr48-1524838017697312]), and delayed disclosures in childhood ([@bibr31-1524838017697312]). This literature review differs by focusing on CSA disclosures in children, youth, and adults from childhood and into adulthood---over the life course. Method {#section2-1524838017697312} ====== [@bibr27-1524838017697312] systematic review framework was utilized to establish what has been investigated in CSA disclosure research, through various mixed methods, to highlight the most convincing findings that should be considered for future research, practice, and program planning. This review centered on the question: What is the state of CSA disclosure research and what can be learned to apply to future research and practice? By way of clarification, the term systematic refers to a methodologically sound strategy for searching literature on studies for knowledge construction, in this case the CSA disclosure literature, rather than intervention studies. The years spanned for searching the literature were 2000--2016, building on previous reviews without a great deal of overlap. Retrieval of relevant research was done by searching international electronic databases: PsycINFO, PsycARTICLES, Educational Resources Information Center, Canadian Research Index, International Bibliography of the Social Sciences, Published International Literature on Traumatic Stress, Sociological Abstracts, Social Service Abstracts, and Applied Social Science Index and Abstracts. This review searched peer-reviewed studies. A search of the gray literature (unpublished literature such as internal agency documents, government reports, etc.) was beyond the scope of this review because unpublished studies are not subjected to a peer-review process. Keyword search terms used were child sexual abuse, childhood sexual abuse, disclosure, and telling. A search of the 9 databases produced 322 peer-reviewed articles. Selected search terms yielded 200 English publications, 1 French study, and 1 Portuguese review. The search was further refined by excluding studies focusing on forensic investigations, as these studies constitute a specialized legal focus on interview approaches and techniques. As well, papers that focused exclusively on rates and responses to CSA disclosure were excluded, as these are substantial areas unto themselves, exceeding the aims of the review question. Review articles were also excluded. Once the exclusion criteria were applied, the search results yielded 33 articles. These studies were subjected to a thematic analysis as described by [@bibr5-1524838017697312]). This entailed (1) multiple readings by the three authors; (2) identifying patterns across studies by coding and charting specific features; (3) examining disclosure definitions used, sample characteristics, and measures utilized; and (4) major findings were extrapolated. Reading of the articles was initially conducted by the authors to identify general trends in a first level of analyses and then subsequently to identify themes through a deeper second-level analyses. A table of studies was generated and was continuously revised as the selection of studies was refined (see [Table 1](#table1-1524838017697312){ref-type="table"}). ###### Child Sexual Abuse (CSA) Disclosure Studies: 2000--2016. ![](10.1177_1524838017697312-table1) Study Purpose Design Sample Findings Summary ------------------------------ --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- [@bibr16-1524838017697312]) To explore disclosure processes for male victims of CSA Phenomenological methodology used to interview male CSA survivors. The Long Interview Method (LIM) guided data collection and analyses. 17 men ranged in age from 19 to 67---average age 47. Purposive sampling strategy was used The majority of the men in the study waited until adulthood to disclose their abuse, with negative stereotypes contributing to their delayed disclosures. Negative stereotypes contributed to delayed disclosure with trying to forget. Breaking isolation was cited as a motivator to disclosure along with the aid of various forms of media on disclosure. Important contextual issues such as negative stereotyping of males, sexuality, and victims were noted. Social media was seen as a facilitator of disclosures All participants had disclosed and received services before participating in the study. Member checking could not be done with the participants to check themes. Small but sufficient size for a qualitative inquiry. Otherwise, high level of rigor in establishing trustworthiness of the data and analysis. Retrospective study could imply recall issues [@bibr6-1524838017697312] To explore the meaning African American women make of their traumatic experiences with CSA and how they disclosed across the life course Collective case study design with using narrative tradition (storyboard) for data collection and analysis. Qualitative interviewing 17 African American women in mid-life between 40 and 63 who experienced intrafamilial CSA. Purposive, snowballing strategy CSA onset was largely between the ages 5 and 9. No one ever talked to them about sex, so they didn't have language to disclose. Barriers: fear of family breakdown and removal, not wanting to tarnish the family's name, and fear of retribution by family members if they disclosed. Pattern of stifled and dismissed disclosures identified over the life course. All 17 participants identified spirituality as a primary source of strength throughout the life course One of few studies to focus exclusively on African American women. Small but sufficient size for a qualitative inquiry. Important cultural and contextual issues were brought forward. Retrospective study that may have been affected by recall issues. Use of a life-course perspective as a theoretical lens for understanding CSA in the middle to later years of life that should be considered in further investigations [@bibr8-1524838017697312]) To provide a mapping of factors that prevent CSA disclosures through an ecological lens from a sample of CSA adult survivors. Qualitative design using LIM. 67 male and female CSA adult survivors (76% identified as female and 24% as male). Age ranges from 19 to 69 years (M = 44.9). Purposive sampling strategy Three broad categories were identified as barriers to CSA disclosure: Barriers from within-internalized victim blaming, mechanisms to protect oneself, and immature development at time of abuse; barriers in relation to others---violence and dysfunction in the family, power dynamics, awareness of the impact of telling, and fragile social network; barriers in relation to the social world labeling, taboo of sexuality, lack of services available, and culture or time period. Half of the participants had not disclosed their CSA experiences before the age of 19. Retrospective aspect of the study could imply recall issues. All participants had disclosed and received counseling at some before participating in the study. High level of rigor in establishing trustworthiness of the data and analysis [@bibr29-1524838017697312] Study objectives investigated the factors that facilitate CSA disclosures Adult male child sexual offenders were interviewed to examine predictors of victim disclosure. Semistructured interviews based on the QID questionnaire. 369 adult males who had been convicted of a sexual offense against a child aged between 1 and 17 years old. Majority were White, uneducated, almost half unemployed before their arrest Disclosure increased with the age of the victim: if penetration had occurred, if the victim was related to the offender, if the victim was not living with the offender at the time of the abuse, or if the victim resisted during the offense. Male victims and victims from dysfunctional backgrounds were less likely to disclose Offender generated data through self-reports could be subject to cognitive distortions---minimization or exaggerations. Perspectives of offenders on vulnerability of victims in relation to disclosure could be important information to inform interventions [@bibr32-1524838017697312]) To investigate the feasibility of using child assessments as data sources of informal CSA disclosure. To assess if these reports provide substantive data on disclosures File reports of children seen for assessment in a child sexual abuse unit in a children's hospital were reviewed Content analysis was completed on 39 files (32 females and 7 males) based on a coding framework. Parents were asked to consent to have their child's file reviewed for the study. Victims assessed were 12--18 years of age Majority of children told their mothers (43%) and peers (33%) first. Three major themes were identified as influencing the disclosure process: (1) feeling distressed, (2) opportunity to tell, and (3) fears for self. Additional themes of being believed, shame/self-blame, and peer influence were also identified The sample size is small but will contribute to a large multisite study in Ireland. Serves as an important exploratory pilot bringing forward disclosure themes for consideration [@bibr10-1524838017697312]) This study aimed to explore how the relationship between the perpetrator and the victim, especially whether these relations are intrafamilial or extrafamilial, impact CSA disclosure File reports of children seen for assessment in a child sexual abuse unit in a children's hospital were reviewed 220 minor victims---78.2% female victims, 41.8% aged between 14 and 18 (most prevalent age range), and 48.2% were abused by a family member Disclosure processes were more complex when it concerned sexual abuse committed by intrafamilial perpetrator: 60% of the victims reveal the facts several years after, and most often to individuals outside the family (78.6% of the disclosures done at school); on the contrary, extrafamilial disclosures take place more spontaneously and quickly: 80% of the victims reveal the facts a few days after, most often to their mother or peers The relationship with the perpetrator has a significant impact on both timing and recipient of disclosure, with intrafamilial abuses less likely to be disclosed promptly and within the family system [@bibr12-1524838017697312] Study focus was on identification of barriers to CSA disclosure with male survivors Using qualitative content analysis, researchers conducted a secondary analysis of online survey data, the 2010 Health and Well-Being Survey, that included men with self-reported CSA histories with an open-ended item on disclosure barriers 460 men with CSA histories completed an anonymous, Internet-based survey. Recruited from survivors' organizations. Age range of 18--84 years. Two thirds of respondents reported clergy-related abuse. Majority of respondents were White Vast majority of participants (94.6%) were sexually abused by another male. Duration of sexual abuse broke down into: 30.2% less than 6 months, 32.3% 6 months to 3 years, and 34.3% more than 3 years. Ten years old was average age of CSA onset. Ten categories of barriers were classified into three domains: (1) sociopolitical: masculinity, limited resources; (2) interpersonal: mistrust of others, fear of being labeled "gay," safety and protection issues, past responses; and (3) personal: internal emotions, seeing the experience as sexual abuse, and sexual orientation. At time of the study, this was the largest qualitative data set to have been analyzed with an explicit focus on adult male survivors' perceptions of barriers to CSA disclosure. Because the sample was limited in terms of the low percentage of racial minorities (9.3%), disclosure differences based on race or ethnicity were not discerned. The majority of abuse reported was by clergy which might present a unique set of barriers to disclosure [@bibr11-1524838017697312] Study purpose was to describe male CSA disclosure processes using a life span approach examining differences based on age. Also, to explore relationships between disclosure attributes and men's mental health Cross-sectional survey design. Eligible participants were screened and completed an anonymous, Internet-based survey during 2010. Measures used: General Mental Health Distress Scale and General Assessment of Individual Needs. Questions related to CSA disclosure and supports were included Purposive sampling of 487 men from three national organizations devoted to raising awareness of CSA among men. Age range: 19--84 years. Mean age for onset of CSA was 10.3 years Older age and being abused by a family member were both related to delays in disclosure. Most participants who told someone during childhood did not receive emotionally supportive or protective responses and the helpfulness of responses across the life span was mixed. Delays in telling were significant periods of time (over 20 years). Approximately one half of the participants first told about the sexual abuse to a spouse/partner (27%) or a mental health professional (20%); 42% of participants reported that their most helpful discussion was with a mental health professional. However, unhelpful responses caused most mental distress. Clinical recommendations included more of a life-course perspective be adopted, understanding impact of unhelpful responses and the importance of expanding networks for male survivors Purposive sampling of men from awareness raising organizations may have attracted particular participants who had already disclosed and received help. Participants needed to have access to Internet which would have eliminated men in lower SES groups and required proficiency in English which would eliminate certain cultural groups. However, the sampling strategy gained access to a predominantly hidden population. Important clinical recommendations are made with an emphasis on a life-course focus [@bibr33-1524838017697312] Qualitative study asked the central research question: "How do children tell?" Objective was to develop theory of how children tell of their CSA disclosure experiences. Parents were interviewed. Grounded theory method study. Interviews were conducted. Line-by-line open and axial coding was conducted on verbatim transcripts Sample of 22 young people; 16 girls and 6 boys; age range: 8--18 years; 22 interviewed in total between the ages of 8 and 18. Mixed sample of some enduring intrafamilial CSA, some extrafamilial CSA, and two endured both forms A theoretical model was developed that conceptualizes the process of CSA disclosure as one of containing the secret: (1) the active withholding of the secret on the part of the child; (2) the experience of a "pressure cooker effect" reflecting a conflict between the wish to tell and the wish to keep the secret; and (3) the confiding itself which often occurs in the context of a trusted relationship. These were derived from eleven categories that were developed through open and axial coding Modest but sufficient sample for an exploratory qualitative inquiry. High level of trustworthiness rigor. A subsample of randomly selected transcripts was independently coded. Very young children and young adults were not captured in this sample. Transferability of findings can only be made to the age range sampled in the context of Ireland [@bibr41-1524838017697312] To investigate the process of CSA disclosure with adolescents from the general population who had experienced CSA. How many disclosed, who did they disclose to, and what were their motives for disclosing Data collection was through face-to-face qualitative interviews. Standardized questions and measures were administered on family situation, sociodemographic data, sexual victimization, general, and mental health. Sexual Assault Module of the Juvenile Victimization Questionnaire was used Convenience sample of 26 sexually victimized adolescents. 23 girls and 3 boys. Age range: 15--18 years. Online advertisements and flyers were used to recruit youth from community and counseling services Less than one third of participants immediately disclosed CSA to another person. In most cases, recipients of both immediate and delayed disclosure were to peers. More than one third of participants had never disclosed the abuse to a parent. Participants reported reluctance to disclose to parents so as not to burden them. Earlier disclosures were related to extrafamilial CSA, single occurrence CSA, age of victim at abuse onset, and parents who were living together. Higher levels of reported guilt and shame were related to delayed disclosures. Peers were viewed by this sample as more reliable confidants Two thirds of the sample did not disclose right away. Strengthening parent--child relationships may be one of the most important ways to increase disclosure to parents. Disclosure to peers has been found a common trend in other research and bears more examination [@bibr21-1524838017697312] Aim of this study was to develop a fuller understanding of CSA disclosures Narrative inquiry methodology. Face-to-face in-depth interviews were conducted with participants. Data were analyzed using Rosenthal and Fischer--[@bibr55-1524838017697312] method. Purposive sampling was employed. Sample consisted of 22 participants aged 25--70 years; 13 women and 9 men. Participants were sexually abused at 15 years or under with someone over the age of 18. Only 5 out of 22 participants told anyone about their early sexual experiences as children. Fear, shame, and self-blame were the main inhibitors to disclosure. These factors are further detailed through subthemes. Telling as a child and as an adult was further expanded upon using [@bibr1-1524838017697312]) framework verifying behavioral indirect attempts to tell and purposeful disclosure as categories. Thematic analysis supported that CSA disclosure should be conceptualized and viewed as a complex and lifelong process Delayed disclosure was common in this qualitative sample. Most participants did not make a selective disclosure until adulthood. These findings support [@bibr1-1524838017697312]) model of disclosure but also highlights the importance of life stage. Modest but sufficient sample size for a qualitative inquiry. Well-designed study with detailed analysis for transferability of findings [@bibr40-1524838017697312]) This study aimed to: (1) add direct inquiry about the process of a child's CSA disclosure; (2) determine if children will discuss process that led them to tell; and (3) describe factors that children identify that led them to tell about or caused them to delay CSA disclosure Study sought to find out if process issues of disclosure could be identified in the context of forensic interviews. Forensic interviewers were asked to incorporate questions about "telling" into an existing forensic interview protocol. Interview content related to the children's reasons for telling or waiting was extracted, transcribed, and analyzed using grounded theory method of analysis 191 interviews of CSA victims aged 3--18 over a 1-year period were used for the study. Inclusion criteria included children who made a statement about CSA prior to referral, reasons for telling or waiting to tell, and those who spoke English. Participants were children who were interviewed at a child sexual abuse clinic. 74% were female and 51% were Caucasian Reasons the children identified for telling were classified into three domains: (1) disclosure as a result of internal stimuli (e.g., the child had nightmares); (2) disclosure facilitated by outside influences (e.g., the child was questioned); and (3) disclosure due to direct evidence of abuse (e.g., the child's abuse was witnessed). The barriers to disclosure identified fell into five groups: (1) threats made by the perpetrator (e.g., the child was told she or he would get in trouble if she or he told), (2) fears (e.g., the child was afraid something bad would happen if she or he told), (3) lack of opportunity (e.g., the child felt the opportunity to disclose never presented), (4) lack of understanding (e.g., the child failed to recognize abusive behavior as unacceptable), and (5) relationship with the perpetrator (e.g., the child thought the perpetrator was a friend) An innovative study to try to assess if formal investigative interviews can facilitate disclosures of CSA. Data were based on a large number of interviews. Detailed analysis produced detailed findings supporting other study findings on CSA disclosure [@bibr3-1524838017697312]) The study aimed to identify factors impeding or promoting CSA disclosures. Overarching research question: What individual, interpersonal, environmental, and contextual influences impede or promote CSA disclosures. A qualitative phenomenological design, LIM, was used to interview adult CSA survivors about their disclosure experiences to provide retrospective accounts of CSA disclosure and meaning-making of these experiences. Thematic analysis was done through a social--ecological lens. Purposive sampling was employed. Snowball sampling was also used to recruit more male survivors. 40 adult survivors of CSA were interviewed: 36% men and 64% women. Age range of 18--65 with a mean age of 40.1 years. Average age of abuse onset was 5.3 years old. 36% of the sample was non-White. Diverse socioeconomic backgrounds Themes fell into four domains: (1) individual and developmental factors, developmental factors as to whether they comprehended what was happening, personality traits also had some bearing on their ability to tell, and anticipating not being believed; (2) disclosure inhibited by family characteristics such as rigidly fixed gender roles with dominating fathers, chaos and aggression, other forms of child abuse, domestic violence, dysfunctional communication, and social isolation; (3) neighborhood and community context, that is, lack of interest from neighbors and teachers not pursuing troubling behavior; and (4) cultural and societal attitudes, media messages and societal attitudes, feeling unheard as kids, gender socialization for males, and cultural attitudes influencing parent's reactions. Purposeful disclosure is higher than reported in other studies because of the sampling attempts to purposefully locate disclosers The study presents a comprehensive social--ecological analysis to CSA disclosure highlighting the multifaceted influences. Of note, 42% had disclosed the abuse during childhood; 26% had not disclosed because they had repressed the memory, or the abuse had occurred in preschool years and they had difficulty with recall. The remainder had attempted some form of disclosure in indirect ways during childhood. A retrospective approach that could be affected by recall issues [@bibr15-1524838017697312] This examination of CSA disclosure explored the ways culture affects processes of CSA disclosure and reporting, both in the United States and internationally Using published literature with clinical data, this article conducted an analysis to provide a culturally competent framework for CSA disclosure questioning Data consisted of published literature on disclosure and culture that was triangulated with clinical case material Cultural and structural factors affecting CSA disclosure are identified in in-depth detail. Recommendations made include (1) disclosure interviewing should be tailored to the child's cultural context, (2) questioning should also take into consideration age and gender factors, and (3) culture stands as an important factor in all cases in which children are considering disclosing or being asked to disclose, and not solely in cases in which children are from noticeable minority groups. Presents a comprehensive interview framework integrating cultural considerations One of the few works that adds knowledge to culturally contextual disclosure interviewing. Unique combination of literature findings with clinical material. Anecdotal accounts may preclude transferability of findings. Overall adds to an impoverished area of CSA disclosure information [@bibr51-1524838017697312] This study explored disclosure strategies with a national sample of youth focusing on (1) What are the hidden experiences of abuse among Canadian youth? (2) What impact does participation in abuse prevention programs have on youth to express their abuse experiences? (3) What disclosure barriers do youth face? (4) What are young people's disclosure patterns? and (5) Who do they tell? Forms were completed by youth following participation in abuse prevention programming by the Canadian Red Cross (RespectED). A series of focus groups and observations of the workshops were used to help contextualize the findings. Evaluation forms were analyzed from two violence prevention programs: (1) It's not your fault and (2) What's love got to do with It? Examination of results from a national sample of 1,621 evaluation forms where youth anonymously disclosed abuse experiences. Respondent's ages: 13 and under (27%), 14--15 (37%), 16--17 (25%), 18 and older (4%), and unknown (7%) Youth who have been abused or witnesses to abuse employ five disclosure strategies: using self-harming behaviors to signal the abuse to others; not talking at all about the abuse to prevent intrusive interventions by others; seeking help from peers; seeking help from informal adult supports; and seeking help from mandated service providers (social workers and police). Results suggest disclosure is an interactive process, with expectations regarding consequences to disclosure. Patterns of incrementally sharing abuse experiences are shaped by young people's interactions with peers, educators, and caregivers. About three-quarters of females previously disclosed; significantly less males disclosed This study highlights that disclosure is an interactive ongoing process. Findings lend support to studies that have identified similarly interactive models of disclosure such as those detailed by [@bibr1-1524838017697312]) and [@bibr44-1524838017697312]). This mixed sample of youth who experienced different forms of abuse and violence exposure were participants---not limited to CSA survivors [@bibr52-1524838017697312] This study explored abuse disclosure strategies with a national sample of Canadian youth who participated in violence prevention programming. One of the goals of the study was to document not previously identified experiences of abuse and youth attitudes toward disclosure of abuse experiences Exploratory design with a nonrepresentative samples. Qualitative analysis of 1,099 evaluation forms completed following Red Cross RespectED violence prevention programming delivered between 2000 and 2003. Forms of anonymous abuse disclosures by youth participants of neglect, emotional, physical, and sexual abuse. Twenty-seven interviews and focus groups were also done to understand contextual issues and engage youth and program facilitators in the interpretation of findings. A coding structure was developed for analysis to synthesize themes across data sources Purposeful sample of 1,099 evaluation forms completed following Red Cross RespectED violence prevention programming delivered between 2000 and 2003 Findings suggest high rates of hidden abuse, with less than one quarter of youth reporting a disclosure. 244 of the 1,099 youth who disclosed abuse on their evaluation forms identified specific individuals they told about their abuse. Disclosure patterns vary with boys, youth aged 14--15, victims of physical abuse, and those abused by a family member being most likely to disclose to professionals or the police. One third of disclosures were directed toward professionals and the least, 5% percent each, were directed toward friends, parents, and others. Participants were most likely to disclose sexual abuse to parents/family, professionals, and the police/courts, with fewer choosing friends. Out of all 1,099 participants, 225 males and 779 females indicated that they had been abused. Out of those, 43 males and 180 females indicated that they had disclosed the abuse. Of those who had disclosed, only a portion of males and females specified who they had disclosed the abuse to ("While 1,099 evaluations with disclosure statements were analyzed, only 22% made mention of people to whom disclosures occurred.") More females specified who they disclosed to compare to males. The data show perceptions among youth of negative consequences following disclosure Innovative design of this study provides insight into young people's perceptions of disclosure experiences. High level of rigor with trustworthiness of the data analysis ensured through use of youth focus groups, interviews, and observational data. The study results are somewhat limited in the thickness of the descriptions it can offer because most of the data are survey based. Regional differences may not have been picked up. Scope of the study is broad and approach is creative [@bibr38-1524838017697312]) This study aimed to investigate disclosure rates and disclosure patterns and examine predictors of nondisclosure in a sample of male and female adolescents with self-reported experiences of sexual abuse Participants completed 65-item questionnaire that included questions about background, consensual sex, sexual abuse experiences (noncontact, contact or penetrating abuse, including peer abuse), disclosure of CSA, own sexual abusive behavior, sexual attitudes, and experiences with pornography and sexual exploitation. The questionnaire included 6 modified items from the SCL-90 and 9 of 25 items from the Parental Bonding Instrument. The data for girls and boys were analyzed separately The sample consisted of 4,339 high school students in Sweden (2,324 girls and 2,015 boys). The mean age of the participants was 18.15 years. This study used a subsample of 1,962 participants who reported CSA and who answered disclosure questions Of the sample, 1,505 girls (65%) and 457 boys (23%) reported CSA. The disclosure rate was 81% (girls) and 69% (boys). Girls and boys disclosed most often to a friend of their own age. Few had disclosed to professionals, and even fewer had reported to the authorities. There were higher rates of disclosure to a professional with more severe abuse (contact abuse with or without penetration) for girls, but lower rates for boys The more severe the sexual abuse was, the less likely both girls and boys had talked to their mother, father, or a sibling. Girls were less likely to disclose if they had experienced contact sexual abuse with or without penetration, less frequent abuse, abuse by a family member, or if they had perceived their parents as less caring and less overprotective and highly overprotective. Boys were less likely to disclose if a family member abused them, they were studying a vocational program (vs. an academic program), lived with both parents or had perceived their parents as less caring and not overprotective. Adolescents who reported CSA perceived their mental health as poorer compared to adolescents without CSA. Nondisclosers reported more symptoms on the Mental Health Scale than those who had disclosed This study highlighted that sexual abuse is largely hidden from adult society, especially from professionals and the legal system. However, time lapsed to disclosure was not reported. Since friends appeared to be the main recipients of sexual abuse disclosures, practice implications of this could be to find ways to give young people better information and guidance about how to support a sexually abused peer. A qualitative component to the study would have provided a broader understanding of disclosure processes. Study limitations include a significant amount of boys who did not complete the questions regarding disclosure on; the timing of disclosures (whether they were delayed or not) was not measured; possibility of recall bias with retrospective studies based on self-reports; and youth participants may not have understood all the questions [@bibr43-1524838017697312] Study focused on disclosure challenges for male survivors of CSA to understand three issues: (1) To whom and in what contexts have they disclosed these experiences? (2) What do they have to say about their disclosure experiences? and (3) What are their perceptions of positive and negative aspects of their disclosure, including incentives and barriers? Male survivors of CSA were interviewed about their disclosure experiences. Analytic techniques included grounded theory method of analysis for coding and development of conceptually clustered matrices. Participants completed two in-depth, semi-structured interviews, lasting between 2 and 3 hr each taking place approximately a week apart The sample consisted of 16 male survivors of childhood sexual abuse; 11 Caucasian, 2 African American, 1 Puerto Rican, 1 part Native American, 1 African Cuban; age range of 24--61 years; 9 identified themselves as heterosexual, 5 as homosexual, and 2 as bisexual Barriers to disclosure were found to be operant in three interrelated domains: (1) personal (e.g., lack of cognitive awareness, intentional avoidance, emotional readiness, and shame); (2) relational (e.g., fears about negative repercussions, isolation); and (3) sociocultural (e.g., lack of acceptance for men to experience or acknowledge victimization). Only 1 of the 16 men in this sample disclosed the full extent of his sexual abuse experiences while he was still a child. The other men reported that they had not disclosed, although some reported attempts to tell that were indirect or incomplete. Several other men disclosed certain experiences or elements of their abuse, but concealed others. By the time of the study, many of these men had disclosed their past experiences in a variety of relationships, including those with family members, partners, therapists, and infrequently friends. Several had only limited discussions of their sexual abuse Since the vast majority of men in the sample had not disclosed in childhood, they may have been predisposed to identifying barriers to disclosure more readily. Retrospective accounts are subject to recall issues. Investigators made significant efforts to gather a diverse sample. High level of rigor was executed in the dependability of the data and iterative process of the interpretation of findings was conducted [@bibr20-1524838017697312] The goal of the present study was to examine how child victims of extrafamilial sexual abuse disclosed the abuse experience Alleged victims of sexual abuse and their parents were interviewed. Children were interviewed using the NICHD Investigative Interview Protocol by experienced youth investigators. Information on disclosure processes was obtained in the first formal interview, before any police investigation or child welfare intervention Thirty alleged victims of CSA; 18 boys and 12 girls. Child sample was 7- to 12-year-olds with an average age of 9.2 years. Twenty mothers and 10 fathers were also interviewed for a total of 30 parent interviews. A content analysis was conducted on child and parent interviews Disclosure categories were identified as follows: (1) delayed 53% of the children delayed disclosure for between 1 week and 2 years; (2) recipient of disclosure: 47% of children first disclosed to siblings or friends, 43% first disclosed to their parents, and 10% first disclosed to another adult. 57% of the children spontaneously disclosed abuse, but 43% disclosed only after they were prompted. 50% of the children reported feeling afraid or ashamed of their parents' responses. Parents' reactions: supportive (37%) and unsupportive (63%). There was a strong correlation between predicted and actual parental reactions suggesting children anticipated their parents' likely reactions accurately. Disclosure processes varied depending on the children's ages (e.g., younger children disclosed to parents), severity and frequency of abuse, parents' expected reactions, suspects' identities, and strategies used to foster secrecy Innovative design to gather disclosure data from young children. Focus is on extrafamilial CSA which may differ than disclosure patterns of intrafamilial CSA. Two thirds of the parents registered unsupportive responses which is high [@bibr4-1524838017697312]) The objectives of the current study were to identify a broad range of factors, including family dynamics that contribute to or hinder a child's ability to disclose CSA. A qualitative phenomenological design---LIM---was used to elicit disclosure experiences; facilitators and barriers; and relevant circumstances. Interviews were transcribed verbatim. Line-by-line open coding was conducted to capture family-level factors. Axial and selective coding facilitated identification of themes Purposive sampling was employed to recruit 20 adult survivors between the ages of 18 and 65 who were sexually abused by a family member. Average age of participants was 40.1 years; 60% of participants were female and 40% male. Average age of onset of abuse was 6.7 years. Mixed clinical and nonclinical sample. The majority had received treatment for CSA at some point in their lives Four major themes emerged suggesting that CSA disclosure can be significantly compromised when certain family conditions exist: (1) rigidly fixed, gender roles based on a patriarchy-based family structure; (2) presence of family violence; (3) closed, indirect family communication patterns; and (4) social isolation of the family as a whole, or specific members, played a part in CSA victims feeling they had no one safe to tell. Family systems formulations through a feminist lens are important in understanding children and families at risk of disclosure barriers Over half the participants had not disclosed the abuse during childhood. Of the nondisclosing participants, six did not disclose because they had repressed or forgotten the memory. Almost one third withheld disclosure intentionally. More data are needed on early disclosures to garner more information on facilitators of disclosure. Retrospective approach implies recall issues. High level of trustworthiness of the data and interpretations were achieved through credibility, dependability, and confirmability through direct quotes [@bibr2-1524838017697312]) The study purpose was to qualitatively explore dynamics that impede or promote disclosure by examining a range of factors including gender as a dynamic---how disclosures of females and males are similar and different, and in what ways gender affects CSA disclosure Survivors of CSA were interviewed about their disclosure experiences using LIM. Analysis of 30 participant narratives was used for theme development regarding impact of gender on disclosure. Interviews were transcribed verbatim for open, axial, and selective coding. Categories and subcategories were collapsed and refining into theme areas Purposive sampling of women and men, along with those who disclosed during the abuse and those who did not. 19 females and 11 males; 18--65 (mean 40.1) years who were sexually abused by a family member or a trusted adult. Average age of abuse onset was 5.3 years, 36% were nonwhite, and 58% had not disclosed during childhood Three themes emerged for men that inhibited or precipitated disclosure for reasons related to gender: (1) fear of being viewed as homosexual; (2) profound feelings of stigmatization or isolation because of the belief that boys are rarely victimized; and (3) fear of becoming an abuser, which acted as a precipitant for disclosure. Two predominant themes with female participants related to difficulties disclosing: (1) they felt more conflicted about who was responsible for the abuse and (2) they more strongly anticipated being blamed and/or not believed One in a dearth of studies that conduct gender analysis. Comparative analysis draws out important practice implications. Retrospective design of the study which implies possible recall issues. High level of trustworthiness of the data and interpretations were achieved through credibility, dependability, and confirmability through direct quotes [@bibr7-1524838017697312]). Study examined patterns of disclosure in a large representative sample of South African CSA victims. Two study objectives to: (1) examine how and when CSA victims disclose their abuse and (2) Identify factors associated with different patterns of disclosure File reviews of all social work and medical case files for CSA victims seen at the crisis center where all cases of CSA reported to the North Durban policing area were referred during the period of January 2001 to December 2003 1,737 cases of CSA reported in the North Durban area of KwaZulu-Natal, South Africa, during January 2001 to December 2003. 1,614 girls and 123 boys; average age of victimized children was 9.9 years. 47% reports were made within 72 hr of the abuse, 31% from 72 hr to 1 month, and 22% more than a month after the abuse Content analysis identified two broad dimensions of disclosure: (1) agency: child-initiated disclosure versus detection by a third party and (2) temporal duration: an event versus a process. These disclosure dimensions defined four discrete categories of disclosure: (1) purposeful disclosure (30% of cases), (2) indirect disclosure (9% of cases), (3) eyewitness detection (18% of cases), and (4) accidental detection (43% of cases). Disclosure independently predicted by victim's age, nature of the victim--perpetrator relationship, offender's age, frequency of abuse, and reporting latency. Mean age of purposeful disclosures (10.67) was higher than the mean age of indirect disclosures (5.84). Explicit forms of disclosure were less likely when the offender was a family member. Shorter reporting latency was more likely with repeated abuse These results fit into [@bibr1-1524838017697312]) disclosure framework. Through data analysis two raters coded disclosure categories using author's disclosure framework, which proved to be both exhaustive and mutually exclusive with the percentage of interrater agreement at 98%. Generalizability of this study is limited to child clients receiving a crisis assessment referred through a police report [@bibr19-1524838017697312]). This study aimed to identify characteristics of suspected child abuse victims that are associated with disclosure and nondisclosure during formal investigations Large database of suspected cases of physical and sexual abuse investigated in Israel between 1998 and 2002 was analyzed. Interviews were also conducted using standardized NICHD Investigative Interview Protocol. Archival data were analyzed The sample was comprised of 26,446 of 3- to 14-year-old alleged victims of sexual and physical abuse interviewed in Israel in the 5-year period from 1998 to 2002. 140 experienced trained youth investigators conducted interviews Overall, 65% of the 26,446 children made allegations when interviewed. Rates of disclosure were greater for sexual abuse (71%) over physical abuse (61%). Children of all ages were less likely to disclose/allege abuse when a parent was the suspected perpetrator. Disclosure rates increased as children grew older: 50% with 3- to 6-year-olds, 67% of the 7- to 10-year-olds, and 74% of the 11- to 14-year-olds disclosed abuse when questioned Overall findings indicated that rates of disclosure varied systematically depending on the nature of the alleged offences, the relationship between alleged victims and suspected perpetrators, and the age of the suspected victims. Analyses only involved cases that had come to the attention of official agencies, making it difficult to determine how many of abuse take place without ever triggering any kind of official investigation [@bibr22-1524838017697312]) This study investigated the context in which children were able to report their child sexual abuse experiences; their views as to what made it difficult to talk about abuse; what helped them in the disclosing process; and their parent's perceptions of their disclosure processes Qualitative approach to data collection and analysis was used. Therapeutic interviews o the children and mostly their mothers were analyzed through a qualitative approach. Follow-up interviews were held 1 year later 20 families with a total of 22 children participated. All children had told about experiences that created concerns for care-givers about CSA. Children's ages ranged between 3 and 16 years (average age 7.5 years); 15 girls and 7 boys. Sexually abused by someone in the family or a close person to the family None of the children told of abuse immediately after it occurred. Children exposed to repetitive abuse kept this as a secret for up to several years; 17 told their mothers first, 3 first told a friend, 1 told their father, and 1 their uncle. Majority of remarks that led to the suspicion of CSA were made in situations where someone engaged the child in a dialogue about what was bothering them, resulting in a referral. The children felt it was difficult to find situations containing enough privacy and prompts that they could share their experiences. When the children did disclose they did it in situations where the topic of child sexual abuse was in some form addressed or activated, where someone recognized the child's cues and probed further. They also were sensitive to others reactions, and whether their disclosures would be misinterpreted. Several of the children perceived negative consequences as major factors contributing to delaying disclosure. They were primarily concerned about negative effects for the mother. The mothers said they were also sensitive to the children's feelings. If their children showed signs of distress and did not want to talk, the mothers would change the subject or not pursue the topic further Evidence for delayed disclosures. The results indicate that disclosure is a fundamentally dialogical process that becomes less difficult if children perceive that there is an opportunity to talk, a purpose for speaking and a connection has been established to what they are talking about. Strengthening parent--child relationships is an important practice implication [@bibr44-1524838017697312]) The purpose of this study was to understand the full process of CSA disclosure and how this unfolded for preadolescent and adolescent girls. Examined what facilitated and hindered disclosure and subsequent consequences Secondary analysis of qualitative focus group data. Original project consisted of four focus groups conducted within the context of ongoing therapy for girls who had experienced CSA. Secondary analysis consisted of written narrative summaries of each session grouping these conceptually, and examining their interconnectedness Sample consisted of 34 participants from four groups. Sessions analyzed were between 60 and 90 min long; audiotaped and later transcribed for content analysis Findings are reported in three major domains: (1) self-phase: where children come to understand victimization internally; (2) confidant selection-reaction phase: where they select a time, place, and person to tell and then whether that person's reaction was supportive or hostile; and (3) consequences phase: good and bad that continued to inform their ongoing strategies of telling. The actions and reactions of adults were significant and informed the girls' decisions. The consequences phase was further subdivided into four aspects: (1) gossiping and news networks, (2) changing relationships, (3) institutional responses and the afterlife of telling, and (4) insider and outsider communities This study provided a contextual examination of the entire disclosure process, closer to the point in time when the abuse and disclosure occurred. Small groups of preadolescent and adolescent girls who had survived sexual abuse also served as consultants and were encouraged to share their knowledge for the benefit of professional practitioners [@bibr1-1524838017697312]) The study sought to examine influences that inhibit or promote children's disclosure of CSA to address gaps in knowledge about how, when, and under what circumstances victims of CSA disclose The study employed LIM---a phenomenological design. Intensive interviewing that were 2 hr long on average generated data for a thematic analysis. The interview guide was developed to probe for individual, interpersonal, environmental, and cultural factors influencing CSA disclosure Using purposive sampling 24 adult survivors of intrafamilial abuse between ages of 18 and 65 (average age 41.2) were recruited from agencies and one university; 57% male and 43% female; average age of abuse onset was 6.5 years; 42% of the participants had disclosed the abuse during childhood; 58% disclosed as adults Through analysis of the interview new categories of disclosure were identified to add to existing types. Three previously identified were confirmed in these data: accidental, purposeful, and prompted/elicited accounted for 42% of disclosure patterns in the study sample. Over half the disclosure patterns described by the study sample did not fit these previously established categories. Three additional disclosure categories emerged: behavioral and indirect verbal attempts, disclosures intentionally withheld, and disclosures triggered by recovered memories This study expanded types of CSA disclosures to more fully understand how children and adults disclose. And under what circumstances. Asking people to recount events that occurred in childhood is susceptible to memory failure, especially when memories were forgotten, delayed, or repressed and later recovered. Distortion and revision of events are also potential problems in recall. High degree of trustworthiness of the data was achieved and quotes provided supported the categories [@bibr9-1524838017697312]) The main goals of this study were to understand impediments that prevent adolescents from disclosing CSA and seeking help from their social network and/or the services In-depth telephone (anonymous) interviews were conducted after informed consent was explained and obtained. Three investigators experienced in counseling CSA counseling conducted the interviews which were recorded with permission. Three researchers independently scored the interviews according to a coding framework The sample was comprised of 36 young people who experienced sexual abuse in adolescence; 35 females and 1 male; aged 12--17. Some of the sample experienced sexual violence in a dating relationship The main impediments to disclose to a family member were: fear of not being believed, shame, and fear of causing trouble to the family. The main impediments for not seeking services were: unaware of appropriate services, wish to keep the secret, lack of awareness of being abused, mistrust of adults and professionals, and fear of the consequences of disclosing sexual abuse. When they did disclose to professionals, teens received very limited support as many professionals were not trained on sexual abuse and could not offer appropriate interventions to victims This study represented the findings of a mixed sample of survivors of child sexual abuse and intimate partner violence. The study was conducted in Italy and it is not clear what sexual abuse response training is available. There may have been a selection bias as the most dissatisfied survivors responded to the research call [@bibr25-1524838017697312] Study purpose was to explore how abuse traits, openness, reactions to CSA disclosure, and social support were related. Differences based on severity of abuse, timing and outcomes of disclosure, social support, and predicting factors of positive and negative reactions were probed Adult women reporting CSA by someone close were interviewed using semi-structured guides together with questionnaires. Data on victimization and current social support were retrieved through the questionnaires, and data on disclosure and reactions were gathered through the interviews with participants 122 adult women between 20 and 60 years old (average age of 41 years) reporting exposure to child sexual abuse by someone close before the age of 18 and had told someone about at least one abuse event. 90% were Swedish subjects. Purposive sampling strategy was used Abuse characteristics: abuse by multiple perpetrators was more common than by a single perpetrator. Age of onset was often before age of 7, with an average duration of 7 years. Severely abused women had talked to more of their social network, especially to professionals. Disclosures: 32% disclosed during childhood (before the age of 18) with an average of 21 years delay. Women who had disclosed in childhood reported more instances of physical abuse, multiple perpetrators, use of violence, and were more likely to have confronted a perpetrator, and had received a negative first reaction. Factors significantly predicting delay were younger age at first event and no use of violence. Disclosure outcomes: of the 26 women who told in childhood during a period with ongoing abuse, 15 women were continuously abused after disclosure 68% delayed disclosure until adulthood. At the time of the study, it was one of the first studies to focus on the interplay between social support networks and disclosure of child sexual abuse. The study results are somewhat limited by an overrepresentation of severely abused women. Retrospective study and self-report of information could imply recall issues and thus limits the accuracy of the information obtained on abuse and disclosure characteristics. Cross-sectional design does not allow for definite conclusions of cause and effect on the relationships found [@bibr28-1524838017697312]) The purpose of this study was to identify factors that influence the disclosures made by female survivors of USE in childhood and adolescence. The predictors of both the timing of disclosure and the recipient of the disclosure were investigated Data were gathered from a subsample of female adolescents that participated in the NSA, which consisted of structured phone interviews. USEs reported in the NSA were assessed using a modified version of the Incident Classification Interview. They were then asked a series of questions about each episode of unwanted sexual contact including event characteristics and perpetrator characteristics A subsample of 263 adolescent females between 12 and 17 years old, mean age of 15.2 years old, who reported at least one experience of unwanted sexual contact in the NSA. Participant characteristics, USE characteristics, and family contextual attributes were explored Children under the age of 7 were at a higher risk for delayed disclosures. Participants whose USE occurred between the ages of 7 and 13 were most likely to tell an adult. Adolescents (14--17) were more likely to tell only peers than children aged 7--10 years. Children under 11 were more likely to tell an adult, but were at risk for delaying disclosure beyond a month. Children aged 11--13 tended to disclose within a month. Closer relationship to the perpetrator or a family member was associated with delayed disclosure. Immediate disclosure was more likely with stranger perpetration. Fear for one's life during and penetration were associated with disclosure to adults. Family factors linked to disclosure were (1) drug abusing household member, which made survivors more likely to disclose more promptly and (2) never living with both parents was associated with nondisclosure This study examined factors including disclosures of USEs in childhood and adolescence in a nationally representative sample of female adolescents who participated in the NSA. Surveys for investigations of victimization experiences may be biased due to underreporting. Adolescents who refused to report or discuss an USE may represent a source of systematic bias and would make the results generalizable only to adolescents who are willing to disclose USE via survey. Although data may be retrospective, recall bias may have been minimized in this study since participants were adolescents, and so the time lag between the USE and the interview were presumably shorter than a study of adult participants recalling CSA experiences [@bibr17-1524838017697312]) The purpose of this study was to investigate variables associated with delay of disclosure of CSA and test a model for factors that influence how quickly children disclose sexual abuse Case file reviews of data obtained from prosecution files, as well as from structured interviews with the children's caretaker and observations of child interviews. Trained graduate students and one victim advocate completed the Sexual Assault Profile questionnaire for child characteristics, the abuse and their disclosure. Children's perception of responsibility and fear of negative consequences were probed. Correlational analyses were conducted with path analyses to test the hypothesized causal relations among variables Sample consisted of 218 children referred to prosecutors' offices for alleged CSA. All children in the sample had disclosed their abuse in some manner. Children ranged in age from 2 to 16 years at the beginning of abuse; 3--16 years at the end of the abuse, and 4--16 years at the time of the initial police report; 77% female, 70% Caucasian, 17% Hispanic, and 11% African American. Predominantly middle to low SES. Approximately 47% intrafamilial abuse 64% disclosed within a month and 29% within 6 months. Five variables for the model were tested. (1) age: children who were older took longer to disclose and older children feared more negative consequences to others than younger children; (2) type of abuse: victims of intrafamilial families took longer to disclose---victims of intrafamilial abuse feared greater negative consequences to others compared to victims of extrafamilial abuse; (3) fear of negative consequences: children who feared negative consequences of disclosure took longer to disclose, children who believed that their disclosure would bring harm to others took longer to disclose, fear of negative consequences to the self or the perpetrator was unrelated to time of disclosure, and girls more than boys feared negative consequences to others; (4) Perceived responsibility: children who felt greater responsibility for the abuse took longer to disclose and older children felt more responsibility for the abuse; and (5) gender was not significantly correlated with time to disclosure This study represents a higher rate of disclosers within a month. These cases had been reported to authorities and were in process of prosecution which may explain higher rate of early disclosures. Legal sample with higher rate of extrafamilial abuse (52%) may also account for earlier disclosures. Model suggests that older children, victims of intrafamilial abuse; felt greater responsibility for the abuse, and perceiving negative consequences to disclosing took longer to disclose. Well-designed study with high level of rigor. Produced a viable model of disclosure for further investigations. However, researchers were not able to interview children directly [@bibr42-1524838017697312]) The study focus was to gather data from a large sample of women about the length of time women who were raped before age 18 delayed disclosure who they disclosed to, and variables that predicted disclosure within 1 month Structured telephone interviews that lasted approximately 35 min were used to collect data using a computer-assisted telephone interview system. All telephone interviews were conducted with each question on a computer screen. The survey consisted of several measures designed to elicit demographic information, psychiatric symptoms, substance use, and victimization history. The present study reports on data from the demographic and child rape victimization questions Two probability samples. Wave 1 was a random sample of 2,009 respondents selected from stratified samples of defined jurisdictions. Random digital dialing was used to solicit households for listed and unlisted telephone numbers. Second random sample of 2,000 women between the ages of 18 and 34 was selected. Both Wave 1 and Wave 2 data were weighted to conform to the 1989 Census statistics 288 (9%) reported experiencing at least one event that met the study's definition of childhood rape. The average age at the time of the first rape was 10.9 years. Of the 288 women who reported a child rape, 28% stated that they had never told anyone about this sexual assault until specifically queried by the interviewer for this study. 58% did not disclose for over 1 year and up to 5 years post-rape. 27% disclosed within a month. Among women who disclosed prior to their NWS interview close friends were the most common person to whom victims made disclosures, followed by mothers and other immediate family members. Fewer than 10% of victims reported making their initial disclosure to social workers or law enforcement personnel. Only 12% of child rape victims stated that their assaults were reported to authorities at some point The time frame of this survey may have had contextual implications. The majority of child rapes reported by this sample occurred prior to the large-scale child assault prevention education programs that were begun in the 1980s that teach children that assaults (including CSA) are wrong and should be disclosed to responsible adults. This information may have influenced (and may currently be influencing) young women's disclosure patterns. For Wave 1, comparison of these data with the population parameters obtained from the U.S. Census Bureau indicated that the sample closely matched the demographic attributes of the population of U.S. women Note. SCL-90 = Symptom Check List-90; SES = socioeconomic status; LIM = long interview method; CSA = child sexual abuse; NICHD = National Institute of Child Health and Human Development; USE = unwanted sexual experiences; NSA = National Survey of Adolescents; NWS = National Women's Study; QIDS = Questionnaire informattsé sur les délinquants sexeuls. Key Findings {#section3-1524838017697312} ------------ First-level analysis of the studies identified key study characteristics. Trends emerged around definitions of CSA disclosure, study designs, and sampling issues. First, in regard to definitions, the term "telling" is most frequently used in place of the term disclosure. In the absence of standardized questionnaires or disclosure instruments, telling emerges as a practical term more readily understood by study participants. Several examples of this usage were found in the research questions, interview guides, and surveys examined: "How and when do people decide to tell others about their early sexual experiences with adults?" ([@bibr21-1524838017697312], p. 161); "Some men take many years to tell someone that they were sexually abused. Please describe why it may be difficult for men to tell about/discuss the sexual abuse" ([@bibr12-1524838017697312], p. 462). "Participants were asked a series of open-ended questions to elicit a narrative regarding their experiences of telling..." ([@bibr33-1524838017697312], p. 1160). "Who was the first person you told?" ([@bibr40-1524838017697312], p. 346). There was sound consistency between studies, defining disclosure in multifaceted ways with uniform use of categories of prompted, purposeful, withheld, accidental, direct, and indirect. However, defining the period of time that would delineate a disclosure as delayed varied widely across studies, wherein some studies viewed 1 week or 1 month as a delayed disclosure (i.e., [@bibr20-1524838017697312]; [@bibr28-1524838017697312]; [@bibr41-1524838017697312]). Other studies simply reported average years of delay sometimes as long as from 20 to 46 years ([@bibr11-1524838017697312]; [@bibr25-1524838017697312]; [@bibr42-1524838017697312]). Second, the number of qualitative studies has increased significantly over the last 15 years. This rise is in response to a previous dearth of qualitative studies. Based on [@bibr24-1524838017697312]) observation that disclosure factors and outcomes had been well documented through quantitative methods; in a widely read editorial, he recommended "Qualitative studies which are able to track the individual experiences of children and their perception of the influences upon them which led to their disclosure of information are needed to complement..." (p. 270). Third, although a few studies strived to obtain representative samples in quantitative investigations ([@bibr19-1524838017697312]; [@bibr28-1524838017697312]; [@bibr42-1524838017697312]), sampling was for the most part convenience based, relying on voluntary participation in surveys and consent-based participation in file reviews ([@bibr7-1524838017697312]; [@bibr38-1524838017697312]; [@bibr41-1524838017697312]; [@bibr51-1524838017697312]). Therefore, generalizability of findings is understandably limited. The qualitative studies used purposive sampling as is deemed appropriate for transferability of findings to similar populations. Some of those samples contained unique characteristics, since they were sought through counseling centers or sexual advocacy groups. These would be considered clinical samples producing results based on disclosures that may have been delayed or problematic. This might presumably produce data skewed toward barriers and bring forward less information on disclosure facilitators. Through an in-depth, second-level analysis, this review identified five distinct themes and subthemes beyond the general trends as noted earlier.Theme 1: Disclosure is viewed as an ongoing process as opposed to a discrete event---iterative and interactive in nature. A subtheme was identified regarding disclosure as being facilitated within a dialogical and relational context is being more clearly delineated.Theme 2: Contemporary disclosure models reflect a social--ecological, person-in-environment perspective to understand the complex interplay of individual, familial, contextual, and cultural factors involved in CSA disclosure. Subthemes include new categories of disclosure and a growing focus on previously missing cultural and contextual factors.Theme 3: Age and gender are strong predictors for delaying disclosure or withholding disclosure with trends showing fewer disclosures by younger children and boys. One subtheme emerged that intrafamilial abuse/family-like relationship of perpetrator has a bearing on disclosure delays or withholding.Theme 4: There is a lack of a cohesive life-course perspective. One subtheme includes the lack of data within the 18- to 24-year-old emerging adult population.Theme 5: Significantly more information is available on barriers than on facilitators of CSA disclosure. Subthemes of shame, self-blame, and fear are uniformly identified as disclosure deterrents. ### Disclosure as an ongoing process: Iterative and interactive in nature {#section4-1524838017697312} Disclosure is now generally accepted as a complex and lifelong process, with current trends showing that CSA disclosures are too often delayed until adulthood ([@bibr8-1524838017697312]; [@bibr11-1524838017697312]; [@bibr21-1524838017697312]). Knowledge building about CSA disclosure has moved in the direction of understanding this as an iterative and interactive process rather than a discrete, one-time event. Since the new millennium, disclosure is being viewed as a dynamic, rather than static, process and described "not as a single event but rather a carefully measured process" ([@bibr2-1524838017697312], p. 455). The catalyst for this view originates from [@bibr47-1524838017697312]) who initially conceptualized CSA disclosures as process based, although this notion was not fully explored until several years later. Examinations of [@bibr47-1524838017697312]) groundbreaking proposition of the CSA accommodation (CSAA) model produced varying results as to whether his five stages of secrecy, helplessness, entrapment and accommodation, delayed, conflicted, and unconvincing disclosures, and retraction or recantation, hold validity (for a review, see [@bibr30-1524838017697312]). However, the idea of disclosure as a process has been carried over into contemporary thinking. Recently, [@bibr33-1524838017697312]) detailed a process model of disclosure wherein they describe an interaction of internal factors with external motivators which they liken to a "pressure cooker" effect, preceded by a period of containment of the secret. Moreover, this and other studies strongly suggest disclosures are more likely to occur within a dialogical context---activated by discussions of abuse or prevention forums providing information about sexual abuse (Hershkowitz et al., [@bibr19-1524838017697312]; [@bibr22-1524838017697312]; [@bibr51-1524838017697312]). The term dialogical simply means to participate in dialogue. Key dialogical vehicles identified in these studies were providing sexual abuse information through prevention programs, being asked about sexual abuse, and being prompted to tell ([@bibr33-1524838017697312]; [@bibr51-1524838017697312]). ### Contemporary models of CSA disclosure reflect a social-ecological perspective {#section5-1524838017697312} Knowledge on CSA disclosure has been steadily advancing toward a holistic understanding of the complex interplay of individual, familial, contextual, and cultural factors ([@bibr4-1524838017697312]; [@bibr6-1524838017697312]; [@bibr15-1524838017697312]). Where at one time factors examined and identified were predominantly of intrapersonal factors of child victims, knowledge construction has shifted to fuller social--ecological, person-in-environment explanations ([@bibr3-1524838017697312]; [@bibr8-1524838017697312]; [@bibr12-1524838017697312]; [@bibr21-1524838017697312]; [@bibr52-1524838017697312]). Social--ecological explanations open up more opportunities to intervene in facilitating earlier disclosures. [@bibr3-1524838017697312]) proposes an ecological mapping of what individual, interpersonal, environmental, and contextual influences impede or promote CSA disclosures based on analysis of in-depth interview data of 40 adult survivors. Subsequently, based on a sample of 67 adult survivors, [@bibr8-1524838017697312]) identified three broad categories, closely aligned with an ecological framework that impede CSA disclosure: (1) barriers from within, (2) barriers in relation to others, and (3) barriers in relation to the social world which can be aligned to intrapersonal, interpersonal, and contextual factors. A summary of knowledge building using a social--ecological framework follows. Knowledge gained in the intrapersonal domain includes expanded conceptualization of disclosure by building on previous categories of accidental, purposeful, and prompted disclosure to also include behavioral and indirect attempts to tell, intentionally withheld disclosure, and triggered and recovered memories ([@bibr1-1524838017697312]). Categories of indirect behavioral disclosure patterns have been further verified in follow-up research by [@bibr21-1524838017697312]), and through an extensive file review that used [@bibr1-1524838017697312]) disclosure framework to analyze their data ([@bibr7-1524838017697312]) for verification. Interpersonal factors have also emerged in regard to certain family characteristics as disclosure barriers. Families with rigidly fixed gender roles, patriarchal attitudes, power imbalances, other forms of child abuse and domestic violence, chaotic family structure, dysfunctional communication, and social isolation have been found to suppress disclosure ([@bibr4-1524838017697312]; [@bibr8-1524838017697312]; [@bibr15-1524838017697312]). In addition, relationship with perpetrator is a factor whereby research indicates that disclosure is made more difficult when the perpetrator is a family member or close to the family ([@bibr10-1524838017697312];[@bibr11-1524838017697312]; [@bibr17-1524838017697312]; [@bibr19-1524838017697312]; [@bibr38-1524838017697312]; [@bibr41-1524838017697312]). This is especially a barrier when the perpetrator lives with the victim ([@bibr29-1524838017697312]). In terms of environmental factors, one study revealed that neighborhood/community conditions can hinder disclosure when there is lack of school involvement in providing a supportive environment, such as in following up on troubling student behavior ([@bibr3-1524838017697312]). Additionally, a child victim's anticipation of a negative response to disclosure, especially that they may not be believed by others outside their family such as neighbors or other community members, has shown to deter disclosure ([@bibr8-1524838017697312]). Cultural factors influencing CSA disclosure have been studied to a much lesser degree. Despite this, a few important studies examining critical sociocultural factors now exist for better understanding CSA disclosure within a cultural context ([@bibr6-1524838017697312]; [@bibr15-1524838017697312]). Among these important contributions, [@bibr6-1524838017697312] research has delineated CSA disclosure processes as "shaped by relational, racial, socio-cultural, historical, and developmental factors" (p. 182). In a unique study using culturally focused research literature as data triangulated with clinical case material, culturally based belief systems in many cultures have been found to foster family climates that can silence children from disclosing abuse ([@bibr15-1524838017697312]). Taboos about sexuality, patriarchal attitudes, and devaluation of women are among some of the cultural barriers that inhibit disclosure ([@bibr15-1524838017697312]). Clearly, disclosure conceptualizations are being integrated into a social--ecological model of individual and developmental factors, family dynamics, neighborhood, and community context as well as cultural and societal attitudes toward better understanding disclosure barriers and facilitators ([@bibr3-1524838017697312]), although more data are needed on cultural and contextual factors. ### Age and gender as predictors of disclosure {#section6-1524838017697312} #### Age {#section7-1524838017697312} Age is consistently found to be an influential factor in CSA disclosure, making the life stage of the victim/survivor a critical consideration. Studies draw distinctions in age-groups falling into either under or over 18 years of age. Eighteen years of age was the common age cutoff point that investigators chose in order to distinguish child/youth populations from adult samples. Sixteen of the studies drew on samples of children and youth, while the other 15 studies sampled adults over the age of 18, and a further two studies used mixed age-groups (refer to [Table 1](#table1-1524838017697312){ref-type="table"}). Among the child and youth samples, the age ranges spanned from preschool to late adolescence (3--17 years of age), with varying methodological approaches implemented across age cohorts. For younger cohorts, file reviews and secondary data analyses of CSA reports were typically undertaken. Adolescents were most often given surveys. Sometimes children and youth were interviewed as part of administering a survey or as a follow-up ([@bibr9-1524838017697312]; [@bibr19-1524838017697312]; [@bibr52-1524838017697312]). In the majority of child and adolescent samples, sexual abuse concerns were already flagged to investigative authorities. However, the work of [@bibr51-1524838017697312], [@bibr52-1524838017697312]) is one exception, whereby their survey elicited new disclosures. Adult studies typically had a mean age between 40 and 50 years. Interviews were the main data collection method with a few exceptions using survey designs (i.e., [@bibr11-1524838017697312]; [@bibr28-1524838017697312]; [@bibr42-1524838017697312]) and case file reviews (i.e., [@bibr7-1524838017697312]; [@bibr17-1524838017697312]). Results show a clear trend toward increased likelihood of disclosure in older youth, and findings from adult samples showing a preponderance of disclosures in adulthood, with the large majority of participants of adults reporting never having had a sexual abuse complaint filed with investigative authorities as a child or an adolescent (i.e., [@bibr21-1524838017697312]; [@bibr16-1524838017697312]; [@bibr43-1524838017697312]; [@bibr52-1524838017697312]). With children and youth under the ages of 18 distinct patterns emerged. First, accidental detection, rather than purposeful disclosure, is more likely to occur with younger children. For example, in one large-scale study of over 1,737 file reviews, over half of the CSA-related cases were identified through accidental and eyewitness detection (61%), while less than one third were purposeful disclosures initiated by the child victim ([@bibr7-1524838017697312]). A second pattern which emerged is that rates of disclosure increase with age, especially into adulthood, which is supported by persistent findings of high rates of delayed disclosure reported later in the life course by adult survivors ([@bibr7-1524838017697312]; [@bibr8-1524838017697312] ; [@bibr11-1524838017697312]; [@bibr25-1524838017697312]; [@bibr28-1524838017697312]; [@bibr29-1524838017697312]; [@bibr43-1524838017697312]). While gender and relationship with the perpetrator are considerable factors in CSA disclosure, age is consistently a stronger predictor of disclosure (or nondisclosure) ([@bibr19-1524838017697312]; [@bibr29-1524838017697312]). Third, younger children who disclose are more likely to do so in an interview situation or other environment that provides prompts or questions about sexual abuse ([@bibr19-1524838017697312]; [@bibr34-1524838017697312]; [@bibr40-1524838017697312]), but this trend can also be seen in older youth ([@bibr51-1524838017697312], [@bibr52-1524838017697312]). #### Gender {#section8-1524838017697312} A number of studies have recently focused on CSA disclosures with male victims, since males have been an understudied population ([@bibr2-1524838017697312]; [@bibr11-1524838017697312]; [@bibr12-1524838017697312]; [@bibr16-1524838017697312]). Most investigations that sampled both sexes show females outweighing male participants. Although women are at double the risk of being subjected to CSA, the ratio of women to men in most disclosure studies has not been representative. This finding may be indicative of male victims more likely delaying disclosing their CSA experiences, leaving male disclosure in child and youth samples underrepresented ([@bibr18-1524838017697312]; [@bibr52-1524838017697312]). [@bibr12-1524838017697312]) have been developing gender-specific modeling of disclosure examining male disclosures. Their proposed model groups male disclosures into barrier categories as determined by individual factors, interpersonal issues, and factors that are sociopolitical in nature. These authors suggest that predominant gender norms around masculinity reinforce the tendency for male victims of CSA to blame themselves for the abuse, resulting in no disclosure. Male participants in a subsequent study also relayed that gender norms and stereotypes contributed to them concealing the abuse because they were abused by a woman ([@bibr16-1524838017697312]). In the one study that compared male and female disclosures, investigator found that men's fears of being viewed as homosexual; profound feelings of stigmatization or isolation because of the belief that boys are rarely victimized; and fear of becoming an abuser acted as disclosure barriers. Whereas females felt more conflicted about who was responsible for the abuse and more strongly anticipated being blamed and not believed ([@bibr2-1524838017697312]). ### Lack of a life-course perspective {#section9-1524838017697312} Given that the study of CSA disclosure draws on age-groups ranging from samples of very young children to retrospective studies of adult survivors, with significant developmental considerations, this area of study lacks an intentional cohesive life-course perspective. Most data are derived from either cross-sectional or retrospective designs, with few longitudinal studies. There are a series of sound, yet disconnected, studies focusing on specific age-groups of children and adolescents, along with adult retrospective studies. Thus, the available knowledge base does not allow for a cohesive picture of CSA disclosure processes and pathways over the life course to emerge. The life-course perspective has long been recommended as a critical lens for the study of child abuse ([@bibr54-1524838017697312]; [@bibr53-1524838017697312]). For example, a life-course perspective has been utilized to understand the immediate- and long-term effects of CSA on the developing child victim ([@bibr54-1524838017697312]). Further, a life-course perspective is important in terms of examining age of onset of CSA to explain the differential effects of sexual victimization and developmental impacts in terms of understanding their ability to disclose---effects that need to be understood within a developmental context, especially for designing appropriate interventions for disclosure at critical transitions from early childhood through to adolescence and into adulthood. In addition, important "turning points" in life may facilitate disclosures. For example, entry into adulthood given that delayed disclosure occurs more often in adulthood. [@bibr1-1524838017697312], [@bibr2-1524838017697312]) found that being in a committed relationship or the birth of children acted as facilitators for some survivors to disclose, especially to their spouses. These significant life events, as contributing to disclosures, bear further examination. ### Summary of barriers and facilitators {#section10-1524838017697312} Research over the past 15 years continues to uncover barriers to CSA disclosure at a higher frequency than that of facilitators. As stated previously, this might be the result of sampling methods whereby participants who volunteer for disclosure research may have had more negative disclosure experiences, especially since many report delays in disclosure. The following section outlines the major trends in both barriers and facilitators (see [Table 2](#table2-1524838017697312){ref-type="table"}). ###### Factors Influencing Child Sexual Abuse Disclosures. ![](10.1177_1524838017697312-table2) Barriers Facilitators ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Age: The younger the child victim, the less likely they will purposefully disclose. Age: Disclosures increase with age, especially in adulthood. Gender: Males may be less likely to disclose in childhood/adolescence, fear of being seen as homosexual and as a victim, females experience more self-blame and anticipation of being blamed and/or not believed Gender: Slight trend toward females who are older (adolescent) to disclose before adulthood Relationship to perpetrator: If the perpetrator is a family member or in a family-like role, disclosure is less likely to happen Relationship to perpetrator: If the perpetrator is not living with the victim, disclosure rates increase Internal: Shame, self-blame, and fear are psychological barriers. In addition, fear of negative consequences on the family and for self-safety inhibits disclosure Dialogical context: Opportunities to disclose through discussion, therapeutic relationship, information sessions on sexuality, and sexual abuse prevention programs Family relations: Families with a patriarchal structure, rigidly fixed gender roles, dysfunctional communication, other forms of abuse (i.e., domestic violence), and isolation inhibit disclosure Family relations: Supportive parent--child relationship. Involvement of others: Eyewitnesses coming forward and reporting; detection through community members, professionals Environmental and cultural context: Lack of discussion about sexuality; passive acceptance that unwanted sexual experiences are inevitable; not wanting to bring shame to the family by admitting sexual abuse; lack of involvement from neighbors, school personnel; and stigma perpetuated by societal perceptions Environmental and cultural context: Promotion of open discussion of sexuality; community member involvement #### Barriers {#section11-1524838017697312} Age and gender were found to contribute to barriers as covered in Theme 3. Disclosures generally increase with age as children gain more developmental capacity, understanding of sexual abuse as victimization, and increased independence. Males are somewhat less likely to disclose, but this is often in interaction with other factors in the environment such as societal attitudes that promote hypermasculinity as desirable, attitudes that perpetuate negative views of boys and men who are victims, and homophobic attitudes ([@bibr3-1524838017697312]; [@bibr12-1524838017697312]; [@bibr16-1524838017697312]). Victims of intrafamilial abuse when the offender is a parent, caregiver, significant family member, or someone in a family-like role are less likely to disclose immediately or at all in childhood/adolescence because of obvious power differentials and dependency needs ([@bibr7-1524838017697312]; [@bibr10-1524838017697312]; [@bibr19-1524838017697312]; [@bibr28-1524838017697312]; [@bibr29-1524838017697312]; [@bibr36-1524838017697312]; [@bibr40-1524838017697312]). Further, the perpetrator residing with their victim(s) increases the likelihood of no disclosure ([@bibr29-1524838017697312]). Internalized victim-blaming, mechanisms to protect oneself (such as minimizing the impact of the abuse), and developmental immaturity at the onset of abuse constituted internal barriers. Further, shame, self-blame, and fear have been identified as significant factors deterring disclosure ([@bibr8-1524838017697312]; [@bibr9-1524838017697312]; [@bibr17-1524838017697312]; [@bibr21-1524838017697312]; [@bibr28-1524838017697312]; [@bibr32-1524838017697312]; [@bibr34-1524838017697312]). However, aspects of shame, self-blame and fear, and have not been fully explored in research. Since these are strong predictors of disclosure suppression, they bear further examination in future research to understand more fully how they operate in disclosure processes. In terms of interpersonal and environmental factors, family dynamics can play a part in deterring disclosure. As previously mentioned, families characterized by rigidly defined gender roles, patriarchal attitudes that perpetuate power imbalances between men and women, parents and children, presence of other forms of child abuse and/or domestic violence, chaotic family structure, dysfunctional communication, and social isolation have been found to suppress disclosure ([@bibr4-1524838017697312]; [@bibr8-1524838017697312]; [@bibr15-1524838017697312]). In regard to broader environmental factors, disclosure can be hindered when involved and supportive community members are not available, or not trained in sensitive responses, or when child victims anticipate not being believed by neighbors and other people outside the family ([@bibr3-1524838017697312]; [@bibr8-1524838017697312]). Further, barriers in relation to the social world were identified as stigmatization, the negative labeling of sexual abuse victims, and taboos surrounding sexuality and talking about sex as driven by cultural norms ([@bibr8-1524838017697312]; [@bibr15-1524838017697312]). Identification of cultural barriers is important recent contribution to understanding disclosure processes---and in particular to the obstacles. Findings related to cultural barriers included themes of children's voices not being heard leading to silencing, the normalization of the sexualization and objectification of girls and women, and the perpetuation of hypermasculinity in men---all acting as barriers in terms of stigma to disclosure ([@bibr2-1524838017697312], [@bibr3-1524838017697312]; [@bibr12-1524838017697312]). [@bibr6-1524838017697312] similarly found that lack of discussions about sex, young age at the onset of sexual abuse, therefore not having the language to express what was happening to them, and preserving the family good name by not talking about abuse in the family were also barriers to disclosure. Finally, it may be the case that more barriers continue to be identified over facilitators of CSA disclosure perhaps because of the methods employed in studies---particularly those drawing on adult populations who delayed disclosure. These samples may not be representative of the overall population of CSA victims, since they may have had more negative disclosure experiences, consequently more readily identifying barriers. On the other hand, these findings may speak to the actual imbalance between facilitating factors and barriers for disclosure, the latter carrying more weight in the victims/survivors' experiences, thus, explaining the high rates of disclosures delayed until adulthood. #### Facilitators {#section12-1524838017697312} Although fewer disclosure facilitators are identified in this review, very important facilitators were nonetheless uncovered---ones that should be noted for professionals in this field of practice. Internal factors that facilitate disclosures include symptoms that become unbearable, getting older with increased developmental efficacy, and realizing that an offence was committed ([@bibr8-1524838017697312]; [@bibr9-1524838017697312]; [@bibr11-1524838017697312]; [@bibr20-1524838017697312]; [@bibr34-1524838017697312]; [@bibr40-1524838017697312]). Circumstantial facilitators are those where the child discloses because there has been evidence provided, eye-witnessing has occurred, and a report has been made. Environmental factors include settings that provide opportunities such as counseling, interviews, information sessions and educational forums/workshops, and prevention programs for children and youth to disclose. To elaborate, dialogical contexts about CSA for children and youth can provide opportunities for discussion that may facilitate disclosures ([@bibr22-1524838017697312]). The research shows creating open dialogue in relationship contexts, to offset the power and influence of the perpetrator, can facilitate earlier disclosure. Among disclosure facilitators is being asked about abuse and given the opportunity to "tell" ([@bibr34-1524838017697312]); workshops on abuse and sexual abuse, in particular, can facilitate disclosures ([@bibr52-1524838017697312]); and using culturally sensitive probes and questions ([@bibr15-1524838017697312]). In [@bibr16-1524838017697312] study, positive disclosure experiences were described by participants as those where they felt that they had been listened to, were safe, were believed, and were not judged by the person they disclosed to. Further, family members and friends (peers) of the child victim can act as key supports to creating an open relational context and fostering positive responses ([@bibr22-1524838017697312]; [@bibr38-1524838017697312]; [@bibr41-1524838017697312]; [@bibr52-1524838017697312]). In particular, as children grow older, they are more likely to disclose to a peer, as shown in a number of studies, and this is an important reality for counselors and educators to be aware of ([@bibr10-1524838017697312]; [@bibr28-1524838017697312]; [@bibr41-1524838017697312]; [@bibr52-1524838017697312]). Discussion {#section13-1524838017697312} ========== Through examination of 33 studies published since the year 2000, this review identified five distinct themes regarding CSA disclosure: (1) Disclosure is best viewed as an iterative, interactive process rather than a discrete event done within a relational context; (2) contemporary models reflect a social--ecological, person-in-environment framework for understanding the complex interplay of individual, familial, contextual, and cultural factors involved in CSA disclosure; (3) age and gender are significant disclosure factors; (4) there is a lack of a life-course perspective; and (5) barriers to disclosure continue to outweigh facilitators. Based on these themes, a number of conclusions are drawn from this review. First, disclosure as a process is emphasized throughout contemporary research. Advances have been made in understanding these complex processes. However, the disclosure process over time---for example, how the first detection of CSA or attempts to disclose in childhood impact later disclosures---are not well understood. This is the result of the absence of a cohesive life-course perspective in investigations, although age consistently surfaces as significantly influencing CSA disclosure. Using a life-course perspective through the use of longitudinal studies is recommended. The use of varied methodological designs, depending on the developmental stage of the victims/survivors, influences the data generated and subsequent findings. For example, most studies on children and youth are based on file reviews of cases that have been brought to the attention of authorities, or surveys, with only a few studies using interviewing of younger children. Therefore, there is less information available on process issues with children and youth. In contrast, research on adult populations largely favors the use of qualitative interview methods for retrospective inquiry producing important process findings. In addition, investigations have not yet captured the disclosure experiences of adults in the "emerging adult" stage given that adult studies have failed to recognized that the age range of 18--24, which is now considered a developmental phase defined by neurobiological developmental uniqueness. As well, late adulthood has not been given attention as shown by the absence of participants representing this age-group in current research (70+). With a swelling geriatric population in North America, issues of historic CSA can be expected to surface and, with that, new disclosures. This trend is also anticipated due to attitudinal shifts that have presumably occurred over the last two generations about revealing such traumas and changing views about discussing sexual victimization. Interview guides used in a number of studies intentionally probed for facilitators, producing notable findings. For example, one such finding focuses on the importance of creating a contextually supportive environment to promote disclosure across the life course. These include developing therapeutic relational contexts for disclosure by providing information about sexuality, sexual abuse, prevention programming, and by asking directly. Disclosures to professionals are positive outcomes of how therapeutic contexts work; however, for forensic purposes prompting such disclosures would be viewed as problematic in legal settings, seriously compromising testimonies for trial proceedings. This is one example that speaks to the structural barriers victims and survivors run up against time and time again. Facilitators that show evidence to promote disclosure in one domain (therapeutic) are seen to work against CSA survivors in another domain---such as legal settings when perpetrators face prosecution. Defense attorneys will use this as evidence that the disclosure was prompted, and therefore the disclosure is potentially seen as not credible. Broadcasting of the frequency of acquitted cases or rulings in favor of the perpetrator through media outlets, often sensationalized, become a further compounding barrier. Given the review findings, we conclude that barriers and facilitators to CSA disclosures are nuanced and clearly embedded within intrapersonal, interpersonal, environmental, contextual, and cultural domains---often interlocked in complex ways. Limitations {#section14-1524838017697312} ----------- Although comprehensive in nature with its life-course coverage, this review may be limited by its qualitative, thematic focus rather than providing an evaluative, quantitative accounting of CSA disclosures. However, because of the recent focus on disclosure processes, the authors chose a suitably compatible approach---qualitative in nature. As well, a traditional checklist approach in rating the studies was not employed for interrater reliability, since two of the authors hold expertise in CSA disclosure research and are well versed with the literature. This expertise, and through closely following a systematic review framework ([@bibr27-1524838017697312]), assures that a thorough adjudication of the research literature was completed. Implications for Research and Practice {#section15-1524838017697312} -------------------------------------- These review findings have implications that can be useful in guiding future research and practice:Solid strides are being made in the use of a social--ecological framework to underpin investigations in the CSA disclosure investigations. Research efforts and practice considerations should continue in this vein. Investigating environmental factors and contextual and cultural forces is understudied, necessitating more research in these areas to more fully fill out understanding of CSA disclosure from a social--ecological perspective.There is good evidence that CSA disclosures are more likely to occur in a dialogical context---formal helping relationships but as well as other relationships such as peers and trusted adults. Providing information and education on topics of sexuality in general, and sexual abuse specifically, can help children and youth to disclose. Raising awareness and prevention programs can promote disclosures of sexual violence committed against children and youth.Goals of therapeutically supported disclosures (i.e., through therapy) may need to take precedence over forensic approaches, if well-being of child victims and adult survivors is to be made paramount. Legal processes may act to facilitate disclosures but can also act as barriers because of the negative outcomes experienced in the court process.Practitioners need to keep in mind that the legal system is lagging far behind in knowledge uptake of recent evidence on CSA disclosures so that victims and survivors continue to be systemically and structurally disadvantaged in legal proceedings.Health-care practitioners (i.e., child abuse pediatricians, family practice doctors, clinical nurse specialists, and public health nurses) should be made aware of the evidence in the CSA disclosure literature to create environments for facilitating therapeutic disclosures.Given that age is a stable predictor of disclosure of CSA, more studies are needed that make use of a life-course perspective. More longitudinal studies are needed to better identify trends over different life stages.The emerging young adult as a developmental age group needs specific investigation. Neuroscience research has established that ages 18--24 is a distinct developmental phase. Late adulthood is another life stage that deserves to be researched.Gender needs to be more fully investigated in relation to impact on disclosure. Awareness that boys and girls have unique challenges and barriers in disclosing CSA should be paramount for practitioners.Intervention planning should take note that disclosures increase when perpetrators no longer reside with victims, and this finding should be heeded by policy and law makers.Shame, self-blame, and fear are intrapersonal factors that persistently emerge as barriers to CSA disclosures and warrant more research to understand how to redress these barriers for earlier disclosures. Conclusion {#section16-1524838017697312} ========== There are still a substantial number of children and youth who are subjected to sexual abuse, despite preventative efforts. Just as concerning is the fact that many victims continue to suffer in silence as evidenced by the high numbers of delayed disclosure. These hidden cases should not be overlooked, and these victims should not be forgotten. Despite significant progress in bringing the issue of CSA to the forefront, improving facilitation of disclosure and increasing positive influences on disclosure processes are still critical in order to protect current and future generations of children and youth from the grave effects of sexual violence. Further, the focus should not be simply on strengthening and shoring up intrapersonal resources of victims to disclose but rather to change environmental conditions to create a more supportive and safer context for CSA victims and survivors to disclose. Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. Ramona Alaggia, MSW, PhD, is an associate professor in social work and the Factor-Inwentash Chair in Children's Mental Health at the University of Toronto. Her teaching and research focuses on gender and violence, sexual abuse disclosures, domestic violence exposure, and resilience processes. Delphine Collin-Vézina, PhD, is an associate professor for School of Social Work, McGill University and director for Centre for Research on Children and Families. She holds the Nicolas Steinmetz and Gilles Julien Chair in Social Pediatrics in Community and the Canada Research Chair (Tier II) in Child Welfare. Her work focuses on research and clinical topics related to child maltreatment, child sexual abuse, and trauma. Rusan Lateef, MSW, is a social worker employed in the criminal justice system with adult male offenders in Ontario, Canada. She specializes in the intersection of health and mental health, child sexual abuse disclosures, and she is a researcher on the "Make Resilience Matter" project examining childhood exposure to domestic violence with Dr. Alaggia at the Factor-Inwentash Faculty of Social Work, University of Toronto.	{ "pile_set_name": "PubMed Central" }	[ "informattsé", "agegroup", "adulthoodov", "defens", "conceal", "ie", "respond", "inquiri", "purpos", "collect", "ten", "longterm", "kind", "nondisclos", "student", "week", "signal", "exploratori", "collinvézina", "durban", "theori", "live", "holist", "socioeconom", "accident", "mistrust", "chair", "caus", "policecourt", "probe", "modul", "unrel", "journal", "phone", "process", "secreci", "norm", "happen", "state", "shift", "attempt", "difficult", "trustworthi", "suspicion", "swiss", "conflict", "women", "longitudin", "context", "dimens", "medic", "hidden", "nation", "higher", "contextu", "advertis", "even", "remov", "convict", "organ", "run", "preval", 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Background {#Sec1} ========== Taenia solium and Taenia saginata, also referred to as the pork and beef tapeworm, respectively, are food-borne parasites of global importance. In 2014, T. solium, for which humans act as final hosts after consumption of undercooked pork, was ranked by an international panel of experts as the food-borne parasite of greatest global concern, affecting millions of individuals every year and causing a substantial economic impact \[[@CR1]\]. In 2016, during a similar exercise carried out by a group of experts at the European level, T. solium was ranked tenth among 27 parasites as the number of human cysticercosis cases is not as high when Europe is considered as a whole. If individual regions were taken into account, the parasite was ranked higher in eastern Europe than in other parts of Europe \[[@CR2]\]. Human cysticercosis is a condition that may arise when humans ingest eggs of T. solium. When the parasite migrates to the central nervous system, the neurological condition is referred to as neurocysticercosis (NCC), which may lead to a number of potentially debilitating neurological manifestations including epilepsy and severe progressive headache \[[@CR3]\]. NCC is a major global public health concern. Recently, T. solium was identified as the number one food-borne parasite contributing to the global burden of disease leading to approximately 28,000 deaths per year and around 2.8 million disability-adjusted life years (DALYs) \[[@CR4]\]. Taenia saginata ranked lower in the multicriteria-based ranking, as symptoms of taeniosis are mild or even absent and generally the disease is not of substantial public health concern \[[@CR1], [@CR2]\]. Nevertheless, the parasite can pose major obstacles for trade and causes a substantial financial burden due to carcass condemnation, freezing and devaluation \[[@CR5]--[@CR8]\]. Taenia solium is highly endemic in pork-consuming poor communities of Asia, Africa and Latin America, while T. saginata is distributed worldwide. Little is known about the situation of T. solium in eastern Europe, and the suspicion of on-going transmission persists \[[@CR9]\]. For T. saginata a recent review on the epidemiology of bovine cysticercosis revealed the presence of the parasite in a large number of countries in western Europe; however, for eastern Europe data were scarce and of poor quality \[[@CR10]\]. In eastern Europe, various socioeconomic and political developments such as the collapse of the Soviet Union and the dissolution of the Socialict Federal Republic of Yugoslavia in 1991 following the wars, led to political and demographic changes. As a result a number of sectors, including the veterinary and public health sectors, were negatively affected. Large numbers of veterinary control officers were replaced by a cheaper working force leading to deteriorated veterinary control systems \[[@CR11]\]. The change to small-scale farms with reduced rearing standards and biosecurity and to small abattoirs with insufficient meat inspection and reduced government and veterinary oversight possibly contributed to an increased prevalence of zoonotic parasites such as T. solium, T. saginata, Trichinella spp. and Toxoplasma gondii \[[@CR11]\]. Backyard slaughtering without meat inspection has also become popular, possibly leading to the perpetuation of zoonotic diseases in parts of the region \[[@CR12]\]. Meat inspection is obligatory at slaughterhouses in the European Union (EU), according to European Regulation 854/2004 \[[@CR13]\]. Eastern Europe consists of "old" and "new" EU Members States as well as states which are still in the pre-accession phase as candidate EU countries. Therefore, adaptation of the national legislation and/or development of implementing regulations which integrate the main principles and features of the EU legislation and institutional structures towards more modern approaches to achieve better surveillance are ongoing \[[@CR11], [@CR14]\]. In the past decade, the number of diagnosed autochthonous NCC cases was increasing in eastern Europe \[[@CR15]--[@CR17]\], leading to the suspicion of on-going T. solium transmission occurring in the area; however, according to experts the numbers have gone down in the last few years. Changes in eating habits might further contribute to the current transmission patterns of the parasite, while imported T. solium tapeworm carriers and increased migration and travels further contribute to the risk \[[@CR18]\]. More recently, increasingly, European integration and introduction of regulatory legislation have influenced the region and, e.g. made it mandatory to have modern sanitation \[[@CR11]\]. Both T. solium and T. saginata have been considered endemic in eastern Europe. Results reported from the region in the past should however be evaluated critically for their representativeness and for the laboratory procedures applied \[[@CR14]\]. In the World Health Organization T. solium endemicity map, updated in 2015, uncertainty on the epidemiological situation in eastern Europe persists \[[@CR19]\]. In order to advance our knowledge on the occurrence of these zoonotic parasites and assess the potential needs for surveillance, we performed a systematic review on the epidemiology of taeniosis/cysticercosis in eastern Europe. This review is part of a series of two systematic reviews: the first one covered western Europe \[[@CR10]\] and the current one covers eastern Europe. Methods {#Sec2} ======= We conducted a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines \[[@CR20]\] (Additional file [1](#MOESM1){ref-type="media"}). To search for information on the epidemiology of T. saginata and T. solium in eastern Europe we used international databases and local, unpublished sources. Epidemiology was defined as the occurrence, prevalence, incidence and geographical distribution of human/porcine/bovine cysticercosis and taeniosis. Eastern Europe was defined based on regional proximity and on gross domestic product/gross national income and included 22 countries: Albania, Belarus, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Estonia, Former Yugoslav Republic of Macedonia, Greece, Hungary, Kosovo, Latvia, Lithuania, Malta, Moldova, Montenegro, Poland, Romania, Serbia, Slovakia and Ukraine \[[@CR10]\]. International databases {#Sec3} ----------------------- The online international bibliographical databases PubMed, ISI Web of Knowledge, CABDirect, OAIster, and OpenGrey were searched for all published data on taeniosis and (neuro)cysticercosis using the following search phrase: (cysticerc\* OR cisticerc\* OR neurocysticerc\* OR neurocisticerc\* OR \"C. bovis\" OR \"C. cellulosae\" OR taenia\* OR tenia\* OR saginata OR solium OR taeniosis OR teniosis OR ténia OR taeniid OR cysticerque) AND (Albania OR Belarus OR Bosnia OR Herzegovina OR Bulgaria OR Croatia OR Cyprus OR Czech Republic OR Estonia OR Former Yugoslav Republic of Macedonia OR Greece OR Hungary OR Kosovo OR Latvia OR Lithuania OR Malta OR Moldova OR Montenegro OR Poland OR Romania OR Serbia OR Slovakia OR Ukraine). The databases were searched for papers published from January 1st 1990 up to January 31st 2017. No language restriction was applied. Papers were excluded when fulfilling one or more of the following criteria: (i) studies concerning a different parasite than T. saginata and/or T. solium; (ii) studies reporting data from outside the specified countries; (iii) studies reporting results out of the scope of the review questions; (iv) duplicates. Papers were first screened for eligibility based upon title and abstract, and in the case of doubt, the full paper was assessed. For each eligible document, a narrative synthesis was made, which were then further digested into a qualitative review. Local sources {#Sec4} ------------- To identify additional, locally published and unpublished data sources, we distributed country sheets (Additional file [2](#MOESM2){ref-type="media"}) to members of the European Network on Taeniosis/Cysticercosis (CYSTINET) COST Action TD1302 and other non-member experts, asking to specify relevant national journals or epidemiological bulletins, MSc/PhD dissertations, national institutes, or registries, and to translate relevant search terms. Due to ethical constraints, unpublished hospital or laboratory data were only presented at an aggregated level. In addition, we searched for information in the proceedings of the CYSTINET (European Network for taeniosis/cysticercosis, COST Action TD1302) and European Network for Foodborne Parasites (COST Action FA1408, Euro-FBP) meetings. Finally, we explored the reference lists of recent topic-specific reviews \[[@CR15]--[@CR17]\] to find additional eligible papers. We applied the same inclusion and exclusion criteria to these additional sources and developed narrative syntheses for all eligible documents. Data collection and analyses {#Sec5} ---------------------------- The data collection was performed by three independent reviewers (CT, BD and SS). For data analysis, we followed the methodology described in \[[@CR10]\]. Briefly, cases reported as case reports providing information on individual characteristics of the patient were defined as individual cases and when no individual information was given, these cases were defined as aggregated. Tables summarizing individual cases included year of diagnosis, age, gender, nationality, and reported risk factors, while for aggregated cases or prevalence, the tables included country, level of data collection, timeframe, number of cases (or prevalence or incidence), Taenia species, risk factors (e.g. immigration/travel history) if available, and reference (i.e. authors and publication year). The following definitions were applied for the description of risk factors: endemic region (Asia, Africa and South and Central America, including Caribbean islands); immigrant from endemic region (any person born in or native from endemic region, or reported to have moved from endemic region); travelled/stayed in endemic region (having travelled, stayed, or resided in endemic region); no history of travels to endemic areas or immigration (autochthonous) (no history of travel/immigration reported). Cases in which the existence of duplicates was probable (e.g. cases included in two retrospective studies on the same area/hospital, covering overlapping time periods, cases diagnosed in the same hospital in the same time-frame but reported in different sources etc.) were included only once, by selecting the one that was published first. Descriptive analyses and maps were performed using the software environment for statistical computing R 3.5.0 and graphs were made in both R and Microsoft Excel \[[@CR21]\]. Results {#Sec6} ======= Search results {#Sec7} -------------- The initial search resulted in 1179 peer reviewed papers identified though international databases. After the screening process, 69 relevant references were identified and included in the review (Additional file [3](#MOESM3){ref-type="media"}: Table S1). In addition to this, researchers of 18 out of 22 eastern European countries were contacted from which additional data (92 relevant sources) from local and unpublished sources were obtained (Additional file [4](#MOESM4){ref-type="media"}: Table S2). Contacts of researchers working within the field from Belarus, Kosovo, Malta and Ukraine were not available; hence local, unpublished data for these countries are missing. The flow diagram of the search strategy steps is presented in Fig. [1](#Fig1){ref-type="fig"}. The countries for which data on T. solium or T. saginata in humans and/or animals were found are presented in Fig. [2](#Fig2){ref-type="fig"}.Fig. 1Flow diagram of the search strategy stepsFig. 2Summary of identified data on taeniosis and cysticercosis (in humans and animals) in eastern Europe (1990--2017): taeniosis (a); human cysticercosis (b); bovine cysticercosis (c); and porcine cysticercosis (d) Taeniosis {#Sec8} --------- In total, 58 unique sources were identified providing information on taeniosis in eastern Europe. Individual (8 records) and/or aggregated cases/prevalence (50 records) of taeniosis were reported in 14 out of 22 eastern European countries (Fig. [2](#Fig2){ref-type="fig"}). No cases of taeniosis were reported in Cyprus, and for 7 countries (Belarus, Bosnia and Herzegovina, Kosovo, Malta, Moldova, Montenegro and Ukraine), no reports on taeniosis could be retrieved for the years between 1990 and 2017. Poland was the country recording most data on taeniosis. Taeniosis case reports {#Sec9} ---------------------- In total only 17 individual cases reported in case reports from Croatia, Greece, Lithuania and Serbia, were available (Additional file [5](#MOESM5){ref-type="media"}: Table S3). Out of the 17 cases, 13 were reported as Taenia spp., three reported as confirmed and one suspected T. saginata using microscopy. No case reports describing T. solium taeniosis cases were identified through the search of published and other unpublished grey literature. Moreover none of all the case reports reported possible risk factors or how the parasite infection was acquired. Aggregated taeniosis cases {#Sec10} -------------------------- Data on aggregated taeniosis cases were obtained from scientific publications, authorities' reports, national registries, epidemiological bulletins, and from hospitals/laboratories (Additional file [5](#MOESM5){ref-type="media"}: Table S4). Aggregated taeniosis cases were reported in 14 eastern European countries and covered different years. Data were obtained from publications ranging from the 1990 up to 2017 (Fig. [3](#Fig3){ref-type="fig"}). The largest proportion of cases was reported as Taenia spp. and at species level the majority of cases were reported as T. saginata. Taenia solium taeniosis cases were a rare finding, reported only in Albania, Estonia, Latvia and Poland. No indication was given on how species identification was performed. For Latvia, the two suspected cases probably originated from abroad (probably Russia) (Deksne, personal communication, 2016). No additional information on the source of infection, potential risk factors or nationality was given for the other reported cases.Fig. 3Taeniosis cases reported in scientific publications, authority reports, epidemiologial bulletins, and laboratories in eastern Europe (published/reported between 1990--2017). Arrow pointing at the number, larger than 300 taeniosis cases. Abbreviation: FYROM, Former Yugoslav Republic of Macedonia The annual number of cases reported for each country varied, with Poland and Romania reporting the highest annual number of aggregated cases. In particular for Poland, a large number of data was available as epidemiological reviews published quarterly in the journal of the National Institute of Public Health - National Institute of Hygiene and the Polish Society of Epidemiology and Infectious Diseases. Taeniosis incidence data {#Sec11} ------------------------ Incidence data were available for seven counties (Bulgaria, Estonia, Lithuania, Poland, Romania, Serbia and Slovakia) and ranged from 0/100,000 people for Bulgaria in the year 2000 to 8.5/100,000 people in Poland in 1991 (Fig. [4](#Fig4){ref-type="fig"}).Fig. 4Taeniosis incidence data reported between the years 1990--2017 in authorities' reports, epidemiological bulletins and national registries in eastern Europe. Abbreviations: BG, Bulgaria (1998 and 2000); EE, Estonia (1990--1999); PL, Poland (1991--2009); RO, Romania (2007--2014); RS, Serbia (1997--2005); SK, Slovakia (1990--2014) Taeniosis prevalence data {#Sec12} ------------------------- In total, five epidemiological studies reporting taeniosis prevalence data published between 1990--2017 were identified (Additional file [5](#MOESM5){ref-type="media"}: Table S5). Within eastern Europe the prevalence of taeniosis ranged between 0--4.9%, with the highest prevalence reported in a study conducted in children of the Roma settlements of Košice and Prešov regions in Slovakia \[[@CR22]\]. A study carried out in Estonia reported a seroprevalence of T. solium of 0.7% (95% confidence interval, CI: 0.3--1.4%) using a commercial NovaLisa IgG enzyme immunoassay ELISA \[NovaTec Immunodiagnostica GmbH, Dietzenbach, Germany (Sp \> 95%, Se 94%)\], and 0.0% (95% CI: 0--0.3%) using the ELISA and a Western Blot IgG (LDBIO DIAGNOSTICS, Lyon, France) in series \[[@CR23]\], while the other four studies used microscopy as a diagnostic tool. Human cysticercosis {#Sec13} ------------------- Relevant information on human cysticercosis was obtained from 63 sources. In total, 40 reports reported individual, and 23 reports reported aggregated cysticercosis cases. Information on human cysticercosis cases was available for 15 out of 22 eastern European countries (Fig. [2](#Fig2){ref-type="fig"}), while no data could be retrieved from Albania, Belarus, Cyprus, Malta, Moldova, Montenegro and Ukraine in the form of either published or unpublished sources. Human cysticercosis case reports {#Sec14} -------------------------------- In total, 58 individual cases of cysticercosis were identified in authorities' reports, from hospital/laboratories or published case reports (Table [1](#Tab1){ref-type="table"}). Out of 58 cases, 45 were diagnosed in 7 eastern European countries, while 13 cases (22%) were originally from eastern Europe but were diagnosed abroad. The average age was 49 years and 49% of the cases were male, 42% female and for 9% the sex was not known (Additional file [5](#MOESM5){ref-type="media"}: Table S6). Of all the individual case reports, 17% were reportedly suspected to be autochthonous cases, in 14% the infection was considered aquired in endemic countries while travelling or living abroad, whereas for the majority of cases (69%) the place of infection was unknown or not recorded. For Lithuania, the cases were possibly connected with travels to Argentina and South Africa (S. Petkevičius, personal communication, 2015).Table 1Number of human cysticercosis cases per country and most likely place of infection (Data from published and unpublished sources between 1990 and 2017)CountryMost likely place of infectionTotal no. of casesEastern EuropeEndemic countryUnknownCroatia1010Czech Republic6410Greece33612Hungary22Latvia112Lithuania55Serbia156Cyprus11Czechoslovakia22Former Yugoslav Republic of Macedonia33Bosnia and Herzegovina55 Aggregated human cysticercosis cases {#Sec15} ------------------------------------ The number of aggregated human cysticercosis cases varied largely across countries, with over 300 cases (most of them reported as autochthonous) reported from Romania. Figure [5](#Fig5){ref-type="fig"} shows the number of human cysticercosis cases identified in eastern Europe in documents published from 1990 to 2017. Information on risk factors and place where the disease might have been acquired were absent for most cases (Additional file [5](#MOESM5){ref-type="media"}: Table S7).Fig. 5Number of identified human cysticercosis cases in eastern Europe - data sources published and unpublished between 1990--2017. Arrow pointing at the number, larger than 300 human cycsticercosis cases. Abbreviation: FYROM, Former Yugoslav Republic of Macedonia Only two serological studies using ELISA and/or western blot techniques were identified. One of the studies was in Croatia, where a prevalence of 1.5% was recorded among people with epilepsy \[[@CR24]\]. The other study was in Estonia, where a western-blot-confirmed T. solium cysticercosis prevalence (commercial ELISA and commercial western blot in series) was of 0.0% in samples from the general Estonian population, children, veterinarians and hunters \[[@CR23]\]. Porcine cysticercosis {#Sec16} --------------------- Relevant information on porcine cysticercosis was obtained from 53 sources, which reported the occurrence of the infection in 8 countries. In Poland the causative agent was specified as being "cysticercus cellulosae" (name used for T. solium cysticercus) in two publications (Additional file [5](#MOESM5){ref-type="media"}: Table S8). The highest number of porcine cysticercosis cases (4487 cases) was recorded in Poland in 2003; however, in the report species were not specified and porcine cysticercosis could refer to "cysticercus cellulosae" or cysticerci of other species like "cysticercus tenuicollis" (name used for Taenia hydatigena cysticercus). Of the porcine cysticercosis cases reported, a molecular method was reportedly used only in Estonia, and T. solium could not be confirmed \[[@CR25]\]. No cases of porcine cysticercosis in pigs were reported from Bosnia and Herzegovina, Croatia, Cyprus, Czech Republic, Former Yugoslav Republic of Macedonia and Hungary, and no information was found for Albania, Belarus, Greece, Kosovo, Malta and Ukraine. Based on routine meat inspection, reported prevalence ranged from 0% to 0.18% in Bulgaria \[[@CR9]\] and varied within and between countries. A single serological study was identified: in Romania, an ELISA-based prevalence of active porcine cysticercosis was reported to be 6.4% \[[@CR26]\]. In Latvia, according to the Food and Veterinary Service, the main slaughtering monitoring authority, in the last 10 years no suspicious cysts have been found in pigs (Food and Veterinary Service Latvia, personal communication, 2015). Bovine cysticercosis {#Sec17} -------------------- Relevant information on bovine cysticercosis was obtained from 41 sources reporting data for 15 out of 22 countries (Additional file [5](#MOESM5){ref-type="media"}: Table S9). Only for Cyprus, no cases were recorded since 1990. For Albania, Hungary, Kosovo, Latvia, Malta and Montenegro, no data were retrieved. Based on routine meat inspection, reported prevalence ranged from 0.0% up to 1.7% and varied within and between countries (Fig. [6](#Fig6){ref-type="fig"}). No studies were found applying more sensitive methods to detect bovine cysticercosis. For Poland in particular, a large amount of data were available, in which the highest annual number of bovine cysticercosis cases (4718 cases) in the country was reported in 1994. The number of reported cases has decreased consistently over the years.Fig. 6Prevalence of bovine cysticercosis based on routine meat inspection detected in eastern Europe (1990--2017). Abbreviations: CZ, Czech Republic; EE, Estonia; HR, Croatia; MD, Moldova; MK, Former Yugoslav Republic of Macedonia; PL, Poland; RO, Romania; RS, Serbia; SK, Slovakia; UA, Ukraine Discussion {#Sec18} ========== Published and unpublished data sources were searched, and contacts with researchers of eastern European countries were established to obtain all possible information on the epidemiology of T. solium and T. saginata in eastern Europe. Individual case reports for taeniosis were only retrieved from four countries, while reports on aggregated cases were available for nearly all the eastern European countries. At a species level, most data were reported as Taenia spp. or T. saginata, leading to uncertainty regarding the true disease epidemiology. In a few countries, T. solium was reported as aggregated taeniosis cases. Given the risk of having T. solium carriers it is essential to improve on differential diagnosis and reporting. A similar scenario was also observed in western Europe, suggesting that diagnosis and case management is not performed adequately \[[@CR10]\]. Moreover, the laboratory procedures and the reporting seem unstandardized and often only a small part of the population is examined; therefore, the data from most countries are incomplete \[[@CR14]\]. Hence, the reported prevalence of human taeniosis from T. saginata and T. solium should be evaluated critically. Also, the origin of infection and risk factors were not reported, leading to continued uncertainty on the real endemic status of eastern Europe. Human cysticercosis reports of individual and aggregated cases were available from nearly one third of the 22 eastern European countries, while for western Europe, information was available from all 18 countries \[[@CR10]\]. Romania and Serbia reported the largest number of diagnosed human cysticercosis cases, highlighting both the reporting and possibly also the epidemiological differences among eastern European countries. Observing the difference in reported cases at a hospital level between countries, either an underestimation or an overestimation might be suspected for eastern Europe. However, several factors such as lack of knowledge, diagnostic capacity and compensation for the farmers, unwillingness or inability to report and logistic difficulties may contribute to the vicious cycle of underreporting and underdiagnosis \[[@CR27], [@CR28]\]. Of all cases, 14% were suspected to have travelled abroad, 17% to be autochthonous cases, while for the majority (69%) it was not specified. This lack of additional information does not allow us to unravel the question of an existing parasite transmission in eastern Europe. The results show that even if the assumption that the infection can be acquired abroad would be correct, then the same happens in Europe as frequently. The automatic assumption that brief visits abroad automatically outrule looking for answers locally, can lead to a barrier for dealing with the actual epidemiological situation. Nearly one quarter of the cases were diagnosed abroad, possibly indicating that either the local health system is not equipped to detect these cases, or that people are actively seeking medical attention elsewhere. For two thirds of the human cases the origin of infection was not given, further contributing to the persisting knowledge and underreporting gap. However, it should be noted that a lowering trend is registered. For instance in Serbia, no new cases were recorded after 2010. Cases of porcine cysticercosis were reported in a number of countries, and as for the human cases, the number of cases and reports per country varied significantly. Notably, in Poland over 4000 cases of porcine cysticercosis were recorded by the veterinary inspection services in 2003; however, no information on the Taenia spp. involved was given and the possibility of (some of) the cases being T. hydatigena cannot be excluded. Also, for most other porcine cysticercosis cases reported in eastern Europe, species identification was never performed, nor molecularly confirmed. Only in one study from Estonia, molecular techniques were applied to confirm a false positive case \[[@CR25]\]. Lack of differential diagnosis highlights the presence of poor meat inspection, due to the possible lack of: qualified meat inspectors, sensitive and specific diagnostic tools, awareness, funding and adequate mandatory reporting systems. Moreover the trend towards smaller-scale pig farming, with outdoor access and home slaughtering (without meat inspection) increases the risk of infection and therefore the need for monitoring. Bovine cysticercosis was reported in over two thirds of the countries in the region. The highest number of cases was reported in Poland, where 4718 bovine cysticercosis cases were recorded in 1994. General meat inspection has a very low sensitivity, hence slaughterhouse figures, even when well-recorded will always underestimate the prevalence \[[@CR29]\]. False positives, e.g. eosinophilic myositis as a consequence of sarcocystis infection or abscessess, can also occur although they usually contribute only a small (negligible) proportion of bovine cysticercosis records. Better, cost-effective and more sensitive diagnostic tools should be developed, and data recording systems should be improved to assure that data from individual slaughterhouses are centralised. Increased availability of data will further allow estimating the overall economic impact of T. saginata which has been shown to be significant in Belgium (858 €/heavily infected carcass and 586 €/lightly infected carcass) \[[@CR7]\], but less so in Catalonia \[northeastern Spain (1140 €/heavily infected carcass and 509 €/lightly infected carcass)\] \[[@CR8]\]. Without reliable data from the surveillance the default assumption should be that the parasite is present, not absent. To overcome case uncertainty and to be able to have more accurate data that will improve our knowledge on the epidemiological situation of T. saginata and T. solium, the possibility to molecularly confirm T. solium findings should be available. Recently, the laboratories across Europe that are running tests for the larval and adult stages of the parasites have been summarised and mapped, facilitating the process when suspected lesions are detected \[[@CR27]\]. The early species-level identification of the tapeworm and subsequent adapted management are crucial to avoid not only human-to-human transmission, but also human-to-pig/cattle transmission. Finally, for nearly all the countries, at least some data and research on the topic could be retrieved and both published and unpublished data sources were available. Particularly the unpublished sources were of added value and contributed to an improved picture of the epidemiological situation in eastern Europe, highlighting the importance of data access and collaborations through networks such as CYSTINET. Nevertheless, for Belarus, Kosovo, Malta and Ukraine a contact could not be established and no information could be retrieved leaving information gaps. In the absence of data the presence of T. saginata and T. solium in these countries cannot be excluded. For western Europe information and a contact was established with all counties of the region, and the amount of published and unpublished literature was more than double that of eastern Europe, which helped to provide a better and more complete overview of the epidemiological situation \[[@CR10]\]. The two zoonotic parasites would merit more attention and research in eastern Europe. Conclusions {#Sec19} =========== There is indication that taeniosis and cysticercosis are present across eastern Europe but information on the occurrence of T. solium and T. saginata across the region remains fragmented. For a number of countries, no information was available and for some, establishing a contact with experts within the field proved challenging. Species identification is not performed in most countries and the findings are not confirmed using molecular methods (especially for taeniosis and porcine cysticercosis), making the endemicity status in the region still unclear. Attention should be paid to: (i) suspected autochthonous human cysticercosis cases to rule out ongoing transmission; (ii) notification of taeniosis and human cysticercosis as these should be implemented to get better data and a clearer overview of the epidemiological situation; (iii) surveillance and reporting systems in animals; and (iv) methods for confirming findings. Additional files ================ {#Sec20} Additional file 1:PRISMA checklist. (PDF 68 kb) Additional file 2:Country sheets template. (PDF 81 kb) Additional file 3:Table S1. List of references included in the review retrieved through online international databases. (XLSX 24 kb) Additional file 4:Table S2. List of references included in the review made available through local sources. (XLSX 71 kb) Additional file 5:Table S3. Individual taeniosis cases identified in case reports in eastern Europe available from 1990 to 2017. Table S4. Aggregated taeniosis cases reported in documents (authorities' reports, epidemiological bulletins, national registries and publications) identified in eastern Europe available from 1990 to 2017. Table S5. Taeniosis prevalence data reported in epidemiological studies published between 1990--2017. Table S6. Individual human cysticercosis cases identified in case reports in eastern Europe (1990--2017). Table S7. Aggregated human cysticercosis cases identified in case reports and publications in eastern Europe (1990--2017). Table S8. Porcine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990--2017). Table S9. Bovine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990--2017). (DOCX 207 kb) CYSTINET : European Network on Taeniosis/Cysticercosis EFSA : European Food Safety Authority GDP/GNI : gross domestic product/gross national income ICD : International Classification of Diseases NCC : neurocysticercosis OIE : World Organization for Animal Health/Office International des Epizooties This work is a collaboration within the framework of CYSTINET, the European network on taeniasis/cysticercosis, COST ACTION TD1302. We acknowledge Dr G. Petrusevska and Dr I. Kuzmanovski, for the unpublished data about NCC in Former Yugoslav Republic of Macedonia, and Prim. Dr E. Miloskoska Blagoeska and Dr G. Kuzmanovska, Former Yugoslav Republic of Macedonia, for their valuable input for the conduction of this work. We also acknowledge Professor Olga Dulović, Clinic for Infectious and Tropical Diseases, Clinical Center of Serbia, Belgrade, Serbia, and School of Medicine, University of Belgrade, Belgrade, Serbia, for the assistance in providing medical records for Serbia. Funding {#FPar1} ======= This work is a collaboration within the framework of CYSTINET, the European network on taeniasis/cysticercosis, COST ACTION TD1302. Availability of data and materials {#FPar2} ================================== All references found eligible in our literature review are included in the supplemental materials (Additional files [3](#MOESM3){ref-type="media"} and [4](#MOESM4){ref-type="media"}). CT, BD and SS conducted the systematic review of the literature and extracted data. CT and BD analysed data and drafted the first version of the manuscript. AA, BD, CT, MLG, SS and VD contributed to the design of the study, interpretation of data and writing of the first draft. The remain co-authors (ZW, AK, AC, ASW, AA, BB, BL, CMC, CV, DA, GD, IB, IK, JK, JS, BK, MJP, MV, MP, MS, MK, OD, PJ, DSJ, VS, ZD, SG and PD) contributed to data collection and writing of the paper. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== This work is a collaboration within the framework of CYSTINET, the European network on taeniasis/cysticercosis, COST ACTION TD1302. Consent for publication {#FPar4} ======================= Not applicable. Competing interests {#FPar5} =================== The authors declare that they have no competing interests. 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Sir, In recent years, we have seen an increasing use of noninvasive ventilation (NIV) in the context of acute respiratory insufficiency (acute respiratory failure). However, adherence to treatment is not perfect; partly because of patient\'s complaints relating to high-pressure levels. This led to envisaging if the use of lower ventilatory pressures could lead to the same clinical results of the higher pressures, on patients with the restrictive pulmonary disease. We have read with great interest study by Kadowaki et al. published in your journal, entitled "Low-intensity noninvasive ventilation: Lower pressure, more exacerbations of chronic respiratory failure," presenting a retrospective study where they investigated the effects of lower NIV pressure on chronic respiratory failure. It concluded that patients can develop more exacerbations, recommending the use of higher initial support pressure levels.\[[@ref1]\] It is an interesting and original article to read, but we would like to comment some parts of it: First, we do not agree with the nondiscrimination of hypercapnic respiratory insufficiency, being defined only by the value of PaCO~2~. There is not any reference to the value of oxygen (or pH) and we think that a patient with severe -- mild hypoxemia and hypercapnia are surely different from a patient with the only hypercapnia, and this may alter the results because these two subtypes are associated with different outcomes\[[@ref2]\]Second, according to the authors, its protocol to start with lower pressure levels (LPLs). We understand that the aim of this work was to study this issue and we also accept this strategy may be an attempt to improve the patients\' adherence; however, we do not agree to be standard starting with low inspiratory positive airway pressure (IPAP). Most recent guidelines report that even patients with restrictive disease should start with initial IPAP 15--20 cmH~2~O\[[@ref3]\]Finally, all patients had heated humidifier and its benefits are well known, although it is not unanimous its routine use.\[[@ref3]\] There are, however, data in literature showing that advantages are more prominent with higher IPAP pressure level.\[[@ref4]\] It is also widely known that use of these devices cause adverse effects such as the risk of infection or poor compliance. Could this have affected patients with LPLs and led to worse outcomes? Financial support and sponsorship {#sec2-1} ================================= Nil. Conflicts of interest {#sec2-2} ===================== There are no conflicts of interest.	{ "pile_set_name": "PubMed Central" }	[ "treatment", "promin", "two", "howev", "lead", "posit", "refer", "work", "origin", "clinic", "accept", "restrict", "financi", "complianc", "well", "seen", "nondiscrimin", "protocol", "part", "lowintens", "affect", "would", "understand", "aim", "data", "risk", "associ", "inspiratori", "hypercapnia", "valu", "partli", "show", "may", "advantag", "conclud", "recent", "ventil", "adher", "effect", "lower", "year", "led", "use", "outcom", "support", "caus", "accord", "literatur", "complaint", "journal", "guidelin", "mild", "interest", "articl", "result", "also", "unanim", "alter", "wors", "kadowaki", "failur", "attempt", "patient", "noninvas", "conflict", "pulmonari", "poor", "devic", "retrospect", "highpressur", "defin", "routin", "ventilatori", "levelref", "context", "envisag", "relat", "lpl", "hypercapn", "higher", "agre", "et", "first", "even", "heat", "low", "ph", "recommend", "sec", "ipap", "wide", "chronic", "exacerb", "develop", "pressur", "author", "hypoxemia", "although", "outcomesrefsecond", "sponsorship", "like", "publish", "humidifi", "sir", "diseas", "respiratori", "investig", "infect", "benefit", "initi", "could", "levelsref", "sure", "present", "insuffici", "standard", "cmhoreffin", "oxygen", "paco", "acut", "useref", "entitl", "differ", "sever", "studi", "subtyp", "airway", "increas", "improv", "report", "issu", "perfect", "comment", "great", "known", "think", "nil", "advers", "read", "al", "level", "strategi", "start", "niv" ]	22,257	[ "Sir", "recent", "years", "seen", "increasing", "use", "noninvasive", "ventilation", "NIV", "context", "acute", "respiratory", "insufficiency", "acute", "respiratory", "failure", "However", "adherence", "treatment", "perfect", "partly", "complaints", "relating", "levels", "led", "envisaging", "use", "lower", "ventilatory", "pressures", "could", "lead", "clinical", "results", "higher", "pressures", "patients", "restrictive", "pulmonary", "disease", "read", "great", "interest", "study", "Kadowaki", "et", "al", "published", "journal", "entitled", "noninvasive", "ventilation", "Lower", "pressure", "exacerbations", "chronic", "respiratory", "failure", "presenting", "retrospective", "study", "investigated", "effects", "lower", "NIV", "pressure", "chronic", "respiratory", "failure", "concluded", "patients", "develop", "exacerbations", "recommending", "use", "higher", "initial", "support", "pressure", "interesting", "original", "article", "read", "would", "like", "comment", "parts", "First", "agree", "nondiscrimination", "hypercapnic", "respiratory", "insufficiency", "defined", "value", "reference", "value", "oxygen", "pH", "think", "patient", "severe", "mild", "hypoxemia", "hypercapnia", "surely", "different", "patient", "hypercapnia", "may", "alter", "results", "two", "subtypes", "associated", "different", "Second", "according", "authors", "protocol", "start", "lower", "pressure", "levels", "LPLs", "understand", "aim", "work", "study", "issue", "also", "accept", "strategy", "may", "attempt", "improve", "adherence", "however", "agree", "standard", "starting", "low", "inspiratory", "positive", "airway", "pressure", "IPAP", "recent", "guidelines", "report", "even", "patients", "restrictive", "disease", "start", "initial", "IPAP", "Finally", "patients", "heated", "humidifier", "benefits", "well", "known", "although", "unanimous", "routine", "however", "data", "literature", "showing", "advantages", "prominent", "higher", "IPAP", "pressure", "also", "widely", "known", "use", "devices", "cause", "adverse", "effects", "risk", "infection", "poor", "compliance", "Could", "affected", "patients", "LPLs", "led", "worse", "outcomes", "Financial", "support", "sponsorship", "Nil", "Conflicts", "interest", "conflicts", "interest" ]
Background ========== Chronic inflammatory demyelinating polyneuropathy (CIDP) is an uncommon manifestation of systemic lupus erythematosus (SLE). To our knowledge, few cases of CIDP and SLE have been previously reported in the literature \[[@b1-amjcaserep-18-980]\]. CIDP is a debilitating clinical condition that is characterized by symmetrical polyneuropathy with histologic findings of demyelination and occasionally remyelination, and is believed to be an acquired autoimmune disorder that targets myelin \[[@b1-amjcaserep-18-980]\]. CIDP continues to progress, or may relapse, for more than eight weeks, a clinical finding that differentiates it from acute inflammatory demyelinating polyneuropathy which is a monophasic sub-acute illness that reaches its nadir within three to four weeks. CIDP is characterized by muscular weakness with or without sensory loss in the extremities and can have a chronic progressive course with remission and repeated relapses \[[@b2-amjcaserep-18-980]\]. The diagnosis of CIDP is more likely when the patient has a predominance of sensory symptoms over motor symptoms. Although the cause of CIDP is unknown, there is evidence to support an autoimmune etiology with multiple immunological triggers \[[@b3-amjcaserep-18-980],[@b4-amjcaserep-18-980]\]. Both the cellular and humoral components of the immune system appear to be involved in the pathogenesis of CIDP and its variants \[[@b3-amjcaserep-18-980],[@b4-amjcaserep-18-980]\]. An estimated 10--20% of SLE patients show peripheral nervous system involvement and patients present with sensorimotor polyneuropathies, with less common syndromes including mononeuritis multiplex or asymmetric polyneuropathy and acute or chronic demyelinating polyneuropathy \[[@b5-amjcaserep-18-980]\]. Multiple factors, including early diagnosis of CIDP and presence of multiple antibodies associated with SLE, predict a good response to intravenous immunoglobulin (IVIG). Case Report =========== A 40-year-old African American woman with a past medical history of SLE, diagnosed at the age of 40 years, and treated with hydroxychloroquine, presented with a three-month history of slowly progressive tingling sensation and weakness in both her lower and upper extremities, and difficulty in walking. She initially presented with fatigue, fever, myalgia and arthralgia at the time of her diagnosis of SLE diagnosis. She had no known complications of SLE and no significant past medical history at the time of presentation. Review of here systems showed worsening fatigue, myalgia, headache, and numbness and some weakness of upper and lower extremities. The sensory and motor symptoms progressed in an ascending fashion resulting in impaired balance without bowel or bladder involvement. On her current admission to hospital, the differential diagnosis of her symptoms was broad and included idiopathic inflammatory myopathy, CIDP, subacute combined degeneration of spinal cord, cervical myelopathy, SLE neuropathy, thyroid myopathy, amyotrophic lateral sclerosis, multiple sclerosis, Eaton-Lambert syndrome, and paraneoplastic syndrome associated with an unknown primary malignancy. Physical examination showed a grading for motor strength of 4/5, decreased temperature of the limb extremities, reduced pinprick and vibration sense and absent reflexes of both the upper and lower extremities, and an unsteady gait. Her sensory symptoms were more prominent when compared to her motor weakness. Her positive and negative autoimmune panel, determined by indirect immunofluorescence, is listed in [Table 1](#t1-amjcaserep-18-980){ref-type="table"}. Other laboratory investigations showed a normal complete metabolic panel, cerebrospinal fluid analysis, thyroid profile, brain magnetic resonance imaging (MRI), an erythrocyte sedimentation rate (ESR) of 75 mm/hr, low C4 complement, leukopenia, and anemia. No paraproteins were identified on serum electrophoresis. Electromyography (EMG) showed axonal demyelinating polyradiculoneuropathy, abnormal peroneal distal latency with very low amplitude and disappearance of F waves consistent with CIDP. The patient was treated with intravenous immunoglobulin (IVIG) 2 gm/kg daily for five days and prednisone 60 mg daily for a total of seven days, while continuing hydroxychloroquine. Following treatment with IVIG and prednisone, the patient showed marked clinical improvement and regained her ability to walk with minor assistance. She was discharged home on a tapering dose of prednisone dose with follow-up in the rheumatology clinic. Discussion ========== Neurological and psychiatric symptoms are reported to occur in between 10--80% of patients with the diagnosis of systemic lupus erythematosus (SLE) during their illness \[[@b6-amjcaserep-18-980],[@b7-amjcaserep-18-980]\]. The range of neurological manifestations of SLE includes central nervous system encephalitis, hemiparesis, brain infarcts, seizures, schizophrenia, atypical trigeminal neuralgia, acute demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy (CIDP) \[[@b8-amjcaserep-18-980]\]. In 1948 Sedgwick and Von Hagen first described the clinical manifestation of a CIDP-like illness in patients with SLE ([@b9-amjcaserep-18-980]\]. Retchland and coworkers stated that CIDP can have an unusual presentation and can precede systemic symptoms of SLE \[[@b10-amjcaserep-18-980]\]. About 50% of the patients with a concomitant diagnosis of SLE and CIDP can achieve a good clinical response to IVIG, and the remainder may have a minimal response. Certain characteristics, including early CIDP diagnosis, the involvement of all four extremities, hyporeflexia or areflexia, slowed peripheral motor nerve conduction velocity, SLE involvement of critical internal organs, and the presence of multiple auto-antibodies associated with SLE, are of a good response to IVIG \[[@b1-amjcaserep-18-980]\]. Patients with SLE and CIDP may present with recurrent episodes of Guillain-Barré syndrome-like symptoms, mononeuritis multiplex, or symmetric polyradiculopathy, over a period of weeks to months. The proposed pathological mechanisms for the clinical symptoms of CIDP in SLE patients include abnormalities in blood vessels that supply the epineurium, resulting in nerve fiber loss, inflammation that involves the interstitial and causes separation of nerve fibers, and reduction in the amount of myelin \[[@b11-amjcaserep-18-980],[@b12-amjcaserep-18-980]\]. CIDP is now believed to be an autoimmune mediated process involving cellular and humoral response that involve T-cells, activated macrophages, cytokines, anti-myelin antibodies, and costimulatory inflammatory molecules, resulting in loss of myelin and axonal loss \[[@b11-amjcaserep-18-980],[@b12-amjcaserep-18-980]\]. The neuropathy of CIDP is usually polyradicular, symmetrical, and involves both proximal and distal muscles. \[[@b13-amjcaserep-18-980]\]. Electromyography (EMG) testing in CIDP may help to determine the extent of sensory and motor deficits and categorize the demyelinating process (prolonged terminal latency, slowing of nerve conduction velocity, dispersion and conduction block) and axonal form of neuropathy (marginal slowing of nerve conduction, small compound muscle or sensory action potential, and denervation on EMG) \[[@b14-amjcaserep-18-980]\]. Hereditary forms of demyelination are suggested by features of uniform demyelination, whereas variable demyelination of nerve segments of the same nerve favor acquired demyelination \[[@b14-amjcaserep-18-980]\]. Demyelination requires slowing of conduction velocities, according to the criteria by American Academy of Neurology (AAN) \[[@b15-amjcaserep-18-980]\]. Most CIDP patients do not meet these AAN criteria, as conduction slowing can be absent when not enough fibers are affected when demyelination is proximal and not affected by distal stimulation, or when there is severe secondary axonal degeneration \[[@b16-amjcaserep-18-980]\]. A study by Haq and colleagues showed that there was only 42% sensitivity of the electrodiagnostic criteria proposed by the AAN in CIDP patients who had sural nerve biopsies \[[@b17-amjcaserep-18-980]\]. The presence of anticardiolipin antibodies in CIDP may also reflect myelin damage \[[@b18-amjcaserep-18-980]\]. For patients with sensorimotor neuropathy, there is a good response to steroid pulse or IVIG treatment, despite age, concurrent illness, and positive anticardiolipin antibodies \[[@b18-amjcaserep-18-980]\]. According to Sindern and colleagues, CIDP is also reported to occur episodically in other autoimmune diseases, including myasthenia gravis, acute glomerulonephritis, Hashimoto's thyroiditis, and multiple sclerosis \[[@b19-amjcaserep-18-980]\]. Very rare associations have been reported with diseases that include hemophagocytic lymphohistiocytosis, which has an estimated prevalence of 0.9--4.6% in patients with SLE \[[@b20-amjcaserep-18-980]\]. CIDP can occur before, after, or simultaneously with the onset of SLE. As shown in this case report, early identification of peripheral limb weakness, areflexia or hyporeflexia, and slowed nerve conduction is crucial before treatment is commenced with steroids, IVIG, or plasmapheresis, all of which may be life-saving. Apart from treatment with steroids and IVIG, other immunosuppressive agents including azathioprine, mycophenolate, rituximab and cyclophosphamide may be considered \[[@b21-amjcaserep-18-980],[@b22-amjcaserep-18-980]\]. Inflammatory neuropathies respond to therapy with glucocorticoids in moderate to higher doses, such as prednisone 30--60 mg/day, but not all patients improve on this treatment. Therapy with glucocorticoids, IVIG, or plasmapheresis may be indicated in CIDP. Evidence of axonal damage on EMG, or vasculitis on nerve biopsy, are indications for initiating more potent immunosuppressive therapy, such as cyclophosphamide. In 2005, Vina and colleagues suggested that more aggressive treatment should be used when more severe systemic manifestation of SLE were in order to suppress the immune-mediated processes involved in the pathogenesis of CIDP \[[@b1-amjcaserep-18-980]\]. Conclusions =========== CIDP is an acquired autoimmune peripheral neuropathy that can be an unusual association seen in patients with SLE. As this case report has shown, early clinical diagnosis of CIDP, supported by serological autoantibody profiles associated with SLE, can predict a good response to steroids. Most patients with CIDP are treated successfully with steroids if the diagnosis is made early. IVIG, plasmapheresis, or immunosuppressive therapy should be considered if there is no response to steroids. Conflict of interest None. ###### Panel of autoimmune serology serological markers tested. Serum markers and antibodies Patient result Normal range ---------------------------------- -------------------- ------------------- ANA Positive Negative ANA titer 1: 1280 \<1: 80 ANA pattern Speckled C4 complement 14.3 mg/dl 18.0--55.0 mg/dl C3 complement 90.5 mg/dl 79.0--152.0 mg/dl Anti-ds DNA antibodies Positive Negative Anti-ds DNA antibodies titer 1: 44 IU \<1: 25 IU Anti SSA antibodies Positive Negative Anti SSA antibodies titer \>1: 25 IU \<1: 20 IU Anti SM antibodies Positive Negative Anti SM antibodies titer \>1: 25 IU \<1: 20 IU Anti SSB antibodies Negative Negative Anti RNP antibodies Negative Negative Anti SCL 70 Negative Negative Anti-histone antibody Negative Negative ANA -- antinuclear antibodies; SSA, SSB -- Sjögren syndrome A and B; SM -- smooth muscle; RNP -- ribonucleoprotein; SCL -- scleroderma. [^1]: Authors' Contribution: [^2]: Study Design [^3]: Data Collection [^4]: Statistical Analysis [^5]: Data Interpretation [^6]: Manuscript Preparation [^7]: Literature Search [^8]: Funds Collection [^9]: Conflict of interest: None declared	{ "pile_set_name": "PubMed Central" }	[ "cord", "amount", "cervic", "treatment", "idiopath", "cidplik", "cours", "decreas", "immunoglobulin", "respond", "impair", "gravi", "collect", "bowel", "biopsi", "emg", "identifi", "discharg", "block", "pathogenesi", "fund", "iu", "mechan", "myasthenia", "week", "indirect", "mri", "commenc", "multipl", "may", "myelopathi", "acquir", "separ", "amyotroph", "amplitud", "denerv", "use", "etiolog", "moder", "areflexia", "support", "caus", "complet", "imag", "accord", "signific", "hagen", "cowork", "temperatur", "asymmetr", "mark", "suggest", "process", "disappear", "cidp", "also", "state", "nerv", "conflict", "ribonucleoprotein", "admiss", "relaps", "remiss", "dispers", "criteria", "normal", "featur", "steroid", 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Introduction {#s1} ============ Wild-type streptavidin is a tetrameric protein with four identical subunits. Each subunit has a biotin binding pocket and can bind biotin tightly with a dissociation constant (K~d~) around 10^−14^ M [@pone.0035203-Green1]. This binding is considered to be irreversible and has been applied in a wide range of biomedical and biotechnological applications [@pone.0035203-Wilchek1], [@pone.0035203-Laitinen1]. However, the tight biotin binding also makes streptavidin not suitable for affinity purification of biotinylated molecules. It would be ideal to develop engineered streptavidin muteins with reversible biotin binding capability. These engineered muteins can be applied to purify biotinylated molecules, develop automated high-throughput protein purification systems, reusable biosensor chips and bioreactors, study protein-protein interactions and design strippable probing agents (e.g. engineered muteins conjugated to horseradish peroxidase) for blot reprobing. To understand the strategies applied in engineering streptavidin with reversible biotin binding ability, it is vital to understand the structural features of streptavidin, its biotin binding pocket and the subunit interfaces. Each streptavidin subunit contains eight antiparallel strands that form a β-barrel structure [@pone.0035203-Weber1], [@pone.0035203-Hendrickson1]. Two of these subunits (A and B as well as C and D in [Fig. 1b](#pone-0035203-g001){ref-type="fig"}) have extensive interfacial interactions to form a relatively stable dimer. Two dimers then assemble into a tetramer via a weaker interface. Although each subunit can bind a biotin molecule, each of the four complete biotin pockets in the tetramer relies on the donation of Trp-120 [@pone.0035203-Sano1], [@pone.0035203-Freitag1], [@pone.0035203-Chilkoti1] located in loop~7--8~ from the neighboring subunit (e.g. subunit A needs Trp-120 from subunit D and vice versa, [Figure 1b](#pone-0035203-g001){ref-type="fig"}). Exceptionally tight biotin binding in streptavidin is mainly contributed by three sets of interactions [@pone.0035203-Stayton1]. The first set involves at least 6 residues (N23, S27, Y43, S88, T90 and D128) in the biotin binding pocket to form an extensive hydrogen bonding network with biotin. The second set involves strong hydrophobic interactions [@pone.0035203-Freitag1] between biotin and four tryptophan residues (79, 92, 108 and 120). Finally, residues in loop~3--4~ (S45, V47, G48, N49 and A50) play a critical role in binding and trapping biotin to the biotin binding pocket [@pone.0035203-Freitag2], [@pone.0035203-LeTrong1], [@pone.0035203-Chu1]. In the absence of biotin, loop~3--4~ has been shown to be flexible and is mainly in an open configuration. However, after biotin binding, loop~3--4~ becomes immobilized and is in a closed position ([Figure 1](#pone-0035203-g001){ref-type="fig"}, panels a and b) because of its interactions with biotin. Furthermore, biotin binding can also strengthen subunit interactions [@pone.0035203-Katz1], in particular, via interactions between biotin in one subunit and Trp-120 in loop~7--8~ from the neighboring subunit [@pone.0035203-Sano1]. In fact, the majority of the streptavidin-biotin complexes are in the tetrameric state in SDS-polyacrylamide gel even if the samples have been boiled before loading [@pone.0035203-Bayer1], [@pone.0035203-Waner1]. ![Biotin entry and exit paths in wild-type streptavidin (panels a and b) and models of the engineered mobile loop~7--8~ in streptavidin (panels c and d).\ Subunits A, B, C and D in wild-type streptavidin are colored in blue, red, yellow and green, respectively. Loop~3--4~ in subunit D is in the open conformation as shown in panel a to allow biotin binding. Trp-120 (blue) from subunit A (blue) is required to form a complete biotin binding pocket in subunit D (green). With biotin binding, loop~3--4~ is in the closed position as shown in panel b. To develop an engineered streptavidin (8-aa-loop-H127C) with reversible biotin binding, loop~7--8~ in wild-type streptavidin was replaced by an engineered mobile loop to function as a dynamic gateway for the exit of the bound biotin. The engineered mobile loop~7--8~ in subunit A is modeled in 10 different positions to illustrate its dynamic nature (panel c). The blue loop (loop~7--8~) in the upper positions will form a wall as part of the biotin binding pocket for the subunit D (green). At these stages, this gate is in the closed state. When loop~7--8~ is in the lower position (panels c and d), the gate is in the open state. With loop~3--4~ in subunit D in the closed conformation, biotin still can possibly escape from the biotin binding pocket at this stage (panel d).](pone.0035203.g001){#pone-0035203-g001} Two approaches have been taken to develop streptavidin muteins with reversible biotin binding ability. The first approach is to replace amino acid residues that are critical in hydrogen bonding to biotin. Although these changes can indeed lower the biotin binding affinities [@pone.0035203-Qureshi1], [@pone.0035203-Kopetzki1], many of the mutations also affect inter-subunit interactions in the streptavidin tetramer. Weakening inter-subunit interactions typically generates a heterogeneous population of streptavidin oligomers which leads to many practical problems. A second approach is to develop recombinant monomeric streptavidin [@pone.0035203-Wu1] which has lower biotin binding affinity. This approach exploits the fact that an individual streptavidin subunit lacks a complete biotin binding pocket since Trp-120 from a neighboring subunit forms a key part of the binding site ([Figure 1](#pone-0035203-g001){ref-type="fig"}, panels a and b). Replacing Trp-120 with alanine (W120A) results in a tetrameric streptavidin mutein with a K~d~ of 3×10^−9^ M for biotin [@pone.0035203-Kopetzki1]. To create monomeric streptavidin, charge repulsion and steric hindrance were introduced at the subunit interface [@pone.0035203-Wu1]. The resulting monomeric streptavidin has a biotin binding constant around 10^−7^ M. However, this mutein can be in the monomeric state only under certain conditions. First, the salt concentration has to be low to maximize electrostatic repulsion between streptavidin subunits. Second, the concentration of the monomers has to be low as exposure of the hydrophobic interface promotes non-specific aggregation. In this study, a new approach was designed to develop an engineered tetrameric streptavidin with reversible biotin binding capability and other desirable features. Results {#s2} ======= Rationale for the design of novel streptavidin muteins {#s2a} ------------------------------------------------------ Our novel approach relies on the effects of two loops (loop~3--4~ with residues 45--52 and loop~7--8~ with residues 114--121) on biotin binding ([Figure 1](#pone-0035203-g001){ref-type="fig"}). Loop~3--4~ forms the lid and Trp-120 from loop~7--8~ forms part of the wall of the biotin binding pocket. With various hydrogen bonding and hydrophobic interactions in the biotin binding pocket and the closure of the lid, biotin can hardly escape from the binding pocket. In this study, an attempt was made to create a dynamic "back door" formed by loop~7--8~ in streptavidin to allow biotin a second route to escape from the binding pocket when the "main door" primarily formed by loop~3--4~ is closed. This objective can potentially be achieved by several approaches. One is to develop a ΔW120 mutein. Since Trp-120 in loop~7--8~ is known to interact strongly with biotin [@pone.0035203-Sano1], [@pone.0035203-Freitag1], [@pone.0035203-Chilkoti1], the deletion of Trp-120 may allow the modified loop~7--8~ to become more flexible. A second approach is to create a mobile 8-amino-acid-loop (8-aa-loop) mutein in which loop~7--8~ is engineered to have a completely different sequence but retains the same length as the original loop in wild-type streptavidin ([Table 1](#pone-0035203-t001){ref-type="table"}). Asparagine and glycine were introduced at the center of the loop since they are known to introduce a turn in the loop structure [@pone.0035203-Wilmot1]. The DSS (aspartate, serine and serine) and SDG (serine, aspartate and glycine) sequences were introduced to form the left and right arms of the loop, respectively ([Table 1](#pone-0035203-t001){ref-type="table"}). These amino acids were selected because they have a high propensity for intrinsic disorder [@pone.0035203-Linding1]. This design minimizes interactions between loop~7--8~ and biotin, and was intended to allow the engineered loop to act as an unlocked mobile door swinging between the open and closed states even in the presence of biotin ([Figure 1](#pone-0035203-g001){ref-type="fig"}, panels c and d). To improve the chance of obtaining a mutein with its engineered "back door" open wide enough for the exit of the bound biotin, a third approach was applied to create a series of muteins (2-aa-loop, 4-aa-loop and 6-aa-loop muteins, [Table 1](#pone-0035203-t001){ref-type="table"}) with both the sequence of loop~7--8~ redesigned and the length of the loop shortened. Muteins with shorter loops were hypothesized to provide bigger openings around the biotin binding pocket which should facilitate the release of biotin. In a preliminary study, the 4-aa-loop mutein was immobilized on agarose matrix. Although this matrix could bind biotinylated proteins, streptavidin mutein was seen leaking from the column during washing and elution. Since the coupling condition allows on average one out of four subunits in the tetrameric streptavidin mutein to be coupled to the matrix to maximize the accessibility of the biotin binding sites, the leakage of streptavidin subunits suggests that changes in the loop~7--8~ structure might result in weakening of the inter-subunit interactions in the streptavidin mutein. To avoid this complication, the H127C mutation [@pone.0035203-Reznik1], [@pone.0035203-Chilkoti2] was introduced to two constructs (8-aa-loop mutein and ΔW120 mutein) to create 8-aa-loop-H127C mutein and ΔW120-H127C mutein, respectively. The H127C mutation has been reported [@pone.0035203-Reznik1], [@pone.0035203-Chilkoti2] to allow crosslink between subunits A and C (and also between subunits B and D) through the formation of a disulfide bond. These disulfide bonds can strengthen inter-subunit interactions. Since the matrix for the 4-aa-loop mutein had a subunit leakage problem, this mutein was not further characterized. The remaining four muteins (ΔW120-H127C, 2-aa-loop, 6-aa-loop and 8-aa-loop-H127C) were used for further analyses. 10.1371/journal.pone.0035203.t001 ###### Features of the loop~7--8~ muteins. ![](pone.0035203.t001){#pone-0035203-t001-1} Streptavidin Loop~7--8~ H127C ----------------- ----------------------------------------------- ------- Wild-type L T S G~113~ T T E A N A W K S~122~ T L V − ΔW120-H127C T T E A N A K \+ 8-aa-loop-H127C D S S N G S D G \+ 8-aa-loop D S S N G S D G − 6-aa-loop D S N G S G − 4-aa-loop D N G G − 2-aa-loop N G − The amino acid sequences in the natural and engineered loop~7--8~ loops are shown in bold. The sequences flanking the loop~7--8~ are shown in black. + or − indicates the presence or absence of H127C in the muteins. Production and purification of streptavidin muteins {#s2b} --------------------------------------------------- To avoid the formation of inclusion bodies and the need to refold the engineered streptavidin, all streptavidin muteins were produced in their soluble state from B. subtilis via secretion [@pone.0035203-Wu2]. The production yield of the muteins in a semi-defined medium [@pone.0035203-Wu3] was 40--60 mg/liter. Each mutein could be affinity purified in one step using the biotin-agarose matrix. The purification of the 8-aa-loop-H127C mutein showed typical results ([Figure 2a](#pone-0035203-g002){ref-type="fig"}). 8-aa-loop-H127C mutein was efficiently captured by the biotin-agarose matrix and could be eluted by buffer containing 4 mM biotin. The recovery was ∼90%. After dialysis to remove biotin from the pooled elution fractions, the purified mutein could rebind the biotin-agarose matrix and could be eluted again using biotin-containing buffer (data not shown). This result demonstrates the reversible biotin binding ability of the mutein. ![SDS-PAGE showing purification of 8-aa-loop-H127C mutein and its oligomeric state.\ In panel a, 8-aa-loop-H127C mutein was affinity purified from B. subtilis culture supernatant using biotin-agarose matrix. S represents the concentrated culture supernatant before purification. FT, W and E are the flow-through, wash and elution fractions, respectively. 5 µl from each fraction (800 µl per fraction, each fraction contained the same volume of sample) was reduced with β--mercaptoethanol and boiled before loading onto the gel. The arrowhead marks the position of the 8-aa-loop-H127C mutein. Panel b shows the tetrameric state of 8-aa-loop-H127C (MH127C). 8-aa-loop (M) and wild-type (wt) streptavidin were included as reference. B and U represent the boiled and unboiled samples respectively. ME represents β--mercaptoethanol (used at 0.7 M final concentration). M shows the position of the molecular weight markers (numbers on the left of both panels are expressed in kDa).](pone.0035203.g002){#pone-0035203-g002} Determination of kinetic parameters {#s2c} ----------------------------------- The kinetic parameters for biotin binding to various muteins were determined by surface plasmon resonance using the BIAcore biosensor with biotinylated IgG proteins as the ligand ([Table 2](#pone-0035203-t002){ref-type="table"}). ΔW120-H127C had a binding affinity for biotin (K~d~ = 8.1×10^−9^ M) that was comparable to that of the W120A mutein (K~d~∼3×10^−9^ M) [@pone.0035203-Kopetzki1]. Although muteins with smaller loops (2- and 6-aa-loop muteins) were expected to have lower biotin binding affinities, their binding affinities (∼1--3×10^−9^ M) were actually comparable to that of the W120A mutein. Streptavidin muteins with nano-molar binding affinity tend to bind biotinylated molecules too tightly for affinity chromatography purification, leading to the poor recovery of target proteins. The mutein with the lowest biotin binding affinity (1.9×10^−8^ M) in this study is the 8-aa-loop-H127C mutein. It was further characterized and its ability to act as an affinity agent for purifying biotinylated proteins was explored. 10.1371/journal.pone.0035203.t002 ###### Kinetic parameters of biotin binding in streptavidin muteins. ![](pone.0035203.t002){#pone-0035203-t002-2} Streptavidin mutein ΔW120-H127C 2-aa-loop 6-aa-loop 8-aa-loop-H127C --------------------------------------------- ------------- ------------- ------------- ----------------- k~a~ or k~on~ (M^−1^s^−1^) 4.96×10^4^ 6.3×10^4^ 2.43×10^4^ 2.21×10^4^ k~d~ or k~off~ (s^−1^) 4.0×10^−4^ 7.02×10^−5^ 7.59×10^−5^ 4.28×10^−4^ K~d~ (M) 8.1×10^−9^ 1.12×10^−9^ 3.13×10^−9^ 1.9×10^−8^ t~½~ (min)[\](#nt103){ref-type="table-fn"} 28.9 165 152 27 Biotinylated IgG proteins were immobilized to the sensor chip, and purified muteins were injected to determine binding affinities. t~½~ is the half-life of biotin in the SAV-biotin complex. It is estimated based on the following equation: t~½~ (min) = 0.693/k~off~/60. Tetrameric state of 8-aa-loop-H127C mutein {#s2d} ------------------------------------------ Since the matrix with 4-aa-loop mutein immobilized showed leakage of streptavidin subunits during chromatography, this observation prompted the examination of the oligomeric state of the 8-aa-loop-H127C mutein. The migration pattern of the 8-aa-loop-H127C mutein resolved by SDS-PAGE was examined in the presence or absence of mercaptoethanol under boiled and non-boiled conditions ([Figure 2b](#pone-0035203-g002){ref-type="fig"}). 8-aa-loop mutein and wild-type streptavidin were included in the study as a comparison. Since the streptavidin inter-subunit interactions are weaker in the absence of biotin [@pone.0035203-Sano1], these analyses were performed in the absence of biotin. Under all conditions, 8-aa-loop mutein migrated like a monomer. By contrast, over 98% of wild-type streptavidin remained as tetramers if the sample was not boiled. This suggests that changes in the amino acid sequence of loop~7--8~ weaken inter-subunit interactions. A significant difference in the oligomeric states of 8-aa-loop and 8-aa-loop-H127C muteins was observed in the absence of reducing agent. Whereas 8-aa-loop mutein remained as monomers whether the sample was boiled or not, ∼80% of 8-aa-loop-H127C mutein remained in the dimeric form even after boiling. This indicates the successful formation of disulfide bonds between subunits. With the unboiled sample, most of 8-aa-loop-H127C mutein adopted the tetrameric state. Since this mutein exists mainly as tetramer even in the presence of detergent in SDS-PAGE, it should be predominantly in the tetramer state under non-denaturing conditions. Thus, the H127C mutation did strengthen inter-subunit interactions in 8-aa-loop-H127C mutein. Purification of biotinylated protein G and biotinylated IgG using 8-aa-loop-H127C mutein-agarose matrix {#s2e} ------------------------------------------------------------------------------------------------------- To explore the feasibility of using 8-aa-loop-H127C mutein for the affinity purification of biotinylated molecules, chemically biotinylated protein G and IgG were captured on the 8-aa-loop-H127C mutein-agarose matrix ([Figure 3](#pone-0035203-g003){ref-type="fig"}, panels a and b). After removal of nonspecifically bound proteins, the bound biotinylated proteins could be eluted from the column using 4 mM biotin. Absence of a 20-kDa streptavidin band in the flow-through, wash and elution fractions suggested that streptavidin subunit leakage was not a problem. Estimation of the amount of IgG molecules captured on the column and quantification of biotinylated IgG in the elution fractions indicated the recovery to be approximately 95%. To demonstrate binding specificity, HeLa cell extracts were applied to the column. Non-specific binding was not observed ([Figure 3c](#pone-0035203-g003){ref-type="fig"}). Non-biotinylated protein G and IgG also could not bind to the matrix (data not shown). When biotinylated IgG was mixed with the HeLa cell extract, biotinylated IgG could be affinity purified to homogeneity in one step ([Figure 3d](#pone-0035203-g003){ref-type="fig"}). ![Purification of biotinylated protein G and IgG using 8-aa-loop-H127C mutein-agarose matrix.\ Excess amounts of biotinylated proteins were loaded to the column. Panel a: Biotinylated protein G with an apparent molecular mass of 37 kDa is marked by arrowhead. Some biotinylated protein G molecules are in oligomeric state with higher molecular masses. Panel b: The 54- and 25-kDa protein bands are the biotinylated heavy (marked by closed arrowhead) and light chains (marked by gray arrowhead) of IgG, respectively. Panel c: HeLa cell extract was applied to the 8-aa-loop-H127C mutein-agarose matrix. Panel d: Biotinylated IgG mixed with HeLa cell extract. S: Sample before purification, FT, W, and E: Flow-through, wash and elution fractions. M: Molecular weight markers.](pone.0035203.g003){#pone-0035203-g003} To regenerate the column for repeated rounds of purification, the matrix was simply washed with 10 column volumes of binding buffer. Using biotinylated BSA as the target protein for purification, the 8-aa-loop-H127C mutein matrix saturated with excess amounts of biotinylated BSA could be regenerated in this manner to purify biotinylated BSA for 10 rounds with no observable loss in binding capacity (data not shown). Structural characterization of the 8-aa-loop-H127C mutein {#s2f} --------------------------------------------------------- To assess the conformation of the 8-aa-loop-H127C mutein, the protein was crystallized in the presence of biotin and its structure was solved using X-ray crystallography. Using data extending to 2.0 Å resolution, the structure clearly shows that the mutein crystallizes as a tetramer with a single subunit in the asymmetric unit ([Figure S1](#pone.0035203.s001){ref-type="supplementary-material"}). Biotin is bound in a manner indistinguishable from that of wild-type streptavidin, but there is very little electron density for loop~7--8~ (residues 114--121) and its neighboring residue 113 ([Figure 4](#pone-0035203-g004){ref-type="fig"}). The lack of electron density suggests that the modified sequence of loop~7--8~ confers a significant degree of dynamic disorder to the loop. The lack of interaction between loop~7--8~ and the biotin molecule bound to the adjacent subunit probably allows a higher level of mobility and dynamic motion in this loop in the mutein. ![Electron density map (2\\|F~o~\\|-\\|F~c~\\| coefficients, contoured at 1.1 sigma) contoured around the region expected to be occupied by residues 114--121 of loop~7--8~.\ The model for residues 110--112 and 122--124 fit the electron density quite well, but there is insufficient electron density to model residues 113--121. The structure of wild-type streptavidin (PDB code 1SWE) is superimposed and drawn in a thinner, gray line representation. Coefficients and phases were calculated using Refmac and the figure was prepared using PyMOL.](pone.0035203.g004){#pone-0035203-g004} Since inter-dimer interactions are critical for stabilizing the tetrameric structure and will likely affect the dynamics of the streptavidin tetramer, changes introduced to the 8-aa-loop-H127C mutein can possibly affect the inter-dimer arrangement. In fact, a significant alteration in the arrangement of subunits in the 8-aa-loop-H127C mutein was observed when compared with wild-type streptavidin. The orientation of the A/B dimer relative to the C/D dimer appears to be very similar in nearly all of the previously reported structures of biotin-bound streptavidin [@pone.0035203-Weber1], [@pone.0035203-Hendrickson1], partly because loop~7--8~ in one dimer interacts with a bound biotin molecule and nearby residues in the other dimer. Even when biotin is not present, Trp-120 and Leu-124 form mostly non-specific van der Waals contacts with Val-47 and Lys-121 from the opposing dimer, respectively. Thus, previously reported structures do not appear to have much difference in inter-dimer orientation or interactions. In this case, the loss of order in loop~7--8~ of 8-aa-loop-H127C mutein allows for a much larger rearrangement of the A/B dimer relative to the C/D dimer. When the A/B dimer of the mutein is superimposed onto the A/B dimer of wild-type streptavidin, the C/D dimer is rotated by 11° relative to the position of the C/D dimer in wild-type streptavidin ([Figure 5](#pone-0035203-g005){ref-type="fig"}). In contrast, the relative positions of the two dimers in other structures of biotin-bound streptavidin crystallized in different crystal forms differ by ∼1--3°. Significantly, an inter-dimer rotation of 5.4° was reported in the first paper comparing the structure of apo-streptavidin with biotin-bound streptavidin [@pone.0035203-Weber1]. The even larger inter-dimer rotation seen in the 8-aa-loop-H127C mutein supports the correlation of a disrupted inter-dimer interface with less order in loop~7--8~ and lower biotin-binding affinity. ![Comparison of quaternary structures of the 8aa-loop-H127C mutein and wild-type streptavidin (1SWE).\ Subunits A, B, C and D are colored blue, cyan, red and orange for both the mutein and wild-type structures. Biotin is drawn in space-filling representation and colored black. In all panels, only subunits A and B have been aligned. As a result, the cartoon diagrams and superimposed ribbon diagram at the far right show how the C/D dimer of the mutein is rotated by ∼10° relative to the C/D dimer of wild-type streptavidin when the A/B dimers of the mutein and wild-type streptavidin are superimposed. Loop~7--8~ (highlighted by the solid, black ovals in the front view) lies at the interface between dimer A/B and dimer C/D of wild-type streptavidin and is fixed in orientation in part by interactions between W120 and the biotin molecule bound to the opposing dimer. Loop~7--8~ is dynamically disordered in the mutein and hence is not shown in the cartoon model; the expected location of loop~7--8~ is denoted by the dashed, black ovals in the front view, and the location of the ends of the ordered parts of both loops (residues 112 and 122) are denoted by small circles. The side chains of Cys-127 from each subunit are drawn in space-filling representation, showing the formation of disulfide bonds between subunits A/C and B/D.](pone.0035203.g005){#pone-0035203-g005} An interesting feature also clearly seen in the crystal structure of the 8-aa-loop-H127C mutein is that a disulfide bond is formed between the Cys-127 residues of adjacent subunits at the A/B and C/D dimer interfaces ([Figure 6](#pone-0035203-g006){ref-type="fig"}). The Cys residue was introduced as a means of stabilizing the A/B and C/D dimers, based on the observed proximity of His-127 residues in adjacent subunits of the structure of wild-type streptavidin [@pone.0035203-Weber1], [@pone.0035203-Hendrickson1], [@pone.0035203-Reznik1], [@pone.0035203-Chilkoti2]. The formation of this disulfide bond in the crystallized mutein is consistent with the presence of disulfide-bonded protein in non-reducing SDS-PAGE analysis as described above ([Figure 2b](#pone-0035203-g002){ref-type="fig"}). ![Electron density map (2\\|F~o~\\|-\\|F~c~\\| coefficients, contoured at 1.1 sigma) contoured around the model of the disulfide bond formed by Cys-127 residues from adjacent subunits (A and C or B and D in the tetramer shown in [Figure 5](#pone-0035203-g005){ref-type="fig"}).\ Coefficients and phases were calculated using Refmac and the figure was prepared using PyMOL.](pone.0035203.g006){#pone-0035203-g006} Discussion {#s3} ========== An idealized streptavidin mutein for affinity chromatography applications should have the following desirable properties. First, it should be tetrameric and all four biotin binding sites are capable of binding biotin. In this state, the hydrophobic interface regions will not be exposed to the surface, thus minimizing non-specific hydrophobic interactions between the engineered streptavidin mutein and proteins in the crude sample to be analyzed. Second, intersubunit interactions should be strong. With one subunit of the tetramer immobilized, the other three subunits should stably associate with this covalently immobilized subunit so that no streptavidin subunits will be stripped off the column during the wash and elution steps. Third, the dissociation constant (K~d~) for biotin should be 10^−7^ to 10^−8^ M. Fourth, the off-rate (k~off~) for the bound biotin in the streptavidin-biotin complex is ideally around 10^−4^ sec^−1^ to allow the estimated half-life of the bound biotin to be around 10--30 minutes. With conditions 3 and 4 combined, the interaction would be both strong and specific enough to allow non-specifically bound molecules to be washed off the matrix without leakage of the specifically bound biotinylated molecules. At the same time, there should be efficient and quantitative elution of biotinylated molecules from the column. A fine balance between affinity towards biotinylated molecules and good recovery is essential for the ideal streptavidin affinity agent. Fifth, the engineered streptavidin muteins immobilized to the matrix should be stable enough to allow the matrix to be used for multiple rounds. Sixth, the engineered streptavidin should be produced with a reasonable production yield in a soluble and functional state without the requirement of refolding via inefficient and expensive denaturation and renaturation processes. The engineered 8-aa-loop-H127C mutein produced from B. subtilis* via secretion meets all of the above requirements. Replacement of residues in loop~7--8~ of wild-type streptavidin allows loop~7--8~ to become flexible and changes the relative orientation of subunits in the tetrameric structure. These structural changes can lead to the lowering of the biotin binding affinity in 8-aa-loop-H127C mutein. Introduction of the H127C mutation is for the objective to stabilize the mutein in the tetrameric state. To confirm that loop replacement is the major factor contributing to the observed lower biotin binding affinity in the 8-aa-loop-H127C mutein, the kinetic parameters of the 8-aa-loop mutein (without the H127C mutation) for binding biotinylated proteins were also determined (data not shown). Its dissociation constant (3.96×10^−8^ M) was found to be comparable to that (1.9×10^−8^ M) of the 8-aa-loop-H127C mutein. In contrary, the streptavidin mutein carrying solely the H127C mutation did not show a significant decrease in biotin binding affinity since it could not be eluted off from the biotin-agarose matrix using a biotin containing buffer. The strength of biotin interactions with wild-type streptavidin and 8-aa-loop-H127C mutein was analyzed using the ligand energy inspector function in the Molegro molecular viewer program [@pone.0035203-Thomsen1] based on X-ray crystallographic data. The binding free energy is estimated by the MolDock scores as shown in [Table 3](#pone-0035203-t003){ref-type="table"}. The more negative values indicate stronger interactions. This analysis suggests that the lower biotin binding affinity in 8-aa-loop-H127C mutein is mainly contributed by both the absence of interactions (i.e. W120 and K121) and weaker interactions (D128, S45, N23 and L25) between biotin and residues in the biotin binding pocket. A previous study also suggests that D128, S45 and N23 play important roles in the biotin exit pathway [@pone.0035203-Freitag3]. Weakening the interactions between biotin and these residues is instrumental in enhancing biotin exit thereby increasing the biotin off rate (k~off~). 10.1371/journal.pone.0035203.t003 ###### Interaction of biotin with residues in the biotin binding pocket of subunit A. ![](pone.0035203.t003){#pone-0035203-t003-3} Protein 8-aa-loop-H127C Wild type SAV (2IZF) Difference (wild type -- mutein) ------------------------------ ----------------- ---------------------- ---------------------------------- MolDock score −125.6 −140.1 −14.4 Trp 120 (from subunit D) 0 −10.5 −10.5 Lys 121 (from subunit D) 0 −1.3 −1.3 Asp128 −4.4 −7.7 −3.3 Ser 45 −6.6 −8.0 −1.4 Asn 23 −4.1 −5.2 −1.1 Leu 25 −3.5 −4.3 −0.8 In addition to the interaction between biotin and specific residues in the biotin binding pocket of streptavidin, the strength of biotin binding appears to depend in part on the flexibility of loop~3--4~ and loop~7--8~. In both wild-type streptavidin [@pone.0035203-Freitag2] and the W120A mutein [@pone.0035203-Freitag1], both loops appear to be rigid in the presence of biotin. In contrast, the flexibility and mobility of loop~7--8~ in 8-aa-loop-H127C mutein creates a new exit path for the release of biotin from the biotin binding pocket even if loop~3--4~ is closed. This effect contributes to increases in the dissociation rate of the bound biotin. During the course of this study, a new streptavidin mutein called traptavidin was reported [@pone.0035203-Chivers1]. Biotin dissociates from traptavidin at least 10 times slower than from wild-type streptavidin and binds with 10 times higher affinity. The crystal structure of traptavidin [@pone.0035203-Chivers2] indicates that loop~3--4~ is well-ordered and adopts a closed conformation in both the presence and absence of biotin. By contrast, loop~3--4~ is well-ordered only when biotin is bound to wild-type streptavidin. Because the conformation of the biotin-bound complex in traptavidin is virtually identical to that seen in wild-type streptavidin, the increase in biotin binding affinity is clearly not due to additional contacts between biotin and traptavidin, as supported by the analysis of binding interactions with Molegro, which assigns similar MolDock scores for biotin binding to traptavidin and wild-type streptavidin (−139.3 and −140.1 respectively). The increased rigidity of loop~3--4~ thus accounts for the higher biotin binding affinity in traptavidin. Conversely, the increased mobility of the engineered loop~7--8~ in 8-aa-loop-H127 mutein may in part account for the observed lower biotin binding affinity. Most of the entire series of streptavidin muteins (ΔW120-H127C, 2- and 6-aa-loop muteins) have biotin binding affinities similar to that of the W120A mutein in the range of 10^−9^ M. Why the 2- and 6-aa-loop muteins do not have lower biotin binding affinities (K~d~\>10^−8^ M) is unclear at present, but the dynamics of loop~7--8~ are likely important. Structural and molecular dynamics studies of these muteins may help to explain relative binding affinities. Two matrices are commercially available to purify biotinylated molecules. One is the monomeric avidin matrix [@pone.0035203-Kohanski1]. Unfortunately, this approach has three drawbacks. First, the production cost is high as three avidin subunits are sacrificed for each avidin subunit immobilized to the matrix. Second, the large hydrophobic surface exposed in the generation of monomeric avidin typically introduces problematic non-specific interactions with unwanted proteins in the crude extract. Third, it is difficult to completely denature the tetrameric avidin because of the strong subunit interactions. Incomplete denaturation of avidin results in the presence of some tetrameric avidin in the matrix which can bind biotinylated molecules irreversibly. The second commercially available matrix contains the engineered streptavidin mutein [@pone.0035203-Kopetzki1]. The main concern with this matrix is the occasional observation of leakage of streptavidin subunits during protein loading, washing and elution. With the above-mentioned limitations, the engineered 8-aa-loop-H127C mutein has substantial advantages over the currently available matrices for affinity purification of biotinylated molecules. Many potential biotechnological applications of this mutein can be developed. In the post-genomic era with the discovery of many novel proteins which can be promising therapeutic targets, biomarkers for diseases, and agents with medical and biotechnological applications, the availability of a user-friendly high-throughput system to purify and immobilize these proteins for structural and functional studies is vital. However, protein purification requires reversibility in binding while immobilization requires ultra-tight interactions. Development of the 8-aa-loop-H127C mutein provides a solution to solve this dilemma. Biotinylated biomolecules can be affinity purified using the 8-aa-loop-H127C mutein matrix and immobilized to the wild-type streptavidin based protein chips. Furthermore, automated high-throughput purification platforms, reusable biosensor chips, protein arrays, bioreactors and magnetic beads can be developed using the 8-aa-loop-H127C mutein. With the reversible biotin binding capability, chemical conjugation or genetic fusion of 8-aa-loop-H127C muteins with other proteins such as alkaline phosphatase can allow blots to be reprobed without worrying masking other probing sites because of steric hindrance imposed by the bulky streptavidin conjugates. Although the 8-aa-loop-H127C mutein in the present format has many desirable features for biotechnological application, two areas need further improvement. First, it would be ideal if its production yield can be increased further (\>60 mg/liter). Since this mutein binds biotin reversibly, production of this mutein does not exert any toxic effect on its expression host by depleting biotin in both the cytoplasm and the culture medium. This is significantly different from the production of wild-type streptavidin which requires the production host to have the ability to produce sufficient quantities of biotin to sustain the cell growth [@pone.0035203-Wu3]. Many other expression systems from prokaryotes to eukaryotes can now be explored. Second, a hybrid tag containing six histidine residues and a single cysteine residue [@pone.0035203-Reznik2] can be fused to either end of the 8-aa-loop-H127C mutein. When this mutein is produced at industrial scale, a single-step purification using biotin-agarose column is usually not sufficient to purify the mutein to homogeneity. The added his-tag can offer another round of affinity purification of this mutein via a different mechanism. The added cysteine residue in the tag can be applied for orientation specific coupling of the mutein to the thiol-based coupling column matrices, biosensor chips or protein arrays. This approach not only improves the accessibility of the biotin binding sites of the immobilized mutein in the affinity matrix [@pone.0035203-Huang1] but also makes chemical coupling of this mutein to various matrices in a user friendly manner. Materials and Methods {#s4} ===================== Construction of expression plasmids for production of streptavidin muteins in B. subtilis {#s4a} ------------------------------------------------------------------------------------------- Plasmid pSSAV [@pone.0035203-Wu2] carrying a B. subtilis levansucrase signal peptide for secretion and a P43 promoter for transcription was used as the expression vector. This vector carries a synthetic gene for wild-type full-length streptavidin. To construct the expression vectors for the loop muteins, the gene encoding the wild-type streptavidin counterpart in pSSAV was replaced by the PstI/BclI synthetic fragments encoding the loop muteins. E. coli pbluescript plasmids containing the synthetic gene fragments were ordered from Epoch Life Science, Inc. Texas, U.S.A. Each E. coli plasmid was digested with PstI/BclI to release the synthetic gene fragment which would then be ligated to the PstI/BclI cut pSSAV to generate the expression vectors. Production and affinity purification of streptavidin muteins {#s4b} ------------------------------------------------------------ B. subtilis WB800 [@pone.0035203-Wu4] cells carrying the expression vectors were cultivated in a semi-defined medium [@pone.0035203-Wu3] at 30°C for 9--12 hours. Culture supernatant was collected by centrifugation and streptavidin muteins were affinity purified using biotin-agarose as described previously [@pone.0035203-Wu1]. X-ray crystallographic studies {#s4c} ------------------------------ Crystals of 8-aa-loop-H127C mutein were obtained using the hanging-drop vapor diffusion technique by combining 2 µL protein (4 mg/mL, 10 mM Tris-Cl pH 7.5, 150 mM NaCl) with 2 µL reservoir solution (50% Tacsimate, pH 6.0 (Hampton Research), 10% (w/v) glycerol). Crystals grew to an approximate size of 0.2×0.2×0.1 mm after ∼2 weeks. They were suspended in a microfabricated loop (Mitegen) and flash-cooled under a nitrogen gas stream (∼100 K, Oxford Cryosystems). Data were measured using X-rays from a rotating anode generator (Rigaku RU-H3R), Multilayer optics (Osmic Blue) and Mar345 imaging plate detector (MarResearch). Diffraction data (200 images×0.5°; 10 minutes/image) were indexed and scaled using DENZO and Scalepack [@pone.0035203-Otwinowski1]. The space group was assigned by autoindexing, inspecting systematic absences and scaling. It was confirmed to be P4~2~2~1~2 by molecular replacement calculations and refinement. PHASER [@pone.0035203-Read1] was used for molecular replacement calculations, yielding a single very strong solution using a single subunit of wild-type streptavidin (1SWE) as the search model. Refinement calculations were performed using REFMAC (v. 5.5.0109) [@pone.0035203-Murshudov1], and model building was performed using COOT (v. 0.6.1) [@pone.0035203-Emsley1]. Geometric parameters were monitored using Procheck [@pone.0035203-Laskowski1] and Molprobity [@pone.0035203-Chen1]. Crystallographic statistics are reported in [Table 4](#pone-0035203-t004){ref-type="table"}. 10.1371/journal.pone.0035203.t004 ###### Crystallographic statistics. ![](pone.0035203.t004){#pone-0035203-t004-4} ----------------------------------------------------------------------------------- ----------------------- Data collection Space Group Mol/A.U. 1 Unit cell a, b, c (Å) 57.1, 57.1, 70.2 Resolution (Å)[a](#nt104){ref-type="table-fn"} 20.0−1.95 (2.02−1.95) R~sym~ [b](#nt105){ref-type="table-fn"} 0.063 (0.496) I/σI 28.4 (2.75) Completeness (%) 96.1 (88.0) Redundancy 6.7 (3.6) Refinement Resolution (Å) 19.39−2.00 Unique reflections 7585 R~work~ [c](#nt106){ref-type="table-fn"}/R~free~ [d](#nt107){ref-type="table-fn"} 0.189/0.232 No. of atoms 1003 Protein atoms 915 Biotin 16 Water 72 r.m.s.d. from ideal[e](#nt108){ref-type="table-fn"} geometry Bond lengths (Å) 0.009 Bond angles (°) 1.26 Ramachandran angles[f](#nt109){ref-type="table-fn"} %-favored 97.5% %-outliers 0% ----------------------------------------------------------------------------------- ----------------------- Values from the outermost resolution shell are given in parentheses. R~sym~ = Σ~i~\\|I~i~−\<I\>\\|/Σ~i~ I~i~ where I~i~ is the ith integrated intensity of a given reflection and \<I\> is the weighted mean of all measurements of I. R~work~ = Σ\\|\\|F~o~\\|−\\|F~c~\\|\\|/Σ\\|F~o~\\| for 95% of reflection data used in refinement. R~free~ = Σ\\|\\|F~o~\\|−\\|F~c~\\|\\|/Σ\\|F~o~\\| for 5% of reflection data excluded from refinement. Root-mean-square deviations from ideal geometry calculated by Procheck [@pone.0035203-Laskowski1]. Ramachandran plot analysis carried out using Molprobity [@pone.0035203-Chen1]. Analysis of interactions between biotin and streptavidin using Molegro viewer {#s4d} ----------------------------------------------------------------------------- The pdb files of the biotin complexes associated with 8-aa-loop-H127C mutein, wild-type streptavidin (2IZF) and traptavidin (2Y3F) [@pone.0035203-Chivers2] were used as the input files. Each of these files was imported to Molegro Molecular Viewer (Version 2.2.0) [@pone.0035203-Thomsen1]. Interaction energy analysis was performed using the ligand energy inspector module in the program. Before analysis, both the ligand and protein hydrogen bonding positions were optimized and the ligand was energy minimized using the action panel in the ligand energy inspector module. The protein-ligand interaction energy (E~inter~, sum of the steric interaction energy, hydrogen bonding energy, short- and long-range electrostatic interaction energies) was expressed in the form of the MolDock score [@pone.0035203-Thomsen1] in arbitrary units. A more negative value reflects a stronger interaction. Other methods {#s4e} ------------- Determination of kinetic parameters using BIAcore biosensor, preparation of streptavidin-agarose matrix and purification of biotinylated proteins were performed as previously described [@pone.0035203-Wu1], [@pone.0035203-Wu5]. The affinity column had a bed volume of 0.1 ml 8-aa-loop-H127C mutein coupled matrix. 200 µl sample containing 0.2 mg of biotinylated protein G or 0.44 mg biotinylated IgG from ThermoFisher was applied to the column. To purify biotinylated IgG from a mixture, HeLa cell extract containing 0.6 mg protein was mixed with 0.44 mg biotinylatyed IgG. This mixture was then applied to the affinity column for purification. The graphic drawings of the structure of the streptavidin-biotin complex shown in [Fig. 1](#pone-0035203-g001){ref-type="fig"} were generated by using the Yasara program [@pone.0035203-Krieger1], (YASARA Biosciences GmbH, Austria). To model the possible positions of loop~7--8~, the pdb file (1SWE) of the streptavidin-biotin complex was used as the input file. The amino acid residues (G113 and S122) at the base of loop~7--8~ were fixed. The possible conformations of loop were searched against the protein data bank using the loop modeling module in Yasara. Two modeled structures with loops in the upper and lower positions were selected as the input pdb files. These two input structures define the boundary of the loop movement. A pdb file containing 10 modelled structures generated from the Morph server [@pone.0035203-Krebs1] website was analyzed by Yasara to generate the animated structures shown in [Fig. 1c](#pone-0035203-g001){ref-type="fig"}. Supporting Information {#s5} ====================== ###### A complete tetramer of the 8aa-loop-H127C mutein is drawn, which each subunit drawn in a different color. The unit cell is drawn as a black box. The two-fold axes running through the origin are drawn according to standard crystallographic conventions. Three orthogonal views are drawn: each view is parallel to one of the three crystallographic axes. For each view, the two remaining axes orthogonal to axis being viewed down are labeled. (DOCX) ###### Click here for additional data file. We gratefully acknowledge access to crystal screening and data collection facilities at the Stanford Synchrotron Radiation Laboratory and the Canadian Light Source at various stages during the course of this project. Competing Interests:Eric Hommema and Greg T. Hermanson are employees of Thermo Fisher Scientific, Inc. Valerie J. O\'Sullivan and Mark Schofield were employees of Thermo Fisher Scientific, Inc. during the study. Mark Schofield is currently an employee of Pall Life Sciences. A US patent application to characterize the mutein has been made through Innovate Calgary, the technology transfer office of the University of Calgary. Thermo Fisher Scientific is interested in developing this product for marketing. This does not alter the authors\' adherence to all the PLoS ONE policies on sharing data and materials. Funding:The work was supported by Natural Sciences and Engineering Research Council of Canada, <http://www.nserc-crsng.gc.ca/Index_eng.asp>. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: VJO EH GTH MS IBN SCW KN SLW. Performed the experiments: VJO EH MS SCW CH IBN. Analyzed the data: VJO EH GTH MS IBN SCW CH KN SLW. Contributed reagents/materials/analysis tools: VJO EH MS GTH IBN KN SLW. Wrote the paper: VJO SCW IBN KN SLW. Manuscript editing: GTH MS. 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INTRODUCTION ============ Spinal cord injury (SCI), which typically results from traffic accidents or long falls, is the most common cause of paralysis \[[@r1]\]. Two key mechanisms play roles in the pathology of SCI: a) primary injury resulting from bruising and compressive lesions, and b) secondary injuries, including inflammation, formation of glial scars, microvascular bleeding, and upregulation of apoptotic factors, all of which inhibit axonal regeneration \[[@r2], [@r3]\]. There are no effective therapies for patients with SCI, and novel treatments are urgently needed. Exosomes are nanosized extracellular vesicles 30--100 nm diameter which are formed as a result of inward budding in multiple cell types \[[@r4]\]. They were first discovered in sheep reticulocytes in 1983 \[[@r5]\]~,~ and the term "exosome" was coined by Johnstone in 1987 \[[@r6]\]. Exosomes may serve as biomarkers in many diseases due to the important role they play in intercellular communication and transport of proteins, mRNAs, and miRNAs into target cells \[[@r7], [@r8]\]. Exosomes can also have therapeutic effects in the nervous and respiratory systems \[[@r9]\]. Other studies demonstrated that exosomes derived from mesenchymal stem cells (MSCs) had therapeutic effects in liver, cardiovascular, and kidney diseases \[[@r10]--[@r12]\]. Furthermore, systemic administration of exosomes derived from MSCs promoted neurovascular reshaping and functional recovery after stroke in rats \[[@r13]\]. Exosomes can also reduce cognitive impairments after traumatic brain injury \[[@r14]\]. Recent research has demonstrated that exosomes containing large amounts of miRNA are pivotal for intercellular communication in the nervous system \[[@r15]\]. The miRNAs contained in MSC-derived exosomes also play important roles in intercellular signaling more generally \[[@r16]\]. Current knowledge indicates that the process by which miRNAs are loaded into exosomes is not random and differs in different cell types \[[@r17]\]. In addition, a recent study suggests that exosomes facilitate the transfer of miR-155 from smooth muscle cells to endothelial cells, which induces endothelial injury and promotes atherosclerosis \[[@r18]\]. Although neural stem cell-derived exosomes attenuate apoptosis and neuroinflammation after traumatic spinal cord injury \[[@r19]\], the mechanism underlying this effect remains unknown. Because insulin-like growth factor-1 (IGF-1) promotes regeneration after neural injury and proliferation in neural cells \[[@r20]--[@r22]\], we hypothesized that it may enhance the protective and regenerative effects of NSC-derived exosomes after SCI. The pathological effects of SCI are primarily a result of neuroinflammation, apoptosis, and oxidative stress injury \[[@r23]--[@r25]\]. We therefore established an H~2~O~2~-induced PC12 cell injury model to mimic SCI in vitro \[[@r26]\] and a rat SCI model using Allen's modified appliance in vivo \[[@r27], [@r28]\]. In this study, we examined the effects of IGF-1-induced (IGF-Exo) and normal exosomes (Nor-Exo) on neural inflammation, apoptosis, and regeneration after SCI and identified an miRNA-dependent mechanism responsible for those effects. RESULTS ======= Preparation and characterization of IGF-Exo ------------------------------------------- Neural stem cells were obtained from E15 fetal rat cerebral cortex and cultured in medium until neurospheres of similar sizes and shapes formed (Fig. 1A). These neurospheres were immunopositive for the NSC marker nestin (shown in red) ([Figure 1B](#f1){ref-type="fig"}). Rat NSCs collected from passage 3 were cultured in two kinds of culture medium: complete medium (DMEM/F12 medium supplemented with 20 ng/mL EGF, 10 ng/mL bFGF, 1× B27 supplement, 100 U/mL streptomycin, and 100 U/mL penicillin) or IGF-1 medium (100 ng/μL IGF-1 in complete medium). Exosomes were isolated from cell supernatants by ultracentrifugation. The shapes and sizes of both types of NSC-derived Exo were examined using transmission electron microscopy. While both the normal (Nor-Exo) and IGF-Exo had diameters of 30--300 nm, and the mean diameter of the IGF-Exo was slightly larger than that of the Nor-Exo ([Figure 1C](#f1){ref-type="fig"}). Western blot analysis indicated that levels of the exosomal markers CD9, CD63, and Alix were high in both Nor-Exo and IGF-Exo ([Figure 1D](#f1){ref-type="fig"}). Nanosight analysis of Exo size distributions revealed that the mean diameters of Nor-Exo and IGF-Exo were 101.1 ± 19.0 nm and 139.3 ± 34.0 nm, respectively ([Figure 1E](#f1){ref-type="fig"}). ![Characteristics of neural stem cells (NSCs) and exosomes derived from NSCs. (A) Morphology of neurospheres with typical shape examined by light microscopy. (B) Nestin immunofluorescence (red), a marker of NSCs, in neurospheres. (C) Exosome morphology examined by transmission electron microscopy. (D) Western blot analysis of exosome surface markers. (E) Particle size distribution of Nor-Exo and IGF-Exo by Nano Sight.](aging-11-102568-g001){#f1} IGF-Exo inhibits apoptosis and promotes regeneration in neural cells -------------------------------------------------------------------- We investigated the protective effect of IGF-Exo in PC12 cells treated with H~2~O~2~ to establish a cellular model of neural injury. CCK-8 assays showed that the 50% lethal dose of H~2~O~2~ was 200 μM for 24h in PC12 cells ([Supplementary Figure 1A](#SD2){ref-type="supplementary-material"}), and the optimal dose for IGF-1 in NSC culture was 200 ng/mL for 24h ([Supplementary Figure 1B](#SD2){ref-type="supplementary-material"}). After 24h of treatment with 200 μM H~2~O~2~, PC12 cells had damaged axons and decreased in number compared with the sham group. Additionally, cell viability was higher and axons were longer in IGF-Exo group PC12 cells than in the injury model group or Nor-Exo group (P \< 0.05) ([Figure 2A](#f2){ref-type="fig"}, [2B](#f2){ref-type="fig"}). In the TUNEL immunofluorescence assay, the ratio of TUNEL-positive cells in the IGF-Exo group was much higher than in the injury model group and slightly higher than in the Nor-Exo group (P \< 0.05) ([Figure 2C](#f2){ref-type="fig"}, [2D](#f2){ref-type="fig"}). To further explore the relationship between IFG-Exo and neural cell apoptosis, we measured Bax, Bcl-2, Beclin-1, and caspase-3 levels in the four PC12 cell groups. Compared with the sham group, Bax, Beclin-1, and caspase-3 expression were increased, while Bcl-2 expression decreased, in the injury group significantly increased (P \< 0.05). Bax, Beclin-1, and caspase-3 expression were also higher in the IGF-Exo group than in the injury group or the Nor-Exo (P \< 0.05) ([Figure 2E](#f2){ref-type="fig"}--[2I](#f2){ref-type="fig"}). ![*IGF-Exo inhibited H~2~O~2~-induced neural apoptosis in PC12 cells invitro.* (A) Morphology for each experimental group examined by light microscopy (control group: PC12 cells without treatment; H~2~O~2~ group: PC12 cells treated with H~2~O~2~ for 24h; Nor-Exo group: PC12 cells pretreated with Nor-Exo for 24h followed by H~2~O~2~ for 24h; IGF-Exo group: PC12 cells pretreated with IGF-Exo for 24h followed by H~2~O~2~ for 24h). (B) Cell viability in each experimental group; (C) TUNEL staining (red) indicating cell apoptosis in each experimental group; nuclear DAPI stain in blue. (D) TUNEL-positive cell numbers per field for each experimental group. (E) Western Blot analysis of apoptotic and anti-apoptotic proteins. (F--I) Relative expression of BAX, Bcl-2, Beclin-1 and Caspase-3. Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls post hoc test). \\P \< 0.01 vs. control group; \#\#P \< 0.01 vs. H~2~O~2~ group.](aging-11-102568-g002){#f2} IGF-Exo reduces lesion size and promotes functional recovery after SCI ---------------------------------------------------------------------- To test the neuroprotective effect of IGF-Exo in vivo, we established a rat model of acute SCI using a modified Allen's weight drop apparatus ([Supplementary Figure 2](#SD2){ref-type="supplementary-material"}). Rats received systemic IGF-Exo, sham, PBS, or Nor-Exo injections via the tail vein ([Figure 3A](#f3){ref-type="fig"}). BBB locomotor scores were markedly lower in SCI group rats than in sham rats at 1, 3, 7, 14, and 28 days after SCI (P \< 0.01). Compared with the SCI group, BBB scores were higher in Nor-Exo and IGF-Exo rats at 7, 14, and 28 days after SCI (P \< 0.05). Moreover, the scores of IGF-Exo rats were higher than those of Nor-Exo rats at 14 and 28 days post-SCI (P \< 0.05) ([Supplementary Figure 3](#SD2){ref-type="supplementary-material"}). MRI and neuroelectrophysiological examinations were also used to evaluate whether IGF-Exo promoted recovery from SCI recovery. DTI indicated that reconnection of the neural fasciculus was increased in the IGF-Exo group compared to the Nor-Exo and SCI groups ([Figure 3B](#f3){ref-type="fig"}, [Supplementary Figures 4](#SD2){ref-type="supplementary-material"}, [5](#SD2){ref-type="supplementary-material"}). Neuroelectrophysiological examination revealed that MEP amplitudes were higher in the IGF-Exo group than in the Nor-Exo and SCI groups ([Figure 3C](#f3){ref-type="fig"}, [3D](#f3){ref-type="fig"}). In longitudinal sections of rat spinal cords collected for HE staining 28 days after SCI, injury areas (cavity) were smaller in the IGF-Exo group than in the SCI and Nor-Exo groups (P \< 0.05) ([Figure 3E](#f3){ref-type="fig"}, [3F](#f3){ref-type="fig"}). Immunostaining analysis of BrdU, a marker of new neurons, and NeuN, a general neural marker, revealed that BrdU and NeuN staining intensity were both lower in IGF-Exo group lesion areas than in the SCI and Nor-Exo groups at 28 days after SCI (P \< 0.05) ([Figure 3G](#f3){ref-type="fig"}). In a TUNEL assay performed 3 days after SCI, the TUNEL-positive cell (red) ratio was higher in the IGF-Exo group than in the injury model group and slightly higher than in the Nor-Exo group (P \< 0.05) ([Figure 3H](#f3){ref-type="fig"}, [3J](#f3){ref-type="fig"}). ![*IGF-Exo inhibited neural apoptosis and neuroinflammation after SCI invivo.* (A) Schematic of tail intravenous injections of Nor-Exo and IGF-Exo in SCI model rats. (B) DTIs constructed for the SCI, Nor-Exo, and IGF-Exo groups at 1 day and 28 days after surgery. (C) Neuroelectrophysiological examination results for each experimental group (control, SCI, Nor-Exo, and IGF-Exo) at 3 days and 28 days after surgery. (D) MEP amplitudes for each experimental group at 3 days and 28 days after surgery. (E) Hematoxylin-Eosin staining of sections containing SCI lesions in each experimental group at 3 days and 28 days after surgery. (F) Cavity space percentages for each experiment group at 3 days and 28 days after surgery. (G) BrdU and NeuN immunofluorescence indicative of neuron regeneration in each experimental group at 28 days after surgery. (H) TUNEL staining (green) indicative of apoptosis after SCI in each experimental group; DAPI in blue. (J) Numbers of TUNEL-positive cells per field for each experimental group. (K--O) Western Blot analysis of pro- and anti-apoptotic proteins (BAX, Bcl-2, Beclin-1, and Caspase-3). Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls post hoc test). \\P \< 0.01, vs. control group; \#\#P \< 0.01, vs. SCI group.](aging-11-102568-g003){#f3} Western blot analysis of Bax, Bcl-2, Beclin-1, and caspase-3 levels in the four groups of SCI rats were used to assess the anti-apoptosis effects of IGF-Exo in vivo. Compared with the sham group, Bax, Beclin-1, and caspase-3 expression increased, while Bcl-2 expression decreased, in the SCI group (P \< 0.05). However, Bax, Beclin-1, and caspase-3 expression was significantly higher in the IGF-Exo group than in the SCI and Nor-Exo groups (P \< 0.05) ([Figure 3K](#f3){ref-type="fig"}--[3O](#f3){ref-type="fig"}). Antiapoptotic miR-219a-2-3p is highly increased in IGF-Exo ---------------------------------------------------------- To illuminate the mechanism underling the neuroprotective effects of IGF-Exo, we characterized exosomal miRNA content though miRNA sequencing analysis. Six miRNAs were upregulated and 2 were downregulated in IGF-Exo compared to Nor-Exo ([Figure 4A](#f4){ref-type="fig"}--[4C](#f4){ref-type="fig"}; [Supplementary Figure 6](#SD2){ref-type="supplementary-material"}). qRT-PCR analysis was then performed to confirm that expression was higher for the 6 upregulated miRNAs identified in the sequencing analysis. All 6 miRNAs, namely rno-miR-138-5p (P \< 0.05), rno-miR-219a-2-3p (P \< 0.05), rno-miR-92b-3p (P \< 0.01), rno-miR-92b (P \< 0.05), rno-miR-25-5p (P \< 0.05), and rno-miR-674-3p (P \< 0.01), were significantly upregulated in IGF-Exo compared with Nor-Exo ([Figure 4D](#f4){ref-type="fig"}). ![Screening and identification of upregulated miRNAs in Nor-Exo and IGF-Exo. (A) Heat map showing the 6 miRNAs for which expression was upregulated ≥ 2-fold in IGF-Exo compared to Nor-Exo. (B) IGF-1-induced miRNA regulation network. (C) Among the 6 upregulated miRNAs, miR-219a-2-3p expression increased the most. (D) qRT-PCR comparison of rno-miR-219a-2-3p, 138-5p, 92b-3p, 92b, 25-5p, and 674-3p expression between Nor-Exo and IGF-Exo from 3 independent experiments. \P \< 0.05, \\P \< 0.01 for IGF-Exo vs. Nor-Exo.](aging-11-102568-g004){#f4} Neuroprotective effects of IGF-Exo after SCI are associated with miR-219a-2-3p-dependent inhibition of YY1 ---------------------------------------------------------------------------------------------------------- We screened possible target genes of miR-219a-2-3p using bioinformatics methods (Target Scan v7.2) and identified YY1 was the potential target ([Figure 5A](#f5){ref-type="fig"}). We therefore constructed a YY1-3′UTR (3′-Untranslated Region) expression vector containing a luciferase gene and transfected it into PC12 cells. A conventional luciferase reporter assay showed that miR-219a-2-3p interacted with YY1 ([Figure 5A](#f5){ref-type="fig"}). Additionally, qRT-PCR showed that YY1 expression decreased when miR-219a-2-3p was upregulated ([Figure 5B](#f5){ref-type="fig"}). Furthermore, YY1 expression increased when miR-219a-2-3p was downregulated ([Figure 5C](#f5){ref-type="fig"}). Blockage of miR-219a-2-3p by Anti-miR-219a-2-3p (the miR-219a-2-3p inhibitor) inhibited IGF-Exo-mediated neuroprotective effects ([Figure 5D](#f5){ref-type="fig"}). CCK-8 assays, TUNEL staining, and Western blots were performed to confirm that miR-219a-2-3p protected against neural injury and inhibited resulting apoptosis ([Figure 5E](#f5){ref-type="fig"}--[5G](#f5){ref-type="fig"}). The results indicated that miR-219a-2-3p expression downregulated, while miR-219a-2-3p knockdown upregulated, the YY1/NF-kB-p65 axis in PC12 cells ([Figure 5H](#f5){ref-type="fig"}--[5J](#f5){ref-type="fig"}). ![Inhibition of miR-219a-2-3p in exosomes reduced the protective effects of IGF-Exo.* (A) Luciferase activity was detected in PC12 cells co-transfected with pGL3 vector, pGL3-YY1, miR-219a-2-3p mimics, or miR-219a-2-3p mutant. (B) Relative YY1 expression after upregulation of miR-219a-2-3p measured by qRT-PCR. B) PC12 cells were transfected with miR-219a-2-3p mimics or miR-NC; relative YY1 expression was analyzed by qRT-PCR. (C) PC12 cells were transfected with Anti-miR-219a-2-3p (miR-219a-2-3p inhibitor) or Anti-NC (control); relative YY1 expression was analyzed by qRT-PCR. (D) Morphology in each experimental group examined by light microscopy (control group: PC12 cells without treatment; H~2~O~2~ group: PC12 cells treated with H~2~O~2~ for 24h; IGF-Exo group: PC12 cells pretreated with IGF-Exo for 24h followed by H~2~O~2~ for 24h); Anti-miR-219a-2-3p-IGF-Exo group: PC12 cells pretreated with miR-219a-2-3p inhibitor-transfected IGF-Exo for 24h followed by H~2~O~2~ for 24h). (E) Cell viability in each experimental group when miR-219a-2-3p was downregulated in IGF-Exo; (F) TUNEL staining (red) indicative of apoptosis in each experimental group; DAPI in blue. (G) Numbers of TUNEL-positive cells per field for each experiment group. (H--J) Western Blot analysis of YY1 and NF-kB-p65 (si-NC: siRNA blank vector; miR-NC: miRNA blank vector; si-YY1: YY1 siRNA; miR-219a-2-3p: miR-219a-2-3p mimics). Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls post hoc test). \\P \< 0.01, vs. control group; \#\#P \< 0.01, vs. SCI group.](aging-11-102568-g005){#f5} DISCUSSION ========== Although basic research and clinical studies have attempted to identify more effective treatments for SCI patients, the prognosis remains very poor \[[@r29]\]. The pathological processes involved in SCI include neuroinflammation, apoptosis, and inhibition of neural regeneration \[[@r30], [@r31]\]. A previous study revealed that exosomes derived from MSC and NSC stem cells suppress inflammation and apoptosis after neural injury. However, the mechanisms underlying these effects remain unknown. Here, we used exosomes derived from NSCs exposed to IGF-1 to treat SCI for the first time and demonstrated that these IGF-Exo enhanced the neuroprotective effects of exosomes by inhibiting apoptosis and neuroinflammation in an miR-219a-2-3p-dependent manner. Several studies have demonstrated that stem cells (especially MSCs and NSCs) administered either systemically or via local transplantation are potential therapies for neural injury and nervous system diseases \[[@r32]\]. However, stem cell therapies face important challenges. Tumorigenesis due to chromosome instability and distal vascular occlusion resulting from the large size of stem cells can be fatal, and the survival rate of stem cells in vivo is relatively low \[[@r33], [@r34]\]. Fortunately, increasing evidence suggests that the biological effects of stem cells can mostly be attributed to molecules they secrete, including those secreted via exosomes \[[@r9], [@r35]\]. Recent studies indicated that exosomes derived from NSCs might aid in the discovery of treatments for central nervous system injury \[[@r36], [@r37]\], but the mechanisms underlying their protective effects are unknown, and NSCs produce relatively small quantities of exosomes. IGF-1 improves proliferation and metabolism in neural cells, and we confirmed this IGF-1-induced increase in NSC cell viability here in a CCK-8 assay. Exosomes play an important role in intercellular communication by transmitting bioactive RNAs and proteins, and exosomes derived from NSCs have therapeutic effects against ischemia and neurodegeneration \[[@r38], [@r39]\]. Because exosomes derived from NSCs can reduce apoptosis and neuroinflammation after SCI, we hypothesized that IGF-Exo could enhance these protective effects and promote regeneration after SCI. In this study, RNA sequencing of Nor-Exo and IGF-Exo identified 8 microRNAs that were differentially expressed between the two exosome types, and miR-219a-2-3p was determined to the most significant among them. The difference in miR-219a-2-3p expression between Nor-Exo and IGF-Exo was verified by qRT-PCR assay. Previous research indicated that miR-219a-2-3p is enriched in human white matter and oligodendrocytes, and may be a potential target in demyelinating disorders \[[@r40]\]. Moreover, bioinformatics screening revealed that miR-219a-2-3p interacts with the downstream gene YY1 \[[@r41]\]. Here, a luciferase assay confirmed that up-regulation of miR-219a-2-3p inhibited YY1 expression, while changes in YY1 expression had no effect on the expression of miR-219a-2-3p. However, YY1 expression is correlated with activity of the inflammatory NK-kB pathway, and up-regulation of YY1 enhanced inflammatory effects. Therefore, IGF-Exo likely inhibits the expression of YY1, and in turn NK-kB pathway activity, through the up-regulation of miR-219a-2-3p, thus inhibiting neuroinflammation and promoting neuroprotective effects. This mechanism was confirmed in a rescue experiment in which miR-219a-2-3p expression was inhibited by transfection of si-miR-219a-2-3p. Some important limitations of this study should be considered when interpreting the results and should be addressed in future studies. Firstly, additional in vitro studies are needed to fully characterize the mechanism underlying the neuroprotective effects of IGF-Exo, and their efficacy should be confirmed in clinical trials. Secondly, IGF-Exo might exert neuroprotective effects and inhibit the apoptosis and inflammation via the action of other miRNAs or pathways, and additional downstream genes of miR-219a-2-3p should be examined further. Finally, additional studies are needed to verify that the effects of miR-219a-2-3p on cell apoptosis and neural regeneration observed here are applicable in other in vitro cell models and under different conditions. In summary, this study demonstrated that exosomes derived from neural stem cells exposed to IGF-1, at least partly, suppress the nerve inflammation, inhibit apoptosis, and promote nerve regeneration via an miR-219a-2-3p-dependent mechanism after the spinal cord injury. This is the first microRNA mechanism to be identified that might explain the effects of NSC-derived exosomes in SCI and may inform the development of novel treatments for spinal cord injury. MATERIALS AND METHODS ===================== Animals ------- Adult female Sprague-Dawley rats (180--200g) and pregnant rats (15d) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal care and experimental protocols were approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force. All animals were housed in individual cages on a 12h light/dark cycle with ad-libitum access to food and water. Isolation and culture of rat embryonic NSCs ------------------------------------------- 15-day embryos were isolated from pregnant rats euthanized with 230 mg/kg sodium pentobarbital. Cerebral cortex tissues were then isolated from the embryos and placed in Hank's solution. After the meninges were removed, the cerebral cortex was washed twice with Hank's solution, cut into pieces, digested with accutase cell detachment solution at 37°C for 10 min, and dispersed by pipetting. The suspension was filtered with a 200-mesh screen and then centrifuged at 800 rpm for 5 min; NSCs were obtained from the pelleted material. The cells were then re-suspended in complete medium (DMEM/F12 medium supplemented with 20 ng/ml EGF, 10 ng/ml bFGF, 1 × B27 supplement, 100 U/mL streptomycin, and 100 U/mL penicillin). After cell density was adjusted to 1 × 10^5^/mL, cells were seeded into culture flasks and incubated at 37°C with 5% CO~2~. After 3-5 days, cells were passaged. All NSCs used in this study were from passage 3. Isolation and purification of NSC-Exo ------------------------------------- Exosomes were isolated from NSC supernatant as previously described \[[@r42]\]. The medium was then collected and centrifuged at 300 × g for 10 min, followed by centrifugation at 2000 × g for 20 min at 4°C. After centrifugation, the supernatant was filtered using a 0.22 μm filter to remove dead cells and large cellular debris. Small cellular debris were then pelleted by centrifugation at 10,000 × g for 30 minutes, and the supernatants were recentrifuged at 100,000 × g for 70 minutes. Finally, pelleted exosomes were resuspended in 100 μL PBS solution in the ultracentrifuge tubes. Exosomes were quantitated by measuring total protein concentration using a bicinchoninic acid assay (BCA; Thermo Fisher). Identification of NSC-Exo ------------------------- The morphology of NSC-derived exosomes (NSC-Exo) was assessed by transmission electron microscopy (TEM, Hitachi HT7700, Tokyo, Japan). Nanosizer^TM^ technology (Malvern Instruments, Malvern, UK) was used to analyze the diameter distribution of NSC-Exo. Western blotting was used to examine the specific surface markers encapsulated in the exosomes, including CD9, CD63, and Alix. In vitro experiment design ---------------------------- In the first in vitro experiment, PC12 cells were divided among the following four groups to verify the protective effect of IGF-Exo: control group (untreated PC12 cells), H~2~O~2~ group (PC12 cells treated with H~2~O~2~ for 24h), Nor-Exo group (PC12 cells pretreated with Nor-Exo for 24h followed by H~2~O~2~ for 24h), and the IGF-Exo group (PC12 cells pretreated with IGF-Exo for 24h followed by H~2~O~2~ for 24h). In the second in vitro experiment, PC12 cells were divided among the following four groups to verify the effects of miR-219a-2-3p: control group (untreated PC12 cells), H~2~O~2~ group (PC12 cells treated with H~2~O~2~ for 24h), NC-IGF-Exo group (PC12 cells pretreated with NC-IGF-Exo derived from IGF-1-induced NSCs transfected with miR inhibitor negative control (Anti-NC) for 24h followed by H~2~O~2~ for 24h), and Anti-miR-219a-2-3p-IGF-Exo group (PC12 cells pretreated with IGF-Exo derived from IGF-1-induced NSCs transfected with miR-219a-2-3p inhibitor (Anti- miR-219a-2-3p) for 24h followed by H~2~O~2~ for 24h). In vivo experiment design --------------------------- Adult female Sprague-Dawley rats were divided among the following four groups (n=8 per group): control group (spinal cord segment T10 was only exposed in a sham operation), SCI group (acute SCI via modified Allen's weight drop apparatus, 0.5 mL PBS systemic administration through tail vein injection after SCI), Nor-Exo group (100 μg of precipitated normal exosome protein in 0.5 mL of PBS was administered via tail vein injection after SCI), and IGF-Exo group (100 μg of precipitated IGF-1-induced exosome protein in 0.5 mL of PBS was administered via tail vein injection after SCI). Cell viability assay -------------------- The viability of PC12 cells and primary neurons was evaluated with a CCK-8 assay (Dojindo, Kumamoto, Japan) to examine the effects of NSC-Exo on neural cell proliferation. The cells were rinsed three times with 1 × PBS after 0, 24, 48, and 72 h of incubation. 10 μL of CCK-8 solution diluted in 90 μL of fresh culture medium was then added followed by incubated for another 2 h at 37°C. Quantitative real-time RT-PCR ----------------------------- Total RNA was isolated from cells and tissues using Trizol reagent (TransGen Biotech, Beijing) according to the manufacturer's instructions. For RNase R treatment, 2 mg total RNA was incubated for 15 min at 37°C with or without 3 U/mg RNase R (Epicentre Technologies, Madison, WI, USA). qRT-PCR was performed using SYBR Green (TransGen Biotech) according to the manufacturer\'s instructions ([Table 1](#t1){ref-type="table"}) and an ABI 7500 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). ###### Primers of miRNA used for qPCR in this study. -------------- ------------------------------------------- Primer Sequence(5′to3′) miR-138-5p Forward: ACACTCCAGCTGGGAGCtGGtGttGTGAATC Reverse: TGGTGTCGTGGAGTCG miR-219-2-3p Forward: ACACTCCAGCTGGGAGAATTGTGGCTGGAC Reverse: TGGTGTCGTGGAGTCG miR-25 Forward: ACACTCCAGCTGGGAGGCGGAGACACGGGC Reverse: TGGTGTCGTGGAGTCG miR-674-3p Forward: ACACTCCAGCTGGGCACAGCtCCCATCTCA Reverse: TGGTGTCGTGGAGTCG miR-92b Forward: ACACTCCAGCTGGGAGGGACGGGACGCGGTGC Reverse: TGGTGTCGTGGAGTCG miR-92b-3p Forward: ACACTCCAGCTGGGTATTGCACtCGTCCCG Reverse: TGGTGTCGTGGAGTCG YY1 Forward: AGTGGGAACAGAAGCAGGTG Reverse: GAGGTCAATGCCAGGTATCC -------------- ------------------------------------------- Transfection ------------ Rat miR-219a-2-3p mimics, anti-miR-219a-2-3p (miR-219a-2-3p inhibitor), si-YY1 (si-YY1), miR mimics negative control (miR-NC), miR inhibitor negative control (miR-NC), and si-YY1 negative control (si-NC) were purchased from GenePharma (Shanghai, China). Sequences are listed in [Table 2](#t2){ref-type="table"}. ###### miRNA mimics, inhibitors, and siRNAs used in this study. ---------------------------------------------- ------------------------ Name Sequence(5′to3′) miR-219a-2-3p mimics AGAAUUGUGGCUGGACAUCUGU miR-219a-2-3p inhibitor (Anti-miR-219a-2-3p) ACAGAUGUCCAGCCACAAUUCU siRNA-YY1(si-YY1) GGUAAUAAGAAGUGGGAACTT miR mimics negative control (miR-NC) UUGUACUACACAAAAGUACUG miR inhibitor negative control (Anti-NC) CAGUACUUUUGUGUAGUACAA si-YY1 negative control(si-NC) UUCUCCGAACGUGUCACGUTT ---------------------------------------------- ------------------------ Cell apoptosis assays --------------------- PC12 cells were incubated with or without NSC-Exo (100 μg/mL) after treatment with H~2~O~2~ (200 μM) for 24 h. A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL; Roche, Basel, Switzerland) was used to detect DNA strand breaks according to the manufacturer's instructions. Images were captured using a fluorescence microscope (Leica, Solms, Germany). Apoptotic neuronal cells were determined by counting total numbers of TUNEL- and DAPI-stained cells. Western blot analysis --------------------- Protein lysates were extracted from cells and 5 mm blocks of injured spinal cord tissue encompassing the lesion collected on days 3 and 28 after SCI by incubating with RIPA lysis and extraction buffer (KeyGEN Biotechnology, China). Protein concentration was determined using BCA. Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (EMD Millipore Corp., USA), incubated overnight at 4°C with primary antibodies, and blocked with bovine serum albumin (5%, v/v). The primary antibodies included Bax (1:2000, rabbit IgG; Abcam, USA), Bcl-2 (1:1000, rabbit IgG; Abcam, USA), Beclin-1 (1:1000, rabbit IgG; Abcam, USA), caspase-3 (1:1000, rabbit IgG; Abcam, USA), GAPDH (1:1000, Abcam, USA), YY1 (1:1000, mouse IgG1; Abcam, USA), and p65 (1:1000, rabbit IgG; Abcam, USA). The membranes were then incubated with secondary antibody (1:2000, Proteintech, USA) for 120 min at room temperature. Protein bands were visualized using enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology, Nanjing, China), and band density was analyzed semi-quantitatively using Image J software. Immunofluorescence staining --------------------------- For immunofluorescence staining, 1.0 x 10^5^ NSCs were cultured in 0.5 mL media unless otherwise specified. Cultured cells were fixed with 4% (wt/vol) paraformaldehyde for 30 min at room temperature, washed with 0.1 M Tris-buffered solution (pH 7.5, TBS), blocked with 10% (vol/vol) normal goat serum in TBS containing 0.3% (vol/vol) Triton X-100 at room temperature for 60 min, and incubated with primary antibodies at 4°C overnight. The primary antibodies were rabbit anti-Nestin (1:1000), rabbit anti-BrdU (1:2000), and donkey anti-NeuN (1:1000). The cells were washed with TBS and incubated with Alexa Fluor 488- and 555-conjugated secondary antibodies (1:1000, Proteintech) at room temperature for 60 min. To visualize nuclei, the cells were counterstained with 2 μg/mL 4\',6-diamidino-2-phenylindole (DAPI). Finally, the cells were mounted with 80% (vol/vol) glycerol, visualized under a fluorescent microscope (IX81, Olympus Corp., Tokyo, Japan), and image data were processed using MetaMorph (Molecular Devices, Sunnyvale, CA, USA) for quantification. Numbers of positive cells were counted in three to five fandom fields per well for three separate wells in each individual experiment. Behavioral tests ---------------- Hindlimb motor function assessments were performed 1, 3, 7, 14, and 28 days after SCI using the Basso Beattie Bresnahan (BBB) locomotor rating scale and slanting board test. The BBB score ranged from 0 (complete hindlimb paralysis) to 21 (normal locomotion). Scores were assigned by assessing the motor capacity of the experimental rats, including the movement of the hindlimbs, weight-bearing, and coordination of forelimb and hindlimb movements. The rats were placed in an open field of wooden models for 4 min and scored by 2 researchers using blinded methods. The slanting board test was performed according to the Rivlin method and involved placing a rat on a rectangular wooden oblique board covered with a rubber pad; the oblique plate was rotated to measure the angle of the inclined plate. When the longitudinal axis of the rat's body was parallel to the longitudinal axis of the inclined plate, the rat was tilted toward the side of the tilted plate to raise it in 5° increments. The maximum angle at which a rat could maintain its position for 5 s was measured. Measurements were performed five times for each animal, and the average values were analyzed. Statistical analysis -------------------- The data are presented as means ± standard deviation (SD) and were analyzed using one-way analysis of variance and Tukey's post hoc multiple comparison tests. All experimental data were analyzed using SPSS 17.0. P-values \< 0.05 were considered statistically significant. Additional experimental materials and methods details, including electrophysiological nerve and MRI assessments, are provided in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. Supplementary Material ====================== AUTHOR CONTRIBUTIONS: Ke Ma and Huiyou Xu performed statistical analysis and wrote the manuscript. Jian Zhang and Fei Zhao were involved in manuscript preparation. Renjie Wang and Xuyi Chen were responsible for study design and data collection. Haiqian Liang, Hongtao Sun, Ping Li, Sai Zhang, and Xuyi Chen were responsible for research funding. All authors approved the final version of the manuscript. CONFLICTS OF INTEREST: None of the authors declare any conflicts of interest. FUNDING: This study was support by the National Key Research and Development Plan of China (No. 2016YFC1101500), the National Natural Science Foundation of China (No. 11672332), the Key Science and Technology Support Foundation of Tianjin City (No. 17YFZCSY00620), the Major Project of Tianjin Science and Technology Military and Civilian Integration (No. 18ZXJMTG00260), and the Project of Tianjin Rescue Medicine Clinical Center (No. 15ZXLCSY00040). 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1. Case Report {#sec1} ============== A 48-year-old male finished Triple-Ironman distance in 41 hours and 18 minutes (11.4 km swimming, 540 km cycling, and 126 km running). At the time of the examinations, he had been practising his current diet of raw vegan diet for 6 years. Prior to this, the vegan athlete had been living as a vegan for 3 years and as a vegetarian for the previous 13 years. All last competitions were performed only based on a raw diet. At the time of both examinations, the vegan athlete was 48 years of age and 1.80 metres in height. In the sporting season he was 79.7 kg in weight, with a body fat index of 12.9%; in the off-season he weighed 80.3 kg with a body fat index of 16.3%. Clinical examination showed a regular heart rhythm at 60 beats/min. Blood pressure was 115/70. The heart sounds were normal. Prior to the spiroergometry, echocardiography was performed based on ASA criteria (1). For comparison purposes, we refer to the values for 10 Ironman triathletes of similar age living on a mixed diet. The results of the spiroergometry are shown in [Table 1](#tab1){ref-type="table"}, those of echocardiography are in [Table 2](#tab2){ref-type="table"}, and blood analysis findings are presented in [Table 3](#tab3){ref-type="table"}. The athletes of the control group were aged 47.4 ± 5.2, weighed 76.2 ± 8.9 kg (with 13.4 ± 2.0% body fat), and were 1.816 metres ±6.6 cm in height. In the active phase the vegan athlete was training on average 18 hours per week, consisting of 2 hours of swimming, 11 hours of cycling, and 5 hours running. This involved covering distances of 5 km (swimming), 330 km (cycling) and 50 km (running). The athletes of the control group were training for a total of 15.9 ± 2.1 hours weekly, involving 2.5 ± 0.7 hours and 5.6 ± 1.5 km swimming, 8.6 ± 1.5 hours and 215.5 ± 53.0 km cycling, and 4.9 ± 0.7 hours and 55.2 ± 6.5 km running. In terms of performance diagnostics, the vegan athlete showed comparable VO~2~max, VO~2~ at VAT, and %VO~2~max at VAT values as compared with the control group (statements regarding significant differences are not possible). VO~2~ and %VO~2~max at RCP were somewhat higher for the vegan athlete. The maximum ergometric performance is higher for the vegan athlete in absolute terms but not relative to the body weight. At RCP the ergometric performance of the vegan athlete is somewhat higher than that of the control group. The vegan athlete had lower pulse rates at rest, at VAT, at RCP, and at the endurance limit. In comparison with the off-season, in the active season the vegan athlete had a higher maximum ergometric performance, VO~2~, and %VO~2~max, as well as cardiac frequency at RCP. In morphological terms, the vegan athlete showed a greater left ventricular end diastolic diameter, with consecutively higher end diastolic volume and stroke volume. The systolic and diastolic functions seem to be similar for the vegan athlete and the control group. Besides a mild thrombopenia, slightly higher CK-NAC levels in the active season and a slight drop of free testosterone (in both examinations) all remaining values were within normal ranges. 2. Discussion {#sec2} ============= It is well known that a vegetarian or vegan diet, when sensibly managed, can make a contribution to the prevention and therapy of illnesses in all phases of life \[[@B1]\]. More and more people are thus adopting a vegetarian (2) or vegan (2) lifestyle. It is equally understandable that such people may want to take part in endurance sports. It follows that the effects of vegan diet on sporting performance are a matter of general interest. This case report is set out to investigate an ultra endurance triathlete, who had been living on a vegetarian diet since 22 years and a purely vegan diet for 9 years, with reference to his ability to perform, cardiac status, and any symptoms of deficiency. It was also a matter of interest for the study to ascertain whether his performance parameters were different from those of mixed diet triathletes. Even competitive athletes can adopt a diet of this kind with a view to the health benefits, without any risk of suffering from dietary deficiencies \[[@B2]--[@B4]\]. With its disciplines of swimming, cycling, and running, the triathlon is a very popular type of endurance sport. As well as competitions involving classic sprint distances (0.5, 20 and 5 km), short distances (1--1.5, 40, and 10 km), and medium distances (ca. 2, 80--90, and 21.2 km), it also offers competitions involving ultra distances: Ironman (3, 8, 180, and 42.2 km) and multiple (2× until 10×) Ironman. As is the case with other types of endurance sport, triathletes undergo various physical adaptations in the course of training. These include an increased cardiac output under stress and increased arteriovenous oxygen extraction with consecutive rises in the maximum oxygen intake (VO~2~max) \[[@B5]\]. In echocardiographic terms, triathletes show physical changes similar to those of cyclists, resembling a mixed state of static/dynamic stress and involving a significant increase in the left ventricular muscle mass and internal left ventricular diameter \[[@B6]\]. In terms of spiroergometry, successful triathletes not only show an increased oxygen intake, but they also demonstrate a capacity for continuous high performance over long periods, as a result of effective internal economy, high fractional extraction of VO~2~max at the given stress level (%VO~2~max), and a high anaerobic threshold \[[@B7]\]. Good indicators of the limit of endurance are the ventilatory anaerobic threshold (VAT) and the respiratory compensation point (RCP) \[[@B7], [@B8]\]. It is not known whether or to what extent triathletes living on a vegan or vegetarian diet differ from triathletes living on a mixed diet. It is known that nonsporting vegans show better metabolic parameters than mixed diet athletes (9). To the best of our knowledge, no data are to be found in the extant literature on the spiroergometric, echocardiographic, or haematological parameters of ultra endurance athletes living on a vegan diet. As a result, this is a ground-breaking case report involving the first ever presentation of the characteristic parameters of a vegan ultra endurance athlete. In view of the equivalence in terms of age and other similar anthropometric features, the data for the vegan athlete are well comparable with those of the control group. On the other hand, we are dealing here with an individual case, so the meaningfulness of the results, in the nature of the case, remains limited. Both the vegan athlete and the control group trained for a comparable number of hours. The vegan athlete\'s cycling training, on the other hand, was more intense and more prolonged. Comparison of the competition results would be a way of checking this, but in view of the fact that the athletes\' preferred competition distances vary from one another, this is not possible. Here, however, we should draw attention to the considerably different age of the athletes. Millet et al. \[[@B8]\] report on athletes of 24.8 ± 2.6 years of age. In comparison with the off-season, in the active season the RCP and the maximum ergometric performance rose moderately, whereas the VAT and VO~2~max remained at a constant level. This study demonstrated a significant rise in the RCP and maximum ergometric performance (with VAT and VO~2~max remaining constant), based on comparison between the pre-competitive and competitive seasons. The echocardiographic parameters of the vegan athlete are different from those of the control group. Essentially the vegan athlete has a greater LVEDD value, with higher EDV and SV, which however do not exceed standard and normal values \[[@B10]\]. Overall the echocardiographic values of the sporting season and the off-season do not differ in any significant degree. Laboratory parameters showed no pathological values. A slight thrombopenia in the sporting season can be explained by the reduced thrombocyte activation and adhesion caused by training. In itself this is not necessarily a pathological manifestation. In conjunction with a threshold value for vitamin B12 and standard iron, ferritin, and folic acid status, the standard haemoglobin, haematocrit, MCH, MCV, and MCHC readings exclude the possibility of manifest or latent anaemia. Raised CK-NAC levels are not unusual with active athletes. Mougious et al. have therefore written about the necessity of separate CK reference values for active sportspersons. Reduced testosterone values in the blood have also been described. An influence on the bone density or the muscle mass, however, cannot be demonstrated. None of the usual laboratory parameters gives grounds for suspecting the presence of any detriment to health caused by sport or by diet. Quite on the contrary, the vegan athlete even shows ideal cholesterol values. 3. Critical Remarks {#sec3} =================== The case report of a vegan triathlete can only provide indications that a vegan diet is at least not detrimental to health, even in connection with a long-distance triathlon, and that a vegan diet results in similar performance as compared with a mixed diet. These conclusions cannot be extrapolated to short distances and Olympic distances, where an explosive performance---above all, a performance in the lactic acid zone---is frequently necessary. Taking place for the most part in the aerobic range, long-distance races may work more in favour of vegan diet than races that approach the anaerobic threshold. In view of the shortage of suitable and willing test subjects, it will not be easy to arrange a group comparison of vegan and traditional triathletes for study purposes. A case study like the present one can supply indications, but the meaningfulness of its findings remains subject to limit. This case report seems to suggest that an ultra triathlete living on a vegan diet has a similar profile, physiologically and in performance terms, to that of triathletes of similar age living on a conventional diet. The cardiac adaptations are likewise similar in both cases. The higher laboratory parameters do not permit conclusions with regard to dietary deficiency or any detriment to health. A well-planned vegan diet can apparently also be adopted by ultra endurance athletes without any risk to health necessarily being incurred. To what extent a vegan diet offers benefits or disadvantages for the performance capacity/health of a triathlete would be a matter for investigation in a future study, though in view of the highly individual training regimes of triathletes, this would be difficult to arrange. Conflict of Interests ===================== There is no data relating to financial interests and relationships (patents, fees, support by companies, etc.). ###### Spiroergometric parameters of the vegan athlete and the control group (values given as a mean value ± standard deviation). Vegan Off-S. Vegan season Control group (n = 10) ----------------------------- --------- -------------- -------------- -------------------------- VO~2~ max L/min 4.8 4.7 4.3 ± 0.6 mL∗min^−1^∗kg^−1^ 60.0 60.0 56.9 ± 7.5 VO~2~ VAT L/min 3.4 3.3 3.3 ± 0.6 mL∗min^−1^∗kg^−1^ 43 42 43.7 ± 7.4 \% VO~2~ max 71 70 76.1 ± 9.3 VO~2~ RCP L/min 3.9 4.4 3.6 ± 0.6 mL∗min^−1^∗kg^−1^ 48 55 48.2 ± 8.3 \% VO~2~ max 80.6 92 84.4 ± 6.9 HR At rest 55 49 58.6 ± 2.9 At VAT 127 123 147.7 ± 11.4 At RCP 141 147 157.0 ± 13.1 Maximal load 163 159 176.7 ± 11.0 W At VAT 280 280 262 ± 42.9 At RCP 340 370 286 ± 56.2 Maximal load 370 400 337 ± 53.8 Maximal load/kg body weight 4.61 5.02 4.46 ± 0.73 HF: heart rate/min, Off-S: off-season, season: active season, vegan: vegan athlete, VO~2~ max: maximum oxygen intake, VO~2~ VAT: oxygen intake at ventilatory anaerobic threshold, VO~2~ RCP: oxygen intake at respiratory compensation point, and W: ergometric performance. ###### Echocardiographic parameters. Vegan athlete, off-season Vegan athlete, active season Control group (n = 10) -------------- ------ --------------------------- ------------------------------ -------------------------- Aortic root cm 3.7 3.4 3.1 ± 0.4 Left atrium cm 3.0 3.0 2.6 ± 0.3 Left atrium ml 36 30 29.6 ± 8.6 LVEDD cm 5.0 5.4 4.64 ± 0.4 IVSD cm 1.2 1.1 1.2 ± 0.1 PWTD cm 1.2 1.0 1.2 ± 0.1 LVMM g 211.3 206.3 203.0 ± 35.3 LVMM g/m² 105.68 103.67 103.1 ± 14.5 EDV mL 119 140 100.2 ± 16.7 ESV mL 48 58 39 ± 6.1 SV mL 72 82 61.2 ± 11.2 EF \% 60 59 61.0 ± 2.1 SF \% 32 31 35.5 ± 9.4 MV E/A ratio 1.45 1.56 1.39 ± 0.20 EDV: end diastolic volume, EF: ejection fraction, ESV: end systolic volume, IVSD: intraventricular septum in diastole, LVEDD: left ventricular end diastolic diameter, LVMM: left ventricular muscle mass, MV E/A ratio: mitral valve E/A ratio, PWTD: posterior wall thickness in diastole, SF: shortening fraction, and SV: stroke volume. ###### Results of blood investigation of the vegan ultra triathlete. Analysis Off-season Active season ------------------------------------------- ------------ --------------- Leukocytes (1000 s/uL) 4.6 4.7 Erythrocytes (Mill·uL) 5.1 4.9 Haematocrit (%) 43 42 Haemoglobin (g/dL) 14.9 14.1 MCV (fl) 83 87 MCH (pg) 29.0 29.1 MCHC (g/dL) 35.1 33.3 Thrombocytes (1000 s/uL) 171 138 Alkaline phosphatase (U/L) 68 70 Glutamyl transpeptidase 8 15 Glutamate oxaloacetate transaminase (U/L) 36 40 Glutamate pyruvate transaminase (U/L) 32 42 Lactate dehydrogenase (U/L) 199 238 Amylase (U/L) 63 61 Lipase (U/L) 35 36 CK-NAC (U/L) 155 209 Protein (g/dL) 6.6 6.7 Cholesterol (mg/dL) 134 113 HDL (mg/dL) 47 51 LDL (mg/dL) 74 50 Triacylglyceride (mg/dL) 148 84 TSH basal (mU/L) 1.23 0.73 Iron (µg/dL) 95.0 82.0 Free testosterone (pg/mL) 5.5 5.7 Vitamin B12 (pg/mL) 329 347 Ferritin (ng/mL) 83.7 82.6 Folic acid (ng/mL) 16.8 14.3 [^1]: Academic Editors: G. Minardi and J. 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Introduction {#sec1-1} ============ Rubella is usually a mild self-limiting illness in children, presenting with fever and rash; however, in pregnant women, infection during the first 16 weeks of pregnancy can result in miscarriage, fetal death, or an infant born with congenital birth defects known as congenital rubella syndrome (CRS).\[[@ref1][@ref2][@ref3][@ref4]\] Rubella is of significant public health importance due to these teratogenic effects. Up to 60% of rubella cases may not present with a rash, many cases are not detected or reported. Seroprevalence surveys have documented widespread circulation of the rubella virus in all parts of the world.\[[@ref4]\] The presence of rubella-specific IgG in an unvaccinated population is a long-term marker of previous rubella infection and immunity status, and the antibodies persist life long, protecting the individual from further infections.\[[@ref1][@ref2]\] A study conducted in Tamil Nadu (South India) among unvaccinated girls aged 10--16 shows the presence of protective antibodies in 86.5%.\[[@ref1]\] A similar study in North India (mean age of 10.7 years) reported that 90% have protective antibodies.\[[@ref5]\] It was estimated that in India, about 50% of children acquire rubella antibodies by the age of 5 years and 80--90% become immune by 15 years by naturally acquired rubella.\[[@ref1][@ref5]\] All these studies were conducted before the widespread use of rubella vaccine in private sectors of India. Childhood exposure renders immunity to majority of women; however, periodic epidemics still occur among children and spread to involve a small portion of susceptible adult women, leading to epidemics of CRS.\[[@ref6]\] Several sero-epidemiological surveys from other countries have reported that a substantial number of women reach childbearing age without acquiring natural immunity to rubella, making them more susceptible to infection.\[[@ref4][@ref7][@ref8]\] Due to the high incidence of subclinical infections during childhood, eliciting a reliable history is difficult making seroprevalence studies using representative sampling from different age groups superior in determining the age groups for vaccination.\[[@ref4][@ref9]\] Indian newborns consist of 20% of annual birth cohorts of the world. Regardless, no country-wide estimates of CRS burden and susceptibility to rubella infection are available due to lack of a national surveillance and registry for rubella.\[[@ref2][@ref3][@ref10]\] There are no systematic reviews or nation-wide studies assessing the susceptibility of Indian population to rubella infection in general or specific to children or adolescent girls.\[[@ref10]\] The WHO evaluation studies validate the inclusion of childhood rubella vaccination as cost-effective in countries having measles vaccine coverage of more than 80%. Countries which can sustain the above coverage level can introduce rubella vaccination in their UIP program for the desired effects.\[[@ref3][@ref5]\] The latest data show that 140 of the 194 WHO member countries have incorporated rubella vaccine in combination with mumps and measles at the age of 12--18 months.\[[@ref11]\] Rubella and CRS control was the goal established in the South-East Asian region as an initial step toward elimination and many developed countries have been successful in doing so.\[[@ref11]\] Rubella vaccine is not yet included in the National childhood vaccination program (UIP) in India and it is given as an optional vaccine by private providers. This results in low coverage and thereby reduced herd immunity since only a small proportion of children from families who can afford the vaccine gets vaccinated. This unregulated use of vaccine may rise the "effective reproduction rate 'R' and ultimately lead to 'age shifting' and paradoxical increase of CRS in future."\[[@ref2][@ref3][@ref12]\] The state of Kerala, with best health indicators in the country, has been facing periodic epidemics of rubella among adult population in recent years. The Social Justice Department, with the help of Health and Education Department, in 2014, introduced the monovalent rubella vaccine among school girls but faced resistance from the community due to less perceived risks and resulted in low coverage (\<25%). Age-specific seroprevalence among girls, which the WHO considers to be a sensitive tool for risk assessment of CRS, has never been done here.\[[@ref3][@ref12]\] This will also help in understanding the degree of impact of private sector/optional vaccine. Rubella IgG estimation in children will provide data for the necessary modification in the immunization strategy. Although rubella and CRS are a public health problem, no studies were done from the state on this subject. With the aim to understand the age-specific susceptibility of acquiring rubella infections and future risk of CRS, estimation of rubella IgG antibodies among girls in the age group of 13--15 was conducted. Participants and Methods {#sec1-2} ======================== The study was conducted at a randomly selected rural area named Mavoor Panchayath of Kozhikode District, Kerala, among adolescent girls. Being the only high school in the area, with 100% school enrollment and zero dropouts, all the adolescent girls from the area were admitted in the selected school, hence representing the population of girls in the community. With an expected rubella-specific IgG prevalence of 80% and for precision of ± 5, the minimum sample size required was estimated using Epi Info software 7 World health organization (WHO) as 250.\[[@ref1]\] The protocol of the study was approved by the Institutional Ethics Committee. The study was conducted in Mavoor Government high school, among students of 8^th^ and 9^th^ standard (ages 13--15) with the permission of the school authorities and the parent--teacher association (PTA). All the girls studying in 8--9^th^ standards in the selected school (n = 280), whose parents have given written informed consent and accent by the girls, were enrolled as participants and they were selected consecutively according to the roll number in the school registers till we got the required sample size of 250. Data and blood sample collection was done by conducting screening camps in the school on fixed days from 10 am to 2 pm. Those who were absent on the fixed day, their data were collected during the subsequent visits. A standardized questionnaire was used by the interviewer to collect the demographic, morbidity, and immunization data (specifically measles-mumps-rubella \[MMR\]). Some of the variables used include history of exanthematous fever and past and recent hospitalizations. The variables include history of any exanthematous fever, recently or past and hospitalization. Medical examination was conducted to detect any disease such as fever/exanthema/lymph node enlargement and anomaly-cataract, deafness, and congenital heart disease to rule out rubella or features of CRS. From every participant, 5 ml venous blood was collected from the antecubital vein under aseptic precautions using vacutainers with gel separators. Serum was separated and brought to the microbiology department at the medical college in vaccine carriers maintained at a temperature of 4°C on the same day and stored at −20°C till further analysis was made. Rubella-specific IgG antibody was measured by quantitative enzyme-linked immunosorbent assay using BEIA Rubella IgG Quant Technogenetics SRL Italy kit, with a relative sensitivity and specificity of 100%. The tests were done using standard procedures by a laboratory technician with adequate experience under the supervision of a microbiologist (author). The results are expressed in IU/ml. As per the product manual (BEIA Rubella IgG Quant Ed. 01-09-2006), IgG titer of \>15 IU is considered positive, 8--15 as equivocal, and \<8 IU as negative. About 10% of the samples were retested at Kasturba Medical College, Manipal Laboratory, a central laboratory accredited by the National Accreditation Board for Testing Laboratories of the Department of Science and Technology, Government of India, and the validity and consistency were assured. The collected data were processed in an excel data sheet with codes, with personal anonymity, and statistical analysis was performed using SSPS program. Chi-square test was applied to find out significant difference and Fisher\'s exact test wherever applicable. Results {#sec1-3} ======= Data and blood sample collection was done from 250 girls. The age ranged from 13 to 15 with a mean age of 14.2 ± 0.7 years. IgG estimation was done on 224 samples (90%), and due to sample lysis, the rest was discarded. Out of which 3 girls had received MMR vaccine who were also excluded from the analysis, so 221 samples were finally included for the analysis. Age wise distribution of girls were 24.5%, 51%, 24.5% respectively in 13, 14 and 15 years. The mean IgG titer was 151.93 ± 128.78 IU, and as per the criteria, 68.3% were positive, 28.5% negative, and 3.2% equivocal \[[Table 1](#T1){ref-type="table"}\]. The class-wise details of IgG values are given in [Table 2](#T2){ref-type="table"}. There is no association between the titer values and the age of the participants (Pearson\'s correlation: 0.043, P = 0.53). ###### Prevalence of IgG antibody titer among girls ![](JFMPC-5-658-g001) ###### Class wise distribution of IgG antibody titer among girls ![](JFMPC-5-658-g002) The childhood immunization details were collected from parents by crosschecking immunization cards and it was found that 61% had given Bacillus Calmette--Guérin, 57% had given three doses of OPV and DPT, and 54% had given measles vaccine. Twenty-four girls had a history of mumps. None of the participants showed features of rubella (fever, rashes, and cervical nodes), congenital anomalies, or features of CRS on clinical examination. The susceptibility to rubella may considerably vary with socioeconomic strata. As a proxy of this, the educational status of both parents was collected, this revealed a 100% literacy rate. Nearly 87% of fathers and 85% of mothers have completed \>10 years of schooling. There was no variation of IgG status between the strata (P = 0.2, 0.4) in both parents meaning that rubella is uniformly transmitting among all income groups \[Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}\]. ![Relation of IgG tire values with father\'s education status\. \10th standard, Plus two, Degree, Postgraduate](JFMPC-5-658-g003){#F1} ![Relation of IgG tire values with mother\'s education status\. \10th standard, Plus two, Degree, Postgraduate](JFMPC-5-658-g004){#F2} Discussion {#sec1-4} ========== Documentation of rubella infection has been difficult due to the challenges in diagnosing the disease due to its subclinical and atypical presentations. Hence, serological surveys play a precise role in defining infectious disease epidemiology, especially in the case of rubella, in determining its potential health impact, and also to help policymakers to decide the vaccination policies to contain the consequences of the disease. In our study, even though girls were not immunized against rubella, 68.3% of the girls demonstrated a protective level of IgG by naturally acquired childhood infection. Since most of them were subclinical or with mild symptoms, their parents could not recall the past infections. The IgG prevalence reported here was less than reported earlier from Tamil Nadu (86.5%) Maharashtra (76%), and New Delhi (90%).\[[@ref1][@ref5][@ref6]\] This may be due to the improved socioeconomic status and higher standard of living in the state. A similar rate of prevalence (67.3%) was reported among girls aged 11--18 from Jammu and Kashmir.\[[@ref13]\] These figures throw light on the widespread reach of rubella among the children of the state, thereby rendering them lifelong immunity.\[[@ref4][@ref12]\] The IgG prevalence among girls did not differ significantly by religion, educational status, or family income, which is consistent with the reports from a hospital-based study previously conducted in the state.\[[@ref14]\] It is seen that more than 30% of the cohort was susceptible to rubella. It is noteworthy that the majority of girls get protection until they attain adolescence; however, not all were protected and therefore some girls may remain susceptible to infection during adulthood and pregnancy. The WHO reports that even when the susceptibility levels in women are below 10%, there is a chance of CRS in the future.\[[@ref15]\] With the buildup of such susceptible cohorts, pregnant women can acquire rubella infection from younger children, leading to epidemics among adult population. Studies from different states of the country on acquired rubella infection among pregnant women during pregnancy period reported 3%, 6.7%, and 8.3% prevalence from Kerala, Andhra Pradesh, and New Delhi, respectively, as evidenced by IgM estimation.\[[@ref14][@ref16]\] With an average annual incidence of 5 lakh deliveries, we could expect about 15,000 infections from Kerala among pregnant women and proportional CRS. Since rubella vaccine was not included in the childhood vaccination program in India only three of our subjects have gave history of previous rubella vaccination. They received it from private providers as an optional vaccine with out-of-pocket expenditure. Currently, about 15--25% of the child population are getting rubella vaccine (RA 27/3) combined with mumps and measles (MMR). Reviews have shown that RA 27/3 vaccine has a duration of protection ranging from 10 to 21 years in children with a seropositivity rate of 95%.\[[@ref9]\] As per the district level household surveys (DLHS I, II. III), the immunization coverage for the Kerala state is 84%, 78.6%, and 83%, with few districts below the state average, and at country average, it is only 65%.\[[@ref17]\] Hence, with the current status of subpopulation immunization leading to "age shifting," oscillating herd immunity, the probability of paradoxical rubella outbreaks among adult population and CRS, as occurred in Greece, cannot be ruled out in India.\[[@ref3][@ref11][@ref12][@ref18]\] This study shows that rubella virus infection is prevalent in Kerala in all socioeconomic strata, and more than 30% girls reach childbearing age without acquiring natural immunity against the disease. Studies conducted across India suggest similar baseline information on the susceptibility profile of women of childbearing age.\[[@ref19]\] Although rubella vaccine is safe and effective, clear policy regarding rubella immunization of children either at 15 months or young girls at 9--12 years has not been outlined in India. For controlling CRS, the "indirect strategy" is to immunize children and reduce transmission of rubella, and the "direct strategy" is to immunize adolescents to prevent rubella infection and CRS.\[[@ref18][@ref19][@ref20]\] The first may pose a risk to adults and the second cannot prevent rubella transmission as described, so both are needed in a combined, coordinated form. Consider a scenario, in which combination MMR vaccine has been introduced in the UIP of India. The state with expected average immunization coverage of 80% and vaccine efficacy of 95% will produce 24% birth cohort without immunity to rubella in each year. Considering the birth rate of 15 per thousand, within a 10--15 year period, it will produce a large number of rubella-susceptible youth population in the state, later leading to CRS. In other states where the immunization coverage is poor, the extrapolation of the data predicts a worse situation. As supported by the WHO, Gavi, Kerala, India, may introduce MMR vaccine in UIP for children in the near future. Many authors have warned that Rubella vaccination: must not be business as usual. The childhood immunization programs against rubella without specifically addressing the countries epidemiology will not achieve the desired results.\[[@ref19][@ref20][@ref21]\] To prevent these negative impacts, efforts should be taken to maintain the "herd immunity threshold" at a level above 83% among the adult population without regional variation within the country.\[[@ref3][@ref20][@ref21]\] Therefore, the best results can be achieved only by a combined immunization policy as adopted by Denmark, Sweden, most of the European countries, and the USA, where the first dose is offered as MMR vaccine at 15--18 months of age and the second dose as MMR vaccine at 6--12 years, or only rubella vaccine exclusively to girls at 12--14 years of age is best for India too to eliminate CRS in the near future along with surveillance of rubella.\[[@ref5][@ref18]\] Conclusion {#sec1-5} ========== Substantial numbers of women reach childbearing age without immunity against rubella and thus are at a risk of passing the infection to their fetuses, who can then develop subsequent congenital defects leading to CRS. An immunization policy recommending rubella-containing vaccine is highly desirable to prevent rubella and CRS. Financial support and sponsorship {#sec2-1} --------------------------------- This study was financially supported by the State Board of Medical Research, Government of Kerala. Conflicts of interest {#sec2-2} --------------------- There are no conflicts of interest. This study was funded by the State Board of Medical Research (SBMR) -- Directorate of Medical Education, Government of Kerala. The authors express thanks to the staff and PTA students of Mavoor, Government high school. Thanks to Dr. Thomas Bina, Head of the Department, Community Medicine, for the help, and Dr. Rema Devi, Former Head of the Department of Microbiology, for providing laboratory support for the study.	{ "pile_set_name": "PubMed Central" }	[ "cervic", "rest", "stateref", "born", "probabl", "two", "unregul", "control", "given", "program", "remain", "household", "strata", "collect", "discard", "thank", "longterm", "pearson", "incom", "risk", "written", "classwis", "select", "fund", "iu", "student", "week", "valu", "fix", "opv", "lysi", "indirect", "may", "therebi", "recent", "fetus", "live", "acquir", "toward", "separ", "use", "gener", "dropout", "socioeconom", "final", "support", "srl", "world", "complet", "accord", "refrefref", "signific", "depart", "establish", "temperatur", "becom", "mother", "exposur", "suggest", "paradox", "process", "excel", "also", "epidemiolog", "shift", "state", "surveil", "difficult", "vaccin", "dlh", "conflict", "women", "kerala", "perform", "popul", "sustain", 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Figure 1Cells lacking some nucleoporins die on FOA because they derepress a telomeric URA3 gene.Telomere association with nuclear pores is critical not only for transcriptional silencing but also for efficient repair of double-stranded breaks in the subtelomeric region of budding yeast chromosomes, according to Therizols et al. (page 189). As in many organisms, yeast telomeres localize to the nuclear periphery. To determine whether nuclear pore proteins are involved in telomere tethering, Therizols et al. looked for telomere localization in cells lacking functional Nup84 complexes, which are essential components of the pore. They found that the telomeres no longer associated with the nuclear periphery in these mutants. As might be expected from previous work on transcriptional silencing, transgenes located in the subtelomeric region were no longer silent in Nup84 complex mutants, indicating that localization of the telomere to the nuclear periphery was functionally important. Surprisingly, when double-stranded breaks were introduced into subtelomeric sites in the mutants, DNA repair efficiency dropped significantly relative to wild-type cells. The efficiency of break repair in central regions of chromosomes did not differ between wild-type and mutant cells. The DNA silencing and repair phenotypes were separated in cells mutant for Esc1p, a protein located at the nuclear periphery but which is not directly involved in the pore. In this case, telomere localization and DNA repair were disrupted, but silencing remained intact. The researchers conclude that anchoring telomeres to the nuclear pore is important for efficient DNA double-stranded break repair in the subtelomeric regions, though it is not yet clear why this is true. Because silencing remained intact in the Esc1p mutants but repair was disrupted, it appears that chromatin structure itself is not the problem. 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All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Small non-coding RNAs (sRNAs) of 20\~30 nucleotides (nt) have gained significant attention in recent years as they are widely involved in various biological processes such as the embryonic, neuronal, muscle, and germline development \[[@pone.0159487.ref001]--[@pone.0159487.ref003]\]. QDE-2-interacting small RNAs (qiRNAs) of typically 20\~21 nt are a new class of sRNAs. qiRNAs are induced by DNA damage \[[@pone.0159487.ref004]\] and mediate gene silencing in the DNA damage response (DDR) pathway in Neurospora crassa by inhibiting protein translation \[[@pone.0159487.ref004]\]. Although qiRNAs were first discovered in 2009, only little is known about their biogenesis and functionality. It has been demonstrated that the biogenesis of qiRNAs requires DNA-damage-induced aberrant RNAs (aRNAs) as precursors \[[@pone.0159487.ref004]\]. Moreover, RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3, as well as dicers have been previously shown to be involved in the production of qiRNAs \[[@pone.0159487.ref004]\]. After DNA damage, aRNAs are highly induced and then specifically recognized by RNA-dependent polymerases to produce double-stranded RNAs (dsRNAs). Subsequently, dsRNAs are converted to qiRNAs by dicers \[[@pone.0159487.ref004], [@pone.0159487.ref005]\]. However, the detailed mechanism for the generation of qiRNAs is largely unknown and requires elucidation in further experimental studies. Previously, novel qiRNAs in Neurospora crassa \[[@pone.0159487.ref004]\] or rice were identified almost exclusively by immunoprecipitation followed by sequencing. However, this conventional approach for the identification of qiRNAs is time-consuming, labor-intensive, and inefficient and even holds the risk of not detecting lowly expressed or issue-specific qiRNAs. Computational methods can overcome these experimental hurdles and extract general information from known qiRNAs to predict novel qiRNAs for further experimental manipulation \[[@pone.0159487.ref006]\]. However, as far as we know, no qiRNA prediction tool has been implemented so far. Therefore, the development of efficient computational approaches for qiRNA prediction is urgently needed and intriguing. Common sequence or structure conservation-based approaches in microRNAs (miRNAs) prediction cannot be directly adopted for qiRNA prediction as no evidence has been reported for a high evolutionary conservation of the sequence and structure of qiRNAs across species. Unlike Piwi-interacting RNAs (piRNAs), the density of plot of qiRNAs does not show a striking clustering characteristic \[[@pone.0159487.ref004]\]. Thus, no clustering characteristic can be used for qiRNA prediction. However, it has been demonstrated that qiRNAs exhibit strong position-specific preferences for uracil (U) at the first nucleotide of the 5' end and for adenine (A) at the first nucleotide of the 3' end \[[@pone.0159487.ref004]\]. In bioinformatics, position-specific nucleotide preferences are usually employed for sRNAs prediction. For example, ping-pong-dependent piRNAs show some position-specific nucleotide preferences, such as for thymine (T) at the first position (1T) and for A at the 10th position (10A) \[[@pone.0159487.ref006]\]. To capture and leverage these sequence features for piRNA prediction, Betel et al. constructed a 21 × 4 feature vector based on a 21-base window around the 5' end (plus 10 nt upstream and 10 nt downstream) and trained a support vector machine (SVM) model for piRNA prediction \[[@pone.0159487.ref007]\]. In 2011, Zhang et al. used a simple k-mer scheme to construct 1364 dimension feature vectors for describing the candidate piRNA sequences and then constructed the software piRNApredictor for piRNA prediction based on an improved Fisher linear algorithm \[[@pone.0159487.ref006]\]. Moreover, position-specific scoring matrix (PSSM) is a commonly used representation of sequence motifs, which has been widely applied to the prediction of significant biological signals such as DNA binding sites \[[@pone.0159487.ref008]\], RNA binding sites \[[@pone.0159487.ref009]\], promoters \[[@pone.0159487.ref010]\], protein secondary structure prediction \[[@pone.0159487.ref011]\], etc. In this work, we developed an ingenious method for the extraction of features based on position probability matrices (PPM) of biosequences and then constructed a novel qiRNApredictor (qiRNA predictor) software package with 80 features. Firstly, the experimentally verified qiRNAs were collected manually from the scientific literatures and then used for training the Random forest (RF) model using the randomForest R package. Subsequently, the performance and robustness of qiRNApredictor were extensively evaluated by n-fold cross-validations (CV) as well as the leave-one-out cross-validation (LOO-CV). Upon LOO-CV, qiRNApredictor exhibits a promising sensitivity of 93.55% and a specificity of 71.61%, which can be used for the prediction of qiRNAs. Materials and Methods {#sec002} ===================== Data preparation {#sec003} ---------------- Herein, only qiRNA sequences in Neurospora crassa were considered (no qiRNA in rice was sequenced in the work by Chen et al. \[[@pone.0159487.ref005]\]). Lee et al. identified 184 individual qiRNA sequences in Neurospora crassa \[[@pone.0159487.ref004]\]. However, 23 qiRNA sequences were directly discarded as they included undefined bases. We further eliminated six individual qiRNA sequences as they were identical to other qiRNA sequences. Finally, 155 experimentally verified qiRNAs were collected as positive samples to train a RF model. Three negative datasets were built. For the convenience of discussion, we called them the "Random", the "sRNA-segment", and "milRNA" negative datasets, according to the ways we collected them or the data origin. The "Random" negative dataset was randomly extracted from NONCODE database. As previously described \[[@pone.0159487.ref006]\], non-coding RNA sequences obtained from the NONCODE database (version 3.0) \[[@pone.0159487.ref012]\] were fragmented into non-overlapping segments. For each of these non-qiRNA segments, we shuffled it 10000 times to destroy any potentially functional structures \[[@pone.0159487.ref006]\]. Then, the same amount of negative samples were randomly selected from the segments under the constraint condition that the length distribution of the selected segments was identical with that of positive qiRNAs. The "sRNA-segement" negative dataset was collected from Rfam database. The sequences of sRNAs of Neurospora crassa were retrieved from Rfam database (release 12.0) \[[@pone.0159487.ref013]\]. Because the lengths of sRNAs are much longer than that of qiRNAs, we fragmented the sequences of sRNAs into non-overlapping segments under the constraint that the length distribution of sRNA segments was similar to that of positive qiRNAs. Then, the same amount of sRNA segments were randomly selected and regarded as the analogue of the degraded fragments of sRNAs. miRNA-like small RNAs (milRNA) are about 19\~25 nt and have a strong preference (51.08%) for U at the 5' end. They were firstly discovered in Neurospora crassa and called milRNAs due to their similarities with miRNAs \[[@pone.0159487.ref014]\]. Furthermore, 325 milRNAs were manually collected from the scientific literatures to construct the "milRNA" negative dataset. Since only 25 milRNAs were obtained from Neurospora crassa, we also collected the putative milRNAs from other fungi species, including Trichoderma reesei \[[@pone.0159487.ref015]\], Sclerotinia sclerotiorum \[[@pone.0159487.ref016]\], Metarhizium anisopliae \[[@pone.0159487.ref017]\], Zymoseptoria tritici \[[@pone.0159487.ref018]\], Fusarium oxysporum \[[@pone.0159487.ref019]\], Fusarium graminearum \[[@pone.0159487.ref020]\], Antrodia cinnamomea \[[@pone.0159487.ref021]\], Aspergillus flavus \[[@pone.0159487.ref022]\], Penicillium chrysogenum \[[@pone.0159487.ref023]\], and Penicillium marneffei \[[@pone.0159487.ref024]\]. Finally, these three negative datasets were incorporated with the positive dataset respectively to construct the "Random", "sRNA-segment" and "milRNA" training datasets. Feature extraction {#sec004} ------------------ The extraction of an appropriate set of features for training a prediction model is one of the vital, yet most challenging issues in machine learning-based prediction approaches. To capture the position-specific preference of nucleotides, the occurrence probabilities of each nucleotide at the first ten positions (1 through 10) and the last ten positions (-1 to -10) of both positive and negative samples were calculated to create four PPMs ([Fig 1](#pone.0159487.g001){ref-type="fig"}). It should be noted that qiRNA sequences of less than 20 nt exhibit some overlaps of the first and last ten positions. The score of nucleotide i at position j can be calculated according to the formula $$\text{S}\left( {\mathbf{i},\mathbf{j}} \right) = \text{log}_{2}\left( \frac{\mathbf{P}_{ij}}{\mathbf{N}_{ij}} \right),\text{ }\mathbf{i} \in \left\lbrack {\text{A},\text{C},\text{G},\text{U}} \right\rbrack,\text{ }\mathbf{j} \in \left\lbrack {- 10,\cdots, - 1;\, 1,\cdots,10} \right\rbrack$$ where *P~ij~ and N~ij~ are the probability of nucleotide i* at position j in PPMs produced by positive and negative samples, respectively ([Fig 1](#pone.0159487.g001){ref-type="fig"}). The score gives an indication how much the position-specific preference of positive samples differs from that of negative samples. For the cases with *P~ij~ = N~ij~, the score equals zero, which means this feature cannot offer any information for the prediction. However, when P~ij~ or N~ij~ is zero, it doesn't mean this feature has great significance. Instead, it may be simply because of the limited sample size. To circumvent the infinite value caused by inadequate sampling, we assigned a zero score for the cases that P~ij~ or N~ij~ is zero. In total, 80 scoring values were directly regarded as features to train a RF model. Then F-score was used to measure the discriminatory power of each feature above \[[@pone.0159487.ref025]\]. The F-score of the ith feature is defined as: $$F\left( i \right) \equiv \frac{\left( {{\overline{x}}_{i}^{( + )} - {\overline{x}}_{i}} \right)^{2} + \left( {{\overline{x}}_{i}^{( - )} - {\overline{x}}_{i}} \right)^{2}}{\frac{1}{n_{+} - 1}{\sum\limits_{k = 1}^{n_{+}}\left( {x_{k,i}^{( + )} - {\overline{x}}_{i}^{( + )}} \right)^{2}} + \frac{1}{n_{-} - 1}{\sum\limits_{k = 1}^{n_{-}}\left( {x_{k,i}^{( - )} - {\overline{x}}_{i}^{( - )}} \right)^{2}}}$$ where n~+~ and n~−~ are the numbers of positive and negative samples, respectively; ${\overline{x}}_{i}$, ${\overline{x}}_{i}^{( + )}$, and ${\overline{x}}_{i}^{( - )}$ are the average of the ith feature of total, positive, and negative samples, respectively. $x_{k,i}^{( + )}$ and $x_{k,i}^{( - )}$ are the ith feature of the kth positive and negative sample, respectively. Larger F-scores indicate better discrimination \[[@pone.0159487.ref025]\]. ![The flowchart of qiRNApredictor.](pone.0159487.g001){#pone.0159487.g001} Random forest {#sec005} ------------- RF is an ensemble learning classifier consisting of a multitude of tree-structured classifiers \[[@pone.0159487.ref026]\]. RF makes use of two powerful machine-learning methods---the bagging and random feature selection in the tree induction \[[@pone.0159487.ref027]\]. In the bagging algorithm, each tree is trained by using a bootstrap sample of the training data, and the prediction results of the ensemble are aggregated by majority vote or averaging rule to give the final prediction results \[[@pone.0159487.ref027]\]. Instead of using all features, only a random subset of features was used here to split at each node when growing a single tree. A type of CV together with the training step using out-of bag (OOB) samples was used to assess the prediction performance of RF. More specifically, a particular bootstrap sample was adopted to grow each tree during the process of training. Since bootstrapping sampling is sampling with replacement, some of the samples were ignored, while others were reused. The ignored samples constitute the OOB sample. On average, 1−e^−1^ ≅ 2/3 of the training samples was used for growing the tree leaving e^−1^ ≅ 1/3 as OOB, which have not been used for tree construction. Therefore, these samples could be used to evaluate the prediction performance \[[@pone.0159487.ref026], [@pone.0159487.ref027]\]. The RF algorithm was implemented by the randomForest R package. Performance evaluation {#sec006} ---------------------- As previously described \[[@pone.0159487.ref028]\], among the predicted positive results obtained by qiRNApredictor, the real positives are called true positives (TP), while the other positive results are called false positives (FP). Among the predicted negative results obtained by qiRNApredictor, real negatives are called true negatives (TN), while the other negative results are called false negatives (FN). The performance is evaluated based on four measurements of specificity (Sp), sensitivity (Sn), accuracy (Ac), and Matthew's correlation coefficient (MCC). These indexes are defined as $$Sn = \frac{TP}{TP + FN},\, Sp = \frac{TN}{TN + FP},\, Ac = \frac{TP + TN}{TP + FP + TN + FN}$$ and $$MCC = \frac{\left( {TP \times TN} \right) - \left( {FN \times FP} \right)}{\sqrt{\left( {TP + FN} \right) \times \left( {TN + FP} \right) \times \left( {TP + FP} \right) \times \left( {TN + FN} \right)}}$$ In this study, we performed 4-, 6-, 8-, and 10-fold CVs as well as the LOO-CV. Receiver Operating Characteristic (ROC) curves were plotted for performance visualization. Results {#sec007} ======= A novel algorithm for qiRNA prediction {#sec008} -------------------------------------- In this work, we collected experimentally identified qiRNAs from the scientific literatures. After eliminating redundancy in sequences and directly removing sequences that include undefined bases, a dataset of 155 experimentally verified qiRNA sequences was obtained ([S1 Table](#pone.0159487.s002){ref-type="supplementary-material"}). As previously described, a negative dataset containing 155 samples was constructed. Finally, a balanced database containing 155 positive and negative samples was used for model construction. qiRNAs are very short of approximately 20\~21 nt in length ([Fig 2](#pone.0159487.g002){ref-type="fig"}), which renders it very difficult to predict qiRNAs with high accuracy. However, the first and last nucleotide of qiRNAs have a high preference for U and A, respectively ([Fig 3](#pone.0159487.g003){ref-type="fig"}). Therefore, we attempted to predict qiRNAs with the position-specific preferences of nucleotides in qiRNA sequences. To this goal, PPMs were firstly constructed for characterizing position-specific properties of qiRNAs. Based on the PPMs, 80 log-likelihood scores were calculated as features for training prediction model (see details in [Materials and Methods](#sec002){ref-type="sec"}). Based on the training datasets, we used the F-score \[[@pone.0159487.ref025]\] to rank 80 features. As expected, nucleotides at the first and last position, such as 1U, 1A, 1C, or -1A, exhibit high F-scores ([Fig 4](#pone.0159487.g004){ref-type="fig"}). This is consistent with the position-specific preferences of nucleotides in qiRNA sequences, which demonstrates that we have succeeded in capturing these characteristics in qiRNA sequences. ![The length distribution of qiRNAs in Neurospora crassa.](pone.0159487.g002){#pone.0159487.g002} ![Position-specific nucleotide preferences of qiRNAs in Neurospora crassa* for (A) the first 15 nt substring from 5' end to 3' end and (B) the last 15 nt substring from 5' end to 3' end.\ The insets are Frequency Plot of qiRNA sequences. The sequence logo analysis was implemented by WebLogo \[[@pone.0159487.ref029]\].](pone.0159487.g003){#pone.0159487.g003} ![The F-scores of PPM features.\ Larger F-scores indicate better discrimination.](pone.0159487.g004){#pone.0159487.g004} To evaluate the performance and robustness of the qiRNApredictor, LOO-CV and 4-, 6-, 8-, and 10-fold CVs were performed. The LOO-CV results based on "Random" training dataset show that our method predicts at 93.55% sensitivity, 71.61% specificity, 82.58% accuracy and 0.6679 MCC value ([Table 1](#pone.0159487.t001){ref-type="table"}). The results of the 4-, 6-, 8-, and 10-fold CVs are also close to those of the LOO-CV. From the ROC curves ([Fig 5A](#pone.0159487.g005){ref-type="fig"}), AUC (area under ROC curves) values were calculated as 0.8779 (LOO-CV), 0.8772 (4-fold CV), 0.8752 (6-fold CV), 0.8761 (8-fold CV), and 0.8765 (10-fold CV), respectively ([Fig 5A](#pone.0159487.g005){ref-type="fig"}). As no qiRNA prediction tool existed to date, the performance of our qiRNA prediction tool could not be compared to any existing tool. To further test the ability of qiRNApredictor in distinguishing qiRNAs among other sRNAs, we also run it through "sRNA-segement" and "milRNA" datasets. milRNA is 19\~25 nt in length with a strong preference (51.08%) for U at the 5' end, which is somewhat similar to qiRNA. As shown in [Fig 5B](#pone.0159487.g005){ref-type="fig"} & [Table 1](#pone.0159487.t001){ref-type="table"}, the results demonstrate that the qiRNApredictor is able to identify qiRNAs among other sRNAs with quite significant AUC, MCC, sensitivity and specificity. Taken together, qiRNApredictor is a promising tool for the prediction of candidate qiRNAs. 10.1371/journal.pone.0159487.t001 ###### The performances of qiRNApredictor on each training dataset by the LOO CV. ![](pone.0159487.t001){#pone.0159487.t001g} Dataset Ac (%) Sn (%) Sp (%) MCC -------------------------------------- ---------- ---------- ---------- -------- "Random" training dataset 82.58 93.55 71.61 0.6679 "sRNA-segement" training dataset 80.97 86.45 75.48 0.6231 "milRNA" training dataset 79.17 70.32 83.38 0.5303 ![The prediction performance of qiRNApredictor.\ (A) The LOO-CV as well as 4-, 6-, 8-, and 10-fold CVs based on "Random" training dataset were calculated. The ROC curves and AUCs were also drawn and analyzed. To increase the specificity, we recommended a stringent cut-off value 0.667 for experimental investigation. The specificity of qiRNApredictor with the cut-off value 0.667 is 92.26%, while the sensitivity is 54.84%. (B) The ROC curves and AUCs were drawn and analyzed for the "Random", "sRNA-segment", and "milRNA" training datasets, respectively, by LOO-CV.](pone.0159487.g005){#pone.0159487.g005} Comparison to k-mer feature classes {#sec009} ----------------------------------- In bioinformatics, k-mers usually refer to k-gram or k-tuples of DNA or protein sequences and can be used to find certain regions within biosequences or be employed as k-mer statistics for giving discrete probability distributions of possible k-mer combinations \[[@pone.0159487.ref006]\]. K-mers can be used to distinguish qiRNA from non-qiRNA based on differences of string usages between the different sequence classes. Here, 1--5 nt strings were used to characterize positive or negative sequences by a vector consisting of the frequencies of k-mer strings. We first adjusted the k parameter in k-mer feature classes to obtain a better performance based on the same training dataset. With increasing k values, the AUC values exhibit first a rapid increase, which slows down in the following ([Fig 6A](#pone.0159487.g006){ref-type="fig"}). Although the increase of features may result in over-fitting, the best performance (AUC = 0.7790) of k-mer feature classes is lower than that of PPM features classes (AUC = 0.8779) ([Fig 6B](#pone.0159487.g006){ref-type="fig"}). This demonstrates that PPM is more suitable for qiRNA prediction. Moreover, we combined PPM and k-mer feature classes for the prediction of qiRNAs. However, the prediction performance of the combined feature classes exhibits to be very close to that of single PPM feature classes ([Fig 6B](#pone.0159487.g006){ref-type="fig"}). ![Comparison to k-mer feature classes.\ (A) Effect of the k parameter in k-mer feature classes on the prediction performance by 5-fold CV; (B) The ROC curves for RFs trained with different feature classes.](pone.0159487.g006){#pone.0159487.g006} Discussion {#sec010} ========== As a new regulatory factor for mediating gene silencing in the DNA damage response, qiRNAs have increasingly attracted considerable interest and investigative efforts. The identification of qiRNAs is fundamental for dissecting regulatory functions and molecular mechanisms. Compared to other expensive and time-consuming experimental methods, the computational prediction of qiRNAs is a conveniently rapid method of getting useful information for subsequent experimental verification. However, no qiRNA prediction tool existed to date. Therefore, developing a novel approach for qiRNA prediction is required and very intriguing. In this work, we designed a novel software named qiRNApredictor based on PPM features and RF algorithm. The performance and robustness of qiRNApredictor were extensively evaluated by n-fold CVs as well as LOO-CV, which gave very promising results. Here, the window size refers to the length of the region of interest at both ends of the sequences. To evaluate the effect of window size on the prediction performance, the window size was adjusted, and the AUC value was calculated based on the results of 5-fold CV. The AUC values first rapidly increase and then slow down with an increasing window size ([Fig 7](#pone.0159487.g007){ref-type="fig"}). There is a qiRNA sequence of only 15 nt, which was simply ignored when testing the window of 16 nt. Interestingly, the combination of two adjacent bases at both ends of sequences obtained a high AUC value of 0.8357. This either demonstrates that the characteristic signal at both ends of the qiRNA sequences is very significant, or it suggests that non-qiRNAs are not very similar to genuine qiRNAs. However, the approach of non-qiRNAs construction is standard according to previous studies \[[@pone.0159487.ref006]\]. ![Effect of the window size in PPM feature classes on the prediction performance by 5-fold CV.](pone.0159487.g007){#pone.0159487.g007} The performance of a classifier system would be improved by selecting the most discriminative set of features due to reducing the complexity of the classifier system. In contrast, the simple combination of PPM and k-mer feature classes does not improve the performance of the classifier system. This suggests that the feature set exhibits redundancies, which leaves room for optimization of the feature set used in qiRNApredictor in the subsequent work. Furthermore, the prediction performance of qiRNApredictor would be improved by finding the secondary structure features of qiRNA precursors. Taken together, our studies provide a novel and promising approach for qiRNA prediction and will facilitate further functional studies of qiRNAs. Supporting Information {#sec011} ====================== ###### The compressed files in ZIP format of the local package of qiRNApredictor. (ZIP) ###### Click here for additional data file. ###### The dataset of 155 experimentally verified qiRNAs in Neurospora crassa obtained from the work by Lee at al \[[@pone.0159487.ref004]\]. (XLS) ###### Click here for additional data file. The authors thank Dr. Ya Jia (CCNU) and Dr. Anbang Li (CCNU) for their helpful suggestions on the project implementation. [^1]: Competing Interests:The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: YY HD QL. Performed the experiments: YY HD. Analyzed the data: YY HD QL MY. Contributed reagents/materials/analysis tools: YY HD QL WC RG CM MY. 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By analyzing SNP array data on "trios" samples including premalignant atypical adenomatous hyperplasias (AAH) of the lung, matched normal and lung adenocarcinoma (LUAD) tissues from 16 patients, Sivakumar et al. \[[@bb0005]\] in this issue investigated the allelic imbalance (AI) events in the premalignant lesions of lung adenocarcinoma. With a modified statistic method to infer the AI events sensitively, they identified AI events in more than 50% of AAH tissues, indicating the importance of chromosomal aberrations and genome instability in the development of AAH. In addition, they identified a number of chromosomal aberrations harboring tumor suppressors and oncogenes, such as 17p loss affecting well-known TP53. It was worth to note that the same group previously reported the somatic mutations and RNA-Seq data from the same samples \[[@bb0010]\]. Thus, they presented a comprehensive molecular landscape of the evolution process from AAH to LUAD, which is important for understanding the etiology of LUAD. First of all, the premalignant data provided evidence that chromosomal aberrations initiated at the early stage of LUAD and emerged even earlier than many driver mutations. Both somatic mutations and copy number aberrations were the hallmarks of cancer. The TCGA study revealed a heavy burden of somatic mutations and copy number alterations in LUAD and most of them are "passengers" that have no effect on the neoplastic process \[[@bb0015]\]. Thus, it is difficult to identify which somatic alterations are drivers that promote a selective growth advantage. Previous studies suggested that genes carried "driver" alterations had a higher alteration rate than other genes and proposed statistical methods to identify driver genes \[[@bb0020]\]. By using the methods, TCGA studies reported that TP53 was a commonly mutated (46%) gene in LUAD \[[@bb0025]\]. In addition, somatic alterations usually affect oncogenes in the receptor tyrosine kinase (RTK)/RAS/RAF pathways in LUAD and tumor suppressor genes including STK11, KEAP1, NF1, RB1, and CDKN2A \[[@bb0025]\]. They also observed copy number amplified regions (3q26, 5p15, 7p11, 8q24, 11q13, 12q13, 12q15, 14q13) and deleted regions (17p13, 9p21, 9p23) in LUAD \[[@bb0025]\]. However, it is difficult to decipher the causal relationship between somatic mutations and copy number alterations in the cancer genomics data. With the evidence from AAH, we can differentiate the initial driver alterations of LUAD like 17p loss involving TP53. Furthermore, in this issue Sivakumar et al. \[[@bb0005]\] reported an important association between AI burden and smoking behavior in AAH. Interestingly, a similar trend was observed in their matched LUAD samples, but the difference was not significant, and at the same time, a higher AI burden was seen in EGFR mutated non-smokers. The results suggested that the underlying mechanisms of AI were quite different in smokers and non-smokers: AI served as a founding event in smoking patients and initiated the development of LUAD, while it was only a bypass product of genome instability in non-smoker patients and therefore occurred only in LUAD tissues. In the phylogenetic tree construction, Sivakumar et al. \[[@bb0005]\] found that 6 of 7 patients with evidence of shared AI between matched AAH and LUAD were smokers, which further supported the conclusion above. Thus, the AI events in smokers and non-smokers should not be considered equally in the following studies. However, the sample size involved in this analysis is relatively small and more well-designed epidemiological studies with larger sample size are warranted to further illuminate the differences. The molecular landscapes of premalignant tissues could also be used to identify potential biomarkers to predict the progress from the prelesions to cancers. Teiseira et al. recently profiled the genomic, transcriptomic, and epigenomic landscape of the prelesion of lung squamous cancer \[[@bb0030]\]. With longitudinally monitoring information, they successfully generated a predictive model to identify which lesions will progress with remarkable accuracy, which offered a new strategy for the early detection of lung cancer. Lastly, the public available multi-omics data from "trios" LUAD patients are a valuable resource for the following studies of LUAD. For instance, we recently conducted whole-genome sequencing and RNA-Seq on 90 lung cancer and observed an association between infiltrating B cells and EGFR mutations in tumor tissues of LUAD \[[@bb0035]\]. We performed a re-analysis of the RNA-Seq data from the previous Sivakumar et al.\'s paper \[[@bb0010]\] and found that the elevation of infiltrating B cells and inflammatory microenvironments preceded the emergence of EGFR mutations \[[@bb0035]\]. However, there are still great challenges to investigate the evolution process from AAH to LUAD. Intratumor heterogeneity of lung cancer was one of them. A recent study conducted whole-exome sequencing on the multi-regions of non-small-cell lung cancer (NSCLC) \[[@bb0040]\]. They observed widespread intratumor heterogeneity for somatic copy-number alterations as well as mutations. A number of known driver alterations occurred in the sub-clones of LUAD. To clearly infer the clonal evolution and trace the origin of these alterations, a multi-region sampling of AAH are necessary for future studies. In addition, the low cell fraction of somatic alterations in AAH also limited the genomic studies and the subsequent evolution analysis. The authors overcome the challenge by jointly modeling the allelic imbalance statistics, but the technology advance may provide new insights into the era. Single-cell RNA-Seq has recently used to decipher the microenvironments and subclones of lung cancers \[[@bb0045]\] and additional methods have been developed to estimate the copy number alterations and copy-neutral loss-of-heterozygosity events in single-cell RNA-Seq data \[[@bb0050]\], which might be used in the future studies of AAH to solve the problems of cell heterogeneity. Conflicts of interest {#s0005} ===================== The authors declare no conflicts of interest.	{ "pile_set_name": "PubMed Central" }	[ "ai", "mutat", "multiom", "pathway", "small", "welldesign", "howev", "tp", "cancer", "origin", "tumor", "smoke", "larger", "resourc", "share", "one", "adenocarcinoma", "well", "seen", "jointli", "trio", "conduct", "trace", "infiltr", "driver", "keap", "causal", "affect", "method", "understand", "gene", "identifi", "differenti", "microenviron", "aberr", "includ", "data", "new", "comprehens", "select", "associ", "preles", "lastli", "mechan", "loss", "heavi", "nsclc", "match", "furthermor", "may", "lossofheterozygos", "biomark", "valuabl", "multiregion", "sensit", "advantag", "burden", "recent", "imbal", "q", "model", "luad", "evolut", "promot", "chromosom", "bypass", "wholegenom", "effect", "tissu", "phylogenet", "warrant", "squamou", "use", "gener", "overcom", "etiolog", "preced", "snp", "fraction", "conclus", "support", "cdkna", "still", "commonli", 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All relevant data are within the manuscript. The measurement data are available from the figshare database (doi: [10.6084/m9.figshare.8088167](https://doi.org/10.6084/m9.figshare.8088167)). Introduction {#sec001} ============ The chance of being struck by lightning increases when humans are outdoors in a thunderstorm. They are especially exposed to lightning strikes directly in the head in open elevated areas \[[@pone.0223133.ref001]--[@pone.0223133.ref003]\]. A direct lightning strike can cause physical damages like burns on the skin, fractures in the skull, or haemorrhage in the brain due to the heat and explosive effects, as described in several pathological diagnosis reports and other literature \[[@pone.0223133.ref004]--[@pone.0223133.ref010]\]. These effects are caused by currents up to 200 kA and voltages of 1 MV and higher in discharges of a few milliseconds \[[@pone.0223133.ref011]--[@pone.0223133.ref013]\]. Overall, the mortality of lightning injury is only about 10% \[[@pone.0223133.ref014],[@pone.0223133.ref015]\] but increases to about 30% when people are struck by lightning outdoors \[[@pone.0223133.ref001],[@pone.0223133.ref002]\]. There are five known mechanisms of lightning injury: direct strikes accounting for 3--5% of the cases, side flashes for 30--35%, contact injury 3--5%, upward streamer 10--15% and ground current 50--55% \[[@pone.0223133.ref003],[@pone.0223133.ref015]\]. A possible mechanism responsible for the relatively large chances of surviving a lightning strike despite the mentioned medical issues could be the formation of a flashover outside the body. In a flashover, most of the current flows outside the body and only a few Amperes flow inside the body \[[@pone.0223133.ref012],[@pone.0223133.ref013]\], as estimated in a theoretical study by Uman \[[@pone.0223133.ref012]\]. This reduced current inside the body during a flashover might be small enough to not be lethal. This might be especially true for the head owing to the assumed additional protective effect of the skull caused by its low conductivity. The head is one of the most important parts of the human body and is most exposed during a direct strike or side flash. However, there are no reports in the literature on measured current distributions inside the head before and during a flashover in a direct lightning strike. Consequently, we aim to establish an experimental setup to measure current distributions for the main compartments of the human head using a physical head phantom. With these measurements, we want to establish normative values that can serve as a basis for later experimental investigations on the possibilities of lightning protection equipment for humans according to the standard DIN EN 1149 \[[@pone.0223133.ref016]\]. To achieve this aim, we have designed human head phantoms comprising scalp, skull, and intracranial compartments, mimicking the geometry, relative permittivity, and electric conductivity of real biological human tissue. In contrary to the homogeneous models used previously to investigate the flashover mechanism \[[@pone.0223133.ref017]\] the nonhomogeneous head phantoms allow to quantify the current distribution in the three compartments. Moreover, the head phantoms allow a qualitative analyses of visible damages per compartment. Consequently, we will be able to experimentally quantify the additional protective effect of the skull caused by its low conductivity. In addition to the currents flowing in the three head compartments, we measured the flashover current with an electrode outside and underneath the head phantom. With these measurements, we strive to obtain an experimental validation of the theoretical values proposed by Uman \[[@pone.0223133.ref012]\]. Materials and methods {#sec002} ===================== Overview {#sec003} -------- We applied pulses to the head phantoms using three different pulse generators with different properties. A Marx generator delivered up to 450 kV and relatively low currents of up to 2 kA with an 1.2/50 μs voltage pulse; a combination wave generator delivered up to 12 kV and 6 kA with low noise (due to the compact design) and 1.2/50 μs voltage pulse or 8/20 μs current pulse; a 10/350 μs current pulse generator delivered up to 12 kV and 42 kA. The various generators emulated different aspects of a lightning flash. For example, the standardized positive first lightning current is defined by front time 10 μs, the maximum amplitude and time to half-value on tail 350 μs, identified as 10/350 μs according to the standard IEC 62305 \[[@pone.0223133.ref018]\]. The equivalent frequency is 25 kHz for the front of this pulse. [Fig 1](#pone.0223133.g001){ref-type="fig"} illustrates the calculation of the equivalent frequency. After the peak, the frequency content decreases to 1 kHz and lower \[[@pone.0223133.ref011]\]. ![Calculation of the equivalent front frequency of a lightning pulse current.\ The front time (T~1~) of the 10/350 μs lightning current is 10 μs. The equivalent frequency (f~eq~) is based on a sine wave and the corresponding equivalent period (T~eq~).](pone.0223133.g001){#pone.0223133.g001} Creation of head phantom {#sec004} ------------------------ We created three-compartment head phantoms based on the computer tomography data of an individual human head similar to Tidswell et al. \[[@pone.0223133.ref019]\] and Sperandio et al. \[[@pone.0223133.ref020]\]. We segmented the inner and outer boundaries of the skull and the outer skin boundary from the CT data \[[@pone.0223133.ref021]\] using Seg3D (SCI Institute, Salt Lake City, UT, United States) and designed forms resampling these boundaries as negative moulds for the head phantom compartments in SolidWorks® (Dassault Systèmes, Vélizy-Villacoublay, France). We produced the moulds of the compartments through a fused deposition modelling process. The moulds are made of the polymer Lignin (GreenTec, Extrudr®, Lauterach, Austria). The casting process of the compartments started with the innermost compartment, the intracranial volume (brain), continued to the central compartment, the neurocranium (skull), and ended with the outermost compartment the scalp; in each case, the preceding compartment provided the base for the next compartment. These head phantoms resampled the geometry of the head and the dielectric properties of biological tissue. We used 2% agarose (Agarose Broad Range, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) in deionized water as the basic phantom material. The dielectric properties of the compartments were set by doping the basic phantom material with different fractions of sodium chloride (NaCl), graphite (Graphite d50, Algin-Chemie e. K., Neustadt-Glewe, Germany), or carbon black (carbon fibre powder, EMV Vega, Recklinghausen, Germany). We used the mixtures (shown in [Table 1](#pone.0223133.t001){ref-type="table"}) for creating head phantoms, in accordance to literature values for the dielectric properties. The dielectric properties were analysed with a Hewlett Packard 4284A 20 Hz to 1 MHz precision LCR meter (HP Inc., Palo Alto, CA, United States) in a two-plate method. 10.1371/journal.pone.0223133.t001 ###### Dielectric properties of the compartments of the two types of head phantoms in the range of 20 Hz to 1 MHz. ![](pone.0223133.t001){#pone.0223133.t001g} ---------------------------------------------------------------------------------------------------------------- Phantom compartment Mixture Electric conductivity in S/m\ Relative permittivity\ 20 Hz--1 MHz 20 Hz--1 MHz -------------------------- ---------------------------- ------------------------------- ------------------------ Head phantom Type I Brain Type I 2 % agarose, 0.17 % NaCl 0.12--0.38 72.5·10^6^--930 Skull Type I 2 % agarose, 0.01 % NaCl,\ 0.024--0.063 9·10^6^--196 4 % graphite Scalp Type I 2 % agarose, 0.17 % NaCl,\ 0.054--0.63 43·10^6^--1290 4 % carbon black Head phantom Type II Brain Type II 2 % agarose, 0.17 % NaCl 0.12--0.38 72.5·10^6^--930 Skull Type II 2 % agarose, 0.01 % NaCl 0.015--0.048 8.1·10^6^--155 Scalp Type II 2 % agarose, 0.17 % NaCl 0.12--0.38 72.5·10^6^--930 ---------------------------------------------------------------------------------------------------------------- Two types of phantoms were created, with (Type I) and without carbon additives (Type II). Type I phantoms had the advantage of better approximating the dielectric properties found in the literature \[[@pone.0223133.ref022]--[@pone.0223133.ref025]\]. The phantoms without carbon additives were transparent, which allowed us to later add dyes for the visualization of micro damages and perforations caused by electric current pulses. For dyeing, all transparent head phantoms were placed completely in an emulsion of 1% ironhexacyanoferrate and deionized water for two days after the tests. [Table 1](#pone.0223133.t001){ref-type="table"} shows the dielectric properties for both types of head phantoms. [Fig 2A](#pone.0223133.g002){ref-type="fig"} shows the scalp from head phantom Type I and [Fig 2B](#pone.0223133.g002){ref-type="fig"} depicts the structure of head phantoms in a CAD model. The scalp compartment appears black due to the carbon additives. The maximum diameter of each phantom is 200 mm and the maximum height is 165 mm. Furthermore, we created a third type of head phantom (head phantom Type III) which was built up like head phantom Type I but with the addition of four silver/silver-chloride microelectrodes (diameter: 1 mm, length: 2.5 mm, BMP1 from MedCat GmbH, Munich, Germany) in the brain compartment. Thus, it was possible to measure voltages inside head phantom Type III during an applied pulse. The microelectrodes were plugged in holders, which were produced in a fused deposition modelling process and were made of Lignin. Each holder had a diameter of 3 mm. Two holders had a length of 50 mm and the other two had a length of 100 mm. We named the anterior microelectrode ME1, the left microelectrode ME2, the posterior microelectrode ME3, and the right positioned microelectrode ME4 ([Fig 2C](#pone.0223133.g002){ref-type="fig"} and [Fig 2D](#pone.0223133.g002){ref-type="fig"}). ![Head phantom geometry and electrode setup of microelectrodes.\ (A) Formed scalp in a lateral view of a head phantom with carbon additives (head phantom Type I). (B) Sagittal cut through the head phantom. The intracranial volume (brain) is coloured blue, the neurocranium (skull) yellow, and the scalp red. (C) Double cut of the head phantom Type III with the microelectrodes ME1 and ME2 and their holders. (D) Double cut of the head phantom Type III with the microelectrodes ME3 and ME4 and their holders. The inset in D shows a photograph of a microelectrode (grey) with a physical cut through a holder (white) and the wire insulation (yellow).](pone.0223133.g002){#pone.0223133.g002} We created an electrode setup with four separate brass electrodes to detect the current distribution across the compartments of the head phantom. These electrodes were grounded in all the experiments. The geometry of each electrode was adapted to the base area of the compartments to minimize the current path from adjacent compartments. When the head phantom was placed on the electrode setup, the innermost electrode (E1) contacted the brain, the second electrode (E2) the skull and the third electrode (E3) the scalp. The fourth electrode (E4) concentrically surrounded the base of the phantom in a distance of 5 mm (E4: inner diameter 205 mm, outer diameter 215 mm). The chosen dimensions of electrode E4 and the distance to E3 allowed to determine the current distribution in the flashover separately since the ends of the flashovers hit only electrode E4. Flashovers are discharges that start from an impact point on the surface of the head phantom and propagate to the electrode ring E4. These events were detected by a single lens reflex (SLR) camera (EOS 5D Mark II, Canon Inc., Tokyo, Japan). [Fig 3](#pone.0223133.g003){ref-type="fig"} shows the electrode setup and its dimensions. ![Electrode setup.\ (A) Schematic of electrode setup with electrodes innermost E1 to outermost E4. (B) Dimensions of the electrode setup in a top view. Each electrode E1--E4 has a thickness of 10 mm.](pone.0223133.g003){#pone.0223133.g003} Experiments with Marx generator {#sec005} ------------------------------- We used a Marx generator to replicate the high-voltage effects on the human head during a lightning strike. The Marx generator (MG, HIGHVOLT Prueftechnik Dresden GmbH, Germany, properties of MG: Energy 6 kJ, max. charging voltage 125 kV per stage) produces a standardized 1.2/50 μs voltage pulse and can reach a total load voltage of 450 kV \[[@pone.0223133.ref026],[@pone.0223133.ref027]\]. An MG can reach a high voltage at the output with one pulse, making it possible to ionize the air over long distances. The discharge is more like a real lightning strike discharge, but the current level of the generated pulse is much lower. In the first test series, one head phantom Type I was placed on the electrode setup E1--E4 underneath the output from the MG. The distance between the high voltage point electrode (output) and the highest point of the head phantom (vertex) was 150 mm. We applied 15 single positive voltage pulses to the head phantom, each pulse with a total load voltage from the MG of 450 kV. An SLR camera documented the expansion of the lightning discharge. In a second test series, we placed a head phantom Type II on the electrode setup (E1--E4) underneath the output of the MG in the same distance of 150 mm. Again, 15 single voltage pulses were applied, each pulse with a total load voltage of 450 kV. The head phantom Type II was dyed before slicing, as explained in Section: Creation of head phantom. We examined and sliced both head phantoms after the test series. For slicing, we used a strained wire with a diameter of 0.5 mm. The slicing always started with a sagittal cut through one of the observed perforations on the surface of the head phantoms. Measurements with combination wave generator {#sec006} -------------------------------------------- A head phantom Type III was tested with an in-house combination wave generator (CWG) to investigate the electrical behaviour of the head phantom and measure the potential distribution in the brain compartment. The used CWG had the following parameters: Energy 350 J, V~max~ 12 kV 1.2/50 μs, I~max~ 6 kA 8/20 μs \[[@pone.0223133.ref026]\]. The head phantom was placed on the electrode setup (E4 was decoupled since no flashovers were expected) and a brass electrode (diameter: 25 mm, height: 24.9 mm) was galvanically coupled to the top of the head phantom, near the vertex. This brass electrode was necessary for applying the pulse to the head phantom, because the voltage of the CWG is not high enough to break through larger air gaps like the Marx generator. Further, the influence of an air gap was avoided in the tests to get the electrical behaviour of only the head phantom. For the measurements, two Pearson current monitors Model 101 and one Pearson current monitor Model 110 (Pearson Electronics Inc., Palo Alto, CA, United States) detected the currents of E1 to E3, while one voltage probe PHV4002-3 (PMK GmbH, Kassel, Germany) detected the voltage drop over the head phantom. Four voltage probes PHV 1000 (PMK GmbH, Kassel, Germany) recorded the potentials of each microelectrode in the brain compartment. All signals were measured simultaneously by an eight-channel oscilloscope DLM4000 (Yokogawa Electric Corp., Musashino, Japan). We applied 10 positive voltage pulses with an amplitude of 5 kV to the head phantom. The measurement setup is shown in [Fig 4](#pone.0223133.g004){ref-type="fig"}. After that, we applied 10 positive voltage pulses with an amplitude of 12 kV to the head phantom. In this test series, the four voltage probes PHV 1000 were disconnected due to their limited admissible voltage of 6 kV. Possible discharges were documented with the SLR camera. ![Measurement setup for the experiments with the combination wave generator (CWG).\ (A) Interconnected measuring equipment with the combination wave generator, head phantom, and oscilloscope. E1 to E3 are the grounded electrodes, each with a current monitor. The four microelectrodes (ME1 to ME4) in the brain compartment were connected by four voltage probes (only two of four probes are symbolized in the schematic). The dashed lines symbolize the measurement wires to the oscilloscope. (B) Photograph of the head phantom Type III placed at the electrode setup and connected via the stimulation electrode to the CWG. A mirror shows the backside of the head phantom Type III.](pone.0223133.g004){#pone.0223133.g004} Measurements with 10/350 μs current pulse generator {#sec007} --------------------------------------------------- We tested four head phantoms on a 10/350 μs current pulse generator to emulate the high-current effects of a direct lightning strike to the head \[[@pone.0223133.ref018]\]. A current pulse generator reaches a higher current level, different from that obtained by combination wave generator or Marx generator. Therefore, a more realistic lightning current level could be applied to the tested head phantoms. Distances between the output of the generator and the head phantoms were restricted due to the lower voltage. Therefore, the experiments replicated the impact point of a lightning discharge on the head without possible upward connecting lightning leaders. The generator (IP176/12S of HIGHVOLT Prueftechnik Dresden GmbH, Dresden, Germany) uses 12 switchable single capacitors (called HV-CAP) IS-Phr 12 (VISHAY Electronic GmbH, Selb, Germany). Each HV-CAP has a capacitance of 175 μF and can be loaded with a voltage of up to 12 kV, which results in an energy of 12.6 kJ per charged capacitor. Each head phantom was placed with the electrode setup in the examination chamber of the current pulse generator. The distance from the power output electrode of the current pulse generator to the surface of the head phantom was 55 mm. Therefore, it was necessary to bridge the distance with a copper ignition wire with a diameter of 0.1 mm, which ended about 4 mm above the surface of the head phantom at the vertex. We used three current monitors with a transmission ratio of 1:1000 (Model 1423 Pearson Electronics Inc., Palo Alto, CA, United States) and two current monitors with a transmission ratio of 1:100 (Model 101) to capture the currents in each compartment of the head phantom. The voltage probe (PHV4002-3) detected the voltage drop from the output of the current pulse generator to the ground. All signals were simultaneous recorded from parallel triggered oscilloscopes 44MXI-A and HDO6054 (Teledyne LeCroy, CA, USA). [Fig 5](#pone.0223133.g005){ref-type="fig"} shows the measurement setup for the tests with the 10/350 μs current pulse generator. ![Measurement setup on the 10/350 μs current pulse generator.\ (A) Schematics of the setup. E1 to E4 are the grounded electrodes. They are connected to the current monitors. The dashed lines indicate the measurement wires to the oscilloscope. (B) Photograph of the head phantom Type II placed in the examination chamber of the current pulse generator.](pone.0223133.g005){#pone.0223133.g005} We performed different measurement series with this setup on the current pulse generator by changing the number of HV-CAPs to vary the resulting current. Four head phantoms were examined and sliced after the measurements in order to investigate the optically visible damages. In addition, the head phantom Type II was dyed with ironhexacyanoferrate to highlight perforations and damages. - Test series I: The series started with 12 HV-CAPs @ 12 kV. The number of the HV-CAPs was reduced by three after three applied pulses to the phantom Type 1. At three used HV-CAPs, the number was reduced to one HV-CAP. The charging voltage was always constant. In the last step, the charging voltage was reduced (one HV-CAP @ 6 kV). The used HV-CAP numbers were: 12 @ 12 kV, 9 @ 12 kV, 6 @ 12 kV, 3 @ 12 kV, 1 @ 12 kV, and 1 @ 6 kV. This test series was performed to determine the electrical behaviour and current distribution of the head phantom at different current levels while the positive 10/350 μs pulse. - Test series II: Repeat of test series I with a newly casted head phantom Type I. - Test series III: A single pulse with 12 HV-CAPs and a charging voltage of 12 kV was applied to one head phantom Type II. This test was performed to find potential damages and perforations at the transparent head phantom Type II which are not visible with the head phantom Type I. - Test series IV: The series was performed with a head phantom Type I similar to I and II, but the sequence for the number of used HV-CAPs was pseudo-randomized, starting with six HV-CAPs @ 12 kV. Results {#sec008} ======= Results of experiments on Marx generator {#sec009} ---------------------------------------- We observed discharges on the surface of the head phantom Type I and head phantom Type II for each 450kV voltage pulse applied from the Marx generator, as shown in [Fig 6A and 6B](#pone.0223133.g006){ref-type="fig"}. It can be seen that the discharge channel hit the head phantoms and branched in discharges. The spilt began either at the surface of the head or in the air gap. After the tests, we examined the head phantoms. The head phantom Type I showed a dried ellipsoidal area around the vertex with a length of 120 mm in the anterior--posterior direction and 100 mm in the lateral direction. Also, we found punctured regions with diameters of 2--5 mm. No further particularities were observed in the brain or skull compartment after the slicing of the head phantom Type I. Several perforations occurred in the vicinity of the optically detected impact points at the head phantom Type II. The four largest ones had a diameter of 3--6 mm (marked with X [Fig 6C](#pone.0223133.g006){ref-type="fig"}). We sliced the head phantom Type II in a sagittal cut through one impact point, as marked in [Fig 6C](#pone.0223133.g006){ref-type="fig"}, and found a dyed channel, which started at the impact point and ended after a length of approx. 50 mm in the brain compartment (highlighted with blue colour in [Fig 6D](#pone.0223133.g006){ref-type="fig"}). This perforation was the only one penetrating the brain compartment---all other observed penetrations ended in the skull compartment. There was no systematic difference in the results from head phantom Type I and head phantom Type II. In a previous study, we measured currents of about 2 kA in a simplified head phantom \[[@pone.0223133.ref028]\] originating from the same pulses of the Marx generator. For the present study, we estimated similar currents in the head phantom Type I and Type II. ![Results of the experiments with the Marx generator.\ (A) Head phantom Type I and a discharge over the surface of the scalp. A mirror behind the head phantom Type I shows possible discharges on the side facing away from the SLR camera. (B) A discharge over the surface of the scalp from the head phantom Type II. (C) Top view on the scalp compartment from the head phantom Type II after 15 applied single voltage pulses. We marked the biggest perforations with X. The cut line for the first slice is marked black. (D) Sagittal cut along the black line in C, the arrows in C show the view direction. We found a tubular perforation, highlighted in blue colour.](pone.0223133.g006){#pone.0223133.g006} Results of measurements on combination wave generator {#sec010} ----------------------------------------------------- No flashover or discharges on the surface of the head phantom Type III were optically detected in the experiments with the CWG and an applied 1.2/50 μs voltage pulse of 5 kV. [Fig 7A](#pone.0223133.g007){ref-type="fig"} shows the average recorded time courses of the voltage and the currents of E1 to E3. The mean values at 2.4 μs are additionally indicated in [Fig 7A](#pone.0223133.g007){ref-type="fig"}. We calculated the relative current distribution of each compartment with the values at 2.4 μs (maximum measured voltage). The brain was exposed to 11.2%, the skull to 8.6% and the scalp to 80.2% of the current. An average voltage of 530 V was detected at the first microelectrode ME1 at 2.4 μs. Further, the voltage reached 1250 V at ME2, 520 V at ME3, and 1220 V at ME4 ([Fig 7B](#pone.0223133.g007){ref-type="fig"}). We calculated the electric field strength (based on the recorded electric potential at 2.4 μs and the distance of the microelectrode to the ground) at these four points: ME1: 10.6 kV/m at ME1; ME2: 12.5 kV/m; ME3: 10.4 kV/m; ME4: 12.2 kV/m. ![Results of measurements on the combination wave generator with an applied voltage of 5 kV.\ (A) Average time courses of recorded voltage and measured currents at the electrodes E1 to E3. (B) Time courses of the recorded voltages for the four microelectrodes (ME) in the brain compartment.](pone.0223133.g007){#pone.0223133.g007} We optically detected multiple discharges at the surface of the scalp compartment with an applied voltage of 12 kV ([Fig 8A](#pone.0223133.g008){ref-type="fig"}). The discharges started from the attached stimulation electrode. [Fig 8B](#pone.0223133.g008){ref-type="fig"} depicts the average recorded time courses of the voltage at the generator output and currents from the electrodes E1 to E3 with labelled mean values at 2.4 μs (maximum measured voltage). The relative current distribution at 2.4 μs was: brain compartment 13.1%, skull compartment 6.1% and scalp compartment 80.8%. ![Results of measurements on the combination wave generator with an applied voltage of 12 kV.\ (A) Registered discharges at the surface of the scalp compartment. The mirror in the background shows the backside of the head phantom. (B) Average time courses of recorded voltage at the generator output and currents from E1 to E3.](pone.0223133.g008){#pone.0223133.g008} Results of measurements on 10/350 μs current pulse generator {#sec011} ------------------------------------------------------------ In each test series, we optically detected a flashover across the head phantoms in the measurements with the 10/350 μs current pulse generator. In almost all tests, the discharge sat on near the highest point of the head phantoms (vertex) and went over the scalp to E4. [Fig 9](#pone.0223133.g009){ref-type="fig"} depicts four different morphology examples. [Fig 9A and 9B](#pone.0223133.g009){ref-type="fig"} show exemplary discharges of the test series I and III with 12 HV-CAPs @ 12 kV with a head phantom Type I and the head phantom Type II, respectively. In both cases, the discharge channel appeared structured in the vicinity of the scalp. However, the discharge channel was surrounded by a corona. The discharge and the resulting flashover were less intensive and the corona around the channel was smaller with one HV-CAP @ 6 kV in contrast to 12 HV-CAPs @ 12 kV. An example of a discharge with one HV-CAP @ 6 kV is shown in [Fig 9C](#pone.0223133.g009){ref-type="fig"}. [Fig 9D](#pone.0223133.g009){ref-type="fig"} depicts the last applied current pulse of the test series IV (after three times six HV-CAPS @ 12 kV and one time 12 HV-CAPS @ 12 kV). The main discharge over the scalp and the discharge channel are difficult to identify. Discharges next to the scalp appeared especially on the right side of the head phantom Type I. The discharges busted the phantom material at several points. ![Detected flashovers across head phantoms for the tests with the 10/350 μs current pulse generator.\ (A) An example of the test series I with 12 HV-CAPs @ 12 kV and with a head phantom Type I. (B) An example of test series III with 12 HV-CAPs @ 12 kV and with head phantom Type II. (C) An exemplary discharge of the test series I with one HV-CAP @ 6 kV and with head phantom Type I. (D) An exemplary discharge of the test series IV at 12 HV-CAPs @ 12 kV with head phantom Type I. The head phantom Type I was destroyed at this discharge.](pone.0223133.g009){#pone.0223133.g009} All recorded voltages and currents showed characteristic time courses. The voltage increased up to the load voltage in about 1 μs; simultaneously, the current increased through the head phantom. Then the voltage decreased rapidly to a value of about 1.5 kV in approximately 50 μs. At the same time, the current in E4 increased and showed an aperiodic pulse behaviour. [Fig 10A and 10B](#pone.0223133.g010){ref-type="fig"} present the average time courses of the test series I and II at 12 HV-CAPs @ 12 kV with the most important key values. It can be seen that simultaneous to the first rise of the voltage, the current in E1, E2, E3 and the total current also increase. The time point t~start~ indicates the beginning of the increasing voltage and current. The discharge process started over the head phantom at this time point. After the increase of the current in E1 and E2 to the maximum, these currents broke down rapidly, along with the voltage and the current in E3 (Case 1). We defined this point as the moment of the beginning of the evolvement of the complete flashover across the head phantom and labelled it t~0~, determined by the highest decrease speed with the first derivation of the voltage. After the rapid collapse of the current in E1 and E2 close to zero ampere, the current in E4 increased from zero ampere to a high amplitude (in [Fig 10A and 10B](#pone.0223133.g010){ref-type="fig"}, up to 41.8 kA or 41.3 kA). Furthermore, we found a second possible case (Case 2) for the current time course in E3. The current in E3 broke in first, increased again, and reached a multiple higher peak, for example from 930 A at t~0~ to 5.7 kA after further 14.4 μs. This case is shown as an example in [Fig 10B](#pone.0223133.g010){ref-type="fig"}. [Fig 10C](#pone.0223133.g010){ref-type="fig"} depicts the time courses from the test series I and II at one HV-CAP @ 6 kV. The duration to t~0~ increases in comparison to the time courses in [Fig 10A and 10B](#pone.0223133.g010){ref-type="fig"}, but the time course shows some similarity in shape. The applied pulses ended about 2.6 ms after t~0~. As a further result, we extracted the time points t~start~ for each test for the test series I and II. The distribution is shown in [Fig 10D](#pone.0223133.g010){ref-type="fig"}. The time of starting of the flashover (t~start~) for the head phantom was in median between 40 μs and 45 μs at an energy level between 151.2 kJ and 75.6 kJ. The time increased from the level of 37.8 kJ at 53.6 μs over 12.6 kJ at 69.5μs to 6.3 kJ at 257 μs. The time t~start~ differed most at 6.3 kJ between 219 μs and 694 μs. ![Time courses from test series I and II with the 10/350 μs current pulse generator.\ (A) Time courses for 12 HV-CAPs @ 12 kV for an example Case 1 at test series I and II. (B) Time courses for 12 HV-CAPs @ 12 kV for an example Case 2 at test series I and II. (C) Time courses for one HV-CAP @ 6 kV. (D) Violin plots of t~start~ for each tested capacitor stage (applied energy). The time t~start~ was defined as latency at which we registered a first appearance of voltages or currents different from zero. The time to start of the flashover was called t~0~. M: mean, Mdn: median, SD: standard deviation.](pone.0223133.g010){#pone.0223133.g010} [Table 2](#pone.0223133.t002){ref-type="table"} provides the calculated relative current distribution for the compartments at t~0~ and at the latency of the maximum of the total current after the fully formed flashover (t~maxtot~), based on the values (test series I and II) of the currents from each tested capacitor stage. The skin compartment was most exposed to the applied current at t~0~ for each used HV-CAP stage, followed by the brain compartment and the skull compartment. After a flashover, the highest currents were detected at t~maxtot~ in electrode E4 for each capacitor stage; we found fractions of current between 98.2% and 99.5% in E4. The next highest part flowed through the scalp compartment. Brain and skull compartments had a smaller exposition, each with a current below 7 A. For the brain and skull compartments, the relative current distribution increased with a lower energy level at t~maxtot~. 10.1371/journal.pone.0223133.t002 ###### Relative current distribution for the test series I and II at the time points before (t~0~) and after a fully formed flashover (t~maxtot~). ![](pone.0223133.t002){#pone.0223133.t002g} -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- HV-CAPS\ Total energy in kJ Applied max. current @ t~0~ in kA Applied max. current @ t~maxtot~ in kA Rel. current distribution for flashover in % Rel. current distribution for scalp in % Rel. current distribution for skull in % Rel. current distribution for brain in % charged up to 12 kV --------------------- -------------------- ----------------------------------- ---------------------------------------- ---------------------------------------------- ------------------------------------------ ------------------------------------------ ------------------------------------------ ----- -------- ------ -------- 12 151.2 1.00 41.5 n/a 99.5 85.8 0.45 5.1 \<0.02 9.1 \<0.02 9 113.4 0.95 31.8 n/a 99.5 87.2 0.32 4.6 \<0.02 8.2 \<0.02 6 75.6 1.40 21.5 n/a 99.5 90.4 0.38 3.5 \<0.02 6 \<0.02 3 37.8 0.77 10.9 n/a 98.2 83.5 1.7 5.9 0.05 10.6 0.06 1 12.6 0.97 3.7 n/a 98.9 87.2 0.8 4.3 0.08 8.5 0.2 1 (6 kV) 6.3 0.48 1.55 n/a 99.2 90.5 0.3 3.1 0.2 6.4 0.3 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- The time courses of the currents in test series III were also aperiodic, as in test series I and II for case 2, but we found a second increase in the current in E3, probably caused by an incomplete flashover. The currents in E1, E2, and E3 decreased and the current increased up to approximately 2.5 kA in E4 after the breakdown of the voltage. After that, we registered an increase of the voltage, an increase of the currents at E1--E3, and a collapse to 0 A in E4 (named collapse in [Fig 11A](#pone.0223133.g011){ref-type="fig"}). After another 5 μs, the voltage broke down again as well as the currents in E1 to E3. The current in E4 increased and followed the typical aperiodic time course. The measurement of the time courses from test series IV with 12 HV-CAPs @ 12 kV are shown in [Fig 11B](#pone.0223133.g011){ref-type="fig"}. The time courses were aperiodic too, but the time to the event points were longer than the measured durations from other test series. We could not record the event t~start~. The moment t~maxtot~ was reached after 131 μs after t~0~. Further, the time course showed waves between t~0~ and t~maxtot~. ![Time courses from test series III and IV with the 10/350 μs current pulse generator.\ (A) Shows the time courses for the test series III, the only one test series with the head phantom Type II and 12 HV-CAPs @ 12 kV. (B) Depicts the recorded time courses for the test series IV at the second repeat with 12 HV-CAPs @ 12 kV.](pone.0223133.g011){#pone.0223133.g011} The maximum current steepness is a parameter to define and compare lightning current pulses. We calculated the maximum current steepness of the measured total currents with the 30%--90%-rule of amplitude. We used the 30%--90%-rule because the current amplitude at 10% was not measurable \[[@pone.0223133.ref018]\]. The maximum current steepness showed a reduction from about 1855 A/μs at an applied energy of 151.2 kJ down to 64 A/μs at 6.3 kJ. Compared to the ideal 10/350 μs current pulses (assumed double exponential function), the measured current pulses had a less current steepness with a factor between 0.95 and 2.08, as shown in [Table 3](#pone.0223133.t003){ref-type="table"}. The difference of current steepness between the measured current pulses and the ideal 10/350 μs current pulses is due to impedances of the head phantom and the discharge channel. The 10/350 μs current pulse generator is not an ideal constant current source; additional impedances extend the pulse form. 10.1371/journal.pone.0223133.t003 ###### Current steepness of measured total currents in test series I and II and comparison with ideal 10/350 μs current pulses. ![](pone.0223133.t003){#pone.0223133.t003g} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ HV-CAPS\ Total energy in kJ Current steepness in A/μs of measured total current pulses Current steepness in A/μs of ideal 10/350 μs current pulses Factor between measured and ideal current pulses charged up to 12 kV --------------------- -------------------- ------------------------------------------------------------ ------------------------------------------------------------- -------------------------------------------------- 12 151.2 1855 3557 1.92 9 113.4 1501 2726 1.82 6 75.6 1260 1843 1.46 3 37.8 655 934 1.43 1 12.6 334 317 0.95 1 (6 kV) 6.3 64 133 2.08 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ We examined and sliced the head phantoms after the test series, with the following results. - Test series I and II: We found similar damages on both head phantoms Type I from test series I and II, which are described for one phantom in the following: 10 impact points were found on the top surface of the scalp ([Fig 12A](#pone.0223133.g012){ref-type="fig"}), ranging from 5 mm to 13 mm in diameter---they are marked with Y. Several smaller perforations are marked with Z in [Fig 12A](#pone.0223133.g012){ref-type="fig"}. One path (outgoing from an impact point) was found in the right parietal occipital region, with a length of about 45 mm (marked with W in [Fig 12A](#pone.0223133.g012){ref-type="fig"}). We sliced the head phantoms with a sagittal cut through an impact point. In the sagittal cut of one head phantom Type I ([Fig 12B](#pone.0223133.g012){ref-type="fig"}), we noticed damages at the upper part of the skull near the impacts in the form of drying and melting, marked as V. The brain compartment seemed to be unaffected ([Fig 12B](#pone.0223133.g012){ref-type="fig"}). - Test series III: This head phantom Type II was dyed with ironhexacyanoferrate to highlight the damages. We found an ellipsoidal impact point on the scalp near the vertex, with an approx. length of 7 mm from anterior to posterior and 10 mm from right to left. Three obvious burn paths were found to be outgoing from the main impact point. The first path had a length of 110 mm to posterior direction, the second path a length of 95 mm to right parietal, and the third path a length of 80 mm to right parietal occipital. Furthermore, we detected perforations in the upper part of the skull near the impact point. We sliced the head phantom Type II with a sagittal cut through the impact point. One perforation had a length of 10 mm down to the skull, marked with L in [Fig 12C](#pone.0223133.g012){ref-type="fig"}. The impact point is marked with O in [Fig 12C](#pone.0223133.g012){ref-type="fig"}. We found melting and blistering points around the impact point (marked with P [Fig 12C](#pone.0223133.g012){ref-type="fig"}). No optical damages were found in the brain compartment. - Test series VI: The head phantom Type I ([Fig 12D](#pone.0223133.g012){ref-type="fig"}) was destroyed after three pulses applied with six HV-CAPs and two pulses applied with 12 HV-CAPs (in the second applied pulse). The scalp was blown up and the skull was broken on the right side of the head phantom. We found a melted area in the brain compartment under the cracked skull with the dimensions of 120 mm anterior to posterior and 95 mm inferior to superior. ![Examined head phantoms after the tests with the 10/350 μs current pulse generator.\ (A) Damaged and dried areas at the top of the scalp compartment for the head phantom Type I after the test series I. The impact points are marked with Y smaller perforations with Z and a current path with W. The black line marks the cut line. The arrows indicate the view direction for the cut. (B) Slice of the head phantom Type I indicated in A. Burned and perforated areas were magnified in the inset (marked with V). (C) Sagittal sliced head phantom Type II from test series III. A perforation was marked with L. Inset shows magnified area around the impact point O and P mark blistering in the scalp compartment. (D) The destroyed head phantom Type I after the test series IV. The scalp and skull compartments were blown open, and the brain compartment was damaged and melted.](pone.0223133.g012){#pone.0223133.g012} Discussion {#sec012} ========== Spatial current distribution in the head phantom {#sec013} ------------------------------------------------ We obtained the relative current distribution in the compartments of the created head phantoms and in discharge channels during various applied voltage and current pulses. In case of no flashover, the scalp compartment was always most exposed to the current (80--90%) in all experiments. The brain compartment was exposed to 6--13% of the current. The skull compartment experienced the smallest amount of current---about 3--6%. In case of flashover formation, the relative current distribution changed and 98--99% of the current flowed in the discharge channel due to its higher electrical conductivity compared to the head phantom. The remaining 1--2% of the current again predominantly flowed in the scalp, followed by the brain and skull compartments. We observed this distribution pattern (most current in the scalp, followed by brain and skull compartments) across all experiments, independent of the applied voltage and current pulse forms. To the best of our knowledge, this is the first time that a study has reported such current distributions that establish normative values to be used in later experimental investigations on the possibilities of lightning protection equipment for humans. Voltage and current time courses {#sec014} -------------------------------- The time courses of the recorded voltages and currents show distinct differences for the cases without and with flashover. For the cases without flashover, we found the expected rise and fall of both voltage and current time courses according to the applied 1.2/50 μs pulse. For cases with flashover, we found three time intervals: a short rising time of the voltage lasting approximately 1 μs after t~start~ marked the initial interval indicating the connecting leader phase \[[@pone.0223133.ref012]\]. Thereafter, the time interval until t~0~, the initial return stroke phase \[[@pone.0223133.ref012]\], is characterized by increasing current in and a constant voltage across the head phantom. We measured a time of approximately 30--700 μs for the second interval. The third interval starts with t~0~ and ends after 2.6 ms and is mostly dominated by the high pulse current; this interval is the denominated surface flashover phase \[[@pone.0223133.ref012],[@pone.0223133.ref013]\]. Contrary to the theoretical predictions by Uman \[[@pone.0223133.ref012]\], our experiments show an almost constant voltage across the head phantom and a nonlinearly increasing current in the head phantom for the first and second time intervals. While Uman \[[@pone.0223133.ref012]\] used an impressed pulse voltage for the first and second time intervals (t~start~ to t~0~), in our experiments, the 10/350 μs current pulse generator provided an impressed constant voltage during the first and second time intervals. In one experiment, the head phantom was destroyed by the impressed pulse. In this specific case, in the third time interval, we found time courses for voltages and currents that exhibited a step-like and spiky behaviour ([Fig 11B](#pone.0223133.g011){ref-type="fig"}). In this interval, the voltage and current irregularly increased and decreased, possibly indicating a change in the electrical conductivity. We assume that these changes in the electrical conductivity might be caused by thermal ionization and plasma processes directly in the head phantom \[[@pone.0223133.ref029]\]. Flashover {#sec015} --------- The flashover is a phenomenon that occurs in technical setups and during lightning accidents with humans \[[@pone.0223133.ref012],[@pone.0223133.ref013],[@pone.0223133.ref029]\]. In our experiments, the flashover took approximately 30--700 μs to form. Differently, a time of 30--150 ns characterizes flashovers for lightning accidents \[[@pone.0223133.ref013]\] and a few 100 ns for technical setups \[[@pone.0223133.ref029],[@pone.0223133.ref030]\]. The time to form a flashover mainly depends on the voltage amplitude and the pulse form. The observed difference in time were likely caused by the voltage differences: our experiments operated with 12 kV while a lightning strike has about 1 MV \[[@pone.0223133.ref011],[@pone.0223133.ref013]\]. Bazelian and Raizer characterize times up to 200 μs for fully formed discharge channels along water surfaces \[[@pone.0223133.ref031]\]. This implies that the time to form a flashover increases with a lower impedance such as biological tissues. The time to form a flashover increased in median from 69.5 μs at 1 CAP at 12 kV to 257 μs for 1 CAP at 6 kV ([Fig 10D](#pone.0223133.g010){ref-type="fig"}). This behaviour is expected for such impulse voltage-time characteristics \[[@pone.0223133.ref029],[@pone.0223133.ref030]\]. Implications for lightning protection {#sec016} ------------------------------------- Our experiments confirmed that most of the current (98--99%) flowed outside of the human head in the discharge channel after a fully formed flashover \[[@pone.0223133.ref012],[@pone.0223133.ref013]\]. Consequently, the faster a flashover is formed over the head, the higher are the chances of survival. Further, the current flowing in the brain compartment is typically a factor of 20 units less in amplitude compared to the current flowing in the scalp. This difference is caused by the protective property of the skull due to its low electrical conductivity and relative permittivity compared to the scalp and brain compartments \[[@pone.0223133.ref022]\]. Electroporation can cause cell damage and death \[[@pone.0223133.ref032]--[@pone.0223133.ref035]\]. An electric field strength of 6 kV/m and above can cause reversible or irreversible electroporation in biological cells \[[@pone.0223133.ref032]--[@pone.0223133.ref035]\]. An electric field strength of about 100--600 kV/m close to the ground are typical values during lightning \[[@pone.0223133.ref011]\]. Our experiments show an electric field strength of about 10.4--12.5 kV/m in the brain compartment during an applied 1.2/50 μs voltage pulse with an amplitude of 5 kV. These values indicate that electroporation can occur even for the lower current and voltage amplitudes in our experiments. Thus, neurological damages during a lightning strike caused by electroporation are likely to occur \[[@pone.0223133.ref006],[@pone.0223133.ref035]\]. We applied high voltage (Marx generator) and high current pulses (10/350 μs current pulse generator) to different head phantoms and optically analysed the damages to the head phantoms. In all setups, we found similar damages, e.g. perforations and burns on the scalp compartment or dried areas throughout the scalp compartment down to the skull. These observations are similar to autopsy reports after lightning accidents \[[@pone.0223133.ref003],[@pone.0223133.ref008],[@pone.0223133.ref015]\]. According to the DIN EN 1149, a personal lightning protection equipment, for example in the form of a coat with a hood consisting of modacrylics (flame retardant synthetic copolymer), can reduce the current amplitudes in the head compartments due to the material's electrostatic discharge behaviour \[[@pone.0223133.ref016]\]. Consequently, this lightning protection equipment can prevent burns and perforation as well. Experimental setup and limitations {#sec017} ---------------------------------- Physical head phantoms allow us to gain insight into head internal field and current distributions. They can provide explanations for observed lightning phenomena and can help to develop novel hypotheses and experiments. However, physical head phantoms are simplifications. Our head phantoms replicate the three main head compartments in terms of their geometric and dielectric properties, but neglect the highly complex interior structures of the human head, like the sulcus/gyrus structure, the anisotropic conductivity of the white matter, according Haueisen et al. \[[@pone.0223133.ref036]\], or hair on the scalp. Future research should include more detailed head phantoms. Another limitation was the measurement setup for the current distribution. The spatial and temporal resolutions of our measurement equipment (oscilloscopes, current monitors, voltage probes) were limited as well. Due to these limitations, we were unable to measure the initial connecting leader phase, in which a negative current typically flows at the start of the discharge. At present, voltage and current generators cannot provide a high voltage and a high current simultaneously similar to a lightning discharge. For this reason, we use three different generators to replicate the voltage and current effects on the head phantoms. We compare the generators in the following paragraph. - The Marx generator (MG) generates a 1.2/50 μs high voltage of up to 450 kV. The high voltage ionizes the air gap over a larger distance between the high voltage point electrode and the head phantom, mimicking the air distance between the cloud and the ground during a lightning discharge \[[@pone.0223133.ref027]\]. The energy and the resulting current (about 2 kA) values are low in comparison to natural lightning. Further, no flashovers are possible due to the low energy and the impedance of the head phantom. The used MG is only suitable for visually investigating the high voltage effects which manifested as perforations on the head phantoms. - The combination wave generator (CWG) produces a 1.2/50 μs voltage up to 12 kV (open-circuit) or an 8/20 μs current up to 6 kA (short-circuit), depending on the impedance of the test object according to IEC 61000--4--5 \[[@pone.0223133.ref026]\]. The voltage amplitude was not high enough to ionize the air over larger distances. For this reason, a stimulation electrode galvanically contacted the scalp compartment in our experiments. The low noise and low interference setup enabled a voltage and current measurement with a high signal noise ratio and a high reproducibility in our experiments. This setup also allowed the measurement of voltages at electrodes integrated in the brain compartment (ME1--ME4). Another advantage of the CWG is the similarity of its voltage pulse form to a lightning voltage pulse form. - The 10/350 μs current pulse generator can be charged up to 12 kV according to IEC 62305 \[[@pone.0223133.ref018]\]. The maximum current in the experiments was about 42 kA. Due to the low voltage, an air gap of only 4 mm between the output of the generator and the test object was possible. We used an ignition wire to increase this distance to 55 mm. Consequently, no connecting upward leader was formed. The used current amplitude and current steepness corresponded to real lightning currents. For the estimation of the current distribution in the head phantom, including the flashover, the 10/350 μs current pulse generator was best suited for comparison to real lightning currents. Conclusion and outlook {#sec018} ====================== We created head phantoms made of agarose to replicate the scalp, the neurocranium, and the intracranial volume of a human head. These head phantoms were tested on pulse generators, which modelled lightning components to investigate the physical and biological effects during a lightning strike. We found: 1. The scalp compartment is most exposed to the current during a lightning strike, with a fraction of approximately three quarters. 2. A fully formed flashover rapidly reduces the current in the brain compartment and the current exposition is reduced in interior structures the faster a flashover is formed. This mechanism can explain why people can survive a lightning strike. 3. The skull has protective properties due to its low electrical conductivity and relative permittivity. 4. The electric field strength in our experiments reaches a level where electroporation is possible. The mechanism of electroporation may explain neurological effects and damages after a lightning strike. We established normative values in our experiments for the human head to lightning discharges without any protective equipment. In the next steps, we will compare the found values with protective equipment applied to the head phantom. Further, we will improve our head phantoms to be more realistic models (e.g. add hair on scalp compartment). [^1]: Competing Interests:The authors have declared that no competing interests exist. [^2]: ‡ These authors also contributed equally to this work.	{ "pile_set_name": "PubMed Central" }	[ "kassel", "amount", "probabl", "two", "almost", "phenomenon", "went", "work", "origin", "remain", "powder", "cours", "decreas", "compris", "eg", "hz", "khz", "penetr", "aμ", "holder", "pariet", "outermost", "colourponegponeg", "exist", "identifi", "pearson", "length", "discharg", "μf", "charg", "mechan", "perfor", "valu", "polym", "threecompart", "bridg", "micro", "multipl", "unaffect", "signal", "suit", "may", "twoplat", "branch", "skull", "collaps", "separ", "use", "gener", "amplitud", "ms", "germani", "deviationponegponeg", "symbol", "extrudr®", "caus", "complet", "accord", "retard", "establish", "probe", "carbon", "tstart", "mark", "relev", "norm", "process", "deposit", "tomographi", "phenomena", "hair", "right", "also", "state", "directli", "difficult", "faster", "adjac", "silversilverchlorid", "cell", "phantom", "perform", "karlsruh", "admiss", "approx", "electrostat", 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Introduction {#s1} ============ Peroxisomes are defined as organelles encasing the metabolism of H~2~O~2~. This highly reactive molecule is generated by several oxidative reactions and degraded inside the organelle by abundant amounts of catalase. In addition, peroxisomes fulfill a number of important metabolic functions for eukaryotic cells requiring an active communication between the peroxisomal lumen and the cytosol or other organelles. Major functions include the β-oxidation of fatty acids, parts of the glyoxylate cycle and parts of the photorespiration (Hu et al., [@B38]; Waterham and Wanders, [@B91]). For β-oxidation fatty acids have to enter peroxisomes, and the resulting acetyl-CoA is distributed throughout the cells. Photorespiration in plants involves mitochondria, chloroplasts and peroxisomes and therefore requires extensive metabolite exchange. A key intermediate shared by the photorespiration and the glyoxylate cycle is glyoxylate, usually generated and metabolized inside peroxisomes. The glyoxylate cycle was originally considered a metabolic process localized to peroxisomes (Breidenbach and Beevers, [@B16]). This localization was rationalized as a means to increase the efficiency of the flux of intermediates. However, the finding that parts of the whole cycle are extra-peroxisomal in the yeast Saccharomcyes cerevisiae and in the plant Arabidopsis thaliana indicated, that a functional glyoxylate cycle requires the transfer of various metabolites across the peroxisomal membrane, too (Minard and McAlister-Henn, [@B51]; Courtois-Verniquet and Douce, [@B20]; Taylor et al., [@B75]; Kunze et al., [@B42]; Pracharoenwattana et al., [@B60]). The nature of the molecules crossing the peroxisomal membrane varies with the organism under study. The single membrane separating the peroxisomal matrix from the surrounding was shown to be permeable only for small molecules such as tri- and dicarboxylates and amino acids (Antonenkov et al., [@B4]). Specific transporters have not yet been identified but features compatible with pore-like structures were demonstrated (Verleur and Wanders, [@B87]; Antonenkov and Hiltunen, [@B2]). In this review we will address the questions how and where glyoxylate is generated and how metabolites of the two most prominent pathways partially localized to peroxisomes, the glyoxylate cycle and the photorespiration, are thought to cross the membrane on their way in and out of the peroxisomes. Peroxisomes and glyoxylate metabolism ===================================== Inside peroxisomes the accumulation of glyoxylate is prevented by conversion into glycine making use of a transaminase reaction or by condensation with acetyl-CoA into malate catalyzed by malate synthase (MLS), one of the key enzymes of the glyoxylate pathway (for reviews see Wanders and Waterham, [@B90]; Theodoulou and Eastmond, [@B76]). Glyoxylate can be produced from different precursor molecules (Figure [1A](#F1){ref-type="fig"}). Cleavage of isocitrate generates glyoxylate and succinate in the glyoxylate cycle (Figure [1B](#F1){ref-type="fig"}). The oxidation of glycolate to glyoxylate is catalyzed by glycolate oxidase being part of the photorespiratory process generating H~2~O~2~ within peroxisomes (Figure [1C](#F1){ref-type="fig"}). Another possible source of glyoxylate is the degradation of purines in those organisms metabolizing the intermediate uric acid (Figure [1D](#F1){ref-type="fig"}). Moreover, in mammals glyoxylate may represent a degradation product of hydroxyproline originally derived from collagen (Salido et al., [@B68]) or alternatively, may be the result of a conversion from glycine catalyzed by D-amino acid oxidase generating H~2~O~2~ (Ohide et al., [@B53]). ![Glyoxylate generation and consumption. (A) Glyoxylate is generated from different precursor molecules and converted into stable metabolites for further utilization. The proteins involved are ureidoglycolate hydrolase (UGH), isocitrate lyase (ICL), glycolate oxidase (GO), malate synthase (MLS), glutamine:glyoxylate aminotransferase (GGAT). (B) In the glyoxylate cycle two acetyl-CoA are condensed to succinate and 2 CoA (not shown). The proteins involved are citrate synthase (CIT), aconitase (ACO), isocitrate lyase (ICL), malate synthase (MLS), malate dehydrogenase (MDH). (C) The generation and consumption of glyoxylate in the photorespiratory process. The stoichiometry of the reaction sequence to obtain three molecules of 3-phospho-glycerate from two oxygenation products of ribulose-1,5-bisphosphate is indicated by the numbers below the molecules (1×, 2×). The proteins involved are ribulose-bisphosphate-carboxylase-oxygenase (RuBisCO), phosphoglycolate phosphatase (PGLP), glycolate oxidase (GO), glutamine:glyoxylate aminotransferase (GGAT), glycine-decarboxylase (GlyDC), serine-hydroxymethyltransferase (SHMT), serine:glyoxylate aminotransferase (SGAT), hydroxypyruvate reductase (HPR), glycerate kinase (GLYK). (D) The reactions in the purine degradation pathway leading to glyoxylate. The proteins involved are urate oxidase (UO), allantoin synthase (HIU-hydrolase + OHCU decarboxylase) (AS), allantoinase (alantoin amidohydrolase) (AL), allantoate amidohydrolase (AAH), ureidoglycine aminohydrolase (UGlyAH), ureidoglycolate amidohydrolase (UAH).](fphys-04-00204-g0001){#F1} The glyoxylate cycle allows the formation of 4-carbon units from 2-carbon units (Figure [1B](#F1){ref-type="fig"}). The resulting succinate serves to replenish the tricarboxylic acid (TCA) cycle representing the major collector and distributor of small carbon units. Alternatively, succinate or its follow-up product oxaloacetate serves as precursor for many biosynthetic processes. This biosynthetic pathway is absent in all animals except nematodes (Kondrashov et al., [@B41]). Acetyl-CoA fed into the glyoxylate cycle can be derived from different sources, such as β-oxidation of fatty acids, degradation of amino acids or in case of microbial organisms from external carbon sources such as ethanol or acetate. The glyoxylate cycle shares a series of three enzymatic activities with the TCA-cycle, namely malate dehydrogenase (MDH), citrate synthase (CIT), and aconitase (ACO) activity. The two unique activities, isocitrate lyase (ICL) and malate synthase (MLS) generate and consume the name-giving metabolite glyoxylate. The cleavage of isocitrate bypasses the decarboxylation reactions and the synthase reaction leads to the net-condensation of acetyl-CoA units. In many organisms these activities can be carried out by two or more isoenzymes with different localization signals and different expression patterns catalyzing the respective reactions. Photorespiration is required in all photosynthetic organisms and serves as carbon recovery system (Maurino and Peterhansel, [@B47]). Oxygenation of ribulose-1,5-bisphosphate by ribulose-bisphosphate-carboxylase-oxygenase (RuBisCO) competes with CO~2~ fixation upon high partial oxygen pressure leading to the formation of 2-phosphoglycolate and 3-phosphoglycerate in the chloroplast (Figure [1C](#F1){ref-type="fig"}). 3-phosphoglycerate is channeled into the Calvin cycle and 2-phosphoglycolate is recycled into 3-phosphoglycerate in a series of reactions involving peroxisomes and mitochondria. In peroxisomes glycolate is oxidized to glyoxylate generating H~2~O~2~, which is immediately decomposed by catalase. Glyoxylate is transaminated to glycine using either serine or glutamine as nitrogen source and further transported to the mitochondria. Here, two glycines are converted into one serine which is now returned into peroxisomes. The peroxisomal serine:glyoxylate aminotransferase and hydroxypyruvate reductase convert serine into glycerate which is transferred to chloroplasts, where it enters the Calvin cycle in the form of 3-phosphoglycerate. In summary, two glycolate molecules are consumed to produce glycerate and CO~2~ and the generation of two H~2~O~2~ is confined to peroxisomes. The degradation pathway of purines leads to uric acid, which in many organisms is further metabolized within peroxisomes to allantoine and allantoic acid (Figure [1D](#F1){ref-type="fig"}). In some animals the allantoicase activity leads to the cleavage of allantoic acid into urea and glyoxylate (Hayashi et al., [@B33]). In plants, some fungi and bacteria allantoic acid is further processed within the endoplasmic reticulum into ureidoglycine and ureidoglycolate, both giving rise to glyoxylate upon degradation (Werner and Witte, [@B92]). The glyoxylate derived from purine degradation is supposedly converted into glycine or condensed with acetyl-CoA catalyzed by a MLS activity providing the versatile metabolite malate. In essence, the heterocyclic core structure is converted into a nitrogen containing product and glyoxylate. The latter is recycled into biosynthetic processes in all organisms, the nitrogen containing product is either excreted (animals) or recycled (plants). Enzymes allocated to the glyoxylate cycle ========================================= Due to the fact that three of the five enzymatic activities contributing to the glyoxylate cycle are shared with the TCA cycle and additional enzymatic activities have been described an assignment of individual proteins to the glyoxylate cycle is not trivial. Some of the encoded proteins can be excluded due to an inappropriate expression pattern, but for some of the proteins only the phenotype of cells lacking the respective activity is a convincing argument for the function in the glyoxylate cycle. As the situation in the yeast S. cerevisiae has been analysed in detail previously (Kunze et al., [@B43]), we will only summarize the most important information and then concentrate on more recent results obtained in the thale cress A. thaliana as the plant model organism and in the human opportunistic fungus Candida albicans. The corresponding proteins are listed in Table [1](#T1){ref-type="table"}. ###### Enzymatic activities required for the glyoxylate cycle. Enzyme *Saccharomyces cerevisiae* *Candida albicans* *Arabidopsis thaliana* ------------------ -------------------------------- ------------------------ ---------------------------- ------ -------------- --------------- ------------- ----------- --------------- Isocitrate lyase [ICL1]{.ul} YER065C Cytosolic ICL1 CaO19.14134 Peroxisomal ICL At3g21720 Peroxisomal ICL2 YPR006C Mitochondrial Malate synthase [MLS1]{.ul} YNL117W Peroxisomal MLS1 CaO19.12296 Peroxisomal MLS At5g03860 Peroxisomal DAL7 YIR031C PTS1 Malate MDH1 YKL085W Mitochondrial MDH1 CaO19.12072 MITO pMDH1 At2g22780 Peroxisomal dehydrogenase [MDH2]{.ul} YOL126C Cytosolic MDH2 CaO19.7481 pMDH2/MDHG1 At5g09660 Peroxisomal MDH3 YDL078C Peroxisomal MDH3 CaO19.1278 PTS1 mDH3/MDHM1 At1g53240 Mitochondrial mDH4/MDHM2 At3g15020 Mitochondrial MDH5/MDHC1 At1g04410 MDH6/MDHC2 At5g43330 MDH7/MDHC3 AT5G56720 cMDH8/MDHP1 At3g47520 Chloroplast Citrate synthase CIT1 YNR001C Mitochondrial CIT1 CaO19.11871 Mitochondrial CSY1 At3g58740 PTS2 [CIT2]{.ul} YCR005C Peroxisomal Q59ZZ5 CSY2 At3g58750 Peroxisomal CIT3 YPR001W Mitochondrial CSY3 At2g42790 Peroxisomal CSY4 At2g44350 Mitochondrial CSY5 At3g60100 Mitochondrial Aconitase [ACO1]{.ul} YLR304C Mitochondrial ACO1 CaO19.13742 MITO ACO1 At4g35830 Mitochondrial ACO2 YJL200C Mitochondrial ACO2 CaOrf19.6632 MITO ACO2 At4g26970 Mitochondrial ACO3 At2g05710 Mitochondrial Proteins, corresponding genes, accession number, and targeting information are shown for enzymes carrying out activities required for the glyoxylate cycle in S. cerevisiae, C. albicans, and A. thaliana. PTS and MITO indicates predicted targeting information (general prediction: <http://wolfpsort.org/>, PTS1-predictor: <http://mendel.imp.ac.at/pts1/>, Mito-predictor: <http://ihg.gsf.de/ihg/mitoprot.html>). Cytosolic, peroxisomal, mitochondrial, and chloroplast indicates experimentally verified localization. Bold underlined are proteins experimentally proven to contribute to the glyoxylate cycle. In the genome of S. cerevisiae the key enzymes ICL and MLS are encoded by two genes each. Based on the high expression levels when cells grow on ethanol, acetate or fatty acids and the inability of cells lacking these proteins to grow on these carbon sources Icl1p and Mls1p have been attributed to the glyoxylate cycle (Fernandez et al., [@B26]; Hartig et al., [@B32]). The two others, Icl2p and Dal7p contribute to the mitochondrial propionate metabolism and the purine degradation, respectively (Hartig et al., [@B32]; Luttik et al., [@B46]). From the three genes coding for MDH, a mitochondrial (Mdh1p; McAlister-Henn and Thompson, [@B48]), a cytosolic (Mdh2p; Minard and McAlister-Henn, [@B51]), and a peroxisomal (Mdh3p; Steffan and McAlister-Henn, [@B72]) gene product are derived. Only the cytosolic and the mitochondrial variants are expressed when cells grow on C~2~-carbon sources (ethanol/acetate) and the cytosolic enzyme was required for growth under these conditions (Minard and McAlister-Henn, [@B51]; McCammon, [@B49]). This experimental evidence indicates that the cytosolic Mdh2p participates in the glyoxylate cycle. Similarly, among the three CIT proteins encoded within the yeast genome only Cit2p is considered to contribute to the glyoxylate cycle, because of its expression pattern on various carbon sources and because of the mitochondrial location of the other two enzymes Cit1p and Cit3p. Interestingly, Cit2p is essential for growth on ethanol or acetate only, when an alternative route probably involving one of the two mitochondrial activities is blocked (Van Roermund et al., [@B82]). Finally, one gene encoding ACO (ACO1; Gangloff et al., [@B28]) gives rise to the cytosolic and the mitochondrial activity (Regev-Rudzki et al., [@B62]) attributing Aco1p to the TCA- and the glyoxylate cycle, whereas Aco2p may be important for fermentation (Van den Berg et al., [@B81]). Even more complex is the situation in A. thaliana. Although only one gene codes for ICL (Eastmond et al., [@B22]) and one for MLS (Cornah et al., [@B19]), eight genes encode NAD^+^ dependent MDHs, five genes code for CITs and three genes code for ACOs (summarized in Table [1](#T1){ref-type="table"}). The localization, expression pattern and phenotype of the corresponding T-insertion mutants may help to discern the respective function of each individual protein. Nevertheless, the attribution of a function in the glyoxylate cycle appears difficult. Three of the five CITs, CYS1-3 harbor peroxisomal targeting information whereas CYS4 and CYS5 include mitochondrial leader peptides (Pracharoenwattana et al., [@B59]). Similarly, among the MDH proteins two are peroxisomal, two mitochondrial, one is observed in chloroplasts and three in the cytosol (Pracharoenwattana et al., [@B60]) \[The Arabidopsis Information Resource (TAIR), <http://www.arabidopsis.org/>, Huala et al., [@B39]\]. The expression pattern of enzymes contributing to the glyoxylate cycle is very characteristic, namely high expression after imbibition during seed germination followed by strong reduction upon postgerminative growth. Isoenzymes that are not expressed in the phase of germination can be excluded from a contribution to the glyoxylate cycle, such as CYS1 (Pracharoenwattana et al., [@B59]). During this developmental phase both, the β-oxidation of fatty acids and the glyoxylate cycle are equally needed and therefore the expression profiles of the corresponding genes are similar (Eastmond and Graham, [@B23]; Rylott et al., [@B67]). A detailed and precise cluster analysis of expression profiles during seedling development in soybean remained inconclusive since the expression pattern of the two key enzymes ICL and MLS did not fall into the same cluster and expression differences of the isoforms of CIT and MDH were not distinguishable (Gonzalez and Vodkin, [@B30]). An unambiguous attribution of a function in the glyoxylate cycle to a distinct protein could be based on the characteristics of cells deficient in the glyoxylate cycle function. A. thaliana seeds lacking ICL are able to germinate, but their seedling establishment is severely impaired in the absence of light or carbohydrates offered as alternative carbon source (Eastmond et al., [@B22]). In other words, gluconeogenesis is compromised but lipid respiration is still active in these mutants. A. thaliana seeds lacking MLS, the other unique enzyme of the glyoxylate cycle, display a similar defect in seedling establishment in the absence of light or carbohydrates. Regarding the compensation of the establishment defect by light the seeds respond differently. In the absence of MLS activity a lower light dose is required to promote seedling establishment than in the absence of ICL activity (Cornah et al., [@B19]). The latter phenotype led to the suggestion that in the absence of MLS glyoxylate, which is produced by ICL can enter gluconeogenesis by hijacking enzyme activities from the photorespiratory pathway. Phenotypes of plants lacking various isoforms of either CYS or MDH are less revealing to discern a specific function. During seedling establishment both peroxisomal MDHs (pMDH1 and pMDH2) and peroxisomal CITs (CYS2 and CYS3) are expressed. Seeds lacking either one of the peroxisomal activities present with disturbed fatty acid degradation (Pracharoenwattana et al., [@B59], [@B60]). Thus, a contribution of these proteins to the glyoxylate cycle cannot be delineated from the block in seedling establishment obtained in these mutant plants, because the lack of fatty acid degradation elicits a similar block in seedling establishment. Conversely, no noticeable defect in establishment was observed for seedlings from plants lacking mitochondrial MDH excluding an essential role of these MDHs in either β-oxidation of fatty acids or the glyoxylate cycle (Tomaz et al., [@B78]). When the metabolic distribution of exogenously added acetate within seedlings is used as indicator for the functionality of the glyoxylate cycle, no differences between seedlings lacking the peroxisomal MDH and wild type seedlings could be observed (Pracharoenwattana et al., [@B60]). However, this assay shows differences in the re-routing of acetate between seedlings from wild-type plants and plants, which are blocked in the glyoxylate cycle due to lack of ICL or MLS, although the differences appear small in the absence of MLS (Cornah et al., [@B19]). Experiments to discern the individual roles of the three cytosolic MDH were not yet carried out. Similarly, among the three ACO proteins a function in the glyoxylate cycle has not been assigned to anyone of them (Peyret et al., [@B56]; Arnaud et al., [@B8]). Altogether, in A. thaliana the assignment of individual ACOs, CITs, and MDHs to the glyoxylate cycle remains open. The situation in the yeast C. albicans is different. Each one of the enzymes ICL, MLS, and CIT is encoded by a single gene (Piekarska et al., [@B58]). In this organism the same enzyme Cit1p seems to contribute to the mitochondrial TCA-cycle and the glyoxylate cycle such as demonstrated for Candida tropicalis (Ueda et al., [@B80]). However, it cannot be excluded that a cytosolic form is derived from alternative translation as two variants are described (C4YLG7 and Q59ZZ5), but none of the described variants contains a peroxisomal targeting signal type 1 (PTS1; C-terminus KYIELVKNINKA). ACO activity is encoded by two genes, MDH activity by three genes (Jones et al., [@B40]). In each case one of the variants has a closer similarity to the respective glyoxylate cycle enzyme in S. cerevisiae, but experimental evidence for their role is missing. Subcellular localization of the enzymes of the glyoxylate cycle --------------------------------------------------------------- In S. cerevisiae the enzymes contributing to the glyoxylate cycle are distributed between the peroxisomal matrix and the cytosol. Icl1p, Mdh2p, and Aco1p were described in the cytosol (Minard and McAlister-Henn, [@B51]; Taylor et al., [@B75]; Regev-Rudzki et al., [@B62]), and Cit2p was found in the peroxisomal fraction (Lewin et al., [@B44]). Mls1p is either targeted to peroxisomes when cells utilize oleic acid or distributed across the cytosol when cells utilize ethanol (McCammon et al., [@B50]; Kunze et al., [@B42]). Upon growth on ethanol or acetate as sole carbon source with all other glyoxylate cycle enzymes in the cytosol the extra-peroxisomal fraction of Cit2p might suffice to generate four-carbon units through the glyoxylate cycle. In A. thaliana the key enzymes ICL and MLS are considered peroxisomal containing a C-terminal PTS1 \[EGTSLVVAKSRM for ICL and IVAHYPINVSRL for MLS, Arabidopsis subcellular database (SUBA), (Heazlewood et al., [@B34])\]. In contrast, ACOs lack peroxisomal targeting information and were allocated to the cytosol and the mitochondria (Arnaud et al., [@B8]). The extra-peroxisomal location of ACO activity was confirmed in castor bean and potato (Courtois-Verniquet and Douce, [@B20]). Which of the other gene products, namely of MDH and of CIT, are contributing to the glyoxylate cycle is unclear. Among the MDHs it can be assumed that one or more of the three cytosolic isoforms participates in the glyoxylate cycle as mutant plants lacking either the peroxisomal (Pracharoenwattana et al., [@B60]) or the mitochondrial (Tomaz et al., [@B78]) isoenzymes show no signs of a glyoxylate cycle defect. In C. albicans both key enzymes of the glyoxylate cycle, ICL and MLS were found in peroxisomes irrespective of the carbon source used (Piekarska et al., [@B58]), rendering this microorganism an alternative yeast model with key enzyme distribution similar to A. thaliana. However, the exclusive presence of one citrate synthase gene (CIT1) and the necessity of a peroxisomal and mitochondrial shuttle mechanism for acetyl-CoA for growth on oleic acid indicates that peroxisomal CIT is not available, but a cytosolic isoform might well be. The similarity of the MDH proteins to the homologs from S. cerevisiae suggests that the cytosolic Mdh2p contributes to the glyoxylate cycle as well. Finally, the cytosolic ACO Aco1p (Jones et al., [@B40]) might be supplemented by a minor peroxisomal subfraction that is caused by a weak PTS1 \[HGSALNFIKSKY, <http://mendel.imp.ac.at/pts1/>, Neuberger et al., [@B52]\]. Briefly summarized, the localization of proteins participating in the glyoxylate cycle on different sides of the peroxisomal membrane in all three model organisms requires the transport of intermediates across the lipid barrier. Transfer of metabolites ======================= Usually, the concentration of free intermediates occurring in metabolic pathways is relatively low, because a local accumulation of participating enzymes, e.g., within an organelle, or a physical interaction between successive enzymes allows a channeling of intermediates. In extreme cases, large protein complexes such as the fatty acid synthase transfer small molecules from one active center to the next. If a membrane separates consecutive steps an efficient metabolic pathway requires a direct shuttling of intermediates either through a transporter protein or through a proteinaceous channel linking enzymes on both sides of the membrane. Alternatively, a comparably high net concentration of intermediates might facilitate diffusion controlled transfer, which appears unlikely for highly reactive compounds such as glyoxylate. Permeability of the peroxisomal membrane ---------------------------------------- To seal certain reactions in a compartment and to restrict the generation of reactive molecules cells seem to spare no effort to translocate the corresponding proteins and metabolic precursors into distinct compartments. The localization of various oxidases to peroxisomes is a typical example for an energy spending activity preventing contamination of the cytosol by H~2~O~2~. In turn, the membrane delimiting a compartment thought to protect the cellular interior from detrimental effects by small, highly reactive molecules could be expected to be impermeable for such substances. However, early studies provided evidence for a permeability of the peroxisomal membrane for small solutes such as urate or amino acids and for density gradient material such as sucrose (De Duve and Baudhuin, [@B21]; Van Veldhoven et al., [@B86]). For cofactors and larger substrates required for peroxisomal enzymes such as NAD or acetyl-CoA the peroxisomal membrane was shown to act as barrier similar to the inner mitochondrial membrane (Van Roermund et al., [@B82]). Shuttle systems were proposed to functionally connect the peroxisomal lumen with the cytosol exchanging substrates and keeping the cofactors in the reduced or in the oxidized state as required (Elgersma et al., [@B24]; Van Roermund et al., [@B82]; Antonenkov and Hiltunen, [@B2]; Visser et al., [@B89]). A concept for the peroxisomal membrane permeability reconciling conflicting data was put forward by Hiltunen and co-workers (Antonenkov et al., [@B6],[@B7]). Pore-like structures permit the free exchange of small solutes (MW \< 300D) across an otherwise impermeable membrane inhibiting the transfer of molecules such as acetyl-CoA, ATP, or NAD. Accordingly, pore-forming activities were reported in peroxisomes isolated from plants, mammalian tissue and yeast (Reumann et al., [@B64]; Antonenkov et al., [@B5]; Grunau et al., [@B31]), but the molecular nature of the channel proteins remained largely unknown. In contrast, specific transporters for adenine nucleotides and NAD were identified corroborating the impermeability of the peroxisomal membrane for such bulky molecules (Palmieri et al., [@B55]; Bernhardt et al., [@B13]; for reviews see Antonenkov and Hiltunen, [@B3]; Hu et al., [@B38]). The localization of enzymatic activities of the glyoxylate cycle on different sides of the peroxisomal membrane requires an efficient transport of intermediates across the lipid barrier (Kunze et al., [@B43]). When acetyl-CoA is generated inside the peroxisomal matrix---usually via β-oxidation of fatty acids---it remains confined to peroxisomes probably because of its size (Van Roermund et al., [@B82]; Antonenkov and Hiltunen, [@B2]). CIT catalyzes the condensation of acetyl-CoA with oxaloacetate into citrate, which in turn is exported and serves as substrate for extra-peroxisomal ACO. The resulting isocitrate is imported into peroxisomes in those organisms in which the corresponding cleavage activity, ICL resides inside peroxisomes (A. thaliana, C. albicans). Thereby, succinate, the net product of the cycle is released within peroxisomes and requires an additional export mechanism. Conversely, glyoxylate is directly handed over to the second acetyl-CoA consuming enzyme, MLS, which is a peroxisomal constituent in all organisms. Some organisms such as S. cerevisiae do not harbor a peroxisomal ICL (Taylor et al., [@B75]), but instead generate the products of this reaction in the cytosol. The disadvantage that a small reactive molecule such as glyoxylate needs to be translocated across a membrane to reach MLS might be balanced by the advantage that succinate is already in the cytosol, which is one step closer to its usual final destination, mitochondria. The interesting observation that the relevant MDH activity for the glyoxylate cycle is exerted by cytosolic isoforms in the yeast S. cerevisiae and supposedly also in A. thaliana calls for an additional export of malate and the subsequent import of oxaloacetate to close the cycle (Minard and McAlister-Henn, [@B51]; Pracharoenwattana et al., [@B60]). All in all a series of transport steps is required to complete a full round of the glyoxylate cycle. If ICL is extra-peroxisomal, e.g., in the yeast S. cerevisiae, citrate and malate are exported, and glyoxylate and oxaloacetate are imported (Figure [2A](#F2){ref-type="fig"}). If ICL resides inside peroxisomes citrate, malate and succinate are exported, whereas isocitrate and oxaloacetate are imported (Figure [2B](#F2){ref-type="fig"}), a situation occurring in the plant A. thaliana. A similar flux of intermediates occurs in C. albicans, although citrate is generated in the cytosol and does not occur in peroxisomes (Figure [2C](#F2){ref-type="fig"}). ![Metabolites of the glyoxylate cycle crossing the peroxisomal membrane. The peroxisomal membrane facilitates the transfer of small metabolites. The transport of glyoxylate cycle intermediates and of C~2~-units is shown for S. cerevisiae (A), A. thaliana (B), and C. albicans (C). Hypothetical pore-forming proteins permitting the export of intermediates are colored orange, hypothetical pore-forming proteins permitting the import of intermediates are colored green. Broken lines are drawn to close the glyoxyate cycle. Intermediates that cross the peroxisomal membrane are indicated bold. Px, peroxisomal side of the membrane, Cyt, cytosolic side of the membrane.](fphys-04-00204-g0002){#F2} The export of malate and import of oxaloacetate as part of the glyoxylate cycle mimics a redox-shuttle system consisting of a peroxisomal and a cytosolic MDH activity that mediates a net-export of reduction equivalents generated by the β-oxidation of fatty acids to the cytosol. Thus, peroxisomal malate generated by MLS might be fueled into the same path to the cytosol as malate generated by the re-oxidation of NADH (peroxisomal ScMdh3p or AtpMDH1/pMDH2). Similarly, the transport of isocitrate might be part of a shuttle mechanism coupling the import of isocitrate and the export of α-oxo-glutarate with the reduction of NADP^+^ inside peroxisomes. Such a hypothetical net-exchange is based on a cytosolic isocitrate dehydrogenase (ScIdp2p) and a peroxisomal isoform (ScIdp3p) converting isocitrate into α-oxo-glutarate and CO~2~ and vice versa. The export of citrate is not restricted to the glyoxylate cycle but also presents a mode of export for C~2~-units that are generated as acetyl-CoA by fatty acid β-oxidation within peroxisomes. In this case the export of citrate is ultimately balanced by the import of oxaloacetate, the precursor for CIT. The export of citrate is considered the only export pathway for C~2~-units in plants since lack of the peroxisomal CITs, CYS2 and CYS3, blocks β-oxidation of fatty acids in A. thaliana (Pracharoenwattana et al., [@B59]), whereas in the yeast S. cerevisiae an additional export system for acetyl-units exists. For the latter, a peroxisomal form of carnitine acetyl-transferase (Cat2p; Elgersma et al., [@B24]) generates acetyl-carnitine that can be translocated across the membrane and rebuilt into acetyl-CoA either in the cytosol or within mitochondria employing either one of three carnitine acetyltransferases, Cat2p, Yat1p, or Yat2p (Swiegers et al., [@B74]). In S. cerevisiae the β-oxidation of fatty acids requires only one mode of export and thus the lack of either the peroxisomal CIT Cit2p or the carnitine acetyltransferase Cat2p localized to the peroxisomes and the mitochondria is tolerated for growth on oleic acid as carbon source, but upon deletion of both genes (ΔCIT2ΔCAT2) cells were not able to utilize oleate (Van Roermund et al., [@B84]). Moreover, under these conditions the cytosolic re-conversion of acetyl-carnitine into acetyl-CoA is also essential, as mutant cells lacking CIT2 and either one of the other carnitine acetyl-transferases (YAT1 or YAT2) were unable to consume oleic acid (Swiegers et al., [@B74]). In C. albicans a peroxisomal condensation of acetyl-CoA with oxaloacetate producing citrate is not possible due to the lack of a peroxisomal CIT. Therefore, the export of acetyl-units depends on the carnitine form and consequently, in the absence of the peroxisomal isoform of Cat2p cells were unable to grow on oleic acid (Strijbis et al., [@B73]). The transfer of the various small intermediates is compatible either with specific transporter proteins or with atypical permeability properties of the membrane. The mitochondrial and chloroplast membranes contain numerous transporter proteins specific for small organic compounds, but corresponding proteins were not identified in peroxisomes. Few examples may illustrate this. In mitochondria of S. cerevisiae a carnitine acylcarnitine carrier protein Crc1p and a citrate-oxoglutarate carrier were identified, but no peroxisomal paralog was discovered yet (Van Roermund et al., [@B84]; Castegna et al., [@B17]). Similarly, a succinate-fumarate transporter was identified in the inner mitochondrial membranes of S. cerevisiae and A. thaliana \[Sfc1p/Acr1p (Bojunga et al., [@B15]) and AtMSFC1 (Catoni et al., [@B18]), respectively\], but no homologous protein was identified to mediate plant peroxisomal succinate export. Furthermore, a glycolate/glycerate transporter required for photorespiration (PLGG1) has been identified in the chloroplast membrane (Pick et al., [@B57]), but no homologous proteins were found in plant peroxisomes. Despite overwhelming evidence for the free exchange of small solutes the molecular nature of pore-forming activities in plant and yeast peroxisomes remained unknown (Reumann et al., [@B63], [@B65]; Grunau et al., [@B31]). Moreover, the yeast peroxisomal membrane pores were shown to conduct solutes of the glyoxylate cycle (Antonenkov et al., [@B4]; Grunau et al., [@B31]). So far, only in mammalian peroxisomes a protein, Pxmp2, was identified that exhibits channel-forming capacities (Rokka et al., [@B66]). Provided that the transport of intermediates is essential for the metabolic activity, the lack of such pores should result in non-functional peroxisomes. Therefore, the corresponding genes should have turned up in various genetic screens searching for mutants with dysfunctional peroxisomes. However, more than one gene could encode redundant functions escaping the detection of these genes in screens. Likewise, such porins should be rather abundant constituents of the peroxisomal membrane and as such should have been identified in various proteomic approaches. The most abundant yeast peroxisomal membrane protein reported is Pex11p, but its localization at the outer surface renders a function in solute transport rather unlikely (Erdmann and Blobel, [@B25]; Van Roermund et al., [@B85]; Opalinski et al., [@B54]). Bipartite enzyme distribution of glyoxylate cycle enzymes: subcellular distribution follows generation of substrates ==================================================================================================================== The hypothesis that substrate availability was the driving force for changes in enzyme localization, has been proposed based on observations in the yeast S. cerevisiae. In this organism Mls1p localization differs between cells grown in medium containing ethanol as sole carbon source, when acetyl-CoA is generated primarily in the cytosol, and cells utilizing oleic acid generating acetyl-CoA within peroxisomes (Kunze et al., [@B42], [@B43]). In other organisms such as the plant A. thaliana ICL and MLS are peroxisomal. In this case an efficient coupling of the glyoxylate cycle to β-oxidation of fatty acids is essential, because both, energy and biomass production for germination and seedling outgrowth rests on the β-oxidation of fatty acid. The resulting acetyl-CoA is used to generate energy via the TCA-cycle and oxidative phosphorylation and to feed the glyoxylate cycle to cover the needs for biosynthetic processes. For the opportunistic fungus C. albicans carbohydrates and fatty acids are the prevalent carbon sources in its natural habitat, the mammalian gut. Thus, acetyl-CoA is expected to be obtained primarily inside mitochondria following glycolysis or inside peroxisomes via β-oxidation under such conditions. Even when these cells were grown in the presence of ethanol or acetate both key enzymes (ICL and MLS) are located in peroxisomes (Piekarska et al., [@B58]). Needless to mention, that neither C. albicans nor plants ever face ethanol or acetate as carbon source in their natural environment. This supports the hypothesis that the prevailing source of acetyl-CoA under natural conditions determines the localization of glyoxylate cycle key enzymes. For both model systems neither evolutionary pressure nor man-made selection forced the organism to change the intracellular location of parts of the glyoxylate cycle. In contrast, strains of baker\'s yeast, S. cerevisiae, have been selected for efficient growth in the presence of ethanol. Consequently, this organism most efficiently utilizes acetyl-CoA generated from this carbon source in the cytosol, which is apparently supported by the potential to relocate the complete glyoxylate cycle to the cytosol. The bipartite localization of enzymes provokes unusual intricacies for a straightforward metabolic pathway consisting of five enzymatic activities. Evolutionary optimization and pressure to increase efficiency demand a physiological advantage to compensate for this complexity. We suggest that the distribution of the glyoxylate cycle enzymes on different sides of the peroxisomal membrane might be due to the combination of (i) the unavoidable provision of its substrate, acetyl-CoA, inside peroxisomes by fatty acid β-oxidation (Kunze et al., [@B43]) and (ii) the incompatibility of some of its enzymes, namely ACO (Verniquet et al., [@B88]) and to a certain extent also ICL (Yanik and Donaldson, [@B93]), with the oxidative milieu within peroxisomes including high H~2~O~2~ concentrations. Thus, during evolution neither compartmentation of the complete pathway within peroxisomes nor the transfer of all enzymes into the cytosol appeared as feasible alternative. However, the tight coupling of peroxisomal acetyl-CoA generation and its fueling into the glyxoylate cycle that appears optimal for growth of A. thaliana and C. albicans in their natural habitats might restrain the incorporation of extra-peroxisomal acetyl-CoA into the glyoxylate cycle. When such organisms utilize the less physiological carbon source acetate as carbon source they satisfy all their energetic and biosynthetic needs from acetyl-CoA that is primarily generated in the cytosol. In this case the peroxisomal membrane could act as barrier that separates cytosolic acetyl-CoA from those peroxisomal enzymes of the glyoxylate cycle that utilize it, namely MLS in both organisms and CIT in A. thaliana. Under these conditions the mitochondrial energy production from cytosolic acetyl-CoA is not expected to be limited, but the biosynthetic capacity of the glyoxylate cycle is restricted by the transfer of acetyl-CoA into peroxisomes involving a specific import mechanism for this intermediate. Such a limitation is avoided in S. cerevisiae, where the glyoxylate cycle can be relocated to the cytosol. Interestingly, A. thaliana and C. albicans can grow under conditions when acetate is utilized as sole carbon source (Hooks et al., [@B36]; Zhou and Lorenz, [@B94]), but they are dependent on specific peroxisomal functions. In A. thaliana a genetic screen for mutants that are unable to utilize exogenous acetate identified two genes encoding peroxisomal proteins (Hooks et al., [@B36]). The peroxisomal transporter protein COMATOSE known to transport fatty acids (Hooks et al., [@B37]) and the intraperoxisomal short chain acyl-CoA/acetyl-CoA synthase (At3g16910, AAE7) (Turner et al., [@B79]; Shockey and Browse, [@B70]) are required for the integration of acetate into organic compounds (Allen et al., [@B1]). Interestingly, neither C. albicans nor S. cerevisiae cells require functional peroxisomes for the utilization of acetate, as cells harboring a deletion of a PEX-gene can grow on medium solely containing this carbon source (Piekarska et al., [@B58]). However, upon selective interruption of fatty acid β-oxidation by the ablation of the enzyme exerting the second step (Fox2p), C. albicans cells cannot utilize acetate any more, but S. cerevisae cells can do so (Hiltunen et al., [@B35]; Piekarska et al., [@B58]). This phentotype of C. albicans cells might be due to their inability to feed cytosolic acetyl-CoA into the glyoxylate cycle, because MLS is enclosed by the peroxisomal membrane. This notion is supported by the observation, that the ability to utilize acetate efficiently can be restored in these cells (ΔFOX2), when the compartmentation of peroxisomal enzymes is prevented by the deletion of the PTS1-receptor PEX5 (ΔFOX2ΔPEX5) (Piekarska et al., [@B58]). Thus, in organisms optimized for an efficient consumption of fatty acids the redirection of cytosolic acetyl-CoA units obtained from other sources such as acetate into peroxisomes is a critical step for its fueling into the glyoxylate cycle. Metabolic fluxes in related metabolic pathways ============================================== Photorespiration represents another example for a metabolic process in which glyoxylate is formed and intermediates traverse the peroxisomal membrane. Considering only the transport reactions between chloroplasts and peroxisomes two molecules of glycolate are transported into peroxisomes and one molecule glycerate is exported. The exchange with the mitochondria includes the export of two molecules of glycines and the import of one molecule serine (Figure [3](#F3){ref-type="fig"}). Already 40 years ago peroxisomes, mitochondria, and chloroplasts were often seen in close vicinity in the electron microscope (Frederick and Newcomb, [@B27]). A direct apposition of the peroxisomal membrane and the outer membranes of chloroplasts (Schumann et al., [@B69]) supports the hypothesis of direct exchange of metabolites thereby avoiding diffusion of intermediates into the cytosol. In leaves of A. thaliana, in which due to a mutation in a peroxisomal membrane protein the close apposition of chloroplasts and peroxisomes is lost, glyoxylate accumulates (Schumann et al., [@B69]). ![Metabolite transport processes for the glyoxylate cycle and the photorespiratory process are considered to be similar. In plant peroxisomes glyoxylate is a key metabolite of the glyoxylate cycle and the photorespiration, the latter involving mitochondria and chloroplasts. Hypothetical pore-forming proteins permitting the export are colored orange, hypothetical pore-forming proteins permitting the import are colored green. The broken lines with arrow heads indicate reactions of the glyoxylate cycle and the conversion of two molecules of glycine into serine inside mitochondria without further details. Reactions of the photorespiration process are encircled.](fphys-04-00204-g0003){#F3} Under normal conditions the glyoxylate cycle and the photorespiration do not overlap although they occur in the same organelle and share glyoxylate as key intermediate. This separation is due to the developmental program, which shows highest glyoxylate key enzyme activities during seed germination \[2--4 days postimbibition (Eastmond and Graham, [@B23])\], whereas the enzymes of the photorespiration are induced by light (Bertoni and Becker, [@B14]) upon cotyledon development. Upon this development the change of glyoxylate cycle containing peroxisomes ("glyoxysomes," see Pracharoenwattana and Smith, [@B61]) into photorespiratory active peroxisomes is considered prototypical for the exchange of enzymatic equipment of such organelles (Titus and Becker, [@B77]; Behrends et al., [@B12]) and is even accompanied by the highly surprising removal of ICL from "glyoxsomes" (Lingard et al., [@B45]). However, employing artificial experimental conditions by exposing outgrowing seedlings to light these developmental programs become blended, and so does the enzymatic equipment. This is demonstrated by the comparison of plants in which the glyoxylate cycle is interrupted either by ICL deficiency or MLS deficiency. The observed growth defect of these plants in the dark can be completely overcome by exposing seedlings lacking MLS to light, but seedlings lacking ICL can only partially recover (Cornah et al., [@B19]). The suggestion that the addition of photorespiratory equipment allows the re-direction of glyoxylate from MLS to serine/glutamine-glyoxylate aminotransferase is supported by the re-organization of the incorporation pattern of radioactively labeled acetyl-CoA into different water soluble intermediates. Thereby, wild type plants and plants lacking MLS display a more similar phenotype than plants lacking ICL. Model for the flux of intermediates across a membrane ===================================================== The localization of enzymes catalyzing successive metabolic reactions on opposite sides of the peroxisomal membrane and the permeability of this membrane for small molecules raises the question how fast small metabolites can cross the barrier membrane. Such a transfer of intermediates might be accomplished either (i) by an unspecific pore or (ii) by a series of specific transporter or facilitator proteins or (iii) by the direct coupling of specific enzymes on both sides of an unspecific pore forming a transmembrane metabolon. A simple diffusion controlled mechanism involving only a pore-like structure appears unlikely, because after reaching the other side of the membrane small metabolites could easily diffuse and the concentration might become (quite) low. This may happen, permitting the adjustment of concentrations between the cytosol and the peroxisomal matrix. However, such a model appears less likely for instable, highly reactive or even toxic intermediates such as glyoxylate. Alternatively, specific transporters or facilitators might close the gap between successive enzymatic reactions and guarantee specificity of transport, however these proteins have not been found so far. An interesting alternative might be a transmembrane metabolon comprised of transiently accumulating metabolic enzymes such as the glyoxylate cycle enzymes on both sides of an unspecific pore. We propose that in this extended form of the classical metabolon (Srere, [@B71]) pore-like channels participate in the assembly of supramolecular complexes, thereby linking proteins on both sides of the membrane enabling the swift transfer or exchange of metabolites. Such structural arrangement could enhance efficiency and ensure sufficient flux of intermediates across the peroxisomal membrane. Importantly, the transiently formed transmembrane metabolon would generate only local concentration peaks of intermediates at the entry sides of the pores. In our model metabolically active proteins transiently acquire a pore leading to a rapid transfer of the corresponding metabolites across a short distance. In this respect the observation seems interesting that polyethylene glycol (PEG), which stabilizes peroxisomal membrane integrity during the isolation procedure (Antonenkov et al., [@B6]) is also known as stabilizing agent for supramolecular protein complexes such as metabolons (Beeckmans and Kanarek, [@B11]; Barnes and Weitzman, [@B9]). Such a model would explain how a variety of substrates could cross the membrane without specific transporter molecules. The selectivity would be maintained via the transient association of proteins on both sides of the pore-like channels providing control and efficiency. According to our model in S. cerevisiae the loosely associated protein complex outside peroxisomes would consist of ACO, ICL, and MDH, on the inside of MLS and CIT, and the channel(s) would allow transferring citrate and malate from the matrix to the cytosol and/or glyoxylate and oxaloacetate from the cytosol to the peroxisomal matrix. When the peroxisomal Mdh3p has no access to the complex formed it is excluded from participation in the glyoxylate cycle. In plants, five metabolites have to cross the membrane. ACO and MDH acting outside peroxisomes require the export of citrate and malate and the import of isocitrate and oxaloacetate for further processing. The product succinate needs to be exported as well to make it available for the cellular metabolism. Since according to our model ICL would loosely associate with CIT and MLS it is reasonable to suggest that the simultaneous export of succinate using the same channel unit represents an additional drive for the cleavage reaction. Furthermore, the peroxisomal membrane would per se not represent a principal barrier for glyoxylate cycle intermediates or any other small metabolite, but controls the velocity of the continuous flux. In addition, this model could also explain how shuttles may function. A local concentration of enzymes on both sides of a membrane-spanning pore could facilitate the shuttling of reduction equivalents in \[isocitrate/α-oxo-glutarate (Van Roermund et al., [@B83])\] or out \[malate/oxaloacetate (Van Roermund et al., [@B82]), lactate/pyruvate (Baumgart et al., [@B10]), G3P/DHAP (Gee et al., [@B29])\] of the peroxisomal matrix (for review see Antonenkov and Hiltunen, [@B2]). The exchange of the reduced and oxidized intermediates would occur due to a local association of the corresponding enzymes, Mdh2p/Mdh3p or Idp2p/Idp3p, on each side of a channel. Conclusions and perspectives ============================ The peroxisomal matrix is surrounded by a single membrane that allows the enclosure of a variety of highly reactive and even toxic compounds. However, in vitro the membrane appears permeable to small molecules and neither a chemical nor an electrochemical gradient has been detected, which represents a major difference to the mitochondrial inner membrane or the thylakoid membrane. The biophysical properties of the peroxisomal membrane demands pore-like structures in addition to the few transmembrane proteins with transporter function for larger molecules. The frequent exchange of intermediates between peroxisomes and the intracellular environment requires directed transport processes with high selectivity, which appears incompatible with pore-like exchange modules. However, a model in which soluble enzymes accumulate in proximity to both ends of such pore-like structures forming a transmembrane metabolon would explain rapid and selective exchange based on a local increase in intermediate concentration. The observation that the enzymes of the glyoxylate cycle are distributed across both sides of the peroxisomal membrane would be prototypical for such transport processes, because an efficient transfer of small organic metabolites across the peroxisomal membrane is essential for this pathway. The high variability in the nature of small organic molecules transported across the peroxisomal membranes between S. cerevisiae, C. albicans, and A. thaliana may reflect the ease of adaptation processes whenever specific transporter proteins are not involved. Similar considerations are applicable to the photorespiration, although the direct exchange of metabolites with other organelles renders the latter apparently more complex. A transport system of such high versatility generates an organelle of high plasticity and allows rapid adjustments to environmental changes and to developmental programs generally considered an important feature of peroxisomes. Altogether, the peroxisomal membrane markedly differs from membranes of other metabolically active organelles, which might represent an intermediate step in the development of an organelle with novel properties. Thus, it can serve as model system to investigate and to understand transport processes across membranes with reduced complexity. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. [^1]: Edited by: Vladimir I. Titorenko, Concordia University, Canada [^2]: Reviewed by: J. Kalervo Hiltunen, University of Oulu, Finland; Vladimir I. Titorenko, Concordia University, Canada [^3]: This article was submitted to Frontiers in Integrative Physiology, a specialty of Frontiers in Physiology.	{ "pile_set_name": "PubMed Central" }	[ "amount", "synthas", "cleavag", "rest", "probabl", "discov", "two", "control", "bean", "origin", "peterhansel", "remain", "program", "eg", "nitrogen", "eukaryot", "opalinski", "compris", "acoul", "compat", "respond", "fuel", "impair", "phosphoglycol", "acylcarnitin", "abund", "ccarbon", "sucros", "exist", "overwhelm", "gene", "gluconeogenesi", "identifi", "put", "employ", "malateoxaloacet", "berg", "block", "round", "select", "rais", "mechan", "poreform", "luttik", "convers", "locat", "enter", "castegna", "signal", "may", "cit", "keep", "pexgen", "taylor", "therebi", "αoxoglutar", "recent", "porelik", "mdhul", "acquir", "pexp", "separ", "gener", "use", "acetyltransferas", "forward", "final", "supposedli", "supplement", "ever", "support", "convinc", "caus", "complet", "accord", "irrespect", 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Methylamine (CH~3~NH~2~) is the simplest primary amine in chemistry. Its CH~3~ group is connected to the NH~2~ group by the C−N bond that exhibits a vibrational stretching motion. As for the two groups themselves, the CH~3~ group has an internal torsional motion while the NH~2~ group has an inversion motion, which makes the CH~3~NH~2~ a prototype in molecular physics for non-rigid molecules having two coupled large-amplitude internal motions. The torsion splits the rotational energy levels into a threefold pattern of E and A symmetry components with an E/A spin statistical weight ratio of 1/2, and inversion produces a further splitting into s and a doublets with spin weight ratio of 1/3. Thus, each vibration-rotation level of CH~3~NH~2~ has Aa, As, Ea and Es sublevels, resulting in a complex vibration-rotation-torsion-inversion energy structures and rich but highly crowded spectra with a wide range of relative line intensities. Therefore, the interesting microwave[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9][@b10], far-infrared[@b11][@b12][@b13][@b14][@b15][@b16][@b17], and infrared[@b18][@b19][@b20][@b21][@b22][@b23] spectra of CH~3~NH~2~ have been extensively studied for many years to explore the splittings and the symmetry species, leading to the valuable application in the detection of interstellar methylamine[@b24][@b25][@b26][@b27]. In contrast, despite the fact that it is of great practical interest related to optically pumped far-infrared laser lines and interstellar detection, spectroscopic study by the sub-Doppler technique on the stretching of the C−N bond that connects the CH~3~ and NH~2~ groups has remained rare until recently[@b28]. Study of the C−N stretching band of CH~3~NH~2~ has been of great spectroscopic, practical, and astrophysical interest for decades. The C−N stretching band of CH~3~NH~2~, earlier studied and reported sixty years ago at the relatively low resolution of 1 cm^−1^ and an accuracy of about 0.1 cm^−1^ by Gray and Lord[@b19], displays strong characteristic absorptions in the infrared region around 1044 cm^−1^. It is characterized by a parallel structure with vibrational P, Q, and R branches, which overlaps well with the CO~2~ laser bands. Thus, CH~3~NH~2~ has been an important member of the class of laser media employed to generate far-infrared laser lines by optically pumping with CO~2~ laser transitions. However, few of the CH~3~NH~2~ lines have so far been identified as to their quantum numbers due to the limited accurate experimental information available on this important band. It is only recently that high resolution spectroscopy of the C−N stretching band of CH~3~NH~2~ has been reported. In 2011, Lees et al.[@b21] and Gulaczyk et al.[@b22] investigated Doppler-limited Fourier-transform (FT) spectra of this band. Numerous transitions were assigned, perturbations from several resonances between the C−N stretching state and high-lying torsional substates of the ground state were analyzed, with both Fermi and Coriolis resonances being observed. Yet to date there are still many overlapped lines and unresolved asymmetry doublet lines in the FT spectrum for this band of CH~3~NH~2~ due to limit on spectral resolution imposed by the Doppler width. It is very desirable to carry out sub-Doppler observations on this band employing a spectrometer with very high resolution and accuracy in order to make confident and reliable measurements and line assignments. In 2010, we observed the precise Lamb-dip spectra of the C−N stretching band of CH~3~NH~2~ at a spectral resolution of 0.4 MHz and determined the transition frequencies with an accuracy of ±0.1 MHz[@b28], which is the first sub-Doppler observation in any spectral range of CH~3~NH~2~. However, the first Lamb-dip spectra of this band have been observed just for 43 saturation dips which are primarily in the C−N stretching Q-branch region and only in two sub-states. There is a clear need to observe many more Lamb-dip signals over a wider range of transitions in P-, Q-, and R-branch to obtain accurate experimental information and parameters for detailed spectroscopic analysis. These precise Lamb-dip measurements are important not only for disentangling overlapped features in the Doppler-limited spectra but also in providing a grid of standard reference frequencies for accurate calibration of the overall FT spectrum. Accordingly, in the present work, using frequency-tunable microwave sidebands of a much larger group of CO~2~ laser lines in our spectrometer[@b28], the Lamb-dip spectroscopy of the C−N stretching band of CH~3~NH~2~ has been systematically studied to cover a greatly expanded set of quantum states. We now report our extended experimental results. Results and Discussion ====================== A frequency-tunable infrared source for Lamb-dip observation ------------------------------------------------------------ Although the C−N stretching band center of CH~3~NH~2~ overlaps well with the CO~2~ laser bands, it is difficult to observe many absorption lines of CH~3~NH~2~ by using just a grating CO~2~ laser, because the available spectral coverage between the CO~2~ laser and CH~3~NH~2~ absorption lines is limited to the overlap in Doppler widths of only about 60--100 MHz for each laser line. This situation was recently improved greatly by the application of a frequency-tunable infrared source in a dual-mode sideband spectrometer to CH~3~NH~2~[@b28]. The schematic of the experimental setup used in the present work for the Lamb-dip spectroscopy of CH~3~NH~2~ is shown in [Fig. 1](#f1){ref-type="fig"}, and has been described in detail previously[@b28]. The tunable radiation is generated by adding microwave sidebands to the CO~2~ laser lines in a GaAs waveguide modulator. In the modulator, the added microwave radiation with a frequency f~MW~ produces a periodic variation of the refractive index of the GaAs crystal and a corresponding small phase change of Δϕ to the incident CO~2~ laser field. Therefore, the laser output from the modulator includes both the input field of the CO~2~ laser plus microwave-modulated field components. The latter are called microwave sidebands of the CO~2~ laser lines. In practice, the output from the modulator shows three spectral signals at f~L~ (frequency of CO~2~ laser carrier), f~L~ + f~MW~ (upper sideband), and f~L~−f~MW~ (lower sideband), respectively. These upper and lower microwave sidebands of CO~2~ laser lines provide a powerful radiation source for precision spectroscopy, operating at room-temperature with narrow linewidths and continuously tunable and precisely controlled frequencies in the 9--11 μm region. Our infrared source has the three main features of 1) a tunability range about 23.6 GHz (±6.7 to ±18.5 GHz) for each CO~2~ laser line by sweeping the microwave frequency f~MW~; 2) a typical microwave-modulated CO~2~ laser power about 10 mW for either the upper or lower sideband; 3) accurate radiation frequency. In operation, the CO~2~ laser is stabilized to the center of a 4.3 μm fluorescence Lamb-dip signal and has an estimated frequency uncertainty of 33 kHz[@b29]. High-resolution observation and precise measurement of the spectral Lamb-dips ----------------------------------------------------------------------------- A Lamb-dip signal has a much narrower spectral linewidth than that of the Doppler-broadened spectral line. The Lamb-dip spectroscopic technique can thus enable blended lines in the Doppler-limited spectrum to be fully resolved and the centers of these resolved absorption lines to be determined very precisely. [Figure 2](#f2){ref-type="fig"} shows a Lamb-dip spectrum for a blended line in the extremely congested Q-branch of the C−N stretching band center of CH~3~NH~2~ around 1044.5930 cm^−1^ in the FT spectrum. A 50 MHz scan of the lower microwave sideband of the 9P22 CO~2~ laser line was recorded at a pressure of 10 mTorr in 2nd derivative (2f) detection mode using a lock-in amplifier time constant of 30 ms. We can see that two spectral lines with an interval of just 30 MHz have been clearly resolved. This shows the usefulness of the high power and wide frequency-tunability of the CO~2~-microwave sidebands for resolving the overlapped features by Lamb-dip observations. Furthermore, in order to precisely measure the absolute transition frequencies for each of the individual line, we narrowed the microwave scanning range down to 3 MHz and swept the microwave sideband both upward and downward 5 times for each line with a frequency step-size of 10 kHz to record their saturation-dip 2f signals. [Figure 3](#f3){ref-type="fig"} displays the result for signal I of [Fig. 2](#f2){ref-type="fig"}, recorded at 14 mTorr pressure with a lock-in time constant of 30 ms. We then fitted the experimental trace (black) to determine the center transition frequency by a least-squares fit to the second derivative of a Gaussian profile[@b29] where G~0~ is a baseline constant, G~1~ is the integrated intensity, ν~0~ is the center frequency, and Δν~pp~ is the frequency separation between positive and negative peaks of the first-derivative G'(ν). The fitting trace is shown as a red solid curve in [Fig. 3](#f3){ref-type="fig"}. From this fit, the microwave frequency at line center was determined to be 12839.477 MHz, which yields an infrared transition frequency as 9P22--12839.477 MHz, giving 31 316 122.019 MHz with an accuracy of 0.1 MHz when the known frequency of the 9P22 CO~2~ laser line[@b30] is added. In the current study, we have measured more than 150 saturated absorption dips for spectral lines which belong to 27 C−N stretching substates. The assignments of these transitions, the measured transition frequencies, and the determined upper-state energy term values are presented in [Table 1](#t1){ref-type="table"}. The observed infrared transitions and assignments of CH~3~NH~2~ --------------------------------------------------------------- In [Table 1](#t1){ref-type="table"}, the transition notation of P/Q/R(υ~t~ S~t−i~ K, J) expresses the assigned quantum numbers for each of the identified spectral lines belonging to the P-, Q-, and R-branch, respectively. Here, υ~t~ is the torsional quantum number, S~t-i~ is the torsion-inversion symmetry (A or E for torsional species and a or s for inversion species), and K is the projection along the molecular near-symmetry a-axis of the rotational angular momentum J. For asymmetry K-doubling levels of A torsional symmetry, we add a superscript + or − to K to indicate the resolved doublet components. The C−N stretching fundamental is a parallel a-type band, the transition selection rules are Δυ~t~ = 0, ΔK = 0, and ΔJ = 0, ±1. Other researchers[@b22] use another common notation of the G~12~ or D~3h~ molecular symmetry group species A, B, E~1~, and E~2~, which corresponds here to As, Aa, Ea, and Es, respectively. The second and third columns list the determined transition frequencies in MHz and in wavenumbers, respectively, according to the specific CO~2~-microwave sideband used. For example, the Lamb-dip signals I and II in [Fig. 2](#f2){ref-type="fig"} observed with the lower microwave sideband of the 9P22 CO~2~ laser line are assigned as transition Q(0 As 8, 8) at a transition frequency of 1044.593391 cm^−1^ and as Q(0 As 5, 5) at 1044.592375 cm^−1^, respectively. The latter was first observed and reported in our previous work[@b28], and has been confirmed here. In [Table 1](#t1){ref-type="table"}, a letter U in the first column denotes a line that is still unassigned in the spectrum. As lines of s inversion symmetry are weak with only 1/3 the intensity of the corresponding a lines due to the relative spin statistical weights, our data are primarily for the a inversion species. For a substantial number of transitions, we could test our assignments and measurement accuracy via application of the Rydberg-Ritz combination principle to closed loops involving the observed lines. As an example for illustration, an energy-level diagram for the (υ~t~ S~t−i~ K) = (0 Aa 3) sub-band is shown in [Fig. 4](#f4){ref-type="fig"}. For each pair of transitions sharing a common upper-state level in [Fig. 4](#f4){ref-type="fig"}, we can form four closed loops involving the eight infrared transitions with labels a to h and the eight microwave transitions in the ground state. By using the predicted microwave frequencies[@b17] (uncertainty of 0.06 MHz) in Loops 1 and 2 and the observed microwave frequencies[@b17] (uncertainty of 0.06 MHz) in Loops 3 and 4, we calculate the loop closure defects (in MHz) as follows: Loop 1: δ~8~^+^ = a − c − ν~1~ − ν~2~ = (9P10 + 14334.992) − (9P36 − 15429.241) − 399010.809 − 354681.521 = − 0.14, Loop 2: δ~8~^−^ = b − d − ν~3~ − ν~4~ = (9P10 + 14339.825) − (9P36 − 15450.205) − 399027.383 − 354690.570 = 0.01, Loop 3: δ~13~^+^ = e − g − α~1~ − α~2~ = (9P2 + 13882.574) − (9P44 − 14507.345) − 620591.113 − 576287.579 = 0.01, Loop 4: δ~13~^−^ = f − h − β~1~ − β~2~ = (9P2 + 13914.907) − (9P44 − 14736.996) − 620746.167 − 576394.504 = − 0.01. Since each loop contains two infrared transitions, the fact that these loop defects are all so close to zero confirms the transition assignments in each of the loops and our experimental uncertainty of 0.1 MHz. Over 30 closed combination loops of transitions have been formed from the present and previous sub-Doppler observations. Line assignments shown with a letter L in [Table 1](#t1){ref-type="table"} are confirmed from these frequency combination closure relations. Such calculations of loop defects are very useful for providing secure assignments for the resolved K-doublet lines and for the component lines in blended features, especially for those spectral lines located around the crowded band center. We noticed that when term values for the ground state (kindly provided by N. Ohashi from his microwave and FIR analyses[@b7][@b14]) are used to calculate the related energy differences involved in the above mentioned loops, we found that the loop defects in MHz from Loop 1 to Loop 4 are −1.05, −2.25, −0.72, and −2.58, respectively. This indicates the estimated uncertainty of those term values to be about 1 MHz, consistent with the estimate from the microwave experimental study by Ilyushin et al.[@b9]. Comparison of the measured transition frequencies with those in the FT spectrum ------------------------------------------------------------------------------- The last two columns of [Table 1](#t1){ref-type="table"} show the frequency differences between the present Lamb-dip measurements and the data in the FT spectrum reported in Ref. [@b21] and Ref. [@b22], respectively. A blank space indicates either a new assignment or an unidentified U line. A histogram illustrating the distribution of these differences is given in [Fig. 5](#f5){ref-type="fig"}. The inset shows the histogram for the FT infrared data reported in Ref. [@b21]. We see that most of the deviations are less than several megahertz, but some of them are tens of megahertz. The mean value of the absolute deviation is about 5 MHz and 8 MHz for the FT data in Ref. [@b21] and Ref. [@b22], respectively, which reflects the frequency accuracy of the FT data estimated in these two works for infrared transitions in the C−N stretching band of CH~3~NH~2~. Power-series expansions of the energy term values in the excited state ---------------------------------------------------------------------- The fourth column of [Table 1](#t1){ref-type="table"} lists energy term values W(υ~t~ S~t − i~ K, J) for the upper levels of the corresponding transitions in the first column, obtained by adding our measured transition frequencies in wavenumbers to the Ohashi calculated ground-state energies, referenced to the (υ~t~ S~t−i~ K, J) = (0 As 0, 0) ground level as the energy zero. When two or three transitions were observed having a common upper level but giving independent values, the average of those term values was taken. In order to determine the J-independent origins of 27 C−N stretching substates, their term values were least-squares fitted to J(J+1) power-series expansions where a~0~ is the J-independent substate origin, a~1~ is an effective rotational constant, and a~2~ and a~3~ are effective centrifugal distortion constants. The obtained expansion coefficients a~0~, a~1~, a~2~, and a~3~ for 27 substates are shown in [Table 2](#t2){ref-type="table"}, in which the 1-σ standard deviations are in units of the last quoted digit. Asymmetry splittings and asymmetry-splitting constants of A symmetry levels in the excited state -------------------------------------------------------------------------------------------------- A rotational level with A symmetry of CH~3~NH~2~ may split due to the higher-order vibration-rotation interactions. For the resolved K-doublet lines, the transition selection rules are K^±^ ↔ K^±^ for ΔJ = ±1 and K^±^ ↔ K ^∓^ for ΔJ = 0. We have observed 57 Lamb-dip signals for resolved K-doublet lines resulting from the asymmetry splittings and precisely determined their transition frequencies. From the calculated level splittings in the ground state, asymmetry splittings for Aa levels with K = 1, 2, and 3 in the υ~t~ = 0 excited state have been determined and are shown in [Table 3](#t3){ref-type="table"}. The observed asymmetry splittings ΔE(υ~t~ S~t−i~ K, J) can be approximately represented by where b~0~ is the principal asymmetry splitting constant and other b~m~ with m ≥ 1 are higher-order corrections. The resulting series expansion coefficients from least-squares fits of the observed asymmetry splittings to [Eq. (3)](#eq3){ref-type="disp-formula"} for the three K-states are presented in [Table 3](#t3){ref-type="table"}. Conclusion ========== In this work for a molecule with the two strongly coupled large-amplitude internal motions of torsion and inversion, by using microwave sidebands of CO~2~ laser lines as frequency-tunable infrared sources in a sub-Doppler spectrometer, Lamb-dip spectroscopy of the C−N stretching band of CH~3~NH~2~ has been systematically studied. Many blended features and unresolved K-doublet lines involving wide variations in relative intensities in dense Doppler-limited spectra have been separated at high resolution. More than 50 K-doublet lines have been observed and the asymmetry-splitting constants for levels with K = 1, 2, and 3 in the excited state have been determined. Over 150 transitions have been assigned and identified into 27 substates and their transition frequencies have been precisely measured with an absolute accuracy of ±0.1 MHz. Energy term values for the upper levels of these assigned transitions have been determined and have been fitted to J(J + 1) power-series expansions for each substate to determine the J-independent C−N stretching substate origins and effective rotational constants. The Rydberg-Ritz combination principle was fully used in calculations of the defects for closed combination loops involving our observed transitions for confirming, revising and extending the transition assignments from previously reported results. Our experiment demonstrates the capabilities of the current experimental setup to precisely study the sub-Doppler infrared spectroscopies of isotopic species of CH~3~NH~2~ and of molecules with more than two internal motions. Our results constitute a high-accuracy database for frequency calibration in the 9--11 μm region, provide more accurate spectral information for excited vibrational states to clearly map the rich and complex energy structures, to reveal the complex interaction mechanisms relevant to the C−N stretching of CH~3~NH~2~, to support further possible astronomical detections of interstellar CH~3~NH~2~, and to assign more energy levels and transition systems for optically pumped far-infrared laser emissions of CH~3~NH~2~. Methods ======= The tunable microwave radiation is generated by a microwave synthesizer and is amplified by a traveling-wave-tube amplifier to a power of about 20W before being fed to the modulator. Under this condition, the typical conversion efficiency into the sidebands from the incident laser beam is about 0.8%. In order to saturate the transitions of CH~3~NH~2~ at low pressures of several mTorr in this work, the full output radiation from the modulator is focused into our multi-reflection absorption cell with a total absorption path of 9.6 m in 16 transits. For observation of the saturation Lamb-dips by generating a counter-propagating beam inside the cell, a mirror M5 is placed at the exit of the cell window to reflect the radiation which has passed through the absorption cell back into the cell. This mirror is adjusted carefully so that the retro-reflected radiation nearly coincides with the incoming radiation but is slightly shifted and passes through a tunable Fabry--Pérot etalon filter for selecting only the desired sideband containing the CH~3~NH~2~ absorption signal. In such an optical arrangement, only those molecules moving at zero velocity parallel to the beam can absorb both the two counter-propagating laser radiations which have the same frequency, creating a saturation dip at the center of the absorption curve. This narrow dip is then detected by a liquid-N~2~-cooled HgCdTe detector as a Lamb-dip signal. In order to increase the probing sensitivity for the spectral signals, we use 1 kHz modulation of the sideband laser frequency for source modulation and demodulate the detected signal with a digital lock-in amplifier operating in the second-derivative (2f) detection mode to display the spectral lines. The signals are then sent to a computer for recording and analysis. A commercial CH~3~NH~2~ sample supplied by BOC Specialty Gases with a stated purity of 99.5% was used in the experiment without further purification. High attention should be taken for avoiding the possible confusion in the spectral analysis from NH~3~ impurity lines. Additional Information ====================== How to cite this article: Sun, Z.-D. et al. Lamb-dip spectroscopy of the C − N stretching band of methylamine by using frequency-tunable microwave sidebands of CO~2~ laser lines. Sci. Rep. 6, 34270; doi: 10.1038/srep34270 (2016). We express our thanks to Prof. N. Ohashi for providing a list of calculated ground-state energies. This research was partially supported by the National Natural Science Foundation of China (Grants No. 11174186, 91536105, and 11374191) and the Tianshan Scholar Program. L.H.X and R.M.L acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada. Author Contributions Z.-D.S. contributed to the experimental setup, conducted measurements, analyzed the data, and wrote the manuscript. S.-D.Q. performed the data analyses. R.M.L. and L.-H.X. conducted the calculation and revised the manuscript. All authors contributed to the scientific discussions and approved the final manuscript. ![Schematic of the sub-Doppler spectrometer using microwave sidebands of CO~2~ laser lines as frequency-tunable infrared sources for the Lamb-dip spectroscopy of CH~3~NH~2~.\ M1--M5, mirrors; BS1, BS2, beam splitters; MW, microwave; TWT Amp., traveling wave tube amplifier; F.--P. etalon, Fabry--Pérot etalon filter; Detector1, InSb detector; Detector2, HgCdTe detector.](srep34270-f1){#f1} ![Observation of two Lamb-dip signals I and II resolved from an overlapped feature in the FT spectrum of CH~3~NH~2~.\ The spectrum was recorded in a scanning range of 50 MHz using the lower microwave sideband of the 9P22 CO~2~ laser line. The sample pressure was 20 mTorr and the lock-in time constant was 30 ms. Signals I and II are assigned as transitions Q(0 As 8, 8) and Q(0 As 5, 5), respectively. The observation of Signal II has previously been reported in Ref. [@b28] and has been confirmed here. This chart demonstrates features of the high power for observation of the saturated absorption spectra and the wide frequency-tunability of the microwave sidebands of CO~2~ laser lines.](srep34270-f2){#f2} ![Lamb-dip signal for the Q(0 As 8, 8) line of the C−N stretching band of CH~3~NH~2~ (signal I in [Fig. 2](#f2){ref-type="fig"}), scanned in a range of 3 MHz with a step-size of 10 kHz and recorded with the lock-in amplifier in 2f detection mode.\ The lock-in time constant was 30ms and the sample pressure of CH~3~NH~2~ was 14 mTorr. The solid curve (red) is a least-squares fit of a second-derivative Gaussian profile to the experimental trace (black). The microwave frequency at line center was determined from the fit to be 12839.477 MHz.](srep34270-f3){#f3} ![K−doublet energy-level diagram for the observed 8 infrared transitions accessing the (υ~t~ S~t − i~ K) = (0 Aa 3) substate of CH~3~NH~2~ in the Lamb-dip sub-Doppler spectrum.\ The measured infrared transition frequencies have been given in [Table 1](#t1){ref-type="table"}. The calculated frequencies (in MHz) for ground-state transitions in Ref. [@b17] are ν~1~ = 399010.809, ν~2~ = 354681.521, ν~3~ = 399027.383, ν~4~ = 354690.570. The measured transition frequencies (in MHz) in the ground states in Ref. [@b17] are α~1~ = 620591.113, α~2~ = 576287.579, β~1~ = 620746.167, β~2~ = 576394.504. The asymmetry splittings of the energy levels are exaggerated for clarity.](srep34270-f4){#f4} ![Histogram of the deviations (in MHz) for transition frequencies between our Lamb-dip measurements presented in [Table 1](#t1){ref-type="table"} and the FT infrared data reported in Ref. [@b22].\ The inset shows the histogram for the FT infrared data reported in Ref. [@b21].](srep34270-f5){#f5} ###### Observed infrared transitions and their upper-state term values for the C−N stretching band of CH~3~NH~2~. [a](#t1-fn1){ref-type="fn"}Transition R/P/Q(υ~t~ S~t−i~ K, J) [b](#t1-fn2){ref-type="fn"}CO~2~ laser line ± microwave req. (MHz) [c](#t1-fn3){ref-type="fn"}ν~obs~. (cm^−1^) Upper-state term value (cm^−1^) [d](#t1-fn4){ref-type="fn"}ν~FT1~ - ν~obs.~ (MHz) [e](#t1-fn5){ref-type="fn"}ν~FT2~ - ν~obs.~ (MHz) ------------------------------------------------------------------------- -------------------------------------------------------------------- --------------------------------------------- --------------------------------- --------------------------------------------------- --------------------------------------------------- R(0 Ea 4, 23) 9R16 − 11898.876 1075.590916 1527.054716 −29.3 −26.3 R(0 Aa 1^−^, 23) 9R16 − 12671.411 1075.565147 1489.380347 −15.5 −14.7 R(0 Ea 4, 20) 9R10 + 10911.802 1072.247745 1426.195845 −13.9 −15.4 R(0 Ea 7, 20) 9R10 + 10894.123 1072.247155 1515.100055 3.8 R(0 Aa 6, 20) 9R10 + 10689.436 1072.240327 1480.052327 1.3 −1.0 R(0 Aa 5, 20) 9R10 + 10553.581 1072.235796 1450.347096 −61.6 −45.4 R(0 Aa 3^+^, 20) 9R10 + 10507.877 1072.234271 1407.452171 −15.9 0.3 R(0 Aa 1^−^, 20) 9R10 + 10487.973 1072.233607 1388.031807 4.0 20.2 R(0 Aa 3^−^, 20) 9R10 + 10453.378 1072.232453 1407.567753 38.6 54.8 R(0 Ea −2, 20) 9R10 + 10305.241 1072.227512 1393.266612 −9.6 −6.7 R(0 Aa 2^−^, 18) 9R8 − 8791.434 1070.169057 1333.969857 −1.7 2.7 R(0 Aa 2^+^, 18) 9R8 − 9899.311 1070.132103 1334.975403 −1.8 −4.0 R(0 Ea −3, 18) 9R8 − 11187.383 1070.089137 1347.673137 9.4 6.9 R(0 Ea 3, 18) 9R8 − 11829.536 1070.067717 1347.269817 6.4 18.5 R(0 Aa 1^+^, 18) 9R8 − 15025.886 1069.961098 1322.810798 1.6 0.5 R(0 Aa 6, 18) 9R8 − 15611.927 1069.941550 1420.150150 −14.7 −25.5 R(0 Aa 3^−^, 18) 9R8 − 15931.388 1069.930894 1347.569494 −15.7 −18.4 R(0 Ea −2, 18) 9R8 − 16465.53 1069.913077 1333.443077 −3.5 0.3 R(0 Ea 2, 18) 9R8 − 16694.656 1069.905434 1334.541634 3.8 0.8 R(0 Aa 7, 18) 9R6 + 11061.409 1069.383062 1454.799162 6.0 −0.7 R(0 Aa 4^+^, 18) 9R6 + 10912.404 1069.378092 1365.464792 −6.9 −8.3 R(0 Aa 4^−^, 18) 9R6 + 10896.661 1069.377566 1365.462566 8.8 7.4 R(0 Ea −1, 17) 9R6 − 8350.046 1068.735565 1295.477665 16.7 R(0 Aa 4^−^, 16) 9R4 − 15504.517 1067.021935 1311.376435 8.6 4.1 R(0 Aa 7, 16) 9R4 − 16034.697 1067.004250 1400.722550 −3.2 −0.5 R(0 Aa 2^+^, 15) 9R2 + 15797.556 1066.564310 1255.648210 R(0 Aa 2^−^, 15) 9R2 + 15795.090 1066.564227 1255.115127 −0.7 2.1 R(0 Ea −5, 15) 9R2 + 13606.873 1066.491236 1311.601836 0.8 0.5 R(0 Ea −3, 15) 9R2 + 12983.316 1066.470437 1268.633737 1.1 0.6 R(0 Aa 8, 15) 9R2 + 11893.876 1066.434097 1416.913897 −25.6 R(0 Ea 3, 15) 9R2 + 11845.906 1066.432497 1268.214197 22.3 19.9 R(0 Ea 1, 15)L 9R2 + 10302.783 1066.381024 1248.412624 2.3 −0.2 R(0 Aa 6, 15) 9R2 + 10069.842 1066.373254 1341.234754 11.0 20.7 R(0 Aa 3^−^, 15) 9R2 + 9488.821 1066.353873 1268.554973 −31.2 −28.9 R(0 Aa 5, 15) 9R2 + 9277.964 1066.346840 1311.482840 −6.5 −4.5 R(0 Ea −2, 15) 9R2 + 8780.626 1066.330250 1254.592450 −24.5 −24.2 R(0 Ea −5, 12) 9P2 + 17768.953 1062.758673 1245.792673 −5.1 −7.1 R(0 Ea −3, 12)L 9P2 + 16567.900 1062.718611 1202.770811 4.3 −1.7 R(0 Aa 8, 12) 9P2 + 16467.650 1062.715267 1351.145967 2.9 5.2 R(0 Ea 1, 12) 9P2 + 15260.579 1062.675003 1182.112603 0.9 −0.3 R(0 Aa 1^−^, 12)L 9P2 + 14932.119 1062.664047 1181.933847 −15.4 −15.5 R(0 Ea 4, 12)L 9P2 + 14173.561 1062.638744 1221.484244 −2.7 −1.2 R(0 Ea 5, 12) 9P2 + 13932.828 1062.630714 1245.862714 −29.1 R(0 Aa 3^−^, 12)L 9P2 + 13914.907 1062.630116 1202.713316 −11.2 −13.7 R(0 Aa 3^+^, 12)L 9P2 + 13882.574 1062.629038 1202.706238 21.1 18.6 R(0 Aa 5, 12) 9P2 + 13705.152 1062.623120 1245.683420 −2.3 0.7 R(0 Ea −2, 12)L 9P2 + 13373.523 1062.612058 1188.822658 −6.7 −4.7 U 9P2 + 13332.331 1062.610684 R(0 Ea 2, 12) 9P2 + 12741.567 1062.590978 1189.173578 −9.7 −5.7 R(0 Ea −1, 12) 9P2 + 12730.848 1062.590620 1179.746320 1.0 5.1 R(0 Aa 1^+^, 12) 9P2 + 11318.384 1062.543506 1179.529006 −3.7 −0.2 R(0 Aa 4, 11) 9P4 + 8971.098 1060.869910 1201.743110 −4.7 −0.1 R(0 Aa 1^+^, 9)L 9P6 − 8477.145 1058.665947 1127.387047 −3.1 0.7 R(0 Aa 2^+^, 8) 9P8 + 9991.249 1057.633433 1122.155833 −4.7 −0.7 R(0 Aa 2^−^, 8) 9P8 + 9671.323 1057.622762 1122.096162 −3.2 −2.1 R(0 Aa 4, 8) 9P8 − 9088.952 1056.996986 1153.512786 −1.5 0.1 R(0 Ea 1, 7) 9P10 + 16357.707 1056.170702 1101.016902 −0.5 2.2 R(0 Ea −3, 7)L 9P10 + 16082.288 1056.161515 1122.270215 −1.5 −5.9 R(0 Aa 6^−^, 7) 9P10 + 15748.817 1056.150392 1195.035492 R(0 Aa 6^+^, 7) 9P10 + 15748.072 1056.150367 1195.035467 −14.7 −12.4 R(0 Aa 1^−^, 7) 9P10 + 15431.349 1056.139802 1100.808202 −6.8 −4.1 R(0 Aa 3^−^, 7)L 9P10 + 14339.825 1056.103393 1122.240693 −7.4 −7.5 R(0 Aa 3^+^, 7)L 9P10 + 14334.992 1056.103232 1122.240332 −2.6 −2.7 R(0 Aa 5^−^, 7) 9P10 + 14226.358 1056.099608 1165.243108 R(0 Aa 5^+^, 7) 9P10 + 14224.595 1056.099549 1165.243049 −10.6 −7.4 R(0 Ea 3, 7)L 9P10 + 14191.512 1056.098446 1121.821546 22.5 25.6 R(0 Ea −2, 7)L 9P10 + 13868.159 1056.087660 1108.348360 1.3 −0.9 R(0 Ea 2, 7)L 9P10 + 12586.227 1056.044899 1108.598499 1.7 −1.8 R(0 Aa 1^+^, 7)L 9P10 + 11458.187 1056.007272 1099.853072 −0.5 −0.1 R(0 Ea −1, 7)L 9P10 + 11298.615 1056.001949 1100.011549 2.6 4.6 R(0 Aa 4, 6) 9P12 + 12380.893 1054.336485 1128.671685 −5.4 −5.4 R(0 Ea 1, 5)L 9P12 − 14021.903 1053.455783 1078.900283 0.7 1.2 R(0 Aa 1^−^, 5)L 9P12 − 15401.199 1053.409774 1078.668574 −5.6 −1.9 R(0 Aa 3, 5) 9P12 − 16245.122 1053.381624 1100.293124 −1.4 −3.2 R(0 Ea 3, 5) 9P12 − 16712.009 1053.366050 1099.863150 11.3 10.2 R(0 Ea −2, 5) 9P12 − 16817.968 1053.362516 1086.392416 5.4 4.2 R(0 Ea 2, 5) 9P12 − 18065.890 1053.320890 1086.642890 −3.4 3.2 Q(0 Aa 3^+^, 4) 9P22 − 10210.545 1044.681083 1084.198083 2.3 Q(0 As 8, 8) 9P22 − 12839.477 1044.593391 1270.813991 −17.5 −19.6 Q(0 Ea −6, 6) 9P22 − 15577.846 1044.502049 1172.979549 −5.4 Q(0 Ea 9, 9) 9P22 − 16084.970 1044.485133 1330.001133 2.0 5.6 U 9P22 − 17775.882 1044.428730 U 9P24 + 17848.480 1043.758600 Q(0 Ea −8, 13) 9P24 + 17816.997 1043.757550 1351.457050 6.9 7.0 Q(0 Ea 9, 13) 9P24 + 17809.443 1043.757298 1397.236398 14.4 14.6 Q(0 Aa 11, 13) 9P24 + 16894.708 1043.726786 1505.045186 −2.3 −2.2 Q(0 Aa 8, 12) 9P24 + 16465.533 1043.712470 1332.143170 −0.3 -0.5 Q(0 Aa 7, 8) 9P24 + 16151.914 1043.702009 1229.649909 −5.1 −0.2 Q(0 Aa 6, 12) 9P24+14714.233 1043.654053 1256.44835 −0.1 1.0 Q(0 Aa 9, 12) 9P24 + 14041.047 1043.631598 1377.577598 −2.7 Q(0 Aa 7, 9) 9P24 + 11963.481 1043.562298 1242.813298 3.0 6.5 U 9P24 + 10809.657 1043.523810 Q(0 Aa 11, 14) 9P24 + 10233.275 1043.504584 1525.496884 −10.1 −8.6 P(0 Aa 1^+^, 6) 9P32 + 10145.824 1035.812044 1069.412640 0.8 0.3 P(0 Ea 1, 7)L 9P34 + 16971.825 1034.054118 1078.900320 2.5 5.0 P(0 Aa 1^−^, 7)L 9P34 + 15354.390 1034.000166 1078.668566 2.3 1.7 P(0 Aa 2^−^, 9) 9P36 − 9648.259 1031.155600 1108.931200 −0.8 0 P(0 Aa 1^+^, 9)L 9P36 − 10356.138 1031.131987 1099.853087 1.7 0.4 P(0 Ea −1, 9)L 9P36 − 10624.323 1031.123042 1100.011642 −1.8 −0.4 P(0 Aa 2^+^, 9) 9P36 − 10809.311 1031.116871 1108.969271 −0.8 0.9 P(0 Ea −3, 9) 9P36 − 13698.547 1031.020497 1122.270197 −5.8 −11.9 P(0 As 3, 9) 9P36 − 14350.507 1030.998750 1122.176450 −3.2 −1.7 P(0 Ea 4, 9) 9P36 − 14830.98 1030.982723 1141.037623 −9.9 −10.6 P(0 Aa 3^+^, 9) 9P36 − 15429.241 1030.962767 1122.240367 −19.0 −11.6 P(0 Aa 3^−^, 9) 9P36 − 15450.205 1030.962068 1122.240768 2.0 9.4 P(0 Ea 3, 9)L 9P36 − 15604.449 1030.956923 1121.821623 −0.3 −0.4 P(0 Ea −2, 9)L 9P36 − 16137.042 1030.939157 1108.348360 3.2 1.1 P(0 Ea 2, 9)L 9P36 − 17653.154 1030.888585 1108.598490 4.1 2.7 P(0 Aa 1^−^, 10) 9P38 − 9484.598 1029.125720 1114.089120 −2.0 1.3 P(0 Aa 4, 10) 9P38 − 16148.642 1028.903431 1153.512831 0.7 0 P(0 Ea −1, 11) 9P40 + 17092.541 1027.952318 1127.560120 1.4 4.1 P(0 Aa 1^+^, 11) 9P40 + 16989.214 1027.948871 1127.387171 −0.2 2.1 P(0 Aa 2^−^, 11) 9P40 + 15993.048 1027.915643 1136.722040 0.9 2.8 P(0 Aa 2^+^, 11) 9P40 + 13721.694 1027.839878 1136.811378 11.0 14.0 P(0 Ea 4, 11) 9P40 + 10281.031 1027.725110 1168.829210 7.0 4.5 P(0 Aa 5, 11) 9P40 + 9887.452 1027.711982 1193.033982 9.9 18.7 P(0 Aa 3^+^, 11) 9P40 + 9730.095 1027.706733 1150.039030 −15.0 −18.6 P(0 Ea −2, 11) 9P40 + 9187.589 1027.688637 1136.157337 2.3 3.7 P(0 Ea 1, 12) 9P42 + 15646.148 1025.819764 1145.257364 −2.1 0.1 P(0 Aa 1^−^, 12) 9P42 + 15032.225 1025.799286 1145.069086 0.2 −0.5 P(0 Ea −1, 13) 9P42 − 16840.998 1024.736110 1160.897510 −1.6 −1.1 P(0 Aa 1^+^, 13) 9P42 − 17569.495 1024.711810 1160.702410 −1.6 −1.1 P(0 Ea −3, 14)L 9P44 − 11971.706 1022.790042 1202.770842 P(0 Aa 2^+^, 14) 9P44 − 12189.339 1022.782782 1189.601080 −2.6 −4 P(0 Aa 6, 14) 9P44 − 12928.003 1022.758143 1275.454543 1.0 2.1 P(0 Ea 3, 14) 9P44 − 13533.332 1022.737952 1202.336550 −1.7 −0.8 P(0 Ea 4, 14)L 9P44 − 13960.687 1022.723697 1221.484200 −9.6 −9.5 P(0 Ea −2, 14)L 9P44 − 14254.030 1022.713912 1188.822610 7.9 26.0 P(0 Aa 3^+^, 14)L 9P44 − 14507.345 1022.705462 1202.706262 −2.3 −4.9 P(0 Aa 3^−^, 14)L 9P44 − 14736.996 1022.697802 1202.713402 0.7 −0.8 P(0 Ea −1, 15) 9P46 + 14919.509 1021.554573 1200.096773 −3.2 P(0 Aa 1^+^, 15) 9P46 + 11162.841 1021.429264 1199.799864 −5.3 −2.1 P(0 Aa 1^−^, 15) 9P46 − 10682.479 1020.700583 1202.570383 1.6 −0.1 P(0 Aa 4, 15) 9P46 − 16527.760 1020.505605 1241.210105 −3.9 −4.9 P(0 Ea −3, 16) 9P48 + 14709.120 1019.391336 1245.215836 0 −1.9 P(0 Ea 3, 16) 9P48 + 13389.423 1019.347316 1244.790816 −34.8 −28.5 P(0 Ea 7, 16) 9P48 + 13353.259 1019.346110 1352.910110 1.4 P(0 Aa 6, 16) 9P48 + 13345.498 1019.345851 1317.848051 9.2 15.4 P(0 Ea −2, 16) 9P48 + 12891.933 1019.330722 1231.214822 0.2 0.6 P(0 Aa 2^+^, 16) 9P48 + 12421.741 1019.315038 1232.157838 −1.4 −2.7 P(0 Ea 4, 16) 9P48 + 12335.332 1019.312155 1263.896255 −21.0 −24.5 P(0 Ea 5, 16) 9P48 + 12295.030 1019.310811 1288.263811 19.3 15.8 P(0 Aa 5, 16) 9P48 + 12229.499 1019.308625 1288.089125 −2.4 3.0 P(0 Aa 3^+^, 16) 9P48 + 11781.408 1019.293678 1245.128078 0.2 4.6 P(0 Aa 3^−^, 16) 9P48 + 11308.036 1019.277888 1245.144388 −0.7 0.2 P(0 Ea 2, 17) 9P50 + 17961.451 1017.320072 1255.207572 −1.3 0 P(0 Ea 1, 17)L 9P50 + 15685.814 1017.244164 1248.412664 1.3 0.8 P(0 Aa 1^−^, 17) 9P50 + 15357.100 1017.233200 1248.244800 −11.2 −7.4 P(0 Aa 4, 17) 9P50 + 9686.673 1017.044055 1286.526755 −24.8 −23.6 P(0 Aa 2^−^, 18) 9P50 − 17254.659 1016.145389 1279.946189 3.9 1.5 P(0 Ea −2, 19) 9P52 − 10876.861 1014.155075 1305.707275 2.5 5.5 P(0 Aa 6, 19) 9P52 − 12143.902 1014.112811 1392.386211 −0.1 −0.4 P(0 Ea −6, 19) 9P52 − 12921.993 1014.086857 1392.296957 −70.7 P(0 Aa 5, 19) 9P52 − 13002.243 1014.084180 1362.650680 9.5 15.1 P(0 Aa 3^−^, 19) 9P52 − 14888.525 1014.021260 1319.767360 −0.4 −0.6 P(0 Aa 2^+^, 19) 9P52 − 16384.082 1013.971374 1307.056674 1.0 2.5 ^a^Letter L indicates that the line assignment has been confirmed from a frequency combination loop; a letter U denotes an unassigned line. ^b^Observed transition frequencies in MHz are expressed by the frequency of the CO~2~ laser line and the determined microwave frequency from the fitting to the recorded Lamb-dip signal. ^c^Observed transition frequencies are converted into wavenumbers using a factor of 29979.2458 MHz/cm^−1^ and CO~2~ laser frequencies from Ref. [@b30]. ^d^Differences (in MHz) between the observed transition frequencies in the present work and values from the FT infrared wavenumbers reported in Ref. [@b21]. A blank space indicates either a new assignment or an unidentified U line to the data in Ref. 21. ^e^Differences (in MHz) between the observed transition frequencies in the present work and values from the FT infrared wavenumbers reported in Ref. [@b22]. A blank space indicates either a new assignment or an unidentified U line to the data in Ref. 22. ###### J(J + 1) power-series expansion coefficients (in cm^−1^) of upper-state term values in 27 substates of the C−N stretching band of CH~3~NH~2~. Substate (υ~t~ S~t−i~ K) a~0~ a~1~ a~2~ × 10^6^ a~3~ × 10^10^ ---------------------------------------- ------------------ --------------- ---------------- ----------------- (0 Aa 0)[\](#t2-fn1){ref-type="fn"} 1044.912526(461) 0.732866(11) −10.72(8) 44.1(16) (0 Aa* 1^+^) 1047.66065(167) 0.725175(4) −3.92(26) 27.3(4) (0 Aa 1^−^) 1047.662408(196) 0.738380(3) −3.34(1) −5.0(1) (0 Aa 2^+^) 1056.257642(711) 0.731710(12) 5.73(6) −29.0(9) (0 Aa 2^−^) 1056.254285(389) 0.731812(7) −2.63(3) 1.1(4) (0 Aa 3^+^) 1069.565927(15) 0.731614(0) −0.31(1) −5.5(0) (0 Aa 3^−^) 1069.566059(76) 0.731610(1) −0.28(1) 5.9(1) (0 Aa 4) 1087.76225(210) 0.730430(40) −1.69(20) −0.2(3) (0 Aa 5) 1112.580226(61) 0.731490(1) −0.84(0) −0.2(0) (0 Aa 6) 1142.383547(391) 0.731342(7) −1.01(3) 0.4(2) (0 Aa 7) 1176.989660(29) 0.7314660(0) −1.02(0) 0 (0 Aa 8) 1218.14342(465) 0.730775(22) (0 Aa 11) 1372.109149(0) 0.730418(0) (0 Ea 0)\* 1045.101654(472) 0.731802(12) −10.24(6) 34.2(9) (0 Ea 1) 1047.93850(858) 0.737074(237) 2.77(18) −107(37) (0 Ea 2) 1055.93689(927) 0.730844(209) 7.42(11) −34(17) (0 Ea 3) 1069.121205(380) 0.731947(11) 78(7) 1.4(0) (0 Ea 4) 1088.374933(240) 0.731462(4) -0.49(2) −9(2) (0 Ea 5) 1112.77336(123) 0.731406(29) −0.8(1) 0 (0 Ea 6) 1142.535800(365) 0.731324(6) (0 Ea 7) 1177.49078(396) 0.730786(131) (0 Ea 9) 1264.227504(0) 0.730818(0) (0 Ea −1) 1047.6819(828) 0.727080(889) −7(2) 0 (0 Ea -2) 1055.62915(546) 0.732554(97) −4.2(4) 10(5) (0 Ea −3) 1069.575107(628) 0.731907(12) −0.49(6) 8.0(1) (0 Ea −5) 1112.674698(42) 0.731554(1) −0.75(0) 0 (0 Ea −6) 1142.275112(0) 0.731058(0) (0 Ea −7) 1177.492590(0) 0.731449(0) (0 Ea −8) 1218.416547(0) 0.730994(0) ^\^First reported in Ref. [@b28] and confirmed in this work. Energy of the level (υ~t~ S~t−i~ K, J) = (0 As* 0, 0) in the ground state is taken as the zero. Errors in parentheses are one standard deviation in units of the last quoted digit. ###### Asymmetry splittings ΔE(υ ~t~ S ~t−i~ K, J) and asymmetry-splitting constants b ~m~ (in MHz) for the resolved K-doublet levels of A torsional symmetry of CH~3~NH~2~. J ΔE(υ~t~ S~t−i~ K, J) (MHz) [a](#t3-fn1){ref-type="fn"}Obs. − Calc. (MHz) K b~0~ b~1~ b~2~ b~3~ b~4~ b~5~ ----- -------------------------------------- ----------------------------------------------- ----- --------------- ---------------- --------------- ---------------- ---------------- ----------------- 7 28634.077 −0.087 1 496.599352 0.533819 −0.004838 12 72095.319 −1.861 15 83058.070 −6.763 8 1788.892 0.035 2 4.325212 −0.107269 9.44 × 10^−4^ −3.35 × 10^−6^ 4.10 × 10^−9^ 9 1141.340 0.068 11 2678.196 0.214 15 15981.426 1.268 18 30145.511 3.581 7 10.823 0 3 8.77 × 10^−4^ −2.91 × 10^−5^ 3.61 × 10^−7^ −2.01 × 10^−9^ 5.09 × 10^−12^ −4.72 × 10^−15^ 9 12.022 0 12 212.193 −0.001 14 214.052 −0.011 16 488.961 −0.078 20 3465.061 −1.431 ^a^Asymmetry splittings were calculated from the asymmetry-splitting constants in Table 3. The K-doublet levels are in the excited states with K = 1, 2, and 3 and in torsional state υ~t~ = 0 with the torsion-inversion symmetry Aa.	{ "pile_set_name": "PubMed Central" }	[ "chemistri", "sideband", "eqreftypedispformula", "two", "control", "refer", "given", "work", "origin", "ediffer", "remain", "program", "ν", "khz", "bs", "previouslyb", "ten", "thank", "asymmetrysplit", "employ", "identifi", "cm−", "incom", "spectrum", "interstellar", "grant", "select", "mechan", "bsrepff", "valu", "groundstat", "infraredbbbbbb", "torsion", "narrow", "locat", "convers", "signal", "may", "back", "move", "split", "branch", "recent", "unresolv", "q", "frequenciesb", "ea", "separ", "gener", "use", "ms", "support", "final", "focus", "accord", "demodul", "fl−fmw", "probe", "cover", "modul", "constitut", "full", "emiss", "relev", "lambdip", "also", "state", "qbranch", "shift", "loop", "difficult", "insb", "cell", "grid", "perform", "commerci", "↔", "numer", "accuraci", "featur", "crystal", "lockin", 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Introduction {#Sec1} ============ Skeletal muscle tissue represent about 40% of total body weight and is responsible for almost all voluntary movements. It has a high regenerative capacity after injury, which is directly linked to the presence of satellite cells (SCs). These cells are the main muscle stem cells and have a key role in muscle development during embryogenesis^[@CR1]--[@CR3]^. SCs reside between the basal lamina and the muscle membrane^[@CR4]^ and although quiescent in normal adult muscles, SCs can be activated by specific signals upon muscle injury. In diseases characterized by chronic degeneration process, such as muscular dystrophies, SCs are constantly activated, leading to the depletion of the SCs pool and consequent failure of the regenerative process^[@CR5]^. Muscular dystrophies are a heterogeneous group of genetic diseases that cause a progressive loss of motor ability. Defects in components of the dystrophin-glycoprotein complex (DGC) are responsible for different forms of muscular dystrophies. The DGC links the myofiber cytoskeleton proteins and the extracellular matrix, providing mechanical support to sarcolemma during myofiber contraction^[@CR6],[@CR7]^. Mutations in the dystrophin (*DMD*) gene can lead to Duchenne/Becker muscular dystrophies^[@CR8]^. DGC proteins are linked to extracellular matrix proteins by post-translational glycosylation of α-dystroglycan (α-DG) protein. Mutations in the *LARGE1* gene, which encodes a glycosyltransferase that acts in the α-DG, lead to congenital muscular dystrophy type 1D^[@CR9]--[@CR11]^. Identified in nature or generated in laboratory, animal models are an important tool to study neuromuscular disorders. These models generally present physiological alterations observed in human patients and can be used for pathophysiological studies and therapy testing^[@CR12]^. A good genetic and biochemical model for Duchenne muscular dystrophy is the *Dmd*^*mdx*^ mouse that bears a nonsense point mutation in exon 23 of the *Dmd* gene, which causes lack of this protein in skeletal muscle. Regardless of total protein deficiency, this mouse shows a mild phenotype, with comparatively moderate muscle pathology. Muscle histopathology analysis shows a great number of regenerating fibers, variation in fiber size and the presence of central nuclei. Differently from human Duchenne muscular dystrophy patients, muscle degeneration in *Dmd*^*mdx*^ mouse is followed by a significant regeneration^[@CR13],[@CR14]^. A model for Congenital muscular dystrophy type 1D is the myodystrophy *Large*^*myd*^ mouse, which harbors a mutation in the *Large1* gene. *Large*^*myd*^ mutant mice develop a progressive severe myopathy, with a maximum lifespan of 39 weeks. Muscle histopathology includes degeneration, loss of striation, variation in fiber size and the presence of central nuclei^[@CR15]^. With the goal of obtaining an animal model for DMD with a severe phenotype, more similar to affected DMD patients, and to better understand the interplay between the DMD protein and LARGE1 glycosyltransferase in muscle function, we generated double-mutant animals deficient in these two proteins^[@CR16]^. The new *Dmd*^*mdx*^*/Large*^*myd*^ mouse model has a severe phenotype with delayed growth and development, and is unable to reproduce. In addition to the important pathophysiological aspects of these two mutations in muscle formation and function, this model has an important application for testing therapies, integrating functional, molecular and protein studies^[@CR16]^. Aiming to understand the behavior of SCs in dystrophic muscles with different degrees of muscle degeneration, we evaluated histological parameters, as well as gene and protein expression of transcription factors related to SCs, in gastrocnemius muscle of adult male mice with different forms of muscular dystrophies. The results were compared regarding regenerating and degenerating features. Our findings indicate that the pool of SCs of the studied dystrophic strains maintains its proliferative capacity and these cells can contribute to muscle regeneration and new fiber formation. Nevertheless, the maturation of these new fibers is incomplete and do not prevent muscle degeneration. Results {#Sec2} ======= Dystrophic muscles retain their SCs pool {#Sec3} ---------------------------------------- The expression of the transcription factor *Pax7*, which is related to the maintenance of SCs pool, was measured to investigate a potential reduction in the SCs pool in the dystrophic mice models as compared to wild-type mice. Statistical analysis with Kruskal-Wallis test revealed similar expression of *Pax7* in the three dystrophic strains as compared to wild-type mice (*Chi-square* = 1.05, *df* = 3, *p* = 0.790), independently of the degree of muscle degeneration (Fig. [1a](#Fig1){ref-type="fig"}). Protein analysis by Western blot also showed similar expression in the dystrophic strains as compared to wild-type animals (Fig. [1b,c](#Fig1){ref-type="fig"}).Figure 1PAX7 Expression in dystrophic muscles: (**a**) Boxplot of mRNA relative Pax7 expression and (**b**) PAX7 protein western blot analysis (full blots figures without contrast adjustments are presented at Supplementary Material). N = 6 animals per strain. (**c**) Boxplot of densitometric quantification. No significant differences in PAX7 were found between WT and the three dystrophic strains using both approaches (Kruskal-Wallis test, Chi-square = 1.05, df = 3, p = 0.790). \*Shows outlier measures. Immunofluorescence analysis using PAX7 antibody in gastrocnemius muscle sections of dystrophic versus wild-type animals showed an apparently higher number of PAX7 positive nuclei per fibers in the three dystrophic models (Fig. [2a](#Fig2){ref-type="fig"}). However, no statistical differences were found among the four groups (p = 0.083, Fig. [2b](#Fig2){ref-type="fig"}). Thus, SCs pool is maintained in spite of the different degrees of degeneration in the dystrophic mouse models.Figure 2Representative images of PAX7-positive SCs in the four mouse strains, magnification 200 × (full immunofluorescence figures are represented at Supplementary Material). (**a**) Immunofluorescence analysis showing PAX7-positive and DAPI-positive cells in each strain, \~3000 fibers were counted for each animal. White arrows are indicating SCs, purple labelling. (**b**) Proportion of SCs per fibers in the four strains. No statistical significance was found among wild-type and the three dystrophic strains. N = 4 animals per strain (One-way ANOVA, F(3, 11) = 2.9, p = 0.084). Number of activated satellite cells is elevated in the dystrophic mice, compatible with active regeneration and pool maintenance {#Sec4} -------------------------------------------------------------------------------------------------------------------------------- Using PAX7 and KI67 antibodies, we identified and counted SCs that remained quiescent and the ones that were activated. While PAX7 staining shows SCs population, KI67 staining is a marker of proliferating cells. Therefore, the double staining for PAX7 and KI67 shows activated SCs^[@CR17]^ (Fig. [3a](#Fig3){ref-type="fig"}). A higher number of activated SCs were found in the three dystrophic models, when compared with wild-type, but no differences among the dystrophic strains (Kruskal-Wallis test, *Chi-square* = 30.248, *df* = 3, *p* \< 0.001). Dystrophic animals showed the following proportions of activated SCs: *DMD*^*mdx*^ 17.4%, *Large*^*myd*^ 25.1% and *DMD*^*mdx*^/*Large*^*myd*^ 16,.7% (Fig. [3b](#Fig3){ref-type="fig"}).Figure 3Representative images of PAX7 and KI67 positive SCs, magnification 200X (full immunofluorescence figures are represented at Supplementary Material). (**a**) Identification of quiescent and activated SCs, using as example the Large^myd^ model. On the merged figure, red arrows indicate quiescent SCs (DAPI+ and PAX7+); green arrows indicate activated SCs (DAPI+, PAX7+ and KI67+). (**b**) Percentage of activated SCs in wild-type and dystrophic models, N = 200 PAX7+ SCs per strain. Statistical difference was observed between the wild type and each one of three dystrophic models (Kruskal-Wallis test, Chi-square = 30.248, df = 3, p \< 0.001, ^\#\#^p \< 0.01, ^\#\#\#^p \< 0.001). Myogenic regulatory genes are activated in the dystrophic muscle {#Sec5} ---------------------------------------------------------------- We also investigated the relative expression of *Myf5*, *Myod*, *and Myog*, which are important genes of the myogenic program. No significant differences were found among the three dystrophic models and the wild-type regarding the expression of *MyoD* (Kruskal-Wallis Test *- Chi-square* = 3.22, *df* = 3, *p* = 0.359), nor *Myf5* (*Chi-square* = 3.21, *df* = 3, *p* = 0.361). In spite of that, we observed a significant elevation (*Chi-square* = 17.93, *df* = 3, *p* \< 0.001) in *Myog* expression among the four mice models. Using the post hoc analysis with Bonferroni method of correction, a significant difference on *Myog* expression was found between wild-type and *Large*^*myd*^ (*p* \< 0.001), wild-type and *DMD*^*mdx*^*/Large*^*myd*^ (*p* = 0.0023), and wild-type and *DMD*^*mdx*^ (*p* = 0.0067). No significant differences were found between the dystrophic strains (Fig. [4a](#Fig4){ref-type="fig"}). High *Myog* expression was confirmed by analysis of protein levels (Fig. [4b,c](#Fig4){ref-type="fig"}).Figure 4Myogenic genes analysis in dystrophic models as compared to wild-type mice: (**a**) mRNA relative expression of Myf5, MyoD, Myog; Significant differences were observed for the Myog (Kruskal-Wallis test, Chi-square = 17.93, df = 3, p \< 0.001) \*Shows outliers animals. (**b**) MYOG western blot protein analysis (full blots figures without contrast adjustments are represented in Supplementary Material). N = 6 animals per strain, (**c**) Densitometric analysis of MYOG protein results. Significant differences were observed for the Myog gene ^\#^p \< 0.05 and ^\#\#^p \< 0.01 (Kruskal-Wallis test, Chi-square = 15.20, df = 3, p = 0.002), both at mRNA and protein levels. As data presented great variance in the expression of the myogenic factors, the non-parametric Levene's test was done to verify the equality of variances in samples. We observed that they were not significantly different (*Myod*, *p* = 0.997; *Myf5*, *p* = 0.973; *Myog*, *p* = 0.190; *Pax7*, *p* = 0.890). Dystrophic muscle retains the ability to form new fibers {#Sec6} -------------------------------------------------------- The number and diameter of new muscle fibers were evaluated by detection of developmental myosin heavy chain isoform (dMyHC) (Fig. [5a,b](#Fig5){ref-type="fig"}). Immunofluorescence analysis showed no dMyHC+ fibers in wild-type muscles, while *Dmd*^*mdx*^ strain presented the greatest number of dMyHC+ fibers (17%). *Large*^*myd*^ and *Dmd*^*mdx*^*/Large*^*myd*^ showed respectively 7% and 10% of new fibers (Kruskal-Wallis Test, *Chi-square* = 13.87, *df* = 3, *p* = 0.003, Fig. [5c](#Fig5){ref-type="fig"}), which is compatible with an intense regeneration in all dystrophic strains. Interestingly, compared to normal mature fibers size (52 µm+/−10 µm), the diameter of these new fibers was smaller, with a mean of 33 µm ± 13.5 µm in *Dmd*^*mdx*^, 30 µm ± 12.1 µm in *Large*^*myd*^ and 20 µm ± 9.0 µm in *Dmd*^*mdx*^*/Large*^*myd*^ (Fig. [5d](#Fig5){ref-type="fig"}), with differences among the dystrophic strains (Kruskal-Wallis test, *Chi-square* = 53.649, *df* = 3, *p* \< 0.001). In addition, the size of these new dMyHC+ fibers was more homogeneous, as shown by the coefficient of variation within normal values (lower than 250) in *Dmd*^*mdx*^ (150), *Large*^*myd*^ (245) and *Dmd*^*mdx*^*/Large*^*myd*^ (155). These results suggest that in the dystrophic models, these new fibers remain small and do not complete the regenerative process.Figure 5Representative images of dMyHC+ positive new regenerated fibers, normal and dystrophic muscles (full immunofluorescence figures are represented at Supplementary Material). dMyHC+ fibers are labeled in red/orange. Magnification 200X. (**a**) Immunofluorescence staining for dMyHC+ in normal WT muscle at time zero, 5 days after induced injury and after 60 days of induced injury, N = 6 animal per time. (**b**) dMyHC+ fibers in the 3 dystrophic models. N = 6 animals per strain (**c**) percentage of dMyHC+ fibers related to total fiber number (n = \~1500 fibers/animal). (**d**) dMyHC+ fiber diameter. Significant differences: ^\#^p \< 0.05 and ^\#\#\#^p \< 0.001 (Kruskal-Wallis test, Chi-square = 53.649, df = 3, p \< 0.001). To confirm our results, the muscles from the different dystrophic strains were compared to regenerating normal muscle subjected to acute lesion by electroporation, at days zero, five, and 60 days post-injury (Fig. [5a](#Fig5){ref-type="fig"}). In wild-type animals, regenerating dMyHC+ fibers appeared five days post-injury and presented variable diameter. The coefficient of variation was very high (484), demonstrating the diversity in fiber diameter. After 60 days, no regenerating fiber was identified, and fiber diameter was more homogeneous^[@CR18]^. Muscle degeneration is persistent in spite of regeneration {#Sec7} ---------------------------------------------------------- The regeneration outcome was evaluated through the quantification of connective tissue substitution, using Sirius red staining (Fig. [6a](#Fig6){ref-type="fig"}). We observed more connective tissue infiltration in dystrophic mice than in wild-type mice (Kruskal-Wallis Test, *Chi-square* = 81.21, *df* = 3, *p* \< 0.001). The differences among the three dystrophic strains were also statistically significant (Kruskal-Wallis Test -- *Chi-square* = 30.31, *df* = 2, *p* \< 0.001), with a higher proportion in the more severely affected muscles: *Dmd*^*mdx*^*/Large*^*myd*^ \> *Large*^*myd*^ \> *Dmd*^*md*.^ (Fig. [6b](#Fig6){ref-type="fig"}).Figure 6Connective tissue replacement in muscle degeneration (full Sirius red images are represented at Supplementary Material): (**a**) Representative images of collagen deposition by Sirius red staining of gastrocnemius muscles of WT, Dmd^mdx^, Large^myd^ and Dmd^mdx^/Large^myd^ mice, N = 6 animals per strain, magnification of 100X. (**b**) Quantification of percentage area of collagen deposition. Significant differences relative to WT muscle: ^\#\#^p \< 0.01 (Kruskal-Wallis test, Chi-square = 30.31, df = 2, p \< 0.001). Histological analysis of muscles by H&E staining also showed a gradual increase in muscle degeneration and variation in fiber size (Fig. [7a](#Fig7){ref-type="fig"}). An increase in the variation coefficient of fiber size in dystrophic strains was observed (normal: up to 250), in agreement with the degree of muscle affection: 635 in *Dmd*^*mdx*^*/Large*^*myd*^ \> 444 in *Large*^*myd*^ \> 420 in *Dmd*^*mdx*^ \> 205 in wild-type (Fig. [7b](#Fig7){ref-type="fig"}).Figure 7Histopathological alterations in the three dystrophic strains and wild-type (full H&E figures, without contrast adjustments, are represented at Supplementary Material). (**a**) HE staining, magnification 200X. N = 6 animals per strain. (**b**) Variation coefficient of muscle fiber size (\>250 is considered pathogenic)^[@CR37]^. Discussion {#Sec8} ========== The reservoir of muscle precursor cells, the SCs, is responsible for after-birth growth and muscle regeneration as a response to injuries, either by exercise or disease. Since the first description in 1961 by Mauro^[@CR4]^, SCs have been the target of a huge number of studies, aiming to understand their behavior and action. SCs are able to self-renew and replenish the stem cell pool, and this role is primordial for muscle regeneration. The regenerative response after muscle injury is marked by the exit of SCs from quiescence and their proliferation and fusion, leading to myofiber repair or new myofiber formation^[@CR19]^. At the same time, there is the activation of an inflammatory response, with the infiltration of immune cells, mainly macrophages^[@CR19]^. The dystrophic muscle is persistently injured due to a specific primary mutation in genes encoding for important muscle proteins, and the regenerative process is incessantly activated, recruiting SCs at higher rates than in normal tissue. Nevertheless, progressive replacement of muscle by fibrofatty tissue in dystrophic muscle has been considered as a factor that impairs the complete regeneration process^[@CR20]--[@CR22]^. Many studies have suggested that the regenerative capacity of SCs would be finite, and exhaustion of the pool of these cells has been considered a significant factor that contributes to progressive muscle deterioration in muscular dystrophies, as studied in DMD^[@CR5],[@CR23]^ and in mdx/utrophin^−/−^ mice^[@CR24]^. SCs repair potential, therefore, would not be sufficient to compensate for the persistent degeneration process^[@CR25]^. A recent study showed that deficiency of DMD protein could lead SCs to divide abnormally, resulting in the depletion of myogenic progenitors. In the long term, this condition can impair muscle regeneration^[@CR26]^. In contrast, several studies have shown an increased number of SCs in human and mice dystrophic muscles^[@CR20]--[@CR22],[@CR27]^. The dystrophic niche seems also to be crucial, since removing SCs from a dystrophic milieu revealed that their regenerative capacity remains both intact and similar to SCs derived from healthy muscle^[@CR28],[@CR29]^. Moreover, as observed in dystroglycanopathies, muscle fibrosis increases laminin disorganization, and this alteration in extracellular matrix composition is suggested to impair SCs function and activity^[@CR30]^. To elucidate the players and mechanisms involved in SCs function is of extreme importance, especially for therapeutic strategies. Here, we studied SCs protein and gene expression in different forms of murine muscular dystrophies, with a specific pattern of muscle histopathology, to correlate the impact of muscle degeneration in the population of SCs in these different microenvironments. The expression of the transcription factor PAX7 by SCs has been correlated with the maintenance of these cells in the undifferentiated state, being an important factor for SCs self-renewal that can be expressed both by quiescent and by early activated SCs^[@CR31]^. We studied *Pax7* mRNA expression in the muscle of dystrophic mouse models, as compared to wild-type, to determine if there was a reduction in SCs population with the dystrophic process. We also identified and counted PAX7-positive SCs in muscle sections and verified the number of activated cells among them in each mouse model. Our analyses, through mRNA expression, protein quantification, and immunohistochemical evaluation showed similar results in all strains, suggesting the maintenance of a normal pool of SCs despite the active but diverse process of regeneration and degeneration that occurs in the *Dmd*^*mdx*^, *Large*^*myd*^ and *Dmd*^*mdx*^*/Large*^*myd*^. These data are in accordance with the hypothesis of the maintenance/increased number of SCs with the dystrophic process^[@CR16],[@CR18],[@CR24]^. The importance of the niche for the regenerative capacity of SCs is well documented in the *Large*^*myd*^ mouse, in which SCs were present in a significantly higher number, but with reduced proliferation capacity when in native muscle fibers. However, when removed from their niche, their proliferative capacity in culture was restored^[@CR30]^. This could also be observed in our models, with activated SCs present in the *Dmd*^*mdx*^, *Large*^*myd*^ and *Dmd*^*mdx*^*/Large*^*myd*^ in a similar proportion, and higher than in wild-type. This self-renewal capacity of SCs, even in the dystrophic muscle, is extremely important for the replenishment of the stem cell pool, mainly in an attempt to regenerate^[@CR32]^. The molecular players of regeneration are the muscle regulatory factors, including MYF5 and MYOD, responsible for muscle-cell type determination and SCs activation, and MYOG, responsible for muscle differentiation^[@CR33],[@CR34]^. Thus, we measured the relative expression of these genes in our three different dystrophic models. At the mRNA level, we observed similar relative expression of *Myf5* and *Myod* in all strains and higher expression of *Myog* in the three dystrophic strains as compared to wild-type, suggesting activation of a later stage of muscle regeneration in all of them, independently of the muscle degeneration degree. MYF5 is expressed in both activated and quiescent SCs, but quiescent cells can maintain their quiescence state using a post-transcriptional regulation by miR-31, which sequester *Myf5* mRNA in messenger ribonucleoprotein (mRNP) granules. Upon activation, miR-31 levels decrease and *Myf5* mRNA is released to translation^[@CR35]^. Therefore, this could explain the similar relative quantification of *Myf5* mRNA in all strains. MYOD can be expressed both in first steps of SCs differentiation and by myocytes in the final steps of differentiation^[@CR1],[@CR33],[@CR36]^. Upregulation of MYOD expression leads SCs to exit the cell cycle and to differentiate and fuse, forming myotubes^[@CR33]^. In spite of the normal expression of MYOD in the muscle of these three dystrophic models, they showed regenerating fibers as observed by dMyHC staining (Fig. [5](#Fig5){ref-type="fig"}). This data confirms the maintenance of SCs capacity to follow a myogenic program in the dystrophic muscle and to be able to form new fibers, as already observed in *Dmd*^*mdx*\ 28^. In the final step of myogenesis, myogenin expression has a key role in the differentiation of SCs into myotubes. High expression of myogenin initiates myoblast maturation to myocytes, myocytes fusion and myotube formation, a process extremely active in dystrophic muscle^[@CR2],[@CR33]^. We studied MYOG expression in the dystrophic strains to investigate whether myotube formation is impaired in these models. Gene expression analysis showed a significant increase of *Myog* expression as well as protein expression in the three dystrophic strains, suggesting the occurrence of myoblast maturation to myocytes, fusion induction, and myotube formation in all the dystrophic muscles^[@CR37]^. Newly regenerated muscle fibers have particular characteristics such as central myonuclei, a basophilic pattern on H&E staining, and expression of developmental myosin heavy chain (dMyHC) isoform^[@CR38]^. In our analysis of regenerating fibers expressing dMyHC, we observed a significant number of new fibers in all the dystrophic strains, including the most severely affected double mutant *Dmd*^*mdx*^*/Large*^*myd*^. The *Dmd*^*mdx*^ strain showed the highest number of regenerating fibers, which is compatible with its pronounced regenerative capacity and milder dystrophic phenotype. *Large*^*myd*^ and *Dmd*^*mdx*^*/Large*^*myd*^ strains also presented a significant amount of regenerating fibers in their muscle, in spite of the more severe dystrophic phenotype, suggesting that the formation of new fibers is possible even when the muscle is already significantly degenerated. Contrarily, all dystrophic strains showed, on average, thinner regenerating fibers, especially the double mutant *Dmd*^*mdx*^*/Large*^*myd*^, as compared to the other strains (Fig. [5d](#Fig5){ref-type="fig"}). These results suggest that in a harsher dystrophic microenvironment, the complete regeneration can be compromised. In addition, the fiber size variation coefficient^[@CR38]^ showed small variation, which suggests the maintenance of these regenerating fibers in an immature state with small diameter, not progressing to a normal mature fiber size. The possibility of fiber branching or splitting fibers cannot be totally ruled out, since it is an alteration commonly found in dystrophic muscles, happening both in degeneration and regeneration conditions. Branched fibers were well described mainly in old fast-twitch EDL muscle approaching the end stage of the dystrophic muscle disease due to dystrophin deficiency^[@CR39]^. During pathological conditions, splitting or branching of large fibers can lead to the formation of many small fibers, increasing fiber diameter variability. The regenerative process can also originate small fibers, but here, we confirmed their immaturity through dMyHC expression. Next, we compared these data with the regeneration process of normal muscle under induced acute lesion by electroporation. At five days post-injury, an intense regeneration was observed with the presence of a high number of dMyHC+ muscle fibers. These new normal fibers presented more variable diameter, leading to a higher variation coefficient. This occurs because these fibers are probably ongoing a normal process of regeneration, reaching an increased size 60 days post-lesion^[@CR18]^. Taken together, these results suggest that the three dystrophic models retain their regenerative capacity, including the maintenance of SCs pool, the proliferation capacity, differentiation marker expression and finally, the formation of new fibers. However, this regenerative process is incomplete, with a possible defect in the maturation characteristics that could be determinant to the dysfunction of these fibers. Remarkably, very interesting data, which are in accordance with our proposal, has been shown by Pipalia *et al*.^[@CR40]^ in a model of wound repair in larval zebrafish. Two different populations of Pax7+ stem cells with different roles were observed: one, expressing Pax7a, which only initiates fiber formation, and the second, formed by Pax7b cells, which contribute to fiber growth. These two myoblast populations suggest a novel cellular complexity in muscle repair, with the possibility of the presence of distinct populations of myogenic cells contributing differentially to the repair also in other vertebrates. The dystrophic process could alter more specifically the second cell type, associated with fiber growth post-regeneration, and explain the presence of smaller regenerating fibers in the mouse models for muscular dystrophies. As to the degenerative process, we studied histopathological alterations through both connective tissue replacement and variation in fiber size, as a correlation with their regenerative potential. Variability in the coefficient of muscle fiber size \>250 is considered pathogenic^[@CR38]^, and this was observed in all dystrophic strains. We also observed more connective tissue infiltration in dystrophic mice, with a crescent increase in muscles with more weakness, such as *Large*^*myd*^ and *Dmd*^*mdx*^*/Large*^*myd*^. Thus, we conclude that the active regeneration in the dystrophic muscle is not indicative of effective improvement of muscle health, but an insufficient attempt to restore muscle fibers. In conclusion, our findings suggest that dystrophic muscles, independently of the degree of degeneration caused by the primary gene defect, retain the pool of SCs with proliferating capacity and are ready to respond to regenerating stimuli. The potential to repair is preserved by the upregulation of myogenic factors like MYOG and by the formation of new fibers expressing dMyHC. Nonetheless, the maturation of these new fibers is incomplete, since these fibers remain small and do not prevent muscle degeneration, as observed by the intense substitution by connective tissue. In this sense, efforts to improve late muscle regeneration should better contribute to therapeutic approaches. Methods {#Sec9} ======= Animals {#Sec10} ------- Histological, immunofluorescence, gene expression and protein analyses, were examined in the gastrocnemius muscle of adult male mice with 12--14 weeks of age. This muscle has about 51.5% of fiber type IID, 22.7% of fiber type IIA, 18.2% fiber type IIB and 7.6% of fiber type I^[@CR41]^. The studied strains were: *DMD*^*mdx−*^ (n = 6), *Large*^*myd*^ (n = 6), *C57Bl/6* (n = 6) and double mutant *DMD*^*mdx*^/*Large*^*myd*^ (n = 6, three males and three females). During the experiments, animals were bred and housed in controlled temperature and light in the animal house of the Human Genome and Stem Cell Research Center (HUG-CELL). All experimental protocols and methodologies were performed in accordance with and were approved by CEUA - COMISSÃO DE ÉTICA NO USO DE ANIMAIS (Ethical committee of the use of animal in experiments - <http://www.ib.usp.br/comissoesib/ceua/>), of the Institute of Biosciences, University of Sao Paulo (protocol number 246/2016), which is the licensing committee of our Institution. In addition, the used methods were carried out in accordance with the relevant guidelines and regulations from the Brazilian National Council for Control of Animal Experimentation (Conselho Nacional de Controle de Experimentação Animal -- CONCEA). Tissue collection {#Sec11} ----------------- After euthanasia, the gastrocnemii muscles were collected and frozen in liquid nitrogen to study gene expression, histopathology, immunofluorescence,and protein quantification. RNA extraction, cDNA synthesis, and qRT-PCR {#Sec12} ------------------------------------------- RNA extraction from frozen muscles was performed using the RNeasy Microarray Tissue Mini Kit (Qiagen). RNA was solubilized in RNase-free water (Ambion™) and quantified in a Nanodrop spectrophotometer (Thermo Fisher Scientific). The cDNA synthesis was performed using 1 µg of total RNA according to the M-MLV protocol (Invitrogen). Samples were amplified in triplicates using the MasterMix containing Sybr Green (Applied Biosystems) and primers (Table [1](#Tab1){ref-type="table"}) on a thermocycler 7500 Fast (Applied Biosystems). The fold-change was obtained by the 2^−ΔΔCT^ method, with *Tbp* gene as normalizer.Table 1Primers sequences.GeneForward sequenceReverse sequence*Pax7*5′ GAGTTCGATTAGCCGAGTGC 3′5′ GAGTTCGATTAGCCGAGTGC 3′*Myf5*5′ GAAGGTCAACCAAGCTTTCG 3′5′ GCTCTCAATGTAGCGGATGG 3′*MyoD*5′ TACAGTGGCGACTCAGATGC 3′5′ TAGGCGGTGTCGTAGCC 3′*Myog*5′ CTGCACTCCCTTACGTCCAT 3′5′ CCCAGCCTGACAGACAATCT 3′*Tbp*5′ TGCACAGGAGCCAAGAGTGAA 3′5′ CACATCACAGCTCCCCACCA 3′ Protein analysis {#Sec13} ---------------- Frozen muscle samples were homogenized in RIPA buffer (Sigma-Aldrich) supplemented with 1% of protease inhibitors (Sigma-Aldrich). Samples were kept on ice for 30 minutes and afterward centrifuged at 16.000 g for 20 min at 4 °C, followed by protein quantification using the Pierce BCA Assay kit (Thermo Scientific). Homogenized samples with 30 µg of protein were separated by SDS-PAGE in 4--15% Mini-PROTEAN® TGX™ Gels (BioRad) and transferred to nitrocellulose membranes using iBlot® Gel Transfer Device (Invitrogen) for 7 minutes. The obtained membranes were probed using the antibodies: PAX7 (1:1000 Novus Biologicals; NBP2-34706) and Myogenin (Abcam ab15232)^[@CR42]^. The membranes were then incubated with the appropriated secondary horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies (Thermo Fisher Scientific) and revealed using the Novex™ ECL Chemiluminescent Substrate Reagent kit (Invitrogen). Images were obtained using photo-documenter (ImageQuant TL, GE Healthcare Life Sciences), all images were collected after 450 seconds of standard exposure. For the reaction with the endogenous control, membranes were stripped with a stripping buffer (Thermo Fisher Scientific) and reprobed with GAPDH antibody (1:1000 Cell Signaling 2118)^[@CR43]^. Bands were quantified by densitometry using the software ImageQuant TL (GE Healthcare Life Sciences). The protein of interest and the endogenous protein were probed and analyzed in the same membrane, both for experimental and control samples. Histology and immunofluorescence analyses {#Sec14} ----------------------------------------- ### Hematoxylin-eosin (H&E) {#Sec15} Transverse cross sections of the right gastrocnemius were stained with H&E and histopathological evaluation was performed by measuring muscle fibers to obtain the fiber diameter utilizing ImageJ software (n = 100 fibers/experimental group). The mean fiber diameter of each strain was obtained, and the variability coefficient was calculated according to the formula: Standard Deviation x 1000 ÷ Diameter Mean. Results were represented as mean variation and variability coefficient. The stained sections were observed under a light microscope (Zeiss). ### Sirius red staining {#Sec16} To identify regions with collagen deposition, indicative of connective tissue replacement and infiltration between muscle fibers, the slides were immersed in Bouin fixative for twenty minutes and washed in water. The sections were stained with Sirius red (0.2 g) dissolved in saturated picric acid (100 mL) for 60 minutes, dehydrated and mounted in Canada balsam. The differences were quantified by the positive area of staining, labeled in red, relative to the total area of the section, using ImageJ software (4--5 fields, n = 6 animals/experimental group). The stained sections were observed and photographed under the same exposure conditions (16 ms), using a light microscope (Zeiss) and the Axiovision software Release 4.8.2 SP3 (2013). ### PAX7 and KI67 immunofluorescence {#Sec17} The presence of SCs was determined by double staining with antibodies against PAX7 and laminin to label the basal lamina. Activated SCs were determined by double staining with antibodies against PAX7 and KI67. Primary antibodies used: PAX7 (1:20; DSHB), laminin (1:100; Dako; Z0097) and KI67 (1:200; Abcam AB15580). Secondary antibodies were Cy3-labeled sheep anti-mouse IgG (1:100; Sigma; C2181) and FITC-labeled donkey anti-rabbit IgG (1:100; Amersham; N1034). The 6 µm muscle sections were fixed with 4% formaldehyde solution and submitted to antigen retrieval with hot citric acid buffer. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for one hour at room temperature. The slides were mounted with Vectashield with DAPI to counterstain nuclei. Immunofluorescence micrographs were obtained with a conventional fluorescence microscope (Zeiss). The proportion of PAX7 positive nuclei to the total number of fiber was quantified (\~3000 fibers, n = 4 animals/experimental group). The proportion of double stained PAX7 and KI67 were analyzed in approximately 200 PAX7 positive nuclei. All images were collected using a Zeiss microscope with epi-fluorescence, using specific filters for DAPI, FITC and for Cy3. The images were photographed using the Axiovision software Release 4.8.2 SP3 (2013), under the same exposure conditions (120--130 ms), standardized for the Cy3 staining for identification of dMyHC expression. The same software created merged figures automatically. ### Developmental myosin heavy chain (dMyHC) {#Sec18} The antibody Clone RNMy2/9D2 recognizes a myosin heavy chain (MHC) present during the embryonic and neonatal period in the development of skeletal muscle. The same MHC is expressed during regeneration of muscle fibres. The presence of dMyHC indicates myogenic activity by newly differentiated fibers labeled in red. The 6 µm muscle sections were double labeled with primary antibodies to dMyHC (1:30; Vector; VP- M664)^[@CR44]^ and laminin (1:50; Dako; Z0097)^[@CR45]^, washed, and then incubated for 1 h with secondary antibodies Cy3-labeled sheep anti-mouse IgG (1:100; Sigma; C2181) and FITC-labeled donkey anti-rabbit IgG (1:100; Amersham; N1034), respectively. After washing, the slides were mounted with Vectashield and DAPI, to counterstain nuclei. Immunofluorescence micrographs were obtained with a conventional fluorescence microscope (Zeiss) and the percentage of dMyHC+/total fibers was quantified (4 fields, n = 6 animals/experimental group). The fiber diameter of dMyHC+ fibers of each strain was measured manually, and the variability coefficient was calculated according to the formula: Standard Deviation x 1000 ÷ Diameter Mean^[@CR38]^. All images were collected using a Zeiss microscope with epi-fluorescence, using specific filters for DAPI, FITC and for Cy3. The images were photographed using the Axiovision software Release 4.8.2 SP3 (2013), under the same exposure conditions (120--130 ms), standardized for the Cy3 staining for identification of dMyHC expression. Merged figures were created automatically by the same software. Statistical analysis {#Sec19} -------------------- Differences among the groups were assessed using one-way ANOVA Test, and Kruskal-Wallis Test, for small and non-normally distributed set of data. Results are reported showing means and standard deviations in bar graphs and medians in boxplot graphs. The statistical analyses were calculated using GraphPad Prism version 7 for Windows (San Diego, CA: GraphPad Software, Inc), Minitab 17 Statistical Software (State College, PA: Minitab, Inc) and IBM SPSS Statistics for Windows version 24.0 (Armonk, NY: IBM Corp) software and a p-value equal or less than 0.05 was considered to be significant. For the expression analysis, additional five animals (males) from each strain were added (in a total of eleven animals), to confirm and strengthen the statistical analysis. Supplementary information ========================= {#Sec20} Supplemental information **Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1038/s41598-019-48156-7. We would like to thank the help from Leticia Feitosa, Heloisa S. Bueno, Patricia S. Kuriki, and the professionals of the Human Genome and Stem Cells Research Center for helping with facilities. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo - Centro de Pesquisa, Inovação e Difusão (FAPESP-CEPID), Conselho Nacional de Desenvolvimento Cientıfico e Tecnológico (CNPq-INCT), Financiadora de Estudos e Projetos (FINEP). CAPES -- Coordenação de aperfeiçoamento de Pessoal de Nivel Superior. Conceived and designed the experiments: A.F.R.J. and M.V. Implementation of the methodologies: A.F.R.J., R.I., C.F.A., S.A.F., D.A.G., A.L.F.S. and L.S.S. Performed experiments: A.F.R.J. and C.F.A. Contributed reagents/materials/analysis tools: R.I., C.F.A., S.A.F., A.L.F.S. and L.S.S. Prepared and/or revised the manuscript. A.F.R.J., L.S.S., P.O.O., C.F.A. and M.V. The authors declare no competing interests.

{ "pile_set_name": "PubMed Central" }

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Glossary: FRET: fluorescent resonance energy transfer; FRAP: fluorescence recovery after photo-bleaching ZAP-70: ζ-associated protein of 70 KD; a pivotal molecule in membrane proximal T cell receptor signaling; Immune synapse: organized structure that forms at the interface of an antigen-presenting cell and antigen-specific T cell; Lipid rafts: cholesterol-rich plasma membrane microdomains that have been implicated in lymphocyte signaling Images of cells are perplexing. Genetically tinted by the jellyfish fluorescent protein painter kit, cells of all colors and shapes turn serious academic journals into modern art catalogues. After decades of abstract thinking and frequently fruitless interpretation of bands, dots, and their graphic derivatives, the attention of many scientists is now attracted by the opportunity to see almost everything in "real" life. The increased communication value of images is a part of a rapidly evolving cultural tendency demanding real-time event documentation. Nowhere could be more remote from this problem than the caught-in-time Certosa di Pontignano, an old castle near Siena, Italy. Maybe that is why we decided to organize a roundtable discussion about imaging during the 2004 EMBO workshop on Lymphocyte Antigen Receptor and Coreceptor Signaling held at this location ([@bib1]). We asked the experts to reflect on the impact of imaging on a field that has been dominated by biochemical approaches. Our aim was to capture some personal viewpoints on the real and virtual power of imaging and the new challenges the technology brings. Can the traditional craft of cell manipulation match the high speeds that can now be visualized? What new reagents are needed to allow us to take full advantage of imaging in the lymphocyte signaling field? Should imaging data be quantified (definitely, yes!) and if so how? (See also the related Commentary on page 501 of this issue \[[@bib2]\].) At the end, we asked the participants to fantasize about how we might be using imaging technology to study lymphocyte signaling in the more distant future. I regret that we could not present all of the lively discussion, but we have included comments and exchanges on some of the key topics that were covered. We thank all the participants for their willingness to share their insight and opinions. PARTICIPANTS ============ **Oreste Acuto:** Institut Pasteur, France **Mark M. Davis:** Stanford University, CA **Christopher C. Goodnow:** The Australian National University, Australia **Claire Hivroz:** Institut National de la Sante et de la Recherche Medicale Unite 520, France **Bernard Malissen:** Centre d\'Immunologie INSERM-CNRS de Marseille-Luminy, France **Anjana Rao:** Harvard Medical School, MA **Michael Reth:** Max Planck Institute of Immunobiology, Germany **Lawrence E. Samelson:** National Cancer Institute, National Institutes of Health, MD **Alexander Tarakhovsky:** Rockefeller University, NY **Salvatore Valitutti:** Institut National de la Sante et de la Recherche Medicale Unite 563, France **Arthur Weiss:** University of California San Francisco, CA **Rose Zamoyska:** National Institute for Medical Research, UK POWER OF IMAGES =============== **Mark M. Davis**: If you ask, "What\'s the value of imaging?" one important thing is that it allows us to escape the tyranny of population biochemistry. We\'ve been forced to indulge in the fantasy that if you have a million cells in a tube, and you hit them with something, that they\'re all going to do exactly the same thing at the same time. However, we know that a lot of stochastic processes are going on. If you just grind the cells up at different time points, you\'re averaging over all those processes. It\'s only when you can see an individual cell that you can see it was stimulated, and then went on a particular pathway. And there\'s a level of quantitation we\'ve never seen with biochemistry. We\'re getting much more precise answers to questions like the amount of calcium that\'s being released during T cell activation. We can see that one peptide gives you X amount of calcium, and two give you 2X, and so forth ([@bib3]). **Lawrence E. Samelson**: With imaging, it\'s not just that you\'re able to overcome the heterogeneity of the cell population; you\'re able to overcome the heterogeneity of the intracellular molecular populations. Many of the molecules that we tag and look at, in terms of intracellular signaling proteins, exist in different pools in the cell: some are cytoplasmic, some are recruited to the membrane, others are associated with the cytoskeleton. It would be impossible to study these different populations without imaging the individual cells and individual molecules. **Bernard Malissen**: We are also feeling out the speed at which signaling processes are working, and it\'s very fast. Neurobiologists have known that for a very long time. We are just putting our finger on the speed at which a T cell is responding to an antigen. So imaging gives you an idea of the timescale we should be thinking of. **Arthur Weiss**: I think that imaging in real time is critical. But in addition, FRAP technology allows us to see things that we never imagined, in terms of the turnover and the dynamics of structures that we think are static. Because you see an immune synapse, you think everything is just sitting there and immobile with no turnover. **MMD**: There are a lot of technical challenges involved in asking questions of a detailed molecular nature, of molecules that are within particular cells, at particular times, and in particular places. But it\'s the future of understanding how these cells work. There are huge challenges in getting markers for events inside the cell. But there are already some that are specifically designed as indicators of a particular kind of phosphorylation. We need many more of those for all the different signals that might be going on. **Rose Zamoyska**: Single molecule imaging is coming, and it\'s not that far off. That really is going to add another dimension to the way we\'re going to be able to analyze signaling events. IMAGING MEETS BIOCHEMISTRY AND GENETICS ======================================= **Oreste Acuto**: Nobody would disagree with all that has been said so far. But we should never forget that biochemistry gives us the fastest way to establish protein--protein interactions, and the highest resolution. **BM**: Ultimately, genetics should allow you to order a pathway without having help of biophysics. If you have powerful genetics, like in the yeast, you can do very nice ordering and epistatic analyses of pathways, and you don\'t need imaging. **RZ**: One of the problems with biochemistry is that you can never look at the consequences of the signals, because the cells are lysed. And one of the advantages of imaging is that we\'re developing techniques where you\'ll be able to look at the differentiation consequences, downstream of particular signals. **Christopher C. Goodnow**: Whenever I hear the biochemistry angle, I always think about Arthur Kornberg and how proud he was of DNA polymerase in the tube, until the geneticists knocked it out in bacteria, and they replicated just fine. It\'s very hard to order pathways and I think the triangulation between biochemistry, genetics, and imaging is going to be essential. You need to know where things are in the cell, and their kinetics and the dynamics, which is what the imaging tells you. You need to know what could happen, which is what the test tube experiment tells you. And then the genetics tells you what is actually needed for a given process. As a community, we don\'t put enough emphasis, in our publication and referee processes, on when the genetics and the biochemistry are disconnected. For example, we have a ZAP-70 catalytic domain point mutant mouse, which has a severe problem with thymic positive selection (unpublished data). The genetics is telling us that there is a real problem. But when we look at tyrosine phosphorylation and do old-fashioned calcium analysis, we can\'t see any problem. So the biochemistry is disconnected from the genetics. The question is if you did high speed imaging, would we suddenly be able to connect the genetics with the biochemistry? **Michael Reth**: The immune synapse is a nice example of how difficult it can be to correlate image analysis data with the biochemical behavior of molecules inside a cell. When the synapse was first discovered, everybody thought that one had finally found the molecular organization of signaling proteins at the activated T cell antigen receptor. Yet when people later on analyzed synapse formation at different time points and used antiphosphotyrosine antibodies ([@bib4]), it became clear that TCR signaling happens much earlier than synapse formation, and that the synapse may have more to do with the secretion of cytokines than with the initiation of the TCR signal. **MMD**: Absolutely, when we first see things we\'re invariably going to misinterpret them. Seeing may be believing, but it isn\'t always understanding. **MR**: Another source of artifacts is the use of GFP-tagged proteins to follow the behavior of molecules in a living cell. This is a powerful tool, but it is important to first test whether the fusion to GFP is not drastically changing the localization or biochemical behavior of the fusion proteins inside the cell. Another problem is the use of fluorescent reagents to visualize structures in living cells like lipid rafts. In this respect, it was interesting to see that when FITC-coupled cholera toxin was used to detect raft structures it resulted in raft aggregation ([@bib5]). So you are creating what you want to observe. QUANTITATING IMAGES =================== **MR**: One problem we have to solve is how to quantify our image analysis. When I see a publication with a beautiful picture of a cell showing the localization of the GFP-tagged protein to a particular membrane structure, I wonder how representative this image is of the behavior of these proteins in the cell population. To overcome the bias generated by our eyes and our brain, we need to apply computer programs for image analysis which, similar to a FACScan, can quantify images and order cells according to the image phenotype. **LES**: Many of these problems are problems that all cell biologists have. You look at a picture in a journal, and you say, "How do I know this is representative?" That gets into the question of honesty, and ethics, and basically good science. But programs for quantitation exist; you can track movement; you can quantitate velocity; you can quantitate dynamics. So a lot of what we need is there. **Salvatore Valitutti**: It\'s very possible to quantitate morphology. It\'s very tough. Very time consuming. But it is possible to ask authors for quantification, or even to show the originals from which the quantitation was made. **Alexander Tarakhovsky**: Do you think that there is an absolute necessity to introduce ways to quantify images? **SV**: I think it will be in the interests of people doing morphology to somehow enforce measurements of intensity, for instance, at the immune synapse. **MMD**: The referees always ask us for quantitation: what does the cell population do? How many cells do this? How many cells do that? On some occasions, when the referees have been skeptical about our interpretation of the images, they ask for all the images, and we send all the images on a CD. So the vetting is actually easier than it used to be, in the sense that you can transmit vast amounts of information very quickly, so that people can be sure that you\'re not being subjective. As to a program that Bill Gates would sell us to do this---fine, when it happens. But there are many different questions you can ask using imaging. I can imagine a very simple kind of software being good for some kinds of imaging analysis, but completely useless for other kinds. **Claire Hivroz**: One of the problems that we have to face is that while these new imaging techniques are really nice, they are sources of artifacts, also. We really need to do the analysis carefully. Biologists are not physicists, and sometimes we don\'t know how to analyze the image, or we don\'t know exactly how to use the software. I think we have to make great efforts to work with physicists, to really understand what we are doing because being sure, for example, that what we are looking at by FRET is really an interaction between two proteins is not that easy. ADVANCING THE TECHNOLOGY ======================== **MR**: By using GFP-tagged proteins we can employ imaging to localize proteins to certain subcellular structures, but what is really lacking are more techniques to not only measure the localization, but also the activity of a protein. Of course, we can measure the intracellular increase of calcium by fluorescent dyes, but these dyes cannot be localized to certain places inside the cell. What we need are more GFP variants that change their fluorescence upon modification, like calcium binding, phosphorylation, or oxidation. With such techniques, you can then see in real time, not only the localization of molecules in the cell, but also at what time point these proteins become active or modified. **Anjana Rao**: I\'m dealing with a program of transcriptional regulation that is sensitive to very low intracellular calcium levels---lower than can be measured reliably by the available technology. So I think, in the case of calcium imaging, we\'re clearly going to need some new technology in the picture. **MMD**: There\'s still a bag of genetic tricks that hasn\'t really been applied to imaging but which could be used. There is development of photo-activatable GFP, for example, and temperature-sensitive mutations. So it might be possible to give cells a burst of a laser, for example, to alter the function of molecules that you had modified. **AT**: Do you think that one can manipulate structures such as the immune synapse? **CCG**: The strength of genetics is in the specificity of the dissection. But the limitation is that you just have no temporal control or spatial control. So, in that respect, it\'s a very limited tool. The ideal scenario would be to replace genetics with pharmacology. To have small molecules that you can add in to inhibit signaling pathways, which are as specific as genetic manipulations. But, of course, where we get into trouble is in knowing how specific a compound is. This is where the neurobiologists, and the cardiologists, have it over us as immunologists. They\'ve got a panoply of quite specific agents to dissect processes such as cardiac contractility. **AT**: Do you think that genetic manipulation could allow conditional protein inactivation at a given time point? **CCG**: Inactivation has to happen very fast. The only thing would be some adaptation of photo-inactivatable or temperature-sensitive alleles, which as Mark \[MMD\] has pointed out, could allow you to eliminate a wild-type signaling protein both rapidly and in a specific region of a cell. **MR**: New in vivo imaging studies can follow the dynamic behavior of single lymphocytes in the tissue of a living mouse. This, in combination with genetics, will allow us to understand better the dynamics of cellular interactions inside the immune system. To follow the behavior and activity of single molecules inside a lymphocyte is much more demanding technically, as these are rapid and very dynamic processes. One needs machines with a very fast kinetic image acquisition if one wants to follow these events in real time. Furthermore, if one wants to track single fluorescent molecules, one has to use a lot of laser light to detect them, and this could be another potential source of artifacts. Laser light not only results in photobleaching, but can also generate reactive oxygen species. As many signaling processes inside the cell are highly sensitive to redox changes, it is possible that shining a lot of laser light on the molecules can change their regulation and behavior. **AW**: One thing I want to stress again is quantitation. And maybe genetics can help there. Chris \[CCG\] talked about a point mutation which doesn\'t show any biochemical difference. We might not be able to validate that our GFP constructs are behaving normally using simple overexpression systems. I would argue that maybe we can use genetic approaches to test the constructs we use to validate the fact that we\'re expressing the right amount of protein, in the right place, at the right time, and that those proteins are regulated properly. Maybe we can harness the power of genetics for proper expression---right place, right time, as well as right level. **AR**: In terms of technology development, there are two things that I would focus on. Microinjection of mutant proteins into cells that don\'t have that protein would be a very good thing to develop. The protein can be tagged in various ways, with small fluorescent probes for instance. And second, I think electron microscopy will be important. Think about the work that Gunther Blobel did, for instance, with the nuclear pore and with nuclear import and export. He had the biochemistry. He had all the different subunits. And then he used EM to localize them in the basket of the nuclear pore. The stable immune synapse is such a structure that could benefit very greatly from EM analysis. IMAGINING THE FUTURE OF IMAGING =============================== **AT**: Now I want you to dream. **AR**: To dream? **AT**: Yes. So, without discussing limitations, tell me how do you think in ten years your dreams will be fulfilled? \[Laughter\] **MR**: My dream is a very powerful computer program that is able to generate a virtual cell where we can test our hypothesis of protein signals inside cells. Using the primary sequence data, this program should be able to generate the tertiary structure of the protein not only in its frozen (crystal) stage, but also its normal dynamic stage in solution. With such a program, one could follow the conformational changes of proteins and better understand the dynamic processes of protein--protein interactions during cell signaling. So that is a dream, but I think one has to wait quite a while for such a program. **MMD**: I don\'t think a computer would be able to know how evolution built a cell. So I think that dream is going to come to a bad end. But in my childhood, there was a movie called "Fantastic Voyage," where there was a tiny, little ship that was built, and miniaturized humans were in it. They cruised around the bloodstream, and they looked at the lymphocytes and other things from this point of view. So a good dream would be one where you miniaturize the ship even more, such that you could go into a cell, through a channel or something, and cruise around the cell and watch different bits of the machinery moving up and down, or doing whatever it does. **AR**: I think you\'d find what Schrödinger predicted in 1944 ([@bib6]). You\'d find probabilistic movements. I think the whole workings of the cell are governed by probability and stochastic movements that, in turn, are governed by the affinity of interaction. My dream would be to apply the imaging techniques we\'re talking about at the membrane, today, to find out what happens in the nucleus. **LES**: Mine is another cautionary tale from early twentieth-century physics, and that is the Heisenberg Uncertainty Principle. We have to watch out for what we do to these systems. Certainly by manipulating systems we create artifacts. So that\'s a nightmare. \[Laughter\] **OA**: My ultimate dream is that we abolish competition. We create a big center and attack very fundamental, basic paradigms in a few systems. It is not strictly necessary to know everything, with everybody working in their own little corner. Why not combine genetics, imaging, and biochemistry in a dedicated center where scientists can perform challenging experiments, like physicists do? That\'s a dream. That\'s something that I think should work. \[Applause\] The editors of the *Journal* would like to thank the organizers of the 2004 EMBO workshop on Lymphocyte Antigen Receptor and Coreceptor Signalling (Certosa di Pontignano, Siena, Italy)---Cosima Baldari, John Telford, Oreste Acuto, and Gary Koretsky---for allowing us to hold the discussion during the meeting and for their assistance with the organization. We also thank Ingrid Mecklenbrauker for helping us document the discussion. The edited transcript was approved by all participants. We apologize to the participants whose contributions we could not include owing to space limitations. [^1]: CORRESPONDENCE A.T.: <[email protected]>

{ "pile_set_name": "PubMed Central" }

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"cultur", "redox", "million", "bill", "youll", "technic", "speed", "opinion", "mutant", "basic", "tough", "share", "one", "seen", "recruit", "fulfil", "attent", "tarakhovski", "francisco", "would", "understand", "quantif", "built", "fusion", "synaps", "dye", "data", "suddenli", "eye", "discuss", "transcript", "christoph", "corner", "tube", "strength", "panopli", "furthermor", "sensit", "specif", "advantag", "exampl", "face", "recoveri", "intracellular", "spatial", "evolut", "system", "kit", "pasteur", "doesnt", "ye", "year", "inhibit", "stress", "machineri", "antigen", "invari", "still", "heterogen", "look", "held", "caughtintim", "affin", "calcium", "frozen", "harvard", "forc", "contractil", "sequenc", "le", "bia", "ζassoci", "itali", "give", "editor", "distant", "earlier", "demand", "fluoresc", "dedic", "edit", "import", "genet", "pharmacolog", "principl", "alreadi", "whatev", "dont", "topic", "alway", "sometim", "repres", "dot", "chang", "antiphosphotyrosin", "proxim", "institut", "relat", "antigenspecif", "fast", "easi", "avail", "remot", "cruis", "see", "quantit", "tyrosin", "receptor", "thing", "isnt", "say", "glossari", "futur", "real", "john", "coreceptor", "owe", "today", "document", "gfptag", "addit", "reliabl", "knock", "drastic", "nowher", "high", "activ", "certosa", "fitccoupl", "limit", "far", "approv", "immobil", "workshop", "gari", "solv", "youd", "rockefel", "phosphoryl", "talk", "membran", "thought", "pontignano", "voyag", "competit", "structur", "yeast", "commentari", "sure", "acquisit", "meet", "case", "deriv", "stochast", "visual", "molecular", "second", "studi", "techniqu", "applaus", "gate", "sit", "said", "space", "shine", "becam", "acuto", "forget", "effort", "expert", "kinet", "mayb", "movi", "toxin", "highli", "ship", "vast", "peptid", "bib", "bit", "earli", "research", "thymic", "mmd", "roundtabl", "find", "valid", "enforc", "cancer", "blobel", "fine", "max", "insid", "that", "band", "creat", "biologist", "absolut", "rapid", "perplex", 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1. Introduction {#sec1} =============== Damage to the cornea from chemical burns is a serious clinical problem, accounting for about 7%--10% of eye injuries, of which, alkali-induced injury to cornea is the most dangerous, due to its quick penetration to the eye surface \[[@B1]\]. It can cause corneal infection, ulceration, perforation, neovascularization, and opacification \[[@B2]\] and often leads to permanent visual impairment. Penetrating keratoplasty has been used successfully to treat many corneal diseases; however, several intractable ocular surface diseases, such as chemical burns, are still untreatable using this procedure \[[@B3]\]. The Boston Type 1 keratoprosthesis (KPro), developed at the Massachusetts Eye and Ear Infirmary and approved by the US Food and Drug Administration (1993), is currently the most commonly used keratoprosthesis worldwide \[[@B4]\] and is used in cases at high risk for penetrating keratoplasty failure \[[@B5]\]. It has been successfully used to restore vision in cases of chronic corneal disease and graft failure, including corneal alkali burn \[[@B6]\]. However, despite multiple advances and improvements in prognosis of patients following KPro surgery, several challenges remain. The postoperative development of RPMs is one of the most common complications of keratoprosthesis surgery, affecting between 25% and 65% of patients, and is poorly understood \[[@B7], [@B8]\]. Matrix metalloproteinases (MMPs), zinc endopeptidase proenzymes responsible for the degradation of extracellular matrix components, play a role in many ocular physiological processes, including embryogenesis, angiogenesis, and wound healing \[[@B9]\]. Breakdown of the basement membrane by MMPs is known to contribute to the pathogenic ulceration and perforation of the corneal stroma \[[@B10]\]. The activity of MMPs is closely controlled by a family of natural antagonists, the tissue inhibitors of matrix metalloproteinases (TIMPs), whether MMPs exert normal or abnormal responses depends upon the balance between MMPs and TIMPs. An imbalance between MMPs and TIMPs in favor of inhibitor might provoke the development of fibrosis and in consequence may lead to tissue remodeling \[[@B11]\]. MMP-9 is the enzyme of major importance in the remodeling and degradation of the corneal stromal collagen in both animals and human beings \[[@B12]\]. It was found to be only expressed in rats which underwent corneal injury, instead of the normal corneal stroma \[[@B13]\]. The expression of MMP-9 and its endogenous inhibitor TIMP-1 in the aqueous humor of alkali-burned rabbit cornea before and after KPro implantation and their role in RPMs formation following KPro surgery have previously received limited attention. In this study, we report in greater detail the MMP-9 and TIMP-1 expressions in aqueous humor before and after KPro implantation into the alkali-burned rabbit cornea, the histopathological characteristics of RPMs developed after KPro implantation, and immunohistochemical expression of MMP-9 and TIMP-1 in this RPM. 2. Material and Methods {#sec2} ======================= 2.1. Animals {#sec2.1} ------------ A total of 60 healthy adult New Zealand White rabbits, weighing between 2.5 and 3.0 kg, were obtained from the Animal Experimental Center of Army General Hospital of Shenyang Military Region (Shenyang, China). This study was approved by the Ethics Committee of Shenyang Military Region General Hospital. All experiments were performed on the left eye of animals and all animals and experimental conditions followed laboratory animal regulations of State Science and Technology Commission. 2.2. Experimental Model of Corneal Alkali Burn {#sec2.2} ---------------------------------------------- Corneal alkali burn model was established as previously described with some modification \[[@B14]\]. To create an alkali burn, 50 rabbits were randomly selected and their left eyes were locally anesthetized with 0.5% Alcaine eye drops (Alcon Ophthalmic Products Co. Ltd., Shanghai, China). Then, a 10 mm in diameter Cellulose nitrate filter paper soaked in 1 mol/L NaOH was applied to the cornea for 45 s, followed by a rinse with 10 mL of balanced salt solution. After the corneal alkali burn was created, animals received chlortetracycline eye ointment (Beijing Shuangji Pharmaceutical Co., Ltd., China) and atropine sulphate eye gel (Shenyang Xingqi Pharmaceutical Co., Ltd., China) daily for one week. The severity of conjunctival and corneal injury was determined according to the Roper-Hall (RHG) grading system \[[@B15]\]. Experimental model of corneal alkali burn was successfully established in 42 New Zealand White rabbits, as demonstrated by the presence of complete corneal opacity, corneal neovascularisation involving more than two quadrants of the cornea, and normal anterior chamber 3 months after severe alkali burn. The 36 model rabbits were randomly selected and divided into three groups (*n* = 12): group I, blank control group, corneal alkali burn without any keratoplasty; group II, control group, cornea alkali burn with penetrating keratoplasty; group III, experimental group, corneal alkali burn with KPro implantation. The 10 untouched rabbits served as allogeneic cornea donors after collection of aqueous humor samples. 2.3. Corneal Implantation {#sec2.3} ------------------------- The surgery was performed as previously described with some modification \[[@B16]\]. The procedures were performed under a surgical microscope (Zeiss, Jena, Germany) by a single investigator (Yinxin Chen), and the surgical time was 45 to 60 min per procedure. Briefly, donor cornea was marked in the periphery with an 8.5 mm diameter biopsy punch (Miltex, Plainsboro, NJ, USA) and then excised with Vannas scissors (Storz Instruments Company, San Damis, CA, USA) after animal euthanasia. The corneas were trephined centrally with a 3 mm diameter punch, slid over the stem of the PMMA front plate, followed by titanium back plate position (KPro device, 8.5 mm in diameter, Boston Keratoprosthesis Operations, MA, USA), and complex was then placed in PBS. Three months after injury, the recipient cornea was marked with an 8.5 mm diameter biopsy punch and excised with Vannas scissors under general and topical anesthesia (120 mg/kg ketamine and 20 mg/kg xylazine). The extracapsular crystalline lens extraction was then performed on eyes of groups II and III and the rabbits were left aphakic with an intact posterior capsule. The lenses of group I eyes were not extracted and the rabbits were left phakic. The donor cornea KPro complex was then positioned in the recipient bed and secured with sixteen interrupted 11.0 nylon sutures (Sharpoint; Angiotech Pharmaceuticals, Vancouver, BC, Canada). A drop of tobramycin and dexamethasone (Tobradex; Alcon, Puurs, Belgium) was applied and a tarsorrhaphy was performed using 8-0 nylon sutures. The tarsorrhaphy was removed 48 h after the procedure, and the same antibiotic and steroid drop was administered once a day for 7 consecutive days following tarsorrhaphy removal. After operation, animals received tobramycin and dexamethasone ophthalmic ointment once daily for three days, atropine sulfate eye gel once daily for one week, chloramphenicol eye drops four times daily for 4 weeks, and chlortetracycline hydrochloride eye ointment once every night for 4 weeks. 2.4. Follow-Up and Clinical Evaluation {#sec2.4} -------------------------------------- RPM referred to membrane growing on the back of the device, all eyes were determined under slit-lamp, and RPMs were diagnosed at the time of explantation. The aqueous humor was obtained by puncturing the anterior chamber using a 45 gauge needle at postoperative days 7, 14, 28, 60, and 90. MMP-9 and TIMP-1 levels in aqueous humor were determined using the commercially available ELISA kits for MMP-9 and TIMP (Shanghai Sunred Biological Technology Co., Ltd., China). Ninety days after implantation, all rabbits were sacrificed by using the air embolism method by injecting 20 mL of air into the ear marginal vein. The presence of complications, like retinal detachment, intraocular infection, and so forth, was assessed by B ultrasound. The RPMs that developed were then excised and histological sections were prepared for further processing. Slit-lamp examination was performed before surgery and at postoperative days 7, 14, 28, 60, and 90, to examine the corneas for signs of inflammation or neovascularization and to assess optical transparency, and photographs were taken. For transmission electron microscopy (TEM), the sections were stained with uranium acetate and lead citrate and observed with JEM-1200 transmission electron microscopy (Tokyo, Japan). Some pieces were fixed in 4% paraformaldehyde in 0.1 mol/L PBS, paraffin embedded, and then sectioned. Hematoxylin and eosin (H&E) staining was performed for routine histopathologic examination. The immunohistochemical staining was performed using rat anti-rabbit polyclonal antibodies against MMP-9 and TIMP-1 and biotin-labeled sheep anti-mouse IgG, all purchased from Beijing Boaosen Biotechnology Co., Ltd., China. Five sections were randomly selected and data were obtained from 50 fields of view (10 fields from each section). 2.5. Statistical Analysis {#sec2.5} ------------------------- Data were expressed as mean ± SD and analyzed using SPSS 19.0 for windows (SPSS Inc., Chicago, Illinois, USA). The One-Way ANOVA procedure was used to determine the difference among groups. A *p* value of \<0.05 was considered statistically significant. 3. Results {#sec3} ========== 3.1. Clinical Results {#sec3.1} --------------------- Three months after the infliction of alkali wounds, the injured corneas exhibited stromal opacities of varying depth; some corneas completely became opaque, when compared with the normal ones (Figures [1(a)](#fig1){ref-type="fig"} and [1(b)](#fig1){ref-type="fig"}). Slit-lamp examination showed RPMs developed 30 ± 10 days after transplantation of KPro ([Figure 1(c)](#fig1){ref-type="fig"}). After corneal transplantation, 1 rabbit accidently died in group II and 2 in group III. The common symptom in early period of postoperation were eyelid margin redness and swelling, conjunctival injection, tearing, photaesthesia and white secretion from the conjunctival sac. The local irritation lasted between 7 and 14 days in group II and between 7 and 21 days in group III. There was a clear boundary of edema on the cornea graft; then the edema reduced gradually at postoperative day 7 and the corneal grafts became clear ([Figure 2](#fig2){ref-type="fig"}). At the postoperative day 14, there was new vessel growth in the upper corneal limbus, and a large amount of new vessels formed 28 days postoperatively ([Figure 2](#fig2){ref-type="fig"}). At 60 and 90 postoperative days, a reduction in the number of vessels and level of haze was noted relative to earlier examinations ([Figure 2](#fig2){ref-type="fig"}). The postoperative complication in group III included RPMs in 5 eyes ([Figure 2](#fig2){ref-type="fig"}, day 14) and vitreous hemorrhage in two eyes. However, in group II, corneal rejection occurred in 5 eyes 14 days after penetrating keratoplasty, with diffuse edema and opacity of the graft ([Figure 2](#fig2){ref-type="fig"}). The shallow anterior chamber and anterior synechiae developed in 1 case and posterior capsule opacification developed in 3 cases ([Figure 2](#fig2){ref-type="fig"}, day 60). There were no postoperative retinal detachment and endophthalmitis in the both groups II and III. 3.2. Aqueous Humour MMP-9 and TIMP-1 Levels following KPro Implantation {#sec3.2} ----------------------------------------------------------------------- The dynamic expressions of MMP-9 and TIMP-1 in aqueous humour were quantified using commercially available ELISA kit; our results showed that there was a small amount of MMP-9 and TIMP-1 in normal rabbit aqueous humor (12.52 ± 3.05 and 33.49 ± 3.39 ng/mL, resp.). The severe alkali burn employed in this study resulted in a significant increase in both MMP-9 and TIMP-1 levels three months later. After corneal transplantation, aqueous humor MMP-9 in group III showed an increasing trend and peaked at postoperative day 90. By contrast, in group II, MMP-9 in aqueous humour reached peak at postoperative day 14 and then significantly decreased thereafter, although the data at postoperative day 90 was still significantly higher than that of preoperative level and of group I (*p* \< 0.05, [Table 1](#tab1){ref-type="table"}). However, unlike the increase in MMP-9 postoperatively, aqueous humor TIMP-1 was significantly decreased following operation in both groups II and III. The data reached valley bottom at postoperative day 90 in group III and at postoperative day 14 in group II and then increased until the postoperative day 90 (*p* \< 0.05 versus preoperative level and blank control, [Table 1](#tab1){ref-type="table"}). The concentration of MMP-9 in group III became higher on the postoperative day 28 when compared with group II, and the difference was statically significant at subsequent points (*p* \< 0.05). By contrast, TIMP-1 level became lower on the postoperative day 28 and the data became statistically significant thereafter when compared with group II (*p* \< 0.05). 3.3. Histopathological and Immunohistochemical Analysis of Retroprosthetic Membrane Specimens {#sec3.3} --------------------------------------------------------------------------------------------- In total, five retroprosthetic membrane specimens were obtained following Boston KPro explantation; our hematoxylin-eosin staining showed a large number of fibroblasts and collagen fibers arranged irregularly and inflammatory cell infiltration. There was ingrowth of new blood vessels in the RPMs (Figures [3(a)](#fig3){ref-type="fig"} and [3(b)](#fig3){ref-type="fig"}). Transmission electron microscopy of the retro-KPro fibrous membrane revealed an abundance of elongated collagenous fibers, separated by fibroblasts and granulocyte infiltration. These cells were spindle-shaped with mitochondria and rough endoplasmic reticulum in the cytoplasm. The extracellular space separating these cells was composed of fibroblasts (Figures [3(c)](#fig3){ref-type="fig"} and [3(d)](#fig3){ref-type="fig"}). Immunohistochemical results showed positive staining of retroprosthetic membrane specimens for MMP-9 and TIMP-1, with yellowish-brown cytoplasm and blue nucleus (Figures [3(e)](#fig3){ref-type="fig"}, [3(f)](#fig3){ref-type="fig"}, [3(g)](#fig3){ref-type="fig"}, and [3(h)](#fig3){ref-type="fig"}). 3.4. Aqueous Humor MMP-9 and TIMP-1 Levels in KPro-Implanted Rabbits with or without RPM {#sec3.4} ---------------------------------------------------------------------------------------- To further explore the role of MMP-9 and TIMP-1 expressions in retroprosthetic membrane formation following the KPro implantation, rabbits with KPro implantation were subdivided into RPM and No-RPM groups. Aqueous humor levels of MMP-9 were significantly higher in the RPM group postoperatively when compared with that in the No-RPM group (*p* \< 0.01 or *p* \< 0.001). By contrast, Aqueous humor TIMP-1 levels in the RPM group were comparatively lower than that of No-RPM group following the operation (*p* \< 0.01). There was no significant difference between the two groups in preoperative aqueous humor MMP-9 and TIMP-1 levels ([Figure 4](#fig4){ref-type="fig"}). 4. Discussion {#sec4} ============= Ocular trauma in the form of an alkali burn to the cornea is among the most serious ocular injuries that may cause severe and permanent visual impairment \[[@B17]\]. It can induce a strong inflammatory reaction characterized by cell infiltration and production of proteolytic enzymes and cytokines \[[@B1]\]. Previous studies have indicated an indispensable role for MMPs in the homeostasis and pathophysiology of the cornea \[[@B18]\]. MMPs and TIMPs are present in the aqueous humor \[[@B19]\], and alkali injury enhanced the expression of MMP-9 in mice \[[@B20]\]. Degradation of the basement membrane by proteolytic enzyme MMP-9 may involve the pathogenic process of alkali injury \[[@B21], [@B22]\]. The presence of MMP-9 has been identified in the tear fluid of patients with corneal alkali burn \[[@B23]\]. Consistent with the results of this study, our results showed that alkali insult of rabbit cornea resulted in a significant increase in aqueous humor MMP-9 levels, while the TIMP-1 levels in aqueous humor were found to decrease to some extent three months after corneal alkali burns. After keratoplasty, MMP-9 levels in aqueous humor were further significantly enhanced in experimental rabbits within the postoperative day 90, whereas TIMP-1 levels were downregulated further when compared with that in group I (blank control). All these results showed that evidence suggested the pathophysiological role of MMP-9 in alkali-induced corneal injury and repair. The Boston keratoprosthesis is by far the most commonly used prosthetic cornea in the world \[[@B24]\]. However, there are a number of significant complications that can occur postoperatively. Among which, retroprosthetic membrane is one of the most common postoperative complications encountered \[[@B25]\], occurring in 25% to 65% of patients within a year after KPro implantation, and up to 45% of cases require eventual treatment \[[@B26], [@B27]\]. The formation of retroprosthetic membrane was shown to correlate with the risk of corneal melt \[[@B28]\]. The etiology of retroprosthetic membrane is still largely unknown. However, the presence of this retrokeratoprosthesis fibrous tissue may act as a physical barrier between the cornea and the aqueous humor, which limits the contact between the aqueous and the corneal graft enough, and ultimately result in stromal necrosis and epithelial degeneration in KPro implanted eyes \[[@B29]\]. In our study, retroprosthetic membrane happened in 5 out of 10 KPro-implanted rabbits (50%), indicating the high incidence of retroprosthetic membrane formation following KPro implantation. Previous study showed that chemical injury seems to be an insignificant risk factor for the development of RPM \[[@B5]\], while other study also indicated that chemical burns have higher incidence of RPM development \[[@B30]\]. However, it is not possible to state whether alkali burn used in this series could be a contributing factor in the higher rate of RPM formation reported herein, since, to the best of our knowledge, there seems to be few researches on KPro implantation in animal model of corneal alkali burns up to now. The RPMs have been previously evaluated in patients with KPro implantation and were suggested to vary according to antecedent clinical events of the patients \[[@B7], [@B29]\]. Therefore, we further evaluated the histopathological characteristics of the RPMs obtained, as well as the immunohistochemical expression of MMP-9 and TIMP-1 in these RPMs. Our results showed a large number of fibroblasts and collagen fibers arranged irregularly with inflammatory cells infiltration, as well as ingrowth of new blood vessels in this retrokeratoprosthesis fibrous tissue. Corroborating the results of MMP-9 and TIMP-1 expressions in aqueous humor, our immunohistochemical analysis showed positive staining of retroprosthetic membrane specimens for MMP-9 and TIMP-1. Moreover, aqueous humor MMP-9 levels were found to be significantly higher in KPro-implanted eyes with RPM developed when compared with that of No-RPM developed ones. By contrast, aqueous humor TIMP-1 levels were comparatively lower than that of No-RPM group. All these results suggested that higher expression of MMP-9 may contribute to retroprosthetic membrane formation following KPro implantation. There are several limitations in our study. The most important one is that the lens status was different among the black control group (group I), control group (group II), and KPro group (group III). Lens status has been reported to be a confounding factor on MMP-9 and TIMP-1 levels \[[@B31], [@B32]\], which may partially contribute to the elevated MMP-9 and decreased TIMP-1 levels observed in our study. However, despite the potential effect of lens status, our study did show the potential pathophysiological role of MMP-9 in RPMs formation, as demonstrated by significantly different aqueous humor MMP-9 and TIMP-1 levels between KPro-implanted eyes with and without RPM development. In conclusion, the finding of our study showed the upregulated expression of MMP-9 and downregulated endogenous MMP-9 inhibitor, TIMP-1, after alkali-induced injury to the rabbit corneas and following KPro implantation, and to the best of our knowledge this study is the first to show the potential pathophysiological role of MMP-9 in RPMs formation following KPro implantation in a rabbit model of corneal alkali burn. MMP-9: : Matrix metalloproteinases 9 TIMP-1: : Tissue inhibitors of matrix metalloproteinase 1 KPro: : Boston Type 1 keratoprosthesis RPM: : Retroprosthetic membrane. Conflict of Interests ===================== The authors declare that there is no conflict of interests regarding the publication of this paper. ![Slit-lamp appearance of anterior segment of the rabbit eyes before alkali burn (a), three months after alkali burn (b), and after implantation of Boston Type 1 keratoprosthesis (KPro), with the retroprosthetic membranes developed (c).](JOPH2016-1094279.001){#fig1} ![Clinical observation of alkali-burned rabbit corneas following Boston Type 1 keratoprosthesis (KPro) implantation or penetrating keratoplasty. Photographs taken immediately after operation and at postoperative days 7, 14, 28, 60, and 90.](JOPH2016-1094279.002){#fig2} ![Histopathology of retroprosthetic membrane extracted from the explanted keratoprosthesis in the corneal alkali-burned rabbits. H&E staining of normal cornea ((a) ×100) and retroprosthetic membrane ((b) ×100); transmission electron microscopy of normal cornea ((c) ×4000) and fibrous retroprosthetic membrane ((d) ×4000); immunohistochemical analysis of MMP-9 ((e) ×200) and TIMP-1 ((f) ×200) expressions in normal cornea (MMP-9: (e) ×200; TIMP-1: (g) ×200) and retroprosthetic membrane (MMP-9: (f) ×200; TIMP-1: (h) ×200).](JOPH2016-1094279.003){#fig3} ![Aqueous humor levels of MMP-9 and TIMP-1 in Boston Type 1 keratoprosthesis (KPro) implanted rabbits with or without RPM formation. Pre: preoperation; Day: postoperative day; RPM: retroprosthetic membrane. ^*∗∗*^ *p* \< 0.01, ^*∗∗∗*^ *p* \< 0.001 versus the No-RPM group.](JOPH2016-1094279.004){#fig4} ###### Dynamic expressions of MMP-9 and TIMP-1 in aqueous humor of alkali-burned rabbit cornea following Boston Type 1 keratoprosthesis (KPro) implantation. Time MMP-9 (ng/mL) TIMP-1 (ng/mL) -------------- --------------------- ------------------ -------------- --------------------- ------------------ -------------- Preoperation 22.85 ± 4.20 22.52 ± 4.03 22.39 ± 3.36 29.42 ± 1.05 29.44 ± 1.08 29.37 ± 1.01 Day 7 33.48 ± 7.29^*∗*\#^ 43.54 ± 4.39^\#^ 22.47 ± 4.13 27.44 ± 1.16^\#^ 26.57 ± 1.94^\#^ 29.46 ± 1.14 Day 14 40.55 ± 8.43^*∗*\#^ 75.48 ± 5.20^\#^ 22.41 ± 3.56 24.51 ± 1.98^*∗*\#^ 21.45 ± 3.59^\#^ 29.51 ± 1.31 Day 28 56.42 ± 3.56^\#^ 52.39 ± 6.83^\#^ 22.40 ± 4.15 21.44 ± 3.57^\#^ 23.50 ± 2.77^\#^ 29.52 ± 1.13 Day 60 69.55 ± 3.52^*∗*\#^ 45.44 ± 6.02^\#^ 22.39 ± 4.16 19.42 ± 1.96^*∗*\#^ 25.41 ± 1.96^\#^ 29.54 ± 1.30 Day 90 83.42 ± 7.63^*∗*\#^ 38.53 ± 3.54^\#^ 22.36 ± 3.55 14.49 ± 4.36^*∗*\#^ 28.46 ± 6.84 29.58 ± 1.37 Group III: corneal alkali-burned rabbits implanted with Boston Type 1 keratoprosthesis (KPro); group II: corneal alkali-burned rabbits with penetrating keratoplasty; group I: corneal alkali-burned rabbits without keratoplasty. ^*∗*^ *p* \< 0.05 versus group II, ^\#^ *p* \< 0.05 versus group I. [^1]: Academic Editor: Sachin Mulik

{ "pile_set_name": "PubMed Central" }

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"photograph", "attent", "biotinlabel", "nineti", "data", "eye", "discuss", "mice", "compon", "describ", "chamber", "stroma", "favor", "last", "factor", "edema", "healthi", "arrang", "left", "be", "sac", "respons", "prosthet", "treat", "instead", "ngml", "system", "kit", "year", "pathogen", "conclus", "central", "still", "commonli", "worldwid", "filter", "month", "vanna", "eyelid", "red", "belgium", "extracellular", "mm", "laboratori", "segment", "window", "declar", "editor", "enhanc", "failur", "igg", "intract", "earlier", "preoper", "import", "excis", "devic", "current", "topic", "paper", "requir", "vision", "routin", "play", "slitlamp", "physiolog", "fibroblast", "obtain", "provok", "margin", "atropin", "ulcer", "sunr", "surgic", "opac", "rhg", "interrupt", "avail", "co", "sign", "melt", "untreat", "kg", "close", "breakdown", "blank", "symptom", "shown", "sec", "total", "high", "activ", "reaction", "limit", "rat", "note", "approv", "granulocyt", "pathophysiolog", "yinxin", "far", "rabbit", "examin", "chicago", "endophthalm", "elev", "surgeri", "peripheri", "quadrant", "secur", "timp", "histolog", "stain", "statist", "membran", "ultrasound", "previous", "microscop", "success", "anesthesia", "sampl", "mulik", "optic", "boundari", "case", "materi", "f", "antagonist", "drop", "ingrowth", "grade", "visual", "boston", "angiotech", "studi", "insult", "blue", "explor", "space", "becam", "character", "xingqi", "adult", "three", "lens", "hydrochlorid", "seri", "usa", "slid", "product", "earli", "specimen", "haze", "neovascular", "research", "nj", "stromal", "recipi", "proenzym", "find", "extent", "figur", "clinic", "sacrif", "tobramycin", "danger", "creat", "endoplasm", "period", "white", "quantifi", "affect", "method", "new", "needl", "form", "encount", "corrobor", "often", "diffus", "procedur", "section", "shuangji", "postoper", "briefli", "contrast", "elong", "correl", "b", "salt", "punch", "best", "lower", "effect", "rins", "postop", "ointment", "bfigreftypefig", "shenyang", "×jophfig", "administ", "within", "∗", "hospit", "problem", "titanium", "poorli", "mean", "number", "degrad", "introduct", "pharmaceut", "tabreftypet", "rate", "patient", "inflict", "uranium", "fluid", "upregul", "human", "academ", "transpar", "advanc", "capsul", "depend", "aphak", "xylazin", "shanghai", "injur", "depth", "followup", "metalloproteinas", "inhibitor", "len", "iii", "epitheli", "subdivid", "n", "understood", "later", "endogen", "challeng", "singl", "develop", "indic", "c", "heal", "intact", "keratoprosthesi", "sulfat", "∗∗∗", "format", "natur", "field", "unlik", "possibl", "figreftypefig", "punctur", "might", "seem", "clear", "confound", "compani", "diseas", "daili", "investig", "kpro", "analysi", "alkali", "complex", "jem", "antimous", "could", "g", "corneal", "retin", "commiss", "limbu", "reticulum", "sulphat", "balanc", "gaug", "afigreftypefig", "diagnos", "region", "grow", "oper", "ketamin", "mmp", "vitreou", "explant", "rpm", "injuri", "increas", "improv", "sharpoint", "report", "euthanasia", "point", "×", "inject", "seriou", "previou", "armi", "trauma", "therefor", "eosin", "swell", "nylon", "opaqu", "photaesthesia", "citrat" ]

22,176

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"retroprosthetic", "membrane", "immunohistochemical", "analysis", "e", "f", "expressions", "normal", "cornea", "e", "g", "retroprosthetic", "membrane", "f", "h", "Aqueous", "humor", "levels", "Boston", "Type", "keratoprosthesis", "KPro", "implanted", "rabbits", "without", "RPM", "formation", "Pre", "preoperation", "Day", "postoperative", "day", "RPM", "retroprosthetic", "membrane", "p", "p", "versus", "group", "Dynamic", "expressions", "aqueous", "humor", "rabbit", "cornea", "following", "Boston", "Type", "keratoprosthesis", "KPro", "implantation", "Time", "Preoperation", "Day", "Day", "Day", "Day", "Day", "Group", "III", "corneal", "rabbits", "implanted", "Boston", "Type", "keratoprosthesis", "KPro", "group", "II", "corneal", "rabbits", "penetrating", "keratoplasty", "group", "corneal", "rabbits", "without", "keratoplasty", "p", "versus", "group", "II", "p", "versus", "group", "Academic", "Editor", "Sachin", "Mulik" ]

Introduction ============ The London Psychosocial Group has been funded by Cancer Research UK to implement and evaluate a psychoeducational intervention to promote early presentation amongst older women with breast cancer. The intervention is designed to be radiographer-delivered to women receiving their final invited mammogram with the National Health Service Breast Screening Programme. Objective ========= To report the experiences of research radiographers in delivering the 10-minute intervention to women in breast screening clinics. Methods ======= Reflective diaries and supervision records were used to collate the experiences of radiographers delivering the intervention as part of a research trial and to discuss emerging issues and challenges for widening the role of diagnostic radiographers across the National Health Service. Results ======= The main challenges included learning and rehearsing a structured interview, delivering complex health messages within time constraints using behavioural change techniques, and dealing with unexpected events, including emotional distress, during interviews. Benefits included enhanced interaction with women attending screening and extending the usual role of the radiographer. Conclusion ========== It was beneficial yet demanding for radiographers to extend their role in this way. Not all radiographers may be motivated or suitable to undertake this work. Training and ongoing supervision are essential to support radiographers who deliver the intervention.

{ "pile_set_name": "PubMed Central" }

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22,177

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INTRODUCTION {#sec1-1} ============ The artificial urinary sphincter (AUS) has been considered the standard of care for men with severe stress urinary incontinence (SUI) and/or radiotherapy-related SUI.[@ref1][@ref2] The hydraulically controlled AMS 800™ AUS (Boston Scientific, Minnetonka, MN, USA) remains the most commercially successful and effective sphincteric device since it was developed in 1972.[@ref3] This ingenious urinary device consists of three components: a control pump placed in the scrotum, an inflatable cuff which is usually implanted around the proximal bulbar urethra, and a pressure-regulating balloon in the retropubic space, which serves dual functions, both a pressure regulator and a fluid reservoir. While long-term clinical outcomes, safety profile, and mechanical durability of the current model of the AMS 800 device are well documented, it is not without its limitations and complications such as prosthetic infection and mechanical failure rate.[@ref4] While the clinical use of AMS 800 AUS in a standard male patient with "uncomplicated" SUI history is well established, there are limited high-quality studies pertaining to AUS outcomes in the selected high-risk groups such as neurogenic bladder dysfunction, revision cases, men who require concurrent penile prosthesis implant for erectile dysfunction, and finally, in female SUI. This article explores the clinical outcomes of AUS in these special populations. MATERIALS AND METHODS {#sec1-2} ===================== A critical review of all relevant publications pertaining to AMS 800 AUS was conducted in PubMed and Embase databases, using keywords "urinary incontinence," "AMS 800," "artificial urinary sphincter," "neurogenic urinary incontinence," "revision," "female stress urinary incontinence," and "concurrent penile prosthesis implant." Full surgical descriptions related to AUS implant and/or revision surgery were excluded in this review. The discussion of each category of special populations also includes a brief review of surgical challenge and a practical action-based set of recommendations on surgical options (**[Table 1](#T1){ref-type="table"}**). ###### Special populations and recommended practical action-based strategies ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- *Special populations* *Main issues to considered* *Surgical strategies* *References* -------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ -------------------------------------------------------------------------------------------------------------------------- Neurogenic Need for self-catheterization (higher risk of erosion with bulbar urethral cuff) Cuff placement at bladder neck [@ref5][@ref6][@ref7][@ref8][@ref9][@ref10][@ref11][@ref12][@ref13] Underlying bladder dysfunction Need for greater intraoperative and postoperative care Higher nonmechanical failure and revision rate Lower continence rate Revision (urethral atrophy) Ischemic changes to previous cuff area\ Replace urethral cuff in a new location Downsizing the urethral cuff size [@ref14][@ref15][@ref16][@ref17][@ref18][@ref19][@ref20][@ref21][@ref22][@ref23][@ref24][@ref25] More difficult surgical dissection Higher risk of infection and nonmechanical failure Tandem or second urethral cuff Transcorporal cuff placement Revising pressure-regulating balloon to high pressure Concurrent erectile dysfunction for penile prosthesis implant Synchronous or staged (delayed) AUS and penile prosthesis implant Synchronous surgery [@ref26][@ref27][@ref28][@ref29][@ref30][@ref31][@ref32][@ref33][@ref34][@ref35][@ref36][@ref37] More difficult surgery and cautious corporal dilatation near the urethral cuff Higher complication rates and the difficulty of manipulating the two scrotal pumping devices Higher risk of infection Staged or sequential (delayed) surgery Cost issue Higher surgery attention to avoid damaging existing implant\ Additional surgical cost Female stress urinary incontinence Usually as salvage option in failed slings Preserving integrity of the vesicovaginal surgical plane during cuff placement at bladder neck [@ref38][@ref39][@ref40][@ref41][@ref42][@ref43][@ref44][@ref45][@ref46][@ref47][@ref48][@ref49][@ref50][@ref51][@ref52] More difficult surgical dissection Higher risk of infection and nonmechanical failure Avoid future vaginal delivery (cuff erosion) ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- AUS: artificial urinary sphincter DISCUSSION {#sec1-3} ========== {#sec2-1} ### Neurologic SUI {#sec3-1} Neurogenic bladder dysfunction due to spinal cord injury (SCI) can result in urinary incontinence, renal impairment, urinary tract infection, bladder or renal stones, and poor quality of life. Most patients will require careful bladder management to ensure a low-pressure bladder, complete bladder emptying, and adequate urinary continence with the use of anticholinergic medications and clean intermittent catheterization (CIC) as first-line therapy, prior to more invasive procedures such as neuromodulation or bladder augmentation. In many SCI patients with persistent urinary incontinence, the AUS provides an ideal continence therapy to address underlying intrinsic sphincter insufficiency, without resorting to urinary diversion. In contrast to the traditional placement of AUS cuff in the proximal bulbar urethra for the treatment of male SUI, the cuff is often placed at the bladder neck or periprostatic tissue to ensure a lower rate of cuff erosion from frequent urethral instrumentations. Furthermore, patients with SCI-related urinary incontinence should be counseled regarding higher risks of nonmechanical device failure and revision surgery and that their overall long-term continence rates may be poorer compared to the nonneurogenic group. Chartier Kastler *et al.*[@ref5] published a 10-year retrospective series on AUS in neurogenic male urinary incontinence and showed that 74% of the patients had no or moderate incontinence between CICs that spanned at least 4 h, at a mean 83-month follow-up period. The majority of these men had SCI and 39% of men with detrusor overactivity underwent concomitant bladder augmentation. Similarly, Guillot-Tantay *et al.*[@ref6] reported the 5-, 10-, 15-, and 20-year explantation-free survival rates of 85.7%, 62.3%, 52.0%, and 39.0%, respectively, in SCI men who underwent AMS 800 implantation. The revision-free survival rates were 78.6% (at 5 years), 42.9% (at 10 years), 28.6% (at 15 years), and 7.1% (at 20 years). Approximately 50% of men were continent at recent follow-up visit (mean: 18.3 years). Three native devices were still in place, eight were revised (four of them were secondarily explanted), and three were explanted due to erosion or infection. Comparing the clinical outcomes between AUS cuff placement at bulbar urethra and bladder neck, Khene *et al.*[@ref7] found a trend favoring bladder neck over peribulbar cuff placement with median revision-free device survival at 14.3 and 11.7 years, respectively (*P* = 0.73), while the median explantation-free device survival was 24.5 and 18.5 years, respectively (*P* = 0.08). On multivariate analysis, CIC was the only predictor of AUS device failure. In a study that directly compares the AUS implantation for neurogenic and nonneurogenic incontinence, Murphy *et al.*[@ref8] found higher revision rates in the neurogenic group (11 out of 13 patients) and only three remained completely dry at a mean 6-year follow-up. In contrast, seven of 17 patients in the nonneurogenic group did not require revision, and eleven were completely dry. The nonmechanical failure rate of the AUS was significantly higher in the neurogenic group. Gonzalez *et al.*[@ref9] published a retrospective study on AUS and bladder augmentation surgery and reported no significant difference in clinical success between the timing of AUS placement and bladder augmentation. However, the two patients who had sphincter erosions had an injury of the augmented bladder during surgery or belonged to the simultaneous surgery group, highlighting the importance of adequate bowel preparation and maintenance of sterile urine during the AUS surgery. Using a modified surgical implant of a port instead of a pump, Bersch *et al.*[@ref10] reported that this technique was effective and safe at the reported cure rate at 90% with a 35% revision rate after 8-year follow-up. Robotic-assisted AUS implantation was reported in the early 2010s and Yates *et al.*[@ref11] showed no complications in six SCI patients. However, longer-term safety and cost efficacy of this novel technique will need to be conducted to compare with traditional open surgical methods. In the younger population, Ruiz *et al.*[@ref12] found that patients with bladder exstrophy and many previous bladder procedures are more exposed to complications such as device erosion compared with patients with epispadias or anorectal malformation. In another series, Spiess *et al.*[@ref13] reported that 19 out of 30 (63%) boys were completely dry, 6 (20%) were slightly wet, and 5 (17%) were incontinent, with the mean lifetime of all AUS devices at 4.7 years with no statistically significant difference in device survival between cuff placed at bladder neck or bulbar urethra (4.6 and 4.9 years, respectively). However, survival analysis revealed a sharp drop after 100 months with only 8.3% of the sphincters implanted lasting beyond this point. There were a total of 32 revisions performed in 17 patients constituting a 0.164 revision rate per patient-years. ### Revision group secondary to urethral atrophy {#sec3-2} A common cause of recurrent or delayed SUI following AUS implantation is tissue atrophy resulting in loss of circumferential urethral compression and luminal occlusion. In fact, tissue atrophy is probably the most common cause of nonmechanical failure and the most common cause for AUS revision. Patients who were initially continent with the device usually complain of gradually increasing urinary incontinence over months or even years and report having to squeeze the pump more often to deflate the cuff to void. Withdrawal urethral pressure profile can be conducted with the cuff in inflated and deflated modes to show the difference in luminal pressure while cystourethroscopic evaluation will show ischemic changes to the bulbar urethra and poor mucosal coaptation when the cuff is fully inflated.[@ref14] Surgical options for AUS revision in urethral atrophy include replacement of the urethral cuff in a new location,[@ref15] downsizing the urethral cuff size,[@ref16] placement of a second (tandem) urethral cuff,[@ref17] transcorporal cuff placement,[@ref18] or revising the pressure-regulating balloon to a higher pressure.[@ref19] In a recent publication comparing primary versus AUS revision cases, Suh *et al.*[@ref20] showed that nonmechanical failure (70.7%) was a dominant etiology of reoperation, and at longer-term follow-up, men with primary AUS performed better than those with AUS revision. While the immediate success rates of primary AUS without reoperation (pAUS) and AUS revision without secondary reoperation (rAUS) groups were 88.6% and 79.2% (*P* = 0.352), respectively, at a median follow-up of 45.1 (range: 9--126) months, the social continence rate was higher in patients with pAUS (92.1%) than with rAUS (62.5%) (*P* = 0.001). In an *ex vivo* culture study, Ziegelmann *et al.*[@ref21] reported that the two most common reasons for AUS revision were urethral atrophy (*n* = 31, 39%) and mechanical failure (*n* = 49, 52%). Excluding patients undergoing revision for infection or erosion, positive culture swabs were identified in 37/200 components (19%), including 25/86 cuffs (29%), 7/56 pumps (13%), and 5/58 reservoirs (9%), with the majority being skin organisms such as *Staphylococcus* species (57%), *Propionibacterium* (10%), and *Aerococcus* (5%). More than a third (39%) of patients had at least one positive component culture, and those patients were more likely to have a history of radiation (65% *vs* 33%, *P* = 0.006). Positive AUS component bacterial swab cultures were found in 39% of patients undergoing AUS revision in the absence of clinical infection. Those patients with positive cultures were more likely to have a history of pelvic radiation. These results suggest that bacterial colonization of organisms with low virulence may not lead to device infection. In another large series, Raj *et al.*[@ref22] showed nonmechanical failure accounted for the majority of AUS revision compared to mechanical failure. Of the 119 patients undergoing secondary implantation, 91 (76.5%) needed no additional surgical intervention, while 28 (23.5%) required a total of 40 surgical revisions for new mechanical (15 \[37.5%\]) and nonmechanical (25 \[62.5%\]) problems. Five-year durability outcomes for primary and secondary AUS implantation were comparable at 80% and 88%, respectively. This study confirmed that outcomes for secondary AUS reimplantation remained comparable to those of primary AUS implantation and that salvage surgery can still produce a good clinical outcome, even following multiple prior revisions and cuff erosion. Linder *et al.*[@ref23] compared the clinical outcomes between cuff downsize versus tandem cuff placement in AUS revision and concluded that there was no significant difference in the overall device survival in patients undergoing single cuff downsizing or tandem cuff placement during AUS revision for urethral atrophy. Of the 69 revision surgeries for urethral atrophy, 56 (82%) were tandem cuff placements, 12 (18%) were single cuff downsizings, and one was the relocation of a single cuff. Furthermore, there was no difference in 3-year overall device survival compared between the single cuff and tandem cuff revisions (60% *vs* 76%, *P* = 0.94). In another retrospective study, O'Connor *et al.*[@ref24] reported a significantly higher rate of complete continence and improvement in the urinary continence score seen in men with double-cuff compared with single-cuff devices. Daily pad use decreased from 7.7 to 1.1 in patients treated with a single-cuff AUS and from 7.8 to 0.7 in patients with a double-cuff AUS (*P* = 0.25) and complete continence was reported in 3 (11%) of 28 men with single-cuff and 12 (43%) of 28 men with double-cuff sphincters (*P* = 0.008). Five complications were reported in the single-cuff recipients and four in the double-cuff patients. In a multicenter study, Eswara *et al.*[@ref25] reported the outcomes after various revision of AUS strategies and found that tandem cuff placement was associated with a lower rate of incontinence failure (*P* = 0.02), whereas cuff repositioning was associated with a higher rate of incontinence failure (*P* = 0.02). An increased rate of mechanical failure was observed with cuff downsizing (*P* = 0.01). ### Coexisting erectile dysfunction for concurrent penile prosthesis implant surgery {#sec3-3} In patients who have both SUI and erectile dysfunction (ED), it may be necessary to counsel these men regarding synchronous or sequential (delayed) AUS and penile prosthesis implantation. Candidates for dual implants are usually patients who have failed conservative management for both conditions and wish to undergo a single surgery. The theoretical concerns of synchronous prostheses implant are potential higher complication rates and the difficulty of manipulating the two scrotal pumping devices in some patients especially in the early stage. On the other hand, a 2-stage procedure requires more attentive surgery in order to avoid damaging the components of the existing implant and possibly operating in less well-defined tissue planes. In either situation, patients need to be counseled on the pros and cons of dual versus staged implants and technical considerations for the device placement will vary depending on the sequence of device implant and the surgeon preferences. Most surgeons usually advocate the dissection and mobilization of the proximal bulbar first as inadvertent urethral injury would require abandoning or modifying the procedure without discarding any of the prostheses. Corporal dilatation must proceed cautiously in the proximal part of corpora near the cuff. At the level of the bulbar urethra, the corporal bodies have already diverged and therefore the likelihood of injuring the cuff or urethra is lower. Single-step dilatation may be a better option than sequential dilatation to avoid injuring the urethra. The publication of trans-scrotal insertion of AUS implant[@ref26] has led to synchronous dual implants performed through a single incision. If the implants are not placed synchronously, the AUS is usually placed first followed by penile prosthesis for the reasons cited above. In a sequential device implant, the greatest concern is accidental damage to the preexisting device and its tubing. Great care needs to be taken to avoid damaging the AUS cuff when subsequently placing the penile cylinders. Some authors proposed that malleable penile prosthesis is utilized in a sequential implant because of lesser components required and that the implant could be performed more distally on the corporal bodies and hence avoid encountering the AUS device. While it is expected that all implants should have ipsilateral tubing and reservoir placement, surgeons should exercise precaution such as reviewing previous surgical records or organizing preoperative imaging study to avoid intraoperative surprises and the placement of the second implant can be planned for the pump and reservoir at the contralateral side. The incision should be performed away from the existing device, with meticulous dissection and the use of cutting currents to avoid damage to any components of the first device. Prosthetic infection is the most dreaded complication and generally occurs due to the inadvertent introduction of skin organisms at the time of surgery and early postoperative bacteremia. Shortening the operative time would seem a logical way of decreasing this incidence, but published literature on synchronous and sequential dual implants has not revealed any higher incidence of prosthetic infection, probably due to the low incidence of prosthesis infection. In the event of an infection, the concern is that it will spread to all components necessitating their removal and making revision surgery more technically challenging. In order to salvage an infected prosthesis, one needs to determine which prosthetic device is infected and whether it is possible to leave the components of the unaffected device intact. Bhalchandra *et al.*[@ref27] argued that in this situation, provided that the infected device is identified early, and if one acts promptly to locate the components affected, it is possible to salvage the components of the unaffected device. While device erosion without infection may make salvaging one of the devices possible, in the context of device infection, it is often difficult to ascertain which device is affected, confidently exclude the possibility of only one device being infected and therefore it would be safer to remove both devices. However, Wilson *et al.*[@ref26] showed that it is possible to preserve one device when the other infected device was removed. The feasibility of salvage surgery was also conferred by Bryan *et al.*[@ref28] where three patients who had simultaneous dual implant had their device successfully salvaged and one patient underwent two dual salvage procedures. In a multi-institutional study on the dual implant, Kendirci *et al.*[@ref29] reported no prosthetic infection postoperatively among the 22 men over a mean follow-up of 17 months following a single upper transverse scrotal approach. The overall revision rate was 14%, mostly due to complications associated with the AUS device such as cuff erosion. In a cost analysis study, Sellers *et al.*[@ref30] showed that dual prosthetic implantation in a single-stage procedure significantly reduced the operative time (24.7%, *P* \< 0.05) and was associated with approximately a \$7000 cost savings compared with individual procedures, with no report of infective complications or device erosion over the 16 months of follow up. Rolle *et al.*[@ref31] showed no difference in the pain score, postoperative hospital stays (2.5 days after the double-implant procedure and 2.4 days after AUS alone), and continence rate (65% *vs* 68%). Furthermore, when given options, all patients stated that they would have preferred synchronous surgery. Mancini *et al.*[@ref32] also reported encouraging outcomes in patient satisfaction, ease of use, and functionality in the dual implantation group that are similar to those observed in the individual prosthesis group. In fact, most patients (94%) stated that they would recommend the dual implantation procedure to others and have the procedure done again. In their series of 55 combined procedures compared to the single insertion of 336 inflatable penile prostheses and 279 artificial urinary sphincters over a 12-year period, Segal *et al.*[@ref33] reported that men treated with combined implantation had a greater mean age and were at greater risk for prostate cancer diagnosis and treatment, and at lesser risk for Peyronie\'s disease than men who received an inflatable penile prosthesis alone (each *P* \< 0.05). Although the operative time was significantly longer for the combined procedure than for the inflatable penile prosthesis alone and the AUS alone (mean: 218.1 *vs* 145.9 and 114.7 min, respectively, *P* \< 0.0001), the rate of device infection, erosion, or malfunction was not increased in combined or staged procedures (*P* \> 0.05). Based on the SPARCS (New York State Department of Health Statewide Planning and Research Cooperative) database for men who underwent inflatable penile prosthesis and/or artificial urinary sphincter insertion between 2000 and 2014, Patel *et al.*[@ref34] found that combined inflatable penile prosthesis and artificial urinary sphincter insertion portends a higher likelihood of inflatable penile prosthesis reoperation at 1 year (OR: 2.08, 95% CI: 1.32--3.27, *P* \< 0.01) and 3 years (OR: 2.60, 95% CI: 1.69--3.99, *P* \< 0.01), while the artificial urinary sphincter outcomes remain comparable. The literature on patients who underwent dual implants after radiotherapy is limited. Kendirci *et al.*[@ref29] reported that the only erosions following synchronous insertion of AUS and PP occurred in the irradiated patients postradical prostatectomy. The higher AUS revision rates in patients who received previous radiation were also confirmed by Martins and Boyd.[@ref35] The poorer surgical outcomes in irradiated population are likely attributed to several factors such as poorer quality of urethral tissue, difficult surgical dissection with delayed recognition of urethral injury as well as improper cuff sizing, and higher rates of cuff atrophy. In a more complex patient cohort such as those with neobladders, Loh-Doyle *et al.*[@ref36] concluded that an AUS and PPI can be performed without an increased risk of device-related complications when compared to a control group of nonneobladder patients. Furthermore, among the five prosthetic infection cases, the infection was confined to a single device and salvage surgery was feasible. Dual implantation of AUS and penile prosthesis is feasible, safe, efficient, and cost-effective for the surgical treatment of PPI and ED. The decision to perform synchronous or delayed surgery for concurrent urinary and penile prosthetic implants is dependent on whether concurrent surgery increases the complication rate and is cost-effective in the longer term. Pertinent points to discuss with prospective patients interested in dual implants include counseling on the complexity of the surgery and potential complications and having enough manual dexterity to recycle either pump as required.[@ref37] In a carefully selected group of patients, there is no doubt that most patients are satisfied with the outcomes and highly appreciative of the improvements in their quality of life. From the surgeon point of view, the dual implant of urinary and penile devices is technically challenging, and the surgeon should be competent at placing either device independently before contemplating dual implantation. Dual implantation of urinary and penile prostheses is feasible and safe provided that strict adherence to prosthetic surgical protocols are adhered to and has been shown to be cost-effective and highly efficacious to treat men with PPI and ED. ### Female stress urinary incontinence {#sec3-4} Synthetic sling surgery has gained widespread acceptance as the standard of care in female SUI, but recent negative press related to synthetic mesh has placed considerable concerns among surgeons and patients. In women with multiple continence surgeries, the placement of AUS potentially offers a better continence rate through circumferential compression and coaptation of the "scarred" urethra and remains a safe and effective salvage option in a carefully selected patient cohort. The limited number of publications on the outcomes of urinary continence following placement of AUS in women is likely related to the complexity of AUS surgery and lack of awareness of AUS as an effective treatment option for female AUS. Among all surgical procedures for female SUI, AUS implantation is thought to have the highest long-term (\>5 years) success rate in a recent web-based survey.[@ref38] However, the responding international urologists and gynecologists also think that AUS implantation has the highest risk of complications and/or revisions. Unfortunately, at present, there is no randomized head-to-head trial between minimally invasive synthetic sling surgery and AUS. The earliest publication on AUS implantation for women with SUI was by Scott,[@ref39] who reported more than 90% of patients with socially acceptable continence and 66% of patients completely dry among the 139 female patients with variable ages and mixed etiologies of urinary incontinence when followed up to 6 years. Over the last three decades, published literature shows that AUS surgery is safe and clinically effective in properly selected women with genuine SUI.[@ref40][@ref41][@ref42][@ref43][@ref44][@ref45][@ref46] In fact, the comparison of the clinical outcomes between AUS in men and women were not too dissimilar in terms of continence rate and patient satisfaction.[@ref45] Similar continence rates were also reported in women who received laparoscopic AUS implantation.[@ref47][@ref48] More recently, robotic-assisted AUS implantation has been shown to be equally effective and safe.[@ref49][@ref50] In a pilot study comparing robotic-assisted and open approaches, Peyronnet *et al.*[@ref51] reported that the change in surgical trend from an open approach (2008--2012) to a robot-assisted approach (2013--2014) was associated with a lower intraoperative complication rate (37.5% *vs* 62.5%; *P* = 0.25), decreased blood loss (17 ml *vs* 275 ml; *P* = 0.22), and shorter length of stay (3.5 *vs* 9.3 days; *P* = 0.09) in the robot-assisted group with comparable continence rates in both groups (75% *vs* 68.8%; *P* = 0.75). Most published literature pertaining to AUS implantation in female patients has a shorter-term follow-up of \<5 years. A longer-term follow-up study by Chung and Cartmill[@ref46] showed that more than 80% of AUS devices remained functioning after 100 months and currently has the longest mean follow-up period of 13.5 years. It is difficult to deduce from most published literature with regard to the role of AUS as a salvage surgical option due to the indiscriminate reporting of the outcomes for patients with or without previous anti-incontinence surgeries; incomplete data collection; and the loose definition of continence rates whether it is pad free or with minimal usage of pads. Most published studies on AUS span over the different generations of AUS and advances in technology such as durable kink-proof tubing, lockout valve, as well as the pressure-regulating balloon may have increased the durability of the AUS device.[@ref52] In recent years, there has been a shift in the treatment paradigm for female stress urinary incontinence toward minimally invasive surgery. Currently, the placement of AUS in female patients is only performed in few major teaching centers. Despite the few existing indications for AUS in women, AUS can potentially be a suitable treatment option for females with SUI secondary to intrinsic sphincter deficiency. Perioperative injury remains the most significant risk factor for AUS explantation. Most studies stressed the importance of preserving the integrity of the vesicovaginal surgical plane as the key to successful implantation. In addition, women of child-bearing age should be warned of the danger of cuff erosion during vaginal delivery with advice ranging from no future pregnancy, elective caesarean delivery, and deactivation of the AUS in the final trimester, as recommended to minimize the risk of cuff erosion. Deactivation during labor and delivery is imperative. With proper selection and strict adherence to surgical techniques, AUS is safe, highly effective, and demonstrated excellent long-term outcomes. Perhaps the role of AUS in distressed and socially restricted women with persistent SUI or recurrent urinary incontinence following anti-incontinence surgeries is underutilized and should be given due consideration. CONCLUSION {#sec1-4} ========== Over the past four decades, advances in mechanical design, applications of new technology, and lessons learned from the past clinical experiences have made AMS 800 AUS the standard of care in men with SUI. While the current AMS 800 device provides an effective, safe, and durable therapeutic option, it is not without its limitations and complications, especially with regard to its utility in some of high-risk populations. Increased understanding of the pathophysiology of various SUI cases, coupled with effective strategies, further improves AUS clinical outcomes. However, the emergence of novel therapies such as a nanotechnology-driven device and stem cell therapy may one day circumvent traditional AUS surgery. COMPETING INTERESTS {#sec1-5} =================== The author declared no competing interests.

{ "pile_set_name": "PubMed Central" }

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22,178

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1. Introduction {#sec1} =============== First synthesized in 1891 and with current production estimated at 4 billion kilograms each year globally \[[@B1]\], bisphenol A (BPA) is a multipurpose compound that is widely used in the modern industrial world. BPA was initially investigated for its potentially therapeutic estrogenic properties in the 1930s; when diethylstilbestrol (DES) was found to be more potent, however, BPA was temporarily cast aside. Its commercial value was reassessed in the 1950s with the introduction of BPA as a fundamental component in the manufacturing of some plastics. As its primary use currently, BPA is a key monomer in the production of the most common form of clear and shatter-proof polycarbonate plastic, but it has also been incorporated into a variety of everyday goods. Questions regarding the safety and side effects of BPA began to emerge in the late 1990s when BPA was found to leech out of plastics and into experimental animal subjects, resulting in an increased incidence of chromosomal anomalies in offspring \[[@B2]\]. There has since been ongoing discussion in both scientific and political spheres about the potential for harm resulting from human BPA exposure and potential bioaccumulation. An overview of the literature regarding the effects of BPA on human health is provided, followed by a presentation of data from 20 subjects whose blood, urine, and sweat were tested for BPA. Results and discussion regarding BPA bioaccumulation and elimination are presented for consideration. 2. Background {#sec2} ============= Currently, BPA is most commonly found as a component in polycarbonates (\~74% of total BPA produced) and in the production of epoxy resins (\~20%). As well as being found in a myriad of products including plastic food and beverage containers (including baby and water bottles), BPA is also commonly found in various household appliances, electronics, sports safety equipment, adhesives, cash register receipts, medical devices, eyeglass lenses, water supply pipes, and many other products. It is also frequently used as an adjunct in the production of brominated flame retardants and brake fluid \[[@B1]\]. Moreover, BPA derivatives, such as bisphenol A-glycidyl methacrylate and bisphenol A-dimethacrylate, have recently been incorporated into the dental industry and used in dental fillings and sealants. The widespread use of this compound is receiving increasing scrutiny as concerns about BPA effects on human health have recently emerged. The main mechanism by which the population is exposed to BPA is through leaching from plastic products. This results from either the release of unpolymerized monomers or the slow decay of polymer bonds in polycarbonates leading to monomer release into proximal foods and liquids. Occupational exposures are also present where plastics are burned and manufactured, and thus BPA may be inhaled by workers \[[@B3], [@B4]\]. An analysis of Chinese employees in factories where BPA and epoxy resins are produced, for example, revealed that over 90% of exposed workers have notable levels of BPA in their serum and urine \[[@B5]\]. A plethora of recent studies affirms that the majority of the population (91--99%) does indeed have detectable levels of BPA, but the level and the toxicological relevance of current exposure levels is a subject of intense academic and public health debate \[[@B6]--[@B13]\]. An extensive review conducted in 2007 concluded that BPA levels in human blood and/or urine are within the range shown to be dangerous in animals and are therefore likely to be biologically active in humans \[[@B6]\]. (As will be discussed, however, blood and urine testing may underestimate the full extent of exposure and bioaccumulation.) Conversely, an industry-sponsored literature review from 2008 declared that daily human consumption was far below dangerous levels and is therefore of minimal concern \[[@B7]\]. Sources of BPA ingestion may vary. In infants and children, baby and beverage bottles used by most individuals in the pediatric population provide ongoing daily sources of BPA \[[@B14]--[@B22]\]. Le et al. found that at room temperature, leaching of BPA occurred into the contained fluid, which increased 55-fold if boiling water was added \[[@B14]\]. Another study found that exposure levels increased not only with temperature, but also with repeated use of a container \[[@B15]\]. Other common sources of ingestion include foods stored in food cans, which are lined with BPA epoxy resin films to prevent corrosion \[[@B23]--[@B28]\], thermal printing paper commonly used in cash register receipts \[[@B29]--[@B31]\], and BPA containing dental composites and sealants \[[@B32]--[@B34]\]. Medical equipment is also raising concerns about BPA levels as a study of newborns found that those who regularly spent time in a neonatal intensive care unit had significantly higher serum BPA levels than the general population---thought to be due to exposure to plastics in medical devices \[[@B35]\]. Likewise, dialysis patients appear to have higher rates of exposure, which may be attributable to circulating solvents which expedite the leaching of BPA from polycarbonate hemodialysis equipment \[[@B36], [@B37]\]. When ingested, unconjugated BPA---the biologically active form of BPA---has historically been thought to be rapidly conjugated in the liver and then excreted through bile or urine, with a half life of approximately 5.3 hours \[[@B38]--[@B40]\]. This rapid excretion has been the basis of reassuring safety evaluations and declarations given by some public health authorities worldwide. However, within many tissues, particularly the lungs, livers and kidneys in rats, and the placenta of animals and humans, *β*-glucuronidase enzyme is present at detectable concentration. This enzyme is able to deconjugate BPA and thus release its active form again \[[@B41]\]. This is of great significance, as it is plausible that, in pregnancy, the conjugated form of BPA will circulate through the placenta, undergo deconjugation, and cause subsequent fetal exposure in utero. This may also result in bioaccumulation of some portion of BPA after exposure. In fact, recent evidence suggests that at low concentrations, while most plasma BPA (about 95%) is bound to serum proteins, BPA has lipophilic affinity with a fat: blood coefficient of 3.3 \[[@B42]\]. Furthermore, BPA appears to have a disproportionate affinity to fat in comparison to other tissues such as kidney, muscle, and other sites; "in fat, the accumulation of BPA was about three times higher than in other tissues" \[[@B42]\]. With evidence of potential bioaccumulation, BPA has the prospect of exerting ongoing metabolic effects. 2.1. Potential Implications of BPA Exposure {#sec2.1} ------------------------------------------- BPA is thought to wield its effects through endocrine disruption, epigenetic modification, cytokine release, and oxidative stress. When first discovered, BPA was investigated for its estrogenic properties, as it is thought to alter the synthesis of estradiol and testosterone and interfere with receptor binding \[[@B43], [@B44]\]. Consequently, exposure to BPA has been linked with a number of developmental and reproductive pathologies in both animal models and human subjects. These include abnormalities in reproductive organ function (irregular cycles, multiple ovarian cysts, reduction in primordial follicles \[[@B45]--[@B49]\]), placental dysfunction \[[@B50]\], increased incidence of miscarriage and neonatal mortality \[[@B50], [@B51]\], precocious puberty \[[@B52]\], and sexual dysfunction such as erectile dysfunction, decreased libido, and ejaculation difficulties \[[@B53]--[@B55]\]. Moreover, interference with the production and signaling of sex hormones has led to neurological impairment \[[@B56]--[@B60]\]. Synapse formation during development is regulated by estrogen and androgens; however with exposure to BPA, a recent study found that levels deemed safe by the US Environment Protection Agency, completely abolish the response of synapses to estrogen in the prefrontal cortex and hippocampus \[[@B61]\]. Epigenetic effects of BPA have been associated with an increased risk of cancer, particularly breast and prostate malignancies \[[@B62]--[@B69]\]. The exposure of breast epithelial cells to BPA was found to alter gene expression of 170 genes and increase their vulnerability to other carcinogens \[[@B62], [@B63]\]. Additionally, there was silencing of lysosomal-associated membrane protein 3, as occurs in ER*α*-positive breast cancer \[[@B62]\]. Similar effects have been seen with respect to prostatic disease, as exposure to BPA has been repeatedly shown to modify methylation of implicated genes \[[@B64]--[@B66]\]. Low dose BPA exposure at weaning during the perinatal period has also been found to increase adipogenesis in female animals \[[@B70]\]. As it is hypothesized that adult body weight may be programmed during early life, these results are noteworthy with regards to the childhood obesity pandemic and the action of endocrine disruptors as determinants of obesity \[[@B70]\]. BPA exposure appears to have widespread impact as it has also been linked by various researchers and studies throughout the world with a whole host of other health problems, including metabolic syndrome, obesity, non-insulin-dependent diabetes mellitus, allergies and asthma, ADHD, autism, cognitive decline, memory impairment, depression, and anxiety \[[@B71]--[@B92]\]. With the rise of sensitivity-related illness, there is also concern that BPA may be a determinant of this recently recognized causative mechanism of disease and source etiology of assorted clinical conditions \[[@B93]\] by stimulating the release of proinflammatory adipokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) from human adipose tissue \[[@B71]\]. As a result of all the emerging attention in the scientific literature, various governments have also embarked on research and policy decisions relating to BPA. In 2010, for example, the Minister of Health for the Canadian Government declared the results of a four-year study indicating, "The Government of Canada is a world leader in chemicals management. Our science indicated that Bisphenol A may be harmful to both human health and the environment" \[[@B94]\]. With emerging information of concern about BPA, the Canadian government became the first to prohibit the sale of BPA-containing polycarbonate baby bottles. France, Denmark and several American states have since implemented similar regulations. 2.2. Limitations of Toxicant Biomonitoring {#sec2.2} ------------------------------------------ It is often assumed that after exposure, BPA is rapidly metabolized to a hormonally inactive metabolite and efficiently excreted in toto from the body \[[@B95]--[@B97]\]. As a result, there has been little concern about bioaccumulation. The question arises, however, that if the compound is rapidly excreted without any accrual, why is there increasing evidence that BPA exposure is anything but innocuous and is able to cause potentially serious problems in animal and human organisms? While some believe that only unremitting ongoing exposure to BPA generates risk, concern has been raised that unrecognized bioaccumulation of some fraction of BPA may occur in some exposed individuals. How does one monitor to determine if toxicant compounds bioaccumulate and thus remain within the human organism? Throughout the world, blood and urine sampling are the general modalities used to biomonitor levels of most toxicant compounds including toxic elements, synthetic compounds, petrochemical compounds, biologic toxicants such as mycotoxins, and xenobiotics sometimes produced as byproducts from processing of parent compounds \[[@B98]\]. There is increasing evidence, however, that relying on blood and urine measurements as indicators of bioaccumulation can be very flawed \[[@B99]\]. Many compounds sequester in tissues and do not remain in blood; testing of whole blood or serum may miss toxicants which have exited the blood compartment and are being stored primarily in tissues such as bone, muscle, or adipose compartments. Levels of toxicant compounds can also rapidly fluctuate with changes in immediate status such as caloric restriction, level of hydration, underlying nutrient status, thermal changes, or exercise \[[@B98], [@B100]\]. Reliance on blood or urine testing for assessment of the body burden of many toxicants may thus be less than reliable clinically or for public health purposes. As a result, attempts to biomonitor toxicant levels by sampling other tissues and bodily excretions have been explored, including hair sampling, salivary testing, stool sampling, perspiration testing, breath analysis, provocation testing, and biopsies of adipose tissue through needle aspiration into fat pads under the skin. It is evident, however, that there are limitations with each of these approaches. Hair samples, for example may only reflect what has been in the blood stream for the last few weeks, while stool samples only assess what is being eliminated though fecal waste---these do not measure the body burden. As detailed toxicokinetics for many xenobiotic compounds are not fully understood, it is difficult to know which proportion of parent compounds and their metabolites accrue within various bodily compartments. Some researchers have recently commenced doing fat biopsies as a tool to biomonitor toxicant levels---this technique involves taking a sample of fat, usually from the abdominal or gluteal area and sending the sample for analysis. Recent evidence, however, confirms that toxicants sequester differently even within specific compartments such as adipose tissue; one adipose tissue site may display toxicant concentrations that are totally different than concentrations at another site \[[@B101]\]. So the toxicant concentration in brain adipose tissue, for example, may be very different than that found in breast or abdominal wall adipose tissue. In review, attempts to biomonitor the levels of toxicant compounds, including BPA, in humans using a single modality such as blood or urine are inadequate at best. This is a challenging realization as most population studies on toxicant compounds reported in the scientific literature as well as most ongoing biomonitoring research is based on blood or urine testing. 2.3. Study Objective {#sec2.3} -------------------- BPA exposure is generally assessed by measuring urine levels of this compound. In this research, we endeavor to determine the relative concentrations of BPA in blood, urine, and sweat. By assessing BPA levels in these three compartments, the possibility of identifying retained BPA will be explored as well as the potential for induced perspiration as a means to eradicate this compound. 3. Methods {#sec3} ========== 3.1. Participant Recruitment {#sec3.1} ---------------------------- 9 males and 11 females with mean ages 44.5 ± 14.4 years and 45.6 ± 10.3 years, respectively, were recruited to participate in the study after appropriate ethical approval from the Health Research Ethics Board of the University of Alberta. 10 participants were patients with various clinical conditions and 10 were otherwise healthy adults. Participants with health issues were recruited from the first author\'s clinical practice by invitation. Each participant in the study provided informed consent and volunteered to give one 200 mL random sample of blood, one sample of first morning urine, and one 100 mL sample of sweat. Demographic and clinical characteristics of all research participants are provided in [Table 1](#tab1){ref-type="table"}. 3.2. Samples Collection {#sec3.2} ----------------------- All blood samples were collected at one Dynalife laboratory site in Edmonton, Alberta, Canada through vacutainer blood collection equipment (BD Vacutainer, Franklin Lakes, NJ, 07417, USA) using 21 gauge stainless steel needles which were screwed into the "BD Vacutainer One-Use Holder" (REF 364815). The 10 mL glass vacutainer was directly inserted into the holder and into the back end of the needle. This process and the use of glass were used to preclude contamination. Blood was collected directly into plain 10 mL glass vacutainer tubes, allowed to clot, and spun down 30 minutes later. After serum was separated off, samples were picked up by ALS Laboratories (about 3 kilometres from the blood collection site) for storage pending analysis. When received at ALS, serum samples were transferred to 4-mL glass vials and stored in a freezer at −20°C, pending transfer to the analytical laboratory. We chose to analyze BPA in serum rather than in whole blood, based on the fact that matrix effect of serum is much lower than whole blood. For urine collection, participants were instructed to collect a first morning urine sample directly into a provided 500 mL glass jar container with Teflon-lined lid on the same day that blood samples were collected. Urine samples were delivered by the participants directly to Edmonton ALS Laboratories. Samples were transferred to 4-mL glass vials and stored in a freezer at −20°C, pending transfer. For sweat collection, participants were instructed to collect perspiration from any site on their body directly into the provided 500 mL glass jar container with Teflon-lined lid---by placing the jar against their prewashed skin when actively sweating or by using a stainless steel spatula against their skin to transfer perspiration directly into the glass jar. (Stainless steel---made up primarily of iron, chromium, and nickel---was chosen as it is the same material as the needles used in standard blood collections and is reported not to off-gas or leach at room or body temperature.) Excess of 100 mL of sweat was provided in all but one case. Each of the glass bottles used for sampling in this study was provided by ALS laboratories and had undergone extensive cleaning and rinsing. The containers were deemed appropriate for sweat collection with negligible risk of contamination: laboratory-grade phosphate-free detergent wash; acid rinse; multiple hot and cold deionized water rinses; oven-dried; capping and packing in quality controlled conditions. Sweat was collected within 1 week before or after doing the blood work. No specifications were given as to how long sweating had commenced before collection. 10 participants collected sweat inside an infrared sauna; 7 collected inside a regular steam sauna, and 3 collected during and immediately after exercise---no specific instruction was given regarding the type or location of exercise. Sweat was delivered by the participants directly to ALS laboratories. Samples were transferred to 4 mL glass vials and stored in a freezer at −20°C, pending analysis. No preservatives were used in the jars provided for sweat and urine collection, nor in the serum storage vials. 3.3. Analytical Methods {#sec3.3} ----------------------- Human serum was analyzed for levels of bisphenol A (BPA) at ALS Canada following the general procedures presented by the Centres for Disease Control and Prevention \[[@B8], [@B102]\]. Briefly, samples were fortified with 12.5 nanograms of isotopically labelled phthalate metabolites, 50 nanograms of labeled bisphenol-A, 250 nanograms of 4-methylumbelliferone glucuronide, 300 microliters of ammonium acetate buffer (pH 6.5), and 10 microliters of *β*-glucuronidase (Escherichia coli K12, Roche Biomedical). The samples were mixed and incubated at 37°C overnight to allow for the deglucuronidation. Following enzymatic hydrolysis, a 20 uL aliquot of the sample is added to 70 uL of HPLC-grade water and 10 ng of labelled 4-methylumbelliferone to determine deglucuronidation efficiency (done once every 100 samples). The remaining sample is loaded onto a Zymark Rapid Trace Station for automated solid phase extraction (SPE). The 60 milligram/3 mL Oasis-HLB cartridges were conditioned with HPLC-grade methanol (2 mL) and 0.1 M formic acid (2 mL). The samples were diluted with 5 mL of 0.1 M formic acid and loaded onto the SPE cartridge at a rate of 1.0 mL/min. The cartridge was washed with water (1 mL) and 10% methanol in water (2 mL) at a flow rate of 1 mL/min. The samples were eluted with 1.0 mL of acetonitrile at a flow rate of 0.5 mL/min. The eluate was evaporated to dryness under a stream of dry nitrogen and the residue was resuspended in 85% methanol in water (200 microliters) and transferred to glass autosampler vials. Quality control of the analysis was maintained by analysing a method blank (calf serum) and two spiked calf serum samples (20 ng/mL, all analytes) along with every 17 samples. The detection limit (0.2 ng/mL) was based upon our lower calibration standard (0.5 ng/mL) which gave an instrument signal to noise response of 3 : 1. Analysis was performed using an API 4000 liquid chromatograph/tandem mass spectrometer. 4. Results and Discussion {#sec4} ========================= Demographic characteristics as well as results for each individual participant are summarized in [Table 1](#tab1){ref-type="table"}. All concentrations are in nanograms per milliliter. The fact that some subjects showed undetectable levels confirms that generalized contamination of these samples is not likely. Furthermore, the levels of BPA were similar to those recently published in studies from Italy and Greece \[[@B103]\] and are comparable with the serum levels found (0.79--7.12 ng/mL) in a recent study by Cobellis et al. in 2009 \[[@B104]\]. The rather low percentage detection among the serum samples in North America is hard to compare as (to our knowledge) there is only one study in the literature that documents BPA levels in blood of North Americans \[[@B105]\]. In that study, using the same extraction method as the methodology used in this study, the authors reported BPA levels in the range of \<0.5 (detection limit) to 22.3 ng/mL in the blood plasma of 40 pregnant American women in the state of Michigan. However, the authors did not report in how many of these 40 women that BPA was detected above their current limit of detection of 0.5 ng/mL. In general, there is major variability in the range of concentrations of BPA detected in blood, and this may be explained by the fact that detection methods vary widely and the specific populations studied also vary considerably \[[@B106]\]. For the urine samples, the percentage detection in the current study (70%) is lower than that of the large scale Canadian biomonitoring study (90.7%) also known as the Canadian Health Measures Survey as reported by Bushnik et al. \[[@B10]\]. However, the geometric mean level of urine BPA in this study is generally higher than those in other biomonitoring studies in North America. Comparative levels for urine BPA as found in the literature are presented in [Table 2](#tab2){ref-type="table"}. For example, Bushnik et al. reported a geometric mean of 1.15 ng/mL urine BPA in Canada for a sample size of 5462 \[[@B10]\], Calafat et al. found a value of 2.4 ng/mL for urine BPA among 950 American adults aged between 20 and 59 \[[@B9]\] and more recently Mendolia et al. reported on 375 American males with a geometric mean value of 1.50 ng/mL of urine BPA \[[@B107]\]. As far as sweat data is concerned, comparison across studies is impossible as to our knowledge this is the first study which attempts to quantify BPA in sweat. One obvious qualitative interpretation of the data from the cohort of 20 study participants is that BPA is rarely detected in blood, and this is probably why most large scale biomonitoring studies, such as the NHANES (National Health and Nutrition Examination Survey) and CHMS (Canadian Health Measures Survey), use urine as the human sample of choice to determine exposure levels in populations. In an attempt to summarize the findings on the distribution of BPA in the 3 different body fluids, we give three 2 × 2 tables in [Figure 1](#fig1){ref-type="fig"}. As discussed earlier, the 2 discordant pairs urine+/serum− and sweat+/serum−, with 12 and 14 in their respective grids show clearly that serum is not the appropriate body fluid to test if BPA biomonitoring in humans is to be characterised. Although there seems to be high correlation between urine and sweat in terms of the presence/absence of BPA in these media, with 12 individuals in the urine+/sweat+ concordant pair, what is more surprising is that there are 4 individuals for which BPA was detected in sweat but undetectable in urine ([Figure 1](#fig1){ref-type="fig"}C). In an attempt to compare the excretion efficiencies of urine and sweat for BPA, we calculated the ratio of BPA concentration in sweat versus urine for those 12 individuals who are in the urine+/sweat+ concordant pair. As shown in [Table 1](#tab1){ref-type="table"}, with the exception of 2 individuals (participants 4 and 11) where urine concentration of BPA is slightly higher than in sweat, in general the ratio is higher than 1, suggesting that induced sweating may be an efficient method for eliminating BPA from the body. This is not surprising in the light of the findings of Csanády et al. whereby they found a preferential partitioning of BPA in adipose tissue compared to blood with a ratio of 3.3 \[[@B42]\]. This suggests that the BPA in blood which is then conjugated and excreted in the urine may only represent about one-third of the body burden of BPA. It is not surprising therefore that induced sweating in saunas can mobilise BPA in adipose tissue thus leading to enhanced excretion in sweat. Given that BPA in body fat is mostly unconjugated, further studies looking at the ratio of free BPA to conjugated BPA in sweat will help to confirm whether the BPA excreted in sweat comes from adipose tissue. Given that in normal circumstances the daily volume of urine is much higher than sweat, urine remains an important mode of elimination of BPA from the human body. An important consideration in response to the results is why there are two participants with evidence of BPA in their urine with no positive level in their sweat. Presumably, the sweat level may be reflective of accrued toxicants in tissue, whereas urine may reflect in part at least recent exposure which the body is endeavoring to eliminate. It may be that BPA begins to bioaccumulate only after a threshold of exposure is reached or once the detoxification mechanisms of the organism are unable to completely eliminate the BPA load that presents after exposure. Further research is required in order to clarify, but this result may indicate that the two individuals have had some recent exposure, but no significant level of stockpiled toxicant. Similarly, the marked variation in the range of the sweat/urine ratio may once again be reflective of different phenomenon: the sweat results may represent transcutaneous excretion of accured BPA toxicant, while the urine results are perhaps reflecting recent BPA exposure in addition to some release of accrued BPA that the body is able to eliminate through renal mechanisms. It is also noteworthy that there was no statistically significant difference (*P*-value \>0.05) in sweat BPA levels depending on the method of sweat collection whether through exercise, infrared sauna, or regular sauna. Although this study sheds light on the importance of sweat as a pathway of elimination for BPA from the body, it has some limitations. First, given the small study size (*n* = 20) it is not possible to extrapolate the findings to the general population. Secondly, samples were analysed for total BPA, instead of unconjugated BPA and conjugated BPA separately. Thirdly, other rare forms of BPA such as chlorinated and sulfated BPA were not analysed for. 5. Conclusion {#sec5} ============= As a result of increased scrutiny of health sequelae associated with human BPA exposure, it is apparent that this endocrine-disrupting compound has potentially negative consequences for the human organism. With new evidence for the possibility of BPA accrual within the body, interventions to facilitate elimination of this toxic compound have clinical relevance with regards to the prevention and treatment of adverse outcomes associated with BPA bioaccumulation. The results of this study suggest that: (i) Sweat testing may be an additional tool for BPA bio-monitoring; and (ii) Induced sweating appears to be a clinically useful tool to facilitate the release of BPA through the skin in order to eliminate this toxicant from the human body. Notable findings and implications of data from this study(i) BPA is excreted in sweat(ii) Sweat BPA concentrations are consistently much higher than urine(iii) Only 2/20 participants had BPA in serum, while 16/20 had BPA in sweat(iv) The data suggests that BPA likely bioaccumulates to some degree in humans(v) The data suggests that BPA retained in tissues (likely adipose) excretes via sweat(vi) The finding in some individuals that little or no BPA is excreted in urine while considerable levels are found in sweat suggests that current biomonitoring via serum (as done in Europe) or urine (as done in North America) may not provide a reliable indication of the BPA toxicant burden(vii) With the recognition that BPA has the potential for hormonal dysregulation, the significance of accrued BPA remains to be conclusively elucidated Conflict of Interests {#sec6} ===================== There is no conflict of interests. No funding has been received for any part of this work. The authors thank Jarrod Roberts for his technical work and assistance with analysis of samples. ![2 × 2 tables indicating the presence of BPA in specific body compartments. Each cell represents number of study participants.](JEPH2012-185731.001){#fig1} ###### Participant results for BPA in three body compartments: serum, urine, and sweat. Participant Gender Age Clinical diagnosis Serum conc. Urine conc. Sweat conc. Sweat/urine ratio Technique used for sweat collection ------------- -------- ----- --------------------------------- ------------- ------------- ------------- ------------------- ------------------------------------- 1 M 61 Diabetes, obesity, hypertension 0 4 82 20.5 Exercise 2 F 40 Rheumatoid arthritis 0 22 24 1.1 Steam sauna 3 M 38 Addiction disorder 0 20 22 1.1 Steam sauna 4 F 25 Bipolar disorder 0 40 22 0.6 Steam sauna 5 F 47 Lymphoma 0 10 24 2.4 Steam sauna 6 F 43 Fibromyalgia 0 32 0 n/a Steam sauna 7 F 48 Depression 0 0 16 n/a Steam sauna 8 F 40 Chronic fatigue 0 0 22 n/a Infrared sauna 9 F 68 Diabetes, fatigue, obesity 0 0 10 n/a Steam sauna 10 M 49 Chronic pain, cognitive decline 0 8 10 1.3 Exercise 11 M 53 Healthy 10 32 20 0.6 Exercise 12 M 23 Healthy 0 30 46 1.5 Infrared sauna 13 M 21 Healthy 30 4 10 2.5 Infrared sauna 14 F 47 Healthy 0 8 12 1.5 Infrared sauna 15 M 53 Healthy 0 4 35 8.8 Infrared sauna 16 F 43 Healthy 0 0 12 n/a Infrared sauna 17 F 51 Healthy 0 0 0 n/a Infrared sauna 18 M 46 Healthy 0 42 0 n/a Infrared sauna 19 M 57 Healthy 0 0 0 n/a Infrared sauna 20 F 50 Healthy 0 8 22 2.8 Infrared sauna ###### Comparison of urine BPA levels across published studies. Urine levels of BPA (ng/mL)-comparison across studies ------------------------------------------------------- --------- ------- ----- ------------------ ------ ---------------------- ----------------------------- This study Canada LC-MS 0.2 20 adults 70 AM:13 Median 8 0--42 Bushnik et al. \[[@B10]\] Canada GC-MS 0.2 5462 (age 6--79) 90.7 GM 1.16 (1.08--1.24) N/A Calafat et al. \[[@B9]\] USA LC-MS 0.4 950 adults N/A GM 2.4 N/A Calafat et al. \[[@B8]\] USA GC-MS 0.1 394 adults 95 GM 1.33 Median 1.28 0.1--5.18 (*95th Centile*) Moors et al. \[[@B108]\] Germany GC-MS 3 15 adults 60 ND-55 Mendiola et al. \[[@B107]\] USA LC-MS 0.4 375 males 90 GM 1.50 \<0.4--6.5 *(95th Centile)* DL: detection limit; AM: arithmetic mean; GM: geometric mean; ND: non detect; N/A: not available. LC-MS: liquid chromatography-mass spectrometry. GC-MS: gas chromatography-mass spectrometry. [^1]: Academic Editor: Robin Bernhoft

{ "pile_set_name": "PubMed Central" }

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"scientific", "political", "spheres", "potential", "harm", "resulting", "human", "BPA", "exposure", "potential", "bioaccumulation", "overview", "literature", "regarding", "effects", "BPA", "human", "health", "provided", "followed", "presentation", "data", "subjects", "whose", "blood", "urine", "sweat", "tested", "BPA", "Results", "discussion", "regarding", "BPA", "bioaccumulation", "elimination", "presented", "consideration", "Background", "Currently", "BPA", "commonly", "found", "component", "polycarbonates", "total", "BPA", "produced", "production", "epoxy", "resins", "well", "found", "myriad", "products", "including", "plastic", "food", "beverage", "containers", "including", "baby", "water", "bottles", "BPA", "also", "commonly", "found", "various", "household", "appliances", "electronics", "sports", "safety", "equipment", "adhesives", "cash", "register", "receipts", "medical", "devices", "eyeglass", "lenses", "water", "supply", "pipes", "many", "products", "also", "frequently", 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Introduction ============ Liver abscesses contain pus and develops due to bacteria or amoebae. In some cases, they can be fatal, so appropriate treatment is required to obtain a favorable prognosis. In the past, this disease had a high mortality rate. It was treated either conservatively by administering antibiotics and observing the course of the disease or by performing laparotomy drainage, and the mortality was 9-80% ([@B1]). However, in the last 40 years, the management of liver abscess has greatly changed. With the progress of imaging techniques, in addition to laparotomy drainage, percutaneous transhepatic drainage or drainage by endoscopic retrograde cholangiopancreatography (ERCP) has been developed. Percutaneous transhepatic drainage can be performed safely under computed tomography (CT) or ultrasound guidance, but it is an invasive procedure. In recent years, broader antibiotics have also been developed and are being used to treat bacterial liver abscess, but no criteria have been established for deciding whether it is possible to rely on conservative treatment with antibiotics alone or whether drainage is required. In this study, we examined the efficacy of contrast-enhanced ultrasonography (CEUS) for deciding whether or not liver abscess can be treated conservatively with antibiotics only. Materials and Methods ===================== We performed a retrospective analysis of 21 patients who had been diagnosed with liver abscess via a biochemical examination of the blood, conventional ultrasonography (US), CEUS, and contrast-enhanced CT (CECT) in the gastroenterology department of our hospital from July 2011 to December 2015. The diagnostic criteria included hypoechoic to hyperechoic lesions and the detection of internal echoes reflecting debris or septation on US/CEUS and round lesions with central hypoattenuation, peripheral rim enhancement or surrounding edema on CECT. US/CEUS and CECT were performed for the initial diagnosis. The ultrasonic devices used for conventional US/CEUS were a Aplio500, Xario (Toshiba, Tokyo, Japan), LOGIQ E9, LOGIQ E9 XD Clear 2.0 (GE Healthcare, Chicago, USA), and Ascendus (Hitachi, Tokyo, Japan). The contrast agent was Sonazoid^Ⓡ^ \[common name perfluorobutane; Dai-Ichi Sankyo Seiyaku (Tokyo, Japan)\]. Sonazoid^Ⓡ^ was administered intravenously at 0.01 mL/kg, and after flushing with 10 mL of saline, an evaluation was made in the arterial-predominant phase (10-30 seconds following administration of the contrast agent) and the post-vascular phase (10 min after injection and lasting for 1 hour or more) ([@B2]). In the conventional US and CEUS examinations, videos were recorded, and the abscess size, stain area rate relative to the whole abscess area in the arterial-predominant phase and defect rate of the whole abscess in the post-vascular phase were calculated ([Fig. 1](#g001){ref-type="fig"}). In cases with multiple liver abscess, we evaluated the largest abscess using CEUS. The stain area rate was evaluated in the maximum parting plane of the abscess. CEUS was performed for the initial diagnosis by a skilled Ultrasound Physician who was a supervisor of the Japan Society of Ultrasonics in Medicine with 20 years of experiences (C.O.). The diagnosis and analysis were performed by two doctors who are board-certified hepatologists of the Japan Society of Hepatology. The strategy in our hospital is to start antibiotics at the time of the diagnosis of hepatic abscess, with drainage treatment added at the judgment of the attending physician if exacerbation was observed in the clinical course. ![Results of an examination with conventional US (a) and CEUS in the arterial-predominant phase (b). CEUS revealed a clear boundary between the necrotic area and normal liver cells.](1349-7235-59-0471-g001){#g001} Patients were evaluated for their age, gender, presence of dementia, diabetes mellitus, cancer, outcome, hospitalization, period, whether or not drainage was performed (including catheter drainage, needle aspiration and surgical operation) and whether or not antithrombotic drugs were being taken. They were divided into two groups: the vascular phase enhancement (VE) group, in which ≥50% or more of the whole abscess was enhanced in the arterial-predominant phase, and the vascular phase non-enhancement (VNE) group, in which \<50% of the whole abscess was enhanced in the arterial-predominant phase ([Fig. 2](#g002){ref-type="fig"}), and the proportion of improvement by the use of antibiotics only and patient characteristics were recorded ([Table 1](#t001){ref-type="table"}). We compared the stain rate in the arterial-predominant phase with the improvement with antibiotics only. We also compared the defect rate in the post-vascular phase with the improvement with antibiotics only. ![Images of the VE and VEN groups. (a) ≥50% of the whole abscess was enhanced in the arterial-predominant phase. (b) \<50% of the whole abscess was enhanced in the arterial-predominant phase.](1349-7235-59-0471-g002){#g002} ###### Characteristics of the Patients with Improvement with Antibiotics Only and Those with No Improvement by Conservative Therapy. ----------------------------------------------------------------------------------------- Improvement by\ No improvement by\ antibiotics only 12 cases conservative therapy 9 cases ------------------------------ --------------------------- ------------------------------ Sex (male/ female) 7/5 3/6 Age 74.4 (57-92) 65.6 (53-83) Comorbidities Dementia 4 1 Diabetes mellitus 4 5 Cancer (colon cancer) 9 (1) 4 (2) Biliary infection 5 3 Antithrombotic drugs 5 2 Laboratory test White blood cell count (/μL) 9,903 (4,300-18,800) 13,951 (5,940-37,700) CRP 12.7 (3.53-21.25) 22.6 (8.7-35.38) Platelet count (×10^4^/μL) 19.8 (10.4-48.9) 20.4 (8-42.2) DIC 0 5 ----------------------------------------------------------------------------------------- All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics. The chi-square or Fisher\' s exact tests were applied to evaluate the differences in the categorical variables. Continuous data were presented as the mean, and Student\' s *t*-test was used to evaluate the difference in continuous variables. The statistical analyses were performed with a two-tailed significance level of 0.05. Results ======= The 21 patients were 10 men and 11 women, the mean age was 70.6±13.0 years old (mean ± standard deviation). The average abscess diameter was 55.2 mm, and in all 21 cases, the cause was bacterial. Amoeba cases were not included in this study. Regarding the outcomes, 12 patients improved with antibiotics only, while 9 did not. Of the 21 patients, the VE group consisted of 12 patients (average diameter 41.1 mm), and the VNE group consisted of 9 patients (average diameter 73.9 mm). In the VE group, 11 of 12 patients (91.7%) improved with antibiotics only, whereas in the VNE group, only 1 of 9 (11.1%) improved with antibiotics only, showing a significantly higher proportion of improvement with antibiotics only in the VE group (p\<0.001). The patient in the VE group who did not improve with antibiotics only had a rupture on the liver surface ([Table 2](#t002){ref-type="table"}). ###### Demographics and Abscess Characteristics in the Patients with Improvement with Antibiotics Only and Those with No Improvement by Conservative Therapy. ---------------------------------------------------------------------------------------------------------- Improvement by\ No improvement by\ antibiotics only 12 cases conservative therapy 9 cases ----------------------------------------------- --------------------------- ------------------------------ Abscess size (mm) 39.5 (8-64.7) 76.1 (45-132) Diameter ≤5cm 8 2 Abscess location(right/left lobe) 9/5 6/4 Single / Multiple 7/5 8/1 Unilocular / Multilocular 10/2 5/4 Stain rate in the arterial-predominant phase\ 1/11 8/1 \<50% (VNE group) / ≥50% (VE group) Hospitalization 25.6 (13-50) 37.6 (2-57) ---------------------------------------------------------------------------------------------------------- The mean hospitalization period in the VE group was 47 days when drainage was performed and 28 days without drainage, whereas that in the VNE group was 41.3 days when drainage was required and 14.5 days when drainage was not required (including 1 case who died of other causes). All seven patients with a stain rate of ≥71% improved with antibiotics only (7 out of 7 patients, 100%). In contrast, patients with a stain rate of ≤ 30% did not improve with antibiotics only \[6 out of 7 (85.7%) underwent drainage, and 1 patient had no drainage but died from other causes\] ([Fig. 3](#g003){ref-type="fig"}). ![The number of patients that showed an improvement by antibiotics alone, and those that showed no improvement by conservative therapy were analyzed regarding the stain area rate in the arterial-predominant phase. All patients who obtained an enhancement of ≥71% for the whole abscess in the arterial-predominant phase were treated by conservative therapy, but those who obtained an enhancement of ≦30% for the whole abscess in the arterial-predominant phase did not demonstrate any improvement by conservative therapy.](1349-7235-59-0471-g003){#g003} Considering the defect rate in the post-vascular phase and improvement rate with antibiotics only, 16 out of 21 patients (76.2%) showed a defect rate of ≥71%. Both groups of patients who demonstrated an improvement whether they took antibiotics or not, showed a high defect rate in the post-vascular phase. As a result, it was considered to be difficult to determine the optimal treatment approach base on the post-vascular phase ([Fig. 4](#g004){ref-type="fig"}). ![The number of patients who showed an improvement by antibiotics alone, and those who demonstrated no improvement by conservative therapy were analyzed regarding the defect area rate in the post-vascular phase. Both groups who improved by antibiotics or not, showed high defect rate in the post-vascular phase. As a result, evaluations using the defect area rate in the post-vascular phase are therefore not considered to be useful.](1349-7235-59-0471-g004){#g004} Of the 21 cases, 33.3% (7 of 21) were receiving antithrombotic drugs, 23.8% (5 of 21) had dementia, and 4.8% had both (1 of 21). The presence of either antithrombotic drug treatment or dementia was noted in 52.4% (11 of 21). A total of 61.9% (13 of 21) of patients had malignant disease, so a substantial proportion of patients required special care when performing drainage. The relationship between the abscess size and improvement rate with antibiotics only was also examined. Eight of 10 patients (80.0%) with an abscess size of ≤ 50 mm and 11 of 13 patients with an abscess size of ≤ 60 mm improved with antibiotics only ([Fig. 5](#g005){ref-type="fig"}). ![Relationship between the abscess size and improvement rate with conservative treatment. The rate of improvement in abscesses measuring ≥61 mm in size was not good.](1349-7235-59-0471-g005){#g005} Discussion ========== Liver abscesses are usually composed of a viscous fluid in an intrahepatic partition caused by inflammation as a defense reaction against infection. Conventional US is used to assess the condition of the normal liver, perform an accurate diagnosis in real time, and perform repeated evaluations at a low cost, and this modality is well tolerated without radiation exposure. However, for the diagnosis of liver abscesses, it is limited. Although CECT is extremely useful for evaluating the extent of abscesses and necrosis, it carries a risk of radiation exposure and contrast agent allergy, and it is difficult to perform in patients with renal dysfunction. It also cannot be used repeatedly to evaluate the treatment effect. CEUS solves the above problems, since it can be used without the risk of affecting the kidney function and can be used in real time without a risk of radiation exposure. The utility of CEUS for examining liver abscesses has been reported to be comparable to that of CECT and magnetic resonance imaging (MRI) ([@B3]-[@B5]). The European Federation for Ultrasound in Medicine and Biology (EFSUMB) and World Federation for Ultrasound in Medicine and Biology (WFUMB) guidelines specify ultrasonic patterns for liver abscesses. Conventional US findings of liver abscess are a low-echoic mass with a thick irregular wall and interior partitions, sometimes including gas (presenting as a bright dotted echo with a shadow behind). These are the most frequently observed findings, but they are not specific. Regarding the findings of CEUS, mature abscesses typically show marginal enhancement in the arterial-predominant phase, occasionally with enhancement of septae followed by venous hypoenhancement ([@B2], [@B6], [@B7]). In the present study, the evaluation of the defect rate in the post-vascular phase was not recognized as a useful marker for deciding whether conservative treatment of liver abscess was possible or whether drainage treatment was necessary. However, even if only a few normal liver cells remain, they are likely to be stained in the arterial-predominant phase, so the evaluation of the arterial-predominant phase, where there is a clear boundary from the necrotic area, where normal liver cells are not present, would be a useful marker for deciding whether conservative treatment is possible or whether drainage treatment is required. Several types of ultrasound contrast agents are currently in use, but Sonazoid^Ⓡ^, a second-generation contrast ultrasonic agent, can evaluate the blood flow dynamics of the post-vascular phase as well as that of the arterial-predominant phase, so a more precise diagnosis has become possible ([@B8]). Sonazoid^Ⓡ^ is used clinically in Japan ([@B9]) and in recent years has come to be used in China, South Korea, Norway and other countries as well; however, in countries where Sonazoid^Ⓡ^ is not approved for use, the evaluation in the post-vascular phase is impossible. Thus far, the treatment approach for liver abscess was determined by the number and size of the abscesses. In cases of a single lesion with a diameter of ≤ 5 cm, percutaneous catheter drainage or needle aspiration is often performed. Drainage catheters should remain in place until drainage is minimal (usually up to seven days). Repeat needle aspiration may be required in up to half of cases if a catheter is not left *in situ*. If the lesion is ≤ 5 cm, the prognosis is expected to be good, regardless of a puncture or catheter being used ([@B10]-[@B13]). For a single, large abscess \>5 cm in diameter, catheter drainage is preferred over needle aspiration. According to Zerem et al., in 60 cases of antibiotics + catheter drainage vs. antibiotic + needle aspiration, in abscesses of ≤ 5 cm, treatment was successful in all patients, irrespective of catheter drainage or needle aspiration. However, in cases with an abscess diameter \>5 cm, catheter drainage was successful in all cases, whereas needle aspiration succeeded in only 50% of cases ([@B13]). Even very large abscesses (\>10 cm) can be successfully managed with catheter drainage, although the risk of treatment failure and other complications is substantial ([@B14], [@B15]). According to Ahmed et al., in Singapore, in 44 cases of hepatic abscesses exceeding 10 cm, 25% of 39 patients who underwent drainage therapy suffered complications such as death, sepsis, and pleural infiltration, and frequent drainage therapy was necessary ([@B15]). However, some reports have claimed that percutaneous transhepatic drainage therapy does not work with abscesses \>5 cm in size ([@B16]). In a retrospective analysis of 80 liver abscesses exceeding 5 cm, the failure rate of percutaneous transhepatic drainage was greater than that of surgical drainage (28% vs. 7%). However, there was no significant difference in the mortality, morbidity, fever period, or incidence of complications. Surgical drainage is usually preferred in the following circumstances: multiple abscesses, loculated abscesses, abscesses with viscous contents obstructing the drainage catheter, underlying disease requiring primary surgical management, and inadequate response to percutaneous drainage within seven days ([@B10], [@B11], [@B17], [@B18]). In our study, 13 of 21 patient had malignant disease. In some reports, liver abscess has been described as occasionally complicated with colon cancer ([@B19]). General screening, especially that of the colon tract, should be performed in hepatic abscess patients. A considerable proportion of pyogenic liver abscesses follow one or more episodes of portal vein pyemia, usually related to bowel leakage and peritonitis. Another important route is the direct spread from biliary infection. Underlying biliary tract disease, such as gallstones or malignant obstruction, is present in 40% to 60% of cases ([@B20]-[@B22]). Occasionally, abscesses arise from surgical or penetrating wounds, including injury from migration of an ingested foreign body ([@B23], [@B24]). Recently, in aging societies, cases with various complications have increased, and patients taking antithrombotic drugs at the diagnosis cannot be punctured. In the present study as well, five patients were taking antiplatelet agents, and two were taking anticoagulants. In the future, the proportion of high-risk patients in whom puncture is difficult will increase. For this reason, it was considered very useful to identify a predictor of conservative treatment based on noninvasive contrast echography at an early stage. In the present study, we found that the stain rate of the abscess in the arterial-predominant phase of contrast ultrasound was useful as a marker for conservative treatment. If the arterial-predominant phase has a high stain rate, there is a strong likelihood that risky drainage treatment can be avoided without prolonging the hospital stay. Although the identification of pathogenic bacteria was not possible partially due to the fact that drainage was not performed, it may nevertheless be possible to successfully detect pathogenic bacteria in blood cultures and thereby choose the optimal sensitive antibiotics. If pathogenic bacteria are not detected in a blood culture and the patient\' s medical condition is getting worse, then drainage treatment should be carefully considered, including the identification of the causative bacteria and the selection of appropriate antibiotics. In addition, regarding the abscess size, 8 of 10 cases with an abscess size of ≤ 50 mm, and 11 of 13 cases with an abscess size of ≤ 60 mm improved with antibiotics only. Since it becomes difficult to form an abscess cavity as the abscess diameter shrinks, this is thought to be correlated with the stain rate in the arterial-predominant phase. Although the diameter of the abscess is useful as a marker, it is thought that the stain rate may be more useful, as in this study, 11 of the 12 patients in the VE group improved with antibiotics only. In the VE group, the hospitalization period was not extended, even when drainage was not performed. Due to the fact that the non-necrotic area was deemed to be indicated for conservative treatment and the area had still not liquefied, the benefits obtainable by drainage were therefore thought to be negligible. In the VE group, one patient who did not improve with conservative therapy had an abscess that burst near the liver surface. When an abscess is near the liver surface, the risk of rupture should always be considered. Our study was limited by the small sample size and the use of only a single contrast agent that is not available in many countries. Conclusion ========== Although decision-making in cases of liver abscess is difficult based on the post-vascular phase of CEUS, the enhancement rate in the arterial-predominant phase can predict the response to conservative treatment. **The authors state that they have no Conflict of Interest (COI).** [^1]: Correspondence to Dr.　Masatoshi Kudo, <[email protected]>

{ "pile_set_name": "PubMed Central" }

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"specify", "ultrasonic", "patterns", "liver", "abscesses", "Conventional", "US", "findings", "liver", "abscess", "mass", "thick", "irregular", "wall", "interior", "partitions", "sometimes", "including", "gas", "presenting", "bright", "dotted", "echo", "shadow", "behind", "frequently", "observed", "findings", "specific", "Regarding", "findings", "CEUS", "mature", "abscesses", "typically", "show", "marginal", "enhancement", "phase", "occasionally", "enhancement", "septae", "followed", "venous", "hypoenhancement", "present", "study", "evaluation", "defect", "rate", "phase", "recognized", "useful", "marker", "deciding", "whether", "conservative", "treatment", "liver", "abscess", "possible", "whether", "drainage", "treatment", "necessary", "However", "even", "normal", "liver", "cells", "remain", "likely", "stained", "phase", "evaluation", "phase", "clear", "boundary", "necrotic", "area", "normal", "liver", "cells", "present", "would", "useful", "marker", "deciding", "whether", "conservative", "treatment", "possible", "whether", "drainage", "treatment", "required", "Several", "types", "ultrasound", "contrast", "agents", "currently", "use", "contrast", "ultrasonic", "agent", "evaluate", "blood", "flow", "dynamics", "phase", "well", "phase", "precise", "diagnosis", "become", "possible", "used", "clinically", "Japan", "recent", "years", "come", "used", "China", "South", "Korea", "Norway", "countries", "well", "however", "countries", "approved", "use", "evaluation", "phase", "impossible", "Thus", "far", "treatment", "approach", "liver", "abscess", "determined", "number", "size", "abscesses", "cases", "single", "lesion", "diameter", "cm", "percutaneous", "catheter", "drainage", "needle", "aspiration", "often", "performed", "Drainage", "catheters", "remain", "place", "drainage", "minimal", "usually", "seven", "days", "Repeat", "needle", "aspiration", "may", "required", "half", "cases", "catheter", "left", "situ", "lesion", "cm", "prognosis", "expected", "good", "regardless", "puncture", "catheter", "used", "single", "large", "abscess", "cm", "diameter", "catheter", "drainage", "preferred", "needle", "aspiration", "According", "Zerem", "et", "cases", "antibiotics", "catheter", "drainage", "antibiotic", "needle", "aspiration", "abscesses", "cm", "treatment", "successful", "patients", "irrespective", "catheter", "drainage", "needle", "aspiration", "However", "cases", "abscess", "diameter", "cm", "catheter", "drainage", "successful", "cases", "whereas", "needle", "aspiration", "succeeded", "cases", "Even", "large", "abscesses", "cm", "successfully", "managed", "catheter", "drainage", "although", "risk", "treatment", "failure", "complications", "substantial", "According", "Ahmed", "et", "Singapore", "cases", "hepatic", "abscesses", "exceeding", "cm", "patients", "underwent", "drainage", "therapy", "suffered", "complications", "death", "sepsis", "pleural", "infiltration", "frequent", "drainage", "therapy", "necessary", "However", "reports", "claimed", "percutaneous", "transhepatic", "drainage", "therapy", "work", "abscesses", "cm", "size", "retrospective", "analysis", "liver", "abscesses", "exceeding", "cm", "failure", "rate", "percutaneous", "transhepatic", "drainage", "greater", "surgical", "drainage", "However", "significant", "difference", "mortality", "morbidity", "fever", "period", "incidence", "complications", "Surgical", "drainage", "usually", "preferred", "following", "circumstances", "multiple", "abscesses", "loculated", "abscesses", "abscesses", "viscous", "contents", "obstructing", "drainage", "catheter", "underlying", "disease", "requiring", "primary", "surgical", "management", "inadequate", "response", "percutaneous", "drainage", "within", "seven", "days", "study", "patient", "malignant", "disease", "reports", "liver", "abscess", "described", "occasionally", "complicated", "colon", "cancer", "General", "screening", "especially", "colon", "tract", "performed", "hepatic", "abscess", "patients", "considerable", "proportion", "pyogenic", "liver", "abscesses", "follow", "one", "episodes", "portal", "vein", "pyemia", "usually", "related", "bowel", "leakage", "peritonitis", "Another", "important", "route", "direct", "spread", "biliary", "infection", "Underlying", "biliary", "tract", "disease", "gallstones", "malignant", "obstruction", "present", "cases", "Occasionally", "abscesses", "arise", "surgical", "penetrating", "wounds", "including", "injury", "migration", "ingested", "foreign", "body", "Recently", "aging", "societies", "cases", "various", "complications", "increased", "patients", "taking", "antithrombotic", "drugs", "diagnosis", "punctured", "present", "study", "well", "five", "patients", "taking", "antiplatelet", "agents", "two", "taking", "anticoagulants", "future", "proportion", "patients", "puncture", "difficult", "increase", "reason", "considered", "useful", "identify", "predictor", "conservative", "treatment", "based", "noninvasive", "contrast", "echography", "early", "stage", "present", "study", "found", "stain", "rate", "abscess", "phase", "contrast", "ultrasound", "useful", "marker", "conservative", "treatment", "phase", "high", "stain", "rate", "strong", "likelihood", "risky", "drainage", "treatment", "avoided", "without", "prolonging", "hospital", "stay", "Although", "identification", "pathogenic", "bacteria", "possible", "partially", "due", "fact", "drainage", "performed", "may", "nevertheless", "possible", "successfully", "detect", "pathogenic", "bacteria", "blood", "cultures", "thereby", "choose", "optimal", "sensitive", "antibiotics", "pathogenic", "bacteria", "detected", "blood", "culture", "medical", "condition", "getting", "worse", "drainage", "treatment", "carefully", "considered", "including", "identification", "causative", "bacteria", "selection", "appropriate", "antibiotics", "addition", "regarding", "abscess", "size", "cases", "abscess", "size", "mm", "cases", "abscess", "size", "mm", "improved", "antibiotics", "Since", "becomes", "difficult", "form", "abscess", "cavity", "abscess", "diameter", "shrinks", "thought", "correlated", "stain", "rate", "phase", "Although", "diameter", "abscess", "useful", "marker", "thought", "stain", "rate", "may", "useful", "study", "patients", "group", "improved", "antibiotics", "group", "hospitalization", "period", "extended", "even", "drainage", "performed", "Due", "fact", "area", "deemed", "indicated", "conservative", "treatment", "area", "still", "liquefied", "benefits", "obtainable", "drainage", "therefore", "thought", "negligible", "group", "one", "patient", "improve", "conservative", "therapy", "abscess", "burst", "near", "liver", "surface", "abscess", "near", "liver", "surface", "risk", "rupture", "always", "considered", "study", "limited", "small", "sample", "size", "use", "single", "contrast", "agent", "available", "many", "countries", "Conclusion", "Although", "cases", "liver", "abscess", "difficult", "based", "phase", "CEUS", "enhancement", "rate", "phase", "predict", "response", "conservative", "treatment", "authors", "state", "Conflict", "Interest", "COI", "Correspondence", "Masatoshi", "Kudo" ]

1.. Introduction ================ Lung cancer is the leading cause of cancer deaths in both men and women in the United States, with an overall 5-year survival rate of 10--15% that has persisted for decades \[[@b1-cancers-03-02975]\]. There are two types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). About 85% of all lung cancers are NSCLC, which can be further divided into three main types: large cell carcinoma; squamous cell carcinoma; and adenocarcinoma of the lung. Adenocarcinoma of the lung is the single most common type of lung cancer; it accounts for about 40% of all lung cancers. Although NSCLC grows more slowly and is less aggressive than SCLC, the most efficient treatment for NSCLC is considered to be surgical resection of the tumor, while SCLC is remarkably sensitive to chemotherapy and radiation treatments. The low survival rate of NSCLC patients is related to the late presentation of patients with an unresectable tumor due to the lack of a validated screening approach for early detection. By early detection of precancerous microscopic lesion, patients can avoid developing clinically diagnosable cancer and high-risk healthy individuals can be prevented from developing cancer by timely chemoprevention. Therefore, early detection of lung cancer, followed by appropriate treatment, will significantly increase the survival rate. Low-dose computerized tomography (CT) scan has suggested a promising possibility to detect lung cancer earlier. Compared to chest X-rays, CT screening significantly reduced mortality in a high-risk population by 20.3% based on the recently reported National Lung Screening Trial (NLST), a large randomized trial funded by the National Cancer Institute (NCI)\[[@b2-cancers-03-02975]\]. Although it is promising, the limited impact of CT screening on mortality may be because it cannot distinguish non-calcified nodules (NCNs) that progress to cancer from the benign ones. It also has limited ability to differentiate between benign and malignant lesions of tumors, especially the centrally located ones \[[@b3-cancers-03-02975]\], demanding a complementary screening tool to increase specificity of detection for cancer. False-positive CT screening results will lead to unnecessary invasive procedures, which can cause stress and economic burden. Therefore, there is still a demand for non-invasive, sensitive biomarkers in order to accurately identify the high-risk population for optimal treatments and minimize unnecessary invasive procedures caused by false-positive CT screening results. These biomarkers will be also useful to increase sensitivity for the detection of centrally located lung cancer and to determine the strategy for the therapeutic management of clinically uncertain nodules that are detected by CT screen. Tobacco smoking is the single, most important risk factor for lung cancer. An estimated 90% of lung cancer is due to cigarette smoking, which also causes at least 18 other types of cancer \[[@b4-cancers-03-02975]\]. It is well accepted that the repeated exposure of the respiratory tracts of smokers to tobacco-related carcinogens causes molecular genetic and epigenetic abnormalities that culminate in lung cancer. Consequently, smoking may create a field of molecular injury throughout the airway epithelium exposed to cigarette smoke and increase the susceptibility of an entire field or area to carcinogenesis (field carcinogenesis) \[[@b5-cancers-03-02975]\]. This suggests that the smoke-induced molecular abnormalities found in the respiratory tract (regardless of its exact location) may mirror the progression of cancer and therefore can be used as potential biomarkers to detect lung tumor at an early stage. Thus, sputum that contains exfoliated airway epithelial cells is an ideal specimen type for the discovery of biomarkers for lung cancer. Molecular analysis of sputum has been an active area for the investigation of lung cancer biomarkers for several reasons. Sputum is the most easily accessible body fluid that contains the pathogenically relevant cell type, namely, bronchial epithelial cells. These cells are typically exfoliated from the central airways, where the CT scan seems to have a blind spot when the cancer arises from here. This also suggests that it is possible to identify tumor cells in sputum. Additionally, the other airway epithelial cells found in sputum may also harbor important molecular information, supported by the field cancerization theory \[[@b5-cancers-03-02975]\], which reflects the local milieu of the lung. High-risk smokers typically produce increased amount of sputum, which makes it feasible to obtain enough material for analysis. Finally, collecting sputum is non-invasive, fast, and economical, which are important characteristics to be an ideal specimen type for large-scale population screening. In this review, we discuss various sputum-based molecular biomarkers and how each one has developed during the last decade. We then explore the strengths and weaknesses of each marker and discuss ways to overcome the limitations. 2.. Sputum-Based Molecular Biomarkers ===================================== 2.1.. Cytology -------------- Sputum cytology is the oldest technique that utilizes sputum in detecting lung diseases. Identified and developed by Saccomannao \[[@b6-cancers-03-02975]\], sputum cytology was shown to detect premalignant changes in high-risk groups several years before a clinical diagnosis of lung cancer. This finding is supported by the concept that changes in the cytology of bronchial epithelial cells reflect the progress from inflammation to cancer \[[@b7-cancers-03-02975]\]. However, it is not considered to be sensitive enough mainly because it requires skills that can identify subtle nuclear changes in cells that often comprise less than 5% of the sputum samples. The "subjectivity" of the pathologist performing the cytopathological classification raises concerns about its reliability and reproducibility due to the lack of standardization. The results of two large, randomized clinical trials support this notion \[[@b8-cancers-03-02975],[@b9-cancers-03-02975]\]. In these studies, they compared annual chest X-ray (CXR) plus sputum cytology with CXR only. After nine years of follow-up, they noticed the mortality due to lung cancer was slightly lower in the double-screened group. However, it is arguable whether it could have been occurred by chance. To overcome this limitation of low sensitivity and reliability, sputum cytology has often been used as a supplement to other screening methods. Lam *et al.* \[[@b10-cancers-03-02975]\] reported that among the high-risk patients with sputum atypia (47%), who were followed by bronchoscopy and CT scans, 15% of these patients had lung cancer. Notably, approximately half of these patients had early-stage cancer. It was reported that the overall sensitivity of sputum cytology in combination with bronchoscopy and CT scan in detecting lung cancer was 71% for all histological types and 100% for squamous cell lung cancer located in the central airways. On the other hand, combining sputum atypia with FISH assay for the detection of gene copy number changes of specific cancer-related genes did not improve the diagnostic performance compared to FISH screening alone \[[@b11-cancers-03-02975]\]. These inconsistent results demand further improvement in the application of sputum cytology for cancer screening. Automated image cytometry for objective and quantitative cytopathological imaging may greatly improve the sensitivity. An automated imaging system quantifying the DNA content had greater sensitivity (80%) than conventional cytology (4.2%) for the diagnosis of lung cancer \[[@b12-cancers-03-02975]\]. Kemp *et al.* \[[@b13-cancers-03-02975]\] performed multicenter validation trial to compare the automated sputum analysis using DNA cytometry to the conventional one and found that the automated method detected lung cancer with 75% sensitivity while conventional cytology only identified 16%. These studies suggest that an objective, automated imaging system in combination with adequate molecular biomarkers may be a promising tool for the screening of lung cancer. 2.2.. Allelic Alteration ------------------------ Microsatellites are stretches of DNA in which a short motif (usually one to five nucleotides long) is repeated ten to hundred times. Microsatellites are prone to mutations during replication due to the transient split of the two helical strands and slippage of the DNA polymerase complex at reannealing, which generates an insertion or deletion loop depending on the slippage direction \[[@b14-cancers-03-02975]\]. Microsatellite instability (MSI) is a situation in which these gained or lost repeat units as a consequence of the mutation result in a somatic change in length. MSI is known to be associated with defective DNA mismatch repair machinery \[[@b15-cancers-03-02975]\]. In hereditary non-polyposis colorectal carcinoma (HNPCC), in which MSI was first observed and is found frequently, MSI is caused by germline mutations in DNA mismatch-repair genes \[[@b16-cancers-03-02975]\], suggesting the phenomenon of MSI is associated with tumorigenesis. Furthermore, changes in the microsatellite repeats are known to correlate with altered gene expression \[[@b17-cancers-03-02975],[@b18-cancers-03-02975]\]. Thus, MSI in tumor is likely to lead to mutations and deregulated gene expression compared to normal tissue, which may be critical for understanding tumorigenesis. Another form of allelic alteration that can be revealed by MSI analysis is the loss of heterozygosity (LOH). LOH represents the loss of one allele of a gene whereby the other allele is already inactivated. LOH is caused by a variety of genetic mechanisms, including physical deletion of chromosome nondisjunction, mitotic nondisjunction followed by republication of the remaining chromosomes, mitotic recombination, and gene conversion \[[@b19-cancers-03-02975]\]. The frequent allelic loss at particular chromosomal regions in tumors is indicative of the presence of a tumor suppressor gene. Consequently, detection of LOH has been used to identify novel tumor suppressor genes. Furthermore, it is shown that some LOH patterns are associated with different tumor types, pathological stages, and progression \[[@b20-cancers-03-02975],[@b21-cancers-03-02975]\]. Allelic imbalance in lung cancer has been actively investigated. Individual LOH or MSI is rarely used as a stand-alone biomarker because there are numerous of them existing through the human genome and the frequency of the occurrence of each marker hardly provides statistical significance. Therefore, the key is to find the right combination of MSI and LOH to increase the sensitivity and specificity. A few commonly chosen regions for assessing MSI and LOH include, but are not limited to, chromosome 3p and 9p \[[@b22-cancers-03-02975]-[@b26-cancers-03-02975]\]. Notably, 3p deletions are detected in almost 100% of SCLC and more than 90% of NSCLC cell lines \[[@b27-cancers-03-02975]\]. Three *bona fide* lung cancer tumor suppressor genes, namely RBSP3 (AP20 region), NPRL2 and RASSF1A (LUCA region), were identified in the 3p21.3 region. In addition, the FHIT gene, of which inactivation is frequently found in various human cancers including lung cancer \[[@b28-cancers-03-02975]\], is located at chromosome 3p14. Chromosome 9p21, which was reported to be most frequently deleted in lung cancer cell lines \[[@b29-cancers-03-02975]\], contains two tumor suppressor genes, CDKN2A and CDKN2B. The frequency of individual or combination of several MSIs or LOHs in lung cancer is typically lower than 70% \[[@b22-cancers-03-02975],[@b23-cancers-03-02975],[@b25-cancers-03-02975]\]. However, this low prevalence may be increased by combining with other types of molecular markers, for example, methylation \[[@b26-cancers-03-02975]\] and sputum cytology \[[@b24-cancers-03-02975]\]. 2.3.. Methylation ----------------- DNA methylation is a common epigenetic mechanism that regulates gene expression. It involves the addition of a methyl group to the 5^th^ position of the cytosine base located 5′ to a guanosine in a CpG dinucleotide \[[@b30-cancers-03-02975]\]. A CpG island, a contiguous stretch of DNA of at least 200 bp up to 4 kb, contains high CpG contents \[[@b31-cancers-03-02975]\] and is often found in the promoter regions. Promoter hypermethylation is accompanied by histone modifications such as acetylation, methylation or phosphorylation of histone tails within the island \[[@b32-cancers-03-02975]\]. This series of epigenetic alterations leads to a conformational change of the chromatin in the promoter regions, which prevents the RNA polymerase and other transcriptional regulatory proteins from accessing this region \[[@b32-cancers-03-02975]\]. As a consequence, transcription is blocked and the gene becomes silenced. About 50% of human gene promoters contain CpG islands, which are generally unmethylated in the normal genome. However, in cancer, it is well established that CpG hypermethylation in the promoter region of genes that function in various pathways (such as DNA repair, cell cycle regulation, apoptosis and RAS signaling) is tightly associated with tumorigenesis \[[@b31-cancers-03-02975]\]. There are several genes that are frequently silenced by promoter hypermethylation in lung cancer. *P16 (CDKN2A)* is a tumor suppressor gene that is hypermethylated at a prevalence of up to 67% in adenocarcinoma and 70% in squamous cell carcinoma of the lung \[[@b33-cancers-03-02975]\]. It regulates the cell cycle and prevents excessive cell growth by regulating the tumor suppressor gene *RB1* and its protein, pRb. pRb prevents the cell from replicating damaged DNA by inhibiting its progression along the cell cycle through G1 into S. Intriguingly, inactivation of P16 has been proposed as an early step to immortalization \[[@b34-cancers-03-02975],[@b35-cancers-03-02975]\], supporting its potential to be a useful biomarker for the early detection of cancer. *MGMT* (*O*^6^-Methylguanine DNA methyltransferase) is a DNA repair enzyme that is inactivated in 24--48% of lung adenocarcinomas \[[@b33-cancers-03-02975]\]. It antagonizes the genotoxic effects of alkylating agents. *MGMT* rapidly reverses alkylation, including methylation, at the *O*^6^ position of guanine by transferring the alkyl group to the active site of the enzyme \[[@b36-cancers-03-02975]\]. Inactivation of *MGMT* in the cell allows the accumulation of O^6^-alkylguanine adducts in the DNA and can lead to transition mutations in genes such as *KRAS* and *TP53* \[[@b33-cancers-03-02975]\]. *RASSF1A* (Ras-association domain family member 1A) is a tumor suppressor that is silenced in 40--60% and 100% of NSCLC and SCLC, respectively \[[@b33-cancers-03-02975]\]. *RASSF1A* lacks apparent enzymatic activity but contains a Ras association (RA) domain and is potentially an effector of the Ras oncoprotein. *RASSF1A* modulates multiple apoptotic and cell cycle checkpoint pathways. Current evidence supports the hypothesis that it serves as a scaffold for the assembly of multiple tumor suppressor complexes and may relay pro-apoptotic signaling byK-Ras \[[@b37-cancers-03-02975]\]. *DAPK* (death-associated protein kinase) is a pro-apoptotic, calcium/calmodulin-regulated serine/threonine protein kinase that is hypermethylated in both adenocarcinoma and squamous cell carcinoma at prevalences ranging from 30-48% \[[@b33-cancers-03-02975]\]. DAPK mediates interferon-γ-mediated apoptosis and participates in a number of additional apoptosis-inducing pathways downstream of CD95 (Fas), tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) \[[@b38-cancers-03-02975]\]. Additionally, DAPK inactivation reduces the induction of p19^ARF^/p53, thus inactivating the p53-dependent pathway for apoptosis, suggesting that attenuation of p53 by loss of DAPK may be an important factor in transformation *in vivo* \[[@b39-cancers-03-02975]\]. These four genes along with a few other genes, including *PAX5* and *GATA*, have been the major focus for examining the sensitivity/specificity of promoter methylation as sputum-based biomarkers for lung cancer detection. Among these, *P16* is reported to be the most frequently hypermethylated gene in sputum samples at prevalence ranging from 25--74% \[[@b40-cancers-03-02975]-[@b42-cancers-03-02975]\]. The combination of promoter methylation of more than one gene and/or with other types of biomarkers such as gene mutations or cytology seems to increase the sensitivity and specificity for cancer detection \[[@b40-cancers-03-02975],[@b41-cancers-03-02975],[@b43-cancers-03-02975]\]. More importantly, hypermethylation of genes, particularly *P16*, may also be useful as biomarker for the prediction of the development of lung cancer among healthy high-risk smokers. Several studies have reported that hypermethylation in a panel of genes can be found in cancer-free smokers and is associated with increased risk of lung cancer \[[@b34-cancers-03-02975],[@b44-cancers-03-02975]-[@b47-cancers-03-02975]\]. The earlier occurrence of epigenetic abnormality than genetic modification such as gene mutation detected in histologically normal cells suggests that hypermethylation may be an early event during tumorigenesis. This may also explain the higher frequency of p16 hypermethylation than mutations in the *TP53* gene and/or the *KRAS* gene in sputum (see below) \[[@b47-cancers-03-02975],[@b48-cancers-03-02975]\]. Gene methylation can be detected by methylation-specific PCR (MSP) \[[@b49-cancers-03-02975]\] using sodium bisulfite, which converts all unmethylated, but not methylated, cytosines to uracil. Thus, bisulfite treatment introduces specific changes in the DNA sequence, which depends on the methylation status of individual cytosine residues. In the subsequent PCR reaction, primer pairs are designed to be "methylation-specific" by including sequences complementing only unconverted 5-methylcytosines. Recently, a modified version of MSP has been reported to increase the sensitivity. It involves a two-step nested PCR, which can improve the sensitivity by \>50-fold compared to the conventional MSP \[[@b46-cancers-03-02975]\]. One advantage of promoter hypermethylation as a biomarker compared to other genetic biomarkers (e.g., gene mutations, microsatellite abnormalities) is its reversibility by demethylating agent. Clinical trials for the treatment of myeloid neoplasms demonstrated that demethylating agents in combination with histone deacetylation inhibitor could demethylate certain genes in responding patients \[[@b50-cancers-03-02975]\]. Thus, the reversibility of promoter hypermethylation in sputum biomarkers can be a powerful monitoring tool for evaluating the efficacy of demethylation therapy for lung cancer patients. 2.4.. Mutation -------------- There has been significant progress in the understanding of the genetic basis underlying lung cancer. Accumulation of mutations in genes that are involved in cell differentiation and growth leads to lung tumorigenesis \[[@b51-cancers-03-02975]\]. Naturally, genetic mutations have been the area of focus for biomarker discovery. *TP53* and *KRAS* are two of the most frequently mutated genes that are found in lung tumors and believed to be important in lung cancer pathogenesis. Thus, they are most well-studied genetic markers for lung cancer. *TP53* is a tumor suppressor and "guardian of the genome" \[[@b52-cancers-03-02975]\]. Hence, mutations in the *TP53* gene are among the commonest in most cancers, including lung cancer \[[@b53-cancers-03-02975]\]. A recent study found that about 65% of lung adenocarcinomas harbor mutations in *TP53*, which identifies *TP53* as the most frequently mutated gene in lung cancer \[[@b54-cancers-03-02975]\]. *TP53* functions as an emergency brake by regulating tumor-preventing apoptosis and cell cycle progression. It modulates the transcription of genes that govern the major defense against tumor growth, which includes cell cycle arrest, apoptosis, maintenance of genetic integrity, inhibition of angiogenesis and cellular senescence \[[@b55-cancers-03-02975]\]. Most mutations are missense and single-base substitutions distributed throughout the core DNA binding domain from exon 5 to exon 8, which abolish its function as a transcription factor \[[@b56-cancers-03-02975]\]. *KRAS* is an oncogene that belongs to the Ras family. *RAS* genes encode a family of membrane-bound guanosine triphosphate (GTP)-binding proteins that regulate cell growth, differentiation, and apoptosis by interacting with multiple effectors, including those in the mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT), and phosphoinositide 3-kinase (PI3K) pathways \[[@b57-cancers-03-02975]\]. RAS proteins acquire transforming potential when an amino acid at position 12, 13, or 61 is replaced as a result of a point mutation in the gene \[[@b58-cancers-03-02975]\]. These activating mutations result in the production of RAS protein that has impaired GTPase activity, leading to constitutive activation of RAS. *KRAS* mutations are frequently found in human malignancies and notably up to 60% in lung adenocarcinoma \[[@b54-cancers-03-02975]\], being the second most frequently mutated gene in lung cancer. The fact that mutations in the *KRAS* gene occur almost exclusively at two hot spots (codons 12 and 13) facilitates the assessment of the presence of mutations at these codons by mutant allele enrichment method (See below). Epidermal growth factor receptor (*EGFR*) belongs to a family of receptor tyrosine kinases (TKs) that transduce important growth factors signaling from the extracellular milieu to the cell. Structurally, each receptor is composed of an extracellular ligand binding domain, a transmembrane domain and an intracellular domain \[[@b59-cancers-03-02975]\]. The receptor, which exists as an inactive monomer, undergoes conformational change upon the binding of a ligand such as EGF and forms a homo- or heterodimer. This event is then followed by autophosphorylation of the key tyrosine residues in the catalytic domain. Subsequently, proteins involved in downstream signaling events that control multiple cellular processes, including proliferation and survival, get recruited to the activated receptor via adaptor molecules \[[@b60-cancers-03-02975]\]. Intracellular signaling is mediated mainly through the RAS-RAF-MEK-MAPK pathway, the PI3K-PTEN-AKT pathway, and the signal transducer and activator of transcription (STAT) pathway \[[@b61-cancers-03-02975]\]. Two independent studies first reported the existence of somatic mutations in the TK domain of EGFR; the mutations are characterized by short deletions in exon 19 and point mutations (G719S, L858R, and L861Q) in exons 19 and 21 \[[@b62-cancers-03-02975],[@b63-cancers-03-02975]\]. The mutations have been classified into three types. Class I mutations include short in-frame deletions that result in the loss of four to six amino acids (E746 to S752) encoded by exon 19. Class II mutations are single-nucleotide substitutions that may occur throughout exons 18 to 21. Class III mutations are in-frame duplications and/or insertions that occur mostly in exon 20 \[[@b64-cancers-03-02975]\]. These mutations destabilize the kinase domain conformation and lead to constitutive activation, followed by uncontrolled activation of downstream signaling pathways including proliferation and survival. The occurrence of mutations in EGFR and KRAS, which encodes a GTPase downstream of EGFR (see above), is mutually exclusive in lung cancers and they exhibit many contrasting characteristics such as clinical background, pathological features of patients harboring each mutation, and prognostic or predictive implications. Clinico-pathological features that correlate with *EGFR*-activating mutations include East Asian ethnicity, adenocarcinoma histology, female gender, and a history of never having smoked \[[@b64-cancers-03-02975]\], while *KRAS* mutations are present in those with significant tobacco exposure. Lung cancers harboring the *EGFR* mutations are remarkably sensitive to *EGFR* tyrosine kinase inhibitors such as gefitinib or erlotinib, which has been a very important discovery impacting the clinical treatment of lung cancer. Because it is tightly associated with never-smokers, *EGFR* mutations are common targets for biomarker discovery for lung cancers in never-smokers but not in smokers. Considering the great heterogeneity of cell types and low number of tumor cells found in sputum, it is crucial to employ a sensitive detection method that will distinguish the true positive signal of tumor cells from the vast majority of contaminating normal cells. Traditionally, to detect *KRAS* mutation at the 12^th^ and 13^th^ codons, the PCR-mutant allele enrichment (PCR-MAE) method was utilized, which has been reported to improve the sensitivity \[[@b65-cancers-03-02975]\] compared to conventional PCR. However, this method is only suitable for the detection of specific known mutations because it requires the use of PCR primers that are complementary to the mutated sequence. For the detection of multiple and unknown mutations in *TP53*, the PCR-single stranded conformation polymorphism (PCR-SSCP) method is commonly used. Although this method may increase the sensitivity compared to conventional PCR, optimal conditions to sensitively separate alleles harboring various mutations have to be determined empirically, which can be tricky and time-consuming. As a result, studies employing similar methods had reported mutation frequencies ranging from lower than 10% frequency \[[@b25-cancers-03-02975],[@b48-cancers-03-02975]\] to about 50% \[[@b65-cancers-03-02975],[@b66-cancers-03-02975]\]. Notably, the rapid development of technology such as high-throughput sequencing now allows the detection of extremely rare mutant alleles in a highly heterogeneous sample. High-throughput sequencing is a method that parallelizes the sequencing process, producing thousands or millions of sequences at once. Choi *et al.* successfully identified two novel mutations in *EML4-ALK* gene in the sputum sample from a lung cancer patient using deep sequencing \[[@b67-cancers-03-02975]\]. The high-throughput nature of this system will also expand the capacity of screening, facilitating the examination of mutations in multiple genes simultaneously. Recently genome-wide screening studies to identify frequently mutated genes in lung cancer (e.g., *STK11*, *LRP1B*, *LPHN3*) have provided an exciting new insight into the discovery of potentially novel gene candidates for lung cancer in addition to *TP53* and *KRAS* \[[@b54-cancers-03-02975],[@b68-cancers-03-02975]\]. The findings of these studies are promising because they offer additional and perhaps better target (or combination of targets) for the early detection of lung cancer. Moreover, identification of the functions of these candidate genes and the consequences of mutations during the early stage of tumorigenesis will provide rationale for the use of mutations of these candidate genes for the early detection of cancer. In summary, genes that harbor limited number of hot spot mutations (e.g., *KRAS* mutations exclusively occur on codon 12 or 13) will be more practical for large-scale screening than genes (e.g., *TP53*) with numerous mutations scattered throughout the gene, which will complicate the screening strategy. In addition, since *TP53* mutations are found in almost every type of human cancer, an ideal biomarker for lung cancer should be a gene with mutation that is specifically associated with lung cancer. Lastly, the use of high-throughput sequencing for evaluating mutations in these new candidate genes in addition to *TP53* and *KRAS* in sputum will make gene mutation screening a powerful tool for lung cancer detection. 2.5.. microRNAs --------------- MicroRNAs (miRNAs) are a class of short (∼22 nucleotides long) and highly conserved non-coding RNAs involved in numerous developmental processes. They regulate gene expression by incomplete base-pairing to a complementary sequence in the 3′ untranslated region of a target mRNA, which leads to mRNA degradation or translation repression \[[@b69-cancers-03-02975]\]. Deregulation of miRNAs has been linked to cancer initiation and progression, indicating that miRNAs may act as tumor suppressor genes or oncogenes and can target apoptosis \[[@b70-cancers-03-02975]\]. It is believed that miRNAs regulate signal transduction pathways and oncogenic pathways leading to lung cancer. Interestingly, several studies have identified differential miRNA expression profiles associated with lung cancers \[[@b71-cancers-03-02975]-[@b74-cancers-03-02975]\], which suggests that miRNA signatures are potential biomarkers for lung cancers. Although miRNA has recently emerged as powerful molecular biomarkers for the detection of cancer, its potential as a sputum-based biomarker has not been fully explored. Jiang *et al.* recently reported two studies evaluating differential miRNA expression in sputum samples from lung cancer patients \[[@b74-cancers-03-02975],[@b75-cancers-03-02975]\]. Although they demonstrated that miRNAs existed stably in sputum and could be amplified robustly \[[@b76-cancers-03-02975]\], the studies are somewhat limited because the candidate miRNA molecules were initially discovered from tissue, not sputum, followed by validation of the selected candidates in sputum. Although this strategy can still provide valuable information, screening the entire miRNA transcriptome in sputum would be more desirable for the discovery of novel sputum-based miRNA biomarkers. However, one of the practical hindrances to the latter is that the normalization methods for miRNA, especially in an extremely heterogeneous sample like sputum, have not been fully established yet, which makes it challenging to assess the validity of miRNA profiling data. In addition, due to the inevitable variability in the sputum cell composition among samples, which is a major confounding factor, interpreting any differential miRNA expression can be challenging. Therefore, although sputum-based miRNAs have great potential as biomarkers for lung cancer, a vigorous system for evaluating the differential miRNA expression in sputum has to be developed. 3.. Conclusions and Future Directions ===================================== Although biomarker discovery in sputum has been promising, the clinical applications have been limited, mainly because of the inconsistent results and low sensitivity. One key to tackling these issues is obtaining more bronchial epithelial cells (BECs) in sputum, which is typically less than 5% \[[@b77-cancers-03-02975],[@b78-cancers-03-02975]\]. Increasing the number of BECs will eventually result in more cancer cells in sputum. One way to achieve this might be using sputum induction instead of spontaneous collection. However, the effect of different collection methods on the resultant percentage of bronchial epithelial cells in sputum has not been actively discussed in the literature. In addition, published studies frequently omit the detailed description of collection methods for sputum, which makes it difficult to deduce the effect collection methods on cell populations from these studies. Therefore, careful assessment of the sputum cell composition collected by different methods is urgently required. More importantly, standardized sputum collection and processing protocols should be established to minimize the inconsistencies resulting from different laboratories using different methods. Enriching the bronchial epithelial cells after collection is another strategy to increase the percentage of bronchial epithelial cells and get rid of the potentially confounding cell types such as macrophages and neutrophils. A few different enrichment methods have been proposed, including laser microdissection and magnetic-assisted cell sorting (MACS). When epithelial cells in lung cancer sputum samples were selectively captured by microdissection, the sensitivity for the detection of mutations in *TP53* and *KRAS* increased about three times (∼46%) compared to using the entire sputum cells \[[@b79-cancers-03-02975]\]. Furthermore, the same research group was able to detect mutations in *TP53* (14.1%) and *KRAS* (1.1%) in lung cancer-free, healthy high-risk group using laser microdissection \[[@b80-cancers-03-02975]\]. MACS is another common cell selection method. Typically, bronchial epithelial cells are negatively selected by using anti-CD14 and anti-CD16 magnetic beads to deplete the macrophages and the neutrophils, respectively, which together account for the majority of the cell populations in sputa. It has been reported that bronchial epithelial cells can be enriched from 1.1% (unsorted) up to 40% after MACS, producing an average of 43% purity in the processed samples and resulting in an average 36-fold enrichment \[[@b81-cancers-03-02975]\]. These studies suggest that by selectively enriching the bronchial epithelial cells, molecular abnormalities in sputum samples could be detected with higher sensitivity. In spite of the efforts to enrich for BECs in sputum, the rare presence of cancer cells in sputum strongly argues for biomarkers that are present in non-cancerous epithelial cells in early stage cancer or in high-risk smokers. Thus, examining molecular abnormalities, which have been identified in tumor tissue, in sputum samples may not be very successful. This requires the discovery of novel sputum-based biomarkers that are caused by lung cancer risk factors, namely tobacco smoking, and are harbored in pre-malignant cells. An exciting example is a recent study by the Backman group, which described abnormal nanostructure architecture in microscopically normal-appearing buccal epithelial cells from lung cancer patients using a novel optical technology, partial wave spectroscopic microscopy, which is exquisitely sensitive to the nanoarchitectural manifestation of the genetic/epigenetic alterations of field carcinogenesis \[[@b82-cancers-03-02975]\]. This suggests the possibility that the histologically normal airway epithelium harbors potentially powerful biomarkers that await discovery. Thus, the combination of epithelium cell enrichment, more sensitive detection methods and novel field cancerization biomarkers will lead to the discovery of more accurate and reliable sputum-based biomarkers that can be used for the early detection of lung cancer.

{ "pile_set_name": "PubMed Central" }

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[ "Introduction", "Lung", "cancer", "leading", "cause", "cancer", "deaths", "men", "women", "United", "States", "overall", "survival", "rate", "persisted", "decades", "two", "types", "lung", "cancer", "small", "cell", "lung", "cancer", "SCLC", "cell", "lung", "cancer", "NSCLC", "lung", "cancers", "NSCLC", "divided", "three", "main", "types", "large", "cell", "carcinoma", "squamous", "cell", "carcinoma", "adenocarcinoma", "lung", "Adenocarcinoma", "lung", "single", "common", "type", "lung", "cancer", "accounts", "lung", "cancers", "Although", "NSCLC", "grows", "slowly", "less", "aggressive", "SCLC", "efficient", "treatment", "NSCLC", "considered", "surgical", "resection", "tumor", "SCLC", "remarkably", "sensitive", "chemotherapy", "radiation", "treatments", "low", "survival", "rate", "NSCLC", "patients", "related", "late", "presentation", "patients", "unresectable", "tumor", "due", "lack", "validated", "screening", "approach", "early", "detection", "early", "detection", "precancerous", 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Introduction {#sec0005} ============ Meningococcal disease may present as meningococcal meningitis, meningococcal meningitis with meningococcemia, and isolated meningococcemia. It is a disease caused by the bacterium *Neisseria meningitidis*. Even though there are 12 bacterial serotypes, invasive meningococcal disease is associated with six, classified based on the polysaccharide capsule (A, B, C, W-135, X, Y) \[[@bib0005]\]. The bacterium colonizes the nasopharynx of asymptomatic carriers up to about 5--10 % of the population, being transmitted from person to person by respiratory secretions \[[@bib0010]\]. In Brazil, meningococcal disease is endemic with periodic outbreaks of epidemics in several municipalities. The incidence coefficients have remained stable in recent years, with approximately 1.8 cases per 100,000 inhabitants \[[@bib0015]\]. In São Paulo city, in 2018, 204 cases were confirmed, with 36 deaths, with incidence coefficient being 1.74 and mortality 0.31 \[[@bib0020]\]. The lethality of the disease in our country has been around 18--20% in recent years, and it may achieve coefficients of almost 50 % in septic presentations \[[@bib0015]\]. Serotypes B and C cause disease predominantly in Europe and the Americas, being the most common in Brazil \[[@bib0015]\]. Although the most common clinical manifestations are fever and acute and rapidly progressive petechial and/or purpuric rash, progressing to septic shock within a few hours, there are some chronic, initially more benign presentations, such as chronic meningococcemia \[[@bib0025]\]. This form represents less than 5% of all cases of meningococcemia, with incidence \<0.05 cases per 100,000 inhabitants per year in developed countries \[[@bib0030]\]. It is a rare manifestation of meningococcal disease without meningitis, of prolonged course of more than one week, with intermittent or continuous fever, arthralgia and cutaneous vasculitis \[[@bib0005]\]. Few cases of this presentation have been reported, both in previously healthy and immunocompromised patients \[[@bib0025],[@bib0035]\]. A case of chronic meningococcemia diagnosed in a vertically infected adolescent with human immunodeficiency virus (HIV) infection will be described, providing a review of the literature on the topic. Its importance lies in the description of a rare disease with atypical manifestation, which is difficult to suspect, and which may be life-threatening if not diagnosed and treated promptly and adequately. This case report was approved by local ethics committee; the patient signed the consent form, with permission to disclose images. Clinical case {#sec0010} ============= Adolescent, 19 years old, male, vertically HIV-infected, followed up since 3 months of age at our Pediatric Infectious Disease Clinical Care Center. Not adherent to the antiretroviral treatment, he has presented several clinical intercurrences during his life. At the time of this event, there was significant viral replication (HIV viral load 87,802 copies/μL) and severe immunosuppression (CD4+ count 17 cells/μL). He came to our medical center with 5 days of prostration, epigastric pain, sparse petechiae, joint pain, ocular pain and conjunctival hyperemia. Fever at the beginning for three days. Initially, he sought another service and the laboratory tests performed detected leukocytosis 19,600/ul (81 % neutrophils), and thrombocytopenia 77,000/ul. He received intravenous hydration and was discharged with symptomatic medication. In our service, he was in good general condition, pale, presenting conjunctival hyperemia, some petechiae on the trunk and limbs, without other signs (see Pictures [1](#fig0005){ref-type="fig"}[--](#fig0010){ref-type="fig"}[3](#fig0015){ref-type="fig"} ). Laboratory tests were performed: leukocytes 4330/ul (53 % neutrophils, 33 % lymphocytes) and improved thrombocytopenia 124,000/uL. As there is epidemiological conditions for arboviruses in our country, serology and blood viral research by polymerase chain reaction (PCR) were done for dengue, zika and chikungunya -- all tested negative; serology tested non-reactive for toxoplasmosis and syphilis; for cytomegalovirus serology tested IgG reagent IgM non-reactive. He received symptomatic treatment with partial clinical improvement, keeping skin lesions unchanged. At this time, he was afebrile.Picture 1Right eye conjunctival hyperemia.Picture 1Picture 2Petechiae on the soles of the feet.Picture 2Picture 3Petechia on the arms.Picture 3 Sixteen days after the onset of symptoms, he presented a return of prostration, fever and disseminated hemorrhagic suffusions (see [Pictures 4 and 5](#fig0020){ref-type="fig"}, [Pictures 4 and 5](#fig0020){ref-type="fig"}). He was hospitalized in septic shock, and during the diagnostic investigation, two blood cultures were positive for *Neisseria meningitidis* C, eighteen days since disease onset.Pictures 4 and 5Hemorrhagic suffusions on the limbs.Pictures 4 and 5 He presented normal cerebrospinal fluid examination with negative bacterioscopy and culture. The patient evolved with acute respiratory distress syndrome and acute renal failure, requiring endotracheal intubation and hemodialysis. He was hospitalized for 46 days, received 10 days of ceftriaxone 2 g 12/12 h. During hospitalization, HIV viral load 382,863 copies/μL and CD4+ count 13 cells/μL. He progressively evolved with improvement, being discharged in good clinical conditions, receiving combined antiretroviral therapy. The vaccine card was checked, he had received two doses of the meningococcal conjugate vaccine C before this clinical complication: 15 years before with HIV viral load 146,000 copies/μL and CD4+ count 762 cells/μL and 3 years before with HIV viral load 130,214 copies/μL and CD4+ count 75 cells/μL. Currently, the patient presents good adherence to the antiretroviral medication, with an improvement in CD4+ cell count (445 cells/μL) and a decrease in viral load (155 copies/μL); he has cutaneous scars derived from meningococcal vasculitis (see [Picture 6](#fig0025){ref-type="fig"})Picture 6Cutaneous scars on the limbs.Picture 6 Discussion {#sec0015} ========== We describe a case of a vertically HIV-infected adolescent with chronic meningococcal disease. Chronic meningococcemia is a disease caused by the bacterium *Neisseria meningitidis*, characterized by a period of more than one week of intermittent or continuous fever, arthralgia and skin lesions without meningitis \[[@bib0005]\]. Initial erythema develops to petechiae and purpura due to dermal microvascular thrombosis and perivascular hemorrhage \[[@bib0040]\]. Other clinical manifestations may occur, such as myalgia, abdominal pain, weight loss, iritis, retinitis \[[@bib0045]\]. It may be a self-limiting disease, but meningitis and death may occur as late complications \[[@bib0050]\]. These manifestations mimic several other infections, namely arboviruses, which was a confounding factor in our patient, delaying the diagnosis. The gold standard diagnostic method is culture isolation of *Neisseria meningitidis* in sterile material \[[@bib0055]\]. Even if the course seems to be self-limited, the fact that the patient may remain as an asymptomatic carrier and thus develop an invasive disease or spread the agent has tended to favor antimicrobial treatment \[[@bib0060]\]. The suggested treatment is based on the use of beta-lactams, namely 3rd generation cephalosporin, used in our patient \[[@bib0005],[@bib0030]\]. The reason why these less severe forms of the disease occur is unknown; the susceptibility of the host and the virulence of the bacterium are possible explanations \[[@bib0005],[@bib0065]\]. Although there seems to be a greater association with serotype B \[[@bib0005]\], serotype C was isolated in our patient. The disease can occur in previously healthy individuals or with some immunodeficiency \[[@bib0025],[@bib0035],[@bib0045]\]. After a literature review, we identified only four cases of chronic meningococcemia in horizontally HIV-infected adult patients since 1990, none with this serious evolution \[[@bib0030],[@bib0045],[@bib0070]\]. A recent study found a substantial increased risk of meningococcal disease among adults with HIV infection that met AIDS criteria; in this study they observed a similar clinical presentation and outcomes of meningococcal disease compared to those without HIV infection \[[@bib0075]\]. The increased relative risk observed for meningococcal disease was similar to those observed for HIV-infected individuals in New York City, South Africa and England, ranged from 3.4--6.6 per 100,000 (relative risk = 5--13 compared with HIV-uninfected persons) \[[@bib0075], [@bib0080], [@bib0085], [@bib0090]\]. Among HIV-infected persons, a low CD4 count or high viral load were associated with an increased risk \[[@bib0085]\]. Considering this increased risk, in 2016 the US Advisory Committees on Immunization Practices approved a recommendation for routine vaccination of HIV-infected person \[[@bib0095]\]. In Brazil, meningococcal conjugate vaccine type C is routinely recommended for HIV-infected patients. With this clinical case we intend to highlight the heterogeneity and low specificity of the symptoms that meningococcal disease may present, especially in immunocompromised hosts, leading to a possible diagnostic delay of a potentially fatal disease. The association between *Neisseria meningitidis* infection and HIV infection is not yet well defined. Due to the potential for progression and the risk of *N. meningitidis* transmission, a better understanding of the association between HIV infection and meningococcal disease is important to prevention strategies. Ethical approval {#sec0020} ================ Written informed consent was obtained from the patient for publication of this case report and accompanying images. This case report was approved by local ethics committee. Funding {#sec0025} ======= This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CRediT authorship contribution statement {#sec00005} ======================================== **Rita S.B. Cardona:** Data curation, Formal analysis, Writing - original draft. **Fabiana Bononi do Carmo:** . **Suenia Vasconcelos Beltrão:** . **Aída de Fátima T. Barbosa Gouvêa:** . **Reinaldo Salomão:** . **Regina Célia de Menezes Succi:** Supervision, Writing - review & editing. **Daisy Maria Machado:** Conceptualization, Writing - review & editing. Declaration of Competing Interest ================================= The authors have no conflict of interests, personal or financial, to declare. The authors would like to acknowledge the entire multidisciplinary team that took care of the patient during hospitalization.

{ "pile_set_name": "PubMed Central" }

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"highlight", "heterogeneity", "low", "specificity", "symptoms", "meningococcal", "disease", "may", "present", "especially", "immunocompromised", "hosts", "leading", "possible", "diagnostic", "delay", "potentially", "fatal", "disease", "association", "Neisseria", "meningitidis", "infection", "HIV", "infection", "yet", "well", "defined", "Due", "potential", "progression", "risk", "meningitidis", "transmission", "better", "understanding", "association", "HIV", "infection", "meningococcal", "disease", "important", "prevention", "strategies", "Ethical", "approval", "Written", "informed", "consent", "obtained", "patient", "publication", "case", "report", "accompanying", "images", "case", "report", "approved", "local", "ethics", "committee", "Funding", "research", "receive", "specific", "grant", "funding", "agencies", "public", "commercial", "sectors", "CRediT", "authorship", "contribution", "statement", "Rita", "Cardona", "Data", "curation", "Formal", "analysis", "Writing", "original", "draft", "Fabiana", "Bononi", "Carmo", "Suenia", "Vasconcelos", "Beltrão", "Aída", "de", "Fátima", "Barbosa", "Gouvêa", "Reinaldo", "Salomão", "Regina", "Célia", "de", "Menezes", "Succi", "Supervision", "Writing", "review", "editing", "Daisy", "Maria", "Machado", "Conceptualization", "Writing", "review", "editing", "Declaration", "Competing", "Interest", "authors", "conflict", "interests", "personal", "financial", "declare", "authors", "would", "like", "acknowledge", "entire", "multidisciplinary", "team", "took", "care", "patient", "hospitalization" ]

Saliva research =============== The need to advance saliva research is strongly recognized by the Strategic Plan of the National Institute of Dental and Craniofacial Research.^[@bib1]^ The National Cancer Institute has also recognized saliva as a promising cancer biomarker source.^[@bib2]^ The ability to monitor health status, disease onset, progression, recurrence and treatment outcome through non-invasive means is highly important to advancing health care management. Saliva (oral fluid) is a perfect medium to be explored, offering the potential for a non-invasive, easy to obtain means for detecting and monitoring disease. The adoption of saliva testing would allow a patient to collect their own specimens at home, yielding savings in health costs, convenience for the patient and facilitating multiple sampling. Specimen collection is less objectionable to patients than in the case of other bodily fluids and easier in children and older individuals. The analysis of saliva can thus provide a cost-effective approach for the screening of large populations. Due to these significant advantages, developing biomarkers in saliva for the detection of serious illnesses such as oral and systemic cancers has been on the national healthcare agenda for several years (Government Performance & Results Act 2008).^[@bib3]^ One mandate formulated in the Government Performance & Results Act report is that by the year 2013 proof of principle will be obtained for the ability of saliva to monitor health and diagnose for one systemic disease. Challenges and opportunities ============================ A vast amount of saliva omics data has been generated by recent studies using high throughput technologies.^[@bib4],[@bib5],[@bib6],[@bib7]^ However, there are still barriers which researchers must overcome before such data can be exploited, such as lack of computationally accessible salivary data and information, and inability to cross-reference the salivaomics data that could potentially be made available through different proteomics, transcriptomics, genomics and metabolomics studies. For these reasons, there is an urgent need to create the Salivaomics Knowledge Base (SKB), a data management system and web resource constructed to support saliva diagnostics research, and we present below the informatics advances brought about through the SKB and through the associated tools and resources. Biomedical ontology =================== Ontologies are controlled structured vocabularies designed to provide consensus-based means to ensure consistent description of data by scientists working in disparate domains. As applied in the biomedical domain, ontology plays a key role in providing consensus-based controlled vocabularies serving the consistent annotation of biological and medical data and information, most conspicuously within the framework of the Gene Ontology^[@bib8]^ and now of its sister ontologies within the Open Biomedical Ontologies Foundry (<http://obofoundry.org>). The Basic Formal Ontology (BFO) is a formal ontological framework developed by Barry Smith, Pierre Grenon and others, which serves as the starting point for some 100 ontology projects primarily in the biomedical domain (<http://www.ifomis.uni-saarland.de/bfo/>). The BFO framework can be readily extended to the treatment of families of ontologies of other types, above all to the treatment of relations between ontologies of different levels of granularity, from genes to species and from a single patient to epidemics at a geographical scale (combining applications of BFO to the medical and to the geographical domain). The framework may also be used as a tool for dealing with the relations between distinct perspectives on the biomedical domain, including culturally generated perspectives of the sort which are studied by linguists and anthropologists.^[@bib9]^ Two BFO-based ontologies of special significance for our work here are the Ontology for Biomedical Investigations (OBI)^[@bib10]^ and the Ontology for General Medical Sciences. OBI addresses the need for a controlled vocabulary to support integration of experimental data. The OBI is an ontology designed to serve the coordinated representation of designs, protocols, instrumentation, materials, processes, data and types of analysis in all areas of biological and biomedical investigation. Ontology for General Medical Science is an ontology of the entities involved in the clinical encounter. Thus, it includes very general terms that are used across medical disciplines, including: 'disease\', 'disorder\', 'disease course\', 'diagnosis\', 'patient\' and 'healthcare provider\'.^[@bib11]^ A dental research ontology consortium ===================================== To advance the consistency of data in the dental research community, Smith *et al.*^[@bib12]^ propose an approach to building a consensus-based ontology to support dental research (ODR). In analogy to efforts in other fields,^[@bib11]^ a consortium of research groups specializing in different areas of study would undertake such an effort, each building different components of ontology to support dental research. Initial efforts in this direction, by scientists in dental research and biomedical ontology at University at Buffalo and University of California, include work on the ontology of oral pathology, oral maxillofacial anatomy, dental disease and dental procedures, and as we discuss below, the Saliva Ontology.^[@bib13]^ Integral to his work is a plan to allow a seamless connection between the use of ontology to support dental research in the dental domain and the use of existing ontology resources developed in other areas of biology and medicine, by reusing elements and strategies from them. The anatomy work is based on the Foundational Model of Anatomy. The ontology of dental procedures use the framework established by OBI.^[@bib14]^ The work on dental diseases is carried out in conjunction with the development of Ontology for General Medical Science.^[@bib11]^ Saliva ontology =============== The Saliva Ontology (SALO) ([Figure 1](#fig1){ref-type="fig"}) is a detailed ontology of this bodily fluid that is optimized to meet the needs of both the clinical diagnostic community and the cross-disciplinary community of omics researchers. The SALO is created through cross-disciplinary interaction with saliva experts, protein experts, diagnosticians and ontologists. To aid development and testing of SALO, we develop a corpus of saliva-relevant literature in SKB to assist in characterizing core terms and synonyms within the ontology and to provide links between SALO content and relevant items in PubMed. SKB will also incorporate the results of experiments in data and text mining using the ontology. SALO will incorporate links to existing ontologies and terminology resources involving treatment of saliva-relevant phenomena. We will also identify and represent within SALO relationships to saliva-relevant types represented in ontologies such as the Gene Ontology, the Protein Ontology^[@bib15]^ and the Chemical Entities of Biological Interest ontology,^[@bib16]^ and also provide links to corresponding SNOMED CT terms where available. SALO is a public domain resource and entirely web-based. Each term in the ontology has its own URL which points to a webpage providing definitions, PubMed sources, references to annotations to SKB and to external databases. SALO and Blood Ontology^[@bib17]^ are the foundation for a unified body fluids ontology resource---Body Fluids Ontology.^[@bib18]^ BioMart ======= BioMart is a free, open-source, federated database system. It is cross-platform and supports many popular relational database managements systems, including MySQL, Oracle, PostgreSQL, SQL Server and DB2. The software is data-agnostic, and can therefore be easily adapted to existing data sets. It is expandable and customizable through a plug-in system, and is open-source so the community can participate in deeper development. Furthermore, BioMart can seamlessly connect geographically disparate databases, facilitating collaboration between different groups. These features have catalyzed the creation of BioMart Central Portal, a first of its kind community-supported effort to create a single access point integrating many different, independently administered biological databases. Anybody can contribute an independently maintained resource to the Central Portal, allowing it to be exposed to and shared with the research community, and linking it with the other resources in the portal. Users can take advantage of the common interface to quickly utilize different sources without learning a new system for each. The system also simplifies cross-database searches that might otherwise require several complicated steps. Several integrated tools streamline common tasks, such as converting between ID formats and retrieving sequences. The combination of a wide variety of databases, an easy-to-use interface, robust programmatic access and the array of tools make Central Portal a one-stop shop for biological data querying.^[@bib19]^ SDxMart---a BioMart portal for salivaomics data =============================================== SDxMart is a BioMart data portal that hosts salivary proteomic, transcriptomic, metabolomic and microRNA data and offers access to the data by using the BioMart interface and querying environment. The SDxMart is designed to provide a variety of queries to facilitate saliva biomarker discovery including complex queries that integrate genomic, clinical and functional information. The SDxMart holds data from projects of oral diseases and systemic diseases including oral cancer, Sjögren\'s syndrome, pancreatic cancer and breast cancer. The types of datasets are: (i) proteomics; (ii) transcriptomics; (iii) microRNA; and (iv) metabolomics. In addition, the SDxMart is imported with several public databases including Ensembl genome database (Ensembl release 37),^[@bib20]^ and the number of resources is continuously growing. Summary ======= The SKB is being created to facilitate researchers using salivary data from multiple perspectives. It is being built in tandem with the SALO and SDxMart which will allow the SKB to interoperate with other omics databases as part of a general strategy to facilitate integration of heterogeneous and disparate data sources that enable system biology approaches. Either SALO or SDxMart is a first and only resource of its kind in the field of dentistry. This work was supported by the US National Institutes of Health (grant number U01DE017790), the Felix & Mildred Yip Endowed Professorship and the Barnes Family Trust Fund. ![**A fragment of the basic Saliva Ontology in its current form.**](ijos201226f1){#fig1}

{ "pile_set_name": "PubMed Central" }

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22,183

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"Ontology", "current", "form" ]

Moriuchi et al. reported a comprehensive reclassification of bacterial strains from the genera *Cupriavidus* and *Ralstonia* based on percentage of conserved proteins (POCP), average nucleotide identity (ANI), multilocus sequence analysis and 16S rRNA gene sequence. In the study, conflicting results were repeatedly observed for the taxonomic classification of strain PBA that was initially identified as *Ralstonia* sp. PBA based on 16S rRNA gene sequence (Gan et al., [@B4]; Moriuchi et al., [@B8]). Strain PBA was isolated as a co-culture with *Hydrogenophaga intermedia* PBC from textile wastewater a decade ago. The co-culture could grow on 4-aminobenzenesulfonate (4-ABS), a recalcitrant dye intermediate (Wagner and Reid, [@B12]), as the sole nitrogen, carbon, and sulfur source to a relatively high cell density (Gan et al., [@B4]). In this syntrophic relationship, strain PBA is the sole provider of *p*-aminobenzoate, an essential vitamin required for the growth of *H. intermedia* PBC, the main 4-ABS degrader (Gan et al., [@B2], [@B3]). In light of new genomic resources, the initial taxonomic assignment of strain PBA has also been previously questioned by Kim and Gan ([@B6]) given its closer phylogenetic affiliation to the genus *Cupriavidus* than to the genus *Ralstonia*. Unfortunately, both recent genome-based taxonomic classifications of strain PBA (Kim and Gan, [@B6]; Moriuchi et al., [@B8]) suffered from incomplete and biased taxon sampling (restricted mostly to members from the genus *Ralstonia* and *Cupriavidus*) that can result in the misinterpretation of evolutionary relationships (Heath et al., [@B5]). The taxonomic affiliation of strain PBA should be inferred from a comprehensive phylogenomic analysis that includes all genera with genome availability from the family Burkholderiaceae. A total of 428 Burkholderiaceae (including strain PBA) and 15 non-Burkholderiaceae genome assemblies were obtained from the NCBI RefSeq database (accessed on 30th May 2019). The genomes were processed using two microbial phylogenomic analysis pipelines e.g., GToTree v1.2.1 (Lee, [@B7]) and PhylophlAN v0.99 (Segata et al., [@B11]) that identify single copy bacterial genes (GToTree: *n* = 203, Betaproteobacteria HMM set; PhylophIAN: *n* = 400) and produce concatenated protein alignment. Maximum likelihood tree construction from the protein alignments used IQTree v.1.6.8 with 1,000 ultrafast bootstrap replicates (Nguyen et al., [@B9]). In both phylogenomic trees, the *Ralstonia* and *Cupriavidus* clusters received maximal support and are sister taxa to the exclusion of strain PBA ([Figures 1A,B](#F1){ref-type="fig"}). The updated phylogenomic placement of strain PBA in light of extensive taxon sampling precludes its genus assignment to the genus *Ralstonia* or *Cupriavidus* and suggests that it is a member of a hitherto undescribed genus within the family *Burkholderiaceae*. Within the Genome Taxonomy Database (Parks et al., [@B10]) that inferred standardized bacteria taxonomy from conserved proteins present in 143,512 bacterial genomes (GTDB release R04-RS89), strain PBA was still assigned to its own genus (g\_\_AKCV01) despite an even more extensive taxon sampling of 4,378 genomes from the family *Burkholderiaceae* (<https://gtdb.ecogenomic.org/tree?r=g__AKCV01> accessed on 1st August 2019). ![Genome-based phylogeny of Burkholderiaceae. Construction of the phylogenomic trees used protein alignments generated from **(A)** PhyloPhlAN and **(B)** GToTree. Numbers in brackets indicate number of taxa in the collapsed clades. GCF codes preceding the species names are RefSeq Assembly numbers. The positions of strain PBA in both trees are shown by black arrows. Branch lengths and labels indicate the number of substitutions per site and IQTree ultrafast bootstrap support values, respectively. The uncollapsed trees are available in the Zenodo database (<http://doi.org/10.5281/zenodo.3258920>).](fmicb-10-02011-g0001){#F1} Given the concordance observed from these independent analyses, the taxonomic assignment of strain PBA has been updated from *Ralstonia* sp. PBA to *Burkholderiaceae* sp. PBA in the NCBI database (Bioproject: PRJNA78957; BioSample: SAMN02471424) (Gan et al., [@B1]) pending future genus description. To facilitate future strain description and comparison, strain PBA has been deposited in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) under the accession number [DSM 106616](DSM106616). Furthermore, the concatenated alignments, uncollapsed phylogenomic trees and genome information are also made available in the Zenodo database (<http://doi.org/10.5281/zenodo.3258920>). Author Contributions {#s1} ==================== HG performed data analysis and wrote the manuscript. Conflict of Interest Statement ------------------------------ The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author is grateful to the Deakin Centre of Integrative Ecology for financial support. **Funding.** This research was supported by the Deakin Centre of Integrative Ecology. [^1]: Edited by: Iain Sutcliffe, Northumbria University, United Kingdom [^2]: Reviewed by: Roberta Fulthorpe, University of Toronto Scarborough, Canada; Zhang Yong, Southwest University, China; Jorgen Johannes Leisner, University of Copenhagen, Denmark [^3]: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology

{ "pile_set_name": "PubMed Central" }

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22,184

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Key messages {#Sec1} ============ What is already known about this subject?o The phenotype of adult-onset Still's disease appears to be dichotomized into systemic or chronic articular forms.o Biological treatments are dominated by IL-1 or IL-6 receptor inhibitors.What are the new lines of research suggested by this study?o The presence of arthritis and a chronic articular phenotype may be associated with a preferential response to tocilizumab, whereas the systemic form may be associated with a preferential response to anakinra.o Tocilizumab increases the likelihood of corticosteroid withdrawal regardless of delays in its initiation or when it is initiated relative to other treatment in the overall therapeutic strategyHow might this impact clinical practice?o These results should help us better codify the treatment strategy for adult-onset Still's disease. Introduction {#Sec2} ============ Adult-onset Still's disease (AOSD) is a rare systemic inflammatory disorder. Its annual incidence is estimated to be between 0.16 and 0.62 per 100,000 persons globally \[[@CR1]\], and its prevalence approximately 1--24 per million \[[@CR2]\]. AOSD is an autoinflammatory disease resulting from multifactorial causes such as genetic factors and immunological imbalances involving macrophage, neutrophil, and inflammasome hyperactivation or dysfunction \[[@CR3]\]. Recent studies suggest that there is a dichotomy between two major phenotypes of AOSD: a systemic pattern that presents with an acute onset associating high fever, skin rash, hematopoietic signs, and sometimes visceral damage; and a chronic articular profile in which polyarthritis prevails \[[@CR4], [@CR5]\]. This phenotypic dichotomy is highlighted by recent studies showing that there are different cytokine imbalances in these two phenotypes. Although results may vary, this imbalance would sometimes favor interleukin (IL)-1β, IL-18, and interferon (IFN)-γ production in the systemic form, whereas in the chronic articular form, IL-6, IL-8, tumor necrosis factor (TNF)-α, and IL-17 production would predominate \[[@CR6]--[@CR12]\]. The prognosis of AOSD is unpredictable as it can manifest with polyarticular erosive disease or life-threatening manifestations such as reactive hemophagocytic lymphohistiocytosis (rHLH). The disease course is also very polymorphic, ranging from a monophasic flare to polycyclic or chronic forms. This clinical and prognostic polymorphism presents a major challenge and makes it difficult to predict the outcome at the time of diagnosis and to determine appropriate management. Therapy is therefore usually empirical, and beside methotrexate, biologics have played an increasing role in the chronic or refractory forms. A recent meta-analysis suggested that IL-1 and IL-6 receptor inhibitors are the most effective biotherapies for AOSD \[[@CR13]\] and have a strong steroid-sparing effect. The systemic phenotype has been suggested to respond preferentially to IL-1 receptor inhibitors, such as anakinra, while the chronic articular form shows better responses to IL-6 receptor inhibitors, such as tocilizumab \[[@CR6], [@CR7], [@CR14]--[@CR17]\]. This exploratory study therefore aimed to identify initial clinical and/or biological factors that were predictive of a preferential response to anakinra or tocilizumab. Secondary objectives were to investigate whether the choice of biotherapy (anakinra or tocilizumab) and their delay in initiation influenced the likelihood of steroid discontinuation and to describe the clinical and biological phenotypes of patients with AOSD. Methods {#Sec3} ======= Study design and data collection {#Sec4} -------------------------------- A multicenter exploratory retrospective study of all patients diagnosed with AOSD at Bordeaux teaching hospital and Pau and Dax peripheral hospitals until 2018 was conducted. Patients included had to be 16 years old or older, to be diagnosed with AOSD, to meet the Yamaguchi and/or Fautrel criteria, to have a chronic or refractory disease course, and to receive or have received a biological disease-modifying antirheumatic drugs (bDMARD). Exclusion criteria were age younger than 16 years old, a diagnosis of systemic-onset juvenile idiopathic arthritis (SoJIA), an exclusion of AOSD diagnosis during disease course, Yamaguchi and Fautrel criteria not met, lack of data, and absence of bDMARD during disease course. Patients with AOSD were identified via ICD-10 code, via a diagnostic grid that automatically references autoimmune and inflammatory diseases at Bordeaux University Hospital, via the bioinformatics services of each hospital, and via direct contact with the specialists treating the patients. Data regarding clinical and biological characteristics at diagnosis and treatment details were collected using a standard data extraction form. All the information was double-checked for any discrepancies. The following demographic and clinical AOSD-related characteristics were collected: sex, date of birth, age at diagnosis, year of diagnosis, duration of disease, duration of disease at first bDMARD initiation, smoking habits, personal or familial history of rheumatic autoimmune inflammatory or neoplastic disease, type of Still's disease-related rash if present, arthralgia, arthritis, hands affected or not, early morning stiffness, myalgia, fever characteristics, general deterioration and weight loss \> 4 kg, lymphadenopathy, pharyngitis, angina, and visceral involvement (pleuritis, pericarditis, pulmonary hypertension). The following biological characteristics were recorded: leukocytosis \> 10,000/mm^3^, neutrophilia \> 8000/mm^3^, eosinophilia \> 500/mm^3^, anemia with hemoglobin \< 12 g/dl, thrombocytosis \> 400,000/mm^3^, increased C-reactive protein (CRP) levels, maximum CRP at diagnosis, decreased albumin, increased liver enzymes, increased creatine kinase, increased lactate dehydrogenase (LDH), increased ferritin, decreased glycosylated ferritin, autoimmunity markers (antinuclear antibodies (ANA), antideoxyribonucleic acid, anti-extractable nuclear antigen, rheumatoid factor, antiβ2GP1, lupus anticoagulant, anticardiolipin), disseminated intravascular coagulation markers, and rHLH markers (cytopenia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis in the bone marrow). Patients were classified as having two distinct disease patterns: the systemic form or the chronic articular form. In the absence of consensual criteria in this regard, this classification was based on expert opinions that was often spelled out in the medical reports and was confronted to disease course outcome, which was available due to the retrospective nature of this study. Outcome measurements {#Sec5} -------------------- The effectiveness of treatment was defined as follows: effective treatment was considered when all initial symptoms and biological anomalies had resolved, partially effective treatment was considered when all but one initial symptom or biological anomaly had resolved, and ineffective treatment was considered when two or more initial symptoms or biological anomaly persisted. The attending doctors assessed disease flares and relapses. Treatment lines were assessed as follows: nonsteroidal anti-inflammatories (NSAIDs) were not considered a treatment modality for chronic and refractory AOSD, but corticosteroids, conventional synthetic disease-modifying antirheumatic drugs (csDMARD) and especially methotrexate (both galenic forms taken together) and cyclosporine, and bDMARD (anakinra, tocilizumab, canakinumab, infliximab, and etanercept) were considered treatment lines. The outcome of interest for the univariate analysis of predictive factors was the full effectiveness of the treatment as defined above, including both systemic and articular manifestations. Statistical analysis {#Sec6} -------------------- Quantitative variables are presented as mean with standard deviation or as median with interquartile range, and qualitative variables are presented as percentages. Analysis was performed on the following variables: all the clinical and biological manifestations included in the Yamaguchi or Fautrel criteria, common demographic characteristics, and all the symptoms or biological abnormalities usually observed during AOSD according to the literature and our personal experience. The nonparametric Mann-Whitney test was used to perform a univariate analysis of quantitative variables, and the Fisher exact test was used for qualitative variables. Calculations were performed with STATA 13.1 SE software. *P* values less than 0.05 were considered significant. Results {#Sec7} ======= Patients' characteristics {#Sec8} ------------------------- Twenty-seven patients were included in this study: 21 from Bordeaux teaching hospital, 4 from Dax hospital, and 2 from Pau hospital. Patient characteristics at diagnosis are described in Table [1](#Tab1){ref-type="table"}.Table 1Characteristics of the patients included in the study All patients fulfilled both the Yamaguchi and Fautrel criteria. No patients had any personal or familial history of rheumatic, autoimmune, inflammatory, or neoplastic disease. All patients presented with a fever above 38 °C, mainly intermittent and vesperal fevers. The next most prevalent symptoms were arthralgia (96%), arthritis (52%), skin rash (81%), general deterioration (72%), and ear-nose-throat (ENT) symptoms (69%). Eleven (40.7%) patients were classified as systemic form of disease, and 16 (59.3%) as chronic articular form. All patients presented with elevated CRP levels. The next most commonly observed anomalies were neutrophilia (96%), low albumin (75%), and increased ferritin (73%). No patients presented with increased creatine kinase (CK), positive extractable nuclear antigen (ENA), positive antineutrophil cytoplasmic antibody (ANCA), positive anticitrullinated protein antibody (ACPA), or positive anticardiolipin. Nine patients were either ANA-positive (five, 1/160), anti-DNA positive (one), or rheumatoid factor (RF)-positive (three) at diagnosis, but these results were judged clinically insignificant because lower than two times the upper normal range and never associated with symptoms concordant with SLE, APLS, or RA and were all controlled to be negative. Furthermore, all patients included in the study met both Yamagushi and Fautrel criteria. The median duration of disease was 5 years (IQR 2--7) and 6.5 years (IQR 5--9) for the systemic and chronic articular forms, respectively. All patients presented a chronic refractory form of the disease, which required the off-label use of bDMARD because of failures of corticosteroids and csDMARD or necessity to reduce or stop corticosteroids. Biological treatment efficacy {#Sec9} ----------------------------- Table [2](#Tab2){ref-type="table"} presents the detailed responses to anakinra and tocilizumab among the patients included in the study. Twenty-six of 27 patients (96.3%) achieved remission with either anakinra or tocilizumab. The median delay in the introduction of the first biotherapy was 5 months (interquartile range (IQR), 1--21 months): this delay was median 1 month (IQR, 0--6) in the systemic form and median 17 months (IQR, 4--38) in the chronic articular form.Table 2Delay in corticosteroids withdrawal and detailed responses to anakinra and tocilizumab among the patients included in the study A total of 15 patients received anakinra; 5 as a first-line treatment, 4 as a second-line treatment, 5 as a third-line treatment, and 1 as a fourth-line treatment. The median delay between diagnosis and the introduction of anakinra was 1.5 months (IQR, 0--14). Thirteen (86.7%) patients were responders. The 2 anakinra nonresponders received anakinra as a third-line treatment: one had previously failed to respond to tocilizumab, and the other later responded to tocilizumab and even managed to stop it. A total of 17 patients received tocilizumab, 2 as a first-line treatment, 6 as a second-line treatment, 5 as a third-line treatment, and 4 as a fourth-line treatment. The median delay between diagnosis and the introduction of tocilizumab was 17 months (IQR, 4--39 months). Fourteen patients (82.4%) were responders. Among the 3 tocilizumab nonresponders, one presented with a systemic form and received tocilizumab as a first-line treatment but later responded to anakinra, one presented with a systemic form and received tocilizumab as a fourth-line treatment after responding to anakinra but was switched because of a suspicion of anakinra-induced toxidermia, and one presented with a chronic articular form and received tocilizumab as a fourth-line treatment after failure of anakinra. The difference of median duration of disease at the introduction of the first bDMARD in the anakinra-treated patients group and in the tocilizumab-treated patients group was statistically significant (*p* = 0.4457): median 2 months (IQR, 0.25--12 months) in the anakinra-treated patients and median 14.5 (IQR, 3.25--38.25 months) in the tocilizumab-treated patients. Eight patients (29.6%) managed to stop their biological treatment without relapse at the last known visit which ranged from 6 to more than 24 months after biological treatment discontinuation: 3 of 13 (23%) anakinra responders and 5 of 14 (35.7%) tocilizumab responders. The difference in delays in initiation of anakinra or tocilizumab was not statistically significant between those who managed to stop their biotherapy without relapse versus those who could not stop their biotherapy or those who relapsed, with a median delay of 3 months (IQR, 1.25--13.75) versus 8.5 months (IQR, 1--23.25), respectively (*p* = 0.61). Five patients received both biotherapies. Two of these patients presented with a systemic form and were tocilizumab nonresponders but anakinra responders, one presented with a chronic articular form and was anakinra nonresponder but tocilizumab responder, one presented with a chronic articular form and did not respond to either biotherapy, and one patient presented with a systemic form and responded to both biotherapies; this patient first responded to anakinra but corticosteroids could not be tapered so he was switched to tocilizumab in order to successfully stop corticosteroids. One patient received canakinumab and failed to respond but was anakinra responder. Three patients received antitumor necrosis factor (TNF) agents but these treatments were not efficacious (2 etanercept, 1 infliximab): two of them presented a chronic articular form and one a systemic form. One of 27 (3.7%) patients did not respond to either biotherapy (anakinra then tocilizumab) and died of a probably infectious-induced AOSD flare. Predictive factors for preferential responses to anakinra or tocilizumab {#Sec10} ------------------------------------------------------------------------ Patients who responded to either biotherapies or no biotherapy were excluded from this analysis (*n* = 2). Table [3](#Tab3){ref-type="table"} describes the univariate analysis for potential predictive factors of a good therapeutic response to anakinra or tocilizumab. The outcome of interest was the full effectiveness of the treatment, defined as the resolution of all initial systemic and articular symptoms and biological anomalies.Table 3Univariate analysis of predictive factors for a good therapeutic response to anakinra or tocilizumab (*n* = 25) A chronic articular phenotype and the presence of arthritis were associated with a substantial response to tocilizumab: 12 of the 14 patients presenting a chronic articular phenotype responded to tocilizumab whereas 3 of the 13 patients presenting a systemic phenotype responded to tocilizumab (*p* = 0.0009, OR 36 \[2.6--1703\]) and 10 of the 14 patients presenting arthritis responded to tocilizumab whereas 3 of the 13 patients without arthritis responded to tocilizumab (*p* = 0.017, OR 10 \[1.22--92.6\]). A systemic form and the absence of arthritis were associated with a substantial response to anakinra: 1 of the 14 patients presenting a chronic articular phenotype responded to anakinra whereas 9 of the 13 patients presenting a systemic phenotype responded to anakinra (*p* = 0.0009, OR 36 \[2.6--1703\]) and 3 of the 14 patients presenting arthritis responded to anakinra whereas 9 of the 13 patients without arthritis responded to anakinra (*p* = 0.017, OR 10 \[1.22--92.6\]). No difference in biological variables was statistically predictive of treatment response, although the presence of thrombocytosis seemed to be preferentially associated with a better response to tocilizumab than to anakinra (*n* = 8/14 vs. *n* = 3/13, *p* = 0.123). Likelihood of steroid withdrawal depending on the specific biotherapy and delays in initiation of biotherapy {#Sec11} ------------------------------------------------------------------------------------------------------------ All patients received corticosteroids at diagnosis. Among the 26 patients in remission, 14 (53.8%) achieved corticosteroid withdrawal within a mean delay of 27 months, 10 (71.4%) were on tocilizumab, and 4 (28.6%) were on anakinra (Table [2](#Tab2){ref-type="table"}). Table [4](#Tab4){ref-type="table"} presents the likelihood of corticosteroid withdrawal depending on treatment with anakinra and tocilizumab: tocilizumab was associated with an increased likelihood of corticosteroid withdrawal (*p* = 0.029), whereas anakinra did not seem to increase the likelihood of corticosteroid withdrawal (*p* = 1.000).Table 4Likelihood of corticosteroid withdrawal stratified by biotherapy A shorter delay before the initiation of anakinra or tocilizumab was not associated with a greater likelihood of corticosteroid withdrawal among biotherapy responders. Among anakinra responders, the difference in the median delay from diagnosis to anakinra initiation between those who achieved steroid withdrawal (*n* = 4) and those who did not (*n* = 10) was not statistically significant (*p* = 0.5767): the median delay was 2.5 months (IQR, 0--8 months) among those who did not stop corticosteroids versus 2 months (IQR, 2--14 months) among those who stopped corticosteroids. Among tocilizumab responders, the difference in the median delay from diagnosis to tocilizumab initiation between those who achieved steroid withdrawal (*n* = 11) and those who did not (*n* = 5) was not statistically significant (*p* = 0.9548): the median delay was 17 months (IQR, 9--21 months) among the tocilizumab responders who did not stop corticosteroids versus 17 months (IQR, 4--38 months) among those who stopped corticosteroids. Initiating tocilizumab as the first-line treatment rather than as a second-line or later treatment did not improve the likelihood of corticosteroid withdrawal (*n* = 1/2 vs. *n*= 11/15, *p* = 1.000). In addition, initiating tocilizumab as a first- or second-line treatment rather than as a third-line or later treatment also did not improve the likelihood of steroid withdrawal (*n* = 6/8 vs. *n* = 5/9, *p* = 0.619); similarly, initiating tocilizumab as a first-, second- or third-line treatment did not improve the likelihood of steroid withdrawal compared to that from tocilizumab used as a fourth-line treatment (*n* = 9/13 vs. *n* = 2/4, *p* = 0.584). Discussion {#Sec12} ========== This retrospective study was conducted to evaluate bDMARD strategies in the management of AOSD, with a special attention to predictive factors of their efficacy and their effect on corticosteroid sparing. All but one patient achieved remission with either anakinra or tocilizumab. Our results support the dichotomy of AOSD in two phenotypes: treatment responses to bDMARD seemed to depend on disease phenotype, the presence of arthritis, and a chronic articular phenotype were associated with a substantial response to tocilizumab, whereas the systemic form was associated with a substantial response to anakinra. In our cohort, anakinra did not increase the likelihood of corticosteroid withdrawal but tocilizumab did, regardless of delays in initiation, or when it was initiated relative to other treatment in the overall therapeutic strategy. The clinical and biological characteristics of the patients included in this study seem similar to those of AOSD patients reported in the literature \[[@CR18]\], with the exception of life-threatening conditions, which have been estimated to affect as many as 15--20% of AOSD patients, while only 8% were affected in this study. Multivariate analysis was not performed because of the sample size, a limitation inherent to rare diseases such as AOSD. As a consequence, we lacked patients who received both biotherapies in each phenotype. But rather than aiming to look for significant differences and to draw definite conclusions, the goal of this study was to establish a reference for future large-scale studies and compile data for registries to confirm the results. The distinction between systemic and chronic articular forms is not always straightforward, and no consensual criteria exist to distinguish these two phenotypes. For example, a systemic form progressing for several years, who received corticosteroids for a long time and who benefited from bDMARD late in disease course, might be confused for a chronic articular form. These results highlight the excellent efficacy of anakinra and tocilizumab in AOSD, regardless of the phenotype, but show some differences between them. However, the retrospective nature of the study is a major limitation to compare these two drugs. The choice of biotherapy may have been different depending on their availability on the year of diagnosis: anakinra was launched on the market several years before tocilizumab and the effects of anakinra and tocilizumab on AOSD were not simultaneously described. One could argue that clinicians may have influenced the choice of biotherapy by preferentially prescribing tocilizumab to patients presenting with arthritis or a chronic articular form, analogous to rheumatoid arthritis, and therefore distorted the results. Comparing these two bDMARDs is impossible outside a randomized controlled trial (RCT), and none ever demonstrated that tocilizumab should be preferentially prescribed to AOSD patients with arthritis \[[@CR5], [@CR19], [@CR20]\]. Yet the following results reinforce our findings. All but three of the patients included in this study were diagnosed in 2009 or later, and these three received anakinra or tocilizumab after both their efficacies were described in AOSD. A major result of our study is that anakinra responders more commonly did not have arthritis, as the absence of arthritis was statistically associated with a better response to anakinra (*p* = 0.017). Tocilizumab also showed less efficiency on the systemic form. Although our results cannot imply definitive conclusions on the fact that one biotherapy is more effective than the other in each phenotypes because of the lack of patients who received both biotherapy in each phenotype, this result supports our clinical feeling and the notion that even if clinicians' choices may have been influenced by analogy to rheumatoid arthritis, their insights seem to be founded. While there is no doubt about anakinra's dramatic efficacy on AOSD in general and particularly on the systemic phenotype \[[@CR2], [@CR18], [@CR19], [@CR21]--[@CR27]\], whether anakinra is effective for joint involvement is a subject of debate as data seem to be inconsistent \[[@CR4], [@CR21]--[@CR24], [@CR28], [@CR29]\]. Some studies reported a substantial efficacy \[[@CR5], [@CR23], [@CR24]\], when others suggest otherwise \[[@CR21], [@CR28], [@CR30]\]. The largest retrospective study investigating anakinra in AOSD showed a significant improvement on articular manifestations (DAS28 falling from 4.7 ± 1.2 to 1.7 ± 0.9 after 12 months) but 14.4% of the patients still presented arthritis after 12 months of treatment \[[@CR24]\]. Notably, they found no difference in delay of response to therapy between the two phenotypes and did not observe any difference in the type of response in the two patterns of the disease. In our study, 5 patients presenting with a chronic articular form received anakinra: 3 responded to anakinra and never received tocilizumab, one was an anakinra nonresponder but responded to tocilizumab, and one responded to anakinra but had to be switched to tocilizumab in order to stop the corticosteroid. Definitive conclusions about anakinra's efficacy on AOSD's joint involvement therefore seem still premature. The absence of demonstrated steroid-sparing with anakinra in this study is questionable as it demonstrated a steroid-sparing effect in several other studies \[[@CR22], [@CR23], [@CR25]\]. First, the evaluation in our study was based on the rate of steroid withdrawal, not on a steroid-sparing effect as prednisone dosage was not available. Secondly, a channeling bias exists: the acute onset of the systemic phenotype and its potential life-threatening complications might cause the clinicians to be more cautious while tapering corticosteroids, and the tocilizumab-induced fall in CRP might reassure them. Furthermore, anakinra was made available before tocilizumab: the progressively known dramatic efficacy of anakinra may have reassured the clinicians and entice them into a faster corticosteroids tapering when tocilizumab was made available. In addition, three patients presenting a systemic form in our study were able to taper corticosteroids down to 5 mg daily but the clinicians opted to maintain this dosage as a long-term therapy. Both the management and the place of corticosteroids in AOSD treatment strategy should maybe differ based on the differences between the two phenotypes. No patients in this study responded to anti-TNF blockers, whatever the phenotype. The efficacy of these medications in AOSD is controversial, but some studies suggest that they have a place in the treatment of the chronic articular form \[[@CR31]--[@CR33]\], while others have showed rather disappointing results \[[@CR19], [@CR20], [@CR34]\]. The excellent efficacy and clear steroid-sparing effect of tocilizumab determined in our study, concurring with the findings of a recent meta-analysis showing that AOSD patients treated with tocilizumab had a pooled remission rate of approximately 85% regardless of the phenotype \[[@CR35]\], and of the first RCT investigating tocilizumab in AOSD \[[@CR36]\], should make clinicians prioritize tocilizumab to anti-TNF blockers for patients with a chronic articular form of AOSD. Canakinumab showed some inconsistent results in AOSD \[[@CR37]--[@CR39]\] but is currently being investigated for its efficacy on AOSD's joint involvement (NCT02204293). No patient in this study received abatacept or rituximab; the data are very limited but some authors reported efficacy in patients who fail to respond to TNF, IL-1, or IL-6 inhibition \[[@CR40]--[@CR45]\]. Physiopathological data may highlight the efficacy of IL-17 or IL-18 blockade in AOSD. A recent study showed that IL-18 inhibition using the recombinant human IL-18-binding protein tadekinig alpha is a therapeutic option in patients with AOSD in a phase 2 open-label trial \[[@CR46]\]. IL-17 \[[@CR47]\] and INF-γ \[[@CR48]\] blockade have not been studied yet but should be in the near future. Janus kinase (JAK) inhibitors may also be of interest, particularly in the chronic articular form of AOSD. The two main goals when initiating a bDMARD in AOSD should be a complete remission and corticosteroids withdrawal or significant tapering. This study does not reveal definitive answers regarding whether an early introduction of biologics improves the general prognosis of AOSD. We believe that the early use of biologics in AOSD patients is worth considering. It is our impression that the earlier biologics are used, the shorter and smoother the disease course is. Similarly, other authors have suggested that biotherapies should be initiated earlier in the course of AOSD \[[@CR13], [@CR18]\], arguing that earlier initiation may improve the prognosis mainly via a steroid-sparing effect. Notably, the results of this study suggest that the steroid-sparing effect of biologics is not influenced by delays in initiation of biological therapy. One-quarter to one-third (29.6%) of our patients managed to stop biologics despite chronic or refractory forms of AOSD, raising the question of how and when to stop biologics in these patients. A channeling bias exists, as no predefined corticosteroids tapering strategy could be set due to the retrospective nature of this study but patients who managed to stop anakinra had all achieved remission for at least 6 to 12 months, had initially stopped corticosteroids, and then all underwent progressive reduction in their biological treatments before definitive discontinuation. Among patients who managed to stop tocilizumab, corticosteroids had always previously been stopped, and tocilizumab dosage was progressively decreased when it had been administered by perfusion or tapered when it had been administered by injections. The high percentage of patients who successfully discontinued biological treatments in our study should encourage clinicians to consider tapering biologics among patients without visceral involvement who are in clinical and biological remission for 6 to 12 months. The first evidence-based clinical practice guidelines for the management of AOSD \[[@CR49]\] emphasized the use of IL-6 inhibitors in the treatment of AOSD, but IL-1 inhibitors were judged to be "weakly recommended as a therapeutic agent against refractory AOSD" although one RCT and several case reports suggest otherwise. A proposal may be to treat a first flare of AOSD with corticosteroids and to introduce biologics in cases of steroid-refractory flares or as early as the second flare of AOSD. In that case, anakinra should be preferred when faced with the systemic form of AOSD and tocilizumab in patients with the articular form. The well-known idea of a "window of opportunity" in rheumatoid arthritis may also be valid in AOSD, with the objective being to prevent the development of a self-perpetuating "cytokine storm." We need large-scale studies, RCTs, and international collaborative registries regarding this multifaceted disease. As emphasized in a recent editorial, these studies will have to be more precise about evaluating the patients' disease course and particularly their phenotypes in order to concentrate research on the subgroup of patients with a chronic or refractory form of the disease \[[@CR50]\]. A national French cohort, the French Adult and Childhood Onset Still Disease Cohort (RaDiCo-ACOSTILL), was recently opened for inclusion and will surely help to shed light on these issues in the near future. Conclusion {#Sec13} ========== This study supports the dichotomy of AOSD in two phenotypes and introduces the idea that treatment response to bDMARD may depend on disease phenotype. The presence of arthritis and a chronic articular phenotype seem to be associated with a substantial response to tocilizumab, whereas the systemic form seems to be associated with a substantial response to anakinra. Although further research is needed, this exploratory study highlights the therapeutic implications of the phenotypic dichotomy of AOSD. These results should help us better codify treatment strategies for AOSD. ANA : Antinuclear antibodies ANCA : Antineutrophil cytoplasmic antibody ACPA Positive anticitrullinated protein antibody Anti-DNA : Antideoxyribonucleic acid Anti-ENA : Anti-extractable nuclear antigen AOSD : Adult-onset Still's disease APLS : Anti-phospholipid syndrome bDMARD : Biological disease-modifying antirheumatic drugs CK : Creatine kinase CRP : C-reactive protein csDMARD : Conventional synthetic disease-modifying antirheumatic drugs ENT : Ear-nose-throat IL : Interleukin INF : Interferon IQR : Interquartile range JAK : Janus kinase LDH : Lactate dehydrogenase NSAIDs : Nonsteroidal anti-inflammatories RCT : Randomized controlled trial RF : Rheumatoid factor rHLH : Reactive hemophagocytic lymphohistiocytosis SLE : Systemic lupus erythematosus SoJIA : Systemic-onset juvenile idiopathic arthritis TNF : Tumor necrosis factor The authors thank the patients who were involved in this study and the individuals from the rheumatology and internal medicine departments who took part in the care of these patients. Funding {#FPar1} ======= This study was not funded. Availability of data and materials {#FPar2} ================================== The authors state that they had full access to all the data in the study and they take responsibility for the integrity of the data and the accuracy of the data analysis as well as the decision to submit them for publication. FV and MT contributed to the conception and design of the study. FV contributed to the acquisition of the data. FV, TB, and MT contributed to the analysis and interpretation of the data. FV, MT, TB, FL, ES, EL, BF, CR, and TS drafted the manuscript. FV, MT, AB, XD, FL, ES, EL, BF, CR, and TS contributed to the critical revision of the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== Not applicable Consent for publication {#FPar4} ======================= The authors state that they had full access to all the data in the study and they take responsibility for the integrity of the data and the accuracy of the data analysis as well as the decision to submit them for publication. Competing interests {#FPar5} =================== François Vercruysse has no relevant relationship conditions or circumstances that present a potential conflict of interest regarding this study. Thomas Barnetche has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Estibaliz Lazaro has received consultancy fees from Roche, Novartis, and UCB. Emilie Shipley has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. François Lifermann has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Alexandre Balageas has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Xavier Delbrel has no relevant relationships conditions or circumstances that present a potential conflict of interest regarding this study. Bruno Fautrel, Marie-Elise Truchetet, Christophe Richez, and Thierry Schaeverbeke have received consultancy fees and/or research funding from Abbvie, Bristol-Myers Squibb, Pfizer, Roche, Lilly, Novartis, and UCB. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

{ "pile_set_name": "PubMed Central" }

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22,185

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Introduction {#s1} ============ Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) [@pone.0061089-Castello1]. The viral genome encodes a single polyprotein precursor of approximately 3000 amino acids, which is cleaved by cellular and viral proteases into three structural proteins (Core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [@pone.0061089-Levrero1]. The HCV core protein, a component of the viral capsid, is initially split from the viral polyprotein between amino acid residues 191 and 192 by a signal peptidase within the endoplasmic reticulum (ER) lumen. Subsequently, this full-length core protein (amino acids 1--191, 191 a.a.) is further cleaved into a mature form (amino acids 1--173, 173 a.a.) by a signal peptide peptidase (SPP) [@pone.0061089-Liu1], [@pone.0061089-Weihofen1]. Further processing of the core protein in the C-terminal region produces a more truncated form, which can only be produced from limited HCV genotypes [@pone.0061089-Lo1]. The full-length core protein localizes in the cytoplasm, whereas truncated core proteins (amino acids 1--173 and 1--151) localize in the nucleus [@pone.0061089-Liu1], [@pone.0061089-Lo1], [@pone.0061089-Suzuki1]. Some cytoplasmic and nuclear proteins have been reported to interact with HCV core protein [@pone.0061089-Jin1]--[@pone.0061089-Tsutsumi1]. Recently, Moriishi *et al.* have reported that HCV core protein binds to the proteasome activator PA28γ in the nucleus, which induces liver steatosis and hepatocarcinogenesis through a PA28γ-dependent pathway in core transgenic mice [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2]. These studies suggest that HCV core as a regulatory protein may be involved in hepatocarcinogenesis during chronic HCV infection. MicroRNAs are small, endogenous non-coding RNA molecules that regulate the expression of at least one-third of human genes by inhibiting mRNA translation or inducing its degradation depending on the degree of complementarity [@pone.0061089-Lewis1], [@pone.0061089-Brodersen1]. Studies have shown that cellular microRNAs play an important role in various physiological and pathological processes including human cancers [@pone.0061089-EsquelaKerscher1], [@pone.0061089-Gaur1]. It has been reported that some cellular microRNAs are misregulated in HCV-related HCC [@pone.0061089-Varnholt1], [@pone.0061089-Jiang1]. Recently, HCV-specific effects on the modulation of cellular microRNAs have been shown in full-length HCV genome-expressing HepG2 cells [@pone.0061089-Braconi1]. These studies suggest that HCV proteins-modulated microRNAs may function as oncogenes or as tumor suppressor genes. The misregulation of *p21^Waf1/Cip1^* gene expression is frequently observed in human cancers [@pone.0061089-Abbas1]. Hepatocarcinogenesis requires continuous cellular stresses such as viral replication, oxidative stress, inflammation, and continued cell death after regeneration, to receive DNA damage in hepatocytes [@pone.0061089-Castello1], [@pone.0061089-Koike1]. The up-regulation of *p21^Waf1/Cip1^* gene expression by cellular stresses may prevent hepatocytes from transformation by inducing a sufficient G1 span to trigger apoptosis or repair DNA damage [@pone.0061089-Abbas1]. Although p21^Waf1/Cip1^ deficiency may not be sufficient to hepatocarcinogenesis, *p21^Waf1/Cip1^* gene misregulation may be involved in multistep hepatocarcinogenesis. Moreover, p21^Waf1/Cip1^ may be a target for cancer therapy [@pone.0061089-Abbas1]. Curcumin, a potential anticancer agent, has been used in preclinical in vitro and in vivo models of HCC [@pone.0061089-Darvesh1]. Curcumin exerts its effect on anticancer, at least in part, by triggering apoptosis. Curcumin may induce apoptosis through p21^Waf1/Cip1^-dependent pathway [@pone.0061089-Srivastava1]. Recently, curcumin has been reported to induce apoptosis in human hepatoma cell lines [@pone.0061089-Wang1]. The HCV core protein is considered to be involved in hepatocarcinogenesis [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2], [@pone.0061089-Moriya1]. HCV has been reported to regulate cellular microRNAs [@pone.0061089-Braconi1]. Moreover, the core protein can regulate p21^Waf1/Cip1^ expression [@pone.0061089-Lee1]--[@pone.0061089-Ohkawa1]. In this study, we analyzed the expression profiles of cellular microRNAs in core-overexpressing human hepatoma cells compared to cells nonexpressing core. The HCV core protein was able to up-regulate microRNA-345 (miR-345) expression in human hepatoma cells. The HCV core-induced miR-345 suppressed endogenous *p21^Waf1/Cip1^* gene expression through targeting its 3′ untranslated region (3′UTR) in HepG2 cells and curcumin-stimulated Huh7 cells. In addition, the core protein inhibited curcumin-induced apoptosis through p21^Waf1/Cip1^-targeting miR-345 in Huh7 cells. Materials and Methods {#s2} ===================== Antibodies, MicroRNA mimics, Mutant microRNA mimic, Small interfering RNA, MicroRNA inhibitor, and Chemicals {#s2a} ------------------------------------------------------------------------------------------------------------ Anti-HA epitope antibody (catalog no. 11583816001) was purchased from Roche Applied Science, anti-p21^Waf1/Cip1^ antibody (catalog no. sc-6246) was from Santa Cruz Biotechnology, and anti-β-actin antibody (catalog no. A1978) was from Sigma-Aldrich. Anti-HCV core (catalog no. ab2740) and anti-HCV NS5B (catalog no. ab35586) antibodies were purchased from Abcam. Human microRNA-345 (hsa-miR-345, MIMAT0000772) and microRNA-93 (hsa-miR-93, MIMAT0000093) mimics were purchased from Thermo Scientific. Mutant microRNA-345 mimic was synthesized by Thermo Scientific. The sequence was 5′- GCU[CUGA]{.ul}CCUAGUCCAGGGCUC-3′. The four mutated sites are underlined. Human p21^Waf1/Cip1^ small interfering RNA (siRNA, catalog no. sc-29427) was purchased from Santa Cruz Biotechnology. Human miR-345 inhibitor (MH12733) was purchased from Applied Biosystems. Curcumin (catalog no. C7727) and dimethyl sulfoxide (DMSO, catalog no. D2650) were purchased from Sigma-Aldrich. Curcumin was dissolved in DMSO to 100 mM and stored at −20°C until use. Plasmid Construction {#s2b} -------------------- Plasmid pT-REx-HA-Core was generated by cloning the N-terminus HA-tagged HCV core coding sequence (genotype 1b strain) [@pone.0061089-Chen1] into pT-REx-DEST30 vector (Invitrogen) according to the manufacturer\'s instructions. HA-Core was amplified by PCR using pcDNA3-HA-Core191 (amino acids 1--191) as a template. Primers used for cloning HA-Core191 were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAAGCGGAAGCTGGGATGG-3′. Primers used for cloning HA-Core173 (amino acids 1--173) were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAAGAGCAACCGGGCAGAT-3′. Primers used for cloning HA-Core153 (amino acids 1--153) were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAATGTGCCAGGGCTCTGG-3′. A control vector, pT-REx-Mock, was created by deleting HCV core coding sequence from the above construct using BsrGI restriction enzyme. Wild-type human *p21^Waf1/Cip1^* 3′UTR was amplified by PCR from human genomic DNA and cloned into pGL3-Control vector (Promega) immediately downstream of luciferase reporter gene but upstream of poly (A) signal using XbaI restriction enzyme to generate pGL3-Control-p21 3′UTR Sense (S) and pGL3-Control-p21 3′UTR Antisense (AS) vectors. Primers used for cloning human *p21^Waf1/Cip1^* 3′UTR were forward 5′-CCC[TCTAGA]{.ul}TCCGCCCACAGGAAGCCTGC-3′ and reverse 5′-CCC[TCTAGA]{.ul}AAAGTCACTAAGAATCATTT-3′. The XbaI site is underlined. The mutant *p21^Waf1/Cip1^* 3′UTR in the seed sequence of hsa-miR-345 was generated by PCR and ligation of two pieces of DNA fragments, and then cloned into pGL3-Control vector to yield a pGL3-Control-p21 3′UTR Mutant vector. Primer pairs used for cloning mutated *p21^Waf1/Cip1^* 3′UTR were forward 5′-CCC[TCTAGA]{.ul}TCCGCCCACAGGAA-3′ and reverse 5′-CCTTGTTCCGCTGCTAATCA-3′, and were forward 5′-TCAGAGACATTTTAAGATGGTGGC-3′ and reverse 5′-CCC[TCTAGA]{.ul}AAAGTCACTAAGAA-3′. The XbaI site is underlined. Full-length HCV Replicon {#s2c} ------------------------ A genotype 1b strain of full-length HCV replicon (HCV-N) [@pone.0061089-Beard1] was kindly provided by Dr. Michael M.C. Lai at Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C.. A control replicon HCV-A357T, which expresses only the initial five amino acids of the core protein due to introduction of a termination codon, was created by the change of one nucleotide at HCV nt 357 in pHCV-N ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, upper panel) using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer\'s instructions. ![HCV core protein up-regulates miR-345 and miR-93 expression in human hepatoma cells.\ (**A**) Huh7 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1--191, 1--173 and 1--153, respectively. At 48 hours after transfection, the expression of HCV core protein was analyzed by immunoprecipitation. (**B**) Huh7 cells were transiently transfected with empty vector and two HCV core gene-expressing vectors, pT-REx-HA-Core191, and pT-REx-HA-Core173, for core protein with amino acids 1--191 and 1--173, respectively. At 48 hours after transfection, cellular microRNA profiling was analyzed by TaqMan low density array. Three microRNAs, miR-21, miR-345 and miR93, of thirty-one microRNAs were indicated. (**C**) Huh7 and HepG2 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1--191, 1--173 and 1--153, respectively. At 48 hours after transfection, relative expression of miR-345 or miR-93 was determined by TaqMan real-time qPCR in Huh7 cells (left upper panel) and HepG2 cells (right upper panel). The expression of HCV core protein was analyzed by Western blotting (left lower and right lower panels). (**D**) The genotype 1b strain of full-length HCV replicon (HCV-N) and control replicon HCV-A357T which expresses only the initial five amino acids of the core protein due to introduction of a termination codon, was created by the change of one nucleotide at HCV nt 357 in pHCV-N (upper panel). HCV core and NS5B gene expression in full-length HCV replicon-expressing cells was analyzed by immunoprecipitation followed by Western blotting and Western blotting only respectively (left lower panel). The relative expressions of miR-345 and miR-93 were determined by TaqMan real-time qPCR in full-length HCV replicon-expressing Huh7 cells (right lower panel). Data was shown as the means ± S.D. from triplicate experiments. \**P*\<0.05, \*\**P*\<0.001.](pone.0061089.g001){#pone-0061089-g001} In Vitro Transcription of HCV RNA {#s2d} --------------------------------- pHCV-N DNA was linearized with XbaI, purified by phenol/chloroform extraction and ethanol precipitation [@pone.0061089-Beard1], and then the linearized DNA was used as template to transcribe full-length HCV RNA using Riboprobe in vitro Transcription Systems (Promega, catalog no. P1440) following the manufacturer\'s protocol. The integrity of RNA transcripts was determined by agarose gel electrophoresis and ethidium bromide staining. Cell Culture and Transfection {#s2e} ----------------------------- Human hepatoma cell lines, Huh7 and HepG2 cells, were cultured at 37°C in a humidified incubator containing 5% CO~2~ in Dulbecco\'s modified Eagle\'s medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone). Plasmid DNA was transfected into cells using GenJet In Vitro DNA Transfection Reagent Ver. II (SignaGen Laboratories) following the manufacturer\'s protocol. To establish the full-length HCV replicon-expressing system in Huh7 cells, HCV RNA was transfected into cells using TransIT-mRNA Transfection Kit (Mirus Bio LLC) according to the manufacturer\'s instructions. The siRNA, microRNA mimic and inhibitor were transfected into cells using GenMute siRNA & DNA Transfection Reagent (SignaGen Laboratories) following the manufacturer\'s protocol. RNA Extraction {#s2f} -------------- Total RNA was extracted using miRNeasy Mini Kit (Qiagen) according to the manufacturer\'s protocol. The trace quantities of genomic DNA were further removed using Turbo DNA-free Kit (Applied Biosystems) following the manufacturer\'s instructions. TaqMan Low Density Array for MicroRNA {#s2g} ------------------------------------- TaqMan low density array was performed using Applied Biosystems 7900HT Fast Real-Time PCR System according to the manufacturer\'s protocol. Briefly, cDNA templates were synthesized using TaqMan MicroRNA Megaplex RT Kit (Applied Biosystems) following the manufacturer\'s instructions. A total reaction mixture containing RT products and TaqMan Universal PCR Master Mix (Applied Biosystems) was added to each line of TaqMan low density array card after gentle vortex mixing. Thermal cycler conditions were as follows: 50°C for 2 minutes, 94.5°C for 10 minutes, and then 40 cycles of 97°C for 30 seconds and 59.7°C for 1 minute. Cycle threshold was automatically given by SDS software v2.2 (Applied Biosystems) using automatic baseline settings and a threshold of 0.2. MammU6 was used as an internal control to normalize the amount of individual microRNA in each sample. Significant difference of relative microRNA expression was determined using the 2^−ΔΔCt^ method [@pone.0061089-Livak1]. TaqMan Real-time qPCR for Single MicroRNA Assay {#s2h} ----------------------------------------------- cDNA template for single microRNA was synthesized using TaqMan MicroRNA RT Kit (Applied Biosystems) according to the manufacturer\'s protocol. Expression of specific microRNA was determined by real-time qPCR using TaqMan MicroRNA qPCR Kit (Applied Biosystems) following the manufacturer\'s instructions. MammU6 was used as an internal control to normalize the amount of specific microRNA in each sample. Significant difference of relative microRNA expression was determined as described above. Real-time qPCR {#s2i} -------------- cDNA template was synthesized using ImProm-II Reverse Transcription System (Promega) according to the manufacturer\'s protocol. Expression of human p21^Waf1/Cip1^ mRNA was determined by real-time qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems) following the manufacturer\'s instructions. The β-actin mRNA was used as an internal control to normalize the amount of human p21^Waf1/Cip1^ mRNA in each sample. Primer sequences for human p21^Waf1/Cip1^ and β-actin mRNAs were described previously [@pone.0061089-Wu1]. Significant difference of relative gene expression was determined as described above. Luciferase Reporter Assay {#s2j} ------------------------- Cells were transfected with luciferase reporter vector in combination with microRNA mimic. All transfections included pRL-TK vector (Promega) for normalization. Luciferase activity was analyzed by Dual-Glo Luciferase Assay System (Promega) according to the manufacturer\'s instructions. Renilla luminescence, expressed from pRL-TK vector, was used as an internal control to normalize the luciferase activity. Immunoprecipitation {#s2k} ------------------- Cell lysates were extracted using RIPA buffer (Sigma-Aldrich) supplemented with Halt Protease Inhibitor Cocktail (Thermo Scientific). The supernatants were incubated for 2 hours at 4°C with anti-HA epitope or anti-HCV core antibody, and then overnight at 4°C with Protein A-agarose beads (Roche Applied Science, catalog no. 11719408001). Following four washes with RIPA buffer, proteins were eluted for 10 minutes at 100°C with 2× Laemmli sample buffer (Sigma-Aldrich), electrophoresed on SDS-PAGE and electroblotted onto PVDF membranes (Millipore). Western Blotting {#s2l} ---------------- Cell lysates were extracted as described above. Cell lysates were electrophoresed on SDS-PAGE and electroblotted onto PVDF membranes. Anti-HA epitope, anti-HCV core, anti-HCV NS5B, anti-p21^Waf1/Cip1^, and anti-β-actin antibodies were used in Western blotting according to the manufacturer\'s protocol. β-actin was used as an internal control. Relative protein expression was quantified by BioSpectrum Imaging System (UVP). DNA Fragmentation Analysis {#s2m} -------------------------- Genomic DNA from cells was extracted using Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer\'s instructions. DNA ladder was determined by agarose gel electrophoresis and ethidium bromide staining. Annexin V-FITC Apoptosis Assay {#s2n} ------------------------------ Apoptotic cells were detected and quantified by fluorescence microscopy and FACS Calibur (Becton Dickinson) respectively, using Annexin V-FITC Apoptosis Detection Kit (BioVision) following the manufacturer\'s protocol. Cell Cycle Analysis {#s2o} ------------------- Cells were subjected to serum starvation for 24 hours to arrest cell growth, and then cultured in fresh serum-containing medium for 24 hours for cell cycle re-entry. Cells were harvested after trypsinization in serum-containing medium, centrifuged and suspended in phosphate buffered saline (PBS). The absolute ethanol was added drop-wise and cells were maintained overnight at −20°C to complete fixation. Cells were centrifuged, resuspended in PBS plus RNase A and propidium iodide (Sigma-Aldrich), and incubated at 37°C for 30 minutes. Fluorescence was measured and analyzed by FACS Calibur. Cell Proliferation Assay (MTT assay) {#s2p} ------------------------------------ Cell proliferation was determined using CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer\'s instructions. To analyze cell proliferation, Huh7 cells were seeded in 96-well culture plates at 3000 cells per well. After 24 hours, the medium was replaced with fresh medium supplemented with increased amount of curcumin. At 24 hours after treatment, Dye Solution was added to each well and cells were incubated at 37°C for 2 hours. Solubilization/Stop Solution was then added to each well and the absorbance was measured at 570 nm using an ELISA reader. Relative cell number was calculated by normalizing the absorbance to untreated cells. Relative cell viability was compared to untreated cells. Statistical Analysis {#s2q} -------------------- Data was shown as the means ± SD from triplicate experiments. The two-sided Student\'s *t*-test was used for comparisons between experimental groups. *P*\<0.05 was considered statistically significant. Results {#s3} ======= MicroRNA-345 and microRNA-93 are overexpressed in HCV core-overexpressing human hepatoma cells {#s3a} ---------------------------------------------------------------------------------------------- It has been reported that the full-length HCV core protein (amino acids 1--191) is further cleaved into truncated forms (amino acids 1--173 and 1--153) [@pone.0061089-Liu1], [@pone.0061089-Lo1]. Although the truncated core protein localizes in the nucleus [@pone.0061089-Liu1], [@pone.0061089-Lo1], [@pone.0061089-Suzuki1], only a small quantity of the core protein localizes to the nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [@pone.0061089-Moriya1], [@pone.0061089-Falcn1], [@pone.0061089-Yamaguchi1]. In this study, three core gene-expressing vectors for core protein with amino acids 1--191, 1--173 and 1--153, respectively, were transfected into Huh7 cells. Immunoprecipitation followed by Western blotting was used to determine HCV core expression. The results showed that three forms of HCV core protein were overexpressed in Huh7 cells ([Fig. 1A](#pone-0061089-g001){ref-type="fig"}). Interestingly, a small quantity of a product with lower molecule weight was observed when the full-length core gene was overexpressed in Huh7 cells ([Fig. 1A](#pone-0061089-g001){ref-type="fig"}, lane 2). This product may suggest a truncated core protein trimmed at the C-terminal region due to the intracellular processing of the full-length core protein. To investigate the effect of the full-length and mature core proteins on the modulation of cellular microRNAs, two core gene-expressing vectors for core protein with amino acids 1--191 and 1--173, respectively, were transfected into Huh7 cells, and then the expression profiles of cellular microRNAs was determined by TaqMan low density array at 48 hours after transfection. The result showed that thirty-one microRNAs exhibited a greater than 2-fold up- or down-regulation in full-length core (191 a.a.) or mature core (173 a.a.)-overexpressing Huh7 cells compared to cells nonexpressing core ([Fig. 1B](#pone-0061089-g001){ref-type="fig"}). The HCV core protein can regulate p21^Waf1/Cip1^ expression [@pone.0061089-Lee1]--[@pone.0061089-Ohkawa1]. Recently, Wu *et al.* have reported that human *p21^Waf1/Cip1^* gene expression can be inhibited by twenty-eight microRNAs in HEK 293 cells [@pone.0061089-Wu1]. In this study, we indicated that two p21^Waf1/Cip1^-targeting microRNAs, microRNA-345 (miR-345) and microRNA-93 (miR-93), were up-regulated in core-overexpressing Huh7 cells ([Fig. 1B](#pone-0061089-g001){ref-type="fig"}). Array data were further confirmed by TaqMan real-time qPCR for miR-345 and miR-93 in Huh7 and HepG2 cells. Three core gene-expressing vectors for core protein with amino acids 1--191, 1--173 and 1--153, respectively, were transfected into cells, and then the relative expression of miR-345 and miR-93 was determined at 48 hours after transfection. The results showed that miR-345 and miR-93 were overexpressed with more than 3.5- and 2-fold changes, respectively, in mature core (173 a.a.) and more truncated core (153 a.a.)-overexpressing Huh7 cells but not in full-length core (191 a.a.)-overexpressing cells compared to cells nonexpressing core ([Fig. 1C](#pone-0061089-g001){ref-type="fig"}, left upper panel). Similar results also indicated in mature core and more truncated core-overexpressing HepG2 cells but not in full-length core-overexpressing cells ([Fig. 1C](#pone-0061089-g001){ref-type="fig"}, right upper panel). To further verify the expressions of cellular microRNAs in full-length HCV replicon-expressing system, the full-length HCV replicon was transfected into Huh7 cells. At 96 hours after transfection, immunoprecipitation followed by Western blotting was used to determine HCV core expression, and Western blotting only for NS5B expression ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, left lower panel, lane 2). The relative expressions of miR-345 and miR-93 were determined by TaqMan real-time qPCR. The results showed that miR-345 induction was not significantly affected in full-length HCV replicon-expressing system in Huh7 cells compared to control cells and untreated cells ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, right lower panel, third bar pair). Indeed, there is no detectable or little, if any, amount of truncated form of HCV core protein expression in full-length HCV replicon-expressing system in Huh7 cells ([Fig. 1D](#pone-0061089-g001){ref-type="fig"}, left lower panel, lane 2). Furthermore, miR-93 overexpression in full-length HCV replicon-expressing cells but not in full-length core-overexpressing cells suggested that other HCV proteins might up-regulate miR-93 expression. Together, these results demonstrate that truncated HCV core proteins (amino acids 1--173 and 1--153) up-regulate cellular miR-345 and miR-93 expression in human hepatoma cells. MicroRNA-345 down-regulates *p21^Waf1/Cip1^* gene expression in human hepatoma cells {#s3b} ------------------------------------------------------------------------------------ It has been reported that human *p21^Waf1/Cip1^* gene expression can be inhibited by miR-345 and miR-93 in HEK 293 cells [@pone.0061089-Wu1]. Because the different cell types might generate different results, the effects of miR-345 and miR-93 on human *p21^Waf1/Cip1^* gene expression were examined in human hepatoma cells. Two luciferase reporter vectors which bear the sense and antisense 3′UTRs from human *p21^Waf1/Cip1^* gene, respectively, were used in luciferase reporter assay ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, upper panel). The relative luciferase activity was determined in Huh7 and HepG2 cells at 24 hours after transfection. The results showed that the treatment with miR-345 mimic led to a significant reduction of luciferase activity in p21 3′UTR Sense construct-transfected Huh7 cells, but treatment with miR-93 mimic had no significant inhibition in luciferase activity ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower panel, second bar cluster). Moreover, treatment with a mixture of miR-345 and miR-93 mimics also had no double reduction of luciferase activity ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower panel, second bar cluster). As expected, miR-345 and miR-93 mimics had no effect in control vector-transfected and p21 3′UTR Antisense construct-transfected Huh7 cells ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower panel, first and third bar clusters). Similar results also indicated in HepG2 cells ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, right lower panel). We further examined human *p21^Waf1/Cip1^* gene expression at protein level when miR-93 mimic was transfected into HepG2 cells. As the results in luciferase reporter assay ([Fig. 2A](#pone-0061089-g002){ref-type="fig"}, left lower and right lower panels), *p21^Waf1/Cip1^* expression at protein level was not suppressed by miR-93 in HepG2 cells ([Fig. S1](#pone.0061089.s001){ref-type="supplementary-material"}). These results demonstrated that human *p21^Waf1/Cip1^* gene may be not a target of miR-93 in human hepatoma cells. We further focused on miR-345 to identify the seed sequence in human *p21^Waf1/Cip1^* 3′UTR by using computational tools, miRanda (<http://www.microrna.org/microrna/home.do>) and TargetScan (<http://www.targetscan.org/>) ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, left panel). To further verify the seed sequence of miR-345, two luciferase reporter vectors which bear the wild-type and mutant (mutated in the seed sequence of miR-345) 3′UTRs from human *p21^Waf1/Cip1^* gene, respectively, were used in luciferase reporter assay ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, left panel, mutated sites are underlined). The results showed that wild-type miR-345 mimic can significantly inhibit the luciferase activity in wild-type p21 3′UTR construct-transfected Huh7 cells but not in mutant p21 3′UTR construct-transfected cells ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, right panel, second and third bar pairs). However, an additional experiment with mutant miR-345 mimic ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, left panel, mutated sites are underlined) and mutant p21 3′UTR with restoring complementarity was able to show a significant inhibition of luciferase activity ([Fig. 2B](#pone-0061089-g002){ref-type="fig"}, right panel, fourth bar pair). These results showed that miR-345 may down-regulate human *p21^Waf1/Cip1^* gene expression through targeting its 3′UTR in human hepatoma cells. To further verify the down-regulation of endogenous *p21^Waf1/Cip1^* gene expression by miR-345, p21^Waf1/Cip1^ mRNA and protein levels were examined in HepG2 cells at 24 hours after transfection with miR-345 mimic. As expected, *p21^Waf1/Cip1^* gene expression at mRNA and protein levels was suppressed with the increased amount of miR-345 mimic in HepG2 cells ([Fig. 2C](#pone-0061089-g002){ref-type="fig"}, left and right panels). Together, these results demonstrate that miR-345 down-regulates *p21^Waf1/Cip1^* gene expression through targeting its 3′UTR in human hepatoma cells. ![MicroRNA-345 down-regulates *p21^Waf1/Cip1^* gene expression through targeting its 3′UTR but not microRNA-93 in human hepatoma cells.\ (**A**) Two luciferase reporter vectors, pGL3-Control-p21 3′UTR Sense (S) and pGL3-Control-p21 3′UTR Antisense (AS) which bear the sense and antisense 3′UTRs from human *p21^Waf1/Cip1^* gene, respectively, were constructed (upper panel). Huh7 cells and HepG2 cells were transfected with p21 3′UTR sense or antisense luciferase reporter vector in combination with miR-345 mimic, miR-93 mimic or a mixture of miR-345 and miR-93 mimics. At 24 hours after transfection, Huh7 cells (left lower panel) and HepG2 cells (right lower panel) were collected for luciferase reporter assay. (**B**) Base-pairing between mature hsa-miR-345 and target site in human *p21^Waf1/Cip1^* 3′UTR is shown (left panel). One luciferase reporter vector, pGL3-Control-p21 3′UTR Mutant (mutated in the seed sequence of miR-345), was constructed. Mutant miR-345 mimic was synthesized. The mutated site is underlined (left panel). Huh7 cells were transfected with wild-type p21 3′UTR sense or mutant p21 3′UTR luciferase reporter vector in combination with wild-type or mutant miR-345 mimic. At 24 hours after transfection, Huh7 cells were collected for luciferase reporter assay. The experiment with mutant miR-345 mimic and mutant p21 3′UTR with restoring complementarity was performed (right panel). (**C**) HepG2 cells were transiently transfected with the increased amount of miR-345 mimic for 24 hours. The *p21^Waf1/Cip1^* gene expression was analyzed by real-time qPCR (left panel) and Western blotting (right panel). β-actin served as an internal control. Data was shown as the means ± S.D. from triplicate experiments. \**P*\<0.05.](pone.0061089.g002){#pone-0061089-g002} MicroRNA-345 inhibits curcumin-induced apoptosis through down-regulation of *p21^Waf1/Cip1^* gene expression in Huh7 cells {#s3c} -------------------------------------------------------------------------------------------------------------------------- Curcumin, a potential anticancer agent, has been used in preclinical in vitro and in vivo models of HCC [@pone.0061089-Darvesh1]. Recently, curcumin has been reported to induce apoptosis in Huh7 cells [@pone.0061089-Wang1]. In this study, we showed that *p21^Waf1/Cip1^* gene expression at protein level was enhanced with increased amount of curcumin in Huh7 cells ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, upper panel). To investigate the functional relevance of *p21^Waf1/Cip1^* up-regulation and curcumin treatment in Huh7 cells, annexin V-FITC apoptosis assay, MTT assay, and cell cycle analysis were performed. The results showed that curcumin induced apoptosis and inhibited cell viability in Huh7 cells in a dose-dependent manner ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, middle and left lower panels). Furthermore, the result also showed that S-phase entry of cell cycle was slightly inhibited in Huh7 cells in response to high doses of curcumin ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, right lower panel). To further verify that the up-regulation of *p21^Waf1/Cip1^* gene expression involved in curcumin-induced apoptosis in Huh7 cells, curcumin-stimulated cells were transfected with increased amount of p21^Waf1/Cip1^ siRNA, and then apoptosis was analyzed by using DNA fragmentation analysis at 24 hours after transfection. The result showed that apoptosis was inhibited as presented in DNA ladder disappeared when *p21^Waf1/Cip1^* gene expression at protein level was suppressed in curcumin-stimulated Huh7 cells ([Fig. 3B](#pone-0061089-g003){ref-type="fig"}), indicating that curcumin induced apoptosis through up-regulation of *p21^Waf1/Cip1^* gene expression in Huh7 cells. Similar results were also showed in [Figure 3C](#pone-0061089-g003){ref-type="fig"}. Curcumin had no effect on the modulation of cellular miR-345 expression in Huh7 cells ([Fig. S2](#pone.0061089.s002){ref-type="supplementary-material"}). As expected, *p21^Waf1/Cip1^* gene expression at protein level was suppressed by miR-345 mimic in curcumin-stimulated Huh7 cells in a dose-dependent manner ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, upper panel). Curcumin-induced apoptosis was inhibited with increased amount of miR-345 mimic ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, left middle panel). Moreover, the introduction of miR-345 mimic enhanced cell viability in curcumin-stimulated Huh7 cells ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, right middle panel). The number of apoptotic cells (early and late apoptotic cells) had a maximum reduction of about 55% when curcumin-stimulated cells were transfected with 50 nM miR-345 mimic ([Fig. 3C](#pone-0061089-g003){ref-type="fig"}, left lower and right lower panels). In HepG2 cells, we also showed that curcumin induced apoptosis, but curcumin had no effect on the modulation of *p21^Waf1/Cip1^* gene expression. The results indicated that curcumin was able to induce apoptosis through a p21^Waf1/Cip1^-independent pathway in HepG2 cells ([Fig. S4](#pone.0061089.s004){ref-type="supplementary-material"}). Together, these results demonstrate that miR-345 inhibits curcumin-induced apoptosis through down-regulation of *p21^Waf1/Cip1^* gene expression in Huh7 cells. ![MicroRNA-345 inhibits curcumin-mediated apoptosis through down-regulation of *p21^Waf1/Cip1^* gene expression in Huh7 cells.\ (**A**) Huh7 cells were treated with different doses (6.25, 12.5, 25 and 50 µM) of curcumin for 24 hours. DMSO served as control (labeled with 0 µM). The *p21^Waf1/Cip1^* gene expression at protein level was determined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was determined by annexin V-FITC apoptosis assay (middle panel). Cell proliferation was analyzed by MTT assay (left lower panel). Cell cycle distribution was examined by cell cycle analysis (right lower panel). (**B**) Huh7 cells were transfected with the increased amount of *p21^Waf1/Cip1^* siRNA in response to curcumin stimulation (50 µM) for 24 hours. Apoptosis was analyzed by DNA fragmentation analysis. The *p21^Waf1/Cip1^* gene expression at protein level was examined by Western blotting. (**C**) Huh7 cells were transfected with the increased amount of miR-345 mimic in response to curcumin stimulation (50 µM) for 24 hours. The *p21^Waf1/Cip1^* gene expression at protein level was examined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was analyzed by fluorescence microscopy (left middle panel) and FACS Calibur (left lower and right lower panels) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Cell proliferation was analyzed by MTT assay (right middle panel). Data was shown as the means ± S.D. from triplicate experiments. \**P*\<0.05, \*\**P*\<0.001.](pone.0061089.g003){#pone-0061089-g003} HCV core-induced microRNA-345 inhibits *p21^Waf1/Cip1^* gene expression in HepG2 cells and curcumin-stimulated Huh7 cells {#s3d} ------------------------------------------------------------------------------------------------------------------------- It has been reported that the mature form (amino acids 1--173) of HCV core protein in the nucleus suppresses *p21^Waf1/Cip1^* gene expression in HepG2 cells [@pone.0061089-Yamanaka1]. To determine that HCV core-induced miR-345 down-regulated endogenous *p21^Waf1/Cip1^* gene expression, core expressing HepG2 cells were transfected with or without miR-345 inhibitor, and then *p21^Waf1/Cip1^* gene expression at protein level was determined at 24 hours after transfection. As expected, the mature form (amino acids 1--173) of the core protein suppressed *p21^Waf1/Cip1^* gene expression ([Fig. 4A](#pone-0061089-g004){ref-type="fig"}, lane 3). However, the suppression of *p21^Waf1/Cip1^* gene expression was attenuated in the presence of increased amount of miR-345 inhibitor ([Fig. 4A](#pone-0061089-g004){ref-type="fig"}, lane 3, lane 6 and lane 7). The full-length core protein (amino acids 1--191) slightly suppressed *p21^Waf1/Cip1^* gene expression ([Fig. 4A](#pone-0061089-g004){ref-type="fig"}, lane 2), this result may be due to the expression of a small quantity of the truncated core protein ([Fig. 1A](#pone-0061089-g001){ref-type="fig"}, lane 2). We have shown that only little amount of p21^Waf1/Cip1^ protein can be detected in Huh7 cells ([Fig. 3A](#pone-0061089-g003){ref-type="fig"}, upper panel, lane 1). Therefore, curcumin was used to induce endogenous *p21^Waf1/Cip1^* gene expression to investigate the inhibition of *p21^Waf1/Cip1^* gene expression by HCV-core-induced miR-345 in Huh7 cells. Similar results were also showed in [Figure 4B](#pone-0061089-g004){ref-type="fig"}. As expected, the mature form (amino acids 1--173) of the core protein suppressed *p21^Waf1/Cip1^* gene expression in curcumin-stimulated Huh7 cells ([Fig. 4B](#pone-0061089-g004){ref-type="fig"}, upper panel, lane 3). Similarly, the suppression of *p21^Waf1/Cip1^* gene expression was attenuated when mature core-expressing cells were transfected with miR-345 inhibitor ([Fig. 4B](#pone-0061089-g004){ref-type="fig"}, upper panel, lane 3, lane 6 and lane 7). Additionally, the suppression of curcumin-induced apoptosis by mature core protein (amino acids 1--173) was significantly attenuated in the presence of increased amount of miR-345 inhibitor ([Fig. 4B](#pone-0061089-g004){ref-type="fig"}, middle and lower panels). Together, these results demonstrate that HCV core-induced miR-345 inhibits endogenous *p21^Waf1/Cip1^* gene expression in HepG2 cells and curcumin-stimulated Huh7 cells. ![HCV core-induced microRNA-345 inhibits *p21^Waf1/Cip1^* gene expression in HepG2 cells and curcumin-stimulated Huh7 cells.\ (**A**) HCV core (amino acid 1--191 or 1--173)-expressing HepG2 cells were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, *p21^Waf1/Cip1^* gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control. (**B**) HCV core (amino acid 1--191 or 1--173)-expressing Huh7 cells in response to curcumin stimulation were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, *p21^Waf1/Cip1^* gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control (upper panel). Apoptosis was analyzed by fluorescence microscopy (middle panel) and FACS Calibur (lower panel) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Data was shown as the means ± S.D. from triplicate experiments. \**P*\<0.05, \*\**P*\<0.001.](pone.0061089.g004){#pone-0061089-g004} Discussion {#s4} ========== In this study, we determine the effect of a mature HCV core protein on miR-345 induction in human hepatoma cells. Moreover, HCV core-induced miR-345 suppresses *p21^Waf1/Cip1^* gene expression in HepG2 cells, and inhibits curcumin-mediated apoptosis through down-regulation of *p21^Waf1/Cip1^* gene expression in Huh7 cells. HCV core protein has an effect on cellular microRNA regulation. HCV core protein is initially separated from HCV polyprotein by a signal peptidase. The full-length core protein (amino acids 1--191) localizes in the cytoplasm, which mainly functions as a component of the viral capsid. HCV core protein is considered to play a crucial role in hepatocarcinogenesis [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2], [@pone.0061089-Moriya1]. Many studies have reported that HCV core protein can interact with cytoplasmic and nuclear proteins [@pone.0061089-Jin1]--[@pone.0061089-Tsutsumi1]. Chen *et al.* has shown that HCV core protein interacts with Dicer, an RNase enzyme that generates mature microRNAs, in the cytoplasm, and then inhibits the function of Dicer [@pone.0061089-Chen2]. This inhibitive effect may contribute to HCV replication [@pone.0061089-Pedersen1]. The mature form (amino acids 1--173) of HCV core protein cleaved by SPP, which lacks the 174--191 peptide for attachment to ER membrane, enables subcellular distribution of the core protein [@pone.0061089-Liu1]--[@pone.0061089-Suzuki1]. Unlike full-length core protein (amino acids 1--191), mature form (amino acids 1--173) has been reported to mainly localize to nucleus [@pone.0061089-Liu1], [@pone.0061089-Lo1], [@pone.0061089-Suzuki1]. Some studies have shown that a small quantity of HCV core protein localizes to the nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [@pone.0061089-Moriya1], [@pone.0061089-Falcn1], [@pone.0061089-Yamaguchi1]. In this study, we observed a small quantity of a product with lower molecule weight when full-length HCV core gene was expressed in cultured Huh7 cells. These studies suggest that the nuclear localization or truncated form of the core protein may play an important role in chronic HCV infection. Moriishi *et al.* has reported that mature (amino acids 1--173) and more truncated (amino acids 1--151) forms of HCV core protein can bind to PA28γ in the nucleus and hence induces liver steatosis and HCC development through a PA28γ-dependent pathway in core gene-transgenic mice [@pone.0061089-Moriishi1], [@pone.0061089-Moriishi2], suggesting that the nuclear localization of the truncated core proteins may function as a transcriptional factor or regulator. In this study, up-regulation of miR-345 expression by mature form (amino acids 1--173) of the core protein may be also associated with its nuclear localization. Indeed, we also demonstrated that more truncated core protein (amino acids 1--153) which deletes more residues of hydrophobic C-terminal region up-regulated miR-345 expression, indicating that the up-regulation of miR-345 expression may be associated with the nuclear localization of HCV core protein. MiR-345 is down-regulated in full-length HCV genome-expressing cells as reported by Braconi *et al.* [@pone.0061089-Braconi1]. Our result was not consistent with the observation from Braconi\'s group, since the different expressing system and different hepatoma cells were used in the experiments. There is no detectable or little, if any, amount of truncated form of HCV core protein expression in our full-length HCV replicon-expressing system. However, truncated form of HCV core protein has been demonstrated to localize in nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [@pone.0061089-Moriya1], [@pone.0061089-Falcn1], [@pone.0061089-Yamaguchi1]. The up-regulation of miR-345 expression by HCV core protein may be associated with the demethylation of its promoter. Tang *et al.* has demonstrated that miR-345 with a CpG island in the promoter is a methylation-sensitive microRNA and is highly induced by demethylating agent in human colorectal cancer cell lines [@pone.0061089-Tang1]. Recently, miR-21 has been identified to indirectly down-regulate DNA methyltransferase 1 (DNMT1) expression by directly targeting human *RASGRP1* gene, a known critical regulator of the upstream Ras-MAPK pathway of DNMT1 [@pone.0061089-Pan1]. In this study, we also determined the up-regulation of miR-21 expression in HCV core-overexpressing Huh7 and HepG2 cells ([Fig. S3](#pone.0061089.s003){ref-type="supplementary-material"}). The up-regulation of miR-21 expression by mature form (amino acids 1--173) of the core protein may contribute to miR-345 promoter hypomethylation. Up-regulation of miR-21 expression has been reported in HCV infectious clone-infected Huh7.5 cells and patients with chronic HCV infection [@pone.0061089-Marquez1]. In our study, the mature form (amino acids 1--173) of the core protein can enhance miR-21 expression, however, the full-length core protein had no effect on miR-21 induction. This finding suggested that the mature form (amino acids 1--173) of the core protein was relevant to the regulation of cellular microRNAs in chronic HCV infection. In fact, we also initially observed the modulation of most of cellular microRNAs by mature form (amino acids 1--173) of the core protein ([Fig. 1B](#pone-0061089-g001){ref-type="fig"}). Further confirmation will be needed. Up-regulation of miR-345 expression may involve in cancer development. Some studies have described the up-regulation of miR-345 expression in human cancers including oral squamous cell carcinomas and malignant mesothelioma [@pone.0061089-Cervigne1], [@pone.0061089-Guled1]. A recent study also reveals that some tumor-related microRNAs including miR-345 are up-regulated from 28 published tumor studies by analyzing microRNA expression microarray datasets, indicating that miR-345 may be an oncomiR in human cancers including HCC [@pone.0061089-Wang2]. MiR-345 has been reported to down-regulate *BAG3* gene expression, an anti-apoptosis gene, as a tumor suppressor microRNA in human colorectal cancer [@pone.0061089-Tang1]. However, in our study, miR-345 down-regulates *p21^Waf1/Cip1^* gene expression as an oncomiR in human hepatoma cells. These two studies suggest that the same microRNA may function as an oncogene or as a tumor suppressor gene depending on cell types and microenvironment. Our study shows that the up-regulation of miR-345 expression may be related to hepatocarcinogenesis during chronic HCV infection. In conclusion, our study demonstrates that mature or more truncated HCV core protein (amino acids 1--173 or 1--153) can up-regulate the expression of miR-345 which then suppresses *p21^Waf1/Cip1^* gene expression in human hepatoma cells. We also show that mature HCV core-induced miR-345 can suppress endogenous *p21^Waf1/Cip1^* gene expression in HepG2 and curcumin-stimulated Huh7 cells. Furthermore, we demonstrate that mature HCV core-induced miR-345 involves in anti-apoptosis through down-regulation of *p21^Waf1/Cip1^* gene expression in curcumin-stimulated Huh7 cells. In conclusion, it is the first time that HCV core protein has ever been demonstrated to inhibit human *p21^Waf1/Cip1^* gene expression through miR-345 targeting. Supporting Information {#s5} ====================== ###### **MicroRNA-93 cannot down-regulate endogenous** ***p21^Waf1/Cip1^*** **gene expression in human hepatoma cells.** HepG2 cells were transiently transfected with the increased amount of miR-93 mimic for 24 hours. The *p21^Waf1/Cip1^* gene expression at protein level was determined by Western blotting. β-actin served as an internal control. (TIF) ###### Click here for additional data file. ###### **Curcumin has no effect on endogenous miR-345 expression.** Huh7 cells were treated with curcumin in dose- (left panel) and time- (right panel) dependent manners. The relative expression of miR-345 was determined by TaqMan real-time qPCR. Data was shown as the means ± S.D. from triplicate experiments. \**P*\<0.05. (TIF) ###### Click here for additional data file. ###### **HCV core protein up-regulates miR-21 expression in human hepatoma cells.** Huh7 and Hepg2 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1--191, 1--173 and 1--153, respectively. At 48 hours after transfection, relative expression of miR-21 was determined by TaqMan real-time qPCR in Huh7 cells (left panel) and HepG2 cells (right panel). Data was shown as the means ± S.D. from triplicate experiments. \**P*\<0.05, \*\**P*\<0.001. (TIF) ###### Click here for additional data file. ###### **Curcumin induces apoptosis through p21^Waf1/Cip1^-independent pathway in HepG2 cells.** HepG2 cells were treated with different doses (6.25, 12.5, 25, and 50 µM) of curcumin for 24 hours. DMSO served as control (labeled with 0 µM). The *p21^Waf1/Cip1^* gene expression at protein level was determined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was determined by annexin V-FITC apoptosis assay (lower panel). Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. (TIF) ###### Click here for additional data file. We thank King-Song Jeng and Michael M.C. Lai at Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C. for reading the manuscript and providing helpful suggestions and the full-length HCV replicon of a Japanese genotype 1b strain (pHCV-N). [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Final approval of the version to be published: SMH TYH. Conceived and designed the experiments: TYS TYH. Performed the experiments: TYS SMH. Analyzed the data: YLS HCC WKC. Wrote the paper: TYS.

{ "pile_set_name": "PubMed Central" }

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22,186

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INTRODUCTION ============ *Helicobacter pylori* infection affects more than 50% of the world's human population and is associated with gastritis, peptic ulcer disease, and gastric cancer \[[@b1-kjim-2019-139]\]. In Korea, the *H. pylori* infection rate in adults is approximately 60% and gastric cancer is the second most frequently diagnosed malignancy \[[@b2-kjim-2019-139],[@b3-kjim-2019-139]\]. Successful eradication of *H. pylori* could be beneficial in alleviating *H. pylori*-related gastroduodenal diseases and reduce the risk of gastric cancer in countries like Korea where the prevalence of both *H. pylori* infection and gastric cancer are high. Until now, the triple therapy combining proton pump inhibitor (PPI) with two antibiotics (clarithromycin and amoxicillin) has been the standard first-line treatment for *H. pylori* eradication. Nonetheless, the eradication rate of this regimen has decreased to \< 80% \[[@b4-kjim-2019-139]-[@b7-kjim-2019-139]\]. The primary factor associated with eradication failure is clarithromycin resistance. In Korea, the clarithromycin resistance rate gradually increased from 17.2% to 23.7% during 2003 to 2008 \[[@b8-kjim-2019-139]\]. The second factor related to eradication failure is increased rates of antibiotic-associated adverse events, which could result in poor patient compliance \[[@b9-kjim-2019-139]\]. In areas with high clarithromycin resistance, bismuth-containing quadruple or non-bismuth quadruple therapies are recommended. Additionally, alternative treatment strategies that increase the eradication rate and reduce adverse events should be developed. Probiotics supplementation with the standard triple therapy might be a candidate to satisfy this purpose. Administration of probiotics have reportedly improved *H. pylori* eradication rates and reduced adverse events associated with the triple therapy \[[@b10-kjim-2019-139]-[@b12-kjim-2019-139]\]. However, the inhibitory effect of probiotics on *H. pylori* infection remains controversial. Recently, two large meta-analyses showed that *Saccharomyces boulardii* reduced overall adverse events and increased eradication rates \[[@b13-kjim-2019-139]\]. Sulforaphane extracted from broccoli is another supplementary candidate for *H. pylori* eradication therapy. Broccoli sprout extract containing sulforaphane (BSES) exhibits cellular anti-oxidative, anti-inflammatory, and anti-cancer effects \[[@b14-kjim-2019-139]-[@b16-kjim-2019-139]\]. Sulforaphane is a potent bacteriostatic agent against *H. pylori* strains and also exhibits bactericidal effects in human epithelial cells \[[@b17-kjim-2019-139]\]. Although no statistically significant effects have been demonstrated in the previous study, we have identified a tendency to show positive results in some urea breath test (UBT) results; further studies have been presented on the effects of using the combination with standard triple therapy \[[@b18-kjim-2019-139]\]. This study excluded patients with cultures resistant to clarithromycin and registered only those subjects with infections not resistant to clarithromycin. Based on our literature review, to our knowledge, this is the first study designed to eliminate clarithromycin resistance as the affecting bias of *H. pylori* eradication failure. We aimed to determine whether *S. boulardii* probiotics or sulforaphane supplementation could increase the *H. pylori* eradication rate and/or reduce antibiotic-associated adverse events in a Korean population. METHODS ======= Patients and study protocol --------------------------- All patients with *H. pylori*-positive chronic gastritis or peptic ulcer disease were recruited at the Kyung Hee University Hospital in Seoul, Korea, from August 2014 to April 2016. All participants were diagnosed with *H. pylori* infection using rapid urease or C^13^-UBTs. Patients with gastric cancer, previous gastrectomy, and severe underlying systemic diseases, or those who had received antibiotics or PPI within the previous month were excluded. Data including age, gender, cigarette smoking, alcohol intake, salty food consumption, family history of gastric cancer, body mass index (BMI), hypertension, and diabetes were collected. All patients underwent clarithromycin resistance testing and subjects with clarithromycin-resistant infections were excluded from the study. Finally, subjects were registered and a computer program was used to randomly assigned the subjects into three treatment groups: triple therapy only (group A), triple therapy plus probiotics (group B), and triple therapy plus sulforaphane (group C) ([Fig. 1](#f1-kjim-2019-139){ref-type="fig"}). Group A received 40 mg of pantoprazole, 1 g of amoxicillin, and 500 mg of clarithromycin twice daily for 7 days. Groups B and C received the same PPI-based triple therapy for 7 days. Additionally, one capsule with either probiotics or sulforaphane was provided three times daily for 4 weeks to patients in groups B and C, respectively. All patients provided written informed consent prior to enrolment. This study was approved by the Institutional Review Board of the Kyung Hee University Hospital (KMC IRB: 1427-01) and registered with ClinicalTrials. gov (NCT03220542). All authors had access to the study data and reviewed and approved the final manuscript. Probiotics and broccoli sprout extract containing sulforaphane -------------------------------------------------------------- *S. boulardii* capsules (3 × 10^10^ colony-forming units/g, Bioflor, Kuhnil Pharm., Seoul, Korea) were administered as the probiotic. BSES capsules containing 250 mg standardized broccoli sprout, yielding 1,000 μg of sulforaphane (Oregon Health, Phoenix, AZ, USA), were administered. The doses from broccoli and probiotics were determined to meet the doses presented in several studies, including previous studies of us \[[@b18-kjim-2019-139]\]. Assessment of *H. pylori* eradication and adverse events -------------------------------------------------------- C^13^-UBTs were performed 4 weeks after the last dose of the study medication. One week after completing treatment, information on adverse events and compliance was collected from the outpatient department through telephone interviews. Compliance was considered satisfactory when patients had taken \> 85% of the study medications. Clarithromycin resistance tests ------------------------------- Mutation of the 23S rRNA results in increased antibiotic resistance \[[@b19-kjim-2019-139]\]. Clarithromycin resistance in *H. pylori* in the present study was determined by evaluating for the presence of the A2142G or A2143G mutations in 23S rRNA. *H. pylori* genomic DNA was extracted from two biopsy specimens from the antrum and body collected during endoscopy. The DNA was amplified using the Seeplex home-brew primer mix from the Seeplex ClaR-*H. pylori* polymerase chain reaction (PCR) kit (Seegene Inc., Seoul, Korea) that was developed using a dual-priming oligonucleotide system to detect the A2142G or A2143G mutations. CYP2C19 genotyping ------------------ The genotyping of exons 4 and 5 in *CYP2C19* was conducted by PCR with restriction fragment length polymorphism (PCR-RFLP). The polymorphism of *CYP2C19* was detected as previously described \[[@b20-kjim-2019-139]\]. The patients were classified into three groups according to *CYP2C19* genotype: rapid metabolizers (RM) had exon 5, wildtype/exon 4, wild-type; intermediate metabolizers (IM) had exon 5, wild-type/exon 4, heterozygote or exon 5, heterozygote/exon 4, wild-type; and poor metabolizers (PM) had both mutated alleles. Data analysis ------------- Eradication was evaluated using intention-to-treat (ITT) and per-protocol (PP) analyses. Continuous variables are expressed as means ± standard deviation, and categorical variables as percentages. Student's *t* test, chi-square test, and two-factor analysis of variation (ANOVA) were used. PASW Statistics for Windows version 22.0 (IBM Co., Armonk, NY, USA) was used for all statistical analyses. All statistical tests were two-tailed and *p* values of \< 0.05 were assumed to indicate statistical significance. RESULTS ======= Baseline characteristics of the study population ------------------------------------------------ A total of 217 patients with *H. pylori*-positive chronic gastritis or peptic ulcer disease were recruited during study periods. Of these, 34 subjects with clarithromycin-resistant infections were excluded from the study. The rate of clarithromycin resistance in this study was 17.3% (34/217 cases). Two cases were dropped. Finally, 183 patients with clarithromycin-sensitive infections were randomly assigned the subjects into three treatment groups: group A (n = 61), B (n = 61), and C (n = 61). The clinical parameters such as age, gender, cigarette smoking, alcohol intake, family history of gastric cancer, salty food consumption, and BMI, were similar among the three groups. Endoscopic finding and *CYP2C19* genotype did not differ significantly between the three groups ([Table 1](#t1-kjim-2019-139){ref-type="table"}). Eradication rates ----------------- The eradication rates according to ITT and PP analyses were 85.2% and 89.6% in group A, respectively. ITT and PP analyses showed 81.9% and 89.2% eradication rates in group B and 86.8% and 96.3% in group C, respectively. The eradication rates according to ITT and PP analyses were not significantly different across the groups based on two-group comparison tests (ITT analysis: A vs. B, *p* = 0.744; A vs. C, *p* = 1.000) (PP analysis: A vs. B, *p* = 0.313; A vs. C, *p* = 0.273). The eradication rate in group C was slightly higher compared to group A. However, the difference between the two groups (A vs. C) was not statistically significant ([Table 2](#t2-kjim-2019-139){ref-type="table"}). Adverse events -------------- In order of frequency, the adverse events included taste disturbance, diarrhea, headache, epigastric pain, nausea, and urticaria. The frequency of overall adverse events in the three groups was not significantly different (A vs. B, *p* = 0.574; A vs. C, *p* = 1.000). The frequencies of gastrointestinal disturbances such as diarrhea, epigastric pain, and nausea were lower in group B compared to group A, but the difference was not statistically significant (*p* = 0.339) ([Table 3](#t3-kjim-2019-139){ref-type="table"}). *CYP2C19* genotypes ------------------- Analysis of *CYP2C19* genotypes revealed the RM genotype in 16.4%, 24.6%, and 21.3% of patients in groups A, B, and C, respectively. The frequency of the RM genotype did not differ significantly across the three groups as per the results of the multiple group comparison test (*p* = 0.532) ([Table 1](#t1-kjim-2019-139){ref-type="table"}). DISCUSSION ========== The Maastricht V/Florence Consensus Report stated that PPI-clarithromycin-amoxicillin triple therapy without prior susceptibility test should be reconsidered in regions with a clarithromycin resistance rate above 15% \[[@b21-kjim-2019-139]\]. Based on our literature review, ours is the first study to exclude patients resistant to clarithromycin, which minimized the most significant bias associated with eradication failure. In our study, 17.3% of the patient infections were clarithromycin-resistant. In another recent Korean study, the clarithromycin resistance rate was \> 20% \[[@b22-kjim-2019-139]\]. This difference may be attributable to different tests used for assessing clarithromycin resistance as well as our study including a large number of relatively heathy subjects. Nonetheless, considering that the clarithromycin resistance rate was \> 15%, alternative regimens are needed for first-line treatment in Korea. Bismuth quadruple (PPI, bismuth, tetracycline, and metronidazole) or concomitant non-bismuth quadruple (PPI, amoxicillin, clarithromycin, and metronidazole) therapies are recommended as first-line treatments \[[@b21-kjim-2019-139]\]. However, these therapies are associated with poor patient compliance and increased adverse events owing to antibiotic overdose. Although not highly evident, supplemental therapy with probiotics or sulforaphane could increase eradication rates and decrease antibiotic-associated adverse events. A meta-analysis of 10 clinical trials on the efficacy of probiotics in *H. pylori* treatment showed that *Lactobacillus*-containing and *Bifidobacterium*-containing probiotic compound preparations increased the overall eradication rate and decreased the overall adverse events \[[@b23-kjim-2019-139]\]. However, another meta-analysis of 33 randomized controlled trials (RCTs) could confirm this finding for only four individual strains (*Lactobacillus acidophilus*, *L. casei*, *L. gasseri*, and *Bifidobacterium infantis*) \[[@b24-kjim-2019-139]\]. This study also performed sub-group analysis according to the effectiveness of eradication regimens. In that study, the less effective the antibiotic therapies, the more useful the probiotic supplementation was: for eradication rates \< 60%, the pooled risk ratio (RR) was 1.28 (1.12 to 1.45), for eradication rates of 60% to 69%, the pooled RR was 1.18 (1.10 to 1.27); for eradication rates of 70% to 79%, the pooled RR was (1.11; 1.06 to 1.17); and supplementation had no effect for eradication rates \> 80% (pooled RR, 1.01; 0.96 to 1.77). The authors also reported a significant difference between the probiotics supplement and triple therapy only groups in the overall incidence of side effects (RR, 0.735; 95% confidence interval, 0.598 to 0.902) \[[@b24-kjim-2019-139]\]. However, the result of side effects was only confirmed for non-blinded trials. One double-blind RCT showed that *Bacillus clausii* significantly reduced gastrointestinal adverse events such as nausea, epigastric pain, and diarrhea but insignificantly increased eradication rates \[[@b25-kjim-2019-139]\]. Recently, a meta-analysis of 11 RCTs showed that *S. boulardii* significantly increased eradication rates but that the rates were still below the desired level of success \[[@b13-kjim-2019-139]\]. Probiotics reduce antibiotic-associated adverse events such as nausea, diarrhea, and abdominal pain, thus helping the completion of eradication therapy. Therefore, probiotics can improve the *H. pylori* eradication rate by reducing adverse events rather than by directly eradicating *H. pylori*. The proposed mechanism of probiotics is to counteract the harmful effects of antibiotics on gut microbiota. However, the opposite conclusions were drawn regarding side effects in three studies reporting no significant reduction in overall side effects with probiotics use \[[@b26-kjim-2019-139]-[@b28-kjim-2019-139]\]. Our study showed that probiotic supplementation did not significantly increase the eradication rate. This may be because of several factors. Firstly, the number of subjects was small. Secondly, the eradication rate was \> 80% and so likely did not contribute an additive advantage, similar to that reported by Dang et al. \[[@b24-kjim-2019-139]\]. Overall, the rate of adverse events was lower in the probiotics supplement group B (triple therapy plus probiotics group) compared to group A (standard triple therapy only group). However, the difference was not statistically significant. Sulforaphane is a molecule within the isothiocyanate group of organosulfur compounds. It can be obtained from cruciferous vegetables such as broccoli, Brussels sprouts, and cabbages. Sulforaphane enhances the protection and repair of the gastric mucosa against oxidative stress *in vitro* and has been shown to have anti-inflammatory effects on *H. pylori*-infected gastric mucosae in mice and human subjects \[[@b29-kjim-2019-139]\]. Sulforaphane has a potent bacteriostatic and bactericidal component against *H. pylori* *in vitro* \[[@b17-kjim-2019-139]\]. Furthermore, it is highly effective against a large number of clinical isolates of *H. pylori*, which are highly resistant to conventional antibiotics \[[@b17-kjim-2019-139]\]. Recently, sulforaphane was reported to reduce colonization and attenuate gastritis in *H. pylori*-infected mice and humans \[[@b30-kjim-2019-139]\]. However, a Japanese epidemiological investigation of the relationship between the prevalence of *H. pylori*-induced chronic atrophic gastritis (CAG) and broccoli consumption found no association between frequent broccoli consumption and a low prevalence of CAG. In addition, our previous study did not show the inhibitory effect of sulforaphane on *H. pylori* colonization density in the gastric mucosa of patients with *H. pylori*-positive gastritis \[[@b18-kjim-2019-139]\]. In the present study, the eradication rate in the sulforaphane supplementation group was slightly higher than that in group A (standard triple therapy only group), but the difference was not statistically significant. The overall rate of adverse events was also similar to group A. Our study has some limitations. First, we included relatively small number of subjects in each group. However, this study was a single center study, and it had limitation to recruit enough patients during study period. Instead, during the study period, all patients who agreed to participate were enrolled by our study. In our study, probiotic supplementation and sulforaphane did not significantly increase the eradication rate nor decrease the adverse events. Nevertheless, the eradication rate in sulforaphane group was slightly higher than that in only triple therapy group. A further study including large number of subjects is needed in the future. Second, we used only *S. boulardii* capsules as the probiotic. The probiotics most commonly used include *Lactobacillus spp*., *Bacillus spp*., and yeast such as *S. boulardii*. The choice of the *S. boulardii* was determined by the effectiveness as well as being the most commonly used in most studies. In conclusion, we found that supplement therapy with probiotics or sulforaphane for *H. pylori* eradication was ineffective at increasing eradication rates and decreasing adverse events. KEY MESSAGE =========== 1\. Probiotic or sulforaphane supplementation with triple therapy for *Helicobacter pylori* infection did not significantly increase the eradication rate nor reduced the adverse events. 2\. An appropriate treatment strategy and new drawn up guidelines on treatment of *H. pylori* are necessary for the effective and safety eradication of *H. pylori*. We appreciate the excellent support of our nursing team at the Kyung Hee University Hospital-Digestive Endoscopy Center. No potential conflict of interest relevant to this article was reported. ![Study enrolment flowchart.](kjim-2019-139f1){#f1-kjim-2019-139} ###### Baseline characteristics of the study population Variable Group A (n = 61) Group B (n = 61) Group C (n = 61) *p* value ------------------------ ------------------ ------------------ ------------------ ----------- Age, yr 53.20 ± 10.97 55.6 ± 10.65 55.77 ± 10.05 0.315 Sex, male/female 33/28 30/31 30/31 0.821 Smoking habits 14 (23.0) 13 (21.3) 10 (16.4) 0.644 Alcohol intake 28 (45.9) 23 (37.7) 19 (31.1) 0.244 Family history of GC 6 (9.8) 8 (13.1) 7 (11.5) 0.851 Salty food consumption 16 (26.2) 27 (44.3) 26 (42.6) 0.076 BMI 24.10 ± 3.56 23.75 ± 2.47 23.77 ± 2.94 0.756 Endoscopic findings Gastric ulcer 3 (4.9) 5 (8.2) 4 (6.6) 0.765 Atrophy 35 (57.4) 32 (52.5) 36 (59.0) 0.749 Intestinal metaplasia 12 (19.7) 12 (19.7) 15 (24.6) 0.746 *CYP2C19* genotype RM 10 (16.4) 15 (24.6) 13 (21.3) 0.532 IM 44 (72.1) 40 (65.6) 44 (72.1) 0.660 PM 7 (11.5) 6 (9.8) 4 (6.6) 0.635 Values are presented as mean ± SD or number (%). GC, gastric cancer; BMI, body mass index; RM, rapid metabolizer; IM, intermediate metabolizer; PM, poor metabolizer. ###### *Helicobacter pylori* eradication rates among the three treatment groups Variable Eradication rate *p* value^[a](#tfn1-kjim-2019-139){ref-type="table-fn"}^ *p* value^[b](#tfn2-kjim-2019-139){ref-type="table-fn"}^ ----------------- ------------------ ---------------------------------------------------------- ---------------------------------------------------------- ------- ------- Eradicated 52 50 53 Failed 6 6 2 Follow-up loss 1 5 6 Dropped out 2 0 0 Poor compliance 0 0 0 ITT analysis 52/61 (85.2) 50/61 (81.9) 53/61 (86.8) 0.744 1.000 PP analysis 52/58 (89.6) 50/56 (89.2) 53/55 (96.3) 0.313 0.273 Values are presented as number (%). ITT, intention to treat; PP, per protocol. *p* was determined by two-group (A vs. B) comparison test. *p* was determined by two-group (A vs. C) comparison test. ###### Adverse events in the three treatment groups Variable Group A (n = 61) Group B (n = 61) Group C (n = 61) *p* value^[a](#tfn3-kjim-2019-139){ref-type="table-fn"}^ *p* value^[b](#tfn4-kjim-2019-139){ref-type="table-fn"}^ --------------------------------------------------------------- ------------------ ------------------ ------------------ ---------------------------------------------------------- ---------------------------------------------------------- Taste disturbance 30 29 28 1.000 1.000 Diarrhea 9 5 8 0.396 1.000 Headache 5 2 6 0.440 0.758 Epigastric pain 4 3 3 1.000 1.000 Nausea 3 2 1 1.000 0.619 Urticaria 3 1 1 0.619 0.619 GI disturbance^[c](#tfn5-kjim-2019-139){ref-type="table-fn"}^ 13 8 12 0.339 0.482 Total 36 31 34 0.574 1.000 GI, gastrointestinal. *p* was determined by two-group (A vs. B) comparison test. *p* was determined by two-group (A vs. C) comparison test. Diarrhea, epigastric pain, and nausea. [^1]: These authors contributed equally to this work.

{ "pile_set_name": "PubMed Central" }

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22,187

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"Statistics", "Windows", "version", "IBM", "Armonk", "NY", "USA", "used", "statistical", "analyses", "statistical", "tests", "p", "values", "assumed", "indicate", "statistical", "significance", "RESULTS", "Baseline", "characteristics", "study", "population", "total", "patients", "pylori", "chronic", "gastritis", "peptic", "ulcer", "disease", "recruited", "study", "periods", "subjects", "infections", "excluded", "study", "rate", "clarithromycin", "resistance", "study", "cases", "Two", "cases", "dropped", "Finally", "patients", "infections", "randomly", "assigned", "subjects", "three", "treatment", "groups", "group", "n", "B", "n", "C", "n", "clinical", "parameters", "age", "gender", "cigarette", "smoking", "alcohol", "intake", "family", "history", "gastric", "cancer", "salty", "food", "consumption", "BMI", "similar", "among", "three", "groups", "Endoscopic", "finding", "genotype", "differ", "significantly", "three", "groups", "Table", "table", "Eradication", "rates", "eradication", "rates", "according", "ITT", "PP", "analyses", "group", "respectively", "ITT", "PP", "analyses", "showed", "eradication", "rates", "group", "B", "group", "C", "respectively", "eradication", "rates", "according", "ITT", "PP", "analyses", "significantly", "different", "across", "groups", "based", "comparison", "tests", "ITT", "analysis", "B", "p", "C", "p", "PP", "analysis", "B", "p", "C", "p", "eradication", "rate", "group", "C", "slightly", "higher", "compared", "group", "However", "difference", "two", "groups", "C", "statistically", "significant", "Table", "table", "Adverse", "events", "order", "frequency", "adverse", "events", "included", "taste", "disturbance", "diarrhea", "headache", "epigastric", "pain", "nausea", "urticaria", "frequency", "overall", "adverse", "events", "three", "groups", "significantly", "different", "B", "p", "C", "p", "frequencies", "gastrointestinal", "disturbances", "diarrhea", "epigastric", "pain", "nausea", "lower", "group", "B", "compared", "group", "difference", "statistically", "significant", "p", "Table", "table", "genotypes", "Analysis", "genotypes", "revealed", "RM", "genotype", "patients", "groups", "B", "C", "respectively", "frequency", "RM", "genotype", "differ", "significantly", "across", "three", "groups", "per", "results", "multiple", "group", "comparison", "test", "p", "Table", "table", "DISCUSSION", "Maastricht", "Consensus", "Report", "stated", "triple", "therapy", "without", "prior", "susceptibility", "test", "reconsidered", "regions", "clarithromycin", "resistance", "rate", "Based", "literature", "review", "first", "study", "exclude", "patients", "resistant", "clarithromycin", "minimized", "significant", "bias", "associated", "eradication", "failure", "study", "patient", "infections", "another", "recent", "Korean", "study", "clarithromycin", "resistance", "rate", "difference", "may", "attributable", "different", "tests", "used", "assessing", "clarithromycin", "resistance", "well", "study", "including", "large", 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"RCTs", "could", "confirm", "finding", "four", "individual", "strains", "Lactobacillus", "acidophilus", "casei", "gasseri", "Bifidobacterium", "infantis", "study", "also", "performed", "analysis", "according", "effectiveness", "eradication", "regimens", "study", "less", "effective", "antibiotic", "therapies", "useful", "probiotic", "supplementation", "eradication", "rates", "pooled", "risk", "ratio", "RR", "eradication", "rates", "pooled", "RR", "eradication", "rates", "pooled", "RR", "supplementation", "effect", "eradication", "rates", "pooled", "RR", "authors", "also", "reported", "significant", "difference", "probiotics", "supplement", "triple", "therapy", "groups", "overall", "incidence", "side", "effects", "RR", "confidence", "interval", "However", "result", "side", "effects", "confirmed", "trials", "One", "RCT", "showed", "Bacillus", "clausii", "significantly", "reduced", "gastrointestinal", "adverse", "events", "nausea", "epigastric", "pain", "diarrhea", "insignificantly", "increased", "eradication", "rates", "Recently", "RCTs", "showed", "boulardii", "significantly", "increased", "eradication", "rates", "rates", "still", "desired", "level", "success", "Probiotics", "reduce", "adverse", "events", "nausea", "diarrhea", "abdominal", "pain", "thus", "helping", "completion", "eradication", "therapy", "Therefore", "probiotics", "improve", "pylori", "eradication", "rate", "reducing", "adverse", "events", "rather", "directly", "eradicating", "pylori", "proposed", "mechanism", "probiotics", "counteract", "harmful", "effects", "antibiotics", "gut", "microbiota", "However", "opposite", "conclusions", "drawn", "regarding", "side", "effects", "three", "studies", "reporting", "significant", "reduction", "overall", "side", "effects", "probiotics", "use", "study", "showed", "probiotic", "supplementation", "significantly", "increase", "eradication", "rate", "may", "several", "factors", "Firstly", "number", "subjects", "small", "Secondly", "eradication", "rate", 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1. Introduction {#sec1-molecules-25-02234} =============== Recently, water has become more and more important subject of studies, as evidenced by the increasing number of water-related publications in various fields of science \[[@B1-molecules-25-02234],[@B2-molecules-25-02234],[@B3-molecules-25-02234],[@B4-molecules-25-02234]\]. Water is fundamental to life---it is one of the essential and widely distributed components of biologic systems, which can be considered as a biomolecule in its own right \[[@B5-molecules-25-02234],[@B6-molecules-25-02234]\]. Different scientific fields have been studying water from different aspects, trying to reach better understanding of its properties, structure and functions, yet still, our picture of water as a substance is rather incomplete. Spectroscopy methods, which are based on interaction of light and matter, have contributed a lot to our understanding of water. Recently, established, scientific discipline of aquaphotomics aims to integrate the knowledge these methods acquired about water based on its interaction with light, into one "omics" discipline which relates the structure of water with its functionality, and whose ultimate objective is better understanding of water as a matrix of aqueous and biologic systems \[[@B7-molecules-25-02234]\]. In this regard, aquaphotomics have made significant novel discoveries, and utilized the properties of water in various application fields ranging from water and food quality monitoring, microbiology, to biomeasurements, biodiagnostics and biomonitoring \[[@B8-molecules-25-02234],[@B9-molecules-25-02234],[@B10-molecules-25-02234]\]. Owing to the wide range of aqueous systems and biologic systems aquaphotomics studied, some novel, surprising insights have been discovered, which place focus on importance of water structure, hydrogen bonding and temperature. The effect of temperature has always been one of the most studied phenomena in spectroscopy with respect to its influence on water structure. The very terms "structure-makers" and "structure-breakers" are coined to describe substances which induce changes in the water structure and consequently its spectrum, comparable to decrease in temperature and increase of temperature, respectively. Effects of salts or sugars are commonly described using those terms \[[@B11-molecules-25-02234],[@B12-molecules-25-02234],[@B13-molecules-25-02234]\]. The known influence of temperature on hydrogen bonding in water is something that can be used as an etalon for better understanding of how substances affect the structure of water in various solutions. Among many others, principal component analysis and two-dimensional correlation spectroscopy \[[@B14-molecules-25-02234]\] or multivariate curve resolution-alternating least squares technique \[[@B15-molecules-25-02234]\] were used to describe the effects of temperature perturbations on the NIR spectra of water in terms of hydrogen bonding either alone or in comparison to salt perturbation. Aquaphotomics studies made some further steps uncovering that various phenomena, related to biomolecules or functionality of living organisms and aqueous systems can be described as related to specific water molecular structure and presented using spectral pattern. For example, in one work, concerned with the detection of UV-induced damage on DNA structure, it was found that the aqueous solutions of UVC-damaged DNA caused increase in hydrogen bonded water---i.e., that damaged DNA was a structure-making element causing changes of water similar to low temperature \[[@B16-molecules-25-02234]\]. In another study, which explored cold tolerance ability of different soybean cultivars, it was found that the water structure in the leaves of those cultivars with higher cold tolerance ability even at low temperatures preserved the water in less-hydrogen bonded state compared to those cultivars who are more susceptible, as if the environmental temperature would have been in fact higher \[[@B17-molecules-25-02234]\]. What these novel insights into the water structure and analogy with temperature influence provided is a novel knowledge and better understanding of the water functionality at different levels of organization of biologic systems. One of the main visualization and analytical tools of aquaphotomics, is the so-called aquagram \[[@B8-molecules-25-02234],[@B18-molecules-25-02234]\] which provides a comprehensible demonstration of the ratios of different water species present in a sample. Aquagrams have been found very useful in many applications, such as diagnosis of estrus in giant pandas \[[@B19-molecules-25-02234]\], orangutans \[[@B20-molecules-25-02234]\] and cows \[[@B21-molecules-25-02234]\] and showing the different water spectral patterns of probiotic and non-probiotic bacteria strains \[[@B22-molecules-25-02234]\], or for example, revealing different types of water in the soft contact lenses, in a completely nondestructive manner \[[@B23-molecules-25-02234],[@B24-molecules-25-02234],[@B25-molecules-25-02234]\]. The cited references demonstrated the usefulness of the presentation of the water spectral patterns in aquagrams. In order to unify the relation between water species depicted by aquagrams and related water functionalities we propose to use temperature as a common denominator to express changes of light absorbance at each of the water vibrational frequencies as changes caused by the most influential perturbation for water---temperature. Considering the advantages of better understanding of the functionality of water structure if the analogy is made with the influence of temperature, the objective of this work was to develop a novel method and a visualization tool, so called temperature-based aquagram which translates the effects of any type of perturbation of water structure in aqueous or biologic systems to the equivalent effects that would have been caused by the temperature changes and expressed in temperature units. The need to introduce the temperature-based aquagrams arose from the experiments on more complex systems, such as previously described, where it was observed that certain phenomena, (caused by solutes for example) have effects on the water molecular structure of the system and contribute to its functionality in the way analog to the changes in temperature. In a study concerned with classification of bacteria based on the probiotic strength it was found that the strong probiotic bacteria, compared to the moderately strong strains or non-probiotic strains, create less hydrogen-bonded water species ([Figure 1](#molecules-25-02234-f001){ref-type="fig"}) \[[@B22-molecules-25-02234]\]. On the other hand, the spectra of pure water at different temperatures show shift of the main band towards the shorter wavelengths with increasing temperature \[[@B14-molecules-25-02234]\], i.e., showing that increase in temperature results in breaking of the hydrogen bonds. If the two cases are compared, it can be seen that probiotic bacteria affect surrounding water similarly to the influence of temperature increase, as if they are a structure-breaking element \[[@B22-molecules-25-02234]\]. This conclusion supports the novel insight into molecular mechanisms of how probiotics work--they increase solubility of substances in water \[[@B26-molecules-25-02234]\], just like the increase in temperature of the water would contribute to better solubilization. From this example, it can be seen that comparisons with effect of temperature can be useful and contribute to better intuitive understanding of the functionality of the studied system. Recalling the soybean cultivars example, the role of many substances that plants accumulate in response to cold stress, becomes more obvious---their function is to provide a certain water molecular structure in the leaves as if the temperature of the environment is different \[[@B17-molecules-25-02234],[@B27-molecules-25-02234]\]. The concept of the novel visualization tool---a temperature-based aquagram---was developed similarly to the one Bernal and Fowler introduced long ago---the so called "structural temperature" concept \[[@B28-molecules-25-02234]\]. The "structural temperature" is the respective temperature at which pure water would have effectively the same molecular structure as the water of the aqueous system under study. At the time of introduction, no adequate method that could describe that structure was found; there were propositions that it can be estimated based on the measurements of viscosity, Raman spectra or X-ray diffraction. In addition, the intended purpose was mainly concerned with the applications in analysis of electrolyte solutions, and since the concept did not offer the possibility to separate effects of the individual ions, the entire idea was more or less abandoned \[[@B29-molecules-25-02234]\]. However, the structural temperature concept fits well into the framework of aquaphotomics, which places the water spectral pattern and the respective water functionality of the system in the central place. In contrast to "reductionistic omics methods" which are focused on isolating the biomolecules and separation of elements of the system, the aquaphotomics views each aqueous or biologic system as a whole, where all the components of the system exert their influence on the water matrix, whose structure is directly related to the function of the system \[[@B9-molecules-25-02234],[@B10-molecules-25-02234]\]. Just like the temperature is the macroscopic, measurable characteristic, arising from the molecular structure of the system, so is the light absorbance at each water specific vibrational frequency, and one can benefit from expressing one in the terms of another. The purpose of this study is to introduce a novel visualization tool, which expresses the effects of any type of perturbation of water molecular structure in aqueous or biologic systems to the equivalent effects that would have been caused by the temperature and expressed in temperature units. To illustrate this, we have chosen a simple salt solution and a concentration as a major perturbation. Salt is chosen as it is not near infrared active substance, hence the changes in absorbance of the solutions are only due to the changes in water molecular structure \[[@B15-molecules-25-02234],[@B30-molecules-25-02234]\]. Following the steps provided in the study, one can easily replicate the experiment, develop the temperature-based aquagrams and use it further for specific purposes. While in this study, the analysis and the results are presented for only 1st overtone of water region, the same methodology is applicable for any region of the water absorbance spectra. This new method provides numerical results on a clearly defined scale with confidence intervals. It enables the comparison of results across time and different experiments and provides information about the statistical significance of the found differences. 2. Results and Discussion {#sec2-molecules-25-02234} ========================= The spectral data in the wavelength interval of 1300 to 1600 nm for the two experiments were separately subjected to principal component analysis (PCA). The PCA score plots demonstrated the multidimensional patterns of the spectral data. Specifically, the highest variations displayed in the first principal components (PC1) were related to temperature and concentration of potassium chloride, representing 99.3% and 99.1% of the spectral variation in case of the temperature and the potassium chloride experiments, respectively. The PCA results did not show outliers either in the temperature or the potassium chloride datasets. The raw and 2nd derivative spectra of the two experiments were analyzed separately to discover the wavelengths exhibiting the largest changes caused by the temperature and salt perturbations. 2.1. Results of Temperature Experiment {#sec2dot1-molecules-25-02234} -------------------------------------- The raw and 2nd derivative absorbance (logT^−1^) spectra in the spectral range of 1300 to 1600 nm of Milli-Q water in the temperature range of 20 to 70 °C are shown in [Figure 2](#molecules-25-02234-f002){ref-type="fig"}. The main feature of the NIR spectrum of water is a broad peak around 1450 nm, comprised of several overlapping bands, described mainly as the 1st overtone of the OH stretching vibration \[[@B31-molecules-25-02234]\]. This observation is confirmed by the second derivative spectra which indicated very intense bands at 1412 and 1462 nm. These bands are well known as bands associated with free water molecules \[[@B32-molecules-25-02234],[@B33-molecules-25-02234],[@B34-molecules-25-02234]\] and strongly hydrogen-bonded water \[[@B14-molecules-25-02234],[@B35-molecules-25-02234]\], respectively. From a spectroscopic point of view, an increase in temperature has been interpreted as a decrease of number of hydrogen bonds \[[@B14-molecules-25-02234],[@B34-molecules-25-02234]\], while others explain it via weakening of hydrogen bonds \[[@B36-molecules-25-02234]\]. There is agreement, regardless of one's preferred theory, that the change of temperature causes alteration in hydrogen-bonding configurations of water as described in more details previously \[[@B37-molecules-25-02234]\]. [Figure 2](#molecules-25-02234-f002){ref-type="fig"} shows a "blue shift", i.e., movement of the main band towards the shorter wavelengths with increasing temperature and an isosbestic (temperature invariant) point. These phenomena are well studied in scientific literature \[[@B14-molecules-25-02234],[@B34-molecules-25-02234],[@B38-molecules-25-02234]\]. Evidence clearly suggest that less hydrogen-bonded water is predominant at higher temperature, while spectra acquired at low temperature are represented mostly in the more hydrogen-bonded area where water molecules form species with one (S1), two (S2), three (S3) and four (S4) hydrogen bonds \[[@B39-molecules-25-02234]\]. Despite the competing theories about the water structure, aquaphotomics identifies water-specific absorbance bands with higher variations caused by respective perturbation for the system of interest and uses them to depict the unique spectral pattern as an integrated marker of the system--perturbation interaction. The presentation of the 12 specific water coordinates \[[@B7-molecules-25-02234]\] of the spectra of Milli-Q water acquired in the temperature range of 20 to 70 °C together with the 95% confidence intervals is given in [Figure 3](#molecules-25-02234-f003){ref-type="fig"}a,b, calculated with the temperature-based aquagram calculation method. In the case of working with more complex system, it would be advisable to follow the protocol of aquaphotomics analysis which can provide more thorough examination of activated water absorbance bands and not necessarily limit the presentation to only the 12 coordinates as is chosen here. The aquagram provides an easy-to-comprehend presentation of the phenomena described above, i.e., the change of the strongly and weakly hydrogen-bonded patterns, based on [Figure 2](#molecules-25-02234-f002){ref-type="fig"}. The movement of the higher absorbance towards shorter wavelengths with increasing temperature is convincing. The scales of the aquagrams ([Figure 3](#molecules-25-02234-f003){ref-type="fig"}a,b) express the effect of perturbation occurring at the 12 coordinates (i.e., in the defined wavelength ranges) in degrees Celsius equivalent. Therefore, in this specific example the radial values show the temperatures corresponding to the signal acquisitions (20 to 70 °C). The plotted dashed and dotted lines represent the upper and lower confidence levels of the aquagram values, respectively, for each single analyzed temperature step. There was no overlapping of the confidence intervals (95%) of the Milli-Q samples measured at different temperature observed, meaning that 2 °C temperature changes caused statistically significant effects on the individual coordinates (i.e., in the defined wavelength ranges). The stability of the temperature-based aquagram calculation is presented in [Figure 3](#molecules-25-02234-f003){ref-type="fig"}b, by depicting only three selected temperature levels (20, 30 and 40 °C). Though the spectral dataset used to calculate the temperature-based aquagram of [Figure 3](#molecules-25-02234-f003){ref-type="fig"}b is only a subset of the dataset used to calculate [Figure 3](#molecules-25-02234-f003){ref-type="fig"}a, the shape and the range are the same in both cases. This type of stability was not possible with the "classic" aquagram ([Figure 3](#molecules-25-02234-f003){ref-type="fig"}c,d). These examples present the applicability and the additional benefits of the newly developed temperature-based aquagram for the evaluation and presentation of the water species in the 12 coordinates (representing defined wavelength ranges) through the evaluation of a well-known perturbation, i.e., the effect of temperature on the water spectral changes. 2.2. Results of Potassium Chloride Experiment {#sec2dot2-molecules-25-02234} --------------------------------------------- The raw and 2nd derivative absorbance (logT^−1^) spectra of the aqueous solutions of 0.001 to 1 M potassium chloride salt in the 1300--1600 nm wavelength range (OH first overtone) are presented in [Figure 4](#molecules-25-02234-f004){ref-type="fig"}. The main component of the aqueous solution, other than water, is KCl, which has no absorption in the NIR region; thus, it is not surprising that the spectra show a broad peak around 1450 nm. The second derivative spectra also provide similar patterns to those of the Milli-Q water acquired at different temperature, indicating bands at 1412 and 1462 nm. The trend of the shift in the peak position was similar to that observed in the temperature-perturbed pure water (i.e., the peak moves towards lower wavelengths as temperature is increased), but the actual peak locations were different from those for the pure water. The effect of low concentrations of salts diluted in water has been illustrated by the changes in the OH bonding of water molecular systems in many experiments \[[@B15-molecules-25-02234],[@B40-molecules-25-02234],[@B41-molecules-25-02234],[@B42-molecules-25-02234]\]. As salts do not absorb NIR light, accurate measurement of even low concentration of salts, published in the above-mentioned papers, means salts change the surrounding water molecular structure according to the number of the solvent molecules in the solution which still can restructure the water molecular system. This phenomena is not new; Bernal and Fowler \[[@B28-molecules-25-02234]\] showed that the addition of electrolytes changes the spectrum of water in the infrared overtone region. Lin and Brown \[[@B43-molecules-25-02234]\] also analyzed the effects of different salts on water spectra, and the authors were able to build accurate regression models using the spectral range of 1490 to 1610 nm for salt content prediction. The spectra shown in [Figure 4](#molecules-25-02234-f004){ref-type="fig"} imply that the increasing concentration of KCl causes "blue shift", i.e., a shift towards the spectral range referring to the band of less hydrogen-bonded water molecules. Our findings, hence show a good agreement with the results of previous research \[[@B15-molecules-25-02234],[@B40-molecules-25-02234],[@B41-molecules-25-02234],[@B42-molecules-25-02234]\]. A more detailed evaluation of the proposed 12 specific water coordinates \[[@B7-molecules-25-02234]\] of the spectra of the aqueous solutions of potassium chloride compared to the spectra of water is provided in [Figure 5](#molecules-25-02234-f005){ref-type="fig"}. The plots show the aquagrams of the single concentration levels together with the respective 95% confidence intervals calculated with the "classic" method ([Figure 5](#molecules-25-02234-f005){ref-type="fig"}a) and the new temperature-based aquagram calculation method ([Figure 5](#molecules-25-02234-f005){ref-type="fig"}b). The relative position of the patterns of the single concentration levels shows some similarity for the two methods. Furthermore, in both methods, spectral patterns of the highest concentration range (100--1000 mM) show the highest difference from the spectra of the lower ranges and Milli-Q samples (the latter one in black). This pattern explains the same phenomena which were found based on the raw spectra, i.e., a higher concentration of salt create water with less hydrogen bonds and decreases the number of water species to more hydrogen bonds. For the higher concentration ranges the aquagrams of the individual concentrations do not overlap, i.e., addition of 0.01 or 0.1 M KCl caused statistically significant change at the individual coordinates. However, the results also show an increasing tendency of the C7 coordinate (1432--1444 nm) with increasing concentration of KCl, which can be assigned to water molecules with one hydrogen bond (S1), meaning that increase in concentration of salt leads to increase in the number of these molecules \[[@B39-molecules-25-02234]\]. The dominantly higher effect of the higher concentrations on the spectral pattern hides the pattern of the lower concentrations; therefore, the calculation of the aquagrams were performed using the spectral data set for the two lowest concentration ranges only ([Figure 6](#molecules-25-02234-f006){ref-type="fig"}). The phenomena of the higher concentrations on the water spectral pattern, namely the higher concentration being more dominantly a structure breaker, can be observed in case of the concentration of samples at the concentration range of 10 to 100 mM. It is interesting to note the similarities to the pattern of the aquagrams calculated on all the three ranges ([Figure 5](#molecules-25-02234-f005){ref-type="fig"}b). The aquagrams of the higher concentrations (10--100 mM) show consistency with the previous findings regarding the structure-breaking characteristic as displayed in [Figure 6](#molecules-25-02234-f006){ref-type="fig"}. More specifically, the higher concentration samples present higher absorbance values in the range between 1342 and 1374 nm, i.e., C01-C03 that refer to the 1st overtone of free OH stretch (OH--(H~2~O)*~n~*, *n* = 1...4) \[[@B44-molecules-25-02234],[@B45-molecules-25-02234]\] and 1440 and 1452 nm, i.e., C07-C08 that are known as the bands of water hydration \[[@B35-molecules-25-02234]\] and water molecule connected to another water molecule (S~1~) \[[@B14-molecules-25-02234],[@B46-molecules-25-02234]\] and the symmetric and asymmetric stretching of first overtone of water \[[@B35-molecules-25-02234],[@B46-molecules-25-02234],[@B47-molecules-25-02234]\]. However, in the range between 1476 and 1512 nm, i.e., C10-C12, the higher concentration samples show lower values and these wavelengths are usually assigned to strongly hydrogen bonded water \[[@B14-molecules-25-02234],[@B35-molecules-25-02234]\] and aqueous protons (\[H^+^•(H~2~O)~6~\]--H~2~O in H~5~O~2~^+^ symmetric stretch) \[[@B48-molecules-25-02234]\]. These findings also mean decreasing concentration of salt causes a shift towards longer wavelengths, i.e., the range referring to the band of more hydrogen bonded water molecules. Using the additional benefit of the temperature-based aquagram calculation method, further results can be achieved from [Figure 6](#molecules-25-02234-f006){ref-type="fig"}. The addition of, for instance, 0.1 M potassium chloride to Milli-Q water results in water structural changes equivalent to changes caused by temperature of about 0.65, 0.6, 0.3, 0.1, 0.2 0.6, 1.8, 1.2, 0.2, −0.1, −0.3 and −0.6 °C at C01, C02, C03, C04, C05, C06, C07, C08, C09, C10, C11 and C12 coordinates, respectively. Furthermore, having calculated the confidence intervals, the statistical significance of the differences is also available. For example, calculations showed that addition of 0.02 M KCl to Milli-Q caused change equivalent to the change due to 0.33 and 0.23 °C temperature increase at coordinates C07 and C08 when compared to pure Milli-Q, which was found significant (*p* = 0.05), too. The aquagrams of the lowest concentration range (1--10 mM) calculated with the temperature-based aquagram method is shown in [Figure 7](#molecules-25-02234-f007){ref-type="fig"} to further evaluate the findings of the effects of decreasing concentrations of salt. The results suggest that we can observe concentration levels of 5 to 10 mM that cause a shift towards longer wavelengths, i.e., the range referring to the band of more hydrogen-bonded water molecules compared to the spectrum of Milli-Q water. These findings imply that salts can also have structure-breaker and structure-maker effects on water structure similar to the behavior of sugars \[[@B13-molecules-25-02234]\]. However, the aquagrams of the even lower concentrations (1 to 5 mM) also show alteration with nearly the same deviation, but towards shorter wavelengths. This may suggest that changing between structure-breaker and structure-maker properties of salt exist at such a low concentration, but the discernment of these minor changes would require very accurate measurements. 3. Materials and Methods {#sec3-molecules-25-02234} ======================== 3.1. Samples {#sec3dot1-molecules-25-02234} ------------ Two experiments with different sources of perturbations were conducted to demonstrate the procedure of temperature-based aquagram development and show the advantages in comparison to the representation of spectral data using classic aquagrams. In both experiments, pure Milli-Q water was used as a sample (Milli-Q purification system (Millipore, Molsheim, France, resistance = 18 MΩ) and in the first experiment perturbation of the sample was caused by changes in temperature, while in the second one, by changes in the concentration of salt. 3.2. The Temperature Experiment {#sec3dot2-molecules-25-02234} ------------------------------- The effect of temperature perturbation on water near-infrared spectra has been well-studied and is thoroughly described in the literature \[[@B14-molecules-25-02234],[@B34-molecules-25-02234],[@B49-molecules-25-02234]\]. Therefore, the experiment was performed on Milli-Q water in the temperature range of 20 to 70 °C to acquire spectra which could be used for the evaluation of the aquagram methods. The Milli-Q water was produced by a Milli-Q purification system (Millipore, Molsheim, France, resistance = 18 MΩ). The spectral acquisition was performed at 2 °C increments, resulting in 26 temperature steps in the range of 20 to 70 °C. 3.3. The Potassium Chloride Experiment {#sec3dot3-molecules-25-02234} -------------------------------------- The addition of salt to water at different concentrations is also an often evaluated perturbation \[[@B42-molecules-25-02234],[@B50-molecules-25-02234]\]. Therefore, an experiment was performed with different concentrations of aqueous solutions of potassium chloride. Potassium chloride (KCl, M = 74.56 g mol^−1^, purity min. 99.0% mass/mass) was purchased from Wako Pure Chemical Industries, Ltd. (Kobe, Japan). Aqueous solutions were prepared in different concentrations of KCl, in the range of 1 to 1000 mM. Three concentration ranges were prepared: Range A, from 100 to 1000 mM concentration, in steps of 100 mM; Range B from 10 to 100 mM, in steps of 10 mM and finally, Range C, in 1 to 10 mM in 1 mM concentration steps. Each dilution was prepared by serial dilution from the stock samples with the highest concentration in a given range (A, B or C) and prepared in two replicates, resulting in two independently prepared sets of samples. Stock solutions were prepared and further serially diluted with added Milli-Q water step-by-step to reach the appropriate concentrations---a solution created in each step was further diluted to prepare the next lower concentration. 3.4. NIR Spectral Acquisition {#sec3dot4-molecules-25-02234} ----------------------------- A FOSS-XDS spectrometer (FOSS NIRSystems, Inc., Hoganas, Sweden) equipped with a Rapid Liquid Analyzer module including a temperature-controlled 1 mm pathlength cuvette holder was used to measure transmittance spectra (logT^−1^) of the Milli-Q samples for the temperature experiment and of the aqueous solutions for the KCl experiment. Spectral acquisition was performed by saving three consecutive spectra in the range of 400--2500 nm at 0.5 nm spectral steps. Each saved spectrum was the average of 32 successive scans. A thermal bath with continuous water circulation was attached to the Rapid Liquid Analyzer module to ensure the required temperature of the sample during scanning in the range of 20 to 70 °C at 2 °C increments. The same apparatus was used to provide a constant temperature of 28 °C where each aqueous solution of potassium chloride was incubated for 90 s to equilibrate to the required temperature before scanning. Milli-Q water samples were measured as every fifth sample during the KCl experiment to provide environmental controls. The total number of spectra for the temperature experiment was 78 (26 temperature steps × 3 consecutive scans) and for the KCl experiment was 330 (30 concentrations × 2 repeats × 3 consecutive scans + 150 Milli-Q control scans) ([Appendix A](#app1-molecules-25-02234){ref-type="app"} Dataset). The FOSS-XDS instrument was operated using VISION 3.5 software (FOSS NIRSystems, Inc., Hoganas, Sweden). In both experiments, reference spectra were recorded before every sample. 3.5. Statistical Data Analysis {#sec3dot5-molecules-25-02234} ------------------------------ Only the wavelength interval of 1300 to 1600 nm, corresponding to the first overtone of the O--H stretching band \[[@B51-molecules-25-02234]\] was used for the evaluations. For the purpose of explaining methodology of how to develop temperature-based aquagrams, in this study, the focus is placed on this particular part of the water absorbance spectra, because it is best understood so far in the terms of the water molecular species whose absorbance bands are well-resolved and their assignments known \[[@B7-molecules-25-02234]\]. In the first overtone of water as a result of systematization of experimental work done on many different systems, not only different aqueous solutions, but also a great variety of biologic systems under different perturbations 12 water absorbance bands termed WAMACs (Water Matrix Coordinates)--each range from 6 to 12 nm width, were discovered \[[@B7-molecules-25-02234]\]. The great body of evidences in scientific literature provided the meaning to these ranges---i.e., it was possible to connect each of these 12 coordinates to specific water molecular species. In aquaphotomics studies the 12 WAMACs are called, coordinates because they represent windows in the spectra through which water structure of the system under study can be observed. In this study, the 12 WAMACs will be used to develop aquagrams, but it should be noted that the methodology explained is applicable for any region of the water absorbance spectra; it is not necessarily limited to the coordinates chosen here. Here, we chose for the reason of simplicity and because they are well-understood to use only those. However, depending on the system under study and the range of the spectra used, the reader is advised to follow the aquaphotomics protocol of analysis for extraction of the "activated water absorbance bands--WAMACs \[[@B8-molecules-25-02234]\] and then follow the further instructions to develop temperature-based aquagrams as described below. Before the development of aquagrams, exploratory analysis was performed. First, Principal component analysis (PCA) \[[@B52-molecules-25-02234]\] was used to describe multidimensional patterns in the spectral data and to discover outliers. The raw and 2nd derivative spectra were plotted to visualize the spectral changes induced by temperature perturbation and by the perturbation of salt concentration on the spectra of Milli-Q water and aqueous solutions of potassium chloride, respectively. The 2nd derivative spectra were calculated using a Savitzky--Golay filter \[[@B53-molecules-25-02234]\] using the 2nd order polynomial and 21 points. ### 3.5.1. Calculation Protocol of "Classic" Aquagram {#sec3dot5dot1-molecules-25-02234} The absorbance values at specific water matrix coordinates (WAMACs) \[[@B7-molecules-25-02234]\] define the water spectral pattern (WASP), which is different for different perturbations. The WASP can be visualized by a chart called the aquagram \[[@B18-molecules-25-02234],[@B19-molecules-25-02234]\]. This representation of the WAMACs was first introduced by Tsenkova \[[@B18-molecules-25-02234]\]. This aquagram (from now on called the "classic" aquagram) displays the multiplicative-scatter-corrected (MSC) (or standard-normal-variate (SNV)) transformed, normalized and averaged absorbance values of different samples or sample groups at 12 specific characteristic wavelengths. As, mentioned, these specific wavelengths were experimentally discovered as absorbance bands of specific water molecular species in previous studies and are later confirmed by overtone calculations of already reported water absorbance bands in the infrared range \[[@B7-molecules-25-02234]\]. The aforementioned water absorbance bands cover various form of water molecular species and are thus useful to depict characteristic spectral patterns in the first overtone region of water. This calculation can be summarized by Equation (1). $$A_{\lambda}^{\prime} = \frac{A_{\lambda} - \mu_{\lambda}}{\sigma_{\lambda}}$$ where, $A_{\lambda}^{\prime}$---value on aquagram for a given wavelength; *A~λ~*---absorbance after MSC applied on 1st overtone region of OH (i.e., 1300--1600 nm); *μ~λ~*---mean of all spectra for the examined group at a given wavelength (after MSC applied); *σ~λ~*---SD of all spectra for the examined group at a given wavelength (after MSC applied); *λ*---12 wavelengths (1342, 1364, 1374, 1384, 1412, 1426, 1440, 1452, 1462, 1476, 1488, 1512 nm) \[[@B7-molecules-25-02234]\]. The classic aquagram shows the relative fingerprint, i.e., the WASPs, in the context of all spectra in the examined group \[[@B18-molecules-25-02234],[@B19-molecules-25-02234],[@B21-molecules-25-02234],[@B54-molecules-25-02234]\]. Recently, this aquagram calculation method was extended by adding the possibility to observe the statistical significance of the differences presented on the aquagrams. Therefore, besides the average spectra of the individual groups used to plot the aquagrams, the respective confidence intervals are also calculated using the so-called Bootstrap method \[[@B55-molecules-25-02234]\]. This improvement makes it possible to plot the aquagrams together with their upper and lower 95% confidence interval limits. ### 3.5.2. Calculation Protocol for Newly Developed (Temperature-Based) Aquagram {#sec3dot5dot2-molecules-25-02234} The newly developed temperature-based aquagram calculation algorithm presents the respective water matrix coordinates in units equivalent to change in temperature and includes the respective confidence intervals. Therefore, it gives rise to the possibility to compare the WAMACs across time and also across different experiments and it provides information about the statistical significance of the differences. The new calculation method is based on the comparison of the areas (under the ranges of the above-mentioned 12 specific water coordinates \[[@B7-molecules-25-02234]\]) of the respective test sample spectra and the spectra of Milli-Q water. This method aims to express the spectral pattern changes in units equivalent to the change of temperature that would cause the observed change. The calculation of this aquagram concept (from now called the temperature-based aquagram) can be summarized in the following steps. Note that the calculation steps are explained for one coordinate (C01 representing spectral range between 1336 and 1348 nm, as an example) out of the 12 coordinates (C01-C12, i.e., defined wavelength ranges) to give an easily understandable description, but the same steps have to be repeated for each of the 12 coordinates. The main calculation steps are summarized in a chart to provide an overview of the developed method ([Appendix B](#app2-molecules-25-02234){ref-type="app"} [Figure A1](#molecules-25-02234-f0A1){ref-type="fig"}). 1. The dataset of the temperature experiment (i.e., the spectra of the Milli-Q water samples acquired during the temperature experiment in the temperature range between 20--70 °C) is defined as the reference dataset. The dataset of the experiment of interest is defined as the experimental dataset. 2. The average spectra of the consecutive scans are calculated for each temperature step, yielding 26 single, unique spectra in the reference dataset. The average spectra of the groups of interest (in this case, salt concentration levels) are calculated in the experimental dataset together with their respective confidence intervals using the Bootstrap method \[[@B55-molecules-25-02234]\], yielding as many single, unique spectra as there are groups are in the experimental dataset (plus their upper and lower 95% confidence interval limits). 3. The area under the spectrum for every single average spectrum---in the reference dataset and in the experimental dataset---at the wavelength range of 1336 to 1348 nm (C01) is calculated taking into account the baseline estimated by linear fitting on the two edges of the first overtone region (i.e., 1300 and 1600 nm). In case of the experimental dataset, the areas of the respective confidence interval limits are also calculated in addition to the area of the average spectrum. [Figure 8](#molecules-25-02234-f008){ref-type="fig"} provides graphical interpretation of the relevant areas and the wavelength regions used for the 12 coordinates. 4. The ratio of the area under the curve for each single coordinate is calculated with respect to the full area under the curve for the first overtone OH region (i.e., the area of C01 is divided by the full area under the spectrum in the range of 1300 to 1600 nm). This is done for every single average spectrum, in the reference dataset and the experimental dataset (together with the respective confidence interval limits for the experimental dataset). This calculation step provides normalized values and avoids possible differences due to scattering and/or pathlength effects. 5. Based on the reference dataset, a continuous array of values for the relative area of C01 (as calculated in Step 1) is calculated for a continuous temperature range from 20 to 70 °C using local polynomial regression. This is an essential step in order to accommodate the data from an experiment performed at specific temperature---see Step 6. 6. The basic principle of the temperature-based aquagram method is to compare the effect of the perturbation used on the system under study which resulted in a certain water spectral pattern to the effect the temperature changes would induce in pure water. Thus, any perturbation can be expressed as an equivalent temperature effect on a Milli-Q water sample. It is necessary to perform a "local calibration" with the reference dataset around the temperature of the experimental dataset. Therefore, in this step, the temperature calibration range is defined. This range is used to express the effect of perturbation in degrees Celsius equivalent. For this, a symmetrical scale is defined from the reference dataset (calculated at Step 5) using two degrees, plus and minus around the temperature of the experiment (hence, a span of 4 °C). For example, if the experiment was performed at 25.0 °C, then the calibration range of 23.0 to 27.0 °C would be used. 7. The temperature calibration equation, the relationship between the change of the temperature and change of the area of C01 at the temperature of the experiment, is determined based on the calculation performed in Step 5 on the reference dataset. (It is known how the area of C01 changes as a function of temperature described by a linear function). Therefore, it is easy to compare the changes for areas for C01 for the experimental dataset (calculated at Step 3) to the changes of the area of C01 caused by temperature, i.e., to express the changes in C01 in units of temperature (degrees Celsius) equivalent. 8. The calculated temperature (degrees Celsius) equivalent value for every group of the experimental dataset is finally visualized together with the respective 95% confidence intervals in a radar chart, where the units of the axes are in degrees Celsius. 9. The calculation and visualization of the results were performed using the R programing language \[[@B56-molecules-25-02234],[@B57-molecules-25-02234]\]. 4. Conclusions {#sec4-molecules-25-02234} ============== Recently, aquaphotomics has been introduced to focus on water as a key component for providing information about the function of the entire system. Non-destructive NIR spectroscopy and aquaphotomics have been applied to explain new phenomena in the field of life sciences. In contrast to reductionistic methods in which biomolecules and other elements are analyzed separately from the system, aquaphotomics studies the aqueous systems through its water matrix. In the present study, a newly developed temperature-based aquagram calculation method is presented as an additional tool to express the changes of water molecular structure in aqueous systems which are caused by perturbations different from temperature. Although the need to introduce temperature-based aquagrams originated from experiments on complex systems, the successful application of the new aquagram calculation method was demonstrated through the evaluation of the results of well-known phenomena. The results of temperature and salt perturbations on Milli-Q water are demonstrated in the present study. The effect of temperature on the spectral pattern of Milli-Q water acquired in the temperature range of 20 to 70 °C is presented with the temperature-based aquagram calculation method. The method provides the presentation of the phenomena demonstrating blue shift in the first OH overtone range with increasing temperature. Furthermore, the scale of the aquagram expressed the effect of perturbation in degrees Celsius equivalent. Aqueous solutions of potassium chloride were chosen for the experiment as KCl has no absorption in the NIR region. Thus, its effect on water spectral patterns can be clearly evaluated and presented as caused by temperature changes at respective wavelengths. Furthermore, it demonstrates that the method is invariably applicable for evaluation the effects of all types of solutes. The new temperature-based aquagram calculation method provided further information about the magnitude of the change. In other words, the results were displayed on a degree-Celsius scale that showed how much a given sample would have needed to have been warmed up or cooled down in each of the single coordinates (C01 to C12) to achieve the same results as the actual measurement, while all measurements were performed at precisely the same temperature. Adding 0.1-M potassium chloride to Milli-Q water resulted in structural changes equivalent to an approximately 0.6 °C temperature increase in the less hydrogen-bonded, and 0.3 °C temperature decrease in the more hydrogen-bonded areas of the OH first overtone spectral region. The examples presented here confirm the applicability and the additional benefits of the temperature-based aquagram calculation method. They provide a demonstration of the ratio of the different water species existing in different aqueous and biologic systems. Additionally, this new type of aquagram calculation displayed the spectral patterns in a meaningful scale and stable pattern independent of any modification of the evaluated dataset, which gives rise to the opportunity to compare results not only within a single chart, but also across time and different experiments. This newly developed tool is especially suitable for visualizing water structure evolution and phase transitions, for example, in the food preservation industry, pharmaceutical development, material science and related applications. The presented new chemometric tool, developed in R-Project, is freely accessible as an R-package from GitHub repository \[[@B58-molecules-25-02234]\] and can be used in various fields of NIR spectroscopy and water research. Authors are grateful to Professor David Funk for his help to revise the manuscript from language editing and from scientific point of view. **Sample Availability:** Samples of the compounds KCl are available from the authors. All authors contributed to this research. The design of the experiments was performed by Z.K., B.P. and R.T., Z.K. and B.P. did the recording and the processing of the NIRS data, as well as the result of the evaluation and implementing the concept in R-project. The manuscript was written by Z.K., B.P., G.B. and J.M., R.T. assisted in writing the study and revised it. Z.K., R.T., G.B. and J.M. contributed to designing the research and revise the manuscript. The work presented in the study was conceived within research projects led by R.T. and Z.K. All authors have read and agreed to the published version of the manuscript. Authors acknowledge the financial support of the ÚNKP-19--4 New National Excellence Program of the Ministry for Innovation and Technology (Z.K.), János Bolyai Research Scholarship of the Hungarian Academy of Sciences (Z.K.) and by the European Union and co-financed by the European Social Fund through project No. EFOP-3.6.3-VEKOP-16--2017--00005. JM gratefully acknowledges the financial support provided by Japanese Society for Promotion of Science (P17406). The authors declare no conflict of interest. Dataset. Raw spectral data used for the calculations presented in this study. Both data of temperature experiment and potassium chloride experiment is included in this dataset where the column experiment describes which spectra belong to which experiment. ![Workflow of the calculation protocol of temperature-based aquagram.](molecules-25-02234-g0A1){#molecules-25-02234-f0A1} ![Water spectral pattern presented on aquagrams shows different water structure in different bacteria strains (Reprinted with permission from Slavchev, A., Kovacs, Z., Koshiba, H., Nagai, A., Bázár, G., Krastanov, A., Kubota, Y. and Tsenkova, R. 2015. \[[@B22-molecules-25-02234]\]).](molecules-25-02234-g001){#molecules-25-02234-f001} ![Raw and 2nd derivative (calculated with Savitzky--Golay filter using 2nd order polynomial and 21 points) absorbance (logT^−1^) spectra in the spectral range of 1300--1600 nm (OH first overtone) of Milli-Q water in the temperature range of 20--70 °C (*n* = 78).](molecules-25-02234-g002){#molecules-25-02234-f002} ![Aquagrams of Milli-Q water in the temperature range of 20--70 °C, (**a**,**b**) with 95% confidence intervals calculated with temperature-based aquagram calculation method, (**c**,**d**) calculated with the classic calculation method, (**a**,**c**) all the 26 temperature steps (*n* = 78) and (**b**,**d**) on three selected temperature steps (*n* = 9) to show the stability of the methods (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g003){#molecules-25-02234-f003} ![Raw and 2nd derivative (calculated with Savitzky--Golay filter using 2nd order polynomial and 21 points) absorbance (logT^−1^) spectra in the range of 1300--1600 nm (OH first overtone) of 0.001--1 M KCl solutions (*n* = 180).](molecules-25-02234-g004){#molecules-25-02234-f004} ![Aquagrams with 95% confidence intervals of Milli-Q water (*n* = 150) and 0.001--1 M KCl solutions (*n* = 180) calculated with the classic (**a**) or the temperature based aquagram (**b**) calculation methods on the individual concentrations (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g005){#molecules-25-02234-f005} ![Aquagrams with 95% confidence intervals of Milli-Q water (*n* = 150) and 0.001--0.1 M KCl solutions (*n* = 120) calculated with the temperature based aquagram calculation method on the individual concentrations (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g006){#molecules-25-02234-f006} ![Aquagrams with 95% confidence intervals of Milli-Q water (*n* = 150) and 0.001--0.01 M KCl solutions (*n* = 60) calculated with the temperature based aquagram calculation method on the individual concentrations (UCL---upper confidence level, LCL---lower confidence level).](molecules-25-02234-g007){#molecules-25-02234-f007} ![Scheme of the calculation for Area under the curve (AUC) aquagram method. Spectrum of pure water with highlighted subranges of the 12 specific water matrix coordinates (WAMACs).](molecules-25-02234-g008){#molecules-25-02234-f008}

{ "pile_set_name": "PubMed Central" }

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22,188

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"n", "calculated", "temperature", "based", "aquagram", "calculation", "method", "individual", "concentrations", "UCL", "confidence", "level", "LCL", "confidence", "level", "Scheme", "calculation", "Area", "curve", "AUC", "aquagram", "method", "Spectrum", "pure", "water", "highlighted", "subranges", "specific", "water", "matrix", "coordinates", "WAMACs" ]

What is your diagnosis ([Figures 1](#f1-dp0504a10){ref-type="fig"}[](#f2-dp0504a10){ref-type="fig"}--[3](#f3-dp0504a10){ref-type="fig"})? ========================================================================================================================================= A. Verruca vulgaris B. Actinic keratosis C. Bowenoid papulosis D. Seborrheic keratosis E. Acanthoma Answer ====== - C\) High-grade squamous intraepithelial lesion of the oral commissure (bowenoid papulosis) Discussion ========== Bowenoid papulosis (BP) is characterized clinically by one or more small, verrucous papules usually located on the genitalia or thighs of younger patients \[[@b1-dp0504a10]\]. Rarely, BP has been reported on extragenital sites with or without concomitant genital lesions \[[@b2-dp0504a10],[@b3-dp0504a10],[@b4-dp0504a10]\]. On the genitalia, BP clinically resembles condyloma acuminatum or lichen planus; however, histopathologically, BP is indistinguishable from squamous cell carcinoma in situ or Bowen's disease, hence, its designation "bowenoid papulosis," a term coined by Wade, Kopf and Ackerman, in 1978 \[[@b1-dp0504a10]\]. BP has been shown to be associated primarily with the high-risk human papillomavirus (HPV) infection subtypes, 16 and 18; however, other high-risk sub-types, such as 31, 32 \[[@b5-dp0504a10]\], 33, 35, 39, 53, and 67, have also been reported. Many lesions of BP resolve with or without therapy and behave in a clinically benign fashion despite their malignant histology \[[@b7-dp0504a10]\]; however, cases of squamous cell carcinoma in-situ and invasive squamous cell carcinoma have been reported to occur in association with lesions of BP, particularly in immunosuppressed patients \[8,9.10\]. Risk of penile squamous cell carcinoma in men may be as high as 30% in patients with BP and increases incrementally with the duration of the disease \[[@b11-dp0504a10]\]. Patients with BP have also been reported to have concurrent HPV-associated dysplasia of the vulva and uterine cervix, of various degrees, including high-grade dysplasia, VIN 3 and CIN 3, respectively. Our patient, a 22-year-old man, presented with verrucous papules centrally located within a central lichenified plaque on the oral commissure. The lesion was clinically thought to be a wart or a lesion of lichen simplex chronicus. Histopathologically, the lesion showed full thickness epithelial atypia demonstrating increased numbers of mitotic figures, loss of polarity, and nuclear pleomorphism compatible with squamous cell carcinoma in situ ([Figures 1](#f1-dp0504a10){ref-type="fig"}[](#f2-dp0504a10){ref-type="fig"}--[3](#f3-dp0504a10){ref-type="fig"}). In situ hybridization was positive for the high-risk subtypes 16/18 ([Figure 4](#f4-dp0504a10){ref-type="fig"}). p16 immunoperoxidase stain demonstrated strong diffuse staining in the lower portion of the lesion with individually positive cells extending into the upper reaches of the epithelium ([Figure 5](#f5-dp0504a10){ref-type="fig"}). Oral BP is exceeding rare with only nine cases, to our knowledge, reported in the medical literature \[[@b11-dp0504a10]--[@b19-dp0504a10]\]. Men were more commonly affected than women, and the ages ranged from 20 to 40 years. Clinically, reported cases of oral lesions of BP in the medical literature are similar to those occurring on genital sites, namely, small verrucous papules \[[@b16-dp0504a10]\]; however, erythematous velvety plaques \[[@b20-dp0504a10]\], raised solitary nodules \[[@b11-dp0504a10]\], leukoplakia or macules resembling candidiasis \[[@b13-dp0504a10]\] have also been described. Histopathologically, lesions of oral BP were indistinguishable from squamous cell carcinoma in situ. A computerized search of the files of the Ackerman Academy of Dermatopathology in New York, NY, from July 1999 through August 31, 2013, yielded 560 biopsies diagnosed as BP; however, only three patients with extragenital BP were identified, including the present case. This finding parallels that of the medical literature and indicates that extragenital BP is exceedingly rare. All three extra-genital lesions from the Ackerman Academy were oral: two occurred on the lip and one, the present case, was located on the oral commissure. There were two males and one female, 22, 64 and 40 years of age, respectively. Clinically, the lesions were thought to be lichen simplex chronicus, verruca vulgaris or pemphigus vulgaris, and bowenoid papulosis, respectively. Histopathologically all lesions were indistinguishable from squamous cell carcinoma in situ. In 2012, a Consensus Panel of the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology recommended a change in terminology for BP from "bowenoid papulosis" to "high-grade squamous intraepithelial lesion." They noted that bowenoid papulosis could be added to the diagnosis in parentheses if it could be verified that the lesion was small and had been excised. If verification could not be accomplished but the clinical setting was one of small papules, they wrote that, "a note stating that the differential diagnosis includes Bowenoid papulosis may be warranted." They also stated that, "Bowenoid papulosis may have a lower risk of progression to cancer than cutaneous HSIL \[high-grade squamous intraepithelial lesion\] found in larger plaques (Bowen disease)." \[[@b21-dp0504a10]\] Treatment for oral BP is similar as that for genital BP and includes intralesional, topical or oral medication as well as surgical excision. Fluoropyrimidine TS-1 (prodrug of 5-FU, gimestat (CDHP), and oteracil potassium (Oxo)), 100 mg daily for three weeks was administered in one elderly woman with HPV-16 positive oral BP and resulted in regression of the lesion \[[@b16-dp0504a10]\]. Intralesional interferon alpha followed by topical imiquimod has also been reported as successful \[[@b17-dp0504a10]\]. Other treatments that have been used for genital BP and may be appropriate in oral lesions include 5-FU, podophylin, retinoic acid, and cidofovir. Surgical modalities include simple excision, cryosurgery, laser vaporization, and electrodessication of small lesions. Podophyllin is toxic in large amounts but has been used successfully for the treatment of oral hairy leukoplakia and could potentially be used to treat refractory BP \[[@b22-dp0504a10],[@b23-dp0504a10]\]. Our patient was treated successfully with 5-fluorouracil cream followed by imiquimod cream each applied five times per week for five weeks with two weeks between each medication. He remains lesion-free five months post treatment. ![Scanning magnification of the lesion on the oral commissure. \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g001){#f1-dp0504a10} ![Higher magnification of the oral lesion. \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g002){#f2-dp0504a10} ![Higher magnification of the slightly verrucous lesion shows hyperplasia with full-thickness atypia of squamous epithelial cells, evidence of loss of polarity, nuclear crowding, nuclear pleomorphism and increased mitotic figures indicative of high-grade squamous intraepithelial lesions. \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g003){#f3-dp0504a10} ![Human papillomavirus in situ hybridization showing positive staining for the high-risk subtypes, 16/18 (red nuclear staining). \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g004){#f4-dp0504a10} ![p16 immunoperoxidase stain shows strong diffuse positive staining of the lower portion of the lesion with individually positive cells extending into the upper reaches of the epithelium (brown staining). \[Copyright: ©2015 Kupetsky et al.\]](dp0504a10g005){#f5-dp0504a10}

{ "pile_set_name": "PubMed Central" }

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22,189

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Plain English summary {#Sec1} ===================== Disordered Eating Behaviours (DEB) start to appear during adolescence and bring serious consequences. Previous studies showed DEB is becoming common in China, and even more prevalent than that is in Western countries. However, limited research has been conducted among adolescents in rural China. The current study aimed to establish the prevalence of DEB and associated psychosocial factors among rural adolescents. Results from the current research showed a high prevalence rate of DEB among rural adolescents and associated psychosocial factors of DEB. Background {#Sec2} ========== Disordered Eating Behaviours (DEB) are abnormal eating attitudes and behaviours which have much in common with diagnosed eating disorders \[[@CR1]\].These can have serious implications which relate to depression, substance abuse, suicidal behaviours, social isolation and which may develop into an eating disorder \[[@CR2], [@CR3]\]. DEB has long been regarded as strongly related to Western cultures but according to a review in the most recent published epidemiologic data relevant to eating disorders in Asia and the Pacific, abnormal eating behaviours found to be highly prevalent in China \[[@CR4]\]. Traditional Chinese culture considers plumpness as beautiful and slimness as reflecting poverty. However, China has been dramatically influenced by Western cultures for the past 39 years due to the 'open-door policy'. The thin-ideal has come to overshadow standards for beauty which contributes to body dissatisfaction and DEB among females \[[@CR5]\]. Accordingly, a number of Chinese studies report a high prevalence of DEB \[[@CR6]--[@CR13]\]. Specifically, female adolescents living in Hong Kong have been found to have the highest prevalence of DEB (10.8%), followed by those from Shenzhen (5.2%) and from Hunan (2.5%) \[[@CR6]\]. Two school-based studies in Taiwan revealed DEB in 17.1% and 10.4% of high school female adolescents \[[@CR7], [@CR8]\]. Further, a school-based study in Hong Kong revealed that DEB were present in 3.9% of adolescent males and 6.5% of adolescent females \[[@CR9]\]. Three school-based studies in Mainland China have also reported that the prevalence of DEB varied from 2.3% to 17% among middle school and college students \[[@CR10]--[@CR12]\]. Western studies have revealed DEB in 29.4% of female adolescents, specifically 14.9% of male adolescents in Germany, and 56% of female adolescents and 28% of male adolescents in US \[[@CR14], [@CR15]\]. However, few studies in DEB have included male adolescents in China \[[@CR9], [@CR13]\]. The variations in measuring instrument, sample characteristics, methodological approaches and study areas, however, may limit the comparability of prevalence across studies. The updated epidemiologic review in 2016 suggested that fat concern and weight control behaviours are prevalent among Chinese young people and replication epidemiological studies in eating disturbances are needed due to ongoing rapid modernization and socio-economic change \[[@CR4]\]. Psychosocial risk factors are strongly associated with a high prevalence of DEB among adolescents \[[@CR13], [@CR16]\]. For instance, longitudinal studies conducted in Southern China found body dissatisfaction, negative affect, perceived sociocultural pressures, and high socioeconomic status (SES) all have significant associations with DEB. \[[@CR13], [@CR17]\]. A comparative study among female adolescents in three different socioeconomically communities of China documented that those in rural areas had fewer concerns about body fat and demonstrated a lower prevalence rate of DEB than those in developed cities \[[@CR6]\]. Socioeconomic development in China is growing very fast nowadays and the rapid ongoing socioeconomic transition has influenced rural China to a large extent. Rural populations now have more opportunities to access the mass media, which may relate to a higher risk of DEB. T Jackson and H Chen have reported that Chinese middle adolescent girls reported higher levels of appearance experiences related to mass media and DEB \[[@CR18]\], and pressure from mass media related to DEB have been observed in Chinese adolescent boys \[[@CR19]\]. There are some studies examining the epidemiology of eating disorders in rural China \[[@CR17], [@CR20], [@CR21]\], but little research has focused on epidemiology of DEB in rural China. In more than 20 years, there has been only one study which has focused on DEB among Chinese female adolescents in a rural area \[[@CR6]\]. Further, given that little attention has been paid to DEB in both genders in rural China, it is necessary to re-investigate the situation under current influences. The present study, therefore, aimed to examine prevalence and associated psychosocial factors of DEB among male adolescents and female adolescents at a Chinese middle school in a rural area. This may contribute to the updated information in the epidemiology of DEB in China and also respond to the updated review's request for replication studies, that is this review suggested further replication epidemiological studies in eating disturbances due to ongoing rapid modernization and socio-economic change \[[@CR4]\]. Methods {#Sec3} ======= Participants and study procedure {#Sec4} -------------------------------- All students from a middle school -- a secondary high school -- in Dongfanghong town (a less economically developed rural area), Heilongjiang province were invited to participate in the study. In 2015, the per capita annual income in our sample area was 30,977 RMB (or \$4509). The school that was studied was the only middle school in this area. Four hundred and sixty-six students aged 11--18 years old were invited to complete an anonymous, self-reported questionnaire covering a wide range of topics including DEB and related psychosocial behaviours on a voluntary basis. Before starting the research, schoolteachers were trained by researchers and schoolteachers explained the study to students and were able to answer students' questions. During the study, the confidentiality and the anonymity of participants were assured. With the assistance of schoolteachers, the questionnaires were administered in September 2015 -- at the beginning of autumn semester. The class teachers decided the time when questionnaires were conducted. They usually selected self-learning class (students studied by themselves) and the time of self-learning class was different among class-rooms. The students used around 1 h -2 h to finish the questionnaires. The class teachers collected the questionnaires following completion and returned them to the researcher. Of 466 questionnaires that were issued, 389 valid questionnaires were returned (a response rate of 83.5%). Outcome variables {#Sec5} ----------------- SCOFF: The SCOFF questionnaire is an effective tool that can screen DEB quickly \[[@CR22]\]. It includes five items addressing the main features of DEB \[[@CR22]\]. See the Additional file [1](#MOESM1){ref-type="media"}: Table S1 for the descriptive statistics of each item. One point for every "yes"; a score of \> = 2 indicates a likely case of DEB. The scale has been validated and has demonstrated effective specificity in identifying DEB \[[@CR23]\]. The Chinese version of the SCOFF questionnaire has been proved to be effective for detecting potential DEB \[[@CR24]\]. Independent variables {#Sec6} --------------------- Body Mass Index (BMI) was computed from self-reported measures of height and weight. Self-reported BMI has been demonstrated to be a reliable measure of BMI \[[@CR25]\]. The classification of BMI was made according to BMI cut-offs by the Working Group on Obesity in China (WGOC) \[[@CR26]\]: ≤ 18.49 kg/m^2^ (underweight), 18.50--23.99 kg/m^2^ (normal weight), 24--27.99 (overweight), ≥28 (obesity). We combined the overweight group and the obesity group into one category due to the small number size in the obesity group. Participants were asked to describe their weight perceptions. Responses were ranged into three groups: underweight, average /normal, and overweight. Compared weight perceptions with actual BMI, weight misconception was ranged into four groups: misconception of underweight, overweight, normal weight and correct weight conception. See the Additional file [2](#MOESM2){ref-type="media"}: Table S2 for the association between BMI and perceived weight categories. Body dissatisfaction was examined using a Body Part Satisfaction Scale with each response from 1 = extremely dissatisfied to 6 = extremely satisfied \[[@CR27]\]. The scale consists of eight items rating the individual's level of satisfaction with the following body areas: face, upper torso, middle torso, lower torso, muscle tone, weight, height and overall appearance. A high score indicates a higher level of body satisfaction. The scale has shown reliable psychometrics \[[@CR27]\]. The Chinese version of this scale has been tested and has strong reliability \[[@CR18]\]. The internal consistency of the scale was 0.90. Depressive symptoms were measured by the Positive and Negative Affect Schedule \[[@CR28]\]. This schedule is a 20-item test with 10 items for negative affect and positive affect, respectively. Participants were asked to describe the extent to which they had varying feelings and emotions in the past month from 1 = very slightly or not at all to 5 = extremely. The scale has shown good validity and acceptable psychometric properties \[[@CR28]\]. The original scale has been tested on Chinese samples \[[@CR19]\]. The Perceived Sociocultural Pressure Scale was used to assess pressures to be thin from family, friends, dating partners and media \[[@CR18]\]. The questionnaire has 10 items that investigate perceived sociocultural pressures to be thin. An example of an item is: "I've noticed a strong message from my family to have a thin body". Three items are for family and friends, respectively. Two items are for dating partners and media, respectively. Responses ranged from 1 = not at all to 5 = extremely. Higher scores indicate more perceived pressure. Internal consistency (α = 0.83), test-retest reliability (*r* = 0.93) and predictive validity of this scale have been documented \[[@CR29]\]. The scale has been tested and used in Chinese samples \[[@CR19]\]. Media use was assessed by a media exposure questionnaire. Participants were asked: whether they watch television and DVDs, whether they use the Internet, whether they use chat rooms and play computer games, as well as how long they spent on media. The questionnaire was developed from a Chinese study \[[@CR5]\]. Parental socioeconomic status (SES) was determined on the adolescents' report of parents' occupation. Occupations were categorized according to the classification by the Chinese National Bureau of Statistics \[[@CR30]\] with nine items to be selected. Four groups were created based on the salary of occupations: professional, service, labourers, and unemployed. The variables of mother's job and father's job were merged into one variable (parents' occupation), recording the higher of the two values. Age and sex were also recorded. Statistical analysis {#Sec7} -------------------- Chi-square analysis was used to compare differences in prevalence of DEB and for comparing perceived weight categories to BMI categories. To examine associated factors of DEB, first, a univariate logistic regression was employed to examine the association between each of the independent variable and DEB, and then multiple logistic analysis was performed. A forward stepwise multiple logistic regression was performed to determine factors significantly associated with DEB, including independent variables based on univariate logistic regression results (cut-off point *p* ⩽ 0.25). Age and gender were controlled. The final model was built based on this forward stepwise multiple logistic regression (*p* ⩽ 0.05). The multi-collinearity among associated factors was examined with *collin* command in Stata (mean variance inflation factor (VIF) = 1.43, correlation matrix = 0.0528). Secondly, Model fit to the data (validity and reliability) was assessed by the likelihood ratio χ^2^ test, Hosmer-Lemeshowgoodness-of-fit test and receiver operating characteristics (ROC) curve. *P* ⩽ 0.05 was regarded as statistically significant and odds ratio (OR) with 95% confidence intervals (95% CI) were calculated. Data analyses were conducted with the Statistical Package for the Social Sciences (SPSS) 22.0 and StataSE 14. Ethical clearance {#Sec8} ----------------- The study was approved by the Regional Committee for Medical Research Ethics in Norway, and the Committee for the middle school in Dongfanghong, China. Results {#Sec9} ======= Descriptive summary of the study participants {#Sec10} --------------------------------------------- Table [1](#Tab1){ref-type="table"} presents the descriptive summary of study participants (*N* = 389).Table 1Descriptive summary of the study participants (*N* = 389)VariablesNPercentMsdFemale19048.8SCOFF total score (five tiems)1.01.1 017244.2 110527.0 26717.2 3328.2 4123.1 510.3Age(year)15.11.7 Early Adolescence \[[@CR11]--[@CR13]\]9524.4 Middle Adolescence \[[@CR14]--[@CR16]\]19850.9 Late Adolescence \[[@CR17], [@CR18]\]9624.7BMI(kg/m^2^)20.54.7 Underweight14036.0 Normal Weight16943.4 Overweight7719.8Perceived Weight Underweight6115.7 Normal Weight21455.0 Overweight11429.3Weight Misconception Misconception of Underweight184.6 Misconception of Normal Weight10226.2 Misconception of Overweight5714.7 Correct Weight Conception20753.2Parents' Occupation Professional11529.6 Service7218.5 Labourers4812.3 Unemployed15439.6With DEB11228.8Pressure Family1.91.1 Friend1.81.0 Dating1.51.0 Media1.61.1Watch TV32383.0Watch DVD7419.0Surfing Online35390.7Chat Online31681.2Play Online25365.0Hours on Media average - 0-4.9 h31581Hours on Media Total14.19.1Body Satisfaction31.18.4 Face4.01.3 Upper torso4.01.3 Middle torso4.01.4 Lower torso4.01.4 Muscle tone3.71.4 Height3.81.4 Weight3.71.5 Overall appearance4.11.2Positive Affect29.67.5Negative Affect21.27.2*N* number, *M* mean, *sd* standard deviation*Pressure* perceived sociocultural pressure to thin body and weight loss*Hours on Media Total* time on Internet working days + time on Internet weekends + time on TV working days + time on TV weekends*Hours on Media average* Hours on Media Total ÷ 4 Prevalence of disordered eating behaviours {#Sec11} ------------------------------------------ Table [2](#Tab2){ref-type="table"} presents the prevalence of DEB according to the participants' general information by using chi-square analysis. The prevalence of DEB among adolescents was 28.8%. The prevalence of DEB among male adolescents and female adolescents was 27.1% and 30.5%, respectively; no significant differences were found based on gender. No significant differences of prevalence of DEB were found across different age groups. Significant differences in the prevalence of DEB were found in different BMI groups, χ^2^ (2) = 14.896, *p* = .001. Overweight adolescents (42.9%) had a significantly higher prevalence of DEB as compared to underweight adolescents (18.6%). Significant differences in the prevalence of DEB were also found in different perceived weight groups, χ^2^ (2) = 51.524, *p* \< .001. Adolescents in the perceived overweight group (54.4%) had a significantly higher prevalence of DEB than those among the perceived underweight group (18.0%). Significant differences in the prevalence of DEB were found in different weight misconception groups, χ^2^ (2) = 43.399, *p* \< .001. Adolescents with overweight misconception (59.4%) had a significantly higher prevalence of DEB than those without overweight misconception. There were no significant differences in the prevalence of DEB among students across different SES groups. Associations between misconception of over−/under-weight and DEB by gender is shown in the Additional file [3](#MOESM3){ref-type="media"}: Table S3.Table 2Prevalence of disordered eating behaviours across participants' general informationWith DEBWithout DEBChi-square% (N)% (N)StatisticGender0.545 Female30.5 (58)69.5 (132) Male27.1 (54)72.9 (145)Age4.919 Early Adolescent \[[@CR11]--[@CR13]\]24.2 (23)75.8 (72) Middle Adolescent \[[@CR14]--[@CR16]\]26.8 (53)73.2 (145) Late Adolescent \[[@CR17], [@CR18]\]37.5 (36)62.5 (60)BMI14.896\*\* Underweight18.6 (26)81.4 (114) Normal Weight30.8 (52)69.2 (117) Overweight42.9 (33)57.1 (44)Perceived Weight51.524\*\*\* Underweight18.0 (11)82.0 (50) Normal Weight18.2 (39)81.8 (175) Overweight54.4 (62)45.6 (52)Weight Misconception41.521\*\*\* Misconception of Underweight22.2 (4)77.8 (14) Misconception of Normal Weight13.7 (14)86.3 (88) Misconception of Overweight61.4 (35)38.6 (22) Correct Weight Conception27.5 (57)72.5 (150)Parents' Occupation2.218 Professional33.9 (39)66.1 (76) Service25.0 (18)75.0 (54) Labourers27.1 (13)72.9 (35) Unemployed27.3 (42)72.7 (112)*N* number of participants\**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001 Psychosocial factors independently predicting disordered eating behaviours {#Sec12} -------------------------------------------------------------------------- Table [3](#Tab3){ref-type="table"} presents results from univariate and multiple logistic regression analyses reporting psychosocial associated factors of DEB. The final multiple regression model included perceived weight, body dissatisfaction, negative affect and watching TV. The risk of having DEB among the perceived overweight group was 2.8 times higher than among those in the perceived underweight group. Students with higher levels of body dissatisfaction were significantly more likely to develop DEB compared to those with lower levels of dissatisfaction. Students with a higher negative affect were significantly more likely to develop DEB compared with those with a lower negative affect. Students watching TV were almost three times more likely to report DEB than students who did not watch TV. The corresponding coefficients and standard errors in the multiple logistic regression model are shown in the Additional file [4](#MOESM4){ref-type="media"}: Table S4.Table 3Multiple logistic regression model showing factors associated with disordered eating behavioursUnivariate Logistic Regression Analysis per Independent VariableForward Multivariate Stepwise Logistic Regression AnalysisOR (95% CI)*P*OR (95% CI)*P*BMI Underweight1---- Normal Weight1.95 (1.14--3.33).015\*\*---- Overweight3.29 (1.77--6.11)\<.001\*\*\*\*----Perceived Weight Underweight11 Normal Weight1.01 (0.48--2.12).9730.79 (0.34--1.86).589 Overweight5.42 (2.56--11.47)\<.001\*\*\*\*2.80 (1.05--7.48).041\*\*Weight Misconception Correct Weight Conception1---- Misconception of Underweight0.75 (0.24--2.38).628---- Misconception of Normal Weight0.42 (0.22--0.80).008\*\*\*---- Misconception of Overweight4.19 (2.27--7.74)\<.001\*\*\*\*----Pressure from family2.17 (1.75--2.70)\<.001\*\*\*\*----Pressure from friends2.48 (1.93--3.18)\<.001\*\*\*\*----Pressure from dating1.86 (1.48--2.34)\<.001\*\*\*\*----Pressure from media1.90 (1.53--2.36)\<.001\*\*\*\*----Watch TV No11 Yes1.80 (0.94--3.46).076\*2.40 (1.11--5.18).026\*\*Watch DVD No1---- Yes1.67 (0.98--2.85).058\*----Chat Online No1---- Yes2.35 (1.21--4.57).011\*\*----Play Online No1---- Yes1.34 (0.84--2.14).23\*----Body Satisfaction0.92 (0.89--0.95)\<.001\*\*\*\*0.96 (0.93--0.99).019\*\*Positive Affect0.96 (0.93--0.99).005\*\*\*----Negative Affect1.10 (1.06--1.14)\<.001\*\*\*\*1.07 (1.03--1.12)\<.001\*\*\*\**OR* odds ratio, *CI* confidence interval*Pressure* perceived sociocultural pressure to thin body and weight loss\**p* ⩽ 0.25, \*\**p* \< 0.05, \*\*\**p* \< 0.01, \*\*\*\**p* \< 0.001 The reliability and the validity of the multiple logistic regression model was assessed by employing post-estimation statistics (likelihood ratio χ^2^ test significant, χ^2^ = 107.925, degrees of freedom (df) = 9, *p* \< 0.001; Hosmer-Lemeshow χ^2^ test = 10.196, df = 8, *p* = 0.252). The Hosmer-Lemeshow test of the goodness-of-fit suggests the model is a good fit to the data (small Chi-squared values with larger *p*-value closer to 1). Area under the ROC curve (AUC) was 0.825 with 95% confidence interval (0.781, 0.869), which is a prediction power for the model. Discussion {#Sec13} ========== The main findings of this school-based study reveal a high prevalence rate of DEB among male adolescents (27.1%) and female adolescents (30.5%). The study also found that perceived weight, negative affect, body dissatisfaction, and watching TV were significantly associated with DEB among adolescents. The prevalence estimates of the current study are among a wide range of previous school-based studies conducted in China (2.26%--58.3%) \[[@CR10], [@CR31]--[@CR33]\]. The available information on DEB in rural China has been reported by Sing Lee and Antoinette M. Lee \[[@CR6]\]. In their study, 266 school female adolescents from Grade 11 from Rural Hunan were included with a prevalence rate of 2.5%. In the present study, prevalence was much higher. This may be attributed to different screening instruments for DEB. The study by Sing Lee and Antoinette M. Lee \[[@CR6]\] used EAT-26 as the screening instrument, while we used SCOFF. The sensitivity and the specificity between the two instruments are different and SCOFF has been reported to have a tendency toward overinclusion \[[@CR34]\]. It is further comparable to reports from Western countries; in spite of variation in measuring instruments, study methodologies and sample compositions: for instance 29.4% of female adolescents and 14.9% of male adolescents in Germany, and 56% of female adolescents and 28% of male adolescents in US \[[@CR14], [@CR15]\]. Given the harmful consequences related to DEB and the high prevalence of DEB, it is vital for public health officials to increase public awareness by introducing relevant and important information about DEB at individual, school and society level. In this way, health organisations can aim at intervening and preventing harmful eating behaviours. So far, there has been no such programs for DEB in the local region. This study did not show significant gender differences in prevalence of DEB. Most Chinese and Western studies report that prevalence of DEB was significantly higher among female adolescents than among male adolescents \[[@CR9], [@CR14], [@CR15]\]. The prevalence of DEB for males is possibly rising and the incidence of DEB for males may have been underrated due to cultural stigma \[[@CR35]\]. It may also due to changing lifestyles: increased access to TV, social media, Internet and gaming. It is possible that female adolescents and male adolescents have a similar risk for developing DEB. More attention, therefore, should be also be paid to male adolescents who are at risk of DEB. The present study found perceived weight as a significant associated factor of DEB. Results are consistent with previous observations on perceived weight status in Chinese adolescents \[[@CR6]\] and Western samples \[[@CR36], [@CR37]\]. It has been suggested that one's perceived weight has a greater influence than one's actual weight when engaging in DEB \[[@CR38]\]. In addition, a considerable number of adolescents in this study misperception with regard to weight status, which is consistent with previous Chinese research \[[@CR39]\]. Furthermore, separate logistical regression in this study showed that students with the misconception of being overweight were more likely to report DEB. This is in line with previous research \[[@CR38]\] and it indicates that one possible avenue for prevention programs may be a focus upon helping students develop accurate weight perception. Along the same lines, body dissatisfaction was a significant associated factor for DEB. Similar results have been reported in both Chinese and Western research \[[@CR13], [@CR16]\]. The thin-ideal image contributes to perfectionism and body dissatisfaction. Male adolescents wish to become more muscular and achieve the male-ideal of V-shaped figure. On the other hand, female adolescents wish to become thinner and achieve the female-ideal of extremely thin. They put much emphasis upon having a perfect body shape, though they have a clinically healthy weight. Perfectionism and body dissatisfaction are both highly correlated with DEB \[[@CR40]\]. Body dissatisfaction can increase dieting behaviours \[[@CR41]\] and negative affect \[[@CR16]\], which can, in turn, lead to poor eating habits and increased eating disturbances. Negative affect was significantly associated with DEB in this study. The result is in agreement with previous Chinese and Western research \[[@CR13], [@CR16]\]. One explanation is that negative urgency (i.e., the tendency to act rashly in response to negative affect) is significantly associated with disordered eating \[[@CR42]\]. Another explanation can be derived from escape theory - people tend to escape negative emotions by abnormal eating (overeating) and they believe or have learned that overeating can relieve negative emotions \[[@CR43], [@CR44]\]. Moreover, negative affect and disordered eating may be interacted mutually, where the feeling of shame and guilty caused by disordered eating may result in negative self-perceptions among female adolescents \[[@CR44]\]. In addition, this study found television access as a significant associated factor of DEB. A small number of adolescents did not have access to TV, possibly related to high academic expectations, family economic situations or personal preference. Media consumption can significantly predict female adolescents' DEB and male adolescents' interest in dieting \[[@CR45]\]. Such an effect of media can also be found in other non-western studies. For example, research in Fiji \[[@CR46]\], Pakistan \[[@CR47]\], Japan \[[@CR48]\], and Korea \[[@CR49]\] have documented significant associations between media exposure and DEB. Becker \[[@CR50]\] has also discussed how Western media introduced by TV has caused thin-ideal internalization among Fijian females. Garfinkel and Garner suggest: "The media have capitalized upon and promoted this image (of thinness) and through popular programming have portrayed the successful and beautiful protagonists as thin. Thinness has thus become associated with self-control and success." (p.145) \[[@CR51]\]. Other media variables in this study did not show significant associations with DEB. It is possible that specific media exposure may contribute to thin body and eating disturbances instead of other general media variables. Although BMI did not contribute to the multiple logistical regression model in this study, BMI was significantly positively associated with DEB from univariate logistical regression analysis. Previous research showed there may exist a U-shaped relation between DEB and BMI \[[@CR52], [@CR53]\]. That is, disordered eating behaviours were more problematic at the two extreme ends of BMI (underweight and obesity). In addition, there is a non-significant trend of increasing prevalence of DEB with age in this study; late adolescents reported highest prevalence of DEB. This may be related to an increased social and emotional stress. Late adolescents, face an exceptional amount of pressure to succeed in college entrance examinations and achieve parental expectations \[[@CR54]\] and experience peer competition, copious homework and reduced social support. These stressors may be associated with an increased risk for DEB \[[@CR55]\]. This study adds to a growing literature on epidemiology of DEB among adolescents in China. It, therefore, provides current knowledge on the epidemiology of DEB in rural China, which helps to capture more attention to DEB in rural China. Since DEB may develop into more severe forms of DEB or eating disorders without appropriate intervention strategies, it is important to design early interventions against DEB. Secondly, this study included both male adolescents and female adolescents which has provided valuable insights into DEB in both genders. Thirdly, this study found risky behaviours associated with DEB among adolescents, which may contribute to better identification of high-risk groups and may be helpful for early prevention and intervention programs. For instance, adolescents with inaccurate weight perception and body dissatisfaction could be a targeted group for eating problems' prevention programs. A school-based program could be carried out by helping students reduce weight-related teasing, improve self-esteem and body satisfaction. This study has some limitations, however. Firstly, the cross-sectional nature of this study could cause difficulty for examining cause-and-effect relationships between DEB and associated factors. Secondly, self-report measures in this study may lead to under- or overestimation of prevalence of DEB and associated factors. For example, self-reported BMI may overestimate measured BMI at the low end of the BMI scale and underestimate measured BMI at the high end \[[@CR56]\]. Thirdly, unknown response bias may exist that can reduce the reliability and validity of the study. For example, students may respond untruthfully or give answers that they felt the researchers wanted to hear. Fourthly, the limitation of the SCOFF questionnaire needs to be considered. Since it has shown low sensitivity in a validation population-based study \[[@CR57]\]. Fifthly, information on students' reading level was not available. Since low reading levels may lead to misunderstanding of the questionnaires, the lack of data on reading levels may reduce the reliability of the study. Lastly, this study was school-based and thus access to participants was limited by those who were attending school. No accurate statistical information about school dropout rate was available. According to a literature review in 2015 \[[@CR58]\], cumulative dropout rate in rural secondary schools was as high as 63%, while dropout rate in cities was less than 10%. There have been no statistics for students attending vocational and technical schools in China. This sample may be biased towards adolescents with higher education, therefore. The results would be more representative by conducting a population- or community-based study. Conclusion {#Sec14} ========== The present study has re-examined DEB among Chinese adolescents in a rural area. It suggests that DEB among rural Chinese adolescents are prevalent. Perceived weight, negative affect, body dissatisfaction and watching TV are significant associated factors for DEB among adolescents. This may provide valuable implications for prevention and intervention strategies in DEB. Furthermore, longitudinal population-based studies are recommended to produce a more comprehensive understanding of causes and risk factors of DEB among Chinese adolescents. Additional files ================ {#Sec15} Additional file 1: Table S1.Descriptive statistics of each item of SCOFF. (DOCX 11 kb) Additional file 2: Table S2.Cross-tabulation of BMI and perceived weight categories among adolescents. (DOCX 11 kb) Additional file 3: Table S3.Associations between misconception of over−/under-weight and DEB by gender. (DOCX 11 kb) Additional file 4: Table S4.Multiple logistic regression model showing factors associated with disordered eating behaviours by coefficient and standard errors. (DOCX 13 kb) AUC : Area under the ROC curve BMI : Body Mass Index CI : Confidence intervals DEB : Disordered eating behaviours df : Degrees of freedom M : Mean N : Number OR : Odds ratio ROC curve : Receiver operating characteristics curve sd : Standard deviation SES : Socioeconomic status SPSS : Statistical package for the social sciences VIF : Variance inflation factor **Electronic supplementary material** The online version of this article (10.1186/s40337-017-0175-x) contains supplementary material, which is available to authorized users. The authors sincerely thank Li Chunying, Zhao Yanhua and Gong Chengzhi for help during research in the first middle school. The authors thank teachers' and students' in the first middle school for their cooperation. Funding {#FPar1} ======= This was a self-financed study. Availability of data and materials {#FPar2} ================================== The data that support the findings of this study are available from the corresponding author upon reasonable request. TF made substantial contributions to study design, acquisition of data, analysis and interpretation of data, and writing the manuscript. DSA made substantial contributions to study design, analysis and interpretation of data, revising the manuscript critically for important intellectual content and general supervision of the research. Both authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== The study was approved by the Regional Committee for Medical Research Ethics in Norway, and the Committee for the first middle school in Dongfanghong, China. Consent for publication {#FPar4} ======================= Not applicable. Competing interests {#FPar5} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

{ "pile_set_name": "PubMed Central" }

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22,190

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Angiogenesis, the development of new blood vessels, is a feature common to many carcinomas and has been proposed as a hallmark of cancer ([@bib6]). However, morphological assessment of lung carcinomas has revealed that they may be subdivided into angiogenic and relatively non-angiogenic tumours ([@bib21]); the latter co-opt pre-existing vasculature to support an alveolar pattern of growth. Nevertheless, the majority of non-small cell lung carcinomas (NSCLCs) are angiogenic and express vascular endothelial growth factor-A (VEGF) ([@bib12]), the predominant pro-angiogenic ligand that is exploited by tumours ([@bib14]). Animal models ([@bib15]; [@bib7]; [@bib18]) and human studies ([@bib28]) have shown that blocking VEGF signalling results in tumour shrinkage, associated with the pruning of angiogenic (proliferative, leaky and immature (i.e., without associated pericytes)) blood vessels. This observation led to the development of a humanised anti-VEGF monoclonal antibody, bevacizumab, which has shown an improved progression-free survival benefit in numerous tumour types ([@bib8]; [@bib19]; [@bib23]). In recurrent or advanced NSCLC, the addition of bevacizumab to carboplatin and paclitaxel chemotherapy (E4599 study) resulted in a 2-month improvement in median overall survival ([@bib24]). When combined with cisplatin and gemcitabine (AVAiL study) in NSCLC, bevacizumab showed no improvement in overall survival, but demonstrated improved progression-free survival ([@bib22]). The E4599 study excluded patients with brain metastases due to concerns that bevacizumab might exacerbate the incidence of tumour-associated brain haemorrhage ([@bib24]). Several analyses now suggest that bevacizumab is safe in patients with brain metastases ([@bib26]; [@bib1]), but there is no evidence regarding the efficacy of bevacizumab in this indication. Indeed, there is little published on the vascular phenotype, VEGF expression, hypoxia and proliferation of NSCLC brain metastases, which may inform efficacy. To date, no valid biomarkers of the survival benefit of bevacizumab have been identified. Objective response rates have been shown not to predict benefit from bevacizumab in colorectal cancer ([@bib5]), and there are concerns that changes in the MRI appearance of glioblastoma following bevacizumab treatment do not predict clinical benefit ([@bib3]). Efforts to identify novel *in situ* biomarkers of efficacy have focused on available tissue samples from primary tumour resections ([@bib9]; [@bib11]), to make inferences concerning the treatment of metastatic disease. However, while there is evidence to support this for metastatic colorectal cancer ([@bib16]), there is little evidence to suppose that this is a valid approach to inform the biology of NSCLC brain metastases. In summary, a greater understanding of the vascular phenotype of NSCLC brain metastases may better inform the use of anti-angiogenic agents in this indication. The aim of this study was to assess the relative expression of VEGF, hypoxia, proliferation, microvessel density, vascular pattern and vascular maturity in a series of matched primary NSCLCs and brain metastases. MATERIALS AND METHODS ===================== Tissue ------ Formalin-fixed paraffin-embedded blocks for 15 cases of primary NSCLC with matched resected brain metastases were retrieved from the pathology archives at the Catholic University Hospital, Rome. Ethical approval for this research was granted by the local research ethics committees. Immunohistochemistry -------------------- Characterisation of the antibodies and immunohistochemical methods for VEGF ([@bib27]), CA9 ([@bib13]), Ki67 ([@bib25]), CD34 ([@bib17]) and CD34 and smooth muscle actin (SMA) (double stain; QBEnd/10 and 1A4; Dako, Ely, UK) ([@bib20]) have been described in detail elsewhere. Scoring ------- The maximum intensity of VEGF expression in \>10% of tumour cells was recorded on a semiquantitative scale from 0 (no expression) to 3 (very strong expression). The percentage of tumour cells with membranous expression of CA9 was estimated by a pathologist. The percentage of tumour cells with nuclear positivity for Ki67 was counted by a pathologist. Microvessel density analysis was performed using the Chalkley method; at low power ( × 40), five vascular hotspots in CD34-labelled sections were selected. Microvessel density was assessed by counting the number of CD34-labelled vessels that overlapped with dots on a 25-point Chalkley eyepiece graticule in each high-power field ( × 200) ([@bib2]). The five counts were then added together to provide the density score. Vascular maturity is defined by the coverage of endothelial cells (CD34 positive) by pericytes (SMA positive) and was calculated by counting the proportion of CD34-labelled vessels that were surrounded, at least in part, by SMA-labelled cells from 10 high-power fields ( × 400). Each case was classified according to the predominant vascular pattern (alveolar, basal, diffuse or papillary) described elsewhere ([@bib21]). All scoring was performed blind to the patients\' identities. Statistical analyses -------------------- Student\'s *t*-test was used to assess the difference between subsets of continuous data and Spearman\'s correlation coefficient was used to assess covariance. The *χ*^2^ test was used to evaluate associations between categorical data and Cohen\'s *κ* statistic was used to determine agreement. RESULTS ======= Chalkley counts in primary NSCLCs and brain metastases were not significantly correlated (*r*=0.148, *P*=0.60) ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). Although, mean Chalkley counts were similar in primary and secondary cancers (42.5 *vs* 43.5, respectively, *P*=0.77). The proportion of mature vessels was on average 63.2% greater in brain metastases than their matched primary NSCLCs (mean 25.7% *vs* 88.9%, respectively, *P*=0.004) ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). The proportions of mature vessels in primary and secondary cancers were not significantly correlated (*r*=−0.46, *P*=0.09). Differences were also observed in vascular patterns, with a predominance of alveolar, basal and diffuse patterns in primary tumours and a predominance of diffuse and papillary patterns in secondary tumours ([Table 1](#tbl1){ref-type="table"}). The percentage of CA9-positive tumour cells was similar to that previously reported ([@bib4]) and was closely correlated between primary NSCLCs and brain metastases (*r*=0.825, *P*=0.0002), suggesting that there were similar levels of hypoxia in matched cases from this series ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). The mean percentage of cells positive for CA9 was not statistically significantly different in primary and secondary cancers (14.0 *vs* 10.7, respectively, *P*=0.27). Vascular endothelial growth factor expression was seen in all primary and secondary NSCLCs; however, there was no agreement between the scores in matched pairs (*κ*=−0.25) and the association was not statistically significant (*P*=0.15) ([Table 1](#tbl1){ref-type="table"}; [Figure 1](#fig1){ref-type="fig"}). The proliferative fraction (percentage of Ki67-labelled cells) was also not significantly correlated between primary and secondary cancers (*r*=0.179, *P*=0.524), though the mean Ki67 was significantly higher in the brain metastases than the primary lung cancers (35.7% *vs* 19.3%, respectively, *P*=0.018) ([Table 1](#tbl1){ref-type="table"}). DISCUSSION ========== This is the first report comparing VEGF expression, CA9 expression, proliferation, microvessel density, vascular pattern and vascular maturity in matched primary NSCLCs and brain metastases. The data show that brain metastases have a significantly greater proliferation rate and vascular maturity than their matched primaries. Neither the proliferation rate, vascular maturity, expression of VEGF, vascular pattern nor microvessel density could be predicted in the brain metastasis from examination of the primary NSCLC. Only CA9 expression showed a strong correlation between primary and secondary NSCLCs. These observations are important for the following reasons: First, anti-VEGF therapies were thought to increase the risk of cerebral haemorrhage in patients with brain metastases, but recent data suggest that bevacizumab is safe in this indication. Our data provide a biological explanation for this observation, showing that the vasculature of brain metastases is stable, and (extrapolating from observations in rectal cancer) ([@bib28]) is unlikely to regress following anti-VEGF therapy, minimising the risk of associated haemorrhage. Second, mature vasculature is less sensitive to anti-VEGF therapy, suggesting that while patients with primary NSCLCs may benefit from anti-VEGF therapy, those with brain metastases may not. Moreover, preclinical data suggest that when endothelial regression is seen in mature vessels targeted by anti-VEGF therapy, a pericyte scaffold remains and permits rapid re-angiogenesis following cessation of therapy ([@bib18]). Thus, a rebound effect may be expected if anti-VEGF therapy is used in brain metastases and continuous, rather than intermittent, therapy might show greater efficacy. No efficacy data exist for bevacizumab in brain metastases, but [@bib26] have stated that 'patients with treated brain metastases will likely derive similar benefit from bevacizumab as patients without brain metastases\' without any biological, pathological or clinical evidence. The data herein are an important counterbalance to this claim. Third, non-angiogenic NSCLCs ([@bib21]) that show an alveolar pattern of growth may metastasize, and do not merely co-opt existing vasculature in the brain, but instead develop a more angiogenic phenotype with haphazardly arranged vessels. Moreover, even non-angiogenic, alveolar tumours express VEGF, are under the influence of hypoxia and are supported by an often immature, dense vasculature. Therefore, one may expect non-angiogenic tumours to potentially respond to anti-VEGF therapy at their site of origin. Finally, retrospective analyses of archived primary tumours from trials of anti-VEGF therapy have thus far failed to identify a biomarker of efficacy ([@bib10]). The data presented herein suggest that the vascular phenotypes of primary and secondary cancers are very different. Therefore, when evaluating anti-angiogenic treatments in the metastatic setting, biomarker studies should also be conducted on metastases and not the primary tumours. The limitations of this study include the sample size, which is small in statistical terms, but it is large for a series of matched brain metastases, provides informative data and reports novel findings. Furthermore, while one may make inferences regarding the applicability of these observations to anti-VEGF therapy using preclinical data ([@bib7]; [@bib18]) and clinical data from other tumours ([@bib28]), only serial biopsies of brain metastases from patients receiving bevacizumab will provide a definitive answer. To date, the two reported series of NSCLC brain metastases treated with bevacizumab in the literature are small, *n*=36 ([@bib1]) and *n*=115 ([@bib26]) and provide no histopathological data. Moreover, it is unlikely that clinicians would want to subject terminally ill patients to repeated invasive procedures to provide such material. In conclusion, brain metastases of NSCLCs have a vascular phenotype that is distinct from the primary tumours. This has implications for the use of anti-VEGF therapy in this setting and should be taken into consideration when designing clinical trials to assess the efficacy of such drugs and biomarkers that may predict benefit. We thank all patients and clinicians who provided material for this research. Dr Adrian Jubb is the recipient of a Career Development Fellowship from the Pathological Society of Great Britain and Ireland. This study was supported by Cancer Research UK, Pathological Society of Great Britain and Ireland and the NIHR Biomedical Research Centre, Oxford. Dr Adrian M Jubb received a speaker\'s honorarium in 2010 from Genentech Inc., which is developing anti-angiogenic therapies. ![(**A**) A scatter plot of CD34 Chalkley counts in matched primary (lung) and secondary (brain) cancers. (**B**) A line plot showing differences in the percentage of blood vessels covered by pericytes in matched primary and secondary cancers. (**C**) A scatter plot of the percentage of matched primary and secondary cancers positive for carbonic anhydrase 9. (**D**) A line plot showing differences in the VEGF score in matched primary and secondary cancers. (**E**, **F**) Double-labelled immunohistochemistry for CD34 (blue) and smooth muscle actin (brown) in a matched primary (**E**) and secondary (**F**) cancer.](bjc2011147f1){#fig1} ###### Scoring frequencies **Case** **VEGF intensity score (0 to 3)** **CA9 (% positive)** **Ki67 (% positive)** **Chalkley count (*n*)** **Vascular maturity (% mature)** **Vascular pattern** ---------- ----------------------------------- ---------------------- ----------------------- -------------------------- ---------------------------------- ---------------------- ---------- -------- -------- ---------- ----------- ----------- 1 2 2 0 20 10 50 34 51 36 96 Basal Papillary 2 2 1 0 0 50 50 45 43 21 88 Basal Basal 3 2 3 0 0 10 30 39 35 23 97 Basal Papillary 4 2 3 0 0 0 15 44 62 4 97 Alveolar Papillary 5 2 2 30 20 60 80 44 47 4 93 Diffuse Basal 6 1 2 5 0 20 15 45 44 14 95 Alveolar Diffuse 7 2 3 0 0 40 30 33 53 27 94 Basal Basal 8 2 2 10 15 0 70 24 18 9 58 Diffuse Diffuse 9 2 2 0 0 40 40 57 45 61 86 Papillary Papillary 10 2 1 0 0 0 20 40 56 30 88 Alveolar Diffuse 11 1 2 30 20 10 5 43 25 30 97 Diffuse Diffuse 12 2 3 80 50 30 10 63 55 28 77 Alveolar Papillary 13 2 1 50 30 5 40 43 39 40 89 Basal Diffuse 14 1 2 5 5 5 40 48 35 12 94 Diffuse Papillary 15 3 1 0 0 10 40 36 44 47 84 Basal Diffuse Median 2 2 0 0 10 40 43 44 27 93 NA NA IQR 2--2 1.5--2.5 0--20 0--20 5--35 17.5--45 37.5--45 37--52 13--33 87--95.5 NA NA Abbreviations: CA9=carbonic anhydrase 9; IQR=interquartile range; NA=not applicable; VEGF=vascular endothelial growth factor.

{ "pile_set_name": "PubMed Central" }

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22,191

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"appearance", "glioblastoma", "following", "bevacizumab", "treatment", "predict", "clinical", "benefit", "Efforts", "identify", "novel", "situ", "biomarkers", "efficacy", "focused", "available", "tissue", "samples", "primary", "tumour", "resections", "make", "inferences", "concerning", "treatment", "metastatic", "disease", "However", "evidence", "support", "metastatic", "colorectal", "cancer", "little", "evidence", "suppose", "valid", "approach", "inform", "biology", "NSCLC", "brain", "metastases", "summary", "greater", "understanding", "vascular", "phenotype", "NSCLC", "brain", "metastases", "may", "better", "inform", "use", "agents", "indication", "aim", "study", "assess", "relative", "expression", "VEGF", "hypoxia", "proliferation", "microvessel", "density", "vascular", "pattern", "vascular", "maturity", "series", "matched", "primary", "NSCLCs", "brain", "metastases", "MATERIALS", "METHODS", "Tissue", "blocks", "cases", "primary", "NSCLC", "matched", "resected", "brain", "metastases", "retrieved", "pathology", "archives", "Catholic", "University", "Hospital", "Rome", "Ethical", "approval", "research", "granted", "local", "research", "ethics", "committees", "Immunohistochemistry", "Characterisation", "antibodies", "immunohistochemical", "methods", "VEGF", "smooth", "muscle", "actin", "SMA", "double", "stain", "Dako", "Ely", "UK", "described", "detail", "elsewhere", "Scoring", "maximum", "intensity", "VEGF", "expression", "tumour", "cells", "recorded", "semiquantitative", "scale", "expression", "strong", "expression", "percentage", "tumour", "cells", "membranous", "expression", "estimated", "pathologist", "percentage", "tumour", "cells", "nuclear", "positivity", "counted", "pathologist", "Microvessel", "density", "analysis", "performed", "using", "Chalkley", "method", "low", "power", "five", "vascular", "hotspots", "sections", "selected", "Microvessel", "density", "assessed", "counting", "number", "vessels", "overlapped", "dots", "Chalkley", "eyepiece", "graticule", "field", "five", "counts", "added", "together", "provide", "density", "score", "Vascular", "maturity", "defined", "coverage", "endothelial", "cells", "positive", "pericytes", "SMA", "positive", "calculated", "counting", "proportion", "vessels", "surrounded", "least", "part", "cells", "fields", "case", "classified", "according", "predominant", "vascular", "pattern", "alveolar", "basal", "diffuse", "papillary", "described", "elsewhere", "scoring", "performed", "blind", "identities", "Statistical", "analyses", "used", "assess", "difference", "subsets", "continuous", "data", "correlation", "coefficient", "used", "assess", "covariance", "χ", "test", "used", "evaluate", "associations", "categorical", "data", "κ", "statistic", "used", "determine", "agreement", "RESULTS", "Chalkley", "counts", "primary", "NSCLCs", "brain", "metastases", "significantly", "correlated", "r", "P", "Table", "table", "Figure", "fig", "Although", "mean", "Chalkley", "counts", "similar", "primary", 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"microvessel", "density", "could", "predicted", "brain", "metastasis", "examination", "primary", "NSCLC", "expression", "showed", "strong", "correlation", "primary", "secondary", "NSCLCs", "observations", "important", "following", "reasons", "First", "therapies", "thought", "increase", "risk", "cerebral", "haemorrhage", "patients", "brain", "metastases", "recent", "data", "suggest", "bevacizumab", "safe", "indication", "data", "provide", "biological", "explanation", "observation", "showing", "vasculature", "brain", "metastases", "stable", "extrapolating", "observations", "rectal", "cancer", "unlikely", "regress", "following", "therapy", "minimising", "risk", "associated", "haemorrhage", "Second", "mature", "vasculature", "less", "sensitive", "therapy", "suggesting", "patients", "primary", "NSCLCs", "may", "benefit", "therapy", "brain", "metastases", "may", "Moreover", "preclinical", "data", "suggest", "endothelial", "regression", "seen", "mature", "vessels", "targeted", "therapy", "pericyte", "scaffold", "remains", "permits", "rapid", "following", "cessation", "therapy", "Thus", "rebound", "effect", "may", "expected", "therapy", "used", "brain", "metastases", "continuous", "rather", "intermittent", "therapy", "might", "show", "greater", "efficacy", "efficacy", "data", "exist", "bevacizumab", "brain", "metastases", "stated", "treated", "brain", "metastases", "likely", "derive", "similar", "benefit", "bevacizumab", "patients", "without", "brain", "without", "biological", "pathological", "clinical", "evidence", "data", "herein", "important", "counterbalance", "claim", "Third", "NSCLCs", "show", "alveolar", "pattern", "growth", "may", "metastasize", "merely", "existing", "vasculature", "brain", "instead", "develop", "angiogenic", "phenotype", "haphazardly", "arranged", "vessels", "Moreover", "even", "alveolar", "tumours", "express", "VEGF", "influence", "hypoxia", "supported", "often", "immature", "dense", "vasculature", "Therefore", "one", "may", "expect", "tumours", "potentially", "respond", "therapy", "site", "origin", "Finally", "retrospective", "analyses", "archived", "primary", "tumours", "trials", "therapy", "thus", "far", "failed", "identify", "biomarker", "efficacy", "data", "presented", "herein", "suggest", "vascular", "phenotypes", "primary", "secondary", "cancers", "different", "Therefore", "evaluating", "treatments", "metastatic", "setting", "biomarker", "studies", "also", "conducted", "metastases", "primary", "tumours", "limitations", "study", "include", "sample", "size", "small", "statistical", "terms", "large", "series", "matched", "brain", "metastases", "provides", "informative", "data", "reports", "novel", "findings", "Furthermore", "one", "may", "make", "inferences", "regarding", "applicability", "observations", "therapy", "using", "preclinical", "data", "clinical", "data", "tumours", "serial", "biopsies", "brain", "metastases", "patients", "receiving", "bevacizumab", "provide", "definitive", "answer", "date", "two", 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"Diffuse", "Diffuse", "Papillary", "Papillary", "Alveolar", "Diffuse", "Diffuse", "Diffuse", "Alveolar", "Papillary", "Basal", "Diffuse", "Diffuse", "Papillary", "Basal", "Diffuse", "Median", "NA", "NA", "IQR", "NA", "NA", "Abbreviations", "anhydrase", "range", "applicable", "endothelial", "growth", "factor" ]

Background ========== The evolutionary arms race between bacteria and their phages, giving rise to resistance and counter resistance mechanisms, shapes the bacterial and phage population size and composition as well as the evolutionary success of these two entities \[[@B1],[@B2]\]. The major phage resistance mechanisms exhibited by bacteria and the strategies adopted by phages to counter these resistance mechanisms have been extensively treated in two recent reviews \[[@B1],[@B2]\]. Understanding phage resistance mechanisms is critical in developing phage therapy applications. *Bacillus anthracis,* a category A biothreat agent, is a spore-forming Gram-positive bacterium of the *Bacillus cereus sensu lato* group. It is a zoonotic soil bacterium that infects animals and occasionally humans, causing the disease anthrax. The October-November 2001 bioterrorism attacks using mail laced with anthrax spores brought about renewed interest in research on *B. anthracis*. The prospect of biothreats using *B. anthracis* and the possibility of naturally emergent or deliberately created antibiotic resistant *B. anthracis* call for highly integrated and enhanced technological platforms for diagnosis and detection of this organism. This need is best illustrated in the case of a bacterial bioterror attack, where timely detection and intervention with countermeasures such as antibiotic therapy are paramount in preventing fatal consequences. Bacteriophages have been and still remain useful tools for bacterial species and strain differentiation \[[@B3]-[@B6]\], although evidence of successful application of phage therapy is still sparse in Western medicine \[[@B7]\]. Recently, the inherent binding specificity and lytic action of bacteriophage-encoded enzymes called lysins have been exploited for the rapid detection and killing of *B. anthracis*\[[@B8]\]. These authors demonstrated that the PlyG lysin isolated from the γ phage of *B. anthracis* specifically kills *B. anthracis* isolates and other members of the *B. anthracis* "cluster" of bacilli *in vitro* and *in vivo*. Both vegetative cells and germinating spores were shown to be susceptible. The lytic specificity of PlyG was also exploited as part of a rapid method for the identification of *B. anthracis*\[[@B8]\]*.* Thus, PlyG is a useful tool not only for the treatment but also for the detection of *B. anthracis*. The standard diagnostic tests for suspected *B. anthracis* as recommended by the Centers for Disease Control and Prevention (CDC) are as follows: presumptive identification to genus level (*Bacillus* family of organisms) requires Gram stain and colony identification. Presumptive identification to species level (*B. anthracis*) requires tests for motility, lysis by γ phage, capsule production and visualization, hemolysis, wet mount and malachite green staining for spores. Confirmatory identification of *B. anthracis* may include lysis by γ phage, capsular staining, and direct fluorescent antibody (DFA) testing on capsule antigen and cell wall polysaccharide. Thus, testing for γ phage sensitivity is an integral part of *B. anthracis* identification \[[@B9]\]. γ phage exhibits a fairly narrow host range but several *B. cereus* strains (e.g., ATCC 4342) have been shown to be sensitive to infection by this phage \[[@B8],[@B10]-[@B12]\]. Several phages (CP51, CP54 and TP21) isolated from *B. cereus* and *B. thuringiensis* strains have been successfully used for transducing chromosomal markers and plasmids between *B. anthracis* strains \[[@B13]-[@B17]\]. However, their utility as *B. anthracis* diagnostic phages is limited because of their broad host range. Bacteriophage AP50 was isolated from soil using *B. anthracis* Sterne as the host \[[@B18]\]. Originally it was thought to be an RNA phage but was later shown to contain double stranded DNA and phospholipid \[[@B19]\]. AP50 was also shown to have a narrow host range; only one third of the 34 *B. anthracis* strains and none of the 52 strains belonging to 6 different *Bacillus* species were susceptible to infection by AP50 \[[@B20]\]. Nine major structural proteins were identified on SDS-PAGE gels. The molecular weight of the phage DNA was estimated to be 9 × 10^6^ daltons \[[@B21]\]. Treatment with organic solvents such as chloroform (5%) and ether (25%) for 30 minutes inactivated the phage to a survival of about 1 × 10^-4^\[[@B22]\]. In a recent study, we described the genetic characterization of AP50c, which is a derivative of AP50 \[[@B23]\]. In contrast to the original report \[[@B20]\] on AP50, AP50c exhibited a much narrower host range, infecting 111 of the 115 *B. anthracis* strains (97%) and none of the 100 *B. cereus sensu lato* strains. We also determined the genome sequence by 454 pyrosequencing and compared it to other *Tectiviridae* phages \[[@B23]\]. The genome size was determined to be 14,398 bp in length and shown to be highly similar to other *Tectiviridae* phages infecting Gram-positive bacteria in genetic organization and encoded proteins. We identified two mutations in the AP50 genome that were responsible for the control of lysogenic/lytic lifestyles and consequently the phenotypic shift from turbid to clear plaque morphology. Based on these properties, we proposed that AP50c could be used as an additional diagnostic and therapeutic tool for *B. anthracis*. Before embarking on phage therapy using AP50c, we sought to characterize the phage resistance mechanisms in *B. anthracis* in response to AP50c infection. In order to address this problem, we took an approach that began with whole genome scanning and concluded with genetic validation. Initially, we isolated a number of *B. anthracis* mutants that were spontaneously resistant to killing by AP50c and observed that these mutants produced an extracellular material that coats the cell surface \[[@B23]\]. We hypothesized that this may be a bacterial adaptive resistance mechanism masking the bacterial receptor of AP50c. In the current study, we have mapped multiple bacterial mutations responsible for spontaneous AP50c resistance. Historically, mapping mutations responsible for specific phenotypes has involved transfer of the mutation with a closely linked selectable marker via one of the three conventional methods: transduction, conjugation or transformation. Physical mapping has involved cloning and sequencing of the genetically mapped gene using traditional cloning vectors and methods. The recent development of rapid, cost-effective sequencing of bacterial genomes using second-generation sequencers or array-based comparative genome resequencing has accelerated the mapping of causal variations in bacterial as well as in other model system genomes \[[@B24]-[@B26]\]. We recently demonstrated the use of 454- generated draft sequences to map mutations conferring various phenotypes, and in that study a putative mutation associated with AP50c resistance was mapped to *csaB*\[[@B27]\], although the link between genotype and phenotype was not verified in that report. CsaB (for cell surface anchoring) protein is involved in pyruvylation of the peptidoglycan-associated polysaccharide, and this modification is necessary for binding of the S-layer homology (SLH) domain. Thus, CsaB is critical for the non-covalent anchoring of SLH-domain containing proteins onto the cell surface \[[@B28]\]. In the current study, we explore the relevance of the preliminary finding of a potential linkage between the *csaB* gene and AP50c phage resistance by isolating and characterizing additional spontaneous mutants and, by using a recently described method \[[@B29]\], validating the WGS data by recreating *csaB* point and deletion mutations in a different genetic background and showing phage AP50c resistance. Furthermore, we restored sensitivity to AP50c infection in the mutants by complementing the defect in the Δ*csaB* mutant *in cis* as well as *in trans,* thereby establishing an essential role of *csaB* in phage infectivity. Results and discussion ====================== Whole genome pyrosequencing and identification of putative variants in AP50^R^ mutants -------------------------------------------------------------------------------------- We took a whole genome sequencing (WGS) approach to map the mutations and identify the gene(s) responsible for the phage resistant phenotype of the AP50^R^ mutants. We have shown previously that the draft sequences generated by Roche/454 shotgun pyrosequencing can be used efficiently to map mutations responsible for various phenotypes \[[@B27]\]. We generated whole genome sequence data of five spontaneous AP50^R^ mutant strains and compared them to that of the AP50c sensitive parent strain. We reasoned that the causal variation of phage resistance might occur in the same gene (for example in the gene encoding the bacterial receptor of the phage) in all five mutants (S-R1, S-R2, S-R3, S-R4, and S-R6), assuming that there is a single pathway to resistance. Hence, we searched for a common variation in all five mutants that is not present in the phage-sensitive parent strain. The phage-sensitive parent strain (Sterne 34F~2~) and the five mutants were sequenced by FLX or Titanium protocol. Some of the basic statistics of the genomic sequences are presented in Table [1](#T1){ref-type="table"}. The estimated average coverage of the genomes ranged from 12X to 46X, and the number of high-quality variations (see Methods) ranged from 46--52 (Table [1](#T1){ref-type="table"}). The number of contigs was generally proportional to the Q39 scores; i.e., the lower the Q39 score the lesser the number of contigs. Among the high-confidence variations, the most likely true positive variations (high-quality) were selected for by applying the following criterion: variations that had a percentage concordance of more than 75% variant base in the reads spanning any given position. Figure [1](#F1){ref-type="fig"} shows the variations as a function of the percentage concordance of the variant reads. There were a total of 242 high-quality variations across all the five AP50^R^ mutants, and of these 215 showed 75% or more concordance. Among these, 200 were identity-by-descent variations. The genomic locations of the 15 unique variations along with their annotations are shown in Table [2](#T2){ref-type="table"}. Among all the genes encoding variations, only two were mutated in all five resistant strains and not in the sensitive strain. These were BAS0840 (*csaB*), which encodes a cell-surface anchoring protein \[[@B28]\] and BAS3946, which encodes a 2, 3-diketo-5-methylthiopentyl-1-phosphate enolase. ###### **Statistics of genome sequence of wild type and mutant*B. anthracis*strains** **Strain** **Runs** **Run type** **Length of assembled genome (bp)** **% ≤ Q39** **Mean read length (bp)** **Number of contigs** **Depth of Coverage** **Number of putative HQ\* variants** ------------ ---------- -------------- ------------------------------------- ------------- --------------------------- ----------------------- ----------------------- -------------------------------------- 34F~2~ 1 FLX 5,356,131 0.06 274 101 23 48 S-R1 1 FLX 5,346,471 0.37 245 227 12 47 S-R2 2 FLX 5,355,072 0.02 274 60 46 50 S-R3 1 FLX 5,355,679 0.04 283 92 28 46 SR-4 1 FLX 5,347,433 0.06 282 102 21 47 SR-6 1 FLX 5,349,716 0.05 277 98 26 52 JB220 1 Titanium 5,349,773 0.39 264 111 23 43 J-R1 1 Titanium 5,348,852 0.59 269 128 22 45 \*HQ, high confidence and high concordance (≥75%). ![**Filtration of true positive variations from false positive variations by percent concordance and coverage.** The graph displays 260 high quality (HQ) variations in the genome sequences (34F~2~ and its derivatives) produced by GS Reference Mapper software as a function of percentage concordance. The graph indicates all the high-quality variations in the six genomic sequences obtained in this work. The variations from the six strains are indicated by different symbols. AP50 R1, AP50 R2, AP50 R3, AP50 R4 and AP50 R6 are S-R1, S-R2, S-R3, S-R4 and S-R6 respectively.](1743-422X-9-246-1){#F1} ###### **Location of true positive variations in genomes of mutant*B. anthracis*compared to reference genome** **Strain** **Start position** **Stop position** **Reference allele** **Sample allele** **Locus description** **Coverage** **Percent Concordance** ------------ -------------------- ------------------- ---------------------- ------------------- --------------------------------------------------- -------------- ------------------------- S-R1 894,910 894,910 \- G CsaB protein 10 100 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 8 100 S-R2 210,679 210,679 T G intergenic 29 97 895,097 895,097 \- G CsaB protein 38 82 2,139,026 2,139,026 G T sensor histidine kinase 34 76 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 24 100 S-R3 895,097 895,097 \- G CsaB protein 23 100 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 19 100 S-R4 895,066 895,066 A \- CsaB protein 18 100 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 19 100 S-R6 160,743 160,743 A \- pX01 58 86 895,083 895,083 \- GCTTA CsaB protein 17 88 1,330,746 1,330,746 G CGGT intergenic 4 75 3,894,626 3,894,626 G A 2,3-diketo-5-methylthiopentyl-1-phosphate enolase 14 100 4,303,652 4,303,652 \- AC solute-binding family 5 protein 20 100 J-R1 68,981 68,981 A \- hypoxanthine-guanine phosphoribosyltransferase 33 97 895,436 895,436 G T CsaB protein 23 96 Mutation in *csaB* is responsible for phage AP50c resistance ------------------------------------------------------------ Of the two mutated genes present in the AP50^R^ strains identified by WGS, BAS0840 (*csaB*) was determined to be the most probable candidate gene for phage resistance based on the following criteria: 1) In each of the five mutants (S-R1, S-R2, S-R3, S-R4 and S-R6) for which WGS was performed, a frame-shift mutation (∇G46 in S-R1, ΔA203 in S-R4, ∇GCTTA219 in S-R6, ∇G234 in S-R2 and S-R3) (Figure [2](#F2){ref-type="fig"}) was identified, which in all cases resulted in truncation of CsaB protein and possibly inactivation of its function. 2) Four additional spontaneous resistant mutants from the same genetic background (34F~2~) were screened by *csaB* PCR followed by Sanger sequencing of the *csaB* amplicons and were found to carry mutations in *csaB* as well (C385T, T274G, ΔC129, C254T). Similar to the findings in the first set of mutants, the C385T and ΔC129 mutations result in a truncated CsaB protein, whereas the T274G and C254T mutations result in a change from a noncharged (tyrosine) to a charged amino acid (aspartic acid) and a polar hydrophilic to a nonpolar hydrophobic amino acid, respectively. 3) A spontaneous AP50^R^ mutant generated in a different genetic background (JB220) was subjected to WGS and found not to carry the enolase (BAS3946) mutation, but it did carry a mutation in *csaB* (G573T), which results in a change from a positively charged (arginine) to a noncharged (serine) residue. 4) Seven additional spontaneous phage resistant mutants in the JB220 background were screened by the *csaB* PCR method described above and also found to carry mutations in *csaB* (G421T, C292T, G796C, T258G, C767A, and ∇G234, the latter of which was found in 2 different mutants). Interestingly, two of these mutations result in a noncharged residue being mutated to a charged residue, and the others result in a truncated *csaB*. These results are summarized in Figure [2](#F2){ref-type="fig"} and Table [3](#T3){ref-type="table"}. 5) Targeted introduction of mutations in *csaB* in a strain (7702) not previously exposed to AP50c resulted in phage resistance (see below and Table [4](#T4){ref-type="table"}). 6) Finally, BAS3946 was deemed unlikely to be involved in phage resistance because the mutation causes a non-synonymous change at G1024A leading to G342S in all five mutants (S-R1, S-R2, S-R3, S-R4 and S-R6) and thus does not result in the loss of the protein. PCR amplification and Sanger sequencing of BAS3946 from all the AP50^R^ mutants showed that the mutation is present only in seven out of nine mutants arising from 34F~2~ background and the parent and not in any of the eight mutants of JB220 background. Additionally, a targeted deletion of BAS3946 or the G1024A point mutation in BAS3946 in a different genetic background (strain 7702) in a strain not exposed to AP50c had no effect on AP50c phage sensitivity (Figure [3](#F3){ref-type="fig"}). ![**Genetic map of*csa*operon, DNA and protein sequences of*csaB*.a**) Genetic map of the *csaB* region on *B. anthracis* chromosome. The block arrows indicate the genes and the direction of transcription of the different genes. **b**) Nucleotide sequence of the wild type *csaB* gene and the location and nucleotide changes in the AP50^R^ mutants. Insertions and deletions are indicated by the red arrowheads and inverted arrowheads respectively. The various strains are indicated next to the mutated sites: derivatives of Sterne 34F~2~ and JB220 are indicated by the prefixes S and J respectively. The mutant loci labeled in black were discovered by WGS and those in green by Sanger sequencing of *csaB* PCR fragments. The altered bases are indicated in blue with the changed base above the wild type. A693C and H270A mutations were genetically engineered mutants. The sequences at the beginning and end of the gene marked in pink are the junctions the *csaB* deletion mutant. **c**) Amino acid sequence of wild type and mutant CsaB proteins. The sites of truncation of CsaB protein due to frame-shift and nonsense mutations are indicated by blue downward arrows. The amino acid changes in non-synonymous mutants are indicated in blue above the wild type residue. The amino acid sequences of the frame-shift and truncated proteins are shown. The residues in red are the additional residues added due to the frame-shift before truncation of the mutant proteins. **d**) DNA and protein sequence of the *csaB* gene in *csaB* deletion mutant.](1743-422X-9-246-2){#F2} ###### **Results of Sanger verification of the various spontaneous AP50**^**R**^**mutants** ----------------------------------------------------------------------------------------------------------------------------------------------- **Sanger run ID** **Strain** **Sanger data** **Mutation** **454 data** **Implication** **BAS3946** ------------------- --------------- ----------------------------------- -------------- -------------- --------------------------- ------------- csaB 1 34F~2~ parent WT None WT \- G1024A csaB 2 S-R1 ∇ G between positions 46 and 47 Frame-shift Same Truncation of CsaB G1024A csaB 3 S-R2 ∇ G between positions 233 and 234 Frame-shift Same Truncation of CsaB G1024A csaB 4 S-R3 ∇ G between positions 233 and 234 Frame-shift Same Truncation of CsaB G1024A csaB 5 S-R4 Δ A at position 203 Frame-shift Same Truncation of CsaB G1024A csaB 6 S-R5 C385T Nonsense ND Truncation of CsaB G1024A csaB 7 S-R6 ∇ GCTTA between position 219-220 Frame-shift Same Truncation of CsaB G1024A csaB 8 S-R7 T274G Y92D ND Noncharged to charged (−) G1024A csaB 9 S-R8 Δ C at position 129 Frame-shift ND Truncation of CsaB WT csaB 10 S-R9 C254 T T85M ND Polar hydrophilic to\ WT nonpolar hydrophobic csaB 11 JB220 parent WT None WT \- WT csaB 12 J-R1 G at position 573 to T R191S Same Charged (+) to noncharged WT csaB 13 J-R2 ∇ G between positions 233 and 234 Frame-shift ND Truncation of CsaB WT csaB 14 J-R3 G421T Nonsense ND Truncation of CsaB WT csaB 15 J-R4 C 292T Nonsense ND Truncation of CsaB WT csaB 16 J-R5 G796C G266R ND Noncharged to charged (+) WT csaB 17 J-R6 T258G S86R ND Noncharged to charged (+) WT csaB 18 J-R7 ∇ G between positions 233 and 234 Frame-shift ND Truncation of CsaB WT csaB 19 J-R8 C767A Nonsense ND Truncation of CsaB WT ----------------------------------------------------------------------------------------------------------------------------------------------- ∇ and Δ indicate insertion and deletion of base(s) respectively at the indicated position from the start of the gene. ND- Not Done. WT- Wild type; G1024A indicates a SNP in BAS3946 at position 1024 from the start of gene from G to A. ###### Strains and plasmids used in this study **Strain** **Description** **Source/Reference** ---------------------------------------- --------------------------------------------- ---------------------- *Escherichia coli* SM10 \- \[[@B39]\] *Bacillus anthracis* 34F~2~ Sterne pXO1^+^pXO2^-^ Hanna lab *Bacillus anthracis* BA663 7702 Sterne pXO1^+^pXO2^-^ Koehler lab *Bacillus anthracis* JB220 7702 ΔBAS2245 Calendar lab *Bacillus anthracis* S-R1 through S-R9 Spontaneous AP50R mutants This study *Bacillus anthracis* J-R1 through J-R8 Spontaneous AP50R mutants This study *Bacillus anthracis* BAP350 BA663 Δ*csaB* This study *Bacillus anthracis* BAP356 BA663 (AP50R-3)-*csaB* ∇G234 This study *Bacillus anthracis* BAP366 BA663 (AP50R-1)-*csaB* ∇G46 This study *Bacillus anthracis* BAP411 BA663 (AP50R-6)-*csaB* ∇GCTTA219 This study *Bacillus anthracis* BAP435 BA663 (AP50R-4)-*csaB* ΔA203 This study *Bacillus anthracis* BAP503 BA663 *csaB* H270A This study *Bacillus anthracis* BAP533 BAP350 *csaB* A693C creating an *XmaI* site This study *Bacillus anthracis* BAP553 BA663 ΔBAS3946 This study *Bacillus anthracis* BAP560 BA663 with BAS 3946 G1024A mutation This study *Bacillus anthracis* BGBN 001 BAP350/pBGBN1003 This study Plasmid pRP1028 Mutant construction This study Plasmid pSS4332 Mutant construction This study Plasmid pSW4 *E. coli*- *B. anthracis* shuttle vector \[[@B37]\] Plasmid pBGBN1001 pSW4 *NotI* This study Plasmid pBGBN1002 pSW4 *oriT* This study Plasmid pBGBN1003 pSW4 *oriT csaB* This study Plasmid pGOv4 pUC19 Amp^R^, Kan^R^ Gene Oracle Plasmid pNV36 pGOv4 ΔBAS3946 Gene Oracle Plasmid pNV37 pGOv4 BAS3946 G1024A Gene Oracle ∇-insertion and Δ-deletion of base (s) at any given position from the start of the gene; e.g., *csaB* ∇G46 indicates insertion of G base after position 46 (between 46 and 47) from the start of the gene, and *csaB* ΔA203 means deletion of A at position 203 from the start of gene. ![**Phage sensitivity spot test on Sterne 7702 and various derivatives.** For each row the left spot was spotted with medium (M), the middle spot received AP50c (A) and the right spot was spotted with γ (G). The clearing in the bacterial spot indicates infection and killing of bacteria. In rows 3 through 6, the γ spots completely cleared the bacterial spots. The various strains are as follows: 1) and 2) BA663 (wild type); 3) BAP350 (Δ*csaB*); 4) BAP356 (*csaB* ∇G234); 5) BAP366 (*csaB* ∇G46); 6) BAP411 (*csaB* ∇GCTTA219); 7) BAP435 (*csaB* ΔA203); 8) BAP503 (*csaB* H270A); 9) BAP533 (*csaB* A693C); 10) BAP553 (ΔBAS3946); 11) BAP560 (BAS3946 G1024A); 12) BGBN 001 (*ΔcsaB*/pBGBN1003)](1743-422X-9-246-3){#F3} Reconstructed *csaB* point mutants and in-frame deletion of *csaB* result in mucoid colony morphology and phage resistance -------------------------------------------------------------------------------------------------------------------------- In order to unequivocally link the phage resistance phenotype to *csaB* and to rule out the involvement of other mutations that may be present in the original AP50^R^ mutants, we reconstructed four of the point mutations in a different genetic background, in Sterne strain 7702, which was not previously exposed to AP50c (see Methods). All four mutants (∇G46, ΔA203, ∇GCTTA219, ∇G234) gave rise to mucoid colony morphology and phage resistant phenotype similar to the original spontaneous mutants, indicating that *csaB* indeed is involved in phage sensitivity. In addition to these mutants, we created a scarless, markerless in-frame deletion of *csaB* in 7702 background, which also showed mucoid and phage resistant phenotypes (Figure [3](#F3){ref-type="fig"}). Functional CsaB is needed for phage sensitivity ----------------------------------------------- Having shown that inactivation of *csaB* results in phage resistance, we wished to rule out the possibility that this result is due to a polar effect of the *csaB* mutations on downstream gene(s) or due to an alteration of regulatory elements in the deletion mutant. *csaA* and *csaB* form an operon upstream of *sap* and *eae* (Figure [2a](#F2){ref-type="fig"})*,* which have been shown to be involved in the formation of the cell surface associated S-layer (Sap) or an alternate S-layer (EA1) respectively \[[@B28]\]. In order to test the hypothesis that a functional CsaB is needed for phage sensitivity, we sought to inactivate the protein by mutating a critical amino acid residue while retaining the full-length protein and all the potential upstream and downstream control elements. A protein sequence alignment of 163 known orthologs revealed several conserved residues within CsaB protein (Figure [4](#F4){ref-type="fig"} and Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1) of which the histidine at position 270 is 100% conserved. We mutated histidine 270 to an alanine, and the resulting mutant was found to be mucoid and phage resistant (Figure [3](#F3){ref-type="fig"}), suggesting that a functional CsaB protein is necessary for phage infectivity, and inactivation of the CsaB protein either by truncation or by mutating a critical amino acid residue gives rise to phage resistance. ![**Amino acid sequence alignment of*csaB*orthologs.** BAS0840 was aligned with the Pfam PF04230 seed alignment, and percent conservation was determined as described in Methods. Only the alignment of the amino acids in the vicinity of histidine 270 of CsaB protein is shown. The full-length alignment is provided in Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1.](1743-422X-9-246-4){#F4} Complementation of *csaB* mutants with wild type copy of *csaB* --------------------------------------------------------------- We took two parallel approaches to test whether complementation of Δ*csaB* mutant with a wild type copy of *csaB* restores phage sensitivity. First, we introduced a copy of *csaB* in its native locus in the *csaB* deletion strain. In this case, however, we engineered the gene to carry a silent mutation (A693C) creating an *Xma*I site in order to distinguish this strain from the phage-sensitive parent strain. The resulting *csaB-Xma*I mutant strain was verified by PCR to carry the *Xma*I mutation (Figure [5a](#F5){ref-type="fig"}) and was found to be non-mucoid and phage sensitive. Thus, restoring the wild type allele in its native locus restored the phage sensitive phenotype. Second, complementation was done *in trans* by cloning the wild type *csaB* on a multicopy plasmid (Figure [5b](#F5){ref-type="fig"}) and transferring the recombinant plasmid (pNBGD1003) into the Δ*csaB* strain via conjugation. In this plasmid, *B. anthracis pagA* promoter was used to drive the constitutive expression of the cloned *csaB*. The resulting recombinant strain was found to be non-mucoid and phage sensitive (Figure [3](#F3){ref-type="fig"}). ![**Complementation of*csaB*mutation.a**) PCR verification of *Xma*I mutant *csaB*. The wild-type sequence of *csaB* was restored in the Δ*csaB* strain with the addition of a silent mutation creating an *Xma*I site (see Methods). The region was amplified by PCR, and PCR products were purified and digested with *Xma*I. The lane labeled BA663 indicates the *csaB* PCR product from wild type strain (7702) after digestion with *Xma*I, whereas the lane labeled BAP533 is the PCR product from the mutant showing the two bands after digestion with *Xma*I. **b**) Genetic map of *csaB* complementing plasmid pNBGD1003. pNBGD1003 is a shuttle vector derived from pSW4. The locations of the Gram positive and Gram negative replicons (from pUB110 and ColE1, respectively), antibiotic resistance genes *aadD* and *bla,* origin of conjugational transfer *oriT*, constitutively expressed *B. anthracis pagA* promoter (labeled PA promoter) and the *csaB* gene downstream of the promoter are indicated.](1743-422X-9-246-5){#F5} Transmission electron microscopic analyses of *csaB* mutants and complemented strains ------------------------------------------------------------------------------------- In an earlier study we found that the spontaneous AP50^R^ mutants produced an extracellular matrix and that AP50c phage failed to attach to the mutant bacterial surface \[[@B23]\]. In the current study, we confirmed these observations by transmission electron microscopy (Figure [6a](#F6){ref-type="fig"}-d). As evident from these images, AP50c phage particles do not attach to the surface of the Δ*csaB* mutant and consequently fail to infect. Phage adsorption and infectivity were regained when Δ*csaB* was complemented *in cis* or *in trans* with the wild type copy of *csaB* (Figure [6e](#F6){ref-type="fig"}-h). The extracellular secretion is seen only in the mutants and is absent in the wild type and the complemented mutants. The TEM images also revealed that there may be a defective or improperly assembled S-layer, or the S-layer may have been masked by the presence of the extracellular matrix. Further confirmation of the adsorption defect and reversion of this defect in complemented strains was obtained in phage adsorption assays. In the *csaB* mutants (BAP350, BAP356, BAP366, BAP411, BAP435, BAP503) \~62-100% of the phage particles were left in the supernatant whereas in wild type (BA663), Δ*csaB* complemented strains (BAP533 and BGBN001) and the BAS3946 mutants (BAP553, BAP560) most of the phage (\>99%) particles were adsorbed and very few particles (\~0.1-0.65%) were left in the supernatant. Thus, the defect in phage adsorption was rectified in the complemented strains. ![**Transmission electron microscopic images of wt and ∆csaB mutants and the complemented strains.a**) BA663 (csaB+); **b**) BA663+AP50c; **c**) BAP350 (∆csaB); **d**) BAP350 (∆csaB)+AP50c; **e**) BAP533 (csaB+XmaI); **f**) BAP533+AP50c; **g**) BAP350 (∆csaB)+ pBGBN 1003 (csaB+); **h**) BAP350 (∆csaB)+ pBGBN 1003 (csaB+)+AP50c. The scale bar is in the bottom right corner of each figure.](1743-422X-9-246-6){#F6} *csaB* mutants are sensitive to γ phage --------------------------------------- Earlier studies have shown that the cell surface protein GamR, which carries a LPXTG motif, is the cellular receptor of the γ phage \[[@B12]\]. We asked whether the cell surface-anchoring defect associated with *csaB* mutants affects GamR in any way, thereby preventing γ adsorption and subsequent infection. Consistent with earlier findings \[[@B30]\], all the mutants and the complemented strains allow infection of γ phage, indicating that mutations in *csaB* do not affect *gamR* expression or bacterial surface localization of GamR protein (Figure [3](#F3){ref-type="fig"}). Thus, *csaB*-mediated resistance is specific to AP50 phage and is not a general phage resistance mechanism. Conclusions =========== Phage resistance mechanisms are an integral part of the evolutionary arms race between phage and bacteria that has been waged for millions of years. Generally two types of bacterial defense mechanisms are recognized: active (restriction/modification systems, CRISPR/*cas*, and abortive infections) and passive host adaptations \[[@B2]\]. In the latter category, at least two, if not three, types of host adaptations interfering with the phage life cycle can be hypothesized: 1) phage entry, 2) phage growth after entry, and 3) potentially lysogeny can be considered to be the first line of defense against phage infection since lysogens acquire resistance rather quickly and at very high percentage upon infection with a turbid plaque AP50t phage \[[@B23]\]. Mutants of phage that make clear plaques arise in the phage population; these mutants were found to carry two mutations within the phage genome---one in a promoter region and another in a gene of unknown function \[[@B23]\]. Although the clear plaque mutants do not seem to regain the infectivity of a lysogen in the case of AP50 (Sozhamannan, unpublished data), spontaneous resistant strains to AP50c phage arise at very low frequencies in bacterial populations. In mutants that are bacterial surface modified, phage resistance could be caused by masking of the bacterial receptor or by bacterial receptor mutations. Other mutations might affect subsequent steps after the phage DNA enters the cell such as DNA replication, particle assembly, or even lysis of the host. In an effort to understand the passive defense mechanisms, we isolated spontaneous phage resistant bacterial mutants. These mutants secreted an extracellular matrix that appeared to protect the bacterium from phage infection. We hypothesized that this extracellular layer might mask the actual cellular receptor, thereby preventing phage adsorption, since it is a gain of new function due to a mutation and is absent in the wild type strain. Genetic mapping of a mutation entails linking the phenotype to a genetic locus. Conventional genetic methods use phage-mediated transduction, transformation or conjugation, by which the mutated gene is co-transferred with a known marker and mapped with reference to the known marker. Recombinant DNA and cloning techniques have been used to isolate and identify the gene responsible for a given phenotype. These conventional techniques are time-consuming and are not feasible in many organisms due to the lack of well-defined genetic systems. Recent developments in second-generation sequencing technologies have allowed us to obtain whole genome sequences of mutants and compare the sequences to that of the wild type strain to determine the causal variations. By taking a whole genome sequencing approach, we have traced the causal variation of the phage resistant phenotype to *csaB*. There are two caveats to this approach. First, the sequencing technology used here (Roche/454) is highly error-prone, resulting in approximately 50--100 false-positive variant calls per bacterial genome sequence. Empirically, we have determined that by applying a filter based on the coverage and percentage of variant reads in a given position, positions with 75% or above tend to be true positive variations. We have used a second filter in this study: a biological replicate strategy for ascertaining causal variations. A second caveat to this approach is that we assumed that the phenotype is due to a monogenic trait and that there is a single pathway to give rise to that phenotype. However, despite these limitations, in the current study, we were able to correctly associate the causal variation of the phage resistance phenotype to *csaB* and to further validate this prediction by reconstructing various *csaB* mutants and showing the linkage between phage resistance and *csaB* mutations. Mesnage et al. have characterized the *csaAB* operon of *B. anthracis* extensively \[[@B28]\]. These authors have proposed a role for *csaB* in cell surface anchoring of S-layer homology (SLH)-containing proteins via pyruvylation of cell wall polysaccharides. Consequently, mutants lacking *csaB* appear to be defective in cell surface anchoring of various SLH proteins including EA1 and Sap. In support of this idea, in *csaB* mutants, Sap and EA1 are conspicuously absent from the cellular fraction and are primarily found in the secretome. These authors also observed additional aberrant cellular morphologies that were replicated in this study, including the accumulation of cell wall material on the cell surface. These authors have attributed this phenotype to the lack of autolysis function in *csaB* mutants \[[@B28]\]. Recently, the role of yet another gene, *tagO,* has been implicated in S-layer protein assembly via binding to secondary cell wall polysaccharide \[[@B31]\]. Given the known function of CsaB in the pyruvylation pathway of polysaccharides, its most probable role in AP50 life cycle may be indirect: in the proper cell surface anchoring of phage receptor protein. The most probable candidates for the bacterial receptor may be Sap and EA1.It is possible that Sap or EA1 is the AP50 receptor, and *csaB* mutants fail to support AP50 adsorption because of the defect in proper cell surface anchoring of the S-layer. The transmission electron micrographs support the idea that the *csaB* mutants lack a well-organized S-layer. The spontaneous phage resistant mutants isolated in this study carry different types of mutations: SNPs and small insertions leading to nonsense codons, frame-shifts and non-synonymous changes. While a vast majority of these mutations cause CsaB truncation, some other mutants carry non-synonymous changes in the full-length protein. The essentiality of these amino acid residues in the overall structure and function of CsaB is not readily apparent since these mutations fall in regions of very low conservation in the protein alignment (Figure [4](#F4){ref-type="fig"} and Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1), but it is possible that the individual amino acid changes may affect protein folding and therefore function. The AP50^R^ mutants derived from 34F~2~ also carried a mutation in BAS3946 besides *csaB*. We have clearly established that the mutation in BAS3946 does not contribute to phage resistance in any way, and it is probably a 'hitch-hiker' mutation that was carried in the immediate parent prior to the selection for phage resistance. This result also highlights the power of the next generation sequencers for unbiased mapping of all variations arising in a given bacterial strain under selection pressure or mutagenesis, unlike conventional genetic techniques, which scan a focused area of the genome for mutations. Another intriguing question that remains is why spontaneous AP50 resistant mutants were not found in the actual phage receptor gene itself, assuming Sap or EA1 is the AP50 phage receptor. It is possible that there are differences in the mutability or essentiality of the genes, although we have already shown that *sap* is not an essential gene for *B. anthracis* viability (Plaut et al., unpublished data). Further characterization of additional genes involved in AP50c life cycle identified by this study is underway. The fact that γ phage and AP50 use different receptors is useful in phage therapy experiments, since development of resistance against both phages would be kept under check when used in combination. Methods ======= Bacterial strains, plasmids and oligonucleotide primers ------------------------------------------------------- Strains and plasmids used in this study are listed in Table [4](#T4){ref-type="table"}. The three Sterne strains used in this study were 34F~2~ (obtained from Philip C. Hanna), 7702 (obtained from Theresa M. Koehler) and JB220 (obtained from Richard Calendar). Strain 7702 is designated as BA663 in this study. Derivatives of 34F~2~ that were spontaneously resistant to AP50 are designated S-R1 through S-R9, and those of JB220 are designated J-R1 through J-R8. When necessary, strains were grown in the presence of spectinomycin (100 μg/ml for *E. coli*; 250 μg/ml for *B. anthracis*) or kanamycin (20 μg/ml for *E. coli*; 25 μg/ml for *B. anthracis*). The primers used in various plasmid constructions and Sanger sequencing of PCR amplicons are listed in Additional file [2](#S2){ref-type="supplementary-material"}: Table S1. Genome sequencing and variation detection ----------------------------------------- Whole-genome sequencing (WGS) was performed in the Genome Sequencer FLX (454 Life Sciences/Roche) using FLX or FLX Titanium reagents according to the manufacturer\'s protocols and instructions \[[@B32]\]. Signal processing of FLX data was performed on the sequencer itself, whereas signal processing of Titanium data was performed off-rig on a Linux cluster of 10 nodes connected via gigabit ethernet. Each node contained eight 64-bit processing cores running at 2.3 GHz with 8 GB of RAM. *De novo* or reference-guided assembly of either FLX or Titanium sequences, preliminary annotation of the genome using the DIYA pipeline \[[@B33]\] and identification of variations were performed using the above-described computer cluster and Sterne (NC 005945) as reference genome as described \[[@B27]\]. A variant flagged by GS Reference Mapper was defined as high confidence if it met the following criteria: a) at least three non-duplicated reads showed the same variation; b) at least one read in each of the forward and reverse orientations showed this variation, unless there are at least five reads with quality scores over 20 (or 30 if the difference involves a 5-mer or higher). A more detailed description of differences is available in the Genome Sequencer Data Analysis Software manual. In this study, a high quality variation is defined as a high confidence (as defined by 454/Roche) and high concordance (≥75% reads have the variation) variation. Culture methods and phage assays -------------------------------- All bacterial strains were grown in Luria Broth or Brain Heart Infusion medium, and the phage assays were performed according to published protocols. Phage spot tests were performed as follows: first, triplicate spots of 50 μl of an overnight culture were made on large phage assay agar plates and allowed to dry. Five μl of phages (\~10^8^ PFU) AP50c and γ was spotted on two bacterial spots while the third spot received 5 μl of medium. The spots were dried at room temperature and the plates were subsequently incubated at 37°C overnight. Phage adsorption assay was performed as follows: One ml of log phase wild type and mutant *B. anthracis* cells was spun and the pellet was resuspended in 100 μl of AP50c phage and incubated for 30 minutes at room temperature. After addition of 900 μl of phage assay broth, the bacteria-phage complex was filtered through a 0.45 μm syringe filter. The plaque forming units (PFU) in the filtrate was titred using 7702 as the indicator bacterium. The percentage adsorption was calculated against the same volume of unadsorbed phage filtered through the syringe filter. Construction of mutants ----------------------- Two plasmids were engineered to facilitate allelic exchange in *B. anthracis*, as improvements to a published system \[[@B29]\]. Details of the improvements will be published elsewhere. Briefly, pRP1028 and pSS4332 serve the functions of pBKJ236 and pBKJ223, respectively, and contain many of the same features. In pRP1028, the erythromycin resistance of pBKJ236 was replaced with spectinomycin resistance, and in pSS4332, the tetracycline resistance of pBKJ223 was replaced with kanamycin resistance. Allelic exchange constructs derived from pRP1028 were transferred to *B. anthracis*, and plasmid integrants were isolated following a temperature shift \[[@B29]\]. Introduction of pSS4332 into the integrant led to I-*Sce*I-mediated cleavage of the integrated plasmid, stimulating the second crossover event. The presence of the desired changes was confirmed by PCR and sequencing. Specifically, to reconstruct the point mutations found in the spontaneous AP50^R^ mutants, the mutations and approximately 500 bp of flanking homology on each side were PCR-amplified from the *B. anthracis* strains and cloned into pRP1028. To delete *csaB*, standard cloning methods were used to engineer a construct with the sequence GTG CGG CCCGGG GGA TCT TAA, representing START+one codon+*Xma*I site+two codons+STOP, along with approximately 500 bp of flanking homology. To restore the wild-type allele to the *csaB* deletion strain, a derivative of pRP1028 was engineered with the *csaB* sequence with the adenosine at position 693 changed to cytosine (creating the *Xma*I site CCCGGG without changing the amino acid sequence), along with approximately 1kb of flanking homology. The change was confirmed by PCR and sequencing, as well as by digestion of the PCR product with *Xma*I (Figure [5a](#F5){ref-type="fig"}). To identify conserved residues of CsaB, the nucleotide sequence (BAS0840) was aligned with the 163 proteins in the seed alignment of Pfam family PF04230 (polysaccharide pyruvyl transferase) \[[@B34]\] using ClustalW \[[@B35]\]. The resulting alignment was processed with WebLogo \[[@B36]\] to calculate the consensus sequence and percent conservation at each position (Figure [4](#F4){ref-type="fig"} and Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1). The histidine at position 270 of BAS0840 was found to be 100% conserved in these proteins. Standard cloning techniques were used to engineer a derivative of pRP1028 in which the histidine codon (CAT) was changed to alanine (GCT) (Figure [2](#F2){ref-type="fig"}), flanked by approximately 850 bp of homology. The presence of the H270A change was confirmed by PCR and sequencing. DNA sequences used to generate ΔBAS3946 and BAS3946 G1024A were synthesized (Gene Oracle, Mountain View, CA). For ΔBAS3946, the sequence included the first 6 codons of the gene, followed by the last 5 codons of the gene and the STOP codon, all flanked by approximately 500 bp of homology. For BAS3946 G1024A, the sequence included the mutation flanked by 500 bp of homology. In each case, DNA was cloned into pRP1028, allelic exchange was performed as described above, and the presence of the desired changes was confirmed by PCR and sequencing. Construction of pNBGD1003 and complementation of ΔcsaB ------------------------------------------------------ The starting plasmid for pNBGD1003 was pSW4 \[[@B37]\] which was modified by inverse PCR to include a *Not*I-*Nde*I site on either side of the *pagA* promoter using primers 1004 and 1005. The *pagA* promoter was amplified using primers 1006 and 1026, and fragments were digested with *Not*I and *Nde*I and cloned, giving rise to pNBGD1001. Plasmid pNBGD1002 was derived from pNBGD1001 by introducing an origin of transfer, *oriT*, as a *Not*I fragment at the *Not*I site. The *oriT* fragment was PCR amplified from pIN297, which is a derivative of pPL2 \[[@B38]\] using primers 1064 and 1065. Plasmid pNBGD1003 was derived from pNBGD1002 by cloning a *Pml*I- *Bgl*II fragment carrying the *csaB* gene sequences into *Nde*I digested, blunted and *Bam*HI digested pNBGD1002. Plasmid pNBGD1003 was electroporated into SM10 and subsequently transferred into *B. anthracis* Δ*csaB* mutant by conjugation, selecting for polymyxinB and kanamycin resistance as described previously \[[@B29]\]. The resulting ex-conjugants were purified and checked for phage adsorption and infection. PCR and Sanger verification of the csaB and BAS3946 genes from AP50^R^ mutants ------------------------------------------------------------------------------ The DNA sequences spanning the *csaB* (1385 bp) and BAS3946 (1445 bp) genes were PCR amplified from wild type and various mutants (both 34F~2~ and JB220 derivatives), and the PCR amplicons were purified and sequenced on both strands using primers indicated in Table S-1 (BGBN14 through BGBN21). The resulting nucleotide sequences were aligned to the wild type sequences of these two fragments to determine the mutational changes. Transmission electron microscopy -------------------------------- Bacterial pellets were fixed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate pH 7.4 overnight at 4°C. Following buffer rinse, samples were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate for 1 hr on ice in the dark. After a brief distilled H~2~O rinse samples were placed in 2% aqueous uranyl acetate (0.22 um filtered) for 1 hour at room temperature in the dark. Following en-bloc staining, cells were dehydrated through a graded series of ethanol to 100% ethanol, transferred through propylene oxide, embedded in Spurr's low viscosity resin (Polysciences), and cured at 60°C for two days. Sections were cut on a Riechert Ultracut E with a Diatome Diamond knife. Eighty nm sections were picked up on formvar coated 1 × 2 mm copper slot grids and stained with uranyl acetate followed by lead citrate. Grids were viewed on a Hitachi 7600 TEM operating at 80 kV and digital images captured with an AMT 1 K × 1 K CCD camera. Competing interests =================== The authors declare no competing interests. Authors' contributions ====================== KABL, KMW, AB, SD, TL, and MG carried out the whole genome sequencing. KABL, PEC, AA, TDR, and SSo conducted nextgen and Sanger sequence analysis. RDP and SSt carried out deletion and complementation studies. CC performed primer design and microbiological work. AM, VM, and RC participated in the study's design and coordination and helped draft the manuscript. SSo conceived of the study, participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ###### Additional file 1 **Figure S1.** Sequence alignment of *csaB* and orthologs. BAS0840 was aligned with the Pfam PF04230 seed alignment and percent conservation was determined as described in Methods. ###### Click here for file ###### Additional file 2 **Table S1.** List of primers used in this study. ^a^ Restriction sites are underlined. ###### Click here for file Acknowledgments and disclaimers =============================== The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. The BDRD, NMRC authors are employees of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. Â§105 provides that "Copyright protection under this title is not available for any work of the United States Government." Title 17 U.S.C. Â§101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person\'s official duties. We would like to thank Michael J Delannoy (JHU EM facility) for help with the electron microscopy.

{ "pile_set_name": "PubMed Central" }

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"obtained", "Richard", "Calendar", "Strain", "designated", "study", "Derivatives", "spontaneously", "resistant", "designated", "designated", "necessary", "strains", "grown", "presence", "spectinomycin", "coli", "anthracis", "kanamycin", "coli", "anthracis", "primers", "used", "various", "plasmid", "constructions", "Sanger", "sequencing", "PCR", "amplicons", "listed", "Additional", "file", "Table", "Genome", "sequencing", "variation", "detection", "sequencing", "WGS", "performed", "Genome", "Sequencer", "FLX", "Life", "using", "FLX", "FLX", "Titanium", "reagents", "according", "protocols", "instructions", "Signal", "processing", "FLX", "data", "performed", "sequencer", "whereas", "signal", "processing", "Titanium", "data", "performed", "Linux", "cluster", "nodes", "connected", "via", "gigabit", "ethernet", "node", "contained", "eight", "processing", "cores", "running", "GHz", "GB", "RAM", "De", "novo", "assembly", "either", "FLX", "Titanium", "sequences", "preliminary", "annotation", "genome", "using", "DIYA", "pipeline", "identification", "variations", "performed", "using", "computer", "cluster", "Sterne", "NC", "reference", "genome", "described", "variant", "flagged", "GS", "Reference", "Mapper", "defined", "high", "confidence", "met", "following", "criteria", "least", "three", "reads", "showed", "variation", "b", "least", "one", "read", "forward", "reverse", "orientations", "showed", "variation", "unless", "least", "five", "reads", "quality", "scores", "difference", "involves", "higher", "detailed", "description", "differences", "available", "Genome", "Sequencer", "Data", "Analysis", "Software", "manual", "study", "high", "quality", "variation", "defined", "high", "confidence", "defined", "high", "concordance", "reads", "variation", "variation", "Culture", "methods", "phage", "assays", "bacterial", "strains", "grown", "Luria", "Broth", "Brain", "Heart", "Infusion", "medium", "phage", "assays", "performed", "according", "published", "protocols", "Phage", "spot", "tests", "performed", "follows", "first", "triplicate", "spots", "μl", "overnight", "culture", "made", "large", "phage", "assay", "agar", "plates", "allowed", "dry", "Five", "μl", "phages", "PFU", "γ", "spotted", "two", "bacterial", "spots", "third", "spot", "received", "μl", "medium", "spots", "dried", "room", "temperature", "plates", "subsequently", "incubated", "overnight", "Phage", "adsorption", "assay", "performed", "follows", "One", "ml", "log", "phase", "wild", "type", "mutant", "anthracis", "cells", "spun", "pellet", "resuspended", "μl", "phage", "incubated", "minutes", "room", "temperature", "addition", "μl", "phage", "assay", "broth", "complex", "filtered", "μm", "syringe", "filter", "plaque", "forming", "units", "PFU", "filtrate", "titred", "using", "indicator", "bacterium", "percentage", "adsorption", "calculated", "volume", "unadsorbed", "phage", "filtered", "syringe", "filter", "Construction", "mutants", "Two", "plasmids", "engineered", "facilitate", "allelic", "exchange", "anthracis", "improvements", "published", "system", "Details", "improvements", "published", "elsewhere", "Briefly", "serve", "functions", "respectively", "contain", "many", "features", "erythromycin", "resistance", "replaced", "spectinomycin", "resistance", "tetracycline", "resistance", "replaced", "kanamycin", "resistance", "Allelic", "exchange", "constructs", "derived", "transferred", "anthracis", "plasmid", "integrants", "isolated", "following", "temperature", "shift", "Introduction", "integrant", "led", "Sce", "cleavage", "integrated", "plasmid", "stimulating", "second", "crossover", "event", "presence", "desired", "changes", "confirmed", "PCR", "sequencing", "Specifically", "reconstruct", "point", "mutations", "found", "spontaneous", "mutants", "mutations", "approximately", "bp", "flanking", "homology", "side", "anthracis", "strains", "cloned", "delete", "csaB", "standard", "cloning", "methods", "used", "engineer", "construct", "sequence", "GTG", "CGG", "CCCGGG", "GGA", "TCT", "TAA", "representing", "Xma", "along", "approximately", "bp", "flanking", "homology", "restore", "allele", "csaB", "deletion", "strain", "derivative", "engineered", "csaB", "sequence", "adenosine", "position", "changed", "cytosine", "creating", "Xma", "site", "CCCGGG", "without", "changing", "amino", "acid", "sequence", "along", "approximately", "flanking", "homology", "change", "confirmed", "PCR", "sequencing", "well", "digestion", "PCR", "product", "Xma", "Figure", "fig", "identify", "conserved", "residues", "CsaB", "nucleotide", "sequence", "aligned", "proteins", "seed", "alignment", "Pfam", "family", "polysaccharide", "pyruvyl", "transferase", "using", "ClustalW", "resulting", "alignment", "processed", "WebLogo", "calculate", "consensus", "sequence", "percent", "conservation", "position", "Figure", "fig", "Additional", "file", "Figure", "histidine", "position", "found", "conserved", "proteins", "Standard", "cloning", "techniques", "used", "engineer", "derivative", "histidine", "codon", "CAT", "changed", "alanine", "GCT", "Figure", "fig", "flanked", "approximately", "bp", "homology", "presence", "change", "confirmed", "PCR", "sequencing", "DNA", "sequences", "used", "generate", "synthesized", "Gene", "Oracle", "Mountain", "View", "CA", "sequence", "included", "first", "codons", "gene", "followed", "last", "codons", "gene", "STOP", "codon", "flanked", "approximately", "bp", "homology", "sequence", "included", "mutation", "flanked", "bp", "homology", "case", "DNA", "cloned", "allelic", "exchange", "performed", "described", "presence", "desired", "changes", "confirmed", "PCR", "sequencing", "Construction", "complementation", "ΔcsaB", "starting", "plasmid", "modified", "inverse", "PCR", "include", "Nde", "site", "either", "side", "pagA", "promoter", "using", "primers", "pagA", "promoter", "amplified", "using", "primers", "fragments", "digested", "Nde", "cloned", "giving", "rise", "Plasmid", "derived", "introducing", "origin", "transfer", "oriT", "fragment", "site", "oriT", "fragment", "PCR", "amplified", "derivative", "using", "primers", "Plasmid", "derived", "cloning", "Pml", "Bgl", "II", "fragment", "carrying", "csaB", "gene", "sequences", "Nde", "digested", "blunted", "Bam", "HI", "digested", "Plasmid", "electroporated", "subsequently", "transferred", "anthracis", "Δ", "csaB", "mutant", "conjugation", "selecting", "polymyxinB", "kanamycin", "resistance", "described", "previously", "resulting", "purified", "checked", "phage", "adsorption", "infection", "PCR", "Sanger", "verification", "csaB", "genes", "mutants", "DNA", "sequences", "spanning", "csaB", "bp", "bp", "genes", "PCR", "amplified", "wild", "type", "various", "mutants", "derivatives", "PCR", "amplicons", "purified", "sequenced", "strands", "using", "primers", "indicated", "Table", "resulting", "nucleotide", "sequences", "aligned", "wild", "type", "sequences", "two", "fragments", "determine", "mutational", "changes", "Transmission", "electron", "microscopy", 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"Authors", "contributions", "KABL", "KMW", "AB", "SD", "TL", "MG", "carried", "whole", "genome", "sequencing", "KABL", "PEC", "AA", "TDR", "SSo", "conducted", "nextgen", "Sanger", "sequence", "analysis", "RDP", "SSt", "carried", "deletion", "complementation", "studies", "CC", "performed", "primer", "design", "microbiological", "work", "VM", "RC", "participated", "study", "design", "coordination", "helped", "draft", "manuscript", "SSo", "conceived", "study", "participated", "design", "coordination", "helped", "draft", "manuscript", "authors", "read", "approved", "final", "manuscript", "Supplementary", "Material", "Additional", "file", "Figure", "Sequence", "alignment", "csaB", "orthologs", "aligned", "Pfam", "seed", "alignment", "percent", "conservation", "determined", "described", "Methods", "Click", "file", "Additional", "file", "Table", "List", "primers", "used", "study", "Restriction", "sites", "underlined", "Click", "file", "Acknowledgments", "disclaimers", "views", "expressed", "article", "authors", "necessarily", "reflect", "official", "policy", "position", "Department", "Navy", "Department", "Defense", "Government", "BDRD", "NMRC", "authors", "employees", "Government", "work", "prepared", "part", "official", "duties", "Title", "provides", "Copyright", "protection", "title", "available", "work", "United", "States", "Government", "Title", "defines", "Government", "work", "work", "prepared", "military", "service", "member", "employee", "Government", "part", "official", "duties", "would", "like", "thank", "Michael", "J", "Delannoy", "JHU", "EM", "facility", "help", "electron", "microscopy" ]

1. Case history {#sec1-1} =============== A 45-year-old married Hindu female, non-smoker, non hypertensive and non diabetic, was diagnosed with paranoid schizophrenia, for which she was maintaining well on tablet haloperidol 20 mg/day for the last 5 years. The total duration of illness was around 7 years, with a drug naive period of approximately 2 years. Around three months ago, the patient's symptoms had become exaggerated as she had not taken her antipsychotic medication for approximately 8-10 days because she was out of town and did not have her medicines with her. Her complaints were sleep disturbance, referential and persecutory ideas with intermittent auditory hallucinations and episodes of irritability with aggressive behavior. The patient presented to the hospital after which haloperidol was started again at 10 mg/day, increased to 20 mg/day and maintained for the next month. However no significant improvement was reported this time using haloperidol. Olanzapine 5 mg/day was added and increased to 10 mg/day within 2 weeks. One month after adding Olanzapine, the patient developed symptoms of nausea, anorexia and weakness. However there were no episodes of vomiting or diarrhea. Also, there was no significant decrement in diet. The symptoms exacerbated over the next 2 weeks along with muscle cramps, unsteady gait and fluctuating orientation. The patient was admitted to emergency services for evaluation. Her physical examination revealed tachycardia (pulse - 118/min), low blood pressure (94/60 mm hg), dehydration, and disorientation to time. Her systemic examination was unremarkable. Her blood tests revealed low serum sodium of 120 mmol/l (135-150 mmol/l). The results of the remaining tests were within normal limits (including renal function tests, liver function tests, serum concentrations of potassium, blood sugar, total proteins and lipids). Treatment was started on the diagnosis of probable normovolemic hyponatremia as physical examination did not reveal any signs of fluid overload. The electrocardiography revealed only sinus tachycardia and plasma osmolarity was reported to be 288 mOsm/kg H~2~O (285--295mOsm/kgH~2~O). She was started on 3% NaCl infusion over the initial few hours until achieving full consciousness followed by normal saline. Fluid restriction and oral salt supplement was advised. The patient recovered in 2 days with serum sodium levels of 140 mmol/l. However, the exact reason for hyponatremia could not be established even after detailed investigations (including thyroid function tests). Olanzapine continued to be administered, except for the day emergency treatment was given. Upon detailed assessment, the patient's diet was found to be consistently good over these years, and even during the last 2 weeks. No specific diet/salt restriction was reported. A week later, the patient again had similar episode of hyponatremia (serum sodium of 116 mmol/l) while continuing on the antipsychotics. She had to be managed in the emergency room with similar procedures and protocols. With no evident organic cause for the hyponatremia, a literature search was carried out and Olanzapine was postulated as a potential agent. It was planned to stop Olanzapine and observe the patient, while continuing medical management for hyponatremia. Within 3 days of stopping Olanzapine, the patient improved and never had a recurrence of hyponatremia over the next 6 months follow up. She was maintained on haloperidol 20 mg/day and risperidone 4 mg/day thereafter. Naranjo algorithm^\[[@ref1]\]^ established a "probable" causal relation between the drug and adverse event, with a total score of 8 (suggestive of a probable relationship). 2. Discussion {#sec1-2} ============= Olanzapine is a commonly used atypical antipsychotic for patients with schizophrenia and other psychotic disorders. In the CATIE study (Clinical Antipsychotic Trials of Intervention Effectiveness), Olanzapine was found to have the lowest discontinuation rate, possibly due to fewer incidences of acute side effects.^\[[@ref2]\]^ Still, life threatening adverse effects such as hyponatremia may limit its safe and effective use. A systematic review of evidence for antipsychotic induced hyponatremia from 1974 to 2003, found more cases of hyponatremia with typical compared to atypical antipsychotics.^\[[@ref3]\]^ Hyponatremia is the commonest electrolyte imbalance encountered in medical emergencies.^\[[@ref4]\]^ It is characterized by a plasma sodium concentration less than 135 mEq/L^\[[@ref5]\]^ and manifests clinically with neurological and systemic symptoms such as nausea, vomiting, anorexia, headache, disorientation, confusion, irritability and lethargy.^\[[@ref6]\]^ Profound hyponatremia may manifest as severe mental status changes including confusion, delirium, seizure, coma, and death. The most common causes of hyponatremia are fluid loss and dehydration, congestive heart failure, liver and renal failure, and Syndrome of inappropriate anti-diuretic hormone. Hyponatremia as a potential adverse event from the use of antipsychotics has not been greatly researched. Only a few case reports/series are available.^\[[@ref7],[@ref8]\]^ Meulendijks and colleagues did a systematic review of evidence for antipsychotic induced hyponatremia from 1974 to 2003 and found that both typical (58 case reports) and atypical antipsychotics (10 case reports) were implicated in hyponatremia and the association was independent of age or gender or dose.^\[[@ref3]\]^ Many antipsychotics (both typical and atypical) like chlorpromazine, fluphenazine, haloperidol, trifluoperazine, amisulpride, olanzapine and risperidone have been implicated.^\[[@ref3],[@ref9]\]^ Hyponatremia is postulated to be caused by blockade of inhibitory effect of dopamine on release of anti-diuretic hormone (ADH) by antipsychotics.^\[[@ref10]\]^ Clozapine, because of its low affinity for D2 receptors, is less prone to causing hyponatremia and may improve associated polydipsia. However on the contrary, it has been demonstrated in rats that dopamine could have a stimulatory effect on ADH release.^\[[@ref11]\]^ Therefore, the exact mechanism of antipsychotic-induced hyponatremia is still unclear. In the index case, hyponatremia and olanzapine were temporally associated, still it is difficult to establish the exact offending medication. Haloperidol is also a known culprit that is described in some case reports. However, haloperidol alone did not produce hyponatremia nor did it in combination with risperidone suggesting that olanzapine may have a stronger causal relation with hyponatremia. Also, Naranjo algorithm established a "probable" causal relationship with olanzapine. Another possible mechanism is that olanzapine precipitated hyponatremia in a predisposed individual taking haloperidol, and the causality may be attributed to the combined effect of both olanzapine and haloperidol. Hyponatremia is a rare yet clinically important adverse reaction to treatment with psychotropic drugs. It is a potentially dangerous medical complication in psychiatric patients. Psychogenic polydipsia is an important differential diagnosis, as it is characterized by similar symptoms of polydipsia and polyuria. Also, psychogenic polydipsia is commonly found in psychiatric patients, more likely in schizophrenia.^\[[@ref12]\]^ Multiple other factors such as diet, salt intake, ageing comorbid conditions (smoking, diabetes) or concurrent medications may also play a role and should be considered.^\[[@ref13]\]^ There is a complete and rapid recovery of drug induced hyponatremia once the offending agent is discontinued, as happened with the index case, without any further recurrences. Clinicians need to be aware of this dangerous complication and exercise caution when prescribing psychotropic drugs. **Funding statement** No funding support was obtained for preparing this case report. **Conflict of interest statement** The authors declare that they have no conflict of interest related to this manuscript **Informed consent** The patient and his guardian signed an informed consent form and agreed to the publication of this case report. ![](sap-29-177-g001.gif) Dr. Ankur Sachdeva obtained his MBBS degree from Vardhman Mahavir Medical College and Safdarjang Hospital, New Delhi in 2009 and an MD (Psychiatry) degree from the Post Graduate Institute of Medical Education and Research, at the Dr. Ram Manohar Lohia Hospital in 2013. He subsequently received the Diplomate of the National Board (D.N.B) in psychiatry in 2014. He is presently working as Assistant Professor (Psychiatry) in ESIC medical college, Faridabad, Haryana, India.

{ "pile_set_name": "PubMed Central" }

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22,193

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1. Introduction {#sec1} =============== South Africa\'s HIV epidemic remains the largest in the world, with an estimated 5.6 million people living with HIV in 2009 \[[@B1]\]. Young people in particular have the fastest-growing infection rates \[[@B2], [@B3]\]. They are expected to delay sexual activity and avoid unplanned pregnancy and sexually transmitted infection to safeguard their sexual health \[[@B4]\]. Yet, the complexity of sexual behaviour has been underscored \[[@B5]\] given that HIV risk behaviour is influenced by various factors. Condom use and negotiation of safer sex are reported to be the most effective means of HIV risk reduction \[[@B6]\]. Often, young people struggle to exert control over their emotions when they engage in sexual activity \[[@B7]\], resulting in risky sexual behaviours that lead to HIV infection. The concept of self-efficacy is defined as having confidence in one\'s ability to perform a particular behavior, and it has been regarded as an important component of health-related behavioral change \[[@B7]\]. The AIDS risk reduction model in which self-efficacy plays an important role \[[@B8]\] emphasize three stages where a young person has knowledge about a particular safer sex behavior (i.e., using condoms), they then have to think that the behavior is socially acceptable (norms/attitudes) and to believe that they would be able to practice the behavior (self-efficacy) before they actually engage in the behavior \[[@B8]\]. Studies of HIV risk behaviours and sexual health have consistently shown that high self-efficacy for condom use is strongly associated with behaviours of condom use with recent partners and consistent use \[[@B6]\]. A study conducted among young people age 15--24 years reported very high levels of self-efficacy when it came to believing that they could discuss condoms with their partners and refusing sex when they did not want to, but this was not always matched with their actual behaviour \[[@B9], [@B10]\]. Even though a decline in HIV prevalence has been reported among South African youth 15--24 from 10.3% in 2005 to 8.6% in 2008, the prevalence remains disproportionately high for females overall in comparison to males \[[@B2]\]. The burden of HIV on women, however, varies considerably by region but is heaviest in sub-Saharan Africa with 1.4 times more adult women than men who were living with HIV in 2010 \[[@B1]\]. In South Africa, for example, young females have 3 to 4 times the prevalence of HIV than their male peers \[[@B1]\]. Thus, it is crucial to take into account the gender differences of HIV prevalence \[[@B11]\] and HIV risk behaviours. There is still a dearth of studies that investigate factors that may influence HIV risk reduction self-efficacy by gender. Therefore, this study examines factors associated with HIV risk reduction self-efficacy among South African youth age 15--24 years as part of an evaluation of the impact of loveLife, a youth focused HIV prevention programme. 2. Method {#sec2} ========= 2.1. Sample and Procedures {#sec2.1} -------------------------- A cross-sectional population-based household survey was conducted using a multi-stage stratified cluster sampling approach. In each household all eligible household members were invited to participate and interviewed. The survey included persons of ages 18 to 24 years living in South African households of the four (out of nine) selected provinces, KwaZulu-Natal, Mpumalanga, Eastern Cape and Gauteng Province, providing an urban-rural representation of South Africa. The selection of the provinces was guided by selecting two provinces with the highest HIV prevalence in the country, KwaZulu-Natal and Mpumalanga, and one most urban province (Gauteng) and one rural province (Eastern Cape). Ethical approval for the study was obtained from the HSRC Research Ethics Committee. Participants signed informed consent forms. 2.2. Measures {#sec2.2} ------------- Age, gender, educational and employment status were assessed. *Contraceptive knowledge* was assessed with 7 items, for example, "Have you heard about the Pill that women can take every day to avoid becoming pregnant?" Response options were "Yes," or "No." Cronbach alpha for this contraception knowledge index was 0.70 in this sample. *HIV knowledge* was assessed with two items: (1) How many people living with HIV do you personally know? And (2) How many people have you personally known (in your lifetime) that have died from AIDS? Responses of the two questions were added up and coded as 0= 0, 1= knows any person living with HIV (PLHIV) or who has died from AIDS, and 2= knows any PLHIV and a person who has died from AIDS. Cronbach alpha for this 2-item HIV knowledge index was 0.73 in this sample. *"Has goals in life"* was assessed with 6 items such as "I have a plan for the future"; response options were agree or disagree. "Has goals in life" was classified as those who indicated to all 6 items to have any goals. Cronbach alpha for this "goals in life" index was 0.63 in this sample. *Partner risk reduction self-efficacy* was assessed with 4 items such as "Would you be able to avoid sex any time you did not want it?" Response options were no, probably no, probably yes, yes. Cronbach alpha for this partner risk reduction self-efficacy index was 0.73 in this sample. *Peer pressure* was assessed with 2 items; (1) "How much pressure you get from your friends to have sexual intercourse, would you say...?" (Response options ranged from 1: "No pressure at all" to 4: "A lot of pressure") and (2) "I feel pressure from friends to do things I do not want to." (Response options ranged from 1: "Very often" to 4: "Never"). Low peer pressure was coded 2, medium 3--5, and high 6--8. Cronbach alpha for this peer pressure index was 0.61 in this sample. *Poverty* was assessed with 6 items on the availability or nonavailability of shelter, fuel or electricity, clean water, medicines or medical treatment, food, and cash income in the past week. Response options ranged from 1: "Not one day" to 4: "Every day of the week" Poverty was defined as higher scores on non-availability of essential items. Cronbach alpha for this poverty index was 0.70 in this sample. *HIV communication* was assessed with 10 items, specifying 10 different source persons or institutions, for example, "Have you ever talked to your mother or female primary care giver/guardian about HIV/AIDS issues" Responses options were 1: Yes or 2: No. Cronbach alpha for this HIV communication index was 0.75 in this sample. Further, 1 item assessed "Did you talk about using a condom with your latest sexual partner in the last 12 months?" Responses options were 1: Yes or 2: No. *Accessibility of condoms* was assessed with the question, "How easy is it for you to get condoms if you needed or wanted them?" Response options ranged from 1: very easy to 4: very difficult. *Relationship control*was assessed with 4 items (for those never in a relationship, they were asked imagine to be) such as "Your partner has more control than you do in important decisions that affect your relationship." Response options ranged from 1: "Strongly disagree" to 4: "Strongly agree." Higher scores on lack of relationship power were defined as lack of relationship control. Cronbach alpha for this relationship control index was 0.81 in this sample. Risk behavior various questions were asked to assess risk behaviour. These included number of lifetime transactional sexual partners, early sexual debut (below 15 years, ever forced to have sex, concurrent sexual relationships, sex with someone who is much older, sexual intercourse frequency, and length of last relationship). *Alcohol use* was assessed with the Alcohol Use Disorder Identification Test (AUDIT-C) questionnaire \[[@B12]\], a measure of consumption of alcohol (i.e., the frequency of drinking, the quantity consumed at a typical occasion), and the frequency of heavy episodic drinking (i.e., consumption of five standard drinks, 60 gram alcohol, or more on a single occasion). Because AUDIT is reported to be less sensitive at identifying risk drinking in women \[[@B13]\], the cutoff points of binge drinking for women (4 units) were reduced by one unit as compared with men (5 units), as recommended by Bush et al. \[[@B14]\]. Using a cut off score of 5 or more hazardous or harmful drinking was defined \[[@B15], [@B16]\]. Cronbach alpha reliability coefficient was in this study for the AUDIT-C 0.91. *HIV status* was assessed by self-report. *Prevention programme exposure* was assessed with the following items. Exposure to ever loveLife face to face programmes was assessed with 24 items, for example, Gone to a loveLife clinic, Participated in a loveLife Community Dialogue, or Gone to a loveLife Youth Centre. LoveLife exposure to face-to-face programmes was summed up and coded as 0, 1-2, 3-4, or 5 or more programme exposures. Further, longer-term participation was assessed with having participated in loveLife programmes for at least one year. In addition, loveLife multi-media exposure was assessed with 9 items, for example, "Have you ever watched a loveLife television show?" "Contacted loveLife on facebook" "Heard a loveLife advert on radio." "Read UNCUT (loveLife) youth magazine." Response options were 1: Yes or 2: No. The 9 multi-media programmes were summed up and coded as 1: 0-1 media exposures, 2: 2--4, and 3: 5--9 media exposures. 2.3. Data Analysis {#sec2.3} ------------------ Both descriptive and inferential statistics were applied in data analysis. Sample weights were calculated after editing the data, and STATA software was used for the analysis. STATA software (svy methods) was used to obtain the estimates of key indicators, significance values (*P* values), and confidence intervals (95% CI) that take into account the complex design and individual sample weights. Computed estimates and odds ratios are reported with 95% confidence intervals and a two-side *P* value of 0.05 used as the cut-off point for statistical significance. Data were checked for normality distribution and outliers. For nonnormal distribution nonparametric tests were used. The Chi-square test was used for comparing nominal variables. Logistic regression analysis was used to study the association between key outcomes (high self-efficacy) and predictor variables. All variables statistically significant at the *P* \< .05 level in bivariate analyses were included in the multivariable models. 3. Results {#sec3} ========== 3.1. Survey Response Rate {#sec3.1} ------------------------- A total of 5 768 households were sampled and approached for the interview. Only 94.8% households were valid and among the valid households 93.6% agreed to be interviewed. Only households that indicated that they had a person aged 18 to 24 years were eligible for an individual questionnaire administration. Of the eligible and valid households 47.2% were eligible for an individual interview, 1.3% refused the individual interview, and 2.3% individuals were absent from the household so that the individual interview response rate was 96.4%. 3.2. Sample Characteristics {#sec3.2} --------------------------- The total samples of young people in the study were 3123, aged 18--24, from four of nine provinces (Eastern Cape, Gauteng, KwaZulu-Natal, and Mpumalanga) in South Africa. From the total sample 1127 and 1007, men and women, respectively, reported to have ever had sex. Overall, among those who had been tested for HIV and indicated their test result 9.3% of the women and 6.2% of the men were HIV positive. Youth programme exposure was assessed in terms of face-to-face participation and media programme exposure, 32.6% had participated in one or more loveLife face-to-face programmes, while more than 80% had been exposed to 2 or more loveLife HIV prevention media programmes (see [Table 1](#tab1){ref-type="table"}). 3.3. Self-Efficacy and Condom Use Consistency in South African Females and Males {#sec3.3} -------------------------------------------------------------------------------- Among males who indicated that they were not able to avoid sex any time they did not want it, 56.5% use condoms consistently. Males who indicated that they are able to avoid sex any time they did not want it, 58.9% use condoms consistently. These findings were statistical significant. For women though, no statistical significance was reported when asked "would you be able to avoid sex any time you did not want it." When asked "would you be able to talk about using condoms with your partner?", of those women who answered no, only 26% are using condoms consistently (versus 41.8% of males) (see [Table 2](#tab2){ref-type="table"}). Generally, across the self-efficacy scale items, more males than females were more likely to use condoms consistently. 3.4. Predictors of Self-Efficacy {#sec3.4} -------------------------------- Univariate analysis found that for males, having been tested for HIV, concurrent sexual partnerships, transactional sex partner, low HIV risk perception, Talked to partner about condom use in the past 12 months, difficulty in getting condoms, acceptable to have coerced sex, high relational control, current tobacco, ever drugs, and participating in few face-face loveLife programmes (1-2) to be associated with high self-esteem. In the case of females, ever forced to have sex, concurrent sexual partnerships, transactional sex partner in lifetime, low HIV risk perception, talked to partner about condom use in the past 12 months, high peer pressure, stigma, ever drugs and having life goals were associated with high self-efficacy in univariate analysis. Multivariate analysis found the following variables to be significantly associated with high self-efficacy for males: having been tested for HIV, concurrent sexual partners, transactional sex partners in life time, low HIV risk perception, difficulty of getting condoms, acceptable to have coerced sex, high relationship control, and participating in 1-2 loveLife face-face programmes. With regards to females, low HIV risk perception, HIV/AIDS stigma, ever drugs and having life goals were associated with high self-efficacy in multivariate analysis (see [Table 3](#tab3){ref-type="table"}). 4. Discussion {#sec4} ============= The findings indicate that, for males, high self-efficacy is significantly associated with knowing your HIV status but only half of the men reported ever testing for HIV. In the case of females, the present study found a significant association between high self-efficacy and having life goals. This finding is similar with a study done among South African youth 15 to 24 years that also found high self-efficacy to be associated with having life goals but in this study it was reported for both young men and women \[[@B6]\]. A significant association was also found for young males finding it acceptable to have coerced sex. This probably reflects the higher percentage reported by females than their male counterparts (7.3% versus 1.1%) ever being forced to have sex against their will. This reflects some sense of disempowerment of young females in preventing themselves against HIV infection \[[@B17]\]. As a result it perpetuates social and cultural forces that shape young people\'s sexual behaviour \[[@B18]\]. Indeed, Varga \[[@B19]\] found that young men seemed to have more freedom to be involved with more than one sexual partner at a time while young women are expected to be faithful to their (one) partner. Thus Varga in her study found that a young man involved with multiple sexual partners was regarded as "*isoka*" (the act of "*ubusoka*") to indicate the young man\'s popularity with women something that seemed to be desirable while a young woman with more than one sexual partner was perceived as lacking self-respect \[[@B19]\]. Jewkes et al. \[[@B17]\] would have argued that HIV prevention programmes which emphasize male condoms and male circumcision may further perpetrate young men\'s freedom in sexual decision making, thus overlooking the vulnerability of young women. This brings into question the effectiveness of existing risk reduction programmes that specifically focus on risk reduction through behavioural interventions for adolescents. Ross \[[@B20]\] paints a rather bleak picture of the efficacy of behavioural intervention. In an update of a recent systematic review of HIV prevention interventions focusing on trials that have included HIV as an outcome, Ross \[[@B20]\] noted that the behavioural interventions conducted through trials were disappointing in terms of the impact on HIV even though some trials showed an impact on other sexual and reproductive health outcomes. The interventions tested were inherently ineffective partly because they were inadequately implemented, or there were problems with the measurement of effectiveness. Perhaps, particular attention needs to be given to the social and sexual norms of the general population among whom the particular target group in this case, young people, live and interact \[[@B20]\]. The present study found that, for males, there was a significant association between high self-efficacy and having concurrent sexual partners as well as having had a transactional sex partner in lifetime. Of grave concern is that high self-efficacy was also significantly associated with those males having difficulty in getting condoms. This is an indication that, even though males reported higher consistent condom use than females, many young men still engage in unprotected sex thus putting themselves and their partners at risk. Varga \[[@B21]\] noted that a predominant theme has been the powerful positive symbolism attached to unprotected sex and negative connotations of condom use. Many women feel stigmatized when insisting on using a condom during sexual intercourse as this is a sign of distrust in their partner \[[@B6]\] or an indication that they are HIV positive. A review done by Marston and King \[[@B18]\] on identifying key themes from various qualitative findings highlighted one theme that specifically relates to this aspect; "condoms are stigmatizing and associated with lack of trust" \[[@B18]\]. Despite an increase in awareness of HIV status in the general population between 15 and 49 year old doubling from 11.9% in 2005 to 24.7% in 2008 \[[@B11]\], unprotected sex with intimate personal partners is nearly always chosen over condom use \[[@B21]\]. A partial explanation for the sexual dynamics perhaps lies in a sociocultural context which has not fostered sexual negotiation skills. A renewed focus on programmes related to self-efficacy programmes in young people is urgently needed. Given the significant finding that amongst males, high self-efficacy is associated with exposure to at least 1 to 2 face-to-face loveLife programmes, evidence do exist that similar intervention strategies focusing on self-efficacy has a positive effect. The results further confirm loveLife\'s emphasis on normative and social factors as being one of the issues that young people have to contend with in their constructions of sexuality, and this seems to be particularly true of young women \[[@B22], [@B23]\]. For instance, more young women report having had sex with a much older partner. This is consistent with the findings from findings in the general population survey \[[@B11]\] where young people reported having partners five or more years older than themselves, and there was a substantive increase from 9.6% in 2005 to 14.5% in 2008. This makes them more vulnerable to HIV infection because they find it difficult to negotiate sex with their older partner \[[@B24]\]. If indeed young women\'s sexual partnerships with older men are solely aimed at economical gain, initiatives aimed at young women\'s economic emancipation need further support. 5. Limitations {#sec5} ============== One of the limitations of that study was that it was a cross-sectional study. Furthermore, it was only done in four provinces; thus, the results cannot be generalized. This paper was a collaborative project between loveLife and the Human Sciences Research Council and was made possible by the Henry J. Kaiser Family Foundation. The authors would like to acknowledge loveLife and the anonymous review committee for their comments and suggestions. They would also like to thank the research participants who took part in the study. ###### Individual, social, structural, contraception, and prevention programme exposure of study sample by gender. ---------------------------------------------------------------------------------------------------------------------------------------------------------------- Male (*N* = 1127) Female (*N* = 1007) -------------------------------------------------------------------------------------------------------------------- --------------------- --------------------- *Sociodemographics* *N* (%) or *M* (SD) *N* (%) or *M* (SD) Poverty index (range 6--24) 8.3 (3.0) 7.9 (2.6) ≤Grade 10 218 (14.7) 171 (18.0) Grade 11 233 (17.3) 201 (18.5) Grade 12 or more 670 (68.0) 632 (63.5) Student 459 (42.3) 361 (44.0) Employed 217 (23.0) 114 (11.1) Unemployed 370 (34.8) 440 (45.0) Knowledge Contraceptive knowledge (range 0--7) 4.7 (1.6) 5.1 (1.5) Knows person living with HIV and/or died from AIDS 0 393 (40.0) 272 (36.3) 1 = knows PLHIV or died from AIDS 252 (23.5) 215 (18.2) 2 = knows PLHIV and died from AIDS 475 (36.6) 514 (45.5) Ever HIV test 556 (48.9) 779 (80.4) *Prior sexual experiences* Early sex (\<15) 145 (17.8) 65 (6.9) Ever forced sex 14 (1.1) 58 (7.3) Sex with much older partner 70 (4.3) 232 (19.3) Concurrent sexual partners 126 (6.8) 53 (3.1) Transactional sex partner in lifetime 103 (7.8) 50 (5.0) Outcome expectancies Low HIV risk perception 294 (23.6) 354 (30.2) Believes that condom use is a sign of not trusting the partner 259 (15.9) 229 (17.5) *Sociostructural factors* Peer pressure Low 350 (41.0) 465 (54.6) Medium 447 (42.3) 398 (37.5) High 206 (16.6) 121 (7.9) Talked with partner about condoms in past 12 months 820 (94.1) 769 (90.7) HIV communication (range 0--10) 5.6 (2.5) 5.6 (2.2) HIV/AIDS stigma 118 (5.5) 75 (3.8) Difficulty of getting condoms (range 1--4) 1.22 (0.6) 1.13 (0.5) Lack of relationship control (range 4--16) 8.5 (2.3) 8.3 (2.5) Agreed with statement "It is acceptable to have sex with my sex partner even though my partner does not want to." 60 (4.0) 43 (2.9) Hazardous or harmful alcohol use 430 (34.0) 160 (16.4) Ever drug use 162 (25.4) 32 (2.6) Current tobacco use 363 (25.4) 69 (5.6) Has goals in life 728 (72.4) 628 (63.6) *HIV status* Diagnosed HIV positive\ 35 (6.7)\ 64 (9.5)\ Diagnosed HIV negative 493 (93.3) 654 (90.5) HIV risk reduction self-efficacy 462 (52.7) 488 (56.6) *Programme exposure* One year or more loveLife participation 315 (22.4) 250 (21.5) loveLife face-to-face participation 0 700 (70.4) 639 (62.6) 2--4 219 (12.2) 193 (22.1) 5--9 100 (8.0) 71 (4.3) 5 or more 98 (9.3) 101 (11.0) loveLife multimedia programme exposure 0-1 174 (18.1) 180 (20.7) 2--4 577 (50.8) 458 (48.2) 5--9 340 (31.1) 331 (31.0) ---------------------------------------------------------------------------------------------------------------------------------------------------------------- ###### Self-efficacy scale responses and condom use consistency. -------------------------------------------------------------------------------------------------------------------------------------- Men Women -------------------------------------------------------------------------------- ----- ------- ------- ------- ------- ------- ------- Would you be able to avoid sex any time you did not want it? No\ 43.5\ 56.5\ 0.000 57.9\ 42.1\ 0.352 Yes 41.1 58.9 54.4 45.6 Would you be able to use a condom every time you have sexual intercourse? No\ 46.1\ 53.9\ 0.000 66.8\ 33.2\ 0.000 Yes 40.4 59.6 51.4 48.6 Would you be able to refuse to have sex if your partner will not use a condom? No\ 47.3\ 52.7\ 0.000 65.7\ 34.3\ 0.000 Yes 38.4 61.6 49.8 50.2 Would you be able to talk about using condoms with your partner? No\ 58.2\ 41.8\ 0.000 74.0\ 26.0\ 0.000 Yes 32.2 67.8 44.0 56.0 -------------------------------------------------------------------------------------------------------------------------------------- ^∗^Scale items response options were No, Probably No, Probably Yes, Yes; "No" was coded "No, Probably No, Probably Yes." ###### Association between individual, social and structural variables, loveLife exposure, and high self-efficacy. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Men Women --------------------------------------------------------------------------------------------------------------------- ------------------------ ------------------------ ------------------------ ----------------------- *Sociodemographics* Education 1.02 (0.84--1.24) 1.07 (0.78--1.46) 1.27 (0.99--1.62) 1.16 (0.82--1.64) Student 1.00 1.00 1.00 1.00 Employed 2.35 (0.79--6.96) 2.03 (0.62--6.01) 0.75 (0.35--1.58) 0.66 (0.22--1.95) Unemployed 0.72 (0.49--1.07) 0.67 (0.33--1.37) 1.13 (0.59--2.18) 1.33 (0.60--2.94) Poverty index 1.04 (0.95--1.13) 1.11 (0.99--1.25) 1.06 (0.86--1.31) 1.15 (0.91--1.45) Knowledge Knows person living with\ HIV and/or died from AIDS 0 1.00 --- 1.00 --- 1 = knows PLHIV or died from AIDS 0.41 (0.12--1.40) 0.97 (0.54--1.77) 2 = knows PLHIV and died from AIDS 0.72 (0.45--1.15) 0.72 (0.37--1.39) Have been tested for HIV ever? 2.38 (1.41--4.02)^∗∗∗^ 2.54 (1.38--4.67)^∗∗^ 0.88 (0.55--1.40) --- Contraceptive knowledge 1.11 (0.75--1.65) --- 1.01 (0.90--1.13) --- *Prior sexual experiences* Early sex (\<15) 2.72 (0.72--10.2) --- 0.94 (0.36--2.44) --- Ever forced sex 0.62 (0.10--3.91) --- 0.31 (0.11--0.86)^∗^ 0.27 (0.03--2.03) Concurrent sexual partners 0.23 (0.12--0.47)^∗∗∗^ 0.28 (0.12--0.65)^∗∗^ 0.30 (0.11--0.84)^∗^ 0.49 (0.16--1.51) Sex with much older partner 0.34 (0.11--1.12) --- 0.72 (0.45--1.17) --- Transactional sex partner in lifetime 0.10 (0.05--0.20)^∗∗∗^ 0.10 (0.03--0.29)^∗∗∗^ 0.09 (0.03--0.27)^∗∗∗^ 0.70 (0.17--2.83) Outcome expectancies Low HIV risk perception 1.41 (1.13--1.76)^∗∗^ 1.45 (1.04--2.02)^∗^ 1.85 (1.47--2.34)^∗∗∗^ 2.26 (1.25--4.09)^∗∗^ Beliefs condom use is a sign of not trusting\ 0.79 (0.42--1.46) --- 0.66 (0.36--1.20) --- the partner *Sociostructural factors* Talked to partner about condom use in the past 12 months 2.50 (1.25--5.00)^∗∗^ 1.21 (0.46--2.75) 2.93 (1.55--5.53)^∗∗∗^ HIV communication 1.07 (0.89--1.30) --- 0.97 (0.83--1.14) --- Peer pressure Low 1.00 --- 1.00 1.00 Medium 0.35 (0.14--0.87) 0.68 (0.40--1.15) 0.93 (0.51--1.67) High 0.23 (0.05--1.13) 0.28 (0.14--0.55)^∗∗∗^ 0.89 (0.33--2.30) Difficulty of getting condoms 0.60 (0.37--0.97)^∗^ 0.60 (0.38--0.98)^∗^ 0.81 (0.63--1.03) --- Agreed with statement "It is acceptable to have sex with my sex partner even though my partner does not want to."\ 0.23 (0.08--0.67)^∗∗^ 0.26 (0.07--0.98)^∗^ 0.76 (0.23--2.49) --- (Acceptable to have coerced sex) HIV/AIDS stigma 0.68 (0.25--1.82) --- 0.24 (0.11--0.50)^∗∗∗^ 0.20 (0.06--0.64)^∗∗^ High relationship control 0.46 (0.27--0.79)^∗∗^ 0.24 (0.10--0.58)^∗∗^ 1.10 (0.72--1.69) --- Hazardous or harmful drinking (AUDIT \> 4) 0.70 (0.44--1.14) --- 0.70 (0.21--2.33) --- Current tobacco use 0.48 (0.27--0.87)^∗^ 0.58 (0.26--1.28) 0.60 (0.13--2.64) --- Ever drugs 0.35 (0.18--0.66)^∗∗∗^ 0.45 (0.17--1.31) 0.10 (0.03--0.39)^∗∗∗^ 0.12 (0.02--0.74)^∗^ *Goals* Has life goals 2.78 (0.98--7.86) --- 1.85 (1.16--2.96)^∗^ 2.98 (1.16--7.63)^∗^ *Programme exposure* One year or more loveLife participation 0.85 (0.43--1.71) --- 1.15 (0.63--2.08) --- loveLife face-to-face participation 0 1.00 1.00 1.00 --- 1-2 0.42 (0.22--0.81)^∗∗^ 0.38 (0.16--0.89)^∗^ 0.48 (0.14--1.72) 3-4 0.87 (0.30--2.47) 0.50 (0.22--1.13) 0.62 (0.22--1.74) 5 or more 1.70 (0.47--6.20) 2.11 (0.46--9.59) 0.81 (0.28--2.33) loveLife multimedia programme exposure 0-1 1.00 --- 1.00 --- 2--4 0.88 (0.40--1.90) 0.63 (0.34--1.17) 5--9 0.70 (0.37--1.34) 1.33 (0.54--3.25) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ^∗∗∗^ *P* \< .001; ^∗∗^ *P* \< .01; ^∗^ *P* \< .05. [^1]: Academic Editors: K. Ikuta and J. Rappaport

{ "pile_set_name": "PubMed Central" }

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"disappoint", "avail", "practic", "easi", "lovelif", "henri", "relationship", "prevent", "disord", "rang", "see", "older", "latest", "thing", "say", "futur", "sign", "classifi", "past", "shown", "reliabl", "sec", "addit", "riski", "total", "high", "activ", "statement", "limit", "note", "concern", "urgent", "approv", "radio", "perhap", "examin", "avoid", "talk", "subsaharan", "statist", "took", "typic", "sampl", "structur", "femal", "ikuta", "identif", "case", "percentag", "grade", "council", "concurr", "predomin", "studi", "belief", "epidem", "sociocultur", "three", "adult", "advert", "unplan", "jewk", "social", "rappaport", "earli", "research", "true", "find", "valid", "sociostructur", "pill", "clinic", "crucial", "pregnant", "conduct", "explan", "debut", "condom", "part", "affect", "method", "educ", "primari", "form", "someon", "often", "heavi", "match", "procedur", "chisquar", "never", "approach", "variou", "predictor", "burden", "feel", "ask", "b", "effect", "pictur", "auditc", 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"urban", "comment", "woman", "gram", "therefor", "rather", "strategi" ]

22,194

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"PLHIV", "died", "AIDS", "knows", "PLHIV", "person", "died", "AIDS", "Cronbach", "alpha", "HIV", "knowledge", "index", "sample", "goals", "life", "assessed", "items", "plan", "future", "response", "options", "agree", "disagree", "goals", "life", "classified", "indicated", "items", "goals", "Cronbach", "alpha", "goals", "life", "index", "sample", "Partner", "risk", "reduction", "assessed", "items", "Would", "able", "avoid", "sex", "time", "want", "Response", "options", "probably", "probably", "yes", "yes", "Cronbach", "alpha", "partner", "risk", "reduction", "index", "sample", "Peer", "pressure", "assessed", "items", "much", "pressure", "get", "friends", "sexual", "intercourse", "would", "say", "Response", "options", "ranged", "pressure", "lot", "pressure", "feel", "pressure", "friends", "things", "want", "Response", "options", "ranged", "often", "Never", "Low", "peer", "pressure", "coded", "medium", "high", "Cronbach", "alpha", "peer", "pressure", "index", "sample", "Poverty", "assessed", "items", "availability", "nonavailability", "shelter", "fuel", "electricity", "clean", "water", "medicines", "medical", "treatment", "food", "cash", "income", "past", "week", "Response", "options", "ranged", "one", "day", "Every", "day", "week", "Poverty", "defined", "higher", "scores", "essential", "items", "Cronbach", "alpha", "poverty", "index", "sample", "HIV", "communication", "assessed", "items", "specifying", "different", "source", "persons", "institutions", "example", "ever", "talked", "mother", "female", "primary", "care", "issues", "Responses", "options", "Yes", "Cronbach", "alpha", "HIV", "communication", "index", "sample", "item", "assessed", "talk", "using", "condom", "latest", "sexual", "partner", "last", "months", "Responses", "options", "Yes", "Accessibility", "condoms", "assessed", "question", "easy", "get", "condoms", "needed", "wanted", "Response", "options", "ranged", "easy", "difficult", "Relationship", "control", "assessed", "items", "never", "relationship", "asked", "imagine", "partner", "control", "important", "decisions", "affect", "relationship", "Response", "options", "ranged", "Strongly", "disagree", "Strongly", "agree", "Higher", "scores", "lack", "relationship", "power", "defined", "lack", "relationship", "control", "Cronbach", "alpha", "relationship", "control", "index", "sample", "Risk", "behavior", "various", "questions", "asked", "assess", "risk", "behaviour", "included", "number", "lifetime", "transactional", "sexual", "partners", "early", "sexual", "debut", "years", "ever", "forced", "sex", "concurrent", "sexual", "relationships", "sex", "someone", "much", "older", "sexual", "intercourse", "frequency", "length", "last", "relationship", "Alcohol", "use", "assessed", "Alcohol", "Use", "Disorder", "Identification", "Test", "questionnaire", "measure", "consumption", "alcohol", "frequency", "drinking", "quantity", "consumed", "typical", "occasion", "frequency", "heavy", "episodic", "drinking", "consumption", "five", "standard", "drinks", "gram", "alcohol", "single", "occasion", "AUDIT", "reported", "less", "sensitive", "identifying", "risk", "drinking", "women", "cutoff", "points", "binge", "drinking", "women", "units", "reduced", "one", "unit", "compared", "men", "units", "recommended", "Bush", "et", "al", "Using", "cut", "score", "hazardous", "harmful", "drinking", "defined", "Cronbach", "alpha", "reliability", "coefficient", "study", "HIV", "status", "assessed", "Prevention", "programme", "exposure", "assessed", "following", "items", "Exposure", "ever", "loveLife", "face", "face", "programmes", "assessed", "items", "example", "Gone", "loveLife", "clinic", "Participated", "loveLife", "Community", "Dialogue", "Gone", "loveLife", "Youth", "Centre", "LoveLife", "exposure", "programmes", "summed", "coded", "programme", "exposures", "participation", "assessed", "participated", "loveLife", "programmes", "least", "one", "year", "addition", "loveLife", "exposure", "assessed", "items", "example", "ever", "watched", "loveLife", "television", "show", "Contacted", "loveLife", "facebook", "Heard", "loveLife", "advert", "radio", "Read", "UNCUT", "loveLife", "youth", "magazine", "Response", "options", "Yes", "programmes", "summed", "coded", "media", "exposures", "media", "exposures", "Data", "Analysis", "descriptive", "inferential", "statistics", "applied", "data", "analysis", "Sample", "weights", "calculated", "editing", "data", "STATA", "software", "used", "analysis", "STATA", "software", "svy", "methods", "used", "obtain", "estimates", "key", "indicators", "significance", "values", "P", "values", "confidence", "intervals", "CI", "take", "account", "complex", "design", "individual", "sample", "weights", "Computed", "estimates", "odds", "ratios", "reported", "confidence", "intervals", "P", "value", "used", "point", "statistical", "significance", "Data", "checked", "normality", "distribution", "outliers", "nonnormal", "distribution", "nonparametric", "tests", "used", "test", "used", "comparing", "nominal", "variables", "Logistic", "regression", "analysis", "used", "study", "association", "key", "outcomes", "high", "predictor", "variables", "variables", "statistically", "significant", "P", "level", "bivariate", "analyses", "included", "multivariable", "models", "Results", "Survey", "Response", "Rate", "total", "households", "sampled", "approached", "interview", "households", "valid", "among", "valid", "households", "agreed", "interviewed", "households", "indicated", "person", "aged", "years", "eligible", "individual", "questionnaire", "administration", "eligible", "valid", "households", "eligible", "individual", "interview", "refused", "individual", "interview", "individuals", "absent", "household", "individual", "interview", "response", "rate", "Sample", "Characteristics", "total", "samples", "young", "people", "study", "aged", "four", "nine", "provinces", "Eastern", "Cape", "Gauteng", "Mpumalanga", "South", "Africa", "total", "sample", "men", "women", "respectively", "reported", "ever", "sex", "Overall", "among", "tested", "HIV", "indicated", "test", "result", "women", "men", "HIV", "positive", "Youth", "programme", "exposure", "assessed", "terms", "participation", "media", "programme", "exposure", "participated", "one", "loveLife", "programmes", "exposed", "loveLife", "HIV", "prevention", "media", "programmes", "see", "Table", "table", "Condom", "Use", "Consistency", "South", "African", "Females", "Males", "Among", "males", "indicated", "able", "avoid", "sex", "time", "want", "use", "condoms", "consistently", "Males", "indicated", "able", "avoid", "sex", "time", "want", "use", "condoms", "consistently", "findings", "statistical", "significant", "women", "though", "statistical", "significance", "reported", "asked", "would", "able", "avoid", "sex", "time", "want", "asked", "would", "able", "talk", "using", "condoms", "partner", "women", "answered", "using", "condoms", "consistently", "versus", "males", "see", "Table", "table", "Generally", "across", "scale", "items", "males", "females", "likely", "use", "condoms", "consistently", "Predictors", "Univariate", "analysis", "found", "males", "tested", "HIV", "concurrent", "sexual", "partnerships", "transactional", "sex", "partner", "low", "HIV", "risk", "perception", "Talked", "partner", "condom", "use", "past", "months", "difficulty", "getting", "condoms", "acceptable", "coerced", "sex", "high", "relational", "control", "current", "tobacco", "ever", "drugs", "participating", "loveLife", "programmes", "associated", "high", "case", "females", "ever", "forced", "sex", "concurrent", "sexual", "partnerships", "transactional", "sex", "partner", "lifetime", "low", "HIV", "risk", "perception", "talked", "partner", "condom", "use", "past", "months", "high", "peer", "pressure", "stigma", "ever", "drugs", "life", "goals", "associated", "high", "univariate", "analysis", "Multivariate", "analysis", "found", "following", "variables", "significantly", "associated", "high", "males", "tested", "HIV", "concurrent", "sexual", "partners", "transactional", "sex", "partners", "life", "time", "low", "HIV", "risk", "perception", "difficulty", "getting", "condoms", "acceptable", "coerced", "sex", "high", "relationship", "control", "participating", "loveLife", "programmes", "regards", "females", "low", "HIV", "risk", "perception", "stigma", "ever", "drugs", "life", "goals", "associated", "high", "multivariate", "analysis", "see", "Table", "table", "Discussion", "findings", "indicate", "males", "high", "significantly", "associated", "knowing", "HIV", "status", "half", "men", "reported", "ever", "testing", "HIV", "case", "females", "present", "study", "found", "significant", "association", "high", "life", "goals", "finding", "similar", "study", "done", "among", "South", "African", "youth", "years", "also", "found", "high", "associated", "life", "goals", "study", "reported", "young", "men", "women", "significant", "association", "also", "found", "young", "males", "finding", "acceptable", "coerced", "sex", "probably", "reflects", "higher", "percentage", "reported", "females", "male", "counterparts", "versus", "ever", "forced", "sex", "reflects", "sense", "disempowerment", "young", "females", "preventing", "HIV", "infection", "result", "perpetuates", "social", "cultural", "forces", "shape", "young", "sexual", "behaviour", "Indeed", "Varga", "found", "young", "men", "seemed", "freedom", "involved", "one", "sexual", "partner", "time", "young", "women", "expected", "faithful", "one", "partner", "Thus", "Varga", "study", "found", "young", "man", "involved", "multiple", "sexual", "partners", "regarded", "isoka", "act", "ubusoka", "indicate", "young", "popularity", "women", "something", "seemed", "desirable", "young", "woman", "one", "sexual", "partner", "perceived", "lacking", "Jewkes", "et", "al", "would", "argued", "HIV", "prevention", "programmes", "emphasize", "male", "condoms", "male", "circumcision", "may", "perpetrate", "young", "freedom", "sexual", "decision", "making", "thus", "overlooking", "vulnerability", "young", "women", "brings", "question", "effectiveness", "existing", "risk", "reduction", "programmes", "specifically", "focus", "risk", "reduction", "behavioural", "interventions", "adolescents", "Ross", "paints", "rather", "bleak", "picture", "efficacy", "behavioural", "intervention", "update", "recent", "systematic", "review", "HIV", "prevention", "interventions", "focusing", "trials", "included", "HIV", "outcome", "Ross", "noted", "behavioural", "interventions", "conducted", "trials", "disappointing", "terms", "impact", "HIV", "even", "though", "trials", "showed", "impact", "sexual", "reproductive", "health", "outcomes", "interventions", "tested", "inherently", "ineffective", "partly", "inadequately", "implemented", "problems", "measurement", "effectiveness", "Perhaps", "particular", "attention", "needs", "given", "social", "sexual", "norms", "general", "population", "among", "particular", "target", "group", "case", "young", "people", "live", "interact", "present", "study", "found", "males", "significant", "association", "high", "concurrent", "sexual", "partners", "well", "transactional", "sex", "partner", "lifetime", "grave", "concern", "high", "also", "significantly", "associated", "males", "difficulty", "getting", "condoms", "indication", "even", "though", "males", "reported", "higher", "consistent", "condom", "use", "females", "many", "young", "men", "still", "engage", "unprotected", "sex", "thus", "putting", "partners", "risk", "Varga", "noted", "predominant", "theme", "powerful", "positive", "symbolism", "attached", "unprotected", "sex", "negative", "connotations", "condom", "use", "Many", "women", "feel", "stigmatized", "insisting", "using", "condom", "sexual", "intercourse", "sign", "distrust", "partner", "indication", "HIV", "positive", "review", "done", "Marston", "King", "identifying", "key", "themes", "various", "qualitative", "findings", "highlighted", "one", "theme", 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Introduction {#s1} ============ The nuclear envelope (NE) delineates the boundary of the nucleus in eukaryotic cells and is composed of two membranes enclosing a luminal space. Because chromosomes are located in the nucleus, cells must enable the access of spindle microtubules to the nucleoplasm in order to segregate the daughter genomes during mitosis. Cells undergoing open mitosis achieve this by breaking down the NE in prophase and reassembling it again in telophase. Alternatively, a number of species segregate chromosomes within the nucleus in the process known as closed mitosis. Like most fungi, the fission yeast Schizosaccharomyces pombe elaborates the bipolar intranuclear mitotic spindle with the minus ends of microtubules anchored at specialized microtubule-organizing organelles, the spindle pole bodies (SPBs). The SPBs are functionally analogous to metazoan centrosomes and undergo a duplication and separation cycle generally correlated with the cell division cycle. The SPB of S. pombe spends most of interphase in the cytoplasm, adjacent to the NE \[[@pbio-0050170-b001],[@pbio-0050170-b002]\]. However, it appears to be securely anchored at the outer NE as suggested by live-imaging studies of the SPB and the NE behavior in interphase \[[@pbio-0050170-b003],[@pbio-0050170-b004]\]. As the SPB duplicates and matures, daughter SPBs remain connected by a bridge. Upon mitotic entry, a portion of the NE underlying the SPB pair invaginates, forming an opening (fenestra), and the SPBs settle into it. Each SPB initiates intranuclear microtubules, and they eventually separate and move to opposite sides of the nucleus while enclosed in their individual fenestrae that are somewhat larger than the SPBs themselves. In anaphase, the fenestrae contract to become about the same size as the SPBs. Late in mitosis, the fenestrae close, and the SPBs are extruded back into the cytoplasm \[[@pbio-0050170-b001]\]. These observations may indicate the existence of specialized and cell cycle--specific structures that function to anchor the SPB at the NE. Interestingly, the NE adjacent to the SPB itself shows some degree of specialization, including local thickening of the NE and presence of electron-dense material between the SPB and the NE \[[@pbio-0050170-b001],[@pbio-0050170-b005]\]. Also, γ-tubulin material localizes to the dark-staining region underlying the SPB in interphase \[[@pbio-0050170-b001]\]. Thus, it seems that there is a complex of molecular associations that defines a structure, including both the SPB and an adjacent portion of the NE. At least one membrane-spanning protein, Cut11p, is known to participate in SPB anchorage during early mitosis \[[@pbio-0050170-b006]\]. An interesting corollary of closed mitosis is the requirement for regulated NE division in order to prevent chromosome entanglement during spindle elongation and to ensure proper NE and endoplasmic reticulum (ER) inheritance. The elongating spindle is a stiff structure that is capable of exerting considerable force on the NE. For instance, assembly of microtubule polymers within liposomes induces striking shape changes, indicating that microtubules are capable of deforming the cellular membranes \[[@pbio-0050170-b007],[@pbio-0050170-b008]\]. The NE of S. pombe is a double-layered protein-containing structure that is likely more rigid than artificial phospholipid membranes. However, the NE is still capable of undergoing severe deformations due to forces exerted by microtubules, as suggested by three-dimensional reconstructions of monopolar spindles in *cut11* ^ts^ mutant cells \[[@pbio-0050170-b006]\] and live NE imaging during the process of spindle assembly (unpublished data) or in interphase \[[@pbio-0050170-b003]\]. Notably, the NE behaves in a radically different manner during mitosis, when it follows an ordered progression of shape changes resulting in accurate division. The NE remains relatively spherical throughout metaphase and anaphase A. As cells proceed to anaphase B, the NE first elongates to become an ellipse with spindle minus ends located at its poles. It eventually assumes a dumbbell shape, after which it undergoes fission resulting in the formation of two daughter nuclei. Unlike microtubules polymerizing within liposomes \[[@pbio-0050170-b007],[@pbio-0050170-b008]\], the elongating mitotic spindle contacts the membrane at specific sites, including the SPBs and, likely, the underlying specialized section of the NE. It is thus possible that the presence of these rigid structures could allow the NE to sustain the pushing forces produced by the elongating spindle. This way, the SPB could be considered not only a microtubule-organizing center, but also a supporting structural feature allowing NE division during closed mitosis. Here, we describe experimental evidence supporting this hypothesis. While investigating mitotic functions of the transforming acid coiled coil (TACC)-related protein, Mia1p, in fission yeast, we found that cells overexpressing Mia1p failed to separate the SPBs, but frequently assembled elongating bipolar spindles. Under these circumstances, spindle poles deformed the NE, and NE division failed. Chromosome segregation was often unsuccessful, and DNA masses were confined within the undivided NE. Laser microsurgery in wild-type cells together with experiments on *dis1*Δ cells that exhibit faulty microtubule/kinetochore attachment, but normal spindle elongation, confirmed that the pole-located SPBs were required for NE division. Strikingly, genetic ablation of the NE during mitosis in Mia1p-overexpressing cells allowed sister chromatid segregation by acentrosomal spindles. Results {#s2} ======= Cells Overexpressing Mia1p Arrest in Mitosis, with Fully Elongated Spindles and Nonsegregated Chromosomes {#s2a} --------------------------------------------------------------------------------------------------------- Previous experiments suggested that the fission yeast TACC-related protein, Mia1p, is required for organizing and anchoring microtubule minus ends in interphase and for proper spindle function during mitosis \[[@pbio-0050170-b004],[@pbio-0050170-b009],[@pbio-0050170-b010]\]. To further our understanding of Mia1p function, we created S. pombe cells carrying a pREP1-based plasmid in which the *mia1*+ open reading frame was expressed under the control of the inducible *nmt1* promoter \[[@pbio-0050170-b011]\]. We found that overexpression of Mia1p in wild-type cells resulted in severe lethality ([Figure S1](#pbio-0050170-sg001){ref-type="supplementary-material"}, see also \[[@pbio-0050170-b010]\]). Overexpression of Mia1p in strains carrying various green fluorescent protein (GFP)-tagged cell cycle protein markers revealed that cells arrested during late mitosis. First, the centromere I marker, Cen1-GFP \[[@pbio-0050170-b012]\], was often found in two distinct and well-separated dots, indicating that anaphase promoting complex (APC) activation occurred and chromosome cohesion was lost ([Figure 1](#pbio-0050170-g001){ref-type="fig"}A and [1](#pbio-0050170-g001){ref-type="fig"}B). We confirmed this by examining the localization of the general centromere/kinetochore marker, Mis6p-GFP \[[@pbio-0050170-b013]\], upon Mia1p overexpression. Mis6p-GFP labeled either two distinct kinetochore clusters, similar to wild-type control, or localized to six closely positioned dots, likely representing nonsegregated anaphase kinetochores ([Figure S2](#pbio-0050170-sg002){ref-type="supplementary-material"}). We also found that the Cdc14p-like phosphatase, Clp1p \[[@pbio-0050170-b014],[@pbio-0050170-b015]\], localized to intranuclear spindles in the majority of cells, consistent with their mitotic state ([Figure 1](#pbio-0050170-g001){ref-type="fig"}A and [1](#pbio-0050170-g001){ref-type="fig"}B). Moreover, most Mia1p-overexpressing cells exhibited actomyosin division rings, as judged by localization of the myosin II light chain, Rlc1p-GFP \[[@pbio-0050170-b016]\] ([Figure 1](#pbio-0050170-g001){ref-type="fig"}A and [1](#pbio-0050170-g001){ref-type="fig"}B). Because the mitotic checkpoint protein, Mad2p-GFP \[[@pbio-0050170-b017]\], largely localized to the NE, it appeared that most Mia1p-overexpressing cells overcame the spindle assembly checkpoint and entered anaphase ([Figure S3](#pbio-0050170-sg003){ref-type="supplementary-material"}) (even though approximately 12% of cells exhibited Mad2p-GFP on kinetochores, indicative of some aspect of spindle malfunction). The component of the septation initiation network, Cdc7p-GFP \[[@pbio-0050170-b018]\], localized to a single SPB, both in control and Mia1p-overexpressing cells (unpublished data). ![Fission Yeast Cells Overexpressing Mia1p Arrest in Mitosis with Fully Extended Spindles\ (A) The centromere markers, Cen1-GFP, Clp1-GFP, and Rlc1p-GFP, exhibit late mitotic localization in cells overexpressing Mia1p. Shown are single maximum-intensity reconstructions of live cells.\ (B) Graph quantifying the proportion of cells exhibiting mitotic localization of marker proteins (*n* = 100).\ (C) Immunofluorescence images of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies and the DNA dye, DAPI.\ (D) Graph quantifying the proportion of mitotic cells exhibiting wild-type or abnormal spindle architecture (*n* = 300). MT, microtubule.](pbio.0050170.g001){#pbio-0050170-g001} We performed immunofluorescence analyses of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies and the DNA dye, DAPI. All anaphase spindles in wild-type cells corresponded to two DNA masses located at the spindle poles, indicative of normal mitotic function ([Figure 1](#pbio-0050170-g001){ref-type="fig"}C and [1](#pbio-0050170-g001){ref-type="fig"}D). Cells overexpressing Mia1p exhibited mitotic phenotypes that could be roughly divided into three groups: 10% of cells showed a wild-type morphology with respect to chromosome segregation; 7% of cells exhibited aster-like monopolar spindles and highly condensed chromosomes, and the rest displayed extended anaphase spindles and nonsegregated DNA masses ([Figure 1](#pbio-0050170-g001){ref-type="fig"}C and [1](#pbio-0050170-g001){ref-type="fig"}D). Taken together, our data indicated that Mia1p-overexpressing cells were predominantly arrested in anaphase, with fully extended spindles and nonsegregated chromosomes. The SPBs Are Displaced from Spindle Poles, and Nuclear Envelope Division Fails in Mia1p-Overexpressing Cells {#s2b} ------------------------------------------------------------------------------------------------------------ The SPBs are thought to be required for mitotic spindle assembly and function in fungi, and are always found at the spindle poles ([Figure 2](#pbio-0050170-g002){ref-type="fig"}A, wild type). We overexpressed Mia1p in S. pombe strains carrying the core SPB marker, Pcp1p-GFP or the peripheral SPB protein, Sad1p-GFP. Strikingly, we found that only 20% of Mia1p-overexpressing Pcp1p-GFP anaphase cells exhibited spindles with pole-located SPBs. Nonseparated SPB pairs located at one, but not the other, spindle pole were found in 25% of cells, whereas the rest of Mia1p-overexpressing cells exhibited duplicated SPBs that were completely mispositioned with respect to either spindle pole ([Figure 2](#pbio-0050170-g002){ref-type="fig"}A and [2](#pbio-0050170-g002){ref-type="fig"}B). Overexpression of Mia1p in Sad1p-GFP cells confirmed that while the SPBs were duplicated and spindles elongated, the SPBs were often displaced from the spindle poles ([Figure S4](#pbio-0050170-sg004){ref-type="supplementary-material"}). ![The SPBs Are Displaced from the Spindle Poles, and NE Division Fails in Cells Overexpressing Mia1p\ (A) Immunofluorescence images of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies, Pcp1p-GFP, and the DNA dye, DAPI.\ (B) Graph quantifying the proportion of mitotic Mia1p-overexpressing cells exhibiting normal or aberrant spindle architecture (*n* = 100). Figures over each bar schematically represent position of the SPBs (black dots) with respect to the spindle (grey line).\ (C) Mia1p overexpression induces panhandle-shaped deformations of the NE. Shown are single maximum-intensity reconstructions of live wild-type and Mia1p-overexpressing cells containing the NE marker proteins Cut11p-GFP and Uch2p-GFP.\ (D) Graph quantifying the proportion of cells exhibiting the panhandle-shaped NE deformations (*n* = 100).\ (E) Time-lapse sequence of Cut11p-GFP--expressing wild-type cells undergoing mitosis. Numbers refer to the time, in minutes.\ (F) Time-lapse sequence of Mia1p-overexpressing Cut11p-GFP cells undergoing mitosis. Note an elongating protrusion of the NE and the SPB pair on the opposite side of the nucleus. Numbers refer to the time, in minutes.\ (G) The SPBs largely localize away from the NE protrusion tips in cells overexpressing Mia1p. Note that in wild-type cells, the SPBs seem to lead the NE division. Shown are single maximum-intensity reconstructions of live wild-type and Mia1p-overexpressing cells containing the NE marker protein, Uch2p-GFP, and the SPB protein, Sad1p-DSRed.](pbio.0050170.g002){#pbio-0050170-g002} Microscopy analyses of the NE markers, Cut11p-GFP \[[@pbio-0050170-b006]\] and Uch2p-GFP \[[@pbio-0050170-b019]\], suggested that the extending intranuclear spindles often deformed the NE, with most cells exhibiting striking panhandle-shaped protrusions with chromosomes "trapped" in the central compartment ([Figure 2](#pbio-0050170-g002){ref-type="fig"}C and [2](#pbio-0050170-g002){ref-type="fig"}D). To visualize NE dynamics, we performed time-lapse analyses of Cut11-GFP behavior in wild-type cells and cells overexpressing Mia1p. In the wild type, the NE went through dramatic changes after the onset of anaphase B. As the spindle elongated, the NE assumed an ovoid shape with the SPBs positioned at its leading edges. The dividing nucleus eventually underwent medial constriction. Finally, the remaining link between the two daughter nuclei was resolved ([Figure 2](#pbio-0050170-g002){ref-type="fig"}E and [Video S1](#pbio-0050170-sv001){ref-type="supplementary-material"}). By contrast, in cells overexpressing Mia1p, we observed the appearance and extension of the NE protrusions at one ([Figure 2](#pbio-0050170-g002){ref-type="fig"}F and [Video S2](#pbio-0050170-sv002){ref-type="supplementary-material"}) or both sides of the nucleus, and eventual failure of nuclear division. Microscopy analysis of Mia1p-overexpressing Uch2p-GFP Sad1p-DSRed cells revealed that the nonseparated SPB pairs were usually found at the base of the protrusions or at the opposite side of the nucleus ([Figure 2](#pbio-0050170-g002){ref-type="fig"}G). To gain insight into the intranuclear architecture upon overexpression of Mia1p, we performed electron microscopy serial-section analyses of rapidly frozen and freeze-substituted cell samples, followed by three-dimensional image reconstruction \[[@pbio-0050170-b020]--[@pbio-0050170-b022]\]. We confirmed that the SPBs were duplicated, but not separated ([Figure 3](#pbio-0050170-g003){ref-type="fig"}A and [3](#pbio-0050170-g003){ref-type="fig"}B, and [Video S3](#pbio-0050170-sv003){ref-type="supplementary-material"}). In the example shown, the spindle is positioned between the SPB pair at the base of the protrusion and the projection tip. The free end of the spindle deforms the double-layered NE ([Figure 3](#pbio-0050170-g003){ref-type="fig"}A and [Video S3](#pbio-0050170-sv003){ref-type="supplementary-material"}). ![The SPBs Are Duplicated but Not Separated in Cells Overexpressing Mia1p\ (A) Electron microscopy images of serial sections of a Mia1p-overexpressing cell showing the abnormal panhandle-shaped protrusion of the NE. Note that microtubules extend throughout the protrusion (indicated by indented arrowheads, panel 4). The SPB pair is located at the base of protrusion. The NE protrusion and position of the SPBs are indicated on panel 4. Scale bar represents 1 μm.\ (B) Higher magnification image of the duplicated SPB pair. Mitochondrion is labeled as M, nucleus as N. Scale bar represents 0.2 μm.](pbio.0050170.g003){#pbio-0050170-g003} Thus, we concluded that (1) the SPBs were duplicated, but not separated; (2) the SPBs were often disassociated from spindle poles; and (3) the SPB-free, or acentrosomal, spindle poles deformed the NE during spindle extension in Mia1p-overexpressing cells. Spindles in Mia1p-Overexpressing Cells Are Bipolar and Require the Microtubule-Crosslinking Protein, Ase1p, for Their Assembly {#s2c} ------------------------------------------------------------------------------------------------------------------------------ Given that the overexpression of Mia1p induced assembly of elongating spindles even though SPBs were not separated, we were interested in determining the polarity of microtubules in these structures. The BimC-related tetrameric kinesin, Cut7p, is essential for spindle formation and exhibits a complex cell cycle--specific pattern of localization \[[@pbio-0050170-b023],[@pbio-0050170-b024]\]. In metaphase wild-type cells, Cut7p-GFP was concentrated at spindle poles and localized along the entire spindle length. As the cell proceeded into anaphase, Cut7p-GFP mostly redistributed along the spindle to eventually concentrate in the broad midzone area, consistent with its function in spindle elongation. It also exhibited a weaker association with the SPBs ([Figure 4](#pbio-0050170-g004){ref-type="fig"}A, ten out of ten cells). Interestingly, Cut7p-GFP exhibited a largely bipolar localization in Mia1p-overexpressing cells, somewhat similar to wild type. Time-lapse analyses of elongating spindles indicated that they were clearly bipolar until early anaphase B (eight out of 11 cells). As spindles elongated, Cut7p-GFP redistributed to the spindle midzone and was eventually almost completely depleted from one of the spindle poles while being enriched on the other one ([Figure 4](#pbio-0050170-g004){ref-type="fig"}B). Fluorescence microscopy analyses utilizing Cut7p-mCherry, Uch2p-GFP, and Pcp1p-GFP marker proteins indicated that Cut7p was enriched on acentrosomal spindle poles in cells overexpressing Mia1p (93% of cells, *n* = 100, see [Figure 4](#pbio-0050170-g004){ref-type="fig"}C). Thus, even though the Cut7p-GFP localization was not symmetric in late anaphase spindles, it defined the spindle midzone, suggesting that orientation of microtubules in Mia1p-induced spindles was antiparallel with plus ends in the center of the spindle, similar to wild type. ![Spindle Structures in Mia1p-Overexpressing Cells Are Bipolar and Antiparallel\ (A) Time-lapse sequence of Cut7p-GFP--expressing wild-type cells undergoing spindle elongation. Numbers refer to the time, in minutes.\ (B) Time-lapse sequence of Mia1p-overexpressing Cut7p-GFP cells undergoing mitosis. Note that Cut7p-GFP clearly localizes to both poles early in anaphase and is progressively depleted from one of them as the cell progresses through mitosis. The midzone localization is similar to wild type. Shown are single maximum-intensity reconstructions of *z*-stacks. Numbers refer to the time, in minutes.\ (C) Cut7p-mCherry is enriched on acentrosomal spindle poles as judged by double fluorescence microscopy analysis with the SPB core marker, Pcp1p-GFP. Shown are single maximum-intensity reconstructions of *z*-stacks.\ (D) The γ-TuRC marker, Alp4p-GFP, is present on acentrosomal poles in Mia1p-overexpressing cells, where it co-localizes with Cut7p-mCherry. Shown are single maximum-intensity reconstructions of *z*-stacks.\ (E) The chromosomal passenger complex protein, Ark1p-GFP, defines the spindle midzone in acentrosomal spindles. Shown are single maximum-intensity reconstructions of *z*-stacks.\ (F) Ase1p-GFP localizes to the spindle poles and spindle midzone in wild-type and Mia1p-overexpressing cells. Shown are single maximum-intensity reconstructions of *z*-stacks.\ (G) Graph quantifying the proportion of cells exhibiting monopolar spindles upon Mia1p overexpressing in wild-type and *ase1*Δ genetic backgrounds (*n* = 300). Inset, immunofluorescence image of the monopolar spindle in a Mia1p-overexpressing *ase1*Δ cell using anti--α-tubulin antibodies and the DNA dye, DAPI. MTs, microtubules.](pbio.0050170.g004){#pbio-0050170-g004} Consistently, the γ-tubulin ring complex (γ-TuRC) component, Alp4p-GFP \[[@pbio-0050170-b025]\], concentrated at both spindle poles in the control and Mia1p-overexpressing cells, as judged by double fluorescence microscopy analyses using Cut7p-mCherry as a spindle marker ([Figure 4](#pbio-0050170-g004){ref-type="fig"}D). Also, although in control cells, the Alp4p-GFP signal always coincided with the SPB marker, Sad1p-DSRed, we observed the presence of Alp4p-GFP-positive, Sad1p-DSRed-negative structures upon Mia1p overexpression indicative of γ-TuRC--containing acentrosomal poles ([Figure S5](#pbio-0050170-sg005){ref-type="supplementary-material"}). We further evaluated spindle polarity by overexpressing Mia1p in cells containing spindle midzone markers. We found that the member of the chromosomal passenger complex aurora kinase, Ark1p-GFP \[[@pbio-0050170-b026]\], localized to kinetochores in metaphase (\~1% of cells) and to the spindle midzone during anaphase (\~5% of cells) in control cells. Ark1p-GFP defined the spindle midzone in 43% of Mia1p-overexpressing cells, and we found it localizing to kinetochores in 15% of cells ([Figure 4](#pbio-0050170-g004){ref-type="fig"}E). The microtubule-binding protein Ase1p is another spindle midzone marker that has been suggested to contribute to proper midzone formation and the structural integrity of the bipolar spindle \[[@pbio-0050170-b027],[@pbio-0050170-b028]\]. Although Ase1p is not essential, spindles in *ase1*Δ cells tend to collapse, but maintain bipolarity, and occasionally break during anaphase elongation \[[@pbio-0050170-b027]\]. Ase1p-GFP localized to the region of the spindle midzone and spindle poles in Mia1p-overexpressing cells ([Figure 4](#pbio-0050170-g004){ref-type="fig"}F). Strikingly, we found that Ase1p was required for the assembly of acentrosomal bipolar spindles upon Mia1p overexpression. The number of cells exhibiting typical aster-like monopolar spindles dramatically increased when Mia1p was overexpressed in the *ase1*Δ genetic background ([Figure 4](#pbio-0050170-g004){ref-type="fig"}G). Control cells lacking Ase1p, but not overexpressing Mia1p, exhibited a very low incidence of monopolar spindles (less than 0.5%). Thus, we concluded that Mia1p-overexpressing cells could assemble bipolar and antiparallel spindle structures despite failure of SPB separation. Elongation of Acentrosomal Spindles Results in Protruding Deformations of the Nuclear Envelope and Failure of Nuclear Division {#s2d} ------------------------------------------------------------------------------------------------------------------------------ Spindle structures assembled in cells overexpressing Mia1p failed to segregate chromosomes and to ensure NE division. A striking feature of Mia1p-induced spindles was a conspicuous absence of the SPBs at one or both spindle poles. We sought to determine whether the SPBs were in fact required for providing a structural rigidity that could allow spindle poles to push against the NE without deforming it. To test this hypothesis, we severed the SPBs from early anaphase spindles in α-tubulin-GFP Cut11p-GFP ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A and [5](#pbio-0050170-g005){ref-type="fig"}B), and in α-tubulin-GFP Uch2p-GFP (unpublished data) expressing wild-type cells using laser microsurgery \[[@pbio-0050170-b029]\]. When both SPBs were cut off, spindles continued elongation due to sliding forces produced in the spindle midzone \[[@pbio-0050170-b029]\]. These spindle remnants readily formed panhandle-shaped protrusions at opposite sides of the nucleus ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A), evocative of the phenotype observed in Mia1p-overexpressing cells ([Figure 2](#pbio-0050170-g002){ref-type="fig"}C), and similar to what has been previously noted \[[@pbio-0050170-b029]\]. When the SPB was severed from one spindle pole, spindles deformed the NE at this location, whereas no NE deformation was observed at the opposite spindle pole still tethered to the other SPB ([Figure 5](#pbio-0050170-g005){ref-type="fig"}B). ![NE Division Fails When the SPBs Are Not Positioned at Elongating Spindle Poles\ (A) Time-lapse sequence of a Cut11p-GFP α-tubulin-GFP--expressing wild-type cell when both SPBs were severed from the elongating anaphase spindle by laser microsurgery.\ (B) Time-lapse sequence of a Cut11p-GFP α-tubulin-GFP--expressing wild-type cell when one SPB was cut off the spindle. Note that the NE is forming the panhandle-shaped protrusions at the sites of contact with the elongating spindle remnants. DIC images at the end of the sequence show normally proceeding septation. Shown are single maximum-intensity reconstructions of *z*-stacks. Numbers refer to the time, in minutes and seconds.\ (C) Time-lapse fluorescence microscopy analyses of the NE and spindle dynamics in Uch2p-GFP α-tubulin-GFP--expressing wild-type, *dis1*Δ, and Mia1p-overexpressing cells. Formation of membrane tethers in *dis1*Δ and Mia1p-overexpressing cells is indicated by arrowheads. Shown are single maximum-intensity reconstructions of *z*-stacks. Numbers refer to the time, in minutes.](pbio.0050170.g005){#pbio-0050170-g005} It is possible that the presence of either the SPB or of chromosomes at the poles, or both, is required for maintaining shape of the nucleus during anaphase movement. To distinguish between these possibilities, we performed time-lapse analyses of cells lacking the MAP215-related protein, Dis1p, and expressing the NE marker, Uch2p-GFP, and the microtubule marker, α-tubulin-GFP. It has previously been shown that at the restrictive temperature of 20 °C, Dis1p is required for kinetochore/microtubule attachment although the spindle elongates to the full extent \[[@pbio-0050170-b030],[@pbio-0050170-b031]\]. We found that initial elongation of the NE proceeded normally in *dis1*Δ cells as compared to wild-type control, and eventually, nuclei of both cell types assumed an ovoid shape ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). However, as the spindle in *dis1*Δ cells elongated further, the NE abruptly collapsed, resulting in the formation of the panhandle-shaped protrusion ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). We then performed imaging of Mia1p-overexpressing cells that also contained Uch2p-GFP α-tubulin-GFP during the same stages of mitosis. In the presence of overexpressed Mia1p, constitutive expression of α-tubulin-GFP failed to generate fluorescent bipolar spindles (unpublished data). Therefore, we simultaneously induced expression of Mia1p from the *nmt1* promoter and α-tubulin-GFP from the weak *nmt81* promoter. We found that although there was still a considerable increase in the number of cells with monopolar spindles, we could observe few cells with properly assembled elongating spindles. The NE in these cells was abnormally deformed immediately after spindle elongation, without going through the initial expansion and elongation phase ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). Based on these experiments, we concluded that the SPBs positioned at spindle poles were essential for NE division and that successful segregation of the NE was sustained by keeping daughter chromosomes attached to the SPBs throughout anaphase B. Genetic Ablation of the Nuclear Envelope Allows Chromosome Segregation in Mia1p-Overexpressing Cells {#s2e} ---------------------------------------------------------------------------------------------------- As shown above, Mia1p overexpression is lethal, largely due to the failure in nuclear division and chromosome segregation. Because most Mia1p-overexpressing cells bypassed the spindle assembly checkpoint and arrested in anaphase, we wondered whether, in part, it was the NE division defect that interfered with chromosome segregation due to entrapment of DNA masses. Thus, we tested whether the Mia1p-induced spindles could segregate DNA at least at some frequency in an experimental situation in which cells underwent mitosis in the absence of the NE. The temperature-sensitive mutant in the Ran-GEF, *pim1--1,* has been reported to induce NE fragmentation upon passage through mitosis \[[@pbio-0050170-b032]\]. Time-lapse analyses of spindle and NE dynamics in α-tubulin-GFP Uch2p-GFP *pim1--1* cells at the permissive ([Figure 6](#pbio-0050170-g006){ref-type="fig"}A and [Video S4](#pbio-0050170-sv004){ref-type="supplementary-material"}) and restrictive ([Figure 6](#pbio-0050170-g006){ref-type="fig"}B and [Video S5](#pbio-0050170-sv005){ref-type="supplementary-material"}) temperatures suggested that lack of Pim1p function induced an irreversible NE fragmentation in early anaphase B. Spindles continued elongating ([Figure 6](#pbio-0050170-g006){ref-type="fig"}B and [Video S5](#pbio-0050170-sv005){ref-type="supplementary-material"}), and cells eventually underwent cytokinesis, arresting at this point in the cell cycle (\[[@pbio-0050170-b032]\] and [Figure 6](#pbio-0050170-g006){ref-type="fig"}D). We induced overexpression of Mia1p in *pim1--1* cells containing the integral SPB marker, Pcp1p-GFP, and the NE marker, Uch2p-GFP, at either permissive or restrictive temperature. Panhandle-shaped NE protrusions could be induced in *pim1--1* cells at 24 °C at a rate similar to control Uch2p-GFP cells ([Figure S6](#pbio-0050170-sg006){ref-type="supplementary-material"}). Only 10% of Pcp1p-GFP Uch2p-GFP *pim1--1* cells overexpressing Mia1p segregated DNA in two masses when the NE was intact ([Figure 6](#pbio-0050170-g006){ref-type="fig"}C), consistent with the above experiments. When the NE was disassembled upon shifting cells to the restrictive temperature, the proportion of binucleate cells increased considerably (32%) ([Figure 6](#pbio-0050170-g006){ref-type="fig"}C). ![DNA Segregation Occurs in Mia1p-Overexpressing Cells When the NE Is Fragmented in Mitotic Cells Harboring the *pim1--1* Mutation\ (A) Time-lapse sequence of an Uch2p-GFP α-tubulin-GFP--expressing *pim1--1* cell undergoing anaphase at the permissive temperature of 24 °C.\ (B) Time-lapse sequence of an Uch2p-GFP α-tubulin-GFP--expressing *pim1--1* ^ts^ cell undergoing anaphase at the restrictive temperature of 36 °C. Cells were shifted to the restrictive temperature 3 h prior to imaging to allow inactivation of Pim1p protein. Note that the NE is fragmented shortly after anaphase B onset. Shown are single maximum-intensity reconstructions of *z*-stacks. Numbers refer to the time, in minutes.\ (C) Graph quantifying the proportion of binucleate Pcp1p-GFP Uch2p-GFP *pim1--1* cells overexpressing Mia1p at 24 °C and 36 °C (*n* = 300).\ (D) Graph quantifying the proportion of uninucleate and binucleate Mia1p-overexpressing Pcp1p-GFP Uch2p-GFP *pim1--1* cells that do not separate the SPBs at 36 °C (*n* = 100). Insets, examples of the scored phenotypes. Epifluorescence images of fixed Mia1p-overexpressing *pim1--1* cells containing the core SPB marker, Pcp1p-GFP, and the outer NE marker, Uch2p-GFP, at 36 °C. DNA is stained with DAPI. The NE is fragmented. Shown are single maximum-intensity reconstructions of *z*-stacks.\ (E) Epifluorescence images of Cen1-GFP Sad1-DSRed--containing *pim1--1* cells, with and without Mia1p overexpression. Shown are single maximum-intensity reconstructions of *z*-stacks. Cartoons outline the phenotypes scored. Circle indicates the SPB; star indicates the centromere I.](pbio.0050170.g006){#pbio-0050170-g006} We found that few cells with spindles lacking the SPBs at least at one spindle pole exhibited two or more closely positioned DNA masses when the NE was intact (4% of cells, [Figure S7](#pbio-0050170-sg007){ref-type="supplementary-material"}). However, when the NE was fragmented, we observed an appearance of divided cells with DNA segregated to daughter compartments. Upon the shift to the restrictive temperature, 27% of cells exhibiting duplicated, but not separated, SPBs on one side of the septum showed presence of chromosomes in both daughter cells (*n* = 100, [Figure 6](#pbio-0050170-g006){ref-type="fig"}D). We repeated this experiment using cells expressing the centromere I marker, Cen1-GFP, and the SPB marker, Sad1p-DSRed. We found that 32% of cells (*n* = 68) with acentrosomal spindles properly segregated sister chromatids when the NE was fragmented, suggesting that failure in NE division could hinder DNA segregation ([Figure 6](#pbio-0050170-g006){ref-type="fig"}E). Taken together, our results suggest that (1) overexpression of the TACC-related protein, Mia1p, results in the assembly of bipolar and moderately functional spindles that lack properly positioned SPBs; (2) one of the essential functions of the SPB in fission yeast could be to facilitate NE division during closed mitosis; and (3) failure to divide the NE could result in restriction of DNA masses movement and, ultimately, in a chromosome segregation defect. Discussion {#s3} ========== In this study, we show that anaphase elongation of acentrosomal spindles during closed mitosis in fission yeast causes NE division failure and associated chromosome segregation defects. Mia1p belongs to a family of microtubule-associated proteins named after the human transforming acid coiled coil protein, TACC, which has been implicated in cancer development (for review, see \[[@pbio-0050170-b033]\]). TACC proteins participate in spindle pole focusing and organization in metazoans \[[@pbio-0050170-b034],[@pbio-0050170-b035]\]. Fission yeast cells lacking Mia1p exhibit a variety of microtubule defects throughout the cell cycle. Mitotic *mia1*Δ cells lack astral microtubules \[[@pbio-0050170-b009]\] and show signs of defective kinetochore/microtubule attachment \[[@pbio-0050170-b010]\]. During interphase, *mia1*Δ cells fail to establish and maintain properly organized microtubule arrays, likely due to a molecular defect in microtubule attachment to the original nucleation sites \[[@pbio-0050170-b004]\]. These functions correlate well with the Mia1p localization pattern. Indeed, Mia1p-GFP is mainly concentrated at the microtubule-organizing centers, though it is also found at plus ends of microtubules \[[@pbio-0050170-b004],[@pbio-0050170-b009],[@pbio-0050170-b010]\]. We found that cells overexpressing Mia1p were capable of organizing ectopic spindle poles and acentrosomal spindles. Although formation of intranuclear microtubule bundles in interphase S. pombe cells has previously been observed \[[@pbio-0050170-b036]\], overexpression of Mia1p led to assembly of mitotic spindles exhibiting normal microtubule arrangement ([Figures 1](#pbio-0050170-g001){ref-type="fig"} and [4](#pbio-0050170-g004){ref-type="fig"}). These spindles contained the γ-TuRC complexes and the BimC kinesin, Cut7p, at acentrosomal spindle poles, and exhibited a well-defined spindle midzone ([Figure 4](#pbio-0050170-g004){ref-type="fig"}). Curiously, unlike the case of normal wild-type spindles, formation of these structures entirely depended on the presence of the microtubule-crosslinking protein, Ase1p ([Figure 4](#pbio-0050170-g004){ref-type="fig"}G). Mia1p-GFP expressed at high levels localized to spindle poles, where it formed large fluorescent structures, and elsewhere in the cytosol (unpublished data). Interestingly, upon overexpression of Mia1p, Ase1p-GFP aggregated at the spindle poles in addition to its normal localization to the spindle midzone ([Figure 4](#pbio-0050170-g004){ref-type="fig"}F). The γ-TuRC component, Alp4p-GFP, also localized to the acentrosomal spindle poles ([Figure 4](#pbio-0050170-g004){ref-type="fig"}D and [Figure S5](#pbio-0050170-sg005){ref-type="supplementary-material"}), suggesting that they could indeed contain a host of microtubule-associated factors. The fact that some proteins required for proper spindle assembly might be sequestered to these abnormal structures could also explain the reason why the SPBs failed to separate in cells overexpressing Mia1p. TACC proteins contain extensive coiled coil regions and can likely form oligomers. This, in combination with their microtubule binding properties, could provide a framework for clustering and focusing of microtubule minus ends observed in vivo. We envisage that accumulation of the γ-TuRC complexes and microtubule-crosslinking proteins near the SPBs, followed by subsequent sliding off through the force produced by the growing spindle, could lead to the formation of acentrosomal spindle poles. A particularly interesting phenotype of Mia1p overexpression was the formation of long, panhandle-shaped NE protrusions upon spindle elongation and the fact that NE division failure was reproducibly accompanied by DNA segregation defects. One possible explanation for such a phenotype could be the lack of microtubule/kinetochore attachment in these cells, which is not particularly surprising given that Mia1p normally functions in this process. We did observe an occurrence of trailing chromosomes in some cells overexpressing Mia1p when the SPBs delineated both spindle poles (for example, see [Figure 2](#pbio-0050170-g002){ref-type="fig"}A). Alternatively, the presence of the SPB inserted into the specialized region of the NE could prevent the formation of membrane tethers during spindle elongation. We found that most acentrosomal spindle poles in Mia1p-overexpressing cells distorted the NE, whereas most poles associated with the SPBs did not exhibit this behavior ([Figure 2](#pbio-0050170-g002){ref-type="fig"}). We also performed a series of laser microsurgery experiments in which we severed either one or both SPBs from the rest of the elongating anaphase spindle ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A and [5](#pbio-0050170-g005){ref-type="fig"}B). We found that, similar to Mia1p overexpression, the free spindle poles deformed the NE, and these cells failed to divide their nuclei despite normally timed septation ([Figure 5](#pbio-0050170-g005){ref-type="fig"}A and [5](#pbio-0050170-g005){ref-type="fig"}B). We sought to distinguish between the possibilities outlined above by following the NE dynamics in cells lacking the MAP215 protein, Dis1p, at the restrictive temperature of 20 °C. *dis1*Δ cells do not attach sister chromatids to the spindle microtubules, but proceed with spindle elongation \[[@pbio-0050170-b030],[@pbio-0050170-b031]\]. Because spindles in *dis1*Δ cells contained the SPBs, we could assess relative contributions of chromosomes and SPBs to NE division. We found that initially, the NE in *dis1*Δ cells went through normal elongation and assumed the ovoid shape typical of wild-type nuclei ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C). However, during subsequent spindle elongation, the NE abruptly collapsed, suggesting that positioning of chromosomes at the poles was essential for maintaining the shape of the dividing nucleus ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C, indicated by an arrowhead). It is possible that once the membrane reservoir (see below) is exhausted during the initial elongation stage, a mass of chromosomes provides a physical block to the NE division. Contrary to the *dis1*Δ phenotype, spindles immediately produced nuclear membrane tethers in Mia1p-overexpressing cells ([Figure 5](#pbio-0050170-g005){ref-type="fig"}C, emerging protrusion is indicated by an arrowhead), and nuclei did not assume the elliptical shape. We concluded that both the SPBs and the spindle pole--positioned chromosomes were essential for successful NE division. The SPB of S. pombe undergoes striking changes in morphology and cellular localization during the cell cycle. Because the SPBs move in and out of the nuclear membrane, depending on the mitotic phase, this process likely relies on a tightly regulated membrane-anchoring system. It has been suggested that Cut11p, a transmembrane protein similar to budding yeast Ndc1p, might participate in SPB anchoring during mitosis. In cells lacking Cut11p function, the SPBs are frequently found floating in the nucleoplasm, in addition to the pronounced SPB separation defect \[[@pbio-0050170-b006]\]. Cut11p localizes to the nuclear pores throughout the cell cycle and is recruited to the SPBs at the onset of mitosis, which would be consistent with its proposed function in the SPB anchorage. Interestingly, at least one computer-generated reconstruction of a defective spindle in Cut11p-deficient cells shows an image very similar to spindles assembled in Mia1p-overexpressing cells. The spindle is bipolar, but only one spindle pole is connected to the SPB. There is no SPB at the other pole that forms a herniated protrusion on the NE \[[@pbio-0050170-b006]\]. However, the SPB localization of Cut11p is lost in mid-anaphase B despite the fact that the SPBs still appear to lead the nuclear movement (\[[@pbio-0050170-b006]\] and unpublished data). Thus, it is possible that there is an additional mechanism executing proper SPB tethering and NE reinforcement at the site of insertion. The integral NE protein and SPB component, Sad1p, could possibly function in this process \[[@pbio-0050170-b037]\]. Sad1p-related proteins from other organisms have been involved in anchoring centrosomes to the NE \[[@pbio-0050170-b038]--[@pbio-0050170-b040]\]. Also, the intranuclear system of Mlp filaments might be a good candidate for reinforcing the site of SPB attachment to the NE. Mlps are coiled coil filamentous proteins localized to the nucleoplasmic side of the NE \[[@pbio-0050170-b041]\]. Both budding yeast Mlp1p and Mlp2p localize to the NE crescent region underlying the SPB, with Mlp2 forming discernible foci. Interestingly, Mlp2p interacts with the core components of the SPB and is required for proper SPB assembly and function \[[@pbio-0050170-b042]\]. It is possible that a large structure such as the SPB has to be anchored into the underlying assembly of Mlp filaments. It is also possible that the Mlp assembly might structurally reinforce the NE in order to maintain the stiffness and the appropriate curvature of the membrane during closed mitosis. It will be of significant interest to address potential roles of Sad1p and Mlp proteins in NE division in future studies. Panhandle-shaped protrusions of the nuclear membrane produced in our experiments can be compared to membrane tethers produced by microtubule polymerization or application of laser tweezers in artificial lipid vesicles \[[@pbio-0050170-b007],[@pbio-0050170-b043]\]. Upon force application, the membrane develops regions of negative curvature and collapses into narrow tubes around the spindle. Interestingly, formation of these long tethers may suggest the existence of a functional membrane reservoir at this stage of the cell cycle \[[@pbio-0050170-b044]\]. We performed time-lapse analyses of wild-type cells (*n* = 7) expressing the NE marker, Uch2p-GFP, and α-tubulin-GFP, and measured the nuclear diameter at metaphase (3.76 ± 0.14 μm) and anaphase (3.12 ± 0.12 μm). Assuming nuclei to be spherical, the combined surface area of the two daughter nuclei is estimated to be approximately 1.37 times larger than that of the mother nucleus. This apparent increase in surface area could result from membrane contribution from the ER or unraveling of nuclear membrane folds. At the same time, it is conceivable that the nuclear envelope behaves like a rigid network of locally deformable components. Studies performed on the NE of mammalian cells suggest that it could be organized as a heterogeneous meshwork consisting of regular lattice domains and interconnecting filaments \[[@pbio-0050170-b045]\]. Under mechanical stress, the meshwork would expand resulting in vacancies of lamin A. Although fungal nuclear envelopes do not contain lamins, it is possible that other proteins underlying the inner membrane, for instance, Mlp filaments, could perform similar function in connecting the network. We reasoned that should the undivided NE restrict the movement of chromosomes in cells overexpressing Mia1p, the removal of this obstacle could enable some degree of chromosome segregation through the action of acentrosomal spindles. Cells harboring a mutation *(pim1--1)* in a gene encoding for the RCC1-related protein, Pim1p, have been shown to undergo NE fragmentation upon passage through mitosis, and arrest with condensed DNA following hyperseptation (\[[@pbio-0050170-b032]\], see also [Figure 6](#pbio-0050170-g006){ref-type="fig"}A and [6](#pbio-0050170-g006){ref-type="fig"}B, and [Videos S4](#pbio-0050170-sv004){ref-type="supplementary-material"} and [S5](#pbio-0050170-sv005){ref-type="supplementary-material"}). We indeed found that a failure of DNA segregation in Mia1p-overexpressing cells was considerably relieved in the *pim1--1* genetic background ([Figure 6](#pbio-0050170-g006){ref-type="fig"}C and [6](#pbio-0050170-g006){ref-type="fig"}D). Interestingly, a significant proportion of these cells could accurately segregate sister chromatids, suggesting that acentrosomal spindles were functional although less efficient than normal ones ([Figure 6](#pbio-0050170-g006){ref-type="fig"}D). It is likely that a combination of factors contributes to the low efficiency of Mia1p-induced acentrosomal spindles in chromosome segregation. First, as outlined above, Mia1p overexpression could interfere with kinetochore attachment to spindle microtubules. Secondly, it is possible that attachment of microtubules to the SPB contributes to the increased accuracy of chromosome segregation, similar to what has been suggested for the case of centrosomes in animal cells \[[@pbio-0050170-b046]\]. There is an enormous structural diversity of spindles in eukaryotes \[[@pbio-0050170-b047],[@pbio-0050170-b048]\]. Apart from the well-established open and closed mitosis categories, spindles can assemble as extra- or intranuclear ones \[[@pbio-0050170-b047]\]. Although in some organisms the NE remains intact, in others, it might partially break down \[[@pbio-0050170-b049],[@pbio-0050170-b050]\] or be forcibly stripped off \[[@pbio-0050170-b051]\]. Similarly, various structural modifications throughout evolution resulted in the appearance of various types of SPBs. Here, we suggest that the highly structured SPB of fission yeast, and likely of other organisms in which chromosome segregation occurs strictly within the nuclear membrane, functions not only as a microtubule-organizing center, but also serves as a structural feature that enables NE division. Materials and Methods {#s4} ===================== S. pombe strains, antibodies, reagents, and constructs. {#s4a} ------------------------------------------------------- S. pombe strains used in this study and their genotypes are listed in [Table S1](#pbio-0050170-st001){ref-type="supplementary-material"}. EMM2 medium with suitable supplements was used for vegetative growth. For live-imaging experiments Mia1p overexpression was routinely induced for 18 h at 24 °C in EMM2 lacking thiamine. Genetic crosses and sporulation were performed on YPD agar plates. Plasmids were constructed using standard techniques of molecular cloning. The anti--α-tubulin antibody, TAT-1, was a gift from Dr. K. Gull. Immunofluorescence techniques. {#s4b} ------------------------------ Cells were fixed with 3.7% formaldehyde for 12 min and then spheroplasted using lysing enzymes and zymolyase in 1.2 M sorbitol in PBS. Permeabilization was performed in 1% Triton X-100 in PBS. Spheroplasts were washed with PBS. PBAL buffer (1 mM sodium azide, 1% BSA, 100 mM lysine hydrochloride, 50 μg/ml carbenicilin in PBS) was used for blocking and for incubation with primary and secondary antibodies. Imaging was done on a Zeiss Axiovert 200M microscope (Carl Zeiss, <http://www.zeiss.com>) with appropriate sets of filters, and images were generated using a CoolSnap camera (Photometrics, <http://www.photomet.com>) and Metamorph software package (Universal Imaging/Molecular Devices, <http://www.moleculardevices.com>). Image processing was done in Adobe Photoshop 7.0.1 (Adobe Systems, <http://www.adobe.com>). Time-lapse fluorescent microscopy. {#s4c} ---------------------------------- Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with CSU10 confocal optical scanner, 488-nm laser illumination source, and CoolSnap digital camera (Photometrics). Alternatively, we performed imaging in epifluorescence mode using a mercury lamp as an illumination source together with appropriate sets of filters, CoolSnap camera (Photometrics), and Uniblitz shutter driver (<http://www.uniblitz.com>) under the control of Metamorph software package (Universal Imaging). Typically, we acquired image stacks that consisted of five or six sections at 0.5-μm spacing. Imaging at 20 °C was performed using a homemade, cooled microscope housing. Laser microsurgery. {#s4d} ------------------- Laser microsurgery was conducted on a custom-assembled microscopy workstation centered around a Nikon TE2000-E2 microscope (Nikon Instruments, <http://www.nikon.com>). Laser pulses (532 nm for 8 ns) were generated by Q-switched Nd:YAG laser (Diva II; Thales Lasers, <http://thales.nuxit.net>) run at a 20-Hz repetition rate. Collimated laser beam was expanded to approximately 8 mm to fill the aperture of a 100× 1.4 NA PlanApo lens and delivered through an dedicated epi-port. Fluorescence images were recorded with a Cascade512B back-illuminated EM-CCD camera (Photometrics) attached to the left microscope port (100% transmission) in confocal mode (spinning disk confocal; Perkin-Elmer, <http://www.perkinelmer.com>) as three-dimensional datasets at 0.25-μm Z steps. Differential interference contrast (DIC) images were recorded with a CoolSnap CF CCD camera attached to the right port of the microscope (modified to 100% transmission). All light sources were shuttered by either fast mechanical shutters (Vincent Associates, <http://www.uniblitz.com>) or AOTF (Solamere Technology Group, <http://www.solameretech.com>) so that cells were exposed to light only during laser operations and/or image acquisition. Electron microscopy. {#s4e} -------------------- Cells were rapidly frozen by high-pressure freezing (BAL-TEC HPM-010; Technotrade International, <http://www.technotradeinc.com/>) and freeze-substituted at −90 °C in 2% glutaraldehyde plus 0.1% uranyl acetate in acetone for 96 h in an EM-AFS device (Leica, <http://www.leica-microsystems.com>). The cells were warmed for more than 3 h to −60 °C and then infiltrated with HM20 (Electron Microscopy Sciences, <http://www.emsdiasum.com>) resin over a period of 3 d. The cells were embedded under UV light at −60 °C in HM20 for 3 d and then warmed to room temperature over a 6-h period. Serial sections (60 nm) of embedded cells were picked up on Formvar-coated slot grids, stained with 1% aqueous uranyl acetate and lead citrate, and imaged in a Philips Tecnai TF20 FEG electron microscope (<http://www.fei.com/>) operating at 80 keV. Serial sections were aligned and modeled using the IMOD software package. Supporting Information {#s5} ====================== ###### Overexpression of Mia1p in S. pombe Is Lethal Cells carrying either an empty pREP1 vector or the pREP1-based plasmid containing *mia1^+^* open reading frame (ORF) were grown overnight in EMM2 medium containing thiamine to the optical density at 595 nm (OD~595~) equal to 0.4. They were spotted onto EMM2 plates with or without thiamine at increasing 10× dilutions and grown for 4 d at 30 °C. (961 KB TIF) ###### Click here for additional data file. ###### Localization of Mis6p-GFP in Wild-Type and Mia1p-Overexpressing Cells The centromere/kinetochore marker, Mis6p-GFP, labeled either two distinct kinetochore clusters (upper panel), similar to wild-type control, or localized to six closely positioned dots, likely representing nonsegregated anaphase kinetochores (lower panel). Shown are single maximum-intensity reconstructions of live cells. (447 KB TIF) ###### Click here for additional data file. ###### The Majority of Mia1p-Overexpressing Cells Exhibit Mad2p-GFP on the NE Graph quantifying the proportion of cells with Mad2p-GFP on kinetochores in wild type and cells overexpressing Mia1p (*n* = 100). Inset displays typical appearance of Mad2p-GFP in Mia1p-overexpressing cells, either on kinetochores or broadly spread around the NE. Shown are single maximum-intensity reconstructions of live cells. (555 KB TIF) ###### Click here for additional data file. ###### The SPBs Are Displaced from the Spindle Poles in Cells Overexpressing Mia1p $A$ Immunofluorescence images of wild-type and Mia1p-overexpressing cells using anti--α-tubulin antibodies, Sad1p-GFP, and the DNA dye, DAPI. $B$ Graph quantifying proportion of mitotic Mia1p-overexpressing cells exhibiting normal or aberrant spindle architecture (*n* = 100). (996 KB TIF) ###### Click here for additional data file. ###### The γ-TuRC Complex Protein, Alp4p-GFP, Localizes to Acentrosomal Spindle Poles in Mia1p-Overexpressing Cells Double fluorescence microscopy images of control and Mia1p-overexpressing Alp4p-GFP Sad1p-DSRed cells. Shown are single maximum-intensity reconstructions of live cells. (489 KB TIF) ###### Click here for additional data file. ###### Overexpression of Mia1p Induces Abnormal NE Deformations in *pim1--1* Cells at the Permissive Temperature $A$ Epifluorescence images of the NE labeled by Uch2p-GFP in wild-type and Mia1p-overexpressing *pim1--1* cells at 24 °C. Shown are single maximum-intensity reconstructions of live cells. $B$ Graph representing the proportion of cells forming the panhandle-shaped protrusions in both genetic backgrounds. (298 KB TIF) ###### Click here for additional data file. ###### Epifluorescence Images of Fixed Mia1p-Overexpressing *pim1--1* Cells Containing the Core SPB Marker, Pcp1p-GFP, and the Outer NE Marker, Uch2p-GFP, at 24 °C Rarely, we could observe Mia1p-overexpressing *pim1--1* cells with acentrosomal spindles that exhibited two or more closely positioned DNA masses at the permissive temperature. DNA is stained with DAPI. Shown are single maximum-intensity reconstructions of *z*-stacks. (492 KB TIF) ###### Click here for additional data file. ###### List of S. pombe Strains Used in This Study (49 KB DOC) ###### Click here for additional data file. ###### Time-Lapse Sequence of a Cut11p-GFP--Expressing Wild-Type Cell Undergoing Mitosis Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with CSU10 confocal optical scanner, 488-nm laser illumination source, and CoolSnap digital camera. Image stacks consisted of six sections at 0.5-μm spacing taken every 30 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (584 KB MOV) ###### Click here for additional data file. ###### Time-Lapse Sequence of a Mia1p-Overexpressing Cut11p-GFP Cell Undergoing Mitosis Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with CSU10 confocal optical scanner, 488-nm laser illumination source, and CoolSnap digital camera. Image stacks consisted of six sections at 0.5-μm spacing taken every 30 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (353 KB MOV) ###### Click here for additional data file. ###### QuickTime Video Was Assembled Based on Electron Microscopy Data Presented in [Figure 3](#pbio-0050170-g003){ref-type="fig"} Serial sections were aligned and modeled using the IMOD software package. The NE is in green, the SPBs are in purple, and microtubules are in yellow. (1.4 MB MOV) ###### Click here for additional data file. ###### Time-Lapse Sequence of an Uch2p-GFP α-Tubulin-GFP--Expressing *pim1--1* Cell Undergoing Anaphase at the Permissive Temperature of 24 °C Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope in epifluorescence mode. Image stacks consisted of five sections at 0.5-μm spacing taken every 60 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (126 KB MOV) ###### Click here for additional data file. ###### Time-Lapse Sequence of an Uch2p-GFP α-Tubulin-GFP--Expressing *pim1--1* Cell Undergoing Anaphase at the Restrictive Temperature of 36 °C Three-dimensional time-lapse images were generated on a Zeiss Axiovert 200M microscope in epifluorescence mode. Image stacks consisted of five sections at 0.5-μm spacing taken every 60 s. Maximum-intensity projection images of each stack were combined into a QuickTime video. (618 KB MOV) ###### Click here for additional data file. Accession Numbers {#s5a} ----------------- The National Center for Biotechnology Information (NCBI) (<http://www.ncbi.nlm.nih.gov>) accession numbers for the proteins discussed in this paper are Alp4p (BAA77269), Ark1p (O59790), Ase1p (CAC21482), Cdc7p (CAA55382), Clp1p (CAB76271), Cut11p (CAB11272), Cut7p (CAA40738), Dis1p (CAA19278), Mad2p (CAA16846), Mia1p (Q9URY2), Mis6p (BAA20458), Pcp1p (CAB03608), Pim1p (CAB60670), Rlc1p (CAB54151), Sad1p (BAA87133), and Uch2p (CAB52608). We are most grateful to M. Balasubramanian for suggesting the NE ablation experiment and allowing us to use strains from his S. pombe collection. E. Makeyev, V. Wachtler, and M. Balasubramanian kindly commented on the manuscript. Many thanks are due to R. Thadani, A. Tolstikov, J. R. McIntosh, and members of the Cell Dynamics group at the Temasek Life Sciences Laboratory for discussions. We would also like to thank Dr. K. Gull for his gift of TAT-1 antibody. **Author contributions.** SO conceived and designed the experiments and wrote the paper. LZ, CS, VM, and SO performed the experiments. All authors analyzed the data. LZ, CS, AK, and SO contributed reagents/materials/analysis tools. **Funding.** This work was supported by National Institutes of Health and Biotechnology Resources grant RR00592 to the National Resource for 3D Microscopy, a National Institutes of Health grant GM59363 to AK, and intramural funds from the Temasek Life Sciences Laboratory. **Competing interests.** The authors have declared that no competing interests exist. GFP : green fluorescent protein NE : nuclear envelope SPB : spindle pole body TACC : transforming acid coiled coil γ-TuRC : γ-tubulin ring complex

{ "pile_set_name": "PubMed Central" }

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22,195

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"SPB", "pombe", "spends", "interphase", "cytoplasm", "adjacent", "NE", "However", "appears", "securely", "anchored", "outer", "NE", "suggested", "studies", "SPB", "NE", "behavior", "interphase", "SPB", "duplicates", "matures", "daughter", "SPBs", "remain", "connected", "bridge", "Upon", "mitotic", "entry", "portion", "NE", "underlying", "SPB", "pair", "invaginates", "forming", "opening", "fenestra", "SPBs", "settle", "SPB", "initiates", "intranuclear", "microtubules", "eventually", "separate", "move", "opposite", "sides", "nucleus", "enclosed", "individual", "fenestrae", "somewhat", "larger", "SPBs", "anaphase", "fenestrae", "contract", "become", "size", "SPBs", "Late", "mitosis", "fenestrae", "close", "SPBs", "extruded", "back", "cytoplasm", "observations", "may", "indicate", "existence", "specialized", "cell", "cycle", "specific", "structures", "function", "anchor", "SPB", "NE", "Interestingly", "NE", "adjacent", "SPB", "shows", "degree", "specialization", "including", "local", 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Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder of the central nervous system, clinically characterized by memory impairment and cognitive dysfunction. The pathological hallmarks of AD in the brain are massive cerebral accumulation of senile plaques largely composed of β-amyloid (Aβ) peptide, intracellular neurofibrillary tangles and neuronal loss[@b1][@b2][@b3][@b4]. Recently, several epidemiological studies have revealed that mutations of particular genes can confer susceptibility to the pathogenesis of AD. APP, PSEN1, PSEN2 gene mutations are considered to be the main causes of familial AD, and play an important role in the early-onset of AD[@b5]. While in late-onset AD, the ε4 allele of the Apolipoprotein E gene (ApoEε4) is known as the major genetic risk factor[@b6]. Moreover, the mutations of genes such as APBB2, GAB2, PRNP and SORL1 have also been reported to be associated with the risk of AD[@b7][@b8][@b9][@b10][@b11]. The ATP-binding cassette subfamily B member 1 (ABCB1) gene in the chromosome 7q21 region spans 209-kb and contains 29 exons[@b12]. To date, several ABCB1 genetic variants have been identified to be involved in various kinds of diseases through re-sequencing, including 3435C \> T (rs1045642), 2677G \> T/A (rs2032582), 1236C \> T (rs1128503) polymorphisms. The 3435C \> T single nucleotide polymorphism (SNP) was the most commonly studied polymorphism, which was previously reported to be related to the susceptibility to the osteonecrosis of the femoral head (ONFH), leukemia, breast cancer and hepatocellular carcinoma[@b13][@b14][@b15][@b16]. The 2677G \> T/A polymorphism is associated with glucocorticoid-induced avascular necrosis of the femoral head (GANFH) and cancer[@b17][@b18]. The 1236C \> T polymorphism may contribute to the tumor response to chemotherapy in cancers in Asians[@b19][@b20]. P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an integral component of the blood-brain barrier (BBB)[@b21]. P-gp pumps various drugs and toxicants out of the brain and Aβ is an endogenous substrate for P-gp, suggesting that P-gp is able to mediate the clearance of Aβ through BBB[@b22]. Therefore, mutations of ABCB1 gene possibly result in an aberrant function of P-gp, and thus promote the pathogenesis of AD. Recently, some epidemiological studies have focused on the association between ABCB1 gene variants and AD susceptibility, and these variants include the 3435C \> T polymorphism, the 2677G \> T/A polymorphism and the 1236C \> T polymorphism[@b23][@b24][@b25][@b26][@b27][@b28][@b29][@b30][@b31]. The 3435C \> T polymorphism of ABCB1, which is a C to T transformation in exon 26 with no change in the amino acid encoded, is not related to AD risk in most studies[@b24][@b25][@b27][@b29][@b30][@b31]. However, Fehér *et al*.[@b23] and van Assema *et al*.[@b26] suggested a positive association between the 3435C \> T polymorphism and AD susceptibility. The 2677G \> T/A SNP is a missense mutation in exon 21, leading to an alanine to threonine or serine substitution. The association between the 2677G \> T/A SNP and the risk of AD is also controversial[@b23][@b24][@b26][@b27][@b31]. The ABCB1 1236C \> T polymorphism is a nucleotide change in exon 12 with no change in the amino acid encoded, and van Assema *et al*.[@b26] suggested 1236C \> T polymorphism might contribute to the progression of Aβ deposition in brain. Because of multiple sites in linkage disequilibrium, haplotypes analysis is necessary to consider the impact of genetic information and guarantee the risk assessment process. In this study, we carried out a meta-analysis to investigate the associations between ABCB1 gene polymorphisms and haplotypes and AD risk. Results ======= Characteristics of eligible studies ----------------------------------- Fourty-nine potential studies were identified after an initial search from the Pubmed, Embase, Web of Science, Medline and Alzgene databases. After further screening, nine studies were finally enrolled in our meta-analysis (2366 cases and 2256 controls). The flow diagram of the search process was shown in [Fig. 1](#f1){ref-type="fig"}. The ethnicities of the populations involved in these studies were Caucasian, Asian and mixed. AD was diagnosed according to the criteria set by the Diagnostic and Statistical Manual of Mental Disorders (DSM) or National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS--ADRDA)[@b32]. Most studies used the Mini Mental State Examination (MMSE) to identify healthy controls[@b33]. The median age of AD cases in these studies ranged from 64 to 82.5 years and the percentage of female patients ranged from 29.4 \~ 74.1%. The methods for gene identification consisted of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), a direct sequencing method and the TaqMan assay. Three common polymorphisms in the ABCB1 gene (3435C \> T, 2677G \> T/A, 1236C \> T) and three haplotypes (1236T/2677T/3435T, 1236T/2677T/3435C, 1236C/2677T/3435T) were assessed. The genotype frequencies of controls were all in Hardy-Weinberg Equilibrium (HWE) (*P* \> 0.05). The key information of the studies on ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms and AD risk is summarized in [Table 1](#t1){ref-type="table"}. The Magliulo's study[@b28] was not included in [Table 1](#t1){ref-type="table"}, because this study only performed the ABCB1 haplotype analysis. Meta-analysis: ABCB1 3435C \> T SNP ----------------------------------- Eight studies investigated the association between the 3435C \> T SNP and AD susceptibility. The pooled summary crude odds ratios (ORs) and corresponding 95% confidence intervals (CIs) in all the genetic models were as follows: TT vs. CC: OR = 1.04, 95% CI = 0.76--1.41, *P* = 0.82; CT vs. CC: OR = 1.18, 95% CI = 0.97--1.45, *P* = 0.10; CT + TT vs. CC: OR = 1.14, 95% CI = 0.91--1.42, *P* = 0.26; TT vs. CT + CC: OR = 0.91, 95% CI = 0.75--1.11, *P* = 0.36; T vs. C: OR = 1.01, 95% CI = 0.87--1.17, *P* = 0.90. A moderate heterogeneity was found between the individual studies (TT vs. CC: χ^2^ = 15.51, *P* = 0.03, I^2^ = 54.9%; CT vs.CC: χ^2^ = 9.56, *P* = 0.22, I^2^ = 26.8%; CT + TT vs. CC: χ^2^ = 12.5, *P* = 0.09, I^2^ = 44%; TT vs. CT + CC: χ^2^ = 11.18, *P* = 0.13, I^2^ = 37.4%; T vs. C: χ^2^ = 14.64, *P* = 0.04, I^2^ = 52.2%). We conducted a sensitivity analysis by excluding each study, and found the Fehér's study[@b23] could interpret the heterogeneity, because the statistical power of this study was higher than the others (OR \> 1.25, P \< 0.05). Moreover, after the exclusion of the Fehér's study[@b23], the I^2^ decreased to 0% with a different but statistically significant pooled estimate in the homozygote comparison model (CT vs. CC: OR = 1.24, 95% CI = 1.06--1.45, *P* = 0.01, [Fig. 2A](#f2){ref-type="fig"}) and the dominant model (CT + TT vs. CC: OR = 1.21, 95% CI = 1.04--1.41, *P* = 0.01, [Fig. 2B](#f2){ref-type="fig"}), indicating that the Fehér's study[@b23] was not robust. These results suggested that there might be an association between ABCB1 3435C \> T SNP and AD susceptibility. Additionally, subgroup analysis by ethnicity showed a significant association between 3435C \> T SNP and an increased risk of AD among Caucasians (CT vs.CC: OR = 1.23, 95% CI = 1.02--1.47, *P* = 0.03, [Fig. 2C](#f2){ref-type="fig"}; CT + TT vs. CC: OR = 1.20, 95% CI = 1.01--1.42, *P* = 0.04, [Fig. 2D](#f2){ref-type="fig"}), but not among Asians or mixed. Pooled ORs and 95% CIs for the association of ABCB1 3435C \> T SNP with AD risk are shown in [Table 2](#t2){ref-type="table"}. Funnel plots were adopted to assess the publication bias, and an evidence of asymmetry was observed (CT vs.CC: *P* = 0.04, [Fig. 3A](#f3){ref-type="fig"}; CT + TT vs. CC: *P* = 0.07, [Fig. 3B](#f3){ref-type="fig"}). This result was further supported by the analysis using Egger's test (TT vs.CC: *P* = 0.28; CT vs.CC: *P* = 0.01; CT + TT vs. CC: *P* = 0.01; TT vs. CT + CC: *P* = 0.61; T vs. C: *P* = 0.39). However, the funnel plots after the adoption of trim and fill method had perfect symmetry and no significant difference was found between the ORs and 95% CIs before and after, suggesting that there were no change within the crude and adjusted estimates ([Fig. 3C,D](#f3){ref-type="fig"} for CT vs. CC model and CT + TT vs. CC model, respectively). These findings indicated that the meta-estimates for 3435C \> T SNP were robust to confounders and methods of exposure assessment. Therefore, there appeared to be an association between the ABCB1 3435C \> T SNP and an increased AD risk according to our analysis. Meta-analysis: ABCB1 2677G \> T/A SNP ------------------------------------- Five studies investigated the association of 2677G \> T/A SNP with the AD risk. No significant association between 2677G \> T/A SNP and AD susceptibility was detected in the total populations (for TT + AA vs. GG: OR = 0.99, 95% CI = 0.59--1.67, *P* = 0.97, for GT + GA vs. GG: OR = 1.20, 95% CI = 0.93--1.56, *P* = 0.16, for GT + TT + GA + AA + TA vs. GG: OR = 1.15, 95% CI = 0.90--1.46, *P* = 0.27, for TT + TA + AA vs. GT + GA + GG: OR = 0.86, 95% CI = 0.57--1.32, *P* = 0.50, [Fig. 4A](#f4){ref-type="fig"}, for T + A vs. G: OR = 1.01, 95% CI = 0.80--1.29, *P* = 0.91). An obvious heterogeneity was observed in most models (for TT + AA vs. GG: χ^2^ = 8.29, *P* = 0.08, I^2^ = 51.8%, for GT + GA vs. GG: χ^2^ = 1.89, *P* = 0.76, I^2^ = 0.0%, for GT + TT + GA + AA + TA vs. GG: χ^2^ = 3.72, *P* = 0.45, I^2^ = 0.0%, for TT + TA + AA vs. GT + GA + GG: χ^2^ = 7.4, *P* = 0.12, I^2^ = 45.9%, for T + A vs. G: χ^2^ = 7.21, *P* = 0.13, I^2^ = 44.6%). However, subgroup analysis according to the ethnicity in the recessive model showed a significant association between 2677G \> T/A SNP and a decreased AD risk among Caucasians (TT + TA + AA vs. GT + GA + GG: OR = 0.68, 95% CI = 0.47--0.98, *P* = 0.04) with no heterogeneity (χ^2^ = 1.33, *P* = 0.72, I^2^ = 0.0%), but not in mixed ([Fig. 4B](#f4){ref-type="fig"}). Begg's funnel plot and Egger's linear regression test were used to assess the publication bias. All *P* values of Begg's funnel plots were more than 0.05 (for TT + AA vs. GG: *P* = 0.81, for GT + GA vs. GG: *P* = 0.46, for GT + TT + GA + AA + TA vs. GG: *P* = 0.81, for TT + TA + AA vs. GT + GA + GG: *P* = 0.81, for T + A vs. G: *P* = 0.81), as well as the Egger's linear regression test (for TT + AA vs. GG: *P* = 0.92, for GT + GA vs. GG: *P* = 0.27, for GT + TT + GA + AA + TA vs. GG: *P* = 0.66, for TT + TA + AA vs. GT + GA + GG: *P* = 0.64, for T + A vs. G: *P* = 0.97), suggesting that there was no publication bias in this meta-analysis. Pooled ORs and 95% CIs for the association of ABCB1 2677G \> T/A SNP with AD risk are shown in [Table 2](#t2){ref-type="table"}, and the funnel plot for the recessive model is demonstrated in [Fig. 5](#f5){ref-type="fig"}. Meta-analysis: ABCB1 1236C \> T SNP ----------------------------------- Four studies were selected to estimate the association of the ABCB1 1236C \> T SNP with the risk of AD. As a result, no statistically significant difference was found in all the genetic models. Subgroup analysis for the 1236C \> T SNP showed no significant differences in overall ORs ([Table 2](#t2){ref-type="table"}). Meta-analysis: ABCB1 haplotypes ------------------------------- Four studies investigated the role of ABCB1 haplotypes in AD, including the 1236T/2677T/3435T (TTT) haplotype, the 1236T/2677T/3435C (TTC) haplotype and the 1236C/2677T/3435T (CTT) haplotype ([Table 3](#t3){ref-type="table"}). The pooling estimates showed the TTC genotype was associated with a significant increase in the risk of AD (OR = 1.99, 95% CI = 1.24--3.18, *P* = 0.00). The TTT and CTT haplotypes exhibited no association with the AD risk (CTT: OR = 0.58, 95% CI = 0.31--1.08, *P* = 0.09; TTT: OR = 0.90, 95% CI = 0.56--1.44, *P* = 0.65, [Table 4](#t4){ref-type="table"}). Discussion ========== As one of the most common forms of dementia, AD affects millions of people worldwide. Recently, several studies have revealed that mutations of ABCB1 can be responsible for the pathogenesis of AD by promoting the accumulation of Aβ via P-gp[@b22][@b23][@b24][@b26]. Although ABCB1 gene variants such as 3435C \> T (rs1045642), 2677G \> T/A (rs2032582), 1236C \> T (rs1128503) polymorphisms have been reported to be associated with AD risk[@b23][@b24][@b26], several researchers have shown opposite results[@b25][@b27][@b28][@b29][@b30][@b31]. To investigate if ABCB1 polymorphisms are associated with the susceptibility to AD, we conducted this meta-analysis to study the relationship between ABCB1 polymorphisms and AD risk. Fehér *et al*.[@b23] reported that the ABCB1 3435C \> T SNP was associated with AD. Van Assema *et al*.[@b26] supported that ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T SNPs were related to changes in P-gp function at the BBB in AD patients, and thus may contribute to the pathogenesis of AD. In this study, we found a positive association between the ABCB1 3435C \> T polymorphism and AD susceptibility using a heterozygous comparison model and a dominant model. Sensitivity analysis was applied to ascertain the potential source of the heterogeneity between the studies. After excluding the Fehér's study[@b23] due to its high OR, homogeneity across studies was achieved, and a statistically significant association between the 3435C \> T SNP and AD risk was found. Moreover, subgroup analysis by ethnicity showed a significant association between the 3435C \> T SNP and an increased risk of AD among Caucasians, but not in Asians or mixed races. Trim and fill analysis for 3435C \> T SNP meta-estimates demonstrated no evidence of publication bias in these studies, suggesting a definite association between the ABCB1 3435C \> T SNP and AD risk. Consistent with the report by Cascorbi *et al*.[@b24], we found that there was an association between the ABCB1 2677G \> T/A SNP and a decreased AD susceptibility among Caucasian, but not in total population, implying that the 2677G \> T/A SNP may play a potential role in the AD pathogenesis. Moreover, our results demonstrated that there was no association between the ABCB1 1236C \> T SNP and AD risk. Due to the existence of multiple genes in linkage disequilibrium, haplotype analysis was useful to identify other indirect SNPs at the same region. As for the ABCB1 gene, the TTT, TTC and CTT haplotypes were most investigated for their roles in AD susceptibility. The studies by Fehér *et al*.[@b23], Cascorbi *et al*.[@b24] and Frankfort *et al*.[@b31] supported that there were no associations between these haplotypes and AD risk. However, Magliulo *et al*.[@b28] found that the TTT haplotype might play a role in donepezil disposition and clinical outcome. In the present study, we found no association between TTT, CTT haplotypes and the risk of AD, consistent with most studies[@b23][@b24][@b31]. In contrast, although no studies have identified a positive association between the TTC haplotype and AD risk, our meta-analysis showed the TTC haplotype was significantly associated with an increased AD susceptibility. In addition, no heterogeneity was found in the overall models, suggesting that the results of this meta-analysis were statistically robust. A recent Genome-Wide Association Study (GWAS) by Lambert *et al*.[@b11] found that no SNPs in the ABCB1 gene are genome-wide significant. In this study, we found that the 3435C \> T SNP and 2677G \> T/A SNP were significantly associated with AD risk. Different ethnicities may contribute to the difference between our study and the GWAS, because the GWAS consisted of individuals of European ancestry, while our study included Caucasians and Asians participants. Moreover, the GWAS focused on the possible susceptibility loci for late-onset AD, whereas our study evaluated the association between ABCB1 SNPs and early-onset and late-onset AD. There are some limitations in this meta-analysis. First, although we collected all the eligible publications, studies included in our meta-analysis were still limited. Randomized controlled clinical trials with larger sample sizes are required to confirm our conclusion. Second, AD is a multifactorial disease, and the potential interactions among gene-gene and gene-environment should take into account. Many other factors such as ApoEε4 may participate in the association between ABCB1 polymorphisms and AD risk. However, we did not carry out a subgroup analysis based on it due to limited data. Third, since some studies have not provided informations such as age, gender, diagnostic criteria and sequencing method, we could not carry out a subgroup analysis to study the effects of these parameters on the association between ABCB1 polymorphisms and AD risk. To better understand the role of ABCB1 polymorphisms or haplotypes in AD susceptibility, large-scale, high-quality studies with multi-ethnic populations are needed. In conclusion, based on the published studies, our meta-analysis confirmed that the ABCB1 3435C \> T SNP and TTC haplotype were significantly associated with an increased AD risk, while the 2677G \> T/A SNP may contribute to a lower susceptibility of AD. Our study, for the first time, found that there were associations between ABCB1 polymorphisms and haplotypes and AD risk. Our findings provide a better understanding of AD pathogenesis, and ABCB1 polymorphisms and haplotypes may serve as important targets for the prevention and diagnosis for AD. Methods ======= Selection of studies -------------------- To identify studies eligible for the meta-analysis, a comprehensive search strategy was applied using the electronic databases including Pubmed, Embase, Web of Science, Medline and Alzgene. The following keywords were used: ABCB1 or ATP-binding cassette subfamily B member 1, and polymorphism or rs1045642 or rs2032582 or rs1128503, and Alzheimer's disease or AD. All selected articles were published from January 2001 to March 2016. Other relevant studies were identified by hand searching the references of included articles. Data extraction --------------- Studies included in this meta-analysis met the following criteria: (1) the study had to be focused on the 3435C \> T, 2677G \> T/A, 1236C \> T SNP or haplotypes within the ABCB1 gene using original data; (2) the study should be case-control-designed or cohort-designed; (3) all patients must meet the diagnostic criteria of AD; (4) both AD patients and controls were included; and (5) the frequencies of individual genotypes in cases and controls were reported or provided from the authors. Exclusion criteria included studies with no relevance to ABCB1 polymorphisms and AD risk, overlapping data, and review articles. Each article was checked with inclusion and exclusion criteria independently by two investigators (X.Z. and X.-H.S.). Statistical analysis -------------------- To assess the association between ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms and AD risk, ORs and corresponding 95% CIs were calculated using homozygote comparison (BB vs. AA), heterozygous comparison (AB vs. AA), dominant model (BB + AB vs. AA), recessive model (BB vs. AB + AA), and allele comparison (B vs. A) (A stands for major allele, B stands for minor allele). The significance of the pooled OR was determined by Z-test, and *P* \< 0.05 was considered as statistically significant. We adopted the random-effects model to calculate the pooled ORs when a significant heterogeneity existed in the genetic models in the initial overall analysis, otherwise a fixed-effects model should be applied. The between-study heterogeneity was estimated using Cochran's Q statistic and the I^2^ statistic. *P* \> 0.10 in Q-test[@b34] and I^2^ \< 25%[@b35] indicated no heterogeneity among studies. The HWE in the controls was measured by the Pearson's Chi-square test, and the significance level was maintained at *P* \< 0.05[@b36]. We assessed publication bias by Begg's funnel plot and Egger's linear regression test[@b37], and adjusted for possible publication bias by means of the trim and fill method[@b38]. Moreover, subgroup analysis was conducted to explain the high heterogeneity in the studies according to the ethnicity. All statistical tests were performed using STATA 12.0 software (Stata Corporation, College Station, TX, USA). Additional Information ====================== **How to cite this article**: Zhong, X. *et al*. Association between ABCB1 polymorphisms and haplotypes and Alzheimer's disease: a meta-analysis. *Sci. Rep*. **6**, 32708; doi: 10.1038/srep32708 (2016). This study was supported by grants from Program for Liaoning Innovation Research Team in University (NO.LT2014016); Liaoning Province Scientific Research Foundation (2014226033); Key Laboratory Foundation from Shenyang S&T Projects (F16-094-1-00). This study was also supported in part by National Natural Science Foundation of China (No. 81501098) and National Science and Technology Major Projects for "Significant New Drugs Development" (2013ZX09103001-003). The funders have no roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript. **Author Contributions** X.Z. and M.-J.W. conceived and designed the experiments; X.Z. and X.-H.S. searched databases and collected full-text papers; X.Z. and M.-Y.L. extracted and analyzed data; X.Z. wrote the manuscript; M.-J.W. reviewed the manuscript. ![Flow diagram of search strategy and study selection for meta-analysis.](srep32708-f1){#f1} ![Forest plots for the association between the ABCB1 3435C \> T SNP and AD risk.\ (**A**) heterozygous comparison model for overall populations; (**B**) dominant model for overall populations; (**C**) heterozygous comparison model for ethnicity subgroup; (**D**) dominant model for ethnicity subgroup.](srep32708-f2){#f2} ![Funnel plot analysis and the trim and fill method to detect publication bias of ABCB1 3435C \> T SNP.\ (**A**) funnel plot analysis in heterozygous comparison model; (**B**) funnel plot analysis in dominant model; (**C**) trim and fill method in heterozygous comparison model; (**D**) trim and fill method in dominant model. Circles represent the weight of the studies and boxes stand for the added studies.](srep32708-f3){#f3} ![Forest plots for the association between ABCB1 2677G \> T/A SNP and AD risk in the recessive model.\ (**A**) for overall populations; (**B**) for ethnicity subgroup.](srep32708-f4){#f4} ![Funnel plot analysis to detect publication bias of ABCB1 2677G \> T/A SNP in the recessive model.](srep32708-f5){#f5} ###### Characteristics of individual studies on ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms and AD risk included in the meta-analysis. First Author Year Country Ethnicity Diagnostic criteria MRI/CT MMSE Genotyping Sample size Mean age Female (%) Case Control P~HWE~ ------------------------------------- ------ ----------------- ----------- --------------------- -------- ------ ------------ ------------- ------------------------- ------------ -------- ----------------- ------------------------------ --------- ----------------- ------------------------------ ------ Fehér, Á. 2014 Hungary Caucasian NINCDS/ADRDA No Yes PCR-RFLP 234/225 75.6 ± 6.8/74.8 ± 7.2 67.9/66.7 66 119 49 43 109 73 0.84 Cascorbi, I. 2013 Germany Caucasian --- No No PCR-RFLP 71/81 78.4 ± 10.7/69.5 ± 11.4 54.9/40.7 9 42 20 17 33 31 0.15 Van Assema, D.M. 2012 Dutch Caucasian NINCDS/ADRDA Yes Yes PCR-RFLP 32/17 64 ± 7/47 ± 17 29.4/43.8 2 9 6 8 15 9 0.73 Liu, H.C. 2012 China Asian --- No No --- 111/114 82.5 ± 8.4/77.3 ± 6.6 63.1/52.6 44 52 15 49 44 21 0.06 Kohen, R. 2011 USA Mixed NINCDS/ADRDA No Yes TaqMan 286/240 68.2 ± 9.4/52.5 ± 19.5 48/50 63 138 83 66 114 60 0.44 Li, H. 2008 Canada Caucasian DSM-IV No Yes Sequencing 753/736 77.8 ± 8.6/73.4 ± 7.9 57.6/64.4 158 352 185 168 339 183 0.66 Reiman, E.M. 2007 USA/Netherlands Caucasian --- No No Sequencing 861/550 74.9 ± 6.6/77.4 ± 7.3 --- 188 426 238 139 257 152 0.15 Frankfort, S.V. 2006 Netherlands Caucasian NINCDS/ADRDA Yes Yes Sequencing 48/41 81 ± 5.5/81.9 ± 5.7 68.8/65.9 5 26 17 9 18 14 0.49 **2677G** \> **T/A**(**rs2032582**) **GG** **GT** + **GA** **TT** + **TA** + **AA** **G/G** **GT** + **GA** **TT** + **TA** + **AA** Fehér, Á. 2014 Hungary Caucasian NINCDS/ADRDA No Yes PCR-RFLP 234/225 75.6 ± 6.8/74.8 ± 7.2 67.9/66.7 69 132 33 62 117 46 0.50 Cascorbi, I. 2013 Germany Caucasian --- No No PCR-RFLP 71/81 78.4 ± 10.7/69.5 ± 11.4 54.9/40.7 25 35 11 27 33 21 0.10 Van Assema, D.M. 2012 Dutch Caucasian NINCDS/ADRDA Yes Yes PCR-RFLP 32/17 64 ± 7/47 ± 17 29.4/43.8 3 8 6 10 10 10 0.07 Kohen, R. 2011 USA Mixed NINCDS/ADRDA No Yes TaqMan 286/240 68.2 ± 9.4/52.5 ± 19.5 48/50 73 147 66 78 119 43 0.84 Frankfort, S.V. 2006 Netherlands Caucasian NINCDS/ADRDA Yes Yes Sequencing 48/41 81 ± 5.5/81.9 ± 5.7 68.8/65.9 13 24 13 12 16 11 0.26 **1236C** \> **T**(**rs1128503**) **CC** **CT** **TT** **CC** **CT** **TT** Fehér, Á. 2014 Hungary Caucasian NINCDS/ADRDA No Yes PCR-RFLP 234/225 75.6 ± 6.8/74.8 ± 7.2 67.9/66.7 56 130 48 54 119 52 0.39 Cascorbi, I. 2013 Germany Caucasian --- No No PCR-RFLP 71/81 78.4 ± 10.7/69.5 ± 11.4 54.9/40.7 23 36 12 26 38 17 0.65 Van Assema, D.M. 2012 Dutch Caucasian NINCDS/ADRDA Yes Yes PCR-RFLP 32/17 64 ± 7/47 ± 17 29.4/43.8 3 8 6 11 11 10 0.08 Frankfort, S.V. 2006 Netherlands Caucasian NINCDS/ADRDA Yes Yes Sequencing 48/41 81 ± 5.5/81.9 ± 5.7 68.8/65.9 12 25 11 12 20 9 0.90 MRI/CT: Magnetic Resonance Imaging/Computerized Tomography; P~HWE~: P value of HWE in control. ###### Meta-analysis of the ABCB1 3435C \> T, 2677G \> T/A, 1236C \> T polymorphisms with AD risk. 3435C \> T(rs1045642) N TT vs.CC CT vs. CC CT + TT vs. CC TT vs. CT + CC T vs. C ------------------------------------- --- ---------------------------- ---------------------------- ------------------------------------------------------- ------------------------------------------------------- ------------------------- ---------------------------- -------------- ------- -------------- ---------------------------- -------------- ------- ------ ------------ ------ ------ ------ ------------ ------ ------ Total 8 1.17 0.97--1.40 0.10 0 **1**.**24** **1**.**06**--**1**.**45** **0**.**01** **0** **1**.**21** **1**.**04**--**1**.**41** **0**.**01** **0** 1.01 0.87--1.16 0.94 0 1.08 0.98--1.18 0.12 0 Ethnicity Caucasian 5 1.15 0.94--1.41 0.17 0 **1**.**23** **1**.**02**--**1**.**47** **0**.**03** **0** **1**.**20** **1**.**01**--**1**.**42** **0**.**04** **0** 0.99 0.84--1.16 0.89 0 1.06 0.96--1.17 0.26 0 Asian 2 0.80 0.37--1.73 0.56 0 1.32 0.74--2.33 0.35 0 1.15 0.67--1.95 0.61 0 0.69 0.34--1.42 0.32 0 0.97 0.66--1.42 0.86 0 Mixed 1 1.45 0.90--2.34 0.13 0 1.27 0.83--1.94 0.27 0 1.33 0.89--1.98 0.16 0 1.24 0.84--1.83 0.28 0 1.21 0.95--1.54 0.12 0 **2677G** \> **T/A**(**rs2032582**) **TT** + **AA vs**. **GG** **GT** + **GA vs**. **GG** **GT** + **TT** + **GA** + **AA** + **TA vs**. **GG** **TT** + **TA** + **AA vs**. **GT** + **GA** + **GG** **T** + **A vs**. **G** Total 5 0.99 0.59--1.67 0.97 51.8 1.20 0.93--1.56 0.16 0 1.15 0.90--1.46 0.27 0 0.86 0.57--1.32 0.50 45.9 1.01 0.80--1.29 0.91 44.6 Ethnicity Caucasian 4 0.73 0.48--1.12 0.15 0 1.13 0.81--1.58 0.48 0 1.00 0.73--1.37 0.98 0 0.68 0.47--0.98 0.04 0 0.88 0.72--1.08 0.22 0 Mixed 1 1.64 1.00--2.70 0.05 0 1.32 0.88--1.97 0.17 0 1.41 0.96--2.05 0.08 0 1.37 0.89--2.11 0.15 0 1.28 1.00--1.63 0.05 0 **1236C** \> **T**(**rs1128503**) **TT vs**.**CC** **CT vs**. **CC** **CT** + **TT vs**. **CC** **TT vs**. **CT** + **CC** **T vs**. **C** Total 4 0.97 0.64--1.47 0.87 0 1.13 0.80--1.60 0.48 0 1.08 0.78--1.50 0.64 0 0.89 0.63--1.25 0.49 0 0.99 0.81--1.21 0.91 0 ###### Characteristics of studies on ABCB1 1236C \> T-2677G \> T/A-3435C \> T haplotypes and AD risk included in the meta-analysis. First Author Year Country Ethnicity Case Control Haplotype distribution ----------------- ------ ------------- ----------- ------ --------- ------------------------ ---- ----- ---- ---- ----- Fehér, Á. 2014 Hungary Caucasian 234 225 12 25 141 23 10 164 Cascorbi, I. 2013 Germany Caucasian 71 81 1 1 28 2 1 30 Magliulo, L. 2011 Italy Caucasian 54 285 0 17 22 3 37 120 Frankfort, S.V. 2006 Netherlands Caucasian 48 41 3 0 43 1 1 33 ###### Meta-analysis of the ABCB1 1236C \> T-2677G \> T/A-3435C \> T haplotypes and AD risk. Haplotype OR 95% CI P I^2^ ----------- ------ ------------ ------ ------ CTT 0.58 0.31--1.08 0.09 0 TTC 1.99 1.24--3.18 0.00 0 TTT 0.90 0.56--1.44 0.65 55.0

{ "pile_set_name": "PubMed Central" }

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ANNOUNCEMENT {#s1} ============ Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV), which leads to mild to severe watery diarrhea, vomiting, and dehydration in pigs of all ages and a high mortality rate in neonatal piglets ([@B1], [@B2]). PEDV is an enveloped single-stranded RNA virus that belongs to the genus *Alphacoronavirus* of the family *Coronaviridae* ([@B3], [@B4]). Although PED has been reported in China since the 1980s, it was sporadic and regional before 2010 ([@B1]). In 2010, a large-scale outbreak of PED with high morbidity and mortality swept across China, leading to enormous economic losses ([@B5][@B6][@B7]). In February 2018, a stool sample obtained from a naturally infected piglet from Zhejiang Province in China was processed and determined to be positive for PEDV by reverse transcription-PCR (RT-PCR) with primers targeting the N gene. A PEDV strain named ZJ/ZX2018-C10 was isolated using Vero cell cultures as previously described ([@B8]). Viral RNA was extracted from the cell supernatant using the Macherey-Nagel viral RNA purification kit (Macherey-Nagel GmbH & Co. KG, Germany) according to the manufacturer's instructions. To determine the complete genomic sequence of the PEDV isolate of strain ZJ/ZX2018-C10, the extreme 5′ and 3′ termini were acquired by rapid amplification of cDNA ends (RACE, Clontech, Japan). Subsequently, a total of 13 overlapping fragments were amplified by RT-PCR using PrimeSTAR hot-start (HS) DNA polymerase (TaKaRa, China) with primers ([Table 1](#tab1){ref-type="table"}) based upon the sequences of PEDV strain CV777 (GenBank accession number [AF353511](https://www.ncbi.nlm.nih.gov/nuccore/AF353511)). The PCR products were analyzed and confirmed on 1% agarose gel and cleaned with a DNA fragment purification kit (TaKaRa, China). Purified PCR products were cloned into the pMD19-T vector and sequenced in both directions with the Sanger sequencing method (Biosune Biotechnology Shanghai Co., China) on the ABI 3730xl DNA analyzer platform (Life Technologies). For each amplification, three to five individual clones were sequenced to determine the consensus sequence of any given genomic region. Sequence contigs with the consensus sequence were assembled into the full-length genome for the PEDV isolate of strain ZJ/ZX2018-C10 using Lasergene version 7.10 (DNAStar, Inc., USA). ###### Primers used for amplifications of the PEDV genomic fragments Primer name Sequence (5′ to 3′) Position[^*a*^](#ngtab1.1){ref-type="table-fn"} Product size (no. of nucleotides) ------------- ------------------------------ ------------------------------------------------- ----------------------------------- 1F GCGTTCCGTCGCCTTCTACA 190--209 2,562 1R CAGGAATCTGGAAGACACTTGCA 2751--2729 2F GTATTATGCCACCAGTGTCCCA 2663--2684 2,295 2R CAGTTGCCAGCAGGCACTGT 4957--4938 3F ACCAGCGGTGCATTGCTTGA 4887--4906 2,589 3R CAATGTGCTCTTGCAATCCTGCA 7475--7453 4F CTGTTAAGTTAGTGGACTCAGCGT 7327--7350 2,549 4R ACTAGCGCCTTCAACTTGCA 9875--9856 5F GCGCTTGTGGTTCACCTGGT 9712--9731 2,547 5R GGATCCACAGCGAAAGCGCA 12259--12240 6F ACGCTTGCAGGCTGGTAAACA 12182--12202 2,281 6R TGGGCAGTGCTCTATCGCACT 14462--14442 7F ATACTAGGGGCGCTTCGGTT 14322--14341 2,459 7R GTCAGGGTGCACAGGAATGAA 16780--16760 8F GTATGTGTGCCCTTAAGCCTGAT 16662--16684 2,341 8R GTAAGTGGACGTTCGGCTTCATA 19002--18980 9F TGTATGCCAAGCGTAAGGTAGGAC 18888--18911 1,816 9R ATCTTGTGGTAGGCTGAGTGTTGG 20703--20680 10F GAAGAATGGTAAGTTGCTAGTGCGTA 20563--20588 2,130 10R GGCTAACAACTGTCCAGAATCAGATG 22683--22658 11F AAAGGTGAGTTGATTACTGGCACG 22498--22521 2,164 11R CTAGTAATGACACAACAAAGATGAGAAC 24664--24637 12F GTGTACGATCCTGCAAGTGGC 23272--23292 2,439 12R TCACCTCATCAACGGGAATAGA 25715--25694 13F TCGTCCAATTGGTTAATCTGTGC 25535--25557 2,306 13R TACCGTTGTGTGCAAGACCAA 27840--27820 5′ RACE TCCACTAGCGGCGGCCTCAGAATA 410--387 185 3′ RACE TCAACGAGATCTTCGATACAGGAA 27671--27694 193 Positions correspond to strain CV777 (GenBank accession number [AF353511](https://www.ncbi.nlm.nih.gov/nuccore/AF353511)). The complete genome sequence of strain ZJ/ZX2018-C10 is 28,035 nucleotide (nt) bases long, excluding the poly(A) tail, with a G+C content of 41.72%. The open reading frames for ZJ/ZX2018-C10 were predicted using Genome Annotation Transfer Utility with PEDV strain CV777 as the reference genome. The genomic organization of the virus is in the following order: 5′ untranslated region (UTR), nucleotide 1 to 292; replicase polyprotein, nucleotide 293 to 12616 for 1a and nucleotide 12616 to 20637 for 1b; spike (S) protein, nucleotide 20634 to 24791; open reading frame 3 (ORF3), nucleotide 24791 to 25465; envelope (E) protein, nucleotide 25446 to 25676; membrane (M) protein, nucleotide 25684 to 26364; nucleocapsid (N), nucleotide 26376 to 27701; and 3′ UTR, nucleotide 27702 to 28035. The complete genome sequence of ZJ/ZX2018-C10 shares 98.4%, 97.1%, and 96.3% nucleotide sequence identities with strain AH2012 (GenBank accession number [KC210145](https://www.ncbi.nlm.nih.gov/nuccore/KC210145)), strain virulent DR13 (GenBank accession number [JQ023161](https://www.ncbi.nlm.nih.gov/nuccore/JQ023161)), and vaccine strain CV777 (GenBank accession number [KT323979](https://www.ncbi.nlm.nih.gov/nuccore/KT323979)), respectively. Interestingly, strain ZJ/ZX2018-C10 possesses one unique deletion site compared to strain AH2012 (nucleotide 24225 to 24227) in the S gene, resulting in a single-amino-acid deletion (^1198^T). Of note, the deletion site is in the S2 domain of the S protein. These findings suggest that ZJ/ZX2018-C10 is a novel PEDV variant. The genome information of strain ZJ/ZX2018-C10 will promote a better understanding of the evolutionary characteristics and molecular epidemiology of PEDV in China. Data availability. {#s1.1} ------------------ The genome sequence of PEDV strain ZJ/ZX2018-C10 has been deposited in GenBank under the accession number [MK250953](https://www.ncbi.nlm.nih.gov/nuccore/MK250953). This work was supported by the Zhejiang Science and Technology Key Research and Development Project (2018C02028) and the National Natural Scientific Foundation of China (31802235). [^1]: **Citation** Su F, Yu B, Li J, Xu L, Yuan X. 2019. Complete genome sequence of variant porcine epidemic diarrhea virus strain ZJ/ZX2018-C10, isolated in Zhejiang, China, in 2018. Microbiol Resour Announc 8:e00048-19. <https://doi.org/10.1128/MRA.00048-19>.

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Correction to: *Scientific Reports* 10.1038/s41598-017-14467-w, published online 25 October 2017 The original version of this Article contained a repeated typographical error in the Abstract and Introduction where, 'Gg' now reads: 'Tg' This has now been corrected in the PDF and HTML versions of the Article.

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Introduction {#Sec1} ============ Medieval Central Asia (ca. AD 2^nd^--16^th^ c.) was a locus of extensive cultural and economic interaction between East Asia, the Middle East, and Europe through a vast network of overland trade routes, commonly called the 'Silk Roads'^[@CR1]--[@CR4]^. Urban centers, located in fertile oases and often described by archaeologists and historians alike as cosmopolitan cities^[@CR5],[@CR6]^, helped anchor Silk Road exchange and foster early globalization across Asia^[@CR7],[@CR8]^. Although settled communities provided a substantial economic foundation through agricultural output and the manufacture of valuable craft commodities such as metals, ceramics, glass, and textiles^[@CR9]--[@CR14]^, mobile pastoralists also had strong influence on the trade system as operators of highland pathways that were based on seasonal movements for herding livestock^[@CR15]^. While transfers of objects and materials have long represented the intensity and scope of Silk Road exchange^[@CR16]^, we still lack detailed data about the way food systems, which reflect sustained engagements with the environment and broader community dynamics, were influenced by these far-reaching economic networks. Medieval Central Asia was defined by unusually diverse multicultural intersections, sudden social upheavals, and frequent demographic movements (Supplementary Information [1](#MOESM1){ref-type="media"}), but fundamental and perhaps durable dealings with food remain unclear. Food is culturally expressive of ecological adaptation, social relations, ideology, and economy^[@CR17]--[@CR21]^. Resolving dietary diversity in ancient Central Asian foodways provides an opportunity to investigate subsistence models of 'nomadic' and 'urban' communities that, together, played key roles in transcontinental interaction across the Silk Roads. In Central Asia, which is characterized by strong seasonal climate and uneven distribution of resources on the landscape^[@CR22],[@CR23]^, there is high potential for dietary diversity among pastoralist communities. Mobile pastoralists heavily subsist on livestock herding but also draw from a variety of food resources beyond domesticated animal products, including cultivated cereals, wild plants, fishes, and hunted game, which are exploited and consumed at varying intensities depending on environment, seasonal availability, mobility, and social networks^[@CR24]--[@CR28]^. On the other hand, medieval urban food systems were strongly invested in cereal agriculture, food storage, and sedentism^[@CR29],[@CR30]^, and were subject to powerful political and religious institutions^[@CR7]^, which may have generated less diverse dietary repertoires. However, dynamic commercial, political, and social activities between population centers and peripheral settlements, in addition to transactions with pastoralists, could have greatly expanded food availability and choice, as people and provisions, such as grains and live animals, regularly moved between urban and nomadic domains^[@CR31]--[@CR35]^. We consider community-level dietary breadth over long periods of human life history to be a marker of dietary connectivity, which represents the cultural integration of food production, distribution, and consumption among individuals. Through globalization processes, which involve growing economic networks between increasingly distant communities, cultural differences diminish as groups cooperate and synchronize their consumption patterns, whether of foods, styles, or ideas^[@CR36]^. In contemporary societies, globalized food economies expand with production standardization and increased dietary uniformity^[@CR37],[@CR38]^. Along these lines, high intra-community dietary variability indicates that community members maintained divergent connections to food resources that express individual dietary preferences and group partitioning. Conversely, low dietary variability within communities signals converged trajectories of foods that reflect shared dietary practices and socio-economic coordination. In order to establish diversity in human dietary intake in Central Asian urban and nomadic communities, we analysed the carbon and nitrogen stable isotopic composition of bone collagen from human remains of 74 individuals (Table [1](#Tab1){ref-type="table"}). Carbon and nitrogen stable isotope ratios (δ^13^C and δ^15^N) provide an integrative measure of dietary intake^[@CR39],[@CR40]^, and human bone collagen reflects a 10--15 year rolling average of protein consumption^[@CR41]--[@CR43]^. We model isotopic niches in bi-variate isotopic space (δ-space) to estimate the breadth and structure of community-level diets based on intra-group variation across both dimensions of isotopic ratios simultaneously^[@CR44],[@CR45]^. Food resources with distinct isotopic content, which are incorporated into individual diets in various proportions, drive community dietary breadth^[@CR46]^. The isotopic niche modelling uses Bayesian inference to fit standard ellipses to data points that are then expressed as probability distributions of area (‰^2^) and position in δ-space^[@CR47]^. Crucially, we performed redundancy analysis to unravel isotopic diversity driven by culturally defined food choices from environmental variables that influence the isotopic composition of food resources.Table 1Geographic and archaeological information about analysed sites, including number of human samples. Detailed archaeological information for each site is provided in Supplementary Information [3](#MOESM1){ref-type="media"}. \*See methods.CountryRegionSiteElevation (m.a.s.l.)ChronologynArchaeological contextReferencesUzbekistanWest Pamir-AlayTashbulak21009th--11th c.4Highland urban complex of the Qarakhanid Empire; citadel, metal workshops, necropolis; 7 ha^[@CR86],[@CR120]^Alyntepe47510th--13th c.1Provincial city with fortified walls and surrounding settlements; industrial scale brick and ceramic production; 40 ha^[@CR121]^Frinkent53010th--13th c.4Fortress complex with cemetery of unique Zoroastrian burials in large ceramic vessels; 14 ha^[@CR122],[@CR123]^Ferghana ValleyChor Dona58011th--13th c.4Fortress mound with associated grain processing facility and ancillary ancient settlement of Andijan; estimated 10--15 ha^[@CR124],[@CR125]^Chartok54512th c.11Context information is not available^[@CR126]^Tashkent OasisUturlik27012th c.9Large city with diverse economic production of crafts; along trade routes with Otrar; 60 ha^[@CR34],[@CR127]^KhoresmTok-kala609th--12th c.9Urban fortress and surrounding settlements that functioned as a regional centre of political and economic influence; estimated 10--15 ha^[@CR128]^KazakhstanOtrar OasisKonyr-tobe I1805th--7th c.9Cemetery platform raised 2.5 m above ground level on the outskirts of a fortress; burials suggest nomadic traditions^[@CR81],[@CR129]^Temirlanovka3202nd--4th c.4Cemetery unassociated with a settlement containing burials of nomadic individualsSI 3Zhetysu (Semirech'ye)Turgen II10402nd--6th c.7Settlement and burial complex with nomadic occupations from the late Bronze Age to medieval period; estimated \< 5 ha^[@CR79]^Butakty II115010th--12th c.6Settlement and burial complex with nomadic occupations from the late Bronze Age to medieval period; estimated \< 5 ha^[@CR80],[@CR130],[@CR131]^Karatal6208th--11th c.3Cemetery complex in use from the late Bronze Age to historical period; numerous nomadic encampment structures^[@CR84]^; SI 3TurkmenistanDehistan PlainGeotchik Depe130Iron Age\*2Urban complex with occupation from the early Iron Age to late Islamic period; 5.5 ha^[@CR102],[@CR132],[@CR133]^Misrijan17011th--12th c.1One of numerous large villages in the Misrijan Oasis; precise site is not reported in the literature^[@CR102],[@CR132],[@CR133]^ Isotopic variation in Central Asia {#Sec2} ---------------------------------- We sampled 14 cemeteries associated with medieval Silk Road communities that span a long transect of Central Asian geography to include present-day Kazakhstan, Uzbekistan, and Turkmenistan (Fig. [1](#Fig1){ref-type="fig"}). The human individuals analysed in this study represent urban and non-urban consumers, who potentially had access to substantial food options that were available through prolific agricultural systems and marketplaces that drew in people and foodstuffs from oasis, desert, steppe, and highland environments (Supplementary Information [2](#MOESM1){ref-type="media"}). Diverse food remains were recovered from the sites represented in this study, which included cereals, legumes, fruits, fish, and livestock (Supplementary Information [3](#MOESM1){ref-type="media"}).Figure 1Map of Central Asia showing sites and regions with human stable isotopic data (δ^13^C and δ^15^N) analysed in this paper. Uzbekistan: 1) Tok-kala, 2) Uturlik, 3) Chor Dona, 4) Chartok, 5) Tashbulak, 6) Altyntepe, 7) Frinkent; Turkmenistan: 8) Geoktchik Depe, 9) Misrijan; Kazakhstan: 10) Konyr-Tobe, 11) Temirlanovka, 12) Turgen, 13) Butakty, 14) Karatal. Map generated with Quantum GIS, version 2.18.2 (<https://www.qgis.org>), using public domain data from Natural Earth (<http://www.naturalearthdata.com>). Pronounced variation in the regional environments and topographies, combined with strong seasonality, in which these sites are situated, confers high isotopic variation in Central Asian foodwebs accessed by people. Vegetation communities range from cool montane meadows and forests consisting of largely C~3~ taxa to hot lowland deserts that support both C~3~ and C~4~ plants^[@CR48]--[@CR51]^, which exhibit (pre-modern^[@CR52],[@CR53]^) δ^13^C values of −25 ± 2--5‰ and −11 ± 1‰, respectively^[@CR54]--[@CR57]^. Food crops exploited in medieval Central Asia included the full spectrum of Eurasian domesticates including C~3~ taxa, such as wheat, barley, rice, nuts, and fruits, and C~4~ taxa, such as millets^[@CR58]^. Although nitrogen isotopic variation in vegetation communities in Central Asia is under-characterized, research in ecosystems comparable to those of Central Asia, such as the Gobi steppe-desert^[@CR59]^, the steppe deserts of the Caspian Depression^[@CR60]^, and semi-arid western Loess Plateau^[@CR61]^, demonstrate wide variation in plant δ^15^N values, ranging from −5 to 14‰, due to differences in local soil nitrogen pools and animal stocking rates^[@CR62]--[@CR65]^. In general, fish exhibit high δ^15^N values relative to terrestrial fauna^[@CR66],[@CR67]^, and in Central Asia, fish exhibit an apparent continuum of δ^13^C values from ca. −11.5‰ to −27‰^[@CR68]^. Results {#Sec3} ======= Human remains in this study represent two chronological intervals of the medieval period. The bulk of the dataset (n = 63) dates to a 'mid-late' period of 6^th^--13^th^ c., which were recovered from sites in southern Kazakhstan, Uzbekistan, and western Turkmenistan. A small sample (n = 11) dates to an 'early' period of nomadic occupation at sites in southern Kazakhstan, which allows for a diachronic comparison of nomadic dietary intake within this region. Overall, human isotopic values range from ca. −20‰ to −10.5‰ for δ^13^C and 9‰ to 15‰ for δ^15^N (Fig. [2a](#Fig2){ref-type="fig"}). Isotope values cluster in both δ^13^C and δ^15^N for urban communities on a regional basis, while pastoralist communities from later periods exhibit wide distributions of δ^13^C values and relatively narrow ranges of δ^15^N values. Subsequent analyses using Bayesian inference clarified isotopic differences among communities while factoring in uncertainty due to small sample sizes. Summary statistics of isotopic data are provided in Supplementary Figure [S1](#MOESM1){ref-type="media"} and Table [S1](#MOESM1){ref-type="media"}; raw isotopic data are provided in Supplementary Tables [S2](#MOESM1){ref-type="media"}--[S4](#MOESM1){ref-type="media"}.Figure 2(**a**) Human carbon and nitrogen isotopic ratios from medieval Central Asia; (**b--c**) Posterior probability distributions of isotopic means obtained by Bayesian bootstrapping (mean~b~) from medieval urban communities in Uzbekistan and Turkmenistan and (**d**-**e**) from medieval nomadic communities in southern Kazakhstan. Human isotopic variation across medieval Central Asia {#Sec4} ----------------------------------------------------- Significant distinctions in the dietary intake of medieval urban and nomadic communities across Central Asia are revealed by Bayesian means (mean~b~) of δ^13^C and δ^15^N values. Sharp dissimilarities are observed between urban communities located south of the Syr-Darya river, which provides a rough environmental boundary between the semi-arid/mountain steppe landscapes in southern Kazakhstan and sandy desert landscapes interspersed with oases and foothill zones to the east. Significant differences in the 95% CIs of isotopic values from each southerly community, with the exception of West Pamir-Alay and Tashkent, are present with mean~b~ δ^13^C values of −19.9‰ for Dehistan, −18.1‰ for West Pamir-Alay, −17.5‰ for Tashkent, −16.3‰ for Ferghana, and −13.3‰ for Khoresm (Fig. [2b](#Fig2){ref-type="fig"}; Information 4). Regional communities in Uzbekistan and Turkmenistan fall into in three trophic groups, each separated by ca. 2‰ in mean~b~ δ^15^N (Fig. [2c](#Fig2){ref-type="fig"}). A diachronic shift in the dietary intake of nomadic communities located in southern Kazakhstan is indicated by a significant 3‰ decrease in mean~b~ δ^13^C values between early and late Otrar (Fig. [2d](#Fig2){ref-type="fig"}; Supplementary Information [4](#MOESM1){ref-type="media"}) and an increase of ca. 2‰ in mean~b~ δ^13^C between early and late Zhetysu. However, mean~b~ δ^13^C for late Zhetysu has a wide 95% CI between −15.9 and −12.3‰, which precludes a reliable estimate of the change. Overall, mean~b~ δ^15^N for communities in southern Kazakhstan are more mutually similar to each other than that in southerly regions (Fig. [2c,e](#Fig2){ref-type="fig"}). Early Otrar displays mean~b~ δ^15^N that is spaced apart by slightly less than ca. 1.7‰ from that of early Zhetysu, while in the later period the regions have identical mean~b~ δ^15^N of ca 12‰. Environment and isotopic variation {#Sec5} ---------------------------------- Isotopic patterns observed at individual archaeological sites are not driven by environmental inputs. Redundancy analysis between mean δ^13^C and δ^15^N values per cemetery site and 25 environmental parameters of ecologically relevant rainfall and temperature variations, in addition to elevation and soil properties, did not result in statistically significant relationships. Multiple linear regressions were also performed, which further failed to generate statistically significant relationships. (See Supplementary Information [5](#MOESM1){ref-type="media"} for methods and results.) Isotopic niche modelling {#Sec6} ------------------------ Central Asian medieval communities display isotopic niches that cluster in two distinct size ranges (Fig. [3](#Fig3){ref-type="fig"}; Supplementary Information [6](#MOESM1){ref-type="media"}). Group 1 exhibits small areas between 0.1 and 3.7‰^2^ for urban communities in Uzbekistan and Turkmenistan, as do nomadic communities in southern Kazakhstan from the early medieval period. Over the next several hundred years, dietary diversity among individuals in Zhetysu and the Otrar Oasis (Group 2) appears to have radically increased, with isotopic niches estimated between 2.7 and 15.7‰^2^. Notably, the greatest intra-community dietary diversity is visible in late Zhetysu, indicated by a 95% CI exceeding that from Group 1 communities, except early Otrar, which slightly overlaps by 0.7‰^2^.Figure 3Community-level dietary diversity of medieval humans represented by posterior distributions of core isotopic niche area (‰^2^) by sites and regions in Central Asia. Isotopic niches were calculated by fitting standard ellipses to cover ca. 39% of the δ^13^C and δ^15^N data points using Bayesian inference. Black dots indicate area means, and the shaded boxes, from dark to light, represent the 50%, 75%, and 95% credible intervals. While all narrow, modelled isotopic niches for medieval urbanites in Uzbekistan are highly unique in orientation and position in δ-space (Fig. [4a](#Fig4){ref-type="fig"}). For late Zhetysu and Otrar, the diffuse spacing of isotopic values indicates exceedingly varied dietary intake. Individuals in late Zhetysu, which represent two archaeological sites, fall within the isotopic niches for Khoresm, Ferghana and Tashkent. Likewise, individuals in late Otrar, which represent one site, span all four urban isotopic niches from Uzbekistan. In contrast, there is substantial proportional overlap between the small isotopic niches from early Zhetysu and Ferghana and also early Otrar and Khoresm, respectively (95% CI: 0.13--0.64 and 0.17--0.69; Supplementary Fig. [S6](#MOESM1){ref-type="media"}), which indicates a high likelihood of dietary parity.Figure 4(**a**) Medieval urban isotopic niches from Uzbekistan are displayed as probability clouds, and individual isotopic values from southern Kazakhstan (nomadic communities) and western Turkmenistan (urban community) are represented as points. (**b**) Isotopic niche overlap analysis for urban communities in Uzbekistan. Standard ellipses covering 95% of δ^13^C and δ^15^N values were modelled using Bayesian inference. Overlapping areas for each pairwise comparisons in δ-space were visualized as probability clouds with underlying isotopic data points superimposed (lower left). Area overlap of total isotopic niche area for each pair was plotted as probability distributions (upper right). Isotopic niches from urban communities in Uzbekistan illustrate strong dissimilarity from each other (Fig. [4b](#Fig4){ref-type="fig"}). The highest proportion of niche overlap occurs between West Pamir-Alay and Ferghana (95% CI: 0.05--0.36) and between West Pamir-Alay and Tashkent (95% CI: 0.01--0.32), suggesting that a maximum of one-third of individuals in these communities had similar dietary intake. However, this niche overlap could be as low as 1--5%. The remaining pairwise comparisons of urban communities show negligible occurrence of overlap (Supplementary Information [7](#MOESM1){ref-type="media"}). Discussion {#Sec7} ========== Taken together, urban and nomadic communities display distinctive but wide-ranging isotopic values that are strongly suggestive of diverse dietary intake across medieval Central Asia. A large overall range of human δ^13^C values from ca. −20 to −11‰ is likely due to individuals consuming mostly C~3~ crops (wheat, barley, and rice) or C~4~ millets at sustained intensities. Among Eurasian cultigens, millets are isotopically distinct with high ^13^C concentration. Millets thrive in hot and arid climate and exhibit fast-growing and drought-tolerant adaptations^[@CR69]^, traits which would have provided Central Asian farmers and mobile pastoralists opportunities for low risk, low-investment cultivation in marginal agricultural areas^[@CR70],[@CR71]^. The natural abundance of C~4~ vegetation is substantially higher in the desert zones of Central Asia^[@CR50]^, where livestock, as part of urban or nomadic subsistence, could have accessed enriched δ^13^C biomass and provided human consumers with protein-dense foods (meat and milk) with high δ^13^C values. A lack of correlations between human isotopic values and site environmental parameters suggests that dietary intake across the region was shaped primarily by food choice. Medieval agriculture in Central Asia, which used complex crop schedules and large irrigation works^[@CR71]--[@CR76]^, likely enabled productivity to overcome environmental constraints on crop variety in order to meet the inter-connected dietary demands of consumers. Modelled isotopic niches indicate that nomadic communities exploited a wide variety of dietary resources, while urban communities engaged in more limited dietary repertoires. The small isotopic niche sizes documented among urban communities in Uzbekistan and Turkmenistan suggest food channels that were shaped via consistent and insular dietary connectivity. On the other hand, large isotopic niches in southern Kazakhstan from a relatively contemporaneous period indicate that a multitude of food acquisition strategies were in use, by which communities tied more closely with pastoral nomadic lifeways comprised individuals with assorted dietary relationships that led to sustained differences in isotopic variability. The dietary connectivity for these nomadic groups may have fostered group partitioning through unsynchronized food interactions among different community members. The small isotopic niches for nomadic communities in early Otrar and Zhetysu, which in this case substantially overlap with that for Khoresm and Ferghana, suggesting more restricted dietary intake by pastoralists, emphasize the subsistence plasticity of pastoral nomads who readily contour their own food production and interaction networks in response to dynamic social and natural landscapes^[@CR24],[@CR26],[@CR77]^. The diachronic shift in isotopic niche size between early and late nomadic communities also highlights two distinct scales of dietary variability that illustrate the importance of multi-resource pastoralism to Silk Road interactions. In southern Kazakhstan, the early medieval period is marked by a growth of urban centres, villages, and agricultural economies^[@CR78]--[@CR81]^, which is also historically associated with frequent conflict among nomadic confederacies that instigated socio-political turmoil^[@CR1],[@CR82]^. In order to mitigate risk and take advantage of economic opportunities presented by these newly founded centres, nomadic communities likely participated in coordinated subsistence interactions with settled populations over short distances, which would have effectively limited access to diverse food resources and thus narrowed their dietary breadth. During the strengthening of Turkic empires several centuries later^[@CR1],[@CR3],[@CR31],[@CR83]^, Silk Road trans-regional trading expanded to include bulk commodities and raw materials^[@CR32]^, and nomadic communities across southern Kazakhstan expressed wide dietary breadth, as indicated by large isotopic niche sizes. One explanation for greater inter-individual dietary diversity during this later medieval period is that nomadic communities tapped into growing trade economies as agents of food exchange and broke out of insular urban subsistence channels. Recent excavations of nomadic encampments in the foothill zones of West Pamir-Alay and Zhetysu illustrate highly variable levels of economic interaction between pastoralists and urban centres. At these sites, cultural materials associated with 8^th^--13^th^ c. radiocarbon chronologies include cotton fabrics^[@CR84]^ and variable mixtures of ceramics ranging between standardized wheel-spun food storage vessels from distant oases communities and locally produced 'handmade' coarsewares, which are rare in urban contexts^[@CR85],[@CR86]^. The presence of hybrid ceramic assemblages and cotton, a woven trade good associated with oasis production centers^[@CR87],[@CR88]^, suggests that complex and non-uniform relationships with urban economies coincided with intra-group dietary diversity in mobile pastoralist communities^[@CR25]^. Accordingly, at the community level, a second scale of dietary variability that is representative of multi-resource pastoralism is observed at late Otrar and Zhetysu, which display wide ranges of δ^13^C values (Fig. [4a](#Fig4){ref-type="fig"}). Some individuals in late Zhetysu have δ^13^C values similar to those commonly observed in humans from prehistoric millet-based farming societies in China, where millets were domesticated^[@CR89]--[@CR91]^, while individuals in late Zhetysu and Otrar had low δ^13^C values, typical of Neolithic and early Bronze Age humans before millet spread to the C~3~-dominant Central Asian steppe^[@CR92]^. Both communities in late Otrar and Zhetysu display similar δ^15^N distributions, though individuals exhibit differences in dietary intake of relative proportions of meat and dairy products (Fig. [2e](#Fig2){ref-type="fig"}). Together, these findings suggest that dietary connectivity at the steppe margins was associated with an ecumene of diverse food consumption, in which individuals maintained separate subsistence strategies as they simultaneously participated in a common nomadic ethos. Compared to urbanites, mobile pastoralists likely maintained closer control of food production and distribution, allowing them to eat according to food preferences, which may have been less important for maintaining social ties than in urban contexts. The combination of narrow dietary niches with isotopic distinction in human remains from medieval urban communities south of the Syr-Darya river is due to trophic-level variation in the intensity of meat and cereal consumption between communities as well as differences in the contribution of millet to urban diets, either eaten directly or indirectly obtained from meat and dairy of animals foddered with millet. Isotopic niche overlap was highest for urban communities in close geographic proximity to one another, a pattern that suggests neighbouring communities either participated in similar food traditions or agricultural practices that were confined to small catchments. These communities may have participated in limited inter-regional trading of staple foods, which likely would have moved through established subsistence channels as if locally produced. There is also the possibility that dietary connectivity in urban contexts was subject to bureaucratic intermediaries, which exerted influence through land tenure and taxation^[@CR1],[@CR32],[@CR93]^. Alternatively, in the absence of top-down control, food exchange networks may have steadily channelled provisions to urbanites as a reflection of other economic networks that inevitably developed to be streamlined towards cultural insiders in cosmopolitan contexts. While the consensus among historians and archaeologists is that urbanites in medieval Central Asia dwelled in rich multicultural settings^[@CR5],[@CR7],[@CR16]^, there appears to be a limit to this diversity in dietary intake as revealed through isotopic niche modelling. Distinctions in food choice and diet between urban communities suggest regional food repertoires were narrowly circumscribed, at least between C~3~ and C~4~ crops and also between animal and plant protein. Regional patterns in diet imply that cultural differences surrounding foods may have been surprisingly diminished within urban communities, which runs counter to the notion of collective cosmopolitanism in medieval Central Asia. Medieval urbanites in Central Asia maintained inward-focused dietary connectivity that likely generated a localized social cohesion through culturally integrated supply chains for consumers. Scholars also associate Silk Road activity with early globalization processes^[@CR7],[@CR8]^, in which urban centres are viewed as the main drivers of cultural influence and outward economic connectivity, while 'nomads' are interpreted as antagonists to ancient civilization^[@CR94]--[@CR96]^. Yet, through multi-resource subsistence strategies, nomadic communities likely wielded flexible economic engagements that traversed open landscapes of contact with people who facilitated far-reaching connectivity. In this sense, nomadic individuals may have been more culturally interoperable and able to participate in, disengage from, and influence cultural spheres more easily than urban populations. Indeed, many of the pan-regional turnovers in religion, language, and political authority that resulted in changes in architecture, technologies, and other commodity classes in the medieval period are historically described as nomadic innovations^[@CR1],[@CR2],[@CR31]^, and essential routes that connected Silk Road sites in the highland regions of Central Asia were likely shaped by nomadic mobility^[@CR15]^. This study takes a new step toward resolving the complex interplay between urban and nomadic societies that are rarely available through archaeological datasets. Establishing dietary diversity provides an emerging understanding of food and connectivity along Central Asia's Silk Roads that highlights the significance of ancient nomadic pastoralists in bridging seemingly insulated urban centres. Materials and Methods {#Sec8} ===================== Human remains {#Sec9} ------------- We performed new analyses on human remains from Uzbekistan and Kazakhstan, which were selected based on 1) medieval chronology from ca. 8^th^--13^th^ c., 2) information on archaeological context, and 3) minimum age estimation of young adult. The majority of human remains analysed from Uzbekistan (n = 38) were excavated at various times over the past 80 years and do not include associated post-cranial elements. Human remains from Tashbulak (n = 4) were excavated in 2015 and are represented by complete inventories of skeletal elements. Human remains from Uzbekistan are stored in the Institute of Archaeology of the Uzbek Academy of Sciences in Samarkand, under the auspices of the Archaeology of the Qarakhanids Project (Co-PIs: Farhod Maksudov and Michael Frachetti). Human remains from Karatal (n = 3) were excavated in 2006 and 2008 and include incomplete skeletal elements due to ancient burial looting and modern erosion (Supplementary Information [3](#MOESM1){ref-type="media"}). Human remains from Karatal are stored at the Central State Museum of Kazakhstan in Almaty, under the auspices of the Dzhungar Mountain Archaeology Project (Co-PIs: Alexei Mar'yahshev and Michael Frachetti). A list of human samples is provided in Supplementary Tables [S2](#MOESM1){ref-type="media"}--[S4](#MOESM1){ref-type="media"}. Isotopic analysis {#Sec10} ----------------- Approximately 1--2 cm^3^ of sample were cut from dense cortical bone. Collagen extraction was performed in the Archaeological Stable Isotope Laboratory of Kiel University following Tuross *et al*.^[@CR97]^. Mass spectrometry for δ^13^C and δ^15^N was performed at the Boston University Stable Isotope Laboratory using a EuroVector Euro EA elemental analyser coupled with a GVI IsoPrime in continuous flow mode with an analytical error of 0.1‰ and 0.2‰ for δ^13^C and δ^15^N, respectively. Isotopic values are reported in permil (‰) relative to the Vienna Pee Dee Belemnite (VPDB) standard for δ^13^C and atmospheric nitrogen (AIR) for δ^15^N. Collagen samples with an elemental C:N ratio less than 2.9 or greater than 3.6 were considered diagenically altered and unsuitable for inclusion^[@CR98]--[@CR100]^. Failed samples (n = 3) are reported in Supplementary Table [S5](#MOESM1){ref-type="media"}. Previously published δ^13^C and δ^15^N values of human bone collagen from southern Central Asia were analysed (Supplementary Tables [S3](#MOESM1){ref-type="media"}-[S4](#MOESM1){ref-type="media"}). Isotopic data from medieval southern Kazakhstan included 13 samples from the Otrar Oasis (3^rd^--7^th^ c.) and 13 samples from southern Zhetysu (2^nd^--12^th^ c.)^[@CR101]^. Data from the Dehistan Plain in western Turkmenistan included two samples from the Iron Age (ca. 1300 BC) and one sample from the medieval period (ca. 11^th^--12^th^ c.)^[@CR102]^. These data were lumped together irrespective of chronology due to low sample size for the region and similar isotopic values, which were unique in our dataset. Human collagen is δ^13^C enriched by ca. 1--3‰ relative to the carbon isotope composition of consumed foods^[@CR103]^, whereas herbivores are enriched in δ^13^C by ca. 5‰ relative to consumed vegetation^[@CR104],[@CR105]^. Human nitrogen isotope values reflect the intensity of cereal versus meat consumption, as there is a 2--5‰ trophic enrichment in δ^15^N with each step up in the food web^[@CR40],[@CR106],[@CR107]^, but also reflect variation in food production systems that introduce exogenous nitrogen, usually in the form of manure, to the floral base of the food web, potentially imparting considerable nitrogen isotopic variation in agricultural and livestock food^[@CR62],[@CR63],[@CR65],[@CR108],[@CR109]^. In attempts to estimate the relative contribution of specific foods in diets, a common approach in archaeological dietary studies is to sample ancient and modern plants and animals to assess possible isotopic variability in food sources that result from various environmental and anthropogenic factors^[@CR110]^. For the purposes of establishing human dietary diversity at the community level, however, knowledge of the isotopic content of ancient food remains is unnecessary. Statistical analysis {#Sec11} -------------------- All statistical analyses were performed using R, version 3.4.0^[@CR111]^. Due to low sample sizes per site (1 ≤ n ≤ 11), δ^13^C and δ^15^N values were analysed by geographic region (3 ≤ n ≤ 15) using Bayesian techniques to quantify uncertainty and overcome issues with non-parametric data distributions, which cannot be reliably analysed with frequentist methods. The means of isotopic values for each region were calculated with a Bayesian bootstrapping method at 5000 iterations using the package bayesboot^[@CR112]^. Posterior distributions of means for each group were considered statistically different from one another if the 95% credible intervals (CI) did not overlap. Isotopic niche modelling {#Sec12} ------------------------ Isotopic niches were analysed using SIBER (Stable Isotope Bayesian Ellipses in R), version 2.1.3^[@CR47]^. The Markov chain Monte Carlo simulation was run with uniform priors 2,000,000 times, with the first 10,000 results discarded (burn-in), followed by a 1:100 thinning. The fitted ellipses express a posterior probability distribution of area (‰^2^) and position in δ-space. This technique is statistically advantageous for analysing communities of consumers, as fitted ellipses are less sensitive to sample size than other spatial metrics, such as convex hulls^[@CR47]^, and uncertainty is factored into estimates, such as sample size, which has previously challenged studies using a finite number of specimens from the archaeological record. Isotopic niches were compared among regional groups by using the Bayesian standard ellipse areas (SEA~b~) and the proportion of SEA~b~ overlap as total SEA~b~ pairwise for each region. Overlapping areas of isotopic niches indicate similarity in isotopic inputs and thus comparable resource exploitation^[@CR113]--[@CR115]^. Environmental modelling and GIS {#Sec13} ------------------------------- Interactive effects between human stable isotope values and environmental parameters at each site in 10 km and 50 km spatial buffers were explored using redundancy analysis (RDA) with the R package vegan, version 2.4--5^[@CR116]^. Environmental parameters included elevation, bioclim data^[@CR117]^, which are derived from monthly temperature and rainfall values from 1970--2000, and soil parameters obtained from the Soil Grid Project^[@CR118]^. (See Supplementary Information [5](#MOESM1){ref-type="media"} for RDA methods and results.) Data were mapped using Quantum GIS, version 2.18.2^[@CR119]^. Base imagery in Fig. [1](#Fig1){ref-type="fig"} was obtained from Natural Earth (<http://www.naturalearthdata.com>). Electronic supplementary material ================================= {#Sec14} Supplementary Information **Electronic supplementary material** **Supplementary information** accompanies this paper at 10.1038/s41598-018-22995-2. **Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. We especially thank Amridin Berdimuradov, the director of the Institute of Archaeology in Samarkand (National Academy of Sciences of Uzbekistan) for his continued support and enthusiasm for this project and international collaborations in general. We also thank Dima Voyakin for providing access to archaeological reports on Temirlanovka and for his help facilitating research in Kazakhstan. Recognition is also due to Marc Meyer and Mellissa Murphy for providing osteological data for the human individuals excavated from the Karatal cemetery complex. A special thank you goes to Malte Rühlemann for assisting with the redundancy analysis. Funding for this research was provided by the Graduate School 'Human Development in Landscapes' at Kiel University for the doctoral research of Taylor Hermes. Research at Tashbulak was funded by the National Geographic Society, the Max van Berchem Foundation, and Washington University in St. Louis. We are grateful for support from the Open Access Publikationsfonds of Land Schleswig-Holstein. Finally, we thank two anonymous reviewers for their suggestions on improving this paper. T.R.H. designed and initiated the research. T.R.H., E.A.B., F.M., and S.M. conducted sample collection. E.A.B. performed the osteological analysis of human remains and, with T.R.H., described sites in Uzbekistan for the supplementary information. T.R.H. wrote the remaining supplementary information. T.R.H. prepared specimens for stable isotope analysis and conducted the data analysis. T.R.H. and C.A.M., and M.D.F. interpreted the results. T.R.H., M.D.F., and C.A.M. wrote the manuscript. Competing Interests {#FPar1} =================== The authors declare no competing interests.

{ "pile_set_name": "PubMed Central" }

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22,199

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"representative", "pastoralism", "observed", "late", "Otrar", "Zhetysu", "display", "wide", "ranges", "values", "fig", "individuals", "late", "Zhetysu", "values", "similar", "commonly", "observed", "humans", "prehistoric", "farming", "societies", "China", "millets", "individuals", "late", "Zhetysu", "Otrar", "low", "values", "typical", "Neolithic", "early", "Bronze", "Age", "humans", "millet", "spread", "Central", "Asian", "communities", "late", "Otrar", "Zhetysu", "display", "similar", "distributions", "though", "individuals", "exhibit", "differences", "dietary", "intake", "relative", "proportions", "meat", "dairy", "products", "fig", "Together", "findings", "suggest", "dietary", "connectivity", "steppe", "margins", "associated", "ecumene", "diverse", "food", "consumption", "individuals", "maintained", "separate", "subsistence", "strategies", "simultaneously", "participated", "common", "nomadic", "ethos", "Compared", "urbanites", "mobile", "pastoralists", "likely", "maintained", "closer", "control", "food", "production", "distribution", "allowing", "eat", "according", "food", "preferences", "may", "less", "important", "maintaining", "social", "ties", "urban", "contexts", "combination", "narrow", "dietary", "niches", "isotopic", "distinction", "human", "remains", "medieval", "urban", "communities", "south", "river", "due", "variation", "intensity", "meat", "cereal", "consumption", "communities", "well", "differences", "contribution", "millet", "urban", "diets", "either", "eaten", "directly", "indirectly", "obtained", "meat", "dairy", "animals", "foddered", "millet", "Isotopic", "niche", "overlap", "highest", "urban", "communities", "close", "geographic", "proximity", "one", "another", "pattern", "suggests", "neighbouring", "communities", "either", "participated", "similar", "food", "traditions", "agricultural", "practices", "confined", "small", "catchments", "communities", "may", "participated", "limited", "trading", "staple", "foods", "likely", "would", "moved", "established", "subsistence", "channels", "locally", "produced", "also", "possibility", "dietary", "connectivity", "urban", "contexts", "subject", "bureaucratic", "intermediaries", "exerted", "influence", "land", "tenure", "Alternatively", "absence", "control", "food", "exchange", "networks", "may", "steadily", "channelled", "provisions", "urbanites", "reflection", "economic", "networks", "inevitably", "developed", "streamlined", "towards", "cultural", "insiders", "cosmopolitan", "contexts", "consensus", "among", "historians", "archaeologists", "urbanites", "medieval", "Central", "Asia", "dwelled", "rich", "multicultural", "appears", "limit", "diversity", "dietary", "intake", "revealed", "isotopic", "niche", "modelling", "Distinctions", "food", "choice", "diet", "urban", "communities", "suggest", "regional", "food", "repertoires", "narrowly", "circumscribed", "least", "crops", "also", "animal", "plant", "protein", "Regional", "patterns", "diet", "imply", "cultural", 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"chronology", "ca", "information", "archaeological", "context", "minimum", "age", "estimation", "young", "adult", "majority", "human", "remains", "analysed", "Uzbekistan", "n", "excavated", "various", "times", "past", "years", "include", "associated", "elements", "Human", "remains", "Tashbulak", "n", "excavated", "represented", "complete", "inventories", "skeletal", "elements", "Human", "remains", "Uzbekistan", "stored", "Institute", "Archaeology", "Uzbek", "Academy", "Sciences", "Samarkand", "auspices", "Archaeology", "Qarakhanids", "Project", "Farhod", "Maksudov", "Michael", "Frachetti", "Human", "remains", "Karatal", "n", "excavated", "include", "incomplete", "skeletal", "elements", "due", "ancient", "burial", "looting", "modern", "erosion", "Supplementary", "Information", "media", "Human", "remains", "Karatal", "stored", "Central", "State", "Museum", "Kazakhstan", "Almaty", "auspices", "Dzhungar", "Mountain", "Archaeology", "Project", "Alexei", "Michael", "Frachetti", "list", "human", "samples", "provided", "Supplementary", "Tables", "media", "media", "Isotopic", "analysis", "Approximately", "sample", "cut", "dense", "cortical", "bone", "Collagen", "extraction", "performed", "Archaeological", "Stable", "Isotope", "Laboratory", "Kiel", "University", "following", "Tuross", "et", "al", "Mass", "spectrometry", "performed", "Boston", "University", "Stable", "Isotope", "Laboratory", "using", "EuroVector", "Euro", "EA", "elemental", "analyser", "coupled", "GVI", "IsoPrime", "continuous", "flow", "mode", "analytical", "error", "respectively", "Isotopic", "values", "reported", "permil", "relative", "Vienna", "Pee", "Dee", "Belemnite", "VPDB", "standard", "atmospheric", "nitrogen", "AIR", "Collagen", "samples", "elemental", "C", "N", "ratio", "less", "greater", "considered", "diagenically", "altered", "unsuitable", "Failed", "samples", "n", "reported", "Supplementary", "Table", "media", "Previously", "published", "values", "human", "bone", "collagen", "southern", 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"region", "calculated", "Bayesian", "bootstrapping", "method", "iterations", "using", "package", "Posterior", "distributions", "means", "group", "considered", "statistically", "different", "one", "another", "credible", "intervals", "CI", "overlap", "Isotopic", "niche", "modelling", "Isotopic", "niches", "analysed", "using", "SIBER", "Stable", "Isotope", "Bayesian", "Ellipses", "R", "version", "Markov", "chain", "Monte", "Carlo", "simulation", "run", "uniform", "priors", "times", "first", "results", "discarded", "followed", "thinning", "fitted", "ellipses", "express", "posterior", "probability", "distribution", "area", "position", "technique", "statistically", "advantageous", "analysing", "communities", "consumers", "fitted", "ellipses", "less", "sensitive", "sample", "size", "spatial", "metrics", "convex", "uncertainty", "factored", "estimates", "sample", "size", "previously", "challenged", "studies", "using", "finite", "number", "specimens", "archaeological", "record", "Isotopic", 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Introduction {#Sec1} ============ Bone age assessment (BAA) is crucial in the evaluation of endocrine disorders and in the prediction of adult height when hormone therapy is the treatment \[[@CR1]\]. Radiographic examination is an easy and cost-effective method for the assessment of bone age. BAAs are performed using various body parts such as the hand, elbow, knee, cervical vertebra or pelvis \[[@CR2]\]. Hand radiography is certainly the most widely used examination \[[@CR3]\]. Several methods have been proposed, including Greulich-Pyle (GP) atlas, Tanner-White (TW) score and Gilsanz-Ratibin (GR) atlas, which are qualitative \[[@CR4]\]. The TW method is based on the level of maturity for 20 regions of interest (ROIs), including the epiphysis, metaphysis and diaphysis in the radius, ulna, first, third and fifth fingers and the carpal bones, so it is complex and time-consuming. The GR method is another technique that uses the new digital atlas that includes reference images of the left hand taken at 6-month intervals from birth to age 6 years, and at 1-year intervals from age 7 to 17 years. However, this subjective method relies on matching of the hand radiograph of the subject with the GR atlas. In the GP method, bone age is determined by matching the left-hand radiograph of the subject with reference radiographs from the atlas. This explains why the GP method is generally easier and quicker to use, so it is more often selected over other methods \[[@CR5]--[@CR8]\]. One disadvantage of the GP method is the characteristics inherent in subjective analysis, and the interobserver reproducibility of BAA with the GP method has been controversial \[[@CR5], [@CR7]\]. A quantitative evaluation method with objective parameters is needed. BAA by using the capitate and hamate, which are the first bones to develop embryologically, has been recommended by a previous study but not performed as of yet \[[@CR9]\]. In addition, validation of BAA across different ethnicities would be necessary for a more reliable method \[[@CR10]\]. Therefore, the aim of this study was to determine if the area of the capitate and hamate bones measured on hand radiography could be a reliable indicator of bone age accuracy, precision, and reproducibility between the planimetry and GP methods. Materials and methods {#Sec2} ===================== Populations {#Sec3} ----------- In this study, all data were collected from the electronic medical records of patients. From January 2014 to December 2015, 423 patients (age 1--180 months) presented to the emergency department. The study population was selected according to the following inclusion criteria: PA radiography of both hands, age younger than 15 years and belonging to a single ethnic group. The following patients were excluded: those with either only right-hand (n=42, 30 boys and 12 girls) or only left-hand (n=33, 24 boys and nine girls) PA radiograph, one with acute leukaemia (one boy) one with idiopathic hypertrophic pyloric stenosis (one boy) and those with other ethnicities (one Chinese, one Japanese and one American) \[[@CR1], [@CR10]\]. Finally, a total of 391 consecutive patients (242 boys and 149 girls) were enrolled in this study. In total, we analysed 782 hands of 391 patients (242 boys and 149 girls). The chronological age, sex and ethnicity of the patients were recorded. The mean patient age was 5 years and 8.52 months (age range 1--180 months). The patients complained of contusion (n=222), laceration (n=175) and/or fracture (finger, 40; humerus, 54). The absence or presence of capitate and/or hamate bones of the hand was registered in all patients. According to the growth chart published by the Korea Centers for Disease Control and Prevention (CDC) in 2007, among patients aged 1 month to 3 years, those whose height was between the 50th percentile of a child who is 1 month younger and the 50th percentile of a child who is 1 month older were selected \[[@CR11]\]. Among patients aged 3--14 years, those whose height was between the 50th percentile of a child who is 6 months younger and the 50th percentile of a child who is 6 months older were selected \[[@CR12]\]. A total of 109 children (mean age 113.07 months; standard deviation 36.07) were included in the 50th percentile group. Equipment and imaging protocol {#Sec4} ------------------------------ Hand radiography was performed using Innovion-SH (DK Medical Systems, Seoul, Korea). The patients were seated alongside or facing a table, and examined in the PA position. The protocols used in this study for taking the PA radiographs of both hands are described in Table [1](#Tab1){ref-type="table"}. Both hands were in neutral position with no flexion, extension or deviation, and were placed palm down on the cassette with the fingers extended \[[@CR13]\].Table 1Imaging protocol of both hands posterior-anterior (PA) x-rayProjectionPosterior-anterior bilateral projectionDetector size24×30 cmOrientationLandscapeExposure50--60Kvp/2--5mAsGridNoSID100 cmCentral rayBetween the two hands at the level of the metacarpophalangeal jointsCollimationTo include one-third of the distal radius and ulna\ to include soft tissues*SID* source to image-receptor distance To assess reliability of measurements, calibration was performed using a coin (real value of the coin diameter, 26.51 mm; measured value of the coin diameter, 26.53 mm; relative error, 0.08 %). In this process, the source to image-receptor distance (SID) was set to 100 cm (Table [1](#Tab1){ref-type="table"}). To prevent projection errors, the x-ray source was directed perpendicular to the cassette (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1(**a**) The diameter of a coin measured using a digital micrometre was 26.51 mm. (**b**) The diameter of the coin on a digital radiograph was measured as 26.53 mm on a Picture Archiving and Communication System (PACS) Image analysis {#Sec5} -------------- Four radiologists (three with 21, 18 and 12 years experience in radiology, respectively, and one first-year resident), blinded to the clinical data, analysed the images on a Picture Archiving and Communication System (PACS; G3, Infinitt Healthcare, Seoul, Korea). For planimetric analysis, the first and second radiologists (with 18 and 12 years experience in radiology, respectively) independently measured the area of the capitate and the hamate on PACS, respectively. ROIs were drawn manually on plain radiographs to encompass the entire capitate and hamate separately. Area measurements of the capitate and hamate were performed for both right and left hands (Fig. [2](#Fig2){ref-type="fig"}).Fig 275-month (6 years and 3 months)-old boy. (**a and b**) The areas of the bilateral capitate bones (solid line) and bilateral hamate bones (dotted line) were measured by drawing two separate regions of interest in the posterior-anterior (PA) radiograph of both hands When the ROI values were \> 10 % different between the two reviewers, another series of measurements were performed by the same two reviewers to reach a consensus. Differences \< 10 % were considered negligible, and the average was reported \[[@CR7]\]. The CH planimetry measurement was calculated by summing the area of the capitate and that of the hamate as follows: CH planimetry = area of capitate + area of hamate. The values of CH planimetry were obtained from both hands. For the analysis of accuracy and reproducibility for BAA, the third and fourth reviewers (with 21 years experience in radiology and the first-year resident, respectively) measured the area of the capitate and hamate of children who were included in the 50th percentile group. Each reviewer independently assessed the bone age of children using the CH planimetry method. On the basis of the GP method, the reviewers estimated the bone age of the 50th percentile group at 2 weeks after the planimetry assessment. Statistical analysis {#Sec6} -------------------- All statistical analyses were conducted using SPSS (version 20; IBM Corporation). Linear regression analysis was used to analyse the relationship between the planimetry measurement of each hand and the chronological age of each sex. Correlation coefficients of ≤ 0.3 were considered to indicate a weak relationship; 0.30--0.70, moderate relationship; and \> 0.70, strong relationship \[[@CR14]\]. A z-test was used to compare two correlation coefficients. Differences in the planimetry measurements between the right and left CH and in the accuracy and precision between two reviewers were analysed by using a paired t-test. A *p*-value of \< 0.05 was considered statistically significant. To compare the two methods, Deming regression analysis and accuracy determination were performed. Accuracy was calculated using the difference percentage between the estimated bone age and the chronological bone age of the 50th percentile group. To assess the reproducibility of the measurements between the third and fourth reviewers, precision, Bland-Altman analysis and Lin's concordance correlation coefficient (ρ~c~) were used. Precision was defined as the percentage difference in the estimated bone ages between two reviewers. The strength of ρ~c~ was categorized as follows: ρ~c~ \< 0.90, poor agreement; 0.9 ≤ ρ~c~ ≤ 0.95, moderate; 0.95 \< ρ~c~ ≤ 0.99, substantial; ρ~c~ \> 0.99, almost perfect agreement. Results {#Sec7} ======= The age and sex distribution of the Korean subjects are listed in Table [2](#Tab2){ref-type="table"}. There were 242 boys (mean age 75.33 months; age range 1--180 months) and 149 girls (mean age 57.46 months; age range 1--178 months).Table 2Age and sex distribution of the individualsAge (months)/SexBoys (n=242)Girls (n=149)1--1113912--23373124--35331736--47212648--59161360--7114672--83171284--956596--107135108--119107120--13143132--143184144--155143156--16795168--17916318010Data represent number of patients For 241 boys (99.59 %) and 149 girls (100 %), irrespective of age, left and right capitate and hamate bones were demonstrated on the radiographs of both hands. The right capitate and bilateral hamates were not demonstrated on the radiograph in only one boy (0.41 %) aged 1 month (Fig. [3](#Fig3){ref-type="fig"}).Fig. 3One-month-old boy. (**a and b**) Left capitate bone (arrow) is demonstrated but right capitate bone and bilateral hamate bones are not demonstrated on the posterior-anterior (PA) radiograph of both hands There were no significant differences in areas between the left and right capitate and hamate of both sexes (Table [3](#Tab3){ref-type="table"}). Additionally, there was also no significant difference in the sum of the area of the capitate and the area of the hamate between right and left hands.Table 3Differences in the values of planimetry of boys and girlsGenderLocationRight (95 % CI)Left (95 % CI)*p*-value\*Boy\ (n=242)Capitate130.52 (117.22--142.64)129.94 (117.23-142.64)0.3049Hamate87.52 (78.48--96.57)87.00 (78.07-95.93)0.1691Capitohamate218.04 (196.32--239.76)216.94 (195.38-238.50)0.1333Girl\ (n=149)Capitate95.70 (84.00--107.41)94.70 (83.17-106.22)0.3611Hamate64.69 (56.66--72.71)64.16 (56.23-72.10)0.2545Capitohamate160.39 (140.75--180.02)158.86 (139.53-178.20)0.1779*CI* confidence interval**\***A paired t-test was used to assess the difference of the means The planimetry curves of the capitate and hamate and the sum of the CH areas demonstrated increased slopes in both sexes (Fig. [4](#Fig4){ref-type="fig"}). There was a strong positive correlation between chronological age and the CH planimetry measurement (Table [4](#Tab4){ref-type="table"}). Moreover, strong correlations were found between chronological age and the capitate area of both sexes, as well as between the chronological age and hamate area (Table [4](#Tab4){ref-type="table"}).Fig. 4High positive relationships between chronological age and planimetries of capitates, hamates and CHs of the right (**a**) and left (**b**) hands of boys and right (**c**) and left (**d**) hands of girls. *CH* capitohamateTable 4Differences in the correlation coefficients between planimetry and chronological age of boys and girlsGenderLocationRight (95 % CI)Left (95 % CI)z-statisticBoyCapitate0.9711 (0.9629--0.9775)0.9729 (0.9652--0.9789)0.9729Hamate0.9607 (0.9496--0.9693)0.9597 (0.9484--0.9686)0.8886Capitohamate0.9702 (0.9618--0.9768)0.9709 (0.9627--0.9774)0.8951GirlCapitate0.9659 (0.9531--0.9752)0.9509 (0.9327--0.9643)0.1118Hamate0.9552 (0.9386--0.9674)0.9526 (0.9351--0.9655)0.8052Capitohamate0.9664 (0.9538--0.9756)0.9580 (0.9424--0.9695)0.3313*CI* confidence interval In the linear equation y = a + bx, y stands for capitate, hamate and CH area; x stands for age in months; and constants (a and b) were calculated. The equations of planimetry for the left hands of boys and girls are as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\displaystyle \begin{array}{l}\mathrm{CH}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{boy}\right)=3.0571\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-13.3391\\ {}\mathrm{CH}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{girl}\right)=2.5856\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-10.2867\\ {}\mathrm{Capitate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{boy}\right)=1.8057\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-6.6278\\ {}\mathrm{Capitate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{girl}\right)=1.5303\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-6.7608\\ {}\mathrm{Hamate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{boy}\right)=1.2514\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-7.2632\kern0.5em \\ {}\mathrm{Hamate}\kern0.5em \mathrm{area}\kern0.5em \left(\mathrm{girl}\right)=1.0552\kern0.5em \mathrm{age}\kern0.5em \left(\mathrm{months}\right)-3.5258\end{array}} $$\end{document}$$ By using the CH planimetry and GP methods, the third and fourth reviewers estimated the bone age of 109 patients who were classified into the 50th percentile group. None of the mean areas of the capitate, hamate and sum of the capitate and hamate were significantly different between the two reviewers. The mean value of chronological age of 109 children (mean age 113.07 months; standard deviation 36.07) was not significantly different from that of the estimated bone age measured by both CH planimetry and GP methods (p ≥ 0.1378). There was no statistically significant difference in the mean value of bone age between the two reviewers using CH planimetry. However, the mean values of bone age were significantly different between the two reviewers assessing the bone age according to the GP atlas (Table [5](#Tab5){ref-type="table"}). The mean value of bone age estimated using the GP method was lower than that measured using CH planimetry (*p* \< 0.0001).Table 5Descriptive statistics of the area and bone age estimated by two reviewers in the 50th percentile groupReviewer 3Reviewer 4*p*-value\*Area (mm^2^) Capitate185.94 ± 65.42184.11 ± 67.970.1128 Hamate121.45 ± 44.63120.25 ± 46.230.2487 Sum307.39 ± 108.92304.36 ± 112.820.0729BAA (months) CH planimetry118.58 ± 39.52117.43 ± 40.960.0687 GP method105.49 ± 39.00109.49 ± 39.380.0003Data are means ± standard deviation\*A paired t test was used to assess the difference of the means*BAA* bone age assessment, *CH* capitohamate, *GP* Greulich-Pyle atlas method When assessing bone age based on the CH planimetry and GP methods, there was no significant difference in the accuracy between the third (CH planimetry, 84.39 % ± 22.33 %; GP method, 85.15 % ± 13.78 %) and fourth (CH planimetry, 84.46 % ± 21.29 %; GP method, 87.66 % ± 12.34 %) reviewers (*p* ≥ 0.0867) (Fig. [5](#Fig5){ref-type="fig"}). Deming regression analysis showed no statistically significant difference in the results between the CH planimetry and GP methods, as the standard errors can be determined from the 95 % confidence intervals (CIs) of intercept (standard error, 4.0028; 95 % CI −4.9522--10.9164) and slope (standard error, 0.03955; 95 % CI, 0.9917--1.1485) including the values 0 and 1, respectively (Fig. [6](#Fig6){ref-type="fig"}).Fig. 5Graph showing accuracy of CH planimetry and GP method in assessing the bone age of 50th percentile group between two reviewers. A paired t-test demonstrated no statistically significant differences between two methods and between two reviewers (*p*-value ≥ 0.0867). Accuracy was defined as the difference percentage between the estimated bone age and the chronological bone age of 50 percentile group. *CH* capitohamate, *GP* Greulich-PyleFig. 6(**a**) Deming regression analysis for comparison of CH planimetry and GP atlas method in assessing bone age. (**b and c**) Bland-Altman plots of two reviewers show the interobserver reproducibility for CH planimetry and GB method for bone age assessment. The 95 % limits of agreement were narrower in CH planimetry than in GP method. *CH* capitohamate, *GP* Greulich-Pyle We analysed the interobserver variability of the CH planimetry and GP methods. The values of ρ~c~ were substantial for both methods for assessing bone age (CH planimetry, 0.9862; GP method, 0.9553) between the two reviewers. However, the precision of CH planimetry (4.42 % ± 3.87 %) was significantly different from that of the GP method (8.45 % ± 8.52 %) (*p* \< 0.001). The Bland-Altman analysis demonstrated that CH planimetry had narrower 95 % limits of agreement (−10.5 to 13.4 months) than the GP method (−29.5 to 21.1 months) (Fig. [6](#Fig6){ref-type="fig"}). These results suggest that the interobserver reproducibility of CH planimetry may be greater than that of the GP method. Discussion {#Sec8} ========== In our study, there were no significant differences noted in the areas between the right and left capitates and between the right and left hamates. Patterson et al. \[[@CR15]\] reported that the volume of carpal bones was not significantly different between the left and right hands, using three-dimensional (3D) computed tomography (CT) volumetry. However, hand PA radiography for bone assessment is usually performed on the left side rather than on the right side, and perhaps because most people are right-handed, the right hand is more prone to get injured and therefore may have deformity. In the early 1900s, a physical anthropologists meeting determined that body measurements should be performed on the left \[[@CR3]\]. The right capitate and bilateral hamates were not demonstrated on the radiograph in only one boy (0.41 %) aged 1 month. The capitate and hamate are known to emerge most frequently and simultaneously between the first and fourth months from birth; however, occasionally, the capitate emerges first, followed by the hamate. As seen in the GP atlas, the nuclei of the capitate and hamate were more likely to be present earlier in girls than in boys \[[@CR16]\]. There was also a strong positive correlation between chronological age and the CH planimetry measurement (Table [3](#Tab3){ref-type="table"}). As shown in Fig. [3](#Fig3){ref-type="fig"}, there were increased slopes in the planimetry curves of the capitates and hamates of boys and girls. Canovas et al. \[[@CR17]\] reported that there was a strong correlation between chronological age and volumes of the carpal bones of the hands measured using 3D CT volumetry. The strongest correlations were found with the triquetrum, capitate and hamate bones, which were present in all 20 hands. There was no significant difference between the two methods in accuracy for estimating bone age (CH planimetry, 84.39--84.46 %; GP method, 85.15--87.66 %). The research performed by Roche et al. showed that the median bone ages estimated using the GP method were similar to that of the chronological ages \[[@CR18]\]. On the contrary, some researchers reported that the difference between chronological age and bone age estimated using the GP method was −0.6 to 2.15 years \[[@CR10], [@CR19]--[@CR21]\]. The accuracy of BAA with the GP method has been reported to vary depending on the patient's age or sex and ethnicity \[[@CR22]\]. To date, few studies have evaluated the percentage accuracy of BAA using the GP method. Lee et al. \[[@CR23]\] reported that a deep learning system for BAA achieved 57.32 % and 61.40 % accuracies for the female and male cohorts when age was precisely matched. The accuracy of matching bone age within 1 year was 90.39 % and 90.39 %, respectively. In our study, the mean bone age measured using the GP method (105.49--109.49 months) was significantly lower than the mean bone age measured using CH planimetry (117.43--118.58 months). This result may be attributed to the difference in growth patterns among different ethnicities because the GP method was established in Caucasians \[[@CR3]\]. Some researchers reported that bone age measured with the GP method tended to be lower than the chronological age or the bone age measured with the TW3 method or the Korean bone standard method, although there was no significant difference between the estimated bone age and chronological age \[[@CR21], [@CR24]\]. These previous studies may have shown no statistically significant difference because of the small number of children or targeting children of a limited age range. In the second part of our study, the interobserver reproducibility of CH planimetry was greater than that of the GP method. The values of ρ~c~ were substantial for both methods (CH planimetry, 0.9862; GP method, 0.9553). In Bland-Altman analysis, the 95 % limit of agreement of CH planimetry was −10.5 to 13.4 months and that of the GP method was −21.1 to 29.5 months (Fig. [6](#Fig6){ref-type="fig"}). The precision of CH planimetry (4.42 % ± 3.87 %) was significantly different from that of the GP method (8.45 % ± 8.52 %) (*p* \< 0.001). To date, there have been many comparative studies on the interobserver reproducibility of BAA with the GP method by using various statistical tests \[[@CR5], [@CR7]\]. Bull et al. \[[@CR7]\] reported that the 95 % CI of the GP method (−2.46 to 2.18 years) was greater than that of the TW2 method (−1.48 to 1.43 years). King et al. \[[@CR5]\] reported that the average spread of intra-observer variation was 0.96 years for the GP method. These results are similar to those of our study. By contrast, the percentage precision of the GP method was 65.5−88.5 % \[[@CR8], [@CR25]\]. Improving the observer variability of manual methods is a motivation for developing automated methods of BAA \[[@CR26]\]. To date, computer-assisted or artificial intelligence-based methods have attempted to make accurate measurements, with discrepancies from 0.39 to 2.41 years \[[@CR27]\]. This study has several limitations. First, it was performed retrospectively at a single institution. We excluded children with chronic illnesses, nutrition deficiency or growth problems from this study population. However, to our knowledge, this planimetric study is the largest series assessing the areas of the capitate and hamate. Second, CT scans provide 3D measurements, whereas radiography demonstrates the 2D projected images of 3D objects; therefore, volumetry can be more accurate than planimetry, and may determine bone age more objectively \[[@CR17]\]. However, CT volumetry has some disadvantages such as high cost and radiation exposure. In contrast, x-ray planimetry has the advantages of lower costs and lower radiation doses \[[@CR28]\]. Moreover, planimetry is an easy-to-use method with an area measurement tool integrated in PACS without any separate software \[[@CR29], [@CR30]\]. Third, manually drawing ROIs along the cortical margin of the capitate and hamate may cause interobserver variation, and problems in planimetric analysis. To minimize this variation, another series of consensus measurements was performed in cases in which the differences between the reviewers exceeded 10 % \[[@CR7], [@CR31]\]. Fourth, quantitative analyses undertaken using digital radiographs displayed on a PACS monitor could be subject to measurement errors \[[@CR32]\]. To avoid this error, to ensure the accuracy of the absolute measurement value, calibration was performed using a coin prior to our study \[[@CR33]\]. Different equipment and protocols among different institutions may cause measurement errors. In the future, a multicentre prospective study should be conducted while avoiding this error. Finally, there is no gold standard for BAA \[[@CR29]\]. We set a reference standard with a range \< 1 standard deviation of the Korea CDC growth chart. Children were enrolled in the reference standard only if their heights were between the 50th percentile of the pervious age stage and the 50th percentile of the later age stage, so that the height ranges do not overlap \[[@CR12]\]. Moreover, as the height of Korean children has changed since the growth chart was published in 2007, it may be necessary to adjust the age range of the reference heights \[[@CR11]\]. In conclusion, the CH planimetry method may be useful for BAA. In the future, if automated CH planimetry can be integrated in PACS with validation for ethnicity and sex, it may save time and allow more precise BAA. CH : Capitohamate GP : Greulich-Pyle TW : Tanner-White GR : Gilsanz-Ratibin PACS : Picture Archiving and Communication System ROI : Region of interest This research was supported by the Hallym University Research Fund (HUFR-2017-27). Guarantor {#FPar1} ========= The scientific guarantor of this publication is Young Chul Kim. Conflict of interest {#FPar2} ==================== The authors of this manuscript declare no relationships with any companies whose products or services may be related to the subject matter of the article. Statistics and biometry {#FPar3} ======================= One of the authors has significant statistical expertise (Young Chul Kim). Ethical approval {#FPar4} ================ Institutional Review Board approval was obtained. Informed consent {#FPar5} ================ Written informed consent was waived by the Institutional Review Board. Methodology {#FPar6} =========== Retrospective, observational, performed at one institution.

{ "pile_set_name": "PubMed Central" }

[ "treatment", "cervic", "roch", "computerassist", "size×", "two", "girlsgenderlocationright", "idiopath", "control", "almost", "refer", "deform", "collect", "timeconsum", "sum", "korea", "healthcar", "written", "select", "fund", "controversi", "leftmathrmboyrightkernem", "take", "valu", "week", "narrow", "firstyear", "knee", "mathrmagekernem", "leukaemia", "may", "cizstatisticboycapit", "fifth", "cipvalueboy", "ρc", "separ", "use", "gener", "cileft", "graph", "moder", "strongest", "score", "final", "support", "easier", "imag", "planimetr", "accord", "caus", "irrespect", "discrep", "prior", "signific", "depart", "establish", "usepackagewasysym", "bull", "exposur", "suggest", "process", "tomographi", "right", "also", "growth", "waiv", "monthsold", "attempt", "equat", "conflict", "ztest", "perform", "ndata", "popul", "patterson", "seoul", "retrospect", "agreement", "criteria", "accuraci", "carpal", "medic", "solid", "monthstabl", "radiographi", "groupreview", "distribut", "expertis", "first", "tool", "leftmathrmmonthsright", "fractur", "capitoham", "gilsanzratibin", "recommend", "rel", "monitor", "wide", "chronic", "capit", "ttest", "inher", "assess", "ci", "base", "achiev", "author", "andor", "interv", "weak", "tend", "physic", "allow", "consecut", "compar", "chart", "center", "dk", "servic", "ethic", "experi", "substanti", "time", "greater", "stage", "significantli", "mathrmareakernem", "boy", "seat", "found", "bodi", "intercept", "gold", "explain", "differ", "tabreftypetablefig", "consid", "vari", "leftmathrmgirlrightkernem", "metaphysi", "provid", "baa", "spss", "known", "draw", "whose", "four", "canova", "level", "frequent", "moreov", "usepackagemathrsf", "king", "wherea", "either", "nine", "x", "small", "cohort", "howev", "radiu", "posit", "inclus", "perpendicular", "arrow", "usepackageamsbsi", "presenc", "alongsid", "respect", "blandaltman", "basi", "without", "well", "mathrmchkernem", "curv", "righthand", "bone", "american", "protocol", "ibm", "aim", 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"present", "place", "intervala", "standard", "mathrmhamatekernem", "commun", "group", "precis", "project", "guarantor", "deviat", "kim", "sever", "threedimension", "matur", "±", "tabl", "fourth", "radiolog", "girl", "sex", "contus", "list", "al", "characterist", "public", "easytous", "evalu", "quicker", "diamet", "posterioranterior", "one", "seen", "xrayprojectionposterioranterior", "soft", "knowledg", "would", "fpar", "data", "pair", "discuss", "young", "prospect", "palm", "describ", "birth", "calcul", "ngirl", "contrari", "strength", "left", "pattern", "advantag", "face", "figreftypefigfig", "atla", "monthssexboy", "version", "system", "year", "across", "comparison", "conclus", "child", "th", "month", "greulichpylefig", "board", "mm", "enddocu", "caucasian", "radiograph", "korean", "articl", "consensu", "plot", "declar", "equip", "sid", "calibr", "earlier", "motiv", "deme", "projectiondetector", "propos", "beginarraylmathrmchkernem", "pvalu", "endocrin", "tw", "qualit", "followsdocumentclassptminim", "repres", "defin", "pelvi", "cmorientationlandscapeexposurekvpmasgridnosid", "dot", "onemonthold", "date", "chang", "independ", "obtain", "leftmathrmmonthsrightkernem", "margin", "infinitt", "institut", "relat", "easi", "object", "therapi", "relationship", "prevent", "disord", "rang", "older", "quantit", "fig", "ill", "setlengthoddsidemarginpt", "futur", "real", "classifi", "hamat", "complain", "digit", "reliabl", "sec", "addit", "shown", "total", "high", "limit", "imagereceptor", "note", "approv", "perhap", "examin", "raybetween", "triquetrum", "diaphysi", "avoid", "statist", "hufr", "stand", "lin", "height", "children", "cortic", "median", "usepackageamssymb", "femal", "meet", "case", "materi", "regist", "percentag", "pvaluearea", "absenc", "concord", "display", "linear", "ulna", "reli", "capitohamatet", "second", "techniqu", "elbow", "studi", "cdc", "line", "flexion", "methodolog", "intraobserv", "perviou", "resid", "perfect", "archiv", "hand", "adult", "three", "exceed", "seri", "pa", "integr", "crtabl", "product", "earli", "learn", "usual", "deep", "research", "valid", "displaystyl", "slope", "clinic", "crucial", "roi", "absolut", "conduct", "individualsag", "anoth", "part", "method", "neutral", "categor", "new", "volumetri", "often", "tannerwhit", "match", "lee", "radiat", "ncapit", "mathrmcapitatekernem", "variou", "subject", "enrol", "hallym", "contrast", "correl", "begindocu", "b", "interobserv", "lower", "pictur", "peopl", "averag", "none", "embryolog", "coin", "within", "artifici", "problem", "encompass", "sourc", "innovionsh", "mean", "number", "introduct", "≥", "tabreftypet", "patient", "jointscollimationto", "poor", "extens", "percentil", "cm", "belong", "male", "depend", "variat", "hormon", "injur", "nuclei", "cr", "vertebra", "scan", "distal", "minim", "overlap", "blind", "et", "spread", "need", "review", "prone", "accur", "meansbaa", "gr", "third", "save", "n", "chul", "cmcentral", "matter", "later", "lefthand", "biometri", "singl", "develop", "exclud", "indic", "defici", "c", "paramet", "pylor", "ethnic", "age", "side", "figreftypefig", "manuscript", "consent", "compani", "epiphysi", "diseas", "bilater", "undertaken", "analysi", "girlcapit", "complex", "onethird", "younger", "g", "could", "lacer", "simultan", "along", "region", "volum", "ct", "acut", "pac", "micrometr", "increas", "softwar", "improv", "report", "similar", "januari", "≤", "usepackageupgreek", "area", "previou", "attribut", "therefor", "rather", "costeffect", "stenosi" ]

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All relevant data are within the paper. Introduction {#sec001} ============ Proper neuronal function necessitates a highly regulated extracellular environment in the brain. Accumulation of interstitial solutes, such as amyloid β and toxic compounds \[[@pone.0229702.ref001]\] may lead to degenerative diseases, such as Alzheimer's disease \[[@pone.0229702.ref002],[@pone.0229702.ref003]\] or even autism \[[@pone.0229702.ref004]\]. Although the Blood Brain Barrier is thought to be the primary mechanism involved in controlling the brain blood-exchange, the existence of a fluid driven transport system (so-called glymphatic system) via cerebrospinal fluid (CSF) or interstitial fluid (ISF) has been proposed recently as a waste clearance system through the perivascular and interstitial spaces in the brain \[[@pone.0229702.ref001],[@pone.0229702.ref005]\]. Hypothetically, CSF crosses the astrocyte end-feet bound to arteries in the perivascular space \[[@pone.0229702.ref006],[@pone.0229702.ref007]\]. After washing the interstitial space, the resulting ISF is flushed back outside the brain via veins in the perivascular space. Several factors play a crucial role in the modulation of this clearance system activity, notably sleep and anesthesia, perhaps via a modulation of brain blood volume and pressure \[[@pone.0229702.ref008]--[@pone.0229702.ref011]\]. This scheme gives astrocytes a crucial role in controlling water movements between the blood and the brain, through a mechanism dependent on Aquaporin-4 (AQP4), a membrane-bound water channel expressed at their end-feet \[[@pone.0229702.ref012],[@pone.0229702.ref013]\]. AQP4 is involved in the rapid volume regulation of astrocytes \[[@pone.0229702.ref014]\] and deletion of the AQP4 gene suppresses the clearance of soluble amyloid β \[[@pone.0229702.ref001]\]. Although most studies have been performed *in vitro*, some studies have shown astrocyte volume changes *in vivo* using 2-photon microscopy. Acute osmotic and ischemic stress induce astrocyte volume changes *in vivo* mice \[[@pone.0229702.ref015]\], and Thrane et al showed that the astrocyte volume change induced by osmotic stimulation was inhibited in AQP4 KO mice \[[@pone.0229702.ref016]\]. Overall, such studies suggest that dynamic volume change of astrocytes, through water flux mediated by AQO-4 channels may be associated with CSF flow regulation. Actually, astrocytes end-feet are involved in the CSF/ISF exchanges in perivascular space during sleep/awake cycle \[[@pone.0229702.ref017]\]. Recently MRI has been proposed as a more versatile approach to investigate the glymphatic system in vivo, using intrathecal or intravenous injections of gadolinium-based contrast agents (GBCAs) as tracers \[[@pone.0229702.ref007],[@pone.0229702.ref018]\]. However, this approach remains invasive and, paradoxically, gadolinium has been shown to deposit in the brain \[[@pone.0229702.ref019],[@pone.0229702.ref020]\] possibly in relation to a lack of brain drainage \[[@pone.0229702.ref010]\]. Therefore, alternative methods are needed to investigate the glymphatic system non-invasively, especially in the human brain. Fluid-dynamics driven and BOLD fast MRI have the potential to evaluate CSF pulsations in the ventricles and hemodynamics \[[@pone.0229702.ref011],[@pone.0229702.ref021],[@pone.0229702.ref022]\], while IVIM and diffusion MRI have been shown as promising methods for the evaluation of the ISF \[[@pone.0229702.ref007],[@pone.0229702.ref023]--[@pone.0229702.ref027]\]. Those considerations led us to investigate whether diffusion MRI was sensitive to astrocyte activity and, in turn, could become a marker of the overall glymphatic system. Diffusion MRI is exquisitely sensitive to changes in tissue microstructure, notably cell swelling \[[@pone.0229702.ref028]\]. Diffusion MRI is, for instance, sensitive to astrocyte swelling induced in rodents \[[@pone.0229702.ref029]\]. Hence, we hypothesized that dynamic changes in astrocytes activity and related volume changes could be monitored directly and non-invasively with diffusion MRI. To test this hypothesis we monitored variations of new diffusion MRI markers, namely the Sindex and the sADC, which have been tailored to increase sensitivity to tissue microstructure through water diffusion hindrance \[[@pone.0229702.ref030],[@pone.0229702.ref031]\] upon acute inhibition of astrocyte AQP4 channels in a mouse brain model using 2-(nicotinamide)-1,3,4-thia-diazole (TGN-020), a compound that blocks AQP4 channels *in vivo* in the mouse brain \[[@pone.0229702.ref032]\]. Material and methods {#sec002} ==================== Animal preparation {#sec003} ------------------ Thirty-two male C57BL6 mice (16--28 g, 4--10 weeks, Charles River, France) were allocated to two groups. The choice of a mouse brain model was motivated by prospect of using our protocol later to AQP-4 knock-out mice. First, for the TGN-020 group, 16 mice received an intra-peritoneal injection of 250mg/kg TGN-020 diluted in a gamma-cyclodextrine solution (10 mM) in order to increase its solubility. Second, for the control group, sixteen mice received an intra-peritoneal injection of the vehicle solution only (10 mM gamma-cyclodextrine in saline). The mice were housed on a 12-hour light-dark cycle and fed standard food ad libitum. Anesthesia was induced using 3% isoflurane in a mix of air and oxygen (air: 2 L/min, O2: 0.5 L/min). Then, 0.015 mg/kg of dexmedetomidine was administered intraperitoneally and followed by a continuous infusion of 0.015 mg/kg/h via subcutaneous catheter and maintained isoflurane at 0.8%. Throughout the acquisition, the animals' body temperature was maintained between 36.5 and 37.0 °C using heated water (Grant TC120, Grant Instruments, Shepreth, UK). To avoid motion-related artifacts the head was immobilized using a bite bar and ear pins. The respiration rate was monitored and stable (60--90 /min) throughout the experiment. All animal procedures used in the present study were approved by an institutional Ethic Committee (Comité d'Ethique en Expérimentation Animale, Commissariat à l'Energie Atomique et aux Énergies Alternatives, Direction des Sciences du Vivant (Fontenay-aux-Roses, France)) and by Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (France) (reference APAFIS\#8462-2017010915542122v2) and were conducted in strict accordance with the recommendations and guidelines of the European Union (Directive 2010/63/EU). This manuscript is in compliance with the ARRIVE guidelines (Animal Research: Reporting in Vivo Experiments) on how to REPORT animal experiments. MRI experiments {#sec004} --------------- The MRI experiments were conducted on a Bruker 11.7T scanner (Bruker BioSpin, Ettlingen, Germany) equipped with a gradient system allowing a maximum gradient strength of 760 mT/m. A cryo-cooled mouse brain coil was used. Animal positioning was performed using multi-slice fast low angle shot imaging (FLASH, TE/TR = 2.3/120 ms). A global first and second order shim was achieved followed by a local second order shim over the brain parenchyma. Structural (anatomical) images were acquired with the following parameters: T2 TurboRARE sequence, TE/TR = 11.15/2500 ms. Diffusion-weighted echo-planar imaging (DW-EPI) data sets were acquired with the following parameters: 150x150x250 μm^3^ resolution, 3 b-values (0, 250, 1750) s/mm^2^ along 6 directions, NA = 4, TE/TR = 36.3/2300 ms, 18 slices, diffusion time = 24 ms, total scan time = 5 min. The area covered by the 18 slices of the scans encompassed 5 mm in the axial plane with the center of the slab positioned at the middle of the brain. Six DW-EPI sets were first acquired (baseline) before TGN-020 or vehicle was injected. Then, after the injection, 12 additional DW-EPI data sets were acquired ([Fig 1](#pone.0229702.g001){ref-type="fig"}) every 5 minutes. ![Schematic figure of MRI experiment protocol.\ The vertical arrow indicates the saline or TGN-020 injection. DWI, diffusion weighted MRI; NR, number of repetitions. Horizontal arrow indicates the timeline.](pone.0229702.g001){#pone.0229702.g001} Data processing {#sec005} --------------- ### Preprocessing {#sec006} Regions of interest (ROIs) were originally created using the Allen Brain Atlas \[[@pone.0229702.ref033]\] and registered to a mouse brain MRI template to create a labeled atlas. We referred to previous studies based on microscopy \[[@pone.0229702.ref034]\] to assess the vascular distribution within the selected brain structures. The atlas was then co-registered with the b = 0 images of the first scans for each subject, and not the other way around to avoid changing raw voxel signals. Those preprocessing steps, also including radiofrequency bias field correction \[[@pone.0229702.ref035]\] and denoising were performed using ANTs (<https://stnava.github.io/ANTs/>) \[[@pone.0229702.ref036]\] ([Fig 2](#pone.0229702.g002){ref-type="fig"}). We assumed geometric distortion to be negligible; this condition was qualitatively checked on several mice for all b values. Furthermore, the standard-deviation of the signal intensity across scans was evaluated for each b value and each voxel (after Gaussian smoothing) was estimated as a marker of motion or instability. A threshold of 4% was used to flag unstable voxels. Data sets were at least one of the ROIs contained flagged voxels were discarded.\[[@pone.0229702.ref037]\]. ![Example of ROI registration with ANTs on a mouse brain.](pone.0229702.g002){#pone.0229702.g002} ### DWI analysis {#sec007} First, a shifted ADC (sADC) was computed using signal acquired at two key b values (instead of the standard values of 0 and 1000s/mm^2^), chosen to optimize signal sensitivity to both Gaussian and non-Gaussian diffusion which is more sensitive to tissue microstructure through hindrance effects \[[@pone.0229702.ref030],[@pone.0229702.ref031]\]. $$sADC = \frac{\ln\left( \frac{S_{Lb}}{S_{Hb}} \right)}{Hb - Lb}$$ where Lb is the low key b value (250s/mm^2^), Hb the high key b value (1750 s/mm^2^), S~Lb~ the signal at low key b value and S~Hb~ the signal at high key b value. Second, data were analyzed using the Sindex method \[[@pone.0229702.ref031]\]. The Sindex diffusion marker has been designed to identify tissue types or conditions based on their microstructure \[[@pone.0229702.ref031]\]. The Sindex was calculated from the direction-averaged, normalized signals, S~V~(*b*) in each voxel, as the algebraic relative distance between the vector made of these signals and those of 2 signature tissue signals S~A~ in condition A, and S~B~ in condition B, at each key b value as \[[@pone.0229702.ref031]\]: $$SI(V) = \left\{ {{\max\left( {\frac{\left\lbrack {{dS}_{V}\left( {Hb} \right) - {dS}_{V}\left( {Lb} \right)} \right\rbrack}{\left\lbrack {{dS}_{B}\left( {Hb} \right) - {dS}_{B}\left( {Lb} \right)\rbrack} \right.},0} \right)} - \left\lbrack {{\max\left( \frac{\left\lbrack {{dS}_{V}\left( {Hb} \right) - {dS}_{V}\left( {Lb} \right)} \right\rbrack}{\left\lbrack {{dS}_{A}\left( {Hb} \right) - {dS}_{A}\left( {Lb} \right)} \right\rbrack} \right.},0} \right)} \right\}$$ with dS~V~,~A,B~(*b*) = \[S~V~,~A,B~(*b*)- S~N~(*b*)\]/S~N~(*b*). S~N~ is taken as an intermediate signal between S~A~ and S~B~. SI was then linearly scaled as *Sindex* = (SI+1)\*25+25 to be centered at 50. A tissue with status similar to condition A has *Sindex* = 75, while for a status similar to condition B one has *Sindex* = 25. The library of the 2 reference diffusion MRI signals was built in advance from previously acquired data, one representing a generic mouse brain tissue (B) and another derived by simulating a moderate increase in diffusion hindrance (A) using the Kurtosis diffusion model \[[@pone.0229702.ref031]\]. For this study, S~A~(Lb) = 0.858; S~A~(Hb) = 0.370; S~B~(Lb) = 0.855; S~B~(Hb) = 0.317. Obviously the Sindex is expected to vary widely across brain regions according to their degree of diffusion hindrance ([Fig 3](#pone.0229702.g003){ref-type="fig"}), for instance white matter regions have higher Sindex values than gray matter regions. However, our focus was on the local changes in Sindex values induced by the injection of TGN-020, reflecting local changes in the degree of diffusion hindrance (a decrease in Sindex reflecting a decrease in hindrance). Sindex and sADC were calculated on a voxel-by-voxel basis to generate parametric maps. Calculation was also performed on a ROI level, averaging signals from all voxels within the ROI. ROIs were placed over the cerebral cortex, the striatum and the hippocampus (whole hippocampus, CA3 region and dentate gyrus, DG). The hippocampus is known to be reached in AQP4 expression \[[@pone.0229702.ref038],[@pone.0229702.ref039]\]. ![**Sindex (first row) maps and sADC (second row) maps for a representative mouse from vehicle group (first column) and from TGN-020 group (right column).** These maps were obtained by averaging the 6 last time points.](pone.0229702.g003){#pone.0229702.g003} Values obtained before (6 first scans) and after injection (6 last scans) were averaged in each ROI for each animal. The parameter time course was also calculated by averaging 3 successively acquired datasets (resulting in six time points with a resolution of 15 minutes) for each animal. Before averaging across individual subjects, we performed an outlier exclusion using a z-score filter (z\>3) calculated over the subject's parameter values for each time point. Statistical analysis {#sec008} -------------------- The statistical tests were performed in python (Python Software Foundation. Python Language Reference, version 3.7. Available at [http://www.python.org](http://www.python.org/)). We performed a paired two sample t-test between pre and post-injection data in each group and a pair-wise t-test with a posthoc correction for multiple comparison to compare the post-injection data between two groups and the pre-injection data between two groups. For the time course data, we performed a two sample t-test between the two groups for each time point. Results {#sec009} ======= Averaged Sindex and sADC following vehicle or TGN-020 injection {#sec010} --------------------------------------------------------------- [Fig 3](#pone.0229702.g003){ref-type="fig"} shows brain maps of Sindex and sADC for a representative mouse following vehicle and TGN-020 injection. Differences between the two conditions are readily visible with a decrease in Sindex and an increase in sADC following TGN-20 injection. Those changes are quantitatively assessed in [Fig 4](#pone.0229702.g004){ref-type="fig"}. [Fig 4A--4C](#pone.0229702.g004){ref-type="fig"} show the Sindex averaged over six scans before and after the injection in different locations, i.e., cerebral cortex, hippocampus and striatum, in vehicle and the TGN-020 group. TGN-020 injection resulted in significant decrease in Sindex in the cortex (p = 0.0061) and the hippocampus (p = 0.00069) but not in the striatum (p = 0.26). No significant change of Sindex was observed following vehicle injection in those locations (p = 0.94 in cortex, p = 0.85 in hippocampus and p = 0.14 in striatum). [Fig 4D--4F](#pone.0229702.g004){ref-type="fig"} show the sADC averaged over six scans before and after the injection in the same locations as for the Sindex, in vehicle and TGN-020 group. TGN-020 injection resulted in significant increase in sADC in the cortex (p = 0.0064) and the hippocampus (p = 0.00068) but not in the striatum (p = 0.26). No significant change of sADC was observed following vehicle injection in any of the ROIs considered (p = 0.91 in cortex, 0.78 in hippocampus and 0.14 in striatum). We also investigated subregions of the hippocampus CA3 and DG ([S1 Fig](#pone.0229702.s001){ref-type="supplementary-material"}). The TGN-020 injection significantly decreased Sindex and increased sADC values both in the DG and the CA3 (Sindex in DG: -11.8, p = 4.8e-7 and in CA3: -4.8, p = 0.00012; sADC in CA3: +10.2, p = 0.00013, sADC in DG: +22.5, p = 7.14e-7). However, the baseline for Sindex and sADC in DG was found noisy and not stable ([S1B and S1F Fig](#pone.0229702.s001){ref-type="supplementary-material"}) due to the smaller size of DG resulting in a very low voxel count compared to the other ROIs (voxel count for DG, CA3, hippocampus, striatum, cortex were 85, 181, 763, 2069 and 8270, respectively). ![Boxplot of Sindex and sADC in the cortex (a: Sindex, d: sADC), the striatum (b: Sindex, e: sADC) and hippocampus (c: Sindex, f: sADC). Left, Vehicle group; right TGN-020 group in each figure. Pre, pre-injection (light blue); post, post-injection (dark blue) of vehicle or TGN-020 group. \*: p\<0.05, \*\*: p\<0.01, \*\*\*: p\<0.001 are the result for the paired t-test between pre and post-injection for each group. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 are the result for a t-test with posthoc correction for multiple comparison between post-injection of the two groups. Diamonds represent outliers, a point is defined as an outlier if its value is below Q1--1.5×IQR or above Q3 + 1.5×IQR, where Q1 is the first quartile, Q3 the third quartile and IQR the interquartile range.](pone.0229702.g004){#pone.0229702.g004} Sindex and sADC time courses following vehicle or TGN-020 injection {#sec011} ------------------------------------------------------------------- We then investigated the time course of the Sindex and sADC for each group. The same ROIs were used as for the averaged Sindex and sADC values. [Fig 5A--5C](#pone.0229702.g005){ref-type="fig"} represents the Sindex time course for each ROI. A decrease of Sindex was observed after the injection of TGN-020 but not with vehicle injection in all ROIs. The Sindex significantly decreased (around 9% in the cortex) following TGN-020 injection in all ROIs and continued until the end of the scanning period. [Fig 5D--5F](#pone.0229702.g005){ref-type="fig"} show the sADC time course with an opposite trend compared to Sindex in all ROIs. ![Time course of Sindex and sADC in the cortex (a: Sindex, d: sADC), the striatum (b: Sindex, e: sADC) and hippocampus (c: Sindex, f: sADC). Blue line is vehicle group and orange line is TGN-020 group. Error bar shows standard deviation. The dashed line represents the injection time. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 by two sample t-test between the two groups for each time point.](pone.0229702.g005){#pone.0229702.g005} Discussion {#sec012} ========== Several studies have underlined the potential of Diffusion MRI to investigate the glymphatic system \[[@pone.0229702.ref007],[@pone.0229702.ref023]--[@pone.0229702.ref025]\]. Astrocytes are considered to play a key role in brain waste clearance through membrane AQP4 channels expressed at their end-feet \[[@pone.0229702.ref012]\]. The ADC has been proposed as a biomarker of AQP gene expression, as earlier studies have demonstrated that the ADC obtained with high b values was correlated with the amount of increasing expression of AQP1 in glioblastoma cell lines \[[@pone.0229702.ref040]\]. In this study, we have investigated whether two diffusion MRI markers more sensitive to tissue microstructure than the ADC, namely the Sindex and sADC, could reveal changes in astrocyte activity induced by TGN-020. Following AQP4 channel inhibition with a TGN-020 solution a decrease in Sindex and increase in sADC were readily observed in the cortex, more in the hippocampus, but not in the striatum, reflecting local differences in astrocyte \[[@pone.0229702.ref041]\] and vascular density \[[@pone.0229702.ref034]\]. In the hippocampus changes in Sindex and sADC were larger in DG than in CA3 layer, presumably in line with differences in AQP4 channels expression on astrocytes \[[@pone.0229702.ref039]\]. However, further validation is required as the baseline Sindex and sADC values were not stable due to the small count in the DG ROI (see [Result](#sec009){ref-type="sec"}). Histological studies would also confirm differences in astrocyte density and volume. Nonetheless, those results suggest that diffusion MRI is sensitive to astrocyte activity and, indirectly, to the status of the glymphatic system. Those diffusion MRI markers provide a higher sensitivity to small changes in tissue features by encompassing in a single marker Gaussian and non-Gaussian diffusion effects. Furthermore they are easy to calculate and are not diffusion signal model dependent, such as the kurtosis model \[[@pone.0229702.ref031]\]. The low key b value used in this study was high enough (250 s/mm^2^) to make perfusion-related IVIM effects negligible. Hence the observed changes in the diffusion markers reflect genuine tissue microstructure related diffusion effects and not perfusion effects (cerebral blood flow) which have already been reported observed with TGN-020 \[[@pone.0229702.ref042]\]. Indeed, acquisition of perfusion-driven IVIM data could be valuable to get information on the vascular distribution which is known to vary across brain structures \[[@pone.0229702.ref034]\], but this was not possible within our protocol timeframe to guaranty animal stability. The Sindex decrease and the sADC increase jointly point out to a decrease in the amount of hindrance for water diffusion in astrocyte rich areas (cortex and hippocampus) under acute AQP4 channel inhibition induced by TGN-20. Based on established diffusion MRI mechanisms \[[@pone.0229702.ref028]\] this hindrance decrease suggests an astrocyte volume reduction \[[@pone.0229702.ref043],[@pone.0229702.ref044]\] associated with an increase of the ISF (were diffusion is tortuous) \[[@pone.0229702.ref045]\] or an increase in astrocyte membrane permeability and water exchange. Indeed, astrocytes rapidly regulate their volume throughout AQP4 channels. Those results obtained by acute AQP4 inhibition contrast earlier reports using chronic models, such as an ADC decrease observed during AQP4 inhibition with interfering RNA \[[@pone.0229702.ref046]\] or change in ADC in AQP4 knockout mice \[[@pone.0229702.ref047]\]. Beside the higher sensitivity to tissue microstructure of the sADC over the ADC, such discrepancy could possibly result from the modified astrocyte phenotype associated with a long-term inhibition of AQP4 expression found in those previous studies \[[@pone.0229702.ref048]\]. The sub-acute or chronic inhibition of AQP4 activity by AQP4-antibodies or small interfering RNA duplexes alter astrocyte morphology and decrease water permeability \[[@pone.0229702.ref049],[@pone.0229702.ref050]\] which could result in an ADC decrease. Also, the effects we observed in baseline conditions should be distinguished from those obtained in conditions of neuronal activation for which AQP4 inhibition by extracellular acidification results in astrocyte swelling, capillary lumen expansion and Virchow-Robin space reduction \[[@pone.0229702.ref051]\]. Further studies should aim at precising the mechanisms involved in the observed effects, however histology would require the brain to be fixed, preventing astrocyte volume changes to be monitored before and after TGN-020/saline injection within the same animal. In vivo fluorescent imaging could be used, but only in the cortex. Clearly, the detailed mechanisms underlying acute AQP4 channel inhibition by TGN-20 and chronic AQP4 knockout mice models are lacking. Diffusion MRI has the potential to clarify those mechanisms, notably through non-Gaussian diffusion markers, such as the Sindex and the sADC. Fractional anisotropy (FA) measurements results were not reported as they are relevant only to areas exhibiting diffusion anisotropy, ie, white matter given the spatial resolution of our images. Beside, due to noise FA values are highly corrupted and often reflect mainly variations in underlying ADC (here sADC) \[[@pone.0229702.ref052]\]. Another issue to consider is that our studies were obviously performed under anesthesia. Anesthetic drugs are known to impact intracranial pressure, which could interfere with CSF-ISF exchanges. For instance, dexmedetomidine and ketamine enhance CSF influx alongside perivascular spaces \[[@pone.0229702.ref053]\]. Moreover, similar to what happens during sleep, anesthesia is associated with a substantial increase in both perivascular and extracellular space volume \[[@pone.0229702.ref054]\]. Thus, it would be interesting to check whether the effects we have observed with TGN-20 persist in awake animals. A recent study has shown that, while the baseline ADC did not change between anesthetic and awake conditions, DOTA-Gd accumulation was significantly suppressed during anesthesia in contrast enhanced MRI \[[@pone.0229702.ref055]\]. Conclusion {#sec013} ========== Work remains to better understand the mechanisms involved in brain waste clearance through a so-called glymphatic system and the contribution of astrocytes to this system. The reference method to investigate the glymphatic system in preclinical settings relies on the intracisternal injection of gadolinium in the cisterna magna \[[@pone.0229702.ref056]\]. Our results show that changes in astrocyte activity thought to regulate CSF-ISF exchanges, an important component of glymphatic system functionality, could be monitored non-invasively with diffusion MRI, especially through its Sindex metric. Further studies will be required to more directly establish the potential of diffusion MRI to monitor the glymphatic system. Due to its complete non-invasiveness, this new approach could be used for clinical studies to confirm or infirm the existence of such a glymphatic system in humans \[[@pone.0229702.ref024]\]. Supporting information {#sec014} ====================== ###### Boxplot and time course of Sindex in the DG (a: boxplot, b: time course) and in the CA3 (c: boxplot, d: time course). Boxplot and time course of sADC in the DG (e: boxplot, f: time course) and in the CA3 (g: boxplot, h: time course). Left, Vehicle group; right TGN-020 group in each figure. Pre, pre-injection (light blue); post, post-injection (dark blue) of vehicle or TGN-020 group in boxplot. \*: p\<0.05, \*\*: p\<0.01, \*\*\*: p\<0.001 are the result for the paired t-test between pre and post-injection for each group. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 are the result for a t-test with posthoc correction for multiple comparison between post-injection of the two groups. Diamonds represent outliers, a point is defined as an outlier if its value is below Q1--1.5×IQR or above Q3 + 1.5×IQR, where Q1 is the first quartile, Q3 the third quartile and IQR the interquartile range. Blue line is vehicle group and orange line is TGN-020 group. Error bar shows standard deviation. The dashed line represents the injection time. +: p\<0.05, ++: p\<0.01, +++: p\<0.001 by two sample t-test between the two groups for each time point. Note: The voxel count in DG was very small (85 versus 181 in CA3) resulting in very noisy data. The apparent significant difference in sADC and Sindex values between the TGN-020 and saline groups one point before baseline (b, f) might result from an underestimation of the standard-deviation. The difference becomes largely more significant after injections, overcoming any uncertainty in standard-deviation estimates. (TIF) ###### Click here for additional data file. 10.1371/journal.pone.0229702.r001 Decision Letter 0 Jiang Quan Academic Editor © 2020 Quan Jiang 2020 Quan Jiang This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 3 Mar 2020 PONE-D-20-03675 Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor. PLOS ONE Dear Pr. Le Bihan, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. How do you know that the observed diffusion changes are related to the glymphatic system?How do you explain the DG change, given the mentioned AQP4 abundant?Do you have the data to identify regions with high vascular presence to look at the Si and sADC changes on those area?In addition to sindex, the authors could use FA for comparison.Response to reviewer's questions. We would appreciate receiving your revised manuscript by Apr 17 2020 11:59PM. When you are ready to submit your revision, log on to <https://www.editorialmanager.com/pone/> and select the \'Submissions Needing Revision\' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: <http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols> Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled \'Response to Reviewers\'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled \'Revised Manuscript with Track Changes\'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled \'Manuscript\'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Quan Jiang, Ph,D. Academic Editor PLOS ONE Journal Requirements: 1\. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE\'s style requirements, including those for file naming. The PLOS ONE style templates can be found at <http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf> and <http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf> 2\. Thank you for including your ethics statement: \"All animal procedures used in the present study were approved by an institutional Ethic Committee and government regulatory agency (reference APAFIS\#8462-2017010915542122v2) and were conducted in strict accordance with the recommendations and guidelines of the European Union (Directive 2010/63/EU). This manuscript is in compliance with the ARRIVE guidelines (Animal Research: Reporting in Vivo Experiments) on how to REPORT animal experiments.\". i\) Please amend your current ethics statement to include the full name of the ethics committee that approved your specific study. ii\) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the "Ethics Statement" field of the submission form (via "Edit Submission"). For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to <http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research> \[Note: HTML markup is below. Please do not edit.\] Reviewers\' comments: Reviewer\'s Responses to Questions **Comments to the Author** 1\. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer \#1: Yes Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 2\. Has the statistical analysis been performed appropriately and rigorously? Reviewer \#1: Yes Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 3\. Have the authors made all data underlying the findings in their manuscript fully available? The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing) requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data---e.g. participant privacy or use of data from a third party---those must be specified. Reviewer \#1: No Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 4\. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer \#1: Yes Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 5\. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer \#1: Authors presented a very interesting study, evaluating the utility of diffusion MRI to map glymphatic function, through the modification of the AQP4 expression. This is of high scientific and clinical value, very timely and of high impact. The experiment is well designed, and the execution is sound. This reviewer has below comments regarding the validity and conclusions. Figures quality/dpi are low! Major comments: 1\. The main limitation is interpreting diffusion measures as glymphatic system function. AQP4 is also involved in brain inflammation and regulation of extracellular space volume. How do you know that the observed diffusion changes are related to the glymphatic system? Even though authors were cautious about the interpretation of their results, they should probably discuss other potential explanations for their findings and adjust their main conclusion accordingly. 2\. Authors reported significant differences in both CA3 and DG of the TGN-020 group. How do you explain the DG change, given the mentioned AQP4 abundant? 3\. Regarding above comment: Do you have the data (for example SWI or high resolution T2) to identify regions with high vascular presence to look at the S_i and sADC changes on those area? That could potentially help identify the underlying AQP4 involvement. 4\. Based on ref \[36\]: "AQP4 protein levels were highest in the cerebellum with lower expression in the cortex and hippocampus." This suggests that the cerebellum should have been a major ROI in this study. Why cerebellum is not included? 5\. Figures have extremely low quality (for example Figures 4 and 5). I could only guess the labels and axes. 6\. It seems rather strange to have (significant) differences prior to injection (e.g. Fig 5.h)? This needs to be addressed. One would expect to see no difference whatsoever. 7\. In favor with reported findings, below paper showed that diffusion MRI is affected by perivascular space fluid presence, which should be cited: Sepehrband, F., Cabeen, R. P., Choupan, J., Barisano, G., Law, M., Toga, A. W., & Alzheimer\'s Disease Neuroimaging Initiative. (2019). Perivascular space fluid contributes to diffusion tensor imaging changes in white matter. NeuroImage, 197, 243-254. 8\. Not in favor of reported finding, below study reported "we failed to detect a significant change in the brain extracellular water volume using diffusion weighted imaging in awake and anesthetized mice." This paper also should be cited/discussed: Gakuba, C., Gaberel, T., Goursaud, S., Bourges, J., Di Palma, C., Quenault, A., \... & Gauberti, M. (2018). General anesthesia inhibits the activity of the "glymphatic system". Theranostics, 8(3), 710. 9\. Another paper that links diffusion changes to glymphatic system is below, which also could be cited: Thomas, C., Sadeghi, N., Nayak, A., Trefler, A., Sarlls, J., Baker, C. I., & Pierpaoli, C. (2018). Impact of time-of-day on diffusivity measures of brain tissue derived from diffusion tensor imaging. Neuroimage, 173, 25-34. A minor comment: 1\. Page 11, line 251: "peculiar to this this brain" -\> "peculiar to this brain" 2\. Please report the age of the mice, in each group (maybe it is reported somewhere, but I couldn't find it). Reviewer \#2: In this paper the dynamic changes of astrocyte activity were investigated using DWI in 32 mice by inhibiting AQP4 channels with a TGN-020 solution. Two novel DWI measures were used to study the results which show a significant decrease in the Sindex, a diffusion marker of tissue microstructure, and a significant increase of the water diffusion coefficient (sADC) in cerebral cortex and hippocampus compared to saline injection. Developing reliable non-invasive biomarkers for the glymphatic system is important for translational studies. This study has done a great job to introduce such biomarkers. However, it may need some more work to improve the study. Major points: 1- In addition to sindex, the authors could use FA for comparison or at least confirming (in a few sentences) that FA is not showing the trend seen in sindex. 2- Why mice were chosen? Rats have bigger brain and therefore the imaging could be easier. 3- Histology analyses of the mice after imaging could be so helpful in confirming the study outcomes. Minor points: Line 16: "We assumed no change of position among the different scans during acquisitions for each mouse and geometric distortion with b values to be negligible; this condition was qualitatively checked on several mice." Why not using motion correction to compensate for animal movement during the imaging? Line 119: "Furthermore, signal instabilities were quantitatively evaluated for each subject and subjects exhibiting instabilities above 4% for most voxels were eliminated." Could you please explain more? Line 156: Please mention in Fig3 caption the time point of the displayed maps? I assume the maps correspond to the averaged 6 time points of the pre injection and the last 6 time points after the injection. Line 251: There is an extra "this". \*\*\*\*\*\*\*\*\*\* 6\. PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. **Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy). Reviewer \#1: No Reviewer \#2: No \[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link \"View Attachments\". If this link does not appear, there are no attachment files to be viewed.\] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, <https://pacev2.apexcovantage.com/>. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at <[email protected]>. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0229702.r002 Author response to Decision Letter 0 10 Apr 2020 Editor comment 1\. How do you know that the observed diffusion changes are related to the glymphatic system? The existence of the glymphatic system is controversial. Our results do not aim at demonstrating the existence or not of a glymphatic system. However, if we admit that such a system exists it has been strongly suggested in the literature (REF XXX) that astrocytes must play a major role, and, in turn, AQP4 channels expressed by astrocytes. The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all. The Discussion and Conclusion have been revised to make this point clear. 2\. How do you explain the DG change, given the mentioned AQP4 abundant? The density of AQP4 receptors in DG is higher than in the other regions we have investigated. Indeed, the larger sADC and Sindex change observed in DG under TGN-020 administration is an indirect proof that TGN-020 acted on astrocyte AQP4 channels. The Discussion has been revised accordingly. 3\. Do you have the data to identify regions with high vascular presence to look at the Si and sADC changes on those area? A relevant parameter could be, indeed, the density of small vessels. Such vessels are not visible even within the high resolution T2-weighted images we acquired using Turbo RARE (see Methods and Figure 2). Such vessels would require the additional acquisition of perfusion MRI data, such as IVIM MRI, which could not be done during the acquisition time window our anesthetic protocol permitted. Hence, we relied on previous studies reporting the vascular distribution across brain structures using microscopy (ref 40). 4\. In addition to sindex, the authors could use FA for comparison. FA is relevant to assess diffusion anisotropy which is present only in whiter matter at the resolution of our images (not in the cortex). The Sindex (and the sADC) is a much more relevant parameter. Furthermore, as explained in a recent article (Iima at al. European Radiology, 2020), FA values are highly sensitive to noise and, in turn, to ADC (or sADC) values. Variations in FA would thus reflect the changes we have observed in sADC values more than genuine changes in diffusion anisotropy. Journal Requirements Please ensure that your manuscript meets PLOS ONE\'s style requirements, including those for file naming. The PLOS ONE style templates can be found at <http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf> and <http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf> 2\. Thank you for including your ethics statement: i\) Please amend your current ethics statement to include the full name of the ethics committee that approved your specific study. ii\) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the "Ethics Statement" field of the submission form (via "Edit Submission"). For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to <http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research> We initially refrained to provide information which could potentially conflict with an anonymous review. Full details have been added in the Methods section. Response to reviewers Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer \#1: Authors presented a very interesting study, evaluating the utility of diffusion MRI to map glymphatic function, through the modification of the AQP4 expression. This is of high scientific and clinical value, very timely and of high impact. The experiment is well designed, and the execution is sound. This reviewer has below comments regarding the validity and conclusions. Figures quality/dpi are low! Major comments: 1\. The main limitation is interpreting diffusion measures as glymphatic system function. AQP4 is also involved in brain inflammation and regulation of extracellular space volume. How do you know that the observed diffusion changes are related to the glymphatic system? Even though authors were cautious about the interpretation of their results, they should probably discuss other potential explanations for their findings and adjust their main conclusion accordingly. The existence of the glymphatic system is controversial. Our results do not aim at demonstrating the existence or not of a glymphatic system. However, if we admit that such a system exists it has been strongly suggested in the literature (Illif et al, Sci Transl Med, 2012,) that astrocytes must play a major role system (Mestre et al, eLife, 2018), and, in turn, AQP4 channels expressed by astrocytes (Ikeshima-Kataoka et al, Int J Mol Sci, 2016). The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all. Further studies will be required to establish the potential of diffusion MRI to monitor the glymphatic system. The Discussion and Conclusion have been revised to make this point clear. 2\. Authors reported significant differences in both CA3 and DG of the TGN-020 group. How do you explain the DG change, given the mentioned AQP4 abundant? The density of AQP4 receptors in DG is higher than in the other regions we have investigated (Hsu et al, Neuroscience, 2011; Hubbard et al, ASN Neuro, 2015). Indeed, the larger sADC and Sindex change observed in DG under TGN-020 administration is an indirect proof that TGN-020 acted on astrocyte AQP4 channels. However, as we explain in the response to comment\#6, the baseline of DG was not stable due to the smaller voxel size. We need higher resolution image for the validation of Sindex changes in DG. The Discussion has been revised accordingly. 3\. Regarding above comment: Do you have the data (for example SWI or high resolution T2) to identify regions with high vascular presence to look at the S_i and sADC changes on those area? That could potentially help identify the underlying AQP4 involvement. We agree that a relevant parameter could be, indeed, the density of small vessels. Such vessels are not visible even within the high resolution T2-weighted images we acquired using Turbo RARE (see Methods and Figure 2). Such vessels would require the additional acquisition of perfusion MRI data, such as IVIM MRI, which could not be done during the acquisition time window our anesthetic protocol permitted. Hence, we relied on previous studies reporting the vascular distribution across brain structures using microscopy (Xiong et al, Front Neuroanat, 2017). 4\. Based on ref \[36\]: "AQP4 protein levels were highest in the cerebellum with lower expression in the cortex and hippocampus." This suggests that the cerebellum should have been a major ROI in this study. Why cerebellum is not included? We did not have a full brain coverage including the cerebellum due to the limitation of the scanning time (scanning time is determined by number of slices). Instead, we investigated the hippocampus because AQP4 expresses abundantly in this region (Hsu et al, Neuroscience, 2011; Hubbard et al, ASN Neuro, 2015). 5\. Figures have extremely low quality (for example Figures 4 and 5). I could only guess the labels and axes. We are sorry for the very low quality of Figure 4 and 5 which probably resulted from the PDF conversion. Revised figures were uploaded after checking quality with the digital diagnostic tool of PLOS: , <https://pacev2.apexcovantage.com/>. 6\. It seems rather strange to have (significant) differences prior to injection (e.g. Fig 5.h)? This needs to be addressed. One would expect to see no difference whatsoever. Because the volume of DG is smaller than cerebral cortex and striatum, the Sindex and sADC in DG were noisier rather than these regions. (The number of voxel size in DG, CA3, hippocampus, striatum, cortex is respectively: 85, 181, 763, 2069 and 8270) This is the reason why baseline of Sindex and sADC in DG was not stable. We decided to move DG and CA3 from the manuscript to supplementary data, instead we have used whole hippocampal region for the main figure. We described the instability in DG and CA3 in the Results section. 7\. In favor with reported findings, below paper showed that diffusion MRI is affected by perivascular space fluid presence, which should be cited: Sepehrband, F., Cabeen, R. P., Choupan, J., Barisano, G., Law, M., Toga, A. W., & Alzheimer\'s Disease Neuroimaging Initiative. (2019). Perivascular space fluid contributes to diffusion tensor imaging changes in white matter. NeuroImage, 197, 243-254. We have added this reference in Introduction (Reference 26). 8\. Not in favor of reported finding, below study reported "we failed to detect a significant change in the brain extracellular water volume using diffusion weighted imaging in awake and anesthetized mice." This paper also should be cited/discussed: Gakuba, C., Gaberel, T., Goursaud, S., Bourges, J., Di Palma, C., Quenault, A., \... & Gauberti, M. (2018). General anesthesia inhibits the activity of the "glymphatic system". Theranostics, 8(3), 710. This article compared the ADC and intracerebral accumulation of DOTA-Gd injected in intracisternal site in awaked and anesthetized state. The suppressed accumulation of DOTA-Gd was observed in anesthetized state compared with awaked state, while ADC was not changed between awaked and anesthetized sate. The DOTA-Gd increased the SNR when they accumulated for long time (30-60 min for accumulation in the study) and acquisition has been done under anesthesia. In contrast, diffusion MRI has been performed in awaked and anesthetized condition. The diffusion MRI could be influenced by the head motion and respiration, which could be more significant in awaked state. This potentially interferes ADC measurement to reduce the sensitivity. Further study to improve the sensitivity is required to assess the potential of diffusion MRI to detect the awaked and anesthetized state. We have described this article in Discussion. 9\. Another paper that links diffusion changes to glymphatic system is below, which also could be cited: Thomas, C., Sadeghi, N., Nayak, A., Trefler, A., Sarlls, J., Baker, C. I., & Pierpaoli, C. (2018). Impact of time-of-day on diffusivity measures of brain tissue derived from diffusion tensor imaging. Neuroimage, 173, 25-34. We thank for these valuable references. We agree that those references would be helpful for the readers to better understand and appreciate our results. This has been cited as reference 27. A minor comment: 1\. Page 11, line 251: "peculiar to this this brain" -\> "peculiar to this brain" We have corrected this typo. 2\. Please report the age of the mice, in each group (maybe it is reported somewhere, but I couldn't find it). We used the mice aged 4-10 weeks in each group. The age of mice has been added into the Material and Methods part of the manuscript. Reviewer \#2: In this paper the dynamic changes of astrocyte activity were investigated using DWI in 32 mice by inhibiting AQP4 channels with a TGN-020 solution. Two novel DWI measures were used to study the results which show a significant decrease in the Sindex, a diffusion marker of tissue microstructure, and a significant increase of the water diffusion coefficient (sADC) in cerebral cortex and hippocampus compared to saline injection. Developing reliable non-invasive biomarkers for the glymphatic system is important for translational studies. This study has done a great job to introduce such biomarkers. However, it may need some more work to improve the study. Major points: 1- In addition to sindex, the authors could use FA for comparison or at least confirming (in a few sentences) that FA is not showing the trend seen in sindex. FA is relevant to assess diffusion anisotropy which is present only in whiter matter at the resolution of our images. The Sindex (and the sADC) is a much more relevant parameter. Furthermore, as explained in a recent article (Iima at al. European Radiology, 2020), FA values are highly sensitive to noise and, in turn, to ADC (or sADC) values. Variations in FA would thus reflect the changes we have observed in sADC values more than genuine changes in diffusion anisotropy. 2- Why mice were chosen? Rats have bigger brain and therefore the imaging could be easier. We chose mice instead of rats because our diffusion MRI protocol could be applicable in the future to investigate AQP-4 KO mice. There is no such transgenic model for rats. Also, our MRI setup includes a cryoprobe dedicated to mouse head imaging, which increases the SNR and contrast. This specific coil is too small for rat heads. 3- Histology analyses of the mice after imaging could be so helpful in confirming the study outcomes. We agree that histology would have been a nice addition to confirm volume changes of astrocytes in the different groups (TGN-020 group and vehicle group). However, such tests require the brain to be fixed, hence they cannot be performed to check volume changes before and after TGN-020 (or saline) injection within the same animal. Another method to monitor astrocyte volume could be in vivo fluorescent imaging, but this method can be applied only in the cortex. Those issues have been added to the Discussion. Minor points: Line 16: "We assumed no change of position among the different scans during acquisitions for each mouse and geometric distortion with b values to be negligible; this condition was qualitatively checked on several mice." Why not using motion correction to compensate for animal movement during the imaging? Motion correction is tricky when considering quantitative diffusion MRI as motion algorithms work on the reconstructed images. Especially, as acquisitions are performed with specific orientations of the diffusion-probing gradient pulses in space any rotation in the reconstructed images would not reflect genuine signal changes which would have occurred if the diffusion-probing gradient pulses had rotated in the same way. Instead, we decided to simply reject data sets were motion was too large (\>4%) using a semi-quantitative index based on signal stability across successive scans. We agree that the text was not clear and we have revised it. Line 119: "Furthermore, signal instabilities were quantitatively evaluated for each subject and subjects exhibiting instabilities above 4% for most voxels were eliminated." Could you please explain more? The standard-deviation of the signal intensity across scans was evaluated for each b value and each voxel (after Gaussian smoothing) was estimated. A threshold of 4% was used flag unstable voxels. . Data sets were one of the ROIs have only flagged voxels were discarded. Line 156: Please mention in Fig3 caption the time point of the displayed maps? I assume the maps correspond to the averaged 6 time points of the pre injection and the last 6 time points after the injection. We have added in the legend of the figure 3 that the maps correspond to the mean of the 6 last time points. Line 251: There is an extra "this". We have corrected this typo. ###### Submitted filename: 200303-responses_to_reviewers-FINAL.docx ###### Click here for additional data file. 10.1371/journal.pone.0229702.r003 Decision Letter 1 Jiang Quan Academic Editor © 2020 Quan Jiang 2020 Quan Jiang This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 12 Apr 2020 PONE-D-20-03675R1 Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor. PLOS ONE Dear Pr. Le Bihan, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the "Comments to the Author" section, enter your conflict of interest statement in the "Confidential to Editor" section, and submit your \"Accept\" recommendation. Reviewer \#1: (No Response) Reviewer \#2: All comments have been addressed \*\*\*\*\*\*\*\*\*\* 2\. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer \#1: Yes Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 3\. Has the statistical analysis been performed appropriately and rigorously? Reviewer \#1: I Don\'t Know Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 4\. Have the authors made all data underlying the findings in their manuscript fully available? The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing) requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data---e.g. participant privacy or use of data from a third party---those must be specified. Reviewer \#1: No Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 5\. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer \#1: Yes Reviewer \#2: Yes \*\*\*\*\*\*\*\*\*\* 6\. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer \#1: Authors responded to the raised issues and addressed reviewer's concern. One point that remained unaddressed is the general assumption. The author states that "The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all." But throughout the paper the text suggests that that's not all. For example, the abstract says "The Glymphatic System (GS) has been proposed as a mechanism to clear brain tissue from waste. Its dysfunction might lead to several brain pathologies, including the Alzheimer'disease. A key component of the GS and brain tissue water circulation is the astrocyte which is regulated by acquaporin-4 (AQP4), a membrane-bound water channel on the astrocytic end-feet." Well, clearly the author is relating this to glymphatic system. Yet, relating this to glymphatic system is not the main concern. If one can measure astrocyte activity, it's not unreasonable to relate it to glymphatic system. The main concern is that AQP-4 is not only involved in modulation of astrocyte activity. It is also involved in the brain inflammatory response and also in the regulation of the extracellular volume. Both of these could affect diffusion signal. Blocking AQP-4 could lead to inflammation or changes in the extracellular fluid which affect the diffusion signal. Therefore, what you observe here may have nothing to do with the astrocyte activity. This limitation should be addressed. PS. Regarding DG, if the data is noisy, it should lead to higher standard deviation not a systematic group mean difference. Reviewer \#2: The authors resolved well my concerns about their work and made the manuscript more clear. I have no other question to add. \*\*\*\*\*\*\*\*\*\* 7\. PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. **Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy). Reviewer \#1: No Reviewer \#2: No \[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link \"View Attachments\". If this link does not appear, there are no attachment files to be viewed.\] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, <https://pacev2.apexcovantage.com/>. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at <[email protected]>. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0229702.r004 Author response to Decision Letter 1 28 Apr 2020 Reviewer \#1: Authors responded to the raised issues and addressed reviewer's concern. One point that remained unaddressed is the general assumption. The author states that "The aim of our work is to show that the modulation of astrocyte activity by TGN-020 which is known as a AQP4 channel blocker can be monitored with diffusion MRI and in particular the Sindex, that's all." But throughout the paper the text suggests that that's not all. For example, the abstract says "The Glymphatic System (GS) has been proposed as a mechanism to clear brain tissue from waste. Its dysfunction might lead to several brain pathologies, including the Alzheimer'disease. A key component of the GS and brain tissue water circulation is the astrocyte which is regulated by acquaporin-4 (AQP4), a membrane-bound water channel on the astrocytic end-feet." Well, clearly the author is relating this to glymphatic system. Yet, relating this to glymphatic system is not the main concern. If one can measure astrocyte activity, it's not unreasonable to relate it to glymphatic system. The main concern is that AQP-4 is not only involved in modulation of astrocyte activity. It is also involved in the brain inflammatory response and also in the regulation of the extracellular volume. Both of these could affect diffusion signal. Blocking AQP-4 could lead to inflammation or changes in the extracellular fluid which affect the diffusion signal. Therefore, what you observe here may have nothing to do with the astrocyte activity. This limitation should be addressed. PS. Regarding DG, if the data is noisy, it should lead to higher standard deviation not a systematic group mean difference � The statement about the Glymphatic System in the abstract and the introduction is only to explain our general motivation to conduct our study of the potential of diffusion MRI to monitor astrocyte activity. It is not a claim that the GS exists (we believe we have been careful in the manuscript about this fact). We only briefly review the literature to shade light on the context of the study and indicate why investigating astrocyte activity might be important. We are aware that TGN-020 effects are not specific to astrocytes, as stated in the manuscript. Effects on the brain inflammatory response can be discarded given the time course of the effects we have observed. Regarding the extracellular space (ISF) effects on the diffusion MRI would result only from changes in diffusion hindrance/tortuosity (fluid flow is well too slow to result in IVIM effects, this has been shown by other groups, furthermore the Sindex was purposely calculated using the lowest key b value 250s/mm² to make residual IVIM effects completely negligible). Hence, variations in extracellular space shape and volume only reflect changes in the local cellular background (a kind of negative imprint). The decrease in Sindex (and sADC increase) points out to decrease in the amount of diffusion hindrance from astrocytes (which is by far the dominant cell type in the regions where we observed the largest response). As stated in the discussion "Based on established diffusion MRI mechanisms \[28\] this hindrance decrease suggests an astrocyte volume reduction \[43,44\] associated with an increase of the ISF (were diffusion is tortuous) \[45\] or an increase in astrocyte membrane permeability and water exchange". We believe that this statement about the mechanism leading to our observation is correct and broad enough, and the manuscript was not changed. � Regarding the DG, the data are, indeed, very noisy given the very small voxel count. The apparent group difference may just be coincidental as the standard-deviation cannot be accurately estimated precisely given the small voxel count. We realize that this might be puzzling, but, for scientific integrity, we decided not to "hide" those results but to show them. A note has been added to the caption of SuppL Fig.1. ###### Submitted filename: 200303-responses_to_reviewers-REV.docx ###### Click here for additional data file. 10.1371/journal.pone.0229702.r005 Decision Letter 2 Jiang Quan Academic Editor © 2020 Quan Jiang 2020 Quan Jiang This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 29 Apr 2020 Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor. PONE-D-20-03675R2 Dear Dr. Le Bihan, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. 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22,201

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"work", "show", "modulation", "astrocyte", "activity", "known", "channel", "blocker", "monitored", "diffusion", "MRI", "particular", "Sindex", "throughout", "paper", "text", "suggests", "example", "abstract", "says", "Glymphatic", "System", "GS", "proposed", "mechanism", "clear", "brain", "tissue", "waste", "dysfunction", "might", "lead", "several", "brain", "pathologies", "including", "key", "component", "GS", "brain", "tissue", "water", "circulation", "astrocyte", "regulated", "water", "channel", "astrocytic", "Well", "clearly", "author", "relating", "glymphatic", "system", "Yet", "relating", "glymphatic", "system", "main", "concern", "one", "measure", "astrocyte", "activity", "unreasonable", "relate", "glymphatic", "system", "main", "concern", "involved", "modulation", "astrocyte", "activity", "also", "involved", "brain", "inflammatory", "response", "also", "regulation", "extracellular", "volume", "could", "affect", "diffusion", "signal", "Blocking", "could", "lead", "inflammation", "changes", "extracellular", "fluid", "affect", "diffusion", "signal", "Therefore", "observe", "may", "nothing", "astrocyte", "activity", "limitation", "addressed", "PS", "Regarding", "DG", "data", "noisy", "lead", "higher", "standard", "deviation", "systematic", "group", "mean", "difference", "Reviewer", "authors", "resolved", "well", "concerns", "work", "made", "manuscript", "clear", "question", "add", "PLOS", "authors", "option", "publish", "peer", "review", "history", "article", "mean", "https", "published", "include", "full", "peer", "review", "attached", "files", "choose", "identity", "remain", "anonymous", "review", "may", "still", "made", "public", "want", "identity", "public", "peer", "review", "information", "choice", "including", "consent", "withdrawal", "please", "see", "Privacy", "Policy", "https", "Reviewer", "Reviewer", "NOTE", "reviewer", "comments", "submitted", "attachment", "file", "attached", "email", "accessible", "via", "submission", "site", "Please", "log", "account", "locate", "manuscript", "record", "check", "action", "link", "View", "link", "appear", "attachment", "files", "revising", "submission", "please", "upload", "figure", "files", "Preflight", "Analysis", "Conversion", "Engine", "PACE", "digital", "diagnostic", "tool", "https", "PACE", "helps", "ensure", "figures", "meet", "PLOS", "requirements", "use", "PACE", "must", "first", "register", "user", "Registration", "free", "login", "navigate", "UPLOAD", "tab", "find", "detailed", "instructions", "use", "tool", "encounter", "issues", "questions", "using", "PACE", "please", "email", "us", "figures", "Please", "note", "Supporting", "Information", "files", "need", "step", "Author", "response", "Decision", "Letter", "Apr", "Reviewer", "Authors", "responded", "raised", "issues", "addressed", "reviewer", "concern", "One", "point", "remained", "unaddressed", "general", "assumption", "author", "states", "aim", "work", "show", "modulation", "astrocyte", "activity", "known", "channel", "blocker", "monitored", "diffusion", "MRI", "particular", "Sindex", "throughout", "paper", "text", "suggests", "example", "abstract", "says", "Glymphatic", "System", "GS", "proposed", "mechanism", "clear", "brain", "tissue", "waste", "dysfunction", "might", "lead", "several", "brain", "pathologies", "including", "key", "component", "GS", "brain", "tissue", "water", "circulation", "astrocyte", "regulated", "water", "channel", "astrocytic", "Well", "clearly", "author", "relating", "glymphatic", "system", "Yet", "relating", "glymphatic", "system", "main", "concern", "one", "measure", "astrocyte", "activity", "unreasonable", "relate", "glymphatic", "system", "main", "concern", "involved", "modulation", "astrocyte", "activity", "also", "involved", "brain", "inflammatory", "response", "also", "regulation", "extracellular", "volume", "could", "affect", "diffusion", "signal", "Blocking", "could", "lead", "inflammation", "changes", "extracellular", "fluid", "affect", "diffusion", "signal", "Therefore", "observe", "may", "nothing", "astrocyte", "activity", "limitation", "addressed", "PS", "Regarding", "DG", "data", "noisy", "lead", "higher", "standard", "deviation", "systematic", "group", "mean", "difference", "statement", "Glymphatic", "System", "abstract", "introduction", "explain", "general", "motivation", "conduct", "study", "potential", "diffusion", "MRI", "monitor", "astrocyte", "activity", "claim", "GS", "exists", "believe", "careful", "manuscript", "fact", "briefly", "review", "literature", "shade", "light", "context", "study", "indicate", "investigating", "astrocyte", "activity", "might", "important", "aware", "effects", "specific", "astrocytes", "stated", "manuscript", "Effects", "brain", "inflammatory", "response", "discarded", "given", "time", "course", "effects", "observed", "Regarding", "extracellular", "space", "ISF", "effects", "diffusion", "MRI", "would", "result", "changes", "diffusion", "fluid", "flow", "well", "slow", "result", "IVIM", "effects", "shown", "groups", "furthermore", "Sindex", "purposely", "calculated", "using", "lowest", "key", "b", "value", "make", "residual", "IVIM", "effects", "completely", "negligible", "Hence", "variations", "extracellular", "space", "shape", "volume", "reflect", "changes", "local", "cellular", "background", "kind", "negative", "imprint", "decrease", "Sindex", "sADC", "increase", "points", "decrease", "amount", "diffusion", "hindrance", "astrocytes", "far", "dominant", "cell", "type", "regions", "observed", "largest", "response", "stated", "discussion", "Based", "established", "diffusion", "MRI", "mechanisms", "hindrance", "decrease", "suggests", "astrocyte", "volume", "reduction", "associated", "increase", "ISF", "diffusion", "tortuous", "increase", "astrocyte", "membrane", "permeability", "water", "exchange", "believe", "statement", "mechanism", "leading", "observation", "correct", "broad", "enough", "manuscript", "changed", "Regarding", "DG", "data", "indeed", "noisy", "given", "small", "voxel", "count", "apparent", "group", "difference", "may", "coincidental", "accurately", "estimated", "precisely", "given", "small", "voxel", "count", "realize", "might", "puzzling", "scientific", "integrity", "decided", "hide", "results", "show", "note", "added", "caption", "SuppL", "Submitted", "filename", "Click", "additional", "data", "file", "Decision", "Letter", "Jiang", "Quan", "Academic", "Editor", "Quan", "Jiang", "Quan", "Jiang", "open", "access", "article", "distributed", "terms", "Creative", "Commons", "Attribution", "License", "permits", "unrestricted", "use", "distribution", "reproduction", "medium", "provided", "original", "author", "source", "credited", "Apr", "Diffusion", "MRI", "reveals", "vivo", "changes", "astrocyte", "function", "induced", "inhibitor", "Dear", "Dr", "Le", "Bihan", "pleased", "inform", "manuscript", "judged", "scientifically", "suitable", "publication", "formally", "accepted", "publication", "complies", "outstanding", "technical", "requirements", "Within", "one", "week", "receive", "containing", "information", "amendments", "required", "prior", "publication", "required", "modifications", "addressed", "receive", "formal", "acceptance", "letter", "manuscript", "proceed", "production", "department", "scheduled", "publication", "Shortly", "formal", "acceptance", "letter", "sent", "invoice", "payment", "follow", "ensure", "efficient", "production", "billing", "process", "please", "log", "Editorial", "Manager", "https", "click", "Update", "link", "top", "page", "update", "user", "information", "billing", "related", "questions", "please", "contact", "Author", "Billing", "department", "directly", "authorbilling", "institution", "institutions", "press", "office", "please", "notify", "upcoming", "paper", "enable", "help", "maximize", "impact", "preparing", "press", "materials", "manuscript", "must", "inform", "press", "team", "soon", "possible", "later", "hours", "receiving", "formal", "acceptance", "manuscript", "remain", "strict", "press", "embargo", "pm", "Eastern", "Time", "date", "publication", "information", "please", "contact", "onepress", "kind", "regards", "Quan", "Jiang", "Ph", "Academic", "Editor", "PLOS", "ONE", "Additional", "Editor", "Comments", "optional", "comments", "Responses", "Questions", "Comments", "Author", "authors", "adequately", "addressed", "comments", "raised", "previous", "round", "review", "feel", "manuscript", "acceptable", "publication", "may", "indicate", "bypass", "Comments", "Author", "section", "enter", "conflict", "interest", "statement", "Confidential", "Editor", "section", "submit", "recommendation", "Reviewer", "comments", "addressed", "manuscript", "technically", "sound", "data", "support", "conclusions", "manuscript", "must", "describe", "technically", "sound", "piece", "scientific", "research", "data", "supports", "conclusions", "Experiments", "must", "conducted", "rigorously", "appropriate", "controls", "replication", "sample", "sizes", "conclusions", "must", "drawn", "appropriately", "based", "data", "presented", "Reviewer", "Yes", "statistical", "analysis", "performed", "appropriately", "rigorously", "Reviewer", "Yes", "authors", "made", "data", "underlying", "findings", "manuscript", "fully", "available", "PLOS", "Data", "policy", "http", "sharing", "requires", "authors", "make", "data", "underlying", "findings", "described", "manuscript", "fully", "available", "without", "restriction", "rare", "exception", "please", "refer", "Data", "Availability", "Statement", "manuscript", "PDF", "file", "data", "provided", "part", "manuscript", "supporting", "information", "deposited", "public", "repository", "example", "addition", "summary", "statistics", "data", "points", "behind", "means", "medians", "variance", "measures", "available", "restrictions", "publicly", "sharing", "data", "participant", "privacy", "use", "data", "third", "party", "must", "specified", "Reviewer", "manuscript", "presented", "intelligible", "fashion", "written", "standard", 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"files", "choose", "identity", "remain", "anonymous", "review", "may", "still", "made", "public", "want", "identity", "public", "peer", "review", "information", "choice", "including", "consent", "withdrawal", "please", "see", "Privacy", "Policy", "https", "Reviewer", "Acceptance", "letter", "Jiang", "Quan", "Academic", "Editor", "Quan", "Jiang", "Quan", "Jiang", "open", "access", "article", "distributed", "terms", "Creative", "Commons", "Attribution", "License", "permits", "unrestricted", "use", "distribution", "reproduction", "medium", "provided", "original", "author", "source", "credited", "May", "Diffusion", "MRI", "reveals", "vivo", "changes", "astrocyte", "function", "induced", "inhibitor", "Dear", "Dr", "Le", "Bihan", "pleased", "inform", "manuscript", "deemed", "suitable", "publication", "PLOS", "ONE", "Congratulations", "manuscript", "production", "department", "institution", "institutions", "press", "office", "please", "notify", "upcoming", "paper", "point", "enable", "help", "maximize", "impact", "preparing", "press", "materials", "manuscript", "please", "inform", "press", "team", "within", "next", "hours", "manuscript", "remain", "strict", "press", "embargo", "pm", "Eastern", "Time", "date", "publication", "information", "please", "contact", "onepress", "questions", "concerns", "please", "email", "plosone", "Thank", "submitting", "work", "PLOS", "ONE", "kind", "regards", "PLOS", "ONE", "Editorial", "Office", "Staff", "behalf", "Quan", "Jiang", "Academic", "Editor", "PLOS", "ONE", "Competing", "Interests", "authors", "declared", "competing", "interests", "exist" ]

Background ========== It is widely acknowledged that breastfeeding provides the ideal nutrition for infants, with authorities worldwide recommending exclusive breastfeeding for the first six months for children living in developed countries \[[@B1],[@B2]\]. Despite such endorsements, studies have shown that physician knowledge on breastfeeding is far from ideal \[[@B3]-[@B8]\], as they receive relatively little formal training on breastfeeding \[[@B5]\], and often fail to follow evidence-based medicine (EBM) guidelines \[[@B9]\]. However, it can be difficult for physicians to keep up-to-date with the high volume of constantly changing, sometimes conflicting, information. It has been suggested that a physician needs to read 17 articles each day to keep up with current medical literature, yet they have limited time to assess the information they receive \[[@B10]\]. Additionally, studies have shown that access to EBM resources is variable \[[@B11]-[@B16]\], and even when resources are available, the ability of physicians to locate relevant information and assess its validity appears limited \[[@B11],[@B12],[@B17]\]. It is therefore unsurprising that physicians may turn to specialist information services for help. Such services include, UKMi (a UK National Health Service \[NHS\] funded medicines information service that has specialist centres taking medicine calls on pregnancy and breastfeeding) \[[@B18]\] and the Motherisk Program (a Canadian-based teratogen information service) \[[@B19]\]. These services tend to be aimed at healthcare professionals, who report high levels of satisfaction with the information provided \[[@B20],[@B21]\]. Equivalent information services for consumers are limited, but the Breastfeeding Network (BfN) (a UK-based charity established in 1997) aims to be an independent source of support and information for breastfeeding women and others, including healthcare professionals \[[@B22]\]. One of the BfN services is the provision of 'Supporterline'; a service where information and support is provided to mothers who are breastfeeding that is complementary to that of health visitors (a health visitor is a UK qualified nurse whose role promotes health in the whole community, particularly involved with families who have children under five), midwives and other healthcare professionals. In 2006 the Supporterline took over 20,000 calls, many specifically related to medicine use. Consequently, in 2007 a national 0800 (free) telephone service for medicine-related enquiries was established, The Drugs in Breastmilk Helpline, which has seen year-on-year increases in enquiry numbers (e.g. 576 in 2007, which had risen to 2215 in 2010). To cope with this increased demand, the service now operates seven days a week and when not manned takes calls via an answer machine. The majority of enquiries are answered by a pharmacist, with support from a trained BfN worker. The Helpline has access to standard medical reference texts (e.g. The British National Formulary \[[@B23]\]) and specialist breastfeeding references, for example Hale\'s *Medications and Mother's Milk*\[[@B24]\], LactMed (an online database from The National Library of Medicine) \[[@B25]\], Briggs et al\'s *Drugs in Pregnancy and Lactation*\[[@B26]\] and *Martindale The Extra Pharmacopoeia*\[[@B27]\]. Given the growth in user numbers, the aim of this study was to assess consumer and healthcare professional opinion, analyse the enquiries taken and to ascertain how information provided was used. Methods ======= Enquirer opinion was surveyed via a structured telephone interview or self-administered survey. A sample size calculator \[[@B28]\] indicated that 90 participants were required to achieve 95% confidence (with a 10% margin for error) so that the study results reflect the results expected from the total enquirer population to the Helpline (in 2009, n = 1388). Analysis of workload data and factoring in a 40% drop-out rate indicated that the study duration would be approximately two months. Participant recruitment began in December 2010. Enquiries were generated either by telephone call or email. All enquirers who contacted the service were eligible to be enrolled in to the study, provided they were English speakers. For telephone enquiries, prior to the BfN call handler concluding the conversation, the enquirer was informed of the study and asked if they would be happy to be contacted in 2 to 4 weeks time to discuss the call further. For those people who agreed to be contacted, their details, along with the enquiry and answer provided by the Helpline, were sent to researchers at the University of Wolverhampton. Each caller was then telephoned and interviewed. Interviews followed a semi-structured format and took approximately 10 min to complete. For email replies, information relating to the study was included at the bottom of the written emailed reply, along with an invitation to complete the self-administered survey. Return of the email was taken as the person giving consent to participate. Questions used for both the interviews and surveys were drawn and adapted from UKMi surveys used to quality assure their service. Questions asked differed depending on whether the enquirer was a member of the public or a healthcare professional. However, the questions asked to each respective group were the same for both the telephone interview and self-administered survey. Data collected fell broadly into three sections: demographic information; evaluation of the Helpline; and usefulness of the information. Questions consisted of a mixture of open, closed and semantic differential scales (e.g. 5 point Likert scales). Prior to data collection starting, a two-week pilot study was undertaken on the study population to determine the data collection tools' content validity and reliability. Feedback from the pilot study showed that no changes were required. Research ethics committee approval was granted by The Behavioural Sciences Research Ethics Committee at Wolverhampton University. NHS ethics approval was not required as the work was assessed as evaluation and not research. Data analysis ============= Responses were collated, stored and analysed using survey software (SNAP for Windows) and spreadsheets (Microsoft Excel 2003 for Windows). Data entry were performed by two research assistants and the validity of data entry assured by a member of the research team (PR) who was not involved in data entry. PR undertook random checks of the data (equating to approximately 20% of all data). Simple descriptive statistics were used to summarise respondents' data. Free text answers were reviewed and categorised by PR. Data were managed using Excel and common themes generated that were agreed upon by both authors. Results ======= One hundred and forty-two enquirers were contacted. On follow up, twenty-one declined to participate and twenty were lost, leaving 101 replies for analysis: 77 from women (64 telephone calls and 13 emails); and 24 from healthcare professionals (10 telephone calls and 14 emails). Data from breastfeeding women ----------------------------- ### Demographic characteristics Most women were under the age of forty (88%, n = 68/77), had either 1 or 2 children (87%, n = 67/77), undertook formal education beyond A levels (84%, n = 63/75) (i.e. further education colleges or university) and were Caucasian (92%, n = 70/76). No calls were received from any person under the age of twenty-five. Demographic data are shown in Table [1](#T1){ref-type="table"}. ###### Characteristics of participants **Demographic characteristics of the breastfeeding women enquirers** ---------------------------------------------------------------------- ------------------------------------------ ------- ------- Age (n = 77) 25-29 18 23 30-34 29 38 35-39 21 27 over 40 9 12 Which child is this in your family? (n = 77) first 46 60 second 21 27 third 8 10 fourth 1 1 fifth 1 1 Ethnic origin (n = 76) white 70 92 mixed 2 3 Asian/Asian British 3 4 other 1 1 School leaving age (n = 75) 16 or under 8 11 17 4 5 18 12 16 19 or over 51 68 **Healthcare professional occupation (n = 24)** **n** **%** Infant feeding co-ordinator 8 33 Health visitor 6 25 Midwife 4 17 Physician 1 4 Pharmacist 1 4 Community staff nurse 1 4 Community breastfeeding leader 1 4 Hospital nurse specialising in lactation 1 4 Not stated 1 4 ### Breastfeeding women's opinion on the helpline Their experience with the service was very positive. Seventy (91%) women were either 'very satisfied' or 'satisfied' with how quickly their call was answered and stated that the answer they received provided sufficient information to enable them to make a decision regarding their enquiry (90%, n = 69/77). These findings are reflected in the level of satisfaction noted, where 72 (94%) were either 'very satisfied' or 'satisfied' with the overall service offered (Table [2](#T2){ref-type="table"}). Only three women felt that phoning the Helpline had not helped them. Analysis of these three calls showed that two involved complex calls, involving persistent nipple and breast pain that were being managed unsatisfactorily by their physician. The third case involved use of a topical nasal decongestant whilst breastfeeding. Advice given was that the woman could use the product, so it is unclear as to why this person said the advice did not provide enough information to make a decision. ###### Questions asked of breastfeeding women and healthcare professionals **Healthcare professionals (n = 24)** **Breastfeeding women (n = 77)** ------------------------------------------------------------------------------------ --------------------------------------- ---------------------------------- ---- ----- **How did you find out about the telephone number?** Birth to Five 0 0 12 16 given it by someone 6 26 23 30 internet 8 35 27 35 leaflet 8 35 16 21 other 1 4 0 0 **Have you called the Drugs in Breastmilk Helpline before?** Yes 17 71 13 17 No 7 30 64 83 **Would you recommend this service to others?** Yes 22 92 77 100 No 2 9 0 0 **Did the information provide you with enough information to make a decision?** Completely 20 87 69 90 Partly 3 13 5 7 Not at all 0 0 3 4 **How satisfied were you with the response to your query, with respect to speed?** Very satisfied N/A N/A 64 83 Satisfied N/A N/A 6 8 Neither N/A N/A 5 7 Dissatisfied N/A N/A 2 3 Very dissatisfied N/A N/A 0 0 **Overall, how satisfied were you with the service?** Very satisfied N/A N/A 69 90 Satisfied N/A N/A 3 4 Neither N/A N/A 4 5 Dissatisfied N/A N/A 1 1 Very dissatisfied N/A N/A 0 0 Despite these enquirers not having been given information to enable them to make a decision, they, as well as all other women, said they would recommend the service to other people (Table [2](#T2){ref-type="table"}). Most women (83%, n = 64) had not contacted the Helpline before and found out about the service primarily through the internet (35%, n = 27) or had been given the telephone number (30%, n = 23). Twelve (16%) had also been made aware by the 'Birth to Five' information booklet (an NHS information booklet distributed to all parents on the health, wellbeing and development of children from birth to five years old) (Table [2](#T2){ref-type="table"}). ### Content of enquiries Seventy enquiries directly related to medicine taking and seven involved giving general advice (Table [3](#T3){ref-type="table"}). Four of the latter involved issues of infant feeding whilst the mother was being treated with antifungal medication. Sixty-five enquiries involved the use of proposed medicines. These were predominantly to manage acute conditions, with self-management of minor illness (n = 11), anti-infectives (n = 14) and pain relief (n = 9) most frequently enquired about. Seventeen enquiries involved medicines that could be used for the management of long-term illness. These covered many medicine classes, with only medicines for depression (n = 4) and Crohn's disease (n = 3) receiving more than one enquiry. ###### Type of medicine-related enquiry received from participants **Proposed medicines** **Currently taken medicinesN (%)** **Advice givenn (%)** ----------------------------------- ------------------------ ------------------------------------ ----------------------- --------- Healthcare professionals (n = 24) 13 (54%) 5 (21%) 2 (8%) 4 (16%) Breastfeeding women (n = 77)\* 50 (65%) 17 (22%) 5 (7%) 7 (9%) \* Two enquiries involved medicines for the treatment of proposed acute and chronic illness. Five calls involved medicines that were currently being taken. Two concerned the effect medicines had on breastmilk (amitriptyline and penicillin), whilst the other three related to continuation of prescribed medication (asthma inhalers, azathioprine and propylthiouracil). The latter three enquirers had already spoken to a healthcare professional. In the calls involving azathioprine and propylthiouracil the women had been told by hospital physicians that they should not take the medicine, but this advice had been contradicted by pharmacists. In both instances the women wanted clarification on what to do. The Helpline advisor agreed with the pharmacist that the medicines could still be taken whilst breastfeeding. The call involving asthma inhalers centred on the woman wanting reassurance that the advice (to continue taking salbutamol and beclometasone inhalers) given by a practice nurse was correct, which it was. The Helpline stated that of the 70 enquiries involving medicines, 68 could be taken whilst breastfeeding. All women followed the guidance given by the Helpline. In the two enquiries where the Helpline did not advise to take the medicine, both involved referral back to the physician on grounds of potential misdiagnosis. Fifty-three women contacted a healthcare professional before phoning the Helpline; 49 of these enquiries were about the suitability of taking a medicine (Figure [1](#F1){ref-type="fig"}). General practitioners (n = 12) and hospital doctors (n = 8) were most frequently asked for advice; other healthcare professionals were infrequently asked (pharmacists, n = 2; midwife n = 2; health visitor n = 2; practice nurse n = 1). In 10 instances women asked more than one healthcare professional for advice (in 7 cases two were asked, and in 3 cases three were asked). The need for reassurance, and receiving conflicting information were two common reasons why women called the Helpline. Typical comments were: ![Handling of medicine-related enquiries from breastfeeding women by healthcare professionals (HCPs) and the Drugs in Breastmilk Helpline.](1746-4358-7-6-1){#F1} 'Conflicting information between different sources, wasn\'t sure which information was correct.' (participant 70, telephone interview) 'Conflicting information between the physician, pharmacist and drug information.' (participant 40, telephone interview) 'Instinctively I felt the drug would be fine, but just wanted to double check.' (participant 5, email survey) 'I had to know whether such an extended period of not breastfeeding was good for my baby. You were the only group offering advice tailored to breastfeeding mums.' (participant 9, email survey) Women also often spoke of distrust of the advice given by a healthcare professional or lack of confidence in their answer: 'Lack of confidence in the doctor's advice as he sounded confused and had to be reminded that I was breastfeeding.' (participant 51, telephone interview) 'Conflicting advice off the internet and didn\'t trust the consultant's advice.' (participant 31, telephone interview) 'Pharmacist wasn\'t sure; wanted to see if ok to use while breastfeeding.' (participant 33, telephone interview) In 11 of the 49 enquiries, the healthcare professional (seven of whom were physicians) was unsure of the answer and unable to tell the person if the medicine could be taken whilst breastfeeding. For 10 of these 11 cases, staff from the Helpline, after consulting reference sources, were able to advise the women that the medicine could be safely taken. In the remaining enquiry, information was supplied to the woman but no definitive answer on whether the medicine could be taken was given. This enquiry was answered by the BfN worker and, following protocol, is only able to provide information and not advice on whether the medication can be taken. Just 17 of the remaining 38 enquiries saw agreement between the healthcare professional and Helpline staff; in all these enquiries both parties agreed that the medicine could be taken. In the 21 enquiries where there was disagreement, healthcare professionals, on 10 occasions, said the medicine should not be taken, but information from the Helpline was that it was safe to take. In one enquiry the healthcare professional stated the medicine could be taken, whereas Helpline staff recommended the mother to speak again to the physician or a breastfeeding expert about her symptoms, even though the medicine was safe to use. In the remaining 10 enquiries women received conflicting advice from two or more healthcare professionals; in all cases the Helpline staff advised that the medicine could be taken. For all enquiries where the healthcare professional was either unsure or provided information contradictory to the Helpline, women chose to follow the Helpline advice. Table [4](#T4){ref-type="table"} highlights examples of the enquiries received during the study. ###### Examples of enquiries received and answers given --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Enquiry** **Helpline response** **Resources used by Helpline** --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 'At around 37 weeks pregnant I had assumed that as I had been told to continue my medication (antidepressant) during pregnancy that it would be ok to breastfeed too, but I raised the query with the registrar at my clinic just to double check. He said he thought it would be fine but would check in a book; he did this while I waited and then told me the book said I should not breastfeed. He said he would check with the hospital pharmacy and let me know. I received a letter informing me it was ok for me to breastfeed whilst taking the medication about 4 weeks after my baby was born. Luckily enough I had contacted BfN rather than wait for the hospital to let me know!!' Information of studies carried out were supplied and an explanation was given that it was important for the patient to continue to take the medication so that she remained well, and that the benefits of breastmilk to her child far outweighed any risk to the baby through her taking medication. The amount of drug passing to the baby was discussed with the mother along with any potential side effects to look out for. Information was provided using: · Hale· Lactmed (<http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?LACT>) 'Got a bad back and wanted to know if it was safe to take naproxen or co-codamol (combination paracetamol/codeine product). Both the doctor and chemist said it was ok to take the medicines'. The mother was provided with information from Hale on the half lives of the drugs and how long the drug would remain in the mother's breastmilk. Naproxen has a half life of 12--15 hours and the amount passing into breastmilk is more than other non steroidal anti-inflammatory drugs such as ibuprofen or diclofenac. This makes it less preferable if the baby is under 6 weeks of age before which time hepatic and renal function is lower. There have been concerns raised about the safety of co-codamol during breastfeeding following the death of a baby in Canada. The mother was counselled to watch for signs of unusual drowsiness and poor feeding in the baby. If she noticed these responses she was advised to stop the drug and seek medical advice. Information was provided using: · Hale · Lactmed (<http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?LACT>) · <http://www.breastfeedingnetwork.org.uk/pdfs/Analgesia_and_breastfeeding_March_2010.pdf>\ <http://www.breastfeedingnetwork.org.uk/pdfs/Codeine-in-breastfeeding-June-08.pdf> Have bleeding cracked nipples, slight inversion so using nipple shields. Want something for pain and also want to know what formula to use as concerned about milk supply. Spoke with GP, pharmacist and nurse but none gave a very clear answer. Discussed moist wound healing and the need to feed or express milk more frequently. Tried to arrange a home visit from a breastfeeding supporter to help with optimising attachment of the baby at the breast, thereby preventing further nipple trauma. Mother was contacted again that evening; she was applying Vaseline to the cracks to prevent scab formation and taking paracetamol. She reported feeling better and will go back to the breast support clinic the following week Used information from:\ · BfN leaflet on moist wound healing plus breastfeeding support\ <http://www.breastfeedingnetwork.org.uk/pdfs/Cracked_Nipples_and_Moist_Wound_Healing_2002.pdf> Patient called about general anaesthetic as she was scheduled to have an ovarian cyst removed (planned operation not emergency). Consultant said to stop breastfeeding for 48 hours. Baby was 5 months old. Mother was informed about short half life of drugs used in general anaesthetic and that some of the drug would remain in fat cells of the body, to be released slowly over the following 48 hours, which might make her baby drowsy. It was suggested that she breastfeed as soon as she felt awake enough to do so. Information on the effect of any anti-emetics, which might be given was also discussed (could increase milk supply). Also discussed how the mother would manage feeds whilst in hospital, and whether she needed to have a breast pump available. Information was provided using:\ ·Hale\ ·Lactmed (<http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?LACT>)\ <http://www.breastfeedingnetwork.org.uk/pdfs/Day_Surgery_and_Breastfeeding_March_2009.pdf> --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Healthcare professional data ============================ Enquiries were taken predominantly from people whose role it is to support new mothers (Table [1](#T1){ref-type="table"}). All (100%, n = 24) healthcare professionals believed the Helpline response either completely or partially answered their enquiry, with 92% (n = 22/24) finding the information 'useful' or 'very useful'; consequently, the majority (92%, n = 22/24) would recommend the service to others. Table [2](#T2){ref-type="table"} shows that, in contrast to enquiries from breastfeeding women, the majority (71%, n = 17/24) of healthcare professionals had used the Helpline previously and preferred to email their enquiry (58%, n = 14/24 compared to only 17%, n = 13/77 of women), even though telephone responses were answered more quickly (100% within 24 hours, compared to 79%). Like women, the internet (35%, n = 8/23) and recommendation by another person (26%, n = 6/23) were common ways that they found out about the Helpline, although healthcare professionals also stated leaflets (35%, n = 8/23) were frequently the source of the information. Analysis of the 24 enquiries showed that 18 were for proposed medicines, 4 involved advice giving, and just 2 were for medicines that were being currently taken (Table [3](#T3){ref-type="table"}). Of the 18 medicines for proposed use, 13 (72%) were to treat acute problems. Medicines involved in these cases were wide ranging, with only anti-infectives (n = 2) and domperidone (n = 2) occurring more than once. The 5 enquiries received for proposed medicines to treat chronic conditions were equally diverse, for example medicines to treat schizophrenia, Attention Deficit Hyperactivity Disorder and Cystic Fibrosis. The two enquiries that concerned medicines being currently taken for chronic conditions were montelukast for asthma and levothyroxine for hypothyroidism. Fourteen (58%) had consulted at least one reference source before contacting the Helpline. The most frequently used reference text was Hale (a specialist text on the safety of drugs in breastfeeding) (n = 9) or the British National Formulary (BNF - a standard text used in the UK for prescribing information) (n = 6). In two cases, a hospital drug information pharmacist was also consulted. Five of the six people that consulted the BNF went on to say that the information it contained was not detailed enough to answer the problem. One of these respondents also stated they did not have access to Hale and thus called the Helpline. Hale was most frequently consulted, yet despite it being more informative than the BNF, 66% of respondents still contacted the Helpline as they believed they would get a more definitive answer based on prior use of the service. Where healthcare staff contacted a specialist drug information pharmacist, the information they provided was deemed insufficient by the enquirer to answer their question. Four of the 10 healthcare professionals stated that previous positive experiences of the Helpline prompted them to contact the service rather than consult reference sources and two called for reassurance (the other enquirers never specified a reason). All enquirers said the information provided by the Helpline either completely (n = 20/23, 87%) or partly (n = 3/23, 13%) provided them with enough information to make a decision. Once in possession of the information, 22 went on to say how they used the information. Thirteen passed on the information to the patient, five gave the information to the relevant prescriber and four used it to make a treatment decision. Discussion ========== The findings from this study show that the predominately pharmacist-led service provided by the BfN is well received by both healthcare professionals and breastfeeding women, and is consistent with user satisfaction surveys of pharmacist-led drug information services \[[@B20],[@B21]\]. Unlike most information services, the BfN Helpline actively encourages the public to contact the service, and this was reflected in a higher proportion of enquiries taken from the public compared to healthcare professionals. Most women were previous non-users of the service in contrast to healthcare professionals, where the majority had contacted the service before. The healthcare professionals predominantly had a specialist role associated with maternal services and this may explain why they were aware of the service. Provision of the Helpline seems well-founded given the level of conflicting information given by two or even three healthcare staff, and the lack of confidence or distrust women had in answers they provided. The disparity seen between the answers given by the Helpline and the responses from healthcare staff further reinforces the decision by their patients to seek out specialist advice. The fact that over 75% of healthcare staff did not know the answer, or gave a conflicting answer compared to that given by Helpline staff, suggests that healthcare staff may not have sufficient knowledge or resources in this field; which echoes previous findings \[[@B2]-[@B7],[@B29],[@B30]\]. Where Helpline staff contradicted healthcare staff advice, breastfeeding women chose to follow Helpline advice. This was not unexpected as many women had contacted the Helpline as a 'last resort'. However, in clinical practice the contradiction of one healthcare professional by another can be difficult, especially when one is perceived as more authoritative than the other (e.g. lactation consultant doctor versus a pharmacist). In these situations, it would seem most prudent for the contradicting person to talk with the person who gave the initial advice, so that a consistent message is given to the breastfeeding women. This did not routinely appear to happen in this study. Findings from this study also raise questions about the suitability of 'generalist' healthcare staff to handle breastfeeding enquiries; in particular, physicians, as they were most consulted. A study by Wallace and Kosmala-Anderson reinforces this view, as they found that general practitioners in the UK saw updates on prescribing for breastfeeding mothers as a low priority, with just 19% highlighting this as an area of need, despite only 34% describing themselves as competent or expert in this field \[[@B31]\]. Only just over half of healthcare professionals had consulted a reference source, and those that did found standard texts, such as the BNF (which almost all UK healthcare staff have easy access to), to be inadequate, as data contained within it lacked sufficient detail. The deficiency of the BNF has been highlighted by the National Institute for Health and Clinical Excellence (NICE) and advocates that healthcare professionals consult supplementary sources of information \[[@B32]\]. Whilst some healthcare staff did consult other sources (i.e. the specialist text by Hale), they still felt unsure on how to interpret the information in a way that was useful to the mother, and sought guidance from the Helpline. This lack of confidence in interpretation of appropriate reference sources suggests a greater need for training. It further supports the use of a specialist, given their access to more resources (as evidenced from Table [4](#T4){ref-type="table"}) and ability to interpret the data to provide informed answers on medicine suitability. A study by Akus also found that healthcare practitioners were using outdated sources for making safety recommendations to their patients \[[@B33]\]. Whilst this cannot be substantiated in this study, other studies \[[@B34]\] have found similar results to Akus and it seems unlikely that healthcare staff in practice are using the most up-to-date reference sources when evaluating medicine suitability for breastfeeding mothers. Enquiries predominantly related to proposed medicines to treat acute self-limiting problems. Advice given by healthcare staff to patients erred on the side of caution; that is they were told to avoid taking the medicine. Whilst this negates any possible adverse events to the child, it does not necessarily equate to appropriate clinical care where decisions should be evidence-based and an assessment of the risk versus benefits to the baby made \[[@B35],[@B36]\]. In defence of healthcare staff, the lack of manufacturer data on the effects of medicines passing in to breastmilk makes recommendation of products problematic. Without safety data, manufacturers generally advocate avoidance. If products are then recommended, prescribers do so outside of the product licence, and liability will fall on that individual and not the company. Amir and colleagues looked at knowledge, attitude and practices of Australian GPs in obtaining information for medicines for breastfeeding women, and found that medico-legal concerns were common, and 76% stated that this was important in their decision-making \[[@B37]\]. What is not in question is that patients will continue to require medication during breastfeeding. Studies have shown that this is relatively commonplace \[[@B38]-[@B40]\], and medicines play an important part in a woman's decision to start and continue breastfeeding, with women frequently hesitant to combine the two, choosing either to stop breastfeeding or attempt to limit exposure \[[@B40]-[@B42]\]. Therefore, given healthcare professional uncertainty and caution in advice-giving, it was interesting to note that the Helpline was able to give definitive answers in almost all cases relating to medicine taking. Given that other countries do experience similar problems with under-skilled healthcare staff in this field, then the establishment of similar Helplines could be considered. The costs of running the BfN Helpline was initially modest, but as call numbers have increased the Helpline has had to rely on greater levels of goodwill from the people that answer the enquiries. From our experience it would be prudent, when starting such a Helpline, to factor in additional resources and money to meet increased demand. Limitations =========== It is acknowledged that this study provides a 'snapshot' of the service, and the number of participants was relatively low. In addition, the study had a high drop-out rate/lost to follow-up, which may have affected the data through non-response bias. Lastly, the findings may not be broadly generalisable to other countries due to differing healthcare structures. Conclusion ========== The Breastfeeding Networks' Drugs in Breastmilk Helpline provides an important service to patients and healthcare staff to make informed decisions on medicine-taking whilst breastfeeding. The relatively high level of healthcare professional uncertainty or incorrect advice given to patients, coupled with patient distrust, signals that healthcare professional education needs improving and that greater use of specialist services should be encouraged. Competing interests =================== The work was supported by the Breastfeeding Network to employ Miss Seera and Miss Matharu to conduct the telephone interviews. Wendy Jones is employed by the Breastfeeding Network to answer calls, but was not involved in data collection or analysis (except for agreeing qualitative themes). Authors' contributions ====================== PR and WJ constructed the data collection tool. WJ answered the majority of the calls. PR oversaw the telephone interviews and verified data capture. PR undertook the analysis. PR and WJ jointly wrote the manuscript. Acknowledgements ================ Thanks to Rinisha Seera and Summet Matharu in data collection and entry. The role of Mrs Ruth Rhodes in answering calls to the Drugs in Breastmilk Helpline and collection of data from callers is gratefully acknowledged, as is the support of the Breastfeeding Network in funding the operation of the Drugs in Breastmilk Helpline.

{ "pile_set_name": "PubMed Central" }

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22,202

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Introduction ============ Dental implants have always undergone different modifications in order to adapt them as much as possible to the morphology of the bone where they will be placed ([@B1]). In this regard, it is important to reduce the diameter of implants placed in areas with limited space for prosthetic reconstruction, such as between adjacent crowns, as conventional-diameter implants may cause damage to the periodontal ligament of adjacent teeth o compromising aesthetics ([@B2]-[@B4]). Narrow implants (those with a diameter less than 3.4 mm) ([@B5]) are particularly useful when the bone crest width does not allow for placement of a conventional diameter implant without the use of regeneration techniques ([@B6]-[@B8]). There is currently no clear scientific evidence regarding the success rate of narrow implants ([@B9]-[@B11]). This study's main purpose is to evaluate the bone loss (BL) around narrow implants after 2 years of implant loading and compare it to that of conventional¬ diameter implants. BL were also evaluated by taking into account where implants were placed, type of loading, whether implants were placed in areas that had previously undergone bone regeneration, or whether patients had previously suffered from periodontal disease. Material and Methods ==================== -A retrospective case-control study was carried out. 82 implants (31 narrow implants and 51 conventional implants) placed in 40 patients were selected. To avoid statistical bias, a cluster of one implant per patient was randomly selected (20 narrow implants and 20 conventional implants). Randomization was carried out by numbering the implants present in the patient and rolling a die to identify the selected implant. The group "Cases" described as patients of either gender or age, carriers exclusively narrow implants (StraumannTM, grade 4 titanium, SLA surface, 3.3 millimeters in diameter, length 10-12 mm, Switzerland) and having a minimum 2 years of follow- up. 20 patients with these characteristics were found from 2009 until 2014. The group "Control", sought by matching for gender and age other 20 patients, who will carry only standard implants of the same brand, with identical lengths and identical tracking system but with 4.1 millimeters in diameter. To evaluate the changes resulting from BL around the implants, a total of 80 panoramic radiographs were used of the 40 patients. The first panoramic image of each patient was taken when the implants were loaded and the second one 2 years after implant placement, using a Planmeca ProMax® orthopantomograph, serial number RDX309857, with a magnification of 1-1. The patients' head and lip position in the orthopantomograph was controlled when taking the radiographs. All BL measurements were taken by a calibrated examiner. This calibration consisted in having a second examiner retake all measurements and compare them. The evaluator calculated a correlation coefficient of 0.925, which implies a coincidence percentage higher than 90%. -Inclusion and exclusion criteria: The patients included in this study had implants (3.3 or 4.1 mm) placed in either the anterior or posterior region of the mandible and maxilla. The patients excluded in this study were treated with 8-mm or smaller implants, whose implants were loaded for less than 2 years, with more than two years of follow-up but no radiography at 2 years (the initial total number of patients with narrow implants was 22, but the final number of patients studied is 20), patients taking medication that affects bone metabolism (corticosteroids, bisphosphonates), patients with inflammatory bone diseases or genetic diseases, patients with active periodontal disease (must be under control after 6 months of periodontal treatment). No early failures were recorded before two years. -Studied variables: In the present study, BL (main variable) was measured (each implant was assigned to a category) using Lagervall & Jansson's index ([@B12]) modified by Corcuera Flores *et al.* ([@B8]) to add a fifth category, using panoramic radiographs of each patient to compare the panoramic images taken after implant loading with the ones taken two years later. The implants used in this study were loaded with single crowns, fixed implant-borne prostheses that require splinting with other implants or overdentures. The variables tobacco smoking, age and gender were collected from the medical records of patients enrolled in the study. Periodontitis was pre-diagnosed when patients exhibited insertion loss and/or a probing depth of 4 mm or more and bleeding on probing ([@B13]). An osteoconductive biomaterial (cancellous bone) and equine collagen membranes (Bio- Gen® and Bio-Teck®, BIOTECK S.p.A., Arcugnano VI Italy) were used when bone grafting was necessary. If this type of bone graft was used, it was noted in the patient's medical record. -Statistical study. All statistical procedures were carried out using SPSS 19.0 for Windows (IBM, USA). Frequency and percentage were used to describe qualitative variables. The correlation coefficient was measured using the Spearman test. Qualitative data were compared using the Chi-squared test and applying Haberman's post hoc test. Quantitative ordinal data (BL) were compared using the Mann-Whitney U test if the independent variable had 2 categories, and the Kruskal-Wallis test was used if the independent variable had more than 2 categories. The statistical significance can be found in the Tables, while the normal ranges are displayed in the text. Regarding the power of the study, this requires an expected difference between groups who was estimated at 0.6 points in relation to the primary endpoint (BL). Under these conditions and considering a margin of error of 5% alpha, the power of the test is higher than 78% (less than 22% beta error). The statistical significance was *p* \< 0.05. Results ======= The study's sample consisted of 40 patients (23 women and 17 men) with an average age of 66.97 years (range 58-72). A total of 82 implants were placed in these patients. Data on the patients studied and implants placed can be seen in [Table 1](#T1){ref-type="table"} and [Table 2](#T2){ref-type="table"}. If the sample was restricted to one implant per patient, only 40 implants were analyzed ([Table 2](#T2){ref-type="table"}). No significant differences in BL were found when comparing narrow implants with conventional implants after 2 years of implants loading. Table 1Descriptive data of the sample. Table 2Data from the selected implants for case-control study. The BL variable was analyzed in addition to its correlation with other variables (bone regeneration, previous periodontal disease, type of prostheses, age...) using one implant per patient, and by analyzing only narrow implants ([Table 3](#T3){ref-type="table"}). No significant differences in BL were found when factoring for age or for patients with previous cases of periodontitis. Significant differences were found when comparing BL with patient tobacco use (*p* \< 0.05), in the sense that all implants placed on patients who used tobacco had some kind of BL. Table 3Comparison of BL (Lagervall & Janson) in selected implants. Data that compare BL of narrow implants, conventional implants or booth using or not bone regeneration didn't show any significant differences. No significant differences were found when comparing bone loss of narrow implants with that of conventional implants. There were also no significant differences found when comparing BL with type of implant prostheses ([Table 3](#T3){ref-type="table"}). Discussion ========== Both periapical ([@B14],[@B15]) and panoramic ([@B8]) radiographs can be used to measure BL. Measurements can be taken directly in millimeters, comparing the position of the peri-implant bone in relation to the implant shoulder ([@B16]), or by dividing the implant into 3 equal parts from the implant shoulder to the apex and observing which third of the implant length the bone resides in ([@B12]). Although measurement in millimeters provide greater accuracy and greater statistical power, the complexity of measuring the BL in panoramic radiographs could subtract accurately measure. The use of Langervall-Jansson's ([@B12]) scale for this study was selected because it had been validated in previous publications ([@B8]). This could be of interest when comparing the results of this study with those of other researchers. Limited bucco-lingual bone crest width has always been one of the main indications for placement of narrow implants ([@B17]-[@B23]). The BL of narrow implants without bone regeneration was compared with the BL of conventional implants that used bone regeneration to gain bucco-lingual crest width. No significant differences in rates of bone loss were found between both types of implants studied. With the limitations of the present study, both procedures appear to present similar predictability, although of course the use of narrow implants is less traumatic for the patient ([@B24],[@B25]). The present study used narrow implants to rehabilitate all types of prosthesis (single crowns, splinted crowns, and overdentures). No significant differences in BL were found. However, it should be noted that given the sample size of the present study, further studies focusing on this point would be necessary. In the present study, no early or late failures were recorded in narrow implants. Regarding the survival rate of narrow implants, implant fracture is a frequent complication when using this type of implants ([@B9],[@B10]). The present study found no incidence of fracture in the implants studied. Wang *et al.* ([@B19]) determined a survival rate of 93.5% based on 31 narrow implants observed over one year. On the other hand, Anitua *et al.* ([@B2]) placed 89 narrow implants, using prostheses similar to those in the present study (overdentures, single crowns and implants splinted) and evaluating the implants after 4 years. The results showed that only one implant was lost, resulting in an overall survival rate of 98.9%. It should be noted that in the study by Anitua *et al.*, the follow-up radiograph carried out after 2 years (similar to the final radiographs in the present study), the average BL was 1.26 mm, or less than one-third of the implant length ([@B2]). The majority of the narrow implants in the present study fell within this range (51%). The present study fails to find statistical differences when comparing the BL of narrow implants placed in patients who had previously suffered periodontitis with implants placed in patients who had not. There is scientific evidence regarding the link between periodontitis and periimplantitis ([@B26]). However, in the present study, this evidence is not conclusive when BL is compared against to prior cases of periodontitis that were successfully treated. Despite this, there are other studies that support the findings indicating a link between the two variables ([@B8],[@B27]). As in other studies, in our data we also found a higher BL in smokers, both narrow implants and standard ([@B27],[@B28]). Within the confines of the present study, no significant differences in BL around implants were found when comparing narrow implants with conventional implants after 2 years of implants loading. In this sense, no differences were found when bone regeneration techniques had been used or not, or between types of prosthesis load supporting the implants. Smoking appears to increase BL around the implants studied, thus, practitioners should take greater precautions when placing implants in smokers. Ackcnowledgements None declared. \${Availability of data and materials} None declared. Authors contributions None declared. Ethics None declared. Conflicts of interest The authors have declared that no conflict of interest exist. Funding None declared.

{ "pile_set_name": "PubMed Central" }

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Introduction {#Sec1} ============ A vast majority of currently published research on microsaccades, as well as almost all public discourse on them, refers to these tiny fixational eye movements, which occur when we attempt to fix our gaze, as involuntary or spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}). However, a multitude of evidence from the literature actually points in the opposite direction. For example, likelihoods of microsaccade direction, amplitude, and frequency are systematically modulated under a variety of conditions^[@CR1]--[@CR17]^, and microsaccade generation is causally affected by activity in the midbrain superior colliculus (SC)^[@CR18]--[@CR21]^ and the cortical frontal eye fields (FEF)^[@CR22]^, both involved in voluntary eye movement control^[@CR23]^. Microsaccades can also be suppressed either voluntarily^[@CR24],[@CR25]^ or when perceptually challenging discrimination stimuli are expected^[@CR7],[@CR8]^, and they precisely re-align gaze on the fixated stimulus^[@CR26]--[@CR29]^, even when behavioral tasks require peripheral-stimulus monitoring^[@CR3]--[@CR5]^. Finally, two highly experienced human subjects were described as being able to generate voluntary horizontal or vertical saccades as small as microsaccades^[@CR30]^. The persistence of microsaccade descriptions as being involuntary and spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}), despite the above evidence, stems from a severe lack of explicit demonstration that these eye movements, like larger saccades, can be generated by naïve subjects at will, based on abstract task-defined instructions, and without visual guidance. The strongest such evidence^[@CR30]^, based on a small number of expert observers and only with cardinal movement directions, has gone without direct follow-up for more than 40 years. However, such explicit demonstration is critically needed, particularly in the present day, given the resurgence of the field^[@CR4],[@CR6],[@CR20]^ (Supplementary Fig. [1](#MOESM1){ref-type="media"}), and given that the occurrence of any individual microsaccade is now known to strongly alter even peripheral visual performance^[@CR3]--[@CR5],[@CR31]--[@CR35]^, providing an almost-deterministic link between covert visual attentional effects and the execution of such tiny eye movements^[@CR3]--[@CR5]^. Here, we provide such demonstration; by adapting, in a novel way, a behavioral task exercising voluntary eye movement control to the realm of microsaccades, we show in both naïve human subjects and monkeys that tiny eye movements overwhelmingly thought to be involuntary (Supplementary Fig. [1](#MOESM1){ref-type="media"}) can be generated at will, based on arbitrary instruction, and without any visual guidance. We also show that these eye movements are governed by highly specific SC neural circuitry. Results {#Sec2} ======= Memory-guided microsaccades are generated at will {#Sec3} ------------------------------------------------- We first asked three male rhesus macaque monkeys to perform a novel memory-guided microsaccade task, which was adapted from the known memory-guided saccade task^[@CR36],[@CR37]^ (Methods). The monkeys were initially trained to maintain fixation while a brief (58 ms) flash was presented at an eccentricity ≥5^o^, greater than an order of magnitude larger than microsaccade amplitudes (most frequently defined as \< 1^o^, ref. ^[@CR38]^, but often significantly smaller, ref. ^[@CR18]^). The monkeys had to remember flash location for 300--1100 ms, after which the fixation spot was removed. The monkeys used fixation spot disappearance as the abstract instruction to voluntarily execute a timely eye movement toward the remembered flash location, and the target only reappeared after the eye movement had ended (Methods); there was no specific instruction to re-fixate the reappearing target, but the monkeys often did so nonetheless. Our task therefore required voluntary eye movement generation based on an arbitrary instruction that we enforced by task design (fixation spot disappearance) and without any visual guidance^[@CR36],[@CR37]^; corrective re-fixation saccades were visually guided. After initial training with large saccades, we tested the monkeys on pseudorandomly chosen stimulus locations, including at small eccentricities typically associated with microsaccades. We specifically interleaved small and large eccentricities across trials within any given session, and we also sampled different visual field locations. We did so in order to avoid overtraining any specific locations or eccentricities. We were struck by the fact that the monkeys very easily generated memory-guided movements even for target eccentricities as low as 6 min arc (0.1^o^), and without any need for special training. They simply generalized the rule that they had learned for eccentricities ≥5^o^. Figure [1a](#Fig1){ref-type="fig"} demonstrates this by way of an example trial from monkey N. The monkey fixated steadily between flash onset (at a location 6 min arc left of and 12 min arc below fixation) and the instruction to trigger a movement (the go signal). The monkey then successfully generated an oblique downward--leftward microsaccade approximately 206 ms after the go signal (fixation spot disappearance). After target reappearance, the monkey corrected the remaining eye position error (due to overshoot in the memory-guided microsaccade) with a second visually guided microsaccade. Note how the reaction time of the corrective, visually guided microsaccade (from target reappearance) was comparable to that of the voluntarily generated memory-guided microsaccade (from fixation spot disappearance), and that the latter movement was directionally accurate (directed towards the remembered foveal flash location). Thus, spatially and temporally accurate microsaccades can be triggered at will, based on arbitrary instruction and without visual guidance (also see Supplementary Movies [1](#MOESM5){ref-type="media"}, [2](#MOESM6){ref-type="media"}).Fig. 1Memory-guided microsaccades in monkeys and humans. **a** Eye position measurements from an example trial in monkey N. Upward deflections in each trace mean rightward or upward eye position displacements, respectively, and the position scale bar denotes 15 min arc. A brief target flash appeared 6 min arc to the left of and 12 min arc below fixation. After a memory interval, the fixation spot disappeared, instructing a memory-guided eye movement towards the remembered flash location. The monkey generated a spatially accurate memory-guided microsaccade after a reaction time (RT) of approximately 206 ms from fixation spot disappearance. When the target reappeared, a smaller, corrective movement (which was now visually guided) was triggered after a shorter reaction time. **b** Similar observations from an example trial in a human subject. In this case, the target flash occurred at 18 min arc to the right of and 6 min arc above fixation. Note that the human data were recorded with a video-based eye tracker; monkey N data were obtained from a scleral search coil (Methods). Figures [2](#Fig2){ref-type="fig"}--[4](#Fig4){ref-type="fig"} and Supplementary Figs. [2](#MOESM1){ref-type="media"}--[4](#MOESM1){ref-type="media"} demonstrate the robustness of the observations in this figure across the population, and Figs. [6](#Fig6){ref-type="fig"}--[7](#Fig7){ref-type="fig"} and Supplementary Fig. [5](#MOESM1){ref-type="media"} highlight underlying neural mechanisms Timely voluntary memory-guided microsaccade generation was consistent in all three monkeys, and also in seven human subjects (Methods; Fig. [1b](#Fig1){ref-type="fig"} shows an example trial from one human subject). For all flash eccentricities \< 1^o^ (the most typically cited amplitude threshold for microsaccades^[@CR18],[@CR38]^; Methods), we plotted histograms of all microsaccade latencies after the go signal (fixation spot disappearance; Fig. [2a, d, g, j](#Fig2){ref-type="fig"}). This allowed us to evaluate the reaction times of instructed, memory-guided microsaccades. We also plotted the histograms of microsaccade latencies after target reappearance (Fig. [2m, p, s, v](#Fig2){ref-type="fig"}), for evaluating the reaction times of corrective, visually guided microsaccades. In all cases, memory-guided microsaccades had reaction times consistent with these movements being successfully triggered after task instruction^[@CR39],[@CR40]^. Moreover, the latency distributions were similar to those of larger memory-guided saccades with flash locations \>1^o^ (Supplementary Fig. [2](#MOESM1){ref-type="media"}). Corrective, visually-guided microsaccades expectedly had shorter reaction times than memory-guided microsaccades (monkey N: 146 ms vs. 203 ms; monkey M: 160 ms vs. 212 ms; monkey P: 189 ms vs. 245 ms; humans: 189 ms vs. 252 ms; all *p* \< 10^−50^, t-test). Therefore, timely, voluntary microsaccades were generated at will after the abstract instruction to do so (fixation spot disappearance).Fig. 2Memory-guided microsaccades had reaction times and success rates consistent with being genuine responses to task instruction. **a** Reaction time distribution for memory-guided microsaccades in monkey N. The data shows all trials with target eccentricities \<1^o^ (Methods). The monkey had a reaction time distribution typical of saccadic responses. Error bars denote 95% confidence intervals, and the histogram was normalized by the total number of trials. **b** Cumulative probability of the same data, demonstrating a plateau of \>80% success rate; the monkey successfully generated a memory-guided microsaccade (that is, within a reasonable reaction time; Methods) on the great majority of trials. Error bars denote 95% confidence intervals. **c** Same data as in **b** but separated by target eccentricity (inset legend). Even the smallest target eccentricities were associated with a majority of successful reactions. **d**--**f** Similar observations from monkey M. **g**--**i** Similar observations from monkey P. **j**--**l** Similar observations from our human subjects (Methods). **m**--**x** We repeated the same analyses above, but now for corrective, visually guided microsaccades occurring after target reappearance. Reaction times (**m**, **p**, **s**, **v**) were faster than for memory-guided microsaccades, and often exhibited express reactions (e.g. arrow in **m**). However, success rates were significantly lower than in the instructed memory-guided microsaccades (**n**, **q**, **t**, **w**; the horizontal blue line in each panel, and associated 95% confidence interval, replicates the plateau success rate from the corresponding panel in **b**, **e**, **h**, **k**). Success rates of corrective, visually-guided microsaccades also depended on target eccentricity (**o**, **r**, **u**, **x**) similarly to memory-guided microsaccades. *n* = 781, 1,602, 2,235, 7,402 memory-guided trials in monkey N, monkey M, monkey P, and humans, respectively. *n* = 2774, 4471, 3461, 10,866 visually guided trials in monkey N, monkey M, monkey P, and humans, respectively It may be argued that while memory-guided microsaccades did occur in a timely fashion, their likelihood of occurrence (what we defined as the success rate) was simply low and random as opposed to reflecting a deterministic, voluntary reaction to an explicit go instruction. After all, irrelevant abrupt onsets can reflexively reset microsaccade generation rhythms^[@CR41]--[@CR43]^, suggesting that some aspects of microsaccade behavior may appear to be reflexive in nature. However, plotting cumulative probabilities of memory-guided microsaccades after the go signal (i.e. all trials with target eccentricity \<1^o^), we found that almost every single instructed trial was associated with a response (Fig. [2b, e, h, k](#Fig2){ref-type="fig"}; erroneous trials were primarily ones with reaction times larger than our upper limit for acceptance, Methods). That is, using solely reaction time as the success criterion (Methods), the great majority of memory-guided microsaccade trials were successful. This was not the case for, say, microsaccades after flash onset at the beginnings of trials when monkeys were instructed to maintain fixation (Supplementary Fig. [3a-k](#MOESM1){ref-type="media"}). These early flash-induced microsaccades were not instructed, and therefore did not happen on every single trial (e.g. they were missing in the example trials of Fig. [1](#Fig1){ref-type="fig"}); their occurrence depended on a variety of other factors related to the quality of fixation control^[@CR3],[@CR5]^. More importantly, success rate for the instructed memory-guided microsaccades (i.e. the fraction of trials in which a movement was successfully generated within a reasonable reaction time; Methods; Fig. [2b, e, h, k](#Fig2){ref-type="fig"}) was equal to or significantly higher than that for the corrective, visually guided microsaccades, which were not instructed (Fig. [2n, q, t, w](#Fig2){ref-type="fig"}) (monkey N: *p* = 0.028; monkey M: *p* = 0.5; monkey P: *p* \< 10^−8^; humans: *p* \< 10^−50^, *χ*^2^-test). This indicates that task instruction was a strong impetus to generate the voluntary microsaccadic eye movements in our task. Expectedly, the lowest success rate for both memory- and visually guided (corrective) microsaccades was for the smallest target eccentricities (Fig. [2c, f, i, l, o, r, u, x](#Fig2){ref-type="fig"}). Overall, these results indicate that memory-guided microsaccades can indeed be generated explicitly following abstract instruction (i.e. one that is learned from task design), and even more successfully than uninstructed corrective, visually-guided movements. These are hallmarks of voluntary behavior. Interestingly, when visually-guided microsaccades were explicitly instructed in a separate condition (the delayed, visually guided saccade task; Methods), their success rates were also less than those of the memory-guided microsaccades (Supplementary Fig. [4a, d](#MOESM1){ref-type="media"}). This is largely due to the fact that the monkeys reflexively fixated the visually persistent target in this task paradigm even before the go signal. Memory-guided microsaccades are spatially accurate {#Sec4} -------------------------------------------------- High success was also evident in the directional accuracy of memory-guided microsaccades and not just in their reaction times (Fig. [2](#Fig2){ref-type="fig"}). For the same data in Fig. [2](#Fig2){ref-type="fig"}, we now plotted the angular directional difference between memory-guided microsaccades and target locations (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}). That is, we plotted the direction error of the movements. We also plotted the same measure for the uninstructed corrective, visually guided microsaccades (Fig. [3c, g, k, o](#Fig3){ref-type="fig"}); in this case, we compared corrective microsaccade direction to the direction of the reappearing target relative to gaze position after the memory-guided microsaccade had ended (Methods). Once again, in both monkeys and humans, memory-guided microsaccades were significantly more directionally accurate than the uninstructed corrective, visually guided movements. We confirmed this statistically by comparing the distribution of absolute directional errors for memory-guided microsaccades to that of absolute directional errors for corrective, visually guided microsaccades. In monkey N, the former was 20.4° ± 5.9° 95% c. i. and the latter was 27.1° ± 1.3° 95% c. i. (*p* \< 10^−4^, Watson-Williams test, circular analog of the two-sampled *t*-test); for monkey M, the results were 23.4^o^ ± 2.5° 95% c. i. and 31.5° ± 1.1° 95% c. i. (*p* \< 10^−10^); and for monkey P, they were 33.8° ± 2.4° 95% c. i. and 51.2° ± 1.4° 95% c. i. (*p* \< 10^−50^). Similarly, for humans, the directional errors were 23.8° ± 0.8° 95% c. i. versus 43.1° ± 0.9° 95% c. i. (*p* \< 10^−50^). When instructed visually guided microsaccades were tested separately (during the delayed, visually guided saccade task; Methods), directional accuracy was restored to the levels of memory-guided microsaccades (Supplementary Fig. [4b, e](#MOESM1){ref-type="media"}), consistent with prior results^[@CR26]^. Therefore, we observed high success in both reaction time (Fig. [2](#Fig2){ref-type="fig"}) and directional accuracy (Fig. [3](#Fig3){ref-type="fig"}) of instructed memory-guided microsaccades.Fig. 3Memory-guided microsaccades had better directional accuracy than either microsaccades during fixation or corrective, visually-guided microsaccades. **a** We plotted the angular distribution of microsaccade direction error (that is, the difference between target direction and microsaccade direction; Methods). In this measure, if a microsaccade has the same direction as the target direction, then the movement has zero direction error. In this panel, this measure is shown for all microsaccades from monkey N occurring in the final 250 ms of fixation before fixation spot disappearance. Microsaccades could occur either towards or away from the remembered target location. The histogram was normalized by the total number of observations. **b** The same monkey exhibited highly accurate microsaccade directions toward the remembered target location when explicitly instructed to do so. **c** After target reappearance, corrective, visually-guided microsaccades were also target-directed, but their direction errors were significantly more variable than the instructed movements in **b** (see statistics in text). Thus, instructed memory-guided microsaccades were highly directionally accurate. **d** Each group of colored arrows shows the directions of 40 randomly selected memory-guided microsaccades when remembered target directions were drawn from the underlying corresponding-colored faint cone. Directionally accurate microsaccades could be generated for all oblique target directions. **e**--**h** Similar observations for monkey M. **i**--**l** Similar observations for monkey P. **m**--**p** Similar observations for our human subjects. In all cases, directional accuracy was the highest for the instructed, memory-guided movements and not for fixational microsaccades (**a**, **e**, **i**, **m**) or corrective, visually guided ones (**c**, **g**, **k**, **o**); also see Supplementary Fig. [3](#MOESM1){ref-type="media"} for flash-induced microsaccades. For the leftmost column, *n* = 1,432, 2,875, 4,870, 2,831 microsaccades for monkey N, monkey M, monkey P, and the human subjects, respectively. The numbers of trials for memory-guided or corrective, visually guided microsaccades are the same as in Fig. [2](#Fig2){ref-type="fig"} It may also be argued that the directional accuracy described above was not necessarily a result of a voluntary effort of will to follow task instruction, but instead an outcome of covert attentional shifts. For example, monkey^[@CR7]^ and human^[@CR1],[@CR2]^ microsaccades exhibit direction biases in covert attentional tasks, and it could be the case that the requirement to remember target location was enough to bias microsaccade directions near the ends of trials (perhaps due to sustained covert attention). However, if so, such a bias should have also appeared during the memory interval itself. We therefore analyzed microsaccade directions in the final 250 ms of the memory interval, during instructed fixation (Fig. [3a, e, i, m](#Fig3){ref-type="fig"}). Directional accuracy, relative to target location, was much worse than in the instructed memory-guided microsaccades (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}) (*p* \< 10^−50^ for comparing absolute directional error in memory-guided microsaccades and microsaccades at the end of the memory interval in each of the monkeys and also in the human data; Watson-Williams test). In fact, the fixational microsaccades (at the end of the memory interval) were biased either away (monkeys) or towards (humans) the target location. This difference in directional bias between species (for the same trial-duration distributions) likely reflects expected differences in the temporal dynamics of microsaccadic oscillations between humans and monkeys^[@CR3],[@CR33],[@CR42]^. We also analyzed microsaccades immediately after target flash onset, to compare potential reflexive stimulus-induced effects (perhaps due to exogenous covert attentional effects after target flash onset) to instructed movements after fixation spot disappearance (Supplementary Fig. [3c, f, i, l](#MOESM1){ref-type="media"}): other than an expected minority of express visually driven reactions immediately after flash onset^[@CR5]^, the directions of flash-induced microsaccades were much more variable than the directions of instructed memory-guided microsaccades. Therefore, even transient, exogenous covert attention shifts^[@CR1],[@CR2]^ are not sufficient to explain the directional accuracy of the instructed memory-guided microsaccades (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}). It is also important to note that voluntary, memory-guided microsaccades could be triggered in all directions. Thus, directional accuracy was not restricted to, say, only horizontal or only vertical directions, one limitation in the original Haddad and Steinman study exploring voluntary triggering of small eye movements^[@CR30]^. To illustrate this, Fig. [3d](#Fig3){ref-type="fig"} shows raw plots of example memory-guided microsaccade direction vectors, color-coded by target locations in each of the four quadrants from monkey N. The other two monkeys and the human subjects also generated directionally accurate memory-guided microsaccades in all directions (Fig. [3h, l, p](#Fig3){ref-type="fig"}). Thus, not only did all subjects and monkeys successfully generate untrained voluntary eye movements for small-eccentricity target locations, but these eye movements were also highly directionally accurate. The directional accuracy was, once again, better than in the uninstructed corrective, visually-guided movements (Fig. [3c, g, k, o](#Fig3){ref-type="fig"}). We next investigated memory-guided microsaccade amplitudes. We found that such amplitudes also scaled with remembered target eccentricity, consistent with the notion that there was voluntary generation of spatially accurate (in both direction and amplitude) microsaccadic eye movements in the task. For example, in Fig. [4a, c, e, g](#Fig4){ref-type="fig"}, we plotted the amplitude of memory-guided microsaccades as a function of remembered target eccentricity (this figure also included larger saccades and eccentricities for comparison). We used logarithmic scaling to magnify the small eccentricities associated with microsaccades^[@CR19]^. There was a small overshoot in movement amplitude for the smallest target eccentricities, but the amplitude of eye movements systematically increased with increasing eccentricity of the remembered target location. That is, despite the initial overshoot, there was scaling of microsaccade amplitudes with remembered target eccentricity. This scaling of memory-guided microsaccade amplitude was true in all 3 monkeys (Fig. [4a, c, e](#Fig4){ref-type="fig"}), as well as in the human subjects (Fig. [4g](#Fig4){ref-type="fig"}). Interestingly, when the monkeys or subjects made uninstructed corrective, visually-guided microsaccades in the same task, the amplitudes of these visually-guided movements (relative to target eccentricity) were worse than for the instructed memory-guided microsaccades (i.e. did not scale appropriately; Fig. [4b, d, f, h](#Fig4){ref-type="fig"}). Larger corrective movements were accurate in amplitude. Similarly, when visually-guided microsaccades were instructed (in the delayed, visually-guided saccade task; Methods), the amplitudes were more accurate (as expected^[@CR26]^), and overshoot did not occur as strongly for the tiniest of eccentricities as in the case of the memory-guided microsaccades (Supplementary Fig. [4c, f](#MOESM1){ref-type="media"}). These results are consistent with all other results above, demonstrating willful generation of individual eye movements of microsaccadic sizes, based on abstract task-defined instruction to do so (fixation spot disappearance).Fig. 4Memory-guided microsaccade amplitudes scaled better with target eccentricity than corrective, visually-guided microsaccades. **a** For all target eccentricities in monkey N, we plotted memory-guided movement amplitude as a function of target eccentricity. The data below 1^o^ target eccentricity (vertical dashed line) are from the same trials described in Figs. [1](#Fig1){ref-type="fig"}--[3](#Fig3){ref-type="fig"} for reaction times, success rates, and directional accuracy. Here, we show their amplitude scaling. Memory-guided microsaccades increased in amplitude with increasing target eccentricity, consistent with them being genuine responses to task instruction, but they showed systematic overshoot (e.g. Figure [1](#Fig1){ref-type="fig"}). The overshoot disappeared for larger eye movements (also see Fig. [5](#Fig5){ref-type="fig"} for a potential explanation). Target eccentricities were binned at the following non-overlapping bin-center values: 0.05, 0.15, 0.25, 0.35, 0.55, 0.75, 1.05, 1.45, 2, 3, 5, 7.5, 10, 15; error bars denote 95% confidence intervals. **b** For similar small target eccentricities, corrective, visually-guided microsaccades overshot the target more than the instructed memory-guided microsaccades, and their amplitudes did not scale with target eccentricity as well as the memory-guided movements (despite the presence of a visual target). Larger corrective movements were more accurate. **c**, **d** Similar observations in monkey M. **e**, **f** Similar observations in monkey P. **g**, **h** Similar observations in the human subjects. In all cases, despite the amplitude overshoot for the smallest memory-guided microsaccades (see Fig. [5](#Fig5){ref-type="fig"}), instructed memory-guided microsaccades exhibited better amplitude scaling with increasing target eccentricity than corrective, visually-guided microsaccades (also see Fig. [5](#Fig5){ref-type="fig"}). *n* = 2,947, 4,931, 3,972, 10,890 for memory-guided movements of all amplitudes in monkey N, monkey M, monkey P, and the human subjects; *n* = 2,528, 4,086, 2,674, 7,002 for corrective, visually-guided movements of all amplitudes in monkey N, monkey M, monkey P, and the human subjects It is also interesting that the latency distributions for the corrective, visually-guided microsaccades in the same task (Fig. [2m, p, s, v](#Fig2){ref-type="fig"}) showed suggestive evidence of the oculomotor system being implicitly aware of residual errors after the previous memory-guided saccades or microsaccades (e.g. potential overshoot). Specifically, there was a distribution of express microsaccades with very short reaction times of \<120 ms after target reappearance. Since express microsaccades are most likely when a movement plan is already well on its way at the time of stimulus onset^[@CR5]^, the occurrence of such express microsaccades in the corrective, visually-guided movements after target reappearance indicates that the previously-occurring memory-guided movements were indeed internally accounted for. This provides further evidence that memory-guided microsaccades are voluntarily triggered, and it is similar to larger saccades^[@CR44]^. Foveal working-memory expansion even with manual responses {#Sec5} ---------------------------------------------------------- Despite the timeliness (Fig. [2a, d, g, j](#Fig2){ref-type="fig"}), success rate (Fig. [2b, e, h, k](#Fig2){ref-type="fig"}), and directional accuracy (Fig. [3b, f, j, n](#Fig3){ref-type="fig"}) of memory-guided microsaccades, their amplitude overshoots for some eccentricities (Fig. [4a, c, e, g](#Fig4){ref-type="fig"}) may be interpreted as a failure to make genuine target-directed voluntary eye movements of small amplitude. However, an alternative possibility is that the overshoots were instead a property of the encoding of foveal locations in spatial working memory, and not an execution limitation by the oculomotor system. For example, this could be a result of foveal magnification of neural tissue, which also exists in the SC to a much larger extent than previously thought^[@CR45]^. To investigate this alternative, we ran our human subjects on two control tasks not explicitly requiring an eye movement to report the perceived location of the remembered target flash (Methods). Rather, we asked the subjects to perceptually report the remembered target location, and they did so via button or computer-mouse presses (Methods). In one variant, the button-press task, subjects were free to move their eyes while responding, and they had up to 20 s to finalize their response; in another variant, the mouse pointer task, subjects pointed to the remembered location (with mouse cursor) while still maintaining fixation. In both cases, amplitude overshoot for the smallest target eccentricities was still clearly evident (Fig. [5](#Fig5){ref-type="fig"}), much like when eye movements were used to perform the task (the memory-guided microsaccade task). We should also note here that directional accuracy was also very high in the memory-guided manual tasks, similar to Fig. [3](#Fig3){ref-type="fig"}. Therefore, memory-guided microsaccade amplitude overshoot (Fig. [4a, c, e, g](#Fig4){ref-type="fig"}) was not a failure of the oculomotor system to voluntarily generate small eye movements. Instead, it reflected an intriguing foveal expansion in the representation of space during working memory (Fig. [5](#Fig5){ref-type="fig"}). Consistent with this, and as stated above, microsaccade amplitude overshoot was less evident with instructed delayed, visually-guided microsaccades (Supplementary Fig. [4c, f](#MOESM1){ref-type="media"}).Fig. 5Overshoot was not restricted to microsaccades; perceptual localization of foveal remembered targets was also expanded in an illusory manner. **a** In humans, we tested perceptual localization of remembered target locations without explicitly requiring an eye movement response. In the button-press task, subjects used a button box to move a displayed cursor to the remembered target location (Methods). We analyzed their perceptual reports similarly to eye movement reports. The same overshoot for small target eccentricities was observed. Error bars denote 95% confidence intervals. **b** We also ran a variant of the task (the mouse pointer task; Methods), in which we also maintained a visual reference at fixation (the fixation spot). The same overshoot for foveal target eccentricities was still observed. Therefore, the overshoot of memory-guided microsaccades in Fig. [4](#Fig4){ref-type="fig"} was likely a general property of representing foveal space in spatial working memory, as opposed to an intrinsic inability to generate small memory-guided microsaccades. *n* = 6,405 and 9,062 trials for the button-press and mouse pointer tasks, respectively Superior colliculus bursts for memory-guided microsaccades {#Sec6} ---------------------------------------------------------- The strongest evidence that we obtained for voluntary control over microsaccades emerged when we recorded SC neural activity. In monkeys N and M, we recorded visual, visual-motor, and motor (saccade-related) SC neurons (Methods) during the memory-guided saccade task. We specifically aimed to sample sites in the rostral SC, in which microsaccade-related activity is expected^[@CR18],[@CR19],[@CR21]^, but we also sampled slightly more caudally (i.e. more eccentrically) for completeness (Methods). We inserted multi-electrode linear arrays into the SC, and we used offline sorting to isolate individual putative single units (Methods). Our first goal was to ask whether SC neurons responded at all during memory-guided microsaccades. For example, some SC neurons do not emit saccade-related responses in the absence of a visual target^[@CR46]--[@CR48]^, and it is not clear whether this can happen for microsaccades or not. Individual SC neurons did indeed emit saccade-related motor bursts for memory-guided microsaccades. For example, in Fig. [6a](#Fig6){ref-type="fig"}, we plotted neural activity from a sample neuron from monkey M. We identified a set of memory-guided microsaccades of amplitude \<1^o^ and direction toward the neuron's movement response field (RF), shown in Fig. [6b](#Fig6){ref-type="fig"} (neural RF's were assessed as described in Methods, and the movements included in Fig. [6a](#Fig6){ref-type="fig"} were directed towards the RF hotspot location and with angular direction within ±90^o^ from the vector connecting gaze center to this location). We plotted radial eye position aligned on microsaccade onset and also firing rate (individual rows of tick marks indicate action potential times, with each row representing an individual movement; this formatting is similar to that in Fig. [1c](#Fig1){ref-type="fig"} of ref. ^[@CR18]^). Memory-guided microsaccade-related SC discharge was clearly present even for memory-guided movements as small as 6 min arc, and similar to expected saccade-related discharge in general. Therefore, individual voluntary, memory-guided microsaccades are associated with SC neural discharge.Fig. 6Superior colliculus (SC) activity during memory-guided microsaccades. **a** The top panel shows radial eye position aligned on microsaccade onset (for memory-guided microsaccades \<1^o^). All traces were aligned to start (on the vertical axis) from the same eye position. Most movements had a radial amplitude \<30 min arc (0.5^o^). These movements were selected relative to the response field (RF) as assessed from the analysis in **b**. The bottom panel shows microsaccade-aligned firing rate. Classic SC microsaccade-related activity^[@CR18]^ was observed for memory-guided microsaccades. Each row of tick marks shows the neuron's discharge for an individual movement from the top panel, and the rows are sorted by memory-guided microsaccade amplitude (black for \<30 min arc; gray for 30--60 min arc). Even the smallest memory-guided microsaccades were associated with SC microsaccade-related discharge^[@CR18]^. The gray bar on the *x*-axis indicates the analysis window used in **b**. Error bars denote 95% confidence intervals. **b** Movement RF of the same neuron. For each movement displacement, plotted in log-polar coordinates^[@CR19]^, we plotted mean firing rate from the measurement interval in **a**. The neuron, from the right SC, exhibited a movement RF on the contralateral side along an oblique upward direction. The neuron clearly responded for memory-guided microsaccades as small as 0.1° (6 min arc). The origin of the log-polar plot denotes 0.03° radial amplitude. The inset shows spike waveforms from every 100^th^ spike recorded in the task (gray; 682 waveforms); red shows the average spike waveform (and 95% confidence intervals) across all recorded spikes Not only did we find microsaccade-related responses for memory-guided microsaccades (Fig. [6](#Fig6){ref-type="fig"}), but there were also neurons that discharged exclusively for memory-guided microsaccades and not for similarly sized corrective, visually-guided microsaccades. Figure [7a](#Fig7){ref-type="fig"} shows an additional example neuron from the same monkey as in Fig. [6](#Fig6){ref-type="fig"}, now demonstrating this unexpected phenomenon. We plotted an estimate of the neuron's movement field on log-polar coordinates up to 1° eccentricity. In the left panel, we plotted the movement field of the neuron when memory-guided microsaccades were generated. Clear discharge for specific movement vectors was evident. In the right panel, we plotted the movement field of the same neuron when corrective, visually-guided microsaccades of similar amplitudes and directions were generated. The neuron now did not emit any movement-related discharge at all (compare to the left panel), even though movements of similar sizes were executed. These results were also obvious when we plotted firing rates as a function of time from microsaccade onset in the two conditions (Fig. [7b](#Fig7){ref-type="fig"}). Activity was present when memory-guided microsaccades were executed, but not when visually-guided movements of the same amplitude and direction range were generated. Activity for these latter movements looked indistinguishable from activity during baseline fixation before trial onset (Fig. [7b](#Fig7){ref-type="fig"}, black curve; Methods define baseline intervals). Therefore, not only was there strong behavioral evidence that naïve monkeys and humans can generate voluntary, memory-guided microsaccades (Figs. [1](#Fig1){ref-type="fig"}--[4](#Fig4){ref-type="fig"}), but there were also individual SC neurons showing exclusive responses only for memory-guided microsaccades but not for similarly sized visually guided ones.Fig. 7Double dissociation of SC microsaccade-related discharge for visually- and memory-guided microsaccades. **a** Movement RF's of an example SC neuron. Left shows peri-movement discharge (like in Fig. [6b](#Fig6){ref-type="fig"}) for memory-guided microsaccades; right shows the same for corrective, visually-guided microsaccades. The neuron was silent for the visually-guided movements, but not for similar-sized memory-guided ones. **b** Peri-movement discharge of the same neuron under the two conditions. The black curve (when visible) shows the neuron's activity during fixation before trial onset; the neuron only discharged for memory-guided, but not visually guided, microsaccades. Error bars denote 95% confidence intervals. **c**, **d** Same as **a**, **b** but for an example neuron only showing microsaccade-related discharge for visually guided movements (similar to visually dependent saccade-related, VDSR, neurons^[@CR46]^). The inset shows (as an example) the analyzed eye movements (like Fig. [6a](#Fig6){ref-type="fig"}) in the two conditions, demonstrating clear overlap (similar overlap was present in **a**, **b**). **e** Distribution of the different neuron types (V = Visual; VM = Visual-motor; M = Motor; VDSR = Visually-dependent saccade-related; ER = Exclusively responding for memory-guided microsaccades). **f** Movement RF hotspots for the neurons. ER-Neurons tended to be more eccentric than other neurons exhibiting microsaccade-related discharge, consistent with a foveal expansion for memory-guided microsaccades (see text and Supplementary Fig. [5](#MOESM1){ref-type="media"}). **g**, **h** Peri-stimulus (**g**) or peri-microsaccadic (**h**) discharge for all of our encountered neuron types during microsaccade generation. Each neuron's response was first normalized before averaging responses across neurons. V- and VM-Neurons exhibited strong visual responses; VM- and M-Neurons showed movement-related activity regardless of stimulus; VDSR-Neurons only showed movement-related activity in the presence of a visual target; ER-Neurons only showed movement-related activity in the absence of a visual target. In all cases, we analyzed neural activity for microsaccades (\<1^o^) toward the RF. Error bars denote 95% confidence intervals We found 41 SC neurons exhibiting exclusive responses for memory-guided microsaccades; these constituted 22% of SC neurons with saccade-related discharge in either memory or visual condition (Fig. [7e, f](#Fig7){ref-type="fig"}). Our criteria for including neurons in this accounting was the observation of visual and/or movement discharge for eccentricities \<1° (Methods). We referred to these neurons as ER-Neurons, for "Exclusively Responding" (Methods). Their average firing rate modulations for foveal visual target onsets and for microsaccades are shown in Fig. [7g, h](#Fig7){ref-type="fig"} (rightmost column), and these modulations are consistent with those in the example neuron of Fig. [7a, b](#Fig7){ref-type="fig"}. Interestingly, for larger saccades, ER-Neurons did exhibit saccade-related activity for visually guided eye movements (Supplementary Fig. [5](#MOESM1){ref-type="media"}), suggesting that memory-guided microsaccades may have recruited additional neural resources from SC saccade-related neurons that would normally be recruited for slightly larger eye movements when visual targets are present (Supplementary Fig. [5](#MOESM1){ref-type="media"}). Consistent with this, RF hotspots for ER-Neurons tended to be more eccentric than other neuron types in our database (Fig. [7f](#Fig7){ref-type="fig"}). Interestingly, we also found all other classic saccade-related SC responses to be present as well for memory-guided microsaccades. For example, we found visual-only neurons responding to foveal flash onset in the beginning of a trial but not during the actual memory-guided (or visually guided) eye movements (Fig. [7g, h](#Fig7){ref-type="fig"}, leftmost column). We also found visual-motor neurons, exhibiting both a visual response and either a memory- or visually-guided microsaccade response (Fig. [7g, h](#Fig7){ref-type="fig"}, second column). Motor neurons that responded for both memory- and visually guided microsaccades, but not to flash or target onset, were also present (Fig. [7g, h](#Fig7){ref-type="fig"}, third column). Finally, in addition to the ER-Neurons (e.g. Fig. [7a, b](#Fig7){ref-type="fig"}), we discovered neurons exhibiting the exact opposite profile: these were visually-dependent microsaccade-related neurons, extending earlier findings of SC visually-dependent saccade-related neurons (VDSR-Neurons^[@CR46]--[@CR48]^) to the realm of microsaccades. One example such neuron is shown in Fig. [7c, d](#Fig7){ref-type="fig"}. The neuron exhibited microsaccade-related activity exclusively during visually-guided microsaccades, but not during similarly sized memory-guided microsaccades. This neuron type (Fig. [7g, h](#Fig7){ref-type="fig"}, fourth column) was not described previously in refs. ^[@CR18],[@CR19]^. Overall, in the memory-guided microsaccade task, the different classes of neurons that we encountered (Fig. [7g, h](#Fig7){ref-type="fig"}) were distributed in our database (Methods) as shown in Fig. [7e](#Fig7){ref-type="fig"}. Our results demonstrate that memory-guided microsaccades are generated at will: based on abstract task-defined instruction, without visual guidance, with high fidelity in spatial accuracy, and with underlying SC circuitry supporting all different aspects of memory- or visually-guided behavior. These results demonstrate that overwhelming descriptions of microsaccadic eye movements as being involuntary and spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}) are too simplistic and can mask the significance of these tiny eye movements in modulating perceptual and attentional performance. Discussion {#Sec7} ========== We were motivated by a frustrating conundrum in the field of microsaccade research. Haddad and Steinman^[@CR30]^ showed in 1973 that two expert human observers were able to voluntarily trigger a microsaccade along one of the cardinal directions. Their study has become virtually forgotten, especially after the resurgence of the field of microsaccade research from its almost-complete death at the end of the previous century^[@CR6]^ (Supplementary Fig. [1](#MOESM1){ref-type="media"}). We decided to revisit this particular study, but avoid some of its methodological constraints (such as the use of only two expert subjects, the limited amount of data, and the lack of neurophysiological insights), and we were able to demonstrate clear voluntary control over individual microsaccades even in naïve human subjects and monkeys. In doing so, we were also able to additionally demonstrate an ability for microsaccades to be driven by foveal visual working memory, in a novel adaptation of the memory-guided saccade task. We were struck by how easily memory-guided microsaccades could be generated, with no need for special training. Monkeys simply generalized the task rule that they had learned for larger saccades, and humans were only verbally instructed to "look at the remembered target location". We believe that our results overturn an overwhelming discourse on microsaccades, even in public understanding of these eye movements, as being involuntary and spontaneous (Supplementary Fig. [1](#MOESM1){ref-type="media"}). Indeed, and as stated above, despite strong supportive evidence like that cited above and in Introduction, a surprisingly large fraction of references to microsaccades in the literature (especially in the last two decades) asserts that these eye movements are involuntary eye movements (Supplementary Fig. [1](#MOESM1){ref-type="media"}). We believe that explicit demonstration of behavioral and neural evidence for voluntary control over individual microsaccades, in our novel adaptation of the memory-guided saccade paradigm, is absolutely necessary, especially in the present day when there is both a renewed interest in microsaccades as well as continued intense debates about their functional roles in influencing brain activity and behavior. Consider, for example, the field of covert visual attention. A long history of research has assumed for several decades that microsaccades either do not happen in these tasks at the time of task-relevant events, or that they happen in a random and spontaneous manner that should not influence the interpretation of results. However, it is now known that this view is too simplistic^[@CR1]--[@CR5],[@CR31]--[@CR34]^. For this important revelation to become appreciated more widely, and in other fields related to visual and cognitive neuroscience, explicit demonstration of voluntary control over individual microsaccades, like we have shown in this study, is firmly needed. The strongest evidence that we found for voluntary control over individual microsaccades is related to how the SC is involved in generating memory-guided versions of these eye movements. We were particularly intrigued by the ER-Neurons (Fig. [7](#Fig7){ref-type="fig"}). These neurons are SC neurons that discharge exclusively for memory-guided microsaccades but not for similarly sized visually-guided microsaccades. It would be highly interesting to investigate the mechanisms associated with these ER-Neurons in more detail in future studies. For example, the fact that these neurons discharge (i.e. exhibit saccade-related activity) for larger visually-guided saccades might suggest an expansion of RF's towards the fovea when spatial working memory is engaged (Supplementary Fig. [5](#MOESM1){ref-type="media"}). Prior work is inconclusive about how SC RF's change during spatial working memory tasks^[@CR47],[@CR49]^, and there is no systematic investigation, to our knowledge, of foveal spatial representation in working memory anyway. Therefore, this issue needs to be investigated in future follow-up studies. This can allow understanding the entire transformation from visual input (flash onset) to memory representation in the memory interval, and finally to movement generation. Relevant questions could include whether RF's change during the memory interval as well, or only at the time of memory-guided microsaccade generation (like in Fig. [7a,b](#Fig7){ref-type="fig"}, Supplementary Fig. [5](#MOESM1){ref-type="media"}). Importantly, studying this in the SC would clarify a sub-cortical involvement in working memory, which is a topic that is generally investigated more strongly from a cortical perspective^[@CR36],[@CR50]^. Studying this in the SC would also link SC activity to mechanisms of spatial updating of memory representations across eye movements^[@CR51],[@CR52]^. We were equally intrigued by the VDSR-Neurons^[@CR46]--[@CR48]^ that we found (Fig. [7c, d, g, h](#Fig7){ref-type="fig"}). In the past approximately four decades since they were first reported on in the primate SC, very few studies have mentioned these neurons, which only exhibit saccade-related discharge if saccades are directed towards a visual target. Moreover, these few mentions were only made from the perspective of larger saccades. Here, we found that VDSR-Neurons also exist in the rostral SC generating^[@CR18]^ microsaccades. That is, we found microsaccade-related neurons that only showed movement-related discharge if there was a visual target guiding microsaccade generation; the same neurons did not discharge when the microsaccades were generated toward a blank. We find these neurons interesting because they suggest that the oculomotor system, even for microsaccades, incorporates information about visual features at the time of saccade execution. It would be interesting to relate such incorporation of visual information in saccade commands to cortical interactions between saccades and visual representations^[@CR53]^ and to shape guidance of saccades^[@CR53]^ and microsaccades^[@CR42]^. Because SC neurons exhibit visual-feature tuning in visual responses^[@CR54]--[@CR56]^, it would also be interesting to explore how these VDSR-Neurons behave during saccades to different visual objects or features. In all, our neural results with visual, visual-motor, and motor neurons all being involved in microsaccade generation confirm views that microsaccades are essentially indistinguishable from larger saccades in terms of neural mechanisms^[@CR20]^. This is consistent with a variety of neurophysiological evidence in multiple areas^[@CR19],[@CR21],[@CR22],[@CR57],[@CR58]^, and it also gives further credence to the notion that these eye movements are indeed voluntary movements, despite the opposite description in a large portion of the literature (Supplementary Fig. [1](#MOESM1){ref-type="media"}). The visual responses, in particular, are worth further investigation, both for understanding SC foveal vision^[@CR45]^ in general, and also for exploring the speeded reactions to target reappearance in the corrective, visually-guided microsaccades (Fig. [2m, p, s, v](#Fig2){ref-type="fig"}). Behaviorally, there was a remarkable difference between instructed and uninstructed movements in general. Most strikingly, the corrective, visually-guided microsaccades in the memory-guided task were both less successful (i.e. less often generated) and also sloppier in direction and amplitude (e.g. Figures [2](#Fig2){ref-type="fig"}--[4](#Fig4){ref-type="fig"}) than the instructed memory-guided movements, even though these latter movements did not have explicit visual guidance. Only when we instructed visually guided saccades (in the delayed, visually guided saccade task) did visually guided microsaccades appear more spatially precise (Supplementary Fig. [4b, c, e, f](#MOESM1){ref-type="media"}). This (perhaps expected) observation supports our interpretation that our memory-guided microsaccades were generated voluntarily. Further support for this conclusion is the presence of some express microsaccades after target reappearance (Fig. [2](#Fig2){ref-type="fig"}). As stated earlier, because express microsaccades are more likely when a movement plan is already under way^[@CR5]^, the presence of these express microsaccades after target reappearance indicates that the oculomotor system was already planning a corrective movement for any residual errors from previous memory-guided microsaccades. Evidence for such rapid correction also exists in larger saccades (see, for example, ref. ^[@CR44]^). Naturally, all of the above may be related to questions of awareness. Specifically, and as stated elegantly by Haddad and Steinman^[@CR30]^, labels of involuntary, spontaneous, and reflexive may have arisen in association with microsaccades exactly because people are generally unaware of making these movements. However, we are also very frequently unaware of making larger saccades. Moreover, these same authors demonstrated^[@CR30]^ that they could be aware of their own self-triggered microsaccades on a surprisingly large fraction of trials. Therefore, awareness on its own is not sufficient to argue that microsaccades are fundamentally different from larger saccades. Finally, our results pave the way for interesting investigations of spatial working memory at the microscopic scale of the fovea. A wealth of research has been performed on the mechanisms of working memory^[@CR36],[@CR50]^ in general, and there were even recent findings that accessing visual working memory engages the oculomotor system^[@CR59]^ (similar to how preparing a manual response in monkeys caused them to generate microsaccades to the four possible previous stimulus locations in ref. ^[@CR7]^). However, just like subcortical mechanisms are less investigated, how foveal space is represented in spatial working memory remains unknown. For example, our perceptual control experiments (Fig. [5](#Fig5){ref-type="fig"}) suggest that the overshoot in microsaccade amplitudes that we observed (Fig. [4](#Fig4){ref-type="fig"}) was not a deficit in oculomotor control per se. Instead, it likely reflected an expansion of foveal space when working memory was engaged (Fig. [5](#Fig5){ref-type="fig"}). This might be a complement of foveal biases in peripheral visual perception^[@CR60]--[@CR62]^. Specifically, because of foveal magnification of neural tissue^[@CR63]^, it could be that engaging spatial working memory in the fovea causes distortions in the representation of space, such that foveal locations get mislocalized outward (i.e. overshoot) whereas peripheral locations get mislocalized inward (i.e. undershoot). Interestingly, perceptual mislocalizations around microsaccades^[@CR31]^ are also consistent with outward distortions in the fovea and inward distortions in peripheral vision. This might suggest that there may be generalized underlying mechanisms of perceptual distortions of different kinds (whether due to eye movements, working memory, attention, or otherwise), likely being caused by how individual brain circuits represent visual space in anatomical tissue, and how multiple such circuits (with potentially different levels and kinds of representational magnification) may interact with each other^[@CR64]^. Methods {#Sec8} ======= Ethics approvals {#Sec9} ---------------- All monkey experiments were approved by ethics committees at the Regierungspräsidium Tübingen. The experiments were in line with European Union directives, and German laws, governing animal research. Human experiments were approved by ethics committees at the Medical Faculty of Tuebingen University. Subjects provided written, informed consent, and they were financially compensated for their participation. Our experiments were in accordance with the Declaration of Helsinki. Laboratory setups {#Sec10} ----------------- Monkey experiments were performed in the same laboratory as that described recently^[@CR54],[@CR55],[@CR65]^. Human experiments were done in the laboratory described in refs. ^[@CR31],[@CR64]^. Eye movements were recorded at 1 kHz using magnetic induction^[@CR66],[@CR67]^ (for monkeys N and M) or video-based eye tracking (for monkey P and the human subjects). Head position in the human subjects was stabilized using a custom head-holder^[@CR31]^. Animal preparation {#Sec11} ------------------ We collected behavioral data from three adult, male rhesus macaques (macaca mulatta). Monkeys N and M (aged 10 and 7 years, respectively, and weighing 11.5 and 8 kg, respectively) were implanted with scleral search coils to allow measuring eye movements using the magnetic induction technique^[@CR66],[@CR67]^. The eye movements of Monkey P (aged 10 years and weighing 8.6 kg) were recorded using a video-based eye tracker (EyeLink 1000, SR Research). All three monkeys were implanted with a head holder to stabilize head position during experiments, with details on all implant surgeries provided earlier^[@CR65],[@CR68]^. We also recorded SC neural activity from monkeys N and M. The SC was approached through recording chambers cranially-implanted on the midline and aimed at a point 1 mm posterior of and 15 mm above the inter-aural line. The chambers were tilted backwards from vertical by an angle of 35° in monkey N and 38° in monkey M^[@CR32],[@CR34],[@CR55]^. Monkey behavioral tasks {#Sec12} ----------------------- Monkeys performed a memory-guided saccade task^[@CR36],[@CR37]^. In each trial, a central white fixation spot (72 cd per m^2^ for monkey N; 86 cd per m^2^ for monkey M; 48.1 cd per m^2^ for monkey P) was presented over a uniform gray background (21 cd per m^2^ for monkey N; 29.7 cd per m^2^ for monkey M; 4.4 cd per m^2^ for monkey P). The fixation spot was a square of 5.3 × 5.3 min arc dimensions. After 300--1,200 ms, a brief target flash (identical to the fixation spot) occurred at some location on the display (duration: \~58 ms), while the fixation spot remained on; this flash represented the location to be memorized by the monkeys for an upcoming saccadic eye movement. After the flash, the fixation spot remained visible for 300--1100 ms; this period constituted the memory interval of the task, after which the fixation spot disappeared. Fixation spot disappearance was the abstract instruction for the monkeys to generate a spatially accurate saccadic eye movement to the remembered flash location. Because of the disappearance of the fixation spot and the flash before it, the saccade that was triggered was generated without any visual guidance. Monkeys had to trigger this saccade within \<700 ms from fixation spot disappearance (reaction times were actually much shorter as shown by the distributions in Fig. [2](#Fig2){ref-type="fig"}, Supplementary Fig. [2](#MOESM1){ref-type="media"}). After gaze entered within a virtual window around the true target location by 300 ms, the target reappeared for 400--500 ms. During this time, a visually-guided saccade, which we also referred to as a corrective saccade, was sometimes triggered to correct for any remaining error between the memory-guided saccade and the actual target position. This corrective movement happened readily, and we did not specifically instruct the monkeys to generate it. It was, however, useful for analysis because individual trials often had both a memory movement as well as a control visually-guided one. The radius of the virtual window around the invisible target position was \<3° and was varied depending on target eccentricity. The monkeys were rewarded with water or apple juice at the end of every trial. If they broke fixation during the fixation interval or failed to remain within the virtual window around target location (after the instruction to make the memory-guided saccade), the trial was aborted and repeated. Across trials, we varied target location (i.e. flash location) between 6 min arc and 16^o^ throughout the entire display. During SC recordings, we made sure to include within our sampling of target locations positions that were in and around the response fields (RF's) of neurons around our recording electrodes, as assessed online using our neurophysiological data acquisition system (see below). We collected a total of 6428 trials in this task from monkey N, 9631 from monkey M, and 14,666 from monkey P. In monkeys N and M, we also compared memory-guided saccades to those obtained in a task with explicit visual guidance. In this delayed, visually-guided saccade task, stimulus events were identical to those described above, except that the target flash was now replaced by target persistence until trial end. In other words, the target remained on during the memory interval (now called the delay interval) and also after fixation spot disappearance. Thus, the instructed saccade (i.e. after the offset of the fixation spot) was visually-guided. We collected 6147 trials in this task from monkey N and 5871 trials from monkey M. Human behavioral tasks {#Sec13} ---------------------- Human subjects performed the same memory-guided saccade task described above. Target locations were chosen, across trials, from a total of 480 possible pre-defined display positions ranging in eccentricity from 6 min arc to 12°. 288 of these 480 possible target positions were within a rectangle of ±0.8^o^ × ±0.8^o^ size in order to allow us to explore memory-guided microsaccades in particular detail; the remaining locations were equally spaced in the rest of the display. Seven subjects (four naïve and three untrained authors) performed this experiment, with each subject completing 480--600 trials per session and 4--5 sessions (total of 16,844 trials overall). Ten practice trials (with target eccentricities \>1^o^) were performed at the beginning of every session. This task allowed us to relate the monkey behavioral and neurophysiological results to human performance. The subjects were aged 23--39, and three were female. We also ran two additional control experiments in the humans, in order to understand whether intricacies of memory-guided microsaccade behavior (e.g. Fig. [4](#Fig4){ref-type="fig"}) were specific to a particular motor modality (eye movements) or more related to the engagement of spatial working memory per se. We therefore required a perceptual assessment of remembered flash location without the use of an instructed eye movement. In the first control task, which we refer to as the button-press task, trials were identical to the memory-guided saccade task described above up until the end of the memory interval. At this point, instead of fixation spot disappearance, the fixation spot was replaced with a cross-shaped cursor of 59 min arc dimensions. Subjects used a response box with four buttons allowing them to move the cursor at individual pixel resolution (1.46 min arc per click) rightward, leftward, upward, or downward. Individual button presses moved the cursor by individual pixels; prolonged button presses moved the cursor at one of two faster speeds depending on the duration of the press. Once subjects were satisfied with the cursor position as reflecting, as accurately as possible, the remembered flash location, they pressed a fifth button confirming their choice. The target then reappeared at its true position with the cursor disappearing simultaneously. Subjects were free to move their eyes at will when the cursor was visible, and they were given up to 20 s to move the cursor and decide on their perceptual localization of the remembered target location. Therefore, this task allowed assessing the quality of target recall independent of making an instructed memory-guided saccade. Seven subjects (ages: 25--39 years; one female) participated in this experiment, four of which had performed the original saccade version of the task. Each subject participated in 5--6 sessions (250--350 trials per session), for an overall total of 11,249 trials across subjects. The second control experiment, called the mouse pointer task, was similar to the button-press task except that the cursor was replaced with a computer mouse pointer, which appeared at a random position 1°--5° away from the true flashed target location in every trial. Critically, in this version of the control task, we maintained the fixation spot on during the manual response interval (i.e. until subjects pressed a mouse button), so that an explicit foveal reference frame was always present when subjects were pointing to the remembered target location (similar to how eye movements presumably relied on the initial reference frame of fixation for localization). Subjects were instructed to fixate the fixation spot throughout the response interval until they pressed a mouse button, and they were given up to 15 s to make their response. This task tested whether an explicit foveal reference frame altered the overall pattern of behavior from the button-press task or from the original memory-guided saccade task (Fig. [5](#Fig5){ref-type="fig"}). Seven subjects (ages: 25--39 years; one female) performed this task, four of which had also participated in the button-press and memory-guided saccade versions of it. We collected 380--500 trials per session for each subject. We ran five sessions per subject, with the exception of one participant who completed only four sessions (for a total of 16,213 trials overall). Monkey neurophysiological recordings {#Sec14} ------------------------------------ We recorded SC activity from monkeys N and M using multi-electrode linear arrays (16-channel V-Probes with 150 μm inter-electrode spacing, Plexon). We performed 25 experiments in monkey N and 16 experiments in monkey M. In each experiment, we advanced either one or two V-probes toward SC surface. We monitored the deepest electrode contact in a probe and compared physiological landmarks picked up on this contact to anatomical landmarks in MRI's obtained from the same animals before implant surgeries. Once the deepest electrode contact was in the SC, we advanced the probes further, such that as many electrode contacts as possible were recording SC activity. We used online spike sorting to guide our approach and maximize the number of channels that were recording SC activity. When we inserted two simultaneous V-Probes (23/25 sessions in monkey N), one probe was in the right rostral SC (rostral being the region where microsaccade-related activity is expected^[@CR18],[@CR19]^) and the other was at a slightly more caudal position in the left SC (to allow sampling multiple neuronal preferred eccentricities from the SC's topographic map). When we inserted a single probe, it could be in the right or left SC and at a variety of rostral and slightly more caudal sites. Once the probes were in the SC, we ran a series of experimental tasks, including the memory-guided and delayed, visually-guided saccade tasks described above. We also ran a variety of other tasks that were not directly relevant to the present study. Our neurophysiology system sampled electrical activity in each contact at 40 kHz. Spike times during online sorting were sampled at 1 kHz. Monkey and human behavioral analyses {#Sec15} ------------------------------------ We detected saccades and microsaccades using established methods in our laboratory^[@CR68],[@CR69]^. We manually inspected all trials to correct for false alarms or misses by the automatic algorithms. We also marked blinks or noise artifacts (which were more likely in video-based eye tracking data) for later removal. We analyzed all eye movements occurring in the interval between fixation onset and trial end in the memory-guided and delayed, visually-guided saccade tasks. In the button-press control task, subjects were free to move their eyes after cross-shaped cursor appearance, so we only analyzed eye movements up to such appearance. Individual subjects had idiosyncratic patterns of cursor trajectories and accompanying eye movements that did not appear related in systematic ways to task performance; we thus elected not to analyze eye movements during the response interval in this task. In the mouse pointer task, we monitored eye movements after cursor appearance to ensure that subjects maintained fixation while responding. In this paper, we report each individual monkey's behavioral performance separately. For the human data, we combined results from all subjects into aggregate measurements. We felt justified in doing so because the human effects exhibited very strong similarities to the monkey behavior, which was a primary reason for performing the human experiments in the first place. Moreover, trial numbers were uniformly distributed across the individual human subjects as much as possible; therefore, no single subject biased the pooled results. In all tasks, we first identified valid trials that we accepted for subsequent analysis. Valid trials did not have any saccades larger than 1^o^ during the memory or delay interval (to ensure that fixation was maintained properly), and also no blinks or noise artifacts within ±100 ms from target flash time (or target onset time in the delayed, visually-guided version of the task). This latter criterion ensured that subjects could see the target flash well in the memory version of the tasks. We also removed trials if there were blinks or noise artifacts from −100 to +400 ms from the instruction to generate a memory- or visually guided saccade. Because microsaccades alter visibility and modulate visual sensitivity in general^[@CR3],[@CR33],[@CR35],[@CR70]^, we wanted to ensure that subjects could detect target flash (or onset) and fixation spot disappearance (or other instruction to respond) with high sensitivity. We therefore required that valid trials also did not have any microsaccades (or large saccades) within ±50 ms from flash (target) onset or fixation spot disappearance (or any other instruction to respond). Finally, memory-guided saccades had to land within 2° of the target; this constraint was irrelevant for memory-guided microsaccades (which were much smaller), but it helped ensure that the eye movements were accurate when large target eccentricities were used. Based on the detected saccades and microsaccades, we computed measures of saccade amplitude, latency (reaction time), direction, starting point, and endpoint. We calculated the starting point as the average eye position in the interval 30 ms before the start of the saccade. The end point was simply the very last sample of the detected saccade. To obtain saccade direction error (i.e. saccade direction relative to target direction from initial fixation spot position), we computed the counter-clockwise angular difference between saccade and target direction. For corrective saccades after target reappearance in the memory-guided saccade task, the target reappeared at a position that was variable relative to gaze position (depending on the landing error of the previous memory-guided movement). We therefore redefined the position of the reappearing target relative to corrective saccade starting eye position. We then calculated saccade direction error as above. We used a similar procedure for target eccentricity. Note that after large memory-guided saccades, target position error for the corrective movements was often small (i.e. large memory-guided saccades were relatively accurate as expected). We therefore had significantly more data for corrective, visually-guided microsaccades than for memory-guided microsaccades (e.g. see the numbers of trials in the legend of Fig. [2](#Fig2){ref-type="fig"}). In the memory-guided saccade task, we defined as the memory-guided saccade (or memory-guided microsaccade) of interest as the first saccade occurring with a latency of at least 50 ms after fixation spot disappearance and less than 50 ms after target reappearance. Similarly, the corrective, visually guided saccade was defined as the first saccade that was executed with latency \>50 ms after target reappearance. Because most studies define microsaccades as smaller than 1° in amplitude and we were interested in comparing these to voluntarily generated movements, we treated trials with target eccentricities less than 1° as memory-guided microsaccade trials. The above analyses were also applied in the delayed, visually-guided saccade task. For the control tasks, we treated button and mouse press locations similarly to how we analyzed saccade endpoints. In all analyses, we defined trials with target eccentricities \<1° as the trials involving microsaccades, as stated above. Corrective, visually-guided saccades were also classified as microsaccades when target eccentricity (relative to eye position at target reappearance) was \<1°. As stated above, we used this threshold because it was a typical threshold used in the literature^[@CR18],[@CR38]^, but our results (also from other studies) confirm that there is indeed a continuum of saccade generation across different amplitudes. Spike sorting for neurophysiological data analyses {#Sec16} -------------------------------------------------- For all neurophysiological data analyses, we performed offline sorting to isolate individual single units. We isolated single units from wide-band (40 kHz) electrode data by adapting the Klusta toolbox^[@CR71]^. As an additional input to the toolbox, we recreated the electrode geometry (16 channels with linear spacing) such that neighboring channels could share spike timing and waveform information (as in, virtual tetrodes). From filtered neural data (750--5000 Hz), the toolbox performed automated spike detection and classification. We inspected the resulting cluster assignments (putative units) using the Kwik-gui interface included in the Klusta toolbox. Based on auto- and cross-correlograms, as well as waveform shapes, we manually curated each cluster in order to obtain well-isolated units. Clusters that showed no characteristic auto-correlogram, or that produced atypical spike waveforms, were clustered together, labeled as noise, and subsequently excluded from further analysis. We estimated single unit isolation quality of all resulting units using established metrics^[@CR72],[@CR73]^. Specifically, we computed an estimate of the false positive rate based on refractory period violations (inter-spike interval violations). Furthermore, we obtained an isolation distance measure for each cluster, by calculating the Mahalanobis distance between any two units. All isolated units that exceeded an estimated false positive rate of 10% or had an isolation distance below 30 were excluded from further analysis; all other units are hereafter termed putative neurons (297 neurons in total; we selected for analysis all 188 neurons that showed clear SC visual and/or saccade-related RF's encompassing microsaccadic amplitude ranges, as assessed by the procedures described below). Spike trains of all putative neurons were then convolved with an appropriately-scaled Gaussian (*σ* = 10 ms) to obtain an estimation of the neuron's firing rate in spikes per s. Neurophysiological data analyses {#Sec17} -------------------------------- We classified SC neurons as visual, visual-motor, or motor depending on visual and saccade-related responses. We defined several 100-ms intervals based on key trial events. The visual response interval comprised the time from 50 to 150 ms after target onset. The motor interval was set to be 100 ms centered around saccade onset. We also measured this saccade-related interval for corrective saccades in the memory-guided saccade task. Since we were especially interested in characterizing microsaccade-related discharge, we classified movement-related aspects of neurons based on responses associated with saccades \<1° in amplitude. To estimate baseline activity during fixation without any target or saccade onset, we used the100-ms steady fixation interval before target onset. We avoided contamination of baseline and visual neural activity by saccades or microsaccades; we excluded all trials in which such movements occurred during the measurement intervals. Similarly, we excluded trials in which there was more than one movement within the motor interval. In all cases, we measured average firing rate within a given measurement interval, which resulted in a population of measurements across trials; we only included neurons with \>10 repetitions per measurement interval. We compared each of visual, memory-guided microsaccade, corrective microsaccade, or visually-guided microsaccade (in the delayed version of the task) activity to baseline activity using one-tailed Wilcoxon-ranksum tests (*α* = 0.05). We then grouped neurons into five categories: (1) Visual (V-Neuron), possessing only a visual increase relative to baseline; (2) Visual-motor (VM-Neuron), possessing a visual increase and either memory- or corrective-(visual) saccade response; (3) Motor (M-Neuron), exhibiting both memory- and corrective-(visual) saccade response but no visual response; (4) Visually dependent (VDSR-Neuron), possessing a saccade-related response only when a visual target was present for the eye movement^[@CR46]--[@CR48]^; (5) Exclusively-responding (ER-Neuron), exhibiting a microsaccade-related response only in the memory-guided microsaccade task but not for similarly sized visually-guided microsaccades. RF hotspot locations (e.g. Fig. [7f](#Fig7){ref-type="fig"}) were estimated based on all of the sampled saccade vector amplitudes (and corresponding flash locations for visual responses) in the memory-guided saccade task (even \> 1°); our estimates may therefore include neurons that may potentially have responded even more strongly had we obtained even larger eccentricities (at the very least, all neurons in Fig. [7f](#Fig7){ref-type="fig"} were related in one form or another to microsaccades). For every isolated neuron that had a motor response, we also estimated the saccadic amplitudes that caused the neuron to discharge, also known as the neuron's movement field. For every neuron, we compared the population of baseline values as described above to the trial-by-trial responses for saccades. We then calculated the movement field by a linear-interpolation of all z-transformed saccade-related responses. Locations with *z*-scores \> 1.96 were considered to be part of the movement field. We plotted movement fields using log-polar coordinates, to magnify small eccentricities associated with microsaccades, as described previously^[@CR19]^. Movement-related hotspots were identified based on the stronger of either the memory-guided or the visually guided movement-related responses. Statistical analyses {#Sec18} -------------------- We performed statistical tests (behavioral and neural) with an *α* level of 0.05 unless otherwise noted. When the assumptions of normality were not met for a two-sampled *t*-test, we used a Wilcoxon ranksum test instead. For tests related to saccade directions, we used the CircStat toolbox^[@CR74]^. When comparing proportions (for example, for success rate), we used a *χ*^2^-test. Literature meta-analysis {#Sec19} ------------------------ We searched for and read articles published between 1965 and November 2017. We used the Web of Science search database, and we employed the keyword "microsaccade\*". We then selected, out of 467 research articles in the global results, a total of 342 articles that were genuinely related to microsaccades and that were also written in English. Any article that referred to microsaccades as being "voluntary" (20/31 articles), "controlled" (6/31 articles), or "suppressed voluntarily" (5/31 articles) was classified in the "voluntary" category (31 articles). Even though "suppressed voluntary" is conceptually different from the voluntary control that we were interested in for the current study, we nonetheless included this description in the voluntary category to give this category the benefit of the doubt in our meta-analysis when comparing to "involuntary" descriptions. In contrast to 31 articles, there were 148 articles in the "involuntary" category (almost 5 times as many). For the "involuntary" category (148 articles), we included any article using the following descriptors for microsaccades: "involuntary" (134/148 articles), "spontaneous" (7/148 articles), "unaware" (3/148 articles), "automatic" (2/148 articles), "unconscious" (1/148 articles), "stochastic" (1/148 articles). Reporting summary {#Sec20} ----------------- Further information on research design is available in the [Nature Research Reporting Summary](#MOESM3){ref-type="media"} linked to this article. Supplementary information ========================= {#Sec21} Supplementary Information Peer Review File Reporting Summary Description of Additional Supplementary Files Supplementary Movie 1 Supplementary Movie 2 **Peer review information**: *Nature Communications* thanks Jochen Braun, Emilio Salinas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. **Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Konstantin F. Willeke, Xiaoguang Tian, Antimo Buonocore. Supplementary information ========================= **Supplementary Information** accompanies this paper at 10.1038/s41467-019-11711-x. We were funded by the Werner Reichardt Centre for Integrative Neuroscience (CIN). The CIN is an Excellence Cluster (EXC307) funded by the Deutsche Forschungsgemeinschaft (DFG). We were also supported by the Hertie Institute for Clinical Brain Research at Tuebingen University, and DFG-funded Research Unit (FOR1847; project: HA6749/2--1). X.T., A.B., Z.M.H. collected neurophysiological data. X.T., J.B., Z.M.H. collected monkey behavioral data. K.F.W., A.R-C. collected human behavioral data. K.F.W., A.B., Z.M.H. analyzed data. Z.M.H. wrote paper. K.F.W., X.T., A.B., A.R-C., Z.M.H. edited paper. All data presented in this paper are stored in institute computers and are available upon reasonable request. The authors declare no competing interests.

{ "pile_set_name": "PubMed Central" }

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"units", "using", "interface", "included", "Klusta", "toolbox", "Based", "well", "waveform", "shapes", "manually", "curated", "cluster", "order", "obtain", "units", "Clusters", "showed", "characteristic", "produced", "atypical", "spike", "waveforms", "clustered", "together", "labeled", "noise", "subsequently", "excluded", "analysis", "estimated", "single", "unit", "isolation", "quality", "resulting", "units", "using", "established", "Specifically", "computed", "estimate", "false", "positive", "rate", "based", "refractory", "period", "violations", "interval", "violations", "Furthermore", "obtained", "isolation", "distance", "measure", "cluster", "calculating", "Mahalanobis", "distance", "two", "units", "isolated", "units", "exceeded", "estimated", "false", "positive", "rate", "isolation", "distance", "excluded", "analysis", "units", "hereafter", "termed", "putative", "neurons", "neurons", "total", "selected", "analysis", "neurons", "showed", "clear", "SC", "visual", "RF", 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"contamination", "baseline", "visual", "neural", "activity", "saccades", "microsaccades", "excluded", "trials", "movements", "occurred", "measurement", "intervals", "Similarly", "excluded", "trials", "one", "movement", "within", "motor", "interval", "cases", "measured", "average", "firing", "rate", "within", "given", "measurement", "interval", "resulted", "population", "measurements", "across", "trials", "included", "neurons", "repetitions", "per", "measurement", "interval", "compared", "visual", "microsaccade", "corrective", "microsaccade", "microsaccade", "delayed", "version", "task", "activity", "baseline", "activity", "using", "tests", "α", "grouped", "neurons", "five", "categories", "Visual", "possessing", "visual", "increase", "relative", "baseline", "possessing", "visual", "increase", "either", "visual", "saccade", "response", "Motor", "exhibiting", "visual", "saccade", "response", "visual", "response", "Visually", "dependent", "possessing", "response", "visual", "target", "present", "eye", "exhibiting", "response", "microsaccade", "task", "similarly", "sized", "microsaccades", "RF", "hotspot", "locations", "Fig", "fig", "estimated", "based", "sampled", "saccade", "vector", "amplitudes", "corresponding", "flash", "locations", "visual", "responses", "saccade", "task", "even", "estimates", "may", "therefore", "include", "neurons", "may", "potentially", "responded", "even", "strongly", "obtained", "even", "larger", "eccentricities", "least", "neurons", "Fig", "fig", "related", "one", "form", "another", "microsaccades", "every", "isolated", "neuron", "motor", "response", "also", "estimated", "saccadic", "amplitudes", "caused", "neuron", "discharge", "also", "known", "neuron", "movement", "field", "every", "neuron", "compared", "population", "baseline", "values", "described", "responses", "saccades", "calculated", "movement", "field", "responses", "Locations", "z", "considered", "part", "movement", "field", "plotted", "movement", "fields", "using", "coordinates", "magnify", "small", "eccentricities", "associated", "microsaccades", "described", "hotspots", "identified", "based", "stronger", "either", "visually", "guided", "responses", "Statistical", "analyses", "performed", "statistical", "tests", "behavioral", "neural", "α", "level", "unless", "otherwise", "noted", "assumptions", "normality", "met", "used", "Wilcoxon", "ranksum", "test", "instead", "tests", "related", "saccade", "directions", "used", "CircStat", "comparing", "proportions", "example", "success", "rate", "used", "χ", "Literature", "searched", "read", "articles", "published", "November", "used", "Web", "Science", "search", "database", "employed", "keyword", "selected", "research", "articles", "global", "results", "total", "articles", "genuinely", "related", "microsaccades", "also", "written", "English", "article", "referred", "microsaccades", "voluntary", "articles", "controlled", "articles", "suppressed", "voluntarily", "articles", "classified", "voluntary", "category", "articles", "Even", "though", "suppressed", "voluntary", "conceptually", "different", "voluntary", "control", "interested", "current", "study", "nonetheless", "included", "description", "voluntary", "category", "give", "category", "benefit", "doubt", "comparing", "involuntary", "descriptions", "contrast", "articles", "articles", "involuntary", "category", "almost", "times", "many", "involuntary", "category", "articles", "included", "article", "using", "following", "descriptors", "microsaccades", "involuntary", "articles", "spontaneous", "articles", "unaware", "articles", "automatic", "articles", "unconscious", "articles", "stochastic", "articles", "Reporting", "summary", "information", "research", "design", "available", "Nature", "Research", "Reporting", "Summary", "media", "linked", "article", "Supplementary", "information", "Supplementary", "Information", "Peer", "Review", "File", "Reporting", "Summary", "Description", "Additional", "Supplementary", "Files", 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"behavioral", "data", "collected", "human", "behavioral", "data", "analyzed", "data", "wrote", "paper", "edited", "paper", "data", "presented", "paper", "stored", "institute", "computers", "available", "upon", "reasonable", "request", "authors", "declare", "competing", "interests" ]

Sir, Basal cell carcinoma (BCC) is the most common malignancy of skin worldwide. Many clinical morphologies of BCC exist. Clinical diagnosis depends on the awareness and knowledge of the varied presentations of BCC. This not only facilitates an early diagnosis of uncommon morphologies of the malignancy, but also ensures prompt intervention and treatment. The morphological variants include nodular, cystic, morpheaform, infiltrative, micronodular, superficial, pigmented, polypoid, pore-like, aberrant, and fibroepithelioma of Pinkus (FEP).\[[@ref1]\] A 60-year-old man presented with a solitary, nonhealing fragile mass over the abdomen of 6 months duration. To start with, the lesion was an asymptomatic pea-sized nodule, which gradually increased in size to attain the present dimension. There was history of bleeding from the lesion---both spontaneously and on minor trauma. Past medical, surgical, and family history were unremarkable. There was no history of trauma, chronic arsenical exposure, or exposure to radiation. Cutaneous examination revealed bright-red, dome-shaped fragile tumor that had a glistening moist surface, covered with hemorrhagic crust \[[Figure 1](#F1){ref-type="fig"}\]. There was no associated lymphadenopathy of inguinal or cervical region. Hairs, nails, and mucosae were absolutely normal, and systemic examination was noncontributory. Pyogenic granuloma, squamous cell carcinoma, and nodular melanoma were considered in clinical differentials. Routine biochemistry panel, including serology for Hepatitis B virus, Venereal Disease Research Laboratory test, and Human immunodeficiency virus (HIV) were nonreactive. The tumor was excised and sent for histopathology. There was basaloid cell proliferation in nodular pattern with peripheral palisading of cells and cleft-like space around it at places. The cells were elongated with an elongated oval nucleus and scanty cytoplasm. The cells showed increased pigmentation in some areas \[Figures [2](#F2){ref-type="fig"}--[4](#F4){ref-type="fig"}\]. Histopathological findings were diagnostic of BCC. ![Nodular growth with moist surface and covered with hemorrhagic crust](IDOJ-7-446-g001){#F1} ![Proliferation of basaloid cells showing peripheral palisading (H and E, 100×)](IDOJ-7-446-g002){#F2} ![Higher magnification (H and E × 400)](IDOJ-7-446-g003){#F3} ![Increased pigmentation in tumor mass (H and E × 400)](IDOJ-7-446-g004){#F4} BCCs are the most common cutaneous tumors, accounting for approximately 70% of all malignant diseases of the skin. It is slow growing, locally invasive malignancy, which arises from pleuripotent cells within the basal layer of the epidermis or follicular structure. The malignancy predominantly affects fair-skinned individuals above the age of 40 years. Usually it is caused by a combination of cumulative ultraviolet (UV) exposure and intense, occasional UV exposure, especially on areas of chronic sun exposure, such as the face, head, and neck. Other known risk factors include chronic exposure to arsenic, chronic inflammation, and genetic conditions such as xeroderma pigmentosum, albinism, and basal cell nevus syndrome. However, occurrence of BCC in covered areas and also in persons without any of the aforementioned predisposing factors, as exemplified in our case indicates that etiopathogenesis is still elusive and more studies are needed to identify other contributing factors. Uncommonly, BCC may develop over atypical locations such as axilla, nipple, scrotum, pubis, and perianal region.\[[@ref2][@ref3]\] According to some authors, patients with truncal BCCs were significantly at a higher risk of multiple BCCs.\[[@ref4]\] There are different clinical and histological variants of BCC. The clinical variants are nodular (most common), pigmented, morpheaform, superficial, and FEP. Some may manifest as nodules, patches, plaques, ulcers, or rarely, a pyogenic granuloma like.\[[@ref5]\] Very few cases of BCCs mimicking pyogenic granuloma have been reported. A unique case of polypoidal BCC on the face of an elderly lady masquerading as pyogenic granuloma, was reported from India.\[[@ref5]\] Besides, Kim *et al*. reported a case of BCC on the dorsolateral side of the ring finger, which clinically simulated pyogenic granuloma.\[[@ref6]\] The treatment in such cases essentially consists of surgical excision and a systemic workup to exclude any metastatic deposit, followed by periodic follow-up. Thus, a rare presentation of BCC along with uniqueness in the site prompted us to report the case. Our report highlights the noteworthiness of taking into consideration BCC in the clinical differentials of lesions, which have a tendency to bleed, even if the classical features of BCC-like pearly border, telangiectasia, and others are absent. Financial support and sponsorship {#sec2-1} ================================= Nil. Conflicts of interest {#sec2-2} ===================== There are no conflicts of interest.

{ "pile_set_name": "PubMed Central" }

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22,205

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Introduction ============ The paraoxonase (PON)[^3^](#FN4){ref-type="fn"} family consists of three members: PON1, PON2, and PON3 ([@B1][@B2][@B3]). PON1 was the first family member described and was named for its ability to degrade the organophosphate paraoxon. PON1 was subsequently found to inhibit low density lipoprotein oxidation and be important for cardiovascular disease ([@B4]). In addition, analysis of PON1 single-nucleotide polymorphisms (SNPs) has linked PON1 to the pathogenesis of numerous other human disorders including atherosclerosis, diabetes, cerebrovascular disease, Alzheimer disease, amyotrophic lateral sclerosis, organophosphate susceptibility, and Parkinson disease ([@B5][@B6; @B7; @B8; @B9; @B10][@B11]). Despite these numerous association studies, the mechanism(s) underlying the role of PON in disease pathogenesis remains to be fully determined, in part because of uncertainty regarding the endogenous or natural substrate of PON. However, the native enzyme activity of PON was recently found to be as a lactonase ([@B12]), suggesting that despite its known enzymatic promiscuity for other substrates including esters and phosphotriesters, its endogenous substrates are lactones. Importantly, all three PON family members have conserved their lactonase activity, but individual PON family members have widely disparate enzymatic activities toward other substrates. One specific lactone-containing molecule that PONs can degrade is the bacterial quorum-sensing molecule, *N*-3-oxododecanoyl homoserine lactone (3OC12-HSL) ([@B13][@B14; @B15][@B16]). 3OC12-HSL is an acyl-homoserine lactone that Pseudomonas aeruginosa, a common cause of hospital-acquired infections and an important cause of pulmonary morbidity and mortality in cystic fibrosis, uses to control biofilm formation and virulence factor production ([@B17], [@B18]). Of the three PON family members, PON2 has the greatest lactonase activity toward 3OC12-HSL ([@B13], [@B19]). In contrast to PON1 and PON3, PON2 is not present in serum, and it has minimal arylesterase and paraoxonase activity. Human airway epithelial cells degrade 3OC12-HSL ([@B19], [@B20]), and murine PON2-deficient airway epithelial cells have an impaired ability to inactivate 3OC12-HSL ([@B19]). In addition, we have recently found that PON1-transgenic Drosophila melanogaster are protected from organophosphate poisoning and P. aeruginosa lethality ([@B21]). PON2 activity in airway epithelial cells may represent a novel antibacterial defense mechanism against invading pathogens that utilize acyl-homoserine lactones for quorum sensing and virulence regulation. Genetic variation in human genes can have a substantial impact on host responses, and SNP analysis may be used in anticipating responses and outcomes or in adjusting drug dosing. In this study we asked whether a common PON2 SNP, a serine to cysteine amino acid change at codon 311 in PON2 ([@B2]), alters PON2 lactonase activity for 3OC12-HSL. This SNP is fairly common in the general population and is strongly conserved evolutionarily. We chose this PON2 SNP because PON2 has the greatest lactonase activity of PON family members, phylogenetic analysis suggests that PON2 is the oldest PON family member ([@B11]), and several PON1 SNPs are in linkage disequilibrium with PON2 SNPs. We found that recombinant PON2 Cys^311^ exhibits an impaired ability to inactivate 3OC12-HSL and that airway epithelial cells from humans homozygous for Cys/Cys at PON2 amino acid position 311 also have an impaired ability to degrade 3OC12-HSL. EXPERIMENTAL PROCEDURES ======================= ### #### Recombinant PON2 Cys^311^ or Ser^311^ Expression Chinese hamster ovary (CHO) cells were used for transfection studies because these cells have no endogenous PON or lactonase activity ([@B13]). CHO cells were cultured as monolayers in plastic dishes and transfected with plasmids expressing hPON2 Cys^311^ or Ser^311^ using Lipofectamine 2000 (Invitrogen), according to the manufacturer\'s protocol, and allowed to express for 24--48 h. #### 3OC12-HSL Degradation by Airway Epithelia and CHO Cells For epithelial cell lysate experiments, epithelia were first washed with cold phosphate-buffered saline (containing calcium and magnesium), and then 50 μl of lysis buffer (50 m[m]{.smallcaps} Tris·HCl, pH 6.9, 150 m[m]{.smallcaps} NaCl, 10 μ[m]{.smallcaps} leupeptin, 10 μ[m]{.smallcaps} aprotinin, 1 μ[m]{.smallcaps} pepstatin A, 1 m[m]{.smallcaps} phenylmethylsulfonyl fluoride, 0.1 mg/ml benzamidine) was added to the apical surface. After 20 min of rocking in lysis buffer at 4 °C, the cells were scraped free from the membrane with a pipette tip and lysed by sonication (10 pulses, pulse duration of ∼1 s) (Branson Sonifier 250, Danbury, CT). The cellular debris was cleared from the lysate by centrifugation (4500 × *g* for 30 s at 4 °C). The relative protein concentrations were determined by the Bio-Rad protein assay (500-0006). Lysate preparations (10--20%) were diluted in phosphate-buffered saline (containing calcium and magnesium) and incubated in the presence of 3OC12-HSL (RTI International, Research Triangle Park, NC) at 37 °C. 3OC12-HSL in acidified ethyl acetate was dried under a nitrogen gas stream and then dissolved in phosphate-buffered saline (containing calcium and magnesium) to achieve a final concentration of 10 μ[m]{.smallcaps} 3OC12-HSL. At various time points, 6-μl aliquots were collected, added to 100 μl of ethyl acetate, and stored in airtight glass vials at −20 °C. #### 3OC12-HSL and 3OC12-HSL Quantitative Assay 3OC12-HSL was obtained from RTI International. 3OC12-HSL was measured in a quantitative bioassay as previously described using Escherichia coli MG4 (pKDT17) ([@B22]). #### Tunicamycin Treatment of Transfected CHO Cells PON2 chemical deglycosylation was performed by first transfecting CHO cells with a plasmid expressing PON2 Cys^311^ or Ser^311^. 30 min later, tunicamycin was added to the cell culture medium at a final concentration of 0.5 μg/ml. 36--48 h later cell lysates were harvested, and SDS-PAGE was performed for PON2. #### Deglycosylation of PON2 with Peptide-N-glycosidase F and Endoglycosidase H Digestion Peptide-*N*-glycosidase F (PNGase F) (Sigma) digestion was performed according to the manufacturer\'s directions. Briefly, cell lysates were collected in lysis buffer (50 m[m]{.smallcaps} Tris·HCl, pH 7.5, 138 m[m]{.smallcaps} NaCl, 5 m[m]{.smallcaps} EDTA, 1 m[m]{.smallcaps} EGTA, 1 m[m]{.smallcaps} NaF, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) and incubated with PNGase F (10 units) for 4 h at 37 °C. The reaction was stopped by incubating at 100 °C for 5 min. Deglycosylation was then assessed with SDS-PAGE. Endoglycosidase H (Endo H) (Sigma) digestion was performed according to the manufacturer\'s directions. The cell lysates were incubated with reaction buffer and denaturation solution and heated for 5 min at 100 °C. Following cooling of the sample, Endo H was added to the samples and incubated for 4 h at 37 °C. Digestion was then determined using SDS-PAGE. #### Western Blot Analysis CHO cell lysates were made by incubation in lysis buffer (50 m[m]{.smallcaps} Tris·HCl, pH 7.5, 138 m[m]{.smallcaps} NaCl, 5 m[m]{.smallcaps} EDTA, 1 m[m]{.smallcaps} EGTA, 1 m[m]{.smallcaps} NaF, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) with 1% Triton X-100 for 20 min at 4 °C on a rocker. The cells were scraped from the tissue culture plates and transferred to a microcentrifuge tube. Sonication was performed for 10 s, and cell debris was cleared by centrifugation at 4,500 × *g* for 30 s at 4 °C. The lysates were then combined with loading buffer and separated by SDS-PAGE. Protein was then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA). Polyvinylidene difluoride membranes were blocked either overnight at 4 °C or for 2 h at room temperature in 5% bovine serum albumin in phosphate-buffered saline. The membranes were then incubated with the PON2 antibody (1:1000; rabbit, polyclonal anti-human PON2; Orbigen, San Diego, CA) for 2 h at room temperature and then washed three times in 1× TTBS (137 m[m]{.smallcaps} NaCl, 2.7 m[m]{.smallcaps} KCl, 2.5 m[m]{.smallcaps} Tris, 0.05% Tween 20). Secondary antibodies were conjugated to horseradish peroxidase (Amersham Biosciences) and used at 1:10,000 for 1 h. Following three washes with 1× TTBS, immunoreactive bands were detected with SuperSignal solution (Pierce) and exposed to film. #### Construction of N-Linked Glycosylation Mutants Site-directed mutagenesis was performed with the Stratagene QuikChange™ kit according to the manufacturer\'s standard protocol (Stratagene, Cedar Creek, TX). Double-stranded DNA template was prepared by a standard megaprep protocol (Qiagen). Mutant strand synthesis was performed with the following primers for N226Q (5′-GATTCAGCAAATGGGATCCAGATTTCACCTGATGATAAG-3′), for N254Q (5′-GTTTTGGAAAAACACACTAATATGCAGTTAACTCAGTTGAAGG-3′), for N269Q (5′-TGGATACACTGGTGGATCAGTTATCTATTGATCCTTCC-3′), for N323Q (5′-ACTACAGTTTATGCCAACCAGGGGTCTGTTCTCCAAGG-3′), for N254A (5′-GTTTTGGAAAAACACACTAATATGGCTTTAACTCAGTTGAAGG-3′), and for N323A (5′-ACTACAGTTTATGCCAACGCTGGGTCTGTTCTCCAAGG-3′). The reaction products were then treated with the restriction endonuclease DpnI to enrich for multiply mutated single-stranded DNA. This reaction mixture was then transformed into XL10-Gold® Ultracompetent cells and spread on Luria-Bertani ampicillin agar plates (containing 80 ng/ml 5-bromo-4-chloro-3-indolyl-β-[d]{.smallcaps}-galactopyranoside (X-gal) and 20 m[m]{.smallcaps} isopropyl β-[d]{.smallcaps}-thiogalactopyranoside). The colonies were picked, and site-specific mutations were confirmed by DNA sequencing (DNA Facility, University of Iowa). #### Immunofluorescence Localization of PON2 in Transfected CHO Cells CHO cells were plated on collagen-coated four-well chamber slides and transfected with a plasmid encoding either human PON2 Cys^311^ or Ser^311^. At 36 h following transfection, the cells were fixed with 1% paraformaldehyde in methanol, blocked in 2% bovine serum albumin in SuperBlock (Pierce), and stained with anti-human PON2 antibody (rabbit polyclonal, Orbigen) and a monoclonal antibody specific for protein-disulfide isomerase to localize the endoplasmic reticulum (Assay Designs, Ann Arbor, MI). Confocal images were acquired with a Bio-Rad MRC-1024 laser scanning confocal system mounted on a Nikon E600 microscope. #### Primary Airway Epithelial Cell Culture Model Primary human airway epithelial cells were isolated from the trachea and bronchi of human donor lungs. The isolated cells were seeded onto collagen-coated, semi-permeable membranes (0.6-cm^2^ Millicell-HA; Millipore, Bedford, MA) and grown at the air-liquid interface as previously described ([@B23]). After genotypic identification of PON2 SNPs in donor fibroblasts, primary or passage 1 cryopreserved airway epithelial stocks were thawed and expanded for the described studies. All of the experiments were performed on well differentiated human airway epithelia (more than 14 days after seeding of cells). The samples were collected with the approval of the University of Iowa Institutional Review Board. #### Identification of PON Polymorphisms and Determination of Genotypic Frequencies DNA samples were genotyped for the presence of several common coding and noncoding polymorphisms, including PON2 Cys^311^ → Ser. PCR primers for amplifying the sequences containing the SNP were designed using Primer 3. The PCR primers are listed in the [supplemental materials](http://www.jbc.org/cgi/content/full/M109.051706/DC1). Sense and antisense probes used for fluorescence polarization-single base extension detection of SNPs were designed using Primer Premier. The sense and antisense probes for genotyping the PON2 Cys^311^ → Ser SNP are: 5′-CTG TAG TCA CTG TAG GCT TCT CA-3′ and 5′-CCG CAT CCA GAA CAT TCT AT-3′, respectively. Each genotyping reaction consisted of a polymerase chain reaction followed by a single base pair extension reaction using either the sense or antisense probe and dideoxynucleotides labeled with carboxytetramethylrhodamine (TAMRA) or R110 for the alternative alleles. The fluorescence signal was detected by fluorescence polarization using the analyst HT (Molecular Devices, Sunnyvale, CA), and genotypes were determined based on the plot of TAMRA *versus* R110 signal values. #### Statistical Analysis All of the experiments were performed in at least triplicate, and the data are presented as the means ± S.E. of the mean (S.E.). Comparisons between two groups were made with Student\'s *t* test. RESULTS ======= ### #### Cysteine Substitution at Amino Acid Position 311 Impairs PON2 Lactonase Activity We cloned PON2 and asked whether mutating the 311 amino acid, from a serine to cysteine, would affect PON2 lactonase activity. Lysates from CHO cells expressing both PON2 Cys^311^ and Ser^311^ inactivated 3OC12-HSL over time, but PON2 Cys^311^ lysates had an impaired ability compared with PON2 Ser^311^ lysates ([Fig. 1](#F1){ref-type="fig"}*A*). These data suggest that cysteine substitution at amino acid 311 impairs PON2 function through mechanisms other than alternative splicing, linkage disequilibrium, or promoter differences. ![**Recombinant PON2 Cys^311^ has impaired lactonase activity.** *A*, 3OC12-HSL degradation by CHO cell lysates following transfection with PON2 Cys^311^ or PON2 Ser^311^. *n* = 3--5 experiments/group. Lysates (20% volume) of CHO cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL and after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of 3OC12-HSL inactivation. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. The data are the means ± S.E. \*, *p* \< 0.05. *B*, CHO cells were transfected with PON2 Cys^311^ or Ser^311^ and 48 h later lysed for gel electrophoresis. Subsequently, Western immunoblot analysis for PON2 was performed as described under "Experimental Procedures." *C*, densitometric analysis of PON2 Cys^311^ and Ser^311^ protein levels in CHO cell lysates at 48 h following transfection. ImageJ software was used for densitometric analysis of PON2 immunoreactive bands. GAPDH levels were used for normalization. *n* = 3. The data are the means ± S.E.](zbc0021098990001){#F1} To test whether the PON2 Cys^311^ variant expresses similar PON2 protein levels as PON2 Ser^311^, PON2 immunoblotting was performed in CHO cell lysates 48 h following transfection. [Fig. 1](#F1){ref-type="fig"}*B* shows that in PON2 Cys^311^ samples we observed a single immunoreactive band migrating slightly higher than 37 kDa, a molecular mass consistent with PON2. In PON2 Ser^311^ samples, we also observed a similarly sized upper band as in PON2 Cys^311^, but noted an additional band with greater electrophoretic mobility several kDa lower than the upper PON2 band. Densitometric analysis and quantification showed no difference in total PON2 protein levels between PON2 Cys^311^ and Ser^311^ groups ([Fig. 1](#F1){ref-type="fig"}*C*). These findings suggest that the 311 amino acid change does not alter PON2 protein stability. The smaller, second band could result from alternative splicing (although this seems highly unlikely to occur using cDNA) ([@B2]), from PON2 aggregation ([@B24]), or most likely from differential post-translational modification or proteolysis. #### An Altered Core Glycosylation Pattern Occurs in the PON2 Cys^311^ Variant PON2 has four putative *N*-linked glycosylation sites at asparagine residues. Purified PON1 is ∼15.8% carbohydrate by weight ([@B25]). To investigate the glycosylation of PON2 and determine whether PON2 Cys^311^ and Ser^311^ are differentially glycosylated, we first expressed PON2 Cys^311^ or Ser^311^ in CHO cells and then treated with tunicamycin, an antibiotic from the bacterium *Streptomyces iysosuperficus* that inhibits GlcNAc phosphotransferase, one of the initial steps required for glycoprotein synthesis. Two days later, cell lysates were harvested, and Western blot analysis showed that, in PON2 Cys^311^ samples, the predominant immunoreactive band following tunicamycin treatment had a greater electrophoretic mobility compared with the PON2 band in untreated cells ([Fig. 2](#F2){ref-type="fig"}*A*). This finding is consistent with *N*-linked core glycosylation of PON2. Similar findings were observed with PON2 Ser^311^ ([Fig. 2](#F2){ref-type="fig"}*B*). After tunicamycin treatment there was no difference in the appearance of the PON2 band between Cys^311^ and Ser^311^ variants. This suggests that the lower band in PON2 Ser^311^ samples represents differential or incomplete glycosylation compared with the upper band in PON2 Cys^311^ and Ser^311^. ![**Effect of chemical and enzymatic deglycosylation of PON2.** CHO cells were transfected with a plasmid expressing human PON2 Cys^311^ (*A*) or PON2 Ser^311^ (*B*). Tunicamycin was added shortly after transfection, and cell lysates were harvested 48 h later. In separate experiments, enzymatic deglycosylation with either PNGase F or Endo H, as described under "Experimental Procedures," was also performed on cell lysates 36--48 h following transfection.](zbc0021098990002){#F2} Because tunicamycin has a number of effects on the cell including induction of the unfolded protein response, we also treated cell lysates with PNGase F at 48 h following transfection. PNGase F is an enzyme that cleaves *N*-linked glycoproteins between the innermost GlcNAc and asparagine residues. *N*-Linked deglycosylation with PNGase F caused both PON2 Cys^311^ and Ser^311^ migration patterns to change as observed with tunicamycin treatment, and after PNGase F treatment there was no difference between the Cys and Ser variants ([Fig. 2](#F2){ref-type="fig"}). PON2 was also sensitive to treatment with Endo H, an endoglycosidase that cleaves the chitobiose core of high mannose and oligosaccharides from *N*-linked glycoproteins. When proteins are correctly processed through the endoplasmic reticulum and Golgi complex, they become resistant to Endo H. Therefore sensitivity to Endo H indicates the presence of proteins that have not been processed beyond the ER. We observed qualitatively similar results when comparing Endo H, tunicamycin, and PNGase F treatments in both PON2 Cys^311^ and Ser^311^ samples ([Fig. 2](#F2){ref-type="fig"}). From these results, we conclude that PON2 undergoes *N*-linked core glycosylation, and incomplete glycosylation is likely responsible for the additional band observed when PON2 Ser^311^ is expressed in CHO cells. #### PON2 Undergoes N-Linked Core Glycosylation at Asparagine Residues 254 and 323 Based on the two different bands of glycosylated PON2, we predicted that at least two of the four putative *N*-linked glycosylation sites are glycosylated in PON2. To determine which asparagine residues of the Asn-Xaa-(Ser/Thr) consensus sequence are *N*-glycosylated in PON2 Cys^311^ and Ser^311^ variants, putative *N*-linked glycosylation sites were disrupted by substituting glutamine for asparagine individually and also in combination (quadruple mutant). Western blotting for PON2 revealed one immunoreactive band in wild-type PON2 Cys^311^ and the N226Q and N269Q mutants. Cell lysates from the N254Q and N323Q mutants also showed one PON2 immunoreactive band, but with a greater electrophoretic mobility than wild-type PON2 Cys^311^ ([Fig. 3](#F3){ref-type="fig"}*A*). Lysate from the quad mutant had a PON2 immunoreactive band with a smaller molecular mass than either N254Q or N323Q. This shows that, for PON2 Cys^311^, two of the four putative *N*-linked glycosylation sites undergo oligosaccharide addition: asparagine residues 254 and 323. ![**Effect of asparagine to glutamine site-specific mutations in predicted *N*-linked glycosylation sites of PON2.** CHO cells expressing wild-type (*WT*) and glutamine site-specific glycosylation mutants of PON2 Cys^311^ (*A*) and PON2 Ser^311^ (*B*) were lysed, electrophoresed, and analyzed by Western blot analysis for PON2 immunoreactive bands. PON2 Cys^311^ (*C*) and PON2 Ser^311^ (*D*) immunoblots following site-directed mutagenesis and PNGase F or Endo H treatment are shown.](zbc0021098990003){#F3} We next hypothesized that the upper band in PON2 Ser^311^ also represents glycosylation at both asparagine 254 and 323, whereas the lower electrophoretic mobility band could be glycosylation at either one of these asparagine residues or a mixture of individually glycosylated PON2 proteins. Alternatively, PON2 Ser^311^ could use different asparagine residues for core glycosylation. Similar to the PON2 Cys^311^, only asparagine 254 and 323 were glycosylated ([Fig. 3](#F3){ref-type="fig"}*B*). The immunoreactive band in both PON2 Cys^311^ and Ser^311^ quad mutants had a similar molecular mass compared with PON2 treated with PNGase F or Endo H ([Fig. 3](#F3){ref-type="fig"}, *C* and *D*). These substitution experiments allowed us to investigate which site is glycosylated in the lower band of PON2 Ser^311^. The N254Q mutant had an unglycosylated band as well as an intermediate glycosylated band, suggesting that the lower PON2 Ser^311^ band is glycosylated at asparagine 254. This is confirmed by the finding that the N323Q substitution results in a single intermediate band ([Fig. 3](#F3){ref-type="fig"}*B*). Substitution at asparagine 323 caused loss of the upper band, but the lower band remained in place. These data show that the upper band in PON2 Cys^311^ and Ser^311^ is glycosylated at asparagine 254 and 323, whereas the lower band in PON2 Ser^311^ is only glycosylated at asparagine 254. #### Glycosylation at Asparagine Residue 323 Is Required for PON2 Lactonase Function We hypothesized that differential glycosylation of PON2 Cys^311^ and Ser^311^ accounts for the differences in lactonase activity between the 311 variants, so we initially asked whether glycosylation is important for PON2 inactivation of 3OC12-HSL. Lysates from CHO cells transfected with the PON2 Cys^311^ or Ser^311^ with the N254G mutation had preserved 3OC12-HSL lactonase activity ([Fig. 4](#F4){ref-type="fig"}, *A* and *B*). We expected that if the lower electrophoretic band, only glycosylated at asparagine 254, in wild-type PON2 Ser^311^ accounts for its enhanced 3OC12-HSL inactivation, then glycosylation at asparagine 323 must inhibit PON2 lactonase activity. In contrast, we found that the N323Q mutant had impaired lactonase activity, showing that glycosylation at asparagine 323 is important for PON2 function. Similarly, lysates from CHO cells transfected with the PON2 Cys^311^ or Ser^311^ quad mutant (N226Q/N254Q/N269Q/N323Q) were unable to degrade 3OC12-HSL, also showing that glycosylation might be important for PON2 3OC12-HSL inactivation. ![**Effect of mutating *N*-linked glycosylation sites on lactonase activity.** *A* and *B*, PON2 Cys^311^ (*A*) or PON2 Ser^311^ (*B*) transfected CHO cell lysates from either wild-type (*WT*), N254G, N323G, or N226/254/269/323G (*Quad*) mutants were tested for 3OC12-HSL inactivating ability. Lysates (20% volume) of CHO cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL and after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of inactivation of 3OC12-HSL. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. \* denotes *p* \< 0.05 for lactonase activity between wild-type samples and N323G and quad mutant. *C*, CHO cells expressing wild-type and alanine site-specific glycosylation mutants of PON2 Cys^311^ and PON2 Ser^311^ were lysed, electrophoresed, and analyzed by Western blot analysis for PON2 immunoreactive bands. *D* and *E*, PON2 Cys^311^ (*D*) or PON2 Ser^311^ (*E*) transfected CHO cell lysates from either wild-type, N254A, N323A, or N254A/N323A mutants were tested for 3OC12-HSL inactivating ability. Lysates (20% volume) of CHO cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL; after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of inactivation of 3OC12-HSL. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. The data are the means ± S.E. \* denotes *p* \< 0.05 for lactonase activity between wild-type samples and N323A and double mutant.](zbc0021098990004){#F4} Mutation of asparagine residues to glutamine could have other effects on PON2 structure and activity besides disrupting *N*-linked glycosylation. Therefore, we constructed additional mutants of the PON2 Cys^311^ and Ser^311^ variants by replacing asparagine with alanine individually at positions 254 and 323, as well as a double mutant, N254A/N323A. Compared with the glutamine mutants, we found similar patterns of PON2 immunoreactive bands in the N254A, N323A, and N254A/N323A mutants ([Fig. 4](#F4){ref-type="fig"}*C*). In addition, the N323A and N254A/N323A mutants had an impaired ability to inactivate 3OC12-HSL ([Fig. 4](#F4){ref-type="fig"}, *D* and *E*). From these data we conclude that: 1) PON2 undergoes *N*-linked core glycosylation at asparagine residues 254 and 323; 2) the upper band in PON2 Ser^311^ is glycosylated at asparagine 254 and 323, whereas the lower band is only glycosylated at asparagine 254; 3) glycosylation at asparagine 323 is required for PON2 lactonase activity; and 4) differential glycosylation between the PON2 Cys^311^ and Ser^311^ variants does not account for greater lactonase activity by PON2 Ser^311^. #### PON2 Localizes to the Endoplasmic Reticulum Our data show that PON2 is Endo H-sensitive and does not undergo complex glycosylation,[^4^](#FN5){ref-type="fn"} suggesting that PON2 resides in the ER. Moreover, recent work by Horke *et al.* ([@B26]) demonstrated PON2 localization to the ER in endothelial cells. To test for changes in PON2 cellular localization as a mechanism of disrupted lactonase activity between PON2 Cys^311^ and Ser^311^ variants, immunohistochemical staining for PON2 was performed in CHO cells following transfection with either PON2 Cys^311^- or Ser^311^-expressing plasmids. A similar pattern of staining for both PON2 Cys^311^ and Ser^311^ was observed with the most intense staining corresponding to an ER location ([Fig. 5](#F5){ref-type="fig"}). ![**Immunohistochemical localization of PON2 Cys^311^ and Ser^311^ variants.** CHO cells were transiently transfected with a plasmid encoding human PON2 Cys^311^ or Ser^311^. 24 h later cells were fixed, stained with human anti-PON2 and an ER-specific antibody, and examined with confocal microscopy. *A*, PON2 Cys^311^-expressing CHO cells. *B*, PON2 Ser^311^-expressing CHO cells. *Scale bar*, 30 μm.](zbc0021098990005){#F5} #### Genotypic Analysis of Human Airway Donor Samples for PON2 S311C SNP To test whether the PON2 311 SNP also affects lactonase activity *in vivo*, we asked whether naturally occurring PON2 mutations at position 311 would affect the ability of primary human airway epithelial cells to degrade 3OC12-HSL. We first isolated DNA from 200 human donor lung samples and performed genotyping by polymerase chain reaction allele-specific oligonucleotide hybridization assays for the PON2 S311C polymorphism and a number of other common PON1, PON2, and PON3 allelic variants including coding (PON1 Q192R, PON1 L55M, PON3 C133A, and PON3 G99A) and promoter (PON1 G-907C) polymorphisms ([supplemental Table S1](http://www.jbc.org/cgi/content/full/M109.051706/DC1)). For PON2, we found that 62% of human donor samples were homozygous for Ser/Ser, 35% heterozygous for Ser/Cys, and 3% homozygous for Cys/Cys at amino acid 311 ([Fig. 6](#F6){ref-type="fig"}*A*). These findings are consistent with the HapMAP-CEU European SNP data base showing 57% of individuals homozygous for Ser/Ser, 38% heterozygous for Ser/Cys, and 5% homozygous for Cys/Cys. ![**Impaired airway lactonase activity in humans homozygous for cysteine at amino acid 311 in PON2.** *A*, percentage of human subjects with genotype of Cys/Cys, Cys/Ser, or Ser/Ser at amino acid position 311 in PON2. *B*, 3OC12-HSL degradation by human airway epithelia. *n* = 5 human donor samples for Ser/Ser, 2 for Ser/Cys, and 4 for Cys/Cys. Lysates (20% volume) of human airway epithelial cell cultures were incubated with 10 μ[m]{.smallcaps} 3OC12-HSL, and after 0, 30, or 60 min of incubation the lysate samples were obtained for measurement of 3OC12-HSL inactivation. The data are expressed as the percentages of active 3OC12-HSL remaining compared with initial levels. The data are the means ± S.E. \*, *p* \< 0.005 comparing lactonase activity between Cys/Cys and Ser/Ser donor epithelia.](zbc0021098990006){#F6} #### Human Airway Epithelial Cells from PON2 Cys/Cys^311^ Donors Have an Impaired Ability to Inactivate 3OC12-HSL To test whether differences in the common PON2 311 variant affect airway epithelia inactivation of 3OC12-HSL, human airway epithelial cells from four individuals homozygous for cysteine, five homozygous for serine, and two heterozygous for cysteine/serine at amino acid position 311 were grown at the air-liquid interface. We chose these donors based upon their PON2 311 SNP and similarities in other described SNPs for PON1 and PON3. Lysates from airway epithelial cells homozygous for serine degraded 3OC12-HSL with ∼25% active 3OC12-HSL remaining after 60 min of exposure ([Fig. 6](#F6){ref-type="fig"}*B*). In contrast, airway epithelial cell samples from donors homozygous for cysteine had an impaired ability to inactivate 3OC12-HSL, and samples from heterozygotes (Ser/Cys) demonstrated an intermediate phenotype for 3OC12-HSL inactivation compared with Ser/Ser and Cys/Cys samples ([Fig. 6](#F6){ref-type="fig"}*B*). These data show that the common PON2 311 polymorphism alters the endogenous ability of human airway epithelia to degrade 3OC12-HSL. DISCUSSION ========== The human PON2 S311C polymorphism is common and has been linked to several disorders including coronary artery disease, abnormal plasma lipoprotein levels, ischemic stroke, and Alzheimer dementia ([@B2], [@B7], [@B27][@B28][@B29][@B30]--[@B31]), but the mechanisms underlying these potential associations remain unknown. The serine allele is ancestral, has relatively little variation across human populations (Single Nucleotide Polymorphism database (dbSNP) data), and is strongly conserved in evolution. We hypothesized that the cysteine for serine substitution at amino acid position 311 in PON2 would disrupt PON2 lactonase activity. We found that the PON2 Cys^311^ variant had an impaired ability to inactivate 3OC12-HSL. More importantly, human airway epithelial cells from Cys/Cys homozygous individuals had an impaired ability to degrade 3OC12-HSL, compared with cells from Ser/Ser homozygous individuals. The impaired lactonase activity by Cys/Cys airway epithelia was not due to changes in promoter activity, alternative splicing, or linkage disequilibrium with other PON SNPs or genes because transfection of CHO cells with a plasmid expressing PON2 Cys^311^ also showed similar results as primary human airway epithelia. Linkage studies of monogenic diseases have resulted in the identification of genes responsible for the pathogenesis of several diseases including cystic fibrosis and Huntington disease. In contrast, association studies of complex genetic diseases often result in the identification of SNPs that may represent false positives or linkage to other genetic mutations. This becomes particularly troubling when the SNPs are found in proteins whose function are poorly understood, when the effect of the SNP on protein function is not known, and when there is no obvious physiologic connection between the gene and the pathogenesis of the studied disease. The identification of a physiologic consequence of a SNP becomes important in understanding association studies. A great example is the work of Richter and Furlong ([@B32]) in their studies determining the "PON1 status" of an individual. These investigators use a functional genomic approach and are able to directly measure, from plasma samples, the PON1 function of an individual based upon hydrolysis of both paraoxon and diazoxon. By using this method, any polymorphisms that might affect PON1 function are taken into account. In addition, hydrolysis of diazoxon quantifies the PON1 levels of an individual, allowing for comparisons in PON1 function to be determined. This assay allows for accurate identification of PON1 phenotype, predicts PON1 E192R status (Glu/Glu, Glu/Arg, or Arg/Arg), and can account for any other yet unidentified SNPs or those changes that may be secondary to a second SNP in linkage disequilibrium with the SNP of interest. This functional genomic approach was utilized by Jarvik *et al.* ([@B33]) to study the effects of PON1 polymorphisms on risk of vascular disease, an association that has been inconsistently linked. Examination of PON1 192 or 55 genotypes alone failed to predict the risk of vascular disease of the carotid arteries. However, when genotype status was combined with the rates of substrate analysis, as described above, vascular disease risk was predicted accurately, thus demonstrating the importance of including a functional analysis in association studies. A similar approach was used to test organophosphate hydrolysis by subjects with sporadic amyotrophic lateral sclerosis (ALS) ([@B34]). As with vascular disease, discrepant results regarding a linkage between PON1 polymorphisms and ALS exist. Wills *et al.* ([@B34]) reported an increased frequency of the PON1 Arg/Arg^192^ variant in ALS patients but similar levels of organophosphate hydrolysis by plasma samples from control and ALS patients. These findings were important because they suggested that if a linkage does exist between the PON1 192 SNP and ALS, etiologies other than organophosphate toxicity are responsible for the association, including disturbances in metabolism of other yet unidentified toxic substances or an effect secondary to linkage disequilibrium with either additional PON variants or other proteins. Application of this functional genetic analysis for PON2 is more difficult because PON2 is primarily an intracellular enzyme, as opposed to PON1, which is found in the serum, and no simple assay exists to detect a functional consequence of the PON2 311 SNP in human samples. Furthermore, the natural substrate and *in vivo* physiological function of PON2 are not yet fully elucidated. However, by performing genetic analysis of donor human lung samples and testing 3OC12-HSL degradation by human airway epithelia, we were able to show that the Cys allele at PON2 311 impairs lactonase activity. The *in vivo* physiological consequence of this effect remains to be determined, but our previous data showing that PON2 deficiency in airway epithelial cells impairs the ability of these cells to inactivate 3OC12-HSL suggest that despite the intracellular location of PON2, it may be important in the host response to quorum sensing-dependent bacteria. Differing molecular masses of PON2 immunoreactive bands between the PON2 311 alloenzymes were observed, so we investigated the *N*-linked glycosylation pattern of PON2. In our study, tunicamycin, Endo H, and PNGase F treatment of PON2 Cys^311^ and Ser^311^ showed that PON2 undergoes *N*-linked core oligosaccharide addition and that following deglycosylation PON2 Cys^311^ and Ser^311^ immunoreactive bands appeared similar in molecular mass. Using directed evolution, Harel *et al.* ([@B35]) reported a crystal structure of PON1 and suggested that of the four corresponding PON1 asparagine residues, 254 and 323 are most likely core glycosylated because of their location on surface loops. Asparagine 226 and 269 are in the central tunnel of the β-propeller and are predicted not to be glycosylated. These predictions are in agreement with our findings. We found that PON2 Cys^311^ has a single molecular mass protein glycosylated at asparagine residues 254 and 323, whereas PON2 Ser^311^ had one isoform identical to PON2 Cys^311^ (glycosylated at asparagine 254 and 323) and another with a smaller molecular mass only glycosylated at 323. Based upon our findings and others ([@B15], [@B36], [@B37]), it appears that differential glycan processing occurs between PON family members and that differential *N*-linked glycosylation occurs between the PON2 311 alloenzymes. Furthermore, even though we showed that glycosylation is important for PON2 3OC12-HSL degradation, it does not explain the differences seen in PON2 Cys^311^ lactonase activity. Although we hypothesized that glycosylation at asparagine 323 would inhibit PON2 activity (based upon the differential glycosylation pattern and lactonase activity between PON2 Cys^311^ and Ser), this was not the case. Several possible explanations exist for both differential glycosylation and lactonase activity between PON2 Cys^311^ and Ser^311^. First, although unlikely, the Cys^311^ mutation may affect PON2 function, which in turn affects the glycosylation pathway of other proteins. Second, changes in glycosylation and PON2 function may be completely unrelated. Finally, whereas differential glycosylation may not account for the impaired ability of the PON2 Cys^311^ airway epithelia to degrade 3OC12-HSL, the two findings might be related. We hypothesize that the most likely underlying mechanism common to both findings is due to differences in disulfide bond formation. This might result from changes in which cysteine residues undergo disulfide bond formation, changing the number of disulfide bonds formed, or changes in which intermolecular disulfide bonds between cysteine residues of different PON2 molecules occur. For example, the free cysteine residue at position 284 in PON1 is required for its lactonase activity ([@B38]), and disruption of PON1 disulfide-linked Cys residues 41 or 352 impaired catalytic activity ([@B39]). Similarly, we found that treatment of both PON2 Cys^311^ and Ser^311^ with the reducing agent dithiothreitol (10 m[m]{.smallcaps}) impaired lactonase activity (data not shown). These data also suggest that disulfide bonds are important for PON2 activity. Alternatively, an altered protein structure or differences in substrate binding to the PON2 active site might account for the phenotype of PON2 Cys^311^ mutants. Therefore, it appears that the altered glycosylation profile observed between the PON2 311 alloenzymes is a marker of altered lactonase activity but is not the mechanism for this difference. In summary, we found that human airway epithelia homozygous for PON2 Cys^311^ have an impaired ability to inactivate 3OC12-HSL. There have been few successes to date in identifying the specific etiologic variant influencing a common disease when an association has been confirmed ([@B40]), mostly because of inadequate functional assay testing. This is the first time for PON2 that a functional consequence for the 311 SNP has been demonstrated in humans. Moreover, these findings suggest three important speculations. First, infections with quorum sensing-dependent bacteria should have a differential phenotype in patients with the PON2 Cys^311^ variant. Second, diseases that associate genetically with this PON2 311 SNP may have an infectious etiology. Third, some of these diseases may be caused by an as yet unidentified metabolic pathway that requires lactonase activity. This work was supported, in whole or in part, by National Institutes of Health Grants DK54759, AI076671, HL61234-09. This work was also supported by the Cystic Fibrosis Foundation. The on-line version of this article (available at <http://www.jbc.org>) contains [supplemental Table S1](http://www.jbc.org/cgi/content/full/M109.051706/DC1). D. A. Stoltz, T. J. Recker, and J. Zabner, unpublished data. The abbreviations used are: PONparaoxonaseSNPsingle-nucleotide polymorphism3OC12-HSL*N*-3-oxododecanoyl homoserine lactonePNGase Fpeptide-*N*-glycosidase FEndo Hendoglycosidase HALSamyotrophic lateral sclerosisCHOChinese hamster ovaryERendoplasmic reticulum. We thank Nick Gansemer, Debbie Hunter, Phil Karp, Peter Kiss, Jan Launspach, Thomas Moninger, Peter Taft, and Geri Traver for excellent assistance and Drs. Jeff Murray and Michael Welsh for insightful discussions. [^1]: Supported by a Parker B. Francis Fellowship.

{ "pile_set_name": "PubMed Central" }

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22,206

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"differential", "glycosylation", "may", "account", "impaired", "ability", "airway", "epithelia", "degrade", "two", "findings", "might", "related", "hypothesize", "likely", "underlying", "mechanism", "common", "findings", "due", "differences", "disulfide", "bond", "formation", "might", "result", "changes", "cysteine", "residues", "undergo", "disulfide", "bond", "formation", "changing", "number", "disulfide", "bonds", "formed", "changes", "intermolecular", "disulfide", "bonds", "cysteine", "residues", "different", "molecules", "occur", "example", "free", "cysteine", "residue", "position", "required", "lactonase", "activity", "disruption", "Cys", "residues", "impaired", "catalytic", "activity", "Similarly", "found", "treatment", "reducing", "agent", "dithiothreitol", "impaired", "lactonase", "activity", "data", "shown", "data", "also", "suggest", "disulfide", "bonds", "important", "activity", "Alternatively", "altered", "protein", "structure", "differences", "substrate", "binding", "active", "site", "might", "account", "phenotype", "mutants", "Therefore", "appears", "altered", "glycosylation", "profile", "observed", "alloenzymes", "marker", "altered", "lactonase", "activity", "mechanism", "difference", "summary", "found", "human", "airway", "epithelia", "homozygous", "impaired", "ability", "inactivate", "successes", "date", "identifying", "specific", "etiologic", "variant", "influencing", "common", "disease", "association", "confirmed", "mostly", "inadequate", "functional", "assay", "testing", "first", "time", "functional", "consequence", "SNP", "demonstrated", "humans", "Moreover", "findings", "suggest", "three", "important", "speculations", "First", "infections", "quorum", "bacteria", "differential", "phenotype", "patients", "variant", "Second", "diseases", "associate", "genetically", "SNP", "may", "infectious", "etiology", "Third", "diseases", "may", "caused", "yet", "unidentified", "metabolic", "pathway", "requires", "lactonase", "activity", "work", "supported", "whole", "part", "National", "Institutes", "Health", "Grants", "work", "also", "supported", "Cystic", "Fibrosis", "Foundation", "version", "article", "available", "http", "contains", "supplemental", "Table", "http", "Stoltz", "Recker", "Zabner", "unpublished", "data", "abbreviations", "used", "N", "homoserine", "lactonePNGase", "N", "FEndo", "Hendoglycosidase", "HALSamyotrophic", "lateral", "sclerosisCHOChinese", "hamster", "ovaryERendoplasmic", "reticulum", "thank", "Nick", "Gansemer", "Debbie", "Hunter", "Phil", "Karp", "Peter", "Kiss", "Jan", "Launspach", "Thomas", "Moninger", "Peter", "Taft", "Geri", "Traver", "excellent", "assistance", "Drs", "Jeff", "Murray", "Michael", "Welsh", "insightful", "discussions", "Supported", "Parker", "Francis", "Fellowship" ]

Introduction ============ Although there is a relatively large body of knowledge assessing postoperative physical therapy (post-op PT),[@b1-jpr-9-493]--[@b6-jpr-9-493] there is limited literature investigating preoperative physical therapy (pre-op PT) on pain intensity and disability after musculoskeletal surgery. The main goal of pre-op PT treatment is to reduce postoperative complications and improve postoperative outcomes.[@b7-jpr-9-493] However, previous literature shows that isolating the real impact of a preoperative rehabilitation is not easy, mainly because of the heterogeneity in the type of the physical therapy (PT) intervention.[@b8-jpr-9-493] In cases, such as cardiac, thoracic, and abdominal surgeries, the main goal of pre-op PT is to improve the physiological capacity of the individual. In this case, it seems that strengthening the inspiratory muscle prior to surgery may result in fewer postoperative complications and shorter postoperative length of stay, enhance functional capacity,[@b9-jpr-9-493],[@b10-jpr-9-493] improve the quality of life, and reduce postoperative pain and complications.[@b11-jpr-9-493] The reported effects of pre-op PT intervention on postoperative outcomes in patients having elective musculoskeletal surgeries are conflicting. A systematic review including functional measures as outcome measures concluded that preoperative exercise programs were not effective on postoperative outcomes on total knee or total hip arthroplasty.[@b12-jpr-9-493] On the other hand, preoperative level of function is predictive of postoperative outcome after lower limb arthroplasty, where patients with better function preoperatively had better function postoperatively.[@b13-jpr-9-493] Moreover, the authors[@b8-jpr-9-493],[@b14-jpr-9-493] have recommended a multidisciplinary preoperative rehabilitation program before total hip and knee arthroplasty to reduce hospital length of stay and modify discharge conditions. Reported evidence on the effects of pre-op PT intervention on general postoperative outcomes for musculoskeletal pain is inconclusive,[@b15-jpr-9-493] and few studies have investigated this issue for shoulder arthroscopy. Arthroscopy of the shoulder is a common musculoskeletal surgery and has become widely accepted as a diagnostic and therapeutic tool in the management of shoulder disorders[@b1-jpr-9-493],[@b16-jpr-9-493],[@b17-jpr-9-493]; however, data describing postoperative outcomes following pre-op PT interventions are scarce. This topic is important because whether pre-op PT intervention influences postoperative outcomes may assist in decision making for optimal timing of rehabilitation in the operative management of shoulder pain. Therefore, the purposes of this prospective study were to 1) identify differences in demographic, clinical, surgical type, and psychological characteristics between patients having and not having pre-op PT treatment and 2) determine whether having pre-op PT influences the length of post-op PT treatment (number of sessions) and 3-month and 6-month postsurgical outcomes (pain intensity and disability). We hypothesized that 1) there would be a difference between patients having and not having pre-op PT in demographic, clinical, surgical types and psychological characteristics and 2) having pre-op PT would favorably influence the length of post-op PT treatment and 3-month and 6-month postsurgical outcomes. Patients and methods ==================== Patients -------- The University of Florida's Institutional Review Board for human participants approved this study. This prospective design includes data from consecutive patients seeking operative treatment of shoulder pain, where procedures were limited to arthroscopy of the glenohumeral joint or distal clavicle. Specific operative procedures included rotator cuff repair, adhesive capsulitis, acromioplasty, and labral repair ([Table 1](#t1-jpr-9-493){ref-type="table"}). Patients were recruited by participating physicians and provided written informed consent before participating in this study. ### Inclusion criteria The inclusion criteria for being a participant were as follows: 1) aged between 18 years and 85 years; 2) complained of pain limited to anterior, lateral, or posterior shoulder; 3) documented or suspected rotator cuff tendinopathy (evidence from clinical examination or imaging studies), including small (\<1 cm), medium (1--3 cm), and large (3--5 cm) tears; 4) documented or suspected adhesive capsulitis (evidence from clinical examination or imaging studies); 5) documented or suspected superior labrum from anterior to posterior lesion (evidence from clinical examination or imaging studies); 6) shoulder instability (anterior or posterior labral lesions); and 7) scheduled for arthroscopic surgery. ### Exclusion criteria The exclusion criteria were as follows: 1) current complaints of pain lasting longer than the past 3 months involving neck, elbow, hand, low back, hip, knee, or ankle; 2) massive rotator cuff tear (\>5 cm); 3) documented shoulder osteoarthritis or rheumatoid arthritis; 4) prior shoulder surgery within the past year or currently complaining of pain from prior shoulder surgery; 5) current shoulder fracture, tumor, or infection; 6) previously diagnosed chronic pain disorder (including, but not limited to, irritable bowel syndrome, fibromyalgia, temporomandibular disorders, and chronic low back pain); 7) current psychiatric management; and 8) current gastrointestinal or renal illness.[@b18-jpr-9-493] Overall procedure ----------------- After signing the informed consent, study participants completed a standard intake information form. Patients underwent baseline assessments, which included psychological questionnaires, and self-reported pain intensity and disability 24--48 hours before shoulder surgery. Patients underwent an arthroscopic procedure affecting the glenohumeral joint and/or distal clavicle. Specific operative procedures included acromioplasty, subacromial bursectomy, labral repair, rotator cuff repair, and others (which included, but was not limited to, capsulorrhaphy, coracoacromial ligament release, stabilization, and Bankart). In addition, the size of the rotator cuff tear was recorded from the operative report, ranging from "no tear" to "massive tear". Four different surgeons operated on all the patients in this cohort. After 3 months and 6 months from the surgical procedure, patients were reassessed on all measures. Measures -------- ### Demographic and historical information Study participants completed a standard intake information form. Demographic data collected at initial evaluation included sex, age, employment status, litigation status, marital status, educational level, and health history. Historical data included the type of onset of symptoms, the length of time of the symptoms, the number of previous episodes of musculoskeletal pain, and previous PT treatment for their shoulder. ### Pre-op PT and post-op PT measurements Study participants were asked if they had had previous PT treatments (under the supervision of a licensed physical therapist) for their shoulder condition in the last 6 months. Participants who attended presurgical treatment were classified as "having pre-op PT treatment", and those who did not attend were classified as "not having pre-op PT treatment". Since the main goal of this study was to pragmatically determine whether having or not having pre-op PT intervention would influence postsurgical outcome, the frequency, intensity, and type of the pre-op PT were not tracked. At 3 months and 6 months, patients were asked for the total number of sessions (and weeks) of post-op PT treatment that they had after shoulder surgery. ### Shoulder pain intensity Shoulder pain intensity was assessed with the Brief Pain Inventory (BPI),[@b19-jpr-9-493] which includes a numerical rating scale for pain intensity. Patients rated their pain intensities over the following three conditions: the present pain intensity, the worst pain intensity over the past 24 hours, and the least pain intensity over the past 24 hours. These three ratings were summed and divided by three for use in data analyses.[@b20-jpr-9-493] Studies have shown that this aggregate measure has sufficient psychometric strengths.[@b20-jpr-9-493],[@b21-jpr-9-493] ### Shoulder disability Disability was assessed with the Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire.[@b22-jpr-9-493] The DASH questionnaire includes 30 items to measure the extent to which patients' pain or limited activity affects their ability to perform certain functions, to sleep, and to carry on routine daily activities and social activities. The DASH questionnaire has been validated for the assessment of shoulder disorders.[@b23-jpr-9-493]--[@b25-jpr-9-493] ### Psychological factors A previous study from our group has shown that pain catastrophizing and depressive symptoms were relevant psychological factors in predicting the postoperative clinical pain intensity in a population with similar characteristics.[@b18-jpr-9-493] Therefore, for our current study, we selected the same psychological factors to explore if pre-op PT has an influence on psychological factors. #### Depressive symptoms Self-reports of depressive symptoms were measured using the patient health questionnaire (PHQ-9).[@b26-jpr-9-493] The PHQ-9 is a 9-item self-report questionnaire designed to evaluate the presence of depressive symptoms during the prior 2 weeks. As a severity measure, scores can range from 0 (absence of depressive symptoms) to 27 (severe depressive symptoms). Each of the nine items, asking for each of the *Diagnostic and Statistical Manual of Mental Disorders*, Fourth Edition (DSM-IV) diagnostic criteria, can be scored from 0 (not at all) to 3 (nearly every day). As a diagnostic measure, major depression is diagnosed if five or more of the nine depressive symptom criteria have been present at least "more than half the days" (a score of 2) in the past 2 weeks, and one of the symptoms is depressed mood or anhedonia. A previous study supports the PHQ-9's validity, feasibility, and capacity to detect changes in depressive symptoms over time.[@b27-jpr-9-493] #### Pain catastrophizing Pain catastrophizing was measured by the pain catastrophizing scale (PCS).[@b28-jpr-9-493] The PCS has 13 descriptions of pain experience assessing catastrophic cognitions, for example, "I feel I can't go on", and "There's nothing I can do to reduce the intensity of the pain". Patients were asked to indicate whether they agreed with these statements by using a five-point rating scale (0, "not at all" to 4, "all the time") to rate the frequency of these cognitions. A PCS sum score was calculated for all items (range, 0--52), with a high score indicating a high level of pain catastrophizing. Data analysis ------------- Data analysis was conducted with SPSS, Version 18.0. Significance levels were set a priori at *P*\<0.05 for all comparisons. Descriptive statistics (mean and standard deviation) were calculated for all variables for baseline, 3 months, and 6 months between groups (patients who had pre-op PT treatment and those who did not). The proportion of patients and the sex distribution between each group were determined. The distributions of variables were tested for normality by visual examination and with Kolmogorov--Smirnov test before conducting analyses. Analysis of variance (ANOVA) models and the Mann--Whitney *U* test were performed to identify differences across all measures for patients who had pre-op PT treatment and those who did not. Multivariate ANOVA models were performed to examine outcome differences at 3 months and 6 months (pain intensity and disability) and the length of post-op PT (number of PT sessions after shoulder surgery) on patient characteristics observed to be significantly different between groups. Results ======= This study included 124 consecutive patients having arthroscopic surgery with a completed 6-month follow-up. Descriptive statistics for the demographic and psychological characteristics, clinical pain, and medical history between patients who had pre-op PT treatment and those who did not is summarized in [Table 1](#t1-jpr-9-493){ref-type="table"}. All continuously dependent variables were found to approximate a normal distribution by visual examination and were deemed appropriate for our planned analyses and ANOVAs. Differences between preoperative groups --------------------------------------- No significant baseline differences (*P*\>0.05) in age, pain intensity, disability, and psychological factors between patients having and not having pre-op PT treatment were observed ([Table 1](#t1-jpr-9-493){ref-type="table"}). However, sex significantly differed (*P*=0.01) between patients having and not having pre-op PT treatment. Proportionally, more men were in the not having pre-op PT treatment group (59%), and more women were in the having pre-op PT treatment group (65%). Size of rotator cuff tear (*P*=0.56) had no significant difference between patients having and not having pre-op PT treatment. Differences in surgical type were further explored between patients having and not having pre-op PT treatment. Significantly, more subacromial bursectomies were performed (37.7%) in patients having pre-op PT treatment compared to patients not having pre-op PT treatment (20.6%) ([Table 1](#t1-jpr-9-493){ref-type="table"}). Influence of pre-op PT on outcomes ---------------------------------- There was no significant difference in the length of post-op PT treatment at 3 months (*F*\[1,96\]=0.37; *P*=0.54) and at 6 months (*F*\[1,81\]=0.26; *P*=0.61) between patients having and not having pre-op PT treatment. Repeated measures of ANOVA examined changes in clinical pain intensity and disability between patients having and not having pre-op PT treatment. Results showed no time × group interaction for clinical pain intensity (*F*\[2,180\]=0.33; *P*=0.72); as expected, there was a significant main effect of clinical pain over time (*F*\[2,180\]=64.41; *P*\<0.001), where pain significantly decreased from baseline (mean =3.33; SD =2.33) to 3 months (mean =1.52; SD =1.61) and from 3 months to 6 months (mean =1.17; SD =1.45) ([Figure 1](#f1-jpr-9-493){ref-type="fig"}). In addition, no time × group interaction for disability was found (*F*\[2,192\] =0.69; *P*=0.49); as expected, there was a significant main effect of disability over time (*F*\[2, 192\]=73.16; *P*\<0.001), where disability significantly decreased from baseline (mean =34.18; SD =17.08) to 3 months (mean =24.67; SD =14.51) and from 3 months to 6 months (mean =13.75; SD =10.39) ([Figure 2](#f2-jpr-9-493){ref-type="fig"}). Exploratory analysis -------------------- Since significant differences at baseline were found for the distribution of sex and subacromial bursectomy between patients having and not having pre-op PT treatment, an exploratory analysis of these results was performed. Differences in demographic, clinical pain, medical history, and psychological characteristics between sex and subacromial bursectomy are summarized in [Table 2](#t2-jpr-9-493){ref-type="table"}. Significant differences were found in baseline clinical pain intensity (BPI) (*t*\[118\]=2.57; *P*=0.01) and baseline disability (DASH) (*t*\[122\]=4.05; *P*\<0.01) between males and females, where females had higher mean in both clinical outcome measures (BPI mean =4.09; DASH mean =42.97) compared to males (BPI mean =2.97; DASH mean =30.05) ([Table 2](#t2-jpr-9-493){ref-type="table"}). Patients who had bursectomy were significantly older (*t*\[122)=--3.37; *P*\<0.01) and predominantly females (41.8% for females vs 22% for males). Repeated measures of ANOVA were used to assess whether pre-op PT treatment differences in sex or subacromial bursectomy affected postsurgical outcomes. No significant interaction was found between pre-op PT treatment, sex, and pain intensity (baseline, 3 months, and 6 months) (*F*\[2,176\]=0.06; *P*=0.93) or between pre-op PT treatment, sex, and disability (*F*\[2,188\]=1.38; *P*=0.26), suggesting that the potential effect of pre-op PT treatment on the length of post-op PT and clinical outcomes did not differ between males and females. The same trend of results was found between pre-op PT treatment, bursectomy, and pain intensity or disability, suggesting that differences in surgical intervention do not influence 3-month or 6-month postsurgical outcomes. Discussion ========== The main goal of pre-op PT treatment is to reduce postoperative complications and improve postoperative outcomes.[@b8-jpr-9-493] This study identified differences in demographic, clinical, and psychological characteristics between patients having and not having pre-op PT treatment and determined that pre-op PT treatment did not influence postoperative outcomes. The only pre-op PT treatment differences observed in this cohort were that males had less participation in pre-op PT treatment than females, and significantly more subacromial bursectomies were performed in patients having pre-op PT treatment. The results of this study also indicated that pre-op PT treatment did not influence the length of post-op PT treatment or postoperative pain and disability outcomes. Our study is a new addition to the small body of literature investigating this topic in elective arthroscopic shoulder surgery and extends the work from different surgical cohorts.[@b29-jpr-9-493]--[@b31-jpr-9-493] In addition, the larger sample size and prospective design are other strengths of this study. The results from several studies have showed the positive effects of pre-op PT treatment on postsurgical outcomes in patients undergoing cardiac surgery and in patients after lung volume reduction surgery.[@b32-jpr-9-493],[@b33-jpr-9-493] However, the results from studies looking at the effect of pre-op PT on postsurgical outcomes after musculoskeletal or joint replacement surgery have been contradictory.[@b8-jpr-9-493] For example, Beaupre et al[@b34-jpr-9-493] showed no differences in knee measurements (range of motion \[ROM\] and strength), pain, function, or quality of life between patients having a preoperative exercise/education program and patients having a total knee arthroplasty. Rooks et al[@b35-jpr-9-493] evaluated the effect of a short preoperative exercise intervention on the functional status, pain, and muscle strength of patients before and after total joint arthroplasty. They found that hip and knee arthroplasty patients responded differently in the preoperative and immediate postoperative periods. D'Lima et al[@b30-jpr-9-493] compared the effects of pre-op PT of general cardiovascular conditioning exercises with the routine procedure of no pre-op PT in patients undergoing primary total knee replacement. All three groups showed significant improvement postoperatively; however, neither type of preoperative exercise added to the degree of improvement after surgery. In contrast, Crowe and Henderson[@b36-jpr-9-493] showed the effectiveness in reducing the length of stay after an individually tailored rehabilitation program for patients undergoing hip or knee arthroplasty. Our results add to this literature by suggesting that in a cohort of patients undergoing shoulder arthroscopy, there appears to be no benefit of pre-op PT treatment on postsurgical pain and disability outcomes. Overall, this finding is in-line with previous studies,[@b29-jpr-9-493]--[@b31-jpr-9-493] indicating that a beneficial effect of pre-op PT treatment is more likely to occur after major surgeries.[@b14-jpr-9-493] One potential explanation for the differences in the benefits of pre-op PT is that the recovery period after invasive and major surgeries involves bed rest and sometimes long periods of physical inactivity, which induces deconditioning and postoperative complications. Therefore, it is reasonable to assume that increasing a patient's physical activity and physiological parameters prior to a surgical intervention will increase the patient's functional capacity with an increase in quality of life. Previous studies have shown that poor fitness before the surgery results in a significantly longer stay in hospital after the surgery[@b37-jpr-9-493],[@b38-jpr-9-493] and greater surgical complications.[@b8-jpr-9-493],[@b37-jpr-9-493] However, the "standard of care" pre-op PT intervention for shoulder conditions would not target the increment in physiological parameters or fitness improvement; therefore, the chance of overall fitness improvement is low. Even though an ambulatory surgery such as a shoulder arthroscopy may not involve bed rest or long periods of inactivity, there is often a period of immobility considered as a "relative rest" that could eliminate any potential benefits from pre-op PT. Looking at sex differences, Charousset et al[@b39-jpr-9-493] described that female sex was negatively associated with the improvement of clinical results. Other studies[@b40-jpr-9-493],[@b41-jpr-9-493] have reported that sex is not associated with postoperative outcomes. Our results are consistent with earlier findings, because even though males had less participation in pre-op PT treatment than females, baseline sex differences did not influence the length of post-op PT treatment, pain intensity, and disability outcomes. Psychological factors are believed to play a role in the patient's response to treatment and final outcome.[@b42-jpr-9-493]--[@b44-jpr-9-493] However, there is little evidence in the literature assessing the potential influence of psychological characteristics on seeking PT care. Our data show that depression and pain catastrophizing did not differ at baseline between patients having or not having pre-op PT treatment. This is an interesting finding because we would expect that patients with greater psychological distress would be more likely to utilize pre-op PT; however, this was not the case for this cohort. Therefore, this is preliminary evidence that pre-op PT is not associated with common measures of psychological distress that have been associated with an increased health care utilization for musculoskeletal pain. A previous study[@b45-jpr-9-493] has shown that after a conservative treatment for subacromial impingement syndrome has failed, there is a greater rate of successful outcome with a specific exercise protocol (strengthening eccentric exercises for the rotator cuff and concentric/eccentric exercises for the scapula stabilizers in combination with manual mobilization) compared to a control exercise group. In addition, a significantly lower proportion of patients in the specific exercise group subsequently chose to undergo surgery. Lee et al[@b5-jpr-9-493] show that pain, range of motion, muscle strength, and function significantly improved after arthroscopic rotator cuff repair, regardless of early postoperative rehabilitation protocols. The goal of this study was to evaluate the broad influence of having pre-op PT on postoperative outcomes and not to test the effectiveness of any specific exercise approaches used during PT. These results still advance the field by showing that pain intensity and disability significantly potentially decrease after shoulder arthroscopy, regardless of having or not having preoperative rehabilitation. Future research using more highly controlled designs is needed, however, to determine if specific exercise approaches have measurable impact on postoperative outcomes. There are some limitations to this study that should be recognized. First, the operational definition of PT treatment was pragmatically determined, so it does not include treatment dosage or type, so we cannot account for the intensity, frequency, or nature of the PT delivered in this study. This is a relevant issue because the nature of the preoperative intervention may be an important consideration, so that future prospective studies can better measure the application of pre-op PT. Furthermore and consistent with the pragmatic nature of the analysis, there was no control over how the pre-op PT was selected or administered, and there could be other relevant factors in the patient decision making (eg, protocol selection, access to care, socioeconomic status, dosage, and content of treatment) that were not included in our analysis. Second, by recruiting patients who are having surgery, we may be missing patients who already succeeded with conservative treatment approaches; therefore, our sample may be less likely to have had a positive response to PT treatment in general. A more comprehensive future study including a wider range of patients to determine whether pre-op PT is preventative of future surgery is needed, as this study was not designed to answer that particular question. Despite these limitations, this study represents a new contribution to the literature by investigating the influence of pre-op PT treatment on 3-month and 6-month postsurgical outcomes. In summary, having pre-op PT was not associated with the length of post-op PT treatment or postsurgical pain and disability. Conclusion ========== This prospective cohort study provides no evidence of benefit for pre-op PT on post-op PT treatment or postsurgical outcomes. Receiving pre-op PT treatment did not influence the length of post-op PT treatment or pain and disability outcomes at 3 months and 6 months. Females or patients receiving certain surgical procedures are more likely to undergo pre-op PT treatment. However, these differences did not influence postsurgical outcomes in this cohort. The authors thank Warren Greenfield III for his assistance with clinical participant screening and recruitment. Doctor Deenesh T Sahajpal provided patients from his clinic. The authors also wish to thank Lindsay Kindler for assistance with testing. This study was supported by Grant \#AR055899 from NIAMS/NIH. **Disclosure** The authors report no conflicts of interest in this work. ![Clinical pain over time between patients having pre-op PT treatment and patients not having pre-op PT treatment.\ **Abbreviations:** pre-op PT, preoperative physical therapy; NRS, numerical rating scale.](jpr-9-493Fig1){#f1-jpr-9-493} ![Disability over time between patients having pre-op PT treatment and patients not having pre-op PT treatment.\ **Abbreviations:** pre-op PT, preoperative physical therapy; DASH, Disabilities of the Arm.](jpr-9-493Fig2){#f2-jpr-9-493} ###### Demographic and psychological characteristics and summary of medical history between patients having and not having pre-op PT treatment Sample characteristics Having pre-op PT (n=61), mean (SD) Not having pre-op PT (n=63), mean (SD) *P*-value ------------------------------------------- ------------------------------------ ---------------------------------------- ----------- Age (years) 44.79 (16.28) 44.32 (19.11) 0.88 Sex Female 28 (45.9%) 15 (23.8%) 0.01 Male 33 (54.1%) 48 (76.2%) Dominant side Right 54 (88.5%) 55 (87.3%) 0.84 Left 7 (11.5%) 8 (12.7%) Ethnicity Hispanic or Latino 5 (8.2%) 5 (7.9%) 0.98 Non-Hispanic or Latino 54 (88.5%) 56 (88.9%) Unknown or not reported 2 (3.3%) 2 (3.2%) Race Asian 0 (0%) 0 (0%) 0.78 Native Hawaiian or others 0 (0%) 1 (1.6%) Black or African 4 (6.6%) 3 (4.8%) American White 52 (85.2%) 55 (87.3%) More than one race 3 (4.9%) 3 (4.8%) Unknown or not reported 2 (3.3%) 1 (1.6%) Surgery side Right 35 (57.4%) 32 (50.8%) 0.46 Left 26 (42.6%) 31 (49.2%) Pain duration (weeks) 68.75 (90.81) 68.14 (103.35) 0.42 BPI baseline 3.47 (2.43) 3.24 (2.23) 0.59 BPI 3 months 1.58 (1.71) 1.45 (1.36) 0.67 BPI 6 months 1.24 (1.31) 1.24 (1.47) 0.99 DASH baseline 35.58 (17.15) 33.51 (18.75) 0.52 DASH 3 months 26.48 (14.34) 22.52 (14.39) 0.15 DASH 6 months 16.04 (11.95) 12.94 (10.76) 0.17 PHQ-9 baseline 4.37 (4.19) 3.34 (4.12) 0.18 PHQ-9 3 months 3.67 (3.89) 2.68 (3.66) 0.19 PHQ-9 6 months 3.21 (4.06) 2.58 (3.07) 0.36 PCS baseline 11.23 (8.57) 10.22 (8.41) 0.51 PCS 3 months 7.53 (7.89) 7.75 (8.28) 0.89 PCS 6 months 6.47 (6.83) 6.82 (7.19) 0.80 Size of rotator cuff tear No tear 32 (52.5%) 30 (47.6%) 0.56 Partial tear \<50% 10 (16.4%) 11 (17.5%) Partial tear \>50% 4 (6.6%) 2 (3.2%) Small 5 (8.3%) 3 (4.8%) Medium 2 (3.3%) 7 (11.1%) Large 2 (3.3%) 4 (6.3%) Massive 6 (9.8%) 5 (7.9%) Unknown 0 1 (1.6%) Acromioplasty 47 (37.9%) 24 (39.3%) 23 (36.5%) 0.75 Subacromial bursectomy 36 (29.0%) 23 (37.7%) 13 (20.6%) 0.03 Labral repair 49 (39.5%) 24 (39.3%) 25 (39.7%) 0.97 Other operative procedures 86 (69.4%) 41 (67.2%) 45 (71.4%) 0.61 Post-op PT sessions at 3 months 18.82 (9.82) 20.92 (22.04) 0.54 Post-op PT sessions at 6 months 7.67 (9.85) 6.56 (9.84) 0.61 Weeks in post-op PT treatment at 3 months 11.18 (2.59) 9.19 (4.26) \<0.01 Weeks in post-op PT treatment at 6 months 4.64 (6.18) 4.77 (6.39) 0.93 **Abbreviations:** BPI, brief pain inventory; DASH, Disabilities of the Arm, Shoulder, and Hand; PCS, pain catastrophizing scale; PHQ-9, patient health questionnaire; post-op PT, postoperative physical therapy; pre-op PT, preoperative physical therapy; SD, standard deviation. ###### Exploratory analyses -- differences in demographic and psychological characteristics between sex and subacromial bursectomy Sample characteristics Females (n=61), mean (SD) Males (n=63), mean (SD) *P*-value Bursectomy (n=36), mean (SD) Not having bursectomy (n=88), mean (SD) *P*-value --------------------------------- --------------------------- ------------------------- ----------- ------------------------------ ----------------------------------------- ----------- Age (years) 48.11 (15.44) 41.71 (18.82) 0.04 52.61 (13.58) 41.25 (18.19) 0.001 Sex Female -- -- -- 18 (50.0%) 25 (28.4%) 0.02 Male 18 (50.0%) 63 (71.6%) BPI baseline 4.14 (2.30) 2.89 (2.23) 0.004 3.67 (2.27) 3.22 (2.35) 0.34 BPI 3 months 1.54 (1.41) 1.51 (1.58) 0.93 1.56 (1.38) 1.50 (1.60) 0.84 BPI 6 months 1.41 (1.66) 1.09 (1.33) 0.33 1.13 (1.39) 1.29 (1.51) 0.62 DASH baseline 42.97 (15.95) 30.32 (17.42) \<0.01 34.91 (16.55) 34.37 (18.56) 0.88 DASH 3 months 28.35 (14.03) 22.07 (14.14) 0.03 23.97 (14.62) 24.64 (14.46) 0.83 DASH 6 months 17.05 (14.26) 12.40 (9.45) 0.08 12.95 (9.57) 15.21 (12.14) 0.38 PHQ-9 baseline 4.16 (4.68) 3.83 (4.65) 0.71 3.86 (4.66) 3.83 (3.98) 0.97 PHQ-9 3 months 2.47 (2.96) 3.46 (4.06) 0.20 3.27 (4.07) 3.12 (3.71) 0.85 PHQ-9 6 months 2.86 (4.31) 3.04 (3.65) 0.82 3.33 (4.27) 2.70 (3.32) 0.43 PCS baseline 10.91 (8.91) 10.72 (8.29) 0.38 10.27 (7.64) 10.90 (8.82) 0.71 PCS 3 months 6.72 (7.85) 8.16 (8.22) 0.61 7.83 (8.58) 7.55 (7.89) 0.87 PCS 6 months 6.23 (7.44) 6.98 (6.78) 0.77 5.79 (6.54) 7.00 (7.16) 0.43 Post-op PT sessions at 3 months 22.25 (15.31) 19.31 (17.05) 0.43 19.92 (16.94) 19.83 (17.06) 0.98 **Abbreviations:** BPI, brief pain inventory; DASH, Disabilities of the Arm, Shoulder, and Hand; PCS, pain catastrophizing scale; PHQ-9, patient health questionnaire; post-op PT, postoperative physical therapy; SD, standard deviation.

{ "pile_set_name": "PubMed Central" }

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22,207

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Chemical context {#sec1} ================== Non-covalent interactions concern a broad range of attractive effects with an equally varied energy contribution to bonding. An interesting group of interactions is one formed by the stabilizing weak polar contacts such C---H⋯ *X* (*X* = O, F, Cl, Br, I), C---H⋯π hydrogen bonds and offset π--π interactions. These interactions are involved in biological, materials, supramolecular chemistry and crystal engineering (Desiraju, 1989[@bb5]; Desiraju & Steiner, 1999[@bb6]; Lehn, 1995[@bb14]; Steed & Atwood, 2000[@bb22]). Naphthalimide is a highly fluorescent moiety that has been used as a construction block in the design of receptors and sensors that recognize charged species and other guests (Landey-Álvarez *et al.*, 2016[@bb13]). Aromatic imides show a highly efficient photo-induced electron transfer (PET) process that can be used as a signaling method in the building of sensors or on--off molecular switches. In this sense, some researchers have proposed one approximation that involves the use of two different fluorescent units linked *via* a suitable spacer group characterized by PET or singlet--singlet energy transfer mechanisms (SSET) called dyads: such units are naphthalimide and dansyl amide. In a former study, these moieties were linked by methylene units as a bridging group and only the photon-induced fluorescence switching was studied (Abad *et al.*, 2005[@bb1]). Later, interactions with different metallic ions were investigated (Shankar & Ramaiah, 2011[@bb18]). Actually, we have studied by single-crystal X-ray diffraction the molecular structure of a naphthalimide-dansyl amide dyad and its interaction in solution with anions and aromatic molecules (Claudio-Catalán *et al.*, 2016[@bb3]). The ability of the dyad to function as a receptor of electron-rich guests and such aromatic compounds and anions are being studied by UV--Vis, fluorescence and NMR experiments. We have found that the dyad could interact with the guests tested through the aryl C---H⋯anion and aryl C---H⋯π interactions. In our ongoing research on naphthalimides as anion receptors, we report herein the synthesis and crystal structure of the title compound, a 1,2-dichlorobenzene solvate, C~56~H~50~N~6~O~8~S~2~·3C~6~H~4~Cl~2~, (I)[](#scheme1){ref-type="chem"}, which has been shown to be inert to the presence of anions or neutral molecules in solution probably due to high stability acquired by the overlap of the aromatic rings. Structural commentary {#sec2} ======================= The asymmetric unit of the title compound (I)[](#scheme1){ref-type="chem"} contains two half-molecules of the parent molecule (*A* and *B*), both having crystallographic inversion symmetry \[(i) −*x*, −*y* + 2, −*z* + 1 for (*A*) and (ii) −*x*, −*y* + 2, −*z* + 2 for (*B*)\], and three 2,3-dichlorobenzene molecules of solvation (Fig. 1[▸](#fig1){ref-type="fig"}). The *N,N*-naphthalenediimide \[N2/C13--C19 (*A*); N5/C41--C47 (*B*)\] and aromatic dansyl groups \[C1--C10 (*A*) and C29--C38 (*B*)\] are almost planar with r.m.s. deviations of 0.0055, 0.0183, 0.0664 and 0.0698 Å, respectively. The two molecules are conformationally similar with dihedral angles between the central naphthalenediimide ring and the peripheral naphthalene and benzyl rings, respectively, of 2.43 (7), 81.87 (7)° (*A*) and 3.95 (7), 84.88 (7)° (*B*). The conformations of *A* and *B* are stabilized by the presence of intramolecular aromatic ring-stacking with distances of 3.5795 (8) and 3.5640 (8) Å for *Cg*1..*Cg*2 and *Cg*3⋯ *Cg*4, respectively \[*Cg*1 and *Cg*3 are the centroids of naphathaleneimides C13--C17/N2 (*A*) and C41--C45/N5 (*B*) and *Cg*2 and *Cg*4 are the centroids of phenyl rings C1--C5/C10 (*A*) and C29--C33/C38 (*B*)\] (Fig. 2[▸](#fig2){ref-type="fig"}). Supramolecular features {#sec3} ========================== In the crystal, four C---H⋯O hydrogen bonds link the molecules into infinite supramolecular chains extending along the *c* axis (Fig. 3[▸](#fig3){ref-type="fig"}, Table 1[▸](#table1){ref-type="table"}). The chains are interconnected through C---H⋯π and offset π--π interactions, generating channels which are filled by solvent molecules (Fig. 4[▸](#fig4){ref-type="fig"}). The C---H⋯π interactions are between the benzyl groups with distances C48⋯*Cg*5′ = 3.6180 (17) and C20⋯*Cg*6′ = 3.6054 (17) Å (*Cg*5′ and *Cg*6′ are the centroids of the phenyl rings C21--C26 and C49--C54, respectively) (Fig. 5[▸](#fig5){ref-type="fig"}). The weak offset π--π interaction is between adjacent phenyl rings with *Cg*6⋯*Cg*6′(−*x*, −*y* + 1, −*z* + 1) = 4.0277 (10) Å (*Cg*6 is the centroid of the C49--C54 phenyl ring). In addition, the dansyl groups show C---H⋯π interactions, with distances C27⋯*Cg*7′ = 3.585 (2) and C55⋯*Cg*8′ = 3.632 (2) Å (Fig. 6, Table 1[▸](#table1){ref-type="table"}) where *Cg*7′ and *Cg*8′ are the centroids of naphthyl ring systems C1--C10 and C29--C38, respectively. In the channel, the N⋯N distance is 12.5 Å. The solvent molecules are interconnected by C71---H71⋯*Cg*(C63--C68)\] and C68---H68⋯Cl1 interactions and are also linked to the channel by C72---H72⋯O6 and C60---H60⋯O2 interactions (Table 1[▸](#table1){ref-type="table"}). In the crystal, there are also short Cl4⋯O4(−*x*, 1 − *y*, 1 − *z*) interactions \[3.0923 (12) Å\] and 22.6 Å^3^ solvent-accessible voids. Database survey {#sec4} ================= A search of the Cambridge Structural Database (Version 5.37; Groom *et al.*, 2016[@bb9]) revealed the existence of 324 deposited naphthalenediimide structures. Amongst those, 94 structures are metal complexes. Supramolecular constructs based on naphthalenediimide moieties with potential applications have been reported; for example PUNPAR (Wu *et al.*, 2015[@bb23]) and NUXJEX (Liu *et al.*, 2014[@bb15]) exhibit the formation of supramolecular nanotubes through cooperative \[C---H⋯O=C\] interactions. In the same way, pseudorotaxanes BALVIU and GUNPEL (Colquhoun, *et al.*, 2010[@bb4]) and catenanes IVUNUI (Fallon, *et al.*, 2004[@bb8]), SUJZIG (Hamilton *et al.*, 1998[@bb10]) and WATYAR (Hansen *et al.*, 2000[@bb11]) have been prepared. Naphthalenediimides have also been used in molecular recognition \[HIRLAX (Schneebeli *et al.*, 2013[@bb17]), MUVJUJ (Shimizu, 2010[@bb21]), PUBPAE (Koshkakaryan *et al.*, 2009[@bb12]) and RULWUS (Ono *et al.*, 2015[@bb16])\]. Synthesis and crystallization {#sec5} =============================== The title compound (I)[](#scheme1){ref-type="chem"} was prepared from 2,7-bis(2-benzylaminoethyl)naphthalenediimide (II), which was synthesized as follows. To a stirred solution of 1,4,5,8-naphthalenetetracarboxylic dianhydride (0.5 g, 1.86 mmol) in toluene (25 mL) was added *N*-benzylethylendiamine (0.56 mL, 0.56 g, 3.73 mmol) followed by the addition of triethylamine (0.52 mL, 0.377 g, 3.73 mmol). The reaction mixture was heated to reflux with azeotropic removal of water using a Dean--Stark trap, for 24 h. The solution was cooled and the solvent was removed under reduced pressure. The resultant oil was purified by column chromatography on silica gel (CH~2~Cl~2~--MeOH 95:05). Compound (II) was obtained as a yellow solid (0.777 g, 78%). M.p. 482--484 K. IR (neat): 3314, 2817, 1700, 1655, 1579, 1454 cm^−1^. RMN ^1^H (400 MHz, CDCl~3~) δ: 1.59 (*s*, 2H, NH), 3.03 (*t*, *J* = 6.4 Hz, 4H, CH~2~NH), 3.84 (*s*, 4H, CH~2~Ph), 4.38 (*t*, *J* = 6.4 Hz, 4H, NCH~2~), 7.16--7.30 (*m*, 10H, H~aromatic~), 8.74 (*s*, 4H, H~aromatic~). RMN ^13^C (100 MHz, CDCl~3~) δ: 40.6 (2C, NCH~2~), 47.0 (2C, CH~2~NH), 53.7 (2C, CH~2~Ph), 126.8 (4C), 126.9 (2C), 127.1 (2C), 128.3 (4C), 128.5 (4C), 131.2 (4C), 140.4 (2C), 163.2 (C=O). MS (FAB^+^): *m*/*z* (%) 533 (37) \[*M*\]; HRMS (FAB^+^): calculated for C~32~H~29~O~4~N~4~ \[*M*\], *m*/*z* 533.2189; found for \[*M*\], *m*/*z* 533.2142. Synthesis of *N*,*N*′-bis\[2-((benzyl){\[5-(dimethylamino)naphthalen-1-yl\]sulfonyl}amino)ethyl\]naphthalene-1,8:4,5-tetracarboximide 1,2-dichlorobenzene trisolvate (I)[](#scheme1){ref-type="chem"}. A mixture of 2,7-bis(2-benzylaminoethyl)naphthalenediimide (II) (0.5 g, 0.937 mmol), dansyl chloride (0.505 g, l.874 mmol) and K~2~CO~3~ (0.259 g, 1.874 mmol) in chloroform/water (4:1) (10 mL) was stirred at room temperature for 20 h. The organic layer was extracted with dichloromethane (2 x 20 mL), dried over anhydrous Na~2~SO~4~, filtered and concentrated under reduced pressure. Further purification was performed by flash chromatography on silica gel (CH~2~Cl~2~--MeOH 95:5). After treatment with diethyl ether, the unsolvated title compound was obtained as a yellow solid (0.936 g, 100%). Crystallization from a chloroform:1,2-dichlorobenzene mixture afforded suitable crystals of the solvated compound (I)[](#scheme1){ref-type="chem"} for the X-ray crystallographic analysis. M.p. 516--517 K. IR (neat): cm^−1^. RMN ^1^H (400 MHz, CDCl~3~) δ: 2.59 \[*s*, 12H, (CH~3~)~2~N\], 3.69 (*t*, *J* = 5.4 Hz, 4H, CH~2~NSO~2~), 4.28 (*t*, *J* = 5.4 Hz, 4H, CH~2~NCO), 4.85 (*s*, 4H, CH~2~Ph), 6.73 (*d*, *J* = 7.2 Hz, 2H, H~aromatic~), 7.18 (*dd*, *J* = 8.4, 7.2 Hz, 2H, H~aromatic~), 7.22--7.29 (*m*, 12H, H~aromatic~), 7.89 (*d*, *J* = 8.4 Hz, 2H, H~aromatic~), 7.97 (*t*, *J* = 8.8 Hz, 2H, H~aromatic~), 8.08 (*dd*, *J* = 7.2, 1.2 Hz, 2H, H~aromatic~), 8.42 (*s*, 4H, H~aromatic~). RMN ^13^C (100 MHz, CDCl~3~) δ: 37.5 (2C, CH~2~NCO), 43.1 (2C, CH~2~NSO~2~), 45.3 (4C, (CH~3~)~2~N), 49.9 (2C, CH~2~Ph), 114.4 (2C), 119.1 (2C), 123.0 (2C), 125.9 (4C),126.3 (2C), 128.1 (2C), 128.3 (2C), 128.9 (4C), 129.1 (4C), 129.2 (2C), 129.7 (2C), 130.1 (2C), 130.5 (2C), 130.6 (4C), 134.9 (2C), 135.6 (2C), 151.3 (2C), 162.9 (C=O). MS (FAB^+^): *m*/*z* (%) 999 (31) \[*M* + H\]^+^; HRMS (FAB^+^): calculated for C~21~H~19~O~2~N~2~ \[*M* + H\]^+^, *m*/*z* 999.3210; found for \[*M* + H\]^+^, *m*/*z* 999.3365. UV/Vis three bands CH~3~Cl: λ nm (∊, M^−1^ cm^−1^): 350 (24698), 362 (25362), 383 (29604). Refinement {#sec6} ============ Crystal data, data collection and structure refinement details are summarized in Table 2.[▸](#table2){ref-type="table"} Aromatic, methylene and methyl H atoms were positioned geometrically and were constrained using the riding-model approximation \[C---H = 0.95--0.98 Å with *U* ~iso~(H) = 1.5*U* ~eq~(C-methyl) and 1.5*U* ~eq~(C) for other H atoms\]. Supplementary Material ====================== Crystal structure: contains datablock(s) I. DOI: [10.1107/S2056989016015188/zs2369sup1.cif](http://dx.doi.org/10.1107/S2056989016015188/zs2369sup1.cif) Structure factors: contains datablock(s) I. DOI: [10.1107/S2056989016015188/zs2369Isup2.hkl](http://dx.doi.org/10.1107/S2056989016015188/zs2369Isup2.hkl) CCDC reference: [1506790](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1506790) Additional supporting information: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?zs2369&file=zs2369sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?zs2369sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?zs2369&checkcif=yes) This work was supported by CONACyT, project CB2011/168952. The authors thank the LANEM Laboratory for the use of spectrometric instruments. Crystal data {#tablewrapcrystaldatalong} ============ --------------------------------------- ---------------------------------------- C~56~H~50~N~6~O~8~S~2~·3C~6~H~4~Cl~2~ *F*(000) = 1492 *M~r~* = 1440.11 *D*~x~ = 1.431 Mg m^−3^ Triclinic, *P*1 Melting point = 516--517 K *a* = 12.17737 (14) Å Cu *K*α radiation, λ = 1.54184 Å *b* = 17.2876 (2) Å Cell parameters from 29431 reflections *c* = 17.8916 (2) Å θ = 2.9--72.6° α = 110.9544 (12)° µ = 3.44 mm^−1^ β = 96.2760 (11)° *T* = 100 K γ = 103.5159 (10)° Prism, orange *V* = 3341.91 (8) Å^3^ 0.20 × 0.10 × 0.05 mm *Z* = 2 --------------------------------------- ---------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== ------------------------------------------------------------------ --------------------------------------- Agilent SuperNova, Dual, Cu at zero, EosS2 diffractometer 13086 independent reflections Radiation source: sealed X-ray tube, SuperNova (Cu) X-ray Source 11914 reflections with *I* \> 2σ(*I*) Mirror monochromator *R*~int~ = 0.026 Detector resolution: 8.0769 pixels mm^-1^ θ~max~ = 73.0°, θ~min~ = 2.7° ω scans *h* = −15→15 Absorption correction: multi-scan (CrysAlis PRO; Agilent, 2014) *k* = −21→21 *T*~min~ = 0.874, *T*~max~ = 1.000 *l* = −21→22 54279 measured reflections ------------------------------------------------------------------ --------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ------------------------------------- ----------------------------------------------------------------------------------------------- Refinement on *F*^2^ Primary atom site location: structure-invariant direct methods Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites *R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.034 H-atom parameters constrained *wR*(*F*^2^) = 0.097 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.056*P*)^2^ + 1.406*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3 *S* = 1.03 (Δ/σ)~max~ = 0.001 13086 reflections Δρ~max~ = 0.74 e Å^−3^ 869 parameters Δρ~min~ = −0.45 e Å^−3^ 0 restraints ------------------------------------- ----------------------------------------------------------------------------------------------- Special details {#specialdetails} =============== -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. 1. Fixed Uiso At 1.2 times of: All C(H) groups, All C(H,H) groups At 1.5 times of: All C(H,H,H) groups 2.a Secondary CH2 refined with riding coordinates: C12(H12A,H12B), C11(H11A,H11B), C20(H20A,H20B), C39(H39A,H39B), C48(H48A, H48B), C40(H40A,H40B) 2.b Aromatic/amide H refined with riding coordinates: C2(H2), C18(H18), C4(H4), C23(H23), C19(H19), C3(H3), C22(H22), C9(H9), C25(H25), C8(H8), C73(H73), C7(H7), C74(H74), C24(H24), C26(H26), C71(H71), C72(H72), C47(H47), C32(H32), C30(H30), C62(H62), C46(H46), C51(H51), C36(H36), C50(H50), C37(H37), C31(H31), C61(H61), C54(H54), C35(H35), C60(H60), C53(H53), C52(H52), C59(H59), C65(H65), C67(H67), C68(H68), C66(H66) 2.c Idealised Me refined as rotating group: C28(H28A,H28B,H28C), C27(H27A,H27B,H27C), C56(H56A,H56B,H56C), C55(H55A,H55B, H55C) -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords} ================================================================================================== ------ --------------- -------------- -------------- -------------------- -- *x* *y* *z* *U*~iso~\*/*U*~eq~ S1 0.19073 (3) 0.75685 (2) 0.24683 (2) 0.01493 (8) Cl6 0.67288 (4) 0.57176 (3) 0.54497 (3) 0.03455 (10) Cl5 0.69686 (4) 0.73661 (3) 0.50338 (3) 0.04428 (13) O3 −0.08861 (9) 0.89558 (7) 0.24342 (6) 0.0210 (2) O1 0.19908 (9) 0.77111 (7) 0.17289 (6) 0.0212 (2) O4 −0.08934 (9) 0.72885 (6) 0.39083 (6) 0.0194 (2) O2 0.26566 (9) 0.71343 (7) 0.27162 (6) 0.0197 (2) N2 −0.08971 (10) 0.81240 (7) 0.31685 (7) 0.0149 (2) N1 0.05830 (10) 0.70359 (7) 0.24087 (7) 0.0152 (2) C13 −0.07211 (12) 0.89229 (9) 0.31046 (9) 0.0157 (3) C1 0.21767 (11) 0.86101 (9) 0.32637 (9) 0.0148 (3) C2 0.22425 (12) 0.92925 (9) 0.30290 (9) 0.0185 (3) H2 0.2112 0.9190 0.2464 0.022\* N3 0.35385 (11) 1.06595 (8) 0.61154 (8) 0.0203 (3) C15 −0.01802 (11) 0.96231 (9) 0.46334 (8) 0.0133 (3) C18 −0.01783 (12) 0.87352 (9) 0.54171 (9) 0.0170 (3) H18 −0.0290 0.8181 0.5437 0.020\* C4 0.27689 (12) 1.02957 (9) 0.44374 (9) 0.0181 (3) H4 0.2967 1.0873 0.4834 0.022\* C16 −0.03523 (11) 0.88034 (9) 0.46729 (9) 0.0139 (3) C23 −0.14700 (13) 0.41817 (10) 0.06594 (10) 0.0226 (3) H23 −0.2258 0.3848 0.0445 0.027\* C17 −0.07277 (11) 0.80072 (9) 0.39066 (9) 0.0148 (3) C19 0.01643 (12) 0.94848 (9) 0.61479 (9) 0.0177 (3) H19 0.0270 0.9435 0.6661 0.021\* C12 −0.13094 (12) 0.73496 (9) 0.23968 (9) 0.0169 (3) H12A −0.1931 0.7419 0.2043 0.020\* H12B −0.1642 0.6838 0.2517 0.020\* C5 0.27561 (11) 0.96116 (9) 0.47012 (9) 0.0148 (3) C10 0.23834 (11) 0.87377 (9) 0.41091 (9) 0.0140 (3) C21 −0.00149 (12) 0.54144 (9) 0.17070 (9) 0.0157 (3) C14 −0.03475 (12) 0.97082 (9) 0.38777 (9) 0.0150 (3) C3 0.25032 (13) 1.01428 (9) 0.36250 (10) 0.0202 (3) H3 0.2495 1.0608 0.3463 0.024\* C6 0.30788 (11) 0.97820 (9) 0.55544 (9) 0.0166 (3) C22 −0.11631 (13) 0.49109 (9) 0.13944 (9) 0.0186 (3) H22 −0.1741 0.5065 0.1683 0.022\* C9 0.22245 (12) 0.80530 (9) 0.43852 (9) 0.0168 (3) H9 0.1960 0.7468 0.4000 0.020\* C11 −0.03476 (12) 0.71921 (9) 0.19387 (8) 0.0155 (3) H11A −0.0676 0.6685 0.1412 0.019\* H11B −0.0020 0.7702 0.1815 0.019\* C25 0.05176 (14) 0.44374 (10) 0.05536 (10) 0.0241 (3) H25 0.1097 0.4273 0.0272 0.029\* C8 0.24521 (13) 0.82367 (10) 0.52047 (9) 0.0202 (3) H8 0.2306 0.7774 0.5384 0.024\* C73 0.43748 (14) 0.73761 (12) 0.62140 (11) 0.0321 (4) H73 0.3867 0.7718 0.6371 0.039\* C7 0.28998 (13) 0.90977 (10) 0.57924 (9) 0.0201 (3) H7 0.3079 0.9205 0.6357 0.024\* C74 0.51894 (16) 0.75901 (11) 0.57842 (11) 0.0300 (4) H74 0.5243 0.8078 0.5647 0.036\* C69 0.59299 (14) 0.70853 (10) 0.55541 (10) 0.0243 (3) C20 0.03168 (12) 0.62007 (9) 0.25087 (9) 0.0163 (3) H20A −0.0326 0.6166 0.2802 0.020\* H20B 0.1002 0.6189 0.2853 0.020\* C70 0.58373 (13) 0.63686 (10) 0.57470 (10) 0.0226 (3) C24 −0.06324 (14) 0.39416 (10) 0.02411 (10) 0.0234 (3) H24 −0.0842 0.3441 −0.0256 0.028\* C28 0.35179 (15) 1.08222 (11) 0.69698 (10) 0.0276 (3) H28A 0.2750 1.0524 0.7009 0.041\* H28B 0.3692 1.1447 0.7286 0.041\* H28C 0.4098 1.0606 0.7189 0.041\* C26 0.08184 (13) 0.51720 (9) 0.12751 (9) 0.0199 (3) H26 0.1603 0.5515 0.1478 0.024\* C71 0.50229 (16) 0.61617 (12) 0.61816 (11) 0.0308 (4) H71 0.4965 0.5673 0.6318 0.037\* C27 0.47087 (14) 1.10772 (12) 0.60510 (11) 0.0308 (4) H27A 0.5270 1.0855 0.6283 0.046\* H27B 0.4904 1.1706 0.6354 0.046\* H27C 0.4729 1.0948 0.5474 0.046\* C72 0.42945 (15) 0.66686 (13) 0.64163 (11) 0.0346 (4) H72 0.3738 0.6530 0.6717 0.042\* S2 0.17486 (3) 0.76401 (2) 0.74465 (2) 0.01531 (8) Cl1 0.74045 (3) 0.73768 (3) 0.03177 (3) 0.03251 (10) Cl2 0.67800 (3) 0.56374 (2) 0.06086 (3) 0.03107 (10) O7 −0.11632 (9) 0.89353 (7) 0.74506 (6) 0.0215 (2) O5 0.17643 (9) 0.78095 (7) 0.67160 (6) 0.0225 (2) O8 −0.10021 (9) 0.72829 (6) 0.89241 (6) 0.0199 (2) O6 0.25383 (9) 0.72127 (7) 0.76537 (7) 0.0212 (2) N5 −0.10660 (10) 0.81110 (8) 0.81829 (7) 0.0157 (2) N4 0.04457 (10) 0.70822 (7) 0.74075 (7) 0.0154 (2) C47 0.03877 (12) 0.94984 (9) 1.11372 (9) 0.0171 (3) H47 0.0585 0.9454 1.1646 0.020\* C41 −0.09349 (12) 0.89094 (9) 0.81190 (9) 0.0163 (3) C38 0.23320 (11) 0.87835 (9) 0.91056 (9) 0.0141 (3) C44 −0.03418 (11) 0.88033 (9) 0.96747 (9) 0.0144 (3) C49 −0.00293 (12) 0.54617 (9) 0.67090 (9) 0.0161 (3) C32 0.26568 (12) 1.03448 (9) 0.94680 (9) 0.0175 (3) H32 0.2866 1.0918 0.9874 0.021\* C29 0.20294 (11) 0.86685 (9) 0.82676 (9) 0.0149 (3) N6 0.36162 (11) 1.06867 (8) 1.11139 (8) 0.0201 (3) C30 0.20231 (12) 0.93567 (9) 0.80535 (9) 0.0184 (3) H30 0.1833 0.9263 0.7493 0.022\* C43 −0.02205 (11) 0.96206 (9) 0.96352 (8) 0.0136 (3) C57 0.61803 (13) 0.70800 (10) 0.06827 (9) 0.0209 (3) C62 0.54740 (14) 0.76180 (10) 0.08531 (10) 0.0250 (3) H62 0.5645 0.8131 0.0750 0.030\* C46 −0.00313 (12) 0.87448 (9) 1.04134 (9) 0.0161 (3) H46 −0.0101 0.8194 1.0432 0.019\* C45 −0.08202 (11) 0.80018 (9) 0.89182 (9) 0.0151 (3) C42 −0.05138 (12) 0.96965 (9) 0.88864 (9) 0.0153 (3) C39 −0.05221 (12) 0.72071 (9) 0.69431 (9) 0.0161 (3) H39A −0.0843 0.6695 0.6419 0.019\* H39B −0.0233 0.7719 0.6814 0.019\* C51 −0.14298 (14) 0.42729 (10) 0.55784 (10) 0.0250 (3) H51 −0.2212 0.3968 0.5306 0.030\* C48 0.02496 (12) 0.62465 (9) 0.75062 (9) 0.0169 (3) H48A −0.0395 0.6180 0.7793 0.020\* H48B 0.0951 0.6266 0.7858 0.020\* C36 0.25694 (13) 0.82636 (9) 1.01796 (9) 0.0196 (3) H36 0.2471 0.7796 1.0351 0.024\* C40 −0.14825 (12) 0.73367 (9) 0.74141 (9) 0.0178 (3) H40A −0.2126 0.7394 0.7069 0.021\* H40B −0.1783 0.6821 0.7536 0.021\* C50 −0.11737 (13) 0.50007 (9) 0.63098 (10) 0.0202 (3) H50 −0.1784 0.5184 0.6538 0.024\* C34 0.31473 (11) 0.98141 (9) 1.05462 (9) 0.0163 (3) C37 0.22504 (12) 0.80914 (9) 0.93661 (9) 0.0168 (3) H37 0.1974 0.7509 0.8976 0.020\* C58 0.59165 (13) 0.63204 (10) 0.08181 (9) 0.0203 (3) C31 0.22989 (12) 1.02016 (9) 0.86658 (9) 0.0191 (3) H31 0.2236 1.0670 0.8521 0.023\* C61 0.45182 (14) 0.74021 (11) 0.11743 (10) 0.0273 (3) H61 0.4038 0.7772 0.1298 0.033\* C54 0.08498 (13) 0.51758 (9) 0.63686 (10) 0.0209 (3) H54 0.1633 0.5485 0.6633 0.025\* C33 0.27227 (11) 0.96537 (9) 0.97068 (9) 0.0148 (3) C56 0.37005 (14) 1.08286 (11) 1.19736 (10) 0.0267 (3) H56A 0.2963 1.0521 1.2048 0.040\* H56B 0.3882 1.1450 1.2306 0.040\* H56C 0.4314 1.0610 1.2146 0.040\* C35 0.30409 (13) 0.91200 (10) 1.07700 (9) 0.0194 (3) H35 0.3288 0.9219 1.1328 0.023\* C60 0.42596 (14) 0.66501 (11) 0.13160 (11) 0.0275 (3) H60 0.3605 0.6507 0.1539 0.033\* C53 0.05946 (15) 0.44438 (10) 0.56466 (10) 0.0251 (3) H53 0.1202 0.4250 0.5425 0.030\* C52 −0.05465 (15) 0.39939 (10) 0.52484 (10) 0.0257 (3) H52 −0.0721 0.3496 0.4751 0.031\* C59 0.49533 (14) 0.61065 (10) 0.11340 (10) 0.0243 (3) H59 0.4770 0.5587 0.1225 0.029\* C55 0.47316 (14) 1.11330 (12) 1.09971 (11) 0.0323 (4) H55A 0.5340 1.0921 1.1187 0.048\* H55B 0.4914 1.1758 1.1312 0.048\* H55C 0.4682 1.1017 1.0415 0.048\* Cl4 0.30187 (3) 0.43343 (3) 0.69251 (3) 0.03212 (10) Cl3 0.40852 (4) 0.62180 (3) 0.83265 (3) 0.03876 (11) C63 0.49571 (14) 0.55818 (10) 0.79349 (10) 0.0242 (3) C65 0.52000 (14) 0.42501 (11) 0.70186 (11) 0.0269 (3) H65 0.4878 0.3685 0.6599 0.032\* C64 0.44887 (13) 0.47549 (10) 0.73244 (10) 0.0226 (3) C67 0.68559 (14) 0.54154 (12) 0.79409 (12) 0.0313 (4) H67 0.7667 0.5641 0.8151 0.038\* C68 0.61463 (15) 0.59036 (11) 0.82303 (11) 0.0302 (4) H68 0.6469 0.6473 0.8641 0.036\* C66 0.63803 (15) 0.45730 (12) 0.73272 (12) 0.0322 (4) H66 0.6870 0.4227 0.7126 0.039\* ------ --------------- -------------- -------------- -------------------- -- Atomic displacement parameters (Å^2^) {#tablewrapadps} ===================================== ----- -------------- -------------- -------------- -------------- -------------- -------------- *U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^ S1 0.01687 (16) 0.01485 (15) 0.01071 (16) 0.00375 (12) 0.00225 (12) 0.00316 (13) Cl6 0.0310 (2) 0.0289 (2) 0.0372 (2) 0.01418 (16) 0.00131 (17) 0.00403 (18) Cl5 0.0503 (3) 0.0319 (2) 0.0516 (3) 0.00476 (19) 0.0321 (2) 0.0162 (2) O3 0.0307 (6) 0.0202 (5) 0.0120 (5) 0.0091 (4) 0.0009 (4) 0.0061 (4) O1 0.0241 (5) 0.0239 (5) 0.0123 (5) 0.0032 (4) 0.0045 (4) 0.0057 (4) O4 0.0258 (5) 0.0134 (5) 0.0168 (5) 0.0037 (4) 0.0036 (4) 0.0050 (4) O2 0.0198 (5) 0.0201 (5) 0.0182 (5) 0.0089 (4) 0.0034 (4) 0.0046 (4) N2 0.0169 (5) 0.0141 (5) 0.0111 (6) 0.0045 (4) 0.0008 (4) 0.0026 (5) N1 0.0160 (5) 0.0129 (5) 0.0151 (6) 0.0031 (4) 0.0000 (4) 0.0053 (5) C13 0.0171 (6) 0.0164 (7) 0.0126 (7) 0.0062 (5) 0.0016 (5) 0.0043 (6) C1 0.0128 (6) 0.0142 (6) 0.0134 (7) 0.0019 (5) 0.0005 (5) 0.0030 (5) C2 0.0194 (7) 0.0205 (7) 0.0145 (7) 0.0039 (5) 0.0002 (5) 0.0079 (6) N3 0.0178 (6) 0.0194 (6) 0.0144 (6) 0.0007 (5) 0.0001 (5) 0.0001 (5) C15 0.0117 (6) 0.0154 (6) 0.0132 (7) 0.0053 (5) 0.0029 (5) 0.0053 (6) C18 0.0202 (7) 0.0145 (6) 0.0178 (7) 0.0059 (5) 0.0042 (5) 0.0077 (6) C4 0.0170 (6) 0.0138 (6) 0.0199 (7) 0.0023 (5) 0.0014 (5) 0.0048 (6) C16 0.0127 (6) 0.0150 (6) 0.0136 (7) 0.0050 (5) 0.0027 (5) 0.0047 (5) C23 0.0232 (7) 0.0163 (7) 0.0226 (8) 0.0006 (6) −0.0023 (6) 0.0066 (6) C17 0.0139 (6) 0.0166 (7) 0.0136 (7) 0.0049 (5) 0.0025 (5) 0.0055 (6) C19 0.0229 (7) 0.0189 (7) 0.0125 (7) 0.0070 (6) 0.0029 (5) 0.0073 (6) C12 0.0188 (7) 0.0148 (6) 0.0123 (7) 0.0036 (5) −0.0007 (5) 0.0016 (5) C5 0.0113 (6) 0.0165 (6) 0.0147 (7) 0.0033 (5) 0.0022 (5) 0.0046 (6) C10 0.0118 (6) 0.0156 (6) 0.0138 (7) 0.0044 (5) 0.0020 (5) 0.0050 (5) C21 0.0211 (7) 0.0130 (6) 0.0136 (7) 0.0058 (5) 0.0022 (5) 0.0060 (5) C14 0.0151 (6) 0.0161 (6) 0.0139 (7) 0.0063 (5) 0.0022 (5) 0.0051 (6) C3 0.0224 (7) 0.0156 (7) 0.0225 (8) 0.0038 (5) 0.0009 (6) 0.0096 (6) C6 0.0122 (6) 0.0192 (7) 0.0139 (7) 0.0031 (5) 0.0013 (5) 0.0028 (6) C22 0.0201 (7) 0.0163 (6) 0.0195 (7) 0.0057 (5) 0.0034 (6) 0.0070 (6) C9 0.0191 (7) 0.0150 (6) 0.0139 (7) 0.0036 (5) 0.0018 (5) 0.0044 (6) C11 0.0192 (6) 0.0144 (6) 0.0111 (6) 0.0046 (5) −0.0002 (5) 0.0041 (5) C25 0.0292 (8) 0.0214 (7) 0.0215 (8) 0.0097 (6) 0.0094 (6) 0.0058 (6) C8 0.0245 (7) 0.0202 (7) 0.0179 (7) 0.0057 (6) 0.0041 (6) 0.0104 (6) C73 0.0235 (8) 0.0393 (10) 0.0249 (9) 0.0132 (7) 0.0023 (7) 0.0013 (8) C7 0.0220 (7) 0.0249 (7) 0.0119 (7) 0.0066 (6) 0.0020 (5) 0.0064 (6) C74 0.0367 (9) 0.0268 (8) 0.0255 (9) 0.0121 (7) 0.0035 (7) 0.0084 (7) C69 0.0245 (7) 0.0234 (7) 0.0206 (8) 0.0029 (6) 0.0062 (6) 0.0058 (6) C20 0.0217 (7) 0.0132 (6) 0.0133 (7) 0.0049 (5) 0.0026 (5) 0.0049 (6) C70 0.0207 (7) 0.0224 (7) 0.0187 (8) 0.0052 (6) −0.0005 (6) 0.0033 (6) C24 0.0339 (8) 0.0147 (7) 0.0160 (7) 0.0048 (6) 0.0017 (6) 0.0021 (6) C28 0.0292 (8) 0.0279 (8) 0.0150 (8) 0.0050 (7) 0.0011 (6) −0.0006 (6) C26 0.0205 (7) 0.0174 (7) 0.0197 (7) 0.0048 (6) 0.0033 (6) 0.0056 (6) C71 0.0371 (9) 0.0308 (9) 0.0239 (9) 0.0053 (7) 0.0069 (7) 0.0131 (7) C27 0.0218 (8) 0.0295 (8) 0.0254 (9) −0.0051 (6) 0.0004 (7) 0.0023 (7) C72 0.0275 (8) 0.0441 (10) 0.0253 (9) 0.0039 (8) 0.0111 (7) 0.0085 (8) S2 0.01726 (16) 0.01544 (16) 0.01142 (16) 0.00420 (12) 0.00325 (12) 0.00358 (13) Cl1 0.02762 (19) 0.0374 (2) 0.0291 (2) 0.00037 (16) 0.01108 (16) 0.01363 (18) Cl2 0.02618 (19) 0.02378 (19) 0.0357 (2) 0.01054 (15) 0.00128 (16) 0.00270 (17) O7 0.0302 (6) 0.0203 (5) 0.0139 (5) 0.0094 (4) 0.0009 (4) 0.0064 (4) O5 0.0268 (5) 0.0241 (5) 0.0130 (5) 0.0027 (4) 0.0052 (4) 0.0060 (4) O8 0.0265 (5) 0.0138 (5) 0.0184 (5) 0.0043 (4) 0.0055 (4) 0.0062 (4) O6 0.0199 (5) 0.0214 (5) 0.0211 (5) 0.0095 (4) 0.0042 (4) 0.0050 (4) N5 0.0172 (5) 0.0146 (5) 0.0140 (6) 0.0051 (4) 0.0020 (4) 0.0043 (5) N4 0.0172 (6) 0.0133 (5) 0.0147 (6) 0.0039 (4) 0.0009 (5) 0.0055 (5) C47 0.0200 (7) 0.0192 (7) 0.0141 (7) 0.0073 (5) 0.0032 (5) 0.0081 (6) C41 0.0170 (6) 0.0169 (7) 0.0154 (7) 0.0066 (5) 0.0027 (5) 0.0060 (6) C38 0.0119 (6) 0.0157 (6) 0.0138 (7) 0.0037 (5) 0.0022 (5) 0.0052 (5) C44 0.0132 (6) 0.0153 (6) 0.0155 (7) 0.0055 (5) 0.0039 (5) 0.0060 (6) C49 0.0216 (7) 0.0132 (6) 0.0141 (7) 0.0049 (5) 0.0024 (5) 0.0065 (6) C32 0.0168 (6) 0.0136 (6) 0.0197 (7) 0.0026 (5) 0.0031 (5) 0.0053 (6) C29 0.0140 (6) 0.0141 (6) 0.0144 (7) 0.0030 (5) 0.0024 (5) 0.0040 (5) N6 0.0185 (6) 0.0192 (6) 0.0152 (6) 0.0015 (5) 0.0011 (5) 0.0017 (5) C30 0.0191 (7) 0.0205 (7) 0.0159 (7) 0.0045 (5) 0.0016 (5) 0.0090 (6) C43 0.0123 (6) 0.0153 (6) 0.0145 (7) 0.0056 (5) 0.0040 (5) 0.0060 (6) C57 0.0193 (7) 0.0250 (7) 0.0135 (7) 0.0009 (6) 0.0015 (6) 0.0060 (6) C62 0.0282 (8) 0.0222 (7) 0.0227 (8) 0.0051 (6) −0.0008 (6) 0.0099 (6) C46 0.0187 (6) 0.0145 (6) 0.0175 (7) 0.0061 (5) 0.0050 (5) 0.0080 (6) C45 0.0144 (6) 0.0167 (7) 0.0159 (7) 0.0059 (5) 0.0054 (5) 0.0070 (6) C42 0.0153 (6) 0.0165 (7) 0.0151 (7) 0.0057 (5) 0.0039 (5) 0.0065 (6) C39 0.0190 (7) 0.0148 (6) 0.0120 (7) 0.0043 (5) 0.0002 (5) 0.0039 (5) C51 0.0270 (8) 0.0178 (7) 0.0230 (8) 0.0012 (6) −0.0033 (6) 0.0059 (6) C48 0.0220 (7) 0.0143 (6) 0.0145 (7) 0.0053 (5) 0.0030 (5) 0.0061 (6) C36 0.0235 (7) 0.0191 (7) 0.0177 (7) 0.0066 (6) 0.0027 (6) 0.0093 (6) C40 0.0188 (7) 0.0158 (6) 0.0138 (7) 0.0038 (5) −0.0008 (5) 0.0022 (6) C50 0.0215 (7) 0.0173 (7) 0.0216 (8) 0.0059 (6) 0.0031 (6) 0.0079 (6) C34 0.0125 (6) 0.0182 (7) 0.0143 (7) 0.0034 (5) 0.0020 (5) 0.0031 (6) C37 0.0192 (7) 0.0146 (6) 0.0150 (7) 0.0043 (5) 0.0022 (5) 0.0050 (6) C58 0.0199 (7) 0.0205 (7) 0.0154 (7) 0.0053 (6) −0.0010 (6) 0.0031 (6) C31 0.0217 (7) 0.0153 (6) 0.0218 (8) 0.0048 (5) 0.0025 (6) 0.0100 (6) C61 0.0243 (8) 0.0297 (8) 0.0248 (9) 0.0115 (7) 0.0003 (6) 0.0065 (7) C54 0.0221 (7) 0.0170 (7) 0.0217 (8) 0.0050 (6) 0.0036 (6) 0.0062 (6) C33 0.0113 (6) 0.0167 (6) 0.0153 (7) 0.0033 (5) 0.0030 (5) 0.0056 (6) C56 0.0289 (8) 0.0264 (8) 0.0150 (8) 0.0059 (6) −0.0005 (6) −0.0002 (6) C35 0.0210 (7) 0.0241 (7) 0.0125 (7) 0.0068 (6) 0.0009 (5) 0.0072 (6) C60 0.0198 (7) 0.0350 (9) 0.0254 (8) 0.0055 (6) 0.0071 (6) 0.0104 (7) C53 0.0325 (8) 0.0192 (7) 0.0247 (8) 0.0101 (6) 0.0118 (7) 0.0067 (6) C52 0.0399 (9) 0.0148 (7) 0.0169 (8) 0.0050 (6) 0.0035 (7) 0.0026 (6) C59 0.0245 (7) 0.0243 (8) 0.0231 (8) 0.0028 (6) 0.0023 (6) 0.0119 (7) C55 0.0239 (8) 0.0291 (8) 0.0276 (9) −0.0054 (7) 0.0026 (7) 0.0017 (7) Cl4 0.01841 (17) 0.0384 (2) 0.0331 (2) 0.00178 (15) 0.00127 (15) 0.01215 (18) Cl3 0.0389 (2) 0.0291 (2) 0.0496 (3) 0.01544 (17) 0.0146 (2) 0.0123 (2) C63 0.0263 (8) 0.0249 (8) 0.0262 (8) 0.0083 (6) 0.0062 (6) 0.0149 (7) C65 0.0296 (8) 0.0228 (8) 0.0301 (9) 0.0058 (6) 0.0090 (7) 0.0130 (7) C64 0.0190 (7) 0.0243 (7) 0.0264 (8) 0.0035 (6) 0.0025 (6) 0.0146 (7) C67 0.0168 (7) 0.0407 (10) 0.0444 (11) 0.0047 (7) 0.0024 (7) 0.0296 (9) C68 0.0305 (8) 0.0249 (8) 0.0304 (9) −0.0012 (7) −0.0021 (7) 0.0138 (7) C66 0.0270 (8) 0.0356 (9) 0.0481 (11) 0.0154 (7) 0.0151 (8) 0.0267 (9) ----- -------------- -------------- -------------- -------------- -------------- -------------- Geometric parameters (Å, º) {#tablewrapgeomlong} =========================== ----------------------------- -------------- ------------------------------- -------------- S1---O1 1.4397 (11) O7---C41 1.2168 (18) S1---O2 1.4361 (10) O8---C45 1.2146 (17) S1---N1 1.6303 (12) N5---C41 1.3991 (18) S1---C1 1.7789 (14) N5---C45 1.4049 (18) Cl6---C70 1.7313 (16) N5---C40 1.4677 (18) Cl5---C69 1.7308 (16) N4---C39 1.4694 (17) O3---C13 1.2181 (18) N4---C48 1.4853 (17) O4---C17 1.2124 (17) C47---H47 0.9500 N2---C13 1.3952 (18) C47---C46 1.404 (2) N2---C17 1.4065 (18) C47---C42^ii^ 1.3808 (19) N2---C12 1.4706 (17) C41---C42 1.478 (2) N1---C11 1.4701 (17) C38---C29 1.433 (2) N1---C20 1.4834 (17) C38---C37 1.4186 (19) C13---C14 1.4812 (19) C38---C33 1.4292 (19) C1---C2 1.376 (2) C44---C43 1.4139 (19) C1---C10 1.432 (2) C44---C46 1.380 (2) C2---H2 0.9500 C44---C45 1.483 (2) C2---C3 1.409 (2) C49---C48 1.5117 (19) N3---C6 1.4189 (19) C49---C50 1.394 (2) N3---C28 1.456 (2) C49---C54 1.391 (2) N3---C27 1.476 (2) C32---H32 0.9500 C15---C15^i^ 1.413 (3) C32---C31 1.367 (2) C15---C16 1.4118 (19) C32---C33 1.4208 (19) C15---C14 1.410 (2) C29---C30 1.376 (2) C18---H18 0.9500 N6---C34 1.4169 (19) C18---C16 1.376 (2) N6---C56 1.456 (2) C18---C19 1.407 (2) N6---C55 1.474 (2) C4---H4 0.9500 C30---H30 0.9500 C4---C5 1.4190 (19) C30---C31 1.411 (2) C4---C3 1.367 (2) C43---C43^ii^ 1.416 (3) C16---C17 1.4841 (19) C43---C42 1.408 (2) C23---H23 0.9500 C57---C62 1.388 (2) C23---C22 1.395 (2) C57---C58 1.391 (2) C23---C24 1.384 (2) C62---H62 0.9500 C19---H19 0.9500 C62---C61 1.386 (2) C19---C14^i^ 1.3787 (19) C46---H46 0.9500 C12---H12A 0.9900 C42---C47^ii^ 1.3808 (19) C12---H12B 0.9900 C39---H39A 0.9900 C12---C11 1.522 (2) C39---H39B 0.9900 C5---C10 1.4285 (19) C39---C40 1.527 (2) C5---C6 1.437 (2) C51---H51 0.9500 C10---C9 1.4194 (19) C51---C50 1.394 (2) C21---C22 1.392 (2) C51---C52 1.384 (2) C21---C20 1.5134 (19) C48---H48A 0.9900 C21---C26 1.393 (2) C48---H48B 0.9900 C14---C19^i^ 1.3787 (19) C36---H36 0.9500 C3---H3 0.9500 C36---C37 1.368 (2) C6---C7 1.374 (2) C36---C35 1.410 (2) C22---H22 0.9500 C40---H40A 0.9900 C9---H9 0.9500 C40---H40B 0.9900 C9---C8 1.365 (2) C50---H50 0.9500 C11---H11A 0.9900 C34---C33 1.437 (2) C11---H11B 0.9900 C34---C35 1.378 (2) C25---H25 0.9500 C37---H37 0.9500 C25---C24 1.390 (2) C58---C59 1.387 (2) C25---C26 1.386 (2) C31---H31 0.9500 C8---H8 0.9500 C61---H61 0.9500 C8---C7 1.412 (2) C61---C60 1.384 (2) C73---H73 0.9500 C54---H54 0.9500 C73---C74 1.382 (3) C54---C53 1.388 (2) C73---C72 1.380 (3) C56---H56A 0.9800 C7---H7 0.9500 C56---H56B 0.9800 C74---H74 0.9500 C56---H56C 0.9800 C74---C69 1.391 (2) C35---H35 0.9500 C69---C70 1.384 (2) C60---H60 0.9500 C20---H20A 0.9900 C60---C59 1.384 (2) C20---H20B 0.9900 C53---H53 0.9500 C70---C71 1.385 (2) C53---C52 1.388 (2) C24---H24 0.9500 C52---H52 0.9500 C28---H28A 0.9800 C59---H59 0.9500 C28---H28B 0.9800 C55---H55A 0.9800 C28---H28C 0.9800 C55---H55B 0.9800 C26---H26 0.9500 C55---H55C 0.9800 C71---H71 0.9500 Cl4---C64 1.7274 (15) C71---C72 1.383 (3) Cl3---C63 1.7278 (17) C27---H27A 0.9800 C63---C64 1.388 (2) C27---H27B 0.9800 C63---C68 1.390 (2) C27---H27C 0.9800 C65---H65 0.9500 C72---H72 0.9500 C65---C64 1.388 (2) S2---O5 1.4391 (11) C65---C66 1.383 (2) S2---O6 1.4347 (11) C67---H67 0.9500 S2---N4 1.6323 (12) C67---C68 1.361 (3) S2---C29 1.7815 (14) C67---C66 1.409 (3) Cl1---C57 1.7341 (15) C68---H68 0.9500 Cl2---C58 1.7293 (15) C66---H66 0.9500 O1---S1---N1 109.76 (6) C39---N4---S2 117.29 (9) O1---S1---C1 106.07 (7) C39---N4---C48 117.42 (11) O2---S1---O1 118.32 (7) C48---N4---S2 120.32 (9) O2---S1---N1 107.61 (6) C46---C47---H47 119.8 O2---S1---C1 108.46 (6) C42^ii^---C47---H47 119.8 N1---S1---C1 105.96 (6) C42^ii^---C47---C46 120.35 (13) C13---N2---C17 125.20 (12) O7---C41---N5 120.01 (13) C13---N2---C12 116.53 (11) O7---C41---C42 122.72 (13) C17---N2---C12 118.27 (11) N5---C41---C42 117.27 (12) C11---N1---S1 117.28 (9) C37---C38---C29 124.03 (13) C11---N1---C20 117.11 (11) C37---C38---C33 118.94 (13) C20---N1---S1 121.14 (9) C33---C38---C29 117.03 (12) O3---C13---N2 120.21 (13) C43---C44---C45 119.78 (12) O3---C13---C14 122.57 (13) C46---C44---C43 120.51 (13) N2---C13---C14 117.22 (12) C46---C44---C45 119.70 (12) C2---C1---S1 116.75 (11) C50---C49---C48 120.56 (13) C2---C1---C10 121.69 (13) C54---C49---C48 120.66 (13) C10---C1---S1 121.45 (10) C54---C49---C50 118.77 (14) C1---C2---H2 119.9 C31---C32---H32 119.3 C1---C2---C3 120.12 (13) C31---C32---C33 121.38 (13) C3---C2---H2 119.9 C33---C32---H32 119.3 C6---N3---C28 115.76 (13) C38---C29---S2 121.66 (10) C6---N3---C27 113.39 (12) C30---C29---S2 116.46 (11) C28---N3---C27 109.56 (13) C30---C29---C38 121.72 (13) C16---C15---C15^i^ 119.37 (16) C34---N6---C56 115.73 (13) C14---C15---C15^i^ 119.12 (15) C34---N6---C55 113.73 (12) C14---C15---C16 121.51 (13) C56---N6---C55 109.64 (13) C16---C18---H18 120.0 C29---C30---H30 120.0 C16---C18---C19 120.10 (13) C29---C30---C31 120.10 (13) C19---C18---H18 120.0 C31---C30---H30 120.0 C5---C4---H4 119.2 C44---C43---C43^ii^ 119.12 (16) C3---C4---H4 119.2 C42---C43---C44 121.49 (13) C3---C4---C5 121.53 (13) C42---C43---C43^ii^ 119.39 (16) C15---C16---C17 119.85 (12) C62---C57---Cl1 119.23 (12) C18---C16---C15 120.56 (13) C62---C57---C58 120.14 (14) C18---C16---C17 119.59 (12) C58---C57---Cl1 120.62 (12) C22---C23---H23 119.8 C57---C62---H62 120.2 C24---C23---H23 119.8 C61---C62---C57 119.58 (15) C24---C23---C22 120.31 (14) C61---C62---H62 120.2 O4---C17---N2 121.06 (13) C47---C46---H46 119.9 O4---C17---C16 122.35 (13) C44---C46---C47 120.15 (13) N2---C17---C16 116.59 (12) C44---C46---H46 119.9 C18---C19---H19 119.9 O8---C45---N5 120.90 (13) C14^i^---C19---C18 120.23 (13) O8---C45---C44 122.48 (13) C14^i^---C19---H19 119.9 N5---C45---C44 116.62 (12) N2---C12---H12A 109.2 C47^ii^---C42---C41 119.85 (13) N2---C12---H12B 109.2 C47^ii^---C42---C43 120.46 (13) N2---C12---C11 112.25 (11) C43---C42---C41 119.68 (12) H12A---C12---H12B 107.9 N4---C39---H39A 109.1 C11---C12---H12A 109.2 N4---C39---H39B 109.1 C11---C12---H12B 109.2 N4---C39---C40 112.29 (11) C4---C5---C10 119.27 (13) H39A---C39---H39B 107.9 C4---C5---C6 121.13 (13) C40---C39---H39A 109.1 C10---C5---C6 119.57 (13) C40---C39---H39B 109.1 C5---C10---C1 117.12 (12) C50---C51---H51 120.0 C9---C10---C1 123.99 (13) C52---C51---H51 119.9 C9---C10---C5 118.88 (13) C52---C51---C50 120.10 (15) C22---C21---C20 120.26 (13) N4---C48---C49 114.11 (11) C22---C21---C26 118.75 (13) N4---C48---H48A 108.7 C26---C21---C20 120.98 (13) N4---C48---H48B 108.7 C15---C14---C13 119.63 (12) C49---C48---H48A 108.7 C19^i^---C14---C13 119.76 (13) C49---C48---H48B 108.7 C19^i^---C14---C15 120.61 (13) H48A---C48---H48B 107.6 C2---C3---H3 120.1 C37---C36---H36 119.3 C4---C3---C2 119.80 (13) C37---C36---C35 121.49 (13) C4---C3---H3 120.1 C35---C36---H36 119.3 N3---C6---C5 117.79 (13) N5---C40---C39 111.48 (11) C7---C6---N3 123.13 (13) N5---C40---H40A 109.3 C7---C6---C5 119.05 (13) N5---C40---H40B 109.3 C23---C22---H22 119.8 C39---C40---H40A 109.3 C21---C22---C23 120.39 (14) C39---C40---H40B 109.3 C21---C22---H22 119.8 H40A---C40---H40B 108.0 C10---C9---H9 120.0 C49---C50---H50 119.8 C8---C9---C10 119.91 (13) C51---C50---C49 120.50 (14) C8---C9---H9 120.0 C51---C50---H50 119.8 N1---C11---C12 112.44 (11) N6---C34---C33 118.05 (13) N1---C11---H11A 109.1 C35---C34---N6 122.96 (13) N1---C11---H11B 109.1 C35---C34---C33 118.94 (13) C12---C11---H11A 109.1 C38---C37---H37 120.0 C12---C11---H11B 109.1 C36---C37---C38 119.94 (13) H11A---C11---H11B 107.8 C36---C37---H37 120.0 C24---C25---H25 120.0 C57---C58---Cl2 121.08 (12) C26---C25---H25 120.0 C59---C58---Cl2 119.07 (12) C26---C25---C24 120.04 (15) C59---C58---C57 119.85 (14) C9---C8---H8 119.2 C32---C31---C30 119.85 (13) C9---C8---C7 121.58 (13) C32---C31---H31 120.1 C7---C8---H8 119.2 C30---C31---H31 120.1 C74---C73---H73 119.8 C62---C61---H61 119.8 C72---C73---H73 119.8 C60---C61---C62 120.36 (15) C72---C73---C74 120.47 (16) C60---C61---H61 119.8 C6---C7---C8 120.58 (14) C49---C54---H54 119.6 C6---C7---H7 119.7 C53---C54---C49 120.74 (14) C8---C7---H7 119.7 C53---C54---H54 119.6 C73---C74---H74 120.2 C38---C33---C34 119.53 (13) C73---C74---C69 119.51 (16) C32---C33---C38 119.41 (13) C69---C74---H74 120.2 C32---C33---C34 121.04 (13) C74---C69---Cl5 119.49 (13) N6---C56---H56A 109.5 C70---C69---Cl5 120.55 (13) N6---C56---H56B 109.5 C70---C69---C74 119.96 (15) N6---C56---H56C 109.5 N1---C20---C21 113.67 (11) H56A---C56---H56B 109.5 N1---C20---H20A 108.8 H56A---C56---H56C 109.5 N1---C20---H20B 108.8 H56B---C56---H56C 109.5 C21---C20---H20A 108.8 C36---C35---H35 119.7 C21---C20---H20B 108.8 C34---C35---C36 120.66 (13) H20A---C20---H20B 107.7 C34---C35---H35 119.7 C69---C70---Cl6 120.85 (13) C61---C60---H60 120.0 C69---C70---C71 120.14 (15) C59---C60---C61 120.07 (15) C71---C70---Cl6 119.01 (13) C59---C60---H60 120.0 C23---C24---C25 119.59 (14) C54---C53---H53 119.9 C23---C24---H24 120.2 C52---C53---C54 120.14 (15) C25---C24---H24 120.2 C52---C53---H53 119.9 N3---C28---H28A 109.5 C51---C52---C53 119.75 (15) N3---C28---H28B 109.5 C51---C52---H52 120.1 N3---C28---H28C 109.5 C53---C52---H52 120.1 H28A---C28---H28B 109.5 C58---C59---H59 120.0 H28A---C28---H28C 109.5 C60---C59---C58 119.98 (15) H28B---C28---H28C 109.5 C60---C59---H59 120.0 C21---C26---H26 119.6 N6---C55---H55A 109.5 C25---C26---C21 120.89 (14) N6---C55---H55B 109.5 C25---C26---H26 119.6 N6---C55---H55C 109.5 C70---C71---H71 120.1 H55A---C55---H55B 109.5 C72---C71---C70 119.83 (16) H55A---C55---H55C 109.5 C72---C71---H71 120.1 H55B---C55---H55C 109.5 N3---C27---H27A 109.5 C64---C63---Cl3 120.99 (12) N3---C27---H27B 109.5 C64---C63---C68 119.41 (15) N3---C27---H27C 109.5 C68---C63---Cl3 119.61 (13) H27A---C27---H27B 109.5 C64---C65---H65 120.2 H27A---C27---H27C 109.5 C66---C65---H65 120.2 H27B---C27---H27C 109.5 C66---C65---C64 119.70 (16) C73---C72---C71 120.08 (16) C63---C64---Cl4 121.09 (12) C73---C72---H72 120.0 C65---C64---Cl4 118.60 (13) C71---C72---H72 120.0 C65---C64---C63 120.31 (14) O5---S2---N4 109.36 (6) C68---C67---H67 120.2 O5---S2---C29 105.88 (7) C68---C67---C66 119.59 (15) O6---S2---O5 118.74 (7) C66---C67---H67 120.2 O6---S2---N4 107.79 (6) C63---C68---H68 119.5 O6---S2---C29 108.16 (6) C67---C68---C63 121.05 (16) N4---S2---C29 106.27 (6) C67---C68---H68 119.5 C41---N5---C45 125.05 (12) C65---C66---C67 119.94 (16) C41---N5---C40 116.49 (12) C65---C66---H66 120.0 C45---N5---C40 118.46 (11) C67---C66---H66 120.0 S1---N1---C11---C12 −132.54 (10) Cl2---C58---C59---C60 179.14 (13) S1---N1---C20---C21 −94.83 (13) O7---C41---C42---C47^ii^ 1.3 (2) S1---C1---C2---C3 −177.24 (11) O7---C41---C42---C43 −177.36 (13) S1---C1---C10---C5 171.10 (10) O5---S2---N4---C39 −29.11 (12) S1---C1---C10---C9 −9.81 (19) O5---S2---N4---C48 125.32 (11) Cl6---C70---C71---C72 −179.28 (14) O5---S2---C29---C38 −165.99 (11) Cl5---C69---C70---Cl6 −1.77 (19) O5---S2---C29---C30 9.43 (13) Cl5---C69---C70---C71 178.36 (13) O6---S2---N4---C39 −159.49 (10) O3---C13---C14---C15 179.55 (13) O6---S2---N4---C48 −5.06 (12) O3---C13---C14---C19^i^ −0.6 (2) O6---S2---C29---C38 −37.72 (13) O1---S1---N1---C11 −32.20 (12) O6---S2---C29---C30 137.70 (11) O1---S1---N1---C20 123.41 (11) N5---C41---C42---C47^ii^ −178.82 (13) O1---S1---C1---C2 8.55 (13) N5---C41---C42---C43 2.53 (19) O1---S1---C1---C10 −167.82 (11) N4---S2---C29---C38 77.78 (12) O2---S1---N1---C11 −162.21 (10) N4---S2---C29---C30 −106.80 (11) O2---S1---N1---C20 −6.60 (12) N4---C39---C40---N5 61.25 (15) O2---S1---C1---C2 136.63 (11) C41---N5---C45---O8 177.01 (13) O2---S1---C1---C10 −39.74 (13) C41---N5---C45---C44 −2.55 (19) N2---C13---C14---C15 0.36 (19) C41---N5---C40---C39 77.33 (15) N2---C13---C14---C19^i^ −179.77 (13) C38---C29---C30---C31 −1.2 (2) N2---C12---C11---N1 61.88 (15) C44---C43---C42---C47^ii^ −179.98 (13) N1---S1---C1---C2 −108.08 (11) C44---C43---C42---C41 −1.3 (2) N1---S1---C1---C10 75.55 (12) C49---C54---C53---C52 0.9 (2) C13---N2---C17---O4 −179.49 (13) C29---S2---N4---C39 84.76 (11) C13---N2---C17---C16 1.17 (19) C29---S2---N4---C48 −120.81 (11) C13---N2---C12---C11 77.16 (15) C29---C38---C37---C36 179.38 (13) C1---S1---N1---C11 81.92 (11) C29---C38---C33---C32 7.81 (19) C1---S1---N1---C20 −122.47 (11) C29---C38---C33---C34 −173.81 (12) C1---C2---C3---C4 4.5 (2) C29---C30---C31---C32 4.9 (2) C1---C10---C9---C8 179.50 (13) N6---C34---C33---C38 174.87 (12) C2---C1---C10---C5 −5.09 (19) N6---C34---C33---C32 −6.8 (2) C2---C1---C10---C9 173.99 (13) N6---C34---C35---C36 −179.89 (13) N3---C6---C7---C8 −179.25 (13) C43---C44---C46---C47 1.3 (2) C15^i^---C15---C16---C18 0.4 (2) C43---C44---C45---O8 −175.86 (13) C15^i^---C15---C16---C17 −179.83 (14) C43---C44---C45---N5 3.70 (18) C15^i^---C15---C14---C13 179.77 (15) C43^ii^---C43---C42---C47^ii^ −0.8 (2) C15^i^---C15---C14---C19^i^ −0.1 (2) C43^ii^---C43---C42---C41 177.83 (15) C15---C16---C17---O4 −179.57 (13) C57---C62---C61---C60 −0.8 (2) C15---C16---C17---N2 −0.24 (18) C57---C58---C59---C60 −0.2 (2) C18---C16---C17---O4 0.2 (2) C62---C57---C58---Cl2 179.84 (12) C18---C16---C17---N2 179.56 (12) C62---C57---C58---C59 −0.8 (2) C4---C5---C10---C1 7.51 (19) C62---C61---C60---C59 −0.3 (3) C4---C5---C10---C9 −171.62 (13) C46---C44---C43---C43^ii^ −0.2 (2) C4---C5---C6---N3 −7.2 (2) C46---C44---C43---C42 178.94 (13) C4---C5---C6---C7 170.94 (13) C46---C44---C45---O8 3.4 (2) C16---C15---C14---C13 0.5 (2) C46---C44---C45---N5 −177.06 (12) C16---C15---C14---C19^i^ −179.38 (13) C45---N5---C41---O7 179.39 (13) C16---C18---C19---C14^i^ −1.2 (2) C45---N5---C41---C42 −0.5 (2) C17---N2---C13---O3 179.57 (13) C45---N5---C40---C39 −102.07 (14) C17---N2---C13---C14 −1.2 (2) C45---C44---C43---C43^ii^ 179.00 (14) C17---N2---C12---C11 −103.74 (14) C45---C44---C43---C42 −1.8 (2) C19---C18---C16---C15 0.6 (2) C45---C44---C46---C47 −177.95 (13) C19---C18---C16---C17 −179.24 (13) C42^ii^---C47---C46---C44 −1.3 (2) C12---N2---C13---O3 −1.41 (19) C39---N4---C48---C49 61.96 (16) C12---N2---C13---C14 177.80 (12) C48---N4---C39---C40 71.72 (15) C12---N2---C17---O4 1.49 (19) C48---C49---C50---C51 −179.45 (13) C12---N2---C17---C16 −177.84 (11) C48---C49---C54---C53 178.52 (13) C5---C4---C3---C2 −1.9 (2) C40---N5---C41---O7 0.03 (19) C5---C10---C9---C8 −1.4 (2) C40---N5---C41---C42 −179.86 (12) C5---C6---C7---C8 2.7 (2) C40---N5---C45---O8 −3.64 (19) C10---C1---C2---C3 −0.9 (2) C40---N5---C45---C44 176.80 (11) C10---C5---C6---N3 174.66 (12) C50---C49---C48---N4 −93.61 (16) C10---C5---C6---C7 −7.2 (2) C50---C49---C54---C53 −0.2 (2) C10---C9---C8---C7 −3.2 (2) C50---C51---C52---C53 −0.3 (2) C14---C15---C16---C18 179.66 (13) C37---C38---C29---S2 −10.89 (19) C14---C15---C16---C17 −0.54 (19) C37---C38---C29---C30 173.93 (14) C3---C4---C5---C10 −4.2 (2) C37---C38---C33---C32 −171.33 (13) C3---C4---C5---C6 177.62 (13) C37---C38---C33---C34 7.05 (19) C6---C5---C10---C1 −174.30 (12) C37---C36---C35---C34 2.9 (2) C6---C5---C10---C9 6.57 (19) C58---C57---C62---C61 1.3 (2) C22---C23---C24---C25 −0.7 (2) C31---C32---C33---C38 −4.4 (2) C22---C21---C20---N1 −105.22 (15) C31---C32---C33---C34 177.27 (13) C22---C21---C26---C25 −0.9 (2) C61---C60---C59---C58 0.8 (2) C9---C8---C7---C6 2.5 (2) C54---C49---C48---N4 87.67 (16) C11---N1---C20---C21 60.82 (16) C54---C49---C50---C51 −0.7 (2) C73---C74---C69---Cl5 −178.72 (13) C54---C53---C52---C51 −0.7 (2) C73---C74---C69---C70 0.8 (3) C33---C38---C29---S2 170.02 (10) C74---C73---C72---C71 −0.7 (3) C33---C38---C29---C30 −5.2 (2) C74---C69---C70---Cl6 178.69 (13) C33---C38---C37---C36 −1.5 (2) C74---C69---C70---C71 −1.2 (2) C33---C32---C31---C30 −2.1 (2) C69---C70---C71---C72 0.6 (3) C33---C34---C35---C36 2.7 (2) C20---N1---C11---C12 70.86 (15) C56---N6---C34---C33 161.71 (13) C20---C21---C22---C23 −179.45 (13) C56---N6---C34---C35 −15.7 (2) C20---C21---C26---C25 178.09 (13) C35---C36---C37---C38 −3.5 (2) C70---C71---C72---C73 0.4 (3) C35---C34---C33---C38 −7.6 (2) C24---C23---C22---C21 1.3 (2) C35---C34---C33---C32 170.71 (13) C24---C25---C26---C21 1.5 (2) C52---C51---C50---C49 1.0 (2) C28---N3---C6---C5 161.65 (13) C55---N6---C34---C33 −70.03 (17) C28---N3---C6---C7 −16.4 (2) C55---N6---C34---C35 112.59 (17) C26---C21---C22---C23 −0.5 (2) Cl3---C63---C64---Cl4 0.69 (19) C26---C21---C20---N1 75.81 (16) Cl3---C63---C64---C65 −179.71 (13) C26---C25---C24---C23 −0.7 (2) Cl3---C63---C68---C67 179.29 (13) C27---N3---C6---C5 −70.49 (17) C64---C63---C68---C67 −1.2 (2) C27---N3---C6---C7 111.47 (17) C64---C65---C66---C67 −0.9 (3) C72---C73---C74---C69 0.1 (3) C68---C63---C64---Cl4 −178.84 (12) S2---N4---C39---C40 −133.10 (10) C68---C63---C64---C65 0.8 (2) S2---N4---C48---C49 −92.44 (13) C68---C67---C66---C65 0.5 (3) S2---C29---C30---C31 −176.58 (11) C66---C65---C64---Cl4 179.88 (13) Cl1---C57---C62---C61 −177.56 (12) C66---C65---C64---C63 0.3 (2) Cl1---C57---C58---Cl2 −1.31 (18) C66---C67---C68---C63 0.5 (3) Cl1---C57---C58---C59 178.02 (12) ----------------------------- -------------- ------------------------------- -------------- Symmetry codes: (i) −*x*, −*y*+2, −*z*+1; (ii) −*x*, −*y*+2, −*z*+2. Hydrogen-bond geometry (Å, º) {#tablewraphbondslong} ============================= Cg, Cg5\', Cg6\', Cg7\' and Cg8\' are the centroids of atoms C63--C68, C21--C26, C49--C54, C1--C10 and C29--C38, respectively. --------------------------- --------- --------- ------------- --------------- *D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A* C8---H8···O5 0.95 2.53 3.1926 (19) 127 C19---H19···O7 0.95 2.55 3.2615 (19) 132 C31---H31···O3^i^ 0.95 2.59 3.3818 (19) 141 C47---H47···O3^iii^ 0.95 2.58 3.2148 (19) 125 C60---H60···O2 0.95 2.54 3.335 (2) 142 C68---H68···Cl1^iii^ 0.95 2.79 3.5922 (19) 142 C72---H72···O6 0.95 2.50 3.293 (2) 141 C71---H71···*Cg* 0.95 2.99 3.813 (2) 145 C55---H55*A*···*Cg*8′^iv^ 0.98 2.94 3.632 (2) 129 C27---H27*A*···*Cg*7′^v^ 0.98 3.03 3.585 (2) 117 C20---H20*B*···*Cg*6′^vi^ 0.99 3.12 3.6054 (17) 111 C48---H48*B*···*Cg*5′^vi^ 0.99 3.13 3.6180 (17) 112 --------------------------- --------- --------- ------------- --------------- Symmetry codes: (i) −*x*, −*y*+2, −*z*+1; (iii) *x*, *y*, *z*+1; (iv) −*x*+1, −*y*+2, −*z*+2; (v) −*x*+1, −*y*+2, −*z*+1; (vi) −*x*, −*y*+1, −*z*+1. ![The molecular structure of (I)[](#scheme1){ref-type="chem"}, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level and H atoms are omitted for clarity. The inversion-related halves of molecules *A* and *B* are generated by symmetry operations (i) −*x*, −*y* + 2, −*z* + 1 and (ii) −*x*, −*y* + 2, −*z* + 2, respectively.](e-72-01503-fig1){#fig1} ![Molecules *A* and *B* showing intramolecular aromatic stacking. Dashed lines indicate the interactions between naphathaleneimide centroids *Cg*1 \[C13--C17/N2 (*A*)\] and *Cg*3 \[C41--C45/N5 (*B*)\] and aryl centroids *Cg*2 \[C1--C5/C10 (*A*)\] and *Cg*4 \[C29--C33/C38 (*B*)\]. Benzyl and methyl groups and H atoms are omitted.](e-72-01503-fig2){#fig2} ![A view of the supramolecular chain extending along the *c* axis, generated by C---H⋯O hydrogen bonds (dashed lines).](e-72-01503-fig3){#fig3} ![A perspective view along the *c* axis of the supramolecular nanotube generated by cooperative C---H⋯π and offset π--π interactions, showing filling by 1,2-dichlorobenzene molecules.](e-72-01503-fig4){#fig4} ![(*a*) A view of the C---H⋯π and offset π--π interactions between adjacent benzyl groups; (*b*) A view of additional C---H⋯π interactions between dansyl amide moieties. Hydrogen atoms not involved in the hydrogen-bonding interactions are omitted.](e-72-01503-fig5){#fig5} ###### Hydrogen-bond geometry (Å, °) *Cg*, *Cg*5′, *Cg*6′, *Cg*7′ and *Cg*8′ are the centroids of atoms C63--C68, C21--C26, C49--C54, C1--C10 and C29--C38, respectively. *D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A* -------------------------- --------- ------- ------------- ------------- C8---H8⋯O5 0.95 2.53 3.1926 (19) 127 C19---H19⋯O7 0.95 2.55 3.2615 (19) 132 C31---H31⋯O3^i^ 0.95 2.59 3.3818 (19) 141 C47---H47⋯O3^ii^ 0.95 2.58 3.2148 (19) 125 C60---H60⋯O2 0.95 2.54 3.335 (2) 142 C68---H68⋯Cl1^ii^ 0.95 2.79 3.5922 (19) 142 C72---H72⋯O6 0.95 2.50 3.293 (2) 141 C71---H71⋯*Cg* 0.95 2.99 3.813 (2) 145 C55---H55*A*⋯*Cg*8′^iii^ 0.98 2.94 3.632 (2) 129 C27---H27*A*⋯*Cg*7′^iv^ 0.98 3.03 3.585 (2) 117 C20---H20*B*⋯*Cg*6′^v^ 0.99 3.12 3.6054 (17) 111 C48---H48*B*⋯*Cg*5′^v^ 0.99 3.13 3.6180 (17) 112 Symmetry codes: (i) ; (ii) ; (iii) ; (iv) ; (v) . ###### Experimental details -------------------------------------------------------------------------- -------------------------------------------------- Crystal data Chemical formula C~56~H~50~N~6~O~8~S~2~·3C~6~H~4~Cl~2~ *M* ~r~ 1440.11 Crystal system, space group Triclinic, *P* Temperature (K) 100 *a*, *b*, *c* (Å) 12.17737 (14), 17.2876 (2), 17.8916 (2) α, β, γ (°) 110.9544 (12), 96.2760 (11), 103.5159 (10) *V* (Å^3^) 3341.91 (8) *Z* 2 Radiation type Cu *K*α μ (mm^−1^) 3.44 Crystal size (mm) 0.20 × 0.10 × 0.05 Data collection Diffractometer Agilent SuperNova, Dual, Cu at zero, EosS2 Absorption correction Multi-scan (*CrysAlis PRO*; Agilent, 2014[@bb2]) *T* ~min~, *T* ~max~ 0.874, 1.000 No. of measured, independent and observed \[*I* \> 2σ(*I*)\] reflections 54279, 13086, 11914 *R* ~int~ 0.026 (sin θ/λ)~max~ (Å^−1^) 0.620 Refinement *R*\[*F* ^2^ \> 2σ(*F* ^2^)\], *wR*(*F* ^2^), *S* 0.034, 0.097, 1.03 No. of reflections 13086 No. of parameters 869 H-atom treatment H-atom parameters constrained Δρ~max~, Δρ~min~ (e Å^−3^) 0.74, −0.45 -------------------------------------------------------------------------- -------------------------------------------------- Computer programs: *CrysAlis PRO* (Agilent, 2014[@bb2]), *SHELXS97* (Sheldrick, 2008[@bb19]), *SHELXL2014/7* (Sheldrick, 2015[@bb20]), *DIAMOND* (Brandenburg, 1999[@bb24]) and *OLEX2* (Dolomanov *et al.*, 2009[@bb7]).

{ "pile_set_name": "PubMed Central" }

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Introduction {#s1} ============ Soybean meal (SBM) is an excellent plant protein source for fish feed [@pone.0058115-Lilleeng1]. However, high levels of soybean protein caused a poor growth rate in juvenile Jian carp (*Cyprinus carpio* var. Jian) [@pone.0058115-Zhang1] and juvenile tilapia (*Oreochromis niloticus×O. aureus*) [@pone.0058115-Lin1]. In addition, SBM has been shown to decrease intestinal protease activity in juvenile tilapia [@pone.0058115-Lin1] and reduce intestinal alkaline phosphatase (AKP) activity in Jian carp [@pone.0058115-Zhang2]. These negative influences were partly due to the anti-nutritional factors present in SBM, such as β-conglycinin, glycinin, and trypsin inhibitor [@pone.0058115-Buttle1]. Among these anti-nutrients, β-conglycinin was suggested as being one of the key anti-nutritional factors [@pone.0058115-Ostaszewska1]. A recent study has reported that β-conglycinin reduced weight gain in pigs [@pone.0058115-Zhao1]. However, little attention has been given to the potential negative effects of β-conglycinin on the growth, digestive and absorptive ability of fish. The reductions in digestive and absorptive ability may be associated with a diminished growth of the intestine; it has been reported that β-conglycinin decreased the villus height in piglet intestines [@pone.0058115-Hao1]. Protein not only provides architectural support for cells but also serves vital roles in maintaining their function and survival, and protein deposition is a result of protein synthesis and degradation [@pone.0058115-Liu1]. It was demonstrated that the target of rapamycin (TOR) signaling pathway plays an important role in protein synthesis and degradation in terrestrial animals, and the eIF4E-binding protein (4E-BP) is one of the major downstream targets of TOR protein [@pone.0058115-Schmelzle1]. Our laboratory was the first to clone the cDNA of TOR (GenBank accession no. FJ899680) and 4E-BP2 (GenBank accession no. HQ010440) of Jian carp and to show that the expressions of these two genes were influenced by choline [@pone.0058115-Wu1] and arginine [@pone.0058115-Chen1]. Accordingly, further investigation is warranted into whether β-conglycinin causes poor growth of digestive organs via changing the TOR and 4E-BP gene expression. The decreased growth, development and function of the digestive organs may be partly due to the fact that β-conglycinin damages the integrity of the intestine. Previous studies have shown that β-conglycinin causes pathological disruption in the intestines of rats [@pone.0058115-Guo1] and piglets [@pone.0058115-Hao1]. Enterocytes are the primary cells lining the intestine [@pone.0058115-AlHussaini1], and increasing levels of β-conglycinin damaged the integrity of mouse enterocytes, leading to LDH releasing into the culture medium [@pone.0058115-Xu1]. Thus, we hypothesize that β-conglycinin may also injure the intestine and its enterocytes in fish, a theory that requires further investigation. It has been shown in pigs that the intestinal injury caused by β-conglycinin results from the effect of this allergen as an inducer of stress [@pone.0058115-Chen2]. Allergies usually cause inflammatory disorders [@pone.0058115-Li1]; for example, it has been reported that β-conglycinin promoted the secretion of inflammatory cytokines (IL-8) in mouse enterocytes [@pone.0058115-Xu1]. In addition, transforming growth factor β (TGF-β) was identified as an important factor in the prevention of intestinal mucosal inflammation in humans and mice [@pone.0058115-Strober1]. A study of Atlantic salmon (*Salmo salar* L.) showed that the TGF-β gene level was significantly decreased by dietary SBM [@pone.0058115-Lilleeng2]. However, little attention has been given to the potential negative effects of β-conglycinin on these inflammatory factors in fish. Inflammation is always accompanied by lipid oxidation, the end products of which are generated during inflammation in mice [@pone.0058115-West1]. Thus, it appears that β-conglycinin-induced intestinal damage may be involved in lipid peroxidation. In addition, lipid peroxidation may bring about protein damage due to its end products [@pone.0058115-Bhor1]. Protein carbonyl content is the most widely used biomarker for oxidative damage to proteins and reflects cellular damage [@pone.0058115-Baltacolu1]. Like other organisms, fish combat elevated levels of oxidative stress with protective enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione-dependent enzymes [@pone.0058115-Bhor1]. However, studies referring to the effects of β-conglycinin on the antioxidant system are scarce. To our knowledge, only this study has examined the activities and mRNA levels of antioxidant enzymes in assessing the antioxidant defenses challenged with β-conglycinin. To this context, we hypothesize that β-conglycinin may reduce fish growth through damaging digestive organ which was in part attributed to antioxidant disturbance. Thus, efficient antioxidant becomes of great important. Glutamine (Gln), a conditionally essential amino acid, appears to be a key nutrient for the gut in mammalian [@pone.0058115-Satoh1]. Our laboratory has demonstrated that dietary Gln supplementation increased the intestinal weight and alkaline phosphosphate activity, and thus improved fish intestinal structure and function in Jian carp [@pone.0058115-Lin2]. Furthermore, Gln serves as a metabolic precursor for glutathione (GSH) which can efficiently reduce ROS [@pone.0058115-Johnson1], [@pone.0058115-Wasa1]. It can protect fish enterocytes against H~2~O~2~-induced oxidative damage [@pone.0058115-Chen3]. Thus, we set up a treatment that administrated Gln along with β-conglycinin to study the hypothesis that Gln can protect fish against β-conglycinin toxicity. Together, this study investigated the hypothesis that β-conglycinin might decrease the growth of fish via dysfunction of digestion and absorption which was in part attributed to the digestive organ damage induced by β-conglycinin-mediated inflammation and oxidative stress. To our knowledge, this study provides the first insight into the underlying mechanism by which β-conglycinin decreases digestive and absorptive ability and damages intestinal integrity in fish. The potential protective effect of Gln against β-conglycinin toxicity was also investigated. Materials and Methods {#s2} ===================== Growth Trial (*in vivo*) {#s2a} ------------------------ Purified β-conglycinin was kindly donated by the China Agricultural University (Patent No. 200410029589·4, China) and was analyzed following the method of [@pone.0058115-Hao1] to be 80% pure. The test diets are presented in [Table S1](#pone.0058115.s001){ref-type="supplementary-material"}. The control diet, devoid of soybean protein, contained fish meal, gelatin and casein as the dietary protein sources. β-conglycinin (80 g β-conglycinin/kg diet) was substituted for the casein in the corresponding experimental diets, following the method of [@pone.0058115-Sun1]. β-conglycinin accounts for about 30% of the total soybean proteins [@pone.0058115-Utsumi1]. Our previous study found that diet with 517.8 g/kg of dehulled soybean meal (CP = 47%) (approximately 73 g/kg of β-conglycinin) significantly depressed the growth, feed efficiency, intestinal weight, length, length index and folds height and the intestinal AKP and γ-GT activities in Jian carp [@pone.0058115-Zhang1], [@pone.0058115-Zhang2]. Therefore, to investigate the toxic effects and potential toxic mechanisms of β-conglycinin in fish, 80.0 g/kg of β-conglycinin was used in this study, which was proved to be enough to induce toxicity in fish by a preliminary experiment (unpublished data). β-conglycinin with 12.0 g/kg of the Gln group was established as a positive control. The concentration of Gln was the optimal dose for the growth of Jian carp [@pone.0058115-Lin2]. All diets were made isonitrogenous with the addition of appropriate amounts of glycine according to the method of [@pone.0058115-Lin2]. Lysine, methionine, threonine, pyridoxine, pantothenic acid, inositol, thiamin, riboflavin, zinc and iron were prepared to meet the nutrient requirements of juvenile Jian carp according to our laboratory studies [@pone.0058115-Zhou1]--[@pone.0058115-Ling1]. The levels of other nutrients met the requirements of common carp (*Cyprinus carpio* L.) according to the NRC (1993) [@pone.0058115-NationalResearch1]. Diets were prepared by thoroughly mixing all the ingredients. Distilled water (approximately 40%, w/w) was added to the premix dry ingredients and further thoroughly mixed. Slow sinking pellets were wet-extruded through a laboratory-scale, single screw extruder. The machine was continually cooled by a cooling wet paper tower. Thus, the temperature of wet-extruded pellets was controlled to be between 55 and 65°C. The noodle-like diets were immediately freeze-dried at −55°C. After freeze-drying, the pellets were stored at −20°C until feeding according to the method described by [@pone.0058115-Salze1]. The Animal Care and Use Committee of Sichuan Agricultural University approved all experimental procedures. Juvenile Jian carp (*Cyprinus carpio* var. Jian) purchased from Tong Wei Hatchery (Sichuan, China) were used in this experiment. Fish were acclimatized to the experimental environment for 4 weeks, and were fed with a carp feed (without soybean protein, prepared in our laboratory, crude protein = 34%, crude lipid = 4.6%). Four hundred and fifty fish, with a mean initial weight of 5.36±0.02 g, were randomly assigned to each of 9 experimental aquaria (90 L×30 W×40 H cm). Aquaria were supplied with flow-through water at a rate of 1.2 L/min; water was drained through biofilters to remove solid substances and reduce ammonia concentration. Water temperature was maintained at 23±1°C. For the feeding trial, each of the diets was fed to a triplicate of fish six times per day for the first 4 weeks and four times per day from the 5^th^ to 6^th^ week, a feeding rhythm that was established in previous study [@pone.0058115-Xiao1]. The fish were fed their respective diets to apparent satiation. Uneaten feed was removed by siphoning thirty minutes after feeding, dried and weighed to calculate feed intake. Fish from each aquarium were counted and weighed at the beginning and end of the experiment. At the end of the experiment, fish were anaesthetized in benzocaine bath (50 mg/L) 12 h after the last feeding as described by [@pone.0058115-Bohne1]; the intestines and hepatopancreas were removed quickly, weighed, frozen in liquid nitrogen and stored at −70°C until analysis. *In vitro* Experiment {#s2b} --------------------- The procedures for cell isolates and cultures were based on those of [@pone.0058115-Jiang2] and [@pone.0058115-Bicho1], with slight modifications. Healthy Jian carp were maintained for approximately 24 h without feeding and were killed by decapitation. The intestines were rapidly removed from the carcass, opened and rinsed with Hanks Balanced Salt Solution (HBSS). The enterocytes were isolated by enzymatic dissociation. Cells were suspended in Dulbecco's Modified Eagle's Media (DMEM) and washed with the same medium 5 times to remove enzymes. Isolated enterocytes were plated in high-glucose DMEM supplemented with 10% FBS, 0.02 mg transferring/mL (Sigma, St. Louis, MO, USA), 0.01 mg insulin/mL (Sigma, St. Louis, MO, USA), 100 IU benzyl penicillin/mL (Sigma, St. Louis, MO, USA) and 100 µg streptomycin sulfate/mL (Sigma, St. Louis, MO, USA). The cells (2×10^3^ cells per well) were seeded in 24-well culture plates (Falcon, Franklin Lake, NJ, USA) that had been previously coated with collagen (Sigma, St. Louis, MO, USA), as previously described by [@pone.0058115-Jiang3]. Cultures were kept at 26±0.5°C. The β-conglycinin treatment was performed by adding fresh medium containing 0, 0.25, 0.6, 0.9, 1.2 and 2.5 mg/mL for 36 h. Four replicate wells were prepared for each concentration of β-conglycinin (n = 4). The second *in vitro* trial was based on the first *in vitro* trial and was performed similarly, except for the concentrations of β-conglycinin (0, 1.2, 2.5, 3.5, 4.5 and 5.5 mg/mL). Analysis and Measurement Methods {#s2c} -------------------------------- ### Investigated indexes {#s2c1} The *in vivo* experiment investigated the hepatopancreas weight (HW), protein content (HPC), hepatopancreas somatic index (HSI) and intestinal weight (IW), protein content (IPC), length (IL), intestinal length index (ILI), intestinal somatic index (ISI) and folds height. In addition, digestive ability (intestinal trypsin, chymotrypsin, lipase and amylase activities) and absorptive function (creatinkinase, Na^+^,K^+^-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase (γ-GT) in the proximal (PI), mid (MI) and distal intestine (DI)) were studied. Furthermore, malondialdehyde (MDA), protein carbonyls (PC), superoxide dismutase (SOD), catalase (CAT), glutathione-*S*-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in the hepatopancreas and intestine were also investigated. Finally, gene expression of TOR and 4E-BP in the PI, MI and DI and interleukin-1 (IL-1), IL-8, tumor necrosis factor-α (TNF-α) and TGF-β genes in the distal intestine (DI) as well as the whole intestinal GPx1a, GPx1b, GR, CuZnSOD, MnSOD and CAT were investigated. The first *in vitro* trial investigated the cell viability (MTT), protein retention (PR), glutamate oxaloacetate transaminase (GOT) in enterocytes and LDH, GOT and glutamate pyruvate transaminase (GPT) activities in culture medium. The second *in vitro* trail investigated the oxidative damage indexes (including LDH and MDA in medium and cellular PC) and the cellular functional indexes (AKP) as well as cell antioxidant indexes (including SOD, CAT, GPx, GST, GR and GSH). ### Digestive and absorptive analysis {#s2c2} At the end of the growth trail, the intestines of 5 fish from each aquarium were sampled and fixed with formalin (10%), sectioned and stained with H-E stain for histological analysis (height of intestinal folds) according to the method described by Lin and Zhou [@pone.0058115-Lin2]. Samples of the intestine and hepatopancreas were each homogenized in 10 volumes (w/v) of ice-cold physiological saline solution and centrifuged at 6,000×***g*** for 20 min at 4°C, respectively. After centrifugation, the supernatant was used for determination of trypsin [@pone.0058115-Hummel1], chymotrypsin [@pone.0058115-Hummel1], lipase [@pone.0058115-Furne1], amylase [@pone.0058115-Refstie1], alkaline phosphatase (AKP) [@pone.0058115-Krogdahl1] and Na^+^, K^+^-ATPase activities [@pone.0058115-Ilenchuk1]. Gamma-glutamyl transpeptidase (γ-GT) activities in the intestine were determined by using a γ-GT Assay Kit (Sigma, St Louis, MO). Protein content was determined following the Bradford method [@pone.0058115-Bradford1]. ### MTT cell viability assay {#s2c3} Cell proliferation was determined by assaying the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to formazan, following the method described by [@pone.0058115-Daly1]. Briefly, at the end of the experiment, the medium was removed and 500 µL of MTT working solution was added to the cultures. After incubation for 4 h, the MTT working solution was removed and replaced with 500 µL of dimethyl sulphoxide (Sigma) to dissolve the formazan precipitates. The amount of formazan was determined by measuring the optical density (OD) at 595 nm on a plate reader (Thermo Labsystems Oy, Helsinki, Finland). ### Cell injury analysis {#s2c4} The amount of LDH released by the cells was measured following the method of [@pone.0058115-Mulier1]. Lipid peroxidation was analyzed as described by [@pone.0058115-Zhang3] and measured in terms of malondialdehyde (MDA) equivalents using the thiobarbituric acid (TBA) reaction. In brief, samples were mixed with trichloroacetic acid and centrifuged. Then, TBA was added to the supernatant. The mixture was heated in water at 95°C for 40 minutes. MDA forms a red adduct with TBA, which has an absorbance of 532 nm. The cellular protein carbonyl (PC) content was determined according to the method described by [@pone.0058115-Baltacolu1], with a minor modification using the 2,4-dinitrophenylhydrazine (DNPH) reagent. The carbonyl content was calculated from the peak absorbance at 340 nm, using an absorption coefficient of 22,000/M/cm. ### Antioxidant enzyme activities and GSH content {#s2c5} The abilities of the anti-superoxide anion (ASA) and the anti-hydroxyl radical (AHR) (OH-scavenging ability) were determined following the instructions of the specific kits (Jiancheng Bioengineering Ltd., Nanjing, China). Briefly, superoxide radicals (O~2~ ^−^) were generated by the action of xanthine and xanthine oxidase. With the addition of an electron acceptor, a coloration reaction (absorbance at 550 nm) was developed using the gross reagent. Vitamin C was used as the standard agent. AHR was then assayed based on the Fenton reaction (Fe^2+^+H~2~O~2~→Fe^3+^+OH^−^+•OH). A coloration reaction (absorbance at 550 nm) was also developed using the gross reagent. CAT activities were measured, as have previously described [@pone.0058115-Jiang4]. The superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were assayed as described by [@pone.0058115-Zhang3]. The glutathione-*S*-transferase (GST) activity was measured by monitoring the formation of an adduct between GSH and 1-chloro-2,4-dinitrobenzene (CDNB) [@pone.0058115-Lushchak1]. GSH content was determined by the method described by [@pone.0058115-Vardi1] with a minor modification. ### Analysis of gene expression {#s2c6} The total RNA of samples was extracted using RNAiso Plus (TaKaRa Biotechnology, Dalian Co. Ltd., China, D9108B), according to the manufacturer's instructions. RNA quantity and quality were assessed by electrophoresis on 1% agarose gels and by spectrophotometry at 260 and 280 nm. Subsequently, cDNA was synthesized using a PrimeScript™ RT reagent Kit (Takara, Dalian, Liaoning, China), according to manufacturer's instructions. Briefly, oligo dT primers (50 µM) were used to reverse transcribe respective RNAs in the presence of PrimeScript™ RT enzyme Mix I, 5×PrimeScript™ buffer, Random 6 mers (100 µM) and RNase free dH~2~O at 37°C for 15 min, following inactivation at 85°C for 5 s. Specific primers for genes were designed with Primer Premier software (Premier Biosoft International, Palo Alto, CA, USA), according to the Jian carp sequences (except for IL-1, IL-8, TNF-α and TGF-β, the other genes were cloned and submitted to NCBI by our laboratory) ([Table S2](#pone.0058115.s002){ref-type="supplementary-material"}). Real-time PCRs were performed with a chromo 4™ continuous fluorescence detector (Bio-Rad Laboratories, Inc.) using a SYBR PrimeScriptTM RT-PCR Kit II, according to standard protocols. The expression levels of these genes were normalized to the expression levels of a housekeeping gene, β-actin ([Table S2](#pone.0058115.s002){ref-type="supplementary-material"}). Each assay was performed with 5 replications. The concentrations of the target genes were calculated based on the threshold cycle number (CT). The CT for each sample was determined by using MJ Option Monitor Software (version 3.1; Bio-Rad, Hemel Hempstead, Herts, UK). In addition, the cDNA concentration in each sample was determined according to gene-specific standard curves. Standard curves were generated for both the target and endogenous control genes based on 10-fold serial dilutions. All standard curves exhibited correlation coefficients higher than 0.99, and the corresponding real-time PCR efficiencies ranged from 0.90--1.10. Statistical Analysis {#s2d} -------------------- All data are presented as the mean ± standard deviation (SD). The data were subjected to a one-way analysis of variance (ANOVA). If significant differences were found (*P*\<0.05), Duncan's multiple range tests were used to rank the groups. TNF-α and TGF-β mRNA levels were evaluated with Student's *t*-test. All statistical analyses were performed using the SPSS 13.0 for Windows (SPSS Inc, Chicago, IL, USA). Results {#s3} ======= *In vivo* Experiment {#s3a} -------------------- Initial body weight (IBW), final body weight (FBW), special growth ratio (SGR), survival rate, feed intake (FI), feed efficiency (FE), hepatopancreas weight (HW), protein content (HPC) and somatic index (HSI) and intestinal weight (IW), intestinal protein content (IPC), intestinal length (IL), intestinal length index (ILI) and intestinal somatic index (ISI) and folds height in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) are presented in [Table 1](#pone-0058115-t001){ref-type="table"}. β-conglycinin alone significantly reduced FBW by approximately 15.1% compared with the control, but co-treatment with Gln completely blocked the reduction of FBW caused by β-conglycinin. Similarly, β-conglycinin alone also significantly reduced SGR compared with the unexposed control (*P\<*0.05). The SGR significantly increased by co-treatment with Gln compared with that of fish exposed to β-conglycinin alone (*P\<*0.05); the value was equivalent to the control. Survival was not affected by dietary treatments. The FI was lowest in fish exposed to β-conglycinin alone, and significantly recovered by co-treatment with Gln. The FE was higher in fish fed the control diet than in fish fed with β-conglycinin alone. HW was highest in fish fed the control diet, followed by fish co-fed with Gln and β-conglycinin, and was lowest in fish fed with β-conglycinin alone. HPC was higher in fish co-treated with Gln and β-conglycinin than in fish fed with β-conglycinin alone. Fish exposed to dietary β-conglycinin experienced a decrease in HSI compared with the control. There was no significant difference between the combinations and the β-conglycinin alone. IPC was lower in fish exposed to dietary β-conglycinin alone than in other groups. IW and IL were significantly reduced by dietary β-conglycinin alone compared with the unexposed control but was partly recovered by supplementation with dietary Gln. In addition, ILI was also significantly reduced by β-conglycinin alone compared with the unexposed control. However, there was no significant alteration in ISI among the treatments. The folds height in the PI was significantly reduced by dietary β-conglycinin alone compared with the unexposed control, but was partly recovered by supplementing the diet with Gln (*P\<*0.05). The folds height in the MI and DI were lower in fish exposed to β-conglycinin alone than in that of the control. 10.1371/journal.pone.0058115.t001 ###### Initial body weight (IBW), final body weight (FBW), special growth ratio (SGR), survival rate, feed intake (FI), feed efficiency (FE), hepatopancreas weight (HW), protein content (HPC) and somatic index (HSI) and intestinal weight (IW), intestinal protein content (IPC), intestinal length (IL), intestinal length index (ILI) and intestinal somatic index (ISI) and folds height (µm) in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of juvenile Jian carp (*Cyprinus carpio* var. Jian) exposed to dietary β-conglycinin for 42 days (*in vivo* experiment). ![](pone.0058115.t001){#pone-0058115-t001-1} Indexes Control β-conglycinin alone β-conglycinin plus Gln ------------------ ----------------- --------------------- ------------------------ IBW (g/fish) 5.37±0.04^a^ 5.36±0.01^a^ 5.36±0.01^a^ FBW (g/fish) 28.68±0.92^a^ 24.34±0.86^b^ 27.15±0.60^a^ SGR 3.99±0.09^a^ 3.60±0.08^b^ 3.86±0.05^a^ Survival rate 100.00±0.00^a^ 100.00±0.00^a^ 100.00±0.00^a^ FI (g/fish) 30.21±0.05^a^ 26.42±0.07^c^ 28.79±0.01^b^ FE 87.69±3.50^a^ 81.60±3.47^b^ 86.01±2.35^ab^ HW (g/fish) 1.18±0.22^a^ 0.60±0.12^c^ 0.73±0.24^b^ HPC 4.15±0.20^ab^ 4.01±0.32^b^ 4.36±0.19^a^ HSI 3.56±0.40^a^ 3.07±0.34^b^ 3.39±0.86^ab^ IW (g/fish) 1.10±0.20^a^ 0.62±0.15^c^ 0.75±0.19^b^ IPC 2.35±0.16^a^ 1.99±0.13^b^ 2.41±0.21^a^ IL (cm/fish) 17.93±2.34^a^ 14.10±1.58^c^ 15.09±2.08^b^ ILI 170.22±21.54^a^ 160.42±13.04^b^ 164.49±18.85^ab^ ISI 3.23±0.27^a^ 3.13±0.39^a^ 3.25±0.36^a^ PI folds heights 700.44±70.72^a^ 536.80±22.42^c^ 633.53±37.97^b^ MI folds heights 443.58±42.94^a^ 378.74±23.12^b^ 425.99±46.47^ab^ DI folds heights 524.14±46.60^a^ 447.93±45.50^b^ 482.20±34.71^ab^ Data represent means ± S.D. Values within the same row with different superscripts are significantly different (*P*\<0.05). SGR = 100× (ln final weight - ln initial weight)/number of days. FE = 100× (g weight gain/g feed intake). HPC = 100×(g hepatopancreas protein/g wet hepatopancreas weight). HSI = 100×(g wet hepatopancreas weight/g wet body weight). IPC = 100×(g intestine protein/g wet intesitine weight). ILI = 100× (cm intestine length/cm total body length). ISI = 100×(g wet intestine weight/g wet body weight). Intestinal trypsin, chymotrypsin, amylase and lipase activities are presented in [Table 2](#pone-0058115-t002){ref-type="table"}. The intestinal trypsin of fish fed with β-conglycinin and Gln was significantly increased compared with that of the group fed β-conglycinin alone (*P\<*0.05); the value was equivalent to fish not exposed to β-conglycinin. The intestinal chymotrypsin and lipase activities reflected the same pattern in regard to intestinal trypsin activity. No significant differences were observed in intestinal amylase activities among the treatments. 10.1371/journal.pone.0058115.t002 ###### Intestinal digestive enzyme activity of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (*in vivo* experiment). ![](pone.0058115.t002){#pone-0058115-t002-2} Indexes Control β-conglycinin alone β-conglycinin plus Gln --------------------------- ------------------ --------------------- ------------------------ Trypsin (U/g tissue) 2.41±0.07^a^ 2.14±0.24^b^ 2.36±0.08^a^ Chymotrypsin (U/g tissue) 2.85±0.26^a^ 1.38±0.15^b^ 2.65±0.30^a^ Lipase (U/g tissue) 373.29±45.57^a^ 176.56±29.76^b^ 322.84±49.43^a^ Amylase (U/g tissue) 1309.52±52.42^a^ 1295.24±71.52^a^ 1323.81±56.18^a^ Data represent means ± S.D. of six replicates. Values within the same row with different superscripts are significantly different (*P*\<0.05). CK, Na^+^, K^+^-ATPase, AKP and γ-GT activities in the PI, MI and DI are presented in [Table 3](#pone-0058115-t003){ref-type="table"}. β-conglycinin alone caused significant decreases in CK and Na^+^, K^+^-ATPase activity in all intestinal segments compared with the unexposed control but was completely recovered by a diet combined with Gln (*P\<*0.05). Similarly, the AKP activity in the PI, MI and DI were lower in fish exposed to β-conglycinin alone than that of the other groups. Conversely, the gamma-glutamyl transpeptidase (γ-GT) activity in all intestinal segments was increased by β-conglycinin. Only the β-conglycinin-induced increase of γ-GT activity in the MI was partly prevented by Gln. 10.1371/journal.pone.0058115.t003 ###### Creatine kinase (CK, µmol of phosphorus released/g tissue/h), Na^+^, K^+^-ATPase (µmol of phosphorus released/g tissue/h), alkaline phosphatase (AKP, mmol of nitrophenol released/g tissue/h) and gamma-glutamyl transpeptidase (γ-GT, mmol of 5-amino-2-nitrobenzoate released/g tissue/min) activity in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (*in vivo* experiment). ![](pone.0058115.t003){#pone-0058115-t003-3} Indexes Intestines Control β-conglycinin alone β-conglycinin plus Gln ------------------------ ------------ ----------------- --------------------- ------------------------ **CK** PI 47.14±4.60^a^ 41.62±2.17^b^ 46.52±3.90^a^ MI 55.77±5.74^a^ 43.36±5.63^b^ 50.68±4.66^a^ DI 22.38±2.37^b^ 16.60±2.49^c^ 30.94±3.12^a^ **Na^+^, K^+^-ATPase** PI 289.06±31.54^a^ 237.63±42.39^b^ 298.83±35.00^a^ MI 220.70±30.23^a^ 158.85±24.29^b^ 195.31±16.01^a^ DI 207.68±36.26^a^ 144.53±54.07^b^ 203.13±45.29^a^ **AKP** PI 26.23±3.19^a^ 21.44±2.52^b^ 25.82±3.38^a^ MI 17.28±2.09^a^ 13.43±2.47^b^ 16.53±1.83^a^ DI 3.30±0.24^a^ 2.83±0.30^b^ 3.28±0.32^a^ **γ-GT** PI 38.37±4.85^b^ 89.77±22.69^a^ 82.91±14.52^a^ MI 65.47±9.02^c^ 214.30±37.74^a^ 134.42±17.60^b^ DI 80.81±11.07^b^ 180.23±26.50^a^ 160.82±23.32^a^ Data represent means ± S.D. of six replicates. Values within the same row with different superscripts are significantly different (*P*\<0.05). The oxidative status of the hepatopancreas and intestines and their antioxidant levels are presented in [Table 4](#pone-0058115-t004){ref-type="table"}. The MDA and PC content in the hepatopancreas and intestine were lower in fish fed the control diet than in the other groups (*P\<*0.05). There was no change between the β-conglycinin group and the Gln plus β-conglycinin group. In addition, except for GST, the antioxidants (SOD, CAT, GPx and GR activities and GSH content) in the hepatopancreas and intestine were reduced by β-conglycinin compared with the unexposed control. The decreases in these antioxidants were completely recovered by supplementation with dietary Gln (*P\<*0.05). There was no significant change in GST activity in the hepatopancreas among the treatments. In addition, GST activity in the intestine was reduced by β-conglycinin (*P\<*0.05), but no significant difference was observed between the β-conglycinin alone group and the Gln plus β-conglycinin group. 10.1371/journal.pone.0058115.t004 ###### Oxidative status (malondialdehyde (MDA, nmol/mg protein), protein carbonyl content (PC, nmol/mg protein) and antioxidative enzyme activities (superoxide dismutase (SOD, U/mg protein), catalase (CAT, U/mg protein), glutathione-*S*-transferase (GST, U/mg protein), glutathione peroxidase (GPx, U/mg protein), glutathione reducase (GR, U/g protein) activities and glutathione (GSH, mg/g protein) content) in the hepatopancreas and intestine of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (*in vivo* experiment). ![](pone.0058115.t004){#pone-0058115-t004-4} Organs Indexes Control β-conglycinin alone β-conglycinin plus Gln -------------------- --------- ---------------- --------------------- ------------------------ **Hepatopancreas** MDA 1.12±0.09^b^ 1.38±0.12^a^ 1.29±0.08^a^ PC 0.71±0.13^b^ 1.08±0.14^a^ 0.96±0.15^a^ SOD 41.66±9.02^a^ 21.25±5.67^b^ 28.91±5.58^b^ CAT 11.40±1.36^a^ 8.78±1.14^b^ 10.76±1.65^a^ GST 32.49±5.13^a^ 27.80±4.31^a^ 29.36±3.79^a^ GPx 404.0±31.3^a^ 313.7±51.0^b^ 358.6±43.0^ab^ GR 39.56±7.13^a^ 25.03±4.48^b^ 34.54±7.57^a^ GSH 24.31±2.66^a^ 19.30±1.75^b^ 20.97±2.95^b^ **Intestine** MDA 2.83±0.31^b^ 3.58±0.40^a^ 3.42±0.18^a^ PC 1.16±0.18^b^ 1.72±0.38^a^ 1.56±0.16^a^ SOD 46.13±7.69^a^ 30.61±4.58^b^ 41.69±2.76^a^ CAT 11.03±0.94^a^ 8.90±1.09^b^ 10.13±0.82^a^ GST 83.42±10.46^a^ 51.98±7.93^b^ 62.84±12.21^b^ GPx 174.9±23.7^a^ 143.1±13.2^b^ 170.8±26.1^a^ GR 91.09±12.86^a^ 67.48±12.26^b^ 82.00±11.08^ab^ GSH 38.21±2.99^a^ 29.82±2.78^b^ 35.49±6.85^ab^ Data represent means ± S.D. of six replicates. Values within the same row with different superscripts are significantly different (*P*\<0.05). The TOR and 4E-BP gene levels in the PI, MI and DI are presented in [Table 5](#pone-0058115-t005){ref-type="table"}. β-conglycinin significantly reduced the levels of TOR mRNA in the PI and MI (*P\<*0.05). However, Gln was found to completely block the reductions in TOR expression caused by β-conglycinin in the PI and partly block them in the MI. The TOR gene expression levels in the DI were not significantly different among the three groups, only showing a tendency to decrease with β-conglycinin. The results indicated that β-conglycinin caused an increase in 4E-BP gene expression in the DI, which could partly be reversed by Gln. However, the levels of 4E-BP gene expression in the PI and MI showed no significant differences among the three groups. 10.1371/journal.pone.0058115.t005 ###### Relative expression of TOR and 4E-BP genes in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of juvenile Jian carp exposed to dietary β-conglycinin for 42 days (*in vivo* experiment). ![](pone.0058115.t005){#pone-0058115-t005-5} Genes Intestines Control β-conglycinin alone β-conglycinin plus Gln ----------- ------------ ------------------ --------------------- ------------------------ **TOR** PI 0.012±0.0032^a^ 0.0075±0.0017^b^ 0.012±0.0022^a^ MI 0.033±0.0047^a^ 0.0086±0.0014^c^ 0.016±0.0026^b^ DI 0.0617±0.0128^a^ 0.0538±0.0152^a^ 0.0573±0.0178^a^ **4E-BP** PI 0.060±0.018^a^ 0.082±0.025^a^ 0.091±0.025^a^ MI 0.0029±0.0005^a^ 0.0024±0.0004^a^ 0.0026±0.0008^a^ DI 0.0041±0.0012^c^ 0.015±0.0036^a^ 0.011±0.0033^b^ Data represent means ± S.D. of five replicates. Values within the same row with different superscripts are significantly different (*P*\<0.05). To investigate the effects of β-conglycinin on the intestinal inflammatory response in fish, the expressions of IL-1, IL-8, TNF-α and TGF-β genes in the DI were determined ([Table 6](#pone-0058115-t006){ref-type="table"}). The highest levels of IL-1, TNF-α and TGF-β genes were observed in the β-conglycinin with Gln group (*P\<*0.05). The relative expressions of IL-8 were reduced by β-conglycinin (*P\<*0.05). However, the mean values of TNF-α and TGF-β mRNA levels among the three groups are largely different. The TNF-α and TGF-β were not significantly affected by β-conglycinin treatment when Duncan's multiple range tests were used ([Table 6](#pone-0058115-t006){ref-type="table"}). However, TNF-α ([Figure 1a](#pone-0058115-g001){ref-type="fig"}) and TGF-β ([Figure 1b](#pone-0058115-g001){ref-type="fig"}) were significantly increased by β-conglycinin treatment when Student's *t* test was used. ![Student's *t-*test of TNF-α (a) and TGF-β mRNA (b) levels.\ Data represent means ± S.D. of five replicates. Values with \* are significantly different (*P*\<0.05).](pone.0058115.g001){#pone-0058115-g001} 10.1371/journal.pone.0058115.t006 ###### Relative expression of interleukin-1 (IL-1), IL-8, tumor necrosis factor-α (TNF-α) and transforming growth factor β (TGF-β) genes in the distal intestine (DI) of juvenile Jian carp (*Cyprinus carpio* var. Jian) exposed to dietary β-conglycinin for 42 days (*in vivo* experiment). ![](pone.0058115.t006){#pone-0058115-t006-6} Genes Control β-conglycinin alone β-conglycinin plus Gln ------- ------------------ --------------------- ------------------------ IL-1 0.0021±0.0004^b^ 0.0014±0.0009^b^ 0.0036±0.0009^a^ IL-8 0.0020±0.0004^a^ 0.00060±0.0002^b^ 0.00040±0.0001^b^ TNF-α 0.0002±0.0001^b^ 0.0012±0.0003^b^ 0.015±0.0021^a^ TGF-β 0.019±0.0045^b^ 0.045±0.012^b^ 0.57±0.19^a^ Data represent means ± S.D. of five replicates. Values within the same row with different superscripts are significantly different (*P*\<0.05). [Table 7](#pone-0058115-t007){ref-type="table"} shows the relative expressions of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR genes in the whole intestine of fish exposed to dietary β-conglycinin. The expressions of CuZnSOD, MnSOD, CAT and GPx1b genes were all increased by β-conglycinin (*P\<*0.05). However, only MnSOD gene expression was completely recovered with Gln supplementation. The levels of GPx1a and GR gene expressions showed similar trends, but there were no significant differences among the three groups. 10.1371/journal.pone.0058115.t007 ###### Relative expression of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR genes in the whole intestine of juvenile Jian carp (*Cyprinus carpio* var. Jian) exposed to dietary β-conglycinin for 42 days (*in vivo* experiment). ![](pone.0058115.t007){#pone-0058115-t007-7} Genes Control β-conglycinin alone β-conglycinin plus Gln --------- ------------------ --------------------- ------------------------ CuZnSOD 0.026±0.0086^b^ 0.037±0.0034^a^ 0.042±0.011^a^ MnSOD 0.0064±0.0020^b^ 0.013±0.0038^a^ 0.0065±0.0016^b^ CAT 0.054±0.0067^b^ 0.095±0.024^a^ 0.075±0.017^ab^ GPx1a 0.32±0.097^a^ 0.36±0.047^a^ 0.34±0.092^a^ GPx1b 0.020±0.0055^b^ 0.057±0.015^a^ 0.050±0.016^a^ GR 0.019±0.0058^a^ 0.020±0.0061^a^ 0.014±0.0041^a^ Data represent means ± S.D. of five replicates. Values within the same row with different superscripts are significantly different (*P*\<0.05). *In vitro* Experiment {#s3b} --------------------- ### First *in vitro* trial {#s3b1} The effects of β-conglycinin on MTT OD, protein retention (PR) and glutamic-oxaloacetic transaminase (GOT) activity in enterocytes, and the activities of LDH, GOT and GPT in medium are presented in [Table 8](#pone-0058115-t008){ref-type="table"}. MTT OD was significantly reduced with higher levels of β-conglycinin, and the lowest MTT OD was observed in cells treated with 2.5 mg/mL of β-conglycinin (*P\<*0.05). Cellular PR was highest in cells incubated in the control medium, and lowest in cells exposed to 2.5 mg/mL of β-conglycinin. Cellular GOT activity was lower in cells exposed to 1.2 and 2.5 mg/mL of β-conglycinin than cells in the other groups. LDH in medium was significantly higher in 2.5 mg/mL of β-conglycinin than LDH in the control group. GOT activities in medium were higher in 1.2 and 2.5 mg/mL of β-conglycinin than in the other treatments. GPT activities in medium were highest in 2.5 mg/mL of β-conglycinin, followed by 1.2 and 0.9 mg/mL in the β-conglycinin groups, and lowest in the 0--0.6 mg/mL of β-conglycinin groups. It should be noted that the cellular PR, cellular GOT, and LDH, GOT and GPT in medium, were not significantly different among the treatments of 0, 0.25, 0.6 and 0.9 mg/mL of β-conglycinin. Thus, we deleted those doses in the second *in vitro* trial. 10.1371/journal.pone.0058115.t008 ###### Effects of β-conglycinin (mg/mL) on MTT OD, protein retention (PR, µg) and glutamic-oxaloacetic transaminase (GOT, U/mg protein) activity in enterocytes and the activities of lactate dehydrogenase (LDH, U/L), GOT (U/L) and glutamic-pyruvic transaminase (GPT, U/L) in medium (the first *in vitro* experiment). ![](pone.0058115.t008){#pone-0058115-t008-8} β-conglycinin MTT OD PR GOT LDH GOT GPT --------------- ----------------- --------------- ------------- ------------ --------------- --------------- 0.00 0.115±0.002^a^ 53.5±2.6^a^ 20.0±3.7^a^ 243±7.6^b^ 6.93±0.53^b^ 6.51±0.92^c^ 0.25 0.108±0.002^b^ 44.3±15.4^ab^ 17.1±4.5^a^ 255±16^ab^ 7.11±1.05^b^ 6.75±0.71^c^ 0.60 0.104±0.002^bc^ 43.9±5.7^ab^ 17.6±6.6^a^ 250±23^ab^ 7.35±1.09^b^ 6.69±0.63^c^ 0.90 0.103±0.002^c^ 40.0±4.6^ab^ 14.4±10^a^ 271±37^ab^ 7.47±0.20^b^ 7.59±0.82^bc^ 1.20 0.101±0.002^c^ 43.6±4.7^ab^ 12.6±4.6^b^ 262±13^ab^ 9.80±1.15^a^ 8.25±0.50^b^ 2.50 0.093±0.007^d^ 35.4±10.7^b^ 12.0±3.4^b^ 280±15^a^ 10.72±0.42^a^ 10.24±0.46^a^ Data represent means ± S.D. of four replicates. Values within the same column with different superscripts are significantly different (*P*\<0.05). ### Second *in vitro* trial {#s3b2} AKP and PC in cells and LDH and MDA in medium are presented in [Table 9](#pone-0058115-t009){ref-type="table"}. AKP activity in cells decreased with higher levels of β-conglycinin, up to 4.5 mg/mL (*P*\<0.05), with no difference shown between 4.5 and 5.5 mg/mL (*P*\>0.05). LDH and MDA content in medium significantly increased with increasing levels of β-conglycinin, up to 4.5 mg/mL (*P*\<0.05), and no differences were found with a further increase in β-conglycinin concentration (*P*\>0.05). Cellular PC increased with higher levels of β-conglycinin, up to 4.5 mg/mL (*P*\<0.05), with no difference found between 4.5 and 5.5 mg/mL of β-conglycinin (*P*\>0.05). 10.1371/journal.pone.0058115.t009 ###### Effects of β-conglycinin (mg/mL) on the activity of alkaline phosphatase (AKP, U/g protein) in enterocytes, lactate dehydrogenase (LDH, U/L) in medium, content of malondialdehyde (MDA, nmol/mL) in medium and protein carbonyls (PC, nmol/mg protein) in enterocytes (the second *in vitro* experiment). ![](pone.0058115.t009){#pone-0058115-t009-9} β-conglycinin AKP in cell LDH MDA PC in cell --------------- --------------- ---------------- --------------- --------------- 0.00 1.97±0.18^a^ 9.13±2.03^d^ 1.30±0.05^d^ 0.50±0.08^a^ 1.20 1.79±0.09^ab^ 10.14±2.34^cd^ 1.39±0.12^d^ 0.53±0.09^a^ 2.50 1.59±0.14^bc^ 18.26±4.06^bc^ 1.58±0.03^c^ 0.66±0.09^b^ 3.50 1.56±0.17^c^ 24.34±6.62^ab^ 1.67±0.16^bc^ 0.85±0.07^c^ 4.50 1.22±0.17^d^ 30.43±7.77^a^ 1.72±0.09^a^ 1.13±0.09^cd^ 5.50 1.02±0.09^d^ 30.42±7.72^a^ 1.81±0.02^a^ 1.15±0.14^d^ Data represent means ± S.D. of four replicates. Values within the same column with different superscripts are significantly different (*P*\<0.05). Cellular SOD, CAT, GPx, GST and GR activities and GSH content are shown in [Table 10](#pone-0058115-t010){ref-type="table"}. SOD activities decreased with higher levels of β-conglycinin, up to 3.5 mg/mL (*P*\<0.05), and plateaued thereafter (*P*\>0.05). A dose-dependent decrease in CAT activities was observed in the presence of β-conglycinin and was lowest when β-conglycinin reached 5.5 mg/mL (*P*\<0.05). GPx activities were highest in the group containing no β-conglycinin, followed by 1.2--4.5 mg/mL, with GPx being lowest in the presence of 5.5 mg/mL of β-conglycinin (*P*\<0.05). GST activity in cells treated with 5.5 mg/mL of β-conglycinin significantly decreased by approximately 34.4%, compared with the cells incubated with no β-conglycinin (*P*\<0.05). GR activity and GSH content also decreased with higher levels of β-conglycinin, up to 2.5 mg/mL (*P*\<0.05), and no differences were found with a further increase in β-conglycinin concentrations (*P*\>0.05). 10.1371/journal.pone.0058115.t010 ###### Activities of SOD (U/mg protein), CAT (U/mg protein), GPx (U/mg protein), GST (U/mg protein), GR (U/g protein) and content of GSH (mg/g protein) in enterocytes exposed to β-conglycinin (mg/mL) (the second *in vitro* experiment). ![](pone.0058115.t010){#pone-0058115-t010-10} β-conglycinin SOD CAT GPx GST GR GSH --------------- -------------- --------------- ----------- --------------- -------------- --------------- 0.00 12.2±1.3^a^ 1.68±0.13^a^ 594±31^a^ 108±13^a^ 63.1±10.4^a^ 12.13±0.52^a^ 1.20 12.0±1.0^a^ 1.63±0.18^a^ 490±19^b^ 91.7±21.6^ab^ 51.8±4.5^ab^ 12.12±0.51^a^ 2.50 9.95±0.94^b^ 1.35±0.20^b^ 461±32^b^ 91.7±7.1^ab^ 47.9±8.7^b^ 11.22±0.50^b^ 3.50 6.04±0.62^c^ 1.26±0.03^bc^ 444±47^b^ 89.2±15.5^ab^ 46.0±8.4^b^ 10.54±0.58^b^ 4.50 5.93±0.73^c^ 1.13±0.07^c^ 443±29^b^ 87.2±10.2^ab^ 46.6±8.5^b^ 10.92±0.51^b^ 5.50 6.02±1.06^c^ 0.87±0.07^d^ 347±25^c^ 70.5±21.0^b^ 48.0±8.7^b^ 10.88±0.25^b^ Data represent means ±S.D. of four replicates. Values within the same column with different superscripts are significantly different (*P*\<0.05). Discussion {#s4} ========== Soybean is a source of high-quality protein due to its relatively well-balanced composition of amino acids [@pone.0058115-Friedman1]. However, soybean is also a dietary allergic source, which threatens individuals [@pone.0058115-Sun1]. β-conglycinin is a primary storage protein and it has been identified as one of the major allergenic proteins in soybean [@pone.0058115-Hao1]. Thus, to define mechanisms whereby excessive soybean induces growth depression in animals; this study investigated the role of purified soybean β-conglycinin, separating its effects from the effects of other anti-nutritional factors. Firstly, the present study observed that fish fed diet with β-conglycinin exhibited poor weight gain, which was also observed in piglets [@pone.0058115-Zhao1], [@pone.0058115-Hao1] and rats [@pone.0058115-Han1]. However, survival was not influenced by the dietary treatments. No reports exist concerning the effects of β-conglycinin on survival rate in fish. Similar findings were observed that replacement of dietary fishmeal with more than 41% of soy protein isolate did not influence the survival, but indeed reduced the growth performance in Pacific white shrimp (*Litopenaeus vannamei*) [@pone.0058115-GamboaDelgado1]. A reduction in feed intake was regarded as the primary factor responsible for the depressed growth observed in rats [@pone.0058115-Jiang1]. In the present study, the feed intake in the β-conglycinin group was significantly lower than that of the control group, which was in accordance with the results for rats [@pone.0058115-Nishi1]. This result suggested that the growth reduction caused by β-conglycinin was most likely attributed to the suppression of feed intake. However, there was no information referring to how β-conglycinin leads to the suppression of feed intake in fish. In rats, it has been reported that β-conglycinin peptone stimulating endogenous cholecystokinin (CCK) release suppresses food intake by inducing a feeling of satiety and by reducing gastric emptying [@pone.0058115-Nishi1]. Besides feed intake, feed utilization efficiency also influences growth performance. In the present study, β-conglycinin depressed feed efficiency, indicating that the reduction of fish growth was partly attributed to the fact that β-conglycinin decreased feed efficiency. Interestingly, when fish were administered Gln with β-conglycinin, growth performances were close to or equal to those in the control group, showing that Gln might mitigate the poor growth caused by β-conglycinin. As an initial step in understanding how β-conglycinin reduced the growth of fish, we examined the digestive and absorptive ability of the fish. In this study, β-conglycinin alone significantly decreased the activities of trypsin, chymotrypsin, lipase, creatine kinase, Na^+^, K^+^-ATPase and alkaline phosphatase in the intestine and its enterocytes. Although no reports exist concerning the effects of β-conglycinin on digestive and absorptive enzyme activity in fish, similar findings were observed that diets containing soybeans might decrease the activity of protease in tilapia [@pone.0058115-Lin1], alkaline phosphatase in Atlantic salmon [@pone.0058115-BakkeMckellep1] and creatine kinase in Atlantic halibut (*Hippoglossus hippoglossus*) [@pone.0058115-Murray1]. In rats, it has been reported reported that β-conglycinin peptone in the lumen stimulating endogenous cholecystokinin (CCK) release [@pone.0058115-Nishi1] which exerts a negative-feedback regulation on pancreatic enzyme secretion. It is well known that feed intake is suppressed by a feeling of satiety and by reducing gastric emptying in rats [@pone.0058115-Nishi1]. Gastric emptying is associated with the digestive and absorptive ability. Those suggested that the depressed digestive ability in this study may be in part contributed to the suppressed feed intake, which needs further investigations. In addition, when compared with those in the control, the digestive and absorptive enzyme activities in the Gln with β-conglycinin group showed no significant differences. These results indicated that dietary Gln exerted a mitigating role against the disruption in intestinal function caused by β-conglycinin. The reduction of digestive and absorptive capacity is usually due to poor growth of the digestive organs. The exocrine pancreas is the main site for digestive enzyme synthesis and secretion in fish [@pone.0058115-Guillaume1]. The present results showed that the weight and index of the hepatopancreas decreased with β-conglycinin. However, there is a lack of information in regard to the effects of β-conglycinin on the hepatopancreas in animals. Similar results were observed in cobia (*Rachycentron canadum*) that were fed diets with soybean products [@pone.0058115-Salze1]. In addition, the disruption of absorptive capacity in fish has been traced to the growth suppression of the intestine, which was reflected by the decrease of the intestinal length index in Atlantic salmon [@pone.0058115-Refstie1]. The present results showed that the intestinal weight, length, length index and folds height in the intestine was decreased with β-conglycinin alone. In the present *in vitro* study, β-conglycinin decreased the MTT OD values of enterocytes in a dose-dependent way. Although there are no reports concerning the effects of β-conglycinin on the growth and development of fish intestine, similar results in regard to decreased jejunum villus height induced by β-conglycinin were observed in rats [@pone.0058115-Han1]. Additionally, when fish were fed with Gln and β-conglycinin, the poor growth of the hepatopancreas and intestine caused by β-conglycinin was partly mitigated. The reduced growth and development of the digestive organs may be partly due to a reduction in protein deposition. This study showed that protein content in the intestine and in its enterocytes was decreased by β-conglycinin. As mentioned above, protein syntheses are regulated by TOR signaling [@pone.0058115-Schmelzle1]. Interestingly, the expressions of the TOR gene in the PI, MI and DI followed similar patterns to the protein content. The patterns of 4E-BP2 mRNA levels showed opposite tendencies to those of the TOR mRNA levels in the PI and DI, indicating that β-conglycinin increased the inhibition of translation. However, this study is the first to investigate the effects of β-conglycinin on the TOR/4E-BP pathway. Thus, the underlying mechanism by which β-conglycinin affects the expression of TOR and 4E-EP genes is largely unknown and needs to be further investigated. Gln can regulate TOR activity in HeLa cells [@pone.0058115-Nicklin1]. Interestingly, fish fed Gln with β-conglycinin presented a partly mitigated reduction of the TOR/4E-BP pathway caused by β-conglycinin. The impairment of intestinal growth and function by β-conglycinin might result from a destruction of intestinal integrity. Damage to the integrity of the intestine is a common phenomenon in animals consuming soybean proteins [@pone.0058115-Sun1]. Recently, β-conglycinin has been shown to result in intestinal epithelium damage in rats [@pone.0058115-Han1]. Moreover, β-conglycinin was shown to cause apoptosis and structural damage to enterocytes in mice [@pone.0058115-Xu1]. When the cell membrane is damaged, the important metabolic enzymes, such as LDH, GOT and GPT are released from the cells [@pone.0058115-Chen3]. Thus, the activity of LDH, GOT and GPT in the culture medium are often used to assess cell damage [@pone.0058115-Xu1], [@pone.0058115-Chen3]. This study showed that when the β-conglycinin level exceeded 2.5 mg/mL, the activities of LDH, GOT and GPT in the culture medium increased, indicating severe damage to the cellular membrane, which agrees with the results of the study on mouse intestinal epithelium cells [@pone.0058115-Xu1]. The intestinal injury caused by β-conglycinin may result from inflammation. It is well known that soybean protein might cause inflammation of the distal intestine in fish [@pone.0058115-Krogdahl1], [@pone.0058115-BakkeMckellep1]. IL-1 and TNF-α are indicated as pro-inflammatory, whereas TGF-β is indicated as an anti-inflammatory cytokine [@pone.0058115-Urn1]. The expression of the TNF-α gene in the β-conglycinin alone group is above the control level ([Figure 1a](#pone-0058115-g001){ref-type="fig"}), suggesting that β-conglycinin induced an inflammatory response in the DI of fish. It is well known that inflammation may lead to increase of water content, which may be an explanation of the decreased intestinal protein content caused by β-conglycinin. However, the expression of IL-8 gene was decreased by β-conglycinin. The reasons for these differences are largely unknown and may be partly associated with the sample period; it was reported that common carp fed SBM sampled at 0, 1, 3 and 5 weeks showed different patterns of IL-1, TNF-α and TGF-β mRNA levels in the DI [@pone.0058115-Urn1]. However, fish fed with Gln and β-conglycinin obtained the highest values of IL-1, TNF-α and TGF-β mRNA levels. TGF-β plays an important role in the prevention of mucosal inflammation [@pone.0058115-Lilleeng2]. Thus, the up-regulation of TGF-β levels by Gln may be involved in protecting against β-conglycinin-induced inflammation. However, these observations need more attention and additional measurements need to be made in other intestinal compartments to explain their significance. Intestinal inflammation may invoke a subsequent peroxidative damage due to excessive ROS production [@pone.0058115-Mahmood1]. In addition, the N-glycan structure of β-conglycinin is essential for the formation of dityrosine bridges, which is a ubiquitous process primarily responsible for oxidative stress [@pone.0058115-Baudry1]. MDA is one of the most easily assayed end products of lipid peroxidation [@pone.0058115-Requena1]. The current results showed that β-conglycinin elevated MDA content in the hepatopancreas and intestine as well as in its enterocytes, supporting the hypothesis that β-conglycinin may induce oxidative damage in these organs. It is known that the end products of lipid peroxidation may bring about protein damage [@pone.0058115-Bhor1]. Protein carbonyls (PC) are the oxidative product of amino acid residue and can be used as a biomarker of oxidative damage to protein [@pone.0058115-Baltacolu1]. In this study, the protein oxidations in the intestine and its enterocytes exhibited a similar trend to those of MDA. The study showed that Gln might protect fish enterocytes against H~2~O~2~-induced oxidative damage [@pone.0058115-Chen3]. However, in this study, lipid peroxidation (MDA) and protein oxidation (PC) in the hepatopancreas and intestine of fish fed with Gln and β-conglycinin only showed reduction trends when compared with those in the β-conglycinin alone group. The reasons for this result are still unknown. It may be partly because the dose of Gln used in this study was not large enough to inhibit β-conglycinin-induced oxidative damage. Several studies had indicated that oxidative damage was often accompanied by the reduction of antioxidant capacity, reflected by GSH content and the inhibition of antioxidant activity, including that of SOD, CAT, GPx, GST and GR [@pone.0058115-Chen3], [@pone.0058115-Jiang2]. Therefore, in the current study, non-enzymatic and enzymatic antioxidant responses were also measured. The current study demonstrated that the content of GSH in the hepatopancreas, intestine and its enterocytes was reduced by β-conglycinin. It has been reported that the gut was one of the major sites for GSH synthesis in rats [@pone.0058115-Cao1]. GSH synthesis in endothelial cells was through a process that required the activity of γ-GT [@pone.0058115-Moellering1]. Thus, in this study, the increase in γ-GT activity may correspond to a first attempt to overcome oxidative stress by producing a high amount of GSH. In agreement with this result, an increase of γ-GT activity was observed in the liver and white muscle of Nile tilapia exposed to ammonia [@pone.0058115-Hegazi1]; however, we do not know why GSH decreased when γ-GT activity increased in this study. We hypothesize that GSH was consumed during the scavenging process for the oxidation induced by β-conglycinin. Moreover, our results showed that with dietary β-conglycinin treatment alone, the activities of SOD, CAT, GPx and GR in the hepatopancreas and intestine and the activity of intestinal GST decreased significantly when compared with those in the control group. Moreover, significant reductions in SOD, CAT, GPx, GST and GR activity were also observed in enterocytes treated with a certain level of β-conglycinin. However, in contrast to the reduced activities of these enzymes, the mRNA levels of the CuZnSOD, MnSOD, CAT and GPx1b genes increased with β-conglycinin. β-conglycinin-induced increases in the mRNA expression of antioxidant enzymes may be related to an adaptive mechanism against stress. Concurring with this hypothesis, an over-expression of cardiac-specific CAT prevents injury to the heart following oxidative damage induced by exposure to the anticancer agent doxorubicin [@pone.0058115-Kang1]. Thus, perhaps these antioxidant enzymes were largely consumed during the defense against the oxidation caused by β-conglycinin. In addition, in the Gln with β-conglycinin group, most antioxidant enzyme activities and gene expressions are close or equal to those values in the control group. Our previous study showed that in enterocytes treated with Gln in the presence of H~2~O~2~, antioxidant enzyme activities were higher than treatment with H~2~O~2~ alone and were close to the values in the control group [@pone.0058115-Chen3], proving that β-conglycinin damages the antioxidant system of fish. Gln is a precursor of nucleotides, glucose, other amino acids and serves as a major fuel for intestinal epithelial cells in mammals [@pone.0058115-Windmueller1]. Recent studies have demonstrated that dietary Gln supplementation could improve growth performance, intestinal structure and function in carp [@pone.0058115-Lin2], as well as enhance the antioxidant defense in hybrid sturgeon (*Acipenser schrenckii ♀×Huso dauricus ♂*) [@pone.0058115-Zhu1]. According to these findings, we investigated the potential protection of Gln against β-conglycinin toxicity in the present study. As results, compared with the β-conglycinin alone group, dietary supplementation with Gln significantly increased SGR, feed intake, feed efficiency, hepatopancreas protein, hepatopancreas weight, intestinal protein, intestinal weight, intestinal length, intestinal length index and foregut folds height, and also significantly increased the activities of intestinal digestive enzymes including trypsin, lipase and chymotrypsin, absorptive enzymes including creatinkinase, Na^+^, K^+^-ATPase and alkaline phosphatase in the PI, MI and DI, as well as antioxidant enzymes including CAT and GR in hepatopancreas and SOD, CAT, GPx in the intestine. Moreover, SGR, intestinal length index, activities of intestinal trypsin, lipase, chymotrypsin, creatinkinase, Na^+^,K^+^-ATPase, alkaline phosphatase and GPx were restored to have no significant difference compared with the control. These results indicated that dietary Gln exerted a protective role against the growth suppression, intestinal integrity damage and function disruption induced by β-conglycinin. Conclusion {#s4a} ---------- The potential action pathway of β-conglycinin-induced poor growth of fish is presented in [Figure 2](#pone-0058115-g002){ref-type="fig"}. Briefly, this study indicates that β-conglycinin can cause oxidative damage (and impair antioxidant system in animals), and thus lead to damage and poor growth of digestive organs, subsequent by dysfunction of digestion and absorption, and finally reduce fish growth, which may provide some information to the mechanism of β-conglycinin-induced negative effects in fish. Moreover, since Gln was observed to mitigate negative influences of β-conglycinin, it is reasonable for us to recommend supplementation with Gln when high levels of SBM (β-conglycinin) are used in fish diets. ![The potential action pathway of β-conglycinin-induced depression of fish growth.](pone.0058115.g002){#pone-0058115-g002} Supporting Information {#s5} ====================== ###### Ingredients and nutrient content of the experimental diets (*in vivo* experiment). (DOC) ###### Click here for additional data file. ###### Real-time PCR primer sequences. (DOC) ###### Click here for additional data file. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Primary responsibility for final content: X-QZ. Read and approved the final manuscript: J-XZ L-YG LF W-DJ S-YK YL KH JJ S-HL LT X-QZ. Conceived and designed the experiments: X-QZ LF. Performed the experiments: J-XZ L-YG W-DJ KH JJ S-HL. Analyzed the data: S-YK YL LT. Wrote the paper: J-XZ W-DJ.

{ "pile_set_name": "PubMed Central" }

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"activity", "GSH", "content", "also", "decreased", "higher", "levels", "P", "differences", "found", "increase", "concentrations", "P", "Activities", "SOD", "protein", "CAT", "protein", "GPx", "protein", "GST", "protein", "GR", "protein", "content", "GSH", "protein", "enterocytes", "exposed", "second", "vitro", "experiment", "SOD", "CAT", "GPx", "GST", "GR", "GSH", "Data", "represent", "means", "four", "replicates", "Values", "within", "column", "different", "superscripts", "significantly", "different", "P", "Discussion", "Soybean", "source", "protein", "due", "relatively", "composition", "amino", "acids", "However", "soybean", "also", "dietary", "allergic", "source", "threatens", "individuals", "primary", "storage", "protein", "identified", "one", "major", "allergenic", "proteins", "soybean", "Thus", "define", "mechanisms", "whereby", "excessive", "soybean", "induces", "growth", "depression", "animals", "study", "investigated", "role", "purified", "soybean", "separating", "effects", "effects", "factors", "Firstly", "present", "study", "observed", "fish", "fed", "diet", "exhibited", "poor", "weight", "gain", "also", "observed", "piglets", "rats", "However", "survival", "influenced", "dietary", "treatments", "reports", "exist", "concerning", "effects", "survival", "rate", "fish", "Similar", "findings", "observed", "replacement", "dietary", "fishmeal", "soy", "protein", "isolate", "influence", "survival", "indeed", "reduced", "growth", "performance", "Pacific", "white", "shrimp", "Litopenaeus", "vannamei", "reduction", "feed", "intake", "regarded", "primary", "factor", "responsible", "depressed", "growth", "observed", "rats", "present", "study", "feed", "intake", "group", "significantly", "lower", "control", "group", "accordance", "results", "rats", "result", "suggested", "growth", "reduction", "caused", "likely", "attributed", "suppression", "feed", "intake", "However", "information", "referring", "leads", "suppression", "feed", "intake", "fish", "rats", "reported", "peptone", "stimulating", "endogenous", "cholecystokinin", "CCK", "release", "suppresses", "food", "intake", "inducing", "feeling", "satiety", "reducing", "gastric", "emptying", "Besides", "feed", "intake", "feed", "utilization", "efficiency", "also", "influences", "growth", "performance", "present", "study", "depressed", "feed", "efficiency", "indicating", "reduction", "fish", "growth", "partly", "attributed", "fact", "decreased", "feed", "efficiency", "Interestingly", "fish", "administered", "Gln", "growth", "performances", "close", "equal", "control", "group", "showing", "Gln", "might", "mitigate", "poor", "growth", "caused", "initial", "step", "understanding", "reduced", "growth", "fish", "examined", "digestive", "absorptive", "ability", "fish", "study", "alone", "significantly", "decreased", "activities", "trypsin", "chymotrypsin", "lipase", "creatine", "kinase", "alkaline", "phosphatase", "intestine", "enterocytes", "Although", "reports", "exist", "concerning", "effects", "digestive", "absorptive", "enzyme", "activity", "fish", "similar", "findings", "observed", "diets", "containing", "soybeans", "might", "decrease", "activity", "protease", "tilapia", "alkaline", "phosphatase", "Atlantic", "salmon", "creatine", "kinase", "Atlantic", "halibut", "Hippoglossus", "hippoglossus", "rats", "reported", "reported", "peptone", "lumen", "stimulating", "endogenous", "cholecystokinin", "CCK", "release", "exerts", "regulation", "pancreatic", "enzyme", "secretion", "well", "known", "feed", "intake", "suppressed", "feeling", "satiety", "reducing", "gastric", "emptying", "rats", "Gastric", "emptying", "associated", "digestive", "absorptive", "ability", "suggested", "depressed", "digestive", "ability", "study", "may", "part", "contributed", "suppressed", "feed", "intake", "needs", "investigations", "addition", "compared", "control", "digestive", "absorptive", "enzyme", "activities", "Gln", "group", "showed", "significant", "differences", "results", "indicated", "dietary", "Gln", "exerted", "mitigating", "role", "disruption", "intestinal", "function", "caused", "reduction", "digestive", "absorptive", "capacity", "usually", "due", "poor", "growth", "digestive", "organs", "exocrine", "pancreas", "main", "site", "digestive", "enzyme", "synthesis", "secretion", "fish", "present", "results", "showed", "weight", "index", "hepatopancreas", "decreased", "However", "lack", "information", "regard", "effects", "hepatopancreas", "animals", "Similar", "results", "observed", "cobia", "Rachycentron", "canadum", "fed", "diets", "soybean", "products", "addition", "disruption", "absorptive", "capacity", "fish", "traced", "growth", "suppression", "intestine", "reflected", "decrease", "intestinal", "length", "index", "Atlantic", "salmon", "present", "results", "showed", "intestinal", "weight", "length", "length", "index", "folds", "height", "intestine", "decreased", "alone", "present", "vitro", "study", "decreased", "MTT", "OD", "values", "enterocytes", "way", "Although", "reports", "concerning", "effects", "growth", "development", "fish", "intestine", "similar", "results", "regard", "decreased", "jejunum", "villus", "height", "induced", "observed", "rats", "Additionally", "fish", "fed", "Gln", "poor", "growth", "hepatopancreas", "intestine", "caused", "partly", "mitigated", "reduced", "growth", "development", "digestive", "organs", "may", "partly", "due", "reduction", "protein", "deposition", "study", "showed", "protein", "content", "intestine", "enterocytes", "decreased", "mentioned", "protein", "syntheses", "regulated", "TOR", "signaling", "Interestingly", "expressions", "TOR", "gene", "PI", "MI", "DI", "followed", "similar", "patterns", "protein", "content", "patterns", "mRNA", "levels", "showed", "opposite", "tendencies", "TOR", "mRNA", "levels", "PI", "DI", "indicating", "increased", "inhibition", "translation", "However", "study", "first", "investigate", "effects", "pathway", "Thus", "underlying", "mechanism", "affects", "expression", "TOR", "genes", "largely", "unknown", "needs", "investigated", "Gln", "regulate", "TOR", "activity", "HeLa", "cells", "Interestingly", "fish", "fed", "Gln", "presented", "partly", "mitigated", "reduction", "pathway", "caused", "impairment", "intestinal", "growth", "function", "might", "result", "destruction", "intestinal", "integrity", "Damage", "integrity", "intestine", "common", "phenomenon", "animals", "consuming", "soybean", "proteins", "Recently", "shown", "result", "intestinal", "epithelium", "damage", "rats", "Moreover", "shown", "cause", "apoptosis", "structural", "damage", "enterocytes", "mice", "cell", "membrane", "damaged", "important", "metabolic", "enzymes", "LDH", "GOT", "GPT", "released", "cells", "Thus", "activity", "LDH", "GOT", "GPT", "culture", "medium", "often", "used", "assess", "cell", "damage", "study", "showed", "level", "exceeded", "activities", "LDH", "GOT", "GPT", "culture", "medium", "increased", "indicating", "severe", "damage", "cellular", "membrane", "agrees", "results", "study", "mouse", "intestinal", "epithelium", "cells", "intestinal", "injury", "caused", "may", "result", "inflammation", "well", "known", "soybean", "protein", "might", "cause", "inflammation", "distal", "intestine", "fish", "indicated", "whereas", "indicated", "cytokine", "expression", "gene", "alone", "group", "control", "level", "Figure", "fig", "suggesting", "induced", "inflammatory", "response", "DI", "fish", "well", "known", "inflammation", "may", "lead", "increase", "water", "content", "may", "explanation", "decreased", "intestinal", "protein", "content", "caused", "However", "expression", "gene", "decreased", "reasons", "differences", "largely", "unknown", "may", "partly", "associated", "sample", "period", "reported", "common", "carp", "fed", "SBM", "sampled", "weeks", "showed", "different", "patterns", "mRNA", "levels", "DI", "However", "fish", "fed", "Gln", "obtained", "highest", "values", "mRNA", "levels", "plays", "important", "role", "prevention", "mucosal", "inflammation", "Thus", "levels", "Gln", "may", "involved", "protecting", "inflammation", "However", "observations", "need", "attention", "additional", "measurements", "need", "made", "intestinal", "compartments", "explain", "significance", "Intestinal", "inflammation", "may", "invoke", "subsequent", "peroxidative", "damage", "due", "excessive", "ROS", "production", "addition", "structure", "essential", "formation", "dityrosine", "bridges", "ubiquitous", "process", "primarily", "responsible", "oxidative", "stress", "MDA", "one", "easily", "assayed", "end", "products", "lipid", "peroxidation", "current", "results", "showed", "elevated", "MDA", "content", "hepatopancreas", "intestine", "well", "enterocytes", "supporting", "hypothesis", "may", "induce", "oxidative", "damage", "organs", "known", "end", "products", "lipid", "peroxidation", "may", "bring", "protein", "damage", "Protein", "carbonyls", "PC", "oxidative", "product", "amino", "acid", "residue", "used", "biomarker", "oxidative", "damage", "protein", "study", "protein", "oxidations", "intestine", "enterocytes", "exhibited", "similar", "trend", "MDA", "study", "showed", "Gln", "might", "protect", "fish", "enterocytes", "oxidative", "damage", "However", "study", "lipid", "peroxidation", "MDA", "protein", "oxidation", "PC", "hepatopancreas", "intestine", "fish", "fed", "Gln", "showed", "reduction", "trends", "compared", "alone", "group", "reasons", "result", "still", "unknown", "may", "partly", "dose", "Gln", "used", "study", "large", "enough", "inhibit", "oxidative", "damage", "Several", "studies", "indicated", "oxidative", "damage", "often", "accompanied", "reduction", "antioxidant", "capacity", "reflected", "GSH", "content", "inhibition", "antioxidant", "activity", "including", "SOD", "CAT", "GPx", "GST", "GR", "Therefore", "current", "study", "enzymatic", "antioxidant", "responses", "also", "measured", "current", "study", "demonstrated", "content", "GSH", "hepatopancreas", "intestine", "enterocytes", "reduced", "reported", "gut", "one", "major", "sites", "GSH", "synthesis", "rats", "GSH", "synthesis", "endothelial", "cells", "process", "required", "activity", "Thus", "study", "increase", "activity", "may", "correspond", "first", "attempt", "overcome", "oxidative", "stress", "producing", "high", "amount", "GSH", "agreement", "result", "increase", "activity", "observed", "liver", "white", "muscle", "Nile", "tilapia", "exposed", "ammonia", "however", "know", "GSH", "decreased", "activity", "increased", "study", "hypothesize", "GSH", "consumed", "scavenging", "process", "oxidation", "induced", "Moreover", "results", "showed", "dietary", "treatment", "alone", "activities", "SOD", "CAT", "GPx", "GR", "hepatopancreas", "intestine", "activity", "intestinal", "GST", "decreased", "significantly", "compared", "control", "group", "Moreover", "significant", "reductions", "SOD", "CAT", "GPx", "GST", "GR", "activity", "also", "observed", "enterocytes", "treated", "certain", 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1. Introduction {#sec1-foods-09-00918} =============== Global interest for natural food colors is on the rise, especially in Asian countries. Historically, food ingredients and spices like yellow turmeric and red pepper powders are easily prone to food adulteration \[[@B1-foods-09-00918]\]. They play very essential roles in traditional cuisines and are valued for their therapeutic properties irrespective of their color. Current innovation and developmental trend in food processing has led to a paradigm interest in food additives and bioactive compounds with particular focus on their nutritional and organoleptic values. Among the bioactive compounds, betalains and phenolics are the most auspicious with plenty of health benefits, making them easily compatible with food fortification and supplementation. The discovery of betanin, betanidin, and indicaxanthin derivatives following their degradation led to the assumption of their unique nature together with betaxanthin (formerly known as flavonoid) and betacyanin (regarded as nitrogenous anthocyanin) \[[@B2-foods-09-00918]\]. This led to them being grouped as betalains in 1968 \[[@B3-foods-09-00918]\]. Among the acknowledged sources of betalains, i.e., beetroot, swiss chard, dragon fruit, and prickly pear \[[@B3-foods-09-00918],[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918]\], beetroot has gained the most attention since, apart from the whole tuber, even their waste parts such as stalk \[[@B7-foods-09-00918]\], peel \[[@B8-foods-09-00918],[@B9-foods-09-00918]\], and pomace \[[@B10-foods-09-00918],[@B11-foods-09-00918]\] are rich in betalains. The ratio of color contents is decisively influenced by specie source, genetic, cultivar, or the environment of cultivation throughout growing, and the stages of harvesting \[[@B4-foods-09-00918],[@B12-foods-09-00918],[@B13-foods-09-00918]\]. Practically, one kilogram of fresh beetroot contains 300--600 mg of betacyanin and 320--420 mg of betaxanthin \[[@B14-foods-09-00918]\]; compared to the flesh, betalain concentration ranges from 40 to 70% higher in the peel and it also depends on genotypes \[[@B15-foods-09-00918]\]. As reducing agents, phenolic compounds can inhibit oxidation reaction catalyzed by enzymes and can stabilize lipid peroxidases \[[@B16-foods-09-00918]\]. Over 10,000 phenolic compounds exist in several kinds of fruits and vegetables, cereals, tea leaves, coffee beans, and their accumulation in the whole parts of the plant including root, stem, bark, flower, and leaf have been noticed \[[@B17-foods-09-00918]\]. Their extraction from various sources has been boosted by emerging technologies these days, particularly microwave radiation. Some of them are twenty cultivars of tomatoes \[[@B18-foods-09-00918]\], six plant species \[[@B19-foods-09-00918]\], four types of spices \[[@B16-foods-09-00918]\], four types of algae (brown macroalgae species) \[[@B20-foods-09-00918]\], and the rest are discussed in the next section with their proper process optimization. Balasundram and coworkers \[[@B21-foods-09-00918]\] reviewed phenolic compound structures found in by-products of fruit and vegetable processing. Extraction of phenolic compounds from these food products, however, remains a developing field as different techniques are continually being explored. Compared to conventional extraction techniques, non-conventional methods are more pronounced for better quality and quantity of desired products due to advantages such as less use of solvent and lower exposure time to high temperature \[[@B22-foods-09-00918]\]. Specifically, major advantages of microwave treatments are the reduction in operation time and solvent consumption (critically possibility of solvent-free processing), less energy consumption as the rays are delivered directly to the matrix, as well as their ease of handling and processing \[[@B23-foods-09-00918]\]. As reported by Cardoso-Ugarte and coworkers \[[@B24-foods-09-00918]\], the same amount of betalain concentration can be achieved by microwave irradiation ten times faster than the conventional ways with betalain yield scaled up to double. However, with dielectric heating, acceleration in the chemical reaction of target compounds such as epimerization, oxidation, and polarization during microwave processing should be considered \[[@B14-foods-09-00918]\]. The aim of this review was to point out the versatility of microwave irradiation in the recovery of bioactive compounds focusing on phenolics and betalains from various kinds of fruits and vegetables for their improved practical application in food and beverages, pharmaceuticals, cosmetics, and other related sectors. 2. Food Color Compounds {#sec2-foods-09-00918} ======================= Functional additives are compulsory in food processing in order to improve or maintain the existing properties of food during processing \[[@B25-foods-09-00918]\]. Bio-colorants have been explored from plants (flower, root, stalk, seed, fruit, peel, leaf, pomace, rhizome, and stigma), insect (cochineal), algae, bacteria, and fungi \[[@B25-foods-09-00918],[@B26-foods-09-00918],[@B27-foods-09-00918]\] and their existence is essential for pollination and seed disposal as insects and birds are attracted by their hues \[[@B28-foods-09-00918]\]. Carotenoids, phenolics, alkaloids, nitrogen-containing compounds, and organosulfur compounds are major classes of bioactive compounds. Among them, carotenoids (E160, E161, E164), chlorophylls (E140, E141), flavonoids (anthocyanin (E163)), and betalains (E162) are commonly used as natural colorants in food and cosmetic industries ([Table 1](#foods-09-00918-t001){ref-type="table"}) \[[@B3-foods-09-00918],[@B25-foods-09-00918],[@B28-foods-09-00918]\]. Since these compounds have characteristic absorption in the visible spectrum, it is possible to quantify them through UV-visible spectrophotometer within 380--700 nm wavelength range following Beer--Lambert's law. - Carotenoids, 40 carbon atoms possessing terpenoids, are derived from the condensation of geranylgeranyl-PP molecules \[[@B29-foods-09-00918]\]. They are lipid-soluble, basically found in cyanobacteria, algae, plants, some fungi and bacteria, and are produced intracellularly by bioproduction of microorganisms \[[@B29-foods-09-00918],[@B30-foods-09-00918]\]. Based on their chemical structure, they can be classified as hydrocarbon carotenoids and xanthophylls, and their extraction can easily be performed with nonpolar solvents \[[@B29-foods-09-00918]\]. - Flavonoids are water-soluble compounds, consisting of 15 carbon atoms (C6-C3-C6) and belong to the class of phenylpropanoids \[[@B21-foods-09-00918],[@B31-foods-09-00918],[@B32-foods-09-00918]\]. Flavonoids furnish intense color, texture, and taste in fruits and flowers, stretching to a wide range of fruit and vegetable parts mostly leaves, flowers, and skin of the fruits \[[@B17-foods-09-00918],[@B27-foods-09-00918]\]. The color variety and classification differ according to the structural groups such as hydroxyl, methyl, glucosyl, and acyl \[[@B32-foods-09-00918]\]. Anthocyanins (glycosylated and acylated) are groups of flavonoids derived from phenylalanine conferring coloration from pale yellow to blue with respect to pH changes \[[@B32-foods-09-00918]\]. Anthocyanins possess several therapeutic properties as they strongly exhibit free radical scavenging capacity \[[@B17-foods-09-00918]\]. They are abundantly found in berries, blackcurrant, and other purple color giving fruits and vegetables with host taste attributes \[[@B17-foods-09-00918],[@B28-foods-09-00918]\]. - Both anthocyanin and betalain (betacyanin) have UV protectable ability for host plant tissues \[[@B28-foods-09-00918]\]. Betalains have a wider range of pH (3--7) with yellow to red coloration within that range though less stable to temperature and light exposure as compared to anthocyanin \[[@B33-foods-09-00918]\]. Slavov and coworkers \[[@B34-foods-09-00918]\] investigated the color pattern resulting from a mixture of betalain and anthocyanin-rich fruit juices accompanying their functional properties as their coexistence has never been explored. 2.1. Betalains {#sec2dot1-foods-09-00918} -------------- Betalains are the main compounds associated with the displayed red color of flowers, fruits, and other plant tissues. Betalains are derivatives of tyrosine which are normally found in plant groups in the order of Caryophyllales including Amaranthaceae (*Beta vulgaris*), Cactaceae (*Opuntia*, *Pitaya*, *or Pitahaya*), Nyctaginaceae (*Bougainvillea*), Phytolaccaceae (*Phytolacca americana*), and Portulacaceae (*Portulaca grandiflora*) \[[@B2-foods-09-00918],[@B3-foods-09-00918],[@B27-foods-09-00918],[@B37-foods-09-00918]\]. In Caryophyllaceae and Molluginaceae families, anthocyanin is more pronounced than betalain \[[@B2-foods-09-00918],[@B28-foods-09-00918],[@B32-foods-09-00918]\]. Their existence in high-class fungi, for example, agaric *Amanita muscaria*, *Hygrocybe*, and *Hygrophorus* have been reported as well \[[@B2-foods-09-00918],[@B27-foods-09-00918],[@B33-foods-09-00918],[@B38-foods-09-00918]\]. Betalain compounds are accumulated in plant cell vacuoles, predominantly located in edible parts of plant tissues (epidermal and subepidermal), accompanied by other phytochemical compounds \[[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918],[@B37-foods-09-00918],[@B39-foods-09-00918]\]. Betalains are nitrogen-containing heterocyclic compounds with high water affinity. Their exhibition of antioxidant activity is mainly based on their functional structure, for example, betalamic acid possesses conjugated double bonds that serve as reducing agents via electrons sharing \[[@B4-foods-09-00918]\]. Additionally, their ability to stabilize even after electron donation is responsible for its antioxidant property \[[@B4-foods-09-00918]\]. Depending on the type of groups (imino compounds or amino acid/derivatives) attached to the chromosphere of betalains, which is betalamic acid \[4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridene-2,6-dicarboxylic acid\], two basic color compounds are derived \[[@B2-foods-09-00918],[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918],[@B14-foods-09-00918],[@B28-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918],[@B38-foods-09-00918]\]. Betaxanthin is obtained by conjugating betalamic acid with amines or amino acids, whereas betacyanin is derived from the condensation of *cyclo*-[l]{.smallcaps}-(3, 4-dihydroxylphenylalanine), known as *cyclo-DOPA,* or its glucosyl derivatives \[[@B2-foods-09-00918],[@B3-foods-09-00918],[@B4-foods-09-00918],[@B5-foods-09-00918],[@B6-foods-09-00918],[@B14-foods-09-00918],[@B27-foods-09-00918],[@B28-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918],[@B38-foods-09-00918]\] ([Figure 1](#foods-09-00918-f001){ref-type="fig"}). In the UV visible region, betaxanthin and betacyanin are detectable at the maximum wavelength absorption of 480 and 540 nm, respectively \[[@B27-foods-09-00918]\]. Betacyanin is responsible for the red color but also more tolerant to heat and stable to processing conditions than the yellow color giving betaxanthin \[[@B3-foods-09-00918],[@B9-foods-09-00918],[@B39-foods-09-00918]\]. Betaxanthin sources are limited, which makes for their low market demand despite their availability in the form of essential dietary amino acids, luckily, *Celosia* species are available as alternative sources of bright yellow color betaxanthin \[[@B40-foods-09-00918]\]. Indicaxanthin with prolin group (abundant in *Opuntia* species) and vulgaxanthin I ([Figure 1](#foods-09-00918-f001){ref-type="fig"}d) with glutamine substituent, mostly found in *Beta vulgaris* species, are two prominent yellow color giving betaxanthins \[[@B4-foods-09-00918],[@B27-foods-09-00918],[@B28-foods-09-00918],[@B37-foods-09-00918]\]. Their presence incites the fluorescence property of betaxanthin \[[@B4-foods-09-00918],[@B38-foods-09-00918]\]. From the findings of Mikołajczyk-Bator and Pawlak \[[@B41-foods-09-00918]\], thermal degradation of red-violet pigments was three times lower than yellow color given compounds despite the antioxidant capacity of the latter, which tends to be higher after heat treatments. Betanin, gomphrenin, amaranthin, and bougainvillein are four major types of betacyanin differed by the substituent groups attached to *cyclo-DOPA* moiety in ortho position ([Figure 1](#foods-09-00918-f001){ref-type="fig"}g) \[[@B3-foods-09-00918],[@B4-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918],[@B38-foods-09-00918]\]. Betanidin is the basic structural unit of most betacyanin derivatives, followed by, betanin (betanidin *5-O-ß*-glucoside) derives from glucosylation and acylation of aglycon betanidine (I) \[[@B3-foods-09-00918],[@B27-foods-09-00918],[@B33-foods-09-00918],[@B37-foods-09-00918]\]. Betanin is the most stable red color compound, and its antioxidant ability is based on its donation of hydrogen and electron \[[@B4-foods-09-00918]\]. Presence of glucosyl substituent in C-6 hydroxyl group position and amine group in the ring system encourages radical scavenging activity of betacyanin \[[@B33-foods-09-00918],[@B41-foods-09-00918]\], attributes of its anti-oxidative stress-related disorders, anti-cancer, and anti-inflammatory properties in specific plants and vegetables \[[@B33-foods-09-00918]\]. Phyllocathin (betanidin *5-O-ß*-malonyl-glucoside) and hylocerenin (betanidin *5-O-ß*-\[3″-hydroxyl-3″-methyl-glutaryl\] glucoside) are acylated forms of betacyanin \[[@B37-foods-09-00918]\]. In addition, isobetanin, betanidin, isobetanidin, prebetanin, neobetanin coexist in a small amount apart from betanin \[[@B4-foods-09-00918],[@B34-foods-09-00918]\]. Those neo-derivatives are of interest due to their yellow color appearance derived from thermal degradation of *Beta vulgaris* and *Opuntia* species betanin \[[@B37-foods-09-00918]\]. Most degradation processes of betacyanin are isomerization, deglycosylation, dehydrogenation, hydrolysis, and decarboxylation \[[@B5-foods-09-00918],[@B37-foods-09-00918],[@B41-foods-09-00918]\]. Color variety of betalains is influenced by yellow color content to some extent \[[@B25-foods-09-00918]\], and their stabilities are determined by pH, light, heat, water activity, chelating agents, antioxidants, enzymatic reactions \[[@B4-foods-09-00918],[@B37-foods-09-00918],[@B43-foods-09-00918]\]. Peroxidases (POX), polyphenol oxidases (PPO), *ß*-glucosidase, and betalain oxidase are the most responsible enzymes for betalain degradation during processing \[[@B14-foods-09-00918],[@B37-foods-09-00918]\]. The disturbance of some endogenous enzymes can be overcome by blanching at 70 °C for 2 min \[[@B13-foods-09-00918],[@B27-foods-09-00918]\]. Betalain colorants are available in the form of concentrates \[[@B44-foods-09-00918]\] and dried powder through air, freeze, or spray drying \[[@B43-foods-09-00918],[@B45-foods-09-00918]\], and are normally used in dairy and confectioneries and even in meat products \[[@B27-foods-09-00918],[@B46-foods-09-00918]\], although addition in cold products is preferable because of their thermal sensitiveness. Furthermore, Sivakumar and coworkers \[[@B47-foods-09-00918]\] investigated their application for leather dying through the sonic extraction method. Coating of betalain pigments extracted from skin and pulp of *Cactus* fruit with acidified mucilage from the pulp of the fruit \[[@B48-foods-09-00918]\] or ionic gelation \[[@B49-foods-09-00918]\] can extend the shelf life as well as the stability of the pigments. Microencapsulation of beetroot pomace extracts and their applications in bakery products have also been explored recently \[[@B50-foods-09-00918]\]. 2.2. Phenolic Compounds {#sec2dot2-foods-09-00918} ----------------------- Phenolic compounds (C~6~H~5~OH), also known as polyphenols, are aromatic metabolites made up of a polar functional (−OH) group bonded directly to an aromatic hydrocarbon ring \[[@B21-foods-09-00918]\]. Their ease of bonding to hydrogen raises their hydrogen bond formation ability for efficient water solubility in aqueous media \[[@B4-foods-09-00918]\]. Phenolic compounds are derivatives of aromatic amino acid phenylalanine ubiquitous in most plant kingdom with different glycosylated forms ranging from a simple phenolic molecule to high complex polymer molecules \[[@B21-foods-09-00918],[@B51-foods-09-00918]\]. They are mostly localized in plant tissues (0.5--5 g in 100 g of dry weight) imparting pigments to their host \[[@B51-foods-09-00918]\]. As aromatic secondary metabolites, phenolics are anti-inflammatory, antimicrobial, and antithrombotic. They are willing to donate their electrons from the outermost shell to fulfil the leakage of free radicals and can extend the shelf-life of food products \[[@B16-foods-09-00918],[@B17-foods-09-00918],[@B21-foods-09-00918],[@B52-foods-09-00918]\]. Consequently, phenoxyl radicals (PhO˙) are developed after transferring the hydrogen atom to free radical, which in turn can conjugate with adjacent hydroxyl or amine groups to enhance their stabilities, making them resistant to further oxidation process \[[@B4-foods-09-00918]\]. Based on carbon atom numbers, phenolics, flavonoids, and tannins upon derivatization possess specific structures \[[@B21-foods-09-00918]\]. Among them, phenolic acids and flavonoids are the most active plant-based phenolic compounds (30% and 60% of total dietary polyphenols content) ([Figure 2](#foods-09-00918-f002){ref-type="fig"}), they can easily be extracted with water, supercritical or subcritical water, and alcoholic solvents \[[@B17-foods-09-00918],[@B53-foods-09-00918],[@B54-foods-09-00918]\]. Benzoic acid derivatives (C~6~--C~1~) and cinnamic acid derivatives (C~6~--C~3~) are the two main classes of phenolic acids \[[@B21-foods-09-00918]\], whereas flavonols, flavones, flavanones, flavanols, isoflavones, flavanonols, and anthocyanidins are unique flavonoid compounds with variation in heterocyclic ring structures \[[@B17-foods-09-00918],[@B21-foods-09-00918],[@B31-foods-09-00918]\]. 3. Generalities of Solid-Liquid Extraction ========================================== Classical extraction of target compounds from different types of matrix can be achieved through several cold or hot extraction methods: distillation; solvent extraction such as percolation, maceration, infusion, enfleurage; and cold compression \[[@B55-foods-09-00918]\]. Solvent selection should be suited with specific properties of compounds of interest. Apart from water, polar solvents such as alcoholic solvents are extensively used to upgrade extraction efficiency, although there are some oppositions of their application in the matter of total solid content increase \[[@B56-foods-09-00918]\]. Between 20%--50% *v*/*v* methanol or ethanol are the fundamentally used solvents for complete extraction of betalains. Being a non-toxic green solvent, extractions with ethanol solvent do not need any further purification step, while methanol is preferable for denaturation of enzymes and for overcoming the interference of water-soluble protein \[[@B14-foods-09-00918],[@B27-foods-09-00918],[@B33-foods-09-00918]\]. Biosynthesis reaction of compounds present in the matrix can be handled by acidifying the extraction medium; for example, enzymatic decolorization of betalain can be retarded by ascorbic acid, which interferes with oxidative activity of polyphenol oxidases or ß-gluconolactone and inhibits ß-glucosidase \[[@B33-foods-09-00918]\]. Moreover, ascorbic acid has some preservative effects on *Amaranthus* betacyanin, improved the stability of red color betalain, and upgraded yield of betanin to the highest as well \[[@B57-foods-09-00918]\]. However, as exogenous antioxidants, care should be taken in the application of ascorbic acids for antioxidant activity determination \[[@B14-foods-09-00918]\]. Chelating agents such as citric acid and EDTA (ethylene-diamine-tetra-acetic acid) are also prominent for acidification extraction of bioactive compounds in order to boost their yield \[[@B4-foods-09-00918]\]. In some cases, inorganic acid like hydrochloric acid is favorable to prevent the activity of endogenous enzymes \[[@B8-foods-09-00918],[@B27-foods-09-00918]\]. 4. Emerging Technology {#sec4-foods-09-00918} ====================== Handling of perishable vegetables is a big challenge for food processing industries and has led to the innovation of processing techniques to assure food quality and at the same time minimize food loss. Minimal processing is the preparation of food with minimal treatment and the smallest changes in food quality through modern processing technology. Attempts have been made to bring practical usage from emerging technologies to minimal food processing in the areas of tempering, vacuum drying, freeze-drying, dehydration, cooking, baking, roasting, pasteurization, sterilization, extraction, blanching, and direct microwave blanching \[[@B58-foods-09-00918]\]. Based on the characteristics of targeted products, thermal treatment methods (ultra-high temperature processing, aseptic or semi-aseptic heat treatment, sous-vid, infrared heating, high frequency or radiofrequency heating, ohmic heating, microwave heating, inductive electrical heating, etc.) and non-thermal treatment methods (low direct current electric fields, ionizing radiation, gamma irradiation, pulsed electric fields (PEF), UV light, pulsed light, laser light heating, ultrasonic wave heating, high-pressure processing, etc.) have been developed \[[@B59-foods-09-00918]\]. Currently used emerging technologies for the extraction of secondary plant metabolites (betalains and phenolic compounds) are microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), high pressure and temperature extraction (HPTE), supercritical fluids extraction (SFE), pressurized liquids extraction (PLE), pulse electric field extraction (PEF), gamma-irradiation-assisted extraction, and extraction by low direct current electrification \[[@B7-foods-09-00918],[@B8-foods-09-00918],[@B16-foods-09-00918],[@B18-foods-09-00918],[@B19-foods-09-00918],[@B20-foods-09-00918],[@B24-foods-09-00918],[@B34-foods-09-00918],[@B42-foods-09-00918],[@B46-foods-09-00918],[@B47-foods-09-00918],[@B52-foods-09-00918],[@B54-foods-09-00918],[@B57-foods-09-00918],[@B60-foods-09-00918],[@B61-foods-09-00918],[@B62-foods-09-00918],[@B63-foods-09-00918],[@B64-foods-09-00918],[@B65-foods-09-00918],[@B66-foods-09-00918],[@B67-foods-09-00918],[@B68-foods-09-00918],[@B69-foods-09-00918],[@B70-foods-09-00918],[@B71-foods-09-00918],[@B72-foods-09-00918],[@B73-foods-09-00918],[@B74-foods-09-00918],[@B75-foods-09-00918],[@B76-foods-09-00918],[@B77-foods-09-00918],[@B78-foods-09-00918],[@B79-foods-09-00918],[@B80-foods-09-00918],[@B81-foods-09-00918],[@B82-foods-09-00918],[@B83-foods-09-00918],[@B84-foods-09-00918],[@B85-foods-09-00918],[@B86-foods-09-00918],[@B87-foods-09-00918],[@B88-foods-09-00918],[@B89-foods-09-00918],[@B90-foods-09-00918]\]. 4.1. Microwave Irradiation {#sec4dot1-foods-09-00918} -------------------------- In green chemistry, MAE is a well known novel technology with comparative advantages of reaction time reduction and lower or no solvent requirement over conventional extraction techniques \[[@B91-foods-09-00918]\]. Electromagnetic waves with frequencies between 300 MHz and 300 GHz are noted as microwave and previously applied for navigation and telecommunication operations \[[@B53-foods-09-00918],[@B92-foods-09-00918]\]. Based on their application purposes, two frequency ranges are classified as 2450 MHz for home usage with adjustable power output \<1000 W and 915 MHz for the industrial one \[[@B23-foods-09-00918],[@B53-foods-09-00918],[@B55-foods-09-00918],[@B91-foods-09-00918]\]. Being a segment of the electromagnetic spectrum, like visible light, the phenomenon of bending, reflection, refraction, and absorption of microwave radiation by the medium through which it passes are unavoidable \[[@B92-foods-09-00918]\]. Likewise, the degree of absorptivity or transmissivity of transmitted waves relies on the electromagnetic properties of the treated object, for instance, dielectric property, polarity, permittivity, and the shape of the object. Another important factor is in situ water content of the matrix as the level of swelling and rupture of the matrix can be varied with it \[[@B93-foods-09-00918]\]. The contradiction of electromagnetic wave is based on its electric and magnetic wave propagation oscillating in a perpendicular direction to each other with wavelength (1 m\~1 mm) \[[@B23-foods-09-00918],[@B53-foods-09-00918],[@B55-foods-09-00918],[@B91-foods-09-00918]\]. Herein, the electric part if only absorbed by natural biological materials \[[@B94-foods-09-00918]\] consequently leads to heating process occurring in the medium with absorption property of microwave \[[@B55-foods-09-00918],[@B91-foods-09-00918]\]. Microwave radiation is non-ionized with photon energy ranging from 3.78 × 10^−6^ eV to 1.01 × 10^−5^ eV, and interaction process occurs by heat conduction mode \[[@B55-foods-09-00918]\]. Moreover, the transformation phenomenon of kinetic to thermal energy is mainly concerned with the polarization potential of the polar molecules and their surroundings \[[@B23-foods-09-00918],[@B92-foods-09-00918]\]. The protocol is that once polar molecules present in a substance are hit by the electromagnetic beams, they become energetic and swing with the alternative movement of electric field, and consequently the alternative action of alignment and realignment of polar molecules creates friction between them, which in turn lead to the heating up of the surroundings \[[@B53-foods-09-00918],[@B92-foods-09-00918]\] ([Figure 3](#foods-09-00918-f003){ref-type="fig"}). Meanwhile, ionic components which are present in the substance migrate according to the electric field \[[@B91-foods-09-00918]\] ([Figure 3](#foods-09-00918-f003){ref-type="fig"}). This intermolecular friction and ionic movements occur several million times (4.9 × 10^9^ times at a frequency of 2450 MHz) per second and raise the internal pressure of cell \[[@B91-foods-09-00918]\]. Consequently, higher internal pressure encountered due to rapid vaporization of in situ water could rupture the cell wall and enclosed substances inside the cell wall could be forced out of the cell at a high rate \[[@B23-foods-09-00918]\]. 4.2. Mathematical Terms {#sec4dot2-foods-09-00918} ----------------------- Electric loss tangent (tan δ) of materials, which is also known as dissipation factor, quantifies the transformation of electric and magnetic energy to thermal energy in the materials; it can be explained in terms of dielectric loss (ε″) and dielectric loss constant (ε′) as given below in (Equation (1)) \[[@B23-foods-09-00918],[@B91-foods-09-00918],[@B92-foods-09-00918],[@B94-foods-09-00918]\]:$$D_{f} = \tan\delta = \frac{\varepsilon^{''}}{\varepsilon^{\prime}}$$ Absorption, transmission, and reflection abilities of different materials, known as electric permittivity, can be tracked in several ways. The absolute permittivity elucidates only how the material interacts with applied electromagnetic waves for nonionizing radiation and nonmagnetic materials \[[@B94-foods-09-00918]\]. Alternatively, it can be said that nonmagnetic materials rely on dielectric permittivity (ε\*) to interact with electromagnetic waves and can be expressed by dielectric constant (ε′) and dielectric loss (ε″) shown in (Equation (2)); \[[@B94-foods-09-00918]\]:$$\varepsilon^{*}~ = ~\varepsilon_{r}^{\prime} - j\varepsilon_{r}^{''}$$ Real and imaginary parts of permittivity represent dipolar oscillation and damping \[[@B92-foods-09-00918]\], and are valued depending on the motion of dipoles, nature of materials, high or low frequency, elevated or reduced temperature, and also the concentration of aqueous ionic solution \[[@B94-foods-09-00918]\]. Commonly used solvents with their loss tangent values are listed in [Table 2](#foods-09-00918-t002){ref-type="table"}. Relative permittivity (ε) can be estimated from electric displacement field of material (*D*, C · m^−2^) which is divided by vacuum (*D~vacuum~*, C · m^−2^) \[[@B92-foods-09-00918]\]. In dielectric material, conversion of electrical energy into thermal energy is defined by its loss factor. Consequently, dipole rotation and electrical conduction are assumed as loss factors in microwave heating, (Equation (3)):$$\varepsilon_{r}^{''}~ = ~\varepsilon_{d}^{''} + \varepsilon_{\sigma}^{''}$$ where ε~d~″ is relative dipole loss and ε~σ~″ is relative ionic loss. If the components are homogeneous, their shape is more responsible for the effective permittivity of the mixtures, especially when the particle size is smaller than the wavelength \[[@B94-foods-09-00918]\]. Literally, ordinary foods possess the penetration depth of electromagnetic waves (10--15 mm) \[[@B94-foods-09-00918]\]. The penetration level of microwave through the substance can be traced by the degree of reflection, transmission, and absorption of the wave by the host substance; for example, pure water has a low degree of reflection and transmission but a high degree of absorption of the wave \[[@B92-foods-09-00918]\]. Dissipated microwave power (*P~D~*, W) in a material can be estimated from electric field strength (*E*, V · m^−1^), and frequency (*f*, Hz) as reported by \[[@B92-foods-09-00918],[@B94-foods-09-00918]\] (Equation (4)):$$P_{D} = k~ \cdot E^{2} \cdot f \cdot \varepsilon^{''}$$ where *k* is a constant value of 55.61 × 10^−14^ C · m^2^ · V^−1^. Due to direct proportionality of dissipated microwave power (P~D~) to the absorptivity of the material, the attenuation of microwave differs according to the depth of penetration (z) and can be estimated as expressed in Equation (5):$$z = \frac{\mathsf{\lambda}}{2\pi} \cdot \sqrt{\frac{2}{\varepsilon^{\prime}\left( {\sqrt{1 + tan^{2}\delta} - 1} \right)}}$$ Frequency of applied radiation (f, Hz) and speed of the wave (c, m · s^−1^) can be used to evaluate wavelength ($\mathsf{\lambda}$, m) \[[@B92-foods-09-00918]\]. 4.3. Microwave-Assisted Extraction (MAE) of Betalains and Phenolic Compounds {#sec4dot3-foods-09-00918} ---------------------------------------------------------------------------- Microwave irradiation is proposed as an effective alternative way of recovering bioactive compounds from agro-industrial wastes with superior benefits such as high reproducibility within short treatment period, minimal solvent and energy consumption, and simple manipulation as opposed to classical solid--liquid-based extraction methods (soxhlet, rotary, maceration, and heat refluxing), supercritical water extraction, and even ultrasonic extraction \[[@B16-foods-09-00918],[@B18-foods-09-00918],[@B20-foods-09-00918],[@B52-foods-09-00918],[@B54-foods-09-00918],[@B60-foods-09-00918],[@B61-foods-09-00918],[@B62-foods-09-00918],[@B63-foods-09-00918],[@B64-foods-09-00918]\]. In advance, MAE has brought 1.7 times greater yield percent of phenolic \[[@B65-foods-09-00918]\], whilst operation time was two times shorter than UAE \[[@B54-foods-09-00918]\]. Alongside, 5 min of microwave irradiation could bring an equivalent amount of phenolic and flavonoid compounds with an hour of ultrasonic treatment time \[[@B61-foods-09-00918]\]. However, UAE was pointed out as the better choice in the investigations of \[[@B22-foods-09-00918],[@B63-foods-09-00918]\], in this case, the discrepancy in applied frequency and power wattage should be taken into account. Somehow, it is undeniable that both MAE and UAE are better in internal or external mass transfer compared to classical methods. In UAE, the formation of tiny gas bubbles in the solvent medium by mechanical wave encourages several micro-injections into the cell wall, which improves the solvent transport. Herein, sonication of water can propagate highly reactive hydroxyl radicals which may have some effect on the extraction of targeted products \[[@B52-foods-09-00918],[@B66-foods-09-00918]\]. Heating mode, duty cycle, power or heat energy, and temperature in an advanced microwave set up are basic operational parametric modes in MAE of betalains and phenolic compounds. Response surface methodology (RSM) is a suitable tool to approach the efficiency of MAE through the interaction of process variables despite the fact that the predicted values estimated by the model are only within the experimental limit \[[@B9-foods-09-00918],[@B18-foods-09-00918],[@B62-foods-09-00918],[@B63-foods-09-00918],[@B67-foods-09-00918],[@B68-foods-09-00918],[@B69-foods-09-00918],[@B70-foods-09-00918],[@B71-foods-09-00918],[@B72-foods-09-00918],[@B73-foods-09-00918]\]. Central composite design was mostly chosen for the investigation of each or parallel effects of process variables on the corresponding response. Based on the response level whether high or low, the weight of the factor on the response could be judged. Here again, the levels of the factors optimized by RSM support to be in between, i.e., neither at most nor lowest level or else the performed limits are regarded as insufficient for the design to fully predict the optimum condition of the variables within those limits \[[@B26-foods-09-00918]\]. For example, it can be said that the model well predicted the optimum condition of the extraction process if the highest amount of total phenolic compounds (39 mg GAE/gDW) was achieved at ethanol concentration (31.33%), solvent to material ratio (32.21 mL/g), temperature (52.24 °C) among the variables which were ethanol concentration (20--40%), solvent to material ratio (20--40 mL/g), temperature (40--60 °C) \[[@B73-foods-09-00918]\]. [Table 3](#foods-09-00918-t003){ref-type="table"} and [Table 4](#foods-09-00918-t004){ref-type="table"} represent some reported optimum conditions, within experimental variable ranges, for the extraction of betalains and phenolic compounds from fruit and vegetable as well as their wastes. In advance, some example of power output and time setting to accomplish MAE of secondary plant metabolites are demonstrated in [Figure 4](#foods-09-00918-f004){ref-type="fig"} and [Figure 5](#foods-09-00918-f005){ref-type="fig"}. In MAE, both the solvent and the sample undergo transformation by electromagnetic energy of high value based on ionic conduction and dipole rotation (either permanent or induced by the electric field). Ionic conduction, which is due to the electrophoretic migration of dissolved ions, increases solvent penetration into the matrix and facilitates the solvation of targeted compounds \[[@B91-foods-09-00918]\]. Influence of solvent type on microwave extraction of desired phenolic compound quantity has been investigated with pure water, pure and aqueous forms of acetone, ethanol and methanol, and of them, 50% (*v*/*v*) ethanol was observed to be most effective \[[@B61-foods-09-00918],[@B64-foods-09-00918],[@B78-foods-09-00918],[@B80-foods-09-00918]\]. Presence of a suitable amount of water in the solvent somehow can improve the swelling of the plant matrix, increasing the contact surface area of the matrix with the solvent \[[@B62-foods-09-00918]\]. However, it is controversial whether inert solvent to microwave radiation is better than the absorbed one or not as acetone performed better in phenolic compounds extraction possibly due to transparent property to microwave radiation subsequently allowing plant cells receive direct contact of the radiation \[[@B19-foods-09-00918]\]. Moreover, it should be noted that microwave can rapidly lose its energy before it reaches the innermost part of the medium if the medium it passed through is in high absorptivity of radiation \[[@B92-foods-09-00918]\]. Applied power output for microwave irradiation influences phenolic compounds \[[@B73-foods-09-00918]\], and it is obvious if irradiation time is short \[[@B59-foods-09-00918]\], which possibly leads to accelerated diffusion of the solvent into the matrix and in turn leaching out of the materials \[[@B23-foods-09-00918]\]. Prolonged heating via elevated temperature treatments can jeopardize pigment retentions; in this case, the acidification method can be considered for decompartmentalization of color compounds after microwave treatment \[[@B24-foods-09-00918]\]. Synergistic effects of processing time and temperature or microwave power, solvent concentration, and matrix-to-solvent ratio on mass transfer rate differ for specific secondary plant compounds, thus process condition adjustment should be focused on targeted compounds in order to distribute radiations uniformly \[[@B17-foods-09-00918],[@B24-foods-09-00918],[@B73-foods-09-00918]\]. Reported SEM (scanning electron microscopy) results showed that cell wall thinning and holes formation were prominent in microwave treatment rather than traditional heating modes \[[@B65-foods-09-00918],[@B80-foods-09-00918],[@B82-foods-09-00918],[@B95-foods-09-00918],[@B96-foods-09-00918]\]. This is because microwave penetrates the matrix and denaturalizes the cell wall in which bioactive compounds are trapped in a short time, subsequently letting them diffuse quickly into the solvent medium. However, the superheating effect can be encountered as a consequence, which is more pronounced with pure water solvent due to its quite low dissipation factor but high dielectric constant, facilitating the absorption of the microwave \[[@B92-foods-09-00918]\]. Since microwave heating is based on the alignment of the polar molecules back and forth, the transformation of kinetic energy to thermal energy is more intensive when their movement cannot catch up the frequency of applied microwave, which is the so-called dumping process \[[@B62-foods-09-00918]\]. That is why intermittent modes of microwave irradiation and cooling in between extraction steps were found to be effective \[[@B21-foods-09-00918],[@B24-foods-09-00918],[@B52-foods-09-00918],[@B61-foods-09-00918],[@B64-foods-09-00918],[@B81-foods-09-00918]\]. 5. Other Applications {#sec5-foods-09-00918} ===================== Apart from betalain and phenolic compounds, MAE has been widely used in the extraction of pectin with some improvements in quantity and quality, degree of esterification, and gel strength \[[@B97-foods-09-00918]\]. Moreover, essential oil extractions from different sources via advanced microwave-assisted techniques have been realized \[[@B93-foods-09-00918],[@B98-foods-09-00918],[@B99-foods-09-00918]\]. The combination of novel extraction methods which are microwave, ultrasonic wave, enzymatic, and also alkali assisted extraction have been complimented as well \[[@B90-foods-09-00918],[@B95-foods-09-00918],[@B100-foods-09-00918],[@B101-foods-09-00918]\] ([Table 5](#foods-09-00918-t005){ref-type="table"}). Other improved MAE methods are in situ extraction of essential oils with microwave oven \[[@B102-foods-09-00918]\], MAE of multi-elements from food materials via perchloric acid and hydrogen peroxide \[[@B103-foods-09-00918]\], MAE and GC-FID quantification of total branched-chain fatty acids in lamb subcutaneous adipose tissue \[[@B104-foods-09-00918]\]. Additionally, some other modified techniques of microwave irradiation such as vacuum microwave-assisted extraction, nitrogen-protected microwave-assisted extraction, dynamic microwave-assisted extraction, pressurized solvent free microwave-assisted extraction, radiation-assisted hydro-distillation, vacuum microwave hydro-distillation, microwave-assisted steam distillation, vacuum microwave-assisted hydro-gravity, microwave dry-diffusion and gravity have been mentioned in the reviews of \[[@B23-foods-09-00918],[@B91-foods-09-00918]\]. Apart from extraction, microwave application has been extended to processing of beetroots which has more effectiveness in oxidative stability of mayonnaise than roasted or boiled ones, thereby preserving betalains and phenolic compounds \[[@B105-foods-09-00918]\]. 6. Conclusions {#sec6-foods-09-00918} ============== As natural bioactive compounds rich in antioxidants, therapeutic properties of betalain and phenolic compounds are numerous. Although they are sensitive to heat, pH, light, etc., not only during processing but also throughout the storage, their presence in food wastes and residues even after processing is noteworthy as well. Currently, their practical usages in food industries, fortification, and supplementation of foods, cosmetics, and pharmaceuticals are thriving. Therefore, further thorough investigations for extraction of these valuable compounds via modern technologies, meantime maintaining their original properties as much as possible, are still needed to be explored. Most of MAE discussed in this review are operated in batch, although semi-continuous or continuous modes are also available. Since microwave irradiation encourages the direct interaction of vast electromagnetic waves and the target material, higher power ensures enough contact of the microwave with the matrix to expedite the isolation of the compounds trapped in the cell wall. Therefore, high temperature or power and short irradiation time is a beneficial combination for MAE of plant materials. Higher extraction time is not necessary once after the equilibrium state between the solvent and the matrix is reached since it can lead to the degradation of most bioactive compounds. All in all, if it is in proper use, microwave can be the best option for bioactive compounds extraction for the sake of simplicity as it can easily be set up or run by the home used microwave oven, high efficiency as the heating is built at the innermost part of the matrix and diffuses to the surrounding, saving energy as the operation can be completed in few minutes, and cleanliness as it is the only way of solvent free or lowest solvent used extraction. We appreciate the Doctoral School of Food Science, Szent Istvan University for the support accomplishing this work as well as the European Union's Horizon 2020 research and innovation programme under the Marie Sklowdowska---Curie grant No: 765860 (AQUAlity). Conceptualization, M.M.Z.; methodology, M.M.Z.; software, M.M.Z., C.B.A.; resources, M.M.Z., C.B.A.; writing---original draft preparation, M.M.Z.; writing---review and editing, M.M.Z., C.B.A.; supervision, S.B. All authors have read and agreed to the published version of the manuscript. This study was financially supported by the European Union and the European Social Fund (grant agreement no.EFOP-3.6.3-VEKOP-16-2017-00005), Tempus Public Foundation, under the Stipendium Hungaricum Scholarship Program. The authors declare no conflict of interest. ![Derivation of betacyanin and betaxanthin color compounds from betalamic acid: (**a**) Betalamic acid which is core structure of betalain, (**b**) Amino acid which conjugates with betalamic acid to derive betaxanthin, (**c**) Yellow color giving betaxanthin, (**d**) Vulgaxanthin I which is a derivative of betaxanthin, (**e**) cyclo-*DOPA* which condenses with betalamic acid to give betacyanin, (**f**) Red-violet color giving betacyanin, and (**g**) Betanin which is a derivative of betacyanin \[[@B2-foods-09-00918],[@B5-foods-09-00918],[@B14-foods-09-00918],[@B27-foods-09-00918],[@B28-foods-09-00918],[@B37-foods-09-00918],[@B42-foods-09-00918]\].](foods-09-00918-g001){#foods-09-00918-f001} ![Basic structures of phenolic compounds: (**a**) Backbone structure of phenolic acid, and (**b**) Structure of flavonoid moiety \[[@B21-foods-09-00918],[@B31-foods-09-00918]\].](foods-09-00918-g002){#foods-09-00918-f002} ![Treatment of microwave heat radiation for secondary plant metabolites extraction: (**a**) Domestic microwave oven heating system, (**b**) Heat diffusion from inside of the matrix to solvent medium and the surroundings, (**c**) Alignment of dipolar molecules and ions with electromagnetic wave, (**d**) Friction and rotation of dipolar molecules which heat up the matrix, and (**e**) Movements of ions with the electromagnetic wave.](foods-09-00918-g003){#foods-09-00918-f003} ![Ranges of power output for extraction of secondary plant metabolites (betalains and phenolic compounds).](foods-09-00918-g004){#foods-09-00918-f004} ![Ranges of microwave irradiation time (min) for extraction of phytochemicals (betalains and phenolic compounds).](foods-09-00918-g005){#foods-09-00918-f005} foods-09-00918-t001_Table 1 ###### Natural colors applied in the food and cosmetic industries, their E numbers, and common sources \[[@B3-foods-09-00918],[@B25-foods-09-00918],[@B28-foods-09-00918],[@B35-foods-09-00918],[@B36-foods-09-00918]\]. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Group Compound/Product Colour Code Common Sources ------------------------------ -------------------------------------------------- --------------------------------------------------------------------------------------------------------------- ------------------- -------------------------------------------------------------------------------------------- \- Curcumin, turmeric oleorisin Yellow-orange E100 Turmeric \- Riboflavin (vitamin B2), riboflavin-5′-phosphate Yellow-orange E101(i), E101(ii) Eggs, green vegetables, milk and other dairy products, meat, mushroom, almond \- Riboflavin-5-sodium phosphate Yellow E106 Eggs, organ meats (kidney and liver), lean meat, milk, green vegetables \- Carminic acid Magenta-red/ crimson E120 Cochineal insect \- Chlorophyll Green E140, E141 Grass, alfalfa leaf, *Tagetes erecta* (marigold flowers), spinach \- Caramel Brown E150 Modified sugar \- Vegetable carbon Black E153 Vegetables Carotenoid α-carotene,\ Orange-red, red, yellow, amber, brown E160a Carrot, pumpkin, apricot, sweet potato, beans, spinach, kale,\ β-carotene,\ collard greens, papaya, bell peppers, tomatoes, green leafy vegetables γ-carotene Carotenoid Annatto Orange-red E160b Fruit of the achiote tree Carotenoid Paprika oleoresin Red E160c *Capsicum annuum or Capsicum frutescens* Carotenoid Lycopene Bright to deep red E160d *Solanum lycopersicum* (tomatoes), watermelon Carotenoid β-Apo-8′-carotenal Orange to orange-red E160e Spinach, citrus fruits Carotenoid Lutein Orange-red to yellow E161b Green leafy vegetables and fruits, yellowish flowers Carotenoid Canthaxanthin Violet E161g Mushroom, crustaceans, fish, eggs Nitrogen containing compound Betalains/ betanin Red violet to yellow (pH \< 3 (more reddish), pH \> 7 (more yellowish)) E162 Mangosteen, beetroot, dragonfruit, red cabbage, swiss chard, *Opuntia* Phenolic Anthocyanin Dark purple (pH 1 (red), pH 4--5 (colorless), pH \< 7 (purple), pH \< 8 (deep blue), pH \< 12 (yellow/brown)) E163 Black currant, berries, cherry, plum, grape, red cabbage, *Opuntia*, hibiscus, rose, onion Carotenoid Saffron Yellow-orange-red E164 *Crocus sativus* ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ foods-09-00918-t002_Table 2 ###### Reported mathematical parametric values associated with commonly used solvents for phytochemical recovery through MAE (Microwave-Assisted Extraction). Solvent Loss Tangent Dielectric Constant (ε′) Dielectric Loss (ε″) Reference ---------- -------------- -------------------------- ---------------------- ----------------------------------------------------------------------- water 0.123 80.4 9.8892 \[[@B23-foods-09-00918],[@B53-foods-09-00918],[@B58-foods-09-00918]\] ethanol 0.941 25.7 24.1837 \[[@B23-foods-09-00918],[@B53-foods-09-00918]\] methanol 0.659 32.7 21.5493 \[[@B23-foods-09-00918],[@B53-foods-09-00918]\] acetone 0.054 20.6 1.1124 \[[@B53-foods-09-00918]\] foods-09-00918-t003_Table 3 ###### Recovery of betalain color compounds through microwave irradiation. Raw Material Solvent Product Process Optimum Conditions Reference ------------------------------- -------------------------------- ------------------------------------ ------------------------------------ ------------------ --------------------- --------------------------- Red beetroot peel acidified water betanin 229.26 mg/L (predicted by RSM) 0.95 min 224.61 W \[[@B8-foods-09-00918]\] ethanol 472.11 mg/L (predicted by RSM) 1.25 min 384.25 W Red beetroot pure water betaxanthin 1.25 mg/g of freeze dried red beet 2.2 min/ 1.7 min 400 W, 100% nominal \[[@B24-foods-09-00918]\] acidified water betacyanin 1.88 mg/g of freeze dried red beet Red beetroot pure water betacyanin 52.2 mg/g of fresh matter 3 min x 4 times 450 W \[[@B34-foods-09-00918]\] betaxanthin 42.8 mg/g of fresh matter *Opuntia* fruit peel 34.6% methanol betalain 201.6 mg/g of extract 2.5 min 400 W \[[@B63-foods-09-00918]\] Dragon fruit peel pure water betalain 9 mg/L (predicted by RSM) 8 min 100 W \[[@B72-foods-09-00918]\] White-fleshed red pitaya peel pure water betacyanin 1.66 mg/g of dry extract 5 min 600 W \[[@B74-foods-09-00918]\] RSM = response surface methodology. foods-09-00918-t004_Table 4 ###### Total phenolic compound (TPC) recovery by microwave radiation treatment. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Raw Materials Extraction Analytical Method Process Optimum Conditions Reference --------------------------------- ---------------------------- ------------------- ------------------------------------- ----------- ---------------------- --------------------------- *Coriandrum sativum* (spice) 50% ethanol FC 0.82 mg GAE/g of DW 18 min 200 W, 50% nominal \[[@B16-foods-09-00918]\] *Cinnamomum zeylanicum* (spice) 16.8 mg GAE/g of DW *Cuminum cyminum* (spice) 11.6 mg GAE/g of DW *Crocus sativus* (spice) 29.4 mg GAE/g of DW *Lessonia trabeculate*\ 70% methanol FC 0.74 mg GAE/g of DW 15 min intermittent \[[@B20-foods-09-00918]\] (Brown algae) *Lessonia nigrecens*\ 1.07 mg GAE/g of DW (Brown algae) *Ascophyllum nodosum*\ 1.4 mg GAE/g of DW (Brown algae) *Laminaria japonica*\ 0.73 mg GAE/g of DW (Brown algae) Rosemary 96% ethanol FC 0.9 mg GAE/g of fresh leaf 7 min 250 W (intermittent) \[[@B52-foods-09-00918]\] pure water 0.1 mg GAE/g of fresh leaf Grape seed methanol FC 67.88 mg GAE/g of DW 60 min 60 W \[[@B60-foods-09-00918]\] Grape skin 7.33 mg GAE/g of DW Eucalyptus leaf 50% ethanol FC 76.6 mg GAE/g of fresh leaf 5 min 600 W (intermittent) \[[@B61-foods-09-00918]\] Olive leaf 50% ethanol FC 88.3 mg TAE/g of DW 15 min intermittent \[[@B64-foods-09-00918]\] Peanut skin 30% ethanol FC 143.6 mg GAE/g of skins\ 0.5 min 950 W, 90% nominal \[[@B67-foods-09-00918]\] (predicted by RSM) Broccoli 72.06% methanol FC 21.39 mg GAE/g of DW 16.94 min 159.33 W \[[@B68-foods-09-00918]\] Wine lee 75% ethanol\ FC 36.4 mg GAE/g of lee extract powder 17 min 200 W \[[@B69-foods-09-00918]\] (acidified) (*Ipomoea Batatas*)\ 53% ethanol FC 61.26 mg GAE/g DW 2.05 min 302 W \[[@B71-foods-09-00918]\] Sweet potato leaf *Melastoma sanguineum* Fruit 31.33% ethanol FC 39.02 mg GAE/g of DW 45 min 500 W \[[@B73-foods-09-00918]\] *Lycium spp.* leaf pure methanol FC 6.65 mg GAE/g of DW 30 min 40 W \[[@B75-foods-09-00918]\] Buckwheat 50% ethanol FC 18.5 mg GAE/g of buckwheat 15 min \- \[[@B76-foods-09-00918]\] Pitaya peel pure water FC 5.8 mg GAE/g of extract 20 min 400 W \[[@B77-foods-09-00918]\] Sour cherry pomace 50% ethanol FC 14.14 mg GAE/g of DW 12 min 700 W \[[@B78-foods-09-00918]\] --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- GAE = gallic acid equivalent, FC = folin-ciocalteu analytical method, TAE = tannic acid equivalent, DW = dry weight. foods-09-00918-t005_Table 5 ###### Some other applications of advanced MAE and their findings. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Sources Methods Advantages References --------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------- Sorghum husks Ultrasonic-microwave assisted extraction of colorant (UMAE) Higher in thermal stabilities and yield percent (3.6 times) with high contents of apigeninidin and luteolinidin than conventional shaking \[[@B90-foods-09-00918]\] Aromatic herbs Enhanced solvent free microwave-assisted extraction (ESFMAE) ESFMAE increased in oxygenated compound content which was more odoriferous than monoterpene hydrocarbons \[[@B93-foods-09-00918]\] Cherry seeds Ultrasonic-microwave-assisted aqueous enzymatic extraction (UMAAEE) Compared to Soxhlet extraction, oil by UMAAEE possessed superior physicochemical properties and higher content of bioactive constituents \[[@B95-foods-09-00918]\] Tunisian cumin (*Cuminum cyminum* L.) seeds Microwave hydrodiffusion and gravity extraction (MHGE) MHGE successfully improved the EO yield with high amount of oxygenated compounds in shorter extraction time, less electrical consumption, lower carbon dioxide emissions, and smaller volume of waste water \[[@B96-foods-09-00918]\] Rosemary plants Microwave hydro-distillation (MHD) MHD was superior in terms of saving energy and extraction time compared to hydro-distillation \[[@B98-foods-09-00918]\] *Foeniculum vulgare* Mill. seeds Enhanced solvent free microwave-assisted extraction using double walled reactor (ESFMAE) ESFMAE method was faster, cleaner, less cost and energy usage, and better essential oil composition than hydro-distillation method \[[@B99-foods-09-00918]\] Arabica coffee beans Ultrasonic-microwave assisted extraction of green coffee oil (UMAE) Extraction yields of two diterpenes (cafestol and kahweol) by UMAE were significantly higher than that of solvent method \[[@B100-foods-09-00918]\] Corn brans Ultrasonic-microwave assisted alkali extraction of arabinoxylan (UMAAE) By UMAAE, water-unextractable\ \[[@B101-foods-09-00918]\] arabinoxylan (WUAX) showed good DPPH radical scavenging activity and strong Fe^2^+ chelating activity *Schisandra chinensis* Baill fruits Ionic liquid-based microwave-assisted extraction (ILMAE) ILMAE method shortened the energy consumption time, improved the extraction efficiency of lignans as to reflux extraction \[[@B106-foods-09-00918]\] Flowers of *Ulex europaeus* L. Microwave hydrodiffusion and gravity extraction (MHGE) MHGE allowed an efficient water removal from the material, and could be suitable for extraction of antioxidant rich aromatic compounds \[[@B107-foods-09-00918]\] Tomatoes Deep eutectic solvent-based microwave-assisted dispersive liquid--liquid microextraction preconcentration of multiclass pesticide residues in tomato samples (DES-MWA--DLLME) DES-MWA--DLLME represented good repeatability,\ \[[@B108-foods-09-00918]\] high (enrichment factors) EFs, low (limit of detection) LODs ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

{ "pile_set_name": "PubMed Central" }

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"rich", "bfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsfoodsgfoodsf", "mainli", "introduct", "quantiti", "prolin", "rate", "will", "fluid", "ipomoea", "highpressur", "advanc", "mhz", "japonica", "beam", "desmwadllm", "methyl", "bfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfood", "adulter", "citric", "realiz", "prone", "bfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfoodsbfood", "oppos", "chard", "δ", "intermolecular", "nitrogenprotect", "betacyanin", "freezedri", "furnish", "tuber", "bran", "biocolor", "peroxidas", "esfma", "way", "cdot", "frequenc", "school", "ordinari", "simpl", "betanidin", "condens", "transform", "agent", "ultrasonicmicrowav", "wavelength", "complex", "hibiscu", "g", "apart", "genotyp", "formerli", "green", "region", "oxygen", "volum", "mode", 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"per", "food", "varieti", "analyt", "ruptur", "phenol", "tini", "via", "isobetanidin", "agroindustri", "canthaxanthin", "infrar", "fc", "environ", "accumul", "transfer", "predict", "free", "parametr", "draft", "motion", "consist", "essenti", "min", "observ", "isoflavon", "microorgan", "reach", "matrix", "vacuol", "gomphrenin", "secdotfood", "fact", "puls", "bright", "bakeri", "occur", "emerg", "extend", "prolong", "better", "neither", "nigrecen", "divid", "consumpt", "propag", "like", "freez", "technolog", "strong", "molluginacea", "umaa", "appear", "histor", "matrixtosolv", "domest", "agar", "tabl", "gammairradiationassist", "induc", "compoundproduct", "influenc", "multiclass", "throughout", "exhibit", "telecommun", "evalu", "portulacacea", "leach", "eb", "brought", "one", "shell", "varepsilonrprim", "batata", "chlorophyl", "favor", "capsicum", "solventfre", "cactu", "tangent", "sensit", "tan", "antimicrobi", "beetroot", "erecta", "alcohol", "treat", "preserv", "thorough", "commonli", "ev", "cycl", "cook", "affin", "sousvid", "mm", "saffron", "industri", "beet", "enhanc", "obviou", "demand", "rise", "steam", "wall", "chang", "size", "practic", "meanwhil", "electrophoret", "rang", "transport", "nitrogencontain", "bake", "oxidas", "mgl", "sanguineum", "w", "metabolit", "pronounc", "fe", "waterunextract", "success", "ε", "simplic", "almond", "sorghum", "ossmalonylglucosid", "watermelon", "second", "glucosyl", "ring", "blue", "harvest", "dark", "phyllocathin", "enclos", "vast", "cba", "percent", "research", "irradi", "prefer", "oscil", "content", "extent", "branchedchain", "conduct", "hungaricum", "incit", "stretch", "shelf", "electromagnet", "meat", "γcaroten", "classic", "lean", "loss", "dairi", "yellowish", "modern", "never", "hydrolysi", "effect", "contain", "mathemat", "ccc", "sourc", "uma", "degrad", "oxid", "phenoxyl", "spray", "oliv", "doubl", "aglycon", "vv", "hydrogen", "wavefoodsgfoodsf", "lod", "socal", "depth", "scan", "composit", "corn", "save", "matter", "ethanol", "possess", "sfe", "challeng", "design", "betalam", "c", "format", "field", "possibl", "shake", "cultivar", "fracmathsflambdapi", "asept", "yellowbrown", "αcaroten", "attract", "hot", "could", "solubl", "raw", "made", "yield", "oper", "reddish", "rhizom", "pass", "foodstreftypet", "microinject", "isol", "big", "therefor", "spinach", "husk", "proper" ]

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"regarded", "nitrogenous", "anthocyanin", "led", "grouped", "betalains", "Among", "acknowledged", "sources", "betalains", "beetroot", "swiss", "chard", "dragon", "fruit", "prickly", "pear", "beetroot", "gained", "attention", "since", "apart", "whole", "tuber", "even", "waste", "parts", "stalk", "peel", "pomace", "rich", "betalains", "ratio", "color", "contents", "decisively", "influenced", "specie", "source", "genetic", "cultivar", "environment", "cultivation", "throughout", "growing", "stages", "harvesting", "Practically", "one", "kilogram", "fresh", "beetroot", "contains", "mg", "betacyanin", "mg", "betaxanthin", "compared", "flesh", "betalain", "concentration", "ranges", "higher", "peel", "also", "depends", "genotypes", "reducing", "agents", "phenolic", "compounds", "inhibit", "oxidation", "reaction", "catalyzed", "enzymes", "stabilize", "lipid", "peroxidases", "phenolic", "compounds", "exist", "several", "kinds", "fruits", "vegetables", "cereals", "tea", "leaves", "coffee", 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"Pitaya", "Pitahaya", "Nyctaginaceae", "Bougainvillea", "Phytolaccaceae", "Phytolacca", "americana", "Portulacaceae", "Portulaca", "grandiflora", "Caryophyllaceae", "Molluginaceae", "families", "anthocyanin", "pronounced", "betalain", "existence", "fungi", "example", "agaric", "Amanita", "muscaria", "Hygrocybe", "Hygrophorus", "reported", "well", "Betalain", "compounds", "accumulated", "plant", "cell", "vacuoles", "predominantly", "located", "edible", "parts", "plant", "tissues", "epidermal", "subepidermal", "accompanied", "phytochemical", "compounds", "Betalains", "heterocyclic", "compounds", "high", "water", "affinity", "exhibition", "antioxidant", "activity", "mainly", "based", "functional", "structure", "example", "betalamic", "acid", "possesses", "conjugated", "double", "bonds", "serve", "reducing", "agents", "via", "electrons", "sharing", "Additionally", "ability", "stabilize", "even", "electron", "donation", "responsible", "antioxidant", "property", "Depending", "type", 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I. Introduction =============== Aortic aneurysm is a serious condition characterized by the tearing of the inner layer of the aorta. It can lead to sudden death caused by cardiac failure and occasionally, aortic rupture[@B1][@B2]. In addition, hypertension can increase the stress on the weakened aortic wall, increasing its susceptibility to tearing. Aortic aneurysms are divided into Stanford type A and Stanford type B dissections. While type B dissections generally are initially treated medically, with surgery reserved for complications, type A aortic aneurysms, which involve a tear in the aorta ascendens, generally require emergency or quasiemergency surgical treatment[@B2]. Dexmedetomidine (DEX) is an anti-anxiety, sedative, and analgesic medication that can stabilize circulatory dynamics[@B3] and minimize blood pressure fluctuations. We report a case of a 45-year-old morbidly obese man with an aortic aneurysm (Stanford types A and B), in whom invasive oral treatment, namely tooth extraction and cyst enucleation, for the removal of intraoral infectious sources, was successfully performed under local anesthesia with DEX sedation before elective cardiac surgery. II. Case Report =============== A 45-year-old morbidly obese man was transported by ambulance to the Emergency Medical Care Center at Fukuoka University Hospital (Fukuoka, Japan) with suspected acute heart failure after developing a sudden difficulty in breathing with pink frothy sputum. According to his wife, the patient had no significant medical history other than hypertension. Detailed examination revealed an aortic aneurysm (Stanford types A and B), aortic regurgitation, aortic valve ring ectasia, ascending thoracic aorta aneurysm, hypertension, paroxysmal tachycardia, atrial fibrillation, primary lung cancer in the left lower lobe, and morbid obesity (weight, 124 kg; height, 170 cm; body mass index \[BMI\], 42.9 kg/m^2^). The aortic aneurysm required emergency surgery. However, because of the patient\'s morbid obesity, elective vascular graft replacement of the thoracic ascending aorta, aortic valve replacement, pulmonary vein isolation surgery, and segmental left lower lobe resection were planned after gradual weight reduction with dietary therapy. On hospital day 16, the patient underwent a preoperative detailed intraoral examination in our department. At the patient\'s first visit to our department, his weight was 109 kg (BMI, 37.7 kg/m^2^). Intraoral examination revealed six teeth with periapical lesions and two pigeon eggsized radicular cysts that could become potential sources of infection after surgery.([Fig. 1](#F1){ref-type="fig"}) The patient was at increased risk of developing severe complications of aortic aneurysm such as cardiac failure and rupture of the aorta through a sudden increase in blood pressure [@B1][@B2].([Fig. 2](#F2){ref-type="fig"}) The use of intravenous sedation was deemed appropriate to avoid sudden changes in blood pressure and to stabilize the patient\'s hemodynamics. The patient\'s respiratory depression due to morbid obesity also required attention. Accordingly, we planned to use DEX[@B4], which is reported to cause mild respiratory depression, for sedation. The surgery and sedation procedure were explained to the patient, and informed consent was obtained. The surgery involved three oral surgeons, one board-certified dental anesthesiologist, and one nurse. The patient was required to fast for six hours before the procedure. We adopted Fowler\'s position during the surgery, and vital signs (peripheral capillary oxygen saturation, heart rate, blood pressure, and electrocardiogram) were continuously monitored. Oxygen was administered at 3 L/min through a nasal cannula. An initial loading dose of DEX was administered at 4.4 µg/kg/hr for 10 minutes, followed by continuous infusion at 0.075 to 0.3 µg/kg/hr. The procedure took 1 hour 27 minutes to complete, with stable hemodynamics throughout and no marked changes, even during local anesthesia with 2% lidocaine and 1:80,000 epinephrine.([Fig. 3](#F3){ref-type="fig"}) Surgery was safely completed without any complications. For postoperative analgesia, oral celecoxib 400 mg/day was administered until 24 hours after surgery. Thereafter, acetaminophen 3,000 mg/day and loxoprofen sodium hydrate 60 mg were administered as rescue analgesics. Postoperative hemostasis was satisfactory, and no perioperative complications were observed. III. Discussion =============== A dental surgeon should have knowledge of multiple systemic diseases and have the ability to plan and perform appropriate treatment procedures in patients requiring whole body management. Herein, we report successful invasive oral treatment in the form of tooth extractions and cystectomies for the removal of intraoral infectious sources before elective cardiac surgery in a 45-year-old morbidly obese male patient with an aortic aneurysm. Aortic aneurysms are a medical emergency and can quickly lead to death. The prime consideration of the medical management for aortic aneurysms is strict blood pressure control[@B1]. Physical and emotional stress such as anxiety or pain increases blood pressure, and patients undergoing dental treatment are generally anxious. Long surgical duration and intraoperative postural maintenance during oral surgical procedures can cause physical and emotional stress. Moreover, the use of local anesthetics with vasoconstrictors during invasive procedures such as tooth extraction and pain due to an inadequate response to infiltration anesthesia can increase blood pressure. In our patient, the stabilization of circulatory dynamics was very important to prevent the progression and rupture of the aortic aneurysm through a sudden increase in blood pressure. Initially, we considered using 3% mepivacaine without a vasoconstrictor for local anesthesia. However, 2% lidocaine with 1:80,000 epinephrine was selected for the following reasons. First, mepivacaine is believed to have weaker anesthetic effects compared to lidocaine. Second, the effects of epinephrine[@B5] added to the local anesthetic are shortlived, lasting only a few minutes. Finally, an increase in endogenous epinephrine secretion induced by pain can increase blood pressure to a greater extent than exogenous epinephrine. DEX is a popular sedative used in the intensive care unit for patients undergoing mechanical ventilation and has been recently approved for use in Japan in non-intubated patients requiring sedation. Since the patient\'s diaphragmatic movement would be impaired with consequent breathing difficulty in the supine position because of his morbid obesity, the procedures were performed with the patient in Fowler\'s position. Furthermore, the patient had a mild respiratory reserve capacity and was highly sensitive to sedatives, raising the concern of sleep apnea. Therefore, it was considered appropriate to use sedatives that caused mild respiratory depression. Aortic regurgitation is widely known to cause infective endocarditis. Montazem[@B6] reported that infective endocarditis can be observed with high frequency in cases of oral infection. Therefore, the elimination of intraoral infectious sources before cardiac surgery, particularly valve replacement, is important[@B7]. However, severe complications are known to possible during oral surgical procedures in patients with cardiac comorbidities. Care must be taken to prevent the development of life-threatening complications in patients undergoing oral surgical procedures in order to improve the success rate of cardiac surgeries in patients with cardiac comorbidities. Intravenous sedation is extremely useful in stabilizing circulatory dynamics in such patients, and midazolam and propofol are commonly used sedatives. The former induces a dose-related settlement of the tongue root and respiratory depression, while the latter exhibits inhibitory effects on the heart and cardiovascular status. In addition to causing respiratory depression, DEX is an agonist of 2-adrenergic receptors in certain parts of the brain[@B5], causing mild respiratory depression and exhibiting myocardial protective effects and analgesic and sedative effects[@B3]. We believe that DEX was the most suitable for our patient, in whom oral surgical procedures were associated with very high risk and might not have been possible without sedation using DEX. In conclusion, our understanding of risk factors associated with systemic diseases enabled us to plan and safely perform oral surgical procedures with appropriate perioperative whole body management in a patient scheduled to undergo subsequent cardiac surgery. **Conflict of Interest:** No potential conflict of interest relevant to this article was reported. ![Imaging findings. A. Original panoramic X-ray findings. Periapical lesions are observed in the apical area of the right mandibular third molar, left mandibular first and second molars, and left maxillary central incisor. B. Computed tomography findings (sagittal section image). Cyst-like images are observed in the apical area of the left maxillary lateral incisor and second molar.](jkaoms-42-162-g001){#F1} ![Problems in the invasive treatment of this patient.](jkaoms-42-162-g002){#F2} ![Summary of the therapeutic procedure. 1⃞: Extraction of the left maxillary lateral second molar and cyst enucleation. 2⃞: Extraction of the left maxillary central incisor and lateral incisor and cyst enucleation. 3⃞: Extraction of the left mandibular first and second molars. 4⃞: Extraction of the right mandibular third molar. Observer\'s assessment of alertness/sedation (OAA/S) scale: Score level 5=responds readily to name spoken in normal tone, score level 4=lethargic response to name spoken in normal tone, score level 3=responds only after name is called loudly and/or repeatedly, score level 2=responds only after mild prodding or shaking, score level 1=does not respond to mild prodding or shaking. × to ×: sedative duration, ㉧ to ㉧: operative duration.](jkaoms-42-162-g003){#F3}

{ "pile_set_name": "PubMed Central" }

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INTRODUCTION {#sec1-1} ============ Visual impairment has significant effects on life quality and leads to serious social and economic implications for family and society. It is also a challenging issue for employment and recruitment in many countries. Visual impairment may affect productivity and may lead to injuries. The consequences of visual impairment are an important public health issue with greater impact in the developing countries, where 80% of the world blindness occurs.[@ref1] The World Health Organization (WHO) encourages all countries to monitor the magnitude and causes of visual impairment in order to scrutinize and eliminate avoidable blindness.[@ref2] According to WHO estimates, every 5 seconds one person goes blind in the world. There were 40 to 45 million blind individuals worldwide in 2004 and about three times this figure suffered from visual impairment.[@ref3] It is estimated that the number of blind people worldwide will reach 76 million in 2020.[@ref3],[@ref4] Several studies reporting the causes and prevalence of blindness and visual loss exist in the literature. However, only a limited number of these reports is based on blindness registries, mainly in developed countries. Visual problems have also significant implications in the military in terms of exemption from the military service.[@ref5] As a developing country, Turkey is one of the biggest countries in Europe. The population of Turkey is approximately 77 million in 2013.[@ref6] Comparing to other European countries, Turkey has a considerably young population. According to Turkish Statistical Institute, the number of people between the age of 20-40 years-old is 25 million (32,5%).[@ref6] The Ministry of Health in Turkey does not have a detailed blindness registry. However, because of the compulsory military service in Turkey, each male at the age of 20-years old is examined in Military Recruiting Offices and in the military hospitals. The health status and diseases of every single young man is stored in the database of National Defense Ministry. Those data give valuable information about the prevalence of diseases and causes of disabilities. Detailed results related to musculoskeletal diseases were published previously.[@ref7] This study has been carried out to explore the prevalence and causes of blindness in young Turkish men who have undergone medical examination for compulsory military service between 1 January 2009 and 31 December 2011. METHODS {#sec1-2} ======= The number of young men examined for compulsory military service between 2009 and 2011 was 1.777.500. The number of people used in prevalence calculation in this study was 579.503 for 2009, 583.299 for 2010 and 614.698 for 2011.[@ref7] Military service is compulsory for men in Turkey. Each male at the age of 19-years old is primarily examined in Recruiting Offices where two physicians are available in accordance with Health Ability Regulation document (HARD) of Turkish Armed Forces (TAF) before they start their military service. In case of obligatory reasons, the examinations are performed after the age of 19-year old. During these examinations, disabilities that will prevent from performing military service according to HARD are analyzed. From ophthalmological point of view, proceedings for cases such as enucleation and evisceration are made in Recruiting Offices, whereas cases requiring ophthalmic examination are referred to primary military hospitals, the number of which is 37. If required, the candidates are referred to one of 6 secondary Military Hospitals. The third referral centers are GATA Military Medical School (Ankara) and Haydarpasa GATA Military Medical School (Istanbul). Health reports belonging to the men not suitable for military service are collected in Health Department Directorate of National Defense Ministry (NDM). Descriptions in the Survey {#sec2-1} -------------------------- Decisions regarding health conditions in TAF are made in accordance with the criteria defined in HARD. All diseases have been classified as those suitable for military service, those unfit for military service (UFMS), and those under treatment of an active disease or in the recovery period of a disease. In this report, only those unfit for military service were evaluated. Those fit for military service and those under treatment or having an active disease have been excluded. Ophthalmic examination methods and criteria for UFMS {#sec2-2} ---------------------------------------------------- From ophthalmological point of view, unilateral ophthalmic pathologies causing a visual acuity (VA) under 0.2 (VA≤0,1) and bilateral ophthalmic pathologies causing a total VA (right eye plus left eye) under 1.0 (VA≤0,9) are classified as UFMS. In addition to VA measurements, some high refractive error and anisometropia data and ocular alignment disorders are categorized as UFMS irrespective of VA criteria. These criteria are (1) The sum of high axis of both eyes over 14.00 diopters, (2) anisometropia greater than 4 diopters in hypermetropics and greater than 6 diopters in myopics (by using spherical equivalents), (3) ocular movement disorders more than 'one gaze position in one eye or equal or more than one gaze position in both eyes, (4) visual field loss greater than 2/3 in one eye or greater than 1/2 in both eyes with regard to Humphrey visual field perimeter using central 30/2 SITA Fast program and (5) any night blinding disorder including retinitis pigmentosa, stationary night blinding disorders, etc. The diagnosis of UFMS in candidates with previous evisceration or enucleation due to any cause could be made in the recruitment offices by general practitioners (in case the candidates documented, the surgical report of the disease and evisceration cause). Most of the diagnosis causing UFMS could be made in secondary or tertiary referral military hospitals. Best corrected visual acuity was obtained with Snellen charts. Refraction data was obtained by using auto refractometers 40 minutes after instilling 3 drops of cyclopentolate (Sikloplejin, Abdi Ibrahim, Istanbul, Turkey) with 5 minutes apart. Vertex distance was set as 12.5 mm in the autorefractometers. Globe position and eye movements, pupillary reactions, biomicroscopic and fundoscopic examinations were performed in all cases. The candidates with nyctalopia and/or photophobia were evaluated by full-field electroretinogram (ffERG) (Roland-Consult, Wiesbaden, Germany). The diagnosis of night blinding disorders (retinitis pigmentosa, fundus albipunctatus, congenital stationary night blindness) and cone dystrophies was made by using ffERG. The candidates with macular dystrophy or macular pigmentary changes were evaluated by multifocal electroretinogram (mfERG), pattern electroretinogram (PERG) and ffERG when indicated. The candidates with suspected malingering were evaluated by pattern visual evoked potentials (PVEP) and clinical malingering examination methods that were explained in detail elsewhere.[@ref8] The candidates with optic disc pallor were evaluated by standard ophthalmologic examination, retinal nerve fiber layer thickness analysis, PVEP and orbital and cranial magnetic resonance imaging (MRI). Administrative and Ethical Permissions {#sec2-3} -------------------------------------- The data for this study was acquired from Surgeon General Office of NDM. Ethical permission of the study was obtained from Ethics Committee of Gulhane Military Medical Academy (GMMA). Evaluating the data and statistical analysis {#sec2-4} -------------------------------------------- In order to examine the characteristics of geographic distribution, the provinces where the recruiting offices of the candidates are located were grouped as west, south, central, northern and eastern zoning system as prepared by Turkish Statistical Institute. Since medical examinations before military service began at the ages of 19-20, the ages of the cases have been grouped as "19, 20, 21-24, and 25 years and above" when examining the demographic characteristics. The ophthalmic pathologies responsible for the UFMS were categorized into 4 groups; (1) refractive errors, (2) strabismus and nystagmus, (3) anterior segment causes, and (4) posterior segment causes. Study data was transferred to computer and analyzed with SPSS 15.0 software. RESULTS {#sec1-3} ======= Health data of 1.777.500 military service recruits whose health examinations have been carried out between the years 2009-2011 has been reviewed. It was found that 14.862 cases had ophthalmologic diseases causing unfitness for military service. The prevalence of ophthalmologic diseases causing unfitness for military service was found 0.746% for 2009, 0.871% for 2010 and 0.889% for 2011. The most populous age group was 19 years old (31.8%). The candidates with 25 or more years old comprised 21.8% of the candidates. The most number of the examinations (25.8%) were performed in the central region of Turkey. Detailed age and region distribution of the candidates in years 2009-2011 are presented in [Table-I](#T1){ref-type="table"}. ###### Demographic data of the candidates for military service. 2009 2010 2011 Total -------------- ------ ------- ------ ------- ------- ------- ------- ------- ------ Age groups 19 1546 35.8 1540 30.3 1632 29.9 4718 31.8 20 648 15.0 926 18.2 1037 19.0 2611 17.6 21-24 1208 28.0 1463 28.8 1602 29.3 4273 28.8 25 and above 919 21.2 1149 22.7 1192 21.8 3260 21.8 Region West 739 17,1 901 17,7 920 16,8 2560 17,2 South 426 9,9 568 11,2 552 10,1 1546 10,4 Central 1177 27,2 1328 26,2 1329 24,3 3834 25,8 North 631 14,6 689 13,6 776 14,2 2096 14,1 East 1348 31,2 1592 31,4 1886 34,5 4826 32,5 Total 4321 100.0 5078 100.0 5463 100.0 14862 100.0 Among ophthalmic causes, refractive errors were the most frequent pathology causing UFMS (40.1%). Among refractive errors, 'high axis over 14.00 diopters' was the most common refractive error type (65.58%). Mean prevalence of refractive errors causing UFMS was 3.35 ‰. Detailed refractive error data is presented in [Table-II](#T2){ref-type="table"}. ###### The distribution of ophthalmic causes of unfitness for military service. Diagnoses 2009 2010 2011 Total -------------------------------------------------------------------------------- ------ ------ ------- ------- ------ ------- ------ ------ ------- ------ ------- ------- **Refractive Errors** 1664 38.5 0.287 2005 39.5 0.344 2292 42.0 0.373 5961 40.1 0.335 High axis over 14 dpt. (OD+OS) (any VA) 1305 30.3 0.225 1607 31.6 0.276 1817 33.3 0.296 4729 31.8 0.266 Anisometropia (VA≤0.1) 359 8.2 0.062 398 7.9 0.068 475 8.7 0.077 1232 8.3 0.069 **Ocular Alignment Disorders** 596 13.8 0.102 851 16.6 0.146 814 14.9 0.133 2261 15.3 0.128 Strabismus 233 5.4 0.040 344 6.7 0.059 338 6.1 0.055 915 6.2 0.052 a\. Strabismic amblyopia (VA≤0.1) 211 4.9 0.037 300 5.9 0.051 303 5.5 0.049 814 5.5 0.046 b\. EOM paralysis (any VA) 22 0.5 0.003 44 0.8 0.008 35 0.6 0.006 101 0.7 0.006 Nystagmus (VA≤0.1) 130 3.0 0.022 163 3.2 0.028 138 2.7 0.023 431 2.9 0.024 **Anterior Segment Pathologies** 859 19.8 0.148 984 19.3 0.169 1133 19.8 0.184 2975 20.1 0.167 Corneal and lens pathologies (VA≤0.1) 532 12.3 0.092 595 11.7 0.102 661 12.1 0.107 1788 12.0 0.101 Pseudophakia (VA≤0.1) 117 2.7 0.020 157 3.0 0.027 162 3.1 0.027 436 2.9 0.025 Aphakia (any VA) 187 4.3 0.032 206 4.0 0.035 199 3.6 0.032 592 4.0 0.033 Previous penetrating keratoplasty (VA≤0.1) 13 0.3 0.002 17 0.4 0.003 105 0.9 0.017 134 1.0 0.007 Previous keratorefractive operations (VA≤0.1) 10 0.2 0.002 9 0.2 0.002 6 0.1 0.001 25 0.2 0.001 **Posterior Segment Pathologies** 1384 31.9 0.294 1540 30.5 0.265 1482 27.1 0.241 4406 29.69 0.249 Previous vitrectomy surgery due to any cause and history of retinal detachment 96 2.2 0.017 116 2.3 0.020 114 2,0 0.018 326 2.19 0.019 Nonsurgical retina, vitreous and optic nerve disease 1194 27.6 0.261 1293 25.6 0.222 1264 23,3 0.206 3751 25,3 0.212 Ocular albinism 71 1.6 0.012 96 1.9 0.017 67 1.2 0.011 234 1.6 0.013 Glaucoma (due to VA ≤0.1 or VF loss) 23 0.5 0.004 35 0.7 0.006 37 0.6 0.006 95 0.6 0.005 Ocular alignment disorders (including strabismus and nystagmus) comprised 9.1% of the ophthalmic causes of UFMS. Strabismic amblyopia was the most common pathology among ocular alignment disorders (60.4%). The prevalence of strabismic amblyopia (blindness, VA≤0, 1 was 0.046%. Nystagmus comprised 2.9% of ophthalmic causes of UFMS. The mean prevalence of nystagmus causing UFMS was 0.024%. Detailed ocular alignment disorders data is seen in [Table-II](#T2){ref-type="table"}. *Cornea and lens pathologies were evaluated under the title of anterior segment pathologies. Corneal and lens pathologies other than pseudophakia, aphakia and penetrating keratoplasty comprised of 12% of ophthalmic causes of UFMS. Although detailed data is not available, most of those causes of anterior segment pathologies are corneal scars and cataract. Mean prevalence of these pathologies was 0.101%. Aphakia comprised 4.0% of ophthalmic causes. The mean prevalence of aphakia was 0.033%. Pseudophakia comprised 2.9% of ophthalmic causes and the mean prevalence of pseudophakia was 0.025%. The mean prevalence of previous penetrating keratoplasty was 0.008%. Detailed anterior segment pathologies causing UFMS is presented in [Table-II](#T2){ref-type="table"}*. Posterior segment pathologies comprised 31.8% of ophthalmic causes of UFMS. The mean prevalence of blindness from posterior segment pathologies was 0.266%. *Posterior segment pathologies causing UFMS is shown in [Table-II](#T2){ref-type="table"}*. DISCUSSION {#sec1-4} ========== To our knowledge, this is the first report using official registries in NDM on the causes of severe visual impairment from Turkey. This report includes the results of ophthalmologic examination of all young men coming up to 19-years-old in Turkey. This is because this report presents the real data-not statistics- about the causes of severe visual impairment in Turkey. Currently, in Turkey, the overall prevalence of severe visual impairment in young men is 0.554%*. This ratio includes visual field losses (over 2/3 in an eye or over 1/2 in both eyes) related to any cause and excludes uncorrected refractive errors. Nonsurgical retina, vitreous and optic nerve diseases are the most frequent cause of visual impairment (0.212%). The detailed causes are not available in this report. The authors involved in the ophthalmic examination of the candidates for military service (FCG, UY) state that most of those causes are related to optic atrophy and retinitis pigmentosa. Corneal and lens pathologies are the second most frequent cause of blindness (0*.101%)*. Detailed causes are not available in this report. The authors also state that most of the corneal and lens pathologies causing visual impairment are related to corneal leucoma, keratoconus and cataract. The prevalence of visual impairment with pseudophakia and aphakia are 0.025% and 0.033%. The prevalence of visual impairment with previous keratorefractive operations is extremely low (0.001%). To our knowledge, there is no data available on the incidence of visual impairment in Turkey to compare with this study*. *Refractive errors are the most prevalent cause of visual impairment in young Turkish men. The prevalence of high refractive errors is 0.335%. Visual impairment due to refractive errors is far more than that ratio as that ratio includes only the persons with refractive errors over 14.00 dpt (high axis OD+high axis OS). This report does not include the ratio of uncorrected refractive errors to all refractive errors. Weih et al. reported that 60% of participants with visual impairment due to refractive error and 82% of participants with VA of less than 20/40 to 20/60 were not wearing distance correction in a sample of Australiens.[@ref9] This ratio may somewhat be higher in Turkey due to lower socioeconomic status. The authors also reported that less than 1% of people aged 40-49 years had visual impairment due to uncorrected refractive error*. *The prevalence of registered blindness was 0.17%-0.22% in Ireland (blindness was defined as BCVA in the better eye of less than 6/60)[@ref10] and 0.26%-0.32% in Israel (using WHO criteria).[@ref11] The incidence of registered blindness was 0.036%-0.029% in Israel (using WHO criteria), 0.02% in Ireland using (US criteria) and 0.012% in Southern Germany (BCVA in the better eye less than 1/50).[@ref11]-[@ref14] The prevalence of blindness in a district of China in male population with an age between 1 to 29-years-old was reported as 0.015% in 2009.[@ref14] The reported blindness increased from 114.7 per 100.000 in 2003 to 145.8 per 100.000 in 2006 to 165.9 per 100.000 in 2009 although the population of the district remained stable. The authors stated that increase in prevalence during 2001-2009 represented increased registration rather than increasing levels of disease. The registries in NDM did not allow us to have the prevalence of blindness in terms of WHO or US criteria. However, from our data, we can state that the prevalence of monocular blindness in Turkey (VA≤0.1) is 0.055%*. This prevalence cannot be directly compared to the prevalence of blindness reported from other countries. This article was designed as a further analysis of data set about disability reasons young adult males in Turkey between 2009-2011. The previous paper was published as "The prevalence of disorders causing disability in young adult males in Turkey between 2009-2011; Pak J Med Sci 2013 Vol. 29 No. 5".[@ref4] ***Funding:*** The authors of this study did not receive any financial support for this study. ***Declaration of interest:*** None. {#sec1-5} Authors' contribution {#sec2-5} --------------------- **FCG:** Substantial contributions to conception and design, revising it critically for important intellectual content, final approval of the manuscript. **NK:** Acquisition of data, drafting and final approval of the manuscript. **UY and AI:** Substantial contributions to conception and design, drafting and final approval of the manuscript. **IA and RA:** Acquisition of data, revising it critically for important intellectual content, final approval of the manuscript. **SK:** Analysis and interpretation of data, drafting and final approval of the manuscript.

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Body ==== In this issue, one case report, one review article, one brief report, and three original papers were published. Tae-Sung Yeum et al. reported a clinical case of a rare neurologic disorder, anti-N-methyl D-aspartate receptor encephalitis without teratoma, which presented psychotic symptoms. Yeeun Lee et al. wrote a review article focused on the mental health and related environmental factors of ethnic minority youths in the Republic of Korea (ROK). Minha Hong et al. presented a brief report on the differences in the practice of psychiatrists for attention-deficit/hyperactivity disorder (ADHD) among adults in the ROK according to the training background of fellowship for the child and adolescent psychiatry. Minha Hong et al. surveyed to examine the differences and the survey questionnaire used in this study was created by the "Adult ADHD Study Group" organized by the Korean Academy of Child and Adolescent Psychiatry (KACAP) as the first step to establish a practice parameter for ADHD in adults \[[@ref1]\]. Minji Lee and Subin Park suggested that sociodemographic variables, familiarity with the disorder, and personality traits are associated with the lay beliefs about tic disorders and Tourette's syndrome from the findings of an online survey. Geon Ho Bahn et al. determined the cut-off scores of the Korean adult ADHD rating scale (K-AARS). The K-AARS is the first scale developed in ROK to diagnose ADHD in adults. Mi Ae Oh et al. article, The Analysis of Self-Mutilation in Adolescence Based on the Theory of Mentalization: From Sukhvinder in the Novel "Casual Vacancy," is a secondary publication in Englishthatwas first publishedin Koreanandinterpreted the self-cutting behaviors of adolescents in literature. Since Eugene Garfield proposed the idea of impact factor (IF) in the journal "Science" in 1955, the publication of scientific treatises has undergone a tremendous transformation \[[@ref2]\]. Although IF is not a perfect tool to measure the quality of articles, as there is no better technique and it has the advantage of already being in existence, IF is considered to be a good technique for scientific evaluation \[[@ref3]\]. However, we tend to overlook two things about IF. First, according to the 80/20 phenomenon, 20% of the articles may account for 80% of the citations. Second, of the 38 million items cited during the period from 1900 to 2005, only 0.5% were cited more than 200 times and half were not cited at all \[[@ref2]\]. One of the commonalities of frequently cited papers is that they are written in English. Does this suggest that an academic paper has to be written in English in order to be quoted more? The billboard chart is an indicator of the popularity of global music markets. BTS ranked first on the Social 50 in the bill-board chart in the last week of June 2019 \[[@ref4]\]. Interestingly, members of the BTS global official fan club ARMY sing BTS songs, even though their lyrics are not in English. Similar to the BTS ARMY, the frequency of citations of academic papers does not depend on whether the papers are written in English or not, but on how interested the readers were in the paper. The editorial committee of the Journal of the Korean Academy of Child and Adolescent Psychiatry (JKACAP) is looking for ways in which the papers can be cited more and is also trying to prevent the papers published in this journal from becoming orphaned articles. Unless the readers are interested in the articles in JKACAP written in English, the articles will be shunned by readers. As one of the better tactics is for the article to be included in popular bibliographic databases, the editorial committee applied for JKACAP to be indexed in the Emerging Sources Citation Index (ESCI) \[[@ref5]\] on December 18, 2017 and received the acceptance mail on June 17, 2019. ESCI journals are being evaluated on a continual basis for inclusion in the Science Citation Index Expanded (SCIE) and Social Science Citation Index (SSCI). As the editor-in-chief of JKACAP, I am extremely grateful to the members of the editorial committee and the people who support publishing in this journal. With JKACAP being indexed in the ESCI, we will now strive harder to contribute to the development of the mental health of children and adolescents not only in Korea, but throughout Asia. Hansen JR. First man: the life of Neil A. Armstrong. New York: Simon and Schuster; 2018

{ "pile_set_name": "PubMed Central" }

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22,213

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Key MessagesAdaptive and adequately resourced health systems are necessary but not sufficient to ensure good health outcomes among adolescent girls in post-conflict settings.Integrating an understanding of the social determinants of health and wellbeing is critical in shaping health service uptake and ultimately outcomes for adolescent girls.Psychosocial outcomes are also central to broader health and wellbeing and need urgent attention in post-conflict developing country settings in particular.Tailored gender- and age-sensitive psychosocial service provisioning is vital in supporting the health and wellbeing of adolescent girls---a cohort often facing complex vulnerabilities in post-conflict settings. Introduction ============ Adaptive and adequately resourced health systems are necessary to ensure good health outcomes in fragile and post-conflict settings. Nevertheless, it is increasingly recognised that domains beyond the health system also significantly impact on broader health and wellbeing. This paper focuses on the importance of psychosocial services for vulnerable populations and specifically adolescent girls in fragile contexts. Adolescence is a pivotal time in the life course when there is considerable opportunity for change. At the same time it is also a period when adolescent girls are especially vulnerable to a range of life-changing experiences including trauma, social isolation, bullying by peers, a lack of supportive adults, and sexual and other forms of violence. The aims of the study are to highlight why current humanitarian and biomedical approaches in the current form are inadequate to address these complexities. It also stresses how critical an understanding of the social determinants of health is to enable adequate and context appropriate responses. This paper argues that it is important to go beyond biomedical approaches and instead investigate the complex social determinants of health critical for achieving broader health and wellbeing, especially amongst the most vulnerable. There is now more evidence that explores the links between gender and adolescence, mental health and development (e.g. [@czx127-B33]; [@czx127-B39]). Yet in many developing countries, health system debates and research have tended to overlook mental health and psychosocial wellbeing generally (e.g. [@czx127-B8]) and particularly the needs of adolescent girls in post-conflict or other fragile settings (for further details of relevant literature see [@czx127-B39]). To contribute to these debates, we draw on qualitative fieldwork from 2014 to 2015 in three countries recently affected by conflict---Gaza, Liberia and Sri Lanka---honing in on the psychosocial vulnerabilities of girls aged 10--19 and the extent to which these are addressed by the existing psychosocial service environment. Situating our study within the wider literature =============================================== Drawing on a large and diverse body of literature, in this section we discuss the conceptual underpinnings of this study, making explicit linkages between the social determinants of health and the mental health and psychosocial wellbeing of adolescent girls. According to the World Health Organization (WHO) 'The social determinants of health (SDH) are the conditions in which people are born, grow, work, live, and age, and the wider set of forces and systems shaping the conditions of daily life. These forces and systems include economic policies and systems, development agendas, social norms, social policies and political systems'[^1^](#czx127-en1){ref-type="fn"}. The ways in which these conditions are experienced by those at risk of psychosocial ill-being are refracted through a number of individual level variables, including age and gender, and family and community level factors, including discriminatory gendered social norms (see e.g. [@czx127-B19] Why the focus on adolescents, particularly girls? ------------------------------------------------- Although definitions vary, the United Nations defines adolescence as the second decade of life between 10 and 19 years ([@czx127-B43]). During this time, girls and boys undergo bodily changes (e.g. the start of menstruation, the development of breasts for girls and deepening voice and hair growth for boys) and begin the transition from childhood to adulthood, when they will assume roles such as parent, spouse, worker and citizen. During adolescence, the constraining role and influence of gendered social norms on girls' lives (in a range of domains, from education and marriage to mobility outside the home) becomes more evident ([@czx127-B20]; [@czx127-B43]). It is also during this period that developmental changes occur and key skills are acquired, such as those that relate to: health, physical and neurological development; social behaviours and attitudes; and education and employment. For girls and young women in many settings, this life stage also heightens deprivation, danger and vulnerability, including an increased risk of sexual and domestic violence. This constrains girls' ability to develop agency and leads to other development deficits, often leading to negative consequences in their adult lives (see e.g. [@czx127-B18] and [@czx127-B14]. Other context-specific factors, such as national laws and local cultural norms and practices also have a major impact on adolescent girls' lives, dictating not only how girls should think and behave but how others expect girls to behave. In this study, the national context (of a fragile or post-conflict state) shapes how girls and their families recover from or continue to face violence, disruption, displacement and loss of livelihoods. These experience are often gendered, as social norms shape how families react to and cope with external shocks and stresses, often with negative outcomes for girls including marrying them off early and pulling them out of school (e.g. [@czx127-B22]). From mental health to psychosocial wellbeing -------------------------------------------- Mental health is now recognised as being crucial to overall health (e.g. [@czx127-B54]) and is defined as including '... subjective well-being, perceived self-efficacy, autonomy, competence, intergenerational dependence, and self-actualization of one's intellectual and emotional potential, among others' ([@czx127-B54], p. 5). Alongside the suffering it causes, mental ill-health can exacerbate household poverty, increase inequality, reduce social capital, and hinder economic growth ([@czx127-B56]). Being exposed to situations common to post-conflict contexts, such as violence, death and difficult living conditions (e.g. lack of food, epidemics, or being forced to flee one's home) is likely to negatively affect the psychosocial wellbeing of individuals and families (see e.g. [@czx127-B4]; [@czx127-B11]). Sexual violence (largely directed against women and girls), a known risk factor for mental ill-health, is also heightened in conflict contexts. Even after a conflict has ended, girls and women often continue to be subject to sexual and other violence. This often reflects a certain 'normalisation' of such violence as well as continuing availability of weapons, lack of punishment for perpetrators, and frustration among men who are no longer able to live up to the role expected of them by traditional norms (see e.g. [@czx127-B2] and [@czx127-B10]). Mental health and psychosocial risks in adolescence --------------------------------------------------- Mental ill-health and psychosocial problems typically start during adolescence; unless young girls or boys receive appropriate treatment, the results of these psychosocial problems will continue to affect them as adults ([@czx127-B55]). One study noted that between 10% and 20% of children and young people have been reported as experiencing diagnosable mental illnesses ([@czx127-B21]). This is leading to rising suicide rates, with young people now the highest-risk group for suicide in around 30% of all countries ([@czx127-B55], [@czx127-B58]); in China, India and the South East Asia region, suicide is the leading cause of death among those aged 15--19 ([@czx127-B32]; [@czx127-B57]). Other negative consequences of mental ill-health and psychosocial problems for young people include poorer school attainment, resorting to substance abuse, greater likelihood of experiencing violence, and increased likelihood of facing reproductive and sexual health-related challenges ([@czx127-B32]; [@czx127-B21]). Despite widespread and increasing awareness of the mental health challenges facing adolescents, their needs in this area are largely unmet, and particularly in developing countries. Reasons for this relative neglect include lack of effective policies targeting adolescents, limited funding and human resources (particularly qualified healthcare professionals), limited training and experience of lower-level health staff, and the ongoing stigma linked to mental ill-health (ibid.). Adolescence is recognised as a troubling time in a person's development, but it brings particular challenges for children and young people in conflict/post-conflict situations. They may experience traumatic events such as the death of (or separation from) their parents; they may be subject to sexual and/or physical violence; they may be abused, abducted, or forced to become involved with fighting forces; and they may miss out on their schooling and on other social and economic opportunities ([@czx127-B7]). All these experiences can bring on serious mental ill-health and emotional trauma, and can have negative and long-term impacts on how adolescents see themselves, interact with each other as well as on their aspirations for the future. These experiences are often gendered---in a study of Sierra Leone's child soldiers [@czx127-B5], for instance, found that girls demonstrated significantly higher scores of depression and anxiety than boys (80% vs 52% and 72% vs 55%, respectively), partly explained by the fact that girls experienced many more cases of rape and sexual abuse than boys (44% vs 5%). Towards a norms-sensitive conceptual framework for understanding psychosocial wellbeing --------------------------------------------------------------------------------------- Humanitarian actors increasingly recognise the impact of conflict and disaster on people's psychosocial wellbeing and in recent years have incorporated mental health and psychosocial support (MHPSS) into their response ([@czx127-B8]; [@czx127-B51]; [@czx127-B52]). The 2004 Indian Ocean tsunami was arguably a turning point, as it required a systematic response to people's mental health as well as physical survival needs, with a focus on building individual and community resilience ([@czx127-B42]; [@czx127-B39]). This has also given rise to new conceptual frameworks that focus on different resource domains that are affected in situations of conflict. These include: human capacity (individuals' mental and physical health, skills and knowledge); social ecology (the relationships that allow people to exist as a community, sometimes called social capital); material and physical environments; and local values and culture ([@czx127-B40]; [@czx127-B15]; [@czx127-B53]). Building on these psychosocial wellbeing frameworks, we apply a social norms lens, positing that social norms affect all domains of adolescents' lives, not just education and marriage but also access to resources (economic, material and human), their health and wellbeing, and their ability to participate in community life (agency and voice) ([@czx127-B35]). Applying a social norms lens helps us to understand and put in context the different paths to wellbeing that are available to adolescents, as well as the challenges faced by girls (and boys), and what works to address or overcome these challenges and barriers. As seen in [Figure 1](#czx127-F1){ref-type="fig"}, this approach puts adolescent girls centre stage, with key areas of wellbeing around them. Girls are situated within their families/households, their wider community and the (fragile) state, characterised by ongoing or recent conflict or other crises related to health, food security or natural disasters. These different areas of girls' lives are in turn influenced by changes in the global context, which include greater learning around adolescents' needs and the rise of MHPSS programming. ![Pathways towards adolescent girls' psychosocial and broader wellbeing](czx127f1){#czx127-F1} The availability of local services also affects girls' psychosocial wellbeing, with tailored services being especially critical to support their evolving needs (e.g. around menstruation, information on family planning, etc.). Such service provisioning needs to be informed by an understanding of the social determinants of health (e.g. gender, ethnicity and fragility) and the importance of integrating a mix of formal, faith-based providers, and informal services. Prevailing gender norms (around education, marriage, sexuality and mobility) also determine girls' vulnerabilities, their access to and uptake of relevant services, and their ability to develop their full capabilities. Services must be carefully targeted and designed to meet girls' needs if they are to maximise girls' psychosocial wellbeing and allow them to develop to their full potential. Methods and case study contexts =============================== The study design combined primary qualitative data collection with a secondary review of literature. This allowed us to situate our research within broader debates on adolescence, fragile states, and mental health and psychosocial services.[^2^](#czx127-en2){ref-type="fn"} The research involved the collection of primary qualitative data, in conjunction with national study partners, in Sri Lanka, Liberia and Gaza in 2014 and 2015. The full research proposal and instruments, which were developed jointly with the national partners, were submitted to and approved by the Overseas Development Institute's ethics committee. The study was also cleared by national research ethics boards in the study countries, i.e. in Liberia the Carter Centre has its own ethics board, in Gaza the study was reviewed by the Helsinki Framework ethics board, and in Sri Lanka the study partners made the necessary arrangements to conduct the study. Phase one of the data collection aimed to map people's perceptions (adults and young people) of the different dimensions of wellbeing (especially those affecting girls) and the factors that affect their wellbeing. The next phase focused on the availability of services, particularly looking at whether MHPSS programmes were sensitive to adolescents' (and especially girls') needs and how services could be made more responsive to girls' needs.[^3^](#czx127-en3){ref-type="fn"}[Table 1](#czx127-T1){ref-type="table"} provides an overview of the study contexts and [Box 1](#czx127-BOX2){ref-type="boxed-text"} provides an overview of the methodology. Table 1.Study sites and relevant country contextsTopicGazaLiberiaSri LankaStudy sitesShajaia neighbourhood *Gaza City*: high level of violence by Israeli forces leading to high numbers of IDPs: out of a total population on 120 000, 25% are refugees (NECC 2014). High rate of unemployment: \>50% of the residents receive social assistance (Ibid.). Conservative community: median marriage age for women is under 20 years old ([@czx127-B1])New Kru town *Montserrado county*: densely populated borough of capital city, Monrovia (∼70 000 people) ([@czx127-B35]). One of the communities that suffered most from Ebola virus and the civil war (Montserrado acted as an entry point for rebel armies entering Monrovia) (Ibid.).Diyagama village *Polonnaruwa District, North Central Province*: Majority of population (total of 7000) belongs Tamil ethnic group ([@czx127-B34]). Until 2006 the area served as a Liberation Tigers of Tamil Eelam stronghold ([@czx127-B12]).Kadalkiraman *Batticaloa District, Eastern Province*: highest level of poverty in the country (poverty head count index of 20.3%) and highest poverty gap index (5.1 compared with a national of 1.7) (UNDP 2012). Majority of the population (total of 2464 are Tamil ([@czx127-B12]). Located in high-security area with large military camps. Good road: easy access to services (Ibid.).Tubmanburg *Bomi county*, is a city of 13 114 people in one of the poorest counties in Liberia ([@czx127-B6]). During the civil war Tubmanburg served as rebel army LURD's headquarters. In total Bomi country authorities reported 175 Ebola related deaths since the Liberian outbreak started in 2014 (MoHSW 2015).Population, including youthPopulation: 1.71 million (1.2 are refugees): nearly half the population is under the age of 15 (PCBS 2012a). Ethnicity: 99% are Palestinian Arabs, 1% other (Ibid.).Religion: Predominantly Muslim: 99.3%, Christian 0.7% (Ibid.).Population: 4.19 million with nearly half under 15 and 20.4% between 10 and 17 years old ([@czx127-B59], [@czx127-B60]). Ethnicity: 2.5% Congo people (descendants of former slaves) and 95% indigenous ([@czx127-B35]). Religion: 40% Christian, 20% Muslims and 40% indigenous beliefs (Ibid.).Population: 20.3 million with 23.2% of the population between 15 and 29 years (UNDP 2014). Ethnicity: The majority is Sinhalese and 16.5% are Tamil (UNDP 2012). Religion: majority are Buddhist and 9.3% Muslim (Ibid.).Fragility context---economic situation and conflict legacyFragility: Gaza is marked as a 'hostile entity' by Israel---characterised by blockades (since 2007 and ongoing) and military operations (MoH 2014). Economy: Growth slowed from 6% in 2012 to 2% in 2013 (in Palestine) ([@czx127-B59]a, [@czx127-B60]). Gaza has an HDI of 0.736 (UNDP 2015). Conflict legacy: Conflict-related chronic stress has negatively impacted the public health of the population of Gaza and has, amongst other things, caused high levels of non-communicable diseases including mental health and psychosocial challenges ([@czx127-B1]).Fragility: Civil conflict (1989--2003) due to ethnic tension causing 150.000 deaths and displaced 850.000 Liberians (UN 2015). Ebola outbreak 2014--2015 (WHO 2015). Economy: Growth rate of 7.5% in 2014. HDI of 0.412 in 2013 (UNDP 2014).Fragility: Violent uprisings between 198 and -2009 caused by socioeconomic and ethnic grievances by Tamils. Country was hit by a tsunami in 2004 killing 35 000 people ([@czx127-B12]).Economy: Gained middle income status in 2010 (Ibid.). HDI increased by 28% between 1980 and 2012 (HDI in 2012: 0.715). Human development indicators lagging in several provinces and rural areas (UNDP 2014).Conflict legacy: 48% of the population living in extreme poverty. High unemployment, food insecurity (UN Liberia 2013).Conflict legacy: Conflict caused loss of thousand lives (likely as many as 330.000 deaths) injuries and disability (∼40.000 surgical procedures and 5.000 amputations during the conflict), destruction and displacement (290.000 Sri Lankans displaced from the battle zone) (UN 2011).Gendered effects of conflict including VAWGGendered vulnerabilities: Conflict limits women's already low mobility and restricts their agency and ability to build up social networks ([@czx127-B1]).Many women are displaced and live in temporary mixed housing arrangements that are not constructed gender-sensitively (mixed living room areas and lack of adequate toilet facilities) (Ibid.).Gendered vulnerabilities: Higher rates of illiteracy for women (adult literacy rates: 37% for girls and 63% for boys (UNICEF 2007). With only 58.2% of all women participating in the labour force, most are forced to engage in vulnerable employment (UNDP 2013). Violence: During the civil conflict between 61% and 77% of all women were raped. Normalisation of GBV including FGM/C, early marriage and polygamy, sexual and domestic violence (UN 2013). Over 34% of women reported domestic violence by their partners ([@czx127-B16]).Gendered vulnerabilities: Conflict restricted movement of women and girls impacting negatively on their education and livelihoods ([@czx127-B17]). Conflict changed traditional gender norms: in some cases girls and women became combatants or were responsible for household livelihoods, leading to a form of female empowerment ([@czx127-B37])Violence: Within a context of conflict, brutalisation and militarisation opportunities for sexual violence have been normalised for women (Ibid.).Violence: High level of domestic violence: 51% of married women experienced violence at the hands of her husband ([@czx127-B30]). Girls in temporary shelter arrangements are extra vulnerable for gender based violence and sexual abuse ([@czx127-B1]).Mental health policy and services contextPolicy: National Health Strategy (2012--2016), Public Health Policy for Children (2012) Service providers: Ministry of Health established 54 Primary Health Care centres and 13 hospitals, but only one psychiatric hospital. Other providers include: UNRWA who established 22 centres that provide mental health services for refugees; NGOs that are providing mostly costly secondary and tertiary services in \> 50 clinics; Private-for-profit operators that are mainly focussed on obstetrics and surgical intervention (MoH 2014).Policy: National Mental Health Policy (2009), Basic Package of Health Services (2010).Policy: National Mental Health Policy (2005--2015).Service providers: Policy established by the National Institute for Mental Health: national strategy aims to reduce stigma and discrimination and calls for mental health legislation ([@czx127-B12]). Government provided mental health training for primary health care doctors (WHO 2013). GBV Desks at hospitals intervene in rape and sexual abuses cases and provide counselling to survivors ([@czx127-B12]). Child protection officers raise awareness on the importance of child protection and offer mental health services for children ([@czx127-B12]).Service providers: Only one hospital (private---with only one trained psychiatrist) in Liberia providing mental health services: Grant Memorial Mental Hospital in Monrovia. Primary health personnel lack training. Other mental health services are provided by NGOs or FBOs. Health systems were further damaged by the Ebola outbreak ([@czx127-B35]). Box 1.MethodologyWe used a range of qualitative tools including: in-depth interview (IDI) guides for adolescent girls, boys and parents; key informant interview (KII) guides for service providers and community leaders; focus group discussion (FGD) guides with parents of adolescents and service providers; and guides for carrying out intergenerational trios--where a grandmother/grandfather, mother/father and daughter/son are interviewed individually to obtain information about stasis and change across the generations (for further information see: <http://www.odi.org/sites/odi.org.uk/files/odi-assets/publications-opinion-files/9826.pdf>). Drawing on our conceptual framework, these tools addressed sets of questions around different wellbeing domains and different levels of the social ecology. The aim was not to explore psychometric measures or properties but rather to understand the ways in which different dimensions of adolescent girls' wellbeing are shaped by household, community, national and global factors and contexts.To encourage respondent participation, we also integrated visual techniques into FGDs, including community timelines and mapping exercises. These techniques helped us to gain insights into what services for adolescents exist in the focal communities, changes over time in service provision and their effects. In all countries, a mapping of mental health and psychosocial services was also carried out alongside a validated Health Facility Assessment tool to assess the appropriateness of the mental health and psychosocial services provided at facilities and to identify gaps in human resources, institutional infrastructure, and other resources.A total of 386 respondents participated in the study across the three countries (see table below). The relatively large sample size was determined by a combination of resource parameters, the importance of triangulating findings across diverse informants as well as the principle of research saturation, i.e. reaching a point where no new insights were being garnered by additional interviews.**Type of respondent, by tool and country** Type of respondentGazaLiberiaSri LankaTotals(1 round of data collection)(2 rounds of data collection)(2 rounds of data collection)Adolescents6 FGDs (44 participants in total---30 girls, 14 boys) 12 IDIs (8 girls, 4 boys)27 IDIs (18 girls, 9 boys) 9 FGDs (5 participants in each)64 IDIs (22 girls, 38 boys)192Adults---parents/caregivers, key informants27 IDIs/KIIs12 IDIs/KIIs 4 FGDS (involving 14 mixed sex adults/community leaders)76 IDIs/KIIs171Service providers1 FGD (6 participants)7 IDIs/KIIs2 FGDs (5 participants in each)23Total respondents89147150386Facility assessments53513In each country local researchers with expertise in mental health and psychosocial wellbeing worked alongside an international researcher. Following an initial 4 day training (which included role plays, discussions on how to deal with sensitive issues, piloting and revision of instruments), the teams spent ∼2 weeks in the field gathering data. The identification and selection of respondents in all countries was guided by local contacts, key stakeholders (e.g. service providers) and NGOs working in the study sites. Following initial interviews, snowball sampling was also used. [Table 1](#czx127-T1){ref-type="table"} provides details of the study sites in each country.After obtaining informed consent from research participants, interviews were recorded, transcribed and translated by in-country research partners. A detailed thematic coding structure was developed jointly by the international and country teams and findings from the interviews were analysed following this set of codes and sub-codes. In terms of quality control, daily debriefing sessions were held to facilitate ongoing analysis and reflection, and a subset of the transcripts (both in local languages and translated) were also read by the in-country study leads to ensure quality and consistency. We also had an external peer reviewer who provided detailed feedback on design and analysis questions at each stage of the study. Finally, in-country consultative meetings were organised with key stakeholders, to share and further examine findings comparing these with existing evidence and tacit knowledge, allowing also for the emergence of alternative interpretations.Limitations of the study included the absence of baseline data prior to conflict episodes and resulting challenges in teasing out conflict-specific drivers shaping the outcomes of the mental health and psychosocial services provided to adolescent girls. Additionally, it was challenging to recruit mental health service users---as originally intended in the study---because of confidentiality requirements and their psychological status. As such, the study teams interviewed both adolescent service users and non-service users and was able to obtain sufficient insights from them, as well as from other respondent (especially parents and service providers), about their perceptions of their own and others' mental health and psychosocial status. Given that we emphasise the importance of context specificity, different research teams gave different emphasis to the study components as appropriate to their settings. For example, in Liberia there was a greater focus on mental health and psychosocial support for sexual violence survivors which is a major part of the post-conflict landscape, whereas in Sri Lanka the team placed greater emphasis on issues of stigma surrounding adolescent experiences and the deeply entrenched gendered social norms associated with pubertal development which permeate both service provider attitudes and uptake of services. Results ======= Understanding adolescent psychosocial vulnerabilities in fragile contexts ------------------------------------------------------------------------- In this section we explore how adolescents in our case study countries perceive the factors underpinning their psychosocial health and the risks to it, linking to a number of key domains mapped in [Figure 1](#czx127-F1){ref-type="fig"}. ### Economic resources Most of the girls in our sample identified access to economic resources (including education and livelihood opportunities) as key to their psychosocial wellbeing. In Sri Lanka, study respondents noted that while adolescent girls and their parents strongly valued education, many girls still dropped out of school due to poverty, romantic relationships or early marriage ('*Definitely, they won't come back to school when they elope'*, 14-year old girl, Diyagama). In Liberia, all our study respondents (girls and their parents/carers) noted that education was vital to young people's future, in spite of substantial challenges around supply (e.g. quality of schooling and educational infrastructure) and demand (e.g. financial constraints):"'. my mother used to advise me... not to go around with boys, that once I had a good education, no man would be able to bluff me' (FGD, adolescent girls, New Kru Town)."In all countries, respondents reported that limited access to economic assets and livelihood opportunities by their parents/carers also affected girls' wellbeing. In Liberia, in particular, girls spoke about this causing both themselves and their parents/carers high levels of stress. It also led them to engage in risky sexual behaviours,[^4^](#czx127-en4){ref-type="fn"} as this quote highlights:"'We engage in sexual activities because at times, the things we wish to have our parents are not able to afford them; ... At times our parents coerce us to get involved in early sex...saying don't you see your friends going out there' (adolescent girl in FGD, New Kru Town)." ### Social connections and close relationships Supportive and caring relationships with family and others played a pivotal role in the psychosocial wellbeing of adolescent girls in our sample. In Sri Lanka, most girls reported one or both parents as their closest relative, providing the most day-to-day support."'I tell everything to amma (mother). I tell amma first and then after the others' (girl, 16, Kadalkiramam)."However, many girls also reported feeling disappointed or frustrated by their parents---for instance, citing 'a father's drunken behaviour' or 'being punished by a parent'. Some girls in our sample were living with an older relative (usually female) either because the family had split or one of her parents had died. While some girls described these relationships as loving and positive, others were treated badly and even traumatised by the actions of these relatives. Girls in Sri Lanka reported that relationships beyond the family---with teachers, neighbours and peers, for example---were also important, especially for obtaining advice, sharing problems and having fun. In Liberia, many girl respondents were not living with their biological families, which was a source of considerable pain and anxiety. Even so, they noted that relationships with family (whether immediate or extended), peers and neighbours were vital to their psychosocial wellbeing."'we also learn from our friends; we learn from other people; how they behave; we learn the good things from our friends not the bad things' (girl, 17, Bomi)."In Gaza, the role of family support was also mixed. While some girl respondents sought advice and support from family members, others were more reluctant, afraid that their parents might not listen or may inadvertently make the problem worse:"'If I share the problem with my mother, the problem could become bigger because I know mom will inform my dad of it' (girl, 14, Gaza)."These problems were also observed by specialists working with adolescents, as the following quote shows:"'*Adolescents feel that they are not adequately valued by the family and the community. Families don't understand the needs of adolescents. There are many communication gaps between adolescents and their families: adolescents don't understand parents' concerns and worries about them and also parents don't understand needs, aspirations and desires that adolescents have. It's a mutual misunderstanding' (social protection specialist, Gaza City).*" ### Competence and self-worth or self-esteem Our findings were mixed regarding self-worth and self-esteem. Despite strong cultural and economic constraints, a significant number of girls in Liberia and Sri Lanka demonstrated a strong sense of self-esteem, describing ambitious plans for the future:"'I wish to study well and be a teacher, build a house and look after my parents' (girl, 14, Diyagama, Sri Lanka).'I want to own house. I want my own money. I want to be working for myself' (girl, 14, Bomi, Liberia)."Study respondents---girls, their parents, and service providers --cited certain factors that were indicative of good self-esteem, including: having the love and support of parents and other relatives; having space to take part in events at school or in the community; and having hopes for a better future. Obstacles to positive self-esteem included: living in a remote area and lack of opportunities for young people to use their skills and talents; lack of places where girls are allowed to meet and socialise; and negative attitudes towards adolescents and young people. In Gaza, however, girls repeatedly complained that their family and community did not value them equally with boys, particularly when it came to sharing their views and thoughts. One girl aged 15 said: '*My grandfather, when we want to speak, says*, *"You shut up, what do you think you know? You keep yourself out of things that aren't your business.*"' ### Physical and mental health A theme common to all three countries (notably Liberia) was the sense of inadequate protection and security, which mapped closely onto the physical and mental health domain. In Liberia, difficult family life and abuse of girls (physical and sexual) were noted as major risks to girls' wellbeing. Nearly all girl respondents in that country reported that parents or guardians regularly beat them as a form of discipline. Moreover, interviews and focus groups revealed that close relatives or teachers often perpetrated the sexual violence girls were subject to. In Tubmanburg, Bomi, one 14-year-old girl explained:"'In school if a girl fails a particular subject and asks the teachers to help her, the teacher will want to sleep with her; she may accept to sleep with the teacher; then the teacher exploits the girl.'"Study respondents in Sri Lanka reported that adolescents' physical and mental wellbeing, and particularly girls' wellbeing, is closely linked to their protection and security. Influences on girls include the experiences of the adults in their lives who have lived through war and conflict. Often, these experiences continue to affect how those adults relate to their children even after the conflict has ended. In Gaza most respondents cited fear of sexual harassment as causing anxiety to girls and young women, especially those living in remote and/or border areas. Other risks cited by respondents in all three countries included experiencing aggression or violence in the home, separation of parents and families, one or both parents migrating to find work, or a parent remarrying. All these circumstances can mean that young girls have to live with their extended family, which typically means taking on more domestic chores, loss of support for them to stay in school, and frequent exposure to harsh discipline and insults. In Gaza, for instance, girls faced the added vulnerability of being displaced from their homes, and reported distress at having to stay in mixed-sex shelters. (These were often schools or community centres temporarily repurposed to support displaced families whose homes were damaged by aerial bombing during the 2014 fighting.) ### Coping responses In all three countries, adolescent girls reported developing strategies to cope with psychosocial vulnerability. Individual-level coping strategies included drawing on 'inner strength' (girls in Sri Lanka); in Gaza, girls would read, draw, paint, write stories, use social media or simply daydream; while girls in all three countries mentioned turning to religion, embracing spirituality or consulting traditional healers."'The temple is the most important place for me. I like the calm environment it has' (girl, 19, Diyagama, Sri Lanka).'I go to church. God saves us from troubles and all the sin we commit. God guards me and protects me from many things (girl, 15, Bomi, Liberia)."Across all three countries, girls cited support from the family (immediate nuclear family as well as extended family) as an important strategy in helping them cope with stresses. Aunts were often attributed with understanding girls more than their mothers did, as the aunts were often closer in age to the adolescent girls."'I'm asking God and my aunt to help me and to take care of my child while I'm in school' (17-year-old adolescent mother, Bomi, Liberia)."However, we found that families can also undermine adolescent girls' psychosocial wellbeing, whether due to violence, neglect and lack of privacy. For example, in Gaza, one adolescent lamented: *'I don't share problems with my mother. For one thing, she tells my father and the problem gets bigger!*' Within the community, adolescent girls mentioned friends, teachers and staff working for service providers as being important in helping them cope with life's challenges. Girls in Sri Lanka described how some teachers inspired them, gave them confidence and were willing to listen to their problems. In Sri Lanka and Gaza, girl respondents cited emotional support from a social worker or participation in psychosocial support programmes as helping them cope. Girls in all three countries described socialising and engaging in recreational activities as important coping strategies. Accessing formal service provision ---------------------------------- In Sri Lanka and Gaza, there is a relatively established system of formal MHPSS service provision, whereas in Liberia such services are in their early stages. While the past decade has seen considerable progress in strengthening MHPSS systems in all three countries, our study respondents note that there are still limitations in how services and programmes meet girls' specific age and gender needs. In Gaza, our findings suggest that despite MHPSS services being available, young people often do not access them due to cultural and other barriers. Whether in 'normal' every-day or crisis situations, services rarely target the needs of adolescents, with many programmes focusing on younger children. Similarly, in Sri Lanka, although there are a wide range of MHPSS services, few are designed to deal specifically with adolescents, though the gender-based violence desks in hospitals (which offer psychosocial support to victims and survivors, children and adults) are a notable exception. Respondents in Sri Lanka also suggested the need to focus on social rather than medical interventions as a response to the actions of vulnerable adolescents, such as self-harm or suicide. Liberia also has some health and social support programmes targeting young people, but they vary widely in terms of effectiveness and impact. In terms of gender-sensitive services and programmes, service user perceptions were mixed. Study respondents in Gaza reported that many service providers were enthusiastic and caring and wanted to help those accessing services. However, some (male) providers made judgements about the girls who sought such services and, in some cases, were not willing to treat the girl unless she had a chaperone (usually a family member). Not only did this undermine patient confidentiality, it also fuelled concerns around mental illness being a heritable issue, which could damage girls' marriage prospects. According to one study respondent in Gaza (a caregiver): '*The general physician stopped following up my daughters, especially the older one, and he asked me to stop treating her because she is now a young lady, and continuing receiving mental health services will affect her reputation and she will be stigmatised forever... he said* "*It is enough. Don't take her to any doctor. This will affect her if people know about her case*.*"'* Drawing on respondents' perspectives in all three countries, our findings highlight some critical gaps and disconnects in current MHPSS systems. First, according to study respondents, few activities focus on prevention or identifying groups of adolescents who may be at greater risk of mental ill-health. In Liberia, for instance, despite the existence of counsellors in schools, their role was limited to intervening only at times of crisis. '*The counsellors we have at school only counsel us when we fail---that's when they call us to talk'* (16-year-old adolescent girl, Bomi, Liberia). Second, in all three countries, key informants noted that poor-quality and fragmented MHPSS services remain a critical challenge to providing a unified response. Thirdly, respondents noted the need for greater strategic direction for MHPSS services, which are hampered by a short-term approach and limited (or a total lack of) follow-up mechanisms. Fourth, lack of evidence-based practice was mentioned by many respondents. Finally, in all three contexts, lack of essential resources (financial and human) remains an important constraint according to all study respondents. A theme that was brought up by the majority of study respondents was the negative attitudes people displayed towards using mental health services in general and in particular to adolescent girls using such services. In all three countries, people experiencing mental health problems can face substantial stigma and discrimination. In Gaza, for example, most study participants felt that although various efforts had been made to reduce stigma (e.g. through carrying out awareness-raising programmes), they have largely proved ineffective; the result is, according to study respondents, that many people delay seeking help from those services and often do so only after using traditional healers. Adolescent girls' psychosocial wellbeing covers many dimensions and sectors, yet the provision of services remains siloed and constrained by weak coordination. In Gaza, respondents were concerned that there was very limited coordination between psychosocial and mental health service providers and programmes, minimal exchange of information and feedback, and hardly any systematic follow-up of referred cases. In Liberia, respondents also noted that there were few synergies across sectors providing specific MHPSS services or programmes. Moreover, despite mental health professionals having received training in child and adolescent services, they have tended to focus their activities on adults and individuals with serious mental illness. In Sri Lanka, vertical integration between key sectors (health, education and child protection), from community-based services to district- and national-level mechanisms (although there remains a strong focus on adolescent girls' protection and medical needs) was identified in policies and approaches by study respondents. However, on the ground, service providers noted that collaboration across sectors is generally low. The situation is similar for horizontal linkages---while there are many non-government organisations (NGOs) working in different sectors, most, according to study respondents, work independently at local level. Discussion ========== Overall, four broad insights emerged from our study that also contribute to literature on the social determinants of health (e.g. [@czx127-B9], [@czx127-B50]). Firstly, it is vital that social determinants are located within specific political economy understandings to take into account the complex and multi-layered challenges that affect access to all services including mental health and psychosocial support services. In our case study countries the political economy is characterised by fragility in terms of governance, the rule of law and institutional capacities and coordination. This fragility will clearly impact the kind, level, comprehensiveness and quality of service provision as well as the extent to which service are accessible and affordable, and particularly for adolescent girls. While all three case study countries are classified as post-conflict, we found that the political economy dynamics vary significantly, with important implications in terms of psychosocial vulnerabilities and the service environment. In Sri Lanka, poverty emerged as the dominant driver of psychosocial vulnerability among adolescent girls, although the conflict persists as a shadow in their lives. By contrast, in Gaza, conflict (or the risk thereof) due to the Israeli blockade and periodic bombings is ever-present for everyone, but with particular challenges for adolescent girls. In Liberia, despite the fact that the conflict formally ended \>10 years ago, levels of sexual violence and abuse of women remain among the highest in the world.[^5^](#czx127-en5){ref-type="fn"} This reflects what others have termed 'hyper-masculinity', whereby social norms around masculinity normalise and reinforce practices of sexual and gender-based violence (SGBV) rather than seeking to reframe acceptable male attitudes and behaviours as part of the broader peace-building process (see e.g. [@czx127-B3], [@czx127-B10], [@czx127-B13], [@czx127-B61]). Secondly, although attention to social and gendered norms within the social determinants of health literature is growing, it remains too often cursory and analysts fail to disentangle the wide-ranging consequences of social and gendered norms, both on health and wider psychosocial wellbeing. Integrating such a perspective is thus critical and what the current study also strives to do. Thirdly, and linked to the second broad insight, our study highlights the particular psychosocial vulnerabilities adolescent girls face and how they are closely linked to and intertwined with a range of gendered social norms that begin to bear heavily on girls' lives as they enter adolescence. Thus informed by our conceptual framework, which outlines adolescent girls' pathways to psychosocial wellbeing, our study suggests that norms around mobility, marriage, sexuality, education, time use and emotional life underlie and explain much of the behaviour of adolescent girls, including how they perceive and experience mental health and psychosocial distress as well the forms of support, both informal and informal, both available to them and which they are able to access. Yet the role that gendered social norms can play in influencing accessibility and update of services is too often neglected in health systems thinking in many developing country contexts. In Sri Lanka, for instance, barriers to psychosocial service uptake for girls include the stigma associated with admitting to psychosocial problems and the fact that a girl cannot voice questions and interest in adolescence, since it is highly associated with taboos around sex and sexuality which are in turn guided by underlying gendered social norms. Similarly, gendered social norms also continue to hamper girls' ability to take up MHPSS services. Among Muslim communities in Sri Lanka religious institutions and leaders typically mediate in cases that involve sexual abuse and early pregnancy, often 'resolving' the matter by ordering the perpetrator to marry the girl. Hence norms around marriage/early marriage in this case override possibilities of girls accessing MHPSS should they be available. Given this context, services were dedicated to addressing 'risks' and providing 'protection' for girls, framing their activities around regulating girls' 'questionable or immoral behaviour' rather than an overriding concern to improve girls' psychosocial wellbeing. In Gaza, gendered social norms which restrict girls' mobility, largely because of fear that this mobility would damage 'family honour' often result in them facing social isolation which can in turn fuel further mental health and psychosocial distress. Similarly the stigma related to MHPSS service uptake in Gaza also inhibits them seeking support since this will likely harm girls' marriage prospects, and given that marriage is one of the most significant life stages in a person's life, and underlies much of the social order, this becomes more important than seeking on MHPSS services. And in Liberia, aggressive masculinities---a legacy of the conflict---and the normalisation of sexual violence, again a key underlying gendered social norm, remain major threats to girls' physical and psychosocial wellbeing, for which services and support remain highly inadequate. Fourthly, although psychosocial wellbeing is arguably subsumed within the social determinants of health framework, our findings highlight that more explicit attention must be paid to psychosocial wellbeing and mental health in conflict-affected contexts, and that these dimensions should be accorded equal weighting to adolescents' physical health concerns. Finally our findings suggest that unless these insights are well understood, efforts to strengthen the human resources needed to support adolescent girls' health and wellbeing may prove ineffective, and may even inadvertently uphold discriminatory gendered social norms that often underpin local health practices. Conclusions =========== Reflecting on the implications of our findings for future policy and practice, we highlight three broad cross-cutting areas that can also be modified for other contexts. First, our study findings highlight that a range of actions are needed to (1) address the factors that contribute to girls' psychosocial vulnerabilities, (2) boost girls' coping strategies, and (3) mitigate against negative coping responses. Such actions include taking a proactive approach to identifying vulnerable groups (including adolescent girls and those at greatest risk of SGBV) and tailoring services and activities to meet their needs. Low-resource measures for mitigating the risk of psychosocial ill-being in fragile contexts could include: encouraging the use of (peer and other) social networks to support adolescents, providing safe spaces for girls, and providing training (to adolescents and their caregivers) on positive coping strategies, including ways to reduce stress. Building on and working with supportive teachers, as well as harnessing the role of the education sector more broadly to help develop adolescents' self-esteem and self-efficacy, also emerged as a critical area for investment. Finally, the generalised stigma that hinders access to psychosocial services needs to be addressed, with substantial efforts for outreach through the media and community mobilisation. Secondly, our findings also indicate that building the capacities of service providers is critical to stimulate an improved service environment. Areas for capacity-building include: early diagnosis of mental health illnesses, provision of SGBV-related services, and treating substance abuse. The service environment, however, should not be limited to these more medicalised approaches; it should include wide-ranging activities, from promoting girls' participation in social activities, to providing spaces for creative expression, as well as providing safe spaces for girls to play and enjoy leisure time. Finally, there is a need for more investment to make services proactive and integrated. In the education sector, for instance, the quality of school-based psychosocial support, counselling services and protocols need to be improved; similarly, in the justice sector, the police need to receive adequate training on SGBV and how to support adolescent girls who are victims or survivors. A third set of actions concerns the need to enhance policy strategies, and to regulate and coordinate actors providing MHPSS services at different levels (community, sub-national and national). There is also a need to strengthen national institutions and ministries so that they become recognised as legitimate regulators of psychosocial services, and can provide, among other things, improved licensing and accreditation processes. Finally, our findings underscore the need to provide more evidence to inform programming, and particularly the need for more robust and disaggregated data to reflect the realities of adolescents' lives. In sum, our findings highlight the importance of recognising adolescents' psychological needs and their gendered patterning, and working to ensure that their needs are integrated into programme design and implementation. Such efforts need to simultaneously tackle the underlying gendered social norms affecting attitudes and behaviours both around psychosocial and mental health service uptake and service provision. This is particularly pertinent in fragile and post-conflict settings if women and girls are to become active agents in change processes, including those related to peace processes and conflict resolution. Ethical approval ================ Ethical approval was sought and obtained through the ODI ethics review board. In addition, the study was also cleared by national research ethics boards in the study countries, i.e. in Liberia, The University of Liberia, the Pacific Institute for Research and Evaluation (PIRE) approved the research, in Gaza the study was reviewed by the Helsinki Framework ethics board, and in Sri Lanka the study was reviewed by a group of practitioners and researchers with relevant expertise. We would like to acknowledge the support of a number of people. In Liberia, The Liberia Center for Outcomes Research led this work supported by Janice Cooper, with Abayomi Cole and Elton Gbollie as research coordinators. In Sri Lanka the work was led by the Good Practice Group and in particular Kusala Wettesinghe, Sarala Emmanuel and Ananda Galappatti. In Gaza the study was led by Bassam Abu Hamad and Nadja Al Bayoumi from Al Quds University. We would also like to thank all the data collectors in the three countries. We also want to acknowledge support from Maria Stavropoulou for her work on the background literature review and Ingrid Gercama for support on data analysis. The Research in Gender and Ethics (RinGs): Building Stronger Health Systems Partnership (funded by DFID) provided invaluable inputs during the development and revision of this paper. Finally, we would like to thank all respondents for giving us their time and sharing their stories with us. Funding ======= This study was funded by the ReBUILD consortium, a multi-year multi-country UK Department for International Development (DFID) funded research programme, and we would like to thank them for their ongoing support and contribution to this research. The views expressed within this paper are not necessarily those of DFID. *Conflict of interest statement*. None declared. <http://www.who.int/social_determinants/en/> For details of the literature review please see [@czx127-B39]. In Gaza only one round of data collection took place since it was added to the study at the time when it thought the team would not be able to continue fieldwork in Liberia due to Ebola; in fact the team was able to carry out a shorter second round of data collection in Liberal post the height of the epidemic. Transactional sex is widespread throughout sub-Saharan Africa and there is a large body of literature on this. For a recent overview article please see [@czx127-B36]. Rape of girls and women persists and accounts for over one-third of sexual violence cases. Victims are mostly young people (10-19), with male perpetrators mostly known to the victim and largely (almost 40%) between the ages of 20-39 (GoL, 2011).

{ "pile_set_name": "PubMed Central" }

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"tended", "focus", "activities", "adults", "individuals", "serious", "mental", "illness", "Sri", "Lanka", "vertical", "integration", "key", "sectors", "health", "education", "child", "protection", "services", "mechanisms", "although", "remains", "strong", "focus", "adolescent", "girls", "protection", "medical", "needs", "identified", "policies", "approaches", "study", "respondents", "However", "ground", "service", "providers", "noted", "collaboration", "across", "sectors", "generally", "low", "situation", "similar", "horizontal", "linkages", "many", "organisations", "NGOs", "working", "different", "sectors", "according", "study", "respondents", "work", "independently", "local", "level", "Discussion", "Overall", "four", "broad", "insights", "emerged", "study", "also", "contribute", "literature", "social", "determinants", "health", "Firstly", "vital", "social", "determinants", "located", "within", "specific", "political", "economy", "understandings", "take", "account", "complex", 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"everyone", "particular", "challenges", "adolescent", "girls", "Liberia", "despite", "fact", "conflict", "formally", "ended", "years", "ago", "levels", "sexual", "violence", "abuse", "women", "remain", "among", "highest", "world", "fn", "reflects", "others", "termed", "whereby", "social", "norms", "around", "masculinity", "normalise", "reinforce", "practices", "sexual", "violence", "SGBV", "rather", "seeking", "reframe", "acceptable", "male", "attitudes", "behaviours", "part", "broader", "process", "see", "Secondly", "although", "attention", "social", "gendered", "norms", "within", "social", "determinants", "health", "literature", "growing", "remains", "often", "cursory", "analysts", "fail", "disentangle", "consequences", "social", "gendered", "norms", "health", "wider", "psychosocial", "wellbeing", "Integrating", "perspective", "thus", "critical", "current", "study", "also", "strives", "Thirdly", "linked", "second", "broad", "insight", "study", "highlights", "particular", "psychosocial", "vulnerabilities", "adolescent", "girls", "face", "closely", "linked", "intertwined", "range", "gendered", "social", "norms", "begin", "bear", "heavily", "girls", "lives", "enter", "adolescence", "Thus", "informed", "conceptual", "framework", "outlines", "adolescent", "girls", "pathways", "psychosocial", "wellbeing", "study", "suggests", "norms", "around", "mobility", "marriage", "sexuality", "education", "time", "use", "emotional", "life", "underlie", "explain", "much", "behaviour", "adolescent", "girls", "including", "perceive", "experience", "mental", "health", "psychosocial", "distress", "well", "forms", "support", "informal", "informal", "available", "able", "access", "Yet", "role", "gendered", "social", "norms", "play", "influencing", "accessibility", "update", "services", "often", "neglected", "health", "systems", "thinking", "many", "developing", "country", "contexts", "Sri", "Lanka", "instance", "barriers", "psychosocial", "service", "uptake", "girls", "include", "stigma", "associated", "admitting", "psychosocial", "problems", "fact", "girl", "voice", "questions", "interest", "adolescence", "since", "highly", "associated", "taboos", "around", "sex", "sexuality", "turn", "guided", "underlying", "gendered", "social", "norms", "Similarly", "gendered", "social", "norms", "also", "continue", "hamper", "girls", "ability", "take", "MHPSS", "services", "Among", "Muslim", "communities", "Sri", "Lanka", "religious", "institutions", "leaders", "typically", "mediate", "cases", "involve", "sexual", "abuse", "early", "pregnancy", "often", "matter", "ordering", "perpetrator", "marry", "girl", "Hence", "norms", "around", "marriage", "case", "override", "possibilities", "girls", "accessing", "MHPSS", "available", "Given", "context", "services", "dedicated", "addressing", "providing", "girls", "framing", "activities", "around", "regulating", "girls", "immoral", "behaviour", "rather", "overriding", "concern", "improve", "girls", "psychosocial", "wellbeing", "Gaza", "gendered", "social", "norms", "restrict", "girls", "mobility", "largely", "fear", "mobility", "would", "damage", "honour", "often", "result", "facing", "social", "isolation", "turn", "fuel", "mental", "health", "psychosocial", "distress", "Similarly", "stigma", "related", "MHPSS", "service", "uptake", "Gaza", "also", "inhibits", "seeking", "support", "since", "likely", "harm", "girls", "marriage", "prospects", "given", "marriage", "one", "significant", "life", "stages", "person", "life", "underlies", "much", "social", "order", "becomes", "important", "seeking", "MHPSS", "services", "Liberia", "aggressive", "masculinities", "legacy", "conflict", "normalisation", "sexual", "violence", "key", "underlying", "gendered", "social", "norm", "remain", "major", "threats", "girls", "physical", "psychosocial", "wellbeing", "services", "support", "remain", "highly", "inadequate", "Fourthly", "although", "psychosocial", "wellbeing", "arguably", "subsumed", "within", "social", 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Introduction {#Sec1} ============ Biotechnological process development, analysis, and control is key to obtain robust processes providing highest product quality attributes as well as a reduced time-to-market latency. Catalyzed by regulatory initiatives for biopharmaceutical products, process analytical technology (PAT) emerged as a major tool that demands for bioprocess analysis and control by frequently measurements ensuring specified final product quality \[[@CR1]\]. Especially in biopharmaceutical production and process development of heterologous protein expression, the physiological state of the cells is highly related to the formation of critical quality attributes \[[@CR2], [@CR3]\]. Therefore, time-resolved knowledge about physiological parameters, such as the specific growth rate or specific substrate uptake rate, is essential in the PAT framework as well as to perform process development, characterization, and validation \[[@CR4]\]. Moreover, those variables frequently serve as targets for control strategies \[[@CR5]--[@CR7]\]. The key to this physiological information is the catalyst concentration---the biomass. However, the required online measurement of biomass is a critical endeavor using hard-type sensors such as dielectric spectroscopy, broth fluorescence or permittivity measurements, each connected to limitations and drawbacks as outlined elsewhere \[[@CR8]\]. Software sensors, or short soft-sensors, provide an elegant, non-invasive way to estimate biomass concentration using different other, easy-accessible measurements \[[@CR9]\]. In this contribution, we want to focus on a dominant biotechnological process mode, the microbial fed-batch fermentation, and on the improvement of one of the most mature soft-sensor implementations using off-gas and substrate-feed measurements. These soft-sensors are established tools for bioprocess control and analysis, which was also frequently shown in practical applications \[[@CR5], [@CR10], [@CR11]\]. Briefly, mass conservation laws are used to calculate turnover rates from online measurements, which might be superimposed with signal errors. In a second step accuracy of turnover rates and constraints, formulated as first-order principles such as mass and energy conservation laws, are used to reconcile the inaccurate turnover rates in order to optimally obey the constraints. Finally, the reconciled turnover rates are used to calculate the biomass formation rate (*r* ~*X*~), which leads after simple integration over time to the biomass concentration. The resulting information can be used to calculate specific turnover rates, such as the specific substrate uptake rate (*q* ~*S*~), which frequently serves as a control variable \[[@CR12]\]. Therefore, *r* ~*X*~ and *q* ~*S*~ are regarded as the most prevailing benchmark entities to evaluate biomass estimation---and physiological control---capability. However, the control quality by soft-sensors is limited by measurement errors of raw signals used to derive the measured turnover rates. When it comes to industrial applicability, the ultimate question is: Which measurement accuracy is required in order to obtain a sufficiently accurate estimation of the reconciled rates and the biomass? This question can only be answered if the error sources, their respective impact, and possible counteractions are understood. We note that we use the definition of errors as deviations to the true values, excellently defined elsewhere \[[@CR13]\]. Random errors leading to a lack of signal precision are caused by small changes within the system, e.g., air movement, temperature, and electrostatic fluctuations. A multitude of algorithms exists to smooth signals with random errors ranging from simple median filters to polynomial filters such as Savitzky-Golay filter up to frequency filters such as the Butterworth filter. While random errors can be minimized quite easily, this is not the case for systematic errors caused by miscalibrations, inaccuracy of analytical devices, or a defective feature in the sensor. Those systematic errors can only be detected and possibly reduced by making use of all available information in terms of first-principle constraints and the accuracy of turnover rates in reconciliation procedures as outlined above. First-order principles can be generically formulated for defined processes, whereas the accuracy of turnover rates, which are input to the reconciliation procedure, are not known *a priori*. They highly depend on the accuracy of the raw signal measurements and dynamically change over time. Previously, this was approximated by propagating the variance of measurement accuracies to the turnover rates \[[@CR19]\]. However, commonly, the expected or maximal error on measurement signals is provided by device manufacturers. Therefore, it is an existing unmet need to establish a methodology that leverages this accuracy information of the raw signals onto the derived turnover rates, which are subsequently used in the reconciliation procedure. Hence, it is the goal of this contribution to develop an error propagation procedure to derive the accuracy of turnover rates from expected measurement errors and demonstrate its benefits in terms of increased physiological accuracy within the soft-sensor framework in microbial fed-batch mode. Moreover, in those previously elaborated methodologies, the impact of measurement error on softsensor accuracies could only be estimated retrospectively, given the process data. Therefore, we want to address the question raised above, and present a novel generic workflow that identifies tolerable measurement errors of combinations of multiple analytical measurements in order to meet the desired accuracy of soft-sensor estimations prior to conducted experiments using mechanistic knowledge. Material and methods {#Sec2} ==================== Aim and relevance of the presented approach {#Sec3} ------------------------------------------- The following study was carried out with in silico generated data. The aim of the in silico data generation was to obtain representative microbial fed-batch fermentation data including an induction phase, the predominant industrial mode for the production of recombinant proteins. The experiments were based on a typical *Escherichia coli* process with oxidative growth and glucose as substrate. As the batch phase is not part of the discussed soft-sensor, only the fed-batch part was considered here. As commonly used in industry, the modeled fed-batch phase started with an exponential feeding profile. After 8 h, the induction phase started with a linear feed rate. The biomass yield coefficient is dynamic. Due to the metabolic load, typically an especially strong decrease can be observed in the induction phase \[[@CR14]\]. This can be measured by the soft-sensor and was also considered in the data generation process (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1**a** Simulated feed profile and biomass concentration. **b** Simulated trajectories of the biomass/substrate yield (*Y* ~X/S~) and the specific growth rate (μ) The advantages of an in silico study are obvious:It is possible to "run" a bioprocess completely without any errors on the signals and to introduce defined errors into the system. This is not possible with real data, as the exact "real" values without errors on the data cannot be determined.A virtually infinite number of experiments with different combinations of errors can be carried out. This enables a systematic study of errors in a high-dimensional "uncertainty space." Computational environment {#Sec4} ------------------------- All calculations were conducted in a MATLAB environment (2015a, The MathWorks, Inc.). The mechanistic model was created in form of a system of ordinary differential equations. As graphical user interface and bioprocessing toolbox inCyght (2016.02, Exputec GmbH) was used. In silico data generation {#Sec5} ------------------------- ### Main mechanistic assumptions {#Sec6} The main mechanistic assumptions behind data generation and soft-sensor are the same. Substrate, ammonia, and oxygen are converted to biomass and carbon dioxide. In this simple case, the extracellular formation of product or metabolites will be neglected. This assumption is true for many biopharmaceutical processes, as the product formation rate often is several order of magnitudes lower than the biomass formation rate \[[@CR15]\]. For processes were this assumption has to be rejected, the soft-sensor framework has to be extended by online product measurement, e.g., by using spectroscopic techniques \[[@CR16]\].$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_S{\mathrm{CH}}_{\mathrm{pH}}{\mathrm{O}}_{\mathrm{pO}}+{r}_{\mathrm{O}2}{\mathrm{O}}_2+{r}_N{\mathrm{N}\mathrm{H}}_3\ \to {r}_X{\mathrm{CH}}_{zH}{\mathrm{O}}_{zO}{\mathrm{N}}_{zn}+{r}_{\mathrm{CO}2}{\mathrm{CO}}_2 $$\end{document}$$ Two first principle assumptions were made; the carbon balance:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_S + {r}_X + {r}_{C{O}_2}=0 $$\end{document}$$ And the degree of reduction balance:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_S{\gamma}_S + {r}_X{\gamma}_X + {r}_{O_2}{\gamma}_{O_2}=0 $$\end{document}$$ The detailed list of equations for the data generation step is shown in the [Electronic Supplementary Material](#Sec27){ref-type="sec"} (ESM) Section 1. ### Addition of noise and errors on the data {#Sec7} To test the original and new soft-sensors with erroneous data, both systematic as well as random errors were introduced into the model. Based on information of off-gas sensor and mass flow controller manufacturers, as summarized in Table [1](#Tab1){ref-type="table"}, realistic amounts of systematic errors were superimposed to the off-gas data which were used as input for the soft-sensors.Table 1Typical measurement errors of off-gas analyzers and mass flow controllersRelative error to measurement valueMeasurement accuracy (zero deviance)Drift/year*ΔF* ~*a*,in~ (mass flow controller)±0.5 % of readout±0.3--1 % of full scale±1 % of full scale$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}} $$\end{document}$ (infrared)n.a.±1 % of full scale±1 % of full scale$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta {y}_{{\mathrm{O}}_2,\mathrm{in}} $$\end{document}$ (paramagnetic)±3 % of readout±0.2 % full scale±2 % value$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta {y}_{{\mathrm{O}}_2,\mathrm{in}} $$\end{document}$ (Galvanic cell)±3 % of readout±0.2 % full scale±2 % value O~2~ and CO~2~ concentrations in the exhaust gas are simply applied on the model output for the off-gas data:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{array}{l}{X}_{{\mathrm{O}}_2,\ \mathrm{measured}}={X}_{{\mathrm{O}}_2,\mathrm{model}}\cdot \left(1+{\upvarepsilon}_{{\mathrm{O}}_2}\right)\kern1em \\ {}{X}_{{\mathrm{CO}}_2,\ \mathrm{measured}}={X}_{{\mathrm{CO}}_2,\mathrm{model}}\cdot \left(1+{\upvarepsilon}_{{\mathrm{CO}}_2}\right)\kern1em \end{array} $$\end{document}$$ As the error on the mass flow controller affects both total oxygen and carbon dioxide input into the system and the resulting final concentrations of O~2~ and CO~2~, the error has to be given as input to the model. The set-points for the MFC are the known values, but the model input and real values are calculated as follows.$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{array}{l}{F}_{{\mathrm{O}}_2,\mathrm{in},\mathrm{model}}=\frac{F_{{\mathrm{O}}_2,\mathrm{in},\mathrm{setpoint}}}{1+{\upvarepsilon}_{MFC}}\hfill \\ {}{F}_{{\mathrm{CO}}_2,\mathrm{in},\mathrm{model}}=\frac{F_{{\mathrm{O}}_2,\mathrm{in},\mathrm{setpoint}}}{1+{\upvarepsilon}_{\mathrm{MFC}}}\hfill \end{array} $$\end{document}$$ For the errors in the feed rate, a relative error on the set-point rate is applied.$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_{S,\mathrm{model}} = \frac{r_{S,\mathrm{setpoint}}}{1+{\upvarepsilon}_{r_S}} $$\end{document}$$ The amounts of systematic errors applied for the different experiments are listed in "[Comparison of soft-sensor estimates to unbiased model data](#Sec13){ref-type="sec"}" section. The model delivered an online value each seven seconds. For the addition of random error, white Gaussian noise was added to the off-gas signals. The noise was generated by using MATLAB's awgn function with a relative standard deviation of 1 % for the off-gas data and 10 % on the feed rate. The noise on the feed rate is typically relatively high, as the signal often is calculated by deriving the scale signal. Quantitative evaluation of bioprocess data and error propagation {#Sec8} ---------------------------------------------------------------- ### Preprocessing {#Sec9} As described in the "[Introduction](#Sec1){ref-type="sec"}" section, random errors can be minimized by using preprocessing methods. We decided to apply a Savitzky-Golay filter with a window size of 30 min and second-degree polygon on the off-gas signals. These parameters in most cases showed a low signal distortion, while on the other hand, the elimination of noise was good. However, it has to be noted that for specific filtering and smoothing problems, better filters and filter parameters may exist. In our experience, most of them are not generically applicable, meaning that if they work very well for a specific problem on a defined signal with specific signal dynamics, they may completely fail on another. ### Data-driven rate calculation {#Sec10} The aim of the next section is to express estimators for those conversion rates derived from measurements. In general, all conversion rates can be formulated using the simple idea, that the conversion rate equals the net accumulation within the reactor minus the inflow into the reactor plus the outflow out of the reactor. For demonstration purpose of the subsequent error propagation, the calculation of the conversion rate for CO~2~ will be shown exemplarily:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {r}_{{\mathrm{CO}}_2}\kern0.5em =\kern0.5em \mathrm{C}\mathrm{E}\mathrm{R}\kern0.5em =\kern0.5em \frac{d\left({\mathrm{CO}}_2\right)}{dt\ }\kern0.5em -\dot{{\mathrm{CO}}_{2,\mathrm{in}}}\kern0.5em +\kern0.5em \dot{{\mathrm{CO}}_{2,\mathrm{out}}} $$\end{document}$$ The term $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \frac{d\left({\mathrm{CO}}_2\right)}{dt\ } $$\end{document}$ can be neglected since it is predominantly a function of pH and temperature, which were kept constant over all in silico simulations. Therefore, the carbon emission rate (CER) formulates to:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{C}\mathrm{E}\mathrm{R}\kern0.5em =\kern0.5em \frac{F_{a,\mathrm{in}}}{V_m}\left({y}_{{\mathrm{CO}}_2,\mathrm{out}}\cdot R{a}_{\mathrm{inert}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}\right) $$\end{document}$$ Where *Ra* ~inert~ is the inert gas ratio, which connects the inflow to the outflow by:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ R{a}_{\mathrm{inert}}=\frac{F_{a,\mathrm{out}}}{F_{a,\mathrm{in}}} $$\end{document}$$ And is defined as:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ R{a}_{\mathrm{inert}}\kern0.5em =\kern0.5em \frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{y_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}} $$\end{document}$$ Here, *y* ~wet~ is the oxygen concentration in the off-gas stream without bio-reaction and indirectly relates to water stripping out of the reactor. Well-known procedures can be applied in order to calculate the substrate- and oxygen- uptake rate for a substrate limited *E. coli* fermentation as described elsewhere \[[@CR15]\]. ### Error propagation {#Sec11} In general, all the input signals for estimating the conversion rates are random variables, associated with a random and systematic error, therefore the estimators itself are random variables, too. As discussed in the "[Introduction](#Sec1){ref-type="sec"}" section, random errors in the raw signals can be minimized using preprocessing methods, whereas systematic errors cannot be removed and propagate directly to the estimated conversion rates. However, via Gaussian error propagation, it is possible to estimate the expected error of the conversion rates. This knowledge will subsequently help us to formulate a much more robust reconciliation procedure and estimation of biomass. The influence of the absolute measurement error (*Δy*) of the signal *y* onto a derived signal *r* can be approximated using a Taylor expansion \[[@CR3]\]:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ r\left(y+\varDelta y\right)=r(y) + \frac{1}{1!}\frac{dr(y)}{dy}\cdotp \varDelta y+\frac{1}{2!}\frac{d{}^2r(y)}{dy{}^2}\cdotp {\left(\varDelta y\right)}^2+\dots $$\end{document}$$ We want to note that the absolute measurement error *Δy* of the measurement signal can be in most cases calculated from technical device data sheets given by their maximal amplitude (e.g., ±3 % of readout). Therefore, the absolute measurement error *Δy* can be seen as worst-case error. For an approximate solution, the Taylor expansion can be terminated after the second term and the resulting absolute deviation of the derived signal (*Δr*) can be written as:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ r\left(y+\varDelta y\right)-r(y) = \varDelta r=\frac{dr(y)}{dy}\cdotp \varDelta y $$\end{document}$$ If the derived signal (here the conversion rate) depends on more than one input variable and the error of the input signal is only known by its boundaries, which is the typical case for biotechnological applications, we can write in analogy:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \varDelta r=\left|\frac{\partial r}{\partial {y}_1}\right|\cdotp \varDelta {y}_1+\left|\frac{\partial r}{\partial {y}_2}\right|\cdotp \varDelta {y}_2+\dots $$\end{document}$$ For the CER, the error propagation formulates to:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{array}{l}\varDelta \mathrm{C}\mathrm{E}\mathrm{R}=\left|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {F}_{a,\mathrm{in}}}\right|\cdot \varDelta {F}_{a,\mathrm{in}}+\left|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {V}_m}\right|\cdot \varDelta {V}_m+\left|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {y}_{{\mathrm{CO}}_2,\mathrm{out}}}\right|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}}+\left|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial R{a}_{\mathrm{inert}}}\right|\cdot \varDelta R{a}_{\mathrm{inert}}+\left|\frac{\partial \mathrm{C}\mathrm{E}\mathrm{R}}{\partial {y}_{{\mathrm{CO}}_2,\mathrm{in}}}\right|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{in}}\hfill \\ {}\varDelta \mathrm{C}\mathrm{E}\mathrm{R}=\left|\frac{1}{V_m}\left({y}_{{\mathrm{CO}}_2,\mathrm{out}}\cdot R{a}_{\mathrm{inert}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}\right)\right|\cdot \varDelta {F}_{a,\mathrm{in}}+\left|\frac{F_{a,\mathrm{in}}}{V_m^2}\left({y}_{{\mathrm{CO}}_2,\mathrm{out}}\cdot R{a}_{\mathrm{inert}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}\right)\right|\cdot \varDelta {V}_m+\left|\frac{F_{a,\mathrm{in}}}{V_m}\cdot R{a}_{\mathrm{inert}}\right|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}} + \left|\frac{F_{a,\mathrm{in}}}{V_m}\cdot {y}_{{\mathrm{CO}}_2,\mathrm{out}}\right|\cdot \varDelta R{a}_{\mathrm{inert}}+\left|\frac{F_{a,\mathrm{in}}}{V_m}\right|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{in}}\hfill \\ {}\varDelta {R}_{a,\mathrm{in}\mathrm{ert}}=\left|\frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{\left({y}_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}\right){}^2}\right|\cdot \varDelta {y}_{{\mathrm{O}}_2,\mathrm{out}}+\left|\frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{{\left({y}_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}\right)}^2}\right|\cdot \varDelta {y}_{{\mathrm{CO}}_2,\mathrm{out}}+\left|\frac{1-{y}_{{\mathrm{O}}_2,\mathrm{in}}-{y}_{{\mathrm{CO}}_2,\mathrm{in}}}{\frac{1}{y_{{\mathrm{O}}_2,\mathrm{in}}}\cdot \left({y}_{{\mathrm{O}}_2,\mathrm{out}}-{y}_{{\mathrm{CO}}_2,\mathrm{out}}-\frac{y_{\mathrm{wet}}}{y_{{\mathrm{O}}_2,\mathrm{in}}}\right){}^2}\right|\cdot \varDelta {y}_{\mathrm{wet}}\hfill \end{array} $$\end{document}$$ This procedure can easily be extended to the OUR and the substrate uptake rate (*r* ~*S*~). Typical results of error propagation to the off-gas rates are shown in Fig. [2a](#Fig2){ref-type="fig"}.Fig. 2**a** Time-resolved profiles for OUR and CER are shown together with their respective accuracy as error *bars*, calculated by error propagation as described in "[Error propagation](#Sec11){ref-type="sec"}" section. **b** Comparison of biomass (*black*) and *r* ~*x*~ (*gray*) soft-sensor prediction to the unbiased signals (*solid lines*). Estimations of traditional soft-sensor implementations, assuming 3 % error of all input rates, are shown *dotted*; the adapted soft-sensor with error propagation for the input rates is shown in *dashed lines*. For this particular simulation, raw signals were superimposed by 2 % error for CO~2~ and O~2~ off-gas concentration, respectively, and 1 % error on the *r* ~*S*~ and the MFC, respectively For the presented in silico study input signals: *F* ~*a*,in~, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{CO}}_2,\mathrm{out}} $$\end{document}$ for CER and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{O}}_2,\mathrm{out}} $$\end{document}$ for the OUR, were regarded as superimposed with considerably relevant systematic measurement error. All other input signals were considered to be perfectly accurate. Typical errors for mass flow controllers and off-gas analytics are given in Table [1](#Tab1){ref-type="table"}. However, the error propagation model could be easily extended to more inputs with systematic error. For the error propagation of *r* ~*S*~, the only considerably source of systematic signal error was the concentration of the substrate, which might vary due to evaporation during sterilization procedures. ### Minimum variance rate reconciliation and biomass soft-sensor estimation {#Sec12} In the following section, we want to briefly summarize an established minimum variance reconciliation and biomass estimation procedure in order to reduce systematic error on measured turnover rates (OUR, CER, and *r* ~*S*~) using first principles as reported in detail elsewhere \[[@CR15], [@CR17], [@CR18]\]. First principles, such as elemental balances (see "[Main mechanistic assumptions](#Sec6){ref-type="sec"}" section), can be seen as constraints to the bioreactor system. We can formulate many of those constraints and thereby connect components with each other. Commonly, a compact matrix formulation is used to connect conversion rates of components with each other using multiple constraints:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ E\cdot r=0 $$\end{document}$$ *E* is the elemental composition matrix \[*e* × *n*\] with *e* being the number of elemental balances and *n* the number of relevant components. *r* is the vector containing the turnover rates. Under real conditions, the elemental balances do not close due to systematic errors of the rates with a residual vector *ε*:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ E\cdot r=\varepsilon $$\end{document}$$ For this in silico example, the elemental *C* balance as well as the degree of reduction (DoR) balance were used as frequently applied elsewhere \[[@CR5]\]. In order to identify gross errors in the system, it is necessary to check if the residual vector (*ε*) differs significantly from zero. Therefore, Reilly and Carpani introduced a statistical measure (*h* value) which weights the residuals by their accuracy (covariance matrix). Using this *χ* ^2^ distributed measure, it is possible to set confidence levels for detecting gross errors \[[@CR18]\]. The presented concept can easily be extended to more elemental balances or energy balances; however, the implementation with C- and DoR-balance is predominantly implemented in industry since additional measurements (e.g., nitrogen or heat transfer) are more complex to realize in practice. The redundancy of the measurable rates (rank of redundancy matrix R \[[@CR15], [@CR17], [@CR18]\]) equals 1, and therefore this redundancy can be used to balance the measured rates in a minimum variance sense and obtain reconciled rates \[[@CR15]\]. After no gross error could be detected with statistical significance and the measured rates are reconciled, those reconciled rates can be finally used to estimate the biomass formation rate, which is the only non-measured rate in this example. For this minimum variance balancing procedure, the covariance matrix of the measured signals is required. A fair assumption is to state that the covariance of the measured rates is diagonal, which assumes non-correlated errors in the measured signals. In current soft-sensor implementations, an empirical approach was chosen and the covariance of all measured rates was assumed to equal 3 % of the readout \[[@CR10], [@CR15]\]. As a unique feature of the presented soft-sensor implementation, we will use at this point the derived error boundaries from above as worst-case estimators for the variances of the signals. Since the herein-derived error boundaries vary dynamically over time, the new approach will be further on called adaptive soft-sensor. Comparison of soft-sensor estimates to unbiased model data {#Sec13} ---------------------------------------------------------- As a methodology to investigate the result of the soft-sensor as a function of the error of the input signals, we investigated 5915 in silico experiments with systematically varied errors on the off-gas measurements, substrate concentration, and mass flow controlled (described in "[Addition of noise and errors on the data](#Sec7){ref-type="sec"}" section). The selected ranges in Table [2](#Tab2){ref-type="table"} were based on technical manufacturer information of MFC and off-gas analyzer (see Table [1](#Tab1){ref-type="table"}).Table 2All errors listed here were combined with each other and applied in 5915 experiments. All those in silico generated data sets were used to test the prediction accuracy of traditional and adaptive soft-sensor approachApplied relative error on in- and outputsStep size$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\varepsilon}_{{\mathrm{CO}}_2,\kern0.75em }{\varepsilon}_{{\mathrm{CO}}_2} $$\end{document}$−3 to +3 %0.5 %$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\varepsilon}_{r_S} $$\end{document}$−3 to +3 %1 %*ε* ~MFC~−2 to +2 %1 % As a final output of the soft-sensor, the estimated biomass formation rate ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\hat{r}}_X $$\end{document}$) was compared to the true, unbiased biomass formation rate from the in silico model (*r* ~*X*,*true*~), which is known. This comparison was done by calculating the median percentage of difference (MPD) over all data points of the time series according to:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{M}\mathrm{P}\mathrm{D}\kern0.75em =\kern0.5em 100\cdot \mathrm{median}\left(\frac{{\hat{r}}_X-{r}_{X,\mathrm{true}}}{r_{X,\mathrm{true}}}\right) $$\end{document}$$ For each of the 5915 simulations, a MPD value for *r* ~*X*~ and *q* ~*S*~ was calculated. Those values are displayed as surface plots as shown in Figs. [3](#Fig3){ref-type="fig"} and [4](#Fig4){ref-type="fig"}.Fig. 3Comparison of *r* ~*X*~ for traditional (*left*) and adapted (*right*) soft-sensor in terms of MPD, showing the deviation between the real biomass formation rate and the soft-sensor values (%) as a function of the errors on the off-gas data. (*a*) Errors on the off-gas data, but no errors on r~S~ and the MFC. (*b*) Errors on the off-gas data and 2 % error on *r* ~*S*~. (*c*) Errors on the off-gas data and 2 % error on the MFC Fig. 4Comparison of *q* ~*S*~ in terms of MPD (*a*), the median of the estimated relative standard deviation on the reconciled *q* ~*S*~ (*b*), and the median *h* values (*c*) for traditional (*left*) and adapted (*right*) soft-sensor, depending on the error level of the off-gas analyzers Results {#Sec14} ======= Comparison of the soft-sensors accuracy {#Sec15} --------------------------------------- ### Biomass formation rate {#Sec16} In the following sections, a comparison between the traditional approach and the adaptive soft-sensor is done. While traditionally, the errors on CER, OUR, and *r* ~*S*~ were estimated to be 3 % for all rates and over the whole process, the adapted soft-sensor calculates the accuracy on the rates through error propagation, by making use of the known uncertainty ranges of the raw signals. The herein dynamically resolved accuracy for OUR and CER, assuming 3 % maximal error on the read out of off-gas analytical measurements of O~2~ and CO~2~, are depicted in Fig. [2a](#Fig2){ref-type="fig"}. The accuracy of CER is much higher than the accuracy of OUR. This information is used by the adapted soft-sensor; therefore, we obtain much more accurate biomass and *r* ~*X*~ estimates than previous implementations without error propagation, compared to unbiased biomass and *r* ~*X*~ signals, as shown in Fig. [2b](#Fig2){ref-type="fig"}. Since Fig. [2b](#Fig2){ref-type="fig"} shows only the results for one particular error combination of errors on O~2~, CO~2~, *r* ~*S*~ and the MFC, we have to resolve the predictions with all other error combinations in order to show superiority of the adapted soft-sensor. Figure [3](#Fig3){ref-type="fig"} shows a comparison of the biomass formation rate (*r* ~*X*~) between the true rates (model) and the estimated rates (left column: traditional soft-sensor, right column: adaptive soft-sensor) by means of MPD. In each of the subfigures, the MPD is shown as a function of the error on the O~2~ and CO~2~ off-gas concentration, varied between −3 and +3 %. The error on the MFC and on *r* ~*S*~ was varied across the rows of the subfigures. When using the traditional approach (subplots on the left side), especially errors on the oxygen signal lead to high errors on the estimated rates as well as much higher MPD values (up to 42 % compared to maximal 19 % for the adaptive soft-sensor) and almost horizontal lines of equivalent MPD lines in Fig. [3](#Fig3){ref-type="fig"}. The adaptive soft-sensor propagates the measurement accuracy to the turnover rates, which makes the OUR less trustworthy than the CER as indicated by the error bars in Fig. [2](#Fig2){ref-type="fig"}. Therefore, also errors on the CO~2~ off-gas measurement have an impact on the MPD values, resulting in a rotation of the equivalent MPD lines to the diagonal direction (subplots on the right of Fig. [3](#Fig3){ref-type="fig"}). This change of influential parameters on the MPD will be observed multiple times throughout this work and is always caused by changing the accuracy of turnover rates to realistic values using the error propagation procedure. In the subplots (a1) and (a2) in Fig. [3](#Fig3){ref-type="fig"}, the MPD values regarding *r* ~*X*~ are shown as a function solely of error on O~2~ and CO~2~ off-gas measurement. In the subplots b1 and b2, we added a relative error of 2 % on *r* ~*S*~ to the true model values and in subplots c1 and c2, we added an error of 2 % on the MFC set-point. The average MPD values as well as the maximal MPD values (up to 40 vs. 18 %) reached throughout all subplots are much lower for the adaptive soft-sensor compared to the traditional approach. ### Control quality for specific substrate uptake rate {#Sec17} As one of the main applications of the soft-sensor is the process control based on physiological parameters, the two versions of the soft-sensors were also compared in terms of prediction accuracy for the specific substrate uptake rate *q* ~*S*~. Here, subplots (a1) and (a2) in Fig. [4](#Fig4){ref-type="fig"} show the MPD for *q* ~*S*~ with varying error on O~2~ and CO~2~ measurement and no error on *r* ~*S*~ and MFC signal. Again, the adaptive soft-sensor shows on average much lower MPD values as well as much lower maximal MPD values (up to 15 % for the traditional and up to 4 % for the adaptive soft-sensor). b1 and b2 show that the estimated relative standard deviation for both soft-sensors is in the area of 3 %. However, when looking at the results in A1 and A2, only the adaptive soft-sensor delivers the estimated standard deviations, since MPD values are in the range of ±3 %. As shown in Fig. [4](#Fig4){ref-type="fig"}(c1 and c2), the *h* values of the traditional soft-sensor quickly exceed levels of 3. In this case, the null hypothesis, that there is no gross error in the system, has to be rejected with a confidence level of 95 %. However, the system had no gross error in reality, and as the *h* values of the adaptive soft-sensor show, the null hypothesis cannot be rejected when using the correctly calculated covariance matrix for the minimum variance reconciliation. Therefore, the *h* values of the traditional soft-sensor have no statistical significance as the covariance matrix, as explained before, is not correctly estimated. ### Integrated comparison over the entire uncertainty space {#Sec18} The goal of this section is to derive a global parameter which we can use to judge which soft-sensor approach leads to generally more accurate estimations. In general, we face a four-dimensional input space consisting of different errors on the O~2~, CO~2~, *r* ~*S*~, and MFC measurement. This space will be subsequently called the uncertainty space. At each point in this uncertainty space, the MPD of the true model value of *r* ~*X*~ and *q* ~*S*~ is compared to the two soft-sensor approaches. Taking the mean of all those MPD values of the uncertainty space for each soft-sensor approach gives us a clear measure which soft-sensor implementation is generally more accurate. This integrated parameter will be called the global average MPD. Table [3](#Tab3){ref-type="table"} summarizes the results of this kind of analysis and shows for each cell the mean MPD of simulations where the O~2~ and CO~2~ error was varied between −3 and +3 %, analogous to one subplot of Fig. [3](#Fig3){ref-type="fig"}. In the columns of Table [2](#Tab2){ref-type="table"}, the error on the MFC is varied, in the rows the error on *r* ~*S*~. The global average MPD regarding *r* ~*X*~ of the adaptive soft-sensor is 8.7 % compared to 15.2 % of the traditional approach. This is a reduction of the MPD by 43 %. For the estimation of the specific substrate uptake rate *q* ~*S*~, the global average MPD can be even lowered from 7.6 to 2.7 which corresponds to a MPD reduction of 64 %.Table 3Comparison of traditional and adaptive soft-sensor for different error levels. Each of the cells show the mean MPD value of simulations in which the error on O~2~ and CO~2~ was varied between −3 and +3 % Generic workflow to ensure appropriate control quality {#Sec19} ------------------------------------------------------ Besides showing superior behavior of the new soft-sensor implementation over state of the art, we want to present a novel generic workflow to obtain a desired soft-sensor estimate or reconciliation quality by adapting accuracy of measurement devices. This workflow includes the following steps as indicated in Fig. [5](#Fig5){ref-type="fig"}:Fig 5Generic workflow for identification of desired measurement error and noise for robust biomass soft-sensor estimation. *Asterisk* indicate variables which were superimposed by random noise and systematic error Use a mechanistic process model to generate time-resolved data which will be used to derive rates. These are used as input to the soft-sensor (here O~2~ and CO~2~ concentration of the off-gas stream, substrate concentration and inflowing air controlled by MFC).Obtain biased signals by superimposing them with representative systematic (see manufacturer specifications) and random noise (estimated process noise).Calculate turnover rates including their accuracy as described in "[Data-driven rate calculation](#Sec10){ref-type="sec"}" section. The rates and their accuracy (covariance) are input to the soft-sensor.Use the soft-sensor's first principles to reconcile measured turnover rates unless gross errors are detected. The reconciled rates can be used to estimate the biomass and all related entities (e.g., *q* ~*S*~ or biomass).The herein obtained *q* ~*S*~ estimate (or biomass estimate) is compared to the true, unbiased model signal. If the estimate does not meet the predefined thresholds (e.g., 5 % global average MPD), more accurate measurement devices and their respective measurement errors are used to continue with step 2 to 5 with reduced systematic error levels. The selection of appropriate measurement devices is driven by technical, manufacturing, and financial constraints, which is not scope of this study.If the estimate meets the predefined thresholds in terms of global average MPD, a robust estimate under industrial relevant process conditions is achieved. As an example, the error ranges of Table [2](#Tab2){ref-type="table"} were taken as a starting position in step 2 of the generic workflow presented in Fig. [5](#Fig5){ref-type="fig"}. It was assumed that the desired control quality could not be reached with the current analytical devices (step 5), therefore, exemplary a higher accuracy of the oxygen sensor and MFC from ±3 to ±0.5 % and ±2 to ±1 %, respectively, was implemented. The results before and after this change are shown in Fig. [6](#Fig6){ref-type="fig"}. After the change, the estimated error surface of *q* ~*S*~ is rotated in a favorable direction to enlarge regions of lower error (0 to 2 % error), as depicted in the non-shaded areas of the two subfigures of Fig. [6](#Fig6){ref-type="fig"}. Overall, this results in 10 % reduced global average MPD.Fig. 6Estimation error of *q* ~*S*~ before (**a** ±3 % maximal error on oxygen measurement and ±2 % maximal error on MFC) and after (**b** ±0.5 % maximal error on oxygen measurement and ±1 % maximal error on MFC) the increase of the input signal accuracies. Not only values outside the uncertainty range can be excluded but also the mean MPD inside the uncertainty space is more accurate Discussion {#Sec20} ========== Superior accuracy for the estimated rates of the adaptive soft-sensor {#Sec21} --------------------------------------------------------------------- As perceived in Figs. [2](#Fig2){ref-type="fig"}, [3](#Fig3){ref-type="fig"}, and [4](#Fig4){ref-type="fig"}(a1 and a2) as well as summarized in Table [1](#Tab1){ref-type="table"}, the adaptive soft-sensor delivers more accurate estimates of *r* ~*X*~, which integrates to biomass, and *q* ~*S*~. Moreover, maximal MPD values for *r* ~*X*~ and *q* ~*S*~ are much lower for the adaptive soft-sensor which implies that the biomass estimate as well as the control of *q* ~*S*~ can be performed much more robust under real process conditions since large deviations to the true values of *r* ~*X*~ and *q* ~*S*~ can be avoided. This is due to the fact that the covariance matrix for the minimum variance reconciliation procedure is arbitrary chosen for the traditional soft-sensor, which assumes a too low uncertainty range for the OUR, as shown exemplarily in Fig. [2](#Fig2){ref-type="fig"}. The adaptive soft-sensor on the other hand dynamically uses all available information (uncertainty ranges of off-gas analysis) to calculate a realistic covariance matrix. This leads to a much more robust estimate of *r* ~*X*~ and *q* ~*S*~. For each subfigure, there are some "sweet spots" for certain error combinations, where the classical soft-sensor shows a better accuracy in the prediction of *r* ~*X*~. Since the exact combination of the present errors on the input signals is not known *a priori*, this is no advantage under real process conditions. In a previous approach, the uncertainty of turnover rates was approximated by propagation of variance \[[@CR19]\]. However, under industrial applications, the maximal expected error is provided or known as an empirical parameter. Moreover, in the previous work, simulations were not used to systematically investigate the true error obtained by softsensor estimations. Therefore, our approach including the generic workflow offers the possibility to pre-access the expected dynamics of the process and influence of measurement errors on soft-sensor predictions using mechanistic modeling. Statistical meaningfulness of standard deviation and *h* value {#Sec22} -------------------------------------------------------------- The covariance matrix is a critical input for the minimum variance reconciliation procedure. As for the traditional soft-sensor, the covariance matrix consists of arbitrary values which do not represent the true and dynamically changing uncertainties; all calculated statistical measures, i.e., standard deviation and *h* value, lose their significance. This is different for the adaptive soft-sensor. As shown in Fig. [4](#Fig4){ref-type="fig"}(b1 and b2), the relative standard deviations of the estimated rates are in the range of 3 % and almost identical for traditional and adaptive soft-sensor. However, when comparing these standard deviations to the actually measured errors in terms of MPD in Fig. [4](#Fig4){ref-type="fig"}(a1 and a2), it becomes clear that the calculated standard deviations do not fit to these errors for the traditional soft-sensor; the measured MPDs go up to 15 % in the considered area. For the adaptive soft-sensor, the standard deviations are meaningful and on the same magnitude as the actually measured MPDs. Under real process conditions, the calculated standard deviation of an estimated rate is the only available measure to evaluate their prediction accuracy and expected uncertainty and is therefore of critical importance. As shown in Fig. [4](#Fig4){ref-type="fig"}(c1 and c2), the *h* values of the traditional soft-sensor quickly exceed levels of 3. As already explained in "[Control quality for specific substrate uptake rate](#Sec17){ref-type="sec"}" section, the calculated *h* values are statistically not meaningful. This means that they cannot be used to detect a gross error in the system with a defined level of significance. They only can be used to relatively compare similar processes or detect gross errors when the *h* values are magnitudes higher than expected. This is not true for the adaptive soft-sensor, as over the whole uncertainty space the *h* values are below 3, and no false positive detection of gross errors occurred with 95 % confidence. Applicability of the generic workflow to set measurement accuracies and ensure desired accuracy of soft-sensor estimations {#Sec23} -------------------------------------------------------------------------------------------------------------------------- The question about the required measurement accuracy of raw signals to meet the desired accuracy of derived variables, such as the soft-sensor estimation for bioprocess control, is equally urged by device manufacturer as by process engineers. This is due to the fact that measurement accuracy is often correlated to higher asset costs of advanced devices or more frequent maintenance intervals of existing devices. In "[Generic workflow to ensure appropriate control quality](#Sec19){ref-type="sec"}" section, we present a generic workflow to answer this question. Since the measurement accuracy of the derived soft-sensor estimate is not only a function of the accuracy of the input signals (step 2 of the workflow) but also of the dynamics of the process, a mechanistic model has to provide this information (step 1 of the workflow). If one has multiple possibilities of exchanging devices or maintenance intervals to increase accuracy of input signals, this can be solved iteratively in the workflow by testing different of those combinations and evaluating if the resulting accuracy of the soft-sensor is sufficient. Moreover, as shown in Fig. [6](#Fig6){ref-type="fig"}, it is thereby possible to not only get rid of areas with high levels of MPD but rather additionally increase the accuracy in the reduced uncertainty space due to the introduction of supplementary knowledge about the accuracy of input signals. Extrapolations of the adaptive soft-sensor and the generic workflow to other application areas {#Sec24} ---------------------------------------------------------------------------------------------- The presented error propagation approach as well as the presented workflow are generically applicable to include additional sources of information. For example, the consideration of energy balances based on the metabolic heat production during a process \[[@CR20]\] or the already mentioned combination of the soft-sensor with spectroscopic techniques \[[@CR16]\] could be included. This would result in an even more robust and diverse applicable package. Conclusion {#Sec25} ========== In this contribution, we aim to present an error propagation procedure increasing the accuracy and robustness of the soft-sensor estimates. Traditionally, the uncertainties for conversion rates (CER, OUR, *r* ~*s*~) were arbitrarily assumed and static over the whole process. Here, we established a novel procedure to obtain meaningful uncertainties, dynamically changing over time, which are used as representative knowledge source together with first principles in the soft-sensor framework. In this in silico case study, the new approach using the adaptive soft-sensor, the error on the estimates could be reduced by 43 % for the estimated biomass growth rate (*r* ~*X*~) compared to traditional soft-sensor implementations. For the estimation of the specific substrate uptake rate *q* ~*S*~, the error on the estimate could even be lowered by 64 %. When using the traditional soft-sensor approach, the resulting *h* values could not be used to statistically reject the null hypothesis of detecting gross errors, since estimations of covariance of the turnover rates were arbitrarily chosen and static over time. The new approach delivers both statistically meaningful *h* values for the detection of gross errors and informative standard deviations on the estimated rates. Latter ones are essential under real process conditions to judge soft-sensor estimation quality, as obviously there exist no possibility to evaluate the control quality by comparing the estimates to unbiased model values. Additionally, we presented a new generic approach to ensure a predefined control quality of the soft-sensor estimate by iteratively evaluating the effect of the different errors on the raw signal measurements. It has been demonstrated that by following this generic workflow, it is possible to additionally significantly increase the adaptive soft-sensor accuracy. The presented approach can be generically applied taking also additional error sources into account. The new methodology is practically applicable to industrial conditions, where maximal errors of measurement devices are used to obtain dynamically changing accuracies of derived turnover rates as shown in Fig. [2a](#Fig2){ref-type="fig"}. Electronic supplementary material ================================= {#Sec27} Below is the link to the electronic supplementary material.ESM 1(PDF 651 kb) CER : Carbon dioxide evolution rate (mol h^−1^) *E* : Elemental composition matrix *F*~*a*,in~ : Air flow in (L min^−1^) *F*~*a*,out~ : Air flow out (L min^−1^) MFC : Mass flow controller MPD : Median percentage of difference OUR : Oxygen uptake rate (mol h^−1^) *q*~*S*~ : Specific substrate uptake rate (mol mol^−1^ h^−1^) *Ra*~inert~ : Inert gas ratio (−) *r*~*i*~ : Consumption/formation rate for species *i* (mol h^−1^) *r*~*X*~ : Biomass formation rate (mol h^−1^) *S* : Substrate, C-normalized (mol) *V*~*m*~ : Molar volume (L mol^−1^) *X* : Biomass, C-normalized (mol) *µ* : Specific growth rate (h^−1^) *ε* : Residual vector for non-closing balances *y*~wet~ : Oxygen fraction in the off-gas without microbial activity (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{O}}_2,\mathrm{out}} $$\end{document}$ : Oxygen fraction in the off-gas stream (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{O}}_2,\mathrm{in}} $$\end{document}$ : Oxygen fraction in the inlet air (−) *γ*~*i*~ : Degree of reduction for species *i* (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{CO}}_2,\mathrm{out}} $$\end{document}$ : Carbon dioxide fraction in the off-gas stream (−) $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {y}_{{\mathrm{CO}}_2,\mathrm{in}} $$\end{document}$ : Carbon dioxide fraction in the inlet air (−) *Y*~*X*/*S*~ : Biomass/substrate yield coefficient *ε*~*i*~ : Applied relative error on species *i* (−) *Δy* : Absolute measurement error of signal *y* Valentin Steinwandter and Thomas Zahel contributed equally to this work. Open access funding provided by TU Wien (TUW). Authors' contributions {#d29e4286} ====================== Thomas Zahel developed and implemented the error propagation procedure within the softsensor framework as well as the design of the generic workflow to identify tolerable measurement accuracy to deliver predefined softsensor accuracy. Valentin Steinwandter developed the in silico simulation environment, consisting of the representative mechanistic model as well as the test environment to systematically investigate the uncertainty space and implemented the generic workflow. Both authors contributed equally to the writing of the presented work. Funding {#FPar1} ======= Financial support was provided by the Austrian research funding association (FFG) under the scope of the COMET program within the research project "Industrial Methods for Process Analytical Chemistry---From Measurement Technologies to Information Systems (imPACts)" (contract \# 843546). Conflict of interest {#FPar2} ==================== The authors declare that they have no conflict of interest.

{ "pile_set_name": "PubMed Central" }

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"raw", "onlin", "cer", "mathrmmmathrmpmathrmdkernem", "balanc", "ydot", "afigreftypefig", "escherichia", "varianc", "especi", "made", "input", "yield", "chosen", "oxygen", "region", "spot", "galvan", "leftupvarepsilonmathrmorightkernem", "volum", "yx", "mode", "behavior", "user", "wellknown", "exemplari", "tu", "residu", "increas", "softwar", "ra", "improv", "access", "reactor", "report", "offer", "similar", "×", "point", "usepackageupgreek", "yrightdot", "balancedocumentclassptminim", "area", "spectroscopi", "ymathrmomathrmout", "termin", "driven", "firstord", "calculationsecreftypesec", "plu", "attribut", "introduc", "therefor", "previou", "defect", "rank", "rather", "strategi", "inert", "detect" ]

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"frequently", "serve", "targets", "control", "strategies", "key", "physiological", "information", "catalyst", "concentration", "biomass", "However", "required", "online", "measurement", "biomass", "critical", "endeavor", "using", "sensors", "dielectric", "spectroscopy", "broth", "fluorescence", "permittivity", "measurements", "connected", "limitations", "drawbacks", "outlined", "elsewhere", "Software", "sensors", "short", "provide", "elegant", "way", "estimate", "biomass", "concentration", "using", "different", "measurements", "contribution", "want", "focus", "dominant", "biotechnological", "process", "mode", "microbial", "fermentation", "improvement", "one", "mature", "implementations", "using", "measurements", "established", "tools", "bioprocess", "control", "analysis", "also", "frequently", "shown", "practical", "applications", "Briefly", "mass", "conservation", "laws", "used", "calculate", "turnover", "rates", "online", "measurements", "might", "superimposed", "signal", "errors", 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Introduction {#s1} ============ Inhibitory control, the ability to suppress planned or ongoing cognitive or motor processes is necessary to ensure flexible and adapted goal-directed behavior in ever-changing environments (Aron, [@B3]; Dillon and Pizzagalli, [@B23]). Converging functional neuroimaging, transcranial magnetic stimulation and lesion data indicate that inhibitory control relies on a cortico-subcortical network involving the right inferior frontal gyrus (rIFG), the pre-supplementary motor area (SMA) and the basal ganglia (Garavan et al., [@B31]; Aron et al., [@B6]; Chambers et al., [@B19]; Majid et al., [@B51]), and manifesting at latencies of 150--400 ms after the onset of the stimuli associated with the inhibition goals (Kaiser et al., [@B39]; Smith and Douglas, [@B72]). Although inhibitory control performance mostly depends on how the stimuli triggering the inhibition are processed, mounting evidence indicates that the cognitive- and brain-states preceding the presentation of these stimuli also contribute to the success of the inhibition. In Eriksen flanker tasks, contrasts between event-related potentials (ERPs) time-locked to the motor responses to trial preceding error vs. accurate trials revealed a specific ERP component peaking 50--100 ms post-response onset over frontal electrode sites \[The "error related positivity" (EPP) Ridderinkhof et al., [@B69]; Allain et al., [@B1]; Hajcak et al., [@B34]\]. Britz and Michel ([@B13]) extended these results by showing that dorsolateral prefrontal cortices were engaged differentially during the 100 ms preceding the stimulus onset in error vs. correct trials during a classical color stroop task. These collective results suggest that errors are foreshadowed by a disruption of prefrontal task monitoring systems before the actual need for inhibitory control (see also Eichele et al., [@B26]; Masaki et al., [@B53]; Steinhauser et al., [@B76]). Additional support for the critical role of pre-stimulus brain states in inhibitory control performance comes from studies in which the occurrence of the inhibition stimuli was predictable. In such cases, proactive inhibitory mechanisms are engaged before the stimulus presentation and interact with stimulus-driven reactive inhibitory mechanisms to eventually enhance inhibitory control (Claffey et al., [@B20]; Jahfari et al., [@B38]; Aron, [@B4]; Cai et al., [@B18]; Duque et al., [@B24]; Majid et al., [@B51]). By manipulating the degree of predictability of inhibition trials, these studies showed that when response inhibition can be prepared, effector-selective proactive inhibition mechanisms, mediated by the dorsolateral prefrontal cortex, are engaged to support fronto-basal reactive inhibition mechanisms (Aron, [@B4] for review). Criaud et al. ([@B21]) further demonstrated that proactive mechanisms persist once established at the beginning of each trial, suggesting that proactive inhibition has not only a transient effect but also modifies the general response mode of the participants. Taken together, these findings suggest that inhibitory control performance does not solely depend on how participants manage the conflict induced by the stimulus but also on the state of the task-monitoring and control systems before the trial. However, the studies conducted so far on the state-dependency of inhibitory control were based on response- or stimulus-evoked ERPs and thus could not reveal whether spontaneous, not-time-locked fluctuations of ongoing brain activity preceding unpredictable inhibition-stimuli impact on inhibitory control performance. The averaging of the EEG signal in ERP studies indeed canceled out the activity not time-locked to the event of interest and thus dismissed this substantial fraction of the variability of the raw electrical brain activity (e.g., Arieli et al., [@B2]). Based on the current evidence for the state-dependency of behavioral and brain responses during various types of perceptual tasks (e.g., Lehmann et al., [@B45]; Ress et al., [@B68]; Fox et al., [@B30]; Fox and Raichle, [@B29]; Britz et al., [@B12], [@B14]), we hypothesize that spontaneous brain states immediately preceding the presentation of inhibition-triggering stimuli might also influence the success or failure to inhibit responses during inhibitory control tasks. To address this question, we used well-established methods of single-trial topographic analyses of EEG to determine whether specific voltage topographies present at the moment of the onset of unpredictable were associated with correct rejection (CR) vs. false alarms (FAs) to NoGo stimuli during a Go/NoGo task (Lehmann et al., [@B45]; Kondakor et al., [@B43], [@B42]; Koenig et al., [@B40]; Muller et al., [@B58]; Britz et al., [@B12]; Brodbeck et al., [@B15]). The current investigation was based on a reanalysis of the data from Manuel et al. ([@B52]) in which EEG was recorded in eleven healthy participants during an auditory spatial Go/NoGo task. Material and methods ==================== Participants ------------ Eleven healthy volunteers participated in the study, all male and right-handed (Oldfield, [@B61]), aged 22--39 years (mean ± *SD*, 29.4 ± 1.6 years). Each participant provided written, informed consent to participate in the study. No participant had a history of neurological or psychiatric illness, and all reported normal hearing. All procedures were approved by the Ethics Committee of the Faculty of Biology and Medicine of the Vaudois University Hospital Center and University of Lausanne. Stimuli ------- Auditory stimuli were 150-ms noise bursts (200--500 Hz bandpass filtered; 5 ms rise/fall), lateralized by means of a right- or left-ear leading interaural time difference of 770 μ s resulting in a perceived lateralization of \~80° from the central midline (Blauert, [@B9]). The sounds were presented via ER-4P Etymotic earphones. Procedure and task ------------------ The current study is based on a reanalysis of the data from Manuel et al. ([@B52]), in which the procedure and task are already detailed; we thus present only the main task parameters here. Participants were seated in an electrically shielded and sound-attenuated booth in front of a 19-in screen. Stimulus delivery and response recording were controlled using E-prime 2.0. The paradigm comprised an auditory spatial Go/NoGo task in which participants had to respond as quickly as possible via a button to left-lateralized sounds (Go stimuli, hereafter termed LG) and to withhold responses to right-lateralized sounds (NoGo stimuli, termed RNG). Each trial started with the presentation of a centrally presented gray cross on a black background for a randomly determined duration ranging from 1000 to 1900 ms. At the same time that the cross disappeared, the LG and RNG sounds were presented and response collection window was opened. In the Go conditions, a feedback on accuracy and response speed was provided immediately after the response. LG and RNG trials were presented with an equal probability of 0.5. The experiment was divided into three sessions. Each session started with a calibration block of 16 randomly presented trials (eight LG and eight RNG), followed by two test blocks of 80 randomly presented trials each (40 LG and 40 RNG). The calibration blocks were used to individually adjust the task difficulty and to maintain time pressure across the whole experiment. During each calibration phase, the mean response time (RT) to LG trials was calculated online and used to determine the individual participant\'s RT threshold (RTt), which was set at 80% of the mean RT from the calibration block. During the test block, a Go response RT was considered as correct if it was below the 80% RTt of the immediately preceding calibration phase. Otherwise, a feedback screen indicating "too late!" was displayed immediately after the Go response (slow hit). On each trial, the mean percentage of correct trials, including fast hit and CR, was displayed. Participants were not informed about this thresholding procedure. Except for the global accuracy, no visual feedback was displayed after fast hits or FAs (see Vocat et al., [@B79] for a similar procedure). The whole Go/NoGo training session included a total of 528 stimuli (160 stimuli in the test block + 16 stimuli in the calibration block × 3 sessions = 528) and lasted for a total of \~ 35 min. After the completion of each session, a rest period of 10 min was provided to participants. EEG acquisition and preprocessing --------------------------------- Continuous EEG was acquired at 1024 Hz through a 128-channel Biosemi ActiveTwo system referenced to the common mode sense/driven right leg ground. All the EEG analyses were conducted with the Cartool software (Brunet et al., [@B16]). Before the single trial analyses, data at artifact electrodes from each participant were interpolated (Perrin et al., [@B65]). EEG epochs of the 50 ms preceding the stimulus onset were extracted from the raw EEG data, for each participant, for RNG CR and for the RNG FAs conditions. Because there was less FA than CR (see the behavioral results), we balanced the number of epochs included in each condition before the analyses to ensure that any potential differences between the two conditions did not follow from difference in statistical power. First, the same number of CR as FA epochs was randomly extracted for each participant separately. On the resulting epochs, a ±80 μV artifact rejection criterion was applied to exclude trials with eye blinks or other artifacts. When necessary, the number of epochs was then again balanced across conditions to eventually result in the inclusion of 25.4 ± 16.8 (mean ± *SD*) epochs in each condition for the single-trial analyses. Behavioral data analyses ------------------------ Behavioral data were analyzed to determine whether FA commission occurred randomly within the sequence of NoGo stimuli (see the Discussion section). We analyzed the pattern of FA occurrence by calculating the temporal auto-correlation function of the FA response type. If there was a relationship between FA occurrence at one trial with FA occurrence at previous or subsequent trials, as could for instance occur during periods of decrease in attention inducing series of FA, it should manifest as an increase in the autocorrelation coefficient (Britz et al., [@B12], [@B14]; Bernasconi et al., [@B8]). We first performed a binary classification on the FAs and the Hits and Misses (respectively responded and missed Go trials), as well as CR of NoGo trials. To determine if the patterns of FA of the participants were different from a random distribution, we permuted 1000 times the sequence of each participant and then compared the autocorrelation coefficients of the sequence of the participant to the distribution of the autocorrelation coefficients of the randomized sequence of the participant. The autocorrelation coefficients of the FA occurrence were computed for each trial n with that in trial *n* + *m* for *m* = 1 − 20. Single-trial topographic analyses --------------------------------- The time-locked averaging of the EEG signal across multiple repetitions of an event (e.g., the presentation of a stimulus or a behavioral response) to build ERPs cancels out all the fluctuations of brain activity not time-locked to the event of interest, because by definition, the phase of these fluctuations varies across trials. To circumvent this problem and to investigate whether non-stimulus-locked variations in the prestimulus activity impact on inhibitory control proficiency, we utilized previously published methods of single-trial EEG analysis (Lehmann et al., [@B45]; Kondakor et al., [@B43], [@B42]; Koenig et al., [@B41]; Mohr et al., [@B57]; Britz et al., [@B12]; see for review Britz and Michel, [@B13]; Eichele et al., [@B26]; Steinhauser et al., [@B76]). This approach is based on evidence that evoked and induced ongoing EEG signal is not random, but rather organized in a succession of quasi-stable topographies of the electric potentials. Since the configuration of the electric potentials at the scalp reflects the sum of all active sources in the brain at each moment in time (Lehmann et al., [@B46]), the observation of stable topographies or "maps" suggest that during these time intervals, the functional state of the brain is stable (e.g., Michel et al., [@B55]). The short periods of functional stability have been referred to as "functional microstate" and typically comprise 80--120 long segments of stable configuration of the scalp-recorded voltage topography of the electric potential (Lehmann and Skrandies, [@B47]; Koenig et al., [@B41]). While EEG topography remains stable during a microstate, the strength of the electric potentials, as indexed, for example, by the Global Field Power (GFP), increases and then decreases. The GFP represents a single-number index of the strength of electric potentials; it is calculated as the spatial standard deviation of the electric potentials: the square root of the sum of all squared potentials divided by the number of electrodes (Lehmann and Skrandies, [@B47]; Murray et al., [@B59]). Compelling relationships have been found between spontaneously occurring microstates preceding the presentation of a stimulus and the behavioral and brain response to this stimulus (Lehmann et al., [@B45]; Kondakor et al., [@B43], [@B42]; Mohr et al., [@B57]; Muller et al., [@B58]; Britz et al., [@B12]), suggesting that microstate-based single-trial EEG topographic analyses enable reliable investigations of the effects of variations in the raw ongoing EEG signal. The single-trial topographic analysis comprises the following processing steps, which mainly consist in reducing the raw EEG data into a limited number of common stable microstates within and then across participants (for similar procedures see also Koenig et al., [@B41]; Mohr et al., [@B57]; Britz et al., [@B12], [@B14]). The first step involved determining the topographies differentiating the FAs and CR conditions for each participant: (1) We extracted from each EEG epoch, for each participant and each condition the topography manifesting at the single time frame when the GFP was maximal (i.e., when the signal-to noise ratio of the microstate was the highest) within the 50 ms pre-stimulus onset. We selected this pre-stimulus time frame because it corresponds to the half of the duration of an average microstate and thus this procedure enables to extract topography at the time frame best representing the microstate present at the moment when the stimulus is presented (the total length of a microstate ranges between 80 to 120 ms; Britz et al., [@B14]); (2) For each participant separately, we applied a spatial *k*-means cluster analysis (e.g., Pascual-Marqui et al., [@B63]) on these topographies to identify the most dominant topographies among all topographies extracted in step (1). (3) The optimal number of clusters, or "template topographies," was determined based on a modified Krzanowski-Lai criterion (Krzanowski and Lai, [@B44]); (4) A fitting procedure, in which each original topography of step (1) was relabeled with the template map of step (2) with which it best correlated (Pegna et al., [@B64]), enabled us to identify the two template topographies that best differentiated the FA and CR conditions: the template map with the highest frequency of occurrence in the FA and in the CR conditions were retained. The second step comprised determining the topographies differentiating the FA and CR across participants: (5) We applied a second *k*-mean cluster analysis on the groups of FA and CR map identified during the first step for each participant and back-fitted the resulting template topographies on the original data using the same procedure as steps (1), (2), and (3). The statistical comparison between the frequency of occurrence of the maps in the FA and CR conditions revealed the topographies differentiating the two conditions at the group level. Electrical source estimations ----------------------------- Electrical sources underlying the template map(s) of interest were estimated using a distributed linear inverse solution based on a local autoregressive average (LAURA) regularization approach (Grave De Peralta Menendez et al., [@B33]; Grave-De Peralta et al., [@B32]; see also Michel et al., [@B56] for a comparison of inverse solution methods) implemented in the Cartool software (Brunet et al., [@B16]). LAURA selects the source configuration that mimics the biophysical behavior of electric potential (i.e., activity at one point depends on the activity at neighboring points). The solution space is based on a realistic head model and included 3005 solution points homogeneously distributed within the gray matter of the average brain of the Montreal Neurological Institute (courtesy of R. Grave de Peralta Menendez and S. Gonzalez Andino, University Hospital of Geneva, Geneva, Switzerland). Results {#s2} ======= Behavior -------- Reaction time to Go stimuli was 251 ± 21 ms (mean ± SEM). The percentage of FAs was 10.9 ± 0.95% (Manuel et al., [@B52]). The autocorrelation coefficients of the sequence of FA of the participants were all between *z* = −0.37 and 0.64 *SD* from the mean of the coefficients from the randomized sequences, indicating no significant (−1.96 \< *z* \< 1.96; *p* \> 0.05) differences between the distributions of FA in the random vs. actual sequences for lags 1--20 (Figure [1](#F1){ref-type="fig"}). ![**Analysis of the sequence of false alarms**. For lags 1--20, the maximal (blue), mean (green), and minimal (red) autocorrelation coefficients are represented, for the sequences of the participants (plain lines) and for randomized sequences (dotted lines). The sequences of FA from the participant did not differ statistically from the random sequences (see the Results section), supporting that random fluctuations in the pre-stimulus period may account for FA occurrence.](fnhum-07-00238-g0001){#F1} Single-trial topographic analyses --------------------------------- Consistent with previous applications of the current single-trial topographic analysis (Britz et al., [@B12], [@B14]), the *k*-means cluster analysis applied during the first step of the analysis (see the Method section) identified on average 5.9 ± 1.6 maps for each participant as optimally explaining the data. These template maps accounted for 72.7 ± 7.3% of the global explained variance. The maps were then grouped for each conditions across participants and a second cluster analysis was applied. The best clustering during the second step explained 93% of the variance with 11 template maps. The resulting template maps were then back-fitted to the original individual subject data for each conditions to determine their frequency of occurrence. Two-tailed pairwise *t*-tests were conducted to compare the frequency of occurrence of each template map between the CR and FA conditions. A single topographic map was found to differ in its frequency of occurrence between the FA and CR conditions (Figure [2](#F2){ref-type="fig"}, violet square): This topography occurred significantly more often in the CR than FA condition \[*t*~(10)~ = 2.83; *p* = 0.018; *d*~*z*~ = 0.9; Figure [2](#F2){ref-type="fig"}\]. To test for the probability of type 1 errors, we generated 1000 randomized permutations of the raw data maps and computed, for each of the permutation, the same *t*-tests as those we applied to the actual data. The results revealed a probability of 1.7% to have the same pattern as in the real data (i.e., one *p*-values \< 0.019 among the 11 tests) in the 1000 permutations, i.e., when there was no structure in the data. ![**Topographies of the prestimulus microstates as revealed by the cluster analysis**. The map that significantly differentiated the correct rejections is framed in violet. The red lines indicate the *t*-test *p* \< 0.05 significance threshold and an asterisk is placed where significance has been reached.](fnhum-07-00238-g0002){#F2} Electrical source estimations ----------------------------- The estimation of the electric sources at the origin of the template topography differentiating the CR from the FA condition showed that it resulted from the activation of a right hemispheric parieto-frontal network extending from the inferior parietal lobule to the frontal gyri (Figure [3](#F3){ref-type="fig"}). The largest hub of activation was centered around the supramarginal gyrus/primary auditory cortex. Importantly, this result is only a visualization of the sources underlying the EEG topography and not the result of a statistical contrast; it should thus be interpreted with caution. ![**LAURA distributed source estimations corresponding to the topography identified as best characterizing the pre-stimulus period before correct rejections identified a right hemispheric fronto-temporal prestimulus activation**.](fnhum-07-00238-g0003){#F3} Discussion {#s3} ========== The present study suggests that performance in a classical Go/NoGo task is influenced by the momentary state of the ongoing brain activity immediately preceding the onset of unpredictable NoGo stimuli. A specific EEG voltage topography manifested more frequently before successful response-inhibition to NoGo stimuli than before FAs. There was no evidence for topographies specifically preceding FA trials. Electrical source estimations localized the source of the topography preceding successful inhibition within a right fronto-temporal network. Our results contribute to current knowledge on the influence of pre-stimulus brain state on inhibitory control by indicating that inhibition performance is modulated by the momentary brain state present when the stimuli are presented. Fluctuations in the ongoing brain activity have been shown to represent almost 90% of the variability in the EEG signal (Eichele et al., [@B26]). This source of variability was mostly dismissed in previous ERPs studies on the effect of pre-stimulus activity because time-locked averaging of the EEG signal were applied to extract ERPs (e.g., Hajcak et al., [@B34]; Masaki et al., [@B53]); in such procedures, the signal not time-locked to the event is cancelled out and usually considered as background physiological noise. In line with our findings, mounting evidence indicate that ongoing spontaneous EEG fluctuations are actually functionally relevant and account for a substantial fraction of the variability in the behavioral and brain responses to stimuli in various experimental contexts (Picton et al., [@B66]; O\'Connell et al., [@B60] for the role of pre-stimulus endogenous modulation in oscillatory activity in task performance; Britz and Michel, [@B13] for discussion). However, the dependency of sensory-cognitive processes to single trial variability in brain activity was so far demonstrated in perceptual but not executive tasks. For instance, Mohr et al. ([@B57]) showed that a specific topographic map with a left anterior-right posterior dipole orientation spontaneously manifesting before word presentation predicted an enhanced discrimination of emotional word when the word was presented in the left visual field but only for men during a bilateral lexical decision task. Lehmann et al. ([@B45]) and Kondakor et al. ([@B43], [@B42]) further demonstrated that the brain responses to identical tones depended on the spontaneously occurring topography preceding their presentation. Corroborating and extending these findings, our results suggest that spontaneous fluctuations of ongoing brain activity not only modulate perceptual processes but also high-order, top-down executive mechanisms as those supporting inhibitory control in conflict tasks. The sources of the scalp topography preceding more frequently CR than FAs were maximal within a right fronto-parietal network. The right rIFG has been repeatedly involved in inhibitory control by functional neuroimaging (Garavan et al., [@B31]; Rubia et al., [@B71]; Aron and Poldrack, [@B7]; Aron et al., [@B5]) and lesion studies (Decary and Richer, [@B22]; Aron et al., [@B6]; Rieger et al., [@B70]; Floden and Stuss, [@B28]; Picton et al., [@B67]). The rIFG is thought to trigger motor inhibition via its connections to the subthalamic nucleus (STN; Inase et al., [@B37]; Aron et al., [@B5]; Aron, [@B4]). Most of these studies, however, involved the rIFG in inhibitory control by contrasting the post-stimulus brain responses to stimuli associated with vs. without response inhibition. In the current study, we showed that random fluctuations in the activity of rIFG before the demand for inhibition also play a critical role in inhibitory control proficiency. In conditions when the onset of the NoGo stimuli cannot be predicted, if the NoGo stimuli are presented when the activity of the rIFG is high, the probability of a correct inhibition increases. Although speculative, an account for this effect could be that a pre-activation of the rIFG increases the speed of inhibition process because less time is needed to reach the elicitation threshold of the inhibitory command from frontal to subcortical structures. Right inferior parietal areas have been previously involved in response inhibition (Garavan et al., [@B31]; Liddle et al., [@B49]; Menon et al., [@B54]; Rubia et al., [@B71]) or response conflict resolution (Braver et al., [@B11]; Van Veen et al., [@B78]). These studies suggest that inferior parietal cortices mediate attention to the task and might thus, in turn, modulate performance (see also Hampshire et al., [@B35]). Increase in the activity of the precentral gyrus in condition of successful inhibition has also been reported in stop-signal task (though more superior as in the current study; Li et al., [@B48]), and have been interpreted as "negative motor areas" (Ikeda et al., [@B36]; Yazawa et al., [@B80]), whose direct stimulation elicits response inhibition (Luders et al., [@B50]). Alternatively, since participants had to respond with their right hand, an activation of ipsilateral (right) motor areas might have inhibited contralateral, homotopic motor areas via interhemispheric inhibition mechanisms and in turn facilitated the rejection of NoGo stimuli (e.g., Ferbert et al., [@B27]). Hand and arm motor representations are, however, higher along the central sulcus as the area found in the current study. The precise role of the right temporal structures in inhibitory control is more difficult to delineate. These areas have been shown to interact with higher level prefrontal regions during inhibitory control tasks (Egner and Hirsch, [@B25]) and to be modulated in inhibition-related disorders (Tamm et al., [@B77]; Solanto et al., [@B73]), but their precise role remains unclear. An alternative account for the role of right temporal areas in FA commission would be their involvement in the processing of the auditory spatial features distinguishing Go from NoGo stimuli in the current task. Go and NoGo goals activation indeed first require discriminating whether the stimulus is presented on the left or right auditory hemifield. Right temporal areas have been repeatedly involved in the early stage of auditory spatial processing (Spierer et al., [@B75], [@B74]) and their pre-activation could have facilitated the detection of NoGo stimuli and in turn response inhibition. In line with this hypothesis, Manuel et al. ([@B52]) suggested an important role of early auditory analyses of the lateralization of the stimuli in inhibitory control proficiency during the current auditory spatial Go/NoGo task. Importantly, however, we would like to emphasize that our source estimations were only visualizations, rather than a statistical analysis, of the network likely underlying the topography preceding FA commission. These results should thus be interpreted with caution. Although our pattern of result is highly consistent with current evidence for a crucial role of the rIGF in stimulus-induced inhibitory control processes, it contrasts with previous data on the brain regions whose pre-stimulus activity modulates inhibition proficiency. Electrophysiological studies on trials preceding error during an Eriksen flankers task identified a specific response-locked ERP component at 100 ms (EPP; Ridderinkhof et al., [@B69]; Allain et al., [@B1]; Hajcak et al., [@B34]). The EPP was specific to trials preceding an error and its amplitude was larger on frontal, central and parietal midline electrodes, compatible with a generator located within the anterior cingulate cortex (ACC). Using a stroop task, Britz and Michel ([@B13]) showed a specific pattern of stimulus-locked fronto-parietal activity before erroneous vs. correct trials. The modulation of prefrontal activity before error commission found in the studies reviewed above was interpreted as reflecting a transient disruption of action monitoring and executive control systems. Our results complement these finding by demonstrating that in addition to the impact of maintaining a minimal level of action monitoring during the task found by response- or stimulus-locked ERP approaches, spontaneous ongoing activity within the networks mediating stimulus-induced response inhibition also influences inhibitory control performance. In line with this assumption, growing evidence indicate that as soon as the need for response inhibition can be predicted, proactive inhibitory control mechanisms are engaged to facilitate the inhibitory control of task-irrelevant responses (Claffey et al., [@B20]; Jahfari et al., [@B38]; see Aron, [@B4] for review; Cai et al., [@B18]; Criaud et al., [@B21]). "Active breaking" mechanisms would be engaged before the stimulus onset to decrease the activity of motor area and in turn facilitate response inhibition (Cai et al., [@B18]; Duque et al., [@B24]). Anticipatory mechanisms have been shown to rely on a fronto-striatal network partly overlapping with the estimated sources of the topography prominently associated with successful inhibition in the current study. We cannot rule out that the frequency of occurrence of the topography preceding successful inhibition was influenced by proactive preparatory mechanisms in the current study. According to this hypothesis, the anticipation of NoGo stimuli would have increased the frequency of occurrence of the "facilitatory" topography or, in other word, pre-activated inhibitory control mechanisms. Speaking against this hypothesis, there was no strategic advantage of intentionally maintaining inhibitory control networks active in our task because such decrease in the response elicitation threshold would have resulted in a general decrease in response speed to Go stimuli. In our paradigm, emphasis was put on response speed: slow Hits (correctly responded Go stimuli but above the RTt determined in calibration blocks) were considered as error and reported explicitly as such by the visual feedback provided after each trial. Although there was a probability of 0.5 for NoGo stimuli occurrence, response prepotency was maintained very high by the recurrent feedback on responses speed. The threshold for considering a response to Go trials to slow was indeed individually adjusted to 80% of the mean response speed calculated during the calibration blocks intervening between experimental blocks (see the Method section). Hence, we interpret our results as indicating that when the fronto-temporal brain network was already (spontaneously) activated before a NoGo stimulus was presented, inhibition performance was improved. Compatible with this hypothesis, the analyses of the distribution of FA occurrence across the experimental session showed that participants committed FA following the same pattern as when FA were randomly distributed within the sequence. The adoption of any strategy by the participant would have most likely resulted in non-random sequences consisting of series of FA or CR because a specific task set could unlikely be maintained constantly during the whole experiment (Palva and Palva, [@B62]). Our results cannot disentangle the relationship between proactive inhibition and the occurrence of specific microstates. To our knowledge, no study has so far investigated whether top-down, strategic implementation of task sets can impact on the frequency of occurrence-specific microstate and thus whether proactive mechanism could have influenced our pattern of result. Further studies focusing on longer epochs and manipulating the engagement of proactive inhibitory mechanisms are necessary to elucidate this question. Of note, we found a specific EEG topography preceding successful trials, but no specific topography preceding errors. This pattern of result suggests that while the engagement of specific cognitive set may enhance motor responses inhibition in conditions of high response prepotency, many different deviations from this optimal brain-state could result in inhibition failure, in turn explaining that we did not identify a specific topographic map preceding errors. In this regard, a limitation of the current study is that by defining our FA and CR conditions only based on RT (and lack thereof), we possibly included in the CR condition some trials in which erroneous motor activation was actually engaged but inhibited before reaching activation threshold. Referred to as "covert" errors, these partial-error trials can only be detected with the recording of electromyography activations and have been suggested to depend on distinct mechanisms as overt errors (Burle et al., [@B17]; Allain et al., [@B1]; Boulinguez et al., [@B10]). In any case, the inclusion of covert error trials in the CR condition would have increased noise in the data and thus most likely increased the probability of type 2 but not type 1 errors. Second, because FAs were quite rare in our data, our single trial topographic analyses included only a limited number of trials. While the capacity to deal with small numbers of EEG epochs is an advantage of this approach and we reach reliable statistical results, how the number of epochs impacts the statistical outcome of our approach remains to be determined. Collectively, our results point out that spontaneous fluctuations within inhibitory control networks at the moment when NoGo stimuli are presented influence inhibition performance. The question remains open, however, as to whether and how prestimulus modulations impact on the processing of the inhibition-related stimuli. It would be notably interesting to link directly the current results with previous literature on pre-target predictor of inhibition performance and to examine potential differences in the early sensory processing of the NoGo stimuli between FA and CR trials. Further studies comparing the pre-stimulus with the post-stimulus activity would be necessary to elucidate this question. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by a grant from the Swiss National Science Foundation to Lucas Spierer (\#320030_143348). The Cartool software has been programmed by Denis Brunet (Functional Brain Mapping Laboratory, Geneva, Switzerland) and supported by the Center for Biomedical Imaging of Geneva and Lausanne. We thank David Magezi, David Souto and the reviewers for their valuable comments. [^1]: Edited by: John J. Foxe, Albert Einstein College of Medicine, USA [^2]: Reviewed by: Redmond O\'Connell, Trinity College Dublin, Ireland; Kevin Whittingstall, Université de Sherbrooke, Canada

{ "pile_set_name": "PubMed Central" }

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"stimul", "reveal", "occur", "equal", "gyru", "extend", "freftypefig", "evid", "topdown", "connect", "lg", "smith", "impact", "set", "dillon", "fa", "binari", "event", "record", "divid", "inhibitori", "although", "channel", "inhibitionrel", "like", "topograph", "publish", "magezi", "largest", "degre", "univers", "einstein", "sinc", "cannot", "prestimulu", "biolog", "demonstr", "present", "cartool", "place", "highest", "standard", "critic", "prepar", "group", "modifi", "psychiatr", "address", "threshold", "precis", "université", "faculti", "deviat", "main", "analyz", "electrophysiolog", "frontobas", "±", "homogen", "difficulti", "precentr", "committe", "peak", "induc", "microstatebas", "spierer", "lesion", "importantli", "responselock", "arm", "grave", "facilit", "singletri", "manuel", "instanc", "influenc", "sensori", "al", "neuroimag", "dougla", "kondakor", "contribut", "raichl", "black", "rule", "engag", "lexic", "volunt", "reject", "speed", "caution", "recurr", "stimuli", 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"motor", "areas", "Ikeda", "et", "Yazawa", "et", "whose", "direct", "stimulation", "elicits", "response", "inhibition", "Luders", "et", "Alternatively", "since", "participants", "respond", "right", "hand", "activation", "ipsilateral", "right", "motor", "areas", "might", "inhibited", "contralateral", "homotopic", "motor", "areas", "via", "interhemispheric", "inhibition", "mechanisms", "turn", "facilitated", "rejection", "NoGo", "stimuli", "Ferbert", "et", "Hand", "arm", "motor", "representations", "however", "higher", "along", "central", "sulcus", "area", "found", "current", "study", "precise", "role", "right", "temporal", "structures", "inhibitory", "control", "difficult", "delineate", "areas", "shown", "interact", "higher", "level", "prefrontal", "regions", "inhibitory", "control", "tasks", "Egner", "Hirsch", "modulated", "disorders", "Tamm", "et", "Solanto", "et", "precise", "role", "remains", "unclear", "alternative", "account", "role", "right", "temporal", "areas", "FA", "commission", "would", "involvement", "processing", "auditory", "spatial", "features", "distinguishing", "Go", "NoGo", "stimuli", "current", "task", "Go", "NoGo", "goals", "activation", "indeed", "first", "require", "discriminating", "whether", "stimulus", "presented", "left", "right", "auditory", "hemifield", "Right", "temporal", "areas", "repeatedly", "involved", "early", "stage", "auditory", "spatial", "processing", "Spierer", "et", "could", "facilitated", "detection", "NoGo", "stimuli", "turn", "response", "inhibition", "line", "hypothesis", "Manuel", "et", "al", "suggested", "important", "role", "early", "auditory", "analyses", "lateralization", "stimuli", "inhibitory", "control", "proficiency", "current", "auditory", "spatial", "task", "Importantly", "however", "would", "like", "emphasize", "source", "estimations", "visualizations", "rather", "statistical", "analysis", "network", "likely", "underlying", "topography", "preceding", "FA", "commission", "results", "thus", "interpreted", "caution", "Although", "pattern", "result", "highly", "consistent", "current", "evidence", "crucial", "role", "rIGF", "inhibitory", "control", "processes", "contrasts", "previous", "data", "brain", "regions", "whose", "activity", "modulates", "inhibition", "proficiency", "Electrophysiological", "studies", "trials", "preceding", "error", "Eriksen", "flankers", "task", "identified", "specific", "ERP", "component", "ms", "EPP", "Ridderinkhof", "et", "Allain", "et", "Hajcak", "et", "EPP", "specific", "trials", "preceding", "error", "amplitude", "larger", "frontal", "central", "parietal", "midline", "electrodes", "compatible", "generator", "located", "within", "anterior", "cingulate", "cortex", "ACC", "Using", "stroop", "task", "Britz", "Michel", "showed", "specific", "pattern", "activity", "erroneous", "correct", "trials", "modulation", "prefrontal", "activity", "error", "commission", "found", "studies", "reviewed", "interpreted", "reflecting", "transient", "disruption", "action", "monitoring", "executive", "control", "systems", "results", "complement", "finding", "demonstrating", "addition", "impact", "maintaining", "minimal", "level", "action", "monitoring", "task", "found", "ERP", "approaches", "spontaneous", "ongoing", "activity", "within", "networks", "mediating", "response", "inhibition", "also", "influences", "inhibitory", "control", "performance", "line", "assumption", "growing", "evidence", "indicate", "soon", "need", "response", "inhibition", "predicted", "proactive", "inhibitory", "control", "mechanisms", "engaged", "facilitate", "inhibitory", "control", "responses", "Claffey", "et", "Jahfari", "et", "see", "Aron", "review", "Cai", "et", "Criaud", "et", "Active", "breaking", "mechanisms", "would", "engaged", "stimulus", "onset", "decrease", "activity", "motor", "area", "turn", "facilitate", "response", "inhibition", "Cai", "et", "Duque", "et", "Anticipatory", "mechanisms", "shown", "rely", "network", "partly", "overlapping", "estimated", "sources", "topography", "prominently", "associated", "successful", "inhibition", "current", "study", "rule", "frequency", "occurrence", "topography", "preceding", "successful", "inhibition", "influenced", "proactive", "preparatory", "mechanisms", "current", "study", "According", "hypothesis", "anticipation", "NoGo", "stimuli", "would", "increased", "frequency", "occurrence", "facilitatory", "topography", "word", "inhibitory", "control", "mechanisms", "Speaking", "hypothesis", "strategic", "advantage", "intentionally", "maintaining", "inhibitory", "control", "networks", "active", "task", "decrease", "response", "elicitation", "threshold", "would", "resulted", "general", "decrease", "response", "speed", "Go", "stimuli", "paradigm", "emphasis", "put", "response", "speed", "slow", "Hits", "correctly", "responded", "Go", "stimuli", "RTt", "determined", "calibration", "blocks", "considered", "error", "reported", "explicitly", "visual", "feedback", "provided", "trial", "Although", "probability", "NoGo", "stimuli", "occurrence", "response", "prepotency", "maintained", "high", "recurrent", "feedback", "responses", "speed", "threshold", "considering", "response", "Go", "trials", "slow", "indeed", "individually", "adjusted", "mean", "response", "speed", "calculated", "calibration", "blocks", "intervening", "experimental", "blocks", "see", "Method", "section", "Hence", "interpret", "results", "indicating", "brain", "network", "already", "spontaneously", "activated", "NoGo", "stimulus", "presented", "inhibition", "performance", "improved", "Compatible", "hypothesis", "analyses", "distribution", "FA", "occurrence", "across", "experimental", "session", "showed", "participants", "committed", "FA", "following", "pattern", "FA", "randomly", "distributed", "within", "sequence", "adoption", "strategy", "participant", "would", "likely", "resulted", "sequences", "consisting", "series", "FA", "CR", "specific", "task", "set", "could", "unlikely", "maintained", "constantly", "whole", "experiment", "Palva", "Palva", "results", "disentangle", "relationship", "proactive", "inhibition", "occurrence", "specific", "microstates", "knowledge", "study", "far", "investigated", "whether", "strategic", "implementation", "task", "sets", "impact", "frequency", "microstate", "thus", "whether", "proactive", "mechanism", "could", "influenced", "pattern", "result", "studies", "focusing", "longer", "epochs", "manipulating", "engagement", "proactive", "inhibitory", "mechanisms", "necessary", "elucidate", "question", "note", "found", "specific", "EEG", "topography", "preceding", "successful", "trials", "specific", "topography", "preceding", "errors", "pattern", "result", "suggests", "engagement", "specific", "cognitive", "set", "may", "enhance", "motor", "responses", "inhibition", "conditions", "high", "response", "prepotency", "many", "different", "deviations", "optimal", "could", "result", "inhibition", "failure", "turn", "explaining", "identify", "specific", "topographic", "map", "preceding", "errors", "regard", "limitation", "current", "study", "defining", "FA", "CR", "conditions", "based", "RT", "lack", "thereof", "possibly", "included", "CR", "condition", "trials", "erroneous", "motor", "activation", "actually", "engaged", "inhibited", "reaching", "activation", "threshold", "Referred", "covert", "errors", "trials", "detected", "recording", "electromyography", "activations", "suggested", "depend", "distinct", "mechanisms", "overt", "errors", "Burle", "et", "Allain", "et", "Boulinguez", "et", "case", "inclusion", "covert", "error", "trials", "CR", "condition", "would", "increased", "noise", "data", "thus", "likely", "increased", "probability", "type", "type", "errors", "Second", "FAs", "quite", "rare", "data", "single", "trial", "topographic", "analyses", "included", "limited", "number", "trials", "capacity", "deal", "small", "numbers", "EEG", "epochs", "advantage", "approach", "reach", "reliable", "statistical", "results", "number", "epochs", 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"Foundation", "Lucas", "Spierer", "Cartool", "software", "programmed", "Denis", "Brunet", "Functional", "Brain", "Mapping", "Laboratory", "Geneva", "Switzerland", "supported", "Center", "Biomedical", "Imaging", "Geneva", "Lausanne", "thank", "David", "Magezi", "David", "Souto", "reviewers", "valuable", "comments", "Edited", "John", "Foxe", "Albert", "Einstein", "College", "Medicine", "USA", "Reviewed", "Redmond", "Trinity", "College", "Dublin", "Ireland", "Kevin", "Whittingstall", "Université", "de", "Sherbrooke", "Canada" ]

Introduction {#section1-2324709613492503} ============ "Pseudo--Ludwig's angina" is a descriptor that has been used for sublingual swelling due to noninfectious causes, as opposed to the true condition, Ludwig's angina, which is a manifestation of regional suppuration in the neck. Multiple case reports have described sublingual hematoma secondary to hemorrhage in patients anticoagulated with warfarin.^[@bibr1-2324709613492503][@bibr2-2324709613492503][@bibr3-2324709613492503][@bibr4-2324709613492503][@bibr5-2324709613492503][@bibr6-2324709613492503][@bibr7-2324709613492503][@bibr8-2324709613492503]-[@bibr9-2324709613492503]^ Sublingual hematoma with airway compromise secondary to brodifacoum, a common long-acting anticoagulant rodenticide, has only been reported in the veterinary literature.^[@bibr10-2324709613492503]^ A large sublingual hematoma can mimic Ludwig's angina; either etiology can compromise the airway. Initial treatment includes either reversal of the bleeding diathesis for the former or appropriate antibiotic coverage for the latter. Angioedema is yet another potential cause of upper airway obstruction due to swelling, but this etiology is not typically invoked as underlying "pseudo--Ludwig's angina." We report a case of massive tongue swelling and impending airway compromise in the context of an intentional ingestion of the long-acting anticoagulant brodifacoum. This presentation was initially suspected to be due to local hemorrhage, consistent with coagulopathy and the previously reported pattern of "pseudo-Ludwig's angina" associated with warfarin misadventure. Ultimately, however, imaging excluded the presence of hematoma and the clinical course was more consistent with angioedema, temporally implicated in response to the brodifacoum ingestion. Case Report {#section2-2324709613492503} =========== A 32-year-old African American male-to-female transgender patient presented to the emergency department, complaining of inability to swallow and difficulty speaking for the previous 2 days. She denied odynophagia, fever, trauma, seizure, or significant respiratory symptoms. Past medical history included depression, chronic pain, and a remote suicide attempt. On physical exam, she was afebrile and had a normal respiratory rate and oxygenation by pulse oximetry. The oral examination was remarkable for a massively swollen tongue, elevation of the sublingual tissue, and fullness of the anterior neck. She could not vocalize. The remainder of the physical examination was unremarkable. The patient was given empiric intravenous clindamycin to cover for possible infection and dexamethasone to reduce swelling. Anesthesia and otolaryngology services were consulted for emergent airway management and the patient was taken directly to the operating room for fiber-optic nasal intubation. While in the operating room, prior to intubation, she communicated by writing that she had swallowed a rat poison 4 weeks prior to presentation in an intentional suicide attempt. Her initial international normalization rate (INR), which returned a short time later, was greater than 13.7. During intubation, she was noted to have tongue, nasopharyngeal, oropharyngeal, left arytenoid, and epiglottic swelling. After intubation, she was admitted to the intensive care unit for management of her airway. To treat her coagulopathy, she was administered high-dose vitamin K and fresh frozen plasma. Dexamethasone and clindamycin were continued. A computed tomography scan of the head was obtained on hospital day 2. This showed diffuse tongue, floor of mouth, submental, and submandibular space edema but no abnormal increased density that would be expected with a discrete hematoma or diffuse interstitial bleeding ([Figure 1](#fig1-2324709613492503){ref-type="fig"}). Her airway swelling improved slowly and she was successfully extubated on hospital day seven. The patient was able to converse with minimal difficulty, being able to describe the brand packaging and price of the rat poison she ingested, which confirmed that it was a brodifacoum-containing product. She denied any co-ingestion or that she was taking any other daily medications at that time. The patient's coagulopathy initially required vitamin K doses of 100 mg every 6 hours per feeding tube as well as intermittent fresh frozen plasma to maintain an INR less than 3. On hospital day 13, she was transferred out of the intensive care unit. Her corticosteroid therapy was tapered without recurrence of swelling. She was ultimately transitioned to vitamin K by mouth, which was safely reduced to a total daily dose of 25 mg daily. A psychiatric consultation determined that she was no longer a danger to herself and she was discharged on hospital day 17 with the plan to further taper her vitamin K by 2.5 mg every week and monitor weekly INR values. A brodifacoum level was not quantified. ![Axial contrast-enhanced computed tomography scan through the oropharynx demonstrates diffuse edema throughout the swollen tongue (\*), which fills the entire oral cavity resulting in severe narrowing of the pharyngeal airway with patency maintained by endotracheal tube and nasogastric tube (arrow)](10.1177_2324709613492503-fig1){#fig1-2324709613492503} Discussion {#section3-2324709613492503} ========== This is the first case report of airway obstruction associated with over-anticoagulation yet without hematoma. Hematoma leading to such obstruction, referred to as "Pseudo--Ludwig's angina," is a well-established warfarin-associated phenomenon (including a case report from our own institution).^[@bibr1-2324709613492503][@bibr2-2324709613492503][@bibr3-2324709613492503][@bibr4-2324709613492503][@bibr5-2324709613492503][@bibr6-2324709613492503][@bibr7-2324709613492503][@bibr8-2324709613492503]-[@bibr9-2324709613492503]^ It has only been documented previously in association with brodifacoum in the veterinary literature.^[@bibr10-2324709613492503]^ The absence of hematoma, coupled with the clinical course, leads us to conclude that the patient's presentation is best explained by angioedema and that this angioedema can be attributed to brodifacoum. Although angioedema is a well-recognized complication of many pharmaceuticals, it has neither been reported with anticoagulants nor is it listed in the package insert for Coumadin.^[@bibr11-2324709613492503]^ Nonetheless, this potential complication of long-acting anticoagulants, even if unusual, is important because such ingestions are common. In 2010, there were 10 488 human exposures to similar long-acting anticoagulant rodenticides reported to US Poison Control centers, of which 2774 were treated in health care facilities.^[@bibr12-2324709613492503]^ Similar to warfarin, brodifacoum inhibits the hepatic synthesis of vitamin K--dependent clotting factors. The addition of the brominated polycyclic hydrocarbon side chain to the basic warfarin structure has several properties that serve to promote anticoagulation: It allows brodifacoum to have a higher affinity for hepatic tissue, it interrupts the vitamin K(1)-epoxide cycle at more than one point, it has greater lipid solubility, and it manifests enterohepatic recirculation.^[@bibr13-2324709613492503]^ Several of these attributes lead to a markedly longer half-life for brodifacoum as compared with warfarin.^[@bibr14-2324709613492503]^ Management of hemorrhage secondary to overdose has been discussed in several studies.^[@bibr15-2324709613492503][@bibr16-2324709613492503][@bibr17-2324709613492503][@bibr18-2324709613492503][@bibr19-2324709613492503][@bibr20-2324709613492503]-[@bibr21-2324709613492503]^ Bromine-containing drugs as a class have certain unusual features. Exposure to bromine and brominated products has been reported to cause a plaque forming dermatitis, termed "bromoderma." The exact mechanism is unknown, but it is postulated to be either an inflammatory or delayed hypersensitivity reaction.^[@bibr22-2324709613492503],[@bibr23-2324709613492503]^ This, however, is not a classic angioedema pattern. Nonetheless, it does underscore that bromine is associated with delayed responses and could signal a different risk profile than that of warfarin. Warfarin is not bromine containing and has been long and widely used without reported angioedema. Indeed, warfarin has been suggested as a potential treatment to prevent angioedema.^[@bibr24-2324709613492503][@bibr25-2324709613492503][@bibr26-2324709613492503]-[@bibr27-2324709613492503]^ Based on our review of the literature, we are unable to establish a clear mechanism of action for angioedema caused by brodifacoum. Angioedema is a well-recognized risk of angiotensin-converting enzyme (ACE) inhibitors.^[@bibr28-2324709613492503]^ The chemical structure of brodifacoum, however, does not appear to be similar to that class of medications and there is no evidence to suggest that it functionally interacts with ACE receptors^[@bibr29-2324709613492503]^ and it is not otherwise known to inhibit the metabolism of ACE. We did not rechallenge the patient to brodifacoum or another bromine-containing substance to support a causal association, nor can we absolutely exclude that another unreported exposure may have been involved or perhaps interacted with the brodifacoum. Conclusion {#section4-2324709613492503} ========== Airway compromise secondary to over anticoagulation, although rare, previously has been well documented from local hematoma, rather than a result of the angioedema we report here. In addition to hemorrhage, airway angioedema should remain in the differential for the etiology of airway compromise, even when coagulopathy from an anticoagulant is present. **Declaration of Conflicting Interests:** The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. **Funding:** The author(s) received no financial support for the research, authorship, and/or publication of this article.

{ "pile_set_name": "PubMed Central" }

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22,217

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"care", "unit", "corticosteroid", "therapy", "tapered", "without", "recurrence", "swelling", "ultimately", "transitioned", "vitamin", "K", "mouth", "safely", "reduced", "total", "daily", "dose", "mg", "daily", "psychiatric", "consultation", "determined", "longer", "danger", "discharged", "hospital", "day", "plan", "taper", "vitamin", "K", "mg", "every", "week", "monitor", "weekly", "INR", "values", "brodifacoum", "level", "quantified", "Axial", "computed", "tomography", "scan", "oropharynx", "demonstrates", "diffuse", "edema", "throughout", "swollen", "tongue", "fills", "entire", "oral", "cavity", "resulting", "severe", "narrowing", "pharyngeal", "airway", "patency", "maintained", "endotracheal", "tube", "nasogastric", "tube", "arrow", "Discussion", "first", "case", "report", "airway", "obstruction", "associated", "yet", "without", "hematoma", "Hematoma", "leading", "obstruction", "referred", "Pseudo", "Ludwig", "angina", "phenomenon", "including", "case", "report", "institution", 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Introduction {#s1} ============ Thyrotropin-releasing hormone (TRH) was originally isolated as the first hypothalamic hormone [@pone.0040437-Boler1], [@pone.0040437-Burgus1] and a major stimulator of the secretion of thyrotropin (TSH) from the anterior pituitary gland [@pone.0040437-Jackson1]. Subsequently, TRH was also found to promote production of TSH in part by stimulating transcription of the TSHβ and α genes. TRH binds to its receptor in the anterior pituitary and activates phospholipase C, leading to calcium mobilization and protein kinase C activation [@pone.0040437-Carr1]--[@pone.0040437-Kiley1] and also stimulation of the MAPK pathway [@pone.0040437-Kanda1], [@pone.0040437-Ohmichi1]. The actions of these intracellular signaling pathways ultimately lead to an increase in transcription of the TSHβ and α genes [@pone.0040437-Shupnik1], [@pone.0040437-Sun1]. However, precisely how TRH mediates transcription of the TSHβ gene *in vivo* still remains unclear. A pituitary-specific transcription factor, Pit1, was first postulated as a candidate protein that influences TRH-induced stimulation of the TSHβ gene. Pit1 which contains two transactivation domains termed the POU-specific domain and POU homeo domain is expressed in somatotrophs, lactotrophs and thyrotrophs, and is critical for the development of pituitary thyrotrophs [@pone.0040437-Andersen1]. In fact, a patient with a mutation of the Pit1 gene exhibited TSH, PRL- and GH- deficiency [@pone.0040437-Tatsumi1], [@pone.0040437-Cohen1]. Pit1 has also been reported to be important for regulation of the TSHβ gene by TRH [@pone.0040437-Andersen1]. TRH-dependent phosphorylation of Pit1 has been suggested to increase Pit1-binding to low-affinity TSHβ promoter-binding sites, and overexpression of a mutant Pit1 containing the DNA-binding domain but lacking the major transactivation domain substantially blocked the TRH-induction of the TSHβ promoter activity in GH3 cells [@pone.0040437-Steinfelder1]. Therefore, TRH may exert its function by changing the state of the Pit1 protein. The second candidate for a protein involved with TRH-induced stimulation of the TSHβ gene is GATA2 [@pone.0040437-Gordon1]. GATA2 belongs to a subtype of transcription factors, the GATA family, that binds through its Zn finger domain with the GATA-responsive element (GATA-RE), which has high homology among all GATA family members [@pone.0040437-Ko1]. GATA2 is expressed in thyrotrophs and gonadotrophs in the pituitary [@pone.0040437-Dasen1]. It has been reported that TRH enhanced GATA2- dependent activation of the TSHβ promoter and that this stimulation was abolished by an amino-acid substitution of the GATA2-Zn finger domain or a mutation of the GATA-responsive element of the TSHβ gene. In addition, an recent EMSA study by Oba et al revealed that TRH increased the DNA-binding capacity of GATA2 on the gene [@pone.0040437-Ohba1]. We generated TRH-deficient mice using homologous recombination in embryonic stem cells [@pone.0040437-Yamada1]. These mice show characteristic phenotypes, including tertiary hypothyroidism and mild hyperglycemia. The basal serum TSH level was unexpectedly elevated, and the result of the TRH test suggested that the secreted TSH had reduced biological activity. An ontogeny based analysis of these mice demonstrated that there was no requirement for TRH in the development of embryonic thyrotrophs in the pituitary, but TRH was required for the maintenance of the normal function of pituitary thyrotrophs [@pone.0040437-Shibusawa1]. NR4A1 (also known as Nur77, NGFI-B or TR3) belongs to a superfamily of orphan nuclear receptors and was originally isolated as an immediate-early response gene induced by a nerve growth factor in a pheochromocytoma cell line, PC12 [@pone.0040437-Milbrandt1]. NR4A1 is also regulated by many physiological stimuli including growth factors, inflammatory signals and hormones, and implicated in a wide range of important biological processes including apoptosis, brain development, glucose metabolism, and vascular remodeling [@pone.0040437-MartnezGonzlez1]--[@pone.0040437-Liu1]. Expression of NR4A1 has also been identified in several endocrine organs including the anterior pituitary, ovary, adrenal gland, and testis [@pone.0040437-Stocco1]--[@pone.0040437-Smith1]. Several investigators have demonstrated that NR4A1 plays an important role in the function of the hypothalamo-pituitary-adrenal (HPA) axis and hypothalamo-pituitary-gonadal (HPG) axis [@pone.0040437-Philips1], [@pone.0040437-Philips2], [@pone.0040437-Inaoka1], [@pone.0040437-Fernandez1]. However, there is no report regarding the involvement of NR4A1 in the hypothalamo-pituitary-thyroid axis, since there has been no appropriate animal model to use. Pit1 is necessary but not sufficient for basal transcription of the TSHβ gene, in fact, the basal activity of the TSHβ promoter was minimally stimulated when co-transfected with a mouse Pit1 in the thyrotroph cell lines αTSH and TtT-97 [@pone.0040437-Gordon2]. In addition, several studies indicated that TRH-induced activation of the TSHß gene requires both PKC and MAPK pathways [@pone.0040437-Carr1]--[@pone.0040437-Ohmichi1], [@pone.0040437-Sun1]. TRH-induced stimulation of GATA2-activation of the TSHβ promoter depends on PKC but not MAPK in GH3 cells [@pone.0040437-Ohba1]. Therefore, there may be additional factors involved with TRH-induced stimulation of the TSHβ gene *in vivo.* In the present study, to elucidate the mechanisms underlying TRH-induced stimulation of the TSHβ gene *in vivo*, we first examined the role of TRH in the secretion and synthesis of TSH by measuring serum TSH and pituitary TSH mRNA levels in different thyroid status in TRH knockout mice. We then attempted to determine factors related to action of TRH on the TSHβ gene through expression profiling of pituitary mRNAs using wild-type and TRH-deficient pituitary supplemented with thyroid hormone. We discovered that an orphan nuclear receptor, NR4A1, played a critical role in TRH-mediated stimulation of the pituitary TSHβ gene *in vivo*. Results {#s2} ======= TRH Regulates the Basal Activity and Responsiveness to Thyroid Hormone of the TSHβ Gene In vivo {#s2a} ----------------------------------------------------------------------------------------------- As previously reported, the serum TSH levels in homozygotes of TRH knockout mice (TRHKO) (143.9±7.9 ng/ml, n = 7) were slightly but significantly higher that those in the wild-type (71.3±4.1 ng/ml, n = 7, *p\<*0.01) and heterozygous mice (81.2±2.5, n = 6, *p\<*0.01). Previous studies suggested that the elevated serum TSH levels in TRHKO reduced the biological activity [@pone.0040437-Shibusawa1], [@pone.0040437-Nikrodhanond1]. In order to examine the feedback system of the hypothalamo-pituitary-thyroid axis in TRHKO, we first measured serum TSH levels in euthyroid TRHKO given daily injections of T4. The decreased serum thyroid hormone level normalized (1.92±0.05 ng/ml, n = 6) following the daily subcutaneous injection of thyroid hormone (T4, 1.5 μg/100 g body weight) for 14 days. In these euthyroid TRHKO, the elevated serum TSH levels reverted to normal (75.8±3.7 ng/ml, n = 6). These results suggested the serum TSH level to be normally regulated by thyroid hormone in the absence of TRH ([Fig. 1A](#pone-0040437-g001){ref-type="fig"}). To determine the mechanism of the normalization of the serum TSH level in euthyroid TRHKO, we next measured TSHβ and α mRNA levels in the pituitary by real-time PCR with TaqMan probes. Although serum TSH levels in the intact TRHKO were significantly elevated, pituitary TSHβ and α mRNA levels were paradoxically decreased to 47.4±16.2% and 71.8±5.0% of those in the control wild-type mice, respectively. In contrast to the change of serum TSH level in the euthyroid TRHKO, both TSHβ and α mRNA levels were further reduced to 10.1±1.1% and 30.1±3.0% of the control values ([Fig.1B and C](#pone-0040437-g001){ref-type="fig"}). Furthermore, a predominant reduction was observed in the TSHβ mRNA level as compared to the TSHα mRNA level. These results suggested that the normalization of the serum TSH level in TRHKO by thyroid hormone may be, at least in part, due to a decrease in the synthesis of TSH in the pituitary. Since there were no reports of thyroidectomy in mice, we first established a technique using dissection microscopy [@pone.0040437-Mori1]. The thyroidectomy for mice was basically the same as that for rats, except for the use of microscopy. The success rate of this procedure was approximately 70%, and the rest of the mice died due to respiratory failure with damage to the phrenic nerve. In the wild-type mice, thyroidectomy caused a significant decrease in the serum thyroid hormone levels from 1.14±0.55 ng/ml (n = 6) to 0.50±0.11 ng/ml (n = 6) within two weeks. Although the serum T4 level in intact TRHKO was approximately 60% of that in the wild-type mice (0.7±0.05 ng/ml, n = 7), thyroidectomy further decreased these levels to 0.28±0.02 ng/ml (n = 5)([Fig. 1D](#pone-0040437-g001){ref-type="fig"}). Reflecting the hypothyroid status, the serum TSH level of the wild-type mice (64.2±4.4 ng/ml) was significantly increased approximately 25-fold of the control (2349.5±255.5 ng/ml) after two weeks. In contrast, although the serum TSH level was higher in the intact TRHKO than wild-type mice, the increase was insufficient (611±240 ng/ml, n = 6) compared to that in the wild-type control ([Fig 1E](#pone-0040437-g001){ref-type="fig"}). ![The hypothalamic-pituitary-thyroid axis in TRH deficient mice.\ A) Serum TSH levels were measured in wild-type mice (Wt), TRH knockout mice (TRHKO) heterozygotes (Hetero) and homozygotes (TRH−/−) and TRHKO supplemented with thyroid hormone (TRH−/− + T4). TRHKO (n = 7) were supplemented with thyroid hormone by the daily injection of T4 (1.5 μg/100 g body weight) for 14 days to be euthyroid (n = 6). The serum TSH level in TRH−/−+T4 mice was similar to that in the wild-type (n = 7) and heterozygous mice (n = 6). Values are represented as the mean ± SEM. \*\*, *p*\<0.01; \*, N.S., not significant. B) TSHβ mRNA levels were measured in pituitaries of the wild-type mice, TRHKO, and TRHKO supplemented with thyroid hormone (TRH−/−+T4). The TSHβ mRNA level in TRH−/− was decreased to about 50% of that in the wild-type, and that of TRH−/−+T4 mice was further decreased to about 10% (n = 3, *p\<*0.01). \*\*, *p*\<0.01. C) TSHα mRNA levels showed a similar profile to the TSHβ mRNA level shown in [Fig 1B](#pone-0040437-g001){ref-type="fig"}. However, the change in TSHβ mRNA was more profound than that in TSHα mRNA. \*\*, *p*\<0.01. D) Two weeks after thyroidectomy, serum free T4 levels were significantly reduced in both the wild-type mice (n = 6) and TRHKO mice (TRH−/−) (n = 5). The serum T4 levels were significantly lower in TRH−/− mice than wild-type mice. \*\*, *p*\<0.01. E) Reflecting hypothyroidism induced by thyroidectomy (TX), the serum TSH level in the wild-type mice showed an approximately 25-fold increase (Wt+TX, n = 6), but TRHKO mice (TRH−/−) showed only about a 6-fold increase (TRH−/−+TX, n = 6). \*\*, *p*\<0.01. F) The TSHβ mRNA levels in thyroidectomized wild-type mice (Wt+TX) were significantly increased after two weeks to approximately 6-fold the control value (Wt) (n = 3). However, the TRHKO mice (TRH−/−) showed a lesser increase (TRH−/−+TX)(n = 3). \*\*, *p*\<0.01; G) A similar profile was observed in TSHα mRNA levels in the pituitary. But a milder effect was observed compared to those of TSHβ mRNA levels. \*\*, *p*\<0.01; n = 3. H) Correlation between serum thyroid hormone levels and the corresponding pituitary TSHβ mRNA levels. Circles represent values for the wild-types and the squares, those for TRH knockout mice (TRH−/−). When comparing the slopes of the curve, it was demonstrated that TRH altered the basal activity and responsiveness of the TSHβ gene to thyroid hormone. The arrow indicates the effect of TRH, taking approximately 80% responsibility for the value of the TSHβ mRNA level in the wild-type pituitary. I) Correlation between serum thyroid hormone levels and the corresponding serum TSH levels. In mild hypothyroidism, the serum TSH levels in TRHKO mice (TRH−/−) were similar to those in the wild-type mice. However a lack of TRH induced a marked impairment of the serum TSH level in severe hypothyroid status.](pone.0040437.g001){#pone-0040437-g001} To evaluate the mechanism of the insufficient increase in the serum TSH levels in TRHKO in response to hypothyroidism induced by thyroidectomy, we measured TSHβ and α mRNA levels in the TRHKO pituitary and compared them to those in the wild-type. As expected thyroidectomy caused a significant increase in the TSHβ mRNA level, to approximately 6-fold (584.9±13.4%) that in the wild-type pituitary. Similarly, the TSHα mRNA level was increased to 199.0±2.4%. However, the increase of the TSHβ mRNA level in TRHKO was only approximately 3 fold (296±7.8%), and similarly, the TSHα mRNA level was increased to 105±1.3% of the control ([Fig 1F, and G](#pone-0040437-g001){ref-type="fig"}). The experimental hypothyroidism induced by MMI treatment with regular chow that contained iodine in a previous study induced a milder hypothyroidism than that due to thyroidectomy [@pone.0040437-Shibusawa1]. The serum thyroid hormone level in the MMI-induced hypothyroid wild-type mice was similar to that in the simple TRHKO. The treatment with MMI increased the TSHβ mRNA level to 300.0±15.5%. To analyze the correlation between the serum thyroid hormone levels and the corresponding TSHβ mRNA levels and serum TSH levels, all the data was plotted in. As shown in [Fig 1H](#pone-0040437-g001){ref-type="fig"}, it is clear that each pituitary TSHβ mRNA level corresponding to the serum thyroid hormone level in the TRHKO pituitary was significantly lower than those of the wild-type controls. Furthermore, when compared to the slope of the correlation curve, the slope for the TRHKO was almost parallel to that in the wild-type mice, suggesting that TRH affected the basal activity and the responsiveness of the TSH gene to thyroid hormone. When the TSHβ mRNA level at the middle of this curve was calculated, approximately 80% (83,7% at serum T4, 0.7 ng/ml) depended on the presence of TRH. It is important to note that although the serum TSH levels in TRHKO with mild hypothyroidism were similar to those in the wild-type mice, the lack of TRH impaired the raising of serum TSH levels, particularly in mice with severe hypothyroidism, as shown in [Fig 1I](#pone-0040437-g001){ref-type="fig"}. cDNA Microarray Analysis of the TRH-deficient Pituitary with and without Thyroid Hormone Replacement {#s2b} ---------------------------------------------------------------------------------------------------- In order to identify the factors involved with regulation of the pituitary TSH gene by TRH, we examined the gene expression in TRH-deficient pituitary with or without thyroid hormone replacement. Total RNAs were extracted from the pituitary of wild-type (WT signal), TRHKO (Homo Signal), and euthyroid TRHKO with thyroid hormone replacement (T4 signal) (TRHKO +T4∶1.5 μg T4/100 g body weight, subcutaneously for 14 days) and subjected to a cDNA microarray analysis using the Affymetrix Mouse Gene Chip 320 2.0 Array. After exclusion of absent signals (less than 100 signals), 10445 probes were expressed out of 45102 probes and subjected to a cluster analysis using a K-means algorithm. As shown in [Fig 2A](#pone-0040437-g002){ref-type="fig"}, it was classified into ten clusters according to profiles of changes among three groups and the expression level of the gene. Most genes belonging to clusters other than cluster 5 showed an increase in expression with thyroid hormone replacement. In contrast, cluster 5 consists of genes showing a significant decrease with both a deficiency of TRH and thyroid hormone replacement. It is important to note that among 10445 probes, the sole gene in the entire genome showing the largest and most consistent decrease in the absence of TRH and by supplement with thyroid hormone was the TSHβ gene showing 63% expression of the wild-type pituitary in the absence of TRH, and a further decrease to 6% of the wild-type ([Fig 2B, C, and D](#pone-0040437-g002){ref-type="fig"}). These findings demonstrated that TRH is a highly specific regulator of the TSHβ gene in the entire genome. Indeed, genes for other anterior-pituitary hormones such as POMC, FSH, LH, GH, and prolactin, and genes reported to regulate expression of the TSHβ gene including Pit1 and GATA2 were found to show no significant change ([Fig. 2C and D](#pone-0040437-g002){ref-type="fig"}). ![Microarray and K-means cluster analyses of genes regulated by TRH and by thyroid hormone in TRH-deficient pituitary.\ RNA samples from pituitaries of wild-type, TRHKO (TRH−/−), and euthyroid TRHKO mice with thryroid hormone replacement (TRH−/−+T4) were subjected to a whole-genome microarray using Affymetrix Mouse Gene chip 320 2.0 Array. After exclusion of absent signals (less than 100 signals), 10445 out of 45102 genes expressed in the TRHKO pituitary were subjected to the cluster analysis using a K-means algorithm. Hierarchical cluster analysis was carried out using a computer program, Genowiz™ 3.2. A) Genes were divided into ten hierarchical clusters according to changes and profiles among three groups, wild-type (WT signal), TRH−/− (Homo signal), and TRH−/− + T4 (T4 signal). B) In addition to the TSHβ genes, cluster 5 contained the Egr-1, NR4A1, Glt2, and Tmsb10 genes. C) mRNA levels of other anterior pituitary hormones including POMC, GH, LHβ, FSHβ, and prolactin (PRL) were not altered among the three groups, suggesting the expression to be TRH-independent. D) Profiles of the NR4A1, NR4A2, Egr-1, Pit1 and GATA2 genes are depicted. Expression of NR4A1, NR4A2 and Egr-1 might be TRH-dependent. E) Confirmation of results of the microarray analysis of NR4A1 mRNA levels in the TRH-deficient pituitary by real-time PCR. TRHKO mice (TRH−/−, n = 3) were supplemented with thyroid hormone by the daily injection of T4 (TRH−/−+T4, n = 3) and with TRH by 1 mg/kg body weight/day with an Alzet pomp for 7 days (TRH−/−+TRH n = 3). Total RNA obtained from these pituitaries was subjected to real-time PCR. The NR4A1 mRNA level in the TRHKO pituitary was decreased to about 25% of that of the wild-type. The decreased NR4A1mRNA level was not altered by thyroid hormone replacement, but completely reversed to the normal level by TRH administration, suggesting expression of NR4A1 mRNA in the pituitary to be TRH-, but not thyroid hormone-dependent.](pone.0040437.g002){#pone-0040437-g002} Other genes classified into cluster 5 were the NR4A1, Egr-1, Gtl2 and Tmsb10 genes as shown in [Fig. 2B](#pone-0040437-g002){ref-type="fig"}. The Gtl2 gene is an imprinted gene which is maternally expressed and appears to function as an RNA molecule; multiple splice variants are observed in the available sequence data, and a pituitary transcript variant has been reported to inhibit cell proliferation [@pone.0040437-Zhao1]. The tymosin-beta 10 gene (Tmsb10) is related to cell growth through proliferation, and its expression is associated with brain development in rats and humans. In addition, previous reports indicated enhanced Tmsb10 expression in a variety of human tumors such as renal and thyroid medullary carcinomas and melanomas [@pone.0040437-Santelli1], [@pone.0040437-Takano1]. Although these two genes were found in the same cluster, cluster 5 to which the TSHβ gene belonged, the signals slightly increased in the absence of TRH, suggesting that these two genes were expressed in a TRH-independent manner. The other two genes, NR4A1 and Egr-1, showed a profile of change similar to that of the TSHβ gene. The NR4A1 mRNA signal in the TRH-deficient pituitary decreased to 70% of that of wild-type mice, and further decreased to 52% of the wild-type level in euthyroid TRH-deficient pituitary (TRHKO+T4). Similarly, the Egr-1 signal in the TRH-deficient pituitary was decreased to 39% of the wild-type value, and reduced to 40% of the wild-type value in the euthyroid TRH-deficient pituitary (TRHKO +T4). Therefore, the reduction of these two genes in TRHKO mice was not recovered by thyroid hormone replacement, indicating that the expression of these two genes was TRH-dependent and probably up-regulated by TRH. The NR4A1 and Egr-1 genes are known as immediate-early response genes. Egr1- knockout mice have been reported to have LHβ deficiency [@pone.0040437-Lee1] and impaired liver regeneration [@pone.0040437-Liao1] but normal thyroid function and intact thyrotrophs in the pituitary, therefore we excluded Egr-1 from the study [@pone.0040437-Topilko1]. In contrast, NR4A1 is known to be an important signal for both the hypothalamo-pituitary-adrenal axis, particularly the pituitary POMC gene and the adrenal Cyp11β2 and HSD3β genes, and the hypothalamo-pituitary-gonadal axis including the 20α--HSD gene in the ovary and the Cyp17 and HSD3β genes in the testis [@pone.0040437-Stocco1]--[@pone.0040437-Smith1], however, its role in the hypothalamo-pituitary-thyroid axis (HPT axis) remains unclear. We therefore examined the importance of NR4A1 in the HPA axis in subsequent experiments. Furthermore, the previous analysis of NR4A1 knockout mice demonstrated normal functioning of the adrenal grand, suggesting other homologous factors such as NR4A2 and NR4A3 to compensate for the function of NR4A1 [@pone.0040437-Crawford1]. However, in the present microarray analysis, NR4A2 mRNA expression was not significantly changed and NR4A3 mRNA was not found in the pituitary ([Fig. 2D](#pone-0040437-g002){ref-type="fig"}), suggesting that no such compensation in the TRH-deficient mice. Furthermore, there was no report regarding the H-P-T axis and thyroid function in either NR4A1 knockout mice or NR4A1/NR4A3 double knockout mice [@pone.0040437-Crawford1]--[@pone.0040437-Mullican1]. NR4A1 mRNA Level was Regulated by TRH In vivo {#s2c} --------------------------------------------- To confirm the results of the microarray analysis, we measured NR4A1 mRNA levels in the pituitary gland by real-time PCR with the TaqMan probe of NR4A1 ([Fig 2E](#pone-0040437-g002){ref-type="fig"}). We first confirmed that the NR4A1 mRNA level in the TRH deficient-pituitary was decreased to about 25% of the wild-type level (1.2±0.1 vs. 3.8±0.2, NR4A1 mRNA/GAPDH mRNA, n = 3, *p*\<0.01). When the TRHKO were treated with thyroid hormone to be euthyroid (1.5 μg T4/100 g body weight, injected subcutaneously for 14 days), the level of NR4A1 mRNA was not significantly changed (1.5±0.1, n = 3). However, when TRH was given to TRHKO mice by Alzet pump subcutaneously for 7 days, the NR4A1mRNA level completely reverted to normal (2.8±0.4, n = 3) ([Fig. 2E](#pone-0040437-g002){ref-type="fig"}), suggesting the expression of NR4A1 mRNA to be TRH-dependent but thyroid hormone-independent. Furthermore, the NR4A2 mRNA level did not differ among the wild-type (0.9±0.1, n = 3), TRHKO (1.0±0.4, n = 3), and TRHKO +T4 (0.8±0.1, n = 3), and NR4A3 mRNA was not detected in the pituitary showing the Ct value of the real-time PCR as 29.5±0.9, while those of NR4A1 and NR4A2mRNA were 24.4±1.65, 27.3±1.0, respectively, suggesting again that compensation of the role of NR4A1 by NR4A2 and 3 might not occur in the anterior pituitary. Expression of NR4A1 in the Thyrotrophs and Regulation by TRH In vivo {#s2d} -------------------------------------------------------------------- We next investigated *in vivo* expression and regulation by TRH of NR4A1 in the pituitary thyrotrophs using double-fluorescent immunohistochemistry. As shown in the left panel of [Fig 3A](#pone-0040437-g003){ref-type="fig"}, in the wild-type pituitary, NR4A1 shown with green signals was observed in the nucleus of most cells in the anterior, intermediate and posterior lobes. We found in numbers of cells, the coexistence of NR4A1 with TSHβ immunoreactivity shown by red signals in the cytoplasm of the anterior lobe. In contrast, in TRHKO pituitary shown in the right panel, a significant decrease in the staining of TSHβ was observed. However, careful observation indicated that in the remaining weakly-stained cells, NR4A1 expression was absent as shown by a black hole, suggesting the suppressed expression of NR4A1 in the nucleus of the thyrotrophs to be due to the absence of TRH. ![Expression and regulation of NR4A1 by TRH in pituitary thyrotrophs in vivo.\ The expression of NR4A1 in pituitary thyrotrophs was confirmed by double fluorescent immunohistochemistry. In numbers of cells in the anterior pituitary NR4A1 were expressed and stained as green signals in the nucleus. A) Numbers of cells expressing NR4A1 were also stained with antibody against TSHβ (red signals) in the wild-type pituitary (left panel). As shown in the right panel, the intensity and number of the TSHβ-immunopositive cells were remarkably decreased in the TRH-deficient pituitary, and at X400, most of the NR4A1 expression was reduced or lost in these cells as seen as black dots (indicated by white arrows). B) As observed in [Fig 4B and C](#pone-0040437-g004){ref-type="fig"}, the expression of ACTH (B) and FSH (C) was also observed as red signals in the cytoplasm in the anterior lobe. As expected, these ACTH- and FSH- positive cells expressed NR4A1, but the staining intensity was not altered in the TRH-deficient pituitary (right panel).](pone.0040437.g003){#pone-0040437-g003} ![Stimulation of the promoter activity of the TSHβ gene by NR4A1 in vitro.\ A) The human TSHβ promoter region between bp --1192 and +37 linked to a luciferase reporter gene (pA3TSHβ(−1192∼+37)-Luc) was transfected into CV-1 cells. Expression of Pit1 and GATA2 was necessary for the basal promoter activity of the TSHβ gene. When an expression vector for Pit1 was co-transfected, the promoter activity of the TSHβ was stimulated by 10-fold, but co-transfection with GATA2 did not significantly alter it. However, synergistic stimulation of the promoter activity was observed when Pit1 and GATA2 were simultaneously expressed. B) Under the above conditions expressing Pit1 and GATA2, the TSHβ promoter activity was stimulated by NR4A1 in a dose-dependent manner. When 1 μg of expression vector of NR4A1/3 wells was co-trasfected, the promoter activity was stimulated about 2-fold, and when 3 μg, it reached a plateau at about 6-fold. Relative luciferase activity against the value without expression of NR4A1 is shown. C) Stimulation of the promoter activity by NR4A1 was specific for the TSHβ gene (TSHB). The promoter activities of the thyrotropin-releasing hormone (TRH), TSHα, and prolactin (PRL) genes was not stimulated by overexpression of NR4A1. Relative luciferase activity against the value without expression of NR4A1 in each promoter is shown. Values are represented as the mean ± SEM from at least three experiments. \*\*, *p*\<0.01; N.S., not significant.](pone.0040437.g004){#pone-0040437-g004} We also identified ACTH- and FSH- producing cells using specific antibodies in the anterior pituitary. As shown in [Fig. 3B and C](#pone-0040437-g003){ref-type="fig"}, many ACTH- and FSH- producing cells expressed NR4A1 in the anterior pituitary. However, the expression of NR4A1 in the ACTH- and FSH- producing cells was not changed by the absence of TRH as shown in the right panel of [Fig. 3B and C](#pone-0040437-g003){ref-type="fig"}, suggesting that NR4A1 in the ACTH- or FSH- producing cells was not regulated by TRH *in vivo*. Activation of the TSHβ Promoter by NR4A1 {#s2e} ---------------------------------------- We next examined whether NR4A1 stimulates the activity of the TSHβ promoter *in vitro*. As previously reported by us and others, for the full expression of the TSHβ promoter Pit1 and GATA2 were required in CV-1 cells ([Fig. 4A](#pone-0040437-g004){ref-type="fig"}) [@pone.0040437-Gordon1], [@pone.0040437-Nakajima1]. As shown in [Fig 4B](#pone-0040437-g004){ref-type="fig"}, in the presence of Pit1 and GATA2, overexpression of NR4A1 significantly increased the TSHβ promoter activity in a dose-dependent manner and when 4.0 μg of expression vector per plate was used, the activity of the promoter was stimulated by 6-fold the basal level. The effect of NR4A1 could be observed only on the TSHβ promoter, not the TRH promoter or prolactin promoter, suggesting that the effect was specific for the TSHβ promoter ([Fig. 4C](#pone-0040437-g004){ref-type="fig"}). Furthermore, the activity of the TSHα promoter was also not stimulated by overexpression of NR4A1. It may be due to the small effect of the absence of TRH on the TSHα mRNA level as compared to the TSHβ mRNA level as shown in [Fig. 1C](#pone-0040437-g001){ref-type="fig"}. In order to determine the region responsible for the effect of NR4A1 on the TSHβ promoter, we first searched for an NR4A1 response element (NurRE)(AAAT(G/A)(C/T)CA) in the TSHβ promoter and found a complete consensus sequence of NurRE (5′-aaatatca-3′) in the region between bp -1091 and -1083 [@pone.0040437-Philips2], [@pone.0040437-Maira1]. However, as shown in [Fig 5A](#pone-0040437-g005){ref-type="fig"}, transient transfection analysis showed that the deletion of this region of the TSHβ promoter (-1078 ∼ +37 construct) caused a similar response to NR4A1, indicating that this element did not act as a NurRE in the TSHβ gene. Furthermore, a series of deletion mutants including the construct containing the region between -138 and +37 showed a significant response to NR4A1, suggesting that the region between -138 and +37 included the area responsible for the NR4A1-induced stimulation of the TSHβ gene. It has been reported by several investigators that Pit1 and GATA2 bound to this region of the TSHβ gene, and this region is also responsible for the TRH-induced stimulation ([Fig. 5A](#pone-0040437-g005){ref-type="fig"}) [@pone.0040437-Gordon1], [@pone.0040437-Gordon3], [@pone.0040437-Kashiwabara1]. ![Characterization of NR4A1-induced stimulation of the promoter activity of the TSHβ\ **gene.** A) To determine the region responsible for the NR4A1-induced stimulation of the TSHβ promoter activity, a series of deletion constructs of the human TSHβ promoter were established. The consensus sequence of the NR4A1 response element (NurRE) was identified in the region between -1091 and -1083 from the transcription start site. The right figure shows the fold-stimulation by NR4A in each deletion mutant of the TSHβ gene. Putative Pit1 and GATA2 binding sites, and the negative thyroid hormone response element (NRE) were indicated as pit1, GATA2 and NRE. All the deletion constructs including pA3TSHβ (−138 ∼ +37) -Luc showed similar stimulation by NR4A1, suggesting the region responsible to be within this area. Fold-increase in activity against that without TRH is shown. B) Chromatin-immunoprecipitation (ChIP) assays were performed with anti-NR4A1 antibody (NR4A1) and normal mouse IgG as a negative control (IgG). GH4C1 cells were transfected with pA3TSHβ (-1192 ∼ +37)-Luc in the presence or absence of 100 n M TRH. Amplified PCR products were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX. Chip assays demonstrated that NR4A1 was recruited to the region between -138 and +13 of the TSHβ promoter (-138/+13), but not the region containing NurRE (NuRE). Addition of TRH did not alter recruitment of NR4A1 on the gene (data not shown). All ChIP assays were repeated at least three times. C) The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE and a fragment containing the human TSHβ bp -123∼-87. There was no binding of NR4A1 to the TSHβ gene in the absence (−) or presence (+) of 100 nM TRH, while the POMC promoter fragment bound to NR4A1. Arrows indicate NR4A1 bound to the POMC promoter as monomers and dimers. NR4A1 was synthesized by the TNT-coupled reticulocyte lysate system (NR4A1). Lysate indicates un-programmed lysate. D) Effect of knockdown of NR4A1 on the TRH-induced stimulation of the promoter activity of the TSHβ gene. D-1. The NR4A1 mRNA levels were measured after transfection with siCONTROL or siNR4A1 in GH4C1 cells. Transfection of siNR4A1 led to an approximately 80% reduction of the mRNA level after 48 hr. Data are presented as the mean ± SEM from three experiments. \*\*, *p*\<0.01 D-2. GH4C1 cells were transfected with siNR4A1 or siCONTROL, and then TSHβ (-1192 ∼ +37)-Luc reporter, Pit1, and GATA2 expression vectors were cotransfected. After incubation with 100 nM TRH for 24 hr, the promoter activity was measured. In the vehicle transfected with siCONTROL, incubation with TRH stimulated promoter activity of the TSHβ gene showing 242±33% of that without TRH stimulation. Knockdown of NR4A1 resulted in significant reduction of TRH-induced stimulation (156% of that without TRH stimulation). Data are presented as the mean ± SEM from three experiments. \*, *p*\<0.05 E) Negative regulation of the TSHβ gene by thyroid hormone was examined in the presence or absence of NR4A1 in CV-1 cells. Expression vector for thyroid hormone receptor β 1 (TRβ1) was also co-transfected. The responsiveness of the TSHβ promoter activity to thyroid hormone (T3) without expression of NR4A1 was significantly reduced as compared to that with expression of NR4A1, suggesting NR4A1 increased the basal promoter activity and responsiveness to T3 of the TSHβ promoter. F) Effect of NR4A1 and Egr-1 on the promoter activity of the TSHβ gene. Egr-1 affected neither the basal promoter activity of the TSHβ gene nor the NR4A1-induced stimulation of the gene. The amount of vector/3 wells transfected is indicated below the graph. \*\*, *p*\<0.01; \*, *p*\<0.05; N.S. not significant.](pone.0040437.g005){#pone-0040437-g005} NR4A1 was Recruited to the Region between −138 and +13 {#s2f} ------------------------------------------------------ There was no consensus sequence for NurRE in the region between bp -138 and +37 of the TSHβ gene, and the EMSA study showed no binding of NR4A1 in the region −123∼−87 of the TSHβ gene, which has been reported to bind to Pit1 and GATA2 and be responsible for TRH-induced stimulation, while the POMC promoter fragment bound to NR4A1 as both dimers and monomers ([Fig. 5C](#pone-0040437-g005){ref-type="fig"}). Therefore, we expected indirect recruitment, and examined whether NR4A1 was recruited to this region by a chromatin-immunoprecipitation (ChIP) assay and a specific antibody against NR4A1. The ChIP assay demonstrated that NR4A1 appeared to be recruited both in the presence or absence of TRH, while the region containing a NurRE sequence (between -1091 and -1083) did not recruit NR4A1 ([Fig. 5B](#pone-0040437-g005){ref-type="fig"}). Knockdown of NR4A1 by siRNA Reduced TRH-induced Stimulation of the TSHβ Gene {#s2g} ---------------------------------------------------------------------------- In order to examine the importance of NR4A1 for activation of the TSHβ promoter by TRH, we next knocked-down NR4A1 by siRNA and examined the effect of NR4A1 on the gene. Treatment with siRNA (siNR4A1) induced a reduction of NR4A1 mRNA to 20% of the control siRNA (siControl) in a rat pituitary cell line, GH4C1 cells ([Fig. 5D](#pone-0040437-g005){ref-type="fig"}-1). The level of TSHβ promoter activity induced by TRH was 2.5-fold high in the control cells transfected with siControl, while activation by TRH was significantly suppressed to about 40% of that of siControl when NR4A1 expression was knocked down ([Fig. 5D](#pone-0040437-g005){ref-type="fig"}-2), suggesting the importance of NR4A1 for TRH-induced stimulation of TSHβ promoter activity. Expression of NR4A1 Shifted the Dose-response Curve of the TSHβ Gene Against Thyroid Hormone {#s2h} -------------------------------------------------------------------------------------------- When compared to the curve of dose-response repression of the TSHβ gene by thyroid hormone in the presence or absence of overexpression of NR4A1 in CV-1 cells, the curve was shifted to the right in the presence of NR4A1, indicating that NR4A1 increased the responsiveness of the TSHβ gene to thyroid hormone *in vitro* ([Fig 5E](#pone-0040437-g005){ref-type="fig"}). This phenomenon was similar to that of the *in vivo* experiment comparing TSHβ mRNA levels between the wild-type mice and TRHKO differing thyroid hormone status shown in [Fig. 1D](#pone-0040437-g001){ref-type="fig"}. Furthermore, another typical early response gene, Egr-1, did not alter the promoter activity of the TSHβ gene in the presence or absence of NR4A1 ([Fig 5F](#pone-0040437-g005){ref-type="fig"}). Immediate-early Response of NR4A1 mRNA via TRH through the PKC and MAPK Pathways {#s2i} -------------------------------------------------------------------------------- We next examined how TRH stimulates the expression of endogenous NR4A1mRNA in GH4C1 cells. Incubation with 100 nM TRH induced a marked and rapid increase in NR4A1 mRNA levels with a peak at 60 mins that was 51-fold the basal level, and then a rapid reduction to 19% of the basal level within 120 mins ([Fig 6A](#pone-0040437-g006){ref-type="fig"}). Furthermore, treatment with TRH increased NR4A1 mRNA levels in a dose-dependent manner with a minimum dose of 0.1 nM and a plateau at 10 nM ([Fig. 6B](#pone-0040437-g006){ref-type="fig"}), suggesting the NR4A1 mRNA to act as an immediate-early response gene for TRH-stimulation. Furthermore, Western blot analysis of GH4C1 cells demonstrated that incubation with 100 nM TRH increased NR4A1 expression at the protein level within 2 h with a peak at 2 h, and then a decrease in 4 h ([Fig 6C](#pone-0040437-g006){ref-type="fig"}). In addition, TRH did not alter the pit1 mRNA level, and GATA2 and POMC mRNAs were not detected in GH4C1 cells (data not shown). ![Immediate-early response of NR4A1 mRNA and protein levels to TRH in GH4C1 cells.\ A) Incubation with 1μM TRH led to a remarkable increase of the endogenous NR4A1 mRNA level with a peak within 1 hr (51-fold) and then an immediate reduction in 120 mins in GH4C1 cells. B) The TRH-induced increase of the NR4A1 mRNA level was observed in a dose-dependent manner. The minimum effect was observed with 0.1 nM TRH and then reached a plateau at 10 nM. C) Western blot analysis in GH4C1 cells demonstrated that incubation with 100 nM TRH led to a significant increase of NR4A1 protein (NR4A1) within 2 h, and NR4A1 was then degradated in 4 h, while the level of GAPDH was not altered.](pone.0040437.g006){#pone-0040437-g006} To explore which signal transduction pathway was involved with TRH-induced NR4A1 mRNA expression, we used several specific inhibitors. As shown in [Fig. 7A](#pone-0040437-g007){ref-type="fig"}, treatment with 100 nM TRH significantly stimulated the expression of endogenous NR4A1 mRNA in 60 mins, however, an prior incubation with a inhibitor for PKC (1 nM), staurosporine significantly inhibited this stimulation by 60%, and 10 nM staurosporine showed about 90% inhibition. Similarly, an inhibitor for MAPK, PD98059 (1--1000 nM), showed significant inhibition of TRH-induced stimulation of NR4A1 mRNA (90% of the control)([Fig. 7B](#pone-0040437-g007){ref-type="fig"}). In contrast, an inhibitor for Ca2+ channels, nimodipine, or for PKA, KT5720, did not affect it ([Fig. 7C and D](#pone-0040437-g007){ref-type="fig"}). These findings suggested that TRH-induced NR4A1 mRNA expression was mediated through both PKC and MAPK pathways. ![Regulation of NR4A1 mRNA by TRH through the PKC and MAPK pathways.\ The signal transduction pathways for the TRH-induced stimulation of NR4A1 mRNA expression were determined using several inhibitors in GH4C1 cells. After overnight incubation in DMEM without serum, GH4C1 cells were treated for 1 hr with a PKC inhibitor, Staurosporine (A), a MAPK inhibitor, PD98059 (B), a Ca channel inhibitor, nimozipine (C), and a PKA inhibitor, KT5720 (D). Significant inhibition of 100 nM TRH-induced stimulation of endogenous NR4A1 mRNA expression was observed with 1.0∼ nM Staurosporine and 1.0 ∼ nM PD98059, but not 0.5 nM nimozipine or 1 μM KT5720. The value relative to that without TRH is shown. Data are presented as the mean ± SEM. from three experiments. \*\*, *p\<*0.01.](pone.0040437.g007){#pone-0040437-g007} Stimulation of the NR4A1 Promoter Activity by TRH through TRH Receptors {#s2j} ----------------------------------------------------------------------- To examine whether TRH stimulates transcription of the NR4A1 gene, we performed a transient transfection assay with a NR4A1 promoter in GH4C1 cells expressing endogenous TRH receptors and CV-1 cells without the expression. In GH4C1 cells, incubation with 100 nM TRH induced a significant stimulation of the NR4A1 promoter activity (257±22% of the control. n = 3, *p\<*0.05). In contrast, no significant stimulation was observed in CV-1 cells ([Fig. 8A](#pone-0040437-g008){ref-type="fig"}). To determine whether TRH-induced NR4A1 mRNA expression is mediated through TRH receptors, we performed a similar experiment in CV-1 cells with and without expression of TRH receptors. As shown in [Fig 8B](#pone-0040437-g008){ref-type="fig"}, incubation with 100 nM TRH significantly stimulated the activity of the NR4A1 promoter with expression of TRH receptors, but, no change was observed without, suggesting the effect of TRH to be mediated through its receptors. Conversely, the POMC promoter activity was significantly stimulated by overexpression of NR4A1 to about 6-fold the basal level in At-T20 cells expressing endogenously POMC gene ([Fig. 8C](#pone-0040437-g008){ref-type="fig"}). However, incubation with TRH did not alter endogenous the POMC mRNA levels in At-T20 cells with and without TRH receptors ([Fig. 8D](#pone-0040437-g008){ref-type="fig"}). In addition, the POMC promoter was not affected by incubation with TRH in the presence or absence of TRH receptors ([Fig. 8E](#pone-0040437-g008){ref-type="fig"}). These findings suggested that the effect of TRH on NR4A1 mRNA and subsequent stimulation of the TSHβ promoter occurred in a receptor- and promoter-specific manner. ![TRH stimulates the promoter activity of the NR4A1 gene through TRH receptors.\ A) A fragment spanning bp −1295 and +152 of the promoter of the human NR4A1 gene fused to a luciferase reporter gene (pA3NR4A1(−1295∼+152)-Luc) was transfected into CV-1 cells (CV-1) that express no endogenous TRH receptors (TRHRs) and GH4C1 cells (GH4C1) expressing endogenous TRHRs. In CV-1 cells, 100 nM TRH did not stimulate the promoter activity of the NR4A1 gene, in contrast, it led to a significant increase in GH4C1 cells (255±33% of the control without TRH, n = 3, *p*\<0.05). B) When TRH receptors were expressed in CV-1 cells (CV-1+TRHR), 10^−7^ M TRH induced a significant increase of NR4A1 promoter activity, suggesting that TRH exhibited stimulation of the NR4A1 gene through TRH receptors. Fold-increase on incubation with 100 nM TRH is represented. The value in GH4C1 cells is also shown as a control. C) The rat POMC promoter containing the region from bp −706 to +64 fused to a luciferase reporter (POMC-Luc) was transfected into At-T20 cells expressing the endogenous POMC gene. The promoter activity was significantly increased about 6-fold by overexpression of NR4A1. D) The level of endogenous POMC mRNA was not altered either by incubation with expression of TR and T3 or by incubation with expression of TRH receptors (TRHRs) and 100 nM TRH in At-T20 cells. E) The POMC promoter activity was not stimulated by incubation with 100 nM TRH even with expression of TRH receptors (TRHRs), suggesting that the effect of TRH occurred in a promoter-specific manner. Data are presented as the mean ± SEM. from three experiments. \*, *p*\<0.05; N.S., not significant.](pone.0040437.g008){#pone-0040437-g008} Discussion {#s3} ========== In the present study, we first demonstrated that TRH altered the basal expression of the TSHβ gene and the responsiveness of the gene to thyroid hormone *in vivo* by using TRHKO differing in thyroid hormone status. When TRH-deficient mice were supplemented with thyroid hormone, the elevated serum TSH level became normal. In contrast, the decreased TSH mRNA levels in the TRH-deficient pituitary were further decreased, and this reduction was found predominantly in the TSHβ subunit. Although thyroidectomy caused severe hypothyroidism in TRH-deficient mice, the increases in serum TSH levels and pituitary TSHβ mRNA levels were insufficient. Analysis of the correlation between the serum thyroid hormone level and the corresponding serum TSH and TSHβ mRNA levels clearly demonstrated that TRH altered the basal activity and responsiveness to thyroid hormone of the gene. In addition, the serum TSH level was more profoundly affected in animals with severe hypothyroidism in the absence of TRH. These findings demonstrate that TRH is a major up-regulator of TSH synthesis, altering the basal activity and the responsiveness to thyroid hormone of the gene, and TRH also affected TSH production by post-transcriptional mechanisms *in vivo*. In hypothyroid status TRH and thyroid hormone receptors (TRs) both may be required for hypothalamic-pituitary-thyroid axis for stimulation of the production and secretion of TSH *in vivo* [@pone.0040437-Chiamolera1]. Unliganded thyroid hormone receptors exert a silencing effect in genes regulated positively by thyroid hormone but, we and others reported that unliganded TRs have a constitutively stimulating effect on the genes regulated negatively by thyroid hormone *in vitro*, such as the TSH gene [@pone.0040437-Satoh1]--[@pone.0040437-Lazar1]. Therefore, one may expect that unliganded TRs function as an up-regulator in hypothyroid animals. However, when animals who possessed no TRs (TRα1−/−β−/−) were rendered to hypothyroidism, their serum TSH and TSHβ mRNA levels in the pituitary were equivalent to those in the wild-type mice, demonstrating that TRs are not required and not involved with the up-regulation of the TSH gene in the hypothyroid status [@pone.0040437-Gthe1]. Furthermore, we have recently reported the predominant action of TRH compared to TRs in the hypothalamic-pituitary-thyroid axis using TRβ/TRH double knockout mice (KO) [@pone.0040437-Nikrodhanond1]. TRβKO mice had an impairment of negative feedback regulation by thyroid hormone, resulting in a significant rise in serum T4 levels and showed resistance to thyroid hormone with elevated serum TSH levels compared with wild-type mice [@pone.0040437-Nikrodhanond1], [@pone.0040437-Shibusawa2]--[@pone.0040437-Forrest1]. Double KO showed a reduced serum thyroid hormone level compared with simple TRβKO and showed only a slight increase in TSH levels compared with wild-type mice. In addition, when TRβKO and double KO were rendered to severe hypothyroid by treatment with PTU, double KO failed to show a significant rise in serum TSH levels as compared to TRβKO. Therefore, TRH is essential for the stimulation of the TSHβ gene under both euthyroid and hypothyroid conditions. How does TRH up-regulate TSHβ gene expression *in vivo*? In the present study, the cDNA microarray and K-means cluster analysis with TRH-deficient pituitary revealed that among whole genes a gene in which most dramatic effect of TRH was observed is the TSHβ gene, and no changes were observed in other pituitary hormones including GH, POMC, PRL, FSH, or LH. In addition, NR4A1 was a gene showing TRH-dependent expression and showed a similar profile to the TSHβ gene. However, expression of Pit1 and GATA2, other candidates for a factor related to TRH-induced stimulation of the TSHβ gene, was not altered by TRH or thyroid hormone replacement *in vivo*. Therefore, we focused on the effect of NR4A1 on the transcription of the TSHβ gene *in vitro.* Overexpression of NR4A1 clearly stimulated the promoter activity of the TSHβ gene in a time- and dose-dependent manner. In addition, we found that incubation with TRH markedly stimulated expression of NR4A1 mRNA by over 50-fold in an early time within 60 minutes. Furthermore, immunocytochemistry studies demonstrated co-expression of NR4A1 with TSHβ in the anterior pituitary, and in addition the expression was significantly decreased in the TRH-knockout mice. To our knowledge, this study is the first demonstration of the presence of NR4A1 even in ACTH- and FSH- producing cells by immunohistochemistry [@pone.0040437-Lavoie1]. Taken together, we demonstrated that NR4A1 is an up-regulator working down-stream of TRH in the anterior pituitary *in vivo.* The analysis with a series of deletions of the promoter region of the TSHβ gene demonstrated that the responsible region of TRH-NR4A1 induced-stimulation of the TSHβ gene was between bp -138 bp and +37 from the transcription start site (TSS). Furthermore, although no consensus sequence for binding site for NR4A1 was found in this region, Chip assay with a specific antibody against NR4A1 revealed that NR4A1 was recruited to this region. Steinfelder et al, also demonstrated with a transient transfection study and deletion analysis of the human TSHβ gene that the region between -128 and +8 in relation to TSS is responsible for TRH-induced stimulation of the gene [@pone.0040437-Steinfelder1]. This region was close to the TSS and contained binding sites for Pit1 and GATA2, and the thyroid hormone inhibitory element (nTRE). Steinfelder et al. reported that the response element of the TSHβ gene to TRH was located in two discrete regions (-128 to -92 base pairs and -28 and +8 base pairs). The upstream site contains a DNA sequence with homology to the DNA-binding site for a pituitary specific transcription factor, Pit1, and the downstream site overlaps with the thyroid hormone inhibitory element [@pone.0040437-Steinfelder1], [@pone.0040437-Gordon1]. Furthermore, a recent study of pituitary-specific GATA2 Knockout mice demonstrated reduced pituitary TSH content and serum TSH levels, and pituitary TSHβ mRNA showed a defective response to hypothyroidism [@pone.0040437-Charles1]. Two regions of the mouse TSHβ gene have a sequence homologous to a consensus motif of the GATA binding site, AGATGC, at the region between -110 and -105 to the TSS, and AGATAA between -98 and -93 [@pone.0040437-Gordon1], and these sequences are perfectly conserved in rodents and humans [@pone.0040437-Ohba1]. Although both Pit1 and GATA2 have been reported to be essential for the expression of the TSHβ gene in thyrotrophs, the present study demonstrated that knockdown of NR4A1 with siRNA significantly reduced TRH-induced stimulation of the promoter activity of the TSHβ gene. Therefore, as shown in [Figure 9](#pone-0040437-g009){ref-type="fig"} as a proposed model of action of TRH on the TSHβ gene, NR4A1 may also be critical and cooperatively work with Pit1 and GATA2 as an up-regulator of TRH-induced stimulation of the TSHβ gene through this region -128 and +8 close to the TSS. Although interactions of NR4A1 with several transcription factors and coregulators, including silencing mediator for retinoid and thyroid hormones (SMRT), steroid receptor coactivator (SRC-1), TRAP220, and SIN3B/KDM5A and Z3 (ZMYND8-containing) complex have been reported, those with Pit1 and GATA2 remain to be studied [@pone.0040437-Gordon3], [@pone.0040437-Kelly1]--[@pone.0040437-Malovannaya1]. ![Proposed models of the TRH-NR4A1-TSHβ pathway.\ TRH stimulates the transcription of the NR4A1 gene through TRH receptors, and PKC and MAPK pathways within 1 hr. The increased amount of NR4A1 stimulates TSHβ promoter activity, which may act cooperatively with Pit1 and GATA2 on the gene.](pone.0040437.g009){#pone-0040437-g009} NR4A1 was originally isolated as a gene induced by nerve growth factor in PC12 cells [@pone.0040437-Milbrandt1]. Subsequently, it has been reported to act as a stimulus-induced transcriptional activator in many tissues and organs, and recently been implicated in apoptosis, cell cycle regulation, inflammation, carcinogenesis and metabolism [@pone.0040437-MartnezGonzlez1]--[@pone.0040437-Liu1]. In addition, NR4A1 plays an important role in the hypothalamo-pituitary-adrenal (HPA) axis [@pone.0040437-Philips1], [@pone.0040437-Philips2], [@pone.0040437-Inaoka1], [@pone.0040437-Fernandez1], [@pone.0040437-Maira1]. Philips et al. reported that the regulation of transcription of the POMC gene by CRH was mediated through activation of NR4A [@pone.0040437-Philips2]. However, NR4A1-deficient mice showed normal adrenocortical function, but subsequent studies demonstrated that other members of a subfamily of nuclear receptors such as NR4A2 (Nurr1) and NR4A3 (Nor-1) might compensate for a loss of NR4A1 and maintain sufficiently normal steroidogenesis *in vivo* [@pone.0040437-Fernandez1], [@pone.0040437-Crawford1]. We also examined expression of NR4A2 and NR4A3 mRNA in GH4C1 cells, however, neither significant expression nor effect of TRH was observed on these mRNA levels. In addition, as mentioned above knockdown of NR4A1 in GH4C1 cells significantly reduced TRH-induced stimulation of the TSHβ gene. Therefore, NR4A1 appeared to be essential for thyrotrophs in the pituitary gland. Furthermore, the pituitary--thyroid--axis in NR4A1-deficient mice has not been characterized, and so will be interesting to study in the future [@pone.0040437-Crawford1], [@pone.0040437-Lee2]. NR4A1 has also been well characterized as a regulator in the hypothalamo-pituitary-gonadal (HPG) axis. GnRH stimulated NR4A1 expression in pituitary gonadotropes and stimulated transcription of the LH gene [@pone.0040437-Kakar1], [@pone.0040437-Wurmbach1]. In addition, in the testis and ovary, expression of the steroidogenic acute regulatory (StAR) gene and steroidogenesis are regulated by the LH-NR4A1 pathway [@pone.0040437-Manna1]--[@pone.0040437-Martin1]. Thus, NR4A1 would be a common transducer of transcriptional signals of hypothalamic hormones for the regulation of targeted pituitary hormone genes in the anterior pituitary gland. Methods {#s4} ======= Reagents and Solutions {#s4a} ---------------------- Inhibitors (Staurosporine, PD98059, nimodipine and KT5720) were purchased from Sigma--Aldrich (St. Louis, MO). Staurosporine, PD98059 and KT5720 were dissolved in DMSO, and nimodipine in methanol. All vehicles were incubated with the same concentration of dissolved solvent. TRH was kindly provided by Tanabe-Mitsubishi Pharmaceutical Company, Japan. Animals {#s4b} ------- Procedures for animal care and use in this study were approved by the Review Committee on Animal Use at Gunma University, Maebashi, Japan. Animals were maintained on a 12-hour light and 12-hour dark schedule (lights on at 6.00 h) and fed laboratory chow and tap water ad libitum. All experiments were performed between 9.00 and 11.00 h. The mice with thyroxin (T~4~) replacement received 1.5 μg T4/100 g body weight, subcutaneously for 14 days before the experiment. TRH replacement was performed with 1 mg TRH/kg body weight/day by Alzet pump (micro-osmotic pomp model 1002, Alzet, U.S.A.) subcutaneously for 7 days before the experiment. Experimental hypothyroid mice were generated by thyroidectomy under microscopy. Thyroidectomized mice were given with 1% CaCl2 in drinking water to prevent hypocalcemia due to the simultaneous removal of the parathyroid gland. A mild hypothyroidism was induced by 0.1% MMI in drinking water for 7 days with regular chow that contained iodine. Radioimmunoassay (RIA) {#s4c} ---------------------- Serum TSH levels were measured by a specific mouse TSH RIA with mouse TSH/LH reference (AFP51718MP), mouse TSH antiserum (AFP98991) and rat TSH antigen (NIDDK-rTSH-I-9), all of which were obtained from Dr. A.F. Parlow (Harbor-UCLA Medical Center, Torrance, USA). Serum free T~4~ levels were determined by RIA (GammaCoat Free T4, DiaSorin Inc. USA). All assays were performed in duplicate and had a detection limit of 0.16 ng/dl. RNA Extraction and Real-time PCR {#s4d} -------------------------------- Total RNA was prepared from mouse pituitary, GH4C1 cells and At-T20 cells using ISOGEN (Nippongene, Toyama, Japan) according to the manufacturer's instructions. Then cDNA was reverse-transcribed from 300 ng of total RNA (TaqMan Reverse Transcription Reagents, Applied Biosystems, Tokyo, Japan), and 0.5 μl was subjected to real-time PCR. All reactions were performed in triplicate using TaqMan probes and an Applied Biosystems 7500 sequence detection system. TaqMan probes for NR4A1 (Mm01300401, Rn01533237), NR4A2 (Mm00443060, Rn00570936), NR4A3 (Mm01354011, Rn00569312), Pit1 (Mm00476852, Rn00564562), GATA2 (Mm00492300, Rn00583735), POMC (Mm00435874, Rn00595020) and GAPDH (Mm99999915, Rn99999916) were purchased from Applied Biosystems. The expression level of each mRNA relative to that of GAPDH was calculated using a standard curve, and the relative quantification method was performed as described in ABI User Bulletin \#2. All experiments were repeated at least twice. cDNA Microarray Analysis and K-means Cluster Analysis {#s4e} ----------------------------------------------------- Total RNA was prepared from each mouse pituitary using ISOGEN (Nippongene, Toyama, Japan) according to the manufacturer's instructions. cRNA was then prepared and hybridized to an Affymetrix Mouse Gene chip 320 2.0 Array. Only genes with raw data greater than 100 signals for all conditions were included in the analysis. Genes after exclusion of absent signals were subjected to a cluster analysis using a K-means algorithm. The hierarchical cluster analysis was carried out using the computer program Genowiz™ (ver. 3.2). Double-fluorescent Immunohistochemistry {#s4f} --------------------------------------- A rabbit polyclonal antibody for NR4A1 (LS-B114) was purchased from LifeSpan BioSciene (Seattle, WA, USA). Antibodies for mouse TSHβ (M-16: sc-7815) and was purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA). An ACTH antibody was purchased from DAKO Corp. (Glostrup, Denmark), and a follicle-stimulating hormone (FSH) antibody (MON5012) was obtained from MONOSAN (MONOSAN, Uden, Netherlands). Fluorescently labeled secondary antibodies used were TSHβ (red), Alexa Fluor 594 donkey anti-goat IgG (Invitrogen, California, USA), NR4A1(green), FITC: polyclonal goat rabbit immunoglobulins (HRP) (DAKO Glostrup Denmark); ACTH (red), Alexa Fluor 594 rabbit anti-mouse IgG (Invitrogen, California, USA), NR4A1(green), FITC: polyclonal goat rabbit immunoglobulins (HRP) (DAKO Glostrup, Denmark); FSH (red, Alexa Fluor 594 donkey anti-mouse-IgG(H+L) (Invitrogen, California, USA), NR4A1 (green), and Alexa Fluor 488 goat anti-rabbit-IgG (H+L) (Invitrogen, California, USA). Pituitaries were excised and fixed with 4% paraformaldehyde at 4°C overnight. Tissues were embedded in paraffin wax and sectioned sagittally in 4 μm-thick slices. The sections were pretreated with 0.3% hydrogen peroxide for 30 min for the quenching of endogenous peroxidase activity and then autoclaved for 10 min at 120°C. They were rinsed in TBS (Tris-buffer saline) and incubated with skim milk for 30 min at room temperature to block non-specific antibody binding, and treated with the above antibodies (TSHβ, ACTH, or FSH) at 4°C overnight. After another three washes in TBS, the sections were incubated with the secondary antibodies, then washed in TBS, and treated with the NR4A1 antibody at 4°C overnight. After three washes in TBS, the sections were incubated with the secondary antibodies, washed in TBS, and treated with Vectashield mounting medium (VECTOR Laboratories, CA, USA). We observed the slides with a BX52 biological microscope and DP70 digital camera (OLYMPUS, Tokyo, Japan). Mammalian Cell Culture {#s4g} ---------------------- CV-1 (a monkey kidney cell line) [@pone.0040437-JENSEN1], GH4C1 (a rat pituitary cell line) [@pone.0040437-Tashjian1] and At-T20 (a mouse pituitary adenoma cell line) [@pone.0040437-Gumbiner1] cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), as described previously [@pone.0040437-Lin1]. In some experiments, GH4C1 cells were starved in DMEM without serum overnight, then pre-treated with several inhibitors for signal transduction at the indicated concentrations 30 min before the treatment with TRH. Plasmid Constructions {#s4h} --------------------- Expression vectors for the mouse NR4A1, Egr-1, GATA2, Pit1 and TRH receptor (TRHR) were prepared by PCR and verified by sequencing of the DNA. The mouse NR4A1, Egr-1, GATA2 and Pit1 cDNAs were subcloned into the vector pcDNA^TM^3.1 D/V5-His-TOPO, and TRHR was subcloned into the vector pCDM8 for *in vitro* transcription/translation and transient expression analyses. The human TRβ~1~ cDNAs were subcloned into the vector pKCR~2.~ The human NR4A1 promoter containing 1,295 bp of the 5′ flanking sequence and 152 bp of exon 1(pA3NR4A1(-1295∼+152)-Luc), the rat POMC promoter containing 706 bp of the 5′ flanking sequence and 64 bp of exon 1, the human TSHβ-Luc containing 1,192 bp of the 5′ flanking sequence and 37 bp of exon 1 (pA3TSHβ(−1192∼+37)-Luc), the human prolactin gene containing 332 bp of the 5′ flanking sequence and 65 bp of exon 1 (pA3PRL-Luc), and the human TSHα-Luc containing 486 bp of the 5′ flanking sequence and 44 bp of exon 1 (pA3TSHα-Luc) [@pone.0040437-Hashimoto1] were fused to a firefly luciferase reporter plasmid (pA3Luc). Cell Transfection and Luciferase Assay {#s4i} -------------------------------------- Twenty-four hours before transfection, cells were split into 6-well plates at subconfluency. The transient transfection was performed using a calcium phosphate precipitation method, as described previously [@pone.0040437-Lin1]. The total amount of transfected plasmid was adjusted by adding an empty expression vector in all experiments. Sixteen hours after transfection, the medium was changed to DMEM supplemented with 10% FBS treated with AG1-X8 resin (Bio-Rad) and activated charcoal (Sigma) to remove thyroid hormones. Cells were further incubated in the presence or absence of TRH at the indicated concentration. To determine luciferase activity, cell monolayers were rinsed twice with PBS, then lysed with 300 μl of 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO~4,~ 4 mM EGTA, 1 mM dithiothreitol, and 1% v/v Triton X-100. Cells were scraped from the dishes and centrifuged at 12.000× g for 5 min at 4°C. Assays for Luc activity were performed using 150 μl aliquots of cell lysate and 210 μl of 25 mM glycylglycine (pH 7.8) containing 15 mM MgSO~4,~ 4 mM EGTA, 3.3 mM KPO~4,~ 1 mM dithiothreitol, and 0.45 mM ATP. The reaction was initiated by addition of 200 μl of 0.2 mM d-luciferin and light emission was measured for 10 seconds using a luminometer. Luciferase activity was expressed as arbitrary light units per microgram of cellular protein. All the transfection experiments were repeated at least twice with triplicate determinants. Western Blot Analysis {#s4j} --------------------- GH~4~C~1~ cell lysates were prepared in RIPA buffer and 100 μg/ml PMSF, 30 μl/ml Aprotinin, and 1 μmol/ml sodium orthovanadate, and then passed through a 23-gauge needle on ice. For cell and tissue extracts, the samples were incubated on ice for 30 min in RIPA buffer. Insoluble cell debris was removed by centrifugation at 10.000 X g for 10 min. Aliquots of protein-containing supernatant were stored at −80°C. Protein concentrations were determined by the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc. Tokyo, Japan). The lysates (40 μg) were resolved by SDS-PAGE gel (10%) and transferred to a polyvinylidene fluoride membrane (Hybond-P, Amersham Biosciences, Tokyo, Japan) with a semidry system (BIO CRAFT, Tokyo, Japan) for detection of NR4A1. The blots were blocked for 1 h with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and probed for 16 h with a primary antibody against NR4A1 (LS-B114, LifeSpan BioScienes, Seattle, WA, USA). After three washes with Tris-buffered saline with 0.1% Tween 20, antigen-antibody complexes were detected using a peroxidase-conjugated secondary rabbit antibody and an enhanced fluoro-chemiluminent system (ECL-plus; Amersham Biosciences). Small Interfering RNA (siRNA) Experiments {#s4k} ----------------------------------------- Pooled siRNA oligonucleotides targeting NR4A1 were designed, synthesized and annealed at Dharmacon Research, Lafayette, CO (siGENOME SMART pool NR4A1, L-100466-01)(siNR4A1). Pooled unrelated siRNA (siCONTROL non-targeting siRNA pool, D-001810-0X)(siControl) was used as a control. These siRNAs were transfected into GH4C1 cells by the lipofection method (Lipofectamine RNAiMAX™, Invitrogen, California, USA). Briefly, in the 6-well format, 100 pmol of siRNA per well was transfected into the GH4C1 cells. After 6 h, the medium was changed to DMEM containing 10% FBS. Twenty-four hours after the first transfection, the transient transfection of TSHβ-Luc, Pit1 and GATA2 was performed using a calcium phosphate precipitation method. Sixteen hours after the second transfection, the medium was changed to DMEM supplemented with 10% FBS treated with AG1-X8 resin and activated charcoal. Cells were further incubated in the presence or absence of TRH. After twenty-four hours incubation, a luciferase assay and RNA extraction were performed. Electrophoretic Mobility Shift Assay (EMSA) {#s4l} ------------------------------------------- The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE (5′-gatcctagtgatatttacctccaaatgccagga-3′) and a fragment containing the human TSHβ bp −123∼−87 (5′-cagtatgaattttcaatagatgcttttcagataagaaa-3′), which has been reported to be responsible for TRH-induced stimulation [@pone.0040437-Ohba1], [@pone.0040437-Philips1]. Double-stranded oligonucleotides were labeled with \[α^32^P\]dCTP by a fill-in reaction using a Klenow fragment of DNA polymerase I. NR4A1 was synthesized by *in vitro* transcription/translation from pcDNA3.1-NR4A1 using T~7~ RNA polymerase and the TNT-coupled reticulocyte lysate system (Promega Corporation). The binding reaction, gel electrophoresis, and autoradiography were performed under conditions described previously [@pone.0040437-Nakajima1]. Chromatin-immunoprecipitation (ChIP) Assay {#s4m} ------------------------------------------ ChIP assays were performed as we previously reported, using a kit from Upstate Biotechnology (Massachusetts, USA) [@pone.0040437-Hashimoto2]--[@pone.0040437-Ishii1]. GH4C1 cells were transfected with pA3TSHβ(−1192∼+37-Luc and incubated in medium containing 10% fetal bovine serum. After incubation overnight, 37% of formaldehyde was directly added to the culture at a final concentration of 1%, and the cells were incubated for 15 min at room temperature to crosslink protein to DNA. The whole cell extracts were pelleted and resuspended in 500 μl of lyses buffer (1% SDS/50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1 mM PMSF and 1 mg/ml aprotinin) for 10 min at 4°C. The lysate was sonicated 3 times with 10-sec pulses using a sonicator set at 70% of maximum power to reduce DNA length to between 200 and 1000 bp. The chromatin solution (500 μl) was used for each ChIP assay with 0.6 mg of a rabbit anti-NR4A1 antibody (LS-B114, LifeSpan BioScienes, Seattle, WA, USA). As a negative control, normal mouse IgG (sc-2025, Santa Cruz Biotechnology, CA, USA) was used. The primers used for the region between bp --138 and +13 were as follows, forward primer, 5′-GGTAAAGATATTGTGAGCTTGTTTGTCTAA-3′, and reverse primer, 5′- GCTGTGGTGACCCAAACTAAAAGC-3′, and the length of the predicted PCR product was 151 bp. The primers used for the region between bp -1192 and -1049 containing putative NurRE sequences (AAATATCA: -1091 bp and -1083 bp) were 5′-GGATCCCTTCCCTACACCATATAGAAAAAT-3′, and 5′-GTCATGAAATCTCTA CCCGGTCCTACATCT-3′, and the predicted size was 143 bp. Conventional PCR was performed in 50 μl with platinum high-fidelity (Invitrogen) for 30 cycles (annealing temperature of 60°C), and signals were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX (Bio-Rad). All ChIP assays were repeated at least three times. Statistical Analysis {#s4n} -------------------- Statistical analyses were performed with ANOVA and Student's t test or the Wilcoxon/Kruskal-Wallis test using JMP (SAS Institute Inc., Cary NC). **Competing Interests:**The authors have declared that no competing interests exist. **Funding:**This study was supported, in part, by grant from Health and Labor Sciences Research Grants, Research on Measures for Intractable Disease Hyptahalamo-Pituitary Dysfunction Research Group (MM), and Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (MY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: YN MY NS. Performed the experiments: YN MY RT NS. Analyzed the data: YN MY RT NS AO TT KH TS TT SO TS MM. Wrote the paper: YN MY.

{ "pile_set_name": "PubMed Central" }

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Background ========== The traditional method for harvesting this vein involves the use of a long incision from ankle to groin. A frequent observation after coronary artery bypass graft surgery is that many patients complain more about their leg wound than they do about their median sternotomy. The long incision is associated with significant intra-operative and postoperative complications \[[@B1]\]. Impaired leg wound healing has been reported to occur in up to 25% of patients \[[@B2]\]. Wound complications including infections, hematoma formation, cellulitis and saphenous neuropathy prolong recovery \[[@B3]\]. The use of minimal invasive saphenous vein harvesting has been advocated in an effort to minimize such wound related problems \[[@B4]-[@B6]\]. There is already some evidence that these methods may reduce leg wound complications \[[@B6]\], although they have not yet gained widespread use. Several techniques are available for minimal invasive vein harvesting, but all necessitate traction on the vein to maximize surgical visibility and enable side branch ligation. Excessive surgical manipulation of saphenous vein impairs endothelial cell function and reduces the bioavailability of nitric oxide \[[@B7]-[@B9]\]. This endothelial injury promotes platelet and leukocyte adhesion that in turn can result in smooth muscle cell proliferation that exacerbates the intimal hyperplasia that is a common cause of vein graft occlusion \[[@B10]-[@B12]\]. Thus, functional integrity of the harvested and prepared saphenous vein has important implications for immediate and long-term graft patency Materials and methods ===================== The patients ------------ From May 2002 to May 2003, 80 patients scheduled for elective first time coronary artery bypass grafting were recruited into the study. Study participants had isolated coronary artery disease that required at least part of their revascularization to be done using the LSV. Exclusion criteria included patients undergoing emergency coronary artery bypass grafting and those with severe varicose veins. The study was approved by the Local Research and Ethics Board at Glasgow Royal Infirmary. Before enrollment and randomization each participant provided written informed consent. Surgical techniques ------------------- Two consultant cardiac surgeons (UN and AM) carried out all of the operative procedures reported in this study. ### Long incision (open) saphenous vein harvesting (Group A) The incision was commenced just above the medial malleolus. The vein was identified and cleared of all adventitia and connective tissue using sharp and blunt dissection. The skin was incised over the whole length of the vein to the required length and careful dissection was used to isolate the vein in situ, with attention given to avoid unnecessary trauma to the vein or its tributaries. Side branches were ligated with 4/0 ethibond ligatures on the vein side and metal clips on the patient side. The leg wound was closed in layers and a full length pressure dressing was applied. ### Minimal invasive technique for long saphenous vein harvest (Group B) A 2 cm longitudinal incision was made above the medial malleolus. The long saphenous vein was identified and cleared of all adventitial and connective tissue using sharp dissection. The distal end of vein was tied. The proximal part was also tied and about 6--8 cms of thread was left with that end. The vein was then divided between the two tied ends. The end with a thread was passed through the ring in the Mayo vein stripper and forward pressure was applied to the vein stripper in the direction of usual vein anatomy while applying traction on the vein through the length of thread. Whenever resistance was felt on the vein stripper, a small incision (2 to 3 cm) at the area where resistance was felt was made. With a combination of sharp and blunt dissection any branch of the saphenous vein at that site was isolated and ligated using a 4/0 ethibond ligature on the vein side and a metal clip on the patient side. The same process was repeated with multiple short incisions until the required length of vein was obtained. The skin incisions were closed with skin staples and full-length pressure dressing applied. In common to both techniques of harvest, the vein was inflated with heparinised blood to check for any unidentified side branches or tears in the vein. Any that were identified were either ligated with a 4/0 ethibond suture or closed with a 6-0 prolene suture. Randomization ------------- Patients were randomized immediately prior to surgery by minimization using a computer program \[[@B13]\]. Minimization has the advantage that differences in important patient variables that might otherwise occur by chance can be avoided. The following patient characteristics were employed for minimization: diabetes, peripheral vascular disease, age and gender. Wound complications ------------------- Wound complications were assessed using the ASEPSIS \[[@B14]\] scoring method (Table [1](#T1){ref-type="table"}). All incisions were carefully assessed by a single observer on each day of the in-patient stay. The ASEPSIS score measures erythema, exudates and wound separation. A numerical score is calculated according to the proportion of the wound affected by each of these characteristics (table [5](#T7){ref-type="table"}). All discharging wound were swabbed and any bacterial growth identified by standard methods. Since some patients were discharged by the 5th postoperative day we calculated the sum of the first five daily scores for each patient. Pain measurement ---------------- A visual analogue score was used to assess postoperative leg pain \[[@B15],[@B16]\]. This consisted of a 10 cm long straight line with extreme limits marked with perpendicular lines and appropriate labels, but with no words or numbers between the endpoints. The patient was asked to mark the line with a cross at a point that represented their current level of pain. The sensitivity of this type of pain evaluation has been previously validated \[[@B15]\]. Pain assessment was carried out daily until hospital discharge. Vasomotor studies ----------------- Of the 79 patients in the study 29 were evaluated for vasomotor studies, 14 from Group A and 15 from Group B. A saphenous vein segment was removed prior to distension and immediately rinsed without pressure, immersed in iced oxygenated Krebs-Henseleit buffer \[[@B17]\] and transported to the laboratory. These vein segments were divided into three or four rings approximately 4 mm long that were mounted in a 25 ml organ bath. The tension was recorded directly onto a computer. Optimal resting tension was determined in baseline studies. Rings were then pre-contracted with phenylephrine (30 mmol/l). Endothelium-dependent relaxation was evaluated by cumulative addition of calcium ionophore A23187 (0.1 to 10 mmol/l) and endothelium-independent relaxation was evaluated by sodium nitroprusside (0.001 to 0.1 mmol/l) \[[@B18]\]. Relaxation to the NAD (P) H oxidase inhibitor apocynin (10 to 1000 mmol/l) was studied to assess the contribution of super oxide to vasoconstriction. Statistics ---------- Statistical analysis were performed using Student\'s t-test to compare parametrically distributed variables, the Mann Whitney test to compare non-parametrically distributed variables and the Chi-squared to compare discrete variables. Results ======= Of eighty patients undergoing coronary artery surgery in the study period, one patient was unavoidably excluded from the study before vein harvest commenced. Two patients who died within the first 48 hours were excluded from further analysis, leaving 38 patients in group A and 39 in group B. There were no differences in the demography of the patients between the two groups (Table [2](#T4){ref-type="table"}). No patient from group B was converted to the traditional open technique. ###### The ASEPSIS wound score Criterion Description Points ---------------------------------------------- -------------------------------------------- -------- **A**Additional treatment Antibiotics 10 Drainage of pus under local anesthetics 5 Debridement of wound (General anesthetics) 10 **S**Serous discharge Daily 0--5 **E**Erythema Daily 0--5 **P**Purulent exudates Daily 0--10 **S**Separation of deep tissues Daily 0--10 **I**Isolation of bacteria 10 **S**Stay in hospital prolonged over 14 days 5 ###### Points scale for the daily wound inspection Wound characteristic Proportion of wound affected (%) ---------------------------- ---------------------------------- --- --- --- --- ---- Serous exudates 0 1 2 3 4 5 Erythema 0 1 2 3 4 5 Purulent exudates 0 2 4 6 8 10 Separation of deep tissues 0 2 4 6 8 10 ###### The total score with the category of infection Total score Category of infection ------------- -------------------------- 0--10 Satisfactory healing 11--20 Disturbance of healing 21--30 Minor wound infection 31--40 Moderate wound infection \>40 Severe wound infection ###### Preoperative demographics Group A Group B ----------------------------- ----------- ----------- Age (years: mean ± 1sd) 63 (8) 65 (8) Male sex 29 29 NYHA class (I/II/III/IV) 0/8/14/15 0/8/15/16 Smoker 14 14 Diabetic Type 1 3 2 Type 2 6 8 Obese (BMI\>30) 10 8 Peripheral vascular disease 9 9 Venous stasis disease 5 9 ###### Intraoperative data Group A Group B ------------------------------------------- ----------- ----------- ------------ CPB time (mins: mean ± 1sd) 85 (23) 78 (15) ns Theatre time (mins: mean ± 1sd) 200 (32) 192 (24) ns Vein harvest site: Calf only 35 35 Calf and thigh 3 4 Total no of grafts (mean ± 1sd) 3.3 (0.7) 3.2 (0.5) ns No of vein grafts (mean ± 1sd) 1.9 (0.7) 1.6 (0.5) ns Length of vein harvested (cm: mean ± 1sd) 31 (13) 25 (9) p = 0.018 Length of wound (cm: mean ± 1sd) 35 (13) 16 (6) p \< 0.001 Number of incisions (mean ± 1sd) 1.2 (0.4) 6.9 (2.7) p \< 0.001 Vein harvest time (mins: median + IQR) Stage 1 16 (10) 11 (14) p = 0.01 Stage 2 9 (6) 2 (1) p \< 0.001 Stage 3 2 (2) 2 (4) p = 0.5 Total 26 (16) 15 (22) p = 0.002 Intra operative findings ------------------------ The total operative time taken to harvest vein was divided into 3 parts; stage 1: start of skin incision to removal of vein, stage 2: incision closure and stage 3: vein preparation. Adding the 3 gives the total vein operation time (Table [3](#T5){ref-type="table"}). Technique B was significantly quicker whilst requiring no increase in the time required to prepare the vein. Time was saved both during removal of the vein and during skin closure. There was no statistical difference in length of vein harvested but total wound length and number of incisions was less in Group B (Table [3](#T5){ref-type="table"}). Postoperative pain ------------------ Although the analogue scale pain scores were less in Group B than in Group A, the differences did not reach statistical significance either for maximum pain score or total pain score over the 5 first postoperative days. (Table [4](#T6){ref-type="table"}) ###### Pain scores in first 5 post operative days Group A Group B Student\' s t-test --------------------------------- ------------ ----------- -------------------- Maximum pain score (mean ± 1sd) 4.4 (2.0) 3.8 (2.1) P = 0.17 Total pain score (mean ± 1sd) 10.2 (5.5) 9.1 (5.8) P = 0.43 ###### Relaxation of long saphenous vein rings in response to calcium ionophore, sodium nitroprusside and apocynin. Agonist Group A Group B ------------------------------------- ------------- ------------- --------- Apocynin (mean ± 1 sem) 22.3 (8.7) 20.6 (8.6) p = 0.7 Calcium Ionophore (mean ± 1 sem) 18.4 (7.0) 17.6 (7.5) p = 0.4 Sodium Nitroprusside (mean ± 1 sem) 37.6 (12.1) 40.1 (13.1) p = 0.6 Infection and healing --------------------- The accumulated ASEPSIS scores for the first five postoperative days are shown in Figure [1](#F1){ref-type="fig"}. When the scores are categorized according to the grades given in Table [1c](#T3){ref-type="table"}, there were 8 patients in Group A with a minor wound infection or worse, compared to only 2 in Group B. This reaches statistical significance using a Chi-squared test (p \< 0.001). ![](1749-8090-1-15-1){#F1} Vasomotor studies ----------------- Long saphenous vein rings harvested from Group A and Group B showed similar relaxation to calcium ionophore, sodium nitroprusside and apocynin. (Table [5](#T7){ref-type="table"}) Discussion ========== Modern management of CABG surgery patients emphasizes an early return to normal activities. In this regard early mobilization after surgery plays an important part in the process of recovery. In turn, any reduction in morbidity from the saphenous vein harvest procedure will promote early mobilization and speed rehabilitation. In this randomized study we found that compared to the traditional open method of harvesting long saphenous vein, a less invasive technique employing a Mayo vein stripper was quicker and provided significantly improved wound healing. There are other instruments available but the Mayo stripper has the advantage of being reusable and is therefore less expensive than disposable devices. Although we only examined endothelial function in a sample of the harvested veins, we found no evidence that the manipulation of the vein, consequent upon using the Mayo stripper, affected endothelial function any more than the traditional open harvest method. In order to eliminate the effect of surgical expertise, we used 2 very experienced surgeons who were equally comfortable with either technique to carry out all of the vein harvesting. However, we see no reason why the less invasive technique cannot be taught to surgical trainees and surgical assistants, or why they should not be able to carry it out to an equally high standard. We failed to demonstrate any significant difference in the perception of pain from the leg wounds. In view of the fact that there is a significant difference in the overall length of the skin incisions between the two patient groups this is a little surprising, and does not reflect our subjective observations. In conclusion, this study supports the notion that harvesting vein through multiple incisions using the Mayo vein stripper is quicker, results in better wound healing and has no deleterious effect on endothelial function compared to the open technique.

{ "pile_set_name": "PubMed Central" }

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"required", "length", "vein", "obtained", "skin", "incisions", "closed", "skin", "staples", "pressure", "dressing", "applied", "common", "techniques", "harvest", "vein", "inflated", "heparinised", "blood", "check", "unidentified", "side", "branches", "tears", "vein", "identified", "either", "ligated", "ethibond", "suture", "closed", "prolene", "suture", "Randomization", "Patients", "randomized", "immediately", "prior", "surgery", "minimization", "using", "computer", "program", "Minimization", "advantage", "differences", "important", "patient", "variables", "might", "otherwise", "occur", "chance", "avoided", "following", "patient", "characteristics", "employed", "minimization", "diabetes", "peripheral", "vascular", "disease", "age", "gender", "Wound", "complications", "Wound", "complications", "assessed", "using", "ASEPSIS", "scoring", "method", "Table", "table", "incisions", "carefully", "assessed", "single", "observer", "day", "stay", "ASEPSIS", "score", "measures", "erythema", "exudates", "wound", "separation", "numerical", "score", "calculated", "according", "proportion", "wound", "affected", "characteristics", "table", "table", "discharging", "wound", "swabbed", "bacterial", "growth", "identified", "standard", "methods", "Since", "patients", "discharged", "postoperative", "day", "calculated", "sum", "first", "five", "daily", "scores", "patient", "Pain", "measurement", "visual", "analogue", "score", "used", "assess", "postoperative", "leg", "pain", "consisted", "cm", "long", "straight", "line", "extreme", "limits", "marked", "perpendicular", "lines", "appropriate", "labels", "words", "numbers", "endpoints", "patient", "asked", "mark", "line", "cross", "point", "represented", "current", "level", "pain", "sensitivity", "type", "pain", "evaluation", "previously", "validated", "Pain", "assessment", "carried", "daily", "hospital", "discharge", "Vasomotor", "studies", "patients", "study", "evaluated", "vasomotor", "studies", "Group", "Group", "B", "saphenous", "vein", "segment", "removed", "prior", "distension", "immediately", "rinsed", "without", "pressure", "immersed", "iced", "oxygenated", "buffer", "transported", "laboratory", "vein", "segments", "divided", "three", "four", "rings", "approximately", "mm", "long", "mounted", "ml", "organ", "bath", "tension", "recorded", "directly", "onto", "computer", "Optimal", "resting", "tension", "determined", "baseline", "studies", "Rings", "phenylephrine", "relaxation", "evaluated", "cumulative", "addition", "calcium", "ionophore", "relaxation", "evaluated", "sodium", "nitroprusside", "Relaxation", "NAD", "P", "H", "oxidase", "inhibitor", "apocynin", "studied", "assess", "contribution", "super", "oxide", "vasoconstriction", "Statistics", "Statistical", "analysis", "performed", "using", "compare", "parametrically", "distributed", "variables", "Mann", "Whitney", "test", "compare", "distributed", "variables", "compare", "discrete", "variables", "Results", "eighty", "patients", "undergoing", "coronary", "artery", "surgery", "study", "period", "one", "patient", "unavoidably", "excluded", "study", "vein", "harvest", "commenced", "Two", "patients", "died", "within", "first", "hours", "excluded", "analysis", "leaving", "patients", "group", "group", "B", "differences", "demography", "patients", "two", "groups", "Table", "table", "patient", "group", "B", "converted", "traditional", "open", "technique", "ASEPSIS", "wound", "score", "Criterion", "Description", "Points", "Additional", "treatment", "Antibiotics", "Drainage", "pus", "local", "anesthetics", "Debridement", "wound", "General", "anesthetics", "Serous", "discharge", "Daily", "E", "Erythema", "Daily", "P", "Purulent", "exudates", "Daily", "Separation", "deep", "tissues", "Daily", "Isolation", "bacteria", "Stay", "hospital", "prolonged", "days", "Points", "scale", "daily", "wound", "inspection", "Wound", "characteristic", "Proportion", "wound", "affected", "Serous", "exudates", "Erythema", "Purulent", "exudates", "Separation", "deep", "tissues", "total", "score", "category", "infection", "Total", "score", "Category", "infection", "Satisfactory", "healing", "Disturbance", "healing", "Minor", "wound", "infection", "Moderate", "wound", "infection", "Severe", "wound", "infection", "Preoperative", "demographics", "Group", "Group", "B", "Age", "years", "mean", "Male", "sex", "NYHA", "class", "Smoker", "Diabetic", "Type", "Type", "Obese", "Peripheral", "vascular", "disease", "Venous", "stasis", "disease", "Intraoperative", "data", "Group", "Group", "B", "CPB", "time", "mins", "mean", "ns", "Theatre", "time", "mins", "mean", "ns", "Vein", "harvest", "site", "Calf", "Calf", "thigh", "Total", "grafts", "mean", "ns", "vein", "grafts", "mean", "ns", "Length", "vein", "harvested", "cm", "mean", "p", "Length", "wound", "cm", "mean", "p", "Number", "incisions", "mean", "p", "Vein", "harvest", "time", "mins", "median", "IQR", "Stage", "p", "Stage", "p", "Stage", "p", "Total", "p", "Intra", "operative", "findings", "total", 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Background {#Sec1} ========== Cancer of the stomach (GC) and the distal esophagus and esophagogastric junction (AEG) is a substantial global health problem with around 1 million new cases and 750,000 deaths per year, accounting for estimated 10% of all cancer-related deaths \[[@CR1], [@CR2]\]. In Europe and North America, the overall 5-year survival for GC is approximately 25% \[[@CR3]\], while superior outcomes with 5-year survival rates of approximately 60% are reported in East Asia \[[@CR4]\]. The optimal medical treatment for advanced GC and AEG is still a source of debate, but after the publication of the randomized "MAGIC Trial" and "ACCORD Trial", neoadjuvant chemotherapy has become first choice for the treatment of locally advanced GC and AEG, and reported improved 5-year survival rates of 36 and 38% respectively \[[@CR5], [@CR6]\]. This situation is similar for esophageal and cardia cancers where the recently published randomized "CROSS Trial", using neoadjuvant chemoradiation, reported 3-year survival rates of 59% \[[@CR7]\]. Objective assessment of the treatment effect after neoadjuvant treatment is only possible by histopathology in the resected specimens. In patients with AEG and GC, the presence of \< 10% of vital residual tumor cells is considered as a major pathologic response, and is associated with a significant survival benefit \[[@CR8]--[@CR10]\]. The difficulty is to identify patients who do not respond or progress under neoadjuvant treatment. Those patients may not profit from neoadjuvant treatment, still suffer from adverse events, and finally risk tumor progression. In the setting of esophageal cancer, measurement of early changes in tumor glucose uptake by use of 18-fluorodeoxyglucose-PET (PET) and later PET-CT yielded promising results for predicting response following neoadjuvant chemotherapy \[[@CR11]\]. Metabolic tumor activity can be quantified by the standardized uptake value (SUV), and it was shown that a drop of ≥35% measured after 2 weeks of induction chemotherapy was an accurate cut-off value to predict response \[[@CR12]\]. This cut-off at ≥35% was prospectively studied in the MUNICON-I trial including 119 patients with AEG I and II \[[@CR13]\]. Metabolic response evaluation by PET-CT accurately identified all non-responding tumors within two weeks of treatment. In addition, there was a significant survival difference between metabolic responders and non-responders. No data are currently available for early metabolic response evaluation by PET-CT in patients with AEG III and GC, and the potential benefit is therefore unclear. We studied patients with AEG II/III and GC in a cohort of patients using the criteria for early metabolic response from the MUNICON-I trial to evaluate the accuracy and feasibility of metabolic response evaluation by early PET-CT following neoadjuvant CTX. Methods {#Sec2} ======= A retrospective cohort of 72 consecutive patients with biopsy proven GC or AEG Siewert type II-III \[[@CR14]\] was included in this study. All patients underwent routine staging, including, laboratory tests, upper GI-endoscopy with endoscopic ultrasound (EUS) and ^18^FDG PET-CT as reported previously \[[@CR15]\]. Patients with stage cT2N+ or cT3--4, Nx by EUS and PET-CT (UICC TNM Classification, 7th edition, \[[@CR16]\] underwent diagnostic laparoscopy to exclude occult peritoneal carcinomatosis prior to neoCTX. All patients were discussed in a multidisciplinary specialized tumor board prior to treatment initiation. The study was approved by the local ethics committee (PB 2016--00769). Imaging by ^18^FDG positron emission tomography-CT {#Sec3} -------------------------------------------------- A baseline PET-CT was performed as part of the staging procedure and an early response PET-CT, 14 days after the first cycle of neoadjuvant CTX. Imaging was performed on an in-line system (Discovery RX or Discovery VCT; GE Healthcare). These systems integrate a state-of-the art full ring PET scanner with a multi-slice helical CT (LightSpeed 16 or VCT 64 slice; GE Healthcare) allowing for acquisition of co-registered CT and PET images in one session. Patients fasted for at least 4 h before scanning, which started 50--60 min after the injection of a standard dose of 340--370 MBq 18F-FDG. A low dose CT (80 mA, 140 kV, 0.5-s tube rotation, 4.25-mm section thickness, 867-mm scan length, and 22.5-s data acquisition time) was performed first. Immediately after CT, the PET emission scan was acquired, with 2 min emission time per cradle position (total PET-CT acquisition time 12--16 min). PET images were reconstructed using a standard 3-dimensional iterative algorithm (ordered-subset expectation maximization). Image reading was done on screen using a commercially available software package (Advantage workstation, version 4.4; GE Healthcare). For quantitative measurement, a circular region of interest was placed over the tumor in the slice with maximum \[18F\]-FDG uptake in the baseline scan. A tumor was defined as negative or non-avid when there was no measurable activity over background \[18F\]-FDG uptake in the tumor area defined by endoscopy and/or demonstrated as mass in the integrated multi-slice CT. In the second PET scan, the region of interest was placed according to the baseline study using the surrounding anatomical landmarks. Patients with a decrease of ≥35% SUV were classified as metabolic responders (12,13). Neoadjuvant chemotherapy {#Sec4} ------------------------ Two neoadjuvant chemotherapy regimens were applied, either 3 cycles ECF according the "MAGIC" regimen \[[@CR5]\], or 3 cycles of FLOT, consisting of biweekly oxaliplatin 85 mg/m2, day1; docetaxel 50 mg/m2, day2; and continuous infusion 5-FU 2600 mg/m2 days 1--2 \[[@CR17], [@CR18]\]. Adverse events were reported according to the National Cancer Institute Criteria, version 3.0. Surgery {#Sec5} ------- Standardized resections were performed including subtotal (80%) gastrectomy for distal GC, gastrectomy for middle or proximal third GC and transhiatal extended gastrectomy for AEG Siewert type II-III tumors \[[@CR19]\]. Systematic D2-lymphadenectomy (LAD) was routinely performed \[[@CR20]\], and additionally LAD of the lower mediastinum for AEG types II/III. In few selected patients, para-aortic lymph node dissection (D3-LAD, \[[@CR20]\]) was performed. Complications were recorded using the Clavien-Dindo classification \[[@CR21]\]. Pathology {#Sec6} --------- Pathologic tumor regression was evaluated using a published validated scoring system \[[@CR8]\]. Patients with less than 10% residual tumor cells were classified as responders. All other patients were classified as non-responders. All specimens were reviewed by an experienced gastrointestinal pathologist (AW). Follow-up {#Sec7} --------- Patients were followed clinically and by contrast CT (local tumor recurrence, lymph node metastases, systemic metastases including peritoneal carcinomatosis) at 4-month intervals during the first year after surgery and at 6-month intervals thereafter, and endoscopy at 6 months and yearly thereafter. Survival was calculated from the day of study inclusion. Statistical analysis {#Sec8} -------------------- Differences in proportions were analyzed using Fisher's exact test. Inter-individual comparisons of quantitative data were done by use of a Wilcoxon signed rank test. Survival was estimated according to Kaplan-Meier. Statistical comparisons between different groups of patients were done with a log-rank test. All tests were two-sided and done at the 5% level of significance with the use of SPSS for Windows, version 11.50 (SPSS Inc., Chicago, IL, USA). Results {#Sec9} ======= Study population {#Sec10} ---------------- From October 1, 2008 to October 31, 2013, 72 consecutive patients with resectable, locally-advanced GC or AEG II/III were included. Among them, 28 patients were finally not eligible for neoadjuvant chemotherapy due to severe comorbidities, patient's decision, or peritoneal carcinomatosis at diagnostic laparoscopy. In 44 patients planned for neoCTX, 14 (32%) did not show FDG uptake and could therefore not be further evaluated. The remaining 30 patients (68%) underwent neoCTX and were restaged by PET-CT 14 days after beginning neoCTX. Patient characteristics are shown in Table [1](#Tab1){ref-type="table"}.Table 1Patient characteristicsParameterMedian (range)*n* = 30%Age (years)57.4 (36.9--78.9)Gender male2273 emale827Body Mass Index (kg/m^2^)23.2 (16.7--30.8)Charlson-comorbidity-Index 22377 3--5413 \> 6310ECOG score 02273 1827Localization AEG Siewert Type II1240 AEG Siewert Type III1034 Gastric cancer826Grading G21447 G31653Laurén's classification Intestinal2480 Mixed310 Diffuse310uT-category uT2413 uT32274 uT4413cN-category cN0310 cN+2790cM-category cM02480 cM1620Clinical TNM staging is based on EUS (uT) and/or CT or PET-CT (cN) Chemotherapy {#Sec11} ------------ Nine patients (30%) received neoCTX with ECF \[[@CR5]\], and 21 (70%) received FLOT \[[@CR17], [@CR18]\]. 8/9 patients pretreated with ECF received the planned 3 cycles and one patient refused the third cycle because of side effects. 20/21 (95%) patients with FLOT received 3 full cycles and 1 patient received 2 cycles due to severe bone marrow depression. Surgery and perioperative complications {#Sec12} --------------------------------------- All but one patient proceeded to surgical resection within 3 weeks, while it had to be postponed to week 5 due to bone marrow depression. Radical lymphadenectomy was performed in all patients with a high median number of resected lymph nodes of 43 (range 23--113). Perioperative morbidity according to the Clavien-Dindo classification was observed in 50% of patients, including major complications grade IIIb in 4 (13%) and grade IV in 1 (4%) patient. There was no in-hospital or 90-day mortality. Surgical details are summarized in Table [2](#Tab2){ref-type="table"}.Table 2Surgical characteristicsParameter*n* = 30%Type of resection Subtotal gastrectomy27 Gastrectomy27 Extended gastrectomy27 Transhiatal extended gastrectomy^a^2272 Esophagectomy27Type of lymphadenectomy D2-lymphadenectomy413 D2+ lower mediastinum2013 D3-paraaortic ± lower mediastinum467 2-field (abdominal and extended mediastinal)27R-category R02583 R1/R2517Postoperative complications (Clavien-Dindo) none1550 Grade I310 Grade II723 Grade IIIa00 Grade IIIb413 Grade IVa14 Grade IVb00 Grade V00^a^Splenectomy: *n* = 1 Pathology {#Sec13} --------- Most patients had locally-advanced ypT3--4 tumors (83%), and positive lymph nodes (67%). Advanced N-categories ypN2--3 were detected in 50% of resected specimens. Major pathologic regression occurred in 11/30 (36.7%) tumors with only 2/30 (7%) showing a pathologic complete remission. Major pathologic response in R0-resected patients was significantly associated with improved median survival rates (not reached) compared to minor response (28.2 months; 95% CI 16.7--39.7 months) by log-rank test (*p* = 0.04). Results are summarized in Table [3](#Tab3){ref-type="table"}.Table 3Histopathology after neoadjuvant chemotherapyParameterMedian (range)*n* = 30%ypT-category ypT027 ypT127 ypT213 ypT32170 ypT4a310 ypT4b13Number of removed lymph nodes43 (21--113)ypN-category ypN01033 ypN1517 ypN2723 ypN3827 ypN3 (AEG)310 ypN3a (GC)414 ypN3b (GC)13Tumor regression grading Complete regression (Ia)27 \< 10% residual tumor (Ib)930 ≥ 10% and \< 50% residual tumor (II)620 ≥ 50% residual tumor (III)827 no regression (IV)516Modified tumor regression grading Minor (grade II - IV, ≥ 10% residual tumor)1963 Major (grade Ia - Ib, \< 10% residual tumor)1137 Metabolic response {#Sec14} ------------------ In 30 patients with PET positive primary tumors, median SUV significantly decreased from 10.4 (range 4.0--29.8) to 5.0 (range 0--25.2) 14 days after the first cycle neoCTX (*p* \< 0.0001). Metabolic response was observed in 20 (66.7%), and no response in 10 (33.3%) patients. Prediction of pathologic response by metabolic response on PET-CT resulted in a sensitivity of 90.9% (95%-CI: 57.1--99.5%), specificity 47.3% (95%-CI: 25.2--70.5%), positive predictive value (PPV) 50% (95%-CI: 27.8--72.1%), negative predictive value (NPV) 90% (95%-CI: 54.1--99.4%) and overall accuracy of 63.3% (95% CI: 38.5--78.6%). Although the overall accuracy is low, the NPV is high with a correct identification in 9/10 true non-responding tumors. Metabolic response and prognosis {#Sec15} -------------------------------- Median follow-up was 22.4 months (range 3.2--61.8) for surviving patients. Median overall survival was significantly better for metabolic responders than for non-responders (median survival not reached and 28.2 months, 95%-CI: 7.2--10.7 months, respectively, log-rank *p* = 0.04). Survival curves are shown in Fig. [1](#Fig1){ref-type="fig"}.Fig. 1Overall survival according response PET-CT Overall survival estimates of *n* = 30 patients according to Kaplan-Meier curves based on their response PET-CT after two cycles of neoCTX. Numbers at risk are shown in 12 month intervals Discussion {#Sec16} ========== This cohort study in patients with locally advanced AEG II/III and GC shows that metabolic response two weeks after starting neoadjuvant therapy evaluation by FDG-PET-CT, using the validated threshold for metabolic responders from the MUNICON-I intervention trial (13) of ≥35% reduction of SUV does not accurately predict overall pathologic response. It does however identify a subgroup of patients that does not respond to neoCTX with a specificity of 90%. In this cohort, this subgroup compromised 14% (10/72) of the study population with an inferior prognosis compared to PET responders. The results of our study compare well to the results of the MUNICON-1 trial in patients with esophageal cancer, where 110 patients with AEG I/II were evaluated by PET-CT \[[@CR13]\]. The authors claim that responders can be identified by early metabolic imaging, however, 42% of the 50 PET responders showed a minor regression like the 50% observed in our study. The NPV for the metabolic response was 100% in the MUNICON trial, which is comparable to the 90% in our study and the 10% difference is likely due to the low patient numbers and different tumor entities. We therefore conclude that metabolic response evaluation by PET-CT does not accurately predict overall response but identifies non-responders in both trials. We also confirmed that PET-responders have a better prognosis than non-responders (*p* = 0.04) with remarkably close survival rates (median survival was not reached in PET-responders in both trials and 26 months and 28.2 months respectively for PET non-responders). In the MUNICON-I intervention trial, chemotherapy was discontinued in metabolic non-responders, thereby saving time, and reducing side-effects and costs without compromising the outcome. Less data is available for patients with gastric cancer. Vallböhmer et al. \[[@CR22]\] found no predictive value for the FDG uptake in 40 gastric cancer patients. Ott and colleagues \[[@CR23]\] prospectively studied 49 GC patients including Siewert type III tumors with a metabolic response (SUV reduction ≥35%) by PET. Overall, 23/49 (47%) patients had non-intestinal type cancers and 38/49 (78%) were in the proximal third. Metabolic response correctly predicted histopathologic regression in 11/16 responding and 27/33 non-responding tumors. This resulted in a sensitivity and specificity of 69 and 82%. PPV and NPV were 65 and 84% and overall accuracy was 78%. Median survival of metabolic responders was not reached, and significantly (*p* = 0.037) better than for non-responders (24.1 months). Again, remarkably similar results were obtained in our study. The lower NPV is likely attributable to the higher proportion of non-intestinal tumors (46.9%) compared to our study (20%) and the significantly lower baseline SUV obtained in non-intestinal-type tumors. In contrast to patients receiving chemotherapy alone, early metabolic response evaluation by PET-CT was not successful in patients receiving neoadjuvant chemoradiation for esophageal and esophagogastric junction cancers. \[[@CR24]--[@CR26]\]. Technically, a higher cut-off value might be better to predict histopathological response. Indeed, a previous study from the Munich group \[[@CR12]\] showed that a 45% or more decrease in SUV would result in higher specificity for histological response (86% versus 75%) but most important, in a lower NPV. This was also true in our study with a cut-off at 50% (data not shown). The FDG uptake is not uniform among the subgroups. Esophageal tumors, AEG I, show about 100% FDG uptake, much higher than AEG III or GC, or diffuse type cancer \[[@CR15]\]. Therefore, the use of PET-CT is not uniformly recommended, particularly in non-intestinal gastric cancer including signet ring cell cancer \[[@CR23], [@CR27]--[@CR29]\]. Distal gastric cancer with low differentiation grades, including diffuse types are less likely to achieve major tumor regression after chemotherapy \[[@CR30]\]. A risk score was evaluated in 410 patients receiving neoCTX for GC. Well-differentiated tumor grading, intestinal tumor type histology and tumor localization in the middle third of the stomach were identified as the significant positive predictive factors for histopathologic response and prognosis. A prognostic index could be created based on tumor localization, grading and type according to Lauren classification that identified 3 risk groups (low, intermediate and high) with significantly different clinical and histopathological response rates and overall survival times. \[[@CR31]\]. Several molecular markers have been investigated in view of characterizing tumor entities and predicting tumor response and prognosis following neoadjuvant treatment \[[@CR32], [@CR33]\], since a variety of novel targeted therapeutic approaches are introduced in cancer treatment \[[@CR34]\]. In HER2-positive advanced GC and AEG, the international phase III trastuzumab for GC (ToGA) study showed a significant improvement in the median overall survival of patients upon the addition of trastuzumab to cisplatin and fluoropyrimidine backbone therapy \[[@CR35]\]. In the MUNICON-II trial, salvage neoadjuvant radiochemotherapy in metabolic non-responders lead to local remissions in a considerable number of patients but was not able to change the clinical course \[[@CR36]\]. Altogether, metabolic non-responders may profit from a therapeutic switch to these novel approaches. Our study clearly has its limitations, mainly attributable to the small study population which does not allow more sophisticated statistical analysis including multivariate testing, and all test results must therefore be interpreted with caution. Despite these limitations, our results are remarkably close to the results of two published comparable studies, the MUNICON PET-CT trial in Siewert type I/II tumors \[[@CR13]\] and the PET-study in gastric cancer including Siewert type III tumors \[[@CR23]\] using early metabolic response evaluation. Furher research could also include assessing survival outcomes in patients classified as non-responders by PET-CT with subsequent discontinuation of neoadjuvant chemotherapy and comparing them with those remaining on treatment. Conclusions {#Sec17} =========== In conclusion, our study in patients with AEG and GC adds further evidence that response PET-CT reliably detects metabolic non-responders and can therefore identify patients who should either immediately proceed to resection or receive a modified multimodality therapy. PET-CT-guided neoadjuvant chemotherapy appears feasible in patients with AEG and GC but important issues remain to be addressed in future trials especially standardization for metabolic imaging as planned by the EORTC GI Group and NEOPEC Trial Group \[[@CR37], [@CR38]\]. AEG : Adenocarcinoma of the esophagogastric junction CT : Computed tomography EUS : Endoscopic ultrasound GC : Gastric cancer MDCT : Multidetector spiral computed tomography neoCTX : neoadjuvant chemotherapy PET : 18-fluorodeoxyglucose (^18^FDG) positron emission tomography PET-CT : Combined positron emission tomography and computed tomography We thank Miss Monika Stentström, Clinical Nurse for assistance with patients follow-up. Availability of data and materials {#FPar1} ================================== The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. PMS: consultant surgeon in all patients, analyzed and interpreted the patient data, manuscript preparation DE: retrieved, analyzed and interpreted the patient data, manuscript editing TR: chemotherapy and oncological follow-up of patients, manuscript editing DV: interpreted the patient data, manuscript editing PV: radiologic response evaluation, interpreted the patient data, manuscript editing AW: histological examination, interpreted the patient data, manuscript editing PB: endoscopic classification of AEG, endoscopic ultrasound, endoscopic follow-up of patients, interpreted the patient data, manuscript editing PS: chemotherapy and oncological follow-up of patients, manuscript editing KL: retrieved, analyzed and interpreted the patient data and was a major contributor in the writing the manuscript. Ethics approval and consent to participate {#FPar2} ========================================== The study was approved by the local ethics committee, Kantonale Ethikkommission Zürich, Stampfenbachstrasse 121, 8090 Zürich, Tel + 41 (0)43259 79 70 and registered under registration number PB 2016--00769. The need for written informed consent form from each patient has been waived by ethics committee due to retrospective manner of the study. Competing interests {#FPar3} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar4} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

{ "pile_set_name": "PubMed Central" }

[ "treatment", "ypna", "sophist", "choic", "two", "induct", "remain", "tumor", "cours", "decreas", "respond", "ethikkommiss", "laparoscopi", "thank", "biopsi", "identifi", "healthcar", "length", "risk", "classif", "written", "select", "mdct", "valu", "week", "maxim", "monika", "may", "cisplatin", "therebi", "emalebodi", "recent", "pb", "acquir", "cancergrad", "use", "gastrectomi", "final", "score", "ppv", "imag", "complet", "accord", "prior", "signific", "multidetector", "full", "ypta", "kaplanmei", "becom", "emiss", "ps", "tomographi", "helic", "also", "waiv", "intervent", "ctn", "multivari", "cmclinic", "iiia", "iva", "cell", "landmark", "dv", "perform", "metastas", "commerci", "popul", "screen", "proven", "remiss", "retrospect", "vct", "criteria", "accuraci", "neoctx", "fisher", "gctumor", "logrank", "medic", "nation", "higher", "prognosi", "dlad", "first", "remov", "h", "uicc", "intestin", "recommend", "multislic", "particip", "major", "middl", "assist", "assess", "ci", "global", 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"experienc", "cm", "advanc", "male", "mediastinum", "chemotherapi", "morbid", "vallböhmer", "fdg", "cr", "iv", "claim", "scan", "anatom", "distal", "magic", "dissect", "followup", "ib", "et", "promis", "iii", "review", "accur", "need", "expect", "session", "third", "save", "n", "background", "europ", "later", "yearli", "ia", "elig", "around", "tnm", "intermedi", "kantonal", "endoscop", "aeg", "plan", "exclud", "colleagu", "nonintestin", "suv", "tomographyct", "alon", "field", "natur", "age", "kl", "esophag", "possibl", "side", "manuscript", "consent", "might", "positron", "remark", "uptak", "r", "investig", "radiochemotherapi", "analysi", "ypnb", "g", "could", "vasplenectomi", "death", "utcncategori", "esophagu", "vital", "east", "region", "yield", "especi", "neg", "neopec", "lad", "ct", "cancerrel", "claviendindo", "residu", "softwar", "improv", "report", "america", "similar", "refus", "issu", "inject", "prognost", "area", "progress", "subtot", "contributor", "advers", "attribut", "previou", "therefor", "lauren", "introduc", "rank", "infus", "depress", "postpon", "detect" ]

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Introduction {#s1} ============ Glioblastoma (GBM) is one of the most common and malignant subtype of brain tumors in adults ([@B1]). Although the incident rate is relatively low, GBM is very invasive that leads to rapid neurological destruction and a disproportionately high mortality. Despite the improvement of new surgical and radiation techniques, the median survival rate of patients with GBM is rather low ([@B2], [@B3]). A blood-brain-barrier permeable DNA alkylating agent temozolomide ([@B4]) is currently the first line chemotherapy for treating GBM. In clinics, combination of radiotherapy with temozolomide significantly prolongs the survival rate for GBM patients ([@B5], [@B6]). Unfortunately, tumor often regrows after radio- and chemotherapies ([@B7], [@B8]) resulting in poor prognosis ([@B9], [@B10]). The recurrence is caused at least in part, by the resistance of GBM to conventional chemo- and radio-therapies ([@B11]--[@B14]), highlighting an urgent necessity of designing new strategies for the treatment of GBM. Sonic hedgehog (Shh) is a member of the hedgehog (Hh) family which functions as a chemical signal in transmitting information to the embryonic cells required for normal development. Shh plays a critical role in the regulation of vertebrate organogenesis and the development of brain and spinal cord including midbrain and ventral forebrain neuronal differentiation and proliferation, and many other parts of the body ([@B15]--[@B18]). Because of its role in embryonic development, aberrant or dysregulation of Shh signaling has been implicated in the initiation and/or maintenance of different types of tumor ([@B19]--[@B21]) including GBM ([@B22]). Consistent with these reports, Shh antagonists have been shown to possess anti-tumor activity in patients with basal cell carcinoma and medulloblastoma ([@B21], [@B23], [@B24]). Cancer stem cells (CSCs) are cancer cells that possess the ability to replenish tumors through the self-renewal and differentiation into multiple cell types ([@B25], [@B26]). CSCs have been identified in the breast, colon, brain and other areas and can differentiate into all the cell phenotypes of the parental tumor ([@B27], [@B28]). CSCs are hypothesized to be associated with chemo- and radio-resistance that lead to recurrence of tumor formation. In this theory, conventional radio- and chemotherapies kill differentiated or differentiating cells which form the bulk of the tumor, while sparing CSCs. Therefore, targeting CSCs offers a promising approach to improve cancer treatment or even cure cancer ([@B29]). In the present study, we isolated glioma stem-like cells (GSCs) from human GBM cell line U87MG (U87) using CD133 as a marker. We found that Shh expression is higher in the CD133^+^ cells than in the CD133^−^ cells. LDE225, a smoothened antagonist ([@B30], [@B31]), delayed GBM growth *in vivo* and significantly reduced the number of tumor spheroids derived from CD133^+^ cells. Furthermore, tumor growth was much slower in *Shh* knockdown mice suggesting that glioma growth may be dependent on a small population of CD133^+^ cells that are regulated by the Shh pathway. Materials and Methods {#s2} ===================== Animals ------- The BALB/cAnN.Cg-Fox*n1*^*nu*^/CrlNarl mice were purchased from the National Laboratory Animal Center (NLAC). Five mice were housed in a cage with controlled temperature (22 ±2°C) and humidity (55 ± 5%), kept on a 12 h light/dark cycle, and were given free access to water and food. Care and use of laboratory animals were in accordance with National Institutes of Health (NIH) guidelines. All the procedures were approved by the Institutional Animal Care and Use Committee of the College of Medicine, NCKU, with project approval number (\#104064 and \#107106). Cell Culture ------------ The human glioblastoma (GBM) cell lines U87MG (U87) was provided by Dr. Michael Hsiao (Genomics Research Center, Academia Sinica, Taiwan). GBM patient-derived cell line P\#5 was developed by Dr. Jian Ying Chuang. Both cells were cultured in Dulbecco\'s Modified Eagle medium-high glucose (DMEM-high glucose, Caisson) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Caisson). All cells were maintained in a humidified incubator with 5% CO~2~ at 37°C. Isolation and Characterization of Cancer Stem Cells From Glioblastoma Cell Line ------------------------------------------------------------------------------- For magnetic-activated cell sorting (MACS) purification, fresh GBM and spheroids were dissociated, washed, and incubated either with PE conjugated CD133/2 or IgG2b (Miltenyi Biotech, 1:11) at a concentration of 10^8^ nucleated cells per ml at room temperature for 15 min. EasySep^?^ PE selection cocktail at 100 μl/ml cells was added and mixed down for more than 5 min. Magnetic cell separation was performed using manual FalconTM polystyrene round-bottom tubes and an EasySep^?^ Magnet machine. Tube was removed from magnet and cells resuspended in an appropriate amount of desired medium. The CD133^+^ cells were incubated in neural stem cells selection medium (NeuroCult^TM^ NS-A Basal Medium, NeuroCult^TM^ NS-A Proliferaction Supplement, bFGF 10 ng/mL, EGF 10 ng/mL, 20 μg/mL Heparin; STEMCELL, Canada) and gave rise to non-adherent spheres on Ultra Low Attachment Multiple Well Plates (CORNING). CD133^+^ cells were initially allowed to form tumor spheroids in suspension culture, dissociated using Accutase (BD Biosciences) at 37°C for 30 min, and then split 1:3 to 1:5. The number of tumor spheroids formed by CD133^+^ cells treated with or without LDE225 (25 μM) for 7 days in culture were determined with an Olympus DP72 image analysis system and Inverted fluorescence microscope Olympus IX71. Cell Proliferation Assay ------------------------ Cell proliferation was measured using WST1 \[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium\] assay (Clontech Laboratories, California, USA). The CD133^+^ cells were seeded at a concentration of 8,000 cells/well in 200 μl culture medium containing various concentrations of LDE225 or cyclopamine (e.g., final concentration of 1--100 μM) into 96-well plates. The cells were incubated for 48 h at 37°C and 5% CO~2~. WST-1 reagent (10 μl/well) was added, and cells were incubated for 1 h at 37°C and 5% CO~2~. The absorbance of the product was measured at 440 nm with a microplate (ELISA) reader. The cell counts were determined by the percentage of the absorption relative to the vehicle-treated control culture. Western Blotting Assay ---------------------- Cell pellets were collected, centrifuged at 4,000 rpm and stored at −80°C. Drugs- or vehicle-treated cell pellets were lysed in a RIPA lysis buffer containing 50 mM Tris--HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor (Roche) and phosphatase inhibitor (Roche). Lysates were shaken at 40 rpm on ice for 1 h and then centrifuged at 12,000 rpm for 30 min at 4°C. The mouse brain tissues were homogenized in a lysis buffer (50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA and protease/phosphatase inhibitor cocktail (Roche, Nutley, USA). Supernatants were collected and then protein concentration was measured by Bradford assay. The protein was resuspended in 5X sample buffer (12.5 mM Tris, 25% glycerol, 4% SDS, 1.54% DTT, and 0.02% Bromophenol blue). The total protein was denatured at 95°C for 5 min. Protein electrophoresis on 15 or 8% SDS-polyacrylamide gel under 120 volt, and the separated protein was transferred to a PVDF membrane (Immunobilon transfer membranes, Millipore) by semi-dry transfer system (BIO-RAD) under 350 mA, 20 volt for 1.5 h. Membranes were incubated with blocking buffer (3% bovine serum albumin in PBS) for 1 h. Membranes were incubated with the following primary antibodies: rabbit anti-Shh (1:3,000, Millipore, Darmstadt, Germany), rabbit anti-P62 (1:2,000, Cell Signaling Technology, Danvers, USA), mouse anti-LC3 (1:2,000, MBL, Japan), mouse anti-CD133 (1:5,000, Millipore, Darmstadt, Germany), rabbit anti-Ptch1 (Protintech, Illinois, USA), rat anti-Gli1 (Sigma, Darmstadt, Germany) antibody overnight at 4°C, followed by treatment with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Lab., West Grove, USA) for 1 h at room temperature. After three rinses with 0.3% Triton X-100 in PBS for 10 min each, the ECL-plus chemical reagents (PerkinElmer) were added to the membrane. Films (Fuji, Japan) were exposed at different time points to ensure the optimum density. The band intensities were analyzed for densitometry by using Windows Image-J version 1.29. The protein levels in all groups were expressed as a percentage of those in controls. Immunofluorescent Staining -------------------------- For immunostaining of tumor spheroids, CD133^+^ cells were initially allowed to form spheroids in suspension culture for 7 days. The cells were then fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature. The cells were then incubated for 1 h in blocking solution (1% bovine serum albumin), and then incubated in primary antibodies at 4°C overnight. The following primary antibodies were used: mouse anti-CD133 (1:100, Millipore, Darmstadt, Germany), rabbit anti-SOX-2 (1:1,000, Abcam, Cambridge, UK) and rabbit anti-Shh (1:200, Millipore, Darmstadt, Germany), rabbit anti-ALDH1A1 (Protintech, Illinois, USA), rat anti-ABCG2 (Santa Cruz, Texas, US), rabbit anti-LC3 (Novus, Colorado, US). After three rinses with PBS containing 0.1% Triton X-100 for 10 min each, the cells were incubated in secondary antibodies at room temperature for 1 h. The following secondary antibodies were used: Alexa Fluor®594-conjugated Goat-anti-rabbit IgG (Jackson ImmunoResearch Lab., West Grove, USA) and Alexa Fluor®488-conjugated Sheep-anti-mouse IgG and Alexa Fluor®488-conjugated Goat-anti-rat IgG (Jackson ImmunoResearch Lab., West Grove, USA). DAPI (4\',6-diamidino-2-phenylindole) (1:1,000, Sigma-Aldrich, St. Louis, USA) dye was used as nuclear counterstain. The cells are washed in PBS containing 0.1% Triton X-100 three times for 10 min. The sections were cover-slipped with Prolong^?^ gold anti-fade reagent (Life Technologies, Carlsbad, USA). Fluorescence signals were detected with a Leica DM 2500 microscope and MetaMorph image software used for counting the cells number. JC-1 Mitochondrial Membrane Potential Assay ------------------------------------------- Early apoptosis was measured by using JC-1 Mitochondrial Membrane Potential assay (Cayman Chemical, Michigan, USA). The CD133^+^ cells were seeded at a concentration of 5,000 cells/well in 100 μl culture medium containing 25 μM LDE225 or 0.1% DMSO as vehicle control into black 96-well plates. The cells were incubated for 48 h at 37°C and 5 % CO~2~. JC-1 Staining solution (10 μl/well) was added, and cells were incubated for 30 min at 37°C and 5% CO~2~. Plates were centrifuged at 300 × g at room temperature. The supernatant was removed, and JC-1 buffer was used to suspend JC-1 stained cells. The fluorescence intensity of J-aggregate and JC-1 monomer was measured with excitation and emission at 535/595 nm and 485/535 nm with a microplate (ELISA) reader. The ratio of J-aggregate and JC-1 was determined as early apoptosis. Caspase-Glo® 3/7 Assay ---------------------- The activity of caspase 3 and 7 was measured by using Caspase-Glo® 3/7 Assay (Promega, Wisconsin, US). The CD133^+^ cells were seeded at a concentration of 1,000 cells/well in 100 μl culture medium containing 25 μM LDE225 or 0.1% DMSO as vehicle control into white 96-well plates. The cells were incubated for 48 h at 37°C and 5% CO~2~. Caspase-Glo®3/7 Reagent (10 μl/well) was added, and cells were incubated for 1 h at 37°C and 5% CO~2~. The luminescence was recorded with a microplate (ELISA) reader. The activity of caspase 3 and 7 was determined by the percentage of the luminescence signal relative to the control culture. *In vivo* Intracranial Xenograft Animal Model and Bioluminescence Imaging ------------------------------------------------------------------------- U87 GBM cells were transduced with lentiviral vector expressing GFP and firefly luciferase. GFP/Luc expressing cells were sorted out for further passages (FACS-Aria, BD Biosciences). For tumorigenesis, luciferase-expressing GBM cells were inoculated intracranially into the 8- to 10-week-old male nude mice (BALB/cAnN-Foxnlnu/CrlNarl mice, National Laboratory Animal Center). Nude mice were anesthetized with chloral hydrate and placed on a stereotaxic device. Subsequently, a hamilton syringe with 30-gauge needle was mounted on a stereotaxic device, and luciferase-expressing GBM cells were injected into the left side of the brains, 1.5 mm caudal and lateral to the bregma, and at a depth of 3.5 to 4 mm. LDE225 (Cayman) was injected intraperitoneally injected at a dose of 20 mg/kg twice weekly. Tumor growth was monitored by IVIS spectrum Live Imaging System (IVIS-200, Xenogen) twice weekly. Before monitoring, mice were injected with 150 mg/kg D-luciferin (PerkinElmer), and simultaneously anesthetized with isoflurane. The results of luciferase radiance were quantitated by Live Imaging Software (Xenogen) and the results were analyzed by using GraphPad Prism software. *Shh* shRNA Lentivirus Production --------------------------------- Production of lentivirus was initiated by triple transfection of HEK293T cells by a Lipofectamine® LTX Reagent (Life Technologies, Carlsbad, USA) method using small hairpin interfering RNA (shRNA) together with pCMV-dR8.91 and pMD2.G. The *Shh*-shRNA or scrambled shRNA conjugated on the vector of pLKO.1 with puromycin-resistant region was provided by National RNAi Core Facility (Institute of Molecular Biology, Academia Sinica, Taiwan). Cells were harvested 48 h later, medium containing lentiviruses filtered with 0.45 μm filters and viral particles were concentrated from the supernatant by Lenti-X™ Concentrator (Clontech Laboratories, Mountain View, USA) and purified to yield 1 × 10^8^ transducing units/ml storing at −80°C until use. The target sequence of *Shh*-shRNA is described as follows: *Shh*-shRNA: 5′-GCGGAAGGTATGAAGGGAAGA-3′. *Shh*-Over-Expression --------------------- To create lentiviruses over-expressing *Shh*, full-length *Shh* open reading frames (ORFs) (NM_000193; GenScript, New Jersey, USA) was amplified by PCR and was inserted into pLVX-IRES-ZsGreen1 expression vector (Clontech Laboratories, California, USA). The pLVX-NES1-IRES-ZsGreens1 vector encoding *Shh* (or empty vector) and the two packaging plasmids (pCMV-dR8.91 and pMD2.G) were co-transfected into HEK293T cells by lipofectamine® LTX Reagent (Life Technologies, Carlsbad, USA). Lentiviruses were harvested at 48 h after transfection, filter lentivirus supernatant through a 0.45 μm PVDF membrane filters, concentrated by Lenti-X™ Concentrator (Clontech Laboratories, Mountain View, USA), purified to yield 1 × 10^8^ transducing units/ml and stored at −80°Cuntil use. Statistical Analysis -------------------- Experiments were performed at least in triplicate. All results were presented as mean ± standard error of the mean (SEM). Independent experiments were analyzed by unpaired *t*-test. Two-way ANOVA was used to analyze the differences in tumor spheroids numbers *in vitro* and intracranial tumor growth *in vivo* at different times of treatment. Levels of *p* \< 0.05 were considered to be of statistical significance. Results {#s3} ======= CD133 has been reported as a marker of human neural and brain tumor stem cells ([@B32], [@B33]). We previously have isolated cancer stem-like cells with CD133 from glioblastoma (GBM) cell lines using magnetic bead cell sorting ([@B34]). Then CD133^+^ and CD133^−^ cell populations were collected and cultured separately. Shh Expression Is Higher in CD133^+^ Cells ------------------------------------------ We examined Shh expression in CD133^+^ and CD133^−^ cells. Western blotting analysis showed that Shh expression was higher in the CD133^+^ cells than in the CD133^−^ cells ([Figure 1A](#F1){ref-type="fig"}). Immunofluorescence staining revealed that most of CD133^+^ cells were positive and co-localized with Shh ([Figure 1B](#F1){ref-type="fig"}). SRY (sex determining region Y)-box 2 (SOX2) is a transcription factor that plays an important role in maintaining embryonic and neural stem cells ([@B35], [@B36]). [Figure 1C](#F1){ref-type="fig"} showed that SOX2 expression was higher in the CD133^+^ cells than in the CD133^−^ cells.These Shh-positive cells were also co-immunolabeled with SOX2 ([Figure 1D](#F1){ref-type="fig"}). ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily. The expression level of ABCG2 has been implicated in multidrug resistance (MDR) in cancer chemotherapy and the ability of self-renewal which correlates with CD133 ([@B37], [@B38]). AlDH1A1 is a member of the highly conserved ALDH family that is observed in several cancer stem cells, and is often used to isolate and functionally characterize cancer stem cells ([@B39]). We also examined the expression of ABCG2 and AlDH1A1 by immunofluorescence in parental and CD133^+^ cells. The expression of ABCG2 and ALDH1A1 is higher in CD133^+^ cells than in parental cells ([Supplemental Figure 1](#SM1){ref-type="supplementary-material"}). ![Tumor spheroids derived from CD133^+^ cells exhibit higher level of Shh expression. **(A)** Expression of Shh in CD133^+^ and CD133^−^ cells determined by Western blotting analysis. The CD133^+^ cells exhibited higher level of Shh expression. **(B)** Immunofluorescence staining of CD133 and Shh in tumor spheroids derived from CD133^+^ cells. CD133 and Shh were co-localized at tumor spheroids. **(C)** Expression of SOX2 in CD133^+^ and CD133^−^ cells determined by Western blotting analysis. The CD133^+^ cells exhibited higher level of SOX2 expression. **(D)** Immunofluorescence staining of CD133, Shh, and SOX2 in tumor spheroids derived from CD133^+^ cells. CD133 and SOX2, Shh, and SOX2 were co-localized at tumor spheroids.](fonc-10-01233-g0001){#F1} We used a potent and selective smoothened antagonist LDE225 ([@B30]) to determine the requirement of Shh signaling in the proliferation and tumor growth of GBM cell lines. We first confirmed the effect of LDE225 on tumor growth by using intracranial injection model. Luc-expressing parental U87 cells were injected intracranially into athymic mice and tumor growth was monitored using IVIS-200 imaging system. At day 7, LDE225 or DMSO as vehicle was injected intraperitoneally twice per week (20 mg/kg) into the mice and tumor growth was observed for 22 more days. Comparing with vehicle control, [Figure 2A](#F2){ref-type="fig"} showed that LDE225 was able to delay tumor growth. A two-way ANOVA revealed a main effect of group (LDE225 vs. control) \[*F*~(1,\ 42)~ =6.126, *p* \< 0.05\], interaction \[*F*~(6,\ 42)~ = 2.297, *p* = 0.0524\] and days after application \[*F*~(6,\ 42)~ = 5.849, *p* \< 0.001\] ([Figure 2B](#F2){ref-type="fig"}). Kaplan--Meier analysis of the survival data of vehicle control and LDE225-treated mice displayed in [Figure 2C](#F2){ref-type="fig"}. We also confirmed whether LDE225 inhibits Shh signal pathway. CD133^+^ cells were treated with LDE225 (25 μM) or vehicle for 48 h and the expression of Patch1 and Gli1 were determined by Western blotting analysis. As shown in [Figure 3A](#F3){ref-type="fig"}, LDE225 downregulated the expression of PATCH1 and GLI1. Next, CD133^+^ and CD133^−^ cells were treated with LDE225 (25 μM) for 48 h and cell viability was assessed using WST-1 assay. Concentration dependent relationship estimated the IC~50~ of about 20 μM for LDE225 to reduce CD133^+^ cell viability ([Figure 3B](#F3){ref-type="fig"}), while IC~50~ of LDE225 was about 50 μM for parental cells ([Supplemental Figure 2A](#SM2){ref-type="supplementary-material"}). As shown in [Figure 3C](#F3){ref-type="fig"}, CD133^+^ cells were more sensitive to LDE225 inhibition than that of CD133^−^ cells. At the concentration of 25 μM, LDE225 inhibited the proliferation of CD133^+^ and CD133^−^ cells by 57.57 ± 3.17% (*n* = 3) and 26.18 ± 6.22% (*n* = 4), respectively \[*t*~(5)~ = 4.013, *p* \< 0.05\]. We also determined the effect of LDE225 on patient-derived cell line P\#5 which displays various characteristics of GBM ([@B40], [@B41]). As illustrated in [Supplemental Figure 2B](#SM2){ref-type="supplementary-material"}, LDE225 inhibited cell viability with IC~50~ \~423 μM. Similar inhibition of CD133^+^ was observed by another Shh inhibitor cyclopamine, which binds to and inactivates smoothened protein ([@B42]) (IC~50~ ≈ 100 μM, [Supplemental Figure 2C](#SM2){ref-type="supplementary-material"}). We determined the effect of Shh inhibition on self-renew capacity of CD133^+^ cells ([Figure 3D](#F3){ref-type="fig"}). CD133^+^ cells were treated with LDE225 (25 μM) or vehicle and CD133^+^-derived tumor spheroids were counted at 1, 3, 7, and 14 days. Treatment with LDE225 significantly reduced the number of tumor spheroids. A two-way ANOVA revealed a main effect of drug (LDE225 vs. vehicle) \[*F*~(1,\ 16)~ = 100.9, *p* \< 0.001\], interaction \[*F*~(3,\ 16)~ = 44.27, *p* \< 0.001\] and days after application \[*F*~(3,\ 16)~ = 44.25, *p* \< 0.001\] ([Figure 3E](#F3){ref-type="fig"}). ![LDE225 delays tumor growth in an intracranial tumor model. **(A)** Luc-expressing parental U87 cells (3 × 10^5^ cells) were injected intracranially into athymic mice and tumor growth was monitored using the IVIS-200 imaging system. **(B)** Tumor growth was slower in LDE225 treated mice than in vehicle control mice. \*\**p* \< 0.001 vs. vehicle control. **(C)** Kaplan--Meier analysis of LDE225 treatment on survival.](fonc-10-01233-g0002){#F2} ![Effects of Shh inhibitors LDE225 on the cell viability of CD133^+^ and CD133^−^ cells. **(A)** CD133^+^ cells were treated with LDE-225 (25 μM) for 48 h and the expression of Patched1 and Gli1 was determined by the Western blotting analysis. LDE225 downregulated Patched1 and Gli1 in CD133^+^ cells. **(B)** Concentration-dependent effect of LDE225 on the cell viability of CD133^+^ cells. \*\**p* \< 0.01, \*\*\**p* \< 0.001 vs. vehicle. **(C)** CD133^+^ and CD133^−^ cells were treated with LDE225 (25 μM) for 48 h and cell viability was assessed using WST-1 assay. CD133^+^ cells were more sensitive to LDE225 inhibition than that of CD133^−^ cells. \**p* \< 0.05 vs. CD133^−^ cells. **(D,E)** Effects of LDE225 (25 μM) on the number of tumor spheroids derived from CD133^+^ cells. Primary tumor spheroids derived from CD133^+^ were dissociated and cultured. They were then treated with LDE225 (25 μM) for the times as indicated in **(E)**. The medium was replaced every 2 days in the presence of LDE225. \**p* \< 0.05, \*\**p* \< 0.01 vs. Vehicle.](fonc-10-01233-g0003){#F3} Inhibition of Shh Induces Autophagy in CD133^+^ Cells ----------------------------------------------------- In LDE225-treated cells, the observation by transmission electronic microscope showed the appearance of large membranous vacuoles in the cytoplasm which is a characteristic feature of cells undergoing autophagy ([Figure 4A](#F4){ref-type="fig"}). In addition, LC3 is distributed in the autophagosome membrane ([@B43]). The conversion of LC3-I to LC3-II is a common biomarker for autophagy activation ([@B44], [@B45]). We determined the conversion of LC3-I to LC3-II with anti-LC3 antibody. Immunoblotting using lysates from LDE225 (25 μM)-treated CD133^+^ cells revealed a significant increase in processed LC3-II \[*t*~(5)~ = 3.39, *p* \< 0.05\] ([Figure 4B](#F4){ref-type="fig"}). This was accompanied by the reduced expression of Mushashi-1, a RNA--binding protein selectively expressed in neural progenitor cells ([@B46]). The intensity of LC3 fluorescence punctate also increased after LDE225 treatment ([Figure 4C](#F4){ref-type="fig"}). Furthermore, LDE225-induced cell death was rescued by autophagy inhibitor 3-methyladenine (3-MA) ([@B47]) ([Figure 4D](#F4){ref-type="fig"}) suggesting that autophagy plays a critical role in LDE225-induced cytotoxicity in CD133^+^ cells. ![LDE225 induces autophagy in the CD133^+^ cells. **(A)** The graphs of transmission electron microscopy represented CD133^+^ cells treated with LDE225 (25 μM) or vehicle for 48 h. Arrows indicate autophagosomes. **(B)** Western blot analysis of LC3-I/LC3-II and Mushashi-1 expression in the CD133^+^ cells treated with LDE225 (25 μM) for 48 h. LDE225 induced conversion of LC3-I to LC3-II which was accompanied by the downregulation of Mushashi-1. **(C)** LC3 expression in CD133^+^ cells treated with LDE225 (25 μM) or vehicle for 48 h. The fluorescence intensity of LC3 punctate increased after LDE225 treatment. **(D)** Block of LDE225-induced cell death by autophagy inhibitor 3-MA. CD133^+^ cells were pretreated with 3-MA (3 μM) 1 h before the exposure to LDE225 (25 μM). Pretreatment with 3-MA attenuated LDE225-induced cell death. \**p* \< 0.05.](fonc-10-01233-g0004){#F4} We performed different apoptosis assays to evaluate whether LDE-induced cell death involves apoptosis. We used JC-1 mitochondrial membrane potential assay which measures the mitochondrial membrane potential as the indicator of cell health. Change in fluorescent property of JC-1 dye can be utilized to evaluate early apoptosis. CD133^+^ cells were treated with LDE225 (25 μM) or 0.1% DMSO as vehicle control for 48 h. [Supplemental Figure 3A](#SM3){ref-type="supplementary-material"} shows that vehicle led to mitochondrial depolarization and decreased the ratio of J-aggregate/JC-1 monomer while that was not different between vehicle and LDE225 (25 μM) treatment. Caspase-Glo assay also revealed that Caspase 3/7 activity was not different between LDE225 and vehicle ([Supplemental Figure 3B](#SM3){ref-type="supplementary-material"}). These results suggest that LDE225-induced cell death likely was not mediated primarily through apoptosis. Knockdown of Shh Slows Tumor Growth in an Intracranial Tumor Model ------------------------------------------------------------------ We clarified whether Shh plays a role in tumor growth under *in vivo* conditions using an orthotopic GBM model. To monitor intracranial tumor growth, we infected Luc-expressing CD133^+^ cells with lentiviruses carrying the expression vector containing *Shh* shRNA. Transduced CD133^+^ cells (8 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using IVIS-200 imaging system. [Figure 5A](#F5){ref-type="fig"} shows that tumor growth was much slower in *Shh* shRNA-knockdown mice than in control RNA-transfected mice. A two-way ANOVA revealed a main effect of group (*Shh* shRNA vs. control) \[*F*~(1,\ 11)~ = 4.980, *p* \< 0.05\], interaction \[*F*~(7,\ 77)~ = 3.110, *p* \< 0.01\] and days after application \[*F*~(7,\ 77)~ = 4.853, *p* \< 0.001\] ([Figure 5B](#F5){ref-type="fig"}). Kaplan--Meier analysis of the survival data demonstrated a statistically significant difference (*p* \< 0.05) in the median survival between vector control and *Shh* shRNA-treated mice ([Figure 5C](#F5){ref-type="fig"}). Brain tumor growth was analyzed in coronal brain slices by H&E staining. As shown in [Figure 5D](#F5){ref-type="fig"}, in comparison with vector control, shRNA-*Shh*-transfection generated significantly smaller intracranial tumor. ![Knockdown of Shh attenuates tumor growth in an intracranial tumor model. **(A)** Luc-expressing CD133^+^ cells were infected with lentiviruses carrying the expression vector containing *Shh* shRNA. Transduced CD133^+^ cells (8 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using the IVIS-200 imaging system. **(B)** Tumor growth was much slower in *Shh* shRNA- transfected mice than in control RNA-transfected mice. \**p* \< 0.05, \*\*\**p* \< 0.001 vs. control RNA-transfected. **(C)** Kaplan--Meier analysis of *Shh* shRNA transfection on survival. **(D)** Brains of mice harboring tumors generated from control RNA-transfected mice and *Shh* shRNA-transfected mice and stained by H&E. Note that tumor in *Shh* shRNA-transfected mice was significantly larger than control RNA-transfected tumor.](fonc-10-01233-g0005){#F5} To examine whether knockdown of Shh affected autophagy and stemness *in vivo*, we examined the expression of LC3-II in *Shh* shRNA-treated mice. [Supplemental Figure 4](#SM4){ref-type="supplementary-material"} shows that the levels of Shh, CD133, mushashi-1 and SOX2 were lower whereas the conversion of LC3-I to LC3-II was higher in mice of Shh knockdown CD133^+^ cells compared to CD133^+^ cells of control mice. These results suggest that knockdown of *Shh* reduces stemness and GBM tumor growth. Over-Expression of Shh Promotes Tumor Growth -------------------------------------------- To over-express Shh, we cloned the mouse *Shh* gene into parental U87 cells with lentiviruses carrying the expression vector containing *Shh* gene. The empty vector (LV-Vehicle) served as control. The LV-Shh or LV-vehicle transfected parental U87 GBM cells (8 × 10^4^ cells) was injected intracranially into athymic mice. As shown in [Figures 6A,B](#F6){ref-type="fig"}, tumor growth was much faster in LV-*Shh*-transfected mice than in LV-vehicle-transfected mice. A two-way ANOVA revealed a main effect of group (LV-*Shh* vs. control) \[*F*~(1,\ 6)~ = 7.665, *p* \< 0.05\], interaction \[*F*~(9,\ 54)~ = 4.022, *p* \< 0.001\] and days after application \[*F*~(9,\ 54)~ = 9.691, *p* \< 0.001\]. Kaplan--Meier analysis of the survival data demonstrated a statistically significant difference (*p* \< 0.05) in median survival between control and LV-*Shh*-treated mice ([Figure 6C](#F6){ref-type="fig"}). Brain tumor size was analyzed in coronal brain slices. As shown in [Figure 6D](#F6){ref-type="fig"}, in comparison with vector control, LV-*Shh*-transfection generated significantly larger intracranial tumor. ![Over-expression of Shh promotes tumor growth. **(A)** Shh overexpression in parental U87 cells was transduced by lentiviruses carrying the expression vector containing mouse *Shh* gene (LV-Shh). The empty vector (LV-Vehicle) served as control. The LV-Shh or LV-Vehicle infected U87 (8 × 10^4^ cells) was injected intracranially into athymic mice. Tumor growth was monitored by IVIS-200 imaging system. **(B)** Tumor growth was much faster in LV-*Shh*-transfected mice than in LV-vehicle-transfected mice. \**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001 vs. vector control. **(C)** Kaplan--Meier analysis of over-expression of Shh transfection on survival. **(D)** Brains of mice harboring tumors generated from LV- Vehicle and LV-*Shh*-transfection and stained by H&E. Note that LV-*Shh*-transfection tumor was significantly larger than vector control tumor.](fonc-10-01233-g0006){#F6} To examine whether over-expression of Shh affected autophagy and stemness *in vivo*, we examined the expression of LC3-II in LV-*Shh*-treated mice. [Supplemental Figure 5](#SM5){ref-type="supplementary-material"} shows that the levels of Shh, CD133, mushashi-1 and SOX2 were higher whereas the conversion of LC3-I to LC3-II was lower in Shh over-expression GBM compared to that of control. These results suggest that over-expression of *Shh* promotes cancer stemness and GBM tumor growth. Additive Effect of Rapamycin and LDE225 on The Viability of CD133^+^ Cells -------------------------------------------------------------------------- Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced by the mTOR inhibitor rapamycin ([@B48]--[@B50]). To confirm our hypothesis, we tested the effect of rapamycin on CD133^+^ cells. Single treatment of CD133^+^ cells with LDE225 (25 μM) or rapamycin (100 nM) inhibited the cell viability by 32.0 ± 7.9% (*n* = 6) and 24.6 ± 9.9% (*n* = 6), respectively. As shown in [Figure 7A](#F7){ref-type="fig"}, combination of LDE225 and rapamycin resulted in 54.6 ± 3.3% (*n* = 6) inhibition (*p* \< 0.05 vs. single treatment). Interestingly, combined treatment resulted in additive conversion of LC3-I to LC3-II. The conversion of LC3-I to LC3-II after LDE225 or rapamycin treatment was 159.8 ± 14.7% (*n* = 7) and 138.8 ± 14.5% (*n* = 7) of control, respectively. Combined treatment resulted in 217.7 ± 19.7% (*n* = 7) of control (*p* \< 0.05 vs. LDE225, *p* \< 0.01 vs. rapamycin) ([Figure 7B](#F7){ref-type="fig"}). ![LDE225 and rapamycin in combination additively reduces cell viability and induces conversion of LC3-I to LC3-II in CD133^+^ cells. **(A)** CD133^+^ cells were treated with LDE225 (25 μM) and rapamycin (100 nM) alone or in combination for 48 h and cell viability was assessed using WST-1 assay. **(B)** CD133^+^ cells were treated with LDE225 (25 μM) and rapamycin (100 nM) alone or in combination for 48 h and the levels of LC3-I and LC3-II were measured by Western blotting analysis. **(C)** CD133^+^ cells were treated with LDE225 (25 μM) and rapamycin (100 nM) alone or in combination for 48 h and the levels of p-mTOR and mTOR were measured by Western blotting analysis. Combined treatment with LDE225 and rapamycin reduced p-mTOR to the level not different from that treated with rapamycin alone (*p* = 0.2797). \**p* \< 0.05, \*\**p* \< 0.01.](fonc-10-01233-g0007){#F7} Autophagy can also be induced through mTOR-independent pathway ([@B50]). We determined the effect of LDE225 (25 μM) on mTOR phosphorylation. LDE225 (25 μM) did not significantly influence the phosphorylated level of mTOR (p-mTOR) whereas rapamycin (100 nM) reduced it. Combined treatment with LDE225 and rapamycin reduced p-mTOR to the level not significantly different from treatment with rapamycin alone (*p* = 0.2797) ([Figure 7C](#F7){ref-type="fig"}). Thus, it is likely that LDE225 induced autophagy through mTOR-independent pathway. Amiodarone Reduces Stem-Like Cell Viability and Inhibits Tumor Formation ------------------------------------------------------------------------ Amiodarone, a clinically used anti-arrhythmic drug, could induce autophagy via mTOR-independent signaling ([@B51]). We determined whether amiodarone reduced cancer stem-like cell viability *in vitro* and exhibited anti-tumor activity *in vivo*. CD133^+^ cells were treated with amiodarone for 48 h and cell viability was assessed using WST-1 assay. As shown in [Figure 8A](#F8){ref-type="fig"}, amiodarone dose-dependently reduced cell viability \[*F*~(7,\ 24)~ = 16.71, *p* \< 0.001\]. Immunoblotting using lysates from amiodarone (5 μM)-treated CD133^+^ cells revealed a significant increase in processed LC3-II \[*t*~(4)~ = 4.3976, *p* \< 0.05\] ([Figure 8B](#F8){ref-type="fig"}). Furthermore, amiodarone-induced cell death was reversed by autophagy inhibitor 3-methyladenine (3-MA) ([Figure 8C](#F8){ref-type="fig"}) suggesting that autophagy plays a critical role in amiodarone-induced cell death of CD133^+^ cells. CD133^+^ cells were treated with amiodarone (5 μM) or vehicle and CD133^+^-derived tumor spheroids were counted at 1, 3, 7, and 14 days. Treatment with amiodarone significantly reduced the number of tumor spheroids \[*t*~(4)~ = 10.09, *p* \< 0.001, at 14 days culture\] ([Figure 8D](#F8){ref-type="fig"}). ![Effects of amiodarone on the cell viability and tumor spheroid formation of CD133^+^ cells. **(A)** Concentration-dependent effect of amiodarone on the cell viability of CD133^+^ cells. \*\**p* \< 0.01, \*\*\**p* \< 0.001 vs. vehicle. **(B)** Western blot analysis of LC3-I and LC3-II expression in the CD133^+^ cells treated with amiodarone (5 μM) for 48 h. **(C)** Block of amiodarone-induced cell death by autophagy inhibitor 3-MA. CD133^+^ cells were pretreated with 3-MA (3 μM) 1 h before the exposure to amiodarone (5 μM). Pretreatment with 3-MA attenuated amiodarone-induced cell death. \*\**p* \< 0.01. **(D)** Effects of amiodarone (5 μM) on the number of tumor spheroids derived from CD133^+^ cells. Primary tumor spheroids derived from CD133^+^ were dissociated and cultured. They were then treated with amiodarone (5 μM) for the times as indicated. The medium was replaced every 2 days in the presence of amiodarone. \**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001 vs. Vehicle.](fonc-10-01233-g0008){#F8} We determined whether amiodarone inhibited tumor formation. Transduced CD133^+^ cells (6 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using IVIS-200 imaging system. At day 11, amiodarone was injected intraperitoneally once per day for 8 days (80 mg/kg) into the mice and tumor growth was observed for 17 more days. [Figure 9A](#F9){ref-type="fig"} shows that tumor growth at day 35 was much slower in amiodarone-treated mice than in control vehicle-treated mice \[*t*~(8)~ = 2.758, *p* \< 0.05\]. Brain tumor size was analyzed in coronal brain slices. As shown in [Figure 9B](#F9){ref-type="fig"}, in comparison with vehicle control, amiodarone-treated mice generated significantly smaller intracranial tumor. ![Amiodarone slows tumor growth in an intracranial tumor model. **(A)** Transduced CD133^+^ cells (6 × 10^4^ cells) were injected intracranially into athymic mice and tumor growth was monitored using the IVIS-200 imaging system. At day 11, amiodarone was injected intraperitoneally once per day for 8 days (80 mg/kg) into the mice and tumor growth was observed for 17 more days. \**p* \< 0.05 vs. vehicle. **(B)** Amiodarone-treated mice generated significantly smaller intracranial tumor by H&E staining.](fonc-10-01233-g0009){#F9} Discussion {#s4} ========== Roles of Shh Pathway in CD133^+^ Cells-Derived Tumor Spheroid Formation and Tumorigenesis ----------------------------------------------------------------------------------------- We have isolated CD133-positive cells from human U87MG (U87) GBM cells using magnetic bead cell sorting ([@B34]). CD133^+^-derived cell clones were able to grow *in vitro* in tumor spheroids and generate a tumor *in vivo* by intracranial cell injection in immunocompromised mice. The developmental signal transduction pathway mediated by Shh plays an important role during embryogenesis by regulating body patterning, cell proliferation and differentiation ([@B52], [@B53]). Activation of Shh has been causatively correlated with initiation and/or maintenance of cancer ([@B54]). In the present study, using Western blotting analysis and immunofluorescent staining, we showed that CD133^+^ cells exhibited higher Shh expression compared to CD133^−^ cells. Treatment with smoothened antagonist LDE225 significantly reduced the number of CD133^+^ cells-derived tumor spheroids suggesting Shh signaling likely contributes to tumor spheroids formation *in vitro*. Silencing *Shh* gene with small hairpin interfering RNA inhibited tumor growth in an intracranial mouse model. Conversely, over-expression of *Shh* gene facilitated tumor growth. Taken together, these results suggest that Shh signaling is involved in *in vitro* tumor spheroid formation and *in vivo* tumor growth. Shh Inhibitor Reduces the Number of CD133^+^-Derived Tumor Spheroids by Inducing Autophagy ------------------------------------------------------------------------------------------ The reports about the relationship between Shh and autophagy pathway in GBM are limited. One study in SHSY5Y cells showed that cyclopamine reduced serum starvation-induced LC3 positive cells but increased the levels of cleaved caspase 3 ([@B54]). They suggested that Shh inhibitor induced cell death by reducing autophagic processes concomitant with facilitating apoptotic cell death. In the present study, we demonstrated that LDE225 induced cell death in CD133^+^ cells in a concentration-dependent manner with IC~50~ of \~20 μM. Similar inhibition of cell survival was observed after application of another Shh inhibitor cyclopamine. Electron microscopy examination revealed that, after exposure to LDE225, CD133^+^ cells exhibited large membranous vacuoles in the cytoplasm that is a characteristic feature of cells undergoing autophagy. Further experiments of the conversion of LC3-I to LC3-II and attenuation of cell death by autophagy inhibitor 3-MA confirmed that LDE225-induced autophagy in the CD133^+^ cells. This study, in contrast to previous results, suggests that Shh inhibitor produces cytotoxicity in CD133^+^ cells by inducing autophagic cell death. To further test this hypothesis, we used rapamycin which is a mTOR inhibitor and is capable of inducing autophagy ([@B49], [@B51]). We found that rapamycin reduced CD133^+^ cell viability. Interestingly, the effects of LDE225 and rapamycin were additive. Furthermore, LDE225 at the concentration that induced autophagy and reduced cell viability of CD133^+^ cells did not affect the phosphorylated level of mTOR. Combined LDE225 and rapamycin failed to show further reduction of p-mTOR compared with rapamycin alone. These results suggest that LDE225 induces autophagy through mTOR-independent pathway. These findings also provide additional support that Shh regulation of autophagy plays an important role in the survival of GSCs. Thus, Shh pathway inhibitor could act as a sensitizer to increase efficiency of conventional chemotherapeutic agents in GBM by inducing cancer stem cell autophagic death. Anti-tumorigenesis of Amiodarone -------------------------------- Amiodarone, a frequently prescribed anti-arrhythmic drugs in clinics, has been identified as a mTOR-independent autophagy enhancer ([@B55], [@B56]). If LDE225-induced cell death was mediated by the mTOR-independent autophagy, then amiodarone should produce similar effects as LDE225. Indeed, this was the case. Amiodarone reduced CD133^+^ cell viability and tumor spheroid formation *in vitro* and exhibited anti-tumor efficacy *in vivo*. In summary, there was a small percentage of human U87 GBM cells which were CD133-positive, exhibited cancer stem cell markers and were capable of forming tumor spheroids *in vitro* and tumor *in vivo*. These CD133^+^ cells showed higher Shh expression and inhibition of Shh pathway with LDE225 reduced their survival and self-renew property. In LDE225-treated CD133^+^ cells, there appeared large membranous vacuoles in the cytoplasm and the conversion of LC3-I to LC3-II. LDE225-induced cell death was reversed by autophagy inhibitor 3-MA, suggesting that LDE225-induced autophagic cell death in CD133^+^ cells. Further, LDE225 did not affect the level of p-mTOR and combined LDE225 and rapamycin failed to show further reduction of p-mTOR when compared with rapamycin alone. Taken together, these results suggest that targeting Shh signal pathway may overcome chemoresistance and provide a therapeutic strategy for the treatment of malignant gliomas. Data Availability Statement {#s5} =========================== All datasets generated for this study are included in the article/[Supplementary Material](#s9){ref-type="sec"}. Ethics Statement {#s6} ================ The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the College of Medicine, NCKU. Author Contributions {#s7} ==================== J-YC, C-LS, and P-WG contributed to the conception and design of the experiments. H-CH and C-CL performed the experiments and statistical analysis. C-LS and P-WG wrote the paper. All authors contributed to the article and approved the submitted version. Conflict of Interest {#s8} ==================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Dr. Yang-Kao Wang for providing lentiviral package plasmids, Dr. Shih-Chieh Lin for providing GFP/Luciferase plasmid, Dr. Min-Der Lai for correcting the grammar and scientific editing of the manuscript, and the Laboratory Animal Center, College ofMedicine, National Cheng Kung University and Taiwan Animal Consortium for the technical support in IVIS. **Funding.** This study was supported by grants MOST 104-2320-B-006-007-MY3 and MOST-108-2320-B-006-008 from the Ministry of Sciences and Technology of Taiwan. Supplementary Material {#s9} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fonc.2020.01233/full#supplementary-material> ###### The differential expression of CD133 and cancer stem cell markers in parental GBM cells and tumor spheroids derived from CD133^+^ cells. **(A)** Immunofluorescence staining of CD133, SOX2, ABCG2, and AlDH1A1 in parental U87 cells. **(B)** Immunofluorescence staining of ABCG2 and AlDH1A1 in tumor spheroids derived from CD133^+^ cells. ###### Click here for additional data file. ###### The inhibitory effects of shh inhibition on cell proliferation. **(A)** Concentration-dependent effect of LDE225 on the cell viability of parental U87 cells. ^\*^*p* \< 0.05, ^\*\*\*^*p* \< 0.001 vs. vehicle. **(B)** Concentration-dependent effect of LDE225 on the cell viability of patient-derived P\#5 cell line. ^\*^*p* \< 0.05, ^\*\*\*^*p* \< 0.001 vs. vehicle. **(C)** Concentration-dependent effect of cyclopamine on the cell viability of CD133^+^ cells. ^\*^*p* \< 0.05, ^\*\*\*^*p* \< 0.001 vs. vehicle. ###### Click here for additional data file. ###### LDE225-induced cell death is not mediated primarily through apoptosis. CD133^+^ cells were treated with LDE225 (25 μM) or vehicle for 48 h. **(A)** JC-1 assay detect the changes of mitochondria potential which is able to measure early apoptosis. Vehicle (0.1% DMSO) led to mitochondrial depolarization with the decreased ratio of J-aggregate/JC-1 monomer. There was not different from LDE225 (25 μM) treatment. **(B)** Caspase/Glo assay revealed the activity of Caspase 3/7. There was not different between LDE225 and vehicle treatment. ###### Click here for additional data file. ###### The conversion of LC3-I to LC3-II was enhanced in *Shh* shRNA transfection CD133^+^-bearing mice. Tumor tissues were collected from *Shh* shRNA or vector-control transfection CD133^+^-bearing mice. **(A)** The efficiency of *Shh* shRNA-mediated knockdown of Shh was confirmed by western blot analysis. **(B--D)** The levels of CD133 **(B)**, mushashi-1 **(C)**, and SOX2 **(D)** were lower in *Shh* shRNA transfection CD133^+^-bearing mice. **(E)** The conversion of LC3-I to LC3-II was enhanced by *Shh* shRNA transfection. ^\*^*p* \< 0.05 vs. control. ###### Click here for additional data file. ###### The conversion of LC3-I to LC3-II was lower in Shh over-expression GBM-bearing mice. Tumor tissues were collected from LV-*Shh* or vector-control transfection GBM -bearing mice. **(A)** The efficiency of LV-*Shh*-mediated over-expression of Shh was confirmed by western blot analysis. **(B--D)** The levels of CD133 **(B)**, mushashi-1 **(C)**, and SOX2 **(D)** were higher in LV-*Shh* transfection GBM-bearing mice. **(E)** The conversion of LC3-I to LC3-II was reduced by LV-*Shh* transfection. ^\*^*p* \< 0.05, ^\*\*^*p* \< 0.01 vs. control. ###### Click here for additional data file. [^1]: Edited by: Liam Chen, Johns Hopkins University, United States [^2]: Reviewed by: Han Shen, Westmead Institute for Medical Research, Australia; Sujatha Venkataraman, University of Colorado Denver, United States [^3]: This article was submitted to Neuro-Oncology and Neurosurgical Oncology, a section of the journal Frontiers in Oncology

{ "pile_set_name": "PubMed Central" }

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"without", "respect", "life", "larger", "antiarrhythm", "inde", "financi", "well", "streptomycin", "five", "amiodaroneinduc", "attenu", "stem", "medicin", "′gcggaaggtatgaagggaaga′", "applic", "lentiviru", "chemotherapeut", "clontech", "antiabcg", "shh", "differenti", "condit", "includ", "multidrug", "error", "associ", "suspens", "de", "orf", "chuang", "show", "plate", "concept", "sd", "hairpin", "academia", "invas", "polystyren", "lab", "desir", "bradford", "model", "encod", "interact", "electron", "jaggregatejc", "intens", "revers", "tissu", "secondari", "overcom", "implic", "led", "per", "materialsreftypesec", "chemoresist", "mountain", "stereotax", "michigan", "food", "seed", "resist", "attach", "kept", "transfect", "p", "lentix™", "e", "scientif", "electrophoresi", "luciferas", "produc", "via", "interest", "bsmreftypesupplementarymateri", "mdr", "scienc", "result", "properti", "test", "concentr", "syring", "express", "chen", "anesthet", "conjug", "efficaci", "ic", "measur", "cassett", "cytoplasm", "incid", "inform", "follow", "dose", "transfer", "mani", "triton", "free", "santa", "capac", "dataset", "estim", "determin", "purchas", "water", "plvxnesireszsgreen", "westmead", "consist", "neuron", "ensur", "humid", "manual", "nuclear", "ministri", "min", "observ", "taken", "lci", "sonic", "caisson", "vehicletr", "vehicl", "vacuol", "diamidinophenylindol", "everi", "sigmaaldrich", "unfortun", "jyc", "prolifer", "consortium", "role", "low", "japan", "kung", "abl", "abfreftypefig", "isofluran", "famili", "cure", "dtt", "film", "target", "reveal", "dissoci", "facil", "goatantirat", "iodophenylnitrophenyldisulfophenylhtetrazolium", "neuroculttm", "blot", "cd", "prolong", "proliferact", "rnatransfect", "record", "fulllength", "inhibitori", "although", "downregul", "like", "sdspolyacrylamid", "marker", "technolog", "transmit", "univers", "proteasephosphatas", "protintech", "phenotyp", "balbcanncgfoxnnucrlnarl", "regul", "accompani", "replenish", "bulk", "heparin", "biolog", "neural", "potent", "demonstr", "appear", "surviv", "cayman", "present", "anim", "place", "semidri", "standard", "sinica", "critic", "serv", "group", "bfreftypefig", "modifi", "project", "optimum", "perkinelm", "stainingfoncgf", "analyz", "puromycinresist", "main", "amiodaron", "han", "patientderiv", "jc", "sever", "±", "homogen", "coloc", "harbor", "committe", "absorpt", "confirm", "induc", "weekold", "cotransfect", "microscopi", "cyclopamin", "click", "hh", "pellet", "sigma", "destruct", "reagent", "illinoi", "facilit", "cfreftypefig", "liam", "vertebr", "influenc", "sex", "gli", "day", "wrote", "ivi", "patch", "characterist", "hsiao", "genom", "contribut", "exhibit", "cultur", "sphere", "black", "autophagi", "absorb", "larg", "mix", "evalu", "−°cuntil", "egf", "reader", "tripl", "technic", "mostb", "ml", "least", "recurr", "mainten", "gel", "plko", "afreftypefig", "sox", "one", "shhoverexpress", "smaller", "reduct", "bd", "olympu", "millipor", "°c", "radiotherapi", "apoptosi", "balbcannfoxnlnucrlnarl", "eclplu", "easysep", "dye", "transcript", "nacl", "data", "slower", "discuss", "mice", "ltx", "carlsbad", "describ", "tube", "dluciferin", "factor", "immunostain", "furthermor", "left", "dp", "kill", "pattern", "sensit", "sri", "illustr", "novu", "treat", "facsaria", "ngml", "version", "midbrain", "system", "parent", "intracrani", "inhibit", "immunoblot", "mac", "densiti", "lvshh", "unpair", "supplementari", "antishh", "dmemhigh", "comparison", "±°c", "weekli", "atpbind", "cruz", "organogenesi", "texa", "bead", "cycl", "wst", "regrow", "filter", "coron", "ad", "abcg", "yangkao", "uml", "bb", "alkyl", "mm", "anticd", "stemcel", "antip", "laboratori", "lucexpress", "clarifi", "articl", "delay", "sequenc", "roundbottom", "window", "declar", "cytotox", "enhanc", "shen", "empti", "igg", "member", "fluoresc", "glioma", "rise", "import", "edit", "bloodbrainbarri", "devic", "bear", "current", "tri", "grove", "mbl", "interestingli", "paper", "requir", "repres", "mediumhigh", "play", "chang", "u", "independ", "lai", "mtor", "institut", "pvdf", "vectorcontrol", "size", "aldh", "surgic", "suspend", "bmi", "western", "invert", "nude", "avail", "httpswwwfrontiersinorgarticlesfoncfullsupplementarymateri", "fresh", "dodecyl", "relationship", "eagl", "temozolomid", "quantit", "co", "lv", "gbm", "primarili", "john", "asmreftypesupplementarymateri", "transport", "shown", "proteas", "intraperiton", "addit", "superfamili", "μl", "total", "rna", "high", "nonidet", "depolar", "activ", "hekt", "limit", "rat", "statement", "shrnatransfect", "note", "metamorph", "urgent", "approv", "radio", "nsa", "malign", "st", "rabbit", "resuspend", "lentivir", "examin", "phosphoryl", "counterstain", "breast", "edta", "effici", "humidifi", "radioresist", "stain", "statist", "membran", "lvshhtransfect", "chemo", "previous", "shsyy", "microscop", "alexa", "lin", "mgml", "correct", "sampl", "goatantirabbit", "median", "frame", "lvvehicl", "cheng", "count", "antilc", "f", "case", "materi", "deriv", "antigli", "cleav", "mortal", "antagonist", "percentag", "darmstadt", "absenc", "display", "shaken", "bregma", "biorad", "gsc", "molecular", "techniqu", "studi", "cocktail", "plasmid", "immunocompromis", "blue", "lcilcii", "line", "amiodaronetr", "loui", "harvest", "open", "character", "dm", "tumorfoncgf", "adult", "three", "plvxireszsgreen", "ix", "immunobilon", "usa", "mitochondri", "highli", "wang", "bfgf", "spinal", "product", "csc", "earli", "conserv", "fail", "neurooncolog", "west", "ldeinduc", "research", "gave", "find", "iggb", "pcr", "concomit", "cancer", "ripa", "hous", "transduc", "figur", "gbmbear", "clinic", "jaggreg", "band", "submit", "dysregul", "creat", "rapid", "autophagosom", "white", "conduct", "venkataraman", "efreftypefig", "supernat", "anoth", "part", "affect", "method", "lentivirus", "aberr", "concentrationdepend", "new", "cellswel", "primari", "danver", "dapi", "needl", "form", "prism", "hopkin", "leica", "often", "transduct", "procedur", "uk", "section", "csmreftypesupplementarymateri", "radiat", "albumin", "hydrat", "california", "lcii", "lysat", "approach", "variou", "gfp", "interf", "insert", "biomark", "μmtreat", "buffer", "contrast", "promot", "correl", "b", "rnabind", "effect", "magneticactiv", "lower", "mammalian", "slice", "rins", "fuji", "cd−", "contain", "sem", "cdderiv", "constru", "nih", "micropl", "−°c", "radianc", "antisox", "ying", "nucleat", "mount", "nutley", "function", "taiwan", "passag", "mean", "number", "lightdark", "introduct", "trishcl", "brain", "cage", "antiptch", "rate", "lvshhmediat", "patient", "manner", "togeth", "poor", "permeabl", "mediat", "australia", "ratio", "human", "prescrib", "cellsthes", "densitometri", "expos", "replac", "male", "depend", "denver", "chemotherapi", "aldha", "dulbecco", "next", "colleg", "depth", "inhibitor", "gfpluciferas", "lyse", "promis", "unitsml", "review", "corn", "neurolog", "firefli", "cambridg", "care", "pe", "hamilton", "falcontm", "n", "luminesc", "later", "chloral", "medium", "possess", "bioscienc", "design", "shrnaknockdown", "singl", "develop", "indic", "survivalfoncgf", "c", "sulfat", "alon", "format", "spheroid", "side", "manuscript", "vivo", "glioblastoma", "hypothesi", "necess", "hrpconjug", "magnet", "dfreftypefig", "carcinoma", "cdbear", "agent", "cellsderiv", "frontier", "analysi", "ldetreat", "g", "could", "simultan", "death", "onlin", "glycerol", "gaug", "dmso", "wisconsin", "unit", "region", "yield", "dna", "neg", "grow", "rapamycin", "apoptot", "rnai", "core", "excit", "rpm", "maintain", "softwar", "access", "improv", "shhposit", "increas", "incub", "report", "sujatha", "offer", "similar", "point", "×", "isol", "inject", "vehiclefoncgf", "summari", "colorado", "area", "lc", "clone", "fb", "previou", "rather", "therefor", "minder", "xenograft", "strategi", "mushashi", "bromophenol", "detect", "amplifi" ]

22,221

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"functions", "chemical", "signal", "transmitting", "information", "embryonic", "cells", "required", "normal", "development", "Shh", "plays", "critical", "role", "regulation", "vertebrate", "organogenesis", "development", "brain", "spinal", "cord", "including", "midbrain", "ventral", "forebrain", "neuronal", "differentiation", "proliferation", "many", "parts", "body", "role", "embryonic", "development", "aberrant", "dysregulation", "Shh", "signaling", "implicated", "initiation", "maintenance", "different", "types", "tumor", "including", "GBM", "Consistent", "reports", "Shh", "antagonists", "shown", "possess", "activity", "patients", "basal", "cell", "carcinoma", "medulloblastoma", "Cancer", "stem", "cells", "CSCs", "cancer", "cells", "possess", "ability", "replenish", "tumors", "differentiation", "multiple", "cell", "types", "CSCs", "identified", "breast", "colon", "brain", "areas", "differentiate", "cell", "phenotypes", "parental", "tumor", "CSCs", "hypothesized", "associated", "lead", "recurrence", "tumor", "formation", "theory", "conventional", "chemotherapies", "kill", "differentiated", "differentiating", "cells", "form", "bulk", "tumor", "sparing", "CSCs", "Therefore", "targeting", "CSCs", "offers", "promising", "approach", "improve", "cancer", "treatment", "even", "cure", "cancer", "present", "study", "isolated", "glioma", "cells", "GSCs", "human", "GBM", "cell", "line", "using", "marker", "found", "Shh", "expression", "higher", "cells", "cells", "smoothened", "antagonist", "delayed", "GBM", "growth", "vivo", "significantly", "reduced", "number", "tumor", "spheroids", "derived", "cells", "Furthermore", "tumor", "growth", "much", "slower", "Shh", "knockdown", "mice", "suggesting", "glioma", "growth", "may", "dependent", "small", "population", "cells", "regulated", "Shh", "pathway", "Materials", "Methods", "Animals", "nu", "mice", "purchased", "National", "Laboratory", "Animal", "Center", "NLAC", "Five", "mice", "housed", "cage", "controlled", "temperature", "humidity", "kept", "h", "cycle", "given", "free", "access", "water", "food", "Care", "use", "laboratory", "animals", "accordance", "National", "Institutes", "Health", "NIH", "guidelines", "procedures", "approved", "Institutional", "Animal", "Care", "Use", "Committee", "College", "Medicine", "NCKU", "project", "approval", "number", "Cell", "Culture", "human", "glioblastoma", "GBM", "cell", "lines", "provided", "Michael", "Hsiao", "Genomics", "Research", "Center", "Academia", "Sinica", "Taiwan", "GBM", "cell", "line", "developed", "Jian", "Ying", "Chuang", "cells", "cultured", "Modified", "Eagle", "glucose", "glucose", "Caisson", "supplemented", "fetal", "bovine", "serum", "FBS", "penicillin", "streptomycin", "Caisson", "cells", "maintained", "humidified", "incubator", "Isolation", "Characterization", "Cancer", "Stem", "Cells", "Glioblastoma", "Cell", "Line", "cell", "sorting", "MACS", "purification", "fresh", "GBM", "spheroids", "dissociated", "washed", "incubated", "either", "PE", "conjugated", "Miltenyi", "Biotech", "concentration", "nucleated", "cells", "per", "ml", "room", "temperature", "min", "PE", "selection", "cocktail", "cells", "added", "mixed", "min", "Magnetic", "cell", "separation", "performed", "using", "manual", "FalconTM", "polystyrene", "tubes", "Magnet", "machine", "Tube", "removed", "magnet", "cells", "resuspended", "appropriate", "amount", "desired", "medium", "cells", "incubated", "neural", "stem", "cells", "selection", "medium", "Basal", "Medium", "Proliferaction", "Supplement", "bFGF", "EGF", "Heparin", "STEMCELL", "Canada", "gave", "rise", "spheres", "Ultra", "Low", "Attachment", "Multiple", "Well", "Plates", "CORNING", "cells", "initially", "allowed", "form", "tumor", "spheroids", "suspension", "culture", "dissociated", "using", "Accutase", "BD", "Biosciences", "min", "split", "number", "tumor", "spheroids", "formed", "cells", "treated", "without", "μM", "days", "culture", "determined", "Olympus", "image", "analysis", "system", "Inverted", "fluorescence", "microscope", "Olympus", "Cell", "Proliferation", "Assay", "Cell", "proliferation", "measured", "using", "assay", "Clontech", "Laboratories", "California", "USA", "cells", "seeded", "concentration", "μl", "culture", "medium", "containing", "various", "concentrations", "cyclopamine", "final", "concentration", "μM", "plates", "cells", "incubated", "h", "reagent", "added", "cells", "incubated", "h", "absorbance", "product", "measured", "nm", "microplate", "ELISA", "reader", "cell", "counts", "determined", "percentage", "absorption", "relative", "control", "culture", "Western", "Blotting", "Assay", "Cell", "pellets", "collected", "centrifuged", "rpm", "stored", "cell", "pellets", "lysed", "RIPA", "lysis", "buffer", "containing", "mM", "Tris", "HCl", "pH", "mM", "NaCl", "Nonidet", "sodium", "deoxycholate", "sodium", "dodecyl", "sulfate", "SDS", "protease", "inhibitor", "Roche", "phosphatase", "inhibitor", "Roche", "Lysates", "shaken", "rpm", "ice", "h", "centrifuged", "rpm", "min", "mouse", "brain", "tissues", "homogenized", "lysis", "buffer", "mM", "pH", "sucrose", "mM", "EDTA", "inhibitor", "cocktail", "Roche", "Nutley", "USA", "Supernatants", "collected", "protein", "concentration", "measured", "Bradford", "assay", "protein", "resuspended", "sample", "buffer", "mM", "Tris", "glycerol", "SDS", "DTT", "Bromophenol", "blue", "total", "protein", "denatured", "min", "Protein", "electrophoresis", "gel", "volt", "separated", "protein", "transferred", "PVDF", "membrane", "Immunobilon", "transfer", "membranes", "Millipore", "transfer", "system", "volt", "Membranes", "incubated", "blocking", "buffer", "bovine", "serum", "albumin", "PBS", "Membranes", "incubated", "following", "primary", "antibodies", "rabbit", "Millipore", "Darmstadt", "Germany", "rabbit", "Cell", "Signaling", "Technology", "Danvers", "USA", "mouse", "MBL", "Japan", "mouse", "Millipore", "Darmstadt", "Germany", "rabbit", "Protintech", "Illinois", "USA", "rat", "Sigma", "Darmstadt", "Germany", "antibody", "overnight", "followed", "treatment", "secondary", "antibodies", "Jackson", "ImmunoResearch", "West", "Grove", "USA", "h", "room", "temperature", "three", "rinses", "Triton", "PBS", "min", "chemical", "reagents", "PerkinElmer", "added", "membrane", "Films", "Fuji", "Japan", "exposed", "different", "time", "points", "ensure", "optimum", "density", "band", "intensities", "analyzed", "densitometry", "using", "Windows", "version", "protein", "levels", "groups", "expressed", "percentage", "controls", "Immunofluorescent", "Staining", "immunostaining", "tumor", "spheroids", "cells", "initially", "allowed", "form", "spheroids", "suspension", "culture", "days", "cells", "fixed", "paraformaldehyde", "PBS", "pH", "min", "room", "temperature", "cells", "incubated", "h", "blocking", "solution", "bovine", "serum", "albumin", "incubated", "primary", "antibodies", "overnight", "following", "primary", "antibodies", "used", "mouse", "Millipore", "Darmstadt", "Germany", "rabbit", "Abcam", "Cambridge", "UK", "rabbit", "Millipore", "Darmstadt", "Germany", "rabbit", "Protintech", "Illinois", "USA", "rat", "Santa", "Cruz", "Texas", "US", "rabbit", "Novus", "Colorado", "US", "three", "rinses", "PBS", "containing", "Triton", "min", "cells", "incubated", "secondary", "antibodies", "room", "temperature", "following", "secondary", "antibodies", "used", "Alexa", "IgG", "Jackson", "ImmunoResearch", "West", "Grove", "USA", "Alexa", "IgG", "Alexa", "IgG", "Jackson", "ImmunoResearch", "West", "Grove", "USA", "DAPI", "Louis", "USA", "dye", "used", "nuclear", "counterstain", "cells", "washed", "PBS", "containing", "Triton", "three", "times", "min", "sections", "gold", "reagent", "Life", "Technologies", "Carlsbad", "USA", "Fluorescence", "signals", "detected", "Leica", "DM", "microscope", "MetaMorph", "image", "software", "used", "counting", "cells", "number", "Mitochondrial", "Membrane", "Potential", "Assay", "Early", "apoptosis", "measured", "using", "Mitochondrial", "Membrane", "Potential", "assay", "Cayman", "Chemical", "Michigan", "USA", "cells", "seeded", "concentration", "μl", "culture", "medium", "containing", "μM", "DMSO", "vehicle", "control", "black", "plates", "cells", "incubated", "h", "Staining", "solution", "added", "cells", "incubated", "min", "Plates", "centrifuged", "g", "room", "temperature", "supernatant", "removed", "buffer", "used", "suspend", "stained", "cells", "fluorescence", "intensity", "monomer", "measured", "excitation", "emission", "nm", "nm", "microplate", "ELISA", "reader", "ratio", "determined", "early", "apoptosis", "Assay", "activity", "caspase", "measured", "using", "Assay", "Promega", "Wisconsin", "US", "cells", "seeded", "concentration", "μl", "culture", "medium", "containing", "μM", "DMSO", "vehicle", "control", "white", "plates", "cells", "incubated", "h", "Reagent", "added", "cells", "incubated", "h", "luminescence", "recorded", "microplate", "ELISA", "reader", "activity", "caspase", 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Introduction ============ Microtubule-targeting agents (MTAs) have been used for a long time against a wide range of malignancies. It is generally believed that MTAs kill cancer cells by causing cell cycle arrest in M-phase, followed by activation of apoptotic pathways and cell death.[@b1-ott-10-5633]--[@b4-ott-10-5633] Many studies have used this rationale to explain the potent radiosensitization effects exerted by MTAs,[@b5-ott-10-5633],[@b6-ott-10-5633] ie, by inducing mitotic arrest, MTAs increase the proportion of tumor cells in a phase of the cell cycle that is very susceptible to DNA damage.[@b7-ott-10-5633],[@b8-ott-10-5633] Currently, chemoradiotherapy regimens including MTAs have been proven effective for the treatment of breast cancer, esophageal cancer and a variety of other neoplasms.[@b9-ott-10-5633],[@b10-ott-10-5633] Support for a mitosis-based mechanism for the therapeutic effects of MTAs has been derived from the characteristic side effects of these drugs, which include hair loss, neutropenia and gastrointestinal upset. These deleterious effects demonstrate the profound sensitivity of rapidly dividing tissues to MTAs. However, the action of MTAs on mitotic cells fails to explain their clinical efficacy against many slow-growing solid tumors with exceptionally low mitotic indices.[@b11-ott-10-5633] Most human tumors have a doubling time of 30--60 days or longer, making it unlikely that mitotic arrest serves as a critical mechanism of MTA-induced therapeutic benefit.[@b12-ott-10-5633] A prime example of this "proliferation rate paradox" is the significant activity of MTAs against adenocarcinoma of the prostate, a highly indolent cancer.[@b13-ott-10-5633],[@b14-ott-10-5633] Thus, a number of interphase-based mechanisms for the efficacy of MTAs in cancer therapy have been proposed, although not without controversy.[@b15-ott-10-5633],[@b16-ott-10-5633] A recent study has shown that the MTAs, vincristine (VCR) and paclitaxel, can delay DNA damage repair.[@b17-ott-10-5633] These MTAs were also shown to interfere with the trafficking of DNA damage response (DDR) proteins, including ATM, ATR and p53, from the cytosol to the nucleus, strongly suggesting that MTAs can sensitize cells to radiation by blocking microtubule-based transport of DDR proteins into the nucleus during interphase. It is challenging to physically separate mitotic from interphase cells in the presence of an MTA, as this results in a steady accumulation of new mitotic cells as long as the MTA is present. Thus, the question remains as to what extent the role of MTAs in radiosensitization is caused by interference with microtubule-facilitated nuclear import. To address this question, we took advantage of the fact that ionizing radiation (IR) treatment induces G2--M cell cycle arrest, thereby transiently eliminating the mitotic cell population and strongly enriching for interphase cells. Using glioblastoma cells and the MTA, mebendazole (MBZ), as a model system, we show that the effect of MBZ in interphase is responsible for the majority of the radiosensitization effect of this MTA. Materials and methods ===================== Cell lines and reagents ----------------------- GL261 (glioma) cells were obtained from the National Cancer Institute (Frederick, MD, USA). Cells were cultured in macrophage serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 1% penicillin/streptomycin and 1 mM L-glutamine. GBM14 cells have been described previously,[@b18-ott-10-5633] and they were obtained by Dr J Sarkaria at Mayo Clinic (Rochester, MN, USA) and cultured in StemPro media (Thermo Fisher Scientific), as directed by the manufacturer. Drug treatment -------------- For each experiment, GL261 and GBM14 cells were cultured without drug or treated with MBZ (Sigma-Aldrich Co., St Louis, MO, USA). MBZ stocks were prepared by dissolving the drug in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co.). About 24 hours after plating, glioma cells were treated with MBZ, while control cells were treated with 0.01% DMSO. Irradiation procedure --------------------- For all experiments requiring radiation, cells were irradiated using a biological irradiator (RS2000; Rad Source Technologies, Buford, GA, USA). Antibodies ---------- Phospho-MPM2 was obtained from EMD Millipore (Billerica, MA, USA), and γH2AX, H2AX, Chk2, Nbs1 and GAPDH were purchased from Cell Signaling (Danvers, MA, USA). Immunofluorescence ------------------ GL261 cells were seeded at a density of 30,000 cells/well in 24-well plates containing cover slips coated with laminin (Sigma-Aldrich Co.). The following day, cells were exposed to 6 Gy of IR. At designated time points post IR, cells were washed in PBS and fixed in 4% formaldehyde in PBS at room temperature. Once the cells were fixed, immunofluorescence was performed using an MPM2 antibody (EMD Millipore) and counterstained with 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI). For each condition, a total of 10 fluorescence micrograph images were taken with a Zeiss Axiovert 200M inverted microscope (Thornwood, NY, USA), running on Axiovision software. For each image field, the total number of cells and the number of MPM2-positive cells were quantified. The mitotic index was calculated at each time point using the number of MPM2-positive cells as a percentage of the total number of cells counted in all 10 fields. Cell viability assays --------------------- GL261 cells were seeded in 96-well plates at a density of 1,000 cells/well. GBM14 cells were seeded at a density of 10,000 cells/well. About 24 hours after seeding, cells were treated with 25--150 nM MBZ and irradiated with 3, 6 or 9 Gy. Drug treatment was applied for a 6-hour time window beginning either 6 hours pre IR or 3 hours post IR. Following the 6-hour window, the drug was washed out and replaced with fresh medium. Control cells were either left nonirradiated or irradiated with 3, 6 or 9 Gy of IR. Cell viability was examined 72 hours post IR using a WST-1 cell viability assay (Abcam, Cambridge, MA, USA) at an absorbance of 450 nm. For any given radiation dose--response, data were normalized by the fraction of viable cells treated with a given dose of drug in the absence of radiation. The radiosensitizing effect of MBZ and VCR was quantified using the dose enhancement factor (DEF) at the point of 50% (DEF~50~) cell viability. The DEF was calculated for each MBZ concentration using the following formula: (surviving fraction with radiation alone)/(surviving fraction with radiation + MBZ). Assessment of the DDR --------------------- GL261 cells were seeded at a density of 800,000 cells/well in 6-cm dishes. About 24 hours after seeding, cells were irradiated with 6 Gy of IR, followed by treatment with 150 nM MBZ during 3--9 hours post IR. Control cells were either left nonirradiated or irradiated with 6 Gy. All cells were harvested in lysis buffer at 0.5, 3, 6 and 9 hours post IR. The lysis buffer was composed of 50 mM Tris--HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% Nonidet P-40, 1× protease inhibitor (Thermo Fisher Scientific), phosphatase inhibitor (Hoffman-La Roche Ltd., Basel, Switzerland) and 0.4 U/mL Benzonase (EMD Millipore). For each condition, 60 μg of cell lysate was diluted in 1× NuPAGE Sample Reducing Agent (Thermo Fisher Scientific) and 1× NuPAGE LDS Sample Buffer (Thermo Fisher Scientific). All samples were loaded onto a NuPAGE, 4%--12% Bis-Tris Protein Gel (Thermo Fisher Scientific) for Western blot analysis. Histone H2AX and γH2AX were detected by Western blot using anti-histone H2AX or anti-γ-H2AX (Ser139) monoclonal antibodies (Cell Signaling). Nuclear and cytoplasmic fractionation ------------------------------------- GL261 cells were seeded at a density of 500,000 cells in 6-cm dishes. The following day, cells were treated with 3 Gy. MBZ treatment (25--250 nM) was performed for a 6-hour time window beginning 3 hours post IR. Irradiated control cells were treated with 3 Gy followed by 0.01% DMSO in medium during the same time period. Nonirradiated control cells were treated with 0.01% DMSO in medium for a total of 6 hours. After the 6-hour period of MBZ treatment, cells were harvested for protein analysis in cytoplasmic (C) and nuclear (N) fractions. The C and N fractions were collected according to the protocol of the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific). For each sample, 20% of the final volume of the C or N fractions was diluted in 1× NuPAGE Sample Reducing Agent and 1× NuPAGE LDS Sample Buffer. All samples were loaded onto a NuPAGE, 4%--12% Bis-Tris Protein Gel for Western blot analysis. Blots were incubated in antibodies: Nbs1, Chk2, GAPDH and H2AX monoclonal antibodies (all from Cell signaling). Cytoplasmic and nuclear fractions were normalized by GAPDH and H2AX levels, respectively. The percentage of cytoplasmic retention of DDR proteins was calculated by the following formula: \[C/(C + N)\] × 100%. Statistical analysis -------------------- All analyses were performed using Graphpad Prism 7 software. Radiosensitization experiments were analyzed using the two-way ANOVA method to compare the mean cell viability between treatment groups. The interaction between the MBZ treatment group and radiation dose was also examined. One-way ANOVA was used to compare the concentration of a drug that gives half-maximal response (EC~50~) values for each effect as outlined in [Table 1](#t1-ott-10-5633){ref-type="table"}. If a significant difference between means was found by ANOVA, then multiple comparisons between treatment groups were conducted. The Tukey--Kramer method was used to adjust for multiple comparisons. Studies examining the effect of MBZ on intracellular transport of DDR proteins were analyzed with unpaired two-tailed Student's *t*-test for direct comparisons of means. The same method was used to compare means in the analysis of H2AX phosphorylation following exposure to IR. For all studies, results were considered statistically significant for values of *P*\<0.05. Results ======= IR transiently eliminates the mitotic cell population ----------------------------------------------------- To select for a population composed entirely of interphase cells, we took advantage of the fact that IR treatment induces G2--M cell cycle arrest and transiently eliminates the mitotic cell population. Glioma cells were treated with 6 Gy of IR. To monitor the mitotic index, we quantified the proportion of MPM2-positive cells by immunofluorescence at several time points post IR. MPM2 is an antibody that recognizes a phosphorylated serine/threonine epitope found in proteins that are phosphorylated at the onset of mitosis.[@b19-ott-10-5633] MPM2 is recognized as a reliable mitotic marker in the literature and has been used to assess the mitotic index in cells exposed to radiation and a number of chemotherapeutic agents.[@b20-ott-10-5633]--[@b22-ott-10-5633] The baseline mitotic index of the glioma cell population was 3.5% and was strongly inhibited during a period of 3--10 hours after exposure to 6 Gy of IR. Recovery of the mitotic cell population begins after 10 hours post IR and completes by 24 hours post IR. To confirm that the mitotic index remained strongly inhibited even in the presence of MBZ, we applied MBZ during 3--9 hours post IR. In the presence of MBZ, the mitotic index remained suppressed at 4 hours (mitotic index =0.58%), 6 hours (mitotic index =0.12%) and 9 hours (mitotic index =0.28%) post IR. Thus, the treatment of GL261 glioma cells with 6 Gy of IR eliminated the mitotic cell population for a time period of \~6 hours ([Figure 1](#f1-ott-10-5633){ref-type="fig"}). MTAs sensitize interphase cells to IR ------------------------------------- In the following experiments, we compared the radiosensitizing effect of MTAs when applied during different time periods with respect to IR, using both murine GL261 cells and primary patient-derived GBM14 cells. A simplified schematic of all three treatment conditions is shown in [Figure 2](#f2-ott-10-5633){ref-type="fig"}. To examine whether MTAs can sensitize glioma cells to IR, we treated GL261 cells with MBZ ([Figures 3](#f3-ott-10-5633){ref-type="fig"} and [4](#f4-ott-10-5633){ref-type="fig"}) or VCR ([Figure 5](#f5-ott-10-5633){ref-type="fig"}). In order to test, whether MTAs can sensitize interphase cells to IR, we treated glioma cells with 25--150 nM MBZ during 3--9 hours post IR ([Figures 3A](#f3-ott-10-5633){ref-type="fig"} and [4A](#f4-ott-10-5633){ref-type="fig"}). The use of MBZ after exposure to IR made it possible to study the impact of MBZ independent of its effect on mitotic cells. Cells were exposed to MBZ after different doses of IR. After this 6-hour time frame, the drug was washed out and replaced with a drug-free medium. The radiosensitizing effect of MBZ on interphase cells was compared with the effect of MBZ on a cell population composed of both interphase and mitotic cells. Thus, cells were treated with MBZ for a 6-hour time window immediately prior to irradiation, and subsequently the drug was either washed out and replaced with drug-free medium ([Figures 3B](#f3-ott-10-5633){ref-type="fig"} and [4B](#f4-ott-10-5633){ref-type="fig"}) or left in the original medium until the end of the assay, 72 hours post IR ([Figures 3C](#f3-ott-10-5633){ref-type="fig"} and [4C](#f4-ott-10-5633){ref-type="fig"}). Treatment with MBZ post IR sensitized GL261 cells to IR with a maximal DEF at 50% viability (DEF~50~) of 1.34 ([Figure 3D](#f3-ott-10-5633){ref-type="fig"}). Treatment for a 6-hour time window pre IR, which increased the mitotic index to 9.2% from a baseline value of 2.8%,[@b23-ott-10-5633] sensitized glioma cells to IR with a maximal DEF~50~ of 1.2 in GL261 cells ([Figure 3E](#f3-ott-10-5633){ref-type="fig"}) and 1.33 in GBM14 cells ([Figure 4E](#f4-ott-10-5633){ref-type="fig"}). In the GL261 cell line, maximal radiosensitization was observed when the drug was applied pre IR and left in the medium for 72 hours, DEF~50~ =1.41 ([Figure 3F](#f3-ott-10-5633){ref-type="fig"}). In the GBM14 cell line, the application of the drug pre IR followed by 72-hour incubation sensitized cells to IR with a DEF~50~ =1.60 ([Figure 4F](#f4-ott-10-5633){ref-type="fig"}). Maximal radiosensitization in the GBM14 cell line was achieved when MBZ was applied post IR, DEF~50~ =1.69 ([Figure 4D](#f4-ott-10-5633){ref-type="fig"}). These observations show a maximal or near-maximal radiosensitization effect of MBZ when applied to cells during interphase. To investigate whether these findings can be generalized to other MTAs, we performed an identical set of experiments in GL261 cells using VCR, an established MTA that is frequently used in clinical practice. The radiosensitizing effect of VCR was greatest when applied post IR, DEF~50~ =1.53 ([Figure 5A and D](#f5-ott-10-5633){ref-type="fig"}). The application of VCR for a 6-hour period pre IR radiosensitized GL261 cells with a DEF~50~ of 1.34 ([Figure 5B and E](#f5-ott-10-5633){ref-type="fig"}). When VCR was applied pre IR and left in the medium for the remaining 72 hours, the magnitude of the radiosensitizing effect was quite similar, DEF~50~ =1.30 ([Figure 5C and F](#f5-ott-10-5633){ref-type="fig"}). Thus, it appears that the radiosensitizing effect of both MBZ and VCR is largely determined by the impact of these agents on interphase cells. MBZ treatment prolongs DDR after irradiation -------------------------------------------- To confirm that MTAs sensitize interphase cells to IR by interfering with the DDR, we examined the level of IR-induced phosphorylated H2AX (γH2AX), which is a highly sensitive marker of DNA damage.[@b24-ott-10-5633],[@b25-ott-10-5633] We exposed glioma cells to 6 Gy of IR, followed by treatment with 150 nM MBZ during 3--9 hours post IR. Cells were harvested at different time points post IR, and γH2AX was quantified by Western blot analysis. As shown in [Figure 6](#f6-ott-10-5633){ref-type="fig"}, treatment with MBZ led to more sustained γH2AX levels in response to IR, indicating a delay in the DDR. This finding demonstrated that MTAs sensitize interphase cells to IR by interfering with the DDR. MBZ interferes with the trafficking of DDR proteins --------------------------------------------------- To investigate whether MTAs synergize with IR by disrupting intracellular transport of DDR proteins, we performed cytoplasmic and nuclear (C/N) fractionation studies with glioma cells that had been exposed to IR followed by treatment with MBZ. Similar to the cell viability studies, MBZ was applied during 3--9 hours post IR (only when the mitotic cell population was absent). Glioma cells were treated with different concentrations of MBZ followed by C/N fractionation. Each fraction was analyzed by Western blot. We selected two DDR proteins, Chk2 and Nbs1, to serve as potential targets of MTA-mediated toxicity. Chk2 is a key protein kinase involved in the DDR that is responsible for cell cycle checkpoint activation and DNA repair following DNA damage induced by IR.[@b26-ott-10-5633] Nbs1 is a crucial component of an enzymatic complex that repairs double strand breaks (DSBs) following irradiation or heat shock.[@b27-ott-10-5633] In control cells, which had not been treated with MBZ, Chk2 and Nbs1 were localized entirely in the nucleus ([Figure 7A](#f7-ott-10-5633){ref-type="fig"}). Radiation treatment did not significantly alter the intracellular distribution of DDR proteins ([Figure 7A](#f7-ott-10-5633){ref-type="fig"}). Treatment of glioma cells with MBZ sequestered Chk2 and Nbs1 in the cytoplasm in a dose-dependent manner ([Figure 7B and C](#f7-ott-10-5633){ref-type="fig"}). These results demonstrate that MBZ, like other MTAs,[@b17-ott-10-5633] inhibits the trafficking of DDR proteins from the cytoplasm to the nucleus. MBZ sensitizes glioma cells to IR by interfering with the trafficking of DDR proteins ------------------------------------------------------------------------------------- To more closely examine the relationship between the effects of MBZ on intracellular trafficking of DDR proteins and IR sensitization, we compared the EC~50~ values for radiosensitization (DEF), cytoplasmic sequestration of DDR proteins and induction of mitotic arrest by MBZ ([Table 1](#t1-ott-10-5633){ref-type="table"}) in GL261 cells. The EC~50~ was defined as the concentration of MBZ required to achieve a half-maximal effect. The EC~50~ of cytoplasmic sequestration for Chk2 (31 nM) and Nbs1 (25 nM) was significantly lower than the EC~50~ of mitotic arrest (192 nM). Most notably, the EC~50~ of radiosensitization (35 nM) was very similar to the EC~50~ for cytoplasmic sequestration of DDR proteins, but significantly lower than the EC~50~ of mitotic arrest (*P*\<0.01). Similarly, the EC~50~ for radiosensitization by VCR (\<0.5 nM) is significantly lower than the EC~50~ for mitotic arrest in GL261 cells (2.5 nM).[@b23-ott-10-5633] These findings support the hypothesis that MBZ indeed radiosensitizes GL261 cells in large part by interfering with intracellular trafficking of DDR proteins during interphase. Discussion ========== Studying the mechanisms of action of MTAs in cell toxicity, and in particular whether MTAs target cells in mitotic phase or interphase, has been hampered by the difficulty in separating these two cell populations. In this study, we transiently eliminated the mitotic cell population by irradiating glioblastoma cells, which allowed us to examine the extent to which the MTA, MBZ, can radiosensitize these cells in a cell population that is essentially made up of interphase cells only. Our results show that in this system, the effect of MBZ in interphase is responsible for the majority of the radiosensitization effect caused by this MTA. For a long time, MTAs have been believed to inhibit tumor growth primarily by targeting mitotic cells, but this hypothesis has come under considerable scrutiny.[@b11-ott-10-5633],[@b12-ott-10-5633],[@b15-ott-10-5633],[@b16-ott-10-5633] We examined the role of interphase microtubules by determining the effect of MBZ as a radiosensitizer, when present during a time window in which mitosis is prevented by G2--M cell cycle arrest. We showed that this regimen is better at radiosensitization of tumor cells than when MBZ is present before IR administration, leading to an increase in the number of mitotic cells, and is very similar to that caused by chronic treatment with MBZ, strongly indicating that interphase microtubules are indeed the targets of MBZ. In addition, the EC~50~ of MBZ for radiosensitization is much lower than that for inducing mitotic arrest, further supporting the notion that the radiosensitizing effect of MBZ is independent of its effect on mitosis. We found essentially the same results for VCR. Thus, most likely this conclusion holds for a wide range of MTAs. We also observed that MBZ, even when applied for only 6 hours, leads to a strong delay in the DDR. Thus, our results strongly support the hypothesis that MTAs prolong DNA damage repair by interfering with the trafficking of DDR proteins from the cytosol to the nucleus.[@b17-ott-10-5633] Interestingly, the EC~50~ for radiosensitization by MBZ (35 nM) is very similar to that of cytoplasmic sequestration of DDR proteins by MBZ (25 nM), which is much lower than the EC~50~ of MBZ for the induction of mitotic arrest (184 nM), further supporting the notion that MTAs radiosensitize by blocking trafficking of DDR proteins to the nucleus. The low EC~50~ of MBZ for the inhibition of DDR protein trafficking is surprising, because it is also much lower than the EC~50~ for microtubule depolymerization (132 nM) that we determined recently.[@b23-ott-10-5633] This EC~50~ largely reflects that of the depolymerization of interphase microtubules, as most of the cells are in interphase. Although the mechanistic basis for the relatively low EC~50~ of MBZ for the inhibition of DDR protein trafficking remains to be determined, it will be of great interest to examine whether our findings with MBZ extend to other MTAs. Our observation that radiosensitization can be accomplished at a concentration of MBZ that is significantly lower than the concentration needed for cell killing on its own also has important clinical implications, as it suggests the possibility to utilize a dose of MBZ that minimizes toxicity. Importantly, our results also have implications for the optimal timing of administration of MTAs when used as radiosensitizers. Indeed, we show that optimal radiosensitization is obtained when inhibition of microtubule formation is achieved during the post-IR DNA repair period. This study also underlines the critical role of microtubule-based transport in the response to DNA damage. Thus, further elucidation of these mechanisms may lead to the identification of novel therapeutic targets for radiosensitization. Conclusion ========== We have shown that MBZ sensitizes cancer cells to IR in a manner that is largely independent of the induction of mitotic arrest by this microtubule inhibitor. We also provide evidence that MBZ-induced radiosensitization is mediated by inhibiting DDR protein accumulation into the nucleus. Thus, this study strongly supports a critical role for DDR protein trafficking in the response to radiation, suggesting that elements of the protein trafficking machinery can be mined for additional radiosensitization targets. GBM14 cells were kindly provided by Dr J Sarkaria, Mayo Clinic, Rochester, MN, USA. All data generated and analyzed during this study are included in this publication. This study was supported by grants to MS from the Swim Across America Foundation and the Project To Cure Foundation and to RR from the Zankel Foundation. **Author contributions** All authors contributed toward data analysis, drafting and critically revising the paper and agree to be accountable for all aspects of the work. **Disclosure** The authors report no conflicts of interest in this work. ![IR temporarily eliminates the mitotic cell population.\ **Notes:** GL261 cells were exposed to 6 Gy of IR, and the proportion of MPM2-positive cells was quantified by immunofluorescence as described in the "Immunofluorescence" section. MPM2 was utilized as a marker of the mitotic index. Data are expressed as the average ± SEM of three independent experiments.\ **Abbreviations:** IR, ionizing radiation; SEM, standard error of the mean.](ott-10-5633Fig1){#f1-ott-10-5633} ![Timing of MBZ application with respect to IR.\ **Notes:** (**A**) MBZ was applied for a 6-hour time frame beginning 6 hours pre IR followed by washout of the drug. During this time period, both mitotic and interphase cells were present. (**B**) MBZ was applied for a 6-hour time frame beginning 3 hours post IR followed by washout of the drug. During this time period, the mitotic cell population was absent (composed entirely of interphase cells). (**C**) MBZ was applied beginning 6 hour pre IR and left in the medium for the duration of the experiment.\ **Abbreviations:** IR, ionizing radiation; MBZ, mebendazole.](ott-10-5633Fig2){#f2-ott-10-5633} ![MBZ sensitizes GL261 cells to IR.\ **Notes:** GL261 cells were exposed to 25--150 nM of MBZ in conjunction with 3--9 Gy of IR. MBZ treatment was applied at different time points with respect to IR, and cell viability was determined by the WST assay as described in the "Cell viability assays" section. All data are expressed as the average ± SEM of four independent experiments. For each radiation dose--response curve, data were normalized by the fraction of viable cells treated with a given dose of MBZ in the absence of IR. DEFs were determined at the point of 50% cell viability. (**A**) Cells were treated with MBZ during 3--9 hours post IR. (**B**) Cells were treated with MBZ for a 6-hour time window immediately prior to irradiation. (**C**) Cells were treated with MBZ starting 6 hours prior to irradiation and until 72 hours post IR. *P*-values were \<0.05 for all treatments points when doses of MBZ ≥50 nM. (**D**--**F**) The respective DEFs were calculated as described in the "Cell viability assays" section. \**P*\<0.05, \*\**P*\<0.01, \*\*\**P*\<0.001.\ **Abbreviations:** DEF, dose enhancement factor; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean.](ott-10-5633Fig3){#f3-ott-10-5633} ![MBZ sensitizes GBM14 cells to IR.\ **Notes:** GBM14 cells were exposed to 50 or 150 nM of MBZ in conjunction with 3--9 Gy of IR. MBZ treatment was applied at different time points with respect to IR, and cell viability was determined by the WST assay as described in the "Cell viability assays" section. All data are expressed as the average ± SEM of three independent experiments. For each radiation dose--response curve, data were normalized by the fraction of viable cells treated with a given dose of MBZ in the absence of IR. DEFs were determined at the point of 50% cell viability. (**A**) Cells were treated with MBZ during 3--9 hours post IR. (**B**) Cells were treated with MBZ for a 6-hour time window immediately prior to irradiation. (**C**) Cells were treated with MBZ starting 6 hours prior to irradiation until 72 hours post IR. (**D**--**F**) The respective DEFs were calculated as described in the "Cell viability assays" section. \*\**P*\<0.01, \*\*\**P*\<0.001.\ **Abbreviations:** DEF, dose enhancement factor; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean.](ott-10-5633Fig4){#f4-ott-10-5633} ![VCR sensitizes GL261 cells to IR.\ **Notes:** GL261 cells were exposed to 0.5--2 nM of VCR in conjunction with 3--9 Gy of IR. Experiments with VCR were performed in an identical fashion to those with MBZ. All data are expressed as the average ± SEM of three independent experiments. For each radiation dose--response curve, data were normalized by the fraction of viable cells treated with a given dose of VCR in the absence of IR. DEFs were determined at the point of 50% cell viability. (**A**) Cells were treated with VCR during 3--9 hours post IR. (**B**) Cells were treated with VCR for a 6-hour time window immediately prior to irradiation. (**C**) Cells were treated with VCR starting 6 hours prior to irradiation until 72 hours post IR. (**D**--**F**) The respective DEFs were calculated as described in the "Cell viability assays" section. \**P*\<0.05, \*\**P*\<0.01, \*\*\**P*\<0.001.\ **Abbreviations:** DEF, dose enhancement factor; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean; VCR, vincristine.](ott-10-5633Fig5){#f5-ott-10-5633} ![MBZ prolongs the DDR after exposure to IR.\ **Notes:** (**A**) Western blot analysis. (**B**) Quantification of the Western blot bands. GL261 cells were treated with 6 Gy of IR followed by treatment with 150 nM of MBZ during 3--9 hours post IR. Control cells were treated only with IR. Following treatment, cell lysates were harvested at different time points (0.5--9 hours) post IR as described in the "Assessment of the DNA-damage response" section. Western blot analysis of γH2AX and H2AX levels was conducted at each time point post IR, and γH2AX levels were used as a measure of DNA damage. Data for each time point are expressed as the average ± SEM of three independent experiments.\ **Abbreviations:** DDR, DNA damage response; IR, ionizing radiation; MBZ, mebendazole; SEM, standard error of the mean.](ott-10-5633Fig6){#f6-ott-10-5633} ![MBZ interferes with the trafficking of DDR proteins from the cytoplasm to the nucleus.\ **Notes:** (**A**) GL261 cell were exposed to IR for 9 hours or left untreated. Cell lysates were collected using cytoplasmic and nuclear fractionation as described in the "Nuclear and cytoplasmic fractionation" section. In untreated GL261 cells, DDR proteins are localized to the nucleus. Exposure to IR did not alter the intracellular distribution of DDR proteins. (**B**) GL261 cells were exposed to IR followed by application of 25--250 nM of MBZ during 3--9 hours post IR. Western blot analysis of the cytoplasmic and nuclear fractions was conducted, and the levels of DDR proteins, Chk2 and Nbs1, were quantified for each fraction. Exposure to MBZ post IR resulted in the dose-dependent sequestration of DDR proteins in the cytoplasm. (**C**) The histogram shows quantification of Chk2 and Nbs1 levels from three independent experiments ± SE. \**P*\<0.001.\ **Abbreviations:** C, cytoplasmic; DDR, DNA damage response; IR, ionizing radiation; MBZ, mebendazole; N, nuclear; SE, standard error.](ott-10-5633Fig7){#f7-ott-10-5633} ###### DEF~50~ for MBZ-mediated radiosensitization, cytoplasmic sequestration of DDR proteins and induction of mitotic arrest Treatment EC~50~(nM) 95% CI ------------------------------ ------------------------------------------------- ---------- Radiosensitization (DEF~50~) 35 9--50 Nuclear trafficking (Chk2) 31 17--45 Nuclear trafficking (Nbs1) 25 18--32 Mitotic arrest 192[\*](#tfn2-ott-10-5633){ref-type="table-fn"} 127--257 **Notes:** Each EC~50~ value represents the average of three independent experiments with the representative 95% CI. DEFs were determined from WST assays conducted in GL261 cells with MBZ treatment during the period of 3--9 hours post IR. The EC~50~ of radiosensitization is defined by the half-maximal DEF at 50% cell viability (DEF~50~). Data used to determine the EC~50~ for induction of mitotic arrest by MBZ were obtained previously, also using GL261 cells.[@b23-ott-10-5633] *P*\<0.01. **Abbreviations:** CI, confidence interval; DDR, DNA damage response; DEF, dose enhancement factor; EC~50~, concentration of a drug that gives half-maximal response; IR, ionizing radiation; MBZ, mebendazole.

{ "pile_set_name": "PubMed Central" }

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"billerica", "suppress", "detect", "gm" ]

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INTRODUCTION {#sec1-1} ============ Vitamin D is a fat-soluble secosteroid required for intestinal absorption of calcium, magnesium, and phosphate. It has direct as well as indirect effects on bone. It is also essential for optimal functioning of various organs and tissues. It exerts its effect by binding to Vitamin D receptors (VDRs) on target cells. Vitamin D is derived from food and ultraviolet B photons. It undergoes hydroxylation in liver to form 25(OH)D3 and further in kidneys by enzyme 1α hydroxylase (1αOHase) to form 1, 25,(OH)2D3 (calcitriol), an active form. This active form is also produced at extrarenal sites. Enzyme 1αOHase (CYP27B1) required for this conversion is expressed by several target cells. In cases of infection, the pathogen-associated molecular patterns such as lipopolysaccharides, interferon gamma, and nitric oxide get attracted to toll-like receptors of macrophages. This regulates cytochrome P27B1 (CYP27B1) genes of the target cells. The expressed enzyme locally hydroxylates the inactive form (25\[OH\] D3) resulting in the production of calcitriol (1,25\[OH\] 2D3).\[[@ref1]\] This calcitriol exerts its effect on target cells which express VDR. Calcitriol enters the target cell through caveolae, containing VDR or by endocytic uptake of 25(OH)D3-D-binding protein, with the help of glycoproteins such as cubilin and megalin.\[[@ref2]\] After entering the cell, 1,25(OH)2D3 ligates with VDR in nucleus and generates vital biological responses. The 1,25(OH)2D3-VDR complex heterodimerizes with retinoid X receptor and shows DNA binding at Vitamin D response element, leading to RNA transcription and biological actions which include modulation of inflammatory response via promoting monocyte differentiation to macrophages; preventing release of inflammatory cytokines; preventing cell surface expression of major histocompatibility complex-II;\[[@ref3]\] downregulating T-cells and monocytes; downregulating the expression and production of several pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-8; and decreasing the nuclear factor-κB activity which is a transcription factor having a key role in immunomodulation.\[[@ref4]\] Vitamin D also regulates the expression of specific endogenous antimicrobial peptides.\[[@ref5]\] Since the discovery of VDR, three decades ago, more than fifty target cells have been identified, involving the receptor-mediated Vitamin D functions.\[[@ref6]\] To the best of our knowledge, very few studies have mentioned the presence of VDR in periodontal ligament (PDL) tissue. Investigations by Andrukhov *et al*.\[[@ref7]\] Tang and Meng,\[[@ref8]\] McMahon *et al*.,\[[@ref9]\] and Liu *et al*.\[[@ref10]\] have shown VDR-mediated activities in PDL tissue cells. These studies were however investigated on cell cultures and according to Bhalla *et al*.\[[@ref11]\] VDRs actually may not be present in tissues but may be acquired in cultures. They also stated that receptors may appear *in vivo* when cells are exposed to vascular or inflammatory cell products. Study by Onishi *et al*. in 2003\[[@ref12]\] and Chen in 2012\[[@ref13]\] elucidated immunolocalization of VDR during and at completion of root formation. VDR was detected in periodontium. However, these investigations do not confirm the presence of VDR in human PDL (hPDL) tissue of completely erupted teeth in adults. The present study was intended to evaluate the presence of VDRs in PDL tissue and study its response to serum 25(OH)D3 levels. Detection of VDR in hPDL tissue would implicate the involvement of Vitamin D-mediated response in periodontal tissue. MATERIALS AND METHODS {#sec1-2} ===================== Study design {#sec2-1} ------------ A total of 19 chronic periodontitis patients of both sexes with age ranging from 35 to 65 years, visiting the outpatient department of periodontology (from February 2015 to March 2017), were recruited for the present study. Their medical history was taken followed by a detailed periodontal examination. Individuals with a history of any systemic disease, medications (such as bisphosphonates, barbiturates, cardiac glycosides, cholestyramine, glucocorticoids, and laxatives), hypersensitivity reactions, Vitamin D or calcium supplements, smokers, pregnant women, and those with a history of antibiotic therapy within the last 3 months were excluded from the study. Periodontal examination {#sec2-2} ----------------------- The selected patients were examined clinically. Participants diagnosed with moderate-to-severe chronic periodontitis requiring extractions of at least two teeth (e.g., teeth with compound or complex pockets having 70%--75% bone loss on the involved surfaces and 50%--60% bone loss on the remaining surfaces; teeth with Grade III furcation involvement with severe vertical bone loss in interradicular area with 50%--60% bone loss on mesial and distal surfaces; teeth with severe recession on one side and 50%--60% bone loss on other sides along with mobility; patients with partially edentulous arches with 50%--60% bone loss on few remaining teeth and indicated for extraction for prosthodontic purposes) were considered for the study. PDL tissue was scraped from the remaining surfaces for evaluation of VDRs in the study. The study was approved by the Institutional Ethics Committee (reference number: Perio-II-1/2015--2016). Detailed informed written consent was obtained from all the selected participants, indicating voluntary participation in the study. Venous blood sample of approximately 2--3 ml was withdrawn in a vacutainer (with clot activator and gel for serum separation) from all the 19 chronic periodontitis patients for assessment of serum 25(OH)D3 levels. ADVIA Centaur Vitamin D Total (Vitamin D) assay (antibody competitive immunoassay) was used for the determination of total 25(OH)D3 in human serum. The reference range\[[@ref14]\] used for the study was as follows: deficiency (\<20 ng/mL), insufficiency (20--30 ng/mL), sufficiency (30--100 ng/mL), and toxicity (above 100 ng/mL). The sensitivity range of ADVIA Centaur Vitamin D assay is 4.2--150 ng/mL. Periodontal Phase I therapy (nonsupportive periodontal therapy) and extraction of tooth were carried out. The participants were then referred to physician for supplementation of Vitamin D3. The dose of cholecalciferol (Torrent Pharmaceuticals Ltd, Ahmedabad, India) was 60,000 IU once a week for 6 weeks. After supplementation, the participants were tested for serum 25(OH)D3 levels. This was followed by extraction of the second tooth. PDL tissue of the extracted tooth was used for the evaluation of VDR concentration. First, the extracted tooth was collected in a vial containing solution of 0.9%W/V sodium chloride, 50 U/mL of penicillin, and 50 μg/mL of streptomycin. The tooth was then washed with the same solution to remove blood completely. PDL tissue was scraped from the root surfaces with sterile curettes and placed in Eppendorf tube containing 1 ml of phosphate-buffered saline. The tubes were centrifuged at 4°C at 5000 gravity for 10 min. The supernatant was removed. Pellet was resuspended in 0.3ml of phosphate buffered saline. The tube was then subjected to three freeze-thaw cycles to facilitate cell lysis. Enzyme-linked immunosorbent assay procedure {#sec2-3} ------------------------------------------- PDL tissue samples were analyzed by using Human VDR ELISA Kit (Elabscience Catalog no E-EL-H2043) for *in vitro* quantitative determination of human VDR concentrations. The minimum detectable dose of human VDR by this kit was 0.38 ng/ml. The detection range was 0.63--40 ng/ml. The enzyme-linked immunosorbent assay (ELISA) procedure was performed as per the manufacturer\'s instructions. After preparing standard stock solution of 40 ng/ml and its serial dilutions, optical density (OD) values were obtained. Standard concentrations and OD values were plotted to get a standard curve. Actual ELISA for samples was then assayed using stock solution of 40 ng/ml as control. 100 μl of PDL tissue samples was added in respective wells of ELISA microplate in duplicate. They were incubated for 90 min at 37°C. 100 μl of diluted biotinylated detection antibody was added to all the wells. After incubation for 60 min at 37°C, the wells were washed with wash buffer for 3 times. 100 μl of horseradish peroxide conjugate was added and incubated for 30 min at 37°C. The wash process was repeated 5 times followed by addition of 90 μl of substrate reagent and incubation for 15 min at 37°C. 50 μl of stop solution was then added to it. ELISA plate was read on microplate reader at 450 nm. The OD readings of the samples were plotted on standard graph to get its VDR concentration. Statistical analysis {#sec2-4} -------------------- Data were statistically analyzed using SPSS version 16.0, IBM Corporation, Armonk, NY, USA. The intragroup comparison was done by paired t- test. To study the predictive reliability (goodness of fit) of the regression models, percentage *R*^2^ as a coefficient of determination was used. Correlation analysis was done using Pearson\'s method (Pearson\'s *r* value). *P* \<0.05 indicated statistically significant results (*P* \< 0.05, \*\**P* \< 0.001, \*\*\**P* \< 0.001), and NS indicated statistically nonsignificant results. Power of the study was 80%. RESULTS {#sec1-3} ======= A total of 19 chronic periodontitis patients were evaluated for 25(OH)D3 levels in serum and VDR concentration in human PDL tissue in the present study. The mean serum level before supplementation was 13.96 ng/mL with standard deviation of 3.36. The participants then underwent Vitamin D3 supplementation as prescribed by the physician. The mean serum 25(OH)D3 levels 6 weeks after supplementation of 60,000 IU Vitamin D3 increased to 35.12 ng/mL with a standard deviation of 5.27. The results are presented in [Table 1](#T1){ref-type="table"} and [Figure 1](#F1){ref-type="fig"} which depict significant increment in serum 25(OH)D3 levels after supplementation of Vitamin D3 (*P* \< 0.001). ###### Comparison of mean serum 25(OH)D3 levels before and after supplementation of Vitamin D3 Before supplementation (*n*=19) After supplementation (*n*=19) *P* ------------------------ --------------------------------- -------------------------------- ------- ------ ------------- Serum 25(OH)D3 (ng/ml) 13.96 3.36 35.12 5.27 0.001\*\*\* *\**Is a standard notation used to determine the level of significance.*P* value by independent sample "*t*-" test. *P*\<0.05 is considered to be statistically significant. \**P*\<0.05, \*\**P*\<0.01, \*\*\**P*\<0.001. SD -- Standard deviation; *n* -- Sample size; NS -- Statistically nonsignificant ![Comparison of mean serum 25(OH)D3 levels before and after supplementation](JISP-23-100-g001){#F1} The present study also involved the investigation of VDR in PDL tissue isolated from 19 participants of the above study group at both time intervals. VDR analysis was done by using Human Vitamin D Receptor ELISA kit. The mean VDR concentration in PDL tissue was calculated from OD values obtained from ELISA readings. The mean VDR concentration in periodontal ligament tissue of Vitamin D deficient, chronic periodontitis subjects was -1.443/ml with standard deviation of 0.604. The values increased to 2.38 /ml with standard deviation of 2.64 after supplementation, indicating highly significant increment (*P*-value \< 0.001) \[[Table 2](#T2){ref-type="table"} and [Figure 2](#F2){ref-type="fig"}\]. ###### Comparison of mean Vitamin D receptor concentrations before and after supplementation of Vitamin D3 Before supplementation (*n*=19) After supplementation (*n*=19) *P* --------------------------- --------------------------------- -------------------------------- ------ ------ ------------- VDR concentration (ng/ml) 3.31 1.73 8.78 3.24 0.001\*\*\* *\**Is a standard notation used to determine the level of significance. *P* value by independent sample *t*-test. *P*\<0.05 is considered to be statistically significant. \**P*\<0.05, \*\**P*\<0.01, \*\*\**P*\<0.001. VDR -- Vitamin D receptor; SD -- Standard deviation; *n* -- Sample size; NS -- Statistically nonsignificant ![Comparison of mean Vitamin D receptor concentration before and after supplementation](JISP-23-100-g002){#F2} The study also points out the correlation between VDR concentration and serum 25(OH)D3. When correlation was studied between VDR concentration and serum 25(OH)D3. The correlation coefficient "*r*" value before supplementation was 0.433. The *p* value obtained was 0.063 which indicated no significant correlation before supplementation. After supplementation the correlation coefficient "*r*" value was 0.480. The *P* value obtained was 0.048 which indicated significant positive correlation \[[Table 3](#T3){ref-type="table"}\]. The Linear relationship, before as well as after supplementation is presented in Figure [3a](#F3){ref-type="fig"} and [3b](#F3){ref-type="fig"}. ###### Correlation analysis of 25(OH)D3 levels and Vitamin D receptor concentrations before and after supplementation Before supplementation (*n*=19) After supplementation (*n*=19) --------------------------------- --------------------------------- -------------------------------- ------- --------- 25(OH)D3 with VDR concentration 0.476 0.039\* 0.480 0.038\* \*\*Is a standard notation used to determine the level of significance. Pearson's *r* value, *P*\<0.05 indicated statistically significant correlation. \**P*\<0.05, \*\**P*\<0.01, \*\*\**P*\<0.001. There was a statistically significant positive correlation between 25(OH)D3 levels and VDR concentrations before supplementation (*P*\<0.05) as well as after supplementation (*P*\<0.05). NS -- Statistically nonsignificant; VDR -- Vitamin D receptors; *n* -- Sample size ![(a) Linear relationship between serum 25(OH)D3 and VDR concentration in PDL tissue in chronic periodontitis subjects during deficiency state. On pearson\'s correlation analysis the "*r*" value before supplementation was 0.433 and the *P* value obtained was 0.063 indicating no significant positive correlation (*P* \> 0.05) between 25(OH)D3 and VDR concentration in PDL tissue when subjects were deficient; (b) Linear relationship between serum 25(OH)D3 and VDR concentration in periodontitis cases after attaining sufficient range. The correlation coefficient "*r*" value on supplementation was 0.480 with *P* value of 0.048. A significant positive correlation was noted (*P*\< 0.05) between serum 25(OH)D3 levels and VDR concentration as subjects became sufficient in D3](JISP-23-100-g003){#F3} The results of present study clearly demonstrated that VDR concentration in periodontal ligament tissue is fully dependent on serum 25(OH)D3 levels. Lower serum concentration of 25(OH)D3 was associated with decreased expression of VDRs and increase in 25(OH)D3 levels in serum was associated with increased expression of VDRs in periodontal ligament tissue. DISCUSSION {#sec1-4} ========== The present study evaluated serum concentrations of 25(OH)D3 and VDR concentration in PDL tissue in chronic periodontitis patients, before as well as after supplementation of Vitamin D3. The study also attempted to correlate serum 25(OH)D3 levels with VDR concentration in PDL tissue. The results of the study revealed that all the chronic periodontic patients were found to be deficient in Vitamin D3. According to Ritu,\[[@ref15]\] Vitamin D3 deficiency is common in the Indian population due to dark skin (increased melanin pigmentation), social and religious norms to cover maximum part of the body, tendency to protect self from sun exposure, vegetarian diet (which lacks Vitamin D), less Vitamin D fortification of food products, and because people live in highly dense areas. Marwaha,\[[@ref16]\] Sahu,\[[@ref17]\] and Khadilkar\[[@ref18]\] have reported high prevalence of Vitamin D deficiency in various groups of the Indian population including healthy School children, adolescents, rural girls, pregnant women, and health-care professionals. The study by Patil\[[@ref19]\] also confirmed Vitamin D deficiency in Indian population. They carried out a study on 48 chronic periodontitis and 37 periodontally healthy controls. In their study, 97.9% of chronic periodonttis patients demonstrated 25(OH)D3 deficiency or insufficiency, whereas only 2.1% of the participants had sufficient levels of 25(OH)D3. Studies by Boggess *et al*.,\[[@ref20]\] Chang *et al*.,\[[@ref21]\] Antonoglou *et al*.,\[[@ref22]\] and Abreu *et al*.\[[@ref23]\] have shown association of low Vitamin D3 levels and periodontitis. Thus, Vitamin D deficiency may be considered a risk factor for periodontitis and therefore its evaluation needs to be considered in periodontic patients. Comparison of 25(OH)D3 levels in the study group determined significant increase after supplementation of Vitamin D3 in all the study participants \[[Table 1](#T1){ref-type="table"} and [Figure 1](#F1){ref-type="fig"}\]. The results corroborated with earlier findings. Harinarayan\[[@ref24]\] showed that Vitamin D of 9572 IU/day for 8 weeks significantly raised the serum levels of 25(OH)D and that supplementation up to upper tolerable intake levels is safe and sufficient. The present study suggests that loading dose of cholecalciferol (60,000 IU weekly)\[[@ref25]\] for 6 weeks helps in increasing the levels of Vitamin D3 to sufficient range (≥30 ng/ml). In the present study, Vitamin D3 supplementation was given instead of Vitamin D2. Evidence suggests that cholecalciferol (D3) is superior to ergocalciferol (D2) in terms of potency, elevating and sustaining 25(OH)D concentrations, and maintaining the storage form of Vitamin D.\[[@ref26][@ref27]\] Studies by Trang *et al*.,\[[@ref28]\] Armas *et al*.,\[[@ref29]\] and Heaney\[[@ref30]\] also found that the D3 supplement was more effective in increasing serum 25(OH)D3 than D2. The dosage used in our study was 60,000 IU/week for 6 weeks as recommended by Vieth *et al*.\[[@ref31]\] and Hathcock *et al*.\[[@ref32]\] Similarly, Gupta *et al*.\[[@ref33]\] and Goswami *et al*.\[[@ref25]\] also used comparable dose and found serum 25(OH)D3 levels to increase. ELISA kit with D6 antibody was used to determine detectable levels of VDR in the PDL tissue. VDR concentration was found to be very less. Lower VDR concentration has also been demonstrated earlier in infectious condition. Marshall in 2008\[[@ref34]\] determined that sulphono-lipid capnine substance produced by some gliding bacteria disabled the VDR. Capnine binds to and inactivates the VDR leading to exaggerated inflammatory responses. Waterhouse *et al*.\[[@ref35]\] found that intracellular bacteria in chronic infectious condition dysregulated Vitamin D metabolism by causing VDR dysfunction within phagocytes. These data suggest the mechanism for reduced VDR levels in chronic infectious condition. In inflammatory conditions, the active macrophages through CYP27B1 vigorously convert 25(OH)D3 to its active form (1,25\[OH\] 2D3), further increasing the expression of VDR.\[[@ref36]\] Conversion of 25(OH)D3 into 1,25(OH)2D3 up to toxic levels has been demonstrated by Marshall in 2008\[[@ref34]\] and Waterhouse *et al*.\[[@ref36]\] In contrast to the above findings, the VDR concentration in the present study was found to be very low. The lower concentration of VDR was probably because of severe deficiency of Vitamin D in chronic periodontitis patients; hence, less 25(OH)D3 was available for conversion to active form, further leading to less expression of VDRs. Moreover, as Phase I therapy was initiated prior to the supplementation of Vitamin D3, there was considerable reduction of plaque-induced inflammation. As inflammation reduced, the response to inflammation was low. The results of the present study thus indicate that in cases of Vitamin D deficiency and infection or inflammation, the concentration of VDR decreases as seen in chronic periodontitis. The decreased levels of VDR would in turn be responsible for reduced effect of Vitamin D-mediated generation of adequate immune and inflammatory response. The VDR concentration in PDL tissue in chronic periodontic patients in the present study showed direct correlation with serum 25(OH)D3 levels after supplementation with Vitamin D3. After supplementation, D3 ligand binds to VDR and brings about conformational changes in VDR, making it less vulnerable to proteolytic attack. The following studies determined the increased stability of VDR with Vitamin D3. Li *et al*.\[[@ref37]\] found that VDR is degraded by ubiquitin/proteosome pathway. However, after supplementation, D3 bound to VDR and inhibited ubiquitination and retarded degradation, resulting in elevated VDR levels. Kongsbak *et al*.,\[[@ref38]\] Costa *et al*.,\[[@ref39]\] Zineb *et al*.,\[[@ref40]\] and Wiese *et al*.\[[@ref41]\] also found that 1,25(OH)2D3 stabilized and upregulated VDR protein expression by approximately 2--2.3 folds, by protecting it from proteasomal degradation, thus increasing half-life and mean lifetime of the VDR protein. In our study also, supplementation of Vitamin D3 after periodontal Phase I therapy may have helped in decreased degradation/increased stability of VDR and increased availability of 25(OH)D3 for conversion to 1,25(OH)2D3, increased binding of 1,25(OH)2D3 to VDR, thus leading to increased expression of VDRs. The results of the present study showed that 25(OH)D3 levels in serum and VDR concentration in periodontal ligament tissue was very less in chronic periodontitis patients before supplementation, which increased significantly after supplementation of Vitamin D3. In addition, a significant positive correlation was seen between serum 25(OH)D3 and VDR concentration in Periodontal Ligament tissue, after supplementation of Vitamin D3. Limitations {#sec2-5} ----------- The limitation of the study was patient compliance regarding supplementation of Vitamin D3. CONCLUSION {#sec1-5} ========== The present article determined the presence of VDRs in PDL tissue which responded to levels of Vitamin D3 in serum. The VDR levels were very low in subjects patients with Vitamin D3 deficiency, which increased significantly after supplementation of Vitamin D3 for 6 weeks. Vitamin D3 through VDR-mediated effect plays a crucial role in generating immune and anti-inflammatory responses (decreased production of pro-inflammatory cytokines such as interferon-γ, IL-17, IL-21, decreased levels of IL-6, IL-1β, and TNF-α, and increased apoptotic mechanisms in pro-inflammatory cells). Low levels of VDR are indicative of low immune response in these patients. Vitamin D supplementation could enhance VDR levels which in turn may contribute to enhanced immune response. The innovative finding of expression of VDRs in PDL tissue in fully erupted teeth has opened a new gate for further research. Mechanisms to understand its effect on periodontal regeneration (PDL fibroblast uniqueness and bone formation) need to be studied. Financial support and sponsorship {#sec2-6} --------------------------------- Nil. Conflicts of interest {#sec2-7} --------------------- There are no conflicts of interest.

{ "pile_set_name": "PubMed Central" }

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"vascular", "would", "understand", "fold", "transcript", "data", "pair", "discuss", "intend", "calcul", "tube", "factor", "last", "rural", "healthi", "dysfunct", "henc", "pattern", "sensit", "specif", "duplic", "antimicrobi", "vdrref", "respons", "intracellular", "instead", "increment", "ngml", "metabol", "version", "system", "kit", "year", "inhibit", "densiti", "tang", "patilref", "comparison", "conclus", "stop", "cycl", "liu", "month", "wv", "ad", "uml", "calcium", "articl", "oh", "plot", "rangeref", "enhanc", "freezethaw", "earlier", "exagger", "pvalu", "proinflammatori", "maximum", "requir", "endocyt", "consider", "chang", "play", "fibroblast", "nitric", "independ", "obtain", "institut", "size", "approxim", "avail", "therapi", "sun", "vacutain", "prevent", "relationship", "iuday", "rang", "quantit", "macrophag", "fifti", "receptor", "glycosid", "edentul", "shown", "reliabl", "sec", "addit", "ubiquitin", "cholestyramin", "rna", "total", "μl", "high", "magnesium", "ligament", 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"chronic", "periodontitis", "subjects", "standard", "deviation", "values", "increased", "standard", "deviation", "supplementation", "indicating", "highly", "significant", "increment", "P", "Table", "table", "Figure", "fig", "Comparison", "mean", "Vitamin", "receptor", "concentrations", "supplementation", "Vitamin", "supplementation", "n", "supplementation", "n", "P", "VDR", "concentration", "standard", "notation", "used", "determine", "level", "significance", "P", "value", "independent", "sample", "P", "considered", "statistically", "significant", "P", "P", "P", "VDR", "Vitamin", "receptor", "SD", "Standard", "deviation", "n", "Sample", "size", "NS", "Statistically", "nonsignificant", "Comparison", "mean", "Vitamin", "receptor", "concentration", "supplementation", "study", "also", "points", "correlation", "VDR", "concentration", "serum", "OH", "correlation", "studied", "VDR", "concentration", "serum", "OH", "correlation", "coefficient", "r", "value", "supplementation", "p", "value", 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"PDL", "tissue", "chronic", "periodontitis", "subjects", "deficiency", "state", "correlation", "analysis", "r", "value", "supplementation", "P", "value", "obtained", "indicating", "significant", "positive", "correlation", "P", "OH", "VDR", "concentration", "PDL", "tissue", "subjects", "deficient", "b", "Linear", "relationship", "serum", "OH", "VDR", "concentration", "periodontitis", "cases", "attaining", "sufficient", "range", "correlation", "coefficient", "r", "value", "supplementation", "P", "value", "significant", "positive", "correlation", "noted", "P", "serum", "OH", "levels", "VDR", "concentration", "subjects", "became", "sufficient", "results", "present", "study", "clearly", "demonstrated", "VDR", "concentration", "periodontal", "ligament", "tissue", "fully", "dependent", "serum", "OH", "levels", "Lower", "serum", "concentration", "OH", "associated", "decreased", "expression", "VDRs", "increase", "OH", "levels", "serum", "associated", "increased", "expression", "VDRs", "periodontal", "ligament", "tissue", "DISCUSSION", "present", "study", "evaluated", "serum", "concentrations", "OH", "VDR", "concentration", "PDL", "tissue", "chronic", "periodontitis", "patients", "well", "supplementation", "Vitamin", "study", "also", "attempted", "correlate", "serum", "OH", "levels", "VDR", "concentration", "PDL", "tissue", "results", "study", "revealed", "chronic", "periodontic", "patients", "found", "deficient", "Vitamin", "According", "Ritu", "Vitamin", "deficiency", "common", "Indian", "population", "due", "dark", "skin", "increased", "melanin", "pigmentation", "social", "religious", "norms", "cover", "maximum", "part", "body", "tendency", "protect", "self", "sun", "exposure", "vegetarian", "diet", "lacks", "Vitamin", "less", "Vitamin", "fortification", "food", "products", "people", "live", "highly", "dense", "areas", "Marwaha", "Sahu", "reported", "high", "prevalence", "Vitamin", "deficiency", "various", "groups", "Indian", "population", "including", "healthy", 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"indicative", "low", "immune", "response", "patients", "Vitamin", "supplementation", "could", "enhance", "VDR", "levels", "turn", "may", "contribute", "enhanced", "immune", "response", "innovative", "finding", "expression", "VDRs", "PDL", "tissue", "fully", "erupted", "teeth", "opened", "new", "gate", "research", "Mechanisms", "understand", "effect", "periodontal", "regeneration", "PDL", "fibroblast", "uniqueness", "bone", "formation", "need", "studied", "Financial", "support", "sponsorship", "Nil", "Conflicts", "interest", "conflicts", "interest" ]

Among Latinas living in the United States, breast cancer is the most common form of cancer and the leading cause of cancer death \[[@CR1]\]. Compared to non-Latina White women, Latinas are more likely to be diagnosed with regional or distant cancer \[[@CR1]\]. They are also more likely to undergo mastectomy (versus breast-conserving surgery) and are more likely to die of breast cancer than non-Latina White women diagnosed at a similar age and stage of cancer \[[@CR1]--[@CR4]\]. Given the higher rates of more advanced cancer and rigorous treatments reported in Latinas, the psychological and physical burden of breast cancer may be particularly grave for this ethnic group. In the large body of research on cancer-related quality of life (QOL), however, relatively few studies have focused on the breast cancer experience of ethnic minority women, with the possible exception of Black women \[[@CR5]\]. A diagnosis of breast cancer might be especially disruptive for Latinas in light of culturally specific causal attributions about cancer, including the notion that cancer represents punishment for one's sins, as well as fatalistic beliefs about cancer \[[@CR6]--[@CR8]\]. The importance of attending to QOL in Latinas is underscored by the observations that the Latino population is currently the largest and fastest growing ethnic minority population in the United States \[[@CR9]\], and Latinos are overrepresented among low-income and underserved groups \[[@CR10]\]. Quality of life is a multidimensional construct that encompasses various areas of functioning, including emotional, physical, sexual, and social domains \[[@CR11]\]. The physical domain pertains to a subjective evaluation of health status and bodily functioning (e.g., pain, fatigue, incontinence, lymphedema), whereas the emotional component encompasses psychological functioning, including positive and negative indicators of mood (e.g., anxiety, depressive symptoms, distress, affect) and perceived limitations due to emotional problems. Sexual quality of life generally refers to perceived sexual functioning, changes in sexual desire, and body image distress. The social domain often includes the impact of the disease on an individual's social role and the perceived utility of social support. These domains are not orthogonal, but rather are inter-related \[[@CR11], [@CR12]\]. For example, the experience of pain or fatigue may limit social and familial role performance. Additional indicators of quality of life include perceived cognitive functioning (e.g., memory) and spiritual well-being. The primary aim of this report is to present a systematic review of the literature to compare the QOL of Latinas diagnosed with breast cancer to QOL in women of other racial/ethnic groups. We consider evidence on socio-demographic factors, treatment-related variables, and other characteristics, such as level of acculturation, that may account for differences in QOL. Finally, we discuss directions for research and application. Method {#Sec1} ====== Defining quality of life {#Sec2} ------------------------ Our operational definition of quality of life was guided by findings from previous empirical studies and reviews that sought to illuminate the QOL of patients with breast cancer \[[@CR5], [@CR12], [@CR13]\]. The current review suggests four primary domains of QOL: mental, physical, social, and sexual. Low quality of life was operationalized as disruption in one of the aforementioned domains, presence of negative mood, or lack of positive mood. To illustrate, assessing the frequency of sexual activity in the absence of collecting information regarding its impact on functioning was not considered an assessment of QOL. Indices of QOL could be general or cancer-specific. Inclusion and exclusion criteria {#Sec3} -------------------------------- The current review included research on samples of women of self-identified Latina/Hispanic ethnic status with any stage of breast cancer. The U.S. Census Bureau \[[@CR14]\] defines Latino as individuals who identify themselves or their heritage as Mexican, Mexican American, Chicano, Puerto Rican, Cuban, or those whose origins are from Spain, the Spanish-speaking countries of Central or South America, and the Dominican Republic. To be included in the review, studies were required to provide a comparison of QOL between Latinas and at least one other ethnic/racial group. Studies were excluded if they did not report the QOL of Latinas as a separate group within the study. Dissertations, book chapters, and reviews were excluded. A systematic review of the literature from 1985 to 2009 was conducted using PubMed and PsycInfo. Studies had to be available online by December of 2009. In light of the advances in medical treatment for breast cancer over the past two decades, studies before 1985 were not considered. Manuscripts were retrieved using primary keywords (i.e., QOL, Latina/Hispanic, breast cancer) in combination with other words (e.g., fatigue, SF-36, depression), amounting to 30 key phrases[1](#Fn1){ref-type="fn"}. Another search was conducted to identify additional publications of all the authors whose manuscripts met entry criteria. References of selected studies were also reviewed to identify additional eligible studies. In total, 375 studies were reviewed. Of these studies, 22 met criteria.[2](#Fn2){ref-type="fn"} Studies were initially coded by the first author on 18 predetermined categories[3](#Fn3){ref-type="fn"} previously employed in a review on quality of life in African American cancer survivors \[[@CR5]\], and 25% of those were coded by the second author. Inter-rater reliability regarding agreement on the coding was 90%. Discrepancies were resolved through discussion among the authors. Results {#Sec4} ======= See Table [1](#Tab1){ref-type="table"} for socio-demographic and cancer-related descriptive data on each study's sample and Table [2](#Tab2){ref-type="table"} for information on research design, measures, and a summary of findings[4](#Fn4){ref-type="fn"}. With regard to research design, two qualitative studies, two longitudinal quantitative reports, and 18 cross-sectional quantitative reports met criteria for inclusion. Of the 20 quantitative reports, eight contained identical or overlapping samples. Most studies were conducted in large urban areas (Miami, Los Angeles, New York). Sample sizes for Latinas in quantitative studies ranged from 16 to 492 women (with the exception of Clauser et al. \[[@CR15]\]). Most women had early-stage disease. Breast cancer diagnosis duration was variable, with some relatively recently diagnosed samples (\<1 year) and some samples of longer-term survivors (\>2 years). Of the few studies that reported statistical tests comparing race/ethnicity by demographic and cancer-related variables, results showed that Latinas reported lower income and were more likely to be younger than other groups of women. Latinas were also more likely to be diagnosed at later stages compared to non-Latina Whites. Table 1Demographic data for studies included in the reviewAuthorsStudy SampleAgeCancer StageTime Since Diagnosis or Surgery^1^Income or EducationAshing-Giwa et al., 2004*n* = 26 LatinaLatina *M* = 56 years, range = 40--730 Latinas stage 0Latina *M* = 4 yearsNot provided*n* = 18 non-Latina White5 Latinas stage 1*n* = 24 Black7 Latinas stage 2*n* = 34 Asian American5 Latinas stage 30 Latinas stage 49 Latinas stage unknownAshing-Giwa et al., 2007^5^*n* = 183 LatinaLatina *M* = 5018 Latinas stage 0Latina *M* = 2.9 years87 Latinas \<25 K*n* = 179 non-Latina White59 Latinas stage 136 Latinas 25--45 K*n* = 135 Blacks75 Latinas stage 227 Latinas 45--75 K*n* = 206 Asian American28 Latinas stage 323 Latinas \>75 KLatinas reported significantly lower income than other groups of womenAshing-Giwa et al., 2009*n* = 183 Latina*\*M* = 55.03 years, range 29--62\*332 women stage 1\**M* = 2.98 years, *SD* = 1.67\*205 women \<25 K*n* = 179 non-Latina White267 women stage 2312 women 25--75 K*n* = 135 Blacks95 women stage 3186 women \>75 K*n* = 206 Asian AmericansLatinas significantly younger at diagnosis than other ethnic groupsLim et al., 2009*n* = 183 LatinaLatinas *M* = 53.45 years, *SD* = 11.518 Latinas stage 0Latinas *M* = 2.93 years, *SD* = 1.690 Latinas \<25 K*n* = 206 Asian59 Latinas stage 139 Latinas 25 K -- 45 K75 Latinas stage 229 Latinas \>45 K--75 KNo significant differences in age between Latinas and Asian women28 Latinas stage 3No significant differences between ethnic groups25 Latinas \>75 KLatinas significantly more likely to be diagnosed at a later stage than AsiansLatinas reported significantly lower income than AsiansBowen et al., 2007*n* = 95 Latina*\*M* = 55.55 years, *SD* = 10.4\*179 women in situ\*6.1, 24, and 35 months\*54 women \< = 10 K*n* = 486 non-Latina White453 women localized86 women \>10 K--20 K*n* = 199 Black172 women regional (unstaged)93 women \>20 K--30 K*n* = 24 Other168 women \>30 K--50 K211 women \>50 K--70 K113 women \>70 KCarver et al., 2003^6^*n* = 60 Latina*\*M*^*2*^ = 53.59 years, *SD* = 12.41\*10 women stage 0\*≤1 year post surgery\**M* = 14.25 years, *SD* = 2.87Study 1*n* = 26 Black135 women stage 1\*\<8 weeks*n* = 149 non-Latina White90 women stage 2Study 2*n* = 16 Latina*\*M*^*2*^ = 50.37 years, *SD* = 9.24\*9 women stage 0\*Years of education for full sample 15.71 years, *SD* = 5.33*n* = 5 Black47 women stage 1*n* = 72 non-Latina White41 women stage 2*n* = 4 OtherPetronis et al., 2003*n* = 62 Latina\**M* = 53.61 years, *SD* = 12.42\*11 women stage 0\*≤1 year post surgery\*Years of education at diagnosis for full sample 14.25 years, *SD* = 2.89*n* = 25 Black135 women stage 1*n* = 150 non-Latina White90 women stage 2Spencer et al., 1999*n* = 48 Latina\**M* = 53.75 years, *SD* = 12.62\*10 women stage 0\*≤1 year post surgery\*Years of education at diagnosis for full sample 14.39 years, *SD* = 2.80*n* = 24 Black128 women stage 1*n* = 151 non-Latina White85 women stage 2Carver et al., 2005^7^*n =* 32 Latina\* *M* = 54.18 years Age at diagnosis, *SD* = 10.61\*5 women stage 0\*5--13 years post surgery\*Years of education at diagnosis for full sample 14.18 years, *SD* = 3.44*n =* 114 non-Latina White101 women stage 1*n =* 17 Black57 women stage 2Carver et al., 2006*n =* 32 Latina\* *M* = 54.18 years Age at diagnosis, *SD* = 10.61\*5 women stage 0\*5--13 years after diagnosis\* Years of education at diagnosis for full sample 14.18 years, *SD* = 3.44*n =* 114 non-Latina White101 women stage 1*n =* 17 Black57 women stage 2Clauser et al., 2008*\*n* = over 5,500Not provided for women with breast cancerNot providedNot providedNot providedCulver et al., 2002Sample 1Sample 1Sample 1\*2 days prior to surgerySample 1*n* = 4 Latina*\*M* = 53.72 years, *SD* = 11.14\* 55 women stage 17 days post surgery*\*M* = 14.63 years, *SD* = 2.56*n* = 1 Black20 women stage 23, 6, 12 months post surgery*n* = 70 non-Latina WhiteSample 2Sample 2Sample 2Sample 2*n* = 49 Latina*\*M* = 56.29 years, *SD* = 10.27\*5 women stage 0*\*M* = 9.04 years, *SD* = 4.24^4^*n* = 7 Black21 women stage 130 women stage 2Eversley et al., 2005*n* = 29 LatinaLatina *M* = 47 years, range 29--687 Latinas stage 1\*All women diagnosed within the past 2 yearsLatina *M* monthly income \$1,119, range \$0--\$3,500*n* = 35 Black13 Latinas stage 2*n* = 35 non-Latina White7 Latinas stage 3Non-Latina Whites reported significantly greater income than all other groups of women.*n* = 17 Other2 Latinas stage 4Whites significantly more likely to be diagnosed with stage 1 than women from all other groups.Fatone et al., 2007*n* = 12 Latina\**M* = 54.10 years, *SD* = 10.54\*9 women stage 1--2\*13 women diagnosed between 1998--2001^3^\*7 women some high school or less*n* = 8 Black3 women stage 3--47 women diagnosed in 1997 or earlier13 women high school graduate or some college8 unknown stageFobair et al., 2006*n* = 45 Latina*\*n* = 107 Age \<40 years\* 88 women in situ\*253 women = 2--4 months\*102 women 12 years or less of school*n* = 389 non-Latina White*n* = 442 Age ≥40 years248 women localized275 women = 5--7 months\*446 women \>12 years of school*n* = 29 Black213 women regional (unstaged)20 women = 8 months or more*n* = 86 AsianFriedman et al., 2006.*n* = 22 Latina\**M* = 52 years, *SD* = 10.2Not provided\**M* = 26 months, *SD* = 33.5\*14 less than 8 years*n* = 45 Black14 some high school*n* = 14 non-Latina White24 high school/GED22 some college7 college graduateFu et al., 2009*n* = 63 Latina*n* = 22 Latinas \<452 Latinas stage 042 Latinas \<2 years29 Latinas \<10 K*n* = 58 non-Latina White*n* = 16 Latinas 45--5417 Latinas stage 118 Latinas 2--5 years11 Latinas 10 K--50 K*n* = 18 Black*n* = 13 Latinas 55--6427 Latinas stage 23 Latinas \>5 years8 Latinas 50 K--100 K*n* = 9 Latinas 65+8 Latinas stage 33 Latinas \>100 K\**M* = 52.5, *range* = 26--909 Latinas stage not reported12 Latinas income not reportedGiedzinska et al., 2004*n* = 78 Latina*M* = 51.4, range 30--74 Latinas\* Women were stage 0, 1, or 2Latina *M* = 3.07 years, *SD* = 1.213 Latinas \<15 K*n* = 233 Black18 Latinas 15 K--30 K*n* = 77 Asian American25 Latinas 30 K--45 K*n* = 233 non-Latina WhiteLatinas significantly younger than non-Latina Whites and Blacks8 Latinas 45 K--60 KNo significant differences between ethnic groups11 Latinas 60 K--75 K4 Latinas 75 K--100 K5 Latinas \>100 KNon-Latina Whites and Asians reported significantly higher income than Latinas.Janz et al., 2009*n* = 344 LatinaLatinas high acculturationLatina high acculturation\*Women diagnosed between 2005--2007Latinas high acculturation*n* = 386 Black*n* = 34 age \<50 years16 stage 022 \< High School diploma*n* = 726 non-Latina White*n* = 50 age 50--70 years41 stage 117 High School diploma*n* = 160 high acculturation Latina*n* = 16 \> 70 years29 stage 261 \> High School diploma*n* = 184 low acculturation LatinaLatina low acculturation14 stage 3Latina low acculturation*n* = 30 age \<50 yearsLatina low acculturation70 \< High School diploma*n* = 58 age 50--70 years21 stage 020 High School diploma*n* = 13 \> 70 years35 stage 110 \> High School diplomaLatinas more likely to be \<50 years than non-Latina Whites33 stage 212 stage 3Maly et al., 2010*n* = 492 Latina\* *M* = 50.8 years, *SD* = 9.5Not provided*\*M* = 180 days, *SD* = 20.8\* 377 women \< High School*n* = 54 Black534 women ≥ High School*n* = 292 non-Latina White*n* = 68 Asian/Pacific Islander*n* = 15 OtherMaly et al., 2008*n* = 99 Latina\* *M* = 68.7 years, *SD* = 8.5\*Only 8% of sample had greater than stage 2\* *M* = 8.20 months, *SD* = 2.751.5% of Latinas \< 20 K*n* = 66 Black10.9% of Whites \< 20 K*n* = 92 non-Latina WhiteNo significant differences between ethnic groupsNo significant differences between racial/ethnic groupsLatinas reported significantly lower income than non-Latina WhitesYoon et al., 2008*n =* 103 Latina English speaker*\*n* = 416 Age 50--59\*173 women in situ\* *M* = 223 days after diagnosis\*288 women \< 20 K*n =* 104 Latina Spanish speaker*n* = 198 Age 60--64487 women stage 1244 women 20 K--39 K*n* = 112 Black*n* = 349 Age 65--74368 women stage 2687 women 40 K+*n* = 834 non-Latina White*n* = 256 Age 75--9950 women stage 3*n* = 66 Other19 women stage 4122 women unknown stage\*Data only for breast cancer patients belonging to all ethnic groups^1^When time since diagnosis was not available time since surgery was provided^2^Data for the study comes from two recruitment samples^3^Study published in 2007 and no other information on time since diagnosis provided^4^Mean values on education were reported for each recruitment sample^5^Ashing Giwa et al., 2007, and Ashing Giwa et al., 2009 are the same data set and Lim et al., 2009 is from an overlapping dataset^6^Spencer et al., 1999, Carver et al, 2003 and Petronis et al., 2003 are overlapping data sets^7^Carver et al. (2005) and Carver et al. (2006) used the same data setTable 2Methodological data for studies included in the reviewAuthorAnalyses of DataMeasures or Interview TopicsResults SummaryAshing-Giwa et al., 2004QualitativeFear and anxiety, body image, intimate relationships.Latinas and Asian Americans discussed how their feelings about their body affected their QOL. Latinas discussed feeling embarrassed, sad, frustrated, ugly, and incomplete. Regarding the major theme of overall stress/effect of breast cancer, Blacks, Asians, and Latinas reported decrements in QOL whereas non-Latina Whites emphasized positive aspects. Latinas emphasized deleterious treatment side effects.Ashing-Giwa et al., 2007QuantitativeFACT-G (all subscales)Latinas were significantly lower on physical, social, emotional, and functional quality of life on FACT-G than non-Latina Whites, Blacks, and Asian Americans. After controlling for covariates, analysis on the FACT-G summary score indicated that ethnic differences were no longer significant. Latinas were significantly lower on SF-36 role limitation and emotional well-being compared to all other groups of women.Cross-sectionalRand/SF-36 (Role Limitation/Emotional Well-being)Ashing Giwa et al., 2009QuantitativeSF-36 (Physical and Mental)Latina ethnicity was significantly related to poorer Mental Health on the SF-36; this disparity was no longer significant when job type and stress were entered into the model. No significant relationship between Latina ethnicity and physical health QOL.Cross-sectionalLim et al., 2008QuantitativeFACT-G (Emotional and Physical)Emotional and Physical Well-being scores for Asian Americans were significantly higher than Latinas' scores.Cross-sectionalBowen et al., 2007QuantitativeSF-36 (Physical and Mental)Adjusting for covariates, Black women reported significantly lower impact of cancer on cognitive/mood and incontinence subscales assessing hormone-related symptoms than Latinas and Latinas reported more fear of recurrence than Black women. In a similar set of analyses examining differences in SF-36 Mental and Physical Health summary scores, also adjusting for covariates, Black women evidenced significantly lower physical functioning scores, compared with Latinas, but higher mental health scores. Two additional models were conducted with non-Latina White entered as the referent group. After controlling for covariates, no significant differences between Latinas and non-Latina Whites emerged on physical health, but Latinas evidenced significantly poorer mental health on the SF-36. Latinas did not significantly differ from non-Latina Whites on the scale to assess the social/emotional impact of breast cancer.Cross-sectionalFear of Recurrence ScaleHormone-related symptomsImpact of breast cancer(Social/Emotional)Carver et al., 2003QuantitativeSIP (Social Activities, Recreation and Pastime Activities subscales)Findings were similar to those of Spencer et al. (1999). Latinas reported more POMS fatigue than non-Latina Whites. Study 2 yielded no racial/ethnic differences in QOL on social disruption, fatigue, and distress.Cross-sectionalPOMS (Fatigue subscale)\*Emotional distressPetronis et al., 2003QuantitativeEmotional distress (CES-D, 11 items selected from Andrews and Withey SQOL 10 items selected from Carver et al., 1993)In models controlling for covariates, Latinas indicated significantly greater distress and more social disruption than non-Latinas, but groups did not differ on the PAIS.Cross-sectionalSIP (Social Activities, Recreation and Pastime Activities subscales)PAIS (Sexual subscale)PCBCSpencer et al., 1999QuantitativeEmotional disruption (CES-D, POMS, 11 items selected from Andrews and Withey SQOL )After controlling for covariates, Latinas reported poorer adjustment than non-Latina White and Black women in all domains of QOL.Cross-sectionalSIP (Social Activities and Recreational Pastimes subscales)PAIS (Sexual relations subscale)Carver et al., 2005QuantitativeCES-DLatinas had higher scores on the CES-D and SIP than non-Latinas (i.e., non-Latina Whites and Blacks). No significant differences between ethnic groups on distress (ABS/POMS) or self-rated QOL. Initial values on the two other dependent variables (SIP, CES-D) were entered simultaneously with demographic /medical factors. Latina ethnic status was not a significant predictor of change in depressive symptoms and social disruption.LongitudinalABS/POMSSIP (Social Activities, Recreation and Pastime Activities subscales)10 questions from Andrews and Withey SQOL (1976)Carver et al., 2006QuantitativeQLACSLatinas reported significant elevations in negative feelings, social avoidance, distress about family's future, and distress about recurrence compared to non-Latinas, even when covariates were controlled. Groups did not differ on cognitive impairment and fatigue subscales.Cross-sectionalClauser et al., 2008QuantitativeSF-36 (Mental and Physical)Asians evidenced the highest physical QOL, followed by non-Latina Whites, Latinas, and Blacks. Asians evidenced the highest mental QOL, followed by non-Latina Whites, Blacks, and Hispanics.Cross-sectionalCulver et al., 2002QuantitativeCES-DAfter controlling for covariates, Latinas did not significantly differ on distress from non-Latinas across time points. However, between 6 and 12 months after surgery, Latinas evidenced a significant increase in distress compared to non-Latina Whites. With covariates controlled, Black women reported significantly lower depressive symptoms than Latinas.Longitudinal\*DistressEversley et al., 2005QuantitativeCES-DLatinas had significantly higher depressive symptoms on the CES-D and fatigue on the Piper Fatigue Scale (PFS) when compared to Black and non-Latina White women. Latinas also evidenced higher rates of pain on the Brief Pain Inventory (BPI) and lymphedema-related swelling (measured by a single item) when compared to non-Latina White women. Latina ethnic status remained a correlate of total number of post-treatment symptoms even when covariates were controlled.Cross-sectionalPFSBPILymphedema-related SwellingFatone et al., 2007QualitativePhysical, Social/Functional, Psychological, Sexual, Cognitive, ExistentialThe primary domain of concern for the Latinas was psychological, whereas the primary domain of concern for Black women was physical. Main psychological themes for Latinas were sadness, crying, anxiety, and irritability. In descending order of importance, the remaining areas of concern for Latinas were physical, social/functional, spiritual/existential, cognitive, and sexual.Fobair et al., 2005QuantitativeMOS (Sexual Problems)After controlling for covariates, multiple regression analysis indicated significantly fewer sexual problems among Latinas compared to non-Latina White women.Cross-sectionalFriedman et al., 2006QuantitativeFACT-G (Emotional, Functional, Social/Family subscales)No significant differences for race/ethnicity on QOL.Cross-sectionalIES (Intrusions)Latinas, non-Latina Whites, and Blacks did not differ significantly on any of the measures.POMS-SF (all 6 subscales)Fu et al., 2009QuantitativeMSAS-SF (Depression, Hormone-related Symptoms, Pain-related Symptoms, Chemotherapy-related Symptoms)Latinas had more chemotherapy- and pain-related symptoms on the Memorial Symptoms Assessment Scale Short Form than non-Latina Whites when controlling for covariates. No significant ethnic differences emerged on the other areas of QOL.Cross-sectionalGiedzinska et al., 2004QuantitativeSF-36 (all subscales), CARES (Sexual Dysfunction, Sexual Interest, Body Image subscales)Black women reported significantly higher SF-36 Emotional Well-Being than Latinas, even after covariates were controlled for. No significant differences on the other SF-36 scales were reported. Mean differences in sexual dysfunction were not significant; however, when covariates were controlled, Black women evidenced less sexual dysfunction than Latinas. Black women reported higher Mental Health on the SF-36 than Latinas, even after controlling for covariates. Latinas scored higher than any other ethnic group on symptoms related to breast cancer treatment (BCPT summary score). After controlling for covariates, Latinas did not significantly differ on the BCPT from non-Latina Whites but remained higher than Black and Asian American women. No significant differences for Latinas emerged on the CES-D and Revised Dyadic Adjustment Scale.Cross-sectionalBCPTCES-DDASJanz et al., 2009QuantitativeFACT-B (Physical, Emotional, Functional, Social/Family, Breast Concerns)Low-acculturated and high-acculturated Latinas reported significantly lower physical well-being and more breast-specific concerns than non-Latina White women. Low-acculturated Latinas also reported significantly lower scores on functional well-being, emotional well-being, and social well-being compared to non-Latina Whites, who did not differ from high-acculturated Latinas. In the final model which included all covariates, Latinas low in acculturation reported significantly lower functional well-being and emotional well-being than non-Latina Whites, and more breast-specific concerns than non-Latina Whites, but did not significantly differ on physical and social well-being. Low-acculturated Latinas reported lower levels of functional and emotional well-being than Latinas high in acculturation and Black women.Cross-sectionalMaly et al., 2010Quantitative\*Self-reported symptoms and self-reported symptom resolution (nausea/vomiting, pain, depressive symptoms)In analyses controlling for covariates, both less-acculturated and more-acculturated Latinas were significantly more likely to report having experienced nausea than non-Latina Whites. Less acculturated Latinas were significantly less likely to report having experienced pain than non-Latina White women. Latinas did not significantly differ from non-Latina Whites on depressive symptoms. Less-acculturated Latinas were significantly less likely to perceive depressive symptom resolution than non-Latina White women in the unadjusted and adjusted models. Latinas were less likely that non-Latina Whites to perceive pain resolution; however, no differences emerged on the resolution of nausea or pain in the adjusted models.Cross-sectionalMaly et al., 2008QuantitativeSF-36 (all subscales)Latinas reported poorer QOL compared to non-Latina Whites. With control variables entered in the structural equation model, non-Latina ethnic status was directly associated with better QOL.Cross-sectionalCES-DSTAIBreast Cancer Specific AnxietyYoon et al., 2008Quantitative\* Physical symptoms (nausea/vomiting, difficulty sleeping, arm problems, vaginal dryness, hot flashes)After controlling for covariates, Spanish-speaking Latinas were approximately 60% less likely to report having any severe cancer-related symptoms than non-Latina White women, who did not differ from English-speaking Latinas. English-speaking Latinas were significantly less likely to report hot flashes and that Spanish-speaking Latinas were significantly less likely to report difficulty sleeping than non-Latina Whites.Cross-sectionalAshing Giwa et al., 2007, and Ashing Giwa et al., 2009 are the same data set and Lim et al., 2009 is an overlapping dataset. Carver et al. (2005) and Carver et al. (2006) used the same data set. Spencer et al., 1999, Carver et al, 2003 and Petronis et al., 2003 are overlapping data sets. Scales with reported alphas ranged from .70 to .96\*Author constructed scale. No other information on scale provided^+^QOL only measured at 1 time point*CES-D* Center for Epidemiologic Studies Depression; *SF-36* Rand Short-Form 36; *CARES* Cancer Rehabilitation Evaluation System; *BCPT* Breast Cancer Prevention Trial Symptom Checklist; *POMS* Profile of Mood States; *MSAS-SF* Memorial Symptoms Assessment Scale -- Short Form; *QLACS* Quality of Life in Adult Cancer Survivors; *FACT* Functional Assessment of Cancer Therapy. FACT-G (general cancer), and FACT-B (breast cancer specific) are the same measure except FACT-B contains an additional measure of breast cancer specific QOL; *IES* Impact of Event Scale; *STAI-S* State-Trait Anxiety Inventory; *ABS* Affects Balance Scale; *SIP* Sickness Impact Profile; *SQOL* Self-Rated Quality of Life; *PCBC* Profile of Concerns about Breast Cancer; *QOL* Quality of Life; *PAIS* Psychological Adjustment to Illness Scale; *PFS* Piper Fatigue Scale; *BCPC* Breast Cancer Problems Checklist; *BPI* Brief Pain Inventory; *DAS* Dyadic Adjustment Scale Results are organized by QOL domain. We reviewed findings by each domain and most-commonly employed measures within domains. In the event that two separate measures were used to assess a QOL domain, we report findings for each measure. We also provide findings by measure subscale if reported in the study's results section. Not all studies in the review disentangled domains of QOL but rather reported total scores on measures of QOL that included some combination of the four domains assessed in this review (e.g., mental, physical, social, sexual). In these cases, the relationships between racial/ethnic status and distinct domains of one of the four QOL domains could not be determined. Therefore, for purposes of this review, an additional section labeled Combined QOL Domains was included. Finally, only QOL findings that pertain to Latinas are reported. In other words, QOL comparisons between African American, Asian American, and non-Latina White women are not reported. Findings related to mental health {#Sec5} --------------------------------- Findings related to mental health are divided into two sections: 1) general emotional functioning which includes mental health QOL, anxiety, distress, fear of recurrence, or a combination of these variables, and 2) depressive symptoms. Despite the variations in measurement, a consistent finding among quantitative studies is that Latinas generally report poorer emotional functioning than non-Latinas. The most commonly used measures of general emotional functioning were the Rand Short-Form 36 (SF-36) and Functional Assessment of Cancer Therapy (FACT). In 12 studies, Latinas demonstrated significantly poorer QOL than non-Latina Whites, Blacks, and/or Asian Americans on at least one measure of emotional functioning compared to non-Latina Whites, Blacks, and/or Asian Americans \[[@CR15]--[@CR26]\]. In general, Latinas reported poorer mental health on the SF-36 and the FACT. They also reported greater fear of recurrence and distress. Eight of the 12 studies assessing mental health controlled for relevant covariates and continued to report significantly poorer QOL among Latinas compared to other women. Four studies that assessed emotional functioning reported no significant differences between groups on at least one measure of general emotional functioning among Latinas, non-Latina Whites, and/or Blacks \[[@CR16], [@CR22], [@CR25], [@CR27]\]. Two of these four studies present both positive and negative findings for Latinas. Janz et al. \[[@CR22]\] reported that both low-acculturated and high-acculturated Latinas evidenced poorer QOL compared to non-Latina Whites. Their findings also revealed that high-acculturated Latinas did not significantly differ from non-Latina Whites on some QOL measures and that disparities in QOL were stronger among low-acculturated Latinas than high-acculturated Latinas \[[@CR22]\]. Although Culver et al. \[[@CR16]\] did not find significant ethnic group differences in distress, they did report a significant increase in distress over time in Latinas compared to non-Latina Whites. Finally, no study reported that Latinas evidenced better emotional functioning than women of other ethnic groups. The most commonly used measure of depressive symptoms was the Center for Epidemiologic Studies Depression scale (CES-D). Four quantitative studies \[[@CR16], [@CR28]--[@CR30]\] reported significantly greater levels of depressive symptoms or less depressive symptom resolution among Latinas than non-Latina Whites and/or Blacks on at least one measure. Three of these studies continued to report significant differences in depressive symptomatology despite controlling for covariates. Three studies reported no significant differences between groups on depressive symptoms compared to non-Latina Whites and/or Blacks \[[@CR17], [@CR30], [@CR31]\] on at least one measure. Of note, Maly et al. \[[@CR30]\] reported that Latinas did not significantly differ from non-Latina Whites on depressive symptoms, but less-acculturated Latinas were less likely to report depressive symptom resolution compared to non-Latina Whites. No study reported that Latinas evidenced significantly lower depressive symptomatology than other groups of women. Taken together, these findings suggest that Latina breast cancer patients report greater depressive symptoms and poorer mental health than non-Latinas. Findings related to physical health {#Sec6} ----------------------------------- Although less consistent than the findings pertaining to mental health, the majority of studies suggested that Latinas evidenced worse QOL related to physical health. The most commonly employed measures of physical health were the SF-36 and FACT. Other measures included treatment-related symptoms such as fatigue pain, nausea, and lymphedema. Ten studies reported that Latinas evidenced poorer QOL compared to non-Latina Whites, Blacks, and/or Asian Americans on at least one measure of physical health \[[@CR15], [@CR17], [@CR18], [@CR20], [@CR22], [@CR23], [@CR25], [@CR29]--[@CR31]\]. In general, Latinas reported poorer general physical QOL on the SF-36 and FACT and more cancer treatment-related symptoms such as fatigue, pain, and lymphedema swelling. Of these 10 studies, six reported significantly poorer QOL among Latinas than other groups of women even after controlling for relevant covariates. Five studies reported no significant differences between Latinas and non-Latina Whites, Blacks, and/or Asian Americans on at least one measure of physical health \[[@CR17], [@CR19], [@CR21], [@CR25], [@CR31]\], and three studies reported that Latinas evidenced superior QOL on at least one measure of physical health compared to non-Latina Whites and Blacks \[[@CR20], [@CR30], [@CR32]\]. Among the studies reporting superior physical QOL, findings indicated that Latinas evidenced better physical functioning on the SF-36, less pain, fewer hot flashes, and less difficulty sleeping. Findings related to social functioning {#Sec7} -------------------------------------- Seven studies assessed social functioning. Commonly used measures of social functioning included the SF-36, FACT, and Sickness Impact Profile. Six studies reported that Latinas demonstrated poorer social functioning than non-Latina Whites, Blacks, and/or Asian Americans \[[@CR18], [@CR21], [@CR22], [@CR24], [@CR26], [@CR28]\]. Of these six studies, three reported significantly poorer QOL among Latinas after controlling for relevant covariates. Five reported no significant differences between Latinas and non-Latina Whites and Black women \[[@CR17], [@CR20], [@CR22], [@CR25], [@CR27]\]. Of note, Janz et al. \[[@CR22]\] reported that low-acculturated Latinas but not high-acculturated Latinas reported significantly lower scores on social well-being. No study reported better social functioning among Latinas compared to other groups of women. The pattern of findings suggests that Latinas are more likely to report poor social functioning compared to non-Latinas. Findings related to sexual health {#Sec8} --------------------------------- Four studies reported data pertaining to sexual health. Two of the four studies reported greater sexual dysfunction among Latinas compared to non-Latina Whites, Blacks, and Asian Americans \[[@CR17], [@CR24]\]. Both of these studies reported significant differences after controlling for relevant covariates. One study reported fewer sexual problems among Latinas \[[@CR33]\] compared to non-Latina Whites, and one study reported no differences in sexual disruption between Latina and non-Latina Whites and Black women \[[@CR26]\]. Given the dearth of studies assessing sexual health and lack of consistent findings, it is difficult draw to any conclusion for this QOL domain. Findings related to combined QOL domains {#Sec9} ---------------------------------------- One study combined several domains of QOL. Using a multiethnic sample of non-Latina White, Latina, and Black breast cancer patients, Maly et al. \[[@CR34]\] employed a structural equation model to examine predictors of QOL. Quality of life was a latent variable consisting of the Medical Outcomes Study 36-Item Short Form (SF-36), the Center for Epidemiologic Studies Depression Scale (CES-D), the State-Trait Anxiety Inventory-State (STAI-S), and a scale on breast cancer emotional health. Findings indicate that Latinas reported poorer QOL compared to non-Latina Whites. In a structural equation model with control variables included, non-Latina ethnic status was directly associated with better QOL \[[@CR34]\]. Maly et al. \[[@CR34]\] primarily assessed mental health, and the study's results are consistent with the Findings Related to Mental Health section, in which the majority of studies reported significantly poorer mental health among Latinas compared to other ethnic groups. Qualitative studies {#Sec10} ------------------- Fatone et al. \[[@CR35]\] conducted semi-structured interviews with a sample of Latina and Black women. The primary domain of concern for the Latinas was psychological, whereas the primary domain of concern for Black women was physical. Main psychological themes for Latinas were sadness, crying, anxiety, and irritability. In descending order of importance, the remaining areas of concern for Latinas were physical, social/functional, spiritual/existential, cognitive, and sexual. Another qualitative study sought to understand the breast cancer experiences of Black, Asian American, non-Latina White, and Latina breast cancer survivors \[[@CR36]\]. Participants were interviewed in a focus group format. Two themes related to QOL were feelings about body and overall stress/effect of breast cancer. Latinas and Asian Americans discussed how the impact of their feelings about their body affected their QOL. Latinas discussed feeling embarrassed, sad, frustrated, ugly, and incomplete. Regarding the major theme of overall stress/effect of breast cancer, Blacks, Asians, and Latinas reported decrements in QOL whereas non-Latina Whites emphasized positive aspects. Latinas emphasized deleterious treatment side effects. Discussion {#Sec11} ========== Summary of findings {#Sec12} ------------------- Twenty-two studies met criteria for this review of QOL in Latinas with breast cancer compared to other racial/ethnic groups, of which eight used identical or overlapping samples \[[@CR18], [@CR19], [@CR21], [@CR23]--[@CR26], [@CR28]\]. Synthesizing the findings is complicated by marked variability in the available studies' sample sizes, operationalizations of QOL, approach to statistical control of demographic and other variables, and cultural diversity within the Latina population. Overall, findings suggest several important conclusions: 1) Latinas appear to be at risk for poor QOL following breast cancer diagnosis relative to non-Latinas; 2) relatively little is known about the mechanisms that explain these health-related discrepancies; and 3) research is needed to determine whether and how psychosocial interventions will improve QOL for this group of women. We begin by first providing a summary in which the results are organized according to study findings: studies that reported no significant racial/ethnic differences pertaining to Latinas on all QOL measures, studies in which Latinas consistently evidence higher QOL, studies in which Latinas consistently evidence lower QOL, and studies that yielded mixed findings on the relationship between race/ethnicity and QOL. The remainder of the discussion focuses on interpretations of study findings, explanatory variables, and future directions. Only one study reported no significant differences between Latinas and other women on all measures of QOL \[[@CR27]\]. Of note, this study had a very small sample of non-Latina Whites. One study reported that Latinas evidenced higher QOL than other groups of women on all measures of QOL \[[@CR33]\]. Six studies found that Latinas reported poorer QOL than other groups of women on all measures \[[@CR15], [@CR18], [@CR23], [@CR24], [@CR29], [@CR34]\]. However, after controlling for relevant covariates Ashing-Giwa et al. reported that some ethnic differences between Latinas and other ethnic groups lost statistical significance \[[@CR18]\]. The majority of studies (12) yielded mixed findings \[[@CR16], [@CR17], [@CR19]--[@CR22], [@CR25], [@CR26], [@CR28], [@CR30]--[@CR32]\] in which Latinas generally demonstrated significantly worse QOL on most, but not all, measures. Carver et al. \[[@CR25]\] reported findings from two separate studies within one manuscript. Findings from Study 1 revealed racial/ethnic disparities in QOL whereas findings from Study 2 revealed no significant differences in QOL by race/ethnicity. Of significance, the sample in Study 2 contained only 16 Latinas. The pattern of results indicates that Latinas diagnosed with breast cancer may be at risk for poorer QOL than non-Latinas. Specifically, Latinas are more likely to report poor mental, physical health, and social disruption than do non-Latinas. Measurement of sexual health was too limited to draw any conclusions. Findings from two qualitative studies emphasized the psychological impact of breast cancer (e.g., sadness, embarrassment, frustration), treatment-related symptoms, decrements in physical functioning, and the impact of cancer on social well-being, which are consistent with findings from quantitative studies. Qualitative studies add to the literature by providing a nuanced understanding of the breast cancer experience. For example, Ashing-Giwa et al. \[[@CR36]\] underscored the importance of attending to the impact of Latinas' embarrassment regarding changes to their bodies and the subsequent impact on relationships, which was not fully captured by quantitative studies. Explanatory mechanisms {#Sec13} ---------------------- Stress-based theories of health posit that minority ethnic status may confer risk for poor health outcomes as a result of fewer economic and social resources, race-based discrimination, and additional sources of stress exposure caused in part by social disadvantage \[[@CR37]--[@CR39]\]. Numerous factors can account for the obtained racial/ethnic differences in QOL outcomes. Socio-demographic factors such as income and education can affect QOL and vary by racial/ethnic status, with Latinas usually of lower SES than their White counterparts \[[@CR27], [@CR34]\]. Treatment-related factors have also been associated with QOL outcomes. Women diagnosed with more advanced stages of cancer have reported lower QOL than women diagnosed at earlier stages \[[@CR40]\]. Stage of diagnosis may be especially relevant for Latinas as they are likely to be diagnosed with more advanced stages of cancer \[[@CR1]\]. Studies included in the review that examined differences in socio-demographic and treatment-related factors indicated that Latinas generally reported lower SES and were diagnosed at a later stage than other groups of women. Even when socio-demographic and/or treatment-related factors were controlled statistically, however, the majority of studies still demonstrated lower QOL among Latinas on at least one indicator, and in several cases the findings did not change \[[@CR24], [@CR25], [@CR29]\]. Based on findings from the reviewed studies, we suggest that socio-demographic and treatment-related variables may account for some of the disparities in QOL, but they are not likely responsible for all of the observed racial/ethnic differences and therefore additional explanatory factors must be investigated. After a breast cancer diagnosis, Latinas may face additional stressors that are culturally-relevant. Only four studies examined language use or level of acculturation and QOL among Latinas. Findings from these four studies suggest that low-acculturated Latinas may be at risk for poor QOL, specifically mental health, relative to high-acculturated Latinas and non-Latina Whites \[[@CR22], [@CR23], [@CR30], [@CR32]\]. Janz et al. \[[@CR22]\] compared low-acculturated Latinas to high-acculturated Latinas and non-Latina Whites and found that that low-acculturated Latinas evidenced the poorest QOL. Low-acculturated Latinas reported poorer emotional, physical, and functional well-being on the FACT and more breast-specific concerns relative to non-Latina Whites. Low-acculturated Latinas also reported lower levels of functional and emotional well-being relative to high-acculturated Latinas. Maly et al. \[[@CR30]\] compared low-acculturated Latinas to non-Latina Whites and reported mixed findings regarding level of acculturation and QOL. Findings indicated that less-acculturated Latinas were more likely to experience nausea than non-Latina Whites and less likely to experience pain compared to non-Latina Whites. Less-acculturated Latinas were also less likely to perceive depressive symptom resolution and pain resolution compared to non-Latina Whites \[[@CR30]\]. Of the studies assessing level of acculturation, one indicated that Spanish-speaking Latinas were consistently less likely to report the presence of cancer-related symptoms compared to their White counterparts \[[@CR32]\]. Although not directly tested, Lim et al. revealed that greater acculturation was indirectly related to better mental health among Latinas diagnosed with breast cancer \[[@CR23]\]. The finding that low-acculturated Latinas generally reported poorer mental health contrasts with research in the general Latino population which suggests that longer residence or birth in the United States, versus foreign nativity, are associated with poorer mental health \[[@CR41], [@CR42]\]. In the context of breast cancer, however, low acculturation might be accompanied by lower levels of breast cancer knowledge, greater misconceptions about breast cancer, fatalistic beliefs about cancer, and less information on health resources \[[@CR6], [@CR7], [@CR43]--[@CR45]\]. Due to a potential lack of familiarity with the U.S. medical system and language barriers, Latinas, especially recently immigrated Latinas, may have difficulty navigating the medical system and interacting with health care providers. Studies in the review emphasize the importance of patient-physician relationships among Latinas. A better quality patient-physician relationship, for example, was associated with better QOL among Latinas \[[@CR18], [@CR23]\]. Thus, the racial/ethnic disparities in QOL seen in the current review may be attributed in part to sociodemographic and cancer-related factors, but also likely influenced by barriers related to language and differences in culturally-relevant beliefs and expectations which may shape various aspects of the cancer experience, from coping with the news of diagnosis to interacting with physicians and adjusting to survivorship. More research is needed to shed light on the association between acculturation and QOL among Latina cancer patients and to establish moderating factors in this relationship. Future directions {#Sec14} ----------------- ### Areas for research growth {#Sec15} Studies included in this review were limited in several ways. Few studies assessed sexual health outcomes. Only a limited number of studies included women with metastatic disease, and findings cannot be generalized to this understudied group. Eight of the 20 quantitative studies reported on identical or overlapping samples, which may have inflated patterns of consistent findings across studies. As studies were primarily conducted in large urban areas, generalization to non-urban Latina populations and to specific sub-populations of Latinas (e.g., Cuban American, Mexican American) also requires study. There is limited, existing evidence to suggest that women living in rural areas are at increased risk for poorer QOL relative to women in urban areas \[[@CR46]\]. More research is needed to examine the QOL among Latinas living in rural areas, especially in light of projections that Latinos will become the largest ethnic minority rural group in the United States \[[@CR47]\]. No study assessed major depressive disorder. As future studies continue to assess the QOL of Latina patients, it will be important to distinguish between symptoms and psychological disorders. Latinos in the general population report more depressive symptoms compared to non-Latino Whites \[[@CR48]\]. However, more research is needed to determine whether Latina breast cancer patients are more likely to meet diagnostic criteria compared to non-Latina Whites. The mechanisms through which ethnicity might influence adjustment have not been comprehensively explored. Ethnicity is an atheoretical construct that in itself provides little insight into psychological and physical phenomena, but rather may best be understood in the context of socio-cultural factors that can inform interventions targeted at improving QOL for Latinas \[[@CR49]\]. Latinas' reports of sadness regarding treatment-related changes to their bodies and feelings of incompleteness may constitute one pathway for their compromised QOL \[[@CR36]\]. Therefore, Latinas might be more likely to benefit from reconstructive surgery or from psychosocial interventions to enhance body integrity than non-Latina whites, but more studies are needed to elaborate Latinas' preferences for reconstructive treatment. As another example, a construct of interest in the psycho-oncology literature of particular relevance to the Latino population is spirituality \[[@CR50]\]. Latinas diagnosed with breast cancer report more religious coping than non-Latina White women \[[@CR16], [@CR51]\] and religious and spiritual beliefs have been associated with higher QOL among Latinas \[[@CR52]\]. ### Research design {#Sec16} More longitudinal studies are required to assess change in QOL over time, which would inform healthcare providers of periods during which Latinas might benefit from interventions. Although some studies in the current review were longitudinal, only two studies in this review assessed QOL across time by race/ethnicity, one of which revealed an increase in distress among Latinas, when compared to non-Latina White and Black women, from 6 to12 months after surgery \[[@CR16]\]. Longitudinal studies also can provide valuable information on the relevance of different indicators of QOL throughout the cancer trajectory. For example, distress may be especially prevalent within the first year after diagnosis for Latinas, whereas fear of cancer recurrence may be more pertinent later in cancer survivorship. Not all studies in the review reported average time since diagnosis, and those that did focused on women within the first few years of diagnosis. With the exception of two studies \[[@CR16], [@CR28]\], no study investigated group differences in change in QOL across time, and few studies assessed QOL within the first months following a diagnosis. Additional research on the trajectories of QOL following cancer diagnosis and treatment may provide valuable information as to when Latinas are at greatest risk for poor QOL and may benefit from interventions. ### Measurement {#Sec17} It is important to consider the appropriateness of the measurement tools used in the assessment of QOL among minorities. Measurement tools that have not been validated among Latino populations may produce findings that are difficult to interpret. For example, Yoon et al. \[[@CR32]\] used author-constructed questions to assess QOL and reported that only Spanish-speaking Latinas (versus English-speaking Latinas) were less likely to report any severe physical symptoms compared to non-Latina Whites \[[@CR32]\]. However, 20% of Spanish-speaking respondents answered that they did not know or declined to answer the question for at least one of the five symptoms compared to 5% of Whites and 6% of Blacks, suggesting that Spanish-speaking Latinas may be less familiar with labeling symptoms or may be more uncomfortable endorsing certain symptoms when being interviewed \[[@CR32]\]. More attention to matching the best approach for measuring QOL among Latinas of varying level of acculturation, education, and language proficiency is required. The most commonly employed measures used in the 20 quantitative reports have been validated in Latino populations (e.g., CES-D, SF-36, FACT). Although the CES-D, FACT, and SF-36 have similar psychometric properties for non-Latino Whites and Latinos \[[@CR53]--[@CR55]\], not all research suggests that established measures of QOL are valid in Latino populations, as differences in the factor structure of the CES-D between Latinos and Whites have been observed \[[@CR56]\]. More research is needed to attend to cross-cultural validity of measures. Latino stigma surrounding mental health and additional life stressors experienced by ethnic minorities, for example, may lead to greater reporting of depressive symptomatology through somatic expression than through affective symptomatology \[[@CR57], [@CR58]\]. Among Latina cancer patients, greater somatic complaints may be mistakenly viewed as sequelae of the cancer treatment (e.g., radiation-induced fatigue) rather than as a reflection of depression. Research is needed to elucidate the presentation of depressive symptoms among Latinas with breast cancer. ### Effect sizes {#Sec18} Although most studies noted that Latinas report worse QOL than non-Latinas, these findings do not provide an indication of the magnitude of the effects. Eleven of the 20 quantitative studies provided an effect size (i.e., correlation coefficient, standardized beta, adjusted or unadjusted odds ratio \[OR\]) which varied across quantitative studies. We discuss effect sizes for one study which provided odds ratios comparing Latinas to non-Latina Whites. Maly et al. \[[@CR30]\] reported that, relative to non-Latina Whites, both less and more acculturated Latinas were significantly more likely to report nausea (adjusted OR = 1.79 and 2.09), that less acculturated Latinas were less likely to report pain (adjusted OR = .62), and that less acculturated Latinas were less likely to perceive depressive symptom resolution (adjusted OR = .35). As the literature expands, quantitative approaches such as meta-analysis will be warranted to determine effect sizes across domains of QOL. ### Clinical implications {#Sec19} A general conclusion we draw from this review is that Latinas are likely to benefit from interventions that seek to improve QOL, and studies are needed to determine the efficacy of culturally-tailored interventions which seek to reduce distress and improve QOL for Latinas. Moreover, there is evidence to indicate that Latinas, specifically Spanish-speaking Latinas, would like to receive cancer survivorship-related information about impact on mental health, relationships, and sexual functioning \[[@CR59]\]. To date, stress management interventions for women with breast cancer have proven to be efficacious among samples of predominantly non-Latina White women \[[@CR60], [@CR61]\]. Additional research is needed to determine whether existing interventions for breast cancer patients are equally efficacious among Latinas or whether modifications to existing interventions would benefit Latinas as there are some findings in the literature to suggest that Spanish-speaking Latinas prefer to work with Spanish-speaking Latino health-care providers and would benefit from community-based interventions that consider Latino traditions and beliefs \[[@CR62], [@CR63]\]. In addition, findings from quantitative and qualitative studies suggest that low-acculturated Latinas may benefit from interventions related to enhancing patient-physician communication \[[@CR18], [@CR23]\] and support groups which provide women the opportunity to process the effects of treatment on body image and relationships \[[@CR36]\]. Research is warranted to develop efficacious, culturally-tailored psychosocial interventions for Latina breast cancer patients to reduce cancer-related distress and enhance well-being. Conclusions {#Sec20} ----------- To the best of our knowledge, this is the first systematic review comparing QOL between Latina and non-Latinas with breast cancer. Mounting evidence suggests that Latinas might be at risk for poor QOL outcomes following a breast cancer diagnosis, especially when compared to non-Latina White women. Enhancing the QOL of Latina breast cancer patients requires a multifaceted approach including efforts aimed at improving cancer screening rates to reduce disparities in stage of diagnosis and in treatment, as well as interventions aimed at reducing life disruption and improving well-being. Continuing research in this area will be crucial to this rapidly growing population and the health-care providers with whom they interact. **Open Access** This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. A list of key phrases is available from the authors. One study by Tomich and Helgeson (2004) reported racial/ethnic differences in QOL. However, given the small subsample of Latinas, approximately 1% (*n* = 4), this study was not included. List of variables: 1) study purpose, 2) number of participants, 3) location of study, 4) participants' heritage, 5) age, 6) stage of cancer, 7) cancer treatment, 8) comorbidities, 9) first or recurrent diagnosis, 10) time since diagnosis, 11) health insurance, 12) language spoken, 13) SES, 14) study design, 15) recruitment setting, 16) measures of QOL and corresponding estimates of internal consistencies, 17) data analytic plan, 18) results. References for measures used in the studies are available from the authors.

{ "pile_set_name": "PubMed Central" }

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22,224

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"QOL", "However", "given", "small", "subsample", "Latinas", "approximately", "n", "study", "included", "List", "variables", "study", "purpose", "number", "participants", "location", "study", "participants", "heritage", "age", "stage", "cancer", "cancer", "treatment", "comorbidities", "first", "recurrent", "diagnosis", "time", "since", "diagnosis", "health", "insurance", "language", "spoken", "SES", "study", "design", "recruitment", "setting", "measures", "QOL", "corresponding", "estimates", "internal", "consistencies", "data", "analytic", "plan", "results", "References", "measures", "used", "studies", "available", "authors" ]

Introduction ============ Community-acquired sepsis at an early stage is common, but haemodynamic alterations remain unclear. The aim of the study was to characterize cardiovascular alterations in patients of our ProFS (monocentric observational) study, which was to characterize patients with sepsis in the emergency department. Methods ======= Systemic vascular resistance (SVR) and cardiac output (CO) were measured non-invasively using a TaskForce monitor (CNSystems, Graz, Austria) after admission, 24 hours and 72 hours. Indexed values were calculated (SVRI (dyn·second/cm^5^/m^2^), CI (l/minute/m^2^)). Procalcitonin (PCT, ng/ml) was measured in serum. Results ======= A sample of 64 patients of 208 included patients received haemodynamic examination. Mean age was 61.8 ± 18.0 years, 62.7% were male. Patients were divided by PCT \<2 and ≥2. Age, gender and previous medical history were comparable in both groups. The heart rate was 99.8 ± 21.6 vs 104.6 ± 23.0/minute (*P*= NS) and the mean artery pressure was 89.5 ± 15.6 vs 81.6 ± 21.5 mmHg (*P*\< 0.01). Mean SVRI in patients with PCT \<2 was 2,934 ± 1,045 vs 2,376 ± 842, *P*\< 0.05 at the time of admission. No difference was found after 24 hours (2,959 ± 1,002 vs 2,924 ± 1,324, *P*= NS) and 72 hours (3,123 ± 931 vs 3,556 ± 1,524, *P*= NS). On the contrary, for patients with PCT ≥2 the increase after 72 hours was significant (*P*\< 0.05). Differences after admission could not be observed for CI between patients with PCT \<2 vs ≥2. Mean values after admission were 2.7 ± 1.0 vs 2.8 ± 0.8, after 24 hours 2.5 ± 0.8 vs 2.5 ± 0.5, and after 72 hours 2.3 ± 0.7 vs 2.3 ± 0.6 (all: *P*= NS). See Figure [1](#F1){ref-type="fig"} ![**Systemic vascular resistance index and cardiac index in sepsis in the emergency department**.](cc8613-1){#F1} Conclusions =========== Patients with community-acquired sepsis in the emergency department had an elevated SVRI. At the time of admission patients with high PCT had a significantly lower SVRI than patients with low PCT. Cardiac index at the time of admission was at a lower limit of normal range in all patients. These findings are in strong contrast to the classic pattern of sepsis on the ICU, where SVRI is keenly reduced and CI elevated. They implicate that patients with sepsis in the emergency department may benefit more from application of fluid and positive inotrope substances than from vasopressor.

{ "pile_set_name": "PubMed Central" }

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22,225

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Introduction ============ Mitochondria are key eukaryotic organelles involved not only in the well-known synthesis of ATP through oxidative phosphorylation (OXPHOS; [@evw187-B109]; [@evw187-B172]), but also in many other biological functions, such as intracellular signaling, cell differentiation, programmed cellular death, fertilization, and aging ([@evw187-B145]; [@evw187-B162]; [@evw187-B170]; [@evw187-B90]; [@evw187-B98]; [@evw187-B157]; [@evw187-B27]). Currently, it is widely accepted that mitochondria originated from a single endosymbiotic event that took place over a billion of years ago ([@evw187-B66], [@evw187-B67]; [@evw187-B148]; [@evw187-B68]). It has been proposed that the putative ancestor of all mitochondria was an alpha-proteobacterium ([@evw187-B114]; [@evw187-B67]; [@evw187-B54]; [@evw187-B176]; [@evw187-B9]; [@evw187-B3]; [@evw187-B165]; [@evw187-B40]). The following evolution of mitochondria is still unclear and a matter of debate. In animals, mitochondrial DNA (mtDNA) is a small (∼16 kb) and compact circular molecule that typically contains 13 OXPHOS-related genes, 2 rRNAs encoding for the two subunits of mitochondrial ribosomes, and an array of tRNAs used for translation within the organelle ([@evw187-B16]; [@evw187-B21]). Most genes of the original endosymbiont were therefore lost or have been transferred to the nucleus during a process called Genome Reductive Evolution (GRE) ([@evw187-B8]; [@evw187-B83]; [@evw187-B58]; [@evw187-B81]). As claimed by [@evw187-B103], in mammals no less than 1,500 proteins are needed to keep mitochondria alive and working. Why then only a small cluster of protein coding genes (PCGs) was spared by GRE---the typical 13 in metazoans, and even down to 3 in apicomplexans ([@evw187-B52]; [@evw187-B134])? Many tentative answers were proposed to this question. It is well known, for example, that mitochondria employ a genetic code slightly different from that of the nucleus, which would possibly lead to erroneous translations of some genes if transferred to nucleus ([@evw187-B4]); however, this is true for animals, but not for plants ([@evw187-B79]), which also underwent mitochondrial GRE. The high hydrophobicity of some of these proteins would hamper the import from the cytosol as well ([@evw187-B171]; [@evw187-B129]; [@evw187-B29]; [@evw187-B124], [@evw187-B123]; [@evw187-B38]; [@evw187-B55]; [@evw187-B4]). [@evw187-B101] also claimed that certain gene products are toxic in the cytoplasm. Finally, it has been proposed that the genes still encoded by mtDNAs must be efficiently and directly regulated by mitochondrial redox conditions, so they need mandatory colocation for redox regulation (CoRR) ([@evw187-B133]; [@evw187-B5], [@evw187-B6]; [@evw187-B91]). The sequencing and characterization of several complete mitochondrial genomes is a key to address GRE of mitochondria. Pioneering comparative works of large mtDNA datasets could recently, e.g., shed light on the reduction and ultimate loss in animals' mitochondria of ribosomal protein genes ([@evw187-B100]) and establish that the mitochondrion-encoded genes of almost all eukaryotes are different subsets of the mitochondrial gene complement of jakobids, a group of free-living, heterotrophic flagellates ([@evw187-B81]). At the same time, mitochondrial genomes of single metazoan groups often exhibit peculiar evolution that deserves a careful examination. In this regard, bivalves (Mollusca, Bivalvia) are among the most notable animal taxa, because of several interesting features. Most strikingly, some bivalves follow a non-canonical way of mitochondrial inheritance, the Doubly Uniparental Inheritance (DUI; [@evw187-B153], [@evw187-B154]; [@evw187-B187], [@evw187-B188]). In these species, two separate sex-linked mitochondrial lineages (and their respective mtDNAs) are present, namely, the F and the M ([@evw187-B20]; [@evw187-B121]; [@evw187-B189]; [@evw187-B21]). F mitochondria are passed from the mother to the complete offspring, and they are typically found in the soma and in the female germline (eggs); the M mitochondria are passed from the father to the male offspring, and they are typically found in the male germline (sperms). However, these general DUI rules may hold imperfectly, thus resulting in a leakage of the M mitochondrial lineage in somatic cells of either sex ([@evw187-B26]; [@evw187-B11]; [@evw187-B89]; [@evw187-B59]; [@evw187-B118]). DUI represents a case of a triple genomic conflict (nucleus vs. F mtDNA; nucleus vs. M mtDNA; F vs. M mtDNAs) ([@evw187-B121]; [@evw187-B21]). There are more outstanding features of bivalves mtDNA that are generally shared across the entire class. In fact, bivalve mitochondrial genomes are often very large, up to the 46,985 bp of *Scapharca broughtonii* ([@evw187-B96]); they present many putative unassigned regions (URs) ([@evw187-B58]); they may encode for supernumerary open reading frames (ORFans) ([@evw187-B18]; [@evw187-B105]); they show an unpaired degree of gene rearrangement ([@evw187-B169]; [@evw187-B150]; [@evw187-B127]); they exhibit strong differences in strand usage (see Additional file 6 in [@evw187-B127]). All this considered, the interest of bivalves in the wider picture of the evolution of animal mitochondria is self-evident. In this work, we present the first meta-analysis of 100 bivalve mtDNAs, corresponding to all the species whose mtDNA was available in GenBank in August, 2014. The polyphyly of bivalves in molecular phylogenetic reconstructions is a well-known artifact ([@evw187-B61]; [@evw187-B60]; [@evw187-B1]; [@evw187-B2]; [@evw187-B147]; [@evw187-B127]; [@evw187-B160]; [@evw187-B14]) and it is probably related to the fact that a small group of bivalves (the protobranchs) separated very early and, regarding mitochondrial markers, before the burst of genomic novelties described above ([@evw187-B127]). In the present work, we do not address the issue of bivalve monophyly: therefore, the only protobranch bivalve whose complete mitochondrial genome is available in GenBank in August, 2014 is used as outgroup for phylogenetic analyses. The comparative mitogenomics of bivalves is critically explored to (i) achieve a deeper knowledge about factors that shaped mitochondrial evolution in these metazoans, and (ii) focus on the specific features of bivalve mitochondrial PCGs. Materials and Methods ===================== The Database ------------ Hundred bivalve complete mitochondrial genomes ([supplementary file S1](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online) were downloaded from GenBank in August, 2014 using CLC Sequence Viewer 7.5 (Qiagen A/S, Aarhus). Annotations and sequences were imported and managed in Microsoft Excel^®^ 2007 through custom functions and VBA macros that were also used to output files in the correct format for downstream analyses. In August, 2014 the annotation of *Tegillarca granosa* ([@evw187-B161]) was already published, but the sequence was not available yet; the sequence of *Nucula nucleus* (GenBank accession number EF211991) is still incomplete and unpublished: as a consequence, these species were included in the overall analysis, but not in alignments presented in this paper. Alignment Algorithms -------------------- The first step of most analyses was a structural alignment and data masking: to this purpose, a custom tool was written in **bash** and R ([@evw187-B132]) environments, loading the package seqinr ([@evw187-B28]). The *atp8* gene and tRNAs were excluded from alignments beacuse of many uncertainties in annotations. This tool was called masking_package and is available with a brief tutorial from the GitHub repository <https://github.com/mozoo/masking_package.git>. It performs: structural alignment using T-Coffee ([@evw187-B117]) and nested packages PSI-BLAST ([@evw187-B7]), Muscle ([@evw187-B49]), ProbconsRNA ([@evw187-B41]), RNAplfold ([@evw187-B99]), and MAFFT ([@evw187-B82]), using the option series PSI-Coffee \> Expresso \> **accurate** for Protein Coding Gene (PCG) amino acids and the MR-Coffee mode for rRNA nucleotides;alignment masking in order to eliminate phylogenetic noise using the four softwares Aliscore 2.0 ([@evw187-B108]), BMGE 1.1 ([@evw187-B35]), Gblocks 0.91b ([@evw187-B25]), and Noisy ([@evw187-B44]), setting adequate options for distantly related sequences;comparison of outputs from different masking strategies in terms of number of sites and percentage of removed sites and output of a final concatenated alignment where only sites kept by at least **k** softwares are present (where **k** is up to the user and was set to 2, 3, or 4);computation of the number of sites selected by all the possible combinations of the four softwares. All the masking_package scripts adapted for amino acid (PCGs) and nucleotide (rRNAs) alignments of the present study are available as [supplementary files S2 and S3](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online, respectively. Split *rrnL* genes of ostreids were concatenated prior to alignment. Moreover, all *Crassostrea* species (with the exception of *C. virginica*) have a duplicated *rrnS* gene. In these cases, the two copies were aligned with MR-Coffee, a consensus sequence was computed using seqinr and the result was inserted into the final alignment. Genomic Features ---------------- Five large-scale features of bivalve mtDNAs were extracted: length, percentage of URs, number of genes (including newly annotated ones; see below), and A-T and G-C skew following [@evw187-B138]. Instead of a typical ANOVA, because of non-normality and non-homoscedasticity of data, differences between subclasses had to be tested using a non-parametric alternative: we used the Kruskal--Wallis test ([@evw187-B88]), while relative pairwise comparisons were carried out using the Dunn\'s test ([@evw187-B48]). Given the poor sample size of Anomalodesmata (*N* = 1) and Opponobranchia (*N* = 2), these subclasses were excluded from these preliminary analyses. Afterwards, single alignments were used as input for several custom **bash** and R scripts that were used to: subsample the original dataset, producing subsets corresponding to largest families (Mytilidae, Ostreidae, Pectinidae, Unionidae, Veneridae) and subclasses (Heterodonta, Palaeoheterodonta, Pteriomoprhia) to be aligned and masked;compute the percentage of cases in which each site of each gene was kept after the masking phase, using the agreement of at least 3 out of 4 masking softwares (across the five families, the three subclasses, and in the complete-dataset analysis);comparing masking results using a one-sided *t*-test;back-translate the original, unmasked amino acid alignment into nucleotides;use EMBOSS ([@evw187-B139]) to compute different metrics of pairwise and overall distances, both on total gene length (using **distmat**) and over a sliding window (using **plotcon**);compare nucleotide and amino acid distances through the Kolmogorov--Smirnov test;test alignments for saturation by plotting uncorrected *p*-distance over the Jin--Nei distance (for nucleotides; [@evw187-B77]) or the Kimura distance (for amino acids; [@evw187-B84]);perform a Principal Component Analysis (PCA) on concatenated distance matrices, using the FactoMineR ([@evw187-B94]) package for computations and ggplot2 ([@evw187-B175]) for graphics. The ratio of non-synonymous vs. synonymous substitutions (d~N~/d~S~) was computed for back-translated alignments using PAML 4.8a ([@evw187-B183], [@evw187-B184]); d~N~/d~S~ was computed both for each pair of sequences and along the phylogenetic tree (see below). In the latter case, we used the Likelihood Ratio Test (LRT) to compare the fitting of single d~N~/d~S~ for the complete tree with the use of 12 different d~N~/d~S~, allowing different d~N~/d~S~ ratios for different clades. All LRTs were carried out in the R environment; to be conservative, we used a chi-square distribution with 11 degrees of freedom ([@evw187-B177]). Phylogenetic Analyses --------------------- Using the original annotations available in GenBank, phylogenetic analysis was carried out on PCGs and rRNAs; given many uncertainties in annotation and orthology, the *atp8* gene and tRNAs were excluded from the analysis. The best-fitting partitioning scheme and molecular evolution models were estimated using PartitionFinderProtein and PartitionFinder 1.1.0 ([@evw187-B92]) under the Bayesian Information Criterion and a greedy approach. Gaps were coded following the simple indel method of [@evw187-B152] as implemented in GapCoder ([@evw187-B185]). As a result, the final alignment spanned over 14 genes and three types of data: amino acids for PCGs, nucleotides for rRNAs, and binary data for coded gaps. The software RAxML 8.2.0 ([@evw187-B159]) was used to infer Maximum Likelihood (ML) phylogeny. The strand usage is variable across different bivalve mtDNAs: while in most cases all genes locate to the same strand, genes are evenly distributed on both strand in *N. nucleus*, *S. velum*, and Unionidae. As a strand usage bias can be associated with a compositional bias, which may in turn lead to phylogenetic artifacts, preliminary analyses with 500 bootstrap replicates were separately conducted on those genes mapping on the "+" and on the "−" strand of unionids. If a significant compositional bias is working, we expect to detect possible phylogenetic artifacts by recovering different topologies from the two analyses. The final tree was computed as follows; explicit RAxML commands are listed in [supplementary file S4](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. five randomized maximum parsimony (MP) starting trees were generated;a maximum likelihood (ML) tree was inferred from each MP starting tree with a fixed rearrangement radius of 10 and with an automatically determined one, choosing the setting yielding the best results;a ML tree was inferred from each MP starting tree with the selected initial rearrangement option under different number of rate categories (10, 25, 40, or 55) for the CAT model accounting for evolutionary rate heterogeneity ([@evw187-B158], choosing the setting yielding the best results;the best-known likelihood tree was inferred from the original alignment under the selected rearrangement and rate categories options calling 10 runs from 10 randomized MP starting trees;500 bootstrap replicates were run; andconfidence values were computed from bootstrap replicates and annotated on the best-known likelihood tree. The software MrBayes 3.2.1 ([@evw187-B140]) was used to carry out Bayesian Inference (BI) using 2 separate runs, 4 chains, and 10,000,000 generations of MC^3^, sampling every 100 trees. Convergence between runs and burn-in were estimated looking to standard deviation of average split frequencies sampled every 1,000 generation and to Potential Scale Reduction Factor (PSRF) ([@evw187-B57]). The best-known likelihood tree computed by RAxML was used to obtain a time-scaled chronogram using r8s 1.70 ([@evw187-B144]) and the r8s bootstrap kit by Torsten Eriksson (downloaded from <https://github.com/TorstenEriksson/r8s-bootstrap> in February, 2015). First appearance data for six calibration point were downloaded from the Paleobiology Database (<http://fossilworks.org>) in February, 2015: the root of all bivalves, Mytilidae, Ostreidae, Pectinidae, Unionidae, and Veneridae ([@evw187-B102]; [@evw187-B166]; [@evw187-B36]; [@evw187-B47]; [@evw187-B115]; [@evw187-B17]; [@evw187-B10]; [@evw187-B65]; [@evw187-B23]; [@evw187-B104]; [@evw187-B87]; [@evw187-B116]). We used the Penalized Likelihood method and the truncated Newton algorithm; the cross-validation approach allowed us to estimate the best smoothing parameter up to the sixth decimal digit and five restarts and five guesses were used each round. Following the r8s bootstrap kit procedure, 163 bootstrap replicates of the original alignment were generated and the original tree was optimized on each of them; node ages were estimated under the same r8s setting, estimating the best smoothing parameter up to the second decimal digit. The 99% confidence intervals of each node age were computed using custom R script loading the package ape ([@evw187-B120]) and following methodological recommendations of [@evw187-B76], i.e., type = 8. Trees were graphically edited using the software PhyloWidget ([@evw187-B78]) and Dendroscope 3.3.2 ([@evw187-B75]). Phylogenetic Informativeness (PI) was investigated on the amino acid dataset using the PhyDesign portal ([@evw187-B97]) and Rate4Site ([@evw187-B130]) to estimate site-specific evolutionary rates. Best-fitting amino acid evolutionary models were selected using ProtTest 3.4 ([@evw187-B39]) and PhyML ([@evw187-B70]). PI was computed over five different epochs, whose boundaries were taken from the International Stratigraphic Chart v2015/01 ([@evw187-B30]): Quaternary, "Cenozoic" (Paleogene + Neogene), Mesozoic, "Paleozoic" (from Ordovician to Permian), and "Cambrian" (from 520 to 485.4 Mya). Following [@evw187-B167] and [@evw187-B151], the phylogenetic signal and noise analysis was carried out in a state space of five. Finally, we investigated the correlation between gene rearrangements and substitution rates, which has been demonstrated for insects by [@evw187-B146] and hypothesized for mollusks by [@evw187-B160]. However, the RGR test of [@evw187-B43] and its modified version of [@evw187-B182] cannot be useful in the case of bivalves to describe gene arrangement variability because it is a relative rate test that involves a comparison with the ancestral gene arrangement. As detailed in [@evw187-B127], this is most likely that of the chiton *Katharina tunicata*, but, with the notable exception of *Solemya velum* and *Nucula nucleus* ([@evw187-B127]), most bivalve gene orders are not comparable with each other and seem to be equally very different from that. Therefore, while there is no remarkable difference in terms of distance from the ancestral state, higher rates of mitochondrial gene rearrangement are straightforward in some clusters with respect to others, but this would not be detected by RGR: for this reason, we quantified rates of mitochondrial rearrangement in the clades obtained in the best-known likelihood tree using the architecture rate (AR) as introduced by [@evw187-B62]. This is given by $$AR = \frac{N_{GA} - 1}{N_{OTU} - 1}$$ where *N*~GA~ is the number of different gene arrangements found in a clade and *N*~OTU~ is its number of OTUs. In fact, this index conservatively estimates the number of gene rearrangement events along a clade\'s evolutionary history, as it must be at least as large as the number of different gene arrangements found in extant taxa, and normalizes it with the number of species, so that clades of different sizes are comparable. AR rate ranges from 0 (the whole clade shares the same gene arrangement) to 1 (each OTU shows a different gene arrangement). The sum of branch lengths for a given clade was computed with the software Phylocom 4.2 ([@evw187-B174]), thus estimating the total number of expected substitution per site in a given cluster; this was divided by the root age in millions of years (see above). The correlation of AR rate and substitution rate was assessed with the Spearman\'s rho and Kendall\'s tau tests using R. Annotation of the atp8 Gene and of ORFans ----------------------------------------- The EMBOSS suite was used to find all the possible Open Reading Frames (ORFs) in all the complete mitochondrial genomes, using all the six possible frames. A Hidden Markov Model (HMM) was constructed for each ORF using HHblits 2.0 ([@evw187-B135]) and the latest PDB70 release. All the HMMs were merged in a custom database, which was called Biv_mtDNA_ORFs. All the annotated *atp8* genes were aligned using the T-Coffee (again following the PSI-Coffee \> Expresso \> **accurate** option series). A Hidden Markov Model (HMM) was constructed using HHblits 2.0 and the latest Uniprot release and a HMM-HMM alignment was run against the Biv_mtDNA_ORFs database to check for homology with *atp8*. Positive results were manually screened and original annotations were consequently updated. Bivalve supernumerary mitochondrial ORFs do not have known homologs and are therefore considered as ORFans ([@evw187-B53]). However, it is possible to find homologies at least within Bivalvia. All the published supernumerary ORFs ([@evw187-B18]; [@evw187-B105]; [@evw187-B127]; [@evw187-B13]; GenBank accession numbers HM856636, HQ283344, KC848655, KF030963, NC_015310, NC_015476, NC_015477, NC_015479, NC_015481, NC_015483, NC_018763, NC_022803, NC_023250, NC_023942) were annotated; a HHblits database of all the known ORFans was created as above using Biv_mtDNA_ORFs and was called Biv_mtDNA_ORFans; finally, HHblits was used for each ORFan against this custom database to investigate putative relationships between known ORFans. Summarizing Data ---------------- A PCA was carried out as above using many different parameters ([supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online): nucleotide composition; AT content; A-T and G-C skews; use of between-genes overlapping nucleotides; length; percentage of Unassigned Regions (URs); total number of genes; the strand usage skew (SU skew); number of truncated T--/TA- stop codons in the genome; the Amount of Mitochondrial Identical Gene Arrangements (AMIGA). SU skew and AMIGA were defined as follows. $$SUskew = \frac{H - L}{H + L}$$ where *H* is the number of genes on the H strand and *L* is the number of genes on the L strand. If the strand usage is perfectly balanced (i.e., *H = L*), the SU skew is equal to 0; a negative SU skew indicates a bias towards the L strand, while a positive SU skew indicates a bias towards the H strand. $$AMIGA = \frac{N_{IGA} - 1}{N - 1}$$ where *N*~IGA~ is the number of taxa in the analyzed sample that share an Identical Gene Arrangement and *N* is the total number of taxa. As a consequence, unique gene arrangements will have an AMIGA score equal to 0. Conversely, when all the taxa in a given sample share the same gene arrangement, each AMIGA score will be equal to 1. An intermediate value expresses an intermediate value of conservation. Given many uncertainties in annotations, rRNAs and tRNAs were excluded from the analysis, therefore the AMIGA index relies solely on PCGs. The HERMES Index ---------------- A widespread method of quantifying molecular evolution of mitochondrial genomes in different species and clusters is still lacking for mitogenomic analyses. We developed a new index in this regard, which relies on maximum likelihood factor analysis to summarize different measures that are typically found to be linked with evolutionary rates; it is intended to be computed *a posteriori*, i.e. after the phylogenetic and genomic analysis. As different empirical measures are merged together in a single score, this is a "hyper-empirical" index. Moreover, a taxon retaining most genomic plesiomorphies of the group (at least following state-of-art knowledge) is selected as a benchmark: thus, it is a relative measure, and that taxon was in our case *N. nucleus*. All this considered, the index was called Hyper-Empirical Relative Mitochondrial Evolutionary Speed (HERMES) index. We explored the use of different subsets of the following variables to compute the HERMES index: the AT content;the genome length;the number of (annotated) genes;the percentage of URs;the absolute value of SU skew;AMIGA;the root-to-tip distance computed on the best-known likelihood tree using Phylocom 4.2;the ML distance from *N. nucleus* computed with RAxML specifying the model as above. The factor analysis was carried out using the psych ([@evw187-B137]) package of R; the plot was prepared using the ggplot2 package. Normalization and varimax rotation were used, factor scores were found using correlation preserving, and correlations were found using the Pearson method; given the possible presence of a missing value (as *T. granosa* was not included in the phylogeny and we were therefore unable to compute either the root-to-tip and the ML distance), missing data were set to be imputed using the median. All the variables were pooled together for each species into the value of a single loading: we define this score as the HERMES score of a given species. The best-performing variable set and the goodness-of-fit of the analysis was assessed following the recommendations of [@evw187-B74]: Tucker--Lewis Index (TLI) ([@evw187-B168]) \> 0.95; root mean square of the residuals (SRMR) \< 0.08; root mean squared error of approximation (RMSEA) \< 0.06; moreover, the Kaiser--Meyer--Olkin index (KMO) ([@evw187-B80]) was taken into account on this regard. A Python script was written to compute the HERMES scores of a given assemblage of species, providing the GenBank annotation, gene alignments and a phylogenetic tree; this software can be downloaded from the GitHub repository at the URL <https://github.com/mozoo/HERMES.git>, along with sample data and a tutorial. Results ======= Overall Genomic Features ------------------------ The 100 mitochondrial genomes (mtDNAs) of the present study range from 14,622 (*Lanternula elliptica*) to 46,985 (*Scapharca broughtonii*) bp in length. Excluding the abnormally large genomes of Arcidae, the longest mtDNA is that of *Placopecten magellanicus* (32,115), followed by the female genome of *Venerupis philippinarum* (22,676 bp). Conversely, the proportion of putatively Unstranslated Regions (URs) span from 1.44% (*L. elliptica*) and 2.13% (*S. velum*) to the high scores of Pectinidae (29.86--51.04%) and Arcidae (52.30--70.76%). The number of annotated genes on the molecule is much more stable: exception made for *Scapharca kagoshimensis* and *S. broughtonii* (55 and 54, respectively), this number span from 30 (*Mizuhopecten yessoensis*) to 46 (*P. magellanicus*), with a mean value of 38.11 ± 3.20. As summarized in [figure 1](#evw187-F1){ref-type="fig"}, mtDNA length and %UR increases in the order Palaeoheterodonta \< Pteriomorphia \< Heterodonta. Palaeoheterodonta are in both cases significantly different from either Pteriomorphia or Heterodonta (*P* \< 0.001\*\*\*; [supplementary file S6](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). The Kruskal--Wallis test detected a significant location difference also for the number of annotated genes, which is due to the Pteriomorphia/Heterodonta comparison only (*P* \< 0.05\*; [supplementary file S6](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Finally, each subclass was significantly different from each other when considering A-T and G-C skews---again, comparisons involving Palaeoheterodonta showed the highest significance values (*P* \< 0.001\*\*\*; [supplementary file S6](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Single-species data used in this study are extensively listed in [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. F[ig]{.smallcaps}. 1.---Main features of mitochondrial genomes. Five large-scale mitochondrial features are compared across four bivalvian subclasses (the fifth subclass, Anomalodesmata, was excluded because only one mitogenome was available): Op, Opponobranchia (purple); Pa, Palaeoheterodonta (blue); Pt, Pteriomorphia (red); He, Heterodonta (green). Length, median length of the genome; %UR, median percentage of Untranslated Regions; Annotated genes, mean number of annotated genes; A-T skew, median A-T skew; G-C skew, median G-C skew. Differences between subclasses were tested using the Krukal--Wallis and the Dunn\'s test; Anomaldesmata and Opponobranchia were excluded because of low sample size (*N* = 1 and *N* = 2, respectively). Asterisks above columns refer to levels of significance in both pairwise comparisons or in a single one (bracketed); \**P* \< 0.05; \*\**P* \< 0.01, \*\*\**P* \< 0.001. Notwithstanding the large variability, especially in length and %UR, the genome content is quite stable: if we do not take tRNAs into account, all the mtDNAs share the same gene content and few duplication events are present. The most evident duplication is that of the *rrnS* gene in all species of the genus *Crassostrea* (with the exception of *C. virginica*), whose genes show a divergence between 0.11% (*C. iredalei*) and 5.05% (*C. ariakensis*), as estimated using uncorrected *p*-distance (mean = 2.92%). Moreover, as already signaled ([@evw187-B106]; [@evw187-B136]), in all Ostreidae species the *rrnL* gene is split in two separate fragments. Finally, a duplication of *cox2* was already signaled in *Musculista senhousia*, male type ([@evw187-B122]), and *V. philippinarum*, female type (GenBank Accession Number AB065375). Alignments ---------- Protein Coding Genes (PCGs) alignment lengths range from 186 amino acids of *nad3* to 1,313 amino acids of *cox2* (before masking). The amount and percentage of sites selected by at least 2, 3, or 4 softwares is detailed in [supplementary file S7](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. The use of three masking softwares out of four is a good compromise between the elimination of noise and the elimination of apparently noisy useful sites, therefore this was taken as our preferred setting. All alignments are available from FP upon request. The masking step affected different genes differently: *cox1* and *cytb* are the genes that were least affected during masking phase (68.63% and 60.13% of their sites were kept, respectively), while *cox2*, *nad4L*, and *nad6* alignments were heavily reduced after this phase (to the 16.45%, 18.32%, and 21.73% of the original size, respectively). As expected, rRNA alignments are much longer (4,210 and 2,891 bp for *rrnL* and *rrnS*) and much more shortened after the masking phase (to the 14.06% and 11.45% of the original size, respectively). The amount of shortening was not reduced when smaller, more-related subsets were analyzed ([supplementary file S8](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Differences in the percentages of kept sites among different subsets were never significantly higher when moving towards a smaller, nested taxon, but rather in some cases they were significantly lower (Bivalvia \> Pteriomorphia, *P* \< 0.001\*\*\*; Palaeoheterodonta \> Unionidae, *P* \< 0.05\*; Pteriomorphia \> Pectinidae, *P* \< 0.01\*\*). Actually, in all the cases, phylogenetically-informative sites are almost the same, being the dataset a single family, a subclass, or the whole class ([fig. 2](#evw187-F2){ref-type="fig"}). The longest conserved domains are those of *cox1* and *cytb*, even if also *nad* genes (with the exception of *nad4L* and *nad6*) show extended well-aligned regions. Interestingly, *nad4* is the only case of a conserved domain that appears with reduced and less variable alignments: about 120 amino acids at the N-terminus of the protein are not conserved when the complete dataset is considered, but are increasingly more conserved at the subclass and family level ([fig. 2](#evw187-F2){ref-type="fig"}). The similarity measure of **plotcon** over a sliding window show highest values for the most conserved regions, as expected ([supplementary files S9 and S10](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). F[ig]{.smallcaps}. 2.---Conserved sites of protein coding genes. Single genes were aligned, masked using four different approaches, and finally only sites selected as phylogenetically informative by at least three softwares were kept. Whenever possible in terms of sample size, the same procedure was carried out for reduced datasets, corresponding to single subclasses (3 datasets) and single families (5 families). Each chart shows the proportion of datasets keeping single sites: this is 0 (discarded site) or 100 (kept site) at the class level, and ranges from 0 to 100 at the subclass and family level, because more datasets are available. Uncorrected (*p*-) distances are slightly, but significantly (*P* = 0), higher for amino acids than for nucleotides; however, when a more complex model is used to account for multiple substitution events at a single site, the situation is the opposite, and nucleotide distances are significantly (*P* = 0) higher than amino acid ones ([fig. 3](#evw187-F3){ref-type="fig"}). Moreover, with the exception of the low-scoring *cox1*, all uncorrected distances are comparable among genes, while using the Jin--Nei/Kimura method (for nucleotides/amino acids, respectively), some genes (namely, *atp6*, *nad2*, *nad4L*, and *nad6*) have higher distance values than others ([fig. 3](#evw187-F3){ref-type="fig"}). A similar scenario is retrieved through saturation plots. The uncorrected distance was plotted on Jin--Nei/Kimura distance: while in many cases uncorrected distances tend to increase with the other model, the same four genes show a plateau, indicating that *p*-distance is not able to uncover multiple hits and that a significant degree of saturation is present. As an example, *cytb* and *nad6* plots are shown in [figure 4](#evw187-F4){ref-type="fig"}, while all genes are detailed in [supplementary file S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. Expectedly, aminocid (aa) plot is farther from the plateau than nucleotide (nt) plot in all cases. F[ig]{.smallcaps}. 3.---Nucleotide and amino acid genetic distances. The mean genetic distance across the complete dataset ± one standard deviation is shown for each PCG; brown, nucleotides; green, amino acids. Differences in distance distributions were tested using the Kolmogorov--Smirnov test; \*\*\**P* \< 0.001. F[ig]{.smallcaps}. 4.---Example of four saturation plots. Pairwise *p*-distance is plotted over Jin--Nei/Kimura distance for nucleotides (above)/amino acids (below), respectively; nt, nucleotides; aa, amino acids. All saturation plots are available as [supplementary file S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. A PCA was carried out using pairwise nucleotide and amino acids distances as variables for each gene; the result is shown in [figure 5](#evw187-F5){ref-type="fig"}. Taken together, the first two principal components explain the 85.64% of the variance, but it has to be noted that the first component alone explains the 77.62%. The PCA (and specifically the first component) clearly separates *atp6*, *nad2*, *nad4L*, and *nad6* from other genes. F[ig]{.smallcaps}. 5.---Distance PCA. Single-gene pairwise distance matrices were used as input data in order to investigate differences between PCGs. Finally, the d~N~/d~S~ ratios for each gene are given in [table 1](#evw187-T1){ref-type="table"}. In all cases, the null hypothesis that a single d~N~/d~S~ applies to all the tree branches was not rejected by the LRT (*P* = 1); the d~N~/d~S~ value computed along the entire tree is one or two orders of magnitude higher than the median of pairwise comparisons ([table 1](#evw187-T1){ref-type="table"}). The highest values are shown by *nad6* (d~N~/d~S~ = 0.344; median of pairwise comparisons = 0.045), while the lowest are shown by *cox1* (d~N~/d~S~ = 0.080; median of pairwise comparisons = 0.004). However, namely in *atp6*, *nad4L*, and *nad6*, a quite large number of high (i.e., greater than 10) pairwise d~N~/d~S~ values were computed ([table 1](#evw187-T1){ref-type="table"}). In the vast majority of cases, these values expectedly come from pairs of distantly related species. Table 1Values of d~N~/d~S~Genesd~N~/d~S~Pairwise mediand~N~/d~S~ \> 10*atp6*0.2550.023 ± 6.03041*cox1*0.0800.004 ± 1.4361*cox2*0.2220.008 ± 1.4361*cox3*0.1550.009 ± 0.0270*cytb*0.1640.008 ± 0.0170*nad1*0.1620.009 ± 0.0260*nad2*0.3100.019 ± 2.9499*nad3*0.2160.009 ± 1.4612*nad4*0.2300.010 ± 0.0220*nad4L*0.3120.032 ± 8.44167*nad5*0.2520.008 ± 0.0200*nad6*0.3440.045 ± 11.223103[^2] Phylogenetic Analysis --------------------- The two subsets of markers (i.e., genes that are on the "+" strand and genes that are on the "−" strand of Unionoidea) yielded the same topology in the preliminary analyses, therefore the final tree was computed on the complete set of genes. The best partitioning scheme selected by PartitionFinder separated *atp6*+*nad* genes, cytochrome subunit genes, and rRNAs (which were taken together; see [supplementary file S12](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online, for details). The best-known likelihood tree annotated with support values is shown in [figure 6](#evw187-F6){ref-type="fig"} over a geological timescale, while original phylograms are shown in [supplementary files S13](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online (best-known likelihood tree with bootstrap proportions) and 14 (Bayesian consensus tree). Both Bootstrap Proportions (BPs) and Bayesian Posterior Probabilities (PPs) are generally high: as BP is typically lower than PP, we conservatively show the consensus tree after collapsing nodes with BP \> 70. After the separation of the outgroup *S. velum*, the tree is divided into two major branches: Palaeoheterodonta (BP = 100, PP = 1.00) and Amarsipobranchia *sensu* [@evw187-B125] (BP = 97, PP = 1.00). Within Palaeoheterodonta, M and F genomes (if non-DUI species genomes are considered as F ones) cluster separately, both with BP = 100/PP = 1.00; the branching order is also the same. Within Amarsipobranchia, relationships between *L. elliptica* (Anomalodesmata), Pteriomoprhia (BP = 91, PP = 1.00), and Heterodonta (BP = 73, PP = 1.00) are not resolved. Within Pteriomorphia, Mytilidae (BP = 100, PP = 1.00) are the sister group of all remaining pteriomorphians (BP = 79, PP = 1.00). With the exception of *M. californianus* M, DUI genomes of *Mytilus* spp. cluster by sex, as in Palaeoheterodonta, while *M. senhousia* genomes cluster by species. Within Heterodonta, Lucinindae (*Loripes lacteus*+*Lucinella divaricata*, BP = 100, PP = 1.00) are the sister group of all remaining heterodonts (BP = 100, PP = 1.00). All heterodont DUI genomes (i.e., *M. lamarckii* and *V. philippinarum*) cluster by species. F[ig]{.smallcaps}. 6.---The mitochondrial phylogeny of bivalves. Ultrametric tree of 98 bivalve species computed using r8s starting from the best-known likelihood tree computed with RAxML and the consensus Bayesian tree. RAxML bootstrap values/MrBayes posterior probabilities are printed at each node (an asterisk means a bootstrap of 100 and a posterior probability of 1.00). Below the tree the phylogenetic informativeness for each PCG is shown. The geological timescale in Mya follows [@evw187-B30]. The main split of the class (Palaeoheterodonta, one side, and Amarsipobranchia, the other side) took place about 500 million years ago (Mya), with a confidence interval (CI) of about 4.5 million years (My). The origin of pteriomorphians is dated at 484 mya (CI = 6 My), while the origin of heterodonts is dated slightly later, 454 mya (CI = 16 My). Oldest families are those of mytilids (421 Mya), pectinids (384 Mya), and venerids (336 Mya). All details about time calibration, including node names, mean estimates across bootstrap replicates, and CIs are shown in [supplementary files S15 and S16](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. Different genes have different Phylogenetic Informativeness (PI) scores for different periods ([fig. 6](#evw187-F6){ref-type="fig"}). Most *nad* genes and the *cytb* gene reach their peak in the Cenozoic, while *cox* genes and *atp6* reach it in the Cretaceous. Notably, the peak of *cox1* informativeness is shifted back to the Jurassic-Cretaceous limit and decreases slowly towards the deeper past. In noise vs. signal analysis ([supplementary file S17](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online) *cox1*, *cytb*, *nad1*, and *nad5* typically outperformed all the other markers, although the complete dataset always shows much higher values than single genes. The AR and substitution rates ([supplementary file S18](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online) showed a good correlation when six clades were used, corresponding to the major clades of the tree (Palaeoheterodonta F, Palaeoheterodonta M, Mytilidae, other Pteriomorphia, Lucinidae, other Heterodonta). The value of Spearman\'s rho was 0.90 (*P* \< 0.01\*\*) and that of Kendall\'s tau was 0.83 (*P* \< 0.05\*). Annotation of the atp8 Gene and of ORFans ----------------------------------------- Twenty *atp8* genes were annotated using the HHblits approach; they are listed in [table 2](#evw187-T2){ref-type="table"}. All the newly annotated *atp8* had a homology probability \> 95%, *E*-value \< 0.01, and *P*-value \< 1 × 10^−6^. The only exceptions to these are the *atp8*s in *Paphia euglypta* (Prob. = 92.7%, *E*-value = 0.071, *P*-value = 4.8 × 10^−6^), *Acanthocardia tuberculata* (Prob. = 86.6%, *E*-value = 0.46, *P*-value = 3.1 × 10^−5^), and *Musculista senhousia* M (Prob. = 80.6%, *E*-value = 0.74, *P*-value = 5.0 × 10^−5^): as the *atp8* gene was not annotated in these species, it is highly probable that the homology is correct, but they anyway should be regarded as only tentatively annotated; the complete list of HHblits scores is available as [supplementary file S19](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. The annotation of *atp8* led us to make small changes to the original GenBank annotations; these, along with other corrections of minor flaws that we detected during the analyses, are listed in [supplementary file S20](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online, where the complete, updated annotations of all the present mtDNAs are given. Table 2Newly annotated *atp8* genesSpeciesStartStopLengthaa*Mytilus galloprovincialis* M8,5308,871345115*Mytilus edulis* M9,78910,133345115*Mytilus californianus* F8,7359,037303101*Mytilus galloprovincialis* F8,8029,06226488*Mytilus edulis* F10,39610,65626488*Mytilus californianus* M8,2628,570312104*Ruditapes philippinarum* M4,6304,75212642*Arctica islandica*10,34310,49315150*Semele scabra*11,96912,09712943*Solecurtus divaricata*11,32111,45213545*Nuttallia olivacea*12,93013,05813244*Soletellina diphos*11,21411,34213244*Mimachlamys nobilis*7,9378,08615351*Moerella iredescens*11,62511,75313244*Meretrix petechialis*8,5328,66914147*Meretrix meretrix*8,5328,66914147*Ruditapes philippinarum* F5,9686,08412040*Paphia euglypta*12,99413,10711739*Acanthocardia tuberculata*12,54612,64810334*Musculista senhousia* M7,4037,59119264[^3] Conversely, ORFans seem to be poorly connectible across the entire class: the complete list of HHblits clusters of homologs shows that any given ORFan shows sharp similarities only with ORFans of strictly related species ([supplementary file S21](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Indeed, ORFans from Unionidae are phylogenetically clumped in the same clusters that appear in the tree ([fig. 6](#evw187-F6){ref-type="fig"}) and, notably, F and M ORFans are never intermingled. The same apply for F ORFans of mytilids, but not for M ORFans, whose homology scores are always unsignificant for all entries of the database. The only exception to this is given by *Musculista senhousia*. Three ORFans were described on the F mtDNA of this species ([@evw187-B69]): while no significant hits were retrieved for one of these, the other two and the single M ORFan are clearly homolog ([supplementary file S21](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online; see also [@evw187-B69]). Finally, *S. velum* ORFans did not show significant homologies with other ORFans. Summarizing Data: PCA and HERMES Score -------------------------------------- All bivalve mitogenomic features are listed in [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online. The resulting PCA is shown in [figure 7](#evw187-F7){ref-type="fig"}; the first two principal components account for the 66.61% of the dataset variability. In the PCA plot, larger taxonomic assemblages are easy identified: Palaeoheterodonta on one side and the wide cluster of Amarsipobranchia on the other side. Most families create also a cluster, like Pectinidae, Ostreidae, and Margaritiferidae. Contrastingly, some points are sharply separated from others: *N. nucleus*, *S. velum*, *Scapharca* spp., *P. magellanicus*, and *L. elliptica*. F[ig]{.smallcaps}. 7.---Bivalve mitogenomics PCA. Input data are shown in [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online (see text for details). Different subclasses are indicated with different shades: shades of blue, Palaeoheterodonta; shades of violet, Opponobranchia; shades of red, Pteriomorphia; shades of green, Heterodonta + Anomalodesmata; species of the same family are indicated with the same color. The best-performing variable set to compute the HERMES score was the following: percentage of URs;absolute value of SU skew;AMIGA;root-to-tip distance; andML distance from *N. nucleus*. For example, when inserting also the AT content, the goodness-of-fit parameters become only slightly better, but AT content was given a communality of 0.25%; therefore, we may conclude that this variable is not highly linked with the other five and thus it not significant in quantifying molecular evolution of mitochondrial genomes. The HERMES factor analysis index ([fig. 8](#evw187-F8){ref-type="fig"}) shows good levels of correlation between the selected variables: TLI = 0.965, SRMR = 0.061, RMSEA 95% CI = 0.025-0.233, KMO = 0.764, which are all within boundaries suggested by [@evw187-B74]. The lowest communality was scored by %UR (13.20%), while the highest was scored by the ML distance from *N. nucleus* (98.66%); the mean communality of the model was 60.62%, meaning that the HERMES index accounts for the 60.62% of the total variability of the source matrix. F[ig]{.smallcaps}. 8.---The HERMES index. To highlight HERMES differences between subclasses, species are horizontally listed by subclass and then in alphabetical order. Discussion ========== To the best of our knowledge, the present study is the first overall and detailed appraisal to the mitogenomics of bivalve mollusks. Many similar studies have been published in the past on similar topics, but generally they focused on a single family, like Mytilidae ([@evw187-B19]), Unionidae ([@evw187-B18]), Pectinidae ([@evw187-B181]), or on the DUI phenomenon ([@evw187-B42]). As stated in the introduction, however, large comparative studies are essential to understand the main pathways followed by the evolution of mitochondrial genomes. At the class level, we decided to especially concentrate on PCGs, as annotations are more trustworthy, homology is certain, and the same set of genes is present in all genomes under study. Actually, we find only two duplication events (*cox2* in *M. senhousia* M and *V. philippinarum* F) and we were able to detect the *atp8* gene in 20 species where this gene was originally described as missing ([table 2](#evw187-T2){ref-type="table"}): some of these *atp8* genes were already signaled ([@evw187-B160]), namely those of *A. tuberculata*, *H. arctica*, and *V. philippinarum* ([@evw187-B45]); *Mytilus* spp. ([@evw187-B22]; [@evw187-B155]); Unionidae ([@evw187-B42]). After our analysis, 71 species out of 100 have an annotated *atp8*. The nucleotide/amino acid sequence of this gene is scarcely conserved, and this probably led to annotation flaws, as already stated ([@evw187-B127]; [@evw187-B189]; [@evw187-B13]; [@evw187-B56]). Structural analyses are needed to recover some homology with known *atp8*, and we may suggest the importance of such analyses in other groups where *atp8* is reported as missing, like nematodes ([@evw187-B119]), platyhelminths ([@evw187-B95]), and chaetognaths ([@evw187-B15]). Contrastingly, the GenBank annotation of rRNAs is still commonly obtained by the boundaries of the upstream and downstream genes, despite accurate ongoing work of re-annotation ([@evw187-B12]; [@evw187-B160]). The use of these genes is therefore problematic for analyses like nucleotide composition, skews, or biases. However, phylogenetic methods should be robust enough to overcome this issue (but a masking phase is required): for this reason, we decided to insert rRNAs in the phylogenetic analysis anyway. Finally, tRNAs are extremely prone to gene rearrangement ([@evw187-B16]; [@evw187-B169]; [@evw187-B182]; [@evw187-B62]) and recruitment ([@evw187-B93]; [@evw187-B178]; Wu, Li, Li, Yu, et al. [@evw187-B179]), and therefore they have to be assessed at a lower taxonomical level ([@evw187-B180]). PCGs in Bivalve Mitochondrial Genomes ------------------------------------- The distance analyses reveal a high degree of divergence for single genes. *p*-Distance, which is an underestimation of the true distance in that it does not account for multiple substitution events, is generally comprised between 40% and 60% ([fig. 3](#evw187-F3){ref-type="fig"}). However, when the correct model of molecular evolution is used, it is clear that most of this variability is found in synonymous mutations ([fig. 3](#evw187-F3){ref-type="fig"}), which is clearly demonstrated by the low values of d~N~/d~S~ ([table 1](#evw187-T1){ref-type="table"}). Pairwise d~N~/d~S~ have very low medians, but a very high standard deviations, probably due to high d~N~/d~S~ computed for very distantly related species; therefore, we consider as the best estimation of d~N~/d~S~ in our dataset the value computed along the phylogenetic tree, which is comprised between 0.080 (*cox1*) and 0.344 (*nad6*; [table 1](#evw187-T1){ref-type="table"}). Such a pattern of overall negative selection was already detected for the species of genus *Mytilus* ([@evw187-B186]; [@evw187-B56]). However, conservation and variability should not be assessed at the general gene level, because our results clearly indicate a sharp contrast between strongly conserved domains and highly variable regions. Those domains are shown in [figure 2](#evw187-F2){ref-type="fig"} (see also [supplementary files S9 and S10](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Concluding, some domains of the mitochondrial PCGs are currently under severe purifying selection in bivalves, while, in most cases, variability is due to indel events typical of some species (see, e.g., the *cox2* gene). If the general pattern is the conservation of specific domains in each PCG, some genes seem to follow different evolutionary pathways. In most analyses, *atp6*, *nad2*, *nad4L*, and *nad6* behave differently from other PCGs: they are more heavily affected by masking phase ([supplementary file S7](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), show higher d~N~/d~S~ values ([table 1](#evw187-T1){ref-type="table"}), are heavily saturated ([fig. 4](#evw187-F4){ref-type="fig"} and [supplementary file S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), and are much more variable ([fig. 3](#evw187-F3){ref-type="fig"}). In a nutshell, these genes are driven by different evolutionary constraints with respect to the others ([fig. 5](#evw187-F5){ref-type="fig"}); although the second principal component accounts for only the 8.02% of the variability, this may indicate that, furthermore, each of these genes follows its own evolutionary pathway. History of Bivalve Mitochondria ------------------------------- The present phylogeny ([fig. 6](#evw187-F6){ref-type="fig"}) corroborates a view of bivalve evolution where most extant families were essentially already present in the Lower Ordovician ([@evw187-B111]). Expectedly, the mitogenomic differentiation of the main clades of extant bivalves predates the paleontological evidences of the well-known Ordovician bivalve radiation ([@evw187-B33]; [@evw187-B51]; [@evw187-B143]; [@evw187-B50]; [@evw187-B128]; [@evw187-B111]). Conversely, the root of most families is placed after the Ordovician, probably because of limited taxon sampling: the same would hold for Palaeoheterodonta, but they appear to be much more recent (Early Triassic) since only members of the superfamily Unionoidea were actually inserted in this phylogenetic analysis. The topology of our phylogenetic tree is in perfect agreement with our previous results ([@evw187-B126]; [@evw187-B125]). It is particularly noteworthy the presence of the Amarsipobranchia clade, i.e., Pteriomorphia + Heterodonta. Our previous analyses were based on four mitochondrial genes (*cox1*, *cytb*, *rrnL*, and *rrnS*), and the use of the complete mitochondrial gene array led to the same result, even with a larger sample. Furthermore, the signal vs. noise analysis indicates that the phylogenetic signal of all genes is suitable ([supplementary file S17](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), and it becomes even stronger for the complete, concatenated dataset. Indeed, the same Amarsipobranchia clade was also retrieved by other studies using mitochondrial markers ([@evw187-B60]; [@evw187-B42]; [@evw187-B160]). On the other side, the use of morphological characters leads generally to the Heteroconchia *sensu* [@evw187-B173] clade (i.e., Palaeoheterodonta + Heterodonta; [@evw187-B60]; [@evw187-B14])---however, some morphological analyses retrieved instead the Amarsipobranchia clade ([@evw187-B33]). Recent analyses based on nuclear genes and transcriptomes ([@evw187-B85]; [@evw187-B156]; [@evw187-B147]; [@evw187-B63]) also recovered the Heteroconchia clade. Thus, the discrepancy between phylogenies based on morphology/nuclear genes vs. mitochondrial genes still holds. The amount of data in mitochondrial genomes is clearly restricted with respect to nuclear genomes. It is also possible that specific mitochondrial features may lead to a wrong relationship between heterodonts and pteriomorphians, thus disrupting Heteroconchia. Nucleotide composition may be one of these features: Amarsipobranchia have all negative A-T and positive G-C skews, while the situation is reversed for Palaeoheterodonta ([supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Another feature which is correlated with nucleotide composition is the substitution rate ([@evw187-B86]; [@evw187-B149]; [@evw187-B64]; [@evw187-B72]), which we show is linked with AR rate ([supplementary file S18](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online), as already hypothesized by [@evw187-B160]. However, our HERMES factor analysis demonstrates that the AT content practically does not explain other phylogenetic parameters like root-to-tip distance and ML distance from *N. nucleus*, and the protobranch *Solemya velum* show the same situation of Palaeoheterodonta, corroborating the idea that this is the plesiomorphic condition of bivalves. The same can be seen from the general PCA ([fig. 7](#evw187-F7){ref-type="fig"}): Amarsipobranchia and Palaeoheterodonta are separated, many features are considered (not only nucleotide composition), and Opponobranchia are shifted towards Palaeoheterodonta. Conclusions =========== The HERMES Index: Tempo and Mode of Mitochondrial Evolution ----------------------------------------------------------- The use of a single score to quantify mitochondrial evolution is a complex task, as many different parameters (that, furthermore, are linked together to different extents) can be considered and have to be summarized into a single number. High goodness-of-fit test results (given the complexity of the dataset) show that HERMES ([fig. 8](#evw187-F8){ref-type="fig"}) is indeed a suitable measure of this evolution. We are planning to apply the HERMES index in other taxa, and this will lead us to the identification of taxon-specific mitochondrial features that are suitable to measure molecular evolution; a Python script was written and made publicly available for this purpose (available at <https://github.com/mozoo/HERMES.git>). Concerning bivalves, there is consistence between all the aforementioned data and the HERMES measure: *S. velum* is the slowest-evolving mitogenome, followed by those of Palaeoheterodonta. Amarsipobranchia have similar HERMES scores, typically higher than Palaeoheterodonta; finally, the position of *L. elliptica* is intriguing and deserves further investigation. Within Palaeoheterodonta, F genomes have HERMES scores that are sharply lower than the M counterparts. The very early onset of DUI in Palaeoheterodonta led to the highest inter-sex distance values for DUI species ([@evw187-B13]) and to the well-known gender-joining pattern in phylogenetic trees ([@evw187-B37]; [@evw187-B42]; [@evw187-B189]; [fig. 6](#evw187-F6){ref-type="fig"}). The divergence of the two lineages started at least from the origin of Unionidae, ∼250 Mya ([@evw187-B166]; [@evw187-B36]; [@evw187-B47]; [@evw187-B116]; [fig. 6](#evw187-F6){ref-type="fig"}; [supplementary file S16](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online): F and M genomes are separately evolving since then. Recall that the organization of the ancestral bivalve mitochondrial genome should have resembled that of *Solemya* and *Nucula* ([@evw187-B127]), we can conclude that, while somehow the F mtDNAs of unionids retain much of this original condition, the M mtDNAs seem to have diverged more quickly on their own. DUI in other bivalve lineages appears to be a much more recent phenomenon ([fig. 6](#evw187-F6){ref-type="fig"}), or masked by masculinization role reversals ([@evw187-B73]; [@evw187-B131]; [@evw187-B189]), therefore the gender-joining pattern of unionids remains essentially unique across the phylogeny of the entire class. It is tempting to explore links between the molecular evolution of bivalve mitochondrial genomes as depicted by the HERMES score (and the mitogenomic feature-based PCA as well; [fig. 7](#evw187-F7){ref-type="fig"}) and the fossil evidences of the class. First known bivalves originated in the Early-Middle Cambrian and slowly faded away during the Late Cambrian; no Cambrian species are known from the Ordovician ([@evw187-B51]; [@evw187-B143]; [@evw187-B50]; [@evw187-B32]; [@evw187-B128]; [@evw187-B110]). However, some of them gave rise to extant bivalve clades in the Ordovician period ([@evw187-B34]; [@evw187-B143]; [@evw187-B128]). Protobranch forms were the first branching clade ([@evw187-B113]; [@evw187-B31]; [@evw187-B85]; [@evw187-B125]; [@evw187-B156]; [@evw187-B50]; [@evw187-B147]; [@evw187-B14]; [@evw187-B63]; and reference therein) before the evolution of the true feeding gill of all remaining autobranchs. The strict similarity of the mitochondrial genome of *Solemya* (and *Nucula* as well) with some gastropods ([@evw187-B127]) strengthens this hypothesis and allows to set a direction in the evolution of bivalve mtDNAs after the original appearance of extant bivalves; the HERMES index detects two main phases of this evolution ([fig. 8](#evw187-F8){ref-type="fig"}). According to the HERMES pattern, a first phase was the split of palaeoheterodonts from Amarsipobranchia. Again, fossil data may shed light on this issue. The most ancient families of Ordovician bivalves (dating to the Lower Ordovician, upper Tremadoc, ∼480 Mya) are Ucumariidae, Modiolopsidae (Goniophorinidae), and Lipanellidae ([@evw187-B142]; [@evw187-B50]): following current revised bivalve systematics, they should be classified within Heterodonta, Pteriomorphia, and Heterodonta, respectively ([@evw187-B24]). However, the state-of-art phylogenetic reconstruction is Lipanellidae + (Modiolopsidae + Ucumariidae) ([@evw187-B142], p. 117; [@evw187-B50], p. 12), and Heterodonta should be therefore considered as polyphyletic at their root. Moreover, Ucumariidae are interpreted as connected to extant anamalodesmatans ([@evw187-B142]; [@evw187-B50]), which are currently considered as nested within heterodonts ([@evw187-B61]; [@evw187-B46]; [@evw187-B60]; [@evw187-B71]; [@evw187-B164], [@evw187-B163]; [@evw187-B147]; [@evw187-B14]; [@evw187-B63]). Indeed, our phylogenetic reconstruction and the Amarsipobranchia hypothesis retrieve the same clade; namely, in the present phylogenetic tree it exhibits a basal tritomy (Heterodonta + Pteriomorphia + Anomalodesmata; [fig. 6](#evw187-F6){ref-type="fig"}). Furthermore, it is worth recalling the fossil record of the family Thoraliidae, which is also known from the Lower Ordovician ([@evw187-B112]; [@evw187-B141]; [@evw187-B32]) and it is currently classified within Palaeoheterodonta ([@evw187-B24]). Thus, in the Lower Ordovician a monophyletic Amarsipobranchia-like clade is hypothesized, while the first palaeoheterodonts have been found from the same epoch: concluding, phylogenetic paleontological reconstructions are not discordant with our molecular phylogentic tree or with the HERMES pattern. Eventually, the second phase of bivalve mitochondrial evolution as depicted by the HERMES score is the diversification of Amarsipobranchia, which definitely lost most plesiomorphic mitogenomic features ([fig. 7](#evw187-F7){ref-type="fig"}): all genes, with rarest exceptions, migrated on the same coding strand; an increase in length was coupled to an increase of the genomic regions not assigned to canonical genes; there was the inversion in A-T and G-C skews ([fig. 1](#evw187-F1){ref-type="fig"}; [supplementary file S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online); most strikingly, the gene rearrangement rate was given an unprecedented boost ([supplementary file S18](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online). Supplementary Material ====================== [Supplementary file S1--S21](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) are available at *Genome Biology and Evolution* online (<http://www.gbe.oxfordjournals.org/>). ###### Supplementary Data We would like to thank Fabrizio Ghiselli, Liliana Milani, Stefano Bettinazzi, and Mariangela Iannello for suggestions and stimulating discussion. Thanks are also due to Davide Guerra for his unvaluable guide to the taming of R and to Liliana Silva for having introduced us to the basics of factor analysis. The original manuscript was greatly improved by comments and suggestions by two anonymous reviewers. This work was financed by the "Canziani Bequest" fund (University of Bologna, grant number A.31.CANZELSEW). [^1]: **Associate editor**: Liliana Milani [^2]: d~N~/d~S~, value of d~N~/d~S~ computed by PAML using the best-known likelihood tree (see text for details) as the given phylogeny; pairwise median, median of all pairwise comparisons ± standard deviation; d~N~/d~S~ \> 10, number of pairwise d~N~/d~S~ ratios greater than 10. [^3]: Start and stop refer to the complete mitochondrial genome as referenced in [supplementary file S1](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online; length, nucleotide length without stop codon; aa, amino acid length. All newly annotated *atp8* are on the "+" strand; ORFs are listed in order of hit, as in [supplementary file S19](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evw187/-/DC1) online.

{ "pile_set_name": "PubMed Central" }

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"noisi", "need", "answer", "satur", "coupl", "alon", "bp", "latter", "nucleotideamino", "investig", "largescal", "death", "balanc", "especi", "input", "neg", "residu", "increas", "softwar", "amarsipobranchialik", "evwbperform", "materialhttpgbeoxfordjournalsorglookupsuppldoigbeevwdc", "clade", "×", "organel", "rarest", "flaw", "unionida", "amount", "probabl", "github", "almost", "given", "origin", "remain", "andml", "decreas", "aarhu", "freeliv", "identifi", "tame", "heterotroph", "written", "herm", "retain", "fund", "nadl", "take", "trna", "locat", "multipl", "viewer", "novelti", "collaps", "offspr", "use", "yessoensi", "timescal", "tritomi", "score", "support", "complet", "purpl", "uniparent", "li", "mother", "suggest", "choos", "also", "clc", "directli", "paml", "lanternula", "criterion", "perform", "lh", "horizont", "null", "featur", "normal", "straightforward", "copi", "bivmtdnaorfan", "unionoidea", "distribut", "longer", "first", "anamalodesmatan", "light", "phydesign", "nc", 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"previou", "therefor", "adequ", "ribosom" ]

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"claimed", "mammals", "less", "proteins", "needed", "keep", "mitochondria", "alive", "working", "small", "cluster", "protein", "coding", "genes", "PCGs", "spared", "GRE", "typical", "metazoans", "even", "apicomplexans", "Many", "tentative", "answers", "proposed", "question", "well", "known", "example", "mitochondria", "employ", "genetic", "code", "slightly", "different", "nucleus", "would", "possibly", "lead", "erroneous", "translations", "genes", "transferred", "nucleus", "however", "true", "animals", "plants", "also", "underwent", "mitochondrial", "GRE", "high", "hydrophobicity", "proteins", "would", "hamper", "import", "cytosol", "well", "also", "claimed", "certain", "gene", "products", "toxic", "cytoplasm", "Finally", "proposed", "genes", "still", "encoded", "mtDNAs", "must", "efficiently", "directly", "regulated", "mitochondrial", "redox", "conditions", "need", "mandatory", "colocation", "redox", "regulation", "CoRR", "sequencing", "characterization", "several", "complete", 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"listed", "table", "table", "newly", "annotated", "homology", "probability", "E", "P", "exceptions", "Paphia", "euglypta", "Prob", "E", "P", "Acanthocardia", "tuberculata", "Prob", "E", "P", "Musculista", "senhousia", "Prob", "E", "P", "gene", "annotated", "species", "highly", "probable", "homology", "correct", "anyway", "regarded", "tentatively", "annotated", "complete", "list", "HHblits", "scores", "available", "supplementary", "file", "http", "Supplementary", "Material", "http", "online", "annotation", "led", "us", "make", "small", "changes", "original", "GenBank", "annotations", "along", "corrections", "minor", "flaws", "detected", "analyses", "listed", "supplementary", "file", "http", "Supplementary", "Material", "http", "online", "complete", "updated", "annotations", "present", "mtDNAs", "given", "Table", "annotated", "genesSpeciesStartStopLengthaa", "Mytilus", "galloprovincialis", "Mytilus", "edulis", "Mytilus", "californianus", "Mytilus", "galloprovincialis", "Mytilus", 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"medians", "high", "standard", "deviations", "probably", "due", "high", "computed", "distantly", "related", "species", "therefore", "consider", "best", "estimation", "dataset", "value", "computed", "along", "phylogenetic", "tree", "comprised", "table", "table", "pattern", "overall", "negative", "selection", "already", "detected", "species", "genus", "Mytilus", "However", "conservation", "variability", "assessed", "general", "gene", "level", "results", "clearly", "indicate", "sharp", "contrast", "strongly", "conserved", "domains", "highly", "variable", "regions", "domains", "shown", "figure", "fig", "see", "also", "supplementary", "files", "http", "Supplementary", "Material", "http", "online", "Concluding", "domains", "mitochondrial", "PCGs", "currently", "severe", "purifying", "selection", "bivalves", "cases", "variability", "due", "indel", "events", "typical", "species", "see", "gene", "general", "pattern", "conservation", "specific", "domains", "PCG", "genes", "seem", "follow", "different", "evolutionary", "pathways", "analyses", "behave", "differently", "PCGs", "heavily", "affected", "masking", "phase", "supplementary", "file", "http", "Supplementary", "Material", "http", "online", "show", "higher", "values", "table", "table", "heavily", "saturated", "fig", "fig", "supplementary", "file", "http", "Supplementary", "Material", "http", "online", "much", "variable", "fig", "fig", "nutshell", "genes", "driven", "different", "evolutionary", "constraints", "respect", "others", "fig", "fig", "although", "second", "principal", "component", "accounts", "variability", "may", "indicate", "furthermore", "genes", "follows", "evolutionary", "pathway", "History", "Bivalve", "Mitochondria", "present", "phylogeny", "fig", "fig", "corroborates", "view", "bivalve", "evolution", "extant", "families", "essentially", "already", "present", "Lower", "Ordovician", "Expectedly", "mitogenomic", "differentiation", "main", "clades", "extant", "bivalves", "predates", "paleontological", "evidences", "Ordovician", "bivalve", "radiation", "Conversely", "root", "families", "placed", "Ordovician", "probably", "limited", "taxon", "sampling", "would", "hold", "Palaeoheterodonta", "appear", "much", "recent", "Early", "Triassic", "since", "members", "superfamily", "Unionoidea", "actually", "inserted", "phylogenetic", "analysis", "topology", "phylogenetic", "tree", "perfect", "agreement", "previous", "results", "particularly", "noteworthy", "presence", "Amarsipobranchia", "clade", "Pteriomorphia", "Heterodonta", "previous", "analyses", "based", "four", "mitochondrial", "genes", "cytb", "rrnL", "rrnS", "use", "complete", "mitochondrial", "gene", "array", "led", "result", "even", "larger", "sample", "Furthermore", "signal", "noise", "analysis", "indicates", "phylogenetic", "signal", "genes", "suitable", "supplementary", "file", "http", "Supplementary", "Material", "http", "online", "becomes", "even", "stronger", "complete", "concatenated", "dataset", "Indeed", 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Introduction {#sec1-1} ============ Nigeria's health sector is a part of its economy, which require critical discourse, especially concerning issues surrounding infrastructure, government funding, health workers motivation and satisfaction, among others. Considering the strategic role that health workers play within this sector, it is imperative that researchers and practitioners give the required attention to exploring and interrogating issues that can enhance their performance, to ensure that they function optimally \[[@ref1]\], \[[@ref2]\]. It is not untrue that where the workers in the Nigerian health sector are unfairly treated, the ailing patients will be at the receiving end of such injustice. Consequently, existing studies have attempted to show the relationships between healthcare workers satisfaction and the motivation towards work \[[@ref3]\]. There have also been studies to demonstrate the factors that influence health workers satisfaction with their work \[[@ref4]\], \[[@ref5]\]. Not withstanding, the case with Nigeria requires more than simply understanding the basic elements that explain job satisfaction of health workers. As \[[@ref6]\] opined, there are insufficient empirical details in the literature to explain health workers' motives for linking their level of job satisfaction with their motives to remain committed, or otherwise, to their organisation. Literature has asserted that employees' perception about treatments of diversity at the workplace is critical to enhancing, or otherwise, their behaviours and commitment to the job \[[@ref7]\]. According to \[[@ref8]\], \[[@ref9]\] it is not sufficient to measure employees' levels of satisfaction, but workplace diversity can influence the type of commitment they show to the organisation. In the Nigerian health sector, employees' perceptions about treatments of diversity are crucial not only to their satisfaction with the job but equally to their commitment to the organisation \[[@ref10]\], \[[@ref11]\]. The importance of this view of the public health sector, is that it could induce political behaviours, result in conflict situations and hence promote tensed work environments, if not properly managed. Despite these facts, there is again, a dearth of existing literature that shows an element of empirical analysis to demonstrate the moderating influence of workforce diversity on job satisfaction and organisational commitment of public healthcare employees in Nigeria. Therefore, this study is focused on investigating the mediating effect of employees' perceived treatments of diversity in the workplace on the relationship between job satisfaction and their commitment to the organisation. Material and Methods {#sec1-2} ==================== The method of survey was used to collect data. This research involved 133 public health workers from the Ministry of State Health of Lagos in Nigeria. Public health workers, especially in the Ministry of Health of Nigeria, are important for this research because of the increased awareness of the diverse workforce of the Ministry and the need to manage this diversity in a way that does not hurt employee satisfaction and commitment to the organisation \[[@ref14]\]. Furthermore, due to the importance of the Ministry of Health for the population and the overall well-being of each country, this research is considered essential to maintaining employee interest and motivation in the provision of quality services \[[@ref15]\]. Sample Size & Sampling Procedure {#sec2-1} -------------------------------- The sample size was determined using a sample size determination formula with a 5 per cent sampling error. Apply the Yamane sample size procedure \[[@ref16]\]. The formula N = N / (1 + N(e)^2^) where n = sampling size e = sampling error (0.05) N= the population (that is 200 employees in the healthcare location) n = 200 / (1+200 (0.05)^2^) n = 200 / 1+ 200 (0.0025) n = 200 / 1+ 0.5 n = 200 / 1.5 n = 133.33 (approximately 133) n = 133 Therefore, the sample size is 133 For this research, the technique adopted is the simple random sampling technique. Simple random sampling which is a subdivision of the probability sampling is used and is applicable as a result of the small number and similarity of the population and all subdivision of the population have an equal chance of being selected. This technique allows every member of the population to be a respondent by selecting the respondents without any form of partiality. Execution of Field Research {#sec2-2} --------------------------- The research used questionnaires as the main tools to perform this research. The questionnaire was used in this study for the following reasons; it allowed the respondents to express themselves more freely and clearly and to gather the answers in a standardized manner; It saved time by allowing information to be collected as soon as possible and also by facilitating the collection of possible information from a large sample of respondents. The research questionnaire consisted of a 5-point Likert scale in which the respondent was expected to agree strongly, agree, disagree and strongly disagree with carefully constructed questions, ranging from very positive to very negative, to measure the influence of diversity in the workforce on the commitment and satisfaction of employees. The questionnaire has been tested by the pilot using the Cronbach reliability test. The investigation questionnaire was divided into two parts. Section A was designed to provide information on the background of respondents, while section B was designed to provide information on the nature of workforce diversity, employee satisfaction and level of commitment (affective, regulatory and continuing) in the Ajeromi General Hospital. Measures {#sec2-3} -------- This research took advantage of the ideas from existing research studies. Workforce diversity issues were developed based on \[[@ref17]\], \[[@ref18]\], work satisfaction items were developed based on \[[@ref19]\], while organisational commitment items were adapted from \[[@ref20]\], \[[@ref21]\]. The data collected was encoded and entered into version 22 of SPSS. The analysis of data was carried out using descriptive statistics and structural equation modelling (SEM). Ethical Consideration {#sec2-4} --------------------- The management of the questionnaire was based on the readiness of respondents to respond to the research instrument. The participants in the study were also assured of confidentiality and anonymity, as their names were not reflected in the questionnaire. Validity and Reliability of Research Instrument {#sec2-5} ----------------------------------------------- The alpha coefficient (Cronbach's alpha) has been used in this research to test the reliability of the measurement scale. The Cronbach alpha is a method to determine the internal consistency of a measuring instrument by identifying all possible ways to split the items in the instrument and then investigate the correlation degree. Values of the coefficient from 0 to 1. A highly reliable instrument has a coefficient value near 1, where a score very close to 0 indicates a very low or no reliability of the instrument. It is widely accepted that the score above 0.7 shows the instrument's reliability. The SPSS (Social Science Statistical Package) was used to test the reliability of the research instrument. The statistics on the reliability of the tools used in this study were 0.747. The validity of the instrument was determined by the validity of the content. Statistical Analysis {#sec2-6} -------------------- The statistical analysis was carried out using SPSS (version 22) and AMOS (version 23) software for this study. Descriptive analysis showed the total number of respondents according to the analysed categories as well as the percentages of each category. The inferential statistics which established the multivariate relationships between public healthcare workers' diversity issues and commitment to their organisation was analysed using structural equation modelling (SEM) at a significant level of p \< 0.000. Results {#sec1-3} ======= Sixty-four (48.1%) were male, and the remaining 69 (51.9%) were female. This research involves more female participants than male respondents. The age distribution of the respondents revealed that 68 respondents were aged between 18 and 34 years; 45 respondents were aged between 35 and 44 years. The range of 13 respondents is 45 to 54 and 7 falls within the range of 55 years. This means that the age group was between 18 and 34 years old, with the majority of respondents and had 68 participants in this research. Based on years of working experience (40.6%) of respondents worked for less than 5 years, the table shows that (36.8%) of respondents worked for 5-10 years and (12.0%) for 11-15 years. The table shows that there were some respondents 16 years ago (10.5%). [Table 4](#T1 T2){ref-type="table"} above shows the background of the students surveyed 24 (18.0%) were O.N.D., 9 (6.8%) were N.C.E., 43 (32.3%) were H.N.D., 35 (26.3%) were Bachelor, and 22 (16.5%) were Masters. This means that the majority of the education background was (32.3%), meaning H.N.D. There were 43 respondents. ###### Socio-demographic Distribution of Respondent Demographic Variable Frequency Percentage --------------------------- ------------------- ------------ ------ Gender Male 64 48.1 Female 69 51.9 Age 18-34 68 51.1 35-44 45 33.8 45-54 13 9.8 55 and above 7 5.3 Years of Work experience less than 5 years 54 40.6 5-10 years 49 36.8 11-15 years 16 12.0 16 years above 14 10.5 Educational Qualification O.N.D 24 18.0 N.C.E 9 6.8 H.N.D 43 32.3 Bachelor's degree 35 26.3 Master's degree 22 16.5 ###### Structural Regression Weights for Job Satisfaction, Workforce Diversity and Employee Commitment Estimate S.E. C.R. P Label ----------- --------- ----------- ---------- ------- -------- -------- -------------------- Education \<\-\-- JobSatisf 0.215 0.098 2.184 \*\*\* Relationship Exist Gender \<\-\-- JobSatisf 0.418 0.067 6.223 \*\*\* Relationship Exist Religion \<\-\-- JobSatisf -0.179 0.078 -2.290 \*\*\* Relationship Exist Ethnicity \<\-\-- JobSatisf 0.301 0.057 5.312 \*\*\* Relationship Exist Position \<\-\-- JobSatisf 0.118 0.069 1.723 \*\*\* Relationship Exist AffectCom \<\-\-- Education 0.092 0.048 1.901 \*\*\* Relationship Exist NormCom \<\-\-- Education 0.110 0.049 2.239 \*\*\* Relationship Exist ContCom \<\-\-- Education 0.121 0.059 2.040 \*\*\* Relationship Exist AffectCom \<\-\-- Gender 0.245 0.067 3.642 \*\*\* Relationship Exist NormCom \<\-\-- Gender 0.341 0.069 4.954 \*\*\* Relationship Exist ContCom \<\-\-- Gender 0.129 0.083 1.553 0.120 No Relationship AffectCom \<\-\-- Religion 0.029 0.059 0.486 0.627 No Relationship NormCom \<\-\-- Religion 0.025 0.060 0.417 0.677 No Relationship ContCom \<\-\-- Religion 0.121 0.072 1.670 \*\*\* Relationship Exist AffectCom \<\-\-- Ethnicity 0.078 0.079 0.983 0.326 No Relationship NormCom \<\-\-- Ethnicity -0.025 0.081 -0.312 0.755 No Relationship ContCom \<\-\-- Ethnicity -0.099 0.098 -1.011 0.312 No Relationship AffectCom \<\-\-- Position 0.005 0.066 0.074 0.941 No Relationship NormCom \<\-\-- Position -0.041 0.068 -0.612 0.540 No Relationship ContCom \<\-\-- Position 0.074 0.081 0.909 0.363 No Relationship ![Regression Path of Job Satisfaction, Workforce Diversity & Employee Commitment](OAMJMS-7-2031-g001){#F1} [Figure 1](#F1){ref-type="fig"} shows the model for analysing the multifaceted relationships between workforce diversity, job satisfaction and commitment of employees. [Table 2](#T2){ref-type="table"} also shows structural regression weights of multi-variable analysis. Chi-square / Degree of Freedom (Cmin / df) = 3.876, Goodness of Fit Index (GFI) = 0.949, Normal Fit Index (NFI) = 0.854, Comparative Fit Index (CFI) = 0.872, Root Mean Square Error Approximation (RMSEA) = 0.148 are used to ensure the model fit of the analysis. The values are important based on the arguments presented in the study \[[@ref28]\], \[[@ref29]\], \[[@ref30]\]. The data supports a relationship between diversity of workforce and job satisfaction, diversity of workforce and organizational commitment and influence on the organizational commitment to work. The results of these data support existing research results \[[@ref21]\], \[[@ref22]\], \[[@ref23]\], \[[@ref24]\]. Specifically, the results from the statistical analysis indicate that job satisfaction significantly relates with all dimensions of workforce diversity in the following ways: education (r = 0.19), gender (r = 0.48), religion (r = -0.20), ethnicity (r = 0.42) and position (r = 0.15). The mediating effects of workforce diversity on the relationship between job satisfaction and employee commitment is also evident from the statistical analysis, especially with respect to education (affective commitment = 0.16, normative commitment = 0.18, continuance commitment = 0.18); gender (affective commitment = 0.32, normative commitment = 0.42); and religion (continuance commitment = 0.14). Discussion {#sec1-4} ========== The study focused on investigating the mediating effects of diversity in the workforce on job satisfaction and the organisational commitment of public health workers in Nigeria. For the analysis of data collected from respondents, the structural equation model was used. This study fills a research gap in the existing literature by empirically demonstrating the moderating influence of diversity in the workforce on the satisfaction of public health workers in Nigeria and their organisational activities. Through the understanding of such relationships, managers and governments in the healthcare sector are ably posed to curbing incidences that could arise as a result of adverse politicking and conflicts. Moreover, the findings of this study has been corroborated by existing literature that argued that diversity at the workplace can be harnessed not only to ensure sustainable satisfaction of employees, but to motivate them into creativity and commitment in the organization \[[@ref13]\], \[[@ref14]\], \[[@ref15]\], \[[@ref16]\], \[[@ref17]\], \[[@ref18]\], \[[@ref19]\], \[[@ref20]\], \[[@ref21]\], \[[@ref22]\], \[[@ref23]\], \[[@ref24]\], \[[@ref25]\]. This study constitutes a significant departure from conventional studies on workforce diversity by adopting a mediating perspective of the concept. In other words, the strength of workplace diversity is projected as a means of achieving higher levels of satisfaction from healthcare workers. The perspective adopted in this study is significant to enhancing passion and emotional affinity of healthcare workers toward caring for patients, hence sustaining a healthy community \[[@ref10]\], \[[@ref11]\], \[[@ref12]\]. Based on the findings from the study, educational diversity at the workplace is a very strategic linkage between job satisfaction and employees' commitment (including affective, normative and continuance commitment). Indeed, the need to appreciate the roles of educational diversity reflects in the capacity of each to function across different cadres, yet achieving the common goal of the organisation \[[@ref26]\], \[[@ref27]\]. In the same way, diversities based on gender and religion has been evidence from the statistical point of view of this study to be credible leverages for stimulating employees' interest and commitment to the organisation. This study concludes that not only is job satisfaction significant to ensuring the commitment of healthcare workers to their organisation, but more critical is the role of workforce diversity as viable leverage for transiting the interest of employees from the level of job satisfaction to organisational commitment. Specifically, three levels of workforce diversity, namely; education, gender and religion, were found to be strategic to enhancing healthcare workers perception about diversity treatments in the healthcare sector. Consequently, this study recommends that policymakers and managers in the Nigerian health care sector must pay adequate attention to creating a balance in the educational, gender and religious diversity of healthcare workers in Nigeria to sustain their satisfaction and stimulate their commitment to the organisation. Authors of this research work express sincere appreciation to the Management of Covenant University for giving full sponsorship to the publication of the research work in this journal. **Funding:** This research was financially supported by the Management of Covenant University, Nigeria **Competing Interests:** The authors have declared that no competing interests exist

{ "pile_set_name": "PubMed Central" }

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1. Introduction =============== Safe and reliable venous access is an essential procedure in modern oncology,^\[[@R1],[@R2]\]^ as long-term venous infusion is occasionally required in cancer patients for chemotherapy, fluid resuscitation, and nutritional treatment.^\[[@R3]\]^ Implantable central venous devices can play an important role in minimizing the discomfort of frequent vein puncture and cannulation.^\[[@R4],[@R5]\]^ Recently, the internal jugular vein (IJV) approach using real-time ultrasonography (USG) has gained popularity and has been performed widely; however, this approach might increase patients' discomfort and might cause inconvenience in maintaining an aseptic occlusive dressing.^\[[@R6]\]^ In this study, we aimed to introduce our surgical technique of cephalic venous access and to identify its safety, feasibility, and long-term functional outcomes compared to those of conventional methods. In addition, we investigated the possible risk factors that might affect the dysfunction of the implanted venous port. 2. Materials and methods ======================== 2.1. Study design ----------------- This study was approved by the institutional review board of Bucheon St. Mary\'s Hospital, College of Medicine, The Catholic University of Korea (HC19RESI0002). We retrospectively reviewed the medical records of patients who underwent implantable central venous access performed by surgeons between January 2012 and December 2014 at our hospital. The cases that were conducted by interventional radiologists were excluded. All patients gave written consent to the procedure and data collection for our database. The data, including patient demographics, cause of catheter insertion, venous approach methods, operation time, length of functional availability, and cause of device removal, were collected and analyzed. We classified our patients into 4 groups according to the venous access routes: IJV approach with real-time USG; external jugular vein (EJV) access with a cut-down method; subclavian vein access with the Seldinger landmark technique; and cephalic vein access. The operation methods were decided randomly according to the surgeons' competence and preference. We compared surgical and clinical outcomes between each group and performed additional analyses to identify risk factors for long-term functional impairment of the implanted venous devices. 2.2. Procedure for the cephalic vein approach --------------------------------------------- The patient was placed in a supine position, and his/her arm on the side of the procedure was extended to fully expose the outline of the deltoid and pectoralis muscles. After local anesthesia using diluted lidocaine was applied, a skin incision was made in the upper anterior chest wall along the deltopectoral groove (Fig. [1](#F1){ref-type="fig"}). Sharp dissection was carried out using electrocautery and curved mosquito forceps through the plane between the deltoid and pectoralis major muscles until the cephalic vein was identified (Fig. [2](#F2){ref-type="fig"}). The cephalic vein was then ligated proximally, and transverse venotomy was performed (Fig. [3](#F3){ref-type="fig"}), through which the venous catheter was inserted (Fig. [4](#F4){ref-type="fig"}). After radiologic examination for accurate placement of the catheter tip between the superior vena cava and right atrium, the reservoir was implanted with a suture on the pectoralis fascia (Fig. [5](#F5){ref-type="fig"}). The final catheter position was checked with a postoperative chest X-ray. ![Skin incision in the upper anterior chest wall along the deltopectoral groove.](medi-98-e18007-g001){#F1} ![Identifying the cephalic vein.](medi-98-e18007-g002){#F2} ![Ligating the cephalic vein at the proximal and peripheral sides.](medi-98-e18007-g003){#F3} ![Catheter insertion after transverse venotomy.](medi-98-e18007-g004){#F4} ![Reservoir implantation.](medi-98-e18007-g005){#F5} 2.3. Statistical analysis ------------------------- Summary statistics are presented as numbers and percentages for categorical variables and as the mean ± standard deviation for continuous variables. The Chi-square test or Fisher exact test was used to compare categorical variables, and the independent *t* test was used for continuous variables. Univariate and multivariate logistic regression analyses were applied to analyze the correlation of the possible risk factors with delayed malfunction of the implanted venous devices. We considered a 2-sided *P*-value \< .05 to be statistically significant. All statistical analyses were performed using the software package SAS 9.4 (SAS Institute, Inc, Cary, NC). 3. Results ========== A total of 508 patients were included in this study: 230 patients were included in the subclavian group; 134 in the IJV group; 25 in the EJV group; and 119 in the cephalic vein group. Regarding demographic characteristics (Table [1](#T1){ref-type="table"}), the cephalic group had a large portion of the colorectal malignancies compared to that of the IJV and subclavian groups (53.7% vs 34.3%, *P* \< .01; 53.7% vs 35.2%, respectively, *P* \< .01), and the IJV group included more female patients than the cephalic group (70% vs 57%, *P* = .03). The body mass index (BMI), patient age, and side of the access were similar between the cephalic and other groups. The mean operation time of the entire cohort was 46.9 minutes. The cephalic group had a longer operation time than the subclavian group (51.5 minutes vs 38.1 minutes, *P* \< .01); however, there was no difference in the operation time between the cephalic and IJV groups (51.5 minutes vs 53.7 minutes, *P* = .59). Comparatively, the operation time was longer in the EJV group than in the cephalic group (69.1 minutes vs 51.5 minutes, *P* \< .01). The length of catheter use and cycles of chemotherapy were comparable between the cephalic and other groups. In addition, the incidence of unscheduled catheter removal caused by late complications was similar between the cephalic and other groups as well. Infection and malfunction of the catheter were found to be late complications causing impairment in cyclic chemotherapy. We found 6 cases (1.2%) of immediate complications after the operation: the 4 cases in the subclavian group were all pneumothorax, and the 2 cases in the cephalic group were pneumothorax and postoperative bleeding. The statistical analysis was difficult for a few complicated cases. ###### Demographic characteristics and clinical outcomes according to the venous access. ![](medi-98-e18007-g006) Univariate analysis for the risk factors of late complications was performed according to the venous access routes, BMI, operation time, and age; however, the results were nonsignificant: odds ratios were 0.959 for the noncephalic approach (*P* = .93), 1.031 for BMI (*P* = .56), 1.004 for operation time (*P* = .64), and 0.979 for age (*P* = .21). In the multivariate logistic regression analysis (Table [2](#T2){ref-type="table"}), no factor was demonstrated to increase the incidence of late complications, including infection and catheter malfunction. ###### Multivariate risk factors analysis for the late complications. ![](medi-98-e18007-g007) 4. Discussion ============= This study revealed that the cephalic vein approach can be safely performed and has acceptable surgical, clinical, and functional outcomes compared to conventional percutaneous techniques. Central venous access devices in cancer patients were first introduced in 1982^\[[@R7]\]^ to reduce complications associated with frequent venous puncture during chemotherapy. There are several approaches for implantable central venous access. While the IJV approach with real-time USG has been widely performed because it has a good success rate without increasing the procedure duration,^\[[@R8],[@R9]\]^ we noticed that the surgical and clinical outcomes of the existing central venous approaches seem to be comparable.^\[[@R10]--[@R12]\]^ Despite its safety and feasibility, the conventional IJV approach requires specialized and expensive medical resources, including percutaneous vascular surgical kits, tunneling instruments, vascular USG, and/or fluoroscopic techniques; therefore, this approach might preclude wide clinical applications in small-volume regional hospitals or private clinics. Comparatively, the subclavian vein approach using the Seldinger landmark technique without a USG can be considered alternatively because it is easily performed with no special equipment; however, this approach might cause life-threatening complications such as hemothorax, pneumothorax, mediastinal hematoma, vascular perforation, and myocardial injury.^\[[@R13]\]^ Consequently, we employed the cephalic vein cut-down approach to reduce the risk of immediate complications^\[[@R14]\]^ while achieving cost-effectiveness and patient comfort. Several studies have reported that the cephalic vein approach can avoid possible immediate fatal complications, including pneumothorax and arterial puncture, although this approach might have a low success rate.^\[[@R15]--[@R19]\]^ In our study, the general surgeons performed central venous catheter insertion, and the failure rate of cephalic vein access was found to be 12.5%, which was superior to that of previous reports, ranging from 12% to 20%.^\[[@R11],[@R17]--[@R21]\]^ This result is similar to that of IJV and subclavian access.^\[[@R9],[@R19]\]^ Of the 136 patients who received the cephalic vein approach, the diameter of the cephalic vein was recorded in 62 cases. The mean diameters were 3.1 mm in the success group and 2.2 mm in the failure group, which were statistically significant (*P* \< .01); therefore, we carefully suggest that identifying the diameter of the cephalic vein would be helpful in successful procedure attempts. Two cases of immediate complications were found in the cephalic vein group (1.7%), and late complications occurred in 7 cephalic vein cases (5.9%). These results were similar to those of cephalic vein access in previous studies.^\[[@R16]--[@R23]\]^ Additionally, the complication rate of cephalic vein access in this study was lower than that of IJV access and subclavian access in other studies.^\[[@R9],[@R18],[@R19]\]^ Accordingly, we suggest that the cephalic vein cut-down approach can be safely performed with a comparable success rate and without increasing acute and late catheter-related complications compared to those of conventional percutaneous approaches. In addition, previous studies have shown that age, underlying cancer type, prior anticoagulant therapy, catheter tip position, and surgeon are independent prognostic factors for catheter complications.^\[[@R9],[@R24]--[@R26]\]^ However, in our study, patient BMI, age, and operation time did not increase the risk of long-term catheter dysfunction in univariate and multivariate logistic regression analyses; therefore, it seems reasonable that a central venous port can be considered in elderly or obese patients. Since our procedures were performed by several surgeons, there might have been interoperator variations as well as other confounding biases associated with the retrospective study design. Moreover, there might have been a reporting bias in analyzing the cephalic vein diameter between success and failure groups because the small size of the cephalic vein tended to be emphasized when the procedure failed. Nevertheless, our study was performed with a sufficiently large sample size, and the surgical records were documented by the specialized medical recorder immediately after the surgery. All surgeries were performed according to identical protocols. We suggest that these features might enhance the level of evidence of our study. 5. Conclusions ============== The present study demonstrates that cut-down central venous catheter insertion through the cephalic vein can be performed safely by nonvascular surgeons with no differences in surgical and clinical outcomes compared to those of conventional percutaneous approaches. Moreover, this approach requires no specialized equipment, including percutaneous vascular kits, tunneling instruments, and intraoperative USG. Therefore, the cephalic vein cut-down technique might incur less medical expenses than conventional approaches and would be helpful for both patients and surgeons. Author contributions ==================== **Conceptualization:** Jinbeom Cho, Kang Woong Jun, Byung Joo Song, Kiyoung Sung. **Data curation:** Jiyoung Rhu. **Writing -- original draft:** Jiyoung Rhu. **Writing -- review and editing:** Jinbeom Cho. Jinbeom Cho orcid: 0000-0002-6329-016X. Abbreviations: BMI = body mass index, EJV = external jugular vein, IJV = internal jugular vein, USG = ultrasonography. How to cite this article: Rhu J, Jun KW, Song BJ, Sung K, Cho J. Cephalic vein approach for the implantable central venous access. *Medicine*. 2019;98:46(e18007). Statistical consultation was supported by the Department of Biostatistics of the Catholic Research Coordinating Center, College of Medicine, The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, 06591, Seoul, Republic of Korea (ID. 1810-1439-01). Statistical consultation was supported by the Department of Biostatistics of the Catholic Research Coordinating Center, College of Medicine, The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, 06591, Seoul, Republic of Korea (ID. 1810-1439-01). The authors have no conflicts of interest to disclose.

{ "pile_set_name": "PubMed Central" }

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Background {#Sec1} ========== Globally, cancers are the second most common cause of death with about one in every six deaths attributable to cancer \[[@CR1]\]. In 2012, there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide \[[@CR1]\]. Over the last few years, despite advances in early diagnosis and treatment, and in the survival rate, there has been an increase in the overall incidence of cancers \[[@CR2]\]. The cost of cancer treatment has also increased with advancements in early diagnosis and treatment \[[@CR2]\]. Among Head and Neck Cancers (HNC), the most commonly occurring are Oro-Pharyngeal Cancers (OPC), which include the lip, oral cavity, and oropharynx. Globally, OPC are the sixth most common malignancy \[[@CR3]\]. Despite the technological advancement, cancer awareness and improvement in survival rates for some cancers \[[@CR2]\], no significant improvement has been reported in the five-year survival rates for OPC \[[@CR4], [@CR5]\]. OPC is one of the few oral diseases encountered by the oral health team that has significant morbidity and premature mortality Tobacco products, alcohol consumption and sun exposure are the most recognized risk factors for OPC \[[@CR6]\]. But the incidence of Human Papilloma Virus (HPV) related OPC has also been on the rise, particularly in younger adults who have never smoked or used other tobacco products \[[@CR7], [@CR8]\]. In Australia, HNC are the seventh most common cancers with an incidence rate of 17 cases per 100,000 persons \[[@CR9]\]. In 2017, estimated deaths due to HNC is well over 1000 individuals (with three fourths of the reported deaths in males), that amounts to roughly 3.7% (*n* = 4956) of all new cancers diagnosed \[[@CR9]\]. A recent effectiveness review on oral cancer screening, demonstrated conventional oral examination to be a feasible and satisfactory option for opportunistic screening in dental settings with sensitivity and specificity similar to breast and cervical cancer screening programs \[[@CR4], [@CR10]--[@CR12]\]. Early diagnosis of OPC can greatly increase the five-year survival rates from 50% to more than 80% \[[@CR6], [@CR13]\]. OPC are known to be amenable to early detection as they primarily occur at sites that are accessible and visible during a non-invasive examination \[[@CR4]\]. They are often preceded by a visible precancerous lesion enabling early detection and treatment \[[@CR14]\]. The tumors' proliferative factor and stage at the time of diagnosis largely determines the prognosis of cancers \[[@CR15]\]. However, only 30% of OPC are identified at an early stage \[[@CR16]\] with the majority (50%) being diagnosed at an advanced stage of metastasis (stage III or IV). This is largely due to late presentation, delayed diagnosis and lack of a clear referral pathway between doctors and dentists \[[@CR17]--[@CR21]\]. This forms the strongest argument for early diagnosis of OPC and initiating early treatment. It is essential that OHPs such as dentists, dental hygienists (DHs), dental therapists (DTs), and oral health therapists (OHTs), understand the importance of conducting a thorough oral screening examination for malignant and potentially-malignant lesions as part of their routine clinical assessments, even in younger populations originally considered at lower risk for oral cancer \[[@CR22], [@CR23]\]. The World Dental Federation and Dental Associations, including the Australian Dental Association, proactively encourage Oral Health Professionals (OHP) to incorporate oral mucosal examinations as part of routine assessment \[[@CR6], [@CR24]--[@CR27]\]. Several studies have assessed dentists' knowledge, attitudes and practices regarding oral cancer \[[@CR28]--[@CR33]\]. However, only a few studies include DH, DT, and OHT. Thus, clinical screening practices regarding oral cancer among the complete range of oral health professionals remain largely unknown. Screening programs should be designed to detect precancerous lesions or malignancies in their asymptomatic phases for all patients, not only in the older population groups, traditionally known to be at higher risk, but also in younger patient groups amongst which there has been a rising incidence of OPC \[[@CR4], [@CR7], [@CR8], [@CR34]\]. Nonetheless, studies among oral health professionals in Australia identified lack of training, lack of confidence and time constraints as the three most important barriers for oral cancer screening \[[@CR4], [@CR21]\]. Expansions of early diagnosis and treatment of oral cancers should be a priority from a public health viewpoint. The purpose of this study was to investigate oral cancer screening practices of OHPs in Victoria, Australia as well as evaluate their oral cancer-related opinions and attitudes; and to identify factors associated with the likelihood of an OHP performing an oral cancer screening examination. This information can be used to identify gaps in oral cancer knowledge among OHPs, and subsequently to inform the development of continuing education programs specifically focused on oral cancer prevention, identification and management of malignant and potentially-malignant lesions of the oral cavity and oropharynx at the state level. Methods {#Sec2} ======= The study was a cross-sectional survey of OHP in Victoria, Australia. As of September 2014, there were 4781 registered OHPs in Victoria, which amounts to 23% of the total Australian dental work force. Of them, 3715 are dentists and another 585 are registered DH/DT/OHTs in Victoria \[[@CR35]\]. With the approval of the Human Research Ethics Committee at the University of Melbourne, a request was submitted to the professional associations representing Victorian OHPs, including the Australian Dental Association, the Dental Hygienists Association of Australia (Victorian Branch) and the Victorian Dental and Oral Health Therapists Association, to distribute the survey to their members. OHPs were initially contacted by mail in September 2014. Two weeks and four weeks after the first mailing, postcards were sent to thank those who had returned the questionnaire and to remind the others of the importance of the study, and encourage them to complete the survey. In an attempt to maximize the response rate, participants were offered a choice between completing either a paper-and-pencil survey or a web-based survey (i.e. invitation-letter with URL of online survey, questionnaire and reply paid envelope). Additionally, after the initial postal contact, the Australian Dental Association indicated a preference that e-mails be sent with the link to the online survey, as a reminder. Two reminders were sent in March and April 2015. According to the literature \[[@CR36]--[@CR38]\] the expected response rate among OHPs is around 30%. However, an Australian study involving medical doctors, reported a mean response rate of 19.7% when using a similar simultaneous mixed-mode survey (a paper questionnaire and login details sent together) \[[@CR39]\]. Therefore, we expected a final sample size of around 611 dentists and 89 dental hygienists/dental therapists/oral health therapists. The data collection period was from September 2014 to March 2015. This sample size would be large enough to conduct the required statistical analyses as the minimum sample size necessary to study the relationship between a dependent variable (e.g., knowledge of risk factors for oral cancers) and a set of independent variables based on the case of 10 independent variables accounting for 5% of the variance (a conservative estimate) in the dependent variable, indicates that a sample size of 335 will yield a power of 0.80, that is, an 80% chance of explaining that proportion of the variance at a *p*-value of 0.05 \[[@CR40]\]. Survey data was derived from a questionnaire consisting of four parts: socio-demographic and work characteristics (6 items); knowledge of risk factors for oral cancers (14 items); level of confidence in discussing health behaviors with patients (5 items); and oral cancer screening practices (11 items). The socio-demographic information included sex, age-range and location of practice. By occupational sub-stratification, participants were classified into three professional groups: 'Dentists'; 'Dental Hygienists (DH)'; and 'Oral Health Therapists and Dental Therapists (OHT/DT)'. Dentists were further sub-divided into General Dental Practitioners (GDP) and 'Dental Specialists'. Professional experience was classified into six categories: '5 years or less'; '6 to 10 years'; '11 to 15 years'; '16 to 20 years'; '21 to 25 years'; and 'More than 25 years'. Employment history information included the postal code(s) of their main geographical location of practice. Using the Australia Post's local delivery service guidelines, work locations were classified as being 'Urban', or 'Rural' \[[@CR41]\]. Participants were asked to self-assess their level of knowledge about risk for oral cancer, utilizing a 10-point numerical scale (0 = 'Very poor' to 10 = 'Excellent'). Knowledge regarding risk factors for oral cancer included 11 factors (smoking, periodontal disease, caffeine consumption, family history of oral cancer, chewing betel nut, human papillomavirus \[HPV\] infections, hepatitis C infection, chewing tobacco, level of alcohol consumption, herpes simplex virus infections and history of oral cancer). Participants were asked to select which of the 11 factors were risk factors for oral cancers utilising a three-option response of 'Yes, 'No' or 'Unsure'. To quantify the degree of knowledge of oral cancer risk factors among OHPs, a score system was developed, which involved adding the weighted answers for each of the 11 questions on risk factors. Scores ranged from 0 to 9. OHPs were also asked two questions regarding their perceived need for more training and education on oral cancers with response categories of 'Yes'; 'No'; or 'Unsure'. Questions related to oral cancer screening attitudes and practices asked: a) whether OHP should routinely screen for oral cancer; b) self-reported frequency of screenings (response options were: 'Very rarely'; 'With less than 50% of patients'; 'With 50% or more of patients'; and 'With every patient'); c) factors that prompt OHPs to initiate discussion of risk factors with patients; d) age groups routinely screened for oral cancer; e) whether OHPs inform the patient about being screened; f) if patients are informed what the screening involves; g) if they have ever used additional diagnostic tools to detect malignant lesions; and h) their course of action when an abnormal lesion was detected. Participants were also asked whether they had ever referred a patient to an oral medicine specialist. Items focusing on OHPs' level of confidence in discussing health behaviors with patients, utilized a numerical 10-point scale (0 = not at all confident to 10 = completely confident). These behaviors included; tobacco use, alcohol consumption, sexual behaviors, oral hygiene practices and diet and nutrition. Data analysis {#Sec3} ------------- The analysis provides descriptive information on the participants' work and various socio-demographics. Bivariate associations were evaluated with Chi-squared analysis for nominal or ordinal variables. For variables on an interval scale, results were analyzed using one-way analysis of variance (ANOVA). A significant ANOVA was followed by post-hoc comparisons using Tukey's Honestly Significant Differences tests. To better understand the association between the combination of socio-demographic, work and psychosocial variables and the probability of conducting a screening exam, a stepwise logistic regression analysis (LRA) was performed. All *p*-values \<0.05 were considered significant. Data manipulation and analysis were conducted using IBM SPSS Statistics (Version 21.0, IBM Corporation, Endicott, NY, USA). Results {#Sec4} ======= A total of 380 OHPs responded, achieving an overall response rate of 9.4% (ranging from 9.3% among dentists to 22.9% among DH/DT/OHTs). Fourteen respondents indicated that they do not practice dentistry anymore while another 31 mentioned that they do not routinely treat adult patients. These 45 OHPs were excluded from further analysis. Thus, a total of 335 were included in the final analysis. Most this group were dentists (72%; *n* = 241), either GDP (63.6%) or Dental Specialists (8.4%). Another 13.7% (*n* = 46) were DHs; 12.2% (*n* = 41) were OHTs, and the remaining 2.1% (*n* = 7) were DTs. By gender, the majority were female (58.2%). Among dentist participants, a marginally higher percentage were male (55.8%), while more than 90% of DH/DT/OHTs were female. By age, more than half the total participants (55.5%) were 45 years or younger; however, the largest age group was the '46 to 55 years old' group (24.8%), with 10.7% of the participants in the '25 years of age or younger' group. Differences in age and gender were statistically significant by oral health profession (*p* \< 0.001). Those working exclusively as OHT were younger than those working as dentists or DHs (See Table [1](#Tab1){ref-type="table"}).Table 1Demographic, work characteristics of oral health professionals in VictoriaDentistsDHsOHT/DTsTotal(*n* = 242)(*n* = 45)(*n* = 48)(*n* = 335)Age of group\* 25 or less7.42.135.510.7 26--3519.011.150.022.4 36--4523.235.66.222.4 46--5525.242.36.224.8 \> 5525.28.92.119.7Gender\* Male55.82.28.341.8 Female44.297.891.758.2Duration of practicing\* 5 or less15.711.180.523.0 6--1012.411.117.112.8 11--1511.613.32.411.0 16--209.915.609.9 21--259.920.00.09.9 \> 2540.528.90.033.4Location of workplace Urban76.677.374.576.4 Rural23.422.725.523.6\*Chi- squared test; *p*-value: 0.001 When participants were asked about the location of their workplace, the majority (76.4%) indicated an 'Urban' location, with no statistically significant difference by professional background. Regarding the length of time practicing as an OHP, 33.4% indicated more than 25 years of practice; 30.8% between 11 and 25 years; 23.0% reported five years or less of practice; and the remaining 12.8% reported between 6 and 10 years of practice. As expected, differences by duration of practice between groups were statistically significant (*P* \< 0.001). Oral cancer screening examination. Almost all OHPs (95.2%) indicated the importance of and need for routine oral cancer screening in their clinical practice. However, only half of those surveyed (51.3%) reported conducting a comprehensive oral cancer screening for all of their patients (Table [2](#Tab2){ref-type="table"}). Another 21.8% reported conducting a comprehensive oral cancer screening for '*50% or more*' of their patients. OHPs who reported performing oral cancer screening in '*less than 50% of patients*' or '*Very Rarely'* comprised 14.0% and 12.8% of respondents, respectively. Differences by professional background for completing oral health screening were not statistically significant. The vast majority of respondents (92.2%) routinely screened patients 40 years of age or older for oral cancer. This proportion was 68.4% for 20--39 years old patients, and 38.5% for patients younger than 20 years of age.Table 2Oral screening behaviors of oral health professionals in VictoriaDentistsDHsOHT/DTsTotal(*n* = 242)(*n* = 45)(*n* = 48)(*n* = 335)How frequently do you complete a comprehensive oral cancer screening? With every patient51.746.754.251.4 With 50% or more of patients21.922.220.821.8 With less than 50% of patients13.217.814.614.0 Very rarely13.213.310.412.8How often do you discuss risk factors for oral cancer with your patients? With every patient5.88.98.46.6 With 50% or more of patients22.740.033.326.6 With less than 50% of patients47.935.541.745.3 Very rarely23.615.616.621.5What factors influence your decision to perform an oral cancer screening examination?\ (Multiple answers) Patient complains of a problem32.231.141.433.6 Age of the patient31.425.243.832.4 Medical history26.931.143.829.9 Other16.526.625.019.1 Among those OHPs who reported not conducting a cancer screening for all their patients (*n* = 193), the decision to screen was influenced by the patient complaining of an oral health problem (65.6%); the patient's age (63.8%); or the patient's medical history (53.3%). Another 35.0% of respondents indicated other factors influencing their decision to conduct an oral cancer screening examination, such as exposure to risk factors (i.e. alcohol consumption and smoking). Of note, only the 'age of the patient' as an influencing variable reached statistical significance (*p* \< 0.05). When excluding those who 'Very rarely' conducted an oral cancer screening examination on their patients, just over half of respondents (51.9%) indicated that they 'Sometimes' informed the patient that they were screening for oral cancer lesions. Another 19.9% 'Always' informed patients that they were screening for potentially malignant or malignant lesions. More importantly, almost one third (28.2%) of the OHPs, 'Never' informed patients that such screening assessments were taking place. No statistically significant differences were found by professional background. After excluding those who reported 'Very rarely' conducting oral cancer screenings, for almost all OHPs (97.6%) the examination involved a visual inspection of the patient's oral cavity and 81.1% also included an extra-oral visual inspection (Table [3](#Tab3){ref-type="table"}). Meanwhile, a screening examination that "Always" included either a visual inspection of the oropharynx or conducting neck palpation was reported at lower rates (52.3% and 28.1, respectively).Table 3Oral cancer screening examination behaviors of oral health professionals who performed screening more frequently than "Very rarely"DentistsDHsOHT/DTsTotal(*n* = 210)(*n* = 39)(*n* = 43)(*n* = 292)Screening examination involved:\ Extra-oral visual inspection of the oral cavity Always80.482.183.781.1 Sometimes2.95.10.02.7 Never16.712.816.316.2A visual inspection of the oral cavity Always98.193.010097.6 Sometimes1.47.00.02.1 Never0.50.00.00.3A visual inspection of the oropharynx Always50.564.150.052.3 Sometimes40.335.942.940.0 Never9.20.07.17.7Neck palpationAlways25.523.744.2\*\ 28.1Sometimes56.952.632.552.6Never17.633.721.319.3\*Chi- squared test; *p*-value: 0.05 Apart from neck palpation, which was significantly more frequently conducted among OHTs than other professional groups (*p* \< 0.05), none of the other screening techniques reached statistical significance levels by OHP background. Thirty-three respondents (11.3%), most of whom were dentists, indicated using additional diagnostic aids/tools to identify potentially malignant lesions. The most commonly utilized additional diagnostic tools included biopsies and radiographs. When asked if respondents had ever referred a patient to an oral medicine specialist to have a lesion investigated, which subsequently was diagnosed as a malignancy, 54.2% of respondents had either never encountered such a clinical scenario, or were unsure. Of those who had referred a patient (*n* = 136), the most common intraoral site of the malignant lesion (*n* = 41) was cancer of the tongue, followed by cancer of the floor of the mouth (*n* = 22); hard palate (*n* = 18); and the lip (*n* = 14). There were also eleven cases of bone cancer. OHP respondents were asked about their clinical decision pathway once an abnormal lesion was detected and the practitioner could not reach a diagnosis. The most commonly reported courses of action included: 'Referral to an oral medicine specialist' (85.7%); 'Follow-up at a later appointment' (53.7%); and 'Consultation with another colleague' (45.4%). Least mentioned courses of action included: 'Ask the patient to monitor the lesion' (38.2%); and 'Referral to the patient's general medical practitioner' (14.3%). Differences by professional background were statistically significant for: 'Consultation with another colleague' (*p* \< 0.001), where DHs and OHT/DTs were more likely to pursue this course of action; and 'Asking the patient to monitor the lesion', whereby OHT/DTs (55.8%) were more likely to ask the patient to monitor the lesion compared with 35.9% and 35.9% for dentists and DHs, respectively (*p* \< 0.05). Regarding knowledge of oral cancer risk factors, 99.4% of participants identified smoking as a risk factor for oral cancer. The second most frequently identified risk factor was chewing betel nut and chewing tobacco products (98.2%). A history of oral cancer was the third most frequently identified risk factor (97.0%), followed by family history of oral cancer (96.1%), and alcohol consumption (94.6%). The oral cancer risk knowledge score ranged from 0 to 88 with a mean of 68.5 (s.d. 10.1). When OHPs were asked how often they discussed risk factors for oral cancer with their patients, the largest group (45.3%) indicated discussing risk factors for oral cancer with less than 50% of their patients (Table [2](#Tab2){ref-type="table"}). Another 21.5% reported they 'Very rarely' discussed risks factors with their patients. Discussion of risk factors in 'More than 50% of patients' or 'With every patient' was reported by 26.6% and 6.6% of OHP respondents, respectively. No significant differences were demonstrated by professional background. Concerning participants' level of confidence in discussing behavioral issues that may affect oral health with their patients, participants generally felt confident discussing oral hygiene practices (Mean 8.9; s.d. 1.6), diet and nutrition (Mean 8.3; s.d. 1.7), tobacco use (Mean 8.2; s.d. 1.8), and to some extent alcohol consumption (Mean 7.3; s.d. 2.2). In contrast, the mean level of confidence in discussing sexual behaviors (i.e. oral sex practices) was comparatively low at 3.6 (s.d. 1.8). The probability of conducting an oral health screening examination ("Very rarely": 0 vs. Other categories \[i.e., 'With every patient'; 'With 50% or more of patients'; and 'With less than 50% of patients'\]: 1) was explored utilizing LRA with age, sex, profession, rurality and number of years of work experience, risk factor knowledge index, self-assessed oral cancer knowledge, and self-confidence items as independent variables (Table [4](#Tab4){ref-type="table"}). After controlling for the other independent variables included in the model, four variables remained statistically significant \[χ^2^(4) = 40.689; *p* \< 0.0001\]. Results indicated that dental specialists, were less likely to perform an oral cancer screening examination (OR = 0.18; 95% CI 0.07 to 0.52). As the level of self-assessed knowledge of oral cancer increased, so too did the likelihood of performing an oral cancer screening examination (OR = 1.35; 95% CI 1.09 to 1.67). Additionally, when the OHP's level of confidence in discussing oral hygiene with the patient increased, the likelihood of conducting an oral cancer screening examination also increased (OR = 1.25; 95% CI 1.03 to 1.52). On the other hand, when the 'Patient complained of a problem' the likelihood of performing an oral cancer screening examination decreased (OR = 0.21; 95% CI 0.10 to 0.44). The variance for oral cancer screening examination, using the full model, was 22.5% (Nagelkerke ^2^r = 0.225).Table 4Regression coefficient, odds ratios and 95% confidence interval for odds ratios for the factors associated with the probability of conducting an oral health screening examination among oral health professionals in Victoria, Australiaβ coefficientOdds ratio95% Confidence intervalDental specialist−1.700.180.07 to 0.52Self-assessed knowledge0.301.351.09 to 1.67Level of confidence (oral hygiene)0.221.251.03 to 1.52Patient complained of a problem−1.560.210.10 to 0.44Constant−1.04Nagelkerke r^2^ = 0.225 Discussion {#Sec5} ========== The present study represents an attempt to explore oral cancer-related practices and attitudes among different OHPs working in the Australian state of Victoria. This study's results show little over 50% of OHPs perform comprehensive oral cancer screening with all patients. Although these results are not directly comparable due to wording of questions and response categories, they would contrast with earlier studies done in Australia where OHPs routinely performed oral mucosal screening on 85--95% of their recall and new patients \[[@CR4], [@CR21]\]. In the same manner, studies done elsewhere, reported that 85--89% of dentists and 66--78% of dental hygienists performed oral cancer examination for their patients, although they did not indicate frequency of performing these examinations \[[@CR29], [@CR30]\]. Apart from this, the present study is largely in line with one other previous study done among DH/DT and OHTs in Australia on the importance and need for routine oral cancer screening \[[@CR33]\]. A high proportion indicated that they did so in almost all patients older than 40 years. However, only about two-thirds of respondents routinely screened patients 20--39 years of age. The study also indicated that OHPs, who screened all patients, were less likely to always include neck palpation, and to a lesser extent, visual inspection of the oropharynx in their oral cancer screening practice. This may be due to lack of confidence in undertaking a head and neck examination as evidenced in other study done in Australia \[[@CR21], [@CR33]\]. These findings are cause for some concern as many oral cancers may not present with visibly detectable signs or symptoms during the pre-malignant or localized stages, which represents a clinical window in which these lesions are most amenable to treatment \[[@CR29]\]. Such early stage lesions could otherwise be detected by a comprehensive and thorough visual examination of high risk sites \[[@CR22], [@CR42]\] and facilitating patient education about oral cancer. Furthermore, a comprehensive oral cancer screening examination would seem essential, particularly given the growing incidence of HPV positive oropharyngeal cancers \[[@CR4], [@CR22], [@CR42], [@CR43]\]. Consequently, it is important for OHPs to maintain and develop competence and confidence in neck palpation techniques and visual examination of the oropharynx as part of their oral cancer screening practices \[[@CR22]\]. Where necessary, appropriate education programs should be developed to address the knowledge and skill gaps in this area \[[@CR4], [@CR21]\]. The present study showed that OHTs performed oral cancer screening somewhat more frequently than other OHP groups. As the incidence of oral cancer continues to rise \[[@CR9]\], the role of oral health professionals in the prevention, early identification and management of oral malignancies will become increasingly relevant to public health. In order to reduce the morbidity and mortality of oral cancer, it is necessary to implement programs aimed at prevention, early identification and diagnosis. Early identification would involve facilitating patient recognition of signs and symptoms and providing education about when to consult a health professional. Patient delays in seeking care could be reduced by self-examination for early clinical features and by population level educational interventions, particularly targeting those in the higher risk groups for oral cancer. In recent years, awareness of cancer risk factors has increased due to wider media coverage of the issue \[[@CR44]\]. However, studies indicate that in contrast to other types of cancers, the majority of the population are ignorant about oral cancer \[[@CR45], [@CR46]\]. The public may have a tendency to interpret oral cancer symptoms and signs as relatively minor, as problems that are likely to resolve on their own. Given the lack of general public understanding of oral cancers and inability to recognise clinical symptoms and signs \[[@CR22]\], it can be inferred that patient presentation to an OHP regarding an oral lesion may be either absent or too late, which would adversely impact on early detection and treatment outcomes. In addition to these issues, some cancers may be asymptomatic and the absence of notable symptoms may further contribute to late diagnosis. For these reasons, opportunistic oral cancer screening examinations conducted by OHPs remain an important mode for early identification and diagnosis. Results from the multivariate analysis indicate that after controlling for other variables, dental specialists tend to perform less oral screening compared with other OHPs. It is possible that dental specialists rely on GDPs to conduct comprehensive dental examinations while they focus solely on the condition for which the patient was referred them; however, this cannot be stated categorically as the relevant information was not collected in the survey. The odds of conducting an oral screening examination also decreased when the patient complained of oral pain. It could be inferred, although once again respondents were not asked about this in the survey, that OHPs addressed the presenting complaint on these occasions (e.g. pain) and did not conduct a more comprehensive oral examination. Unsurprisingly, the data indicated that OHPs with a higher self-evaluated confidence level in oral cancer-related knowledge had greater confidence advising patients and were more likely to perform oral cancer screening examinations \[[@CR21]\]. The ultimate goal would be to have the whole oral health team collaborate with an integrated approach to oral health care, particularly in the context of oral cancer knowledge, screening practices and patient and community education. When considering these results, certain limitations must be acknowledged. The low response rate may have introduced non-response bias, and combined with the self-reported nature of the data means it is not possible to generalize findings to all OHPs working in Victoria, Australia. Furthermore, response rates to online surveys have been demonstrated to be less than for surveys administered using a paper-based approach \[[@CR47], [@CR48]\]. However, the response rate is also related to the size of the population under study with larger populations requiring smaller response rates \[[@CR48]\]. Thus, although the number of responses received was not as high as was hoped, the achieved overall response rate was within the expected range for online surveys among oral health professionals (2.5% to 26%) \[[@CR49], [@CR50]\]. Despite some minor differences, this study group is largely representative of the Victorian OHP population in terms of age, profession, gender and distribution. There was an overrepresentation of DH/DT/OHTs in the study sample, who make up 28% of total respondents but only 17% of all registered dental practitioners in Victoria \[[@CR35]\]. When considering the socio-demographic profile, there is no available data to determine the age range of each dental practitioner group in the wider profession in the state of Victoria. However, the age profile of survey respondents (55.5% aged 45 years or younger) is very similar that for all dental practitioners in Australia at the time of the survey (56.7% aged less than 45 years old) \[[@CR35]\]. The proportion of female dentists in the study also mirrored figures reported for the state of Victoria (57%) in 2015. Most dentists worked in urban areas (80.0%) \[[@CR51]\]. Conclusions {#Sec6} =========== Together with risk behavior change, oral cancer screening has been shown to contribute to reductions in oral cancer mortality rates among high-risk individuals \[[@CR22], [@CR52]\]. It is highly probable that OHPs will see patients with oral cancer and many more with potentially malignant lesions in their professional lives \[[@CR4], [@CR21]\]. Present results suggest that continued efforts to enhance the quality and consistency of oral cancer screening practices are required, which should include education and training of OHPs in up-to-date and evidence-based screening methods. The recommendation to see an oral health professional at least once a year would only be beneficial if patients are routinely screening for oral cancer. The results from this study may assist in the development of oral cancer-related education and training programs for OHPs. Increased emphasis on regular training in oral cancer screening protocols and in patient education and counselling practices are imperative to improve oral cancer prevention and rates of early detection, to reduce the mortality-burden resulting from oral cancers. DHs : Dental Hygienists DT : Dental Therapists GDP : General Dental Practitioners HNC : Head and Neck Cancer HPV : Human Papilloma Virus OHP : Oral Health Professional OHTs : Oral Health Therapists OPC : Oro-Pharyngeal Cancers Not applicable Funding {#FPar1} ======= This study was partially funded by a grant received from DENTSPLY Research & Innovation Grants. Availability of data and materials {#FPar2} ================================== Ethics approvals were granted on the basis that only researchers involved in the study could access the de-identified data. Raw data have been stored securely at the Melbourne Dental School, The University of Melbourne. The minimum retention period is 5 years from publication or public release of the results. Supporting documents are available upon request to the corresponding author. RM1 contributed with the conception of the study, design, draft of the manuscript, read and approved the final manuscript. SH carried out the fieldwork, contributed with the data analysis and draft of the manuscript, and read and approved the final manuscript. RM2 contributed with the conception of the study, and the design and draft of the manuscript, and read and approved the final manuscript. DB contributed with the conception of the study, and the design and draft of the manuscript, and read and approved the final manuscript. MM1 contributed with the conception of the study, interpretation of the data and the design and draft of the manuscript. RM3 contributed with interpretation of the data and final drafting of the manuscript. BK carried out part of the fieldwork, interpretation of the data and final drafting of the manuscript. AC contributed with interpretation of the data and drafting of the manuscript. All the authors revised and approved the manuscript. MM2 contributed with the conception of the study, and the design and draft of the manuscript, and read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== This study was approved by The Human Research Ethics Committee of The University of Melbourne, Australia (\#1442325.1). All participants conseted to participate in this study. It was explained to potential participants that reception of completed surveys, was assumed as consent for participation in the study. Consent for publication {#FPar4} ======================= Not Applicable Competing interests {#FPar5} =================== The authors declare that they have no competing interest. RM is a Section Editor for BMC Oral Health. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

{ "pile_set_name": "PubMed Central" }

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"agerang", "cr", "iv", "claim", "followup", "site", "workplac", "iii", "review", "need", "subdivid", "survey", "care", "expect", "enabl", "anymor", "therapistsor", "third", "n", "background", "answer", "mortalityburden", "later", "hpv", "respectivelyt", "around", "counsel", "design", "feder", "elsewher", "develop", "exclud", "indic", "unsur", "colleagu", "c", "amongst", "natur", "age", "perceiv", "possibl", "frequenc", "manuscript", "consent", "school", "seem", "clear", "aidstool", "diseas", "dh", "investig", "r", "analysi", "innov", "regular", "younger", "g", "could", "asymptomat", "simultan", "apart", "action", "raw", "death", "onlin", "papillomaviru", "pain", "diagnos", "varianc", "yield", "grow", "mode", "behavior", "extraor", "cancerrel", "releas", "somewhat", "increas", "awar", "access", "improv", "maintain", "revis", "report", "prioriti", "therapist", "offer", "similar", "paperandpencil", "point", "lra", "tukey", "issu", "emphasi", "urban", "area", "floor", "tongu", "recept", 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22,229

[ "Background", "Globally", "cancers", "second", "common", "cause", "death", "one", "every", "six", "deaths", "attributable", "cancer", "million", "new", "cancer", "cases", "million", "cancer", "deaths", "worldwide", "last", "years", "despite", "advances", "early", "diagnosis", "treatment", "survival", "rate", "increase", "overall", "incidence", "cancers", "cost", "cancer", "treatment", "also", "increased", "advancements", "early", "diagnosis", "treatment", "Among", "Head", "Neck", "Cancers", "HNC", "commonly", "occurring", "Cancers", "OPC", "include", "lip", "oral", "cavity", "oropharynx", "Globally", "OPC", "sixth", "common", "malignancy", "Despite", "technological", "advancement", "cancer", "awareness", "improvement", "survival", "rates", "cancers", "significant", "improvement", "reported", "survival", "rates", "OPC", "OPC", "one", "oral", "diseases", "encountered", "oral", "health", "team", "significant", "morbidity", "premature", "mortality", "Tobacco", "products", "alcohol", 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1. INTRODUCTION {#sec1} =============== Calcifying Epithelial Odontogenic Tumor (CEOT) is an uncommon jaw lesion with a benign and slow-growing pattern but locally aggressive course \[[@r1]\]. The lesions are most prevalent among patients aged 30 to 50 years, and no sex predilection has been observed \[[@r1], [@r2]\]. Clinically, the CEOT appears as a slowly asymptomatic expansion with radiolucent honeycomb appearance in the posterior areas of the mandible \[[@r2]\]. The CEOT is predominantly an intraosseous tumor in approximately 94% of cases \[[@r3]\]. The peripheral variant is a rare tumor (6%) with less aggressive behavior that was described by Pindborg in 1966 \[[@r4]\]. The classical histopathological aspects include sheets and islands of eosinophilic polyhedral epithelial cells in association with homogeneous pink amyloid-like deposits and areas of calcification \[[@r5]-[@r22]\]. One unusual histological finding is the presence of clear cells, as reported in 14 cases of peripheral CEOT in the English literature \[[@r2], [@r5]-[@r19], [@r22], [@r23]\]. CEOT is an odontogenic tumor; the consensus regarding the best therapeutic approach is surgical excision with safe margins recommended for the intraosseous variant \[[@r3], [@r4]\]. However, the peripheral lesions are commonly treated through conservative surgery with no review of this approach \[[@r3]\]. The present study reports a case of a 21-year-old Caucasian female patient with a mandibular peripheral CEOT rich in clear cells. An additional critical literature review focuses on the treatment protocols for the peripheral variant of the tumor. 2. CASE DESCRIPTION AND RESULTS {#sec2} =============================== A 21-years-old Caucasian woman presented to a private dental clinic with a chief complaint of asymptomatic swelling in the gingiva observed four years prior. A gradual increase in size and no history of previous treatment were also reported during the anamnesis. The patient signed the informed consent, which represents the ethical approval of the faculty committee. Her medical and socio-economic histories were not contributory. The extra-oral evaluation did not reveal changes. The intraoral examination revealed a sessile nodule with a color similar to that of the mucosa and a focal erythematous area with a fibro-elastic consistency measuring 1.5 cm in the largest diameter extending from the inferior right lateral incisor to the inferior right first premolar. The lesion involved the vestibular and lingual gingiva, causing displacement of the inferior right canine (Fig. **[1](#F1){ref-type="fig"}**). Panoramic reconstruction and parasagittal slices of the Cone Beam Computed Tomography (CBCT) showed a slightly superficial hypodense area between the inferior right lateral incisor and inferior right canine with reabsorption of the alveolar crest (Fig. **[2](#F2){ref-type="fig"}**). Based on the clinical and immunological aspects, the main diagnosis hypotheses included peripheral ossifying fibroma, peripheral giant cell lesion, and ancient pyogenic granuloma. The peripheral odontogenic tumors were also included as a differential diagnosis. An excisional biopsy was performed and a clear separation was noted between the lesion and mandible bone during the trans-surgical approach. The histopathological analysis revealed a well-circumscribed proliferation comprising numerous islands and strands of epithelial polyhedral cells with well-defined borders and marked round nucleus in the connective tissue under the mucosal epithelium. Numerous nests, cords, and small islands of polyhedral cells with clear and vacuolated abundant cytoplasm were observed interspersed with the amorphous eosinophilic deposits (Fig. **[3](#F3){ref-type="fig"}**). Immunohistochemistry was performed, which yielded positive results for CK-19 in the epithelial cells, except for the clear cells. Congo red staining showed the presence of amyloid-like deposits with apple-green birefringence under polarized light (Fig. **[4](#F4){ref-type="fig"}**). A final diagnosis of a peripheral CEOT rich in clear cells was reached. No complications were observed in the postoperative appointment and a follow-up schedule was established. The patient has had no recurrence after 22 months (Fig. **[5](#F5){ref-type="fig"}**). 3. PERIPHERAL CEOT CASES {#sec3} ======================== Long-term follow-up of the central variant of CEOT has shown the best results, with no recurrence following block resection with safe margins; however, well-documented cases are scarce \[[@r1], [@r24]\]. Additionally, there is discussion regarding aggressive behavior involving clear cells in the CEOT \[[@r1], [@r21], [@r24]\]. Other odontogenic lesions with clear cells including ameloblastoma and clear cell odontogenic tumors have been reclassified as malignant, in which a more aggressive course can be expected \[[@r24], [@r25]\]. Nonetheless, this premise has not been confirmed for CEOT because the latest review of all central variants showed no conclusive data regarding the worse course for this type of lesion \[[@r1], [@r24], [@r25]\]. Peripheral CEOT is considered a harmless lesion, but the recommended approach for peripheral cases in the presence or lack of clear cells is not emphasized in previous reports \[[@r2], [@r4]-[@r6], [@r8]-[@r20], [@r22]\]. In this context, a search of the English literature was performed in the PubMed database using the keywords "calcifying epithelial odontogenic tumor" with "Pindborg," "peripheral," "clear cell," and "treatment." All manuscripts on peripheral CEOT published until May 2018 were considered. Cross-references were included. Studies involving mixed odontogenic tumors in association with CEOT, CEOT associated with other conditions, no exclusive extraosseous tumors, absence of treatment modality, and no full-text database were excluded. The anatomic sites, duration, clinical and imaging aspects, types of treatment, recurrence, and follow-up are summarized in Table **[1](#T1){ref-type="table"}**. 4. DISCUSSION {#sec4} ============= The peripheral variant is a rare presentation of CEOT, with a differential diagnosis including gingival reactive lesions such as ancient pyogenic granuloma, peripheral giant cell lesion, and peripheral ossifying fibroma in addition to other peripheral odontogenic tumors. The histopathological aspects of the extraosseous are similar to those of the intraosseous CEOT counterparts and Congo red staining confirms the presence of amyloid-like material such as immunohistochemistry keratin markers confirm the odontogenic epithelial origin \[[@r21], [@r22]\]. In addition, clear cells have been observed in some odontogenic lesions and, although previous authors have speculated their relationship with more aggressive CEOT, no role has yet been shown for the relationship between the behavior and this histopathological presentation \[[@r21], [@r22]\]. Extraosseous CEOT is considered a less aggressive tumor and conservative surgery is performed in most cases. However, no previous articles have evaluated the best approach for peripheral CEOT based on recurrence and follow-up data. The current review identified no clinical significance in relation to the aggressive aspects. This finding is supported by the observation that approximately 80% of cases were treated with conservative management, with only one recurrent case without a clear cell component \[[@r2], [@r4]-[@r6], [@r8]-[@r18], [@r22]\]. A detailed evaluation of all articles on peripheral CEOT found no reported recurrence during follow-up in 96% of cases \[[@r2], [@r5], [@r6], [@r8]-[@r20], [@r22], [@r23], [@r26]\]. A conservative soft tissue excision was the main approach in the literature and was also sufficient for the complete resolution of the current case. These findings suggest an indolent, local, non-infiltrative course of peripheral lesions when compared to intraosseous CEOT. The discrete cupping or erosion of superficial bone may be caused by compression rather than by invasive behavior, supporting the non-aggressive behavior of the lesion. Other peripheral odontogenic tumors, including ameloblastoma, ameloblastic fibroma, calcifying cystic odontogenic tumor, and adenomatoid odontogenic tumor, present similar characteristics \[[@r27], [@r28]\]. An isolated case with recurrence was observed with posterior intraosseous involvement; however, it was a unique case with bilateral peripheral lesions \[[@r18]\]. Clear cells were also not observed and there was no recurrence after the second conservative surgery \[[@r18]\]. CONCLUSION ========== Our review confirms that conservative enucleation is the most appropriate management for peripheral CEOT. Moreover, we identified no association between clear cells and clinical aggressiveness in peripheral lesions as was previously suggested for the central counterparty. Declared none. CONSENT FOR PUBLICATION ======================= Informed consent was obtained from all the patients prior to being enrolled in the study. CONFLICT OF INTEREST ==================== The authors declare no conflict of interest, financial or otherwise. ![](TODENTJ-12-856_F1){#F1} ![](TODENTJ-12-856_F2){#F2} ![](TODENTJ-12-856_F3){#F3} ![](TODENTJ-12-856_F4){#F4} ![](TODENTJ-12-856_F5){#F5} ###### Previous articles about peripheral calcifying epithelial odontogenic tumor including clear cell variant with emphasis in the treatment. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Authors** **Anatomic Site** **Duration** **Clinical Presentation** **Imaging**\ **Treatment** **Recurrence** **Folow-up** **Presentation** ----------------------------------- ------------------- -------------- -------------------------------- --------------------- ------------------------------------------------- ---------------- -------------- Pindborg, 1966^5^ Max.\ 5 years Painless firm\ NCI Simple excision No NCI gingiva mass Abrams and Howell, 1967^10^\*\* Mand. gingiva NA Painless firm\ Crest\ Simple excision No 3 years mass resorption Decker and Laffitte, 1967^11^ Mand.\ 5years Painless firm\ NCI Simple excision No NCI gingiva mass Patterson *et al*, 1969^12^ Mand. gingiva 1 year Painless firm\ NCI Simple excision No NCI mass Krolls and Pindborg, 1974^13^ Mand. gingiva NA Painless firm\ NCI Simple excision No NCI mass Wherteimer *et al*., 1977^14^\*\* Max. gingiva NA Painless firm\ No Simple excision No NA mass Ai-ru *et al*., 1982^15^\*\* Mand. gingiva 10 years Painless firm\ NCI Ressection No 2 years mass Ai-ru *et al*., 1982^15^\*\* Mand. gingiva 2 years Painless firm\ No Partial\ No 10 years mass ressection Takeda *et al*., 1983^16^ Max\ NA Painless firm\ NCI Excision underlying bone No NCI gingiva mass KH Ng *et al*., 1996^17^ Max.\ 1 year Painless firm\ Erosion Excision No NA gingiva swelling Houston & Fowler, 1997^18^\*\* Max.\ 5 months Ulcerated\ No Simple excision No 4 years gingiva mass Houston & Fowler, 1997^18^\*\* Mand.\ 5 months Ulcerated\ Erosion\* Simple excision No 4 years gingiva mass Orsini *et al*., 2000^9^\*\* Max. gingiva 6 months Painless red\ Not\ Simple\ No 4 years mass performed excision Mesquita *et al*., 2003^6^\*\* Max. gingiva 10 months Painless firm\ No Excision No 2.5 years nodule de Oliveira *et al*., 2009^2^\*\* Max. gingiva NA Painless exophitic mass No Excision No 1 year de Oliveira *et al*., 2009^2^\*\* Mand. gingiva NA Painless exophitic mass Superficial cupping Excision No 1 year Abrahão *et al*., 2009^19b^ Mand\ 3 months Painfull erithematous swelling No^a^ Simple excision^c^ Yes^d^ 3.5 years gingiva Habibi *et al*., 2009^20^\*\* Max.\ 11 years Ulcerated\ NCI Excisional biopsy with 5-mm safety margins No NA gingiva mass Marino *et al*., 2013^21^ Max. gingiva NA Painless\ Bone\ Conservative surgery including teeth extraction No 2 years swelling resorption Afrogheh\ Mand. gingiva 6 months Painless\ Erosion Complete excision No 1.5 years *et al*., 2014^7^\*\* swelling Shetty *et al*., 2016^22^\*\* Mand. gingiva 8 months Painless swelling No Excisional biopsy No 6 months de Carvalho *et al*., 2016^26^ Mand.\ 1 month Painless swelling No Excisional\ No 1 year gingiva biopsy Bajpai M,\ Max.\ 9 months Pink-coloured\ Loss of lamina dura Complete\ No 6\ 2018^23^\*\* gingiva swelling excision months Flores *et al*., 2018\*\*¡ Mand. gingiva 4 years Painless\ Superficial\ Conservative surgery No 22 months swelling hypodense\ cupping --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Max.= maxillary Mand. = mandible NA = Not available NCI = Not clearly identified a = osseous resorption and calcifications (recurrent lesions) b = bilateral c = soft tissue excision followed bone curettage d = after 1 year \*Observed only during the surgical procedure \*\*Clear cell variant ¡ Current case

{ "pile_set_name": "PubMed Central" }

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22,230

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Introduction {#sec1_1} ============ Adrenal myelolipomas are benign tumors which are usually asymptomatic and diagnosed incidentally on imaging or during autopsy. They were first described by Gierke in 1905 and were termed as myelolipomas by Oberling in 1929 \[[@B1], [@B2]\]. They are uncommon benign tumors of the adrenal cortex constituting approximately for 3--5% of all primary adrenal tumors and are increasingly being diagnosed due to improved imaging techniques \[[@B3], [@B4]\]. Adrenal myelolipomas are most frequently detected after the fifth decade and are seen almost equally in both genders. They are commonly found as unilateral masses, but occasional bilateral masses have been documented \[[@B5]\]. A right-sided predominance in unilateral tumors has also been reported \[[@B6]\]. They are usually asymptomatic but myelolipomas may attain large sizes and present as abdominal masses \[[@B7], [@B8]\]. Patients may also present with symptoms of hematuria and hypertension \[[@B9]\]. Their size may also cause mass effect by compressing other intra-abdominal organs \[[@B10]\]. Malignant transformation has not been documented in these tumors. Diagnosis of giant adrenal myelolipomas in a timely manner may prevent complications due to hemorrhage and can be lifesaving. Case Description {#sec1_2} ================ A 52-year-old man with a past medical history significant for atrial fibrillation, diabetes type 2 and hypertension for 30 years was admitted with early satiety for the past 3 months and progressive dyspnea on exertion for 3 weeks. He also complained of gradual unintentional weight loss of approximately 100 pounds over the last 10 years. He had been a non-smoker and a moderate alcohol user for the last 30 years. On initial presentation to the hospital, he denied shortness of breath at rest or chest discomfort and was in no acute distress. His blood pressure was 204/93 mm Hg on the left arm and 194/88 mm Hg on the right arm with an irregular heart rate of 54 beats/min and oxygen saturation of 96% on room air. The lungs were clear to auscultation, jugular venous distension was noted at 13 cm above the sternal angle and 3+ bilateral leg edema with scrotal edema. Cardiac auscultation was pertinent for 3/6 systolic ejection murmur over the left sternal border. His abdomen felt firm and lacking in tympanicity, but it was non-tender in all four quadrants. Bowel sounds were audible but faint. Laboratory values were notable for a brain natriuretic peptide of 980 ng/l, creatinine of 1.2 mg/dl, BUN of 20 mg/dl, HCO~3~ of 31 mEq/l, INR of 1.23, Hb of 12.5 g/dl, Na of 140 mEq/l, K of 3.7 mEq/l and negative cardiac troponins. He was monitored in the Intensive Care Unit for decompensated heart failure with uncontrolled hypertension. To promote diuresis and reduce afterload, he was administered intravenous furosemide 80 mg, oral metolazone 2.5 mg, oral hydralazine 75 mg every 6 h, oral clonidine 0.3 mg, lisinopril 40 mg, isosorbide mononitrate 120 mg and losartan 100 mg. Overnight, he had an episode of moderate self-limited epistaxis with minimal improvement in blood pressure control. He remained in slow atrial fibrillation with a lowest recorded heart rate of 38 beats/min. However, he denied dizziness at any time despite a negative fluid balance of 1.8 liters. Renal function worsened with an increase of creatinine to 1.6 mg/dl. An echocardiogram showed a left ventricular ejection fraction of 30% with four-chamber dilatation, moderately reduced right ventricular systolic function, severe mitral regurgitation and moderate pulmonary hypertension. He underwent right heart catheterization showing a right atrial pressure of 12 mm Hg, right ventricular pressure (systolic/diastolic) of 60/12 mm Hg, pulmonary artery pressure of 60/23 mm Hg and pulmonary capillary wedge pressure of 20 mm Hg and a cardiac index of 2.3 liters/min. A left heart catheterization revealed clean coronaries, and alcohol was thought to be the likely etiology of non-ischemic cardiomyopathy in this patient. Urine catecholamine and vanillylmandelic acid were measured and a magnetic resonance angiography of the renal arteries was performed to evaluate for secondary causes of hypertension. This showed non-obstructed renal arteries bilaterally. A minimally enhancing 22 × 16 × 23 cm mass was visualized in the left upper and mid-abdomen displacing the spleen and stomach posterosuperiorly, the pancreas anteriorly and the left kidney inferiorly (fig. [1b](#F1){ref-type="fig"}). The left adrenal gland was not visible. A contrast-enhanced CT scan of the abdomen to further characterize the mass showed no infiltration of the surrounding vasculature or organs, and the left adrenal gland was visualized although it could not be confirmed if the mass was arising from the adrenals (fig. [1a](#F1){ref-type="fig"}). The suggested radiological differential diagnoses were: liposarcoma, teratoma and myelolipoma. A needle biopsy of this mass showed adipose tissue with foci of extramedullary hematopoiesis consistent with myelolipoma without any evidence of malignancy, confirming this mass to be a benign myelolipoma (fig. [2a, b](#F2){ref-type="fig"}). Urinary epinephrine, metanephrine and vanillylmandelic acid were within normal limits. A renal perfusion scan showed a small left kidney with reduced perfusion contributing 22% to the overall tubular function without obstructive uropathy. The lower extremity edema was partly ascribed to be due to inferior vena cava compression, and diuretics were decreased due to worsening renal function. Given the bradycardia and severe congestive heart failure, an electrophysiologist was consulted for evaluation and need for pacemaker implantation. Bradycardia was ascribed to be partly due to the Cushing reflex secondary to elevated blood pressure, and as the patient had no symptoms secondary to a low heart rate, pacemaker implantation was not advised. Despite maximum doses of clonidine, isosorbide, hydralazine, losartan and lisinopril, at best a moderate blood pressure control was achieved preoperatively. The patient was taken for surgical removal of the mass. During surgery, the mass was found to be soft, highly vascular but well encapsulated and attached only to the left adrenal gland via a small portion that was resected along with the mass (fig. [3a, b](#F3){ref-type="fig"}). There was minimal blood loss during the surgery, and the patient had an uneventful recovery in the postoperative period. A renal perfusion scan done 8 days after surgical removal showed 36% contribution to the overall tubular function. Blood pressure control improved after surgery and the patient was gradually titrated off clonidine, hydralazine and lisinopril. Bradycardia resolved with few episodes of atrial fibrillation with rapid ventricular response that were controlled with β-blockers and digoxin. The antihypertensive medications that the patient was discharged on were carvedilol 40 mg, valsartan 320 mg and isosorbide mononitrate 240 mg. Discussion {#sec1_3} ========== Myelolipomas are composed of fat and hematopoietic tissue and are usually biochemically inactive. They are thought to arise from the reticuloendothelial cells of the adrenal gland in response to repeated stimulation by stress, inflammation and ACTH \[[@B11], [@B12], [@B13]\]. Both the adipose and the hematopoietic cells have been noted to have the same pattern of X-chromosome inactivation and have been postulated to arise by clonal proliferation from a common mesenchymal stem cell \[[@B14]\]. Moreover, the chromosomal translocation (3;21)(q25;p11) has been detected in myelolipomas in patients with hematological malignancies, which has led to the speculation of bone marrow origin of these tumors \[[@B15]\]. Extra-adrenal myelolipomas have been documented in the mediastinum, lung, thoracic spine, liver mesentery, spleen, kidney and pelvis \[[@B16], [@B17], [@B18], [@B19], [@B20], [@B21]\]. They have been noted to be more common in women \[[@B22]\]. Extra-adrenal myelolipomas may mimic extramedullary hematopoietic tumors especially since bone spicules have been reported \[[@B23]\]. Extramedullary hematopoietic tumors generally have a higher content of erythroid cells and less fat tissue compared to extra-adrenal myelolipomas which have a relatively higher number of lymphoid cells \[[@B24]\]. Adrenal myelolipomas are best imaged by CT due to the high negative attenuation value. However, calcification is seen in approximately 27% of the cases and the presence of a higher proportion of hematopoietic tissues may mimic an adrenal adenoma \[[@B4], [@B25], [@B26]\]. Ultrasonography is not a very useful diagnostic modality, especially if the tumors are smaller in size (\<2 cm). An MRI has a similar diagnostic utility as a CT scan in adrenal myelolipomas with a high fat content \[[@B27]\]. The gold standard for diagnosis is tissue biopsy which typically shows hematopoietic cells admixed with mature adipose cells \[[@B6]\]. Adrenal myelolipomas can be associated with hyperaldosteronism, congenital adrenal hyperplasia and Cushing\'s syndrome \[[@B28]\]. They are known to be biochemically inactive but a case of adrenal myelolipoma secreting cortisol has been reported \[[@B8]\]. Adrenal myelolipomas \<5 cm are conservatively managed with sequential imaging, but larger masses are preferably resected due to the risk of hemorrhage \[[@B29]\]. Laparoscopic resection has been successfully used to reduce hospital stay and improve perioperative outcomes, but it is not preferable for tumors \>10 cm or in tumors with infiltration or extensive adhesions \[[@B30], [@B31]\]. Our patient was fortunate not to have any hemorrhagic complications. His clinical presentation was compatible with symptoms related to mass effect of the giant myelolipoma, which resolved after surgical resection. Conclusion {#sec1_4} ========== Adrenal myelolipomas are benign tumors with favorable outcomes when diagnosed in a timely manner. Their insidious clinical course becomes apparent only when mass effects are manifested in the form of an abdominal mass, flank pain, hypertension and pedal edema. It is important to differentiate them from other malignancies, such as liposarcoma, and to ensure that the myelolipoma is not biochemically active. Adequate follow-up of small masses and surgical resection of large masses are the standard of care which provide for lesser morbidity and mortality. Disclosure Statement {#sec1_5} ==================== The authors declare that there was no funding for this study. ![**a** CT scan of the abdominal mass seen in transverse view. **b** MRI scan of the abdominal mass seen in coronal view.](cro-0007-0182-g01){#F1} ![**a** Low-power magnification of the biopsy showing capsule (red arrowhead), adrenal tissue (black arrowhead) and an admixture of adipose and hematopoietic cells. **b** High-power magnification of the biopsy showing adipocytes (black arrowhead), erythroid precursors (cyan arrowhead), lymphoid precursors (blue arrowhead) and megakaryocytes (green arrowhead). Bony spicules and sinusoids are conspicuously absent from the specimen confirming it is a myelolipoma.](cro-0007-0182-g02){#F2} ![**a** Resected specimen showing a well-encapsulated tumor. **b** Resected specimen cut surface revealing adipose tissue with interspersed areas of hemorrhage.](cro-0007-0182-g03){#F3}

{ "pile_set_name": "PubMed Central" }

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**Core tip:** Recognizing patterns of subcutaneous sarcoidosis is important for hand surgeons and other surgical specialties that do not commonly see this patient population in order to rapidly identify and diagnose a disease that has extra-cutaneous manifestations and can lead to greater morbidity and mortality when not diagnosed or treated early. INTRODUCTION ============ Sarcoidosis is a chronic systemic granulomatous disease of unknown etiology characte-rized by the presence of non-caseating granulomas in affected organs\[[@B1]\]. he non-caseating granulomas of sarcoidosis can be found anywhere on the body and involve many different organs. In the head and neck, sarcoidosis typically impacts the cervical lymph nodes, globe, parotid, and larynx with up to 5% of patients demonstrating facial nerve involvement\[[@B2]\]. Ocular involvement is also seen, presenting as uveitis, scleritis, and chorioretinitis\[[@B2]\]. Typically, the lungs are the primary site of disease with cutaneous manifestations being the second most common site. Cutaneous manifestations of Sarcoidosis are seen in up to 9%-37% of patients\[[@B3],[@B4]\]. One particular manifestation of cutaneous involvement, subcutaneous sarcoid nodules, is a rare finding. Previous estimates of the frequency of subcutaneous sarcoidosis ranged from 1.4%-6%, with more recent studies suggesting an occurrence rate of 11.8%-16% among patients presenting with cutaneous sarcoid involvement\[[@B3],[@B5]-[@B7]\]. This variant of Sarcoidosis is defined clinically by asymptomatic, non-tender, flesh colored nodules usually ranging between 0.5-2.0 cm\[[@B6],[@B8]\]. Histologically, subcutaneous sarcoidosis is defined by the presence of non-caseating granulomas present in the subcutaneous tissue\[[@B6]\]. On ultrasound imaging these lesions present as an irregularly defined mass with hyper and hypoechoic areas\[[@B9]\]. Fludeoxyglucose (FDG) positron emission tomography (PET) / computed tomography (CT) has also been used to identify sarcoid lesions and presents as increased uptake in subcutaneous areas\[[@B10]\]. While helpful, FDG PET/CT may not be able to differentiate between connective tissue diseases as Sjrogen's Syndrome also presents as increased uptake\[[@B11]\]. Additionally, many soft tissue diseases can look similar on magnetic resonance imaging (MRI) making defining imaging characteristics of each disease important to diagnosis. On MRI imaging Sarcoid lesions involving the face and neck will appear with high signal intensity on T2-weighted images and enhancement on contrast-enhanced images\[[@B2]\]. Given that Wegener's Granulomatosis can mimic Sarcoidosis in the head and neck, MRI imaging helps differentiate these two soft tissue diseases as Wegener's Granulomatosis will appear as hypodense on T1 and T2-weighted images with variable degrees of enhancement with contrast\[[@B2]\]. Similarly, both sarcoidosis and Scleroma can impact the cervical lymph nodes making diagnosis difficult. MR imaging is again a useful tool in differentiating these two diseases in the lymph nodes as Scleroma will appear as low signal intensity on T1 and high signal intensity on T2 imaging with homogeneous pattern of contrast enhancement, and Sarcoidosis will have a foamy appearance on T1 weighted imaging\[[@B2],[@B12]\]. Lupus Both Sarcoidosis and another soft tissue disease like scleroderma can impact the lymph nodes in the neck Recent literature has suggested a strong correlation between subcutaneous sarcoidosis and evidence of systemic sarcoid involvement, and that sarcoid lesions may be an early finding indicative of systemic disease\[[@B7],[@B13],[@B14]\]. Given its correlation with systemic illness, the ability to correctly identify subcutaneous sarcoid lesions is an important diagnostic tool for physicians in the early stages of sarcoidosis. We describe here a case of subcutaneous sarcoidosis and review the literature to determine if there are any commonalities in the presentation of this disease among patients, and to better assist clinicians with diagnosing this rare disorder. A literature search was performed of the MEDLINE and PubMed database using keywords as "Subcutaneous sarcoidosis" and "Subcutaneous Sarcoidosis", combined with "hand", "hand surgery", "Upper extremity surgery", yielding 202 results. The search includes all articles published since 2000. The search was limited to studies published in English and performed on humans. Cases without either a serum angiotensin-converting enzyme (ACE) level or a chest imaging study for all patients reported were excluded. Ultimately 29 articles were selected using the diagnostic criteria of subcutaneous sarcoidosis first proposed by Vainsencher et al\[[@B6]\]. These 29 articles represent 82 cases of subcutaneous sarcoidosis dating back to 1966. CASE PRESENTATION ================= Chief complaints ---------------- A 38-year-old Caucasian female was referred to Plastic Surgery from Dermatology after presenting with an 8-mo history of 4 firm, asymptomatic, skin-colored nodules on her left and right upper extremities and neck. History of past illness ----------------------- A past medical history of arthritis and recurrent nephrolithiasis. Personal and family history --------------------------- She denied any family history of soft tissue masses or autoimmune disorders. Physical examination upon admission ----------------------------------- The mass on the posterior aspect of her neck measured 0.5 cm x 5 cm. The nodule on her left forearm measured 2 cm x 3 cm (Figure [1A and B](#F1){ref-type="fig"}). On the extensor surface of her right forearm were two masses measuring 1 cm x 1.4 cm and 3.5 cm x 4.5 cm respectively (Figure [1C and D](#F1){ref-type="fig"}). The overlying skin was normal. At the time of the plastic surgery consult, the patient was compliant with her medication regimen of dextroamphetamine-amphetamine 30 mg by mouth once daily, and acetaminophen 500 mg Tab as needed for arthritic pain. ![Subcutaneous nodule with normal overlying skin on the left forearm (A and B), subcutaneous nodules with normal overlying skin on the right forearm (C and D).](WJCC-7-2505-g001){#F1} Laboratory examinations ----------------------- The complete blood count with differential was normal. Comprehensive metabolic panel was normal. Erythrocyte sedimentation rate, C-Reactive Protein, Anti-SCL, Rheumatoid Factor, and antinuclear antibody were all within normal limits. Her Serum ACE level was also within normal limits with a value of 53 U/L (normal 9-67 U/L). While chest radiograph was normal, chest computed tomography revealed mild mediastinal lymphadenopathy. FINAL DIAGNOSIS =============== Based on the histopathological findings of the cutaneous nodules and the computed tomography finding of mediastinal lymphadenopathy, a diagnosis of subcutaneous sarcoidosis was made. TREATMENT ========= Patient was advised on various treatment options and chose to have the lesions surgically excised by a plastic surgeon. OUTCOME AND FOLLOW-UP ===================== Needle biopsy and post-excisional pathology report both revealed the presence of well-formed, dense, non-caseating granulomas located in the subcutaneous tissue. There was no evidence of organisms with Acid-Fast and Grocott's Methamine Silver staining. Among the articles selected for this literature review, 4 were hospital based retrospective chart reviews. DISCUSSION ========== The largest study was conducted by Ahmed et al\[[@B14]\] in 2006 out of the Mayo Clinic\[[@B14]\]. The authors reviewed all cases filed under the diagnosis of sarcoidosis, nonspecific granulomas and granulomatous panniculitis between 1966-2001. Ultimately 21 cases of subcutaneous sarcoidosis were reviewed. Among the 21 patients diagnosed with subcutaneous sarcoidosis, 15 were female and 6 were male. The mean age was 46.3. In 20/21 patients, lesions were located on more than one anatomical site. The most common anatomical site was the upper extremity with all 21 patients presenting with a lesion in this area. Lower extremity lesions were also common as they were found in 16/21 patients. In 15/21 patients, other types of cutaneous lesions of sarcoidosis co-existed with the subcutaneous lesion with plaques presenting in 6 patients, papules in 4 patient, erythema nodosum in 4 patients, and scar sarcoidosis in 1 patient. Out of the 20 patients who were evaluated for systemic involvement, 16 had pulmonic involvement evidenced by an abnormal chest radiograph. 15/16 patients exhibited bilateral hilar lymphadenopathy, with 6 of these cases exhibiting an additional finding of paratracheal and pulmonary infiltrates. The most common systemic involvements, other than the lung, included arthritis, peripheral neuropathy and renal dysfunction. Out of the 11 patients tested for Serum ACE, 3 patients had elevated levels. In 2016, Ando et al\[[@B15]\] reviewed the charts of 130 patients diagnosed with systemic sarcoidosis between 2000-2012 out of Oita University of Japan medical center. 37/130 patients presented with cutaneous sarcoid lesions with 9/37 presenting with subcutaneous sarcoidosis. Among their cohort were 8 female patients and 1 male patient with an average age of 52.5 years. Six of the patients only had lesions on their lower extremities. The other 3 patients had lesions on their upper extremity and trunk, upper and lower extremities, and hip respectively. Two patients presented with sarcoid plaques and scars in addition to their subcutaneous nodules. All 9 patients were found to have lung involvement with 4 of these patients presenting with an additional involvement of their eyes, and 3 patients with an involvement of their muscles. On chest radiograph 3 patients had lymphadenopathy, and 6 patients had lymphadenopathy with pulmonary infiltrates. In 2005, Marcoval et al\[[@B7]\] conducted a retrospective chart review analyzing 480 patients admitted with systemic sarcoidosis from 1974-2002 at the University Hospital of Bellvitge in Barcelona, Spain. A total of 85/480 patients demonstrated sarcoid cutaneous involvement with 10/85 demonstrating subcutaneous sarcoidosis. 9/10 of the patients were female, and the average age of presentation was 52.6 years. All of the patients presented with nodules on their upper extremities with 5 patients presenting with additional nodules on their lower extremities. In addition to subcutaneous nodules, 4 patients presented with erythema nodosum, and 1 patient presented with sarcoid plaques and papules. 8 patients presented with lymphade-nopathy on chest radiograph, and 1 patient presented with lymphadenopathy and pulmonary infiltrate. In 2011 the same lead author, Marcoval et al\[[@B3]\], conducted a similar retrospective chart review analyzing 86 patients with systemic sarcoidosis who presented with cutaneous involvement to the Sarcoid Clinic of Bellvitge University Hospital in Barcelona, Spain. A total of 14/86 patients presented with subcutaneous nodules. Among the 14 patients, 11 were female and 3 were male. All 14 patients had nodules limited to the upper and lower extremities with 6 patients presenting with lesions on their arms, 1 patient presenting with lesions on their legs, and 7 patients presenting with lesions on both their upper and lower extremities. 13/14 patients were found to have abnormal chest radiograph findings with 11 patients exhibiting hilar lymphadenopathy, and 2 exhibiting hilar lymphadenopathy and lung infiltrate. Among the 14 patients, 12 had systemic involvement with arthritis being the most common presentation in 6 of the patients (Table [1](#T1){ref-type="table"}). ###### Subcutaneous sarcoidosis retrospective chart reviews **Authors** **Sex** **Average age** **Serum angiotensin-converting enzyme** **Chest X-ray** **Most common site of lesion** **Most common site of systemic involvement other than the lungs** ------------------------------- --------- ----------------- ----------------------------------------- ------------------------------------------------------------------------- -------------------------------- ------------------------------------------------------------------- Ahmed et al\[[@B14]\], 2006 15F/6M 46.3 3/11 Elevated 9/16 Lymphadenopathy, 6/16 Pulmonary infiltration Upper extremities Joints-arthritis Ando et al\[[@B15]\], 2016 8F/1M 52.5 7/9 Elevated 3/9 Lymphadenopathy, 6/9 Lymphadenopathy with pulmonary infiltration Upper extremities Eyes Marcoval et al\[[@B7]\], 2005 9F/1M 52.6 NA 8/10 Lymphadenopathy, 1/10 Lymphadenopathy with pulmonary infiltration Upper extremities NA Marcoval et al\[[@B3]\], 2011 11F/3M N/A NA 11/14 Lymphadenopathy, 2/14 Lymphadenopathy with pulmonary infiltration Upper and lower extremities Joints-arthritis In our literature review we found 25 case reports representing 28 unique cases of subcutaneous sarcoidosis between 2000-2019. Notable features of these cases are listed in Table [2](#T2){ref-type="table"}. The average age of patients among all reports was 53 years old and 21/28 of the patients were female. ###### Features of 25 case reports from 2000-2019 **Authors** **Sex** **Average age** **Serum angiotensin-converting enzyme** **Chest computed tomography** **Chest X-ray** **Site of lesion** **Extracutaneous involvement other than the lungs** ----------------------------------------- --------- ----------------- ----------------------------------------- ---------------------------------------------------------------------------------- --------------------- ----------------------- ----------------------------------------------------- Barnadas et al\[[@B16]\], 2000 F 38 Normal Normal Normal Upper and lower limbs Malaise, joint pains Girão et al\[[@B17]\], 2000 M 37 Elevated Lymphadenopathy Lower limb Hands and feet arthralgia Dalle Vedove et al\[[@B18]\], 2011 1F/1M 75 2/2 Elevated 1/2 Mediastinal Lymphadenopathy, 1/2 Lymphadenopathy with pulmonary infiltration 2/2 Normal Upper and lower limbs 1/2 Uveitis Kim et al\[[@B19]\], 2014 M 61 Elevated Normal Trunk Renal Fichtel et al\[[@B20]\], 2006 F 42 Elevated Normal Upper and lower limbs None Bosnic et al\[[@B21]\], 2010 F 51 Elevated Normal Face None Kim et al\[[@B22]\], 2017 M 33 Lymphadenopathy with pulmonary infiltration Face, toe None Won et al\[[@B23]\], 2016 F 54 Normal Lower limb None Marcoval et al\[[@B24]\], 2008 F 49 Elevated Lymphadenopathy Upper and lower limbs None Dulgueroy et al\[[@B25]\], 2015 F 34 Elevated Lymphadenopathy with pulmonary infiltration Face None Ruangchaijatuporn et al\[[@B26]\], 2016 M 56 Normal Lower limb None Watanbe et al\[[@B27]\], 2007 F 70 Elevated Lymphadenopathy Lower limb Polyneuropathy of limbs Janegova et al\[[@B28]\], 2016 F 59 Lymphadenopathy with pulmonary infiltration Foot None Yamaguchi et al\[[@B29]\], 2013 F 85 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Upper and lower limbs Joints arthralgia Mori et al\[[@B30]\], 2018 F 72 Elevated Lower limb Renal and cardiac dysfunction Kwan et al\[[@B31]\], 2015 F 53 Elevated Lymphadenopathy Upper and lower limbs None Miida et al\[[@B32]\], 2009 F 62 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Upper limbs Uveitis, renal dysfunction, splenic nodules Bianchini et al\[[@B33]\], 2010 F 38 Elevated Normal Normal Face None Kerner et al\[[@B34]\], 2008 F 53 Lymphadenopathy Lymphadenopathy Upper and lower limbs Facial nerve palsy, arthralgia Kim et al\[[@B35]\], 2013 F 52 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Upper and lower limbs None Guccione et al\[[@B36]\], 2017 M 40 Lymphadenopathy Upper limbs, trunk None Meyer-Gonzalez et al\[[@B37]\], 2011 3F 52.6 1/3 Normal, 2/3 Elevated 1/3 Lymphadenopathy, 2/3 Lymphadenopathy with pulmonary infiltration 3/3 Lymphadenopathy Upper and lower limbs Dactylitis, lower limb weakness Moscatelli et al\[[@B38]\], 2011 M 41 Lymphadenopathy with pulmonary infiltration Normal Hand None Shigemitsu et al\[[@B39]\], 2008 F 65 Lymphadenopathy Upper extremity None Celik et al\[[@B40]\], 2010 F 53 Elevated Lymphadenopathy with pulmonary infiltration Lymphadenopathy Foot None Out of the 20 reported cases that checked for serum ACE, elevated levels were found in 18 patients. Abnormal Chest Computed Tomography results were found in 16/21 patients. Abnormal Chest Radiograph results were found in 12/19 patients. The most common site of lesion seemed to be the upper and lower limbs with 17/25 patients presenting with subcutaneous sarcoid nodules in one or both of these locations. Most reports did not comment on extracutaneous involvement other than the lung, but those did reported a range of systemic findings including arthritis, renal dysfunction, uveitis, dactylitis, and limb weakness. Sarcoidosis is a chronic systemic granulomatous disease of unknown etiology\[[@B1]\]. Although the lungs are typically the primary site of disease, cutaneous manifestations of the Sarcoidosis can be seen in up to 9-37% of patients\[[@B3],[@B4]\]. Subcutaneous sarcoid nodules, is a rare cutaneous sarcoidosis finding that typically presents as asymptomatic, non-tender, flesh colored nodules ranging in size between 0.5-2.0 cm\[[@B6],[@B8]\]. Histological examination of a subcutaneous sarcoid nodule reveals the presence of non-caseating granulomas present in the subcutaneous tissue\[[@B6]\]. Our patient, a 38 year old female, presented with an 8 month history of 4 subcu-taneous nodules ranging in size from 0.5cm-5cm. Her past medical history was significant for arthritis and recurrent nephrolithiasis. Upon testing we found that she had a normal chest radiograph and her serum ACE levels were within normal limits. On chest computed tomography we found evidence of mild mediastinal lymphadenopathy, pathognomonic for sarcoidosis. In analyzing the 29 publications from 2000-2019 along with our own case, we reviewed 83 cases of subcutaneous sarcoidosis. Among the 83 patients, 65 (78.3%) were female and the average age of presentation was 51.1 years old. The upper and lower extremities were the most common site of subcutaneous sarcoidosis development with 76/83 (91.6%) patients presenting with at least one lesion in these anatomical areas. In our analysis we learned that findings of lymphadenopathy or lymphadenopathy with pulmonary infiltrate was a very common chest radiograph finding among patients presenting with subcutaneous lesions. In total, 58/69 (84.1%) patients had abnormal chest radiograph findings. Among the 22 patients that received a chest computed tomography scan, abnormal findings of lymphadenopathy or pulmonary infiltrate were found in 17/22 (77.2%) patients. Elevated levels of serum ACE is also a common finding, although not as prevalent as lung involvement. In total, among the cases that measured serum ACE, 28/41 (68.3%) patients presented with elevated levels. Instances of sarcoidosis organ involvement other than the lung seems to be a rarer finding presenting in only 29/49 (59.1%) patients. Assessment of the number of patients with systemic involvement other than the lung, however, was difficult as some articles did not include this information within their study. In conclusion, our literature review shows that subcutaneous sarcoidosis primarily impacts middle-aged women, is most frequently found on the upper or lower limbs, and commonly presents with abnormal findings of lymphadenopathy or pulmonary infiltration on chest imaging as well as elevated levels of serum ACE. These patterns and findings are important for hand surgeons and other surgical specialties that do not commonly see this patient population to be able to rapidly identify and diagnose a disease that has extra-cutaneous manifestations and can lead to greater morbidity and mortality when not diagnosed and treated early. Informed consent statement: Informed consent was obtained from the patient prior to the writing of this case report. Conflict-of-interest statement: The authors declare there is no conflict of interest CARE Checklist (2016) statement: This paper is in accordance with guidelines from CARE Checklist 2016. Manuscript source: Invited Manuscript Peer-review started: March 28, 2019 First decision: June 17, 2019 Article in press: July 27, 2019 Specialty type: Medicine, research and experimental Country of origin: United States Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: El-Razek AA S-Editor: Cui LJ L-Editor: A E-Editor: Xing YX [^1]: Author contributions: All authors added work and value to the manuscript. Corresponding author: Raman Mehrzad, MD, Academic Fellow, Doctor, Staff Physician, Department of Plastic and Reconstructive Surgery, Rhode Island Hospital, the Warren Alpert Medical School of Brown University, 235 Plain St, Providence, RI 02903, United States. <[email protected]> Telephone: +1-774-2400060

{ "pile_set_name": "PubMed Central" }

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"sarcoidosi", "sex", "list", "help", "joint", "characterist", "shigemitsu", "public", "oita", "cardiac", "countri", "contribut", "exhibit", "evalu", "dens", "mehrzad", "dactyl", "lymph", "extensor", "angiotensinconvert", "uptakeb", "least", "recurr", "one", "seen", "soft", "erythema", "infiltr", "silver", "eye", "discuss", "english", "juli", "forearm", "describ", "alpert", "ramanmhotmailcom", "press", "factor", "bosnic", "write", "dysfunct", "left", "renal", "polyneuropathi", "pattern", "occurr", "june", "face", "metabol", "treat", "system", "rare", "year", "barnada", "uveiti", "outcom", "conclus", "commonli", "look", "month", "compliant", "ad", "complaint", "caucasian", "laboratori", "radiograph", "articl", "declar", "pet", "enhanc", "import", "propos", "excis", "paper", "repres", "defin", "date", "miida", "obtain", "vainsench", "size", "surgic", "kerner", "meyergonzalez", "granuloma", "nontend", "disord", "neuropathi", "see", "rang", "petct", "ill", "dermatolog", "past", "primarili", 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"male", "palsi", "morbid", "fdg", "extrem", "anatom", "followup", "scan", "site", "et", "cmcm", "review", "need", "care", "fellow", "overli", "develop", "exclud", "indic", "globe", "c", "keyword", "yamaguchi", "age", "frequenc", "consent", "manuscript", "school", "positron", "seem", "scleroderma", "search", "uptak", "cui", "diseas", "magnet", "dfreftypefig", "daili", "telephon", "creactiv", "bilater", "mimic", "analysi", "asymptomat", "along", "pain", "dall", "pubm", "diagnos", "antinuclear", "made", "unit", "yield", "scar", "yx", "ct", "mg", "core", "increas", "report", "aspect", "similar", "checklist", "comment", "mori", "wellform", "facial", "treftypet", "area", "plaqu", "previou", "granulomat", "antiscl", "reson", "clinician" ]

22,232

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"manifestation", "cutaneous", "involvement", "subcutaneous", "sarcoid", "nodules", "rare", "finding", "Previous", "estimates", "frequency", "subcutaneous", "sarcoidosis", "ranged", "recent", "studies", "suggesting", "occurrence", "rate", "among", "patients", "presenting", "cutaneous", "sarcoid", "variant", "Sarcoidosis", "defined", "clinically", "asymptomatic", "flesh", "colored", "nodules", "usually", "ranging", "Histologically", "subcutaneous", "sarcoidosis", "defined", "presence", "granulomas", "present", "subcutaneous", "ultrasound", "imaging", "lesions", "present", "irregularly", "defined", "mass", "hyper", "hypoechoic", "Fludeoxyglucose", "FDG", "positron", "emission", "tomography", "PET", "computed", "tomography", "CT", "also", "used", "identify", "sarcoid", "lesions", "presents", "increased", "uptake", "subcutaneous", "helpful", "FDG", "may", "able", "differentiate", "connective", "tissue", "diseases", "Sjrogen", "Syndrome", "also", "presents", "increased", "Additionally", 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"neuropathy", "renal", "dysfunction", "patients", "tested", "Serum", "ACE", "patients", "elevated", "levels", "Ando", "et", "reviewed", "charts", "patients", "diagnosed", "systemic", "sarcoidosis", "Oita", "University", "Japan", "medical", "center", "patients", "presented", "cutaneous", "sarcoid", "lesions", "presenting", "subcutaneous", "sarcoidosis", "Among", "cohort", "female", "patients", "male", "patient", "average", "age", "years", "Six", "patients", "lesions", "lower", "extremities", "patients", "lesions", "upper", "extremity", "trunk", "upper", "lower", "extremities", "hip", "respectively", "Two", "patients", "presented", "sarcoid", "plaques", "scars", "addition", "subcutaneous", "nodules", "patients", "found", "lung", "involvement", "patients", "presenting", "additional", "involvement", "eyes", "patients", "involvement", "muscles", "chest", "radiograph", "patients", "lymphadenopathy", "patients", "lymphadenopathy", "pulmonary", "infiltrates", "Marcoval", "et", "conducted", "retrospective", "chart", "review", "analyzing", "patients", "admitted", "systemic", "sarcoidosis", "University", "Hospital", "Bellvitge", "Barcelona", "Spain", "total", "patients", "demonstrated", "sarcoid", "cutaneous", "involvement", "demonstrating", "subcutaneous", "sarcoidosis", "patients", "female", "average", "age", "presentation", "years", "patients", "presented", "nodules", "upper", "extremities", "patients", "presenting", "additional", "nodules", "lower", "extremities", "addition", "subcutaneous", "nodules", "patients", "presented", "erythema", "nodosum", "patient", "presented", "sarcoid", "plaques", "papules", "patients", "presented", "chest", "radiograph", "patient", "presented", "lymphadenopathy", "pulmonary", "infiltrate", "lead", "author", "Marcoval", "et", "conducted", "similar", "retrospective", "chart", "review", "analyzing", "patients", "systemic", "sarcoidosis", "presented", "cutaneous", "involvement", "Sarcoid", "Clinic", "Bellvitge", "University", "Hospital", 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"Lymphadenopathy", "Lymphadenopathy", "pulmonary", "infiltration", "Upper", "extremities", "Eyes", "Marcoval", "et", "NA", "Lymphadenopathy", "Lymphadenopathy", "pulmonary", "infiltration", "Upper", "extremities", "NA", "Marcoval", "et", "NA", "Lymphadenopathy", "Lymphadenopathy", "pulmonary", "infiltration", "Upper", "lower", "extremities", "literature", "review", "found", "case", "reports", "representing", "unique", "cases", "subcutaneous", "sarcoidosis", "Notable", "features", "cases", "listed", "Table", "table", "average", "age", "patients", "among", "reports", "years", "old", "patients", "female", "Features", "case", "reports", "Authors", "Sex", "Average", "age", "Serum", "enzyme", "Chest", "computed", "tomography", "Chest", "Site", "lesion", "Extracutaneous", "involvement", "lungs", "Barnadas", "et", "F", "Normal", "Normal", "Normal", "Upper", "lower", "limbs", "Malaise", "joint", "pains", "Girão", "et", "Elevated", "Lymphadenopathy", "Lower", "limb", "Hands", "feet", "arthralgia", "Dalle", "Vedove", "et", "Elevated", "Mediastinal", "Lymphadenopathy", "Lymphadenopathy", "pulmonary", "infiltration", "Normal", "Upper", "lower", "limbs", "Uveitis", "Kim", "et", "Elevated", "Normal", "Trunk", "Renal", "Fichtel", "et", "F", "Elevated", "Normal", "Upper", "lower", "limbs", "None", "Bosnic", "et", "F", "Elevated", "Normal", "Face", "None", "Kim", "et", "Lymphadenopathy", "pulmonary", "infiltration", "Face", "toe", "None", "et", "F", "Normal", "Lower", "limb", "None", "Marcoval", "et", "F", "Elevated", "Lymphadenopathy", "Upper", "lower", "limbs", "None", "Dulgueroy", "et", "F", "Elevated", "Lymphadenopathy", "pulmonary", "infiltration", "Face", "None", "Ruangchaijatuporn", "et", "Normal", "Lower", "limb", "None", "Watanbe", "et", "F", "Elevated", "Lymphadenopathy", "Lower", "limb", "Polyneuropathy", "limbs", "Janegova", "et", "F", "Lymphadenopathy", "pulmonary", "infiltration", "Foot", "None", "Yamaguchi", "et", "F", "Elevated", "Lymphadenopathy", "pulmonary", "infiltration", "Lymphadenopathy", "Upper", "lower", "limbs", "Joints", "arthralgia", "Mori", "et", "F", "Elevated", "Lower", "limb", "Renal", "cardiac", "dysfunction", "Kwan", "et", "F", "Elevated", "Lymphadenopathy", "Upper", "lower", "limbs", "None", "Miida", "et", "F", "Elevated", "Lymphadenopathy", "pulmonary", "infiltration", "Lymphadenopathy", "Upper", "limbs", "Uveitis", "renal", "dysfunction", "splenic", "nodules", "Bianchini", "et", "F", "Elevated", "Normal", "Normal", "Face", "None", "Kerner", "et", "F", "Lymphadenopathy", "Lymphadenopathy", "Upper", "lower", "limbs", "Facial", "nerve", "palsy", "arthralgia", "Kim", "et", "F", "Elevated", "Lymphadenopathy", "pulmonary", "infiltration", "Lymphadenopathy", "Upper", "lower", "limbs", "None", "Guccione", "et", "Lymphadenopathy", "Upper", "limbs", "trunk", "None", "et", "Normal", "Elevated", "Lymphadenopathy", "Lymphadenopathy", "pulmonary", "infiltration", "Lymphadenopathy", "Upper", "lower", "limbs", "Dactylitis", 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"presentation", "years", "old", "upper", "lower", "extremities", "common", "site", "subcutaneous", "sarcoidosis", "development", "patients", "presenting", "least", "one", "lesion", "anatomical", "areas", "analysis", "learned", "findings", "lymphadenopathy", "lymphadenopathy", "pulmonary", "infiltrate", "common", "chest", "radiograph", "finding", "among", "patients", "presenting", "subcutaneous", "lesions", "total", "patients", "abnormal", "chest", "radiograph", "findings", "Among", "patients", "received", "chest", "computed", "tomography", "scan", "abnormal", "findings", "lymphadenopathy", "pulmonary", "infiltrate", "found", "patients", "Elevated", "levels", "serum", "ACE", "also", "common", "finding", "although", "prevalent", "lung", "involvement", "total", "among", "cases", "measured", "serum", "ACE", "patients", "presented", "elevated", "levels", "Instances", "sarcoidosis", "organ", "involvement", "lung", "seems", "rarer", "finding", "presenting", "patients", "Assessment", "number", 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"Manuscript", "source", "Invited", "Manuscript", "started", "March", "First", "decision", "June", "Article", "press", "July", "Specialty", "type", "Medicine", "research", "experimental", "Country", "origin", "United", "States", "report", "classification", "Grade", "Excellent", "Grade", "B", "good", "Grade", "C", "Good", "C", "Grade", "Fair", "Grade", "E", "Poor", "AA", "Cui", "LJ", "Xing", "YX", "Author", "contributions", "authors", "added", "work", "value", "manuscript", "Corresponding", "author", "Raman", "Mehrzad", "MD", "Academic", "Fellow", "Doctor", "Staff", "Physician", "Department", "Plastic", "Reconstructive", "Surgery", "Rhode", "Island", "Hospital", "Warren", "Alpert", "Medical", "School", "Brown", "University", "Plain", "St", "Providence", "RI", "United", "States", "Telephone" ]

Introduction {#s1} ============ According to the 'efficient-coding hypothesis' [@pcbi.1000053-Barlow1], the sensory neurons are adapted to the statistical properties of the signals to which they are exposed. Because not all signals are equally likely, sensory systems should best encode those signals that occur most frequently. This idea was first tested by Laughlin [@pcbi.1000053-Laughlin1] in a pioneering study of first order interneurons in the insect compound eye, the large monopolar cells, which code for contrast fluctuations. He showed that the response function of these graded potential cells, measured by intracellular recording, approximates the cumulative probability distribution function of contrast levels measured in the natural fly\'s habitat with a photodiode. The efficient coding hypothesis has been much studied in the visual system [@pcbi.1000053-Laughlin1]--[@pcbi.1000053-Laughlin2]; reviewed in [@pcbi.1000053-Simoncelli1] and to a lesser extent in the auditory system [@pcbi.1000053-Lewicki1],[@pcbi.1000053-Smith1]. However, it has been rarely discussed in the context of olfactory sensory neurons [@pcbi.1000053-Kostal1],[@pcbi.1000053-Rospars1]. With a nonlinear stimulus-response function, the neuron encodes differently an equal change in stimulus intensity depending on the actual concentration ([Figure 1A](#pcbi-1000053-g001){ref-type="fig"}). The key question is, how should a neuron weigh its input so as to transfer as much information as possible? Information theory [@pcbi.1000053-Cover1],[@pcbi.1000053-Dayan1] provides the solution. In the simplest scenario (with no other constraints on the response range), the inputs should be encoded so that all responses are used with the same frequency [@pcbi.1000053-Laughlin1]. The optimal stimulus statistics is given by the stimulus probability distribution ([Figure 1B](#pcbi-1000053-g001){ref-type="fig"}), which is obtained directly from the stimulus-response curve. This simple solution, however, does not hold in the case of olfaction because of the large differences in reaction time at different stimulus concentrations. This is a major difference with respect to Laughlin\'s approach, in which all response states were assumed to be equiprobable. ![Amount of information transferred by a neuron in the case where all response states are equiprobable.\ (A) Stimulus-response function. The amount of transferred information is limited by the finite range of possible response states. Due to the non-linearity of the stimulus-response function, each response state encodes different relative changes in stimulus intensity. (B) Corresponding probability density function (pdf). Maximum information is transferred if all response states are used equally, i.e., if the area under the stimulus pdf is equal for each response state, as shown. In the limit of vanishingly small response states, the optimal stimulus CDF corresponds to the (normalized) stimulus-response function (adapted from [@pcbi.1000053-Laughlin1]).](pcbi.1000053.g001){#pcbi-1000053-g001} In this paper, we paralleled Laughlin\'s approach [@pcbi.1000053-Laughlin1], adapting his method to suit the specificity of olfaction. We chose a well studied olfactory receptor neuron, the pheromone receptor neuron of male moths, to investigate its adaptation to the natural signal it processes, the sexual pheromone emitted by conspecific females. To our knowledge this neuron and its stimulus provide the only example in olfaction for which enough data are available on the odorant plume and the neuron transduction mechanisms to make a quantitative comparison possible between the predicted optimum signal and the natural signal. Flying male moths rely on the detection of pheromone molecules released by immobile conspecific females for mating. The atmospheric turbulence causes strong mixing of the air and creates a wide spectrum of spatio-temporal variations in the pheromonal signal ([Figure 2](#pcbi-1000053-g002){ref-type="fig"}). The largest eddies are hundreds of metres in size and may take minutes to pass a fixed point, while the smallest spatial variations are less than a millimetre in size and last for milliseconds only [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis1]. Due to inhomogeneous mixing, a very high concentration of pheromone can be found in a wide range of distances from the source, though their frequency decreases with distance [@pcbi.1000053-Jones1]. Because of its complicated and inhomogeneous structure, the description of the plume must rely on statistical methods, notably the histogram of the fluctuations in pheromone concentration [@pcbi.1000053-Jones1]--[@pcbi.1000053-Mylne2]. These fluctuations are essential for the insect to locate the source of the stimulus. Experiments in wind tunnels showed that moths would not fly upwind in a uniform cloud of pheromone [@pcbi.1000053-Kennedy1]--[@pcbi.1000053-Willis1]. Characteristics like the frequency and intensity of the intermittent stimulation play a key role in maintaining the proper direction of flight [@pcbi.1000053-Vickers1]. ![Visualization of a pheromone plume.\ The figure is extracted and adapted from a digitized image of a smoke plume filmed in a wind tunnel 1 m across and 2 m long with source on the left side [@pcbi.1000053-Belanger1]. Though the average pheromone concentration in the air decreases with distance, high pheromone concentrations can be found relatively far from the source due to the imperfect mixing of odorant with air. The signal detected by both moving and stationary detectors is therefore always intermittent, consisting of pulses of relatively undiluted pheromone.](pcbi.1000053.g002){#pcbi-1000053-g002} The goal of this paper is to present arguments specifying in which sense the perireception and reception processes occuring in pheromone olfactory receptor neurons (ORNs) can be considered as optimally adapted to their natural stimulus. Although, in the light of previous studies on similar sensory neurons, the ORN may be considered *a priori* as adapted to the pheromone plume, the exact nature of this adaptation and its proof are more challenging questions. Despite widespread agreement that environmental statistics must influence neural processing [@pcbi.1000053-Lungarella1], precise quantification of the link proved difficult to obtain [@pcbi.1000053-Simoncelli1]. So, the main aim of this paper was to identify the specific characteristics to which the pheromone ORN is adapted and to provide quantitative evidence for their adaptation. We proceeded in two steps. First, using the statistical theory of information, we predicted the characteristics of the optimal pheromonal signal that the ORN is best capable of encoding based on the properties of the initial steps of signal transduction. Second, we compared these theoretically-derived properties with statistical characteristics most often determined in experimental measurements, i.e., the probability distribution function of the fluctuations in pheromone concentration, the spectral density function of the stimulation course and the intermittency of the odorant signal. Results {#s2} ======= Model of Pheromone Reception {#s2a} ---------------------------- Pheromone components are detected by specialized ORNs located in the male antenna. We considered a specific ORN type of the moth *Antheraea polyphemus* detecting (E,Z)-6,11-hexadecadienyl acetate, the major component of the sexual pheromone in this species, for which a wealth of precise information is available (reviewed in [@pcbi.1000053-Kaissling1]). The pheromone molecules are adsorbed on the cuticle, diffuse inside the sensory hair to the neuron membrane and are thought to be enzymatically deactivated [@pcbi.1000053-Kaissling1] then degraded. The initial cell response is triggered by the binding of the pheromone molecules to the receptor molecules borne by the dendritic membrane and the ensuing receptor activation. A cascade of events follows, amplifying this initial response and finally leading to the generation of a train of action potentials conveyed to the brain. The pheromone concentration at each instant determines the ORN response. However the extreme temporal variability of pheromone concentration in plumes prevents a full description of stimulus-response relationships by direct electrophysiological measurements. For this reason we based our study on a model of perireception and reception processes describing how any stimulus (concentration of pheromone in the air) is transformed into the receptor response (concentration of activated receptors). This model, based on extensive biochemical, radiochemical and electrophysiological experiments, was developed by Kaissling and coworkers [@pcbi.1000053-Kaissling1],[@pcbi.1000053-Kaissling2]. It involves the following system of chemical reactions: The network includes (1) the translocation of the ligand from the air (input pheromone signal L~air~) to the hair lumen (L); (2) the reversible binding of L to receptor R and the reversible change of the complex R~L~ to an activated state R^\*^ (output signal); (3) the reversible binding of L to a deactivating enzyme N and its deactivation to product P which is no longer able to interact with the receptor. The concentrations of individual components in the network 1--3 are denoted by square brackets and the concentration values are functions of time. For simplicity we omit here the explicit dependence on the time variable *t* and adopt the following notation for the individual concentrations: *L* ~air~ = \[L~air~\](*t*), = \[L\](*t*), *R* = \[R\](*t*), *R* ~L~ = \[R~L~\](*t*), *R* ^\*^ = \[R^\*^\](*t*), *N* = \[N\](*t*), *P* = \[P\](*t*) and *N* ~L~ = \[N~L~\](*t*). The evolution of the system 1--3 in time given the external signal *L* ~air~ is fully described by five first order ordinary differential Equations 4--8 and two conservation Equations 9 and 10: Equations 9 and 10 follow from the fact that the total concentration of the receptor molecules, *R* ~tot~ = *R*+*R* ~L~+*R* ^\*^, as well as the total concentration of the deactivating enzyme, *N* ~tot~ = *N*+*N* ~L~, do not change over time. We assume that at *t* = 0 the concentrations *L, R* ~L~, *R* ^\*^, *N* ~L~ and *P* are zero. The parameter values, derived from extensive experimental investigations, are given in [Table 1](#pcbi-1000053-t001){ref-type="table"}. 10.1371/journal.pcbi.1000053.t001 ###### Parameters of the perireceptor and receptor model. ![](pcbi.1000053.t001){#pcbi-1000053-t001-1} Parameter Value Unit Parameter Value Unit -------------- ------- ------------- -------------- -------- ------- *k* ~3~ = 0.209 s^−1^µM^−1^ *k* ~−3~ = 7.9 s^−1^ *k* ~4~ = 16.8 s^−1^ *k* ~−4~ = 98 s^−1^ *k* ~5~ = 4 s^−1^µM^−1^ *k* ~−5~ = 98.9 s^−1^ *k* ~6~ = 29.7 s^−1^ *k~I~* = 29,000 s^−1^ *R* ~tot~ = 1.64 µM *N* ~tot~ = 1 µM From [@pcbi.1000053-Kaissling1],[@pcbi.1000053-Kaissling2]. Basic Stimulus-Response Properties {#s2b} ---------------------------------- The efficiency of information transfer in the system 1--3 depends critically on its stimulus-response relationship under single and repeated stimulus pulses. For transferring as much information as possible the response states must be optimally utilized. The actual amount of information transferred is limited by biological constraints. In the system studied, information transfer from *L* ~air~ (stimulus) to *R* ^\*^ (response) presents three main limitations. First, it is limited by the finite number of receptor molecules per neuron which places an upper bound on the range of responses. Whatever the pheromone concentration (height of the step) the concentration of activated receptors cannot exceed at any time [@pcbi.1000053-Kaissling2]. Second, temporal details in the stimulus course shorter than a certain lower limit Δ*t* cannot be analyzed by the system. The smallest period of stimulation of the model studied here is 0.4 s [@pcbi.1000053-Kaissling2],[@pcbi.1000053-Rospars2], in agreement with experimental measurements [@pcbi.1000053-Kodadov1],[@pcbi.1000053-Rumbo1]. With smaller periods, at higher frequencies, the amplitude of the oscillations of *R* ^\*^ becomes too small to be effective. Therefore we set Δ*t* = 0.4 s. Two successive pheromone pulses separated by a time shorter than Δ*t* cannot be distinguished. Third, information transfer in time is also limited by the response duration, which depends on the deactivation rate of the activated receptors. The time course of *R* ^\*^ in response to stimulations of different heights *L* ~air~ and limited duration (0.4 s) is shown in the inset of [Figure 3A](#pcbi-1000053-g003){ref-type="fig"}. The concentration of activated receptors rises at first, reaches *R* ~Δ~ ^\*^ at the end of the stimulus pulse, i.e., *R* ~Δ~ ^\*^ = *R* ^\*^(*t* = Δ*t*), and finally decreases. We consider *R* ~Δ~ ^\*^ as the "response" of the system and for the sake of simplicity in the following, we omit index Δ. The duration of the falling phase (receptor deactivation) gets progressively longer for higher pheromone concentrations. This deactivation takes typically much longer than the time resolution parameter Δ*t*. The falling phase is often described by the half-fall time, *τ*(*R* ^\*^), which is the time required for *R* ^\*^(*t*) to decrease from *R* ^\*^ to *R* ^\*^/2. The relationship between *R* ^\*^ and *τ*(*R* ^\*^) is shown in [Figure 3A](#pcbi-1000053-g003){ref-type="fig"}. A unique value of *R* ^\*^ corresponds to each value *L~air~*, which defines the stimulus-response curve ([Figure 3B](#pcbi-1000053-g003){ref-type="fig"}). The fact that the deactivation of activated receptors is relatively slow suggests that the reception system cannot encode a long sequence of pheromone pulses in arbitrarily quick succession. This observation plays an important role in the definition of the optimal stimulus course. ![Response properties of the olfactory reception model.\ (A) Temporal properties. Inset: concentration of activated receptors, *R* ^\*^(*t*), as a function of time for single pulses of pheromone of fixed duration (0.4 s) and different intensities *L* ~air~ (1, 5, 10 and 20 nM). The maximum of *R* ^\*^(*t*) is reached slightly after the end of the stimulation. The prolongation of the falling time with increasing intensities is quantified by the half-fall time, *τ*, as a function of *R* ^\*^ at the end of stimulation. (B) Stimulus-response function *R* ^\*^(*L* ~air~) for single pulses of the same duration as in (A). This curve depends on the temporal resolution and the choice of the response intensity. (C) Optimal cumulative distribution function of the responses, *F~R~*(*R* ^\*^), determined by maximizing the information transfer per average half-time (see [Methods](#s4){ref-type="sec"}). The functions *R* ^\*^(*L* ~air~) and *F~R~*(*R* ^\*^) were used for calculating the optimal stimulus probability distribution (shown in [Figure 5B](#pcbi-1000053-g005){ref-type="fig"}).](pcbi.1000053.g003){#pcbi-1000053-g003} Optimal Stimulus Course {#s2c} ----------------------- In the simplest scenario (with no other constraints on the response range and stimulus-independent additive noise), the inputs should be encoded so that all responses are used with the same frequency [@pcbi.1000053-Laughlin1],[@pcbi.1000053-Brunel1]. The optimal stimulus is thus described by its probability distribution function, which is obtained directly from the stimulus-response curve. Due to the large differences in reaction times at different stimulus concentrations, all response values *R* ^\*^ from 0 to 0.24 µM cannot be considered as equally "usable" (the long falling phases decrease the efficacy of the information transfer). Therefore, the longer the half-fall time of a given response *R* ^\*^ (i.e. the greater concentration *R* ^\*^ is) the less frequent it must be. The particular form of the optimal response cumulative probability distribution function (CDF), *F~R~*(*R* ^\*^), which was determined by maximizing the information transferred and minimizing the average half-fall time (see [Methods](#s4){ref-type="sec"}), is shown in [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}. Then, based on the three factors mentioned (stimulus-response curve, [Figure 3B](#pcbi-1000053-g003){ref-type="fig"}; time resolution Δ*t* = 0.4 s; and optimal response probability distribution, [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}), an optimum stimulus course in time can be predicted as explained in the [Methods](#s4){ref-type="sec"} section. Examples of predicted temporal fluctuations in pheromone concentration are shown in [Figure 4](#pcbi-1000053-g004){ref-type="fig"} at various time scales and compared to experimental observations. Even though the time resolution of the system studied here is only 0.4 s, it seems sufficient to capture the main bursts of pheromone (see the 10 s sample in [Figure 4A](#pcbi-1000053-g004){ref-type="fig"}). The comparison can be made more precise by describing statistically the heights and occurences in time of the pulses. ![Qualitative comparison of reconstructed optimal pheromone stimulations *L* ~air~ with experimentally-measured fluctuations in concentration of tracers (in arbitrary units), at various time scales.\ 10 s (A), 50 s (B) and 350 s (C). Temporal positions of pulses in experiments and simulations do not need to coincide. Quantitative comparisons are done in [Figures 5](#pcbi-1000053-g005){ref-type="fig"} and [6](#pcbi-1000053-g006){ref-type="fig"} and in [Table 2](#pcbi-1000053-t002){ref-type="table"}. (A) Ion signal measured using Langmuir probe in the field, 2.5 m from the source (top, from [@pcbi.1000053-Murlis2]); theoretical prediction (bottom) shows reasonable correspondence: the temporal resolution Δ*t* = 0.4 s is sufficient to capture the main bursts of pheromone. (B) Ion signal, averaged over 330 ms, distance up to 30 m from the source (top, from [@pcbi.1000053-Murlis3]); the predicted signal (bottom) captures the overall character of the natural stimulation. (C) Propylene source, 67 m from the source (top, from [@pcbi.1000053-Mylne1]); the longest pauses (over 1 minute) are caused by the global meandering of the plume: they are absent in the prediction (bottom) because moths are assumed to stay within the pheromone plume.](pcbi.1000053.g004){#pcbi-1000053-g004} 10.1371/journal.pcbi.1000053.t002 ###### Comparison of statistical characteristics of optimal and actual plumes. ![](pcbi.1000053.t002){#pcbi-1000053-t002-2} Characteristics[a](#nt102){ref-type="table-fn"} Predicted Values[b](#nt103){ref-type="table-fn"} Experimental Values -------------------------------------------------------------------- -------------------------------------------------- ---------------------------------------------------------------------------------- Concentration CDF ([Figure 5](#pcbi-1000053-g005){ref-type="fig"}) Exponential Exponential [@pcbi.1000053-Mylne1],[@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna1] Spectra ([Figure 5](#pcbi-1000053-g005){ref-type="fig"}) Approx. flat to 0.2 Hz, Approx. flat to 0.1 Hz or 0.5 Hz Close to −2/3 slope after −2/3 slope to 1 Hz [@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2]) Intermittency 20% 10--40% [@pcbi.1000053-Murlis2],[@pcbi.1000053-Murlis3] 10--20% [@pcbi.1000053-Jones1] Total mean *L* ~air~ 1.0×10^−4^ µM -- Total std. dev. of *L* ~air~ 3.0×10^−4^ µM -- Peak value of *L* ~air~ 3.8×10^−3^ µM -- Peak/mean ratio 37 \>20 [@pcbi.1000053-Murlis2],[@pcbi.1000053-Murlis3] 30--150 [@pcbi.1000053-Jones1] Peak/std.dev. ratio 13 \>3 [@pcbi.1000053-Mylne1] The mean concentration, standard deviation and their ratios are calculated from the complete stimulus course, including parts of zero concentration (see [Methods](#s4){ref-type="sec"}). Based on a simulated sample 4000 s long. Predicted Temporal Pattern of Pulses {#s2d} ------------------------------------ Concerning temporal aspects, the bursts of non-zero signal do not occur at periodic intervals but appear randomly. An important descriptor of the temporal structure is the intermittency [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis1], which is the fraction of total time when the signal is present. The intermittency of the predicted optimal stimulus is 20%, which is in relatively good agreement with experimental data. It has been shown using various types of ion detectors [@pcbi.1000053-Murlis2],[@pcbi.1000053-Mylne2] as well as electroantennogram responses [@pcbi.1000053-Murlis2],[@pcbi.1000053-Baker1], that the natural signal is always present less than 50% of the total time, and usually smaller values are found. The average intermittency values reported are 10--20% [@pcbi.1000053-Jones1] and 10--40% [@pcbi.1000053-Murlis1],[@pcbi.1000053-Murlis2], depending on the experimental conditions, such as the detector size or the global meandering of the plume (see [Discussion](#s3){ref-type="sec"}). Predicted Concentrations of Pheromone Pulses {#s2e} -------------------------------------------- Concerning pulse height, the overall character of the predicted stimulus course is that pulses of high concentration are much rarer than those of low concentration. This feature of the predicted stimulus can be best quantified by the CDF, *P*(*L* ~air~), of the stimulus. The shape of the CDF is one of the most important properties for comparing theoretical predictions to experimental measurements because it describes the relative distribution of odorant concentrations throughout the plume. In fact, because measuring pheromone concentration in the field is not presently feasible [@pcbi.1000053-Murlis2], pheromone molecules must be replaced by measurable tracers. Relative quantities are valid for both pheromones and tracers (see [Discussion](#s3){ref-type="sec"}). They are the only quantities known experimentally for pheromone plumes. So, although our model predicts them, we cannot compare values of *L* ~air~ to actual measurements. Given the definition of the optimal stimulus, function *P*(*L* ~air~) can be directly computed (see [Methods](#s4){ref-type="sec"}). [Figure 5](#pcbi-1000053-g005){ref-type="fig"} shows a comparison between experimentally measured (A) and predicted (B) concentration CDF. The optimal pheromone concentration CDF ([Figure 5B](#pcbi-1000053-g005){ref-type="fig"}, solid line) is not known in analytical form but it can be well approximated by an exponential CDF ([Figure 5C](#pcbi-1000053-g005){ref-type="fig"}, dashed line). The differences between the predicted and true exponential shape can be considered as non-significant, namely, very high values of *L* ~air~ are predicted to be less frequent than in the exponential model. The exponential CDF is in agreement with experimental CDF ([Figure 5A](#pcbi-1000053-g005){ref-type="fig"}), [@pcbi.1000053-Mylne1],[@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna1],[@pcbi.1000053-Hanna2] and holds well especially for observations closer to the source (less than 100 m). Although the precise form of the CDF varies with distance from the plume centerline [@pcbi.1000053-Mylne2] and may be affected by the measurement technique, the shape is always highly skewed. ![Experimental and theoretical cumulative probability distribution functions (CDF) *P*(*C*) of dimensionless odorant concentration *C/*\<*C*\> (concentration divided by the total mean concentration).\ (A) Experimental CDF (solid) as measured at 75 m from a propylene (passive tracer) source and its best exponential fit (dashed) plotted on a logarithmic scale (taken from [@pcbi.1000053-Mylne2]). The intermittency is included in the plots in the non-zero value of *P*(*C*) for zero concentrations (see [Methods](#s4){ref-type="sec"}). The experimental data clearly follow the exponential CDF, except close to *C* = 0, which is caused by technical issues in the measurement process [@pcbi.1000053-Mylne2]. The relatively high value of measured intermittency (close to 47%) is caused mainly by initial data processing [@pcbi.1000053-Mylne2]. (B) CDF predicted by the pheromone reception model together with its best exponential fit, the scales correspond to panel (A) After correcting (see [Methods](#s4){ref-type="sec"}) for the fact that the intermittency predicted by the pheromone reception model (20%) is lower than that measured in [@pcbi.1000053-Mylne2] (as explained in the [Discussion](#s3){ref-type="sec"}), the predictions correspond well to the measured data in (A), except at very high values of *L* ~air~ where they are less frequent than expected. Since this deviation is apparent only for events occurring with probability *P*\<0.01, it can be considered as non-significant.](pcbi.1000053.g005){#pcbi-1000053-g005} Other predicted relative quantities (peak-to-mean ratios, dimensionless concentrations *L* ~air~/〈*L* ~air~〉) were compared with their experimental counterparts. The results, summarized in [Table 2](#pcbi-1000053-t002){ref-type="table"}, show that the predicted statistical properties of the stimulus are not contradicted by the experimental observations. Spectral Density Functions of the Stimulus Course {#s2f} ------------------------------------------------- Spectral density functions of the concentration time course, which analyze the contribution of various frequencies to the overall stimulus course, characterize other properties of the plume which are independent on the nature of the odorant (pheromone or ion source) [@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2]. Furthermore, spectral density function represents a point of view different from the concentration probability distribution. Several spectral density functions, shown in [Figure 6](#pcbi-1000053-g006){ref-type="fig"}, were calculated from the predicted optimal pheromone stimulation (see [Methods](#s4){ref-type="sec"}). The spectral shapes seem to be almost flat from 0.02 Hz to 0.2 Hz with a decreasing slope close to −2/3 above 0.2 Hz. The same slope −2/3, which is theoretically predicted by the inertial subrange theory [@pcbi.1000053-Mylne2], was reported in the spectral densities obtained from measurements close to the source (less than 100 m), in the range 0.1 Hz (or 0.5 Hz, depending on records) to 1 Hz [@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2], although the precise range may depend on the technique of measurement. ![Spectral density functions of the predicted fluctuations in time of pheromone concentration.\ Several spectral density functions were calculated from the predicted optimal pheromone stimulations (such as shown in [Figure 4](#pcbi-1000053-g004){ref-type="fig"}, bottom panels), for different initial random seeds. Calculated spectral shapes are usually almost flat from 0.02 Hz to 0.2 Hz, although exceptions are sometimes observed at lower frequencies, which are also found in experimental data [@pcbi.1000053-Mylne2]. Above 0.2 Hz there is a decreasing slope close to −2/3. Flat spectrum up to 0.2 Hz and true −2/3 slope beyond are shown for comparison (thick line). Spectra from experimental measurements (not shown) on propylene plume obtained close to the source are reported to exhibit a similar flat region followed by −2/3 slope [@pcbi.1000053-Mylne2].](pcbi.1000053.g006){#pcbi-1000053-g006} Discussion {#s3} ========== The goals of this study were to determine to which extent early olfactory transduction in olfactory receptor neurons can be considered adapted (in the evolutionary sense) to odorant plumes and to specify the plume characteristics to which it is adapted. The formulation and resolution of this problem benefited from successful studies of efficient sensory coding undertaken in the field of vision and audition. However, transposition from these sensory modalities to olfaction is not straigthforward, which may explain in part why it has not been attempted earlier. Specificities of olfaction concern both the odorant plume and the sensory system. Odor Plumes {#s3a} ----------- In theory and in practice, the quantitative description of odor plumes and their spatiotemporal distribution is less straightforward than that of visual or auditory scenes. Contrary to light and sound, for which the physical description is essentially complete, the turbulent phenomena which underlie the plume characteristics are still an incompletely mastered domain of physics [@pcbi.1000053-Ruelle1]. In Laughlin\'s classical experiment in vision a single time-independent variable, the contrast level, was measured [@pcbi.1000053-Laughlin1] and directly compared with experimental data. In olfaction, however, the odorant concentration (an analogue to the contrast level) is essentially time dependent which results in a complex optimal stimulus course ([Figure 4](#pcbi-1000053-g004){ref-type="fig"}). Complexity and time dependence make a meaningful direct comparison between predictions and experimental records, but also between different experimental records, impossible. Instead, the comparison must rely on global, statistical descriptors [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis2],[@pcbi.1000053-Mylne2],[@pcbi.1000053-Hanna2]. We identified 5 such descriptors of odor plumes, actually measured and usable in the present context (see [Table 2](#pcbi-1000053-t002){ref-type="table"}), which summarize the present knowledge on odor plumes. Moreover, there are no easy-to-use instruments to measure odor plumes in the field, comparable to luxmeters and microphones. For example, the absolute pheromone concentration cannot be easily known in field experiments [@pcbi.1000053-Murlis2]. This explains why no experimental values were given for this descriptor in [Table 2](#pcbi-1000053-t002){ref-type="table"}. In practice, only ratios of concentrations are presented because they are independent of the dispersed molecules. The pheromone is often substituted by an ion or a passive tracer (polypropylene for example) whose concentration can be measured [@pcbi.1000053-Jones1],[@pcbi.1000053-Murlis2],[@pcbi.1000053-Mylne2]. Because both pheromone and tracer compounds in the air are governed by the same physical laws, the relative (dimensionless) values are conserved, as confirmed by independent experiments with different sources [@pcbi.1000053-Jones1]--[@pcbi.1000053-Murlis2],[@pcbi.1000053-Hanna2]. More generally, this limitation explains why we compared only relative quantities (i.e. shape of probability distributions, spectral density functions, peak-to-mean ratios, dimensionless concentrations *L* ~air~/〈*L* ~air~〉 and intermittency values). Other limitations of plume measurements are discussed below. Model of Early Transduction {#s3b} --------------------------- The essentially multidimensional and stochastic nature of the odor stimulus has a profound influence on the analysis of olfactory transduction system in its natural context, as undertaken here. Indeed to investigate the problems at hand, the kinetic responses of the system to a very large number of stimuli, varying in intensity, duration and temporal sequence must be known in order to simulate the diversity of stimuli encountered in a natural plume. This task is difficult, if not impossible, to manage in a purely experimental approach. However, this difficulty can be overcome with an exact dynamic model of the system because its response to the diverse conditions mentioned can be computed, provided it includes all initial steps from molecules in the air to the early neural response. This is the case of the perireception and reception stages of the moth pheromonal ORN and the reason why it was chosen in the present study. This choice brings about two questions, one about the validity of the model, the other on its position within a larger context. The computational model employed has been thoroughly researched and improved over the last three decades [@pcbi.1000053-Kaissling1], [@pcbi.1000053-Kaissling3]--[@pcbi.1000053-Minor1]. It describes perireceptor and receptor events in the ORN cell type sensitive to the main pheromone component of the saturniid moth *Antheraea polyphemus*. At the time of writing it represents the most completely researched computational models of its kind, agreeing with extensive experimental data from various authors and a wide range of experimental techniques. This model is the best description presently available for early events in any ORN and it summarizes in a nutshell a wealth of dispersed knowledge. This model is based on ordinary differential equations 4--8, following the law of mass action for chemical reactions, and is therefore purely deterministic. This approximation is acceptable when the concentrations of reactants are high enough above single-molecular levels, so that the stochastic fluctuations can be neglected. In this paper, the concentration of *R* ^\*^ is always well above that corresponding to one activated receptor molecule per neuron (approximately 10^−6.2^ µM) because we do not investigate the effect of extremely small pheromone doses. Then, the response of the system can be considered as deterministic, in accordance with the efficient coding hypothesis [@pcbi.1000053-Simoncelli1]. The system studied here constitutes only a small part of the whole pheromonal system, although its role is absolutely essential and all other parts depend on it. First, in ORNs, post-receptor mechanisms modify the receptor signal, primarily by a large amplification factor and by sensory adaptation. Second, the ORN population includes cell types with different properties, e.g. the ORN type sensitive to the minor pheromone components can follow periodic pulses up to 10 Hz [@pcbi.1000053-Rumbo1], a performance not yet accounted for in present models [@pcbi.1000053-Rospars2]. Third, in the brain antennal lobe, convergence of a large number of ORNs on a few projection neurons (PNs) provides another amplification and supports the ability of some PNs to follow periodic signals at 10 Hz or greater [@pcbi.1000053-Christensen1]. Evolutionary adaptation of an integrated ORN response is difficult to study at the present time because no complete model of the ORN from receptors to the generation of the receptor potential and the ensuing spike train, is yet available, at least with the required degree of precision. The same argument holds a fortiori for higher order processes. Notwithstanding, the study of the early sensory events is not as restrictive as it may seem because any incoming odor signal must be first transduced in the population of membrane receptors. No information can be extracted by the post-receptor transduction system which has not been encoded by the receptors in the first place. For this reason it is essential to investigate the nature of the adaptation of the initial events (pheromone interaction with receptors) to the pheromone signal. Determination of the Optimal Stimulus {#s3c} ------------------------------------- Different response states of the pheromone reception system have different efficacies from the coding point of view: the "high" states, with large concentrations of activated receptors, take much more time to deactivate than the "low" states, so that for some time after its exposition to a large concentration of pheromone the system is "dazzled". It means that in the optimal stimulus the low pheromone concentrations must be more frequent than the high ones. This is a difference with respect to the classical problem where the efficacy of all response states at transferring information is considered the same, as in the vision of contrasts for example. The problem to solve is to find the right balance between two conflicting demands: to use all response states (including the high ones) and to react rapidly (the short transient responses must be as frequent as possible), i.e. to maximimize the information transferred *per time unit*. The solution to this optimization problem is provided by information theory as detailed in the [Methods](#s4){ref-type="sec"} section. The optimal balance derives from Equation 19 which relates the average half-fall time and the maximum response entropy distribution. The key factor to consider in the optimization is the average half-fall time, which characterizes globally the "swiftness" of the system -- smaller average half-fall time means faster stimulation rate. In other words, the average half-fall time characterizes the bias towards "low" response states. Simultaneously, the condition of maximum response entropy guarantees that the temporal dynamics of the system is as varied as possible and that during the course of stimulation every possible response state is used (with appropriate frequency). By taking into account only the average half-fall time, and not the precise sequence of its individual values, we therefore do not neglect or limit the temporal dynamics of receptor molecules activation. It is important to note, that the average half-fall time is not a free parameter of the problem; it is not set a priori: its optimal value follows from the optimization procedure (Equation 20). The resulting optimal response CDF is highly biased towards low response states, as expected (see [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}). Nature of the System Adaptation {#s3d} ------------------------------- The main achievement of the present investigation was to predict the characteristics of the stimulus optimally processed by the receptor system based on its biochemical characteristics and an information theoretic approach. The predicted optimal plume was shown to be close to the actual plumes for a series of characteristics, namely intermittency, peak/mean ratio and peak/standard deviation ratio of pheromone pulses, probability distribution of dimensionless pheromone concentration and spectral density function of pheromone concentration ([Table 2](#pcbi-1000053-t002){ref-type="table"}, [Figures 4](#pcbi-1000053-g004){ref-type="fig"}--[](#pcbi-1000053-g005){ref-type="fig"} [6](#pcbi-1000053-g006){ref-type="fig"}). The correspondence between the predictions and measurements is very good for the last two characteristics (probability distributions) and fair for the first three (numerical values). These differences in precision of the predictions may be interpreted by taking into account technical factors. Increasing the noise rejection threshold leads to a decrease of the measured intermittency [@pcbi.1000053-Jones1],[@pcbi.1000053-Mylne2], while increasing the detector size or averaging the signal over longer time windows has the opposite effect [@pcbi.1000053-Murlis3]. So, for example, the small size of olfactory sensilla with respect to detectors may explain in part why in [Figure 4B](#pcbi-1000053-g004){ref-type="fig"}, the predicted intermittency seems lower than that in the corresponding experimental record sample, and also why the peak-to-mean ratio and peak-to-standard deviation ratio are relatively higher. The immobility of the measurement devices, in contrast with the active movements of the moths, is another significant factor. For example, long pauses (of the order of minutes) of zero signal are missing in the prediction but visible in the longest available field record (350 s, [Figure 4C](#pcbi-1000053-g004){ref-type="fig"}). They are caused simply by the plume being blown away from the immobile field detector. First, this loss of signal is clearly an extraneous effect, which cannot be included in our optimal signal predictions and therefore cannot be seen in our results. Second, the moth is not subjected to this extraneous effect, or at least not to the same extent, because, in case of signal loss, it actively seeks the pheromone plume, whereas the fixed detector must passively wait for its return. This difference of mobility may substantially affect the intermittency values, but does not affect the shape of probability distributions (see [Methods](#s4){ref-type="sec"}), hence the better quality of the fits in the latter case. In conclusion, the results obtained suggest that the perireceptor and receptor system investigated here is evolutionary adapted to the pheromone plumes. Even if one considers that the pheromone olfactory system must be *a priori* adapted to the average characteristics of the pheromone plumes, it does not logically follow that the system studied is itself necessarily well adapted. Indeed, it is conceivable that the global adaptation results mainly, not from perireception and reception processes but from other downhill intra- and intercellular processes involved in higher signal processing. The respective importance of the former and latter processes in global adaptation cannot be decided *a priori*. Therefore, the relatively close correspondence between predicted and observed plume characteristics presented here is not trivial. It suggests that the adaptation at the level of receptors is already substantial, and consequently that the global adaptation is not predominantly the result of post-receptor mechanisms involving amplification, sensory adaptation, convergence of different ORN types in the antennal lobes etc. The role of these mechanisms in the global adaptation of the animal remains to be established, as well as the relative importance of the various components of the olfactory system (receptor population, ORN as a whole, population of pheromonal ORNs in the antenna, projection neurons in the antennal lobes, etc.). The response characteristics of these other subsystems, e.g. their various temporal resolutions, will have also to be interpreted, maybe in relation with changing plume characteristics with distance to the source and other factors yet to be identified. Methods {#s4} ======= Optimal Response Probability Distribution Function {#s4a} -------------------------------------------------- As mentioned in the Results section, information transfer in the pheromone reception system is limited by the finite response range, (), and by the deactivation rate of the activated receptors for each concentration value *R* ^\*^. This deactivation rate is described by the half-fall time *τ*(*R* ^\*^). The optimal performance of the system is thus reached by a trade-off between two conflicting demands: to employ full response range (maximum information) vs. to employ only the "fastest" responses (minimum average half-fall time). In other words we need to maximize the information transferred per average half-fall time. In the following we provide the mathematical framework that enabled us to find the probability distribution function over the response states *R* ^\*^ that realizes this trade-off. ### Information transferred {#s4a1} The information transferred by the pheromone reception system in a selected time window (*t,t*+Δ*t*) is described by the relation between all possible stimulus values, *L* ~air~, and the corresponding response values, *R* ^\*^. This relation is explicitly quantified by the mutual information, *I*(*L* ~air~; *R* ^\*^) (see [@pcbi.1000053-Cover1] for details)where *H*(*R* ^\*^) is the entropy of the response probability distribution function and the conditional entropy *H*(*R* ^\*^\|*L* ~air~) measures the uncertainty in the output given the input, or equivalently, the amount of noise in the information transduction [@pcbi.1000053-Cover1],[@pcbi.1000053-Dayan1]. The model of pheromone reception employed here is deterministic and therefore *H*(*R* ^\*^\|*L* ~air~) = 0. Thus maximizing the mutual information corresponds to maximizing the response entropy *H*(*R* ^\*^). (Note that in the usual setting of signal independent and additive noise the term *H*(*R* ^\*^\|*L* ~air~) is constant and then maximization of *I*(*L* ~air~;*R* ^\*^) again corresponds to maximization of *H*(*R* ^\*^).) The available response range, (), is naturally discrete, since it is comprised of individual receptor molecules. The expression of *H*(*R* ^\*^) is ([@pcbi.1000053-Cover1], p.14)where *p*(*R* ^\*^) is the probability of having *R* ^\*^ (expressed as a number of molecules). (In the following we use the base of logarithm 2 only to express all information-related quantities in the usual units of "bit"). The value of *R* ^\*^ corresponding to one activated receptor molecule per neuron is approximately Δ*R* ^\*^ = 10^−6.2^ µM [@pcbi.1000053-Kaissling2], which gives a total of different response states. Since *N* is so large, the impractical Equation 12 can be replaced by a continuous approximation based on differential entropy, *h*(*R* ^\*^), defined as ([@pcbi.1000053-Cover1], p.243)where *f*(*R* ^\*^) is the response probability density function. An approximative relation between *H*(*R* ^\*^) and *h*(*R* ^\*^) is given in ([@pcbi.1000053-Cover1] p.248)In the present case the approximation is excellent because the discretization step Δ*R* ^\*^ is very small compared to the whole response range (). From relation 14 the mutual information 11 can be expressed in terms of differential entropyMaximizing the information transferred is thus achieved by maximizing the differential entropy *h*(*R* ^\*^). The advantage of employing differential entropy is that it lends itself to an elegant approach for entropy maximization in terms of integrals. ### Information optimization {#s4a2} We adopt the standard procedure for maximizing the differential entropy of a continuous probability distribution constrained by a known function *τ*(*R* ^\*^). "Constraining" means that the average value 〈*τ*〉 of *τ*(*R* ^\*^) is under our control (see [@pcbi.1000053-Cover1], p.409)The task is to find a probability density function, *f~R~*(*R* ^\*^), which (i) maximizes the value of *h*(*R* ^\*^) (Equation 13) and (ii) is such that the average 〈*τ*〉 (Equation 19) taken over *f~R~*(*R* ^\*^) is equal to the value we set. The well known solution to this problem (see [@pcbi.1000053-Cover1], p.410 or [@pcbi.1000053-Jaynes1] for its derivation) iswhereIt depends on new parameter, *λ*, called Lagrange multiplier. In the standard setting of maximum-entropy problems ([@pcbi.1000053-Cover1] p.409 or [@pcbi.1000053-Simoncelli1],[@pcbi.1000053-Jaynes1]) the mean value of the constraint function, 〈*τ*〉, is known *a priori*. The value of *λ* is then determined by substituting *f*(*R* ^\*^) = *f* ~R~(*R* ^\*^) in Equation 16, so that the following equation between 〈*τ*〉 and *λ* holdsIn the case of pheromone reception, however, the value of 〈*τ*〉 and consequently of *λ* is unknown. The value of *λ* must be determined by finding a compromise between maximum information transferred (Equation 15) and minimum average half-fall time (Equation 19). This compromise is made explicit by a simple requirementIn other words we maximize the information transfer per half-fall time. ### Application to pheromone reception {#s4a3} In order to simplify practical calculations we substitute *f*(*R* ^\*^) = *f~R~*(*R* ^\*^) into the definition of differential entropy 13 so that Equation 15 reduces toNow we have all the necessary information to calculate (i) the mutual information *I*(*L* ~air~;*R* ^\*^) from Equation 21 (shown in [Figure 7A](#pcbi-1000053-g007){ref-type="fig"}), (ii) the mean half-fall time from Equation 19 ([Figure 7B](#pcbi-1000053-g007){ref-type="fig"}) and (iii) their ratio from Equation 20 ([Figure 7C](#pcbi-1000053-g007){ref-type="fig"}) in dependence on the Lagrange multiplier *λ*. [Figure 7A](#pcbi-1000053-g007){ref-type="fig"} shows that the mutual information is maximized (18.5 bits) for *λ* = 0 which corresponds to the uniform probability distribution function over the whole response range. Generally, since *τ*(*R* ^\*^) is a monotonously increasing function of *R* ^\*^, the optimal probability density function *f~R~*(*R* ^\*^) (Equation 17) is either monotonously increasing (*λ*\<0), monotonously decreasing (*λ*\>0), or constant (*λ* = 0). The multiplier *λ* thus decides whether *f~R~*(*R* ^\*^) puts more weight on the "slow" response states (*λ*\<0) or on the "fast" response states (*λ*\>0). These observations are confirmed in [Figure 7B](#pcbi-1000053-g007){ref-type="fig"} where the mean half-time monotonically decreases with increasing *λ*. [Figure 7C](#pcbi-1000053-g007){ref-type="fig"} shows the information transferred per average half-time, i.e., it shows the compromise between the "slowness" or "reactivity" of the system and the transferred information. Clearly, there cannot be a maximum for *λ*\<0 where the system is both "slow" and below its information capacity (note the sharp decrease of mutual information in [Figure 7A](#pcbi-1000053-g007){ref-type="fig"} for *λ*\<0). The optimal balance between reactivity and information transfer is reached for *λ*≈6 at 8 bits/s. By substituting *λ* = 6 into formula 17 we obtain the desired optimal response probability density function, *f~R~*(*R* ^\*^), which maximizes the information transfer per average half-time. The corresponding CDF *F~R~*(*R* ^\*^), shown in [Figure 3C](#pcbi-1000053-g003){ref-type="fig"}, is given by ![Determination of the optimal Lagrange multiplier *λ* giving the optimal response probability distribution.\ (A) Mutual information for the pheromone reception model in dependence on *λ* (Equation 21). (B) Mean half-fall time in dependence on *λ* (Equation 19). (C) Information transferred per average half-time representing the balance between the reactivity of the system and information transferred (Equation 20). Maximum occurs for *λ*≈6.](pcbi.1000053.g007){#pcbi-1000053-g007} The maximum of information transferred per average half-time ([Figure 7C](#pcbi-1000053-g007){ref-type="fig"}) is not sharply defined, namely, the transfer of 7--8 bits/s persists with values of *λ* greater than the optimal value. At the same time, both mutual information ([Figure 7A](#pcbi-1000053-g007){ref-type="fig"}) and average half-time ([Figure 7B](#pcbi-1000053-g007){ref-type="fig"}) decrease slowly in the corresponding region, indicating that the shape of the optimal response probability distribution changes slowly with respect to *λ*. Indeed, as we verified numerically, varying *λ* within reasonable limits (so that information transferred stays close to 8 bits/s) has no impact on the results presented in this work. Optimal Stimulus Course {#s4b} ----------------------- The optimal stimulus course in time was calculated as follows. First, at time *t* ~0~ = 0 a random value *p* ~0~ is drawn randomly from a uniform probability distribution function over the range \[0,1\]. The concentration corresponding to probability *p* ~0~ is obtained by solving the equationwhere *F~R~*(*R* ^\*^) is the optimal CDF given by formula 22 ([Figure 3C](#pcbi-1000053-g003){ref-type="fig"}). The predicted optimal concentration *L* ~air,0~ for a pheromone pulse of duration Δ*t* = 0.4 s which corresponds to is obtained by solving the equationwhere *R* ^\*^(*L* ~air~) is the stimulus-response function ([Figure 3B](#pcbi-1000053-g003){ref-type="fig"}). The value *L* ~air,0~ is plotted at *t* ~0~ ([Figure 4](#pcbi-1000053-g004){ref-type="fig"}). Second, the concentration *L* ~air,1~ and time of appearance *t* ~1~ of the next pulse are determined. Time *t* ~1~ follows from the falling phase of activated receptors: optimality requires that no pheromone pulse appears before *R* ^\*^ returns to its resting level. In practice it is considered that the resting level is reached when *R* ^\*^ falls below 0.01 µM (less than 5% of the coding range). The concentration *L* ~air,1~ of the pulse at *t* ~1~ is determined in the same way as for the pulse at *t* ~0~ by drawing a new random number *p* ~1~ from the uniform probability distribution function over \[0,1\]. The same process can be repeated as many times as needed to create an optimal pheromone pulse train of arbitrary length. Optimal Stimulus Probability Distribution Function {#s4c} -------------------------------------------------- It is common in the literature on the statistical analysis of plumes [@pcbi.1000053-Jones1],[@pcbi.1000053-Mylne1],[@pcbi.1000053-Mylne2] to define two types of mean concentrations. The total mean concentration, 〈*L* ~air~〉, describes the "true" mean concentration obtained from the whole record of concentration fluctuations in time, i.e., including the parts where no signal was available. On the other hand, the conditional mean concentration, 〈*L* ~air~〉~cond~, describes the mean concentration inside the plume, i.e., with zero concentrations excluded. The intermittency, *γ*, relates the two means as [@pcbi.1000053-Mylne2] (Analogously, the total variances and total standard deviations are calculated by taking into account also the parts where no signal is available [@pcbi.1000053-Mylne2].) By combining Equations 23 and 24 we may symbolically express the optimal CDF of the stimulus, *P*(*L* ~air~), asThough *P*(*L* ~air~) cannot be expressed in a closed form, it can be well approximated by the exponential CDFwhere *ξ* = (5.24±0.01)×10^−4^ µM is the estimated value of 〈*L* ~air~〉~cond~ by least-squares fitting of *F* ~exp~(*L* ~air~) to *P*(*L* ~air~). In order to compare concentration probability distribution functions from different measurements meaningfully, authors [@pcbi.1000053-Mylne2] plot the CDF for a dimensionless concentration *L* ~air~/〈*L* ~air~〉. (In the [Figure 5A](#pcbi-1000053-g005){ref-type="fig"} *C*/〈*C*〉 is used, since the data plotted were obtained using a propylene source, not pheromone), see [Figure 5A](#pcbi-1000053-g005){ref-type="fig"}. The scale of such plots is affected by intermittency due to the presence of the total mean in the ratio. Furthermore, information about intermittency is included explicitly in the plots by letting the probability *P*(*L* ~air~ = 0) of zero concentration beConsequently the CDF *P*(*L* ~air~) must be renormalized [@pcbi.1000053-Mylne2]. Intermittency affects only the dimensionless scale, *L* ~air~/〈*L* ~air~〉, and the value of *P*(*L* ~air~ = 0) but not the overall shape of CDF [@pcbi.1000053-Mylne2]. Therefore we can use formulas 25 and 28 to compare our predictions with experimentally measured data by correcting for different intermittency values. Spectral Density Function of the Stimulus Course {#s4d} ------------------------------------------------ The optimal stimulus course is represented by pulses of different pheromone concentrations, *L* ~air~, occurring in time intervals 0.4 s long. In order to calculate the spectral density function of such stimulation course we sample the time axis with step Δ*t* = 0.4 s. Thus we obtain a series of pheromone concentrations at these time points, {*L* ~air,*j*~}, *j* = 1*...n*, where *n* should be even. The discrete Fourier transform, *φ~k~*, of {*L* ~air,*j*~} is defined for *k* = 1,*...,n* values as [@pcbi.1000053-Champeney1] where *i* is the complex unit. The zero-frequency term is thus at position *k* = 1. The spectral density, *S*(*f*), of the complete time course of the stimulus can be calculated for a total of *n*/2+1 values of frequency *f* (given in Hz) [@pcbi.1000053-Oppenheim1] where *m* = 0, 1, 2,*..., n*/2−1, *n*/2 and *f* = *m*/(*n*Δ*t*) are the frequency values. The function Π(*f*) is the Fourier transform of a pulse of unit height, 0.4 s long and starting at *t* = 0 [@pcbi.1000053-Champeney1],where *a* = 2.5 and *δ* = −0.5. The function Π(*f*) appears in formula 30 because the whole stimulus course (such as shown in [Figure 4](#pcbi-1000053-g004){ref-type="fig"}, bottom panels) can be reconstructed by convolving the discrete series {*L* ~air,*j*~} with such a pulse of unit height in the time domain [@pcbi.1000053-Champeney1]. The authors are grateful to Christine Young for linguistic corrections. The authors have declared that no competing interests exist. This work was supported by Marie-Curie fellowship HPMT-CT-2001-00244 to LK, by ECO-NET 12644PF from the French Ministere des Affaires Etrangeres, by Research project AV0Z50110509, Centre for Neuroscience LC554, and by Academy of Sciences of the Czech Republic grants 1ET400110401 and KJB100110701. [^1]: Contributed reagents/materials/analysis tools: PL. Wrote the paper: LK JR. Performed the research: LK. Designed the research: PL JR.

{ "pile_set_name": "PubMed Central" }

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Introduction {#s1} ============ The kisspeptins are a family of peptides essential for the onset of puberty and the regulation of reproductive function. The absence of a functional kisspeptin receptor (KiSS1r) or *Kiss-1* gene results in low gonadotrophin levels and failure to undergo pubertal development in both mice and humans [@pone.0097611-deRoux1]--[@pone.0097611-dAnglemontdeTassigny1]. Administration of exogenous kisspeptin potently stimulates gonadotropin secretion in rodents and man [@pone.0097611-dAnglemontdeTassigny1]--[@pone.0097611-Dhillo1]. Kisspeptin is the endogenous ligand of the G-protein coupled receptor Kiss1r [@pone.0097611-Muir1]--[@pone.0097611-Ohtaki1]. The stimulatory effects of kisspeptin on the reproductive axis appear to be mediated by a direct activation of KiSS1r-expressing gonadotrophin releasing hormone (GnRH) neurons [@pone.0097611-Irwig1]--[@pone.0097611-Kirilov1]. In humans, *KISS1* encodes a 145 amino acid precursor peptide which is cleaved to form a 54-amino-acid peptide, known as kisspeptin-54 or metastin [@pone.0097611-Ohtaki1], and shorter fragments 14, 13 and 10 amino acids long [@pone.0097611-Kotani1]. All kisspeptin share a common 10 amino acid C-terminal section, which is required for kisspeptin receptor activation. Kisspeptin-54, -14, -13 and -10 all bind to Kiss1 with equal affinity and efficacy *in vitro* [@pone.0097611-Kotani1]. The largest proteolytic product in rodents is a 52 amino-acid peptide. Although this peptide shares only relatively low overall homology (52%) with human kisspeptin-54, the C-terminal 10 amino-acid signalling sequences is highly conserved between mouse and human *KiSS1* proteins, varying by only one amino acid \[Tyr 10 (Y) in rodents to Phe 10 (F) in humans\] [@pone.0097611-Stafford1]. Expression of *Kiss1* mRNA has been observed in tissues including the placenta, pancreas, small intestine, and brain in humans [@pone.0097611-Muir1]--[@pone.0097611-Ohtaki1]. Within the human CNS, kisspeptin immunoreactive cell bodies are predominantly located in the infundibular nucleus of the hypothalamus [@pone.0097611-Hrabovszky1]. In rodents, kisspeptin perikarya are located in two major populations in the hypothalamus, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) [@pone.0097611-Gottsch1], [@pone.0097611-Mikkelsen1]. Kisspeptin mRNA expression has been quantified using *in situ* hybridisation (ISH) and quantitative polymerase chain reaction (qPCR). This provides valuable information on gene transcription, but it is widely recognised that changes in mRNA are not always mirrored by protein levels [@pone.0097611-Schwanhausser1], [@pone.0097611-Vogel1]. In humans, circulating kisspeptin levels have been quantified by radioimmunoassay, and immunocytochemical (ICC) methods have been used to localise kisspeptin expression in humans and rodents [@pone.0097611-Dhillo1], [@pone.0097611-Franceschini1]. However, to date, kisspeptin peptide levels in the rodent have not been measured. Western blotting can be used to measure protein levels, but it can be challenging to detect small changes in kisspeptin--IR levels using this technique. In the current study we have developed a sensitive and specific radioimmunoassay (RIA) which enables the quantification of kisspeptin levels within rodent tissues. Materials and Methods {#s2} ===================== Materials {#s2a} --------- Synthetic kisspeptin-10 was obtained from the Advanced Biotechnology Centre, Imperial College, London, UK. Kisspeptin-52 was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). The kisspeptin polyclonal antibody was purchased from Millipore (Watford, UK). Rat Tissue {#s2b} ---------- Female Wistar rats (220--250 g) obtained from Charles River (Margate, UK) were housed in single cages (specific pathogen free, Imperial College London, UK) and maintained in a controlled environment (temperature 21--23°C, 12-h light--dark cycle, lights on at 07∶00) with *ad libitum* access to food (RM1 diet; SDS UK, Witham, Essex, UK) and water. All animal procedures were approved by the British Home Office Animals (Scientific Procedures) Act 1986. Estrus cyclicty was monitored daily by vaginal lavage, slides were examined under a light microscope to determine the dominant cell type. On the morning of the diestrus phase of the cycle, animals were killed by decapitation, and either whole brain or hypothalamus dissected and snap frozen then stored at −80°C. Pregnant female rats were obtained from Charles River. At pregnancy day 20 rats were killed and the placenta removed, snap frozen and stored at −20°C. An additional cohort of female Wistar rats were bilaterally ovarectomized (OVX) and implanted with an estradiol (E~2~) filled (150 ug E~2~/ml) silicone capsule. This model has previously been shown to mediate changes in kisspeptin expression [@pone.0097611-Roa1]. Two weeks later animals were killed by decapitation and whole brain dissected, snap frozen and stored at −80°C`.` Radioimmunoassay {#s2c} ---------------- An RIA directed against rodent kisspeptin was developed. The rabbit kisspeptin-10 polyclonal antibody used has been demonstrated to detect markedly diminished immunoreactivity in *Kiss1* ^−/−^ mice compared to wild type mice when used for immunocytochemistry [@pone.0097611-Lapatto1], and to have minimal cross-reactivity with other RF-amide peptides [@pone.0097611-Desroziers1]. ^125^I-kisspeptin-10 label was prepared using the chloramine-T method and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column (Waters, Milford, MA) over a 32--38% 90-min gradient of acetonitrile (AcN)/water/0.1% trifluoroacetic acid (TFA). The specific activity of kisspeptin label was 56 Bq/fmol. The assay was set up as previously described [@pone.0097611-Bloom1]. The final antibody dilution used was 1∶87 000. Kisspeptin-10 label was used at 20 Bq/tube. The assay was performed in 0·06 M phosphate buffer (pH 7·2) containing 0·3% (v/v) bovine serum albumin (BSA) and 0·02% (v/v) Tween 20. After 3 days of incubation at 4°C, antibody-bound and free fractions were separated by charcoal (2.4 g charcoal, 0.24 dextran in 100 ml phosphate buffer) adsorption of the free fraction. Unless stated, all tissues were homogenised using a Kimble Kontes pellet pestle (Sigma-Aldrich, Dorset, UK) in 450 µl of acid ethanol extraction buffer (0.15% hydrochloric acid in 25% ethanol) [@pone.0097611-Murphy1]. To assess parallelism of the assay, standard curves of 1, 2, 3, 5, 10, 15, 20, 30, 50 and 100 fmol/tube of kisspeptin-10 and kisspeptin-52 were set up in duplicate in conjunction with dilution curves of hypothalamus tissue extracts (5, 10, 20 and 30 µl of extract, also in duplicate) (n = 5). To assess the recovery efficiency of the assay concentrations from 50 to 200 fmol/tube kisspeptin-10 were added to 450 µl rat cerebellum tissue extract and assayed. For the quantification of kisspeptin-IR in tissues, a standard curve consisting of 1, 2, 3, 5, 10, 15, 20, 30, 50 and 100 fmol/tube kisspeptin-10 in duplicate was used. Chromatography {#s2d} -------------- Synthetic kisspeptin-52 (2 nmol in 150 ul water) was fractionated using RP-HPLC Phenomenex Jupiter 4 µm Proteo 90 Å column and eluted with a 30--35% gradient of ACN plus 0·05% (v/v) water/0·1% (v/v) 50 min at a flow rate of 1 ml/min per fraction. Fractions were collected every min. An additional set of hypothalamic tissue samples (*n* = 4) was extracted using the protocol described above, centrifuged at 15 000 g for 3 min, and the supernatants filtered through 0·2 µm hydrophilic membranes (Sartorius, Göttingen, Germany). Samples of 150 ul from each extract were then loaded separately onto the HPLC column and eluted under the same conditions described above. Fractions from all runs were freeze-dried, reconstituted in assay buffer and the kisspeptin content determined by RIA. Quantification of AVPV, ARC and Placenta Kisspeptin {#s2e} --------------------------------------------------- Several groups have confirmed kisspeptin expression in the placenta and the hypothalamic AVPV and ARC [@pone.0097611-Mark1], [@pone.0097611-Smith1]. Brain sections (300**µ**m) were cut on a cryostat, and bilateral punches (1mm diameter) of the AVPV were taken from Bregma +0.2 to −0.4mm, single midline punch (1mm diameter) that included both ARC was taken from bregma −1.7 to −3.9 according to the rat brain atlas [@pone.0097611-Paxinos1], following the micropunch method of Palkovits [@pone.0097611-Palkovits1]. Hypothalamic punches, cerebellum and placental tissue (350 mg) were homogenised and extracted in acid ethanol buffer as described above, and kisspeptin content measured by radioimmunoassay. A separate set of tissues were also extracted by boiling for 15min in 0·5 M acetic acid [@pone.0097611-Murphy1]. Statistical Analysis {#s2f} -------------------- All data are presented as mean ± standard error of the mean (SEM). Levels of kisspeptin expression were analysed using an unpaired t-test (GraphPad Prism version 5 for Windows; GraphPad Software, San Diego, CA). Results {#s3} ======= Radioimmunoassay {#s3a} ---------------- The assay demonstrated 100% cross-reactivity to both rodent kisspeptin-10 and rodent kisspeptin-52. The assay had a sensitivity of 0.81±0·12 fmol/tube (mean ± SEM), *n* = 3 with 95% confidence. The least detectable tissue concentration was \<1.21 fmol/tube, and the midrange was 18.14 fmol/tube. Inter- and intra-assay variation were established to be 8·2%±0·7 and 6·8%±1·7 respectively, *n* = 5. Recovery of kisspeptin from tissue homogenate was between 78% and 96%. The dilution curve for hypothalamic extract was almost parallel to that of kisspeptin-10 and kisspeptin-52 standard, *n* = 5 ([Figure 1](#pone-0097611-g001){ref-type="fig"}). ![HPLC profile of kisspeptin-immunoreactivity from hypothalamic extracts.\ Solid lines- Kisspeptin concentration; broken lines-% acetonitrile (ACN). ↓KP-52 represents the elution position of Kisspeptin-52 standard. The recovery of Kisspeptin-IR in the tissue extract from each column run was above 80% (means S.E.M.; n = 4).](pone.0097611.g001){#pone-0097611-g001} Chromatography {#s3b} -------------- HPLC of hypothalamic extracts resulted in a distinct kisspeptin-IR peak at the same point at which synthetic kisspeptin-52 peptide eluted ([Figure 2](#pone-0097611-g002){ref-type="fig"}). The recovery of kisspeptin-IR for each column run of the tissue extracts was \>80%. ![Parallelism of standard and tissue extract for novel rodent Kisspeptin RIA.\ Kisspeptin **−**10 (KP-10) and hypothalamus (Hypo) (*n* = 5).](pone.0097611.g002){#pone-0097611-g002} Quantification of AVPV and ARC Punches and Placenta {#s3c} --------------------------------------------------- Kisspeptin-IR in the acid ethanol extracted AVPV and ARC of female rat was determined. Significantly more kisspeptin-IR was detected in the ARC than in the AVPV (47.1±6.0 vs. 7.6±1.3 fmol/punch, P\<0.001, n = 15, [Figure 3](#pone-0097611-g003){ref-type="fig"}). As anticipated, a significant increase in AVPV kisspeptin-IR was observed in OVX+E~2~ compared with the OVX rats (16±0.9 vs. 12.5±1 fmol/punch, mean ± SEM P\<0.05, n = 6**--**8, [Figure 4A](#pone-0097611-g004){ref-type="fig"}), and a significant decrease observed in ARC kisspeptin-IR in OVX+E~2~ compared with OVX rats (43.3±2.9 vs. 66.2±2.1 fmol/punch, mean ± SEM, P\<0.001, n = 6**--**8, [Figure 4B](#pone-0097611-g004){ref-type="fig"}). We detected 1.26±0.15 fmol/mg (n = 5) kisspeptin-IR in acid ethanol extracted placental tissue. No kisspeptin-IR was detected in the cerebellum tissue. No measurable kisspeptin-IR levels were detected in tissues extracted by boiling for 15min in 0·5 M acetic acid (data not shown). ![Quantification of kisspeptin-IR in the rat brain (A) Diagram of a rat brain coronal section showing the position of the punch microdissections for the Hypothalamic anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC).\ AVPV at bregma −0.26mm and the ARC at bregma −3.30mm according to the rat brain atlas of Paxinos and Watson. Black circles denote punch positions. Ac, anterior commissure; 3v, third cerebral ventricle. (B) Quantification of kisspeptin-IR in the AVPV and ARC of female rats by RIA. Significantly more kisspeptin-IR was detected in the ARC than in the AVPV. (Mean S.E.M, n = 15**--**16, \*\*\*P\<0.001, Student's t-test).](pone.0097611.g003){#pone-0097611-g003} ![The effect of E~2~ on kisspeptin-IR in the hypothalamus of OVX female rats.\ (A) E~2~ significantly increased kisspeptin-IR in the AVPV. (B) E~2~ significantly decreased kisspeptin-IR in the ARC. Quantification of kisspeptin-IR by RIA. (Mean S.E.M, n = 6**--**8, \*P\<0.05, \*\*\*P\<0.001, Student's t-test).](pone.0097611.g004){#pone-0097611-g004} Discussion {#s4} ========== In the present study, we have characterised the development of a novel kisspeptin RIA and demonstrated its utility in quantifying kisspeptin-IR within the AVPV and ARC of female rats. We have demonstrated that this assay can detect changes in kisspeptin expression in response to E**~2~** feedback. In accord with studies using other methods, we observed suppression of kisspeptin-IR in the ARC and increased kisspeptin-IR in the AVPV in response to E**~2~** in ovariectomised female rats [@pone.0097611-Smith2]. Numerous studies have demonstrated a wide distribution of kisspeptin expression throughout the rodent brain [@pone.0097611-Brailoiu1], [@pone.0097611-Clarkson1]. Initial studies examining the distribution of kisspeptin-IR rodents were hampered by the limited specificity of available antibodies [@pone.0097611-Brailoiu1]. However, the development of reliable antibodies which recognise rodent, sheep [@pone.0097611-Franceschini1] and human [@pone.0097611-Dhillo1] kisspeptin has enabled the distribution of kisspeptin-IR to be fully mapped. Furthermore, we observe a single IR peak following HPLC of hypothalamic extracts, at the point we would expect kisspeptin-52 to elute, thus supporting previous findings regarding antibody specificity [@pone.0097611-Franceschini1]. In addition to mapping kisspeptin distribution, ICC methodologies have been utilised to quantify changes in expression under different experimental and physiological conditions [@pone.0097611-Clarkson1], [@pone.0097611-Clarkson2]. Kisspeptin-IR has been successfully quantified in the AVPV, and also in the ARC, though the ARC has presented difficulties due to the dense plexus of kisspeptin fibres present [@pone.0097611-Clarkson1], [@pone.0097611-Castellano1], [@pone.0097611-MittelmanSmith1]. The limitations of ICC preclude the quantification of the absolute number of kisspeptin molecules or its concentration in a sample. Further, though semi-quantitative methodologies provide information about relative quantities of kisspeptin-IR within each study, it is difficult to compare the levels observed between different studies and models. Kisspeptin expression can be quantified using ISH [@pone.0097611-Gottsch1] or qPCR [@pone.0097611-Yamada1]. However, it is becoming increasingly evident that examination of mRNA levels may be an unreliable proxy for protein concentrations [@pone.0097611-deSousaAbreu1], [@pone.0097611-Maier1], with only ∼40% protein levels being explained by mRNA abundances in mammalian cell lines [@pone.0097611-Schwanhausser1], [@pone.0097611-Vogel1]. Differences in the distribution of kisspeptin mRNA and immunoreactivity have previously been reported. For example, kisspeptin-IR cell bodies and fibres were described in the dorsomedial nucleus of mice [@pone.0097611-Clarkson1], though ISH failed to identify kisspeptin mRNA within this region [@pone.0097611-Smith1]. A subsequent study suggested that the IR detected within the dorsomedial nucleus may be the result of cross-reactivity of the antibody used with neuropeptide FF [@pone.0097611-Iijima1], though it is interesting to note that a recent study detected kisspeptin-immunoreactivity in some DMN cells in the mouse using a different antibody (Franceschini et al 2013). In the current investigation, the higher level of kisspeptin-IR in the ARC than the AVPV is in accord with the relative levels predicted by assessment of mRNA levels. Kisspeptin-IR was also present in the placenta, albeit at relatively low levels. Previous studies have suggested that the expression of kisspeptin mRNA in the placenta varies during pregnancy, and it may be that higher levels would have been detected at another time point [@pone.0097611-Mark1]. Our data also suggest that the majority of hypothalamic kisspeptin-IR is in the form of kisspeptin-52, though it is possible that our extraction methods favour this particular form, and that other kisspeptin forms are present at lower levels. Further studies are thus necessary to conclusively demonstrate the relative quantities of the different kisspeptins in the hypothalamus. In summary, we have developed a novel radioimmunoassay for the measurement of kisspeptin-IR within rat tissue. When combined with the Palkovits punch micro-dissection methodology, this assay enables quantification of kisspeptin-IR within specific brain nuclei. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: KGM SRB. Performed the experiments: JSKJ KEB JC KTO XFL. Analyzed the data: JSKJ KGM. Wrote the paper: JSKJ KGM.

{ "pile_set_name": "PubMed Central" }

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"monitor", "se", "purifi", "offic", "wide", "characteris", "major", "slide", "ttest", "assess", "onset", "ug", "diminish", "proteo", "author", "cerebellum", "ir", "reversephas", "boil", "common", "ponederouxponedanglemontdetassigni", "micropunch", "ponecastellano", "feedback", "thu", "regard", "compar", "polymeras", "load", "imperi", "novel", "experi", "act", "protein", "type", "time", "kont", "å", "initi", "util", "antibodi", "significantli", "bodi", "centrifug", "extract", "british", "explain", "differ", "kimbl", "perikarya", "vitro", "vari", "sb", "provid", "undergo", "known", "store", "domin", "experiment", "subsequ", "palkovit", "level", "rphplc", "bind", "either", "utilis", "vs", "small", "cohort", "howev", "dorsomedi", "posit", "serum", "respect", "combin", "xfl", "↓kp", "albeit", "curv", "göttingen", "metastin", "protocol", "midlin", "chromatographi", "intraassay", "condit", "v", "includ", "ikisspeptin", "error", "show", "sa", "sd", "ponegottsch", "valuabl", "encod", "model", 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"biotechnolog", "ponebloom", "analyz", "sever", "ponemittelmansmith", "jc", "±", "ff", "homogen", "sf", "difficulti", "peak", "flow", "confirm", "gradient", "polyclon", "pellet", "denot", "margat", "poneschwanhauss", "microdissect", "day", "al", "wrote", "throughout", "black", "dens", "cn", "aminoacid", "diamet", "ml", "least", "implant", "share", "one", "acet", "millipor", "°c", "overal", "ponepaxino", "would", "quantif", "gonadotropin", "transcript", "data", "bponegreftypefig", "discuss", "mice", "describ", "onto", "rm", "furthermor", "localis", "kill", "sensit", "specif", "duplic", "highperform", "exampl", "recoveri", "respons", "atla", "version", "gonadotrophin", "kisspeptinimmunoreact", "pathogen", "ac", "unpair", "hypo", "conclus", "ponemark", "coron", "ponehrabovszki", "reproduct", "cycl", "filter", "ad", "acid", "affin", "adsorpt", "mm", "frozen", "fragment", "increasingli", "sequenc", "ponemurphi", "window", "puberti", "declar", "man", "phe", "failur", "map", "compet", "kiss", "watford", "kissr", "phenomenex", "wild", "current", "essex", "paper", "requir", "alway", "profil", "repres", "date", "chang", "physiolog", "obtain", "milford", "placent", "ctermin", "cyclicti", "western", "avail", "dextran", "ponemai", "quantit", "receptor", "pancrea", "shown", "aponegreftypefig", "tfa", "reliabl", "addit", "μl", "activ", "reaction", "limit", "rat", "note", "approv", "poneohtaki", "examin", "anteroventr", "rabbit", "semiquantit", "effici", "periventricular", "statist", "membran", "ponebrailoiu", "previous", "vagin", "microscop", "unless", "success", "recognis", "diestru", "hplc", "amino", "unreli", "sampl", "femal", "f", "materi", "cleav", "gnrh", "absenc", "keb", "bregma", "conceiv", "techniqu", "studi", "phoenix", "mlmin", "line", "methodolog", "poneiijima", "ponedesrozi", "hybridis", "kp", "home", "ponepalkovit", "hamper", "diet", "highli", "usa", "peptid", "trifluoroacet", "product", "conserv", "proxi", "−−", "fail", "poneclarkson", "hypothalam", "find", "content", "elut", "hous", "cerebr", "figur", "jupit", "pregnant", "absolut", "burlingam", "tween", "supernat", "quantifi", "rodent", "bqfmol", "fmolmg", "anoth", "method", "form", "pestl", "acn", "prism", "procedur", "uk", "section", "chloraminet", "−mm", "albumin", "distinct", "exogen", "circl", "poneroa", "neuropeptid", "hydrochlor", "buffer", "b", "punch", "effect", "lower", "stimulatori", "mammalian", "fraction", "hydrophil", "contain", "river", "fibr", "sem", "ria", "circul", "ponemuirponeohtaki", "broken", "−°c", "within", "ponedanglemontdetassignyponedhillo", "function", "mean", "number", "lightdark", "introduct", "axi", "brain", "pharmaceut", "cage", "quantiti", "rate", "kto", "centr", "synthet", "mediat", "human", "wistar", "advanc", "capsul", "molecul", "vv", "variat", "hormon", "chain", "placenta", "ponestafford", "colleg", "nuclei", "minim", "dissect", "et", "cryostat", "ponekotani", "ovarectom", "expect", "enabl", "third", "n", "later", "ethanol", "ventricl", "ponedesousaabreu", "endogen", "challeng", "coupl", "arc", "freezedri", "design", "ttestponegponeg", "singl", "develop", "fill", "c", "crossreact", "possibl", "hypothalamu", "rfamid", "nmol", "·±·", "kisspeptinir", "kissrexpress", "infundibular", "daili", "dmn", "investig", "precursor", "bilater", "column", "analysi", "∶", "ponefranceschini", "g", "region", "phosphat", "mg", "releas", "maintain", "softwar", "access", "increas", "incub", "diagram", "report", "estradiol", "point", "summari", "poneirwigponekirilov", "plu", "previou", "libitum", "suppress", "detect" ]

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"proteolytic", "product", "rodents", "peptide", "Although", "peptide", "shares", "relatively", "low", "overall", "homology", "human", "signalling", "sequences", "highly", "conserved", "mouse", "human", "proteins", "varying", "one", "amino", "acid", "Tyr", "rodents", "Phe", "F", "Expression", "mRNA", "observed", "tissues", "including", "placenta", "pancreas", "small", "intestine", "brain", "humans", "Within", "human", "CNS", "kisspeptin", "immunoreactive", "cell", "bodies", "predominantly", "located", "infundibular", "nucleus", "hypothalamus", "rodents", "kisspeptin", "perikarya", "located", "two", "major", "populations", "hypothalamus", "anteroventral", "periventricular", "nucleus", "AVPV", "arcuate", "nucleus", "ARC", "Kisspeptin", "mRNA", "expression", "quantified", "using", "situ", "hybridisation", "ISH", "quantitative", "polymerase", "chain", "reaction", "qPCR", "provides", "valuable", "information", "gene", "transcription", "widely", "recognised", "changes", "mRNA", "always", "mirrored", "protein", "levels", "humans", "circulating", "kisspeptin", "levels", "quantified", "radioimmunoassay", "immunocytochemical", "ICC", "methods", "used", "localise", "kisspeptin", "expression", "humans", "rodents", "However", "date", "kisspeptin", "peptide", "levels", "rodent", "measured", "Western", "blotting", "used", "measure", "protein", "levels", "challenging", "detect", "small", "changes", "kisspeptin", "IR", "levels", "using", "technique", "current", "study", "developed", "sensitive", "specific", "radioimmunoassay", "RIA", "enables", "quantification", "kisspeptin", "levels", "within", "rodent", "tissues", "Materials", "Methods", "Materials", "Synthetic", "obtained", "Advanced", "Biotechnology", "Centre", "Imperial", "College", "London", "UK", "purchased", "Phoenix", "Pharmaceuticals", "Burlingame", "CA", "USA", "kisspeptin", "polyclonal", "antibody", "purchased", "Millipore", "Watford", "UK", "Rat", "Tissue", "Female", "Wistar", "rats", "g", "obtained", "Charles", "River", "Margate", "UK", "housed", "single", "cages", "specific", "pathogen", "free", "Imperial", "College", "London", "UK", "maintained", "controlled", "environment", "temperature", "light", "dark", "cycle", "lights", "ad", "libitum", "access", "food", "diet", "SDS", "UK", "Witham", "Essex", "UK", "water", "animal", "procedures", "approved", "British", "Home", "Office", "Animals", "Scientific", "Procedures", "Act", "Estrus", "cyclicty", "monitored", "daily", "vaginal", "lavage", "slides", "examined", "light", "microscope", "determine", "dominant", "cell", "type", "morning", "diestrus", "phase", "cycle", "animals", "killed", "decapitation", "either", "whole", "brain", "hypothalamus", "dissected", "snap", "frozen", "stored", "Pregnant", "female", "rats", "obtained", "Charles", "River", "pregnancy", "day", "rats", "killed", "placenta", "removed", "snap", "frozen", "stored", "additional", "cohort", "female", "Wistar", "rats", "bilaterally", "ovarectomized", "OVX", "implanted", "estradiol", "filled", "ug", "silicone", "capsule", "model", "previously", "shown", "mediate", "changes", "kisspeptin", "expression", "Two", "weeks", "later", "animals", "killed", "decapitation", "whole", "brain", "dissected", "snap", "frozen", "stored", "Radioimmunoassay", "RIA", "directed", "rodent", "kisspeptin", "developed", "rabbit", "polyclonal", "antibody", "used", "demonstrated", "detect", "markedly", "diminished", "immunoreactivity", "mice", "compared", "wild", "type", "mice", "used", "immunocytochemistry", "minimal", "peptides", "label", "prepared", "using", "method", "purified", "liquid", "chromatography", "column", "Waters", "Milford", "gradient", "acetonitrile", "AcN", "trifluoroacetic", "acid", "TFA", "specific", "activity", "kisspeptin", "label", "assay", "set", "previously", "described", "final", "antibody", "dilution", "used", "label", "used", "assay", "performed", "phosphate", "buffer", "pH", "containing", "bovine", "serum", "albumin", "BSA", "Tween", "days", "incubation", "free", "fractions", "separated", "charcoal", "g", "charcoal", "dextran", "ml", "phosphate", "buffer", "adsorption", "free", "fraction", "Unless", "stated", "tissues", "homogenised", "using", "Kimble", "Kontes", "pellet", "pestle", "Dorset", "UK", "µl", "acid", "ethanol", "extraction", "buffer", "hydrochloric", "acid", "ethanol", "assess", "parallelism", "assay", "standard", "curves", "set", "duplicate", "conjunction", "dilution", "curves", "hypothalamus", "tissue", "extracts", "µl", "extract", "also", "duplicate", "n", "assess", "recovery", "efficiency", "assay", "concentrations", "added", "µl", "rat", "cerebellum", "tissue", "extract", "assayed", "quantification", "tissues", "standard", "curve", "consisting", "duplicate", "used", "Chromatography", "Synthetic", "nmol", "ul", "water", "fractionated", "using", "Phenomenex", "Jupiter", "µm", "Proteo", "Å", "column", "eluted", "gradient", "ACN", "plus", "min", "flow", "rate", "per", "fraction", "Fractions", "collected", "every", "min", "additional", "set", "hypothalamic", "tissue", "samples", "n", "extracted", "using", "protocol", "described", "centrifuged", "g", "min", "supernatants", "filtered", "µm", "hydrophilic", "membranes", "Sartorius", "Göttingen", "Germany", "Samples", "ul", "extract", "loaded", "separately", "onto", "HPLC", "column", "eluted", "conditions", "described", "Fractions", "runs", "reconstituted", "assay", "buffer", "kisspeptin", "content", "determined", "RIA", "Quantification", "AVPV", "ARC", "Placenta", "Kisspeptin", "Several", "groups", "confirmed", "kisspeptin", "expression", "placenta", "hypothalamic", "AVPV", "ARC", "Brain", "sections", "µ", "cut", "cryostat", "bilateral", "punches", "diameter", "AVPV", "taken", "Bregma", "single", "midline", "punch", "diameter", "included", "ARC", "taken", "bregma", "according", "rat", "brain", "atlas", "following", "micropunch", "method", "Palkovits", "Hypothalamic", "punches", "cerebellum", "placental", "tissue", "mg", "homogenised", "extracted", "acid", "ethanol", "buffer", "described", "kisspeptin", "content", "measured", "radioimmunoassay", "separate", "set", "tissues", "also", "extracted", "boiling", "acetic", "acid", "Statistical", "Analysis", "data", "presented", "mean", "standard", "error", "mean", "SEM", "Levels", "kisspeptin", "expression", "analysed", "using", "unpaired", "GraphPad", "Prism", "version", "Windows", "GraphPad", "Software", "San", "Diego", "CA", "Results", "Radioimmunoassay", "assay", "demonstrated", "rodent", "rodent", "assay", "sensitivity", "mean", "SEM", "n", "confidence", "least", "detectable", "tissue", "concentration", "midrange", "variation", "established", "respectively", "n", "Recovery", "kisspeptin", "tissue", "homogenate", "dilution", "curve", "hypothalamic", "extract", "almost", "parallel", "standard", "n", "Figure", "fig", "HPLC", "profile", "hypothalamic", "Solid", "Kisspeptin", "concentration", "broken", "acetonitrile", "ACN", "represents", "elution", "position", "standard", "recovery", "tissue", "extract", "column", "run", "means", "n", "Chromatography", "HPLC", "hypothalamic", "extracts", "resulted", "distinct", "peak", "point", "synthetic", "peptide", "eluted", "Figure", "fig", "recovery", "column", "run", "tissue", "extracts", "Parallelism", "standard", "tissue", "extract", "novel", "rodent", "Kisspeptin", "Kisspeptin", "hypothalamus", "Hypo", "n", "Quantification", "AVPV", "ARC", "Punches", "Placenta", "acid", "ethanol", "extracted", "AVPV", "ARC", "female", "rat", "determined", "Significantly", "detected", "ARC", "AVPV", "n", "Figure", "fig", "anticipated", "significant", "increase", "AVPV", "observed", "compared", "OVX", "rats", "mean", "SEM", "n", "Figure", "fig", "significant", "decrease", "observed", "ARC", "compared", "OVX", "rats", "mean", "SEM", "n", "Figure", "fig", "detected", "n", "acid", "ethanol", "extracted", "placental", "tissue", "detected", "cerebellum", "tissue", "measurable", "levels", "detected", "tissues", "extracted", "boiling", "acetic", "acid", "data", "shown", "Quantification", "rat", "brain", "Diagram", "rat", "brain", "coronal", "section", "showing", "position", "punch", "microdissections", "Hypothalamic", "anteroventral", "periventricular", "nucleus", "AVPV", "arcuate", "nucleus", "ARC", "AVPV", "bregma", "ARC", "bregma", "according", "rat", "brain", "atlas", "Paxinos", "Watson", "Black", "circles", "denote", "punch", "positions", "Ac", "anterior", "commissure", "third", "cerebral", "ventricle", "B", "Quantification", "AVPV", "ARC", "female", "rats", "RIA", "Significantly", "detected", "ARC", "AVPV", "Mean", "n", "Student", "effect", "hypothalamus", "OVX", "female", "significantly", "increased", "AVPV", "B", "significantly", "decreased", "ARC", "Quantification", "RIA", "Mean", "n", "Student", "Discussion", "present", "study", "characterised", "development", "novel", "kisspeptin", "RIA", "demonstrated", "utility", "quantifying", "within", "AVPV", "ARC", "female", "rats", "demonstrated", "assay", "detect", 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"physiological", "conditions", "successfully", "quantified", "AVPV", "also", "ARC", "though", "ARC", "presented", "difficulties", "due", "dense", "plexus", "kisspeptin", "fibres", "present", "limitations", "ICC", "preclude", "quantification", "absolute", "number", "kisspeptin", "molecules", "concentration", "sample", "though", "methodologies", "provide", "information", "relative", "quantities", "within", "study", "difficult", "compare", "levels", "observed", "different", "studies", "models", "Kisspeptin", "expression", "quantified", "using", "ISH", "qPCR", "However", "becoming", "increasingly", "evident", "examination", "mRNA", "levels", "may", "unreliable", "proxy", "protein", "concentrations", "protein", "levels", "explained", "mRNA", "abundances", "mammalian", "cell", "lines", "Differences", "distribution", "kisspeptin", "mRNA", "immunoreactivity", "previously", "reported", "example", "cell", "bodies", "fibres", "described", "dorsomedial", "nucleus", "mice", "though", "ISH", "failed", "identify", "kisspeptin", "mRNA", "within", "region", "subsequent", "study", "suggested", "IR", "detected", "within", "dorsomedial", "nucleus", "may", "result", "antibody", "used", "neuropeptide", "FF", "though", "interesting", "note", "recent", "study", "detected", "DMN", "cells", "mouse", "using", "different", "antibody", "Franceschini", "et", "al", "current", "investigation", "higher", "level", "ARC", "AVPV", "accord", "relative", "levels", "predicted", "assessment", "mRNA", "levels", "also", "present", "placenta", "albeit", "relatively", "low", "levels", "Previous", "studies", "suggested", "expression", "kisspeptin", "mRNA", "placenta", "varies", "pregnancy", "may", "higher", "levels", "would", "detected", "another", "time", "point", "data", "also", "suggest", "majority", "hypothalamic", "form", "though", "possible", "extraction", "methods", "favour", "particular", "form", "kisspeptin", "forms", "present", "lower", "levels", "studies", "thus", "necessary", "conclusively", "demonstrate", "relative", "quantities", "different", "kisspeptins", "hypothalamus", "summary", "developed", "novel", "radioimmunoassay", "measurement", "within", "rat", "tissue", "combined", "Palkovits", "punch", "methodology", "assay", "enables", "quantification", "within", "specific", "brain", "nuclei", "Competing", "Interests", "authors", "declared", "competing", "interests", "exist", "Conceived", "designed", "experiments", "KGM", "SRB", "Performed", "experiments", "JSKJ", "KEB", "JC", "KTO", "XFL", "Analyzed", "data", "JSKJ", "KGM", "Wrote", "paper", "JSKJ", "KGM" ]

INTRODUCTION ============ Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent chronic liver disease. NAFLD encompasses a wide spectrum of liver pathologies ranging from simple liver steatosis to nonalcoholic steatohepatitis (NASH), the latter of which is characterized by steatosis with features of cellular injury, including inflammation, and hepatocyte apoptosis.^[@R1]^ Simple steatosis was originally thought to be a benign condition; however, increasing evidence indicates that fatty livers are more vulnerable to factors that promote inflammation and fibrosis.^[@R2]^ In a long-term follow-up study in the United States, up to 5.4% of NAFLD cases ultimately progress to cirrhosis, liver failure, and hepatocellular carcinoma.^[@R3]^ Alarmingly, Chinese patients with NAFLD frequently exhibit significant hepatic necroinflammation and progressive liver fibrosis.^[@R4],[@R5]^ NAFLD is now believed to be a major cause of liver-related morbidity and mortality; therefore, early treatment for NAFLD should be widely considered. Insulin resistance is a key pathogenic factor in both the initiation and development of NAFLD. Several insulin sensitizers, including thiazolidinediones and metformin, have been tested in pilot studies and have been shown to significantly reduce liver inflammation and steatosis in NAFLD patients^[@R6],[@R7]^; however, these compounds are associated with weight gain and raise concerns about cardiovascular safety.^[@R8]^ Thus, the side effects of insulin sensitizers are underestimated, particularly for simple cases of NAFLD in the absence of other complications. Dietary and lifestyle changes are recommended as the primary treatment for NAFLD.^[@R9]^ Increased intakes of fruits and vegetables are likely to have important health benefits for NAFLD patients.^[@R10]^ In addition to vitamins, minerals, and fiber, fruits and vegetables contain high concentrations of bioactive compounds, such as carotenoids, tocopherols, and polyphenols. Among them, anthocyanin polyphenols are of particular interest since several epidemiological studies demonstrated their potential to reduce the risk of insulin resistance-related diseases, such as type 2 diabetes mellitus (T2DM) and cardiovascular disease.^[@R11]--[@R13]^ In a cross-sectional investigation of female adults in England, higher anthocyanin intake was associated with improvements in insulin resistance and inflammatory status.^[@R14]^ The beneficial effects of anthocyanins (either anthocyanin extracts from different plants or pure anthocyanin) have been demonstrated in several animal studies, with respect to oxidative stress, insulin resistance, hepatic steatosis, and inflammation, which are key features of NAFLD.^[@R15]--[@R17]^ Recent clinical trials have investigated the impact of consuming anthocyanins-rich foods on the development and progression of NAFLD. Consumption of purple sweet potato beverages was associated with favorable effects in lowering serum γ-glutamyl transferase and alanine aminotransferase (ALT) levels in healthy men with borderline hepatitis.^[@R18]^ Our previous study demonstrated that bayberry juice can protect against NAFLD in young adults by improving plasma antioxidant status as well as by inhibiting the inflammatory and apoptotic responses involved in this disease,^[@R19]^ but the effect of purified anthocyanin on NAFLD in humans has not been reported. Given the potentially protective effects of anthocyanin on insulin resistance and liver injury and the need for effective therapies for NAFLD, the present study was designed to assess the effects of anthocyanin supplementation in NAFLD patients in a double-blinded placebo-controlled trial. Our objective was to evaluate the effect of anthocyanin purified from berries on selected biomarkers of liver injury. We also examined the effects of anthocyanin on body mass, blood pressure, lipid profile, and glucose homeostasis. SUBJECTS AND METHODS ==================== Subjects -------- The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Ethics Committee of Sun Yat-Sen University prior to initiation of recruitment. Participants diagnosed with NAFLD ranging in age from 25 to 65 were recruited at the Shaoguan Railway Hospital, Shaoguan, China. A diagnosis of fatty liver was indicated based on ultrasound (Voluson E8, GE healthcare, Waukesha, WI) if 2 of the following criteria were met: increased hepatic echogenicity compared with cortical of the right kidney, blurring of liver vasculature, and deep attenuation of the ultrasonographic signal^[@R20]^; and plasma ALT \>30 U/L in men and 19 U/L in women. Criteria were added after recruitment began, as a large fraction of recruited NAFLD patients had normal or even very low ALT levels that would reduce the likelihood of observing significant changes. Hepatic ultrasonography scanning was performed independently by 2 trained radiologists who were blinded to the participants' details and to each other\'s diagnosis. Subjects were included if they were consistently classified as hepatic steatosis by both radiologists. Subjects with excessive alcohol consumption (ethanol \>140 g/wk for men and \>70 g/wk for women), cirrhosis, viral hepatitis, cardiovascular disease, cancer, and any consumption of nonsteroidal anti-inflammatory drugs, corticosteroids, or prescriptive medicines that affect liver function, lipid, or glucose metabolism were excluded. Recruitment began on June 30, 2013, with the first participant identified on July 5, and ended with the last visit on October 30, 2013. A total of 116 subjects were screened, and 74 subjects (39 men and 35 women) were enrolled in the study. Written informed consent was obtained from each patient included in the study. All participants were compensated for their participation in the study. This trial was registered at clinicaltrials.gov as NCT01940263. Study Design ------------ This pilot study was designed according to the CONSORT 2010 statement.^[@R21]^ Participants were assigned in a 1:1 ratio into anthocyanin or placebo groups in a random and double-blinded manner. Randomization was performed using a computer-generated list of random numbers. Randomization was stratified by gender to ensure equal numbers of males and females in each group. Study nurses or clinical physicians enrolled participants. Scheduled participants were consecutively assigned by a medical technologist, who was unaware of enrolment status, to treatment codes that corresponded to labels on otherwise identical concealed containers. Participants, investigators, and outcome assessors were blinded to the treatment for the duration of the study. Treatment assignments were not revealed prior to data collection and analysis. Anthocyanin and placebo capsules (Biolink AS, Sandnes, Norway) were identical in appearance and packaging. Anthocyanin capsules contained 80 mg of anthocyanins that were extracted from bilberry (blueberry) (*Vaccinium myrtillus*) and black currant (*Ribesnigrum*), as detailed previously.^[@R22]^ Placebo capsules consisted of maltodextrin and blue color. Two capsules were self-administered each morning and evening, yielding a daily intake of 320 mg anthocyanin. Our previous studies indicated beneficial effects of anthocyanin intervention on lipid profile and inflammatory status in dyslipidemic subjects when treated for 12--24 weeks^[@R22],[@R23]^; consequently we carried out a 24-week intervention in this study. However, during the follow-up period, considering the high dropout rate and the coming Chinese New Year, it was difficult for subjects to remain on the prescribed diet during the Spring Festival, and the trial was terminated at the interim. The patients attended the clinic at weeks 4 and 8 for anthropometric assessments, and returned the remaining capsules and received the capsules for the next interval. Patient compliance was evaluated by counting the remaining capsules. Use of pharmacological agents, for example, metformin, thiazolidinediones, statins, and probiotics, or any medicines that may affect body weight, insulin resistance, lipid profile, or fatty liver was not allowed during the trial. Subjects were asked to maintain their normal lifestyle and not to use any anthocyanin-rich nutritional supplements, red wine, grapes, blueberry, etc, during this period. Adverse events were defined as injuries related to or caused by the study treatment. At each visit, patients were specifically asked about adverse events, and the physician checked for any association between treatment and adverse events. Clinical and Laboratory Evaluation ---------------------------------- Before and after the 12-week intervention, height, weight, waist, and hip circumference, and blood pressure were measured. Body mass index (BMI) was calculated as follows: body weight (kg)/\[body height (m)\]^2^. Waist circumference was measured at a level midway between the lower rib margin and iliac crest with the measuring tape encircling the body in a horizontal position. In addition, a short form (International Physical Activity Questionnaire \[IPAQ\]) and a 3-day dietary record were used to evaluate physical exercise and dietary intake. Plasma glucose, lipid profile, and liver enzymes were tested using a fully automated biochemical analyzer (AU680, Beckman Coulter, Krefeld, Germany). Additional blood variables (hemoglobin, platelets, and white blood cells) were evaluated using an automated hematology analyzer (Sysmex 9000, Toa Medical Electronics, Kobe, Japan). Plasma insulin was determined by enzyme-linked immunosorbent assay (Human insulin ELISA kit, EMD Millipore Corporation, Billerica, MA). Insulin resistance was evaluated by homeostasis model assessment (HOMA-IR) and calculated as (fasting insulin \[mU/L\] × fasting glucose \[mmol/L\])/22.5.^[@R24]^ Plasma cytokeratin-18 fragment M30 (CK-18 M30) was measured using the M30 Apoptosense ELISA kit (PEVIVA, Bromma, Sweden) according to the manufacturer\'s protocols. Plasma myeloperoxidase (MPO) concentrations were measured using a commercially available ELISA kit (Hycult Biotec, Uden, The Netherlands). All plasma samples were analyzed in duplicate in the same run. According to the willingness of participants, a 2-hour oral glucose tolerance tests (OGTT) was performed before and after intervention. Briefly, patients fasted for 10 hours, after which the first blood samples were drawn at 8:00 [am]{.smallcaps} to test fasting glucose. Patients were then given 75 g glucose dissolved in 300 mL water, and the second blood samples were drawn 2 hours after their first drink. The participants were instructed to avoid strenuous exercise, eat any food, or drink any beverage except water for 2 hours. Impaired glucose tolerance (IGT) was defined as 2-hour glucose \>7.8 mmol/L, but \<11.1 mmol/L, according to the Chinese Type 2 Diabetes Prevention Guide 2010.^[@R25]^ A noninvasive scoring system was used to assess the severity of liver injury in NAFLD patients. The score for each test was calculated according to the following formulas derived from the original studies: NAFLD fibrosis score (NFS) = −1.675 + 0.037 × age (years) + 0.094 × BMI (kg/m^2^) + 1.13 × impaired fasting glucose/diabetes (yes = 1, no = 0) + 0.99 × aspartate aminotransferase (AST)/ALT ratio − 0.013 × platelet (×10^9^/L) −0.66 × albumin (g/dL).^[@R26]^ Study Outcomes -------------- The primary outcome measure was the difference in plasma ALT concentration. Secondary outcome measures included anthropometric measurements, fasting plasma levels of insulin, glucose, lipids, MPO, CK-18 M30, and NAFLD fibrosis score. Statistical Analysis -------------------- Sample size calculations were performed using the nQuery Advisor 7.0 software (Statistical Solutions Ltd., Cork, Ireland) and were based on data from a preliminary study in Japan showing decreased ALT levels (from 51.3 to 42.8 U/L) in subjects consuming an anthocyanin-rich purple sweet potato beverage for 12 weeks.^[@R18]^ Assuming that a 15% decrease of ALT in the intervention group and a 5% increase of ALT in the control group would be detected, a sample size of 30 patients per group was necessary to detect a difference with 83% power at a 5% significance level using the 2-sided test. To account for dropouts, 74 participants were included in our study. Data were analyzed according to the intention-to-treat principle. Patients missing laboratory measurements of the primary and secondary outcome measures were imputed using estimating-equation methods.^[@R27]^ SPSS 19.0 software (IBM-SPSS Inc, Chicago, IL) was used for statistical analysis of resulting data. The Kolmogorov--Smirnov test was performed to test normal distributions. Variables that were not normally distributed were logarithmically transformed to achieve normal distribution. Variables followed normal distributions and were described as means ± standard deviation. Data not exhibiting a normal distribution after transformation were expressed as medians with interquartile ranges. The percent change was calculated as follows: (value at 12 weeks -- value at baseline)/value at baseline × 100. Percent changes expressed as medians and interquartile range for nonnormal distributions or the means and 95% confidence interval for normal distributions. For primary or transformed data with a normal distribution, an independent Student *t* test was used to compare the differences at baseline between groups, and a paired Student *t* test was used to compare the differences before and after the intervention within groups. Otherwise, a Wilcoxon matched pairs signed rank test and a Mann--Whitney U test were used to compare characteristics in each group before and after intervention and differences between the 2 groups, respectively. Pearson correlation coefficients (*r*) were used to determine the association between the changes in the plasma CK-18 M30 concentrations and the changes in ALT in the 12-week study. RESULTS ======= Patient Characteristics ----------------------- A total of 74 volunteers were recruited. During the follow-up periods, 8 subjects dropped out by declining to return and 3 subjects were withdrawn by their physicians. In total, 63 participants (34 in the anthocyanin group and 29 in the control group) finished the whole procedure (Figure [1](#F1){ref-type="fig"}); however, all randomized patients were included in the intention-to-treat analysis. Among these subjects, an OGTT was administered to 33 patients (20 in the anthocyanin group, 13 in the control group). Based on the count of the recalled capsules at every visit, the level of compliance was very good, with capsule intake rates of 91.3% and 89.5% in the anthocyanin and placebo groups, respectively. None of the subjects experienced any serious adverse events resulting from the consumption of either the anthocyanin or placebo capsules throughout the trial period. ![Consolidated standards of reporting trials flow diagram of the study participants.](medi-94-e758-g001){#F1} At baseline assessment, no significant differences in anthropometric characteristics were observed between the 2 groups. The 12-week intervention with purified anthocyanin had no impact on BMI, waist and hip circumference, or blood pressure in anthocyanin group compared to the placebo control group (Table [1](#T1){ref-type="table"}). ###### Anthropometric Characteristics Across Groups at Baseline and 12 wk ![](medi-94-e758-g002) Based on the 3-day dietary record and the IPAQ, there were no significant differences in daily nutrient intake or physical activity between the 2 groups at baseline and after 12 weeks' intervention (Supplementary Table 1, <http://links.lww.com/MD/A272>). Primary Outcome --------------- At baseline, there were no significant differences in plasma liver enzymes between the 2 groups (Table [2](#T2){ref-type="table"}). After intervention, although no difference was observed between 2 groups after male-to-male or female-to-female comparisons, the total mean reduction of ALT levels in the anthocyanin group was greater than that of the placebo group (−19.1% vs −3.1%, *P* = 0.02). Compared to baseline, ALT levels were decreased significantly in both genders in the anthocyanin group; however, only females in the placebo group exhibited a significant drop in ALT levels (Figure [2](#F2){ref-type="fig"}). In addition, the mean value of both genders did not change significantly in the placebo group, which reflected a favorable effect of anthocyanin on chronic liver injury in the study participants. ###### Effects of Anthocyanin Supplementation on Biomarkers of Liver Injury ![](medi-94-e758-g003) ![Effect of the 12-week anthocyanin intervention on ALT levels in NAFLD patients. The columns and error bars indicate medians with quartiles. After intervention, no differences were observed between the 2 groups across intragender comparisons; however, the total mean reduction of ALT levels in the anthocyanin group was greater than that in the placebo group (a), *P* compared difference between 2 groups by Mann--Whitney U test. Compared with baseline, there is a remarkable decline in ALT after a 12-week intervention in males, females, and total patients in the anthocyanin group (b, c), but only in females in the placebo group (b, d). *P* compared with the basal values in each group by Wilcoxon matched pairs signed rank test.](medi-94-e758-g004){#F2} Secondary Outcomes ------------------ ### Lipid Profile and Insulin Resistance As shown in Table [3](#T3){ref-type="table"}, ingestion of anthocyanin for 12 weeks significantly decreased plasma triglyceride (TG) levels; however, the mean percentage changes were not significantly different from placebo controls. No effects on total plasma cholesterol or low-density lipoprotein-cholesterol were observed. Plasma levels of high-density lipoprotein-cholesterol increased moderately in both the groups, but this trend did not reach statistical significance across the 2 groups. ###### Effects of Anthocyanin Supplementation on Fasting Plasma Lipid Profile and Glucose and Insulin Concentrations ![](medi-94-e758-g005) Fasting blood glucose and HOMA-IR decreased significantly from baseline in the anthocyanin group, but did not differ from the placebo values (Table [3](#T3){ref-type="table"}). Insulin levels after 12 weeks was comparable to baseline in both anthocyanin and placebo groups. To examine insulin sensitivity and β-cell function, an oral glucose tolerance test was performed in 33 subjects, including 20 in anthocyanin group and 13 in the control group. Among these patients, 10 (50.0%) in the anthocyanin group and 6 (46.2%) in the control exhibited IGT. The anthropometric characteristics, dietary intake, physical activities, fasting blood glucose and insulin, and HOMA-IR were comparable between the 2 groups at baseline (Supplementary Table 2, <http://links.lww.com/MD/A272>). Anthocyanin supplementation significantly decreased the 2-hour OGTT glucose levels compared to the placebo control (−18.7% vs −3.8%, *P* = 0.02; Figure [3](#F3){ref-type="fig"}). ![Changes in glucose concentration measured 2 h after a 75-g oral glucose load in the 2 treatments during the study period. *P* compared difference between 2 groups by independent-sample *t* test.](medi-94-e758-g006){#F3} ### Other Liver Injury Biomarkers Anthocyanin supplementation did not affect the release of AST, another important enzyme in the liver (Table [2](#T2){ref-type="table"}). Plasma levels of CK-18 M30, a predictor of steatohepatitis,^[@R28]^ decreased by 8.8% after anthocyanin treatment and increased by 5.6% in the placebo group (*P* = 0.04). Inflammation and oxidative stress are involved in NAFLD progression, and MPO is linked to both inflammation and oxidative stress based on its location in leukocytes and role in catalyzing the formation of oxidizing agents.^[@R29]^ We next evaluated plasma levels of MPO and observed a significant difference in the effects of the 2 capsules on circulating MPO levels (−75.0% vs −44.8%, *P* \< 0.01). Compared with baseline, the NAFLD fibrosis score increased by 22.3% in the placebo group (*P* \< 0.05) but only 8.4% in the anthocyanin group (not significant), respectively. Furthermore, a positive correlation (*r* = 0.49, *P* \< 0.01) between the plasma ALT and CK-18 M30 levels was observed in our study subjects (Figure [4](#F4){ref-type="fig"}). ![Correlation between changes in plasma ALT activities and the levels of CK-18 M30; n = 37 in each group. The data were evaluated by using Pearson correlation coefficients (*r*).](medi-94-e758-g007){#F4} DISCUSSION ========== The results of this pilot study suggest that purified anthocyanin supplements can have favorable effects on insulin resistance and several plasma biomarkers of liver injury in patients with NAFLD. Although the patients in our study had relatively mild disease, they represent the spectrum of patients who are commonly seen in a primary care setting. The participants were able to maintain a constant body weight throughout the study (Supplementary Table 3, <http://links.lww.com/MD/A272>), excluding weight changes as a confounding factor that contributed to the improved features of NAFLD. Furthermore, participants were instructed not to alter their physical activity during the study. Therefore, in light of these findings, we suggest that an increase of dietary anthocyanin intake may help slow or reverse the clinical evolution of fatty liver disease, especially in early-stage NAFLD patients. Previous studies of foods rich in anthocyanin have yielded positive findings on whole-body inflammatory status and insulin sensitivity in obese subjects.^[@R30],[@R31]^ In the current study, a significant improvement or normalization of ALT, along with a marked improvement of glucose homeostasis, occurred in NAFLD patients after 12 weeks of anthocyanin treatment. Increasing evidence provided by cohort studies suggests that fatty liver may be a predictor of T2DM and cardiovascular disease,^[@R3]^ and our results are consistent with the observed association between higher anthocyanin intake and lower incidence of T2DM or cardiovascular disease.^[@R11],[@R13]^ However, in contrast to the results in certain trials,^[@R22],[@R31],[@R32]^ we did not observe any effect of anthocyanin on plasma lipid profile. It is possible that the efficacy of anthocyanins on lipid profile responses depends, to some extent, on the population studied and the duration of the intervention. Another important finding of this study is the marked decrease in plasma levels of caspase-cleaved CK-18 fragments in participants receiving anthocyanin treatment. CK-18 is the predominant keratin expressed in the liver and is a well-known substrate of caspases during apoptotic hepatocyte cell death.^[@R33]^ Other investigators have shown that CK-18 M30 has high overall accuracy in differentiating NAFLD from control subjects and moderate accuracy in differentiating NASH from simple steatosis.^[@R34]^ Our previous in vitro study showed that anthocyanin cyanidin 3-*O*-β-glucoside improved hepatocyte function and cell survival in response to high glucose stress.^[@R35]^ In this study, we observed a positive correlation between changes in plasma ALT activities and the levels of CK-18 M30 in anthocyanin-treated patients. These results suggest that CK-18 M30 has potential as a marker for predicting NAFLD progression. Oxidative stress and inflammation are important mechanisms in the pathogenesis of NAFLD. MPO is a heme-containing peroxidase abundantly expressed in neutrophils and to a lesser extent in monocytes.^[@R29]^ Upon cell activation, MPO is released and MPO-derived oxidants have been associated with tissue damage in many diseases, particularly those characterized by acute or chronic inflammation.^[@R36]^ Increased numbers of MPO-expressing cells and accumulation of HOCl-modified and nitrated proteins in steatotic livers enhance progression of simple steatosis to steatohepatitis. In contrast, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in mice on a high fat diet.^[@R37]^ A recent study demonstrated that plasma MPO was positively associated with insulin resistance and inflammation parameters in overweight subjects.^[@R38]^ In NASH patients, both hepatic MPO expression and plasma MPO levels were elevated compared with patients with similar BMI but without NASH.^[@R39]^ Our results suggest that anthocyanin supplementation reduced plasma MPO and showed a trend toward improvement in NAFLD fibrosis score. It is tempting to speculate that MPO may be a factor that directly links the hepatoprotective effects of anthocyanin to its well-documented anti-inflammatory and anti-oxidative properties.^[@R40]^ The majority of studies investigating anthocyanin intervention in humans have used foods containing multiple types of polyphenols. To the best of our knowledge, this is the first study comparing the effects of purified anthocyanin on blood-based biomarkers of NAFLD using a randomized double-blind design with placebo controls. Nevertheless, this study has some intrinsic limitations that must be acknowledged. First, as liver biopsies were not performed, NAFLD features such as steatosis and necroinflammation could not be evaluated. Second, our study was mildly underpowered by small sample size and a relatively short intervention period. We experienced a significant number of screen failures in the enrolment, and patient dropout rates were higher than anticipated, resulting in a lower number of evaluable patients than is estimated to be necessary to detect differences between groups, which may have led to a type II error. Finally, our patients had relatively low BMIs compared to those reported in Western studies; however, Asians generally develop visceral obesity and NAFLD at a lower BMI.^[@R5]^ In summary, our findings above linking the dietary anthocyanin supplement to improved characteristics of NAFLD are particularly important in light of the increasing incidence of NAFLD worldwide. Furthermore, this supplementation is an inexpensive, nontoxic therapy that results in overall health improvement, reducing the risk not only of chronic liver disease, but also T2DM and cardiovascular disease. Future large, prospective, well-designed, multicenter studies are needed to explore the durability of the benefits of anthocyanin. The authors would like to express our gratitude to Dr Yichun Zhong (Shaoguan Railway Hospital) for his kind support of this work. Abbreviations: ALT = alanine aminotransferase, BMI = body mass index, CK-18 M30 = cytokeratin-18 fragment M30, HOMA-IR = homeostasis model assessment of insulin resistance, MPO = myeloperoxidase, NAFLD = nonalcoholic fatty liver disease, NASH = nonalcoholic steatohepatitis, OGTT = oral glucose tolerance test, T2DM = type 2 diabetes mellitus. This work was funded by grants from the National Basic Research Program (973 Program, 2012CB517506), National Natural Science Foundation (81372994, 81172655), and Guangdong Industry-University Research Foundation (2013B090600138). All authors have read and approved the submitted version of the manuscript. Study concept: H-HG; data acquisition: P-WZ; data analysis and interpretation: F-XC, DL; drafting of the manuscript: P-WZ; critical revision of the manuscript for important intellectual content: W-HL; study supervision: H-HG. The authors have no conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal\'s Website ([www.md-journal.com](www.md-journal.com)).

{ "pile_set_name": "PubMed Central" }

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"Insulin", "resistance", "key", "pathogenic", "factor", "initiation", "development", "NAFLD", "Several", "insulin", "sensitizers", "including", "thiazolidinediones", "metformin", "tested", "pilot", "studies", "shown", "significantly", "reduce", "liver", "inflammation", "steatosis", "NAFLD", "however", "compounds", "associated", "weight", "gain", "raise", "concerns", "cardiovascular", "Thus", "side", "effects", "insulin", "sensitizers", "underestimated", "particularly", "simple", "cases", "NAFLD", "absence", "complications", "Dietary", "lifestyle", "changes", "recommended", "primary", "treatment", "Increased", "intakes", "fruits", "vegetables", "likely", "important", "health", "benefits", "NAFLD", "addition", "vitamins", "minerals", "fiber", "fruits", "vegetables", "contain", "high", "concentrations", "bioactive", "compounds", "carotenoids", "tocopherols", "polyphenols", "Among", "anthocyanin", "polyphenols", "particular", "interest", "since", "several", "epidemiological", "studies", 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"improvement", "normalization", "ALT", "along", "marked", "improvement", "glucose", "homeostasis", "occurred", "NAFLD", "patients", "weeks", "anthocyanin", "treatment", "Increasing", "evidence", "provided", "cohort", "studies", "suggests", "fatty", "liver", "may", "predictor", "cardiovascular", "disease", "results", "consistent", "observed", "association", "higher", "anthocyanin", "intake", "lower", "incidence", "cardiovascular", "However", "contrast", "results", "certain", "trials", "observe", "effect", "anthocyanin", "plasma", "lipid", "profile", "possible", "efficacy", "anthocyanins", "lipid", "profile", "responses", "depends", "extent", "population", "studied", "duration", "intervention", "Another", "important", "finding", "study", "marked", "decrease", "plasma", "levels", "fragments", "participants", "receiving", "anthocyanin", "treatment", "predominant", "keratin", "expressed", "liver", "substrate", "caspases", "apoptotic", "hepatocyte", "cell", "investigators", "shown", "high", "overall", "accuracy", "differentiating", "NAFLD", "control", "subjects", "moderate", "accuracy", "differentiating", "NASH", "simple", "previous", "vitro", "study", "showed", "anthocyanin", "cyanidin", "improved", "hepatocyte", "function", "cell", "survival", "response", "high", "glucose", "study", "observed", "positive", "correlation", "changes", "plasma", "ALT", "activities", "levels", "patients", "results", "suggest", "potential", "marker", "predicting", "NAFLD", "progression", "Oxidative", "stress", "inflammation", "important", "mechanisms", "pathogenesis", "NAFLD", "MPO", "peroxidase", "abundantly", "expressed", "neutrophils", "lesser", "extent", "Upon", "cell", "activation", "MPO", "released", "oxidants", "associated", "tissue", "damage", "many", "diseases", "particularly", "characterized", "acute", "chronic", "Increased", "numbers", "cells", "accumulation", "nitrated", "proteins", "steatotic", "livers", "enhance", "progression", "simple", "steatosis", "steatohepatitis", "contrast", "MPO", "deficiency", "attenuates", "development", "NASH", "diminishes", "adipose", "tissue", "inflammation", "mice", "high", "fat", "recent", "study", "demonstrated", "plasma", "MPO", "positively", "associated", "insulin", "resistance", "inflammation", "parameters", "overweight", "NASH", "patients", "hepatic", "MPO", "expression", "plasma", "MPO", "levels", "elevated", "compared", "patients", "similar", "BMI", "without", "results", "suggest", "anthocyanin", "supplementation", "reduced", "plasma", "MPO", "showed", "trend", "toward", "improvement", "NAFLD", "fibrosis", "score", "tempting", "speculate", "MPO", "may", "factor", "directly", "links", "hepatoprotective", "effects", "anthocyanin", "majority", "studies", "investigating", "anthocyanin", "intervention", "humans", "used", "foods", "containing", "multiple", "types", "polyphenols", "best", "knowledge", "first", "study", "comparing", "effects", "purified", "anthocyanin", "biomarkers", "NAFLD", "using", "randomized", "design", "placebo", "controls", "Nevertheless", "study", "intrinsic", "limitations", "must", "acknowledged", "First", "liver", "biopsies", "performed", "NAFLD", "features", "steatosis", "necroinflammation", "could", "evaluated", "Second", "study", "mildly", "underpowered", "small", "sample", "size", "relatively", "short", "intervention", "period", "experienced", "significant", "number", "screen", "failures", "enrolment", "patient", "dropout", "rates", "higher", "anticipated", "resulting", "lower", "number", "evaluable", "patients", "estimated", "necessary", "detect", "differences", "groups", "may", "led", "type", "II", "error", "Finally", "patients", "relatively", "low", "BMIs", "compared", "reported", "Western", "studies", "however", "Asians", "generally", "develop", "visceral", "obesity", "NAFLD", "lower", "summary", "findings", "linking", "dietary", "anthocyanin", "supplement", "improved", "characteristics", "NAFLD", "particularly", "important", "light", "increasing", "incidence", "NAFLD", "worldwide", "Furthermore", "supplementation", "inexpensive", "nontoxic", "therapy", "results", "overall", "health", "improvement", "reducing", "risk", "chronic", "liver", "disease", "also", "cardiovascular", "disease", "Future", "large", "prospective", "multicenter", "studies", "needed", "explore", "durability", "benefits", "anthocyanin", "authors", "would", "like", "express", "gratitude", "Dr", "Yichun", "Zhong", "Shaoguan", "Railway", "Hospital", "kind", "support", "work", "Abbreviations", "ALT", "alanine", "aminotransferase", "BMI", "body", "mass", "index", "fragment", "homeostasis", "model", "assessment", "insulin", "resistance", "MPO", "myeloperoxidase", "NAFLD", "nonalcoholic", "fatty", "liver", "disease", "NASH", "nonalcoholic", "steatohepatitis", "OGTT", "oral", "glucose", "tolerance", "test", "type", "diabetes", "mellitus", "work", "funded", "grants", "National", "Basic", "Research", "Program", "Program", "National", "Natural", "Science", "Foundation", "Guangdong", "Research", "Foundation", "authors", "read", "approved", "submitted", "version", "manuscript", "Study", "concept", "data", "acquisition", "data", "analysis", "interpretation", "DL", "drafting", "manuscript", "critical", "revision", "manuscript", "important", "intellectual", "content", "study", "supervision", "authors", "conflicts", "interest", "disclose", "Supplemental", "digital", "content", "available", "article", "Direct", "URL", "citations", "appear", "printed", "text", "provided", "HTML", "PDF", "versions", "article", "Website" ]

**Suggested citation:** EFSA GMO Panel (EFSA Panel on genetically modified organisms) , Naegeli H, Bresson J‐L, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogué F, Rostoks N, Sánchez Serrano JJ, Savoini G, Veromann E, Veronesi F, Álvarez F, Ardizzone M, Papadopoulou N, Paraskevopoulos K, 2019 Scientific Opinion on the assessment of genetically modified soybean A2704‐12 for renewal of authorisation under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐RX‐009). EFSA Journal 2019;17(1):5523, 12 pp. 10.2903/j.efsa.2019.5523 **Requestor:** European Commission (DG SANTE) **Question number:** EFSA‐Q‐2017‐00721 **Panel members:** Hanspeter Naegeli, Jean‐Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michele Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Francisco Javier Moreno, Ewen Mullins, Fabien Nogué, Nils Rostoks, Jose Juan Sánchez Serrano, Giovanni Savoini, Eve Veromann and Fabio Veronesi. **Acknowledgements:** The Panel wishes to thank the members of its standing Working Groups on Molecular Characterisation, Food/Feed and Environmental Risk Assessment for the preparatory work on this scientific opinion, and the EFSA staff members Andrea Gennaro, Sylvie Mestdagh, Claudia Paoletti and Irene Muñoz Guajardo for the support provided to this scientific opinion. Adopted: 29 November 2018 Summary {#efs25523-sec-0001} ======= Following the submission of application EFSA‐GMO‐RX‐009 under Regulation (EC) No 1829/2003 from Bayer CropScience N.V., the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel) was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the herbicide‐tolerant genetically modified soybean A2704‐12. The scope of renewal application EFSA‐GMO‐RX‐009 is for placing on the market of products containing, consisting of, or produced from soybean A2704‐12, excluding cultivation within the European Union. In delivering its scientific opinion, the GMO Panel took into account application EFSA‐GMO‐RX‐009, additional information provided by the applicant, scientific comments submitted by the Member States and relevant scientific publications. The data received in the context of the renewal application EFSA‐GMO‐RX‐009 contained: post‐market environmental monitoring reports, an evaluation of the literature retrieved by a systematic search, updated bioinformatics analyses, and additional studies performed by or on behalf of the applicant. In addition, the applicant provided sequence data on the soybean A2704‐12 event using material from a commercial variety currently on the market and intended to be marketed in the coming years. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. The GMO Panel concludes that there is no evidence in renewal application EFSA‐GMO‐RX‐009 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean A2704‐12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). 1. Introduction {#efs25523-sec-0003} =============== 1.1. Background {#efs25523-sec-0004} --------------- On 3 November 2017, the European Food Safety Authority (EFSA) received from the European Commission (DG SANTE) application EFSA‐GMO‐RX‐009 by Bayer CropScience N.V. for the renewal of authorisation of genetically modified (GM) soybean A2704‐12 (Unique Identifier ACS‐GMØØ5--3) for the placing on the market of products containing, consisting of, or produced from this GM soybean for import and processing submitted within the framework of Regulation (EC) No 1829/2003[1](#efs25523-note-1006){ref-type="fn"}. Before sending the application to EFSA, the European Commission confirmed whether the data submitted in the context of this renewal application were in line with the legal requirements laid down in Articles 11 and 23 of Regulation (EC) No 1829/2003. After receiving application EFSA‐GMO‐RX‐009, and in accordance with Articles 5(2)(b) and 17(2)(b) of Regulation (EC) No 1829/2003, EFSA informed Member States and made the summary of the application available to the public on the EFSA website.[2](#efs25523-note-1007){ref-type="fn"} On 9 March 2018, EFSA declared the application valid in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003. EFSA made the valid application available to Member States and the European Commission, and consulted nominated risk assessment bodies of Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC following the requirements of Articles 6(4) and 18(4) of Regulation (EC) No 1829/2003, to request their scientific opinion. Member States had three months after the opening of the Member State commenting period (until 9 June 2018) to make their opinion known. Following the submission of application EFSA‐GMO‐NL‐2005‐18 and the publication of the EFSA scientific opinion (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}), the placing on the market of soybean A2704‐12 for products containing, consisting of, or produced from this GM soybean, excluding cultivation in the European Union, was authorised by Commission Decision 2008/837/EC[3](#efs25523-note-1008){ref-type="fn"}. A copy of this authorisation was provided by the applicant.[4](#efs25523-note-1009){ref-type="fn"} EFSA requested additional information on 26 March 2018 and 8 May 2018. The applicant submitted their replies on 27 April 2018 and 19 June 2018, respectively. In giving its scientific opinion to the European Commission, the Member States and the applicant, and in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003, EFSA has endeavoured to respect a time limit of six months from the acknowledgement of the valid application. As additional information was requested by the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel), the time limit of 6 months was extended accordingly, in line with Articles 6(1), 6(2), 18(1), and 18(2) of Regulation (EC) No 1829/2003. According to Regulation (EC) No 1829/2003, this scientific opinion is to be seen as the report requested under Articles 6(6) and 18(6) of that Regulation and thus will be part of the EFSA overall opinion in accordance with Articles 6(5) and 18(5). 1.2. Terms of Reference as provided by the requestor {#efs25523-sec-0005} ---------------------------------------------------- The GMO Panel was requested to carry out a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the placing on the market of products containing, consisting of, or produced from GM soybean A2704‐12, in accordance with Articles 11 and 23 of Regulation (EC) No 1829/2003. Where applicable, any conditions or restrictions which should be imposed on the placing on the market and/or specific conditions or restrictions for use and handling, including post‐market monitoring requirements based on the outcome of the risk assessment and, in the case of GMOs or food and feed containing or consisting of GMOs, conditions for the protection of particular ecosystems/environment and/or geographical areas should be indicated in accordance with Articles 6(5)(e) and 18(5)(e) of Regulation (EC) No 1829/2003. The GMO Panel was not requested to give an opinion on information required under Annex II to the Cartagena Protocol. Furthermore, the GMO Panel did not consider proposals for labelling and methods of detection (including sampling and the identification of the specific transformation event in the food and feed and/or food and feed produced from it), which are matters related to risk management. 2. Data and methodologies {#efs25523-sec-0006} ========================= 2.1. Data {#efs25523-sec-0007} --------- The data for application EFSA‐GMO‐RX‐009 provided by the applicant at the time of submission, or in reply to requests for additional information, are specified below. The applicant submitted sequence data on soybean A2704‐12 single event[5](#efs25523-note-1010){ref-type="fn"} derived from material of an early generation (T4) of soybean A2704‐12 (fourth generation after the original transformant) and from material of a commercially available soybean A2704‐12 variety currently on the market and indicated that these sequences are identical. The applicant clarified that the soybean A2704‐12 commercial variety has been available on the market since 2016 and is intended to be marketed in the coming years. The applicant also clarified that the A2704‐12 event sequence reported in this renewal application is the sequence submitted in the original application EFSA‐GMO‐NL‐2005‐18 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}), corrected for eight nucleotides, two of which affect newly created open reading frames (ORFs) within the insert and spanning the junction sites (EFSA, [2018](#efs25523-bib-0006){ref-type="ref"}). ### 2.1.1. Post‐market monitoring reports[6](#efs25523-note-1011){ref-type="fn"} {#efs25523-sec-0008} Based on the outcome of the initial food and feed risk assessment, a post‐market monitoring plan for monitoring of GM food and feed was not required by the authorisation decision. The implementation of a post‐market environmental monitoring (PMEM) plan, consisting of a general surveillance plan to check for any adverse effects on the environment arising from soybean A2704‐12, was a condition for the authorisation. As no potential adverse environmental effects were identified in the environmental risk assessment of soybean A2704‐12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}), case‐specific monitoring was not considered necessary by the GMO Panel. The applicant provided eight annual PMEM reports covering a reporting period from September 2008 to June 2016. The annual PMEM plans submitted by the applicant included (1) the description of a centralised system established by EuropaBio for the collection of information recorded by various operators (federations involved in soybean seeds import and processing) on any observed adverse effect(s) on human health and the environment arising from handling of soybean possibly containing soybean A2704012; (2) the reports of the surveillance activities conducted by such operators; and (3) the review of relevant scientific peer‐reviewed studies retrieved from literature searches. ### 2.1.2. Systematic search and evaluation of literature[7](#efs25523-note-1012){ref-type="fn"} {#efs25523-sec-0009} In addition to the eight separate literature searches provided as part of the annual PMEM reports, the applicant performed a systematic literature search for soybean A2704012 and the newly expressed phosphinothricin *N*‐acetyltransferase (PAT) protein covering the period from 1 September 2007 till 10 January 2018, in accordance with the recommendations on literature searching outlined in EFSA ([2010](#efs25523-bib-0002){ref-type="ref"}, [2017a](#efs25523-bib-0004){ref-type="ref"}). Searches against electronic bibliographic databases and internet searches to specialist databases were performed to identify relevant publications. Altogether, 564 publications were retrieved. After applying the eligibility/inclusion criteria defined *a priori* by the applicant, six publications were identified as relevant for food and feed safety assessment, molecular characterisation and environmental safety assessment. The list of relevant publications is provided in Appendix [A](#efs25523-sec-1001){ref-type="sec"}. ### 2.1.3. Updated bioinformatic data[8](#efs25523-note-1013){ref-type="fn"} {#efs25523-sec-0010} At the time of submission of the renewal dossier, the applicant provided a complete bioinformatic dataset for the soybean A2704‐12 event (using the corrected sequence) including an analysis of the insert and flanking sequences, an analysis of the potential similarity to allergens and toxins of the newly expressed protein and of all possible ORFs within the insert and spanning the junction sites, and an analysis of possible horizontal gene transfer (EFSA, [2017b](#efs25523-bib-0005){ref-type="ref"}). The outcome of the updated bioinformatic analyses is presented in Section [3.3](#efs25523-sec-0018){ref-type="sec"}. On 8 May 2018, EFSA requested supplementary information on the partial deletion of the endogenous gene previously reported in the frame of the original application EFSA‐GMO‐NL‐2005‐18. On 19 June 2018, the applicant provided the supplementary information which included information on the function of the interrupted endogenous gene and a risk assessment of its interruption with respect to the agronomic--phenotypic characteristics and composition of soybean A2704‐12. ### 2.1.4. Additional documents or studies provided by the applicant[9](#efs25523-note-1014){ref-type="fn"} {#efs25523-sec-0011} The applicant provided an overview on the worldwide approvals of soybean A2704‐12 and a list containing the summaries of all studies performed by or on behalf of the applicant over the course of the authorisation period and not previously submitted to the EU (Appendix [B](#efs25523-sec-1002){ref-type="sec"}). The relevance of the listed studies for molecular characterisation, human and animal safety and the environment was assessed by the applicant. On 26 March 2018, the GMO Panel requested the applicant to provide the full study reports of five of these studies considered potentially relevant for safety assessment. The applicant submitted the requested information on 27 April 2018. ### 2.1.5. Overall assessment as provided by the applicant[10](#efs25523-note-1015){ref-type="fn"} {#efs25523-sec-0012} The applicant provided an overall assessment concluding that information provided in the application for renewal of authorisation of soybean A2704‐12 for food and feed use and processing in the EU does not change the outcome of the original risk assessment (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). ### 2.1.6. Monitoring plan and proposal for improving the conditions of the original authorisation[11](#efs25523-note-1016){ref-type="fn"} {#efs25523-sec-0013} The applicant indicated in the dossier that the environmental monitoring plan is appropriate and does not need any changes. 2.2. Methodologies {#efs25523-sec-0014} ------------------ The GMO Panel assessed the application for renewal of the authorisation of soybean A2704‐12 for food and feed uses, import and processing in accordance with Articles 11 and 23 of Regulation (EC) No 1829/2003. The GMO Panel took into account the requirements described in its guideline for the risk assessment of renewal applications of GM food and feed authorised under Regulation (EC) No 1829/2003 (EFSA GMO Panel, [2015](#efs25523-bib-0003){ref-type="ref"}). The comments raised by Member States are addressed in Annex G of EFSA\'s overall opinion[12](#efs25523-note-1017){ref-type="fn"} and were taken into consideration during the scientific risk assessment. 3. Assessment {#efs25523-sec-0015} ============= 3.1. Evaluation of the post‐market monitoring reports {#efs25523-sec-0016} ----------------------------------------------------- During the general surveillance activities covering the authorisation period of soybean A2704‐12, no adverse effects were reported by the applicant. 3.2. Evaluation of the systematic search and evaluation of literature {#efs25523-sec-0017} --------------------------------------------------------------------- The GMO Panel assessed the applicant\'s literature searches on soybean A2704‐12. Although the overall quality of the performed literature searches is acceptable, the GMO Panel considers that future searches could be improved. The GMO Panel therefore recommends the applicant for future searches to: ensure that enough search term variation is used (covering possible synonyms, related terms, acronyms, spelling variants, old and new terminology, brand and generic names, lay and scientific terminology, common typos, translation issues);include trait terms;include controlled vocabulary (subject indexing) in the searches when available (in addition to/combination with text words);use wider proximity operators (5W). The GMO Panel acknowledges that no publications raising a safety concern for human and animal health and the environment which would change the original risk assessment conclusions on soybean A2704‐12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}) have been identified by the applicant. 3.3. Evaluation of the updated bioinformatic data {#efs25523-sec-0018} ------------------------------------------------- The results of the updated bioinformatic analyses of soybean A2704‐12 using the corrected event sequence (EFSA, [2018](#efs25523-bib-0006){ref-type="ref"}) confirm the previous conclusions on the partial deletion of an endogenous gene. Based on the information from updated databases submitted in the frame of this renewal application, this gene is identified as *SEOe*, a member of the sieve element occlusion (SEO) multigene family encoding for *Glycine max* SEO proteins. The *SEOe* gene is one of four genes in subgroup 5 of the SEO gene family, thereby indicating that functional redundancy for this gene in the soybean genome is likely. In addition, the original agronomic, phenotypic and compositional analyses of soybean A2704‐12 (including endpoints that may be relevant to the reported biological role of SEO proteins in photoassimilate translocation via the phloem network such as plant height, yield and fiber content) did not indicate relevant differences as compared to the non‐GM comparator. Analyses of the amino acid sequence of the newly expressed PAT protein reveal no significant similarities to toxins or allergens. In addition, bioinformatic analyses of the newly created ORFs (using the corrected event sequence) within the insert or spanning the junctions with genomic DNA reveal no significant similarities to toxins and allergens. Updated bioinformatic analyses of the recombinant DNA present in soybean A2704‐12 reveal sufficient sequence identity at both ends of the inserted cassette by the disrupted *bla* gene. This could facilitate double homologous recombination with *bla* genes as they can occur in environmental bacteria. The recombination event, however, would result in the disruption of such genes in the recipients. In addition, the insertion of a *pat* gene codon‐optimised for plants regulated by a plant virus promoter would probably lack PAT activity in potential recipient bacteria. Therefore, bacteria in which the described double homologous recombination would occur would lose their antibiotic resistance and acquire a plant codon‐optimised *pat* gene for which a selective advantage, compared to natural variants of *pat* genes as they occur in environmental bacteria, cannot be expected. The results of updated bioinformatic analyses confirm the previous conclusions that the unlikely, but theoretically possible, horizontal transfer of recombinant genes from soybean A2704‐12 to bacteria does not raise any environmental safety concern (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). 3.4. Evaluation of the additional documents or studies provided by the applicant {#efs25523-sec-0019} -------------------------------------------------------------------------------- The GMO Panel evaluated the summary and/or full study reports of the additional studies provided and (Appendix [B](#efs25523-sec-1002){ref-type="sec"}). This new information does not raise any concern for human and animal health and the environment, which would change the original risk assessment conclusions on soybean A2704‐12. 3.5. Evaluation of the overall assessment as provided by the applicant {#efs25523-sec-0020} ---------------------------------------------------------------------- The GMO Panel evaluated the overall assessment provided by the applicant and confirms that there is no evidence in renewal application EFSA‐GMO‐RX‐009 indicating new hazards, relevant changes in exposure or scientific uncertainties that would change previous conclusions on soybean A2704‐12. 3.6. Evaluation of the monitoring plan and proposal for improving the conditions of the original authorisation {#efs25523-sec-0021} -------------------------------------------------------------------------------------------------------------- The PMEM plan covers general surveillance of imported GM plant material, including soybean A2704‐12. This general surveillance is coordinated by EuropaBio and implemented by selected operators (federations involved in soybean seeds import and processing). In addition, the applicant reviews relevant scientific publications retrieved from literature searches on an annual basis. The GMO Panel is of the opinion that the scope of the plan provided by the applicant is consistent with the scope of soybean A2704‐12, but reminds that monitoring is related to risk management, and thus, the final adoption and implementation of the PMEM plan falls outside the mandate of EFSA. 4. Conclusions {#efs25523-sec-0022} ============== Based on the data provided, the GMO Panel concludes that there is no evidence in the renewal application EFSA‐GMO‐RX‐009 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean A2704--12 (EFSA, [2007](#efs25523-bib-0001){ref-type="ref"}). Documentation provided to EFSA {#efs25523-sec-0023} ============================== Letter from the European Commission to EFSA received on 3 November 2017 for the continued marketing of genetically modified soybean A2704‐12 in accordance with articles 11 and 23 of Regulation (EC) No 1829/2003 by Bayer CropScience N.V. (EFSA‐GMO‐RX‐009).Acknowledgement letter, dated 7 November 2017, from EFSA to the European Commission.Letter from EFSA to applicant dated 15 December 2017 requesting additional information under completeness check.Letter from applicant to EFSA received on 16 February 2018 providing additional information under completeness check.Letter from EFSA to applicant dated 9 March 2018 delivering the 'Statement of Validity' for application EFSA‐GMO‐RX‐009.Letter from EFSA to applicant dated 26 March 2018 requesting additional information and stopping the clock.Letter from applicant to EFSA received on 27 April 2018 providing additional information.Letter from EFSA to applicant dated 30 April 2018 re‐starting the clock from 27 April 2018.Letter from EFSA to applicant dated 8 May 2018 requesting additional information and stopping the clock.Letter from applicant to EFSA received on 19 June 2018 providing additional information.Letter from EFSA to applicant dated 19 June 2018 re‐starting the clock from 19 June 2018. Abbreviations {#efs25523-sec-0024} ============= GMgenetically modifiedGMOgenetically modified organismsGMOPanel EFSA Panel on Genetically Modified OrganismsORFsopen reading framesPATphosphinothricin *N*‐acetyltransferasePMEMpost‐market environmental monitoringSEOsieve element occlusion Appendix A -- List of relevant publications identified by the applicant through the systematic literature search (1/9/2007--10/1/2018) {#efs25523-sec-1001} ====================================================================================================================================== {#efs25523-sec-0025} ReferenceFard NA et al., 2013. *In silico* allergenicity assessment of novel proteins derived from GMHR crops. Ortuno F and Rojas I \[Eds\]. Proceedings IWBBIO.Fard NA, Minuchehr Z and Mousavi A, 2013. Allergenicity study of genetically modified herbicide resistant crops (bioinformatics assessment). Bulletin of Environment, Pharmacology and Life Sciences, 2, 24--32.Nagy A, Pauk J, Takacs K and Gelencser E, 2008. Nutritional evaluation of the proteins of broad range herbicide resistant spring wheat (*Triticum aestivum* L.) lines. II. Resistance to digestion of marker proteins in rat model. Acta Alimentaria, 37, 159--166.Oh J, Ko M and Lee H, 2009. Evaluation for allergenicity for genetically modified organic foods. Journal of Allergy and Clinical Immunology, 123, S244.Schafer BW, Embrey SK and Herman RA, 2016. Rapid simulated gastric fluid digestion of in‐seed/grain proteins expressed in genetically engineered crops. Regulatory Toxicology and Pharmacology, 81, 106--112.Verma AK, Misra A, Subash S, Das M and Dwivedi PD, 2011. Computational allergenicity prediction of transgenic proteins expressed in genetically modified crops. Immunopharmacology and Immunotoxicology, 33, 410--422. Appendix B -- List of additional studies performed by or on behalf of the applicant over the course of the authorisation period and not previously submitted to the EU with regard to the evaluation of the safety of the food and feed for humans, animal or the environment from soybean A2704‐12 {#efs25523-sec-1002} =================================================================================================================================================================================================================================================================================================== {#efs25523-sec-0026} Study identificationTitleM--413199‐01‐1Agronomic characteristics of glufosinate‐tolerant soybean A2704‐12, USA, 2006M‐534302‐01‐1Structural stability analysis of early and recent generations of *Glycine max* A2704‐12M‐540738‐01‐1A2704‐12 soybean ‐ Inheritance of the insert over two generationsM‐131221‐04‐1[a](#efs25523-note-1018){ref-type="fn"}PAT/pat protein: Heat stability studyM‐214021‐03‐1PAT/pat protein: *in vitro* digestibility study in human simulated intestinal fluidM‐214025‐05‐1[a](#efs25523-note-1018){ref-type="fn"}PAT/pat protein ‐ *In vitro* digestibility study in human simulated gastric fluidM‐344158‐01‐1[a](#efs25523-note-1018){ref-type="fn"}PAT protein (encoded the *pat* gene) ‐ Acute toxicity (48 hours) to rainbow trout (*Oncorhynchus mykiss*) by intraperitoneal injectionM‐395296‐01‐1Measurement of PAT protein in soybean oil samples using the QualiPlate Kit for PAT from EnviroLogixM‐475440‐01‐1[a](#efs25523-note-1018){ref-type="fn"}PAT/pat protein ‐ Acute toxicity by oral gavage in miceM‐500889‐01‐1[a](#efs25523-note-1018){ref-type="fn"}The effect of temperature on microbially‐produced PAT/pat as assessed by ELISA[^1] Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, 18.10.2003, p. 1--23. Available online: <http://registerofquestions.efsa.europa.eu/roqFrontend/questionDocumentsLoader?question=EFSA-Q-2017-00721> COMMISSION DECISION of 8 September 2008 authorising the placing on the market of products containing, consisting of, or produced from genetically modified soybean A2704‐12 (ACS‐GMØØ5‐3) pursuant to Regulation (EC) No 1829/2003 of the European Parliament and of the Council. Official Journal of the European Union L 247/50, 16.9.2008. Dossier: Soybean A2704‐12 renewal -- Annex 1. Dossier: Soybean A2704‐12 renewal -- Section 3. Dossier: Soybean A2704‐12 renewal -- Section 2. Dossier Soybean A2704‐12 renewal -- Section 3a; additional information: 27/4/2018. Dossier: Soybean A2704‐12 renewal -- Section 3b; additional information: 19/6/2018. Dossier: Soybean A2704‐12 renewal -- Section 3c; additional information: 27/4/2018. Dossier: Soybean A2704‐12 renewal -- Section 4. Dossier: Soybean A2704‐12 renewal -- Section 5. Available online: <http://registerofquestions.efsa.europa.eu/roqFrontend/questionLoader?question=EFSA-Q-2018-00992> [^1]: Studies for which the full report was requested by the GMO Panel.

{ "pile_set_name": "PubMed Central" }

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It is widely accepted that magnetic reconnection plays an important role in plasmas, particularly in solar eruptive events, such as flares[@b1][@b2][@b3][@b4][@b5], filament eruptions[@b6], coronal mass ejections[@b7][@b8] and jets[@b9]. In the models of magnetic reconnection, magnetic field lines with an oppositely directed component approach each other in a current sheet or at a magnetic null point, break up and reconnect to form new magnetic lines. Magnetic energy is thereby released into thermal and kinetic energy of the plasma, potentially leading to large-scale phenomena. Evidence of reconnection on the Sun has mostly been limited to single aspects and has been indirect, showing changes of magnetic connections[@b10], reconnection inflows[@b11][@b12][@b13] and outflows[@b14][@b15][@b16][@b17], hot cusp-shaped structures at their interface[@b11][@b13][@b14], supra-arcade downflows[@b15][@b18], loop shrinkage[@b19][@b20], sudden brightenings[@b21][@b22], current sheets[@b15][@b20][@b23], plasmoid ejections[@b24][@b25], loop-top hard X-ray sources[@b20][@b26], pulsating radio emissions[@b27], and coronal heating in the interface between emerging and ambient magnetic flux[@b28]. In recent years, direct and more comprehensive evidence of reconnection has been discovered in a couple of energetic events on the Sun. Two very clear cases of reconnection, including inflowing cool loops, outflowing hot loops and plasma heated to \>10 MK, were observed in solar flares through extreme ultraviolet (EUV) and X-ray data[@b12][@b13]. The reconnection inflow and outflow velocities could be inferred in these flares to lie in the ranges of 10--90 and 90--460 km s^−1^, respectively, constraining the reconnection rate to the range of 0.05--0.5. Inflows, outflows and the formation of new loops could also be studied in a well-observed case of reconnection between two sets of small-scale chromospheric loops, imaged at high resolution in H-alpha[@b22]. This study showed a transition from slow to fast reconnection. The three-dimensional (3D) topology of reconnecting loops and their heating has been inferred using the combined perspectives and multiple EUV channels of two spacecraft[@b29]. Here we present comprehensive observational evidence of reconnection between a set of chromospheric fibrils and the threads of an erupting filament, which gave rise to a small flare. We observe the in- and outflows and hot cusp-shaped structures at the ends of a small-scale reconnecting current sheet, as well as newly formed loops that demonstrate the change of magnetic connectivity. We estimate that the reconnection is fast. The intriguing rotational motion of the erupted filament is found to show the untwisting of a flux rope, enabled by the reconnection with the chromospheric fibrils, which extend to essentially current-free magnetic flux in the corona. Our detailed study is made possible using H-alpha images from the New Vacuum Solar Telescope (NVST)[@b30] and EUV images from the Solar Dynamics Observatory (SDO)[@b31] (see the Methods section for details). Hot coronal plasma is also imaged using the X-Ray Telescope (XRT) on board Hinode[@b32] and the Soft X-ray Imager (SXI) on board the Geostationary Operational Environmental Satellite (GOES)[@b33]. Photospheric vector magnetograms taken by the Helioseismic and Magnetic Imager (HMI)[@b34] on board SDO allow us to obtain the 3D field structure of the reconnection region, independently demonstrating the change of topology. The dynamics of this source region model are studied in a data-constrained magnetohydrodynamic (MHD) simulation, which reproduces the observed features of the eruption and confirms that the untwisting of the erupted flux is enabled by reconnection with ambient current-free flux. Results ======= Confined partial eruption of the filament ----------------------------------------- On 3 October 2014, a filament is observed in active region (AR) 12178 (as designated by the National Oceanic and Atmospheric Administration \[NOAA\]) near the disk centre. The filament is composed of a western and an eastern section, which join smoothly at a bend point near solar (*x*,*y*)=(−70,−185). Many thin threads extending along the filament spine make up its fine structure (see the red arrows in [Fig. 1a](#f1){ref-type="fig"}). The threads in the western section show indications of twist of approximately one turn, consistent with the widely but not universally adopted assumption that the magnetic structure of filaments is that of a weakly twisted magnetic flux rope[@b35][@b36][@b37]. [Figure 1b](#f1){ref-type="fig"} shows the position of the filament in the magnetogram. Positive photospheric polarity is given at the western footpoint and southern side of the filament, and negative polarity is given at the eastern footpoint and northern side, so the filament has sinistral chirality[@b38]. This corresponds to positive (right handed) magnetic helicity, because the axial current of the filament must also point eastward for a force-free equilibrium to exist in the given ambient flux distribution. [Figure 1c--h](#f1){ref-type="fig"} and the related [Supplementary Movie 1](#S1){ref-type="supplementary-material"} show the eruption of the filament in H-alpha images taken by the NVST. The eruption commences near the bend point at ∼07:26 UT, as a motion of the dark threads with a southwestward projected direction, which later turns more southward. Associated brightenings, indicating heating due to reconnection, appear soon but only after the onset of the motion. The eruption comprises the whole western filament section and part of the eastern section. Some of the threads in the western section follow the main eruption with a small delay. The threads in the eastern section\'s part adjacent to the bend point (−100≲*x*≲−70) connect smoothly to the threads in the western section and move jointly with them (see the blue-dotted lines in [Fig. 1c,d](#f1){ref-type="fig"}). Subsequently, other threads become visible in their place; these, as well as all other threads in the eastern filament section, remain stable (see the red arrows in [Fig. 1f--h](#f1){ref-type="fig"}). We conclude that part of the magnetic flux in the filament experiences an instability, which causes the motion and subsequently triggers reconnection. The unstable flux comprises the whole western section and part of the eastern section, where unstable flux lies on top of the stable flux. The unstable flux extends into the eastern section at least up to approximately *x*=−100 (the apparent end region of the erupting threads), but possibly up to approximately *x*=−120 (the extension of the triggered brightenings); the stable flux extends from the bend point to the edge of the active region. Because the indicated twist of approximately one turn lies below the threshold of the helical kink instability[@b39][@b40] and the erupted filament does not build up a clear helical shape as a whole (see the blue-dotted line in [Fig. 1c--h](#f1){ref-type="fig"}), the torus instability[@b41] is the primary candidate mechanism for the onset of eruption. All southward motions end by 07:50 UT, and all material is subsequently seen to slide down towards the western end point of the filament in this confined eruption, which does not produce a coronal mass ejection. Obviously, the southward end region of the erupted filament threads is the highest part of the visible erupted structure. The data do not definitely reveal whether the erupted flux is still magnetically rooted in the eastern filament section or if it now connects to other negative flux in the photosphere. However, the erupted threads point approximately towards the neighbouring AR 12179 in the southeast, away from the direction of the eastern filament section, but similar to pre-existing interconnecting loops between the two ARs. This is suggestive of reconnection between the erupting filament and these loops. After ∼09:30 UT, a new filament forms along the original western filament section, initially separate from the surviving filament in the eastern section, but 2 h later beginning to join it. The eruption gives rise to two signatures of magnetic reconnection. The two prominent brightenings that develop at the sides of the eastern filament section ([Fig. 1c--h](#f1){ref-type="fig"}) represent an indirect signature, which agrees perfectly with the reconnection assumed to release the energy in the so-called standard flare model[@b1][@b2][@b3][@b4][@b5]. Ambient flux passing over the filament is lifted; subsequently, its legs approach each other and reconnect in a vertical current sheet that is known to form under the erupting flux, but not imaged in the present event. The released energy is channelled downward along the field lines, producing the chromospheric brightenings where the reconnecting ambient flux is rooted. This can likewise be seen under the western filament section ([Fig. 1d,e](#f1){ref-type="fig"}), but it is much weaker there, in agreement with the much smaller amount of ambient flux in this area ([Fig. 1b](#f1){ref-type="fig"}). After ∼07:38 UT, a second, direct signature of reconnection is revealed. Chromospheric fibrils south of the bend point reconnect with the trailing threads of the erupting western filament section on the north side of the bend point. The observations of the reconnecting current sheet are analysed in detail in the following section, where we find that it also forms under the erupting flux. However, here it is the erupting flux that reconnects with ambient flux, a process not envisioned in the standard flare model. In addition, the erupted filament displays an intriguing motion that is highly suggestive of a rotation about its main direction ([Supplementary Movie 1](#S1){ref-type="supplementary-material"}). The motion is also seen in the Atmospheric Imaging Assembly (AIA)[@b42] data ([Supplementary Movie 2](#S1){ref-type="supplementary-material"}). At least one full turn is indicated. The motion of the better visible threads on the upper side is also shown in a time-slice plot (white-dotted lines in [Fig. 1i](#f1){ref-type="fig"}). When looking along the filament toward the western footpoint, the rotation is clockwise. Erupted flux is generally assumed to have the structure of a flux rope[@b5][@b6][@b7][@b8]. The positive helicity inferred above implies that the twist of the rope is right handed. The clockwise rotation thus represents an untwisting that is equivalent to the relaxation of magnetic tension and supports the conjecture of a flux rope structure for the erupted filament. Erupting flux ropes show an apparent untwisting simply as the result of their expansion. While the total number of field line turns is preserved (in the absence of reconnection), the twist per unit length decreases. The stretching can appear as a propagation of twist if only a part of the rope displays a twist pattern. Although the threads of the erupting filament do not display a pronounced twist pattern in images ([Fig. 1c--h](#f1){ref-type="fig"}), the animation in [Supplementary Movie 1](#S1){ref-type="supplementary-material"} is consistent with this effect in the southward expanding end region of the threads. The stretching can also mimic a rotation in a time-slice plot, as in [Figure 1i](#f1){ref-type="fig"}. However, it cannot explain two effects visible in the present event: many threads shift nearly completely to the other side of the rope (cyan lines in [Fig. 1g,h](#f1){ref-type="fig"}) and their southern end points appear to rotate about the rope axis. True untwisting results from the conversion of twist into writhe of the flux rope axis and from reconnection with less twisted flux. The first effect is negligible here because a clear helical shape does not develop ([Fig. 1c--h](#f1){ref-type="fig"}). Following reconnection with less twisted flux, the twist tends to equilibrate along the new structure. Again, the total number of turns present after the reconnection is preserved, but the equilibration occurs as a result of a true propagation of twist from the more twisted to the less twisted part (as a torsional Alfvén wave packet) and involves a true rotation about the axis of the new structure. Hence, the observed rotational motion indicates that the erupted filament flux reconnects with ambient flux, which usually has no twist. The H-alpha observations reveal only a small part of that flux (the chromospheric fibrils south of the bend point of the filament channel), but a consistent whole picture is provided below by our models for the coronal field of the AR and by the evolution of one of these models in an MHD simulation. Imaging small-scale magnetic reconnection ----------------------------------------- The reconnection process and formation of the associated current sheet are shown in [Fig. 2](#f2){ref-type="fig"}. Their full evolution can be better seen in [Supplementary Movie 1](#S1){ref-type="supplementary-material"}. [Figure 2a](#f2){ref-type="fig"} shows the positions of the original structures in an H-alpha image overlaid by a line-of-sight magnetogram. The reconnection occurs between two sets of magnetic loops with opposite directions (see the arrows in [Fig. 2a](#f2){ref-type="fig"}). One set of magnetic loops, that is, the filament threads whose eruption is delayed, is indicated by the red arrow before the reconnection. The other set, that is, the chromospheric fibrils, is indicated by the white arrow. The reconnection occurs in a small region (marked by the black rectangle in [Fig. 2b](#f2){ref-type="fig"}). In [Fig. 2c](#f2){ref-type="fig"}, two typical loops (red- and white-dotted lines with red and white arrows, respectively) are indicated just before their reconnection. They further approach each other, and their interaction results in a reconnection at ∼ 07:38 UT. During the reconnection process, two cusp-shaped structures are formed (blue-dotted lines in [Fig. 2d](#f2){ref-type="fig"}). Simultaneously, new loops appear on the other side of the cusp structure northeast of the reconnection region (the black arrows in [Fig. 2d,e](#f2){ref-type="fig"}). Subsequently, these loops move away from the cusp. With the two sets of outer loops continuing to move towards each other, the reconnection continues from ∼07:38 to ∼07:50 UT. After the reconnection ceases, the newly formed loops can be seen more obviously (marked by black arrows in [Fig. 2d--h](#f2){ref-type="fig"}). They have accumulated in the northeast reconnection outflow region. The new loops, which form early in the southwest of the reconnection region (at the right cusp), follow the filament eruption ([Supplementary Movie 1](#S1){ref-type="supplementary-material"}), while those that form there later do not rise further and can be clearly seen near the end of the event (indicated by the yellow arrow in [Fig. 2h](#f2){ref-type="fig"}). During the eruptive process the reconnection region is stretched, apparently to form a current sheet, which is visible in H-alpha as a bright linear structure extending between the tips of the two cusps ([Fig. 2f](#f2){ref-type="fig"}). The average length and width of the current sheet are estimated to be 4.3 × 10^3^ and 1.06 × 10^3^ km, respectively. At the same time, we find that the plasma at the tip of the northeast cusp structure is significantly heated, causing the brightening ([Fig. 2g](#f2){ref-type="fig"}). The cusp and current sheet can be seen in multiple channels ([Fig. 3a--d](#f3){ref-type="fig"}; [Supplementary Movie 2](#S1){ref-type="supplementary-material"}). The northeastern bright cusp appears very clearly in all EUV channels of AIA (*T*≈0.02−10 MK). The southwestern cusp structure is weaker, as is to be expected from lower plasma densities at the upper end of a current sheet that is formed and stretched out upward by the eruption. This also shows up only intermittently owing to absorption by the moving threads of the erupted filament, which demonstrates that the current sheet forms under the erupting flux. The current sheet appears as a bright linear structure in all AIA channels, most clearly at the highest temperatures (131 Å; *T*=11 MK). The hot cusp structure can also be clearly seen in the XRT and SXI images during the reconnection process ([Fig. 3e,f](#f3){ref-type="fig"}). The unusual simultaneous visibility of the current sheet and cusp in H-alpha and EUV emissions ([Figs 2f](#f2){ref-type="fig"} and [3a--d](#f3){ref-type="fig"}) is probably due to the fact that cool and dense threads and fibrils are embedded in the reconnection inflow. The major brightening of the current sheet and cusp in H-alpha immediately follows the inflow and fading of a major filament thread (prominent in the current sheet in [Fig. 2c--e](#f2){ref-type="fig"}), suggesting heating of the thread by the reconnection. The emission measure (EM) maps at the different temperature bins ([Fig. 3g--i](#f3){ref-type="fig"}) show that the northeastern, newly formed loops are dominated by EM at temperatures of 4--8 MK, several times higher than the unperturbed corona. On the other hand, the tips of the two cusp structures (circles A and B) and the current sheet between them are most prominent at even higher temperatures, that is, *T*\>10 MK ([Supplementary Movie 3](#S1){ref-type="supplementary-material"}). This is similar to earlier observations of a pair of hard X-ray sources located at the two ends of the conjectured current sheet in flares[@b20]. The EUV images at high temperatures ([Fig. 3d](#f3){ref-type="fig"}) and the EM maps ([Fig. 3i](#f3){ref-type="fig"}) also show a diffuse area of enhanced temperature in the northwest inflow region after ∼07:35 UT, which begins to cool down ∼10 min later and then transiently brightens in H-alpha ([Fig. 1h](#f1){ref-type="fig"}). The magnetic field inhibits heat conduction from the current sheet into the inflow region. Guided by the MHD simulation, which shows the formation of additional weak current layers north of the erupting filament, we interpret these enhancements as a signature of additional energy release in the complex ambient coronal field when it is perturbed by the eruption, followed by downward heat conduction. To quantitatively investigate the motions of the plasma in the reconnection region, we construct the three time-slice plots in [Fig. 4a--c](#f4){ref-type="fig"}, which yield projected velocities along the lines marked in [Fig. 2](#f2){ref-type="fig"}. [Figure 4a](#f4){ref-type="fig"} shows the inward motions of loops, that is, the reconnection inflow. The apparent velocities of these loops are in the range of 3.7--25.0 km s^−1^ for the various filament threads on the northwest side and 6.3--16.8 km s^−1^ for the various fibrils on the southeast side. [Figure 4b](#f4){ref-type="fig"} displays outward motions of bright (hot) plasma, that is, the reconnection outflow, seen immediately downstream of the northeastern cusp. The outflow velocities lie in the range of 41.7--43.7 km s^−1^, with an average value of 42.7 km s^−1^. At the same time, two newly formed loops containing cooled plasma, dark in H-alpha (the white-dotted lines), can be seen in [Fig. 4b](#f4){ref-type="fig"}. These shrink slowly, with an average velocity of 2.6 km s^−1^. In addition, a mixture of bright and dark plasma moves down the legs of the cusp structure, that is, in the interface between the reconnection inflow and outflow ([Fig. 4c](#f4){ref-type="fig"}). This apparent mix of inflowing and outflowing plasma, as well as the intermittently irregular structure of the cusp legs ([Supplementary Movie 1](#S1){ref-type="supplementary-material"}), whose observation is made possible by the high resolution of the NVST, provide support for the recent numerical finding of turbulence in reconnection developing at the separatrices[@b43]. The downflows at the cusps show velocities in the range of 17.6--80.2 km s^−1^ with a decreasing trend. The brightenings at the end of the current sheet were observed in multiple channels. To investigate their evolution, [Fig. 4d](#f4){ref-type="fig"} displays the intensities in the H-alpha, 304, 171 and 335 Å channels, integrated over the area of the circle in [Fig. 2g](#f2){ref-type="fig"}. The early brightenings are caused by the eruption of the filament. The onset of reconnection, as marked by the rise of the EUV emissions at 07:38 UT, coincides with the arrival of the first inflow trace at the forming current sheet ([Fig. 4a](#f4){ref-type="fig"}) and with the onset of outflows from the current sheet ([Fig. 4b,c](#f4){ref-type="fig"}). The bright structures and the outflows show some degree of intermittency, which is a characteristic of plasmoid-mediated reconnection in very long current sheets, but here is probably caused mainly by the inhomogeneity of the inflowing plasma. All tracers of the reconnection process decay after ∼07:45 UT, by which time the length of the current sheet has shortened considerably because of the accumulation of new loops in the downward (northeast) outflow region and the approximate stationarity of the upper tip of the sheet under the erupted but no longer rising filament. 3D NLFFF configuration of the reconnection region ------------------------------------------------- To investigate the 3D magnetic field structure of the reconnection region, we carry out extrapolations of the HMI vector magnetograms at 07:00 and 08:24 UT based on the nonlinear force-free field (NLFFF) assumption for the coronal field ([Fig. 5](#f5){ref-type="fig"}). In the eastern section of the filament, an incoherent flux rope structure is obtained, similar to several recent extrapolation results for active regions before an eruption[@b44][@b45]. In the western section, the strong axial flux expected in a filament is found, but the poloidal (twist) field component is largely missing. We expect that the latter results from the low signal-to-noise ratio in the extended area of the weak field around the western filament section ([Fig. 1b](#f1){ref-type="fig"}). The overall shape of the filament is nevertheless well matched. The extrapolation also correctly indicates that the field in the area of the western filament section has a similar structure sometime after the eruption, providing the prerequisite for the observed formation of a new filament in the western section. These findings yield confidence in the large-scale structure of the extrapolated NLFFF. The extrapolations demonstrate the change of magnetic connections in agreement with the observed change. They further show that the ambient field rooted in the southeast reconnection inflow region (grey field lines in [Fig. 5a](#f5){ref-type="fig"}) first follows the low-lying reconnecting fibrils seen in H-alpha in the westward direction, but then bends strongly upward, forming high-reaching connections to the neighbouring AR 12179 (see [Fig. 6](#f6){ref-type="fig"}). These field lines exchange footpoints, that is, reconnect, with a set of lower field lines running in the northwest inflow region and the western section of the filament channel (also coloured grey). The resulting low-lying loop ([Fig. 5b](#f5){ref-type="fig"}) corresponds very well to the newly formed H-alpha loops downstream of the observed cusp ([Fig. 2d--g](#f2){ref-type="fig"}). The other resulting set of field lines indicates that flux in the erupting western part of the filament reconnects with the high-reaching field lines that extend to the neighbouring active region. This corresponds very well to the southward bending of the erupting flux (blue-dotted lines in [Fig. 1c--h](#f1){ref-type="fig"}) and supports our conjecture above that the strong untwisting motion is realized by a torsional Alfvén wave packet propagating to ambient, essentially untwisted flux. MHD simulation -------------- To further substantiate the occurrence of reconnection triggered by the erupting filament, we model the event in an MHD simulation whose initial and boundary conditions are constrained by the HMI data. Because the extrapolation largely fails to reproduce the twist in the western part of the filament, we construct a new NLFFF model of the AR from the pre-eruption magnetogram using the Flux Rope Insertion method, as detailed in the Methods section. On the basis of the observation that the western filament section erupts fully and the eastern section only partly, two flux ropes are inserted along the path of the filament. A low-lying rope is inserted along the eastern section. A higher-lying rope models the western section and extends into the eastern section as far as the moving threads at the onset of the event ([Fig. 6a](#f6){ref-type="fig"}). Numerical relaxation partly merges them into a smooth configuration, which is essentially a flux rope split into two branches in the eastern part. From models for a range of values of the inserted fluxes, we select the one that yields an unstable western section, with approximately one field line turn, and a stable eastern section to serve as the initial condition of the MHD simulation ([Figs 6b](#f6){ref-type="fig"} and [7a--d](#f7){ref-type="fig"}). The model also includes the high-reaching field lines anchored in positive flux southeast of the filament (grey in [Fig. 5a](#f5){ref-type="fig"} and left set of open field lines in [Fig. 7c](#f7){ref-type="fig"}), the bottom part of which represents the reconnecting chromospheric fibrils (white arrows in [Fig. 2a,c,e](#f2){ref-type="fig"}). [Figure 6b](#f6){ref-type="fig"} shows that these field lines connect to the neighbouring AR 12179. Similarly, the low-lying field lines north of the filament bend point (grey in [Fig. 5a](#f5){ref-type="fig"}) are here also seen to be part of the flux in the western filament section (right set of weakly twisted field lines in [Fig. 7c](#f7){ref-type="fig"}). This flux models the trailing threads of the erupting western filament section (red arrows in [Fig. 2a,c,e](#f2){ref-type="fig"}), which reconnect with the chromospheric fibrils. A wide range of agreement with the observations is obtained. The simulation shows a confined eruption in the southern direction, comprising the full western and part of the eastern filament section ([Fig. 7](#f7){ref-type="fig"}; [Supplementary Movie 4](#S1){ref-type="supplementary-material"}). Magnetic reconnection is triggered very similarly to that shown by the NVST and SDO data. A current sheet is dragged upward into the corona between the rising and remaining flux along the filament section eastward of the original bend point ([Fig. 7](#f7){ref-type="fig"}, fourth column). The ambient flux on the southeast side of the filament moves towards the sheet to reconnect at the observed position with approaching flux from the western section of the flux rope ([Fig. 7](#f7){ref-type="fig"}, third column). As shown above, while being in agreement with the NVST observations, this differs partly from the standard view of reconnection in solar eruptions, which supposes that only ambient flux reconnects under a rising flux rope[@b5][@b6][@b7][@b8][@b46]. The reconnection forms small loops that accumulate under the current sheet at the position of the newly formed H-alpha loops ([Figs 2d--g](#f2){ref-type="fig"}, [5b](#f5){ref-type="fig"} and [7o](#f7){ref-type="fig"}). The other parts of the reconnected field lines yield new, high-reaching magnetic connections from the western footpoint of the erupted flux to AR 12179 ([Fig. 7](#f7){ref-type="fig"}, left two columns; compare with the evolution of the blue-dotted line in [Fig. 1c--h](#f1){ref-type="fig"}). The untwisting of the erupted flux via propagation of twist along these new connections is very clear. These results are similar to the indications from the dynamics of the erupted filament in the H-alpha data (the draining of filament material in the westward direction along the whole length of the erupted filament and the indication of a rotational motion). They are also in agreement with the new connections in the extrapolated field ([Fig. 5b](#f5){ref-type="fig"}). The accumulation of reconnected flux causes a gradual rise of the cusp that forms at the bottom tip of the current sheet ([Fig. 7h,l,p](#f7){ref-type="fig"}). As the rise of the erupted flux is stalled, but the bottom cusp continues to rise as a result of the reconnection, the current sheet weakens and eventually decays. The simulation shows further reconnection in a current sheet forming in the western filament section in the interface between rising and overlying flux. This rebuilds connections from the positive footpoint area of the flux rope to the strong negative photospheric flux patches adjacent to the eastern filament section ([Fig. 7i,m](#f7){ref-type="fig"}). The current sheet is not imaged by NVST and SDO because it is not aligned with the line of sight; however, the resulting connections match the filament that reforms in the western section after 09:30 UT. Discussion ========== In this paper, we study in detail a small-scale magnetic reconnection event triggered by a filament eruption and obtain a comprehensive set of solid observational evidence for the occurrence of reconnection which is unprecedented. This includes the reconnection inflows at both sides of the current sheet at projected velocities of 3.7--25.0 km s^−1^; the reconnection outflow at one side of the current sheet, moving downward at projected velocities of 41.7--43.7 km s^−1^; the formation of a long, thin structure suggestive of a current sheet and of two cusp-shaped structures at its end points, both occurring simultaneously with the onset of reconnection; newly formed loops in both outflow regions, demonstrating the change of magnetic connectivity, those on the downward side shrinking with an average projected velocity of 2.6 km s^−1^; and fast downward motion at projected velocities of 17.6--80.2 km s^−1^ of cool and hot plasma at the cusp-shaped boundary between inflow and outflow. Moreover, the newly formed loops, current sheet and cusps show enhanced emission measure in a broad temperature range, with the loops being dominated by plasma at 4--8 MK and the current sheet and cusps by plasma at *T*\>10 MK. The length, *L*, and width, *d*, of the current sheet are estimated to be 4.3 × 10^3^ and 1.06 × 10^3^ km, respectively. The reconnection rate is an important physical parameter in the theory of reconnection and can be estimated from our observations. It can be expressed as the Alfvénic Mach number, *M*~A~, of the inflow velocity because the reconnection outflow generally approaches the Alfvén velocity, *V*~A~ (ref. [@b47]). Assuming that the observed outflow velocity is of the order of *V*~A~ and neglecting projection effects, *M*~A~≈*V*~in~/*V*~out~, which lies in the range of ∼0.08−0.60 for our measurements. In a steady-state reconnection, the reconnection rate also equals the inverse aspect ratio of the current sheet[@b47], which is obtained as *d*/*L*≈0.25, consistent with the estimated range for *M*~A~. Both indicate that the reconnection is fast. Our estimate tends to be relatively high, but is consistent with the range of previous estimates for solar events, for example, 0.001--0.03 (ref. [@b11]), 0.01--0.23 (ref. [@b48]), 0.055--0.20 (ref. [@b12]), 0.16 (ref. [@b17]) and 0.05--0.5 (ref. [@b13]). Because the measured reconnection outflow in our event hits the accumulating newly formed loops after only a short distance ([Fig. 2](#f2){ref-type="fig"}), it may not have reached the Alfvén velocity. Furthermore, the current sheet width obtained from the images, although resolved by the NVST, is considered an upper limit for the actual width of the field reversal, which tends to occur at microscopic scales[@b47]. Therefore, the estimated reconnection rate is also considered an upper limit. Finally, the confinement of the eruption may be due to a strong restraining force of the overlying flux or due to the cancelation of the upward Lorentz force in the filament by its reconnection with ambient flux. The former effect is equivalent to saturation of the torus instability in an ambient field that decreases too slowly with height, which can be quantified by the decay index, *n*(*z*)=−*d* log* B*~p,\ hor~(*z*)/*d *log *z*, where *B*~p~,~ hor~(*z*) is the horizontal component of the potential field at the *x*--*y* position of interest[@b41]. The decay index profile at the observed position of the current sheet indicates instability (*n*\>3/2) in a small interval around *z*=0.03*R*~⊙~, where the upper flux rope is located in our NLFFF model, and at *z*\>0.13*R*~⊙~, with the intermediate height range being stable. In this stable range, the flux rope reconnects in our MHD simulation. Thus, both potential reasons for confinement may be relevant in the event. Methods ======= Differential emission measure calculation ----------------------------------------- We calculate the differential emission measure (DEM) using the almost simultaneous observations of six AIA EUV lines (131, 94, 335, 211, 193 and 171 Å) formed at coronal temperatures. The DEM is determined by where *I*~*i*~ is the observed intensity of the waveband *i*, *R*~*i*~(*T*) represents the temperature response function of waveband *i*, and DEM(*T*) is the DEM of coronal plasma, which is computed using the routine xrt_dem_iterative2.pro in the Solar Software package. This code was first written for the Hinode/X-ray telescope data[@b49][@b50], and then modified for the SDO/AIA data[@b51]. In this work, log *T* is set in the range of 5.5--7.5, where the DEM is generally well constrained[@b52][@b53]. To obtain the emission measure in the temperature range (*T*~min~,*T*~max~), we evaluate NLFFF extrapolation ------------------- The 3D NLFFF structures of the reconnection region and associated filament ([Fig. 5](#f5){ref-type="fig"}) are reconstructed using an optimization algorithm[@b54][@b55]. A preprocessing procedure is first used to deal with the bottom vector data to remove most of the net force and torque that usually results in an inconsistency between the photospheric magnetic field and the force-free assumption in the NLFFF models[@b56]. The visualization of 3D magnetic field is realized by the software Paraview. The field lines shown are traced from nearly identical points in the photosphere. Flux rope insertion method and MHD simulation --------------------------------------------- The magnetic field model used as the initial condition of the MHD simulation is constructed through Flux Rope Insertion. This method involves computing the potential field in the volume of interest by extrapolation, inserting a magnetic flux rope along the observed path of the filament and numerically relaxing the configuration to an NLFFF[@b36][@b57][@b58]. The active region of interest ([Fig. 6a](#f6){ref-type="fig"}) is modelled with high spatial resolution (0.002*R*~⊙~), and the more distant regions are modelled with a lower-resolution (1°) global potential field. The high-resolution region is derived from the line-of-sight photospheric magnetogram taken by SDO/HMI on 3 October 2014 at 07:26 UT, and the global field is constructed based on the HMI synoptic map. The high-resolution computational domain extends about 25° in longitude, 15° in latitude and up to 1.75 *R*~⊙~ from Sun centre. Two flux ropes are inserted along the path of the filament (blue lines) and anchored in the photosphere (at the blue circles), with path 1 chosen to run higher than path 2. The axial and poloidal fluxes along paths 1 and 2 are 4 × 10^20^ Mx and 10^11^ Mx cm^−1^, and 10^20^ Mx and 10^10^ Mx cm^−1^, respectively, giving the western section higher free magnetic energy and twist. The numerical relaxation partly merges the flux ropes into a smooth configuration and changes the end positions slightly ([Fig. 6b](#f6){ref-type="fig"}). All flux connecting to photospheric sources outside AR 12178 corresponds well to the large-scale coronal loops imaged in the AIA 171-Å channel (which are not shown here, but can be accessed at the public source <http://helioviewer.org/>). The MHD simulation code[@b59] is used with numerical settings analogous to an earlier data-constrained simulation of a solar eruption[@b60]. In particular, the velocity in the bottom plane is set to zero, to model the inertia of the photosphere. The vertical component of the HMI magnetogram thus remains invariant. Software availability --------------------- The Solar Software package is available at <http://www.lmsal.com/solarsoft/>. Data availability ----------------- The NVST is a ground-based telescope with a 986-mm clear aperture in the Fuxian Solar Observatory of the Yunnan Observatories, Chinese Academy of Sciences. It is designed to observe the fine structures on the Sun and their activities in multiple layers (photosphere and chromosphere) with high spatial resolution (0.165 arcsec per pixel) and high temporal resolution (∼12 s). We use data from the H-alpha line-centre channel at 6,562.8 Å, corresponding to chromospheric temperatures. These data can be accessed at <http://fso.ynao.ac.cn/dataarchive_ql.aspx>. EUV and far-ultraviolet images obtained by the AIA on board SDO, including the 304, 171, 193, 335, 211, 94, 131 and 1,600 Å channels, display magnetic reconnection and the associated current sheet at higher temperature, that is, primarily in the corona. Photospheric magnetograms are provided by the HMI Instrument on SDO. The SDO data are publicly available at <http://jsoc.stanford.edu/ajax/lookdata.html> and at [http://helioviewer.org/](http://helioviewer.org/.%20Soft%20X-ray%20images%20of%20the%20Sun%20by%20Hinode/XRT%20and%20GOES/SXI). Soft X-ray images of the Sun by Hinode/XRT and GOES/SXI are available at <http://darts.isas.jaxa.jp/solar/hinode/query.php?A01=Go%20to%20Search/> and <http://sxi.ngdc.noaa.gov/sxi/servlet/sxisearch/>, respectively. Additional information ====================== **How to cite this article:** Xue, Z. *et al.* Observing the release of twist by magnetic reconnection in a solar filament eruption. *Nat. Commun.* 7:11837 doi: 10.1038/ncomms11837 (2016). Supplementary Material {#S1} ====================== ###### Supplementary Movie 1 The NVST H-alpha movie. (a) The general appearances of the filament and its eruption in which the red rectangle indicates the field of view (FOV) of panel b. (b) The process of magnetic reconnection. ###### Supplementary Movie 2 The EUV channel movie observed by SDO/AIA. (a-h) 304, 1600, 171, 193, 211, 335, 94, and 131 Å channel movies respectively. ###### Supplementary Movie 3 The EM map movie. In each panel, the EM maps at the different temperatures are shown. ###### Supplementary Movie 4 MHD simulation of the filament eruption. Field lines of the two flux ropes representing the unstable (rainbow-colored) and stable (green) filament sections are shown in vertical and perspective views (snapshots of which are shown in the left two columns in Fig. 6. The HMI line-of-sight magnetogram is included in the bottom plane. We thank the NVST, SDO, GOES/SXI, Hinode and BBSO teams for providing the data. SDO is a mission of NASA\'s Living With a Star Program. Hinode is a Japanese mission developed and launched by ISAS/JAXA, with NAOJ as a domestic partner, and NASA and STFC (UK) as international partners. It is operated by these agencies in co-operation with ESA and NSC (Norway). This work is sponsored by the National Science Foundation of China under the grant numbers 11503080, 11373066, 11573012, 11373065, 11303016, 11533008 and 11473071, Key Laboratory of Solar Activity of Chinese Academy of Sciences (CAS) under numbers KLSA201412, KLSA201407, Yunnan Science Foundation of China under number 2013FB086, CAS 'Light of West China\' Program, Youth Innovation Promotion Association CAS (no. 2011056), and the national basic research program of China (973 program, 2011CB811400). B.K. is supported by the CAS under grant no. 2012T1J0017 and by the DFG. Y.N.S is supported by the One Hundred Talent Program of Chinese Academy of Sciences. **Author contributions** Z.K.X. developed the ideas, performed analysis of the data, discussed the results and wrote the first manuscript. X.L.Y. developed the ideas and lead the discussion of the manuscript. X.C. and K.Y. carried out the NLFFF extrapolation, DEM analysis and discussion. L.H.Y. carried out the analysis of the XRT and GOES data, and contributed to the discussion. Y.N.S. performed NLFFF modelling of the AR to provide the initial condition for the MHD simulations carried out by B.K.; Y.N.S. and B.K. also contributed to the discussion. B.K. and X.L.Y made major revisions of the manuscript. J.Z. developed ideas and contributed to the discussion. Z.L., Y.B., Y.Y.X and L.Z. contributed to the original observational data. All authors reviewed the manuscript. ![Structure and eruption of the filament.\ (**a**) NVST H-alpha image showing the general appearance of the filament (indicated by the red arrows) at the onset of the eruption. (**b**) HMI line-of-sight magnetogram of AR 12178, with positive (negative) photospheric flux shown in white (black) and the red line representing the position of the filament. (**c**--**h**) The process of filament eruption in H-alpha images. The original position is marked by the red-dotted line. The erupting part is indicated by the blue-dotted lines and blue arrows. Cyan-dotted lines in **f**--**h** indicate some of the filament threads that rise with a delay. The stable eastern filament section is marked by the red arrows in **f**--**h**. (**i**) Time-slice plot acquired at the position A--B, with the white-dotted lines marking the filament threads. The motion of these threads indicates the untwisting of the filament during its eruption.](ncomms11837-f1){#f1} ![Evolution of reconnection.\ (**a**,**h**) NVST H-alpha images in the early and late phases of the reconnection event, overlaid by red (blue) contours representing positive (negative) photospheric polarity from near-simultaneous HMI data. (**b**--**g**) NVST H-alpha images of the reconnection process in which the red- and white-dotted lines indicate the magnetic loops before the reconnection (with red arrows marking the filament threads and white arrows marking the chromospheric fibrils), the blue-dotted lines mark the cusp-shaped structures, and the black (yellow), cyan and pink arrows point to newly formed loops, current sheet and brightening, respectively. The three time-slice plots shown in [Fig. 4](#f4){ref-type="fig"} are made along the white solid line C--D, the blue solid line E--F and the black solid line G--H, respectively.](ncomms11837-f2){#f2} ![EUV/X-ray images and EM maps.\ (**a**--**d**) AIA EUV images at 304, 171, 335 and 131 Å channels, in which the cusp-shaped structures are marked by the dotted lines and the current sheet is indicated by the white arrows. (**e**,**f**) Cusp structure seen in X-ray images observed by Hinode/XRT and GOES/SXI, respectively. (**g**--**i**) EM maps in three temperature ranges showing the dominant temperatures of the inflow region, current sheet, cusps (encircled and marked 'A\' and 'B\' in **i**), and reconnected loops.](ncomms11837-f3){#f3} ![Time-slice plots and EUV brightening.\ (**a**--**c**) H-alpha time-slice plots made by stacking the slits along the slices C--D, E--F and G--H in [Fig. 2](#f2){ref-type="fig"}, respectively. The red-, blue-, white- and pink-dotted lines represent reconnection inflows, outflows, shrinking loops, and downward motions, respectively. (**d**) Temporal evolution of the brightenings in the region marked by the black circle in [Fig. 2g](#f2){ref-type="fig"}, using H-alpha, 304, 171 and 335 Å images, where each light curve is normalized by its maximum. The vertical black-dotted line in each panel indicates the onset of the observed fast reconnection.](ncomms11837-f4){#f4} ![3D configuration of the reconnection region from NLFFF extrapolation.\ (**a**,**b**) 3D NLFFF configuration before (07:00 UT on 3 October 2014) and after (08:24 UT on 3 October 2014) the reconnection. The background images indicate the radial component of the vector magnetic field in the photosphere. The grey lines represent the magnetic loops involved in the reconnection. The pink lines show the magnetic structures of the filament.](ncomms11837-f5){#f5} ![Magnetic field model constructed using the Flux Rope Insertion method.\ (**a**) Line-of-sight magnetogram observed by SDO/HMI at 07:26 UT on 3 October 2014. The two blue curves with a circle at each end show the paths of the inserted flux ropes. (**b**) H-alpha image taken by Big Bear Solar Observatory at 23:00 UT on 2 October 2014, overlaid with red (positive) and green (negative) contours representing the magnetic field shown in **a**. The colour lines show selected field lines from the magnetic field model, and the horizontal size of the MHD computation box (height of 0.75*R*~⊙~ and fully within the high-resolution region) is indicated by the black square.](ncomms11837-f6){#f6} ![MHD simulation of the confined filament eruption and magnetic reconnection.\ The left two columns show field lines of the fully erupting western flux rope section (rainbow colours) and the largely stable eastern flux rope section (green) and the line-of-sight magnetogram in a cube of 0.25*R~⊙~* per side. The inset at *t*=0 indicates the area shown in the third column, and the yellow line shows the position of the vertical cut plane in the fourth column. The third column shows selected field lines in the area of the observed reconnection in a cube of 0.06*R*~⊙~ per side. Field lines rooted in positive photospheric flux (white) initially mostly extend to AR 12179 eastward of AR 12178 ([Fig. 6b](#f6){ref-type="fig"}), while field lines rooted in negative flux (black) initially mostly join the unstable western flux rope section. The fourth column displays the current density and in-plane velocity vectors in a vertical cut through the reconnecting current sheet (0.06*R*~⊙~ per side). At *t*=0, the inserted, unstable flux rope is seen, with velocities set to zero. Times are given in Alfvén time, which corresponds to ∼3 min when scaled to the observations.](ncomms11837-f7){#f7}

{ "pile_set_name": "PubMed Central" }

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]

INTRODUCTION ============ LPA (lysophosphatidic acid) is a potentially bioactive phospholipid that mediates a number of physiological processes, including cell adhesion, proliferation, differentiation, survival and migration. GPCRs (G-protein-coupled receptors) in the EDG (endothelial differentiation gene) and P2Y families are specific receptors for LPA in the plasma membrane, and intracellular signalling via these GPCRs has been well characterized \[[@B1]\]. However, less is known about the physiological regulation of LPA production or how LPA-specific receptors in the plasma membrane are stimulated *in vivo*. At least two plausible enzymatic pathways for the production of LPA have been described. One of these is the deacylation of PA (phosphatidic acid) by activated PLA (phospholipase A) \[[@B2]\]. In support of this idea, a PA-selective PLA~1~ (phospholipase A~1~) called mPA-PLA~1~α/LIPH (membrane-associated phosphatidic acid-selective phospholipase A1α/lipase member H) has been identified that hydrolyses acyl residues at the sn−1 position of PA and enhances release of LPA from the plasma membrane \[[@B3],[@B4]\]. The other pathway for LPA production is direct production of equimolar amounts of LPA and choline from LPC (lysophosphatidylcholine), as catalysed by lysoPLD (lysophospholipase D) \[[@B5],[@B6]\]. The physiological importance of LPA production in serum by lysoPLD was suggested by Tokumura et al. \[[@B7]\] approximately 25 years ago. In circulating blood, LPA modulates blood pressure \[[@B8],[@B9]\], maintenance of pregnancy \[[@B10]\] and maturation of embryonic vessel systems \[[@B11]\]. Previously, a secreted protein with lysoPLD activity was purified from plasma and serum, and was found to be identical with a molecule known as autotaxin \[[@B12],[@B13]\]. Autotaxin \[ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2)\] was originally cloned as a tumour cell motility-stimulating factor and was recognized as a nucleotide pyrophosphatase on the basis of its primary structure and *in vitro* enzymatic characteristics \[[@B14]\]. LPC, which can bind albumin and lipoproteins, is released into circulation from the liver and may be generated by lecithin/cholesterol acyltransferase. Autotaxin might hydrolyse these LPCs as substrates, and LPA accumulated to levels greater than 50 μM in plasma, presumably due to very low activity of LPA-degrading enzymes in that context \[[@B7]\]. When cells are stimulated by hormones and proliferation factors, equimolar amounts of free fatty acid and LPC are produced from PC (phosphatidylcholine) by the activated PLA~2~ (phospholipase A~2~) \[[@B15]\]. The free fatty acids that are released, including arachidonic acid, which is present at position sn−2 of PC prior to cleavage, are converted into eicosanoids. Whereas the metabolism of arachidonic acid has been well characterized, the metabolism of LPC is less well understood. Because the accumulation of LPC in cells induces cell lysis \[[@B16]\], it has been thought that LPC was easily hydrolysed by lysophospholipases we cloned in a previous study \[[@B17]\], followed by conversion into a non-bioactive saturated free fatty acid and GPC (glycerophosphorylcholine). However, LPC itself was recognized recently as a bioactive phospholipid that can induce chemotaxis \[[@B18]\], signal-responsive kinase activities \[[@B19]\] and atherosclerosis \[[@B20]\]. The first enzyme with lysoPLD activity was isolated from rat brain microsomes by Wykle and Schremmer \[[@B5]\]. The characteristics of this intracellular enzyme, such as substrate specificity, cation requirement and optimal pH, are different from those of autotaxin. We purified a novel lysoPLD from the rat brain that is a Mg^2+^-dependent enzyme and utilizes lysoPAF (1-*O*-hexadecyl-*sn*-glycero-3-phosphocholine) as a substrate \[[@B21]\], similar to the enzyme reported by Wykle and Schremmer \[[@B5]\]. This enzyme might be involved in the production of LPA from LPC, which is produced from PC hydrolysed by PLA~2~ in cells. Previously, it has been reported that LPA receptors are expressed in perinuclear areas \[[@B22],[@B23]\] and, furthermore, that the transcription factor PPARγ (peroxisome proliferation-activator receptor γ) is bound to LPA \[[@B24]\]. Based on these observations, it seems reasonable to propose that intracellular lysoPLD is important for cellular responses. In the present study, we have identified a purified protein with lysoPLD activity from the rat brain as the heterotrimeric G protein subunits Gα~q~ and Gβ~1~. Mutations predicted to affect the binding of nucleotide phosphates or Mg^2+^ to the Gα~q~ subunit resulted in a dramatic reduction of lysoPLD activity. Moreover, several types of Gα subunits, including Gα~q~ and Gα~11~, exhibit lysoPLD activity when expressed and purified as tagged proteins. EXPERIMENTAL ============ Materials --------- LPC (1-palmitoyl, 16:0; stearoyl, 18:0; and oleoyl, 18:1) was obtained from Sigma--Aldrich. LysoPAF was from Alexis Biochemicals. 1-\[^14^C\]Palmitoyl-2-lyso-phosphatidylcholine was purchased from GE Healthcare. HPPA \[3-(4-hydroxyphenyl) propionic acid\], peroxidase and choline oxidase were purchased from Wako Chemicals. Silica Gel 60 plates and tag-purified G(β~1~ and γ~2~) complex overexpressed in *Spodoptera frugiperda* insect cells were purchased from Merck, and CHAPS was purchased from Dojindo. Antibodies against Gα~q/11~, Gβ~1--4~, and Gγ~5~ were obtained from Santa Cruz Biotechnology, and anti-FLAG antibody, anti-FLAG affinity gels, FLAG peptides, anti-actin antibody and GTP were purchased from Sigma. The monoclonal anti-autotaxin antibody was obtained from Dr Junken Aoki (Graduate School of Pharmaceutical Sciences, Tohoku University, Japan). Identification of purified lysoPLD ---------------------------------- Purification of lysoPLD from rat brain tissue was performed exactly as described previously \[[@B21]\]. All steps in enzyme purification were carried out at 4°C. Briefly, rat brains were minced and homogenized with a Teflon homogenizer in 9 volumes of buffer A \[0.3 M sucrose in 50 mM Tris/HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol and 0.1 mM PMSF). Nuclear fractions were obtained by centrifugation at 600 ***g*** for 10 min and resuspended in buffer A. To dissolve lysoPLD in the nuclear pellets, 10 mM CHAPS was added and the suspension was sonicated with a Microson Ultrasonic cell disruptor (Misonix). The suspensions were then centrifuged at 10000 ***g*** for 10 min and the supernatants were used as a source of enzyme. The supernatants were applied to a DEAE Cellulofine A-500 column equilibrated with buffer B \[20 mM Tris/HCl (pH 7.5), 0.1 mM PMSF and 5 mM CHAPS), and the enzyme was eluted with a NaCl gradient. Active fractions were sequentially applied to three different types of columns. The enzyme fractions were concentrated using Microcon centrifugal filter devices (Millipore) and then applied to a Superdex 200 10/300 GL column. As a final step, the eluted active fractions were applied to a HiTrap DEAE FF column using a Waters 650 (Millipore). The final eluted fraction was then subjected to SDS/PAGE (10% gel) and the gel was stained with EZ Stain Silver (Atto). The major protein bands were excised for in-gel digestion with MS grade trypsin (Wako Chemicals). The mass spectra of extracted peptides were analysed by MALDI--TOF MS (matrix-assisted laser desorption ionization--time-of-flight MS) (Voyager Elite/STR Perspective, Life Technologies), and the proteins were subsequently identified using MS-Fit (<http://prospector.ucsf.edu>). SDS/PAGE and immunoblotting --------------------------- Proteins from rat brains and the FLAG tag affinity gel-purified fractions (10 μl) were separated on 10% acrylamide gels, and stained with a silver staining kit (Wako Chemicals) or transferred on to PVDF membranes using a semi-dry transfer apparatus (Nippon Eido) and blocked with 5% skimmed milk in TBS-T buffer \[150 mM NaCl, 20 mM Tris/HCl (pH 8.0) and 0.05% Tween 20\]. The membranes were incubated overnight at 4°C with primary antibody diluted 1:1000 (anti-Gα~q/11~, -Gβ~1--4~, -Gγ~5~, -actin or -autotaxin) or 1:5000 (anti-FLAG) in TBS-T and then for 1 h at room temperature (20°C) with a peroxide-conjugated secondary antibody diluted 1:5000 in TBS-T. Reactive bands were detected by chemiluminescence using the Bio-Rad Laboratories ChemiDoc XRS+ system. Plasmid construction -------------------- N-terminally FLAG-tagged Gα~q~ (GenBank® accession number NM_031036.1), Gα~11~ (GenBank® accession number NM_031033.1) or Gβ~1~ (GenBank® accession number NM_030987.2) cDNAs were amplified from cDNAs from a rat brain cDNA library using a forward primer containing the FLAG tag sequence, then subcloned into the pcDNA3.1 V5/His TOPO TA vector (Invitrogen). PCR was performed with the following pairs of primers: Gα~q~, 5′-GGAAGAATGGACTACAAGGACGACGATGACAAGACTCTGGAGTCCATCATGG-3′ and 5′-TCACACCAGATTGTACTCCTTCAGG-3′; Gα~11~, 5′-GCGACGATGGACTACAAGGACGACGATGACAAGACTCTGGAGTCCATGATG-3′ and 5′-TCACACCAGATTGTACTCCTTCAGG-3′; and Gβ~1~, 5′-GTGAAGATGGACTACAAGGACGACGATGACAAGAGTGAACTTGACCAGCTGC-3′ and 5′-GTTCCAGATCTTGAGGAAG-3′. The Gα~i~ (GenBank® accession number NM_010305.1) and Gα~s~ (GenBank® accession number AK168996.1) cDNAs were amplified from a mouse brain cDNA library with the primer pairs 5′-AAAGAATTCGCCACCATGGGCTGCAC-3′ and 5′-AAACTCGAGTTCGAAGAGACCACAGTCTTT-3′, and 5′-AAAGAATTCGCCGCCATGGGCTGCCTC-3′ and 5′-AAACTCGAGTTAGAGCAGCTCGTATTGGCG-3′ respectively. The amplicons were cloned into pcDNA3.1 His/V5 after digestion with EcoRI and XhoI. The FLAG tag was added via a site-directed mutagenesis approach using the following primers paired with their complimentary sequences: Gα~i~, 5′-GAATTCGCCACCATGGACTACAAGGACGACGATGACAAGGGCTGCACATTGAG-3′; and Gα~s~, 5′-GAATTCGCCGCCATGGACTACAAGGACGACGATGACAAGGGCTGCCTCGGC-3′. Mutations were introduced by site-directed mutagenesis using the following primers paired with complimentary sequences: Gα~q~ (G48V), 5′-CTGCTGCTGGGGACAGTCGAGAGTGGCAAG-3′; Gα~q~ (G48A), 5′-CTGCTGCTGGGGACAGCGGAGAGTGGCAAG-3′; Gα~q~ (K52A), 5′-GACAGGGGAGAGTGGCGCGAGTACCTTCATAAG-3′; Gα~q~ (T186A), 5′-GTTCGAGTCCCCGCCACAGGGATCATTG-3′; Gα~q~ (D205A), 5′-TCTTCAGAATGGTCGCTGTAGGAGGCCAAAGG-3′; Gα~q~ (Q209L), 5′-TGTAGGAGGCCTAAGGTCAGAGA-3′; and Gβ~1~ (H311A), 5′-GTCCTAGCTGGAGCTGACAACCGAGTCAGC-3′. The sequences of the constructs were verified by direct DNA sequencing (ABI PRISM 377-XL, Applied Biosystems). LysoPLD assay ------------- The isotopic lysoPLD activity assay was performed as described previously \[[@B21]\]. Briefly, 30 μl of the source of enzyme was incubated at 37°C for 6 h in a reaction mixture containing 0.15 mM 1-\[^14^C\]palmitoyl-GPC (6000 d.p.m./nmol), 20 mM Tris/HCl (pH 7.0), 1 mM Na~3~VO~4~, and 50 mM MgCl~2~ with or without 1 mM GTP in a final volume of 75 μl. The reactions were terminated by the addition of 15 μl of 2 M HCl and 187.5 μl of chloroform/methanol/HCl \[100:200:1 (v/v)\]. The lipids were extracted by the addition of 93.75 μl each of chloroform and 2 M KCl, followed by centrifugation at 500 ***g*** for 10 min at 20°C. The extracted lipids were subjected to two-dimensional TLC \[chloroform/methanol/28% ammonia at 65:35:5 (v/v) for the first dimension and chloroform/acetone/methanol/acetic acid/water at 45:20:10:13:5 (v/v) for the second dimension\]. The LPA spots were visualized and quantified using the Fuji BAS2000 system (Fujifilm). For the colorimetric assay to detect lysoPLD activity, the amount of choline released from choline lysophospholipids was used as a measure of enzyme activity. Purified proteins (40 μl) were incubated at 37°C with 0.15 mM (0.0375--0.6 mM) choline lysophospholipids in the presence of 20 mM Tris/HCl (pH 7.0) and 1 mM Na~3~VO~4~ with or without 50 mM MgCl~2~ and/or 100 mM EDTA in a total reaction volume of 200 μl. After incubation for the given lengths of time, the reactions were terminated by boiling. To determine the amount of choline released, a second reaction was performed at 37°C for 15 min in a 500 μl reaction mixture containing the first assay mixture plus 50 mM Tris/HCl (pH 8.5), 0.5 mM HPPA, 0.033 unit/ml horseradish peroxidase and 1 unit/ml choline oxidase. The fluorescence intensity of each mixture was determined by excitation at 320 nm and collection at 404 nm using an RF5300 instrument (Shimadzu). Purification of FLAG-tagged proteins ------------------------------------ Mouse hepatocytoma cells (Hepa-1 cells), COS-7 cells or Neuro2A cells were seeded into 100 mm dishes in DMEM (Dulbecco\'s modified Eagle\'s medium; Wako Chemicals) with 10% fetal bovine serum (Gibco) 1 day prior to transfection. A total of 24 μg of empty vector or FLAG-tagged protein expression vector were transfected into cells using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer\'s instructions. At 1 day (24 h) post-transfection, the cells in one or five dishes were collected in 800 μl/dish of buffer C \[0.25 M sucrose in 50 mM Tris/HCl (pH 7.4), 1 mM EDTA and 1 mM PMSF\] and disrupted by sonication (three to eight pulses of 10 s each) with a Microson Ultrasonic cell disruptor. The homogenates were then centrifuged at 105000 ***g*** for 1 h at 4°C and the supernatants were removed or used for FLAG-tagged protein purification as indicated below. The pellets were resuspended in 400 μl/dish of buffer C with 10 mM CHAPS, and homogenized with an Ultrasonic cell disruptor. The suspensions were diluted to 1600 μl/dish of buffer C, then centrifuged at 21500 ***g*** for 10 min at 4°C and the supernatants were mixed with 250 μl of anti-FLAG M2 affinity gel (Sigma--Aldrich) suspended in 1.6 ml/dish of TBS buffer \[50 mM Tris/HCl (pH 7.4), 150 mM NaCl and 1mM PMSF\]. After incubation with gentle agitation at 4°C for several hours, the gel was washed with 5 ml of wash buffer (TBS with 0.1% CHAPS), and then tagged proteins were eluted with 250 μl of 100 μg/ml FLAG peptide in wash buffer four times. The viability of cells transfected with control or FLAG-tagged protein expression vectors did not change over 24 h and nearly equal amounts of protein were obtained in each sample. Effect of mouse Gα~q~ and Gα~11~ siRNAs (small interfering RNAs) on lysoPLD activity in Hepa-1 cells ---------------------------------------------------------------------------------------------------- Stealth siRNA, a 25-base pair duplex oligoribonucleotide against mouse Gα~q~ or Gα~11~, was purchased from Invitrogen. The target sense sequences were: Gα~q~, 5′-CCCUUUGACUUACAAAGUGUCAUUU-3′; and Gα~11~, 5′-CCAAGUUGGUGUACCAGAACAUCUU-3′. Hepa-1 cells (1×10^5^) were cultured in 60 mm dishes for 24 h, and then transfected with both siRNAs by using Lipofectamine™ 2000 (Invitrogen) according to manufacturer\'s instructions. After 48 h, the cells were collected in 400 μl/dish of buffer C and disrupted by sonication (three to eight pulses of 10 s each) with a Microson Ultrasonic cell disruptor. The homogenates were then centrifuged at 105000 ***g*** for 1 h at 4°C and the supernatants were removed. The pellets were resuspended in 300 μl/dish of buffer C with 10 mM CHAPS, and homogenized with an Ultrasonic cell disruptor. The suspensions were centrifuged at 21500 ***g*** for 10 min at 4°C, and the supernatants were used as microsomal fractions for lysoPLD assay with 10 μg of the protein for a 30 min incubation. Statistical analysis -------------------- All values are expressed as the means±S.D. (*n*≥3). Group means were compared using the Student\'s *t* test or ANOVA to determine the significance of the differences among the individual means. Statistical significance was assumed at *P*\<0.05. Each experiment was repeated at least twice with similar results. RESULTS ======= A protein with lysoPLD activity is highly associated with heterotrimeric G protein ---------------------------------------------------------------------------------- In a previous study, we purified a novel protein with lysoPLD activity from rat brain tissue and analysed its enzymatic characteristics, which were different from those of the secreted lysoPLD autotaxin. Initially, a 35 kDa protein was the main band in purified lysoPLD from rat brain and did not react with an anti-autotaxin antibody \[[@B21]\]. However, as shown in [Figure 1](#F1){ref-type="fig"}(A), two bands (faint 42 kDa and 35 kDa bands, indicated by arrows on the right-hand side) were observed following repeated purifications as reported previously \[[@B21]\], separation by SDS/PAGE and silver staining. The 42 kDa protein was present at lower levels and was not recognized as potentially important at first. To identify their molecular species, the bands were analysed by PMF (peptide mass fingerprinting). The 42 kDa and 35 kDa proteins were identified as Gα~q~ ([Figure 1](#F1){ref-type="fig"}D) and Gβ~1~ ([Figure 1](#F1){ref-type="fig"}E) respectively. ![Identification of G protein subunits Gα~q~ and Gβ~1~ in purified lysoPLD fractions from the rat brain\ (**A** and **B**) Fractions eluted after HiTrap DEAE FF purification, the final step in a previously reported protocol for purification of a lysoPLD activity from rat brain tissue, were subjected to SDS/PAGE and detected by silver staining (**A**) and immunoblotting (**B**). The right-hand side arrows in (**A**) indicate protein bands at 42 kDa (upper) and 35 kDa (lower). (**C**) LysoPLD activities of the same purified samples obtained from rat brain tissue as determined using a colorimetric assay. (**D** and **E**) Identification of the purified 42 kDa and 35 kDa proteins from rat brain tissues by PMF. The 42 kDa (**D**) and 35 kDa (**E**) proteins were identified as Gα~q~ and Gβ~1~ respectively. Amino acid sequences identified by PMF analysis are indicated with underlining. Fr., fraction.](bic536i001){#F1} To confirm this result, the fractions obtained from the final step of enzyme purification with HiTrap DEAE FF ([Figure 1](#F1){ref-type="fig"}C) were subjected to immunoblot analysis with antibodies specific against Gα~q/11~ and Gβ~1--4~. As shown in [Figure 1](#F1){ref-type="fig"}(B), both Gα~q~ and Gβ~1~ proteins were detected in those fractions and the levels of those proteins correlated well with lysoPLD activities in each fraction. On the basis of these results, we speculated that the heterotrimeric G protein complex is highly associated with lysoPLD activity. Tag-purified G protein exhibits lysoPLD activity ------------------------------------------------ FLAG-tagged Gα~q~ or Gβ~1~ proteins were overexpressed in the mouse hepatocytoma cell line Hepa-1 and then purified via immune-affinity chromatography with an anti-FLAG antibody. When overexpressed, FLAG--Gα~q~ was purified with FLAG peptides, endogenous Gβ co-purified and, similarly, endogenous Gα~q/11~ co-purified with FLAG-tagged Gβ~1~ ([Figure 2](#F2){ref-type="fig"}A). Gγ~5~ also co-purified with FLAG--Gα~q~ ([Figure 2](#F2){ref-type="fig"}A). These results suggest that heterotrimeric G protein complexes can form with overexpressed FLAG--Gα~q~. The density of the immunoreactive bands with anti-Gα~q/11~ or anti-Gβ~1--4~ antibody and lysoPLD activities coincided well ([Figures 2](#F2){ref-type="fig"}A and [2](#F2){ref-type="fig"}B); thus we used fraction 4 for subsequent analyses. The significant increase in lysoPLD activity was not observed in fractions prepared from cells transfected with empty vector. The lysoPLD activity was confirmed by choline production from lysoPAF ([Figure 2](#F2){ref-type="fig"}C) and LPA production from \[^14^C\]palmitoyl-labelled LPC, ([Figures 2](#F2){ref-type="fig"}D and [2](#F2){ref-type="fig"}E). Significant increases in both choline and/or LPA production via lysoPLD activity were detected in the eluates prepared from cells overexpressing FLAG--Gα~q~ or FLAG--Gβ~1~. Autotaxin was not detected in the tag-purified fraction by immunoblot analysis ([Figure 2](#F2){ref-type="fig"}A, indicated by the arrow), and lower bands were non-specific bands. LysoPLD activity of autotaxin is not dependent on Mg^2+^ supplementation \[[@B25]\]. These results suggest that the lysoPLD activities were attributable to Gα~q~ or Gβ~1~ themselves, or to a protein(s) in a complex with these proteins. ![LysoPLD activity of tag-purified G protein subunits Gα~q~ and Gβ~1~\ Empty vector (Cont) or a vector encoding FLAG-tagged Gα~q~ or Gβ~1~ was transfected into Hepa-1 cells and FLAG-tagged proteins (F-Gα~q~ or F-Gβ~1~) were purified from one dish. (**A**) Immunoblot analysis of tag-purified G protein subunits Gα~q~ and Gβ~1~. A total of 10 μl of each sample prior to purification (lane 1), eluted with a wash buffer (lane 2) or eluted with elution buffers containing FLAG-peptides (lanes 3--6) was separated by SDS/PAGE (10% gel) and transferred on to PVDF membranes. The immunoreacted Gα~q~, Gβ~1~, Gγ~5~, or autotoxin proteins (ATX) were detected with anti-Gα~q/11~, -Gβ, -Gγ~5~ or -autotoxin antibodies. As expected, the molecular masses of tagged proteins were approximately 2 kDa larger than endogenous proteins. The immunoreacted Gγ~5~ in lane 1 and other lanes were determined in separated lanes, but on the same membrane. The experiments were repeated several times and similar results were obtained each time. (**B**) A total of 40 μl of each fraction (lanes 2--6 in **A**) from Hepa-1 cells overexpressing empty vector (open square) or FLAG-tagged Gα~q~ (closed diamond) was incubated with 0.15 mM lysoPAF and then lysoPLD activities were calculated using a colorimetric assay. (**C**) A total of 40 μl of the second fractions eluted with FLAG peptides (lane 4 in **A**) obtained from Hepa-1 cells overexpressing empty vector (white bar), F-Gα~q~ (black bar) or F-Gβ~1~ (grey bar) was incubated with 0.15 mM lysoPAF for 6 h and then lysoPLD activities were calculated using a colorimetric assay. Results are means±S.D. (*n*=6). \**P*\<0.001 compared with the vector control. (**D** and **E**) LPA production from \[^14^C\]-labelled LPC by incubation with the purified enzyme from Hepa-1 cells overexpressing empty vector (Cont) (white bar), FLAG-tagged Gα~q~ (F-Gα~q~) (black bar) or FLAG-tagged Gβ~1~ (F-Gβ~1~) (grey bar) for 6 h was visualized after lipids were extracted and subjected to TLC analysis. Results are means±S.D (*n*=3). \**P*\<0.012 compared with the vector control. Arrows (**E**) indicate the positions of LPA. The experiments were repeated more than three times with similar results.](bic536i002){#F2} Time course, substrate specificity and Mg^2+^ cation requirement of lysoPLD activity of Gα~q~ --------------------------------------------------------------------------------------------- To examine the time course of choline production from lysoPAF hydrolysed by purified FLAG-tagged Gα~q~ subunit, the enzyme was incubated with 0.15 mM lysoPAF for 0--6 h and activities were tested using a colorimetric assay. As shown in [Figure 3](#F3){ref-type="fig"}(A), the levels of the hydrolysed product, choline, significantly increased in the presence of purified FLAG-tagged Gα~q~ for up to 6 h of incubation. ![Time dependence, substrate specificity and requirement of Mg^2+^ cations of the lysoPLD activity of tag-purified Gα~q~\ (**A**) A total of 40 μl of the tag-purified fractions obtained from Hepa-1 cells overexpressing empty vector (open squares, *n*=6) or FLAG-tagged Gα~q~ (F-Gα~q~) (closed diamonds, *n*=6) was incubated with 0.15 mM lysoPAF for the indicated times and then subjected to a colorimetric lysoPLD assay. \**P*\<0.01 compared with the vector control. (**B**) The purified F-Gα~q~ (40 μl) was incubated with 0.15 mM of various choline lysophospholipids \[16:0 (black bar, *n*=7), 18:0 (grey bar, *n*=3), 18:1 (horizontally striped bar, *n*=6) or lysoPAF (white bar, *n*=5)\] for 6 h. Relative choline production as compared with lysoPC (16:0) was analysed using a colorimetric assay. \**P*\<0.01 compared with 16:0; \*\**P*\<0.001 compared with the other substrates. (**C**) The lysoPLD activities of the fractions (40 μl) obtained from Hepa-1 cells overexpressing empty vector (white bar) or F-Gα~q~ (black bar) were incubated with or without Mg^2+^ and/or EDTA (*n*=3). Relative choline production as compared with F-Gα~q~ and Mg^2+^ was analysed using a colorimetric assay. \**P*\<0.001 compared with F-Gα~q~ and Mg^2+^~.~ (**D**) The supernatants of homogenates from Hepa-1 cells overexpressing FLAG--Gα~q~ after centrifugation at 105000 ***g*** were applied on to immune-affinity chromatography with anti-FLAG antibody, and immunoblot analysis of tag-purified Gα~q~ (F-Gα~q~) was performed (top panel). The enzymatic activity in the purified fraction was assayed (bottom panel). Results are means±S.D. The experiments were repeated several times with similar results. Cont, control.](bic536i003){#F3} To identify a preferred substrate, purified FLAG-tagged Gα~q~ was incubated for 6 h with several different choline lysophospholipid substrates ([Figure 3](#F3){ref-type="fig"}B). The preferred substrate identified in this assay is lysoPAF (*P*\<0.01). The enzyme was more efficient at hydrolysis of 1-palmitoyl-2-lyso-GPC (16:0) than hydrolysis of 1-oleoyl-2-lyso-GPC (18:1) (*P*\<0.01). The substrate specificity of Gα~q~ for lysophospholipids is very similar to what was observed for the enzyme activity previously purified from the rat brain \[[@B21]\]. Choline production from lysoPAF by the purified FLAG--Gα~q~ was dependent on incubation with Mg^2+^ ([Figure 3](#F3){ref-type="fig"}C), similar to what was observed for the enzyme purified from rat brain tissue \[[@B21]\]. Although after centrifugation at 105000 ***g*** high levels of FLAG--Gα~q~ could be purified from the supernatants of homogenates from cells overexpressing the protein, enzymatic activity was not detected ([Figure 3](#F3){ref-type="fig"}D). The lysoPLD activity of Gα~q~ and Gβ~1~ mutant proteins ------------------------------------------------------- As FLAG--Gα~q~ purified from Hepa-1 cells overexpressing the protein co-purified with endogenous Gβ and purified FLAG-Gβ~1~ co-purified with endogenous Gα~q/11~ ([Figure 2](#F2){ref-type="fig"}A), we next tried to clarify which subunit is responsible for the lysoPLD activity we observed. Autotaxin had been identified previously as a member of the ecto-nucleotide PDE (pyrophosphatase/phosphodiesterase) family due to similarity in its primary structure \[[@B14],[@B26]\]. Later, it became clear that the ester bond between phosphate and choline in LPC was also hydrolysed by autotaxin \[[@B12],[@B13]\]. Therefore we speculated that Gα~q~ subunit might exhibit lysoPLD activity because it hydrolyses GTP to GDP via its intrinsic GTPase activity. The Gβ~1~ subunit would be the candidate for the protein with lysoPLD activity, as a histidine acid phosphatase consensus motif is present in its C-terminus (V^305^GILSGHDNAVSCLGV^320^, analysed by GENETYX). To test this, we introduced several mutations at amino acid residues thought to be important for Gα~q~ or Gβ~1~ enzyme activities. The mutant proteins were then purified and their lysoPLD activities were assayed. Tag-purified Gβ~1~--Gγ~2~ complex overexpressed in *S. frugiperda* insect cells was also obtained, and its lysoPLD activity was determined. As shown in [Figure 4](#F4){ref-type="fig"}(A), the K52A, T186A, D205A, G48A and Q209L mutant forms of Gα~q~ showed a significant reduction of lysoPLD activity, whereas G48V did not. The decrease in lysoPLD activity of G48A and Q209L was low. Based on amino acid sequence similarity to p21^ras^, the amino acid sequence motif G^46^TGESGKS^53^ is predicted to form a loop in which main-chain amide hydrogens of several amino acids and the ϵ-amino group of Lys^52^ might form bonds with the β- and γ-phosphate of GTP \[[@B27]\]. This site also corresponds to the GTP-binding site. Therefore we speculated that the side chain of Lys^52^ might be important for the binding of phosphate at the sn−3 position of LPC, whereas the side chain of Gly^48^ is not likely to be important for this binding. This suggests that the GTP- and LPC-binding sites or interacting amino acids of Gα~q~ seem to be not completely the same. Moreover, the side chain of Thr^186^ may point away from the bound nucleotide in the GDP-bound form, but flip towards the nucleotide in the GTP-bound form, where it might directly interact with a Mg^2+^ ion coordinated with oxygen molecules in the β- and γ-phosphates of GTP. On the basis of crystal structure analysis, it has been suggested that the side chain of Asp^205^ in Gα~q~ might bind catalytic Mg^2+^ via an intervening water molecule \[[@B27],[@B28]\]. The well-known dominant negative Gα~q~ (Q209L) construct was prepared and its activity was assayed. Only slight reduction of lysoPLD activity of Q209L was detected. The Gβ~1~ mutant H311A, which disrupts a conserved amino acid residue in the histidine acid phosphatase consensus, did not show a significant change in lysoPLD activity as compared with the wild-type Gβ~1~ subunit ([Figure 4](#F4){ref-type="fig"}B). Tag-purified Gβ~1~--Gγ~2~ complex overexpressed in insect cells did not exhibit lysoPLD activity. These results strongly suggest that Gα~q~ exhibits lysoPLD activity and, furthermore, that the predicted nucleotide phosphate and Mg^2+^-binding sites of the protein are important for lysoPLD enzymatic activity. When we purified lysoPLD from rat brain or culture cells, Gα~q~ and Gβ were always co-purified. It is possible that lysoPLD activity of Gα~q~ is dependent on the presence of Gβ protein. ![LysoPLD activity of mutant forms of the G protein subunits Gα~q~ and Gβ~1~, and tag-purified Gβ~1~--Gγ~2~ complex\ Empty vector (Cont), FLAG-tagged wild-type Gα~q~ (**A**, F-Gα~q~) or Gβ~1~ (**B**, F-Gβ~1~), or mutant forms (**A**, T186A, K52A, D205A, G48V, G48A, Q209L mutations of Gα~q~, and **B**, F-Gβ~1~m for Gβ~1~) were expressed in Hepa-1 cells and purified from one dish. Top panels: the levels of unmodified or mutant FLAG-tagged Gα~q~ and Gβ~1~ subunits in 10 μl of the purified fractions, and tag-purified Gβ~1~--Gγ~2~ complex (3 ng). The immunoreacted wild-type Gα~q~ and mutant forms in (**A**) were not determined in the same experiments, but all experiments were repeated several times and almost equivalent levels of proteins compared with F-Gα~q~ were obtained each time. (**A**) Bottom panel: relative choline production by tag-purified enzymes (40 μl) from cells overexpressing empty vector (Cont; white bar, *n*=6), T186A (vertically striped bar, *n*=5), K52A (horizontally striped bar, *n*=3), D205A (left oblique striped bar, *n*=3), G48V (grey bar, *n*=4), G48A (right oblique striped bar, *n*=3) or Q209L (crosswise striped bar, *n*=3) as compared with unmodified FLAG-tagged Gα~q~ (black bar, *n*=8) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 6 h. \**P*\<0.001 compared with FLAG-tagged Gα~q~. (**B**) Bottom panel: relative choline production by tag-purified enzymes from cells overexpressing empty vector (Cont; white bar, *n*=6), F-Gβ~1~m (horizontally striped bar; *n*=6) or tag-purified Gβ~1~--Gγ~2~ complex (12 ng) \[G(β~1~ and γ~2~); black bar, *n*=3\] as compared with F-Gβ~1~ (grey bar; *n*=6). \**P*\<0.001 compared with F-Gβ~1~. Results are means±S.D. The experiments were repeated more than three times with similar results.](bic536i004){#F4} LysoPLD activity of Gα~q~ protein and a mutant form, T186A, highly purified from COS-7 cells -------------------------------------------------------------------------------------------- To eliminate the possibility that the detection of lysoPLD activity from purified tagged Gα~q~ protein is cell-line dependent, we performed the same experiment with COS-7 cells, which do not exhibit autotaxin activity and express relatively low endogenous levels of G proteins. As shown in [Figure 5](#F5){ref-type="fig"}(A), highly purified wild-type FLAG--Gα~q~ protein and a mutant form, T186A, could be detected following ectopic expression in COS-7 cells. The levels of the hydrolysed product, choline, significantly increased over time in the presence of purified FLAG-tagged Gα~q~ ([Figure 5](#F5){ref-type="fig"}B). The activity was dependent on incubation with Mg^2+^ cations ([Figure 5](#F5){ref-type="fig"}C). The presence of lysoPLD activity was confirmed by the significant production of choline from lysoPAF ([Figures 5B--5](#F5){ref-type="fig"}D) and LPA from LPC ([Figures 5](#F5){ref-type="fig"}E and [5](#F5){ref-type="fig"}F). The T186A mutant form of Gα~q~ showed significantly less lysoPLD activity than the wild-type FLAG--Gα~q~ protein. These results clearly show that Gα~q~ has lysoPLD activity. As shown in [Figure 5](#F5){ref-type="fig"}(A) (bottom panel), a small amount of endogenous Gβ in COS-7 cells was also co-purified with intact or mutated FLAG-tagged Gα~q~. ![LysoPLD activity of highly purified Gα~q~ protein and a mutant form, T186A, from COS-7 cells\ Empty vector (Cont), FLAG-tagged wild-type Gα~q~ (F-Gα~q~) or a FLAG-tagged mutant form (T186A) was expressed in COS-7 cells and purified from five dishes. (**A**) Silver staining (top panel) or immunoblot analysis (bottom panel) with anti-Gα~q/11~ and Gβ of vector control (Cont), tag-purified wild-type Gα~q~ (F-Gα~q~) or T186A in the purified fractions. (**B**) A total of 40 μl of the tag-purified fractions obtained from COS-7 cells overexpressing empty vector (Cont) (open squares, *n*=3) or FLAG-tagged Gα~q~ (F-Gα~q~) (closed diamonds, *n*=3) was incubated with 0.15 mM lysoPAF for the indicated times and then subjected to a colorimetric lysoPLD assay. \**P*\<0.001 compared with the vector control. (**C**) The lysoPLD activities of the purified fractions (40 μl) obtained from COS-7 cells overexpressing empty vector (open bars) or F-Gα~q~ (closed bars) were incubated with or without Mg^2+^ and/or EDTA (*n*=3). Relative choline production as compared with the vector control was analysed using a colorimetric assay. \**P*\<0.05 compared with the vector control and F-Gα~q~ with or without Mg^2+^ and EDTA. (**D**) Choline production by tag-purified enzymes (40 μl) from cells overexpressing unmodified FLAG-tagged Gα~q~ (F-Gα~q~) (black bar, *n*=3) or T186A (vertically striped bar, *n*=3) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 6 h. \*\**P*\<0.001 compared with F-Gα~q~. (**E** and **F**) LPA production by tag-purified enzymes (30 μl) from cells overexpressing unmodified FLAG-tagged Gα~q~ (F-Gα~q~) (black bar, *n*=3) or T186A (vertically striped bar, *n*=3) was analysed with an isotopic assay after incubation with 0.15 mM \[^14^C\]-labelled LPC for 6 h. LPA production was visualized after lipids were extracted and subjected to TLC analysis. (**E**) \**P*\<0.03 compared with F-Gα~q~. (**F**) Arrows indicate the positions of LPA. Results are means±S.D. The experiments were repeated at least twice with similar results.](bic536i005){#F5} LysoPLD activity of Gβ~1~ protein highly purified from COS-7 cells ------------------------------------------------------------------ To examine the possibility that purified FLAG--Gβ~1~ obtained from COS-7 cells also has lysoPLD activity, we repeated the experiment described above with these cells. A small amount of endogenous Gα~q/11~ in COS-7 cells was co-purified with FLAG--Gβ~1~ ([Figure 6](#F6){ref-type="fig"}A). Almost the same lysoPLD activity as for purified FLAG--Gα~q~ protein was observed, in FLAG--Gβ~1~ with the production of choline from lysoPAF ([Figure 6](#F6){ref-type="fig"}B) and LPA from LPC ([Figure 6](#F6){ref-type="fig"}C). When purified FLAG--Gα~q~ and FLAG--Gβ~1~ proteins were incubated together *in vitro*, an additive effect of lysoPLD activity was observed, as specific activity did not change ([Figure 6](#F6){ref-type="fig"}B). ![LysoPLD activity of highly purified Gβ~1~ protein from COS-7 cells\ FLAG-tagged wild-type Gα~q~ (F-Gα~q~) or a FLAG-tagged wild-type Gβ~1~ (F-Gβ~1~) was expressed in COS-7 cells and purified from five dishes. (**A**) Immunoblot analysis with anti-Gα~q/11~ (top panel) and anti-Gβ (middle panel), or silver staining (bottom panel) of tag-purified wild-type Gα~q~ (F-Gα~q~) or Gβ~1~ (F-Gβ~1~) in the purified fractions. (**B**) A total of 40 μl of the tag-purified fractions obtained from COS-7 cells overexpressing FLAG-tagged Gα~q~ (F-Gα~q~) (black bar, *n*=3), FLAG-tagged Gβ~1~ (F-Gβ~1~) (grey bar, *n*=3) or equal amounts of both F-Gα~q~ and F-Gβ~1~ (white bar, *n*=3) was incubated with 0.15 mM lysoPAF for 6 h and then subjected to a colorimetric lysoPLD assay. (**C**) The lysoPLD activity of the purified fraction (30 μl) obtained from COS-7 cells overexpressing F-Gα~q~ (black bar, *n*=3) or F-Gβ~1~ (grey bar, *n*=3) was analysed with an isotopic assay after incubation with 0.15 mM \[^14^C\]-labelled LPC for 6 h. LPA production was visualized after lipids were extracted and subjected to TLC analysis. Results are means±S.D. The experiments were repeated at least twice with similar results.](bic536i006){#F6} Effect of substrate concentration and GTP on the activity of FLAG-purified lysoPLD from Neuro2A cells overexpressing FLAG--Gα~q~ -------------------------------------------------------------------------------------------------------------------------------- Because the lysoPLD enzyme that we have purified previously was from the brain, we also tested overexpression and tag-affinity purification of FLAG--Gα~q~ using Neuro2A cells. As shown in [Figures 7](#F7){ref-type="fig"}(A) and [7](#F7){ref-type="fig"}(B), the level of lysoPLD activity of purified FLAG-Gα~q~ was dependent on cell type (COS-7\<Hepa-1\<Neuro2A cells). Notably, the enzyme activity of purified FLAG-tagged Gα~q~ expressed in COS-7 cells was approximately 4 nmol/min per mg, whereas the activity from Neuro2A cells was much higher, i.e. 137.4 nmol/min per mg. The calculated *K*~m~ and *V*~max~ values for lysoPAF using Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively ([Figures 7](#F7){ref-type="fig"}B and [7](#F7){ref-type="fig"}C). The *K*~m~ and *V*~max~ values of lysoPLD obtained from rat brain for lysoPAF were 26.7 μM and 0.29 μmol/min per mg respectively. These results using Neuro2A cells and rat brain were almost equivalent with each other. The slightly lower *V*~max~ value in Neuro2A cells is probably due to the purity of the enzyme or the disturbance of enzyme activity by N-terminal FLAG peptides. ![Effect of substrate concentration and GTP on the activity of FLAG-purified lysoPLD obtained from Neuro2A cells overexpressing FLAG--Gα~q~\ Empty vector (Cont) or FLAG-tagged wild-type Gα~q~ (F-Gα~q~) was expressed in Hepa-1, COS-7 or Neuro2A (N2A) cells and purified from five dishes. (**A**) Immunoblot analysis with anti-Gα~q/11~ antibody (upper panel) of tag-purified Gα~q~ (F-Gα~q~) obtained from Hepa-1, COS-7 or Neuro2A cells overexpressing FLAG-Gα~q~. Choline production (lower panel) by tag-purified enzyme (40 μl) obtained from cells overexpressing unmodified FLAG-tagged Gα~q~ (F-Gα~q~) (*n*=3) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 6 h. Results are means±S.D. The experiments were repeated at least two times with similar results. Tag-purified enzyme from Neuro2A cells overexpressing unmodified FLAG-tagged Gα~q~ was incubated with various concentrations from 0.0375 to 0.6 mM of lysoPAF for 6 h, and choline production was analysed with a colorimetric assay. An untransformed plot (**B**) and Lineweaver--Burk (double-reciprocal) plot (**C**) are shown. (**D**) The lysoPLD activity of the purified fraction (30 μl) obtained from Neuro2A cells overexpressing empty vector or F-Gα~q~ was analysed with an isotopic assay after incubation with 0.15 mM \[^14^C\]-labelled LPC for 6 h with (black bar, *n*=3) or without (white bar, *n*=3) 1 mM GTP. Relative LPA production was determined as compared with FLAG-tagged Gα~q~ without GTP. LPA production was visualized after lipids were extracted and subjected to TLC analysis. Results are means±S.D. The experiments were repeated at least two times with similar results.](bic536i007){#F7} As shown in [Figure 7](#F7){ref-type="fig"}(D), 1 mM GTP did not disturb the lysoPLD activity (0.15 mM LPC) of FLAG--Gα~q~ obtained from Neuro2A cells. As the mutation at the GTP-binding region (Gly^48^) did not decrease lysoPLD activity of Gα~q~ much ([Figure 4](#F4){ref-type="fig"}A), the GTP- and LPC-binding sites seem to not be completely the same. Moreover, the exchange of GDP to GTP and activation of GTPase activity of Gα~q~ possibly rarely occurred in this experiment. As for most of the GTPases, the rate of exchange of GDP to GTP and GTPase activity of Gα~q~ are quite low without ligand-stimulated receptor and GAPs (GTPase-activating proteins) \[[@B29]--[@B31]\]. LysoPLD activity of autotaxin may be inhibited by incubation with GTP because autotaxin catalyses the hydrolysis of GTP as well as ATP. LysoPLD activity of other Gα subfamily members and the effect of Gα~q~ and Gα~11~ siRNAs ---------------------------------------------------------------------------------------- In the human genome, 16 different Gα subunits have been identified and these can be divided into four subfamilies based on their amino acid similarities and the machinery for their signal transduction. Thus we were interested in the possibility that other Gα protein family members might also exhibit lysoPLD activity. To test this, we first constructed plasmids encoding FLAG-tagged Gα~11~, Gα~i~ and Gα~s~. The plasmids were introduced into Hepa-1 cells and the activities of the purified overexpressed proteins were analysed. As shown in [Figure 8](#F8){ref-type="fig"}(A), purified FLAG--Gα~11,~ which has 89% sequence similarity with Gα~q~ and belongs to the same subfamily, exhibited a high lysoPLD activity (*P*\<0.01), whereas FLAG--Gα~i~ and FLAG--Gα~s~ did not. However, a slight increase in lysoPLD activity of FLAG--Gα~s~ was observed. ![Testing LysoPLD activity of other Gα subfamily members and effect of Gα~q~ and Gα~11~ siRNAs\ (**A**) FLAG-tagged Gα subtypes F-Gα~q~ (black bar, *n*=18), F-Gα~11~ (grey bar, *n*=6), F-Gα~i~ (horizontally striped bar, *n*=6), F-Gα~s~ (vertically striped bar, *n*=6) or empty vector (Cont; white bar, *n*=12) were overexpressed in Hepa-1 cells and tagged proteins were purified from one dish. Levels of tag-purified Gα subfamily proteins (top panel) and co-purified Gβ proteins (middle panel) detected using anti-FLAG and anti-Gβ~1--4~ antibodies respectively~.~ Note that Gα~s~ migrates at a higher molecular mass than the others. The bands of the same size present in the control and experimental samples are non-specific. Bottom panel: relative choline production by 40 μl of purified enzyme incubated with lysoPAF as compared with FLAG-tagged Gα~q~, detected with a colorimetric assay. \**P*\<0.001 compared with F-Gα~q~. Results are means±S.D. The experiment was repeated several times with similar results. (**B**) Hepa-1 cells were pretreated with control siRNA or mouse Gα~q~ and Gα~11~ siRNAs for 48 h. Levels of endogenous Gα~q~ and Gα~11~ (top panel) and actin (middle panel) detected using anti-Gα~q/11~ and anti-actin antibodies respectively~.~ Bottom panel: lysoPLD activity in a crude microsomal fraction (10 μg) from Hepa-1 cells pretreated with control siRNA (white bar, *n*=3) or Gα~q~ and Gα~11~ siRNAs (black bar, *n*=3) was analysed with a colorimetric assay after incubation with 0.15 mM lysoPAF for 30 min.](bic536i008){#F8} As shown in [Figure 8](#F8){ref-type="fig"}(B), pretreatment of Gα~q~ and Gα~11~ siRNAs clearly decreased the endogenous Gα~q~ and Gα~11~ in Hepa-1 cells. However, lysoPLD activity in the crude microsomal fraction did not changed compared with the control. In the crude microsome, we speculated that other undetermined G proteins or contaminated autotaxin may be responsible for the high endogenous lysoPLD activity and that the lysoPLD activity of the interested protein may be masked. DISCUSSION ========== We were interested to molecularly identify a lysoPLD activity that had been reported previously. Two proteins from the rat brain exhibiting lysoPLD activity (faint 42 kDa and 35 kDa bands by gel electrophoresis) can be observed following a method reported previously for purification \[[@B21]\]. As shown in [Figure 1](#F1){ref-type="fig"}(A), the 42 kDa protein was present at lower levels and, at first, was not recognized as potentially important. The enzymatic properties of the fractions containing both proteins, including optimum pH, substrate specificity and cation requirement, were different from those of a secreted protein with lysoPLD activity, autotaxin. In the present study, we analysed the 42 kDa and 35 kDa proteins by MS and identified them as the heterotrimeric G protein subunits Gα~q~ and Gβ~1~ respectively. When FLAG-tagged Gα~q~ or Gβ~1~ was overexpressed in Hepa-1 cells and purified, both purified subunits exhibited lysoPLD activity ([Figure 2](#F2){ref-type="fig"}). Mutations in conserved amino acid residues of Gα~q~ known to be important for GTPase activity and interaction with nucleotide phosphates or Mg^2+^ significantly reduced lysoPLD activity ([Figure 4](#F4){ref-type="fig"}). Furthermore, highly purified FLAG--Gα~q~ protein ectopically expressed in COS-7 cells clearly hydrolysed LPC to LPA, whereas a mutant form, T186A, showed significantly less lysoPLD activity ([Figure 5](#F5){ref-type="fig"}). The calculated *K*~m~ and *V*~max~ values for lysoPAF of the FLAG-purified enzyme obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively ([Figures 7](#F7){ref-type="fig"}B and [7](#F7){ref-type="fig"}C). These values were similar to the enzyme purified from the rat brain (*K*~m~=26.7 μM and *V*~max~=0.29 μmol/min per mg) (21). Although Gβ~1~ detected by silver staining was the main protein of purified lysoPLD from rat brain, Gβ~1~ is not likely to be the main component for lysoPLD activity because of following four reasons: (i) FLAG--Gβ~1~ mutant (H311A) exhibited significant lysoPLD activity ([Figure 4](#F4){ref-type="fig"}B); (ii) FLAG--Gα~11~ co-purified with lower amounts of Gβ~1~ exhibited lysoPLD activity comparable with Gα~q~ ([Figure 8](#F8){ref-type="fig"}A); (iii) FLAG--Gα~i~ and FLAG--Gα~s~ did not exhibit as high a lysoPLD activity as FLAG--Gα~q~ when they were co-purified with Gβ ([Figure 8](#F8){ref-type="fig"}A); and (iv) tag-purified Gβ~1~ and Gγ~2~ overexpressed in insect cells did not exhibit lysoPLD activity ([Figure 4](#F4){ref-type="fig"}A). However, the potential importance of Gβ~1~ for lysoPLD activity of Gα~q~ may be possible because they were always co-purified with respect to lysoPLD activity. These results reveal a new function for Gα~q~ and Gβ~1~, which are well known as multifunctional signal transduction proteins, as an enzyme with lysoPLD activity. Although FLAG-purified Gα~q~ obtained from the microsomal fraction exhibited significant lysoPLD activity, the purified enzyme from supernatants after 105000 ***g*** centrifugation did not have lysoPLD activity ([Figure 3](#F3){ref-type="fig"}D). The level of lysoPLD activity of purified FLAG--Gα~q~ from the microsomal fraction was dependent on cell type (COS-7\<Hepa-1\<Neuro2A cells) ([Figure 7](#F7){ref-type="fig"}A). These results suggest that post-translational modification(s), or some kind of activator(s) present in the microsome, is important for lysoPLD activity of Gα~q~. However, it is also possible that Gα~q~ proteins in supernatants were misfolded after translation. Agonist-activated GPCRs enhance GDP/GTP exchange on the G protein α subunit, thereby generating an active GTPbound Gα subunit, followed by dissociation from the receptor and Gβγ subunit. The onset and termination of G protein signalling is determined by the length of time spent in the active GTP-bound state. GTP is hydrolysed to GDP by the intrinsic GTPase activity of the Gα subunit, resulting in re-association of the Gα subunit with the Gβγ subunit and termination of signalling \[[@B29]\]. As for most of GTPases, the rates of exchange of GDP to GTP and GTPase activity of Gα~q~ are quite low. However, the GTPase activity of Gα~q~ can be dramatically increased by *in vitro* incubation with ligand-stimulated cholinergic receptor and GAPs, such as the RGS (regulator of G protein signalling) proteins \[[@B30]\] or phospholipase C \[[@B31]\]. In the present study, we found evidence that the lysoPLD activity of FLAG-tagged Gα~q~ is exhibited in the presence of Gβ and Gγ~5~, as endogenous Gβ and Gγ~5~ co-purified with FLAG--Gα~q~ ([Figure 2](#F2){ref-type="fig"}). We speculate that the heterotrimeric form of G protein subunits may exhibit lysoPLD activity. Previously, GTPγS was found to be bound to the purified activators \[[@B32]\] and, additionally, that purified PI-PLC (phosphoinositide phospholipase C) from the bovine brain could be activated by purified Gα~q~ in the presence of GTPγS (0.1--1 mM) \[[@B33]\]. On the basis of our results, we propose that a possible role of the novel lysoPLD identified in the present study for Gα~q~ and Gβ~1~ is the production of LPA inside cells in response to ligand stimulation, which is then followed by sequential activation of PI-PLC (Ca^2+^ influx) and PLA~2~s that hydrolyse PC to free fatty acid and LPC \[[@B15]\]. It was reported previously that LPA receptors can be detected on the nuclear membrane \[[@B22],[@B23]\] and that the transcription factor PPARγ acts as a LPA receptor \[[@B24]\]. It seems reasonable to speculate that the Gα~q~ and Gβ~1~ subunits mediate the signal to these intracellular receptors via lysoPLD activity. After testing several family members as shown in [Figure 8](#F8){ref-type="fig"}(A), Gα~q~ and Gα~11~ exhibit a high lysoPLD activity, but Gα~i~ and Gα~s~ do not. Therefore we conclude that the lysoPLD activity of the Gα subunit is strictly dependent on its subfamily. However, treatment of Hepa-1 cells with Gα~q~ and Gα~11~ siRNAs did not affect lysoPLD activity in the microsomal fraction ([Figure 8](#F8){ref-type="fig"}B). It may be possible that other undetermined G proteins and/or different kinds of lysoPLD, such as autotaxin, are responsible for the high endogenous lysoPLD activity and that they masked the reduced activity. Furthermore, our preliminary experiments showed that the stimulation of radio-labelled cells by carbachol that activates Gα~q/11~ through the muscarinic receptor did not affect the basal amount of LPA (results not shown). We speculate that the degradation of LPA in cells was rapid or that the stimulation system was inadequate. Further investigation is required to help elucidate the importance of LPA production by Gα~q~ in signal transduction. AUTHOR CONTRIBUTION =================== The project strategy was devised by, and most of the experiments were performed by, Chieko Aoyama and Hiroyuki Sugimoto. Purification of proteins was helped by Hiromi Ando, Satoko Yamashita and Sayaka Sugimoto. siRNA experiments and PMF analysis were performed by Yasuhiro Horibata and Motoyasu Satou respectively. ACKNOWLEDGEMENTS ================ We thank Dr Takashi Namatame of the Medical Research Center for help with DNA sequencing and the Research Support Center for allowing us to use the facilities at Dokkyo Medical University School of Medicine. We also thank Dr Junken Aoki (Graduate School of Pharmaceutical Science, Tohoku University, Japan) for providing the monoclonal anti-autotaxin antibody. FUNDING ======= This work was supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan. [^1]: ^1^These authors contributed equally to this work.

{ "pile_set_name": "PubMed Central" }

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"production", "direct", "production", "equimolar", "amounts", "LPA", "choline", "LPC", "lysophosphatidylcholine", "catalysed", "lysoPLD", "lysophospholipase", "physiological", "importance", "LPA", "production", "serum", "lysoPLD", "suggested", "Tokumura", "et", "al", "approximately", "years", "ago", "circulating", "blood", "LPA", "modulates", "blood", "pressure", "maintenance", "pregnancy", "maturation", "embryonic", "vessel", "systems", "Previously", "secreted", "protein", "lysoPLD", "activity", "purified", "plasma", "serum", "found", "identical", "molecule", "known", "autotaxin", "Autotaxin", "ectonucleotide", "originally", "cloned", "tumour", "cell", "factor", "recognized", "nucleotide", "pyrophosphatase", "basis", "primary", "structure", "vitro", "enzymatic", "characteristics", "LPC", "bind", "albumin", "lipoproteins", "released", "circulation", "liver", "may", "generated", "acyltransferase", "Autotaxin", "might", "hydrolyse", "LPCs", "substrates", "LPA", "accumulated", "levels", 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"high", "lysoPLD", "activity", "P", "whereas", "FLAG", "FLAG", "However", "slight", "increase", "lysoPLD", "activity", "FLAG", "observed", "Testing", "LysoPLD", "activity", "Gα", "subfamily", "members", "effect", "Gα", "subtypes", "black", "bar", "n", "grey", "bar", "n", "horizontally", "striped", "bar", "n", "vertically", "striped", "bar", "n", "empty", "vector", "Cont", "white", "bar", "n", "overexpressed", "cells", "tagged", "proteins", "purified", "one", "dish", "Levels", "Gα", "subfamily", "proteins", "top", "panel", "Gβ", "proteins", "middle", "panel", "detected", "using", "antibodies", "Note", "migrates", "higher", "molecular", "mass", "others", "bands", "size", "present", "control", "experimental", "samples", "Bottom", "panel", "relative", "choline", "production", "μl", "purified", "enzyme", "incubated", "lysoPAF", "compared", "detected", "colorimetric", "assay", "P", "compared", "Results", "experiment", "repeated", "several", "times", "similar", "results", "B", "cells", "pretreated", "control", "siRNA", "mouse", "siRNAs", "Levels", "endogenous", "top", "panel", "actin", "middle", "panel", "detected", "using", "antibodies", "Bottom", "panel", "lysoPLD", "activity", "crude", "microsomal", "fraction", "μg", "cells", "pretreated", "control", "siRNA", "white", "bar", "n", "siRNAs", "black", "bar", "n", "analysed", "colorimetric", "assay", "incubation", "mM", "lysoPAF", "min", "shown", "Figure", "fig", "B", "pretreatment", "siRNAs", "clearly", "decreased", "endogenous", "cells", "However", "lysoPLD", "activity", "crude", "microsomal", "fraction", "changed", "compared", "control", "crude", "microsome", "speculated", "undetermined", "G", "proteins", "contaminated", "autotaxin", "may", "responsible", "high", "endogenous", "lysoPLD", "activity", "lysoPLD", "activity", "interested", "protein", "may", "masked", "DISCUSSION", "interested", "molecularly", "identify", "lysoPLD", "activity", "reported", "previously", "Two", "proteins", "rat", "brain", "exhibiting", "lysoPLD", "activity", "faint", "kDa", "kDa", "bands", "gel", "electrophoresis", "observed", "following", "method", "reported", "previously", "purification", "shown", "Figure", "fig", "kDa", "protein", "present", "lower", "levels", "first", "recognized", "potentially", "important", "enzymatic", "properties", "fractions", "containing", "proteins", "including", "optimum", "pH", "substrate", "specificity", "cation", "requirement", "different", "secreted", "protein", "lysoPLD", "activity", "autotaxin", "present", "study", "analysed", "kDa", "kDa", "proteins", "MS", "identified", "heterotrimeric", "G", "protein", "subunits", "respectively", "overexpressed", "cells", "purified", "purified", "subunits", "exhibited", "lysoPLD", "activity", "Figure", "fig", "Mutations", "conserved", "amino", "acid", "residues", "known", "important", "GTPase", "activity", "interaction", "nucleotide", "phosphates", "significantly", "reduced", "lysoPLD", "activity", "Figure", "fig", "Furthermore", "highly", "purified", "FLAG", "protein", "ectopically", "expressed", "cells", "clearly", "hydrolysed", "LPC", "LPA", "whereas", "mutant", "form", "showed", "significantly", "less", "lysoPLD", "activity", "Figure", "fig", "calculated", "K", "V", "values", "lysoPAF", "enzyme", "obtained", "cells", "μM", "per", "mg", "respectively", "Figures", "fig", "B", "fig", "C", "values", "similar", "enzyme", "purified", "rat", "brain", "K", "μM", "V", "per", "mg", "Although", "detected", "silver", "staining", "main", "protein", "purified", "lysoPLD", "rat", "brain", "likely", "main", "component", "lysoPLD", "activity", "following", "four", "reasons", "FLAG", "mutant", "exhibited", "significant", "lysoPLD", "activity", "Figure", "fig", "B", "ii", "FLAG", "lower", "amounts", "exhibited", "lysoPLD", "activity", "comparable", "Figure", "fig", "iii", "FLAG", "FLAG", "exhibit", "high", "lysoPLD", "activity", "FLAG", "Gβ", "Figure", "fig", "iv", "overexpressed", "insect", "cells", "exhibit", "lysoPLD", "activity", "Figure", "fig", "However", "potential", "importance", "lysoPLD", "activity", "may", "possible", "always", "respect", "lysoPLD", "activity", "results", "reveal", "new", "function", "well", "known", "multifunctional", "signal", "transduction", "proteins", "enzyme", "lysoPLD", "activity", "Although", "obtained", "microsomal", "fraction", "exhibited", "significant", "lysoPLD", "activity", "purified", "enzyme", "supernatants", "g", "centrifugation", "lysoPLD", "activity", "Figure", "fig", "level", "lysoPLD", "activity", "purified", "FLAG", "microsomal", "fraction", "dependent", "cell", "type", "cells", "Figure", "fig", "results", "suggest", "modification", "kind", "activator", "present", "microsome", "important", "lysoPLD", "activity", "However", "also", "possible", "proteins", "supernatants", "misfolded", "translation", "GPCRs", "enhance", "exchange", "G", "protein", "α", "subunit", "thereby", "generating", "active", "GTPbound", "Gα", "subunit", "followed", "dissociation", "receptor", "Gβγ", "subunit", "onset", "termination", "G", "protein", "signalling", "determined", "length", "time", "spent", "active", "state", "GTP", "hydrolysed", "GDP", "intrinsic", "GTPase", "activity", "Gα", "subunit", "resulting", "Gα", "subunit", "Gβγ", "subunit", "termination", "signalling", "GTPases", "rates", "exchange", "GDP", "GTP", "GTPase", "activity", "quite", "low", "However", "GTPase", "activity", "dramatically", "increased", "vitro", "incubation", "cholinergic", "receptor", "GAPs", "RGS", "regulator", "G", "protein", "signalling", "proteins", "phospholipase", "C", "present", "study", "found", "evidence", "lysoPLD", "activity", "exhibited", "presence", "Gβ", "endogenous", "Gβ", "FLAG", "Figure", "fig", "speculate", "heterotrimeric", "form", "G", "protein", "subunits", "may", "exhibit", "lysoPLD", "activity", "Previously", "GTPγS", "found", "bound", "purified", "activators", "additionally", "purified", "phosphoinositide", "phospholipase", "C", "bovine", "brain", "could", 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1. Background {#sec61617} ============= Laparoscopic cholecystectomy (LC) is an established, universally accepted procedure for cholelithiasis treatment, to decrease patient discomfort. This procedure is well tolerated by patients, however, an increased incidence of postoperative nausea and vomiting (PONV) predisposes patients to psychological distress increase and delayed recovery and discharge times ([@A16710R1], [@A16710R2]). Although the PONV has multifactorial etiology, one its main predisposing factors is postoperative pain ([@A16710R1]). Opioids are routinely used for pain relief and control, but they increase the incidence of PONV by opioid receptor stimulation in the trigger zone ([@A16710R3]). It seems that some surgical and anesthetic interventions can decrease postoperative pain and subsequently the opioid use, leading to a reduced level of PONV. Pain following LC can be divided into three major groups: 1. Parietal pain caused by surgical incision; 2. Visceral pain originating from gallbladder bed and caused by visceral manipulation during the operation and finally 3. Shoulder pain secondary to distension of diaphragm by neuropraxia of phrenic nerve ([@A16710R4], [@A16710R5]). Due to the different pathophysiological mechanisms of these pain components, different local anesthetic interventions have been developed to reduce postoperative pain, like peritoneal infiltration, instillation into the peritoneal cavity or subdiaphragmatic area, intraperitoneal spraying above the gallbladder and combined peritoneal-peritrocal ropivacaine ([@A16710R6]-[@A16710R12]). However, there is controversy about the characteristics of the pain components and also about the pain reducing the effects of intraperitoneal or peritrocal local anesthetics. 2. Objectives {#sec61618} ============= The present study aims to evaluate the efficacy of intraperitoneal local anesthetic bupivacaine on PONV, after managing the visceral and shoulder pains. To avoid any confusion in different components of pain, we decided to eliminate parietal pain by injection bupivacaine into the abdominal wall, in both groups. So that we could fully concentrate on visceral and shoulder pain and to see if IP bupivacaine injection could affect the opioid demand by influencing visceral and shoulder pain. Intra-abdominal dull pain that cannot exactly be located is considered as visceral pain, while the sharp pain felt in the abdominal wall is deemed as the parietal pain. 3. Patients and Methods {#sec61619} ======================= This was a double-blind, randomized clinical trial conducted on 66 patients of both sexes (aged 20 to 60 years), September 2010 to April 2011. All patients were registered for LC operation, according to the American Society of Anesthesiologists physical status I and II. All studies were performed in accordance with the ethical guidelines, set by the ethical committee of Faculty of Medical Sciences, Kermanshah University of Medical Sciences (Iran). The study was registered in Iranian Registry of Clinical Trials (IRCT13880112946N1). All patients were well informed of the study and all signed a written consent. The exclusion criteria were obesity (body mass index higher than 30 kg/m^2^), history of opioids abuse, antiemetic and steroids administrations within 24 hours prior to the surgery, history of motion sickness or PONV and patients with migraine whose surgery changed to open cholecystectomy. Patients were randomly assigned to two groups using a computer based randomization method. All patients received the same anesthetic agent and protocol. General anesthesia was induced by 0.2 µg/kg sufentanil, followed by 3-5 mg/kg thiopental and 0.6 mg/kg atracurium to facilitate tracheal intubation. Anesthesia was maintained with propofol infusion (200 µg/kg/minute) and injection of 0.1 µg/kg sufentanil, every 15 minutes. Patients in bupivacaine group received 20 mL of bupivacaine in the gallbladder bed, after abdominal CO~2~ insufflation, as well as 20 mL of bupivacaine 0.25% in the gallbladder bed, after resection of gallbladder, whereas control group (n = 33) did not get such injections. During operation, patients were placed in the reverse Trendelenburg position. Pneumoperitoneum was created with a closed Veress needle technique and LC was performed using four trocars, placed in the standard position. The gallbladder was retracted via a supraumbilical trocar port. During laparoscopy, intra-abdominal pressure was maintained at 12 mmHg. CO~2~ was carefully evacuated at the end of the operation by manual compression of the abdomen with open trocars. For elimination of the parietal pain and concentration on the visceral and shoulder pains, both groups received local administration of 5 mL bupivacaine 0.25% at incision of each trocar and different layers of abdomen. For reversal of muscle relaxation, 40 µg/kg neostigmine and 20 µg/kg atropine were administered and patients were transferred to the post anesthesia care unit (PACU) after tracheal extubation. The incidence and severity of nausea and postoperative pain intensity at rest, when coughing and changing positions from supine to sitting were measured, using the visual analog scale (VAS) at one, two, three and four hours after the operation. The incidence of vomiting was evaluated by a "yes" or "no" question at the same time. At the first, second, third and fourth hours after surgery, when examining the intensity of pain and nausea, the patients were asked if they had vomiting. If the answer was positive, the frequency was recorded. All assessments were recorded by trained nurses who were blinded to the study and group assignments. Patients could request for rescue analgesia and antiemetic at any time after operation. Fifty milligram tramadol and 10 mg metoclopramide was intravenously administered as a rescue analgesic and antiemetic, respectively (rescue antiemetic treatment in VAS \> 3). The statistical analysis was performed using SPSS package (SPSS, Chicago, IL, USA, version 16). For statistical analysis of the demographic data and for comparison of the two groups, Chi square, Mann-Whitney U-test and the student t-test analyses were performed. 4. Results {#sec61620} ========== A total of 73 patients were assessed for eligibility, of which 66 patients were enrolled into the study. There were no differences among the groups with respect to patients\' characteristics and operative data. Bupivacaine and control groups did not differ significantly in mean age (39.7 ± 11.3 vs. 42.39 ± 12 years; P = 0.368) and intraoperative opioid usage (32.61 ± 7.92 vs. 30.88 ± 7.20 microgram sufentanil; P = 0.381). Female patients comprised 97.1% of the bupivacaine group and 85.5% of the control group, indicating no significant difference in this regard (P = 0.166). The means values for the pain VAS during resting, coughing and changing positions for four hours following operation are presented in [Figure 1](#fig12236){ref-type="fig"}. ![Comparison of Pain at Rest in the Two Groups](aapm-04-03-16710-i001){#fig12236} The highest pain levels were experienced during the first postoperative hour and these levels were gradually diminished with time. No significant difference was found between the two groups in terms of incidence of nausea ([Figure 2](#fig12237){ref-type="fig"}) and vomiting ([Figure 3](#fig12238){ref-type="fig"}). Vomiting occurred for six patients in the bupivacaine group and seven in the control group, who were then treated with 10 mg metoclopramide. Furthermore, both groups were similar with respect to opioid consumption, during the first postoperative four hours. ![Comparison of Postoperative Nausea in Bupivacaine and Control Groups](aapm-04-03-16710-i002){#fig12237} ![Comparison of Postoperative Vomiting in Bupivacaine and Control Groups](aapm-04-03-16710-i003){#fig12238} There was no significant difference in parietal pain between the two groups. Visceral and shoulder pains showed significant differences between the control and bupivacaine groups only in 4th post-operative hours. Although the viseral and shoulder pain intensity at the other time was lower in Bupivacaine group. That was not statistically significant ([Table 1](#tbl15741){ref-type="table"}). ###### Pain Component Data in the Two Groups Post-Operative Pain Control Group, (n = 33) Bupivacaine Group, (n = 33) P Value ---------------------- ------------------------- ----------------------------- --------- **Shoulder pain, h** 1 11 8 0.587 2 14 8 0.191 3 10 8 0.783 4 18 10 0.046 **Visceral Pain** 1 22 23 0.792 2 24 19 0.301 3 23 23 0.999 4 22 14 0.048 **Parietal Pain** 1 2 0 0.492 2 1 0 0.999 3 2 0 0.492 4 1 0 0.999 Variables related to analgesic consumption in the bupivacaine and control group is shown in [Table 2](#tbl15742){ref-type="table"}. ###### Comparison of Variables Related to Analgesic Consumption in the Bupivacaine and Control Group ^[a](#fn12277){ref-type="table-fn"}^ variables Control Group Bupivacaine Group P Value ---------------------------------------------------------------- --------------- ------------------- --------- **First analgesic request, h** 1.68 ± 0.98 1.27 ± 0.90 0.126 **Pain intensity based on VAS in first analgesic request** 7.40 ± 3.49 7.04 ± 3.24 0.711 **Mean dose of received analgesic after surgery, mg tramadol** 57.14 ± 29.55 64.1 ± 45.69 0.435 ^a^ Data are presented as Mean ± SD. 5. Discussion {#sec61621} ============= This randomized, double-blind, placebo-controlled trial aimed to investigate the effects of IP administration of bupivacaine on visceral and shoulder pain, as well as its effects on PONV after LC. Our results showed that IP administration of 20 mL bupivacaine 0.25% in the gallbladder bed after abdominal CO~2~ insufflation and also after resection of gallbladder did not reduce the patient's demand for extra opioids and had no effect on PONV. The origin of pain and PONV, following LC is not entirely cleared, but it is probably multifactorial. Insufflation of CO~2~, resulting in stretching of the peritoneum, residual pneumoperitoneum after CO~2~ insufflation, peritoneum distension, diaphragm irritation and visceral organ irritation and manipulation have been reported to influence the severity of pain and PONV ([@A16710R5], [@A16710R13]). Although, in several studies using local anesthetics in peritoneal cavity reduced postoperative pain and PONV, the use of different pain control techniques, including the application of intraperitoneal and/or peritrocar local anesthetics has still remained controversial ([@A16710R6], [@A16710R12], [@A16710R14]). Visceral and shoulder pain were significantly different at the 4th post-operative hour between the two groups. Some studies have shown that parietal pain can be blocked by infiltration of local anesthetic agents at incisions and visceral and shoulder pains by IP administration of local anesthetics ([@A16710R5]-[@A16710R9]). A meta-analysis performed by Boddy et al. showed the efficacy of IP local anesthetics in relieving post-laparoscopic cholecystectomy pain, without side effect of analgesic toxicity ([@A16710R15]). Chou YJ et al. demonstrated that IP bupivacaine administration, is effective on the visceral pain reduction, both after trocar incision and at the end of the surgery whereas, with no efficient reduction in abdominal and right shoulder pain and opioid demand ([@A16710R16]). In this study, parietal pain in both groups could be diminished through trocar bupivacaine administration to assess the effects of visceral and shoulder pains on PONV following LC. Our findings showed that IP administration of bupivacaine reduced both visceral and shoulder pains but had no effect on reduction of rescue analgesic. On the other hand, IP bupivacaine had not any impact on reduction of PONV and the incidence of nausea and vomiting were similar in both groups. Based on the findings of this study, it can be concluded that use of opioids has more effects than pain itself to increase PONV. It has been shown that opioid use increases the incidence of PONV by stimulation of opioid receptor in chemoreceptor trigger zone (CTZ) ([@A16710R3]). Due to the presence of the blood-brain barrier at the CTZ, neurons at this region are stimulated by opioids present in the systemic circulation ([@A16710R17]). Furthermore, opioid receptors are involved in reduction of bowel motility. This effect results in bowel distension, increased GI emptying time and constipation, leading to visceral chemoreceptors and mechanoreceptors stimulation. This effect is often responsible for nausea and vomiting in patients receiving opioid drugs ([@A16710R17]-[@A16710R20]). Findings of this study demonstrated that 20 mL intraperitoneal bupivacaine 0.25% administration at the beginning and also at the end of the laparoscopic cholecystectomy process, reduced only visceral and shoulder pains at the 4th postoperative hour, but had no effect on reducing neither PONV, nor opioid requirement, during the first four postoperative hours. However, further studies are needed on larger populations with different doses and concentrations of bupivacaine and also combination of bupivacaine and other analgesic drugs, to gain the maximum benefits of intraperitoneal analgesic in postoperative pain treatment. We would like to sincerely thank Shokoufe Khodakarami, Saeedeh Pourchamani, Shiva Azami and Parastu Yari kia for their help and kind cooperation. **Authors' Contributions:**Mitra Yari took part in study concept and design and participated in manuscript preparation. Parisa Golfam prepared the manuscript and helped with study design and submission of the manuscript. Bahman Roshani helped in manuscript preparationand Nahid Nazari collected the data. **Funding/Support:**Kermanshah University of Medical Sciences, Kermanshah, Iran

{ "pile_set_name": "PubMed Central" }

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22,239

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"originating", "gallbladder", "bed", "caused", "visceral", "manipulation", "operation", "finally", "Shoulder", "pain", "secondary", "distension", "diaphragm", "neuropraxia", "phrenic", "nerve", "Due", "different", "pathophysiological", "mechanisms", "pain", "components", "different", "local", "anesthetic", "interventions", "developed", "reduce", "postoperative", "pain", "like", "peritoneal", "infiltration", "instillation", "peritoneal", "cavity", "subdiaphragmatic", "area", "intraperitoneal", "spraying", "gallbladder", "combined", "ropivacaine", "However", "controversy", "characteristics", "pain", "components", "also", "pain", "reducing", "effects", "intraperitoneal", "peritrocal", "local", "anesthetics", "Objectives", "present", "study", "aims", "evaluate", "efficacy", "intraperitoneal", "local", "anesthetic", "bupivacaine", "PONV", "managing", "visceral", "shoulder", "pains", "avoid", "confusion", "different", "components", "pain", "decided", "eliminate", "parietal", "pain", "injection", "bupivacaine", "abdominal", "wall", "groups", "could", "fully", "concentrate", "visceral", "shoulder", "pain", "see", "IP", "bupivacaine", "injection", "could", "affect", "opioid", "demand", "influencing", "visceral", "shoulder", "pain", "dull", "pain", "exactly", "located", "considered", "visceral", "pain", "sharp", "pain", "felt", "abdominal", "wall", "deemed", "parietal", "pain", "Patients", "Methods", "randomized", "clinical", "trial", "conducted", "patients", "sexes", "aged", "years", "September", "April", "patients", "registered", "LC", "operation", "according", "American", "Society", "Anesthesiologists", "physical", "status", "II", "studies", "performed", "accordance", "ethical", "guidelines", "set", "ethical", "committee", "Faculty", "Medical", "Sciences", "Kermanshah", "University", "Medical", "Sciences", "Iran", "study", "registered", "Iranian", "Registry", "Clinical", "Trials", "patients", "well", "informed", "study", "signed", "written", "consent", "exclusion", "criteria", "obesity", "body", "mass", "index", "higher", "history", "opioids", "abuse", "antiemetic", "steroids", "administrations", "within", "hours", "prior", "surgery", "history", "motion", "sickness", "PONV", "patients", "migraine", "whose", "surgery", "changed", "open", "cholecystectomy", "Patients", "randomly", "assigned", "two", "groups", "using", "computer", "based", "randomization", "method", "patients", "received", "anesthetic", "agent", "protocol", "General", "anesthesia", "induced", "sufentanil", "followed", "thiopental", "atracurium", "facilitate", "tracheal", "intubation", "Anesthesia", "maintained", "propofol", "infusion", "injection", "sufentanil", "every", "minutes", "Patients", "bupivacaine", "group", "received", "mL", "bupivacaine", "gallbladder", "bed", "abdominal", "insufflation", "well", "mL", "bupivacaine", "gallbladder", "bed", "resection", "gallbladder", "whereas", "control", "group", "n", "get", "injections", "operation", "patients", "placed", "reverse", "Trendelenburg", "position", "Pneumoperitoneum", "created", "closed", "Veress", "needle", "technique", "LC", "performed", "using", "four", "trocars", "placed", "standard", "position", "gallbladder", "retracted", "via", "supraumbilical", "trocar", "port", "laparoscopy", "pressure", "maintained", "mmHg", "carefully", "evacuated", "end", "operation", "manual", "compression", "abdomen", "open", "trocars", "elimination", "parietal", "pain", "concentration", "visceral", "shoulder", "pains", "groups", "received", "local", "administration", "mL", "bupivacaine", "incision", "trocar", "different", "layers", "abdomen", "reversal", "muscle", "relaxation", "neostigmine", "atropine", "administered", "patients", "transferred", "post", "anesthesia", "care", "unit", "PACU", "tracheal", "extubation", "incidence", "severity", "nausea", "postoperative", "pain", "intensity", "rest", "coughing", "changing", "positions", "supine", "sitting", "measured", "using", "visual", "analog", "scale", "VAS", "one", "two", "three", "four", "hours", "operation", "incidence", "vomiting", "evaluated", "yes", "question", "time", "first", "second", "third", "fourth", "hours", "surgery", "examining", "intensity", "pain", "nausea", "patients", "asked", "vomiting", "answer", "positive", "frequency", "recorded", "assessments", "recorded", "trained", "nurses", "blinded", "study", "group", "assignments", "Patients", "could", "request", "rescue", "analgesia", "antiemetic", "time", "operation", "Fifty", "milligram", "tramadol", "mg", "metoclopramide", "intravenously", "administered", "rescue", "analgesic", "antiemetic", "respectively", "rescue", "antiemetic", "treatment", "VAS", "statistical", "analysis", "performed", "using", "SPSS", "package", "SPSS", "Chicago", "IL", "USA", "version", "statistical", "analysis", "demographic", "data", "comparison", "two", "groups", "Chi", "square", "student", "analyses", "performed", "Results", "total", "patients", "assessed", "eligibility", "patients", "enrolled", "study", "differences", "among", "groups", "respect", "characteristics", "operative", "data", "Bupivacaine", "control", "groups", "differ", "significantly", "mean", "age", "years", "P", "intraoperative", "opioid", "usage", "microgram", "sufentanil", "P", "Female", "patients", "comprised", "bupivacaine", "group", "control", "group", "indicating", "significant", "difference", "regard", "P", "means", "values", "pain", "VAS", "resting", "coughing", "changing", "positions", "four", "hours", "following", "operation", "presented", "Figure", "fig", "Comparison", "Pain", "Rest", "Two", "Groups", "highest", "pain", "levels", "experienced", "first", "postoperative", "hour", "levels", "gradually", "diminished", "time", "significant", "difference", "found", "two", "groups", "terms", "incidence", "nausea", "Figure", "fig", "vomiting", "Figure", "fig", "Vomiting", "occurred", "six", "patients", "bupivacaine", "group", "seven", "control", "group", "treated", "mg", "metoclopramide", "Furthermore", "groups", "similar", "respect", "opioid", "consumption", "first", "postoperative", "four", "hours", "Comparison", "Postoperative", "Nausea", "Bupivacaine", "Control", "Groups", "Comparison", "Postoperative", "Vomiting", "Bupivacaine", "Control", "Groups", "significant", "difference", "parietal", "pain", "two", "groups", "Visceral", "shoulder", "pains", "showed", "significant", "differences", "control", "bupivacaine", "groups", "hours", "Although", "viseral", "shoulder", "pain", "intensity", "time", "lower", "Bupivacaine", "group", "statistically", "significant", "Table", "table", "Pain", "Component", "Data", "Two", "Groups", "Pain", "Control", "Group", "n", "Bupivacaine", "Group", "n", "P", "Value", "Shoulder", "pain", "h", "Visceral", "Pain", "Parietal", "Pain", "Variables", "related", "analgesic", "consumption", "bupivacaine", "control", "group", "shown", "Table", "table", "Comparison", "Variables", "Related", "Analgesic", "Consumption", "Bupivacaine", "Control", "Group", "variables", "Control", "Group", "Bupivacaine", "Group", "P", "Value", "First", "analgesic", "request", "h", "Pain", "intensity", "based", "VAS", "first", "analgesic", "request", "Mean", "dose", "received", "analgesic", "surgery", "mg", "tramadol", "Data", "presented", "Mean", "SD", "Discussion", "randomized", "trial", "aimed", "investigate", "effects", "IP", "administration", "bupivacaine", "visceral", "shoulder", "pain", "well", "effects", "PONV", "LC", "results", "showed", "IP", "administration", "mL", "bupivacaine", "gallbladder", "bed", "abdominal", "insufflation", "also", "resection", "gallbladder", "reduce", "patient", "demand", "extra", "opioids", "effect", "PONV", "origin", "pain", "PONV", "following", "LC", "entirely", "cleared", "probably", "multifactorial", "Insufflation", "resulting", "stretching", "peritoneum", "residual", "pneumoperitoneum", "insufflation", "peritoneum", "distension", "diaphragm", "irritation", "visceral", "organ", "irritation", "manipulation", "reported", "influence", "severity", "pain", "PONV", "Although", "several", "studies", "using", "local", "anesthetics", "peritoneal", "cavity", "reduced", "postoperative", "pain", "PONV", "use", "different", "pain", "control", "techniques", "including", "application", "intraperitoneal", "peritrocar", "local", "anesthetics", "still", "remained", "controversial", "Visceral", "shoulder", "pain", "significantly", "different", "hour", "two", "groups", "studies", "shown", "parietal", "pain", "blocked", "infiltration", "local", "anesthetic", "agents", "incisions", "visceral", "shoulder", "pains", "IP", "administration", "local", "anesthetics", "performed", "Boddy", "et", "al", "showed", "efficacy", "IP", "local", "anesthetics", "relieving", "cholecystectomy", "pain", "without", "side", "effect", "analgesic", "toxicity", "Chou", "YJ", "et", "al", "demonstrated", "IP", "bupivacaine", "administration", "effective", "visceral", "pain", "reduction", "trocar", "incision", "end", "surgery", "whereas", 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"results", "bowel", "distension", "increased", "GI", "emptying", "time", "constipation", "leading", "visceral", "chemoreceptors", "mechanoreceptors", "stimulation", "effect", "often", "responsible", "nausea", "vomiting", "patients", "receiving", "opioid", "drugs", "Findings", "study", "demonstrated", "mL", "intraperitoneal", "bupivacaine", "administration", "beginning", "also", "end", "laparoscopic", "cholecystectomy", "process", "reduced", "visceral", "shoulder", "pains", "postoperative", "hour", "effect", "reducing", "neither", "PONV", "opioid", "requirement", "first", "four", "postoperative", "hours", "However", "studies", "needed", "larger", "populations", "different", "doses", "concentrations", "bupivacaine", "also", "combination", "bupivacaine", "analgesic", "drugs", "gain", "maximum", "benefits", "intraperitoneal", "analgesic", "postoperative", "pain", "treatment", "would", "like", "sincerely", "thank", "Shokoufe", "Khodakarami", "Saeedeh", "Pourchamani", "Shiva", "Azami", "Parastu", "Yari", "kia", "help", "kind", "cooperation", "Authors", "Contributions", "Mitra", "Yari", "took", "part", "study", "concept", "design", "participated", "manuscript", "preparation", "Parisa", "Golfam", "prepared", "manuscript", "helped", "study", "design", "submission", "manuscript", "Bahman", "Roshani", "helped", "manuscript", "preparationand", "Nahid", "Nazari", "collected", "data", "Kermanshah", "University", "Medical", "Sciences", "Kermanshah", "Iran" ]

Background ========== Type 2 diabetes (T2D) is a chronic disorder of carbohydrate, fat and protein metabolism. The prevalence of diabetes is increasing worldwide. According to the recent World Health Organization (WHO) estimates, more than 180 million people worldwide have diabetes. This number is likely to be more than double by 2030. There will be 300 million people with diabetes by the year 2025 \[[@B1]\]. It is estimated that the developing countries will bear the brunt of diabetes epidemics in the 21st century \[[@B1],[@B2]\]. Mounting evidence indicates that obesity along with increased rates of other chronic inflammatory diseases, such as hypertension (HT) and cardiovascular diseases (CVD), are highly associated with T2D \[[@B3]\]. How all theses complications arise and manifest together is yet to be solved. Thus, understanding the underlying molecular mechanisms of T2D is essential for developing more targeted and effective therapies and preventive approaches against diabetes-related complications. The integration of computational biology and \"omics\" science facilitates an understanding of the mechanisms of diseases in an integrative way, including elucidation of complex networks of genes and proteins along with their regulatory networks \[[@B4]-[@B7]\]. Protein-protein interactions (PPI) regulate nearly every living process. Availability of high-throughput genomic data has enabled unprecedented views of gene and protein co-expression, co-regulations and interactions in cellular systems. The approach of this present study was to build an integrated network model system comprised of high-throughput genomic data along with associated computational analysis that would enable predictions of systems level view of normal and aberrant genes, their connections and their functions- to elucidate the underlying molecular mechanisms. Herein we mined genes differentially regulated through T2D and their \"wiring\" which will provide a more complete understanding of the overall gene network topology and their roles in disease progression. By elucidating these networks, exploring how they have been linked and characterizing how they have evolved- novel disease markers can be identified. In this present study, by analyzing the genes related to T2D, HT and OBS, a highly regulated gene-disease integrated network model was developed. PPI sub-networks containing the hub genes involved in T2D-associated signaling pathways revealed that the oxidative stress mediated regulation cascade, is the link, associated with the mechanism of pathogenesis of type 2 diabetes, hypertension and other inflammatory diseases such as obesity. Methods ======= The present work has developed gene interaction network models, comprised of high-throughput genomic and proteomic data for type 2 diabetes (T2D). The overall strategy of the integrated network model generation has been illustrated in Figure [1](#F1){ref-type="fig"}. The network model has been built in three successive stages. The first stage consisted of the \"parts list\" generation using biomedical literature mining. The second stage was the topological model generation by combining all the pooled genes and their interactions from all available interaction databases. The co-regulated and co-expression data fetched helped to identify the involved genes, proteins and their interactions. In third and final stage the fetched interaction data were filtered against experimentally validated interaction (e.g., yeast 2 hybrid, Chip- chip interaction, immuno-coprecipitation, arrays co-expression) data sets and the functional annotations were analyzed using computational approaches. By identifying the genes, the pathways they are involved in and the interactions they are part of, using a systems approach; the integrated networks of T2D have been generated considering all experimentally verified PPI data of the regulating genes. Finally from the integrated network systems the \'hubs\' or key genes that play central roles in these diseases have been identified. Interestingly along the way of building up the network for T2D, gene networks of other related diseases like obesity and hypertension were also generated. The aim of this study was to identify the key switches of regulation and from that interpreting how these chronic diseases correlate and co-occur. ![**Concept and overall strategy for generating the integrated network of T2D**.](1755-8794-3-45-1){#F1} Stage 1: Initial disease related gene pool generation through extensive text mining ----------------------------------------------------------------------------------- The most important collection of scientific publications is PubMed <http://www.ncbi.nlm.nih.gov/pubmed>, which currently contains more than 19 million citations of biomedical articles from MEDLINE and Life Science journals. Citations include links to full-text articles from PubMed Central (PMC) <http://www.ncbi.nlm.nih.gov/pmc/> or publisher web sites. Currently the number of publications is growing by more than 500K documents per year. PMC is the U.S. National Institutes of Health\'s (NIH) free digital archive of biomedical and Life Sciences journal literature serving the critical role of providing access to published literature toward the first step in the synthesis and translation of genomic research in an easy and comprehensive way. PubMed basically supports keyword based searching and information Retrieval (IR). There have been a significant number of web-based text mining tools available to biologists to discover hidden relationships among biological entities. Some of them are quite effective in generating annotated relationships among graphs in the form of networks. AliBaba <http://alibaba.informatik.hu-berlin.de> is a tool that is capable of automatically and interactively extracting the most valuable information and graphically summarizing search results from PubMed \[[@B8]\]. It uses a dictionary-based approach for recognizing biomedical objects. Dictionaries consist of regular expressions depicting terms and spelling variations. The dictionaries are collected from different sources. To find associations among entities, AliBaba uses two different techniques in parallel: pattern matching and co-occurrence filtering. It mines relationships among cells, diseases, drugs, proteins, species and tissues based on a user query involving text key terms. Thus, AliBaba can be used to turn unstructured text into structured data records \[[@B8]\]. Specifically, for increasing the accuracy and efficiency for discovering relationships between important biological entities, e.g., protein-to-disease associations- the present study combines manually locating and querying information about disease genes entity from the biomedical literature together with automated computational tools to identify the genes and gene products by computationally capturing the related knowledge embedded in textual data. Stage 2: Using the pooled disease genes protein-protein interaction (PPI) maps construction ------------------------------------------------------------------------------------------- PPI maps have considerable impact on the discovery and synthesis of molecular networks. Thus, generating human protein interaction maps has become an important tool in biomedical research for the elucidation of molecular mechanisms and the identification of new modulators of disease processes. There have been a number of web-based tools available to biologists. The Unified Human Interactome database (UniHI, <http://www.unihi.org>) provides a comprehensive platform to query and access human protein-protein interaction (PPI) data \[[@B9]\]. The latest update of UniHI includes over 250,000 interactions between \~22,300 unique proteins collected from 14 major PPI sources \[[@B9]\]. However, this amount of data has its challenges. Even searches with a small number of query proteins can lead to large, highly connected and often unstructured networks (frequently referred to as \'hairballs\'). UniHi has integrated separate PPI resources to provide a comprehensive platform for querying the human interactome (Figure [2](#F2){ref-type="fig"}). UniHI is not intended to replace single databases, but to offer a convenient single portal access to human protein interaction data for the biomedical research community. Additionally, it allows the identification of network topologies which would not be detectable if PPI resources were examined separately. ![**Steps of network model generation**.](1755-8794-3-45-2){#F2} Pathway information can provide highly useful clues about the functions and dynamics of interactions. Especially for the elucidation of local network structures, knowledge of interrelated pathways can be of crucial importance. UniHI provides the possibility to examine the intersection of canonical pathways from Kyoto Encyclopaedia of Genes and Genomes (KEGG) with the extracted networks \[[@B10]\]. UniHI Scanner does not only show the proteins included in the pathway but also the KEGG annotation of the interactions (e.g. phosphorylation, activation or inhibition) between nodes \[[@B11]\]. In this way, it enables researchers to detect possible modifiers of pathways as well as proteins involved in the cross-talk between pathways and users can switch between the graphical display of the complete network and the intersection with selected pathways \[[@B11]\]. Stage 3: Filtering and validation of the PPI data ------------------------------------------------- Advances in recent genome-wide interactome projects have generated a wealth of PPI data. In order to understand the complexity, it is essential to extract meaningful information in the context of physiological systems. This necessitates identification of not only the function of individual proteins, but also to validate the physical interactions and biological processes in which they participate. The emergence of large scale protein-protein interaction maps has opened up new possibilities in systematically surveying and studying the underlying biological system. UniHI (the Unified Human Interactome database, <http://www.unihi.org>) integrates protein interaction data with pathway data from the Human Gene Expression Atlas \[[@B11]\]. In UniHI, PPI maps have been assembled using eight publicly available large-scale interaction maps: three literature-based, three orthology-based and two Y2H-based. Interactions in different maps are compared. For normalization of the intersections however, only the number of interactions between common proteins is used. Thus, the *interaction overlap*is defined as the average percentage of shared interactions between common proteins \[[@B12]\]. In UniHI, maps are subsequently clustered based on the interaction overlap. For all comparisons, it is notably larger than zero, which is the expected value for comparison of random maps. The observed concurrence of interaction maps does not occur merely by chance as it is validated by two permutation tests for pair-wise comparisons of graphs, where the observed overlap of interactions generated is highly significant for all comparisons (*P*\< 0.01) \[[@B12]\]. To measure the conservation of connectivity between pairs of networks, UniHI correlates the number of interactions of proteins in the two networks using Spearman correlation for the set of common proteins \[[@B12]\]. A high correlation between two maps signifies that the interaction-rich (interaction-poor) proteins in one map are also interaction-rich (interaction-poor) in the other map. Finally, for the functional coherency of maps, UniHI employs the gene annotations available in GO \[[@B12]\]. Using UniHI, users can filter interacting proteins by requiring a minimum expression threshold, and the PPI network retrieved from UniHI can be reduced to include only highly expressed proteins or extended to include lowly expressed proteins. Additionally, the PPI resource to be queried can be specified. In this work, the fetched interactions data have been filtered against experimentally validated interaction (yeast 2 hybrid, Chip-chip interaction, immuno-precipitation, arrays co-expression) datasets. Integrated network visualization and identifying the \'hubs\' ------------------------------------------------------------- Flexible visualization is a crucial prerequisite for the display and evaluation of network structures. UniHI \[[@B9]\] provides users to switch between the graphical display of the complete network and the intersection with selected pathways. ***Cytoscape***<http://www.cytoscape.org/> is another open source bioinformatics software platform for visualizing molecular interaction networks and biological pathways and integrating these networks with annotations, gene expression profiles and other state data \[[@B13]\]. Although Cytoscape was originally designed for biological research, now it is a general platform for complex network analysis and visualization. Cytoscape *core*distribution provides a basic set of features for data integration and visualization. A variety of layout algorithms are available, including cyclic, tree, force-directed, edge-weight, and yFiles organic layouts. Additional features are available as *plugin**s***which are available for network and molecular profiling analyses, new layouts, additional file format support, scripting, and connection with databases \[[@B13]\]. Cytoscape supports a lot of standard network and annotation file formats including: SIF, GML, XGMML, BioPAX, PSI-MI, SBML, OBO, and Gene Association. In this work along with UniHI \[[@B11]\], the interactions among the differentially expressed genes were visualized by using Cytoscape 2.5.1 software \[[@B13]\]. Candidate genes, which were common within various diseases, were identified. Network-neighbours around the fetched genes of T2D, HT, OBS and ROS been studied extensively to identify the \'hubs\'. Genes with thirty or more gene degree in the PPI network were considered as hubs. Results ======= Gene pool generation through extensive text mining -------------------------------------------------- Candidate genes, responsible for the diseases (T2D, HT, OBS, ROS) were extracted and sorted out both manually (through literature review) and by using text-mining soft wares (Figure [3](#F3){ref-type="fig"}). At the beginning, the present work only focused on to understand the correlation between type 2 diabetes and hypertension (HT). But later on, ROS (Reactive Oxygen Species) and obesity (OBS) were also considered as they were found to be highly related to first two diseases. So, at the end candidate genes, responsible for all the diseases were pooled through text-mining. A number of genes for T2D, HT, OBS and ROS were identified. Among them the candidate genes were selected based on the following criteria\'s: i) Genes experimentally proved to be associated with any one of the above mentioned diseases, ii) Genes experimentally proved to be associated with two or more concerned diseases, iii) Genes predicted computationally to be associated with any individual disease, iv) Genes predicted computationally to be associated with two or more concerned diseases and v) Genes found in KEGG pathways database to be associated with any individual disease. So basically, a gene-disease network is generated by defining two genes as \'connected\' if they have been studied for association with the same disease(s). The identified genes were further verified through literature review to get insight about their functions and also with KEGG for annotation in different biological pathways. These genes were also confirmed with Phenopedia (HuGEpedia: an integrated, searchable knowledge base of genetic associations and human genome epidemiology) \[[@B14]\] which provides the total number of reported genes that have genetic association with T2D, HT and other related diseases (Additional file [1](#S1){ref-type="supplementary-material"}, [2](#S2){ref-type="supplementary-material"}). After this first stage of text mining using literature databases, the number of genes identified and pooled for: Type 2 Diabetes (T2D) \~ 257, Hypertension \~100, Obesity \~239 and ROS \~55. ![**Text mined data and the initial gene pool list**.](1755-8794-3-45-3){#F3} Finding Protein-Protein Interaction partners (PIPs) --------------------------------------------------- Genes do not act as individual units; they collaborate in overlapping pathways, the co-regulation of which is a hallmark for the disease pathogenesis. A simple enrichment analysis has been applied in order to characterize the T2D set on the network level including pathways and protein-protein interactions. The computational approach used in this study was based on functional links derived from co-expression and co-regulation profiles. The co-expressed and co-regulated genes generally have a higher likelihood of having a direct physical interaction. The gene interaction data used to build the network was based on direct physical interactions that are either experimentally derived (e.g., Y2H or co-affinity purification) or computationally predicted. In order to integrate pathway information and to derive cellular network information on the selected genes, functional annotation from pathway databases such as KEGG, Reactome, BioCyc \[[@B15]-[@B17]\], protein-protein interaction databases such as BIND, DIP, REACTOME, COCIT, HPRD BIN, CCSB, MDC and IntAct was added \[[@B18]\]. To facilitate the findings, UniHi (*United Human Interactome*) \[[@B9]\], a web based PIP finding tool was used. The present work tried to find significantly interconnected sub-networks to interrogate whether the specific pattern formed by a pre-specified list of genes is significant (Figure [4](#F4){ref-type="fig"}). At this stage the number of significant candidate genes of inflammation related diseases were T2D \~1231, HT \~1000, OBS \~1845 and ROS \~417. ![**Mined PPI data and graphical visualization using UniHI**.](1755-8794-3-45-4){#F4} Compiling of data and network visualization ------------------------------------------- Network subgraphs can be network modules, motif clusters, or other network neighborhoods. A network module can be defined as a subgraph consisting of highly interconnected nodes that may fulfill a particular biological function \[[@B19]-[@B21]\]. At this stage of network building, the pooled important common genes for various diseases were integrated into network models and visualized using UniHI \[[@B18]\], Cytoscape \[[@B13]\]. The models have been generated through several stages of screening where in subsequent steps the pooled data have been filtered, verified for authentication and most importantly, validated with experimentally derived data to make the output graph generated for different diseases meaningful and interpretable. This work constructed four disease networks (Additional file [2](#S2){ref-type="supplementary-material"}), each containing a set of proteins that are associated with the diseases. The hub nodes form the backbone of a network, they are considered to be an important measurement for the similarity between protein interaction networks. Moreover, backbone network has also been shown to be highly conserved in maintaining biological function of the cell \[[@B22]\]. Within the networks the \"hubs\" are defined as the protein nodes with degrees ≥ 15 in a disease network \[[@B22]\]. The generated model of T2D has predicted the highest number of hub proteins among the four diseases we studied so far, probably due to the fact that it has the highest number of genes that were used to construct the disease network (Additional file [3](#S3){ref-type="supplementary-material"}). Molecular linkage among T2D, HT, OBS and Inflammation ----------------------------------------------------- To investigate the molecular cross-talk within these interrelated disease mechanisms, the individual disease networks were looked thoroughly at their \'hubs\' to understand the underlying connections between genes and the disease mechanism. This was done in three phases. First, we developed the gene-disease network using the text miner Ali-Baba \[[@B8]\] (Figure [5](#F5){ref-type="fig"}). Thorough analyses of the gene patterns and their relatedness within different diseases, the way they were connected, the cross talk - a gene list was generated containing the connections in all four diseases. In the second phase, the PPI patterns around the selected hub genes were studied extensively using UniHI \[[@B9]\], validated the connections using Gene Ontology (GO) \[[@B23]\] and Genopedia and Phenopedia \[[@B14]\] databases for annotation (Figure [6](#F6){ref-type="fig"} and Additional file [4](#S4){ref-type="supplementary-material"}). And finally in the third phase, the selected genes were cross-validated and clustered using the pathway database KEGG \[[@B10]\] (Figure [7](#F7){ref-type="fig"}). During the whole process a list of 42 co-regulated genes were analysed and these candidate genes were significantly clustered in 19 pathways. The candidate genes were looked for common interaction partners and the highly integrated gene regulatory network was generated (Figure [8](#F8){ref-type="fig"}). ![**Cross-linkage between various diseases**.](1755-8794-3-45-5){#F5} ![**Main \'hub\' genes and connections**.](1755-8794-3-45-6){#F6} ![**Important signaling pathways integration with gene network**.](1755-8794-3-45-7){#F7} ![**Gene regulatory network around the highly inter-connected key hubs**.](1755-8794-3-45-8){#F8} Discussion ========== In this study, a text mining approach was used to identify the disease-related genes of T2D and HT. Along the way of querying, genes associated with oxidative stress (reactive oxygen species, ROS) and OBS was also included based on- the two diseases are \'connected\' if they have been studied for association with the same gene(s) hypothesis. These data were further analysed using various novel bioinformatics tools and resources to identify the key candidate hub genes associated with various diseases. Literature-based annotation analyses showed that all these diseases T2D, HT, OBS are somehow correlated with oxidative stress and inflammation (Figure [5](#F5){ref-type="fig"}). Further PPI studies, generated the disease-gene network within T2D, HT, OBS, ROS and Inflammation. In this work, the genes for inflammation were never pooled separately but from the cross-talk between the networks of T2D, HT and OBS, the strong co-regulated inflammation genes were identified. Although at this point it was not completely understandable how ROS or inflammation plays role in disease mechanism. Further pathway-based investigation, using both Genopedia \[[@B14]\] and KEGG \[[@B15]\] provided more insights about the roles of these genes in association with different disease phenotypes. This work predicted the involvement of about 20 enriched pathways, some of which are very commonly involved in diabetes and/or obesity like the insulin signaling, PPAR signaling, Adipocytokine signaling, MAPK signaling, Jak-STAT signaling pathways. But along them, the involvement of Wnt signaling, p53 signaling, ErbB signalling, TGF-beta signalling, Focal adhesion, toll-like receptor signaling pathways were predicted, whose associations are more widely linked with cancer metastasis (Figure [8](#F8){ref-type="fig"} and Additional file [4](#S4){ref-type="supplementary-material"}). From pathway analysis (Figure [7](#F7){ref-type="fig"}), the involvement of p53 or Wnt signaling pathway shed new light to focus at the PPI networks. After further filtering of the data (through network and/or PubMed interaction partners, IPs) along with cross validation through literature annotation, the most interesting 46 candidate \'hub\' genes were identified. Careful investigation around these \'hubs\' provide more meaningful insights about the cross- talk within gene-disease networks in terms of disease phenotype association with ROS and inflammation (Figure [6](#F6){ref-type="fig"}). From network analysis, common IPs revealed that all the diseases T2D, HT, OBS are linked via two common gene regulatory cascades: (i) EP300/TP53/MYC/CDKN1A/STAT3/CXCR4 and (ii) PPARG/PPARA/IGF1R/AKT1/LEP/LEPR. Basically, these regulatory routes connect insulin signaling pathway through oxidative stress and inflammation. From investigating the \'hub\' gene network connections and broadly studying the interconnection, one hypothesis could be, the components of the second routes can only be regulated through the first or vise-versa (Figure [8](#F8){ref-type="fig"}). To further investigate the hypothesis, and to address how the connections regulated within the network that could be associated with genesis of inter-connections between various diseases, the \'gene-links\' associating two or more hubs were identified. In course of the analysis, regulations around EP300, PPARG, PPARA, STAT3, IGF1R, CXCR4, MYC, TP53, CDNK1A, CDKN2A, PCAF, HIF1A, CREBBP, CEBPB, RELA, HSP90AA1, MAPK3, MAPK8, JAK2, JAK3, PTPN11, TFAP2A, LEP, TCF7L2, CTNNB1, FOXO1A, FOXO3A and GSK3B seem to play crucial roles in the regulation of T2D and other related disorders (Figure [8](#F8){ref-type="fig"}). Interestingly, the genes like GSK3B, PCAF, HIF1A, CREBBP, CEBPB, RELA, TCF7L2, CTNNB1, MYC, TP53, CDNK1A are known as important role players in Wnt and p53 signaling pathways, which would provide a putative link between T2D and certain types of cancer. The molecular events leading to β-cell failure in the diabetic environment, in particular high levels of glucose and free fatty acids, exert toxic effects on the β-cell \[[@B24]\]. A number of signaling pathways have been implicated in β-cell failure, including insulin signaling \[[@B25]\] and oxidative stress \[[@B26]\]. Ectopic overexpression of the Wnt target gene c-myc in mice has been shown to cause β-cell apoptosis and diabetes \[[@B27]\]. Also extensive recent investigations have revealed the existence of molecular crosstalk between insulin and Wnt signaling pathway \[[@B28]-[@B32]\]. A number of genetic studies have confirmed that TCF7L2, a direct downstream target of β-catenin of the Wnt pathway, to be involved in β-cell dysfunction and the etiology of T2D \[[@B24],[@B33]\]. There is clear evidence that TCF7L2 regulates insulin secretion rather than insulin action. A central feature of the Wnt/β-catenin pathway is the regulation of cytosolic β-catenin protein levels via a destruction complex containing glycogen synthase kinase-3b (GSK-3b). GSK-3B has long been known as an important mediator for impaired insulin action on peripheral tissue and in the development of insulin resistance \[[@B34]\]. However, only recently, studies have shed light on the growth regulatory properties of GSK-3B in β-cells. Two elegant studies from the same group reported recently that GSK-3B overexpression in mice induces β-cell mass restriction and the development of diabetes \[[@B35]\] and that the genetic disruption of GSK-3B in β-cells results in increased β-cell mass in those transgenic mice \[[@B36]\]. Importantly, β-catenin is able to interact with the FoxO family of transcription factors, which defend against oxidative stress by stimulating the transcription of oxidant scavenging enzymes such as superoxide dismutase and catalase \[[@B37]\]. Since FOXO and TCF proteins compete for a limited pool of β-cat, enhanced FOXO activity during ageing and oxidative stress may attenuate Wnt-mediated activities. The decrease of β-catenin not only attenuates Wnt signaling but also unleashes the expression of PPARγ, which is normally suppressed by β-catenin \[[@B38]-[@B40]\]. Oxidative stress activates the FoxO family of transcription factors, which in turn attenuate β-catenin/TCF-mediated transcription, leading to derepression of PPARγ transcription. The increase in PPARγ levels serves as an additional β-catenin sink by sequestering it and activating its proteasomal degradation \[[@B41]\]. Adipogenesis is also affected by oxygenation, low oxygen tensions activate HIF (hypoxia inducible factor)-1α, inhibiting differentiation by repressing PPARγ (peroxisome proliferator-activated receptor γ) \[[@B42]\] whereas higher oxygenation tends to induce differentiation \[[@B43]-[@B45]\]. HIF-1α has been shown to compete with TCF4/TCF7L for direct binding to β-catenin \[[@B46]\]. A recent work showed that Wnt signaling (which is up-regulated in high oxygen) has been associated with increased β-cell proliferation \[[@B47]\]. Thus, the increase in Wnt activity associated with higher oxygen availability might have the twofold effect of increasing (a) endocrine cell mass by self replication and (b) exocrine cell mass by progenitor cell expansion \[[@B48]\]. Another study showed the involvement of p53 expression in adipose tissue is crucial in the development of insulin resistance. The work showed in animal model with T2D-like disease, excessive calorie intake led to the accumulation of oxidative stress in the adipose tissue and promoted increased activity of senescence-associated β-galactosidase, increased expression of p53 and increased production of pro-inflammatory cytokines. Conversely, up-regulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance \[[@B49]\]. It has also been reported that production of reactive oxygen species (ROS) is selectively increased in the adipose tissue of obese mice and that increased oxidative stress in fat is a key mechanism underlying the occurrence of insulin resistance related to obesity \[[@B50]\]. The ROS-induced p53 activation causes NF-κB-dependent induction of inflammatory cytokines and thus accelerates the development of diabetes. Once oxidative stress is initiated it affects multiple systems. By reaction of ROS with NO, oxidative stress is increased while NO is diminished, thus promoting inflammation and endothelial dysfunction \[[@B51]\]. Specifically, ROS were implicated in mitogen-activated protein kinase (MAPK) pathways, which induce activation of various nuclear transcription factors, such as nuclear factor (NF)-κB, activator protein (AP)-1, hypoxia-inducible factor (HIF)-1a, sterol regulatory element binding proteins (SREBPs) and GATA-4 \[[@B52]-[@B54]\]. NF-κB participates in obesity and the metabolic syndrome; it induces inflammatory and atherosclerotic consequences. Obesity increases the formation of reactive oxygen species in fat cells, and ultimately results in activation of the p53 tumor suppressor, inflammation and the promotion of insulin resistance \[[@B55]\]. Also, insulin resistance is affected by oxidative stress and, when combined with up regulated NF-κB activity, may promote type 2 diabetes. Conversely, hyperglycaemia was also shown to trigger increased formation of ROS via glucose auto-oxidation. Accordingly, consumption of a high free-glucose diet promoted the development of oxidative stress \[[@B56]-[@B58]\]. HIF-1a, is also associated co morbidities such as hypertension \[[@B59],[@B60]\] and hyperlipidaemia \[[@B61]\]. High glucose concentration up-regulates SREBP-1c and insulin resistance \[[@B62]\]. However, none of these mechanisms are yet fully elucidated \[[@B63]\]. Proposed hypothetical model of mechanism ---------------------------------------- Based on an elaborate study of the key \'hubs\' regulations, their association, along with detailed literature reviewing - in this work we put the puzzle pieces together and proposed a hypothetical mechanism for co-regulation of various inflammatory diseases like T2D, HT and OBS (Figure [9](#F9){ref-type="fig"}). From the generated regulatory networks around key \'hub\' genes it is very much visible that via EP300 the main pathways like insulin signaling, PPAR signaling, calcium signaling, adipocytokine signaling, Jak-STAT signaling pathway, MAPK signaling pathways that are well recognised in association with disease like T2D, OBS and HT. Interestingly, the analyses of the regulatory cascades showed that the genes involved in main disease pathways are all connected and regulated via the genes involved in Wnt-signaling and p53 signaling pathways. ![**Proposed hypothetical model of T2D mechanism**.](1755-8794-3-45-9){#F9} In detailed study of the gene-disease network and the regulation around the \'hubs\', this work suggests EP300/TP53/CDKN1A/STAT3/TCF7L2/MYC/CXCR4 is the main regulatory cascade that generates the highly connected, co-regulated network of insulin signaling which in turn, could be responsible for the genesis of molecular cross-talk within various diseases. It has been suggested that insulin signaling crosstalk with Wnt signaling, due to the existence of common downstream target genes and the shared negative mediator GSK-3 \[[@B33]\]. However, mechanisms underlying this potential crosstalk still remain unclear \[[@B33]\]. β-catenin, an intracellular signaling molecule is essential for Wnt activation. Without Wnt signal, β-catenin, is constantly phosphorylated and thereby inactivated by GSK3B, is eventually degraded to prevent its accumulation. Inactivation of GSK3B, in turn can no longer phosphorylate β-catenin. This leads to nuclear translocation of β-catenin and subsequently, which coactivates TCF/LEF transcription factors \[[@B64]\]. Our regulatory gene- network could fit all those observations and explain a bit more these underlying molecular mechanisms. Wnts induce glucose-stimulated insulin secretion and β-cell proliferation \[[@B65]\]. This signaling system might senses insulin sensitivity via the regulator gene GDK3B inactivation/activation (phosphorylation/dephosphorylation) by increasing insulin effects through AKT1/IGF1R/STAT3/MYC/CDKN1A/TP53/EP300 cascade via activating Akt/PKB and inhibiting the MAPK pathways (Figures [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"} and [9](#F9){ref-type="fig"}). In the absence of insulin, FOXOs upregulate the expression of a set of target genes via AKT1/IGF1R/CXCR4/STAT3/EP300/PPARG cascade, thereby promoting cell cycle arrest, stress resistance and apoptosis \[[@B65]\]. In the presence of insulin, FOXOs are phosphorylated by AKT/PKB protein kinase and stay in the cell cytosol \[[@B66]\]. In contrast to the effect on insulin signaling, oxidative stress (ROS) induces the activation of FOXO signaling \[[@B67]\]. This might be due to the activation via the Jak/STAT signaling pathway \[[@B68]\]. T2D, age, obesity- all increases ROS that in turn increases lipid oxidation, via ROS/FOXO/PPARG/PPARA/β-catenin regulation leads to PPARG activation and repression of Wnt-signaling. So, when PPARG activates it attenuates Wnt-signaling, which induces lipid oxidation that in turn increases ROS. Increased oxidative stress diminished Wnt signaling that may leads to β-cell destruction via FOXO apoptosis \[[@B69]\]. Regulatory gene network (Figure [8](#F8){ref-type="fig"}) suggests that one possibility could be via CREBBP regulation on MYC/TP53/EP300/TCF7L2 cascade, the system might sense signal to FOXO. Since FOXO and TCF compete for limited pool of β-cat, enhanced FOXO activity during oxidative stress may attenuate WNT- mediated activation \[[@B70]\]. Thus, oxidative stress lead to FOXO mediated gene transcription and reduced TCF mediated gene transcription. Conversely, activation of Wnt pathway through inactivation of GSK3B, stabilise β-catenin, that regulates β-cell proliferation via TCF7L2, a down stream effector of this cascade \[[@B69]\]. This in turn coactivates PPARG mediated transcription on the glucokinase gene promoter. Glucokinase is the key regulator of glucose-sensing in pancreatic beta-cells, thereby offering a model for the adipocyte-induced hyper secretion of insulin \[[@B71]\]. Although TCF7L2 expression was positively correlated with the expression of INS, which encodes insulin, it was inversely correlated with glucose-stimulated insulin release \[[@B72]\]. There is clear evidence that TCF7L2 regulates insulin secretion rather than insulin action \[[@B65]\]. Thus, β-catenin interaction with TCF7L2 with nuclear co-activators, such as EP300, results in the stimulation of Wnt or β-cat/TCF downstream target gene transcription \[[@B73]-[@B75]\]. From the gene regulation network, it can be seen that insulin may also stimulates the transcription via MYC, a known downstream target of Wnt signaling, via a PI3K-dependent but Akt/GSK3B-independent mechanism, via IGF1R/MYC/TP53/EP300 regulatory cascade. Indeed, several other studies have also reported the stimulatory effect of insulin or IGF1 on β-cat nuclear localization and cat/TCF mediated gene transcription \[[@B29],[@B33],[@B76],[@B77]\]. Thus, fitting all these evidences in the proposed hypothetical model (Figure [9](#F9){ref-type="fig"}), it can be suggested that insulin signaling may crosstalk with Wnt signaling, due to the existence of common downstream target genes, the shared negative mediator GSK3B, and insulin resistance are capable of explaining the association of T2D co-occurrences with other inflammatory disease like HT, and OBS. T2D and/or OBS can develop independently, due to genetic, behavioral or lifestyle-related variables but both T2D and OBS lead to oxidative stress generation \[[@B78]\]. The pathogenic mechanisms by which diabetes and oxidative stress induce inflammation are not certain at the present time \[[@B79]\]. But the predicted model, at least provided much more logical explanation to fit what so far been known about the inflammation mediated consequences. Excessive calorie intake led to the accumulation of oxidative stress with T2D that can promote increased activity of senescence-associated β-galactosidase, increased expression of p53 and increased production of proinflammatory cytokines. Conversely, upregulation of p53 in adipose tissue can cause an inflammatory response that led to insulin resistance \[[@B79]\]. Also oxidative stress through TCF7L2/NF-kB/MAPK/HIF1A/SREBP regulatory cascade via insulin, MAPK and Calcium signaling pathways, may activate inflammatory responses than in turn causes insulin resistance. Hyperglycemia could lead to increase lipid oxidation and endothelial dysfunction regulated through PPARG/STAT3/CXCR4/ICAM/VCAM cascade via Chemokine, Adipocytokine and Jak-STAT signaling pathways that could cause increase expression ICAM1, VCAM1 levels which in turn increases inflammation and obviously, increases oxidative stress \[[@B80],[@B81]\]. Once oxidative stress is initiated it affects multiple systems-via AKT1/NOS3 regulation. Increased oxidative stress can diminish NO production, thus promoting inflammation and endothelial dysfunction \[[@B51]\]. Inflammatory pathway activation will lead increased expression of adhesion molecules, cytokines and associated with increased risk of HT. Obesity, insulin resistance and hypertension commonly cluster with other risk factors for CDV or chronic kidney disease to form the metabolic syndrome \[[@B65]\]. Conclusion ========== It can be hypothesised from the generated model (Figure [9](#F9){ref-type="fig"}) that oxidative stress is a direct cause for the Wnt pathway activation via the high glucose-induced β-catenin activation through a highly inter-connected gene regulatory cascade, where EP300 plays a role as a key regulator in T2D. Even though these predictions need further experimental validation, the hypothetical model presented in this study could be a starting point to visualize in a systems approach understanding the molecular cross- talk between diseases. The findings provide a novel integrated approach for understanding the pathogenesis of various co-associated diseases by combining the power of pathway analysis with gene regulatory network evaluation. The integrated network model developed by this work provides useful functional linkages among groups of genes and thus addressing how different inflammatory diseases such as obesity, diabetes, and hypertension are connected and propagated at genetic level. It is anticipated that derived models will be of great benefit to a wide research audience, including those involved in disease biomarker identification and drug development. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= Jesmin, MSMR and HJ coordinated the text mining and information extraction, Jesmin, MSMR mined and computed the interaction data, Jesmin, HJ, JB-R, and RH analysed and interpreted the result, drafted and revised the manuscript. All authors have read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1755-8794/3/45/prepub> Supplementary Material ====================== ###### Additional file 1 **Statistics of the reported genes associated with T2D, HT, OBS and ROS**. ###### Click here for file ###### Additional file 2 **Complete \'text-mined\' data set**. ###### Click here for file ###### Additional file 3 **Predicted integrated network model of the four diseases (using Cytoscape)**. ###### Click here for file ###### Additional file 4 **Gene annotation using GO, Genopedia and Phenopedia**. ###### Click here for file Acknowledgements ================ We thank Dr Zaheed Husain (Division of Interdisciplinary Medicine & Biotechnology, BIDMC, Department of Medicine, Harvard Medical School, Boston) for valuable comments and suggestions that helped improve the manuscript.

{ "pile_set_name": "PubMed Central" }

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Organ formation and modifications in tissue architecture occurring during development involve deconstructing both cell adhesion and the extracellular matrix[@b1][@b2][@b3], thereby converting cells into a migratory state with a new tissue identity; this is known as epithelial--mesenchymal transition[@b4][@b5]. Although epithelial cells and mesenchymal cells have contrasting properties, the molecular mechanisms behind these properties are not entirely mutually exclusive. Some epithelial cells acquire the ability to migrate while retaining cell--cell adhesion, allowing these cells to migrate as a group[@b6][@b7][@b8][@b9]. This is possible due to the polarized nature of the epithelia; the apical side retains the cell--cell interface, while migratory protrusions form on the basal cell surface. On reaching their destination, these migratory epithelia are integrated into the target tissues. However, how migratory epithelia find their target tissues, establish new cell--cell interfaces and integrate with the target tissue are poorly understood. Studies of the formation of adhesive interfaces in cultured cells showed that contact with actin-enriched filopodia initiates the assembly of the adherens junction[@b10][@b11] and suggested that microtubules are involved in this process[@b12]. As the formation of cell junctions involves coordinating the assembly of the cell adhesion complex and the generation of tension in each side, the cells forming the junction must be observed simultaneously to understand how mechanical coupling is controlled at the tissue level. Here, to examine how cell adhesion and cell contractility are coupled to establish a new cell interface, we examined the anastomosis formation in the *Drosophila* tracheal system. In this process, a pair of tip cell (hereafter called fusion cell, FC) forms an adherens junction *de novo* ([Fig. 1a,b](#f1){ref-type="fig"}) and deposit extracellular matrix materials into newly forming lumen. Each FC is converted into a torus shape by plasma membrane fusion via the inner leaflet[@b16][@b17][@b18], under the control of endosome and microtubule-associated protein Arl3 (refs [@b8], [@b19], [@b20]). Interestingly, plasma membrane fusion always occurs simultaneously in each FC, suggesting cell shape change and membrane trafficking in each FC pair is highly coordinated. However, how cell adhesion, cell-shape change and membrane trafficking are regulated coordinately in pairs of FCs is poorly understood. We show that FCs form a *de novo* adherens junction at the contact site through stabilization by a mechanism requiring actomyosin and microtubules; this mechanism applies a balanced pulling force to flatten the FCs. In addition to this cell-intrinsic contractile force, the preferential deposition of molecules via the apical secretion pathway promotes maturation of the matrix in the lumen that forms in the FC contact interface and helps to fuse the plasma membranes. Results ======= F-actin and microtubule dynamics in migrating FCs ------------------------------------------------- FCs expressing the F-actin markers GFP-moesin ([Fig. 1c](#f1){ref-type="fig"}; see also [Supplementary Movie 1](#S1){ref-type="supplementary-material"}) or lifeact-GFP ([Supplementary Fig. 1a](#S1){ref-type="supplementary-material"}), each driven by an FC-specific enhancer (*esg_FC* enhancer; see Methods), were closely associated with the basal surface of the dorsal epidermis and showed numerous F-actin-enriched filopodia extending towards the dorsal midline[@b18][@b21]. We also observed invasive filopodia that extended vertically from these FCs into the epidermal layer ([Fig. 1c](#f1){ref-type="fig"} open triangles, [Fig. 1c′](#f1){ref-type="fig"} and see also [Supplementary Fig. 1a](#S1){ref-type="supplementary-material"}); their location did not correspond to epidermal cell junctions, suggesting that the filopodia penetrated the epidermal cells. While migrating, the FCs appeared flat with numerous forward-extending filopodia ([Fig. 1c](#f1){ref-type="fig"}, time 0:00 h), but on reaching their target, their shape became more compact, with the lumen extending from the side of the stalk ([Fig. 1c](#f1){ref-type="fig"}, time 1:54 h). Microtubule labelling with GFP-tau revealed extensive microtubule arrays emanating from the microtubule-organizing centre (MTOC) at the proximal side of the cell ([Fig. 1d](#f1){ref-type="fig"}, time 0:00 h; see also [Supplementary Fig. 1b,c](#S1){ref-type="supplementary-material"} and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}) and F-actin labelling revealed many, F-actin-rich protrusions extending forward to reach the target cell ([Fig. 1e](#f1){ref-type="fig"} and see also [Supplementary Movie 3](#S1){ref-type="supplementary-material"}). On FC contact with its partner FC, the MTOC moved to the contact site ([Fig. 1d](#f1){ref-type="fig"}, time 1:25 h). The vertical protrusions revealed by F-actin marking were not observable with microtubule labelling (compare [Fig. 1c′](#f1){ref-type="fig"} and [Supplementary Fig. 1b](#S1){ref-type="supplementary-material"} for another microtubule marker GFP-Jupiter). A polarized flux of E-Cadherin to the new cell junction ------------------------------------------------------- When two FCs came within a proximity of 15.2 μm (on average), fluorescent markers for E-cadherin ([Fig. 2a](#f2){ref-type="fig"})[@b22] and its associated protein p120ctn ([Supplementary Fig. 3a](#S1){ref-type="supplementary-material"})[@b23], accumulated at the contact site. Immunostaining revealed endogenous E-cadherin localized to the extending edge of forward-reaching filopodia on the tip cells ([Fig. 2b](#f2){ref-type="fig"} top). This punctate E-cadherin localization was not observed on the filopodia of terminal cells or stalk cells ([Fig. 2b](#f2){ref-type="fig"} bottom). To monitor the dynamics of E-cadherin transport, we imaged E-cadherin-GFP fluorescence during the fusion process. We found that the E-cadherin-GFP level remained fairly constant at the junction between the FC and stalk cell ([Fig. 2a](#f2){ref-type="fig"}, position and plot marked with 'L\' and 'R\'), but increased steadily at the FC interface, surpassing the intensity at the FC--stalk cell interface within 20 min after contact ([Fig. 2a](#f2){ref-type="fig"}). We next monitored the E-cadherin-GFP turnover rate at each cell interface by fluorescence recovery after photobleaching (FRAP) analysis. All three E-cadherin-GFP foci (L, R and C) were photobleached simultaneously and their rate of fluorescence recovery was measured ([Fig. 2c](#f2){ref-type="fig"} arrowheads). The fluorescence recovery rate and the mobile E-cadherin-GFP fraction were significantly higher at the FC interface than at the L and R foci ([Fig. 2d,e](#f2){ref-type="fig"}). Imaging with high spatiotemporal resolution revealed that E-cadherin-GFP appeared on the free surface of the plasma membrane, with occasional streams from the cytoplasm to the membrane surface, and then accumulated at the FC contact site ([Fig. 2e](#f2){ref-type="fig"} and [Supplementary Movie 4](#S1){ref-type="supplementary-material"}). In addition, Golgi apparatus in FCs became progressively enriched near the contact site ([Fig. 2g](#f2){ref-type="fig"}). The low E-cadherin-GFP turnover at the FC--stalk cell interface suggests that the exchange rate of the E-cadherin complex at this junction, which was already present at the time of dorsal branch (DB) migration, is low. To analyse E-cadherin\'s contribution, we reduced its synthesis by double-stranded RNA-mediated gene knockdown ([Supplementary Fig. 3b](#S1){ref-type="supplementary-material"}). Expressing E-cadherin double-stranded RNA in tracheal cells with the trachea-specific *btl-Gal4* driver reduced the level of E-cadherin in DB stalk cell junction by ∼27% (Methods), but did not compromise the overall tracheal morphology or the migration speed of the DB before the FCs came into contact with each other ([Fig. 2h,i](#f2){ref-type="fig"}), suggesting that the amount of E-cadherin gene product in this experimental condition was sufficient to sustain the tracheal tissue architecture. However, the FC contraction was significantly delayed after contact and the interface between the FCs failed to mature ([Fig. 2h,i](#f2){ref-type="fig"}). These results suggest that newly synthesized E-cadherin is preferentially partitioned to the distal side, to form the new cell interface. FC contraction by a myosin-dependent pulling force -------------------------------------------------- We next examined the role of myosin II in the contractile force within the FC. When expressed in FCs, a green fluorescent protein (GFP) fusion protein of the myosin heavy chain Zipper formed longitudinal bundles at the time of FC--FC contact, to connect the adherens junctions on each FC ([Fig. 3a](#f3){ref-type="fig"} and [Supplementary Movie 5](#S1){ref-type="supplementary-material"}). These myosin bundles became shorter as the FCs contracted and became more compact ([Fig. 3a](#f3){ref-type="fig"}). Similar myosin bundles were observed in the dorsal-trunk FCs ([Supplementary Fig. 4a](#S1){ref-type="supplementary-material"}). To determine whether FC contraction is driven by a cell-autonomous force, we assessed the contractile state of laser-perturbed FCs. A microwave-length infrared laser (1,440 nm) effectively penetrated the epidermal layer and produced sufficient heat at the focal point to induce a heat-shock response[@b24][@b25]. Infrared laser illumination targeted to one of the paired FCs caused a cell-specific relaxation of cell contractility ([Fig. 3b](#f3){ref-type="fig"}), with a simultaneous excessive contraction in the intact FC. The progression of the fusion process, assessed by shortening of the L--R length, was also arrested ([Fig. 3c](#f3){ref-type="fig"} and [Supplementary Movie 6](#S1){ref-type="supplementary-material"}, *N*=3). This result supports the idea that FC pairs normally exert nearly equal pull on each other to maintain their symmetric appearance, and that this balanced pulling force is required to pull FCs closer together. Furthermore, expressing a dominant-negative form of the myosin heavy chain interfered with FC contraction ([Fig. 3d](#f3){ref-type="fig"} and [Supplementary Movie 7](#S1){ref-type="supplementary-material"}, 100% *N*=9, 3 embryos) and fusion ([Supplementary Fig. 4e,f](#S1){ref-type="supplementary-material"}). Polarized microtubule organization in the FC -------------------------------------------- We next studied the detailed organization of microtubules. The microtubule minus-end marker Nod-LacZ (ref. [@b26]) and the centriole marker GFP-PACT (ref. [@b27]) were concentrated near the FC--stalk cell interface ([Fig. 4a,b](#f4){ref-type="fig"}). From this proximal MOTC, microtubules extended in a fan-shaped pattern to the migrating FC\'s leading edge ([Fig. 1d](#f1){ref-type="fig"} and [Supplementary Fig. 1c](#S1){ref-type="supplementary-material"}). We also imaged the microtubule plus-end marker EB1-GFP in FCs ([Fig. 4c](#f4){ref-type="fig"} and [Supplementary Movie 8](#S1){ref-type="supplementary-material"}); particle image velocimetry analysis revealed rapid, comet-like EB1-GFP signals with a mode velocity of 5 μm min^−1^ moving towards the FC\'s leading edge ([Fig. 4c](#f4){ref-type="fig"}) and an EB1-GFP population moving at even higher velocities (\>10 μm min^−1^) in the periphery ([Fig. 4c](#f4){ref-type="fig"}). Dual-colour imaging of EB1-GFP and the cell membrane revealed that the microtubule plus ends projected to the cortical region of the leading edge and extended repeatedly into the filopodial protrusions ([Fig. 4d](#f4){ref-type="fig"} and [Supplementary Movie 9](#S1){ref-type="supplementary-material"}). The microtubule-filled protrusions were found most frequently in the leading edge, which made contact with the partner FC at the midline and accumulated E-cadherin-GFP ([Fig. 4e](#f4){ref-type="fig"} and [Supplementary Movie 10](#S1){ref-type="supplementary-material"}). Requirement of microtubules for tracheal fusion ----------------------------------------------- To reveal the role of polarized microtubule arrays in tracheal-tube fusion, we perturbed the FC microtubule organization by expressing Katanin p60 (Kat60), a catalytic subunit of the microtubule-severing factor Kat60/p80 (refs [@b28], [@b29], [@b30]). Microtubules in control FCs, visualized by the marker GFP-tau, formed dense central bundles directed towards the leading edge, with diffuse fractions filling the rest of the cell ([Fig. 4f](#f4){ref-type="fig"} top and [Supplementary Movie 11](#S1){ref-type="supplementary-material"}). Analysis of the signal variance in a 200-s interval (20 frames) revealed stable central microtubule bundles and an unstable diffuse fraction ([Fig. 4f](#f4){ref-type="fig"} bottom). In FCs expressing Kat60, the microtubule bundle was misdirected and became unstable (high signal variance, [Fig. 4f](#f4){ref-type="fig"}), indicating that the ectopic Kat60 could destabilize the microtubules. In this condition, endogenous Zip cables connecting the adherence junctions of FCs was reduced ([Supplementary Fig. 2](#S1){ref-type="supplementary-material"}). DBs expressing Kat60 migrated towards the dorsal midline and maintained normal-shaped stalk cells and the FCs contacted each other using filopodia. However, the fusion process was arrested, because the filopodia contact site failed to accumulate E-cadherin or mature into a tight cell contact[@b18] ([Fig. 4g,h](#f4){ref-type="fig"}). In control trachea, signals from the adherens junction marker p120ctn-mRFP increased steadily in the contact site. However, in cells expressing Kat60, the p120ctn-mRFP accumulation was unstable, showing a 'blinking\' pattern ([Fig. 4I](#f4){ref-type="fig"} and see also [Supplementary Movie 12](#S1){ref-type="supplementary-material"}). These results indicate that microtubule destabilization inhibited the accumulation of the E-cadherin complex during fusion[@b18]. To further elucidate the role of microtubules in forming the adherens junctions, we expressed Spastin (Spas), another microtubule-severing factor[@b31], in FCs. Spas was previously demonstrated to inhibit tracheal branch migration and fusion[@b15][@b18]. We found Spas to be less disruptive to the fusion process than Kat60 (Methods), making it suitable for quantifying the E-cadherin-GFP accumulation at the contact site over time. The rate of E-cadherin-GFP accumulation was estimated by the slope of a fitted line ([Fig. 4j,k](#f4){ref-type="fig"}) and the coefficient of determination (*R*^2^-value) was used as a measure of fluctuation ([Fig. 4l](#f4){ref-type="fig"}). Spas reduced the E-cadherin-GFP accumulation rate and increased the fluctuation (lower *R*^2^-value; [Fig. 4l](#f4){ref-type="fig"}). Despite these defects, FCs expressing Spas eventually accumulated E-cadherin-GFP and completed branch fusion. Taken together, these results indicate that proper microtubule organization is required for stable, persistent E-cadherin accumulation to the new cell-adhesion sites in tracheal-branch fusion. Microtubules coordinate deformation of paired FCs ------------------------------------------------- We next analysed the dynamics of cell-shape change during the fusion process by monitoring the distance between the FC contact site (C) and the FC--stalk cell boundaries at the left (L) and right (R) side branch. In control embryos, FC pairs maintained their symmetry, with nearly equal C--L and C--R lengths ([Fig. 5a--c](#f5){ref-type="fig"} and [Supplementary Movie 13](#S1){ref-type="supplementary-material"}). However, FCs that expressed Spas deformed more slowly, as measured by the speed at which the L--R length decreased ([Fig. 5d](#f5){ref-type="fig"}), and failed to maintain a balanced bilateral cell contraction ([Fig. 5a](#f5){ref-type="fig"}) as measured by the L/R balance; the L/R balance was calculated as (*a*−*b*)/(*a*+*b*), where *a*=C--L and *b*=C--R ([Fig. 5b](#f5){ref-type="fig"}). The degree of fluctuation, based on the s.d. of the L/R balance, remained low in control cells. However, imbalances in cell length began appearing in *esg_FC\>Spas* cells 5 min after the first contact between FC cells ([Fig. 5c](#f5){ref-type="fig"}). These findings indicated that proper microtubule organization is required for balanced cell contraction. We further analysed the mechanism of the balanced cell contraction. We hypothesized that asymmetry in the length of paired FCs is caused by an imbalance in contractile forces between the cells, creating a back-and-forth tug-of-war (ToW) situation. To demonstrate this concept, we calculated the contraction speed of each FC at every time interval after contact. We used the contraction speed of viscoelastic cell materials, which is proportional to the contractile force[@b32], as a surrogate of cellular force. Negative and positive values indicate contractile and relaxed state of each FC, respectively. If both FCs contracted or relaxed simultaneously at a given time point after contact, the contractile force was considered balanced. If one cell contracted while the other relaxed, the event was considered imbalanced and in the ToW state. We pooled a number of such cell-shape change events and calculated the percentage of ToW imbalances ([Fig. 5e](#f5){ref-type="fig"}; ToW ratio, solid vertical bar). The ToW ratio was higher in the *esg_FC\>Spas*-expressing cells (62.2%, 7 time series) than in control cells (46.8%, 4 time series). To determine whether this deviation might be caused by random drift, we ran a simulation to calculate the ToW ratio; this simulation assumed that FCs contracted independently with the deformation rate in normal distribution ([Fig. 5e](#f5){ref-type="fig"}; mean displacement, −0.22±0.65 μm min^−1^ for control and −0.17±0.79 μm min^−1^ for *esg_FC\>Spas* cells; see Methods). The calculated probability distributions of control and *esg_FC\>Spas* conditions were similar to the control value ([Fig. 5e](#f5){ref-type="fig"}, thin curves, 10^4^ trials each), but deviated greatly from the measured *esg_FC\>Spas* value. Therefore, the ToW ratio deviation observed in *esg_FC\>Spas* cell pairs was due to an imbalance of forces in the paired FCs, leading to ToW-type contractions. Directed apical Serp secretion to the FC interface -------------------------------------------------- Based on our observation that the Golgi apparatus moved towards the site of contact between FCs ([Fig. 2g](#f2){ref-type="fig"}), we reasoned that the localization of secretory machineries is probably influenced by microtubule polarity. Rab9 is required for the endosome-to-Golgi trafficking of a key luminal component, the chitin deacetylase Serpentine (Serp)[@b33][@b34][@b35]. RFP-Rab9\'s localization in FCs was similar to that of the Golgi apparatus and of Arl3, another small GTPase that is essential for tracheal-branch fusion ([Fig. 6a,b](#f6){ref-type="fig"})[@b19][@b20]. We therefore examined the secretion of luminal components into the new lumen formed between FCs. Fusion points of dorsal trunk were observed for this study, because detailed analysis of lumen formation is possible with their large tube diameter. In *arl3* mutants, isolated chitin-filled lumen was found at the contact site of dorsal-trunk FCs ([Fig. 6c](#f6){ref-type="fig"}) and the lumen contained secreted luminal proteins: the chitin deacetylase Verm[@b33][@b34], the ZP-domain protein Pio[@b36] and the chitin-binding protein Gasp[@b37] ([Fig. 6d--f](#f6){ref-type="fig"}). However, Serp was specifically missing in the isolated lumen ([Fig. 6c](#f6){ref-type="fig"}). To determine whether FCs can secrete Serp, we expressed Serp-GFP by the FC driver and found that Serp-GFP was secreted and filled the entire tracheal lumen ([Fig. 6g](#f6){ref-type="fig"}). In contrast, in an *arl3*-mutant background, Serp-GFP was absent at the FC contact site but filled the rest of the tracheal lumen ([Fig. 6h](#f6){ref-type="fig"}). These results indicate that Arl3 is specifically required for the directed secretion of Serp into the lumen in the interface between paired FCs, but not for its secretion into FC--stalk cell interfaces. Rab9 overexpression overrides the requirement for Arl3 ------------------------------------------------------ The aforementioned results suggested that Rab9-dependent Serp trafficking might be an important function of Arl3. To test this idea, we examined whether Rab9 overexpression could rescue the *arl3*-mutant phenotype. We found that GFP-Rab9 expressed by *btl-Gal4* or *esg_FC-Gal4* rescued the fusion defect of *arl3* mutants ([Fig. 7a--d,g](#f7){ref-type="fig"}). However, GFP-fusion proteins of other small GTPases, Rab11 and Rab6, or of Serp failed to rescue the *arl3* phenotype ([Figs. 7e--g](#f7){ref-type="fig"} and [6h](#f6){ref-type="fig"}). These results indicate that Rab9-dependent recycling and apical secretion is a crucial component of the Arl3-dependent conversion of paired FCs into a torus shape. Discussion ========== Previous studies showed that F-actin-enriched cell protrusions form in the tip of migrating tracheal branches[@b16][@b17][@b18][@b38]. Here we showed that tracheal FCs form polarized microtubule bundles oriented towards the leading edge of the migrating cells. The function of these microtubules is twofold: to concentrate E-cadherin to the newly contacted cell interface and to initiate the formation of new adherens junctions. We found that the E-cadherin that accumulated at the new cell interface is not recycled from the cell surface, but is instead drawn from a newly synthesized pool and recruited preferentially to the FC contact site, and not to existing adherens junctions between FCs and stalk cells. We speculate that the forward reorientation of the MTOC and the polarization of the microtubule plus ends towards the leading edge underlie the preferential deposition of E-cadherin at the FC contact site[@b39]. We considered one possible mechanism of preferential deposition, in which microtubules transport endosomes containing E-cadherin towards the contact site and we attempted to test this model by imaging vesicular trafficking of the complex containing E-cadherin-GFP and other adherens junction components. We did not find definitive evidence for this model. However, we observed that the Golgi apparatus shifted forward, towards the FC contact site, and that an E-cadherin-GFP signal increased in the plasma membrane before becoming concentrated at the contact site. Based on these observations, we favour a model in which the relocalization of the Golgi apparatus near the leading edge of the FC provides a source for E-cadherin that is deposited locally in the plasma membrane and the *trans* association of E-cadherin between the FCs nucleates a further concentration of E-cadherin via *cis* clustering[@b40]. Brodu and colleagues[@b15][@b41] have shown that MTOC components are located apically in stalk cells and the microtubule function is required for the apical assembly of adherens junction proteins Par-3 and E-cadherin through regulation of recycling endosomes. This mechanism appears different from FCs, as assembly of new adherens junction occurs in the cell interface enriched with microtubule plus ends opposite to the centriole located in the proximal side. A second microtubule function was discovered in this study, which was to equalize the contraction in FC pairs after contact. The coordinated contraction in FC pairs pulls two FC--stalk cell junctions simultaneously towards the FC contact site. The contractile force comes from a myosin-driven process; the microtubules may serve as a 'ratchet\' to fix the length of the FCs after each round of contraction. When microtubules were inhibited, the FCs relaxed to their original length after contracting, which delayed the overall fusion process. When microtubules were destabilized, branch fusion proceeded, albeit with delays and imbalances, and fusion was eventually completed. Even in this condition, the conversion of the FC cells into a torus shape occurred simultaneously, suggesting that there is a mechanism to coordinate the fusion event in FC pairs. The proposed ratchet-like function of microtubule must in some way be linked to the contractile activity of myosin. One good candidate molecule for coupling the actomyosin contraction to the microtubule function is Short-stop (Shot), which belongs to the conserved spectraplakin family of cytoskeletal proteins[@b42], and was shown to be required for tracheal branch fusion[@b43]. The involvement of microtubules and Shot in the ratchet-like mechanisms observed in several contraction-dependent morphogenetic events[@b44][@b45][@b46] should be tested in the future. It is interesting to note that balancing of the force applied to the E-cadherin conjugated cell interface via microtubule plus ends is similar in configuration to the mitotic spindle, where microtubule plus ends attached to the kinetochore of each of paired sister chromosomes applies pulling force to each spindle pole[@b47]. The equal number of cadherin-catenin complex in each side of the FC interface associated with *trans*-conjugated E-cadherin pairs in the FC interface may provide a platform for microtubule plus end attachment for generating balanced contractile force. When FCs are fully contracted, the plasma membrane of the two adherens junctions in each FC are connected in a single burst and cell pairs are converted simultaneously into a torus shape. This process requires Arl3 GTPase, which associates with the microtubules and intracellular vesicles concentrated at the FC contact site[@b19][@b20]. Here we showed that Arl3 is required for directed Serp trafficking, and that GFP-Rab9 overexpression overrides the requirement for Arl3. We propose that the microtubule-dependent transport of the Golgi apparatus and endosomes facilitates the concentration of Rab9 and Arl3 at the FC contact site, where they act together to increase the concentration of Serp in the lumen. The deacetylation of chitin converts it to the more hydrophilic form chitosan. The increase in water absorption by chitosan would cause the luminal matrix gel to swell, simultaneously pushing the plasma membranes of the FC interface closer to the plasma membrane of the FC--stalk cell interface so that the membrane-fusion machinery triggers the conversion of the paired FCs into a torus shape. A number of issues remain to be explained. Lumen formation in FCs was clearly detected *arl3* mutants, but not in the normal context. This is probably because very small lumen is sufficient to trigger fusion of wild-type FCs. Although Arl3 is absolutely required for fusion, Rab9 and Serp are not, suggesting that the proposed luminal-matrix swelling due to chitin deacetylation is not the sole mechanism of plasma membrane fusion, and additional Arl3-regulated process of fusion control must exist. Moreover, additional Rab9 cargo that acts together with Serp to rescue the *arl3* mutants is predicted. To uncover the entire fusion process, it will be necessary to search for additional Arl3 and Rab9 targets, and to analyse FC-specific membrane trafficking and secretion. Methods ======= *Drosophila* strains -------------------- The *esg_FC-Gal4* strains used here were constructed by injecting a plasmid containing a genomic fragment upstream of the *esg* gene into the pGaTB vector[@b48] and by isolating insertions in the second and third chromosomes. The fusion-cell enhancer was identified during a study of *esg* regulatory elements[@b49][@b50]. The *esg_FC-Gal4* strain were constructed by inserting the 7,259 bp of genomic DNA (position 15313739--15320998 of NT_033779) upstream of *esg* into the P{CaSpeR-hs} vector (FBtp0000164), whereas YY, MT-M and SH were associated with the Genetic Resource Center, National Institute of Genetics in Mishima, Japan. This construct was described as *pAs-Gal4* expressed in the peripheral amnioserosa[@b51]. *UAS-p120ctn-tagRFP* was constructed with a p120ctn sequence amplified from the complementary DNA library[@b52] and tagRFP (Evrogen) was constructed as described elsewhere[@b23]. *UAS-3xtagRFP* was constructed by cloning three copies of tagRFP as a tandem fusion. UAS-tagRFP-tau was constructed by fusing tau\'s microtubule-binding domain (amino acids 165--327) to the carboxy terminus of tagRFP. *UAS-TagRFP-Jupiter* was constructed by fusing Jupiter open reading frame spanning codon 2 to terminator sequence to the C-terminal end of Tag-RFP. *UAS-EB1-GFP* was constructed by Masako Kaido and Leo Tsuda. All of the fluorescent-protein constructs were cloned into pUAST vector and transformed into germ lines[@b48][@b53]. Kat60\'s microtubule-severing activity was originally discovered in a screen of Gene Search strains[@b54] for effectors interfering with tracheal morphogenesis (Kenzi Oshima, personal communication; P{w^+mC^=GSV6}GS10360 insertion). The following fly strains were used: *btl-Gal4* (ref. [@b55]), *UAS-GFP-moe*[@b56], *UAS-Spas*[@b57], *UAS-GFP-PACT*[@b27], *UAS-DE-cad-GFP*[@b58], *UAS-GFP-Zip* and *UAS-GFP-DN-Zip*[@b59]; the UAS-IR strain for DE-cad RNA interference (RNAi) (NIG Stock Center, 3327R-2), *UAS-tau-GFP*[@b60], *UAS-Nod-lacZ*[@b26], *UAS-GFPS65T* inserted in 2L (FBti0013280), UAS-Grasp65-GFP.2 (FBti0040816) and *UAS-Serp-GFP*[@b34]; *rab9*^*56*^, *rab9*^*199*^ and *UAS-TagRFP-Rab9* (ref. [@b35]); and *arl3*^*1*^ and *UAS-mCherry-CAAX*[@b19]. To compare the effect of Spas and Kat60 overexpression on the tracheal branch fusion, we imaged DB migration under condition of each effector expressed by *btl-Gal4*. DB migration was delayed by Kat60 and fusion failed frequently (31% *n*=26), whereas success rate was higher in Spas (85%, *n*=26) or control (100%, *n*=11). Staining -------- The following antibodies and dilution were used for staining: rabbit anti-GFP (Medical & Biological Laboratories Co., Ltd, \#598, 500 × ), anti-beta-galactosidase (MP Biomedicals, \#0855976, 1000 × ), rat anti-DE-cad (DCAD2, 20 × )[@b61], mouse anti-Gasp (2A12, DSHB, 2 × ), anti-Serp (300 × ) and Verm (300 × )[@b34], anti-Pio (50 × )[@b36], anti-Arl3 (500 × )[@b19] and CBP-Alexa 488 (50 × )[@b35]. Embryos were dechorionated with 1/2 diluted commercial bleach and were fixed by mixing vigorously in the mixture of 2 ml 4% paraformaldehyde in PBS and 2 ml heptane in liquid scintillation vial for 30 min at room temperature. After removal of heptane, embryos were devitelinized by addition of 2 ml methanol and vigorous mixing. Devitellinized embryos were washed two times with methanol and rehydrated with PBS. Embryos were stained as described previously[@b56]. Blocking, antibody incubation and washing were performed with 1% BSA and 0.2% Triton X-100 in PBS. The anti-Katanin 60 antibody (100 × ) was generously donated by Naoyuki Fuse and Fumio Matsuzaki. Microscopy ---------- Time-lapse imaging of living embryos was performed with Olympus FV1000 with GaAsP detectors[@b19]. FRAP analyses were performed on tracheal cells expressing DE-cadherin-GFP using a confocal microscope equipped with a second scanner and a × 60 oil-immersion lens (FV-1000, Olympus). The sampling rate was every 10 s and specific locations were bleached with a 50-mW, 405-nm diode laser. EB1-GFP was imaged using a TCS SP5 II (Leica) equipped with a × 63 oil-immersion lens (numerical aperture=1.4). Optical sections (0.46 μm × 13) were scanned at 200 frames every 0.098 s. Infrared laser illumination was applied as a 1-s pulse at 60 mW and the embryos were imaged in 30-s intervals[@b25]. Measurement and data processing ------------------------------- All imaging data were processed and measured with applications developed in-house; these data were written in C and Objective-C and compiled as figures with ImageJ (Rasband, <http://imagej.nih.gov/ij/>). Measurements were processed with Ruby (<http://www.ruby-lang.org/>) and R (R Development Core Team, <http://www.R-project.org/>). The spatial coordinates (*x*--*y*--*z*--*t*) of areas with high signal intensity (E-cadherin-GFP, p120ctn-RFP or GFP-PACT) were tracked using software that searches for the local maximum within a three-dimensional volume manually specified by operators. Because of its concentrated localization, the E-cadherin fluorescence intensity at FC contact sites was estimated by taking the mean of the pixel intensities within a circle centred at the peak coordinate with a radius of 10 pixels (0.65 μm). The mobile fractions for FRAP analyses were estimated by fitting the fluorescence intensities to the following exponential equation: FC deformation was assessed by the shortening of the distance between the two FC--stalk cell interfaces (L--R) or between the FC contact site and the stalk-cell contact site (C--L and C--R). The particle image velocimetry analysis of EB1-GFP was performed on maximal *z*-projections of a confocal stack. In this analysis, 0.52-μm square windows were placed on the image for every 0.38 μm of *x*--*y* coordinates and the shift of the EB1-GFP signal in each window was estimated by searching within the next time point for the adjacent pixels by shifting original window up to 8 pixels along both *x* and *y* axes. The shift value showing the highest correlation of image pattern to the original window was recorded as vector. Tracks computed along 200 frames were mapped on a 25 × 25 grid of 0.38-μm squares and their direction and velocity were represented as bars. Numerical calculation of tug-of-war frequency --------------------------------------------- To compare the frequency of tug-of-war occurrence between wild-type and MT perturbed conditions, we measured the distance from FC contact site to FC/SC contact site (left and right) in every frames and calculated their change per time frame (displacement). Pairs of displacement value at each time point are then multiplied and the time points of which resulted in negative products were defined as ToW situation. Frequency of ToW situation was 72/154 (46.8%, 6 time series) and 112/180 (62.2%, 7 time series) for FC pairs in control and microtubule destabilized conditions, respectively. This result showed the frequencies of ToWs were significantly increased in MT-destabilized condition (Fisher\'s exact test, *P*=0.006). To simulate ToWs *in silico*, 300 pairs of random numbers were generated from Gaussian distribution with sample mean and s.d. of experimentally measured FC deformations (−0.22±0.65 μm min^−1^ for control and −0.17±0.79 for spas). Probability distributions of simulated ToW frequencies mimicking both conditions were estimated by repeating this trial for 10,000 times. Quantifying the effect of E-cadherin RNAi in the trachea -------------------------------------------------------- UAS-*shg* RNAi constructs were expressed with *btl-Gal4* and embryos were fixed, stained with anti-E-cadherin and confocal image stacks were taken from stage-15 embryos. E-cadherin signal intensity in the epidermis and trachea (stalk cells of DB) was measured by setting region of interest with line tool (ten cell boundaries for each tissue per embryo), background subtracted and the intensity ratio of trachea and epidermis was calculated. Signal intensity ratio was 1.62±0.33 for control (*N*=6) and 1.18±0.14 for E-cad RNAi (*N*=4). This is ∼27% reduction of E-cadherin protein level (*P*=0.0397, Student\'s *t*-test, two tailed). Additional information ====================== **How to cite this article:** Kato, K. *et al*. Microtubule-dependent balanced cell contraction and luminal-matrix modification accelerate epithelial tube fusion. *Nat. Commun.* 7:11141 doi: 10.1038/ncomms11141 (2016). Supplementary Material {#S1} ====================== ###### Supplementary Figures Supplementary Figures 1-4 ###### Supplementary Movie 1 Time course of dorsal branch migration and fusion and their relationship to the epidermis. GFP-moe was expressed in tracheal FCs and pAs by esg_FC-Gal4, and in the sqh-GFP-moe. Lower panel corresponds to a stacked x-z section in the boxed area of dorsal view in top panel. Scale bar in lower right is 10 μm and time stamp is shown in top-right. Original movie used to prepare Fig. 1c. ###### Supplementary Movie 2 Microtubule dynamics in tracheal FC and pAs revealed by tau-GFP marker expressed by the esg_FCGal4 driver. Original movie used to prepare Fig. 1d. ###### Supplementary Movie 3 Double labeling of tau-RFP (top, red) and GFP-moe (middle, green) driven by btl-Gal4. Related to Fig. 1e. ###### Supplementary Movie 4 High-speed imaging of E-cadherin trafficking during the first 3 minutes after contact of FCs. Related to Fig. 2f. ###### Supplementary Movie 5 Dynamics of myosin localization in DB fusion site revealed by esg_FC \> GFP-Zip. Related to Fig. 3a. ###### Supplementary Movie 6 Laser perturbation of fusion cell contraction. Embryos carrying btl-Gal4, UAS-GFP-moe and UASp120ctn-RFP was illuminated with 1440 nm laser (60mW) for 1 second at one of fusion cells (left) of DB5 (t=0). The embryo was imaged with PlanApo 60x NA1.40 oil IR lens with 4x zoom. Then 512 x 512 pixel images of 1-mm-thick sections were captured every 30 sec for 20 minutes. Original movie used to prepare Fig. 3b. ###### Supplementary Movie 7 Effect of myosin inhibition on fusion of DB. GFP-DN-Zip was co-expressed with p120ctn-RFP by btlGal4. Top panel; x-y view, bottom panel: y-z view. Most of FCs reached the midline and accumulated p120ctn-RFP at the interface. But p120ctn-RFP was not concentrated into a spot and FC failed to contract. Original movie used to prepare Fig. 3d. ###### Supplementary Movie 8 Dynamics of microtubule plus end. FC and TC of btl \> EB1-GFP embryo were imaged. The branch was directed toward 10 o\'clock direction. Left: original movie. Right: overlay of PIV tracks. Original movie used to prepare Fig. 4c. ###### Supplementary Movie 9 Double labeling of EB1-GFP (green) and membrane marker (red) imaged in a btl \> EB1-GFP, mRFPCAAX embryo. Dorsal branch was directed toward 2 o\'clock direction. EB1 comet repeatedly entered into filopodia. Original movie used to prepare Fig. 4d. Scale bar: 10μm. ###### Supplementary Movie 10 Double labeling of tau-RFP (top, megenta) and E-cadherin-GFP (middle, green). Horizontal view of the focal plane covering FC. Related to Fig. 4e. ###### Supplementary Movie 11 Effect of Kat60 on microtubule stability visualized by tau-RFP imaging. Left: control, right: Kat60 expressed by btl-Gal4. Related to Fig. 4F. ###### Supplementary Movie 12 Effect of Kat60 on adherens junction formation. p120ctn-RFP expression in FCs was imaged in control (top) and Kat60 (bottom) expressing trachea. Central signal correspond to FC contact site. Frame rate 18 sec. Original movie used to prepare Fig. 4i. ###### Supplementary Movie 13 Microtubule inhibition caused unbalanced FC contraction. Control (top) and Spas expressing FCs are labeled with E-cadherin-GFP and tagRFP. esg_FC-Gal4 driver used here additionally labeled pAs. Each panel consists of x-y and x-z stacks of dorsal views of fusion event in one metamere. Vertical lines mark fusion cell adhesion site (L, C, R). Original movie used to prepare Fig. 5a. We thank the Kyoto and Bloomington Drosophila Stock Centers and the Developmental Studies Hybridoma Bank, and Andrea Dega, Hiroki Oda, Yukiko Yamashita, Leo Tsuda, Masako Kaido, Naoyuki Fuse and Fumio Matsuzaki for fly stocks and antibodies. We thank Kenzi Oshima for suggesting the use of Katanin 60 to perturb microtubules and Stefan Luschnig for communication of unpublished work. We also thank the members of the Hayashi laboratory for helpful discussions. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (22111007 to S.H.) from The Ministry of Education, Culture, Sports, Science and Technology, Japan, by the RIKEN Special Postdoctoral Researcher Program (K.K.) and by the RIKEN Foreign Postdoctoral Researcher Program (B.D.). **Author contributions** K.K. performed the microtubule and cadherin experiments and analysed data with help from H.W. B.D. analysed the functions of myosin, Arl3 and Serp. Y.Y. identified the FC enhancer and M.T.-M. constructed the driver strain. K.K., B.D. and S.H. designed experiments, analysed the data and wrote the manuscript. ![Dorsal-branch migration and fusion of the *Drosophila* trachea.\ (**a**) A stack of confocal GFP images shows the tracheal system of a stage-15 embryo carrying *btl\>GFP-moe*. (**b**) Spatial relationship of the migrating tracheal branch and the epidermis; FCs, terminal cells and stalk cells of the dorsal branch and peripheral amnioserosa (pAs) are indicated. (**c**) Time course of dorsal-branch migration and fusion, shown by F-actin labelling with *esg_FC-Gal4* and *UAS-GFP-moe* (FC and pAs), and with sqh-GFP-moe (epidermis). Each panel shows dorsal (top half) and transverse views (bottom half) of images at the indicated time points (top left of the image). Filled triangles indicate F-actin-rich denticles of the apical surface of the epidermis; open triangles indicate FC filopodia penetrating vertically into the epidermis and nearly reaching the apical surface. Red dotted line indicates the basal epidermal surface. (**c\'**) Enlarged view of the vertical filopodia (1:30). (**d**) Microtubule distribution during tracheal-branch fusion, revealed by expressing the tau-GFP marker in FCs and pAs by the *esg_FC-Gal4* driver. Magenta triangles indicate MTOCs. (**d\'**) Enlargement of **d**, 0:30 time point. (**e**) Double labelling of microtubules (GFP-tau) and F-actin (GFP-moe). Scale bar, 10 μm.](ncomms11141-f1){#f1} ![E-cadherin accumulation and trafficking.\ (**a**) E-cadherin accumulation during formation of the cell junction (C) between FCs is shown by time-lapse movie (left) and by quantification of the accumulated E-cadherin-GFP (right). L and R, left and right FC--stalk boundaries. (**b**) E-cadherin accumulated at two sites in FC filopodia: in FCs making contact (arrow) and at the tip of forward-extending filopodia (white arrowheads). Red arrowhead, FC--stalk boundary; S, stalk cell; T, terminal cell. FRAP analysis (**c**) and quantification (**d**) of E-cadherin-GFP accumulation. Error bar indicate s.d. (**e**) A scatter plot of C versus L/R signals (Fc) after recovery showing the preferential addition of E-cadherin-GFP to the newly forming junction between FCs. (**f**) Intracellular E-cadherin-GFP trafficking: the GFP signal accumulated progressively in the FC contact site (C, white arrow) over a period of ∼3 min. Yellow arrows in the central panel show cytoplasmic E-cadherin-GFP streaming towards the plasma membrane. (**g**) Relocalization of the Golgi apparatus to the front end of FCs during new junction formation, visualized by imaging Grasp65-GFP driven by *esg_FC_Gal4*. (**h**) RNAi inhibition of *de novo* E-cadherin synthesis interfered with branch fusion. (**i**) Velocity measurements demonstrated that branch migration before branch contact was normal, but the cell deformation after contact was significantly delayed in the E-cadherin RNAi embryo (\*Student\'s *t*-test, *P*\<0.05). Scale bar, 10 μm.](ncomms11141-f2){#f2} ![FC contraction.\ (**a**) GFP-myosin heavy chain formed a longitudinal track connecting two adherens junctions in an FC. (**b**) Laser perturbation of FC contraction: the left-side cell (asterisk) in a new FC pair was illuminated by infrared laser at the time of contact. (**c**) The location of each cell-adhesion site (L, C and R) was tracked and the distance between sites was plotted. Laser perturbation caused significant deviations in L/R balance values (*N*=3, a representative case is shown). (**d**) Fusion was inhibited by a dominant-negative form of myosin. White arrow indicates the FC contact site. Scale bar, 10 μm.](ncomms11141-f3){#f3} ![Microtubule dynamics in FCs.\ (**a**) The polarization of FC microtubules during migration. The FC is outlined with a dotted line. The microtubule minus-end marker Nod-LacZ was concentrated at the FC--stalk cell boundary, which was marked with E-cadherin and lumen (chitin) staining. (**b**) The centriole marker GFP-PACT localized near the FC--stalk cell boundary; the lower panel shows the relative GFP-PACT localization along the axis connecting the FC--FC and FC--stalk cell boundaries during fusion. (**c**) Localization of the microtubule plus-end marker EB1-GFP and the flux and velocity distribution deduced by particle image velocimetry (PIV) analysis. (**d**) Multiple EB1-GFP comets entered a single filopodia. (**e**) E-cadherin-GFP accumulated on microtubule tracks at the FC contact site. (**f**) Kat60 destabilized the microtubule lattice in FCs. The distribution of tau-RFP (top, averaged for 20 frames of 10-s intervals) and its pixel-wise coefficient of variation in a 100-s period (bottom, averaged as in the top panel) are shown. (**g**) Kat60 inhibited E-cadherin accumulation at the FC contact site. (**h**) Kat60 inhibited tracheal-branch fusion. FCs contacted each other using filopodia, but did not form a stable adhesion interface. (**i**) Time course of p120ctn-RFP accumulation at the FC contact site. The region of interest (ROI) was set at the FC contact site (yellow box in inset); its intensity profile over a 15-min period is shown. (**j**) Time course of E-cadherin-GFP accumulation (arbitrary unit) in control and *esg_FC-Gal4\>Spas* FCs. (**k**) The rate of E-cadherin-GFP accumulation. (**l**) Fluctuation of E-cadherin-GFP accumulation (*R*^2^ value). Error bar indicates s.d. Scale bar, 10 μm.](ncomms11141-f4){#f4} ![The balanced contraction of paired FCs depends on microtubules.\ (**a**) Microtubule inhibition disrupted the balanced contraction: *x*--*z* views show FC pairs in control and Spas-expressing embryos (esg_FC-Gal4). Green, E-cadherin-GFP; magenta, tagRFP. Arrowheads and lines indicate cell-adhesion sites. (**b**) Plot of the L/R balance values. Spas expression caused greater deviations from a balanced condition, which was represented by a value of 0. The onset of E-cadherin-GFP accumulation at the FC--FC interface was set as 0 min in the time series of each embryo. (**c**) Fluctuations in contraction: the s.d. of the L/R balance value was plotted against time. (**d**) A contraction rate plot based on the L--R length decrease (\*Student\'s *t*-test, *P*\<0.05). (**e**) Evaluation of ToW conditions: vertical bars indicate the frequency of contraction/relaxation in FC pairs for each time interval measured in the two genotypes. Curves indicate the simulated distribution of numerically calculated frequencies of paired contraction/retraction events, assuming that contraction and retraction occur randomly in each FC (10,000 trials). The value calculated for control FCs was slightly smaller than that for Spas-expressing FCs due to the difference in contraction rate (**d**). The ToW value for Spas-expressing FCs (62.2%) was far greater than the value expected from the random hypothesis, suggesting that uncoordinated contraction/retraction events occurred more frequently in the FCs expressing Spas. Scale bar, 10 μm.](ncomms11141-f5){#f5} ![RFP-Rab9 vesicle trafficking in dorsal-branch FCs.\ (**a**) In dorsal-branch FCs (indicated by arrowheads and dotted outlines), RFP-Rab9 vesicle clusters (asterisks) were localized near the FC contact site. Anterior, left. (**a\'**) Clustering of RFP-Rab9 vesicles was inhibited when microtubule was disrupted by Kat60. (**b**) Arl3 was also concentrated adjacent to the apical cell membrane (labelled with Uif) and co-localized with GFP-Rab9 at the FC contact site of the dorsal trunk (yellow arrowheads). (**c**--**f**) A specific secretion defect in *arl3*-mutant embryos: fusion points of stage-16 dorsal trunk were stained for CBP and Serp (**c**), Verm (**d**), Pio (**e**) or Gasp (**f**). Chitin, Verm, Pio and 2A12 were present in the lumen isolated from the FC contact site (blue arrowheads), whereas Serp (yellow arrowheads) was missing. (**g**,**h**) Test of directed Serp trafficking: Serp-GFP was expressed in FCs (blue arrowheads) using the *esg_FC-Gal4* driver in control (*arl3*^*1*^*/+*, **g**) and *arl3*^*1*^ mutant (**h**) embryos, respectively. Secreted Serp-GFP was present throughout the lumen in control embryos (yellow arrowheads in **g**) but absent from the FC contact site in *arl3*^*1*^ embryos (blue arrowheads in **h**). (**g\'**, **h\'**) Enlargement of the boxed regions in **g** and **h**, respectively. Scale bar, 10 μm.](ncomms11141-f6){#f6} ![GFP-Rab9 overexpression rescued the *arl3*-mutant phenotype.\ (**a**,**b**) Control and *arl3*-mutant embryos were stained with CBP. Yellow arrowheads indicate a failure of branch fusion in the dorsal trunk of *arl3*-mutants. (**c**,**d**) GFP-Rab9 expressed specifically in the whole tracheal system by the *btl*-GAL4 driver or in FCs by the *esg_FC-Gal4* driver rescued the branch-fusion defect in *arl3* mutants. Yellow arrowheads indicate fusion-failure points remaining in the rescued embryos. GFP-Rab11 (**e**) and Serp-GFP (**f**) expression failed to rescue the *arl3* phenotype. (**g**) Quantification of the dorsal trunk fusion rate in different genotypes; 100% corresponds to nine successful fusion events in the ten tracheal metameres on one side of the embryo. Rescue activities of GFP-Rab9, GFP-Rab11, GFP-Rab6 and Serp-GFP were tested. Error bars indicate s.d. \*\*The fusion rate was significantly higher in *arl3* mutants overexpressing GFP-Rab9 than in *arl3* mutants without GFP-Rab9 overexpression, by Student\'s *t*-test (*P*\<0.01). Scale bar, 50 μm (**a**--**f**).](ncomms11141-f7){#f7} [^1]: Present address: Department of Imaging Science, Center for Novel Science Initiatives, National Institutes of Natural Sciences, Myodaiji-Cho, Okazaki, Aichi 444-8787, Japan [^2]: Present address: College of Marine Life Sciences, Ocean University of China, No. 5 Yushan Road, Qingdao 266003, China

{ "pile_set_name": "PubMed Central" }

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"esgfcspasexpress", "arldepend", "creat", "antigfp", "rapid", "absolut", "period", "white", "driver", "efreftypefig", "quantifi", "junctionbb", "anoth", "confoc", "method", "random", "kyoto", "educ", "lifeactgfp", "new", "andrea", "form", "vial", "fanshap", "leica", "pctnmrfp", "heavi", "diffus", "rvalu", "uaspctnrfp", "suitabl", "ministri", "mesenchym", "catalyt", "section", "architectur", "circl", "retract", "trunk", "insert", "gfp", "interf", "gasp", "gfppact", "arlmut", "stalk", "egfreftypefig", "contrast", "rasband", "promot", "surpass", "steadili", "b", "detect", "tau", "effect", "lower", "correl", "fraction", "hydrophil", "contain", "hiroki", "megenta", "uasgfpzip", "diod", "call", "averag", "filopodia", "proport", "recycl", "vertic", "came", "within", "masako", "nucleat", "hfreftypefig", "gfpzip", "sourc", "btlgfpmoe", "span", "cadherin", "function", "poorli", "peripher", "mean", "number", "preferenti", "axi", "fcsc", "rate", "taurfp", "doubl", "branchesbbbb", "centr", 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"transform", "bilater", "cellcel", "analysi", "fcfc", "complex", "hybridoma", "thin", "innov", "symmetr", "edg", "g", "could", "embryo", "simultan", "along", "fbtp", "genotyp", "green", "uasspasb", "dominantneg", "pcasperh", "balanc", "made", "varianc", "unit", "region", "dna", "neg", "antigasp", "volum", "spot", "oper", "hifreftypefig", "rnai", "doi", "mode", "okazaki", "core", "focal", "polar", "fbti", "increas", "fcspecif", "maintain", "softwar", "membranefus", "protrus", "incub", "similar", "shape", "point", "issu", "isol", "inject", "×", "mutual", "cytoskelet", "forwardreach", "area", "progress", "actomyosin", "driven", "termin", "foreign", "coexpress", "foci", "axe", "plu", "factinrich", "previou", "therefor", "swell", "defect", "clone", "uasnodlaczb", "contractionretract", "inner", "stabl", "mmthick", "fluctuat", "proper" ]

22,241

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"interfaces", "integrate", "target", "tissue", "poorly", "understood", "Studies", "formation", "adhesive", "interfaces", "cultured", "cells", "showed", "contact", "filopodia", "initiates", "assembly", "adherens", "junction", "suggested", "microtubules", "involved", "process", "formation", "cell", "junctions", "involves", "coordinating", "assembly", "cell", "adhesion", "complex", "generation", "tension", "side", "cells", "forming", "junction", "must", "observed", "simultaneously", "understand", "mechanical", "coupling", "controlled", "tissue", "level", "examine", "cell", "adhesion", "cell", "contractility", "coupled", "establish", "new", "cell", "interface", "examined", "anastomosis", "formation", "Drosophila", "tracheal", "system", "process", "pair", "tip", "cell", "hereafter", "called", "fusion", "cell", "FC", "forms", "adherens", "junction", "de", "novo", "Fig", "b", "fig", "deposit", "extracellular", "matrix", "materials", "newly", "forming", "lumen", "FC", "converted", "torus", 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"fig", "Despite", "defects", "FCs", "expressing", "Spas", "eventually", "accumulated", "completed", "branch", "fusion", "Taken", "together", "results", "indicate", "proper", "microtubule", "organization", "required", "stable", "persistent", "accumulation", "new", "sites", "fusion", "Microtubules", "coordinate", "deformation", "paired", "FCs", "next", "analysed", "dynamics", "change", "fusion", "process", "monitoring", "distance", "FC", "contact", "site", "C", "FC", "stalk", "cell", "boundaries", "left", "L", "right", "R", "side", "branch", "control", "embryos", "FC", "pairs", "maintained", "symmetry", "nearly", "equal", "C", "L", "C", "R", "lengths", "Fig", "c", "fig", "Supplementary", "Movie", "However", "FCs", "expressed", "Spas", "deformed", "slowly", "measured", "speed", "L", "R", "length", "decreased", "Fig", "fig", "failed", "maintain", "balanced", "bilateral", "cell", "contraction", "Fig", "fig", "measured", "balance", "balance", "calculated", "b", "b", "L", "b", "R", "Fig", 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"pairs", "time", "interval", "measured", "two", "genotypes", "Curves", "indicate", "simulated", "distribution", "numerically", "calculated", "frequencies", "paired", "events", "assuming", "contraction", "retraction", "occur", "randomly", "FC", "trials", "value", "calculated", "control", "FCs", "slightly", "smaller", "FCs", "due", "difference", "contraction", "rate", "ToW", "value", "FCs", "far", "greater", "value", "expected", "random", "hypothesis", "suggesting", "uncoordinated", "events", "occurred", "frequently", "FCs", "expressing", "Spas", "Scale", "bar", "μm", "vesicle", "trafficking", "FCs", "indicated", "arrowheads", "dotted", "outlines", "vesicle", "clusters", "asterisks", "localized", "near", "FC", "contact", "site", "Anterior", "left", "Clustering", "vesicles", "inhibited", "microtubule", "disrupted", "b", "also", "concentrated", "adjacent", "apical", "cell", "membrane", "labelled", "Uif", "FC", "contact", "site", "dorsal", "trunk", "yellow", "arrowheads", "c", "f", "specific", "secretion", "defect", "embryos", "fusion", "points", "dorsal", "trunk", "stained", "CBP", "Serp", "c", "Verm", "Pio", "e", "Gasp", "f", "Chitin", "Verm", "Pio", "present", "lumen", "isolated", "FC", "contact", "site", "blue", "arrowheads", "whereas", "Serp", "yellow", "arrowheads", "missing", "g", "h", "Test", "directed", "Serp", "trafficking", "expressed", "FCs", "blue", "arrowheads", "using", "driver", "control", "g", "mutant", "h", "embryos", "respectively", "Secreted", "present", "throughout", "lumen", "control", "embryos", "yellow", "arrowheads", "g", "absent", "FC", "contact", "site", "embryos", "blue", "arrowheads", "h", "Enlargement", "boxed", "regions", "g", "h", "respectively", "Scale", "bar", "μm", "overexpression", "rescued", "b", "Control", "embryos", "stained", "CBP", "Yellow", "arrowheads", "indicate", "failure", "branch", "fusion", "dorsal", "trunk", "c", "expressed", "specifically", "whole", "tracheal", "system", "btl", "driver", "FCs", "driver", "rescued", "defect", "mutants", "Yellow", "arrowheads", "indicate", "points", "remaining", "rescued", "embryos", "e", "f", "expression", "failed", "rescue", "phenotype", "g", "Quantification", "dorsal", "trunk", "fusion", "rate", "different", "genotypes", "corresponds", "nine", "successful", "fusion", "events", "ten", "tracheal", "metameres", "one", "side", "embryo", "Rescue", "activities", "tested", "Error", "bars", "indicate", "fusion", "rate", "significantly", "higher", "mutants", "overexpressing", "mutants", "without", "overexpression", "P", "Scale", "bar", "μm", "f", "Present", "address", "Department", "Imaging", "Science", "Center", "Novel", "Science", "Initiatives", "National", "Institutes", "Natural", "Sciences", "Okazaki", "Aichi", "Japan", "Present", "address", "College", "Marine", "Life", "Sciences", "Ocean", "University", "China", "Yushan", "Road", "Qingdao", "China" ]

Chemical context {#sec1} ================== Naphthalene and its derivatives are known for their wide range of applications in the field of pharmaceuticals. They are also used in the manufacturing of colorants, surface-active agents, resins, disinfectants and insecticides. These derivatives play a vital role in the control of microbial infection (Rokade & Sayyed, 2009[@bb12]) and in the chemical defence against biological enemies (Wright *et al.*, 2000[@bb18]). Compounds with a naphthalene moiety have been shown to exhibit significant anti-TB activity (Upadhayaya *et al.*, 2010[@bb17]). Structural commentary {#sec2} ======================= The molecular structure of the title compound is illustrated in Fig. 1[▸](#fig1){ref-type="fig"}. The benzene ring (C9--C14) of the naphthalene moiety is substituted by a dimethylphenyl ring (C2--C4/C6--C8) and a methoxyphenyl ring (C19--C24) *para* to each other. The naphthalene ring system is slightly bent with the two aryl rings being inclined to each other by 3.06 (15)°. Its mean plane makes dihedral angles of 65.24 (12)° with the dimethylphenyl ring (C2--C4/C6--C8) and 55.82 (12)° with methoxyphenyl ring (C19--C24). The latter two rings are inclined to each other by 59.28 (14)°. The methoxy group (C22/O1/C25) lies out of the plane of the benzene ring (C19--C24) to which it is attached by 11.3 (3)°. The bond lengths and bond angles are similar to those reported for 1,4-diphenylnaphthalene, which crystallized with two independent molecules in the asymmetric unit (Lima *et al.*, 2012[@bb7]). Supramolecular features {#sec3} ========================== In the crystal, there is only one significant intermolecular interaction present, *viz*. a C---H⋯π interaction linking adjacent molecules to form chains propagating along the *a*-axis direction (Table 1[▸](#table1){ref-type="table"} and Fig. 2[▸](#fig2){ref-type="fig"}). Database survey {#sec4} ================= A search of the Cambridge Structural Database (Version 5.39, last update February 2018; Groom *et al.*, 2016[@bb5]) for the aromatic skeleton of the title compound yielded ten hits. They include 1,4-diphenylnaphthalene itself, which crystallized with two independent molecules in the asymmetric unit (CSD refcode ZAXJEP: Lima *et al.*, 2012[@bb7]). There are also a number of copper(II) complexes (LAYQOU: Chen *et al.*, 2017[@bb3]; BOSHIC: Cai *et al.*, 2014[@bb2]; PUBSOV: Lin *et al.*, 2009[@bb8]) of the tetracarboxylic acid derivative, 5,5′-(naphthalene-1,4-diyl)diisophthalic acid, all of which are metal--organic frameworks. Analysis of the Hirshfeld surfaces, interaction energies and energy frameworks {#sec5} ================================================================================= The Hirshfeld surfaces and two-dimensional fingerprint plots were generated in order to explore and quantify the weak intermolecular interactions using the program *CrystalExplorer* 17.5 (Turner *et al.*, 2017[@bb16]). The electrostatic potentials were calculated using *TONTO*, integrated in the program *CrystalExplorer* (Spackman *et al.*, 2008[@bb15]; Jayatilaka *et al.*, 2005[@bb6]). The Hirshfeld surfaces of the title compound were mapped over *d* ~norm~, electrostatic potential, curvedness and shape index (Fig. 3[▸](#fig3){ref-type="fig"} *a*--3*d*); depending upon the closeness to the adjacent molecules, the colour patches are mapped differently on the Hirshfeld surface (Fig. 3[▸](#fig3){ref-type="fig"} *e*). Two-dimensional fingerprint plots showing the result of all intermolecular contacts (McKinnon *et al.*, 2007[@bb11]) are presented in Fig. 4[▸](#fig4){ref-type="fig"} *a*; *d* ~i~ (*x* axis) and *d* ~e~ (*y* axis) are the closest internal and external distance from a given point on the Hirshfeld surface. The fingerprint plot of H⋯H contacts, which represent the largest contribution to the Hirshfeld surface (64.6%), are shown as a distinct pattern with a minimum value of *d* ~e~ = *d* ~i~ ≃ 1.2 Å (Fig. 4[▸](#fig4){ref-type="fig"} *b*). The C⋯H/H⋯C interactions appear as the next largest region of the fingerprint plot, highly concentrated at the edges, having almost the same *d* ~e~ + *d* ~i~ ≃ 2.7 Å (Fig. 4[▸](#fig4){ref-type="fig"} *c*), with on overall contribution of 27.1%. The O⋯H/H⋯O interactions on the fingerprint plot, which contribute 5.2% of the total Hirshfeld surfaces, with *d* ~e~ + *d* ~i~ ≃ 2.8 Å (Fig. 4[▸](#fig4){ref-type="fig"} *d*) are shown as two symmetrical wings. The C⋯C contacts, which are the measure of π--π stacking interactions, occupy 3.1% of the Hirshfeld surfaces and appear as a unique triangle at about *d* ~e~ = *d* ~i~ ≃ 1.8 Å (Fig. 4[▸](#fig4){ref-type="fig"} *e*). These weak interactions mostly contribute to the packing of the title compound. The interaction energies between the molecules are obtained using monomer wavefunctions at the B3LYP/6-31G(p,d) level. The total interaction energy, which is the sum of scaled components, was calculated for a 3.8 Å radius cluster of molecules around the selected molecule (Fig. 5[▸](#fig5){ref-type="fig"} *a*). The scale factors used in the CE-B3LYP benchmarked energy model (Mackenzie *et al.*, 2017[@bb9]) are given in Table 2[▸](#table2){ref-type="table"}. The energies calculated by the energy model reveals that the dispersion energy contributes significantly to the interactions in the crystal (Table 3[▸](#table3){ref-type="table"}). The energy framework calculations were performed for a cluster of molecules present in 2 × 2 × 2 unit cells using the CE-B3LYP energy model. Energies between molecular pairs are represented as cylinders joining the centroids of pairs of molecules with the cylinder radius proportional to the magnitude of the interaction energy. Energy frameworks were constructed for *E* ~elec~ as red cylinders, *E* ~dis~ as green and *E* ~tot~ as blue (Fig. 5[▸](#fig5){ref-type="fig"} *b*--5*d*) and these cylinders represent the relative strength of molecular packing in different directions. Thus the supramolecular architecture of the crystal structure is visualized uniquely by energy frameworks. Synthesis and crystallization {#sec6} =============================== A reaction scheme for the synthesis of the title compound is illustrated in Fig. 6[▸](#fig6){ref-type="fig"}. To a solution of *m*-xylyl-*p*-anisyl tethered benzo\[*c*\]furan (0.16 g, 0.49 mmol) in dry xylenes (15 ml) was added tetrathiafulvalene (TTF) (0.10 g, 0.49 mmol) and the mixture was refluxed until the consumption of benzo\[*c*\]furan was complete; monitored by the disappearance of the fluorescent colour after 6 h. After the removal of xylenes *in vacuo*, the crude adduct was dissolved in dry CH~2~Cl~2~ (15 ml) and then kept at 273 K. To this, triflic acid (0.02 g, 0.13 mmol) was added and the mixture stirred at room temperature for 10 min. After completion of the reaction (monitored by TLC), it was poured into ice--water (20 ml) and then extracted with CH~2~Cl~2~ (2 × 10 ml). The organic layers were combined and washed with aq. NaHCO~3~ (2 × 10 ml) and then dried (Na~2~SO~4~). Removal of the solvent followed by column chromatographic purification (silica gel, 5% ethyl acetate in hexane) afforded the title compound as a yellow solid (0.20 g, 79%). Yellow block-like crystals of the title compound, suitable for X-ray diffraction analysis, were obtained by slow evaporation of a solution in CHCl~3~ (m.p. 351--353 K). Refinement {#sec7} ============ Crystal data collection and structure refinement details are summarized in Table 4[▸](#table4){ref-type="table"}. All H atoms were positioned geometrically and refined using a riding model: C---H = 0.93--0.96 Å with *U* ~iso~(H) = 1.5 *U* ~eq~(C-methyl) and 1.2*U* ~eq~(C) for other H atoms. Supplementary Material ====================== Crystal structure: contains datablock(s) I, Global. DOI: [10.1107/S2056989018008332/su5445sup1.cif](https://doi.org/10.1107/S2056989018008332/su5445sup1.cif) Structure factors: contains datablock(s) I. DOI: [10.1107/S2056989018008332/su5445Isup2.hkl](https://doi.org/10.1107/S2056989018008332/su5445Isup2.hkl) ###### Click here for additional data file. Supporting information file. DOI: [10.1107/S2056989018008332/su5445Isup3.cml](https://doi.org/10.1107/S2056989018008332/su5445Isup3.cml) CCDC reference: [1841351](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1841351) Additional supporting information: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?su5445&file=su5445sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?su5445sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?su5445&checkcif=yes) The authors thank the Central Instrumentation Facility (DST--FIST), Queen Mary's College (A), Chennai-4, for the computing facilities and the SAIF, IIT, Madras, for the X-ray data collection facility. Crystal data {#d1e138} ============ ------------------------ ---------------------------------------- C~25~H~22~O *Z* = 2 *M~r~* = 338.42 *F*(000) = 360 Triclinic, *P*1 *D*~x~ = 1.223 Mg m^−3^ *a* = 9.1670 (9) Å Mo *K*α radiation, λ = 0.71073 Å *b* = 10.4566 (10) Å Cell parameters from 19154 reflections *c* = 11.2499 (11) Å θ = 2.3--22.7° α = 64.707 (4)° µ = 0.07 mm^−1^ β = 71.312 (4)° *T* = 296 K γ = 77.032 (4)° Block, yellow *V* = 918.75 (16) Å^3^ 0.15 × 0.10 × 0.10 mm ------------------------ ---------------------------------------- Data collection {#d1e264} =============== ---------------------------------------------------------- -------------------------------------- Bruker Kappa APEXII CCD diffractometer 1777 reflections with *I* \> 2σ(*I*) Radiation source: fine-focus sealed tube *R*~int~ = 0.060 ω and φ scan θ~max~ = 26.6°, θ~min~ = 2.1° Absorption correction: multi-scan (SADABS; Bruker, 2012) *h* = −11→11 *T*~min~ = 0.900, *T*~max~ = 0.945 *k* = −13→13 19120 measured reflections *l* = −14→14 3828 independent reflections ---------------------------------------------------------- -------------------------------------- Refinement {#d1e377} ========== ------------------------------------- ------------------------------------------------------------------------------------------------- Refinement on *F*^2^ Primary atom site location: structure-invariant direct methods Least-squares matrix: full Secondary atom site location: difference Fourier map *R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.057 Hydrogen site location: inferred from neighbouring sites *wR*(*F*^2^) = 0.193 H-atom parameters constrained *S* = 1.00 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0922*P*)^2^ + 0.0768*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3 3828 reflections (Δ/σ)~max~ \< 0.001 238 parameters Δρ~max~ = 0.27 e Å^−3^ 0 restraints Δρ~min~ = −0.19 e Å^−3^ ------------------------------------- ------------------------------------------------------------------------------------------------- Special details {#d1e533} =============== ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#d1e552} ================================================================================================== ------ ------------- ------------ ------------ -------------------- -- *x* *y* *z* *U*~iso~\*/*U*~eq~ C1 0.7907 (4) 0.8345 (4) 0.2984 (3) 0.0771 (10) H1A 0.7017 0.8047 0.2937 0.116\* H1B 0.7750 0.9350 0.2771 0.116\* H1C 0.8053 0.7849 0.3885 0.116\* C2 0.9307 (3) 0.8015 (3) 0.1987 (3) 0.0509 (7) C3 1.0610 (3) 0.8748 (3) 0.1542 (3) 0.0578 (8) H3 1.0583 0.9411 0.1898 0.069\* C4 1.1927 (3) 0.8530 (3) 0.0602 (3) 0.0573 (8) C5 1.3309 (4) 0.9324 (4) 0.0196 (4) 0.0863 (11) H5A 1.4237 0.8679 0.0169 0.129\* H5B 1.3205 0.9724 0.0848 0.129\* H5C 1.3365 1.0071 −0.0686 0.129\* C6 1.1935 (3) 0.7571 (3) 0.0065 (3) 0.0598 (8) H6 1.2803 0.7415 −0.0585 0.072\* C7 1.0668 (3) 0.6835 (3) 0.0480 (3) 0.0541 (7) H7 1.0699 0.6198 0.0093 0.065\* C8 0.9353 (3) 0.7015 (3) 0.1453 (3) 0.0446 (7) C9 0.8049 (3) 0.6158 (3) 0.1869 (3) 0.0437 (6) C10 0.7284 (3) 0.6328 (3) 0.0942 (3) 0.0537 (7) H10 0.7534 0.7037 0.0079 0.064\* C11 0.6138 (3) 0.5472 (3) 0.1247 (3) 0.0527 (7) H11 0.5648 0.5633 0.0581 0.063\* C12 0.5716 (3) 0.4408 (3) 0.2488 (3) 0.0426 (6) C13 0.6437 (3) 0.4220 (3) 0.3514 (2) 0.0393 (6) C14 0.7621 (3) 0.5075 (3) 0.3195 (2) 0.0403 (6) C15 0.8360 (3) 0.4807 (3) 0.4219 (3) 0.0507 (7) H15 0.9173 0.5323 0.4017 0.061\* C16 0.7907 (3) 0.3810 (3) 0.5493 (3) 0.0602 (8) H16 0.8414 0.3649 0.6147 0.072\* C17 0.6690 (3) 0.3029 (3) 0.5823 (3) 0.0612 (8) H17 0.6361 0.2374 0.6706 0.073\* C18 0.5983 (3) 0.3221 (3) 0.4860 (3) 0.0518 (7) H18 0.5180 0.2682 0.5092 0.062\* C19 0.4540 (3) 0.3483 (3) 0.2743 (2) 0.0432 (6) C20 0.3108 (3) 0.4064 (3) 0.2491 (3) 0.0504 (7) H20 0.2877 0.5046 0.2182 0.060\* C21 0.2007 (3) 0.3235 (3) 0.2682 (3) 0.0525 (7) H21 0.1051 0.3658 0.2507 0.063\* C22 0.2331 (3) 0.1787 (3) 0.3130 (3) 0.0510 (7) C23 0.3750 (3) 0.1177 (3) 0.3379 (3) 0.0601 (8) H23 0.3977 0.0195 0.3676 0.072\* C24 0.4841 (3) 0.2011 (3) 0.3193 (3) 0.0543 (7) H24 0.5794 0.1581 0.3371 0.065\* C25 −0.0236 (4) 0.1430 (4) 0.3343 (4) 0.0834 (11) H25A −0.0854 0.0667 0.3644 0.125\* H25B −0.0284 0.2070 0.2437 0.125\* H25C −0.0624 0.1932 0.3942 0.125\* O1 0.1324 (2) 0.0867 (2) 0.3352 (2) 0.0742 (7) ------ ------------- ------------ ------------ -------------------- -- Atomic displacement parameters (Å^2^) {#d1e1180} ===================================== ----- ------------- ------------- ------------- -------------- -------------- -------------- *U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^ C1 0.076 (2) 0.079 (2) 0.081 (2) −0.0173 (19) −0.0011 (18) −0.044 (2) C2 0.0503 (17) 0.0521 (17) 0.0505 (17) −0.0106 (14) −0.0132 (14) −0.0171 (14) C3 0.068 (2) 0.0485 (17) 0.0622 (19) −0.0158 (15) −0.0235 (16) −0.0169 (15) C4 0.0534 (18) 0.0526 (18) 0.0532 (18) −0.0149 (15) −0.0173 (15) −0.0013 (15) C5 0.069 (2) 0.091 (3) 0.088 (3) −0.040 (2) −0.0228 (19) −0.007 (2) C6 0.0523 (18) 0.0585 (19) 0.0533 (18) −0.0127 (15) −0.0040 (14) −0.0106 (16) C7 0.0547 (17) 0.0528 (18) 0.0498 (17) −0.0128 (14) −0.0061 (14) −0.0169 (14) C8 0.0491 (16) 0.0419 (15) 0.0427 (15) −0.0124 (13) −0.0133 (13) −0.0113 (13) C9 0.0458 (15) 0.0475 (16) 0.0415 (15) −0.0104 (13) −0.0101 (12) −0.0186 (13) C10 0.0621 (18) 0.0550 (18) 0.0421 (16) −0.0192 (15) −0.0150 (14) −0.0093 (14) C11 0.0593 (18) 0.0571 (18) 0.0424 (17) −0.0159 (15) −0.0180 (14) −0.0113 (14) C12 0.0412 (15) 0.0454 (15) 0.0438 (16) −0.0070 (12) −0.0088 (12) −0.0197 (13) C13 0.0393 (14) 0.0416 (15) 0.0365 (15) −0.0044 (12) −0.0067 (11) −0.0165 (12) C14 0.0404 (14) 0.0416 (15) 0.0408 (15) −0.0055 (12) −0.0091 (12) −0.0178 (13) C15 0.0515 (16) 0.0574 (18) 0.0482 (17) −0.0131 (14) −0.0149 (14) −0.0197 (15) C16 0.069 (2) 0.070 (2) 0.0467 (18) −0.0158 (17) −0.0230 (15) −0.0174 (16) C17 0.073 (2) 0.067 (2) 0.0392 (17) −0.0215 (17) −0.0110 (15) −0.0115 (15) C18 0.0526 (17) 0.0563 (18) 0.0454 (17) −0.0149 (14) −0.0063 (13) −0.0185 (15) C19 0.0435 (15) 0.0456 (16) 0.0436 (15) −0.0065 (13) −0.0088 (12) −0.0206 (13) C20 0.0522 (17) 0.0465 (16) 0.0551 (17) −0.0068 (14) −0.0183 (13) −0.0179 (14) C21 0.0458 (16) 0.0576 (19) 0.0610 (18) −0.0057 (14) −0.0174 (13) −0.0261 (15) C22 0.0521 (18) 0.0548 (19) 0.0544 (18) −0.0148 (15) −0.0075 (14) −0.0285 (15) C23 0.060 (2) 0.0463 (17) 0.076 (2) −0.0056 (16) −0.0136 (16) −0.0280 (16) C24 0.0451 (16) 0.0518 (18) 0.0666 (19) −0.0019 (14) −0.0130 (14) −0.0256 (15) C25 0.060 (2) 0.102 (3) 0.108 (3) −0.029 (2) −0.0172 (19) −0.051 (2) O1 0.0641 (14) 0.0687 (14) 0.1023 (18) −0.0233 (12) −0.0140 (12) −0.0415 (13) ----- ------------- ------------- ------------- -------------- -------------- -------------- Geometric parameters (Å, º) {#d1e1774} =========================== ----------------------- ------------ ----------------------- ------------ C1---C2 1.495 (4) C13---C18 1.413 (3) C1---H1A 0.9600 C13---C14 1.421 (3) C1---H1B 0.9600 C14---C15 1.416 (3) C1---H1C 0.9600 C15---C16 1.360 (4) C2---C8 1.399 (4) C15---H15 0.9300 C2---C3 1.401 (3) C16---C17 1.390 (3) C3---C4 1.375 (4) C16---H16 0.9300 C3---H3 0.9300 C17---C18 1.357 (4) C4---C6 1.373 (4) C17---H17 0.9300 C4---C5 1.510 (4) C18---H18 0.9300 C5---H5A 0.9600 C19---C20 1.380 (4) C5---H5B 0.9600 C19---C24 1.391 (4) C5---H5C 0.9600 C20---C21 1.383 (3) C6---C7 1.381 (3) C20---H20 0.9300 C6---H6 0.9300 C21---C22 1.370 (4) C7---C8 1.385 (3) C21---H21 0.9300 C7---H7 0.9300 C22---O1 1.373 (3) C8---C9 1.488 (3) C22---C23 1.373 (4) C9---C10 1.366 (3) C23---C24 1.381 (3) C9---C14 1.430 (3) C23---H23 0.9300 C10---C11 1.397 (3) C24---H24 0.9300 C10---H10 0.9300 C25---O1 1.420 (4) C11---C12 1.363 (4) C25---H25A 0.9600 C11---H11 0.9300 C25---H25B 0.9600 C12---C13 1.430 (3) C25---H25C 0.9600 C12---C19 1.488 (3) C2---C1---H1A 109.5 C18---C13---C12 121.9 (2) C2---C1---H1B 109.5 C14---C13---C12 119.8 (2) H1A---C1---H1B 109.5 C15---C14---C13 118.0 (2) C2---C1---H1C 109.5 C15---C14---C9 121.8 (2) H1A---C1---H1C 109.5 C13---C14---C9 120.2 (2) H1B---C1---H1C 109.5 C16---C15---C14 121.4 (3) C8---C2---C3 118.4 (2) C16---C15---H15 119.3 C8---C2---C1 122.1 (2) C14---C15---H15 119.3 C3---C2---C1 119.5 (3) C15---C16---C17 120.4 (3) C4---C3---C2 122.9 (3) C15---C16---H16 119.8 C4---C3---H3 118.5 C17---C16---H16 119.8 C2---C3---H3 118.5 C18---C17---C16 120.1 (3) C6---C4---C3 117.8 (3) C18---C17---H17 120.0 C6---C4---C5 121.9 (3) C16---C17---H17 120.0 C3---C4---C5 120.3 (3) C17---C18---C13 121.6 (3) C4---C5---H5A 109.5 C17---C18---H18 119.2 C4---C5---H5B 109.5 C13---C18---H18 119.2 H5A---C5---H5B 109.5 C20---C19---C24 117.0 (2) C4---C5---H5C 109.5 C20---C19---C12 120.9 (2) H5A---C5---H5C 109.5 C24---C19---C12 122.1 (2) H5B---C5---H5C 109.5 C19---C20---C21 122.2 (3) C4---C6---C7 120.7 (3) C19---C20---H20 118.9 C4---C6---H6 119.7 C21---C20---H20 118.9 C7---C6---H6 119.7 C22---C21---C20 119.7 (3) C6---C7---C8 122.1 (3) C22---C21---H21 120.2 C6---C7---H7 119.0 C20---C21---H21 120.2 C8---C7---H7 119.0 C21---C22---O1 124.4 (3) C7---C8---C2 118.1 (2) C21---C22---C23 119.5 (3) C7---C8---C9 118.8 (2) O1---C22---C23 116.0 (3) C2---C8---C9 123.1 (2) C22---C23---C24 120.5 (3) C10---C9---C14 117.6 (2) C22---C23---H23 119.8 C10---C9---C8 120.0 (2) C24---C23---H23 119.8 C14---C9---C8 122.3 (2) C23---C24---C19 121.2 (3) C9---C10---C11 122.3 (3) C23---C24---H24 119.4 C9---C10---H10 118.9 C19---C24---H24 119.4 C11---C10---H10 118.9 O1---C25---H25A 109.5 C12---C11---C10 122.1 (3) O1---C25---H25B 109.5 C12---C11---H11 119.0 H25A---C25---H25B 109.5 C10---C11---H11 119.0 O1---C25---H25C 109.5 C11---C12---C13 118.0 (2) H25A---C25---H25C 109.5 C11---C12---C19 120.0 (2) H25B---C25---H25C 109.5 C13---C12---C19 122.0 (2) C22---O1---C25 117.3 (2) C18---C13---C14 118.4 (2) C8---C2---C3---C4 0.2 (4) C12---C13---C14---C9 2.4 (3) C1---C2---C3---C4 −178.4 (3) C10---C9---C14---C15 179.2 (2) C2---C3---C4---C6 1.4 (4) C8---C9---C14---C15 3.5 (4) C2---C3---C4---C5 −178.2 (3) C10---C9---C14---C13 0.0 (3) C3---C4---C6---C7 −1.2 (4) C8---C9---C14---C13 −175.7 (2) C5---C4---C6---C7 178.4 (3) C13---C14---C15---C16 −3.2 (4) C4---C6---C7---C8 −0.7 (4) C9---C14---C15---C16 177.6 (2) C6---C7---C8---C2 2.3 (4) C14---C15---C16---C17 −0.3 (4) C6---C7---C8---C9 −178.6 (2) C15---C16---C17---C18 2.4 (4) C3---C2---C8---C7 −2.0 (4) C16---C17---C18---C13 −0.9 (4) C1---C2---C8---C7 176.6 (3) C14---C13---C18---C17 −2.6 (4) C3---C2---C8---C9 178.9 (2) C12---C13---C18---C17 178.8 (3) C1---C2---C8---C9 −2.5 (4) C11---C12---C19---C20 −53.8 (3) C7---C8---C9---C10 −63.5 (3) C13---C12---C19---C20 126.4 (3) C2---C8---C9---C10 115.5 (3) C11---C12---C19---C24 123.9 (3) C7---C8---C9---C14 112.1 (3) C13---C12---C19---C24 −56.0 (3) C2---C8---C9---C14 −68.9 (3) C24---C19---C20---C21 0.5 (4) C14---C9---C10---C11 −1.1 (4) C12---C19---C20---C21 178.2 (2) C8---C9---C10---C11 174.7 (2) C19---C20---C21---C22 −0.3 (4) C9---C10---C11---C12 −0.3 (4) C20---C21---C22---O1 −179.9 (2) C10---C11---C12---C13 2.7 (4) C20---C21---C22---C23 −0.2 (4) C10---C11---C12---C19 −177.2 (2) C21---C22---C23---C24 0.6 (4) C11---C12---C13---C18 174.9 (2) O1---C22---C23---C24 −179.7 (2) C19---C12---C13---C18 −5.2 (3) C22---C23---C24---C19 −0.4 (4) C11---C12---C13---C14 −3.7 (3) C20---C19---C24---C23 −0.1 (4) C19---C12---C13---C14 176.2 (2) C12---C19---C24---C23 −177.8 (2) C18---C13---C14---C15 4.5 (3) C21---C22---O1---C25 −11.4 (4) C12---C13---C14---C15 −176.8 (2) C23---C22---O1---C25 168.9 (3) C18---C13---C14---C9 −176.2 (2) ----------------------- ------------ ----------------------- ------------ Hydrogen-bond geometry (Å, º) {#d1e2748} ============================= Cg is the centroid of the C13--C18 ring. ------------------------ --------- --------- ----------- --------------- *D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A* C25---H25*C*···*Cg*^i^ 0.96 2.78 3.597 (5) 144 ------------------------ --------- --------- ----------- --------------- Symmetry code: (i) *x*−1, *y*, *z*. ![The molecular structure of the title compound, with the atom labelling. Displacement ellipsoids are drawn at 50% probability level.](e-74-00939-fig1){#fig1} ![The crystal packing of the title compound, viewed along the *b* axis. The C---H⋯π interactions (see Table 1[▸](#table1){ref-type="table"}) are shown as dashed lines, and only the H atom H25*C* (grey ball) has been included.](e-74-00939-fig2){#fig2} ![Hirshfeld surfaces mapped over (*a*) *d* ~norm~, (*b*) electrostatic potential, (*c*) shape index and (*d*) curvedness and (*e*) fragment patches.](e-74-00939-fig3){#fig3} ![Two-dimensional fingerprint plot for the title compound showing the contributions of individual types of interactions: (*a*) all intermolecular contacts, (*b*) H⋯H contacts, (*c*) C⋯H/H⋯C contacts, (*d*) H⋯O/O⋯H contacts, (*e*) C⋯C contacts. The outline of the full fingerprint is shown in grey. Surfaces to the right highlight the relevant surface patches associated with the specific contacts with *d* ~norm~ mapped.](e-74-00939-fig4){#fig4} ![(*a*) Interaction between the selected molecule and the molecules present in a 3.8 Å cluster around it, (*b*) Coulombic energy, (*c*) dispersion energy and (*d*) total energy.](e-74-00939-fig5){#fig5} ![Reaction scheme.](e-74-00939-fig6){#fig6} ###### Hydrogen-bond geometry (Å, °) *Cg* is the centroid of the C13--C18 ring. *D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A* ----------------------- --------- ------- ----------- ------------- C25---H25*C*⋯*Cg* ^i^ 0.96 2.78 3.597 (5) 144 Symmetry code: (i) . ###### Scale factors for the benchmarked energy model Energy model *k* ~elec~ *k* ~pol~ *k* ~energy-dispersive~ *k* ~rep~ ----------------------------------------------- ------------ ----------- ------------------------- ----------- CE-B3LYP⋯B3LYP/6--31G(d,p) electron densities 1.057 0.740 0.871 0.618 ###### Interaction energies (kJ mol^−1^) *R* is the distance between molecular centroids (mean atomic position) in Å and *N* is the number of molecules at that distance. Colour *N* symop *R* *E* ~elec~ *E* ~pol~ *E* ~energy-dispersive~ *E* ~rep~ *E* ~total~ ------------- ----- ------------------ ------- ------------ ----------- ------------------------- ----------- ------------- Red 2 *x*, *y*, *z* 15.38 −2.2 −0.6 −11.2 6.2 −8.6 Orange 1 −*x*, −*y*, −*z* 15.99 −4.3 −0.8 −11.5 4.2 −12.5 Yellow 1 −*x*, −*y*, −*z* 7.45 −6.2 −1.3 −39.2 19.3 −29.7 Lime 2 *x*, *y*, *z* 9.17 −10.0 −1.8 −44.0 26.8 −33.6 Green 2 *x*, *y*, *z* 10.46 −0.1 −0.1 −6.6 1.5 −5.0 Aquamarine 1 −*x*, −*y*, −*z* 6.86 −6.8 −0.9 −39.9 18.7 −31.0 Cyan 1 −*x*, −*y*, −*z* 10.11 −0.3 −0.4 −19.8 6.7 −13.7 Blue 1 −*x*, −*y*, −*z* 5.37 −3.3 −1.9 −69.2 32.2 −45.2 Violet 1 −*x*, −*y*, −*z* 9.31 −6.5 −0.8 −36.0 20.7 −26.0 Orchid 2 *x*, *y*, *z* 14.01 0.1 0.0 −2.0 0.0 −1.7 Magenta 1 −*x*, −*y*, −*z* 11.61 −3.3 −1.0 −41.6 20.4 −27.9 ###### Experimental details -------------------------------------------------------------------------- ------------------------------------------- Crystal data Chemical formula C~25~H~22~O *M* ~r~ 338.42 Crystal system, space group Triclinic, *P* Temperature (K) 296 *a*, *b*, *c* (Å) 9.1670 (9), 10.4566 (10), 11.2499 (11) α, β, γ (°) 64.707 (4), 71.312 (4), 77.032 (4) *V* (Å^3^) 918.75 (16) *Z* 2 Radiation type Mo *K*α μ (mm^−1^) 0.07 Crystal size (mm) 0.15 × 0.10 × 0.10 Data collection Diffractometer Bruker Kappa APEXII CCD Absorption correction Multi-scan (*SADABS*; Bruker, 2012[@bb1]) *T* ~min~, *T* ~max~ 0.900, 0.945 No. of measured, independent and observed \[*I* \> 2σ(*I*)\] reflections 19120, 3828, 1777 *R* ~int~ 0.060 (sin θ/λ)~max~ (Å^−1^) 0.631 Refinement *R*\[*F* ^2^ \> 2σ(*F* ^2^)\], *wR*(*F* ^2^), *S* 0.057, 0.193, 1.00 No. of reflections 3828 No. of parameters 238 H-atom treatment H-atom parameters constrained Δρ~max~, Δρ~min~ (e Å^−3^) 0.27, −0.19 -------------------------------------------------------------------------- ------------------------------------------- Computer programs: *APEX2*, *SAINT* and *XPREP* (Bruker, 2012[@bb1]), *SHELXT2014* (Sheldrick, 2015*a* [@bb13]), *SHELXL2014* (Sheldrick, 2015*b* [@bb14]), *ORTEP-3 for Windows* (Farrugia, 2012[@bb4]) and *Mercury* (Macrae *et al.*, 2008[@bb10]).

{ "pile_set_name": "PubMed Central" }

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"Farrugia", "Mercury", "Macrae", "et", "al" ]

In the past decades, surface plasmons (SPs) have been extensively investigated in the optical frequencies[@b1]. Due to their extremely strong and confined optical fields, SPs have shown various potential applications in the photonic circuits[@b2][@b3], near-field microscopy[@b4][@b5], biological sensors[@b6][@b7][@b8], photovoltaics[@b9][@b10], etc. Since the spoof (or designer) surface plasmon polaritons (SPPs) allow a route to imitate the optical SPPs[@b11][@b12], a variety of advanced plasmonic researches have been put forward and experimentally realized at low frequencies ranging from the microwave to infrared spectra, such as conformal surface plasmons waveguide[@b13], negative-index waveguide[@b14], and terahertz SPPs[@b15][@b16][@b17][@b18]. Recently, an important discovery showed that periodically textured perfectly electrical conducting (PEC) particles either in two or three-dimensions is able to mimic the natural localized surface plasmons (LSPs) at lower frequencies, which is called as spoof LSPs[@b19]. More recently, an ultrathin plasmonic spoof LSP resonator has been proposed, which is easy for fabrication and integration[@b20]. Based on the design of these plasmonic metamaterial particles, the conventional LSPs at optical frequencies can be directly scaled down to lower frequencies[@b21]. Apart from the features of conventional LSPs shown in the optical frequencies, many phenomena have been predicted and experimentally verified for spoof LSPs, such as the vertical transportation[@b22], unusual electric and magnetic LSPs resonances[@b23] and high-order electric LSPs resonances[@b24][@b25]. However, all earlier investigations and experiments on spoof LSPs are limited to the microwave frequency. With the rapid development of terahertz technology in recent years, the implementation of terahertz spoof LSPs resonators may become possible, which will facilitate the device miniaturization and system integration in the terahertz band. In this article, we propose the LSP particle in the terahertz frequency using ultrathin spirally corrugated metallic disk and generate the LSP resonances. We demonstrate that both spoof electric and magnetic LSPs are supported by the proposed ultrathin structure in the terahertz band, which is in deep-subwavelength scale and highly relies on the incident angle of terahertz waves. By slightly tuning the geometrical parameters of the proposed structure, we could change the resonant frequencies and further increase the subwavelength level. Besides the geometrical parameters, arrangements and separations have also significant effects on the resonance frequency[@b26][@b27]. These approaches extends the capabilities of spoof LSPs, and can be applied to the terahertz integrated circuits. Using the proposed magnetic LSPs, we further construct an electromagnetically induced transparency (EIT) metamaterial in the terahertz spectrum[@b28][@b29]. Numerical simulations, experimental measurements are conducted to evaluate and validate the performance of the proposed spoof LSPs, promising in a number of potential terahertz applications such slow lights, filters, and sensing. Results ======= The structure of the designed LSPs particle is schematically illustrated in [Fig. 1(a)](#f1){ref-type="fig"}, which is composed of four gold spiral arms and one internal gold disk with ultrathin thickness 0.2 μm. The radius *r* of the inner disk and length *R* of the outer spiral arms are 6.5 μm and 33 μm, respectively. The four spiral arms have the same linewidth *w* = 5 μm, and are separated by a gap *g* = 5 μm. The designed LSPs particle is fabricated on a polyimide substrate with 20 μm thickness (*ε* = 3, *tan* = 0.03). We first consider the case when the particle is normally illuminated with an *x*-polarized plane wave. The extinction cross sections (ECSs) are calculated using commercial software, the CST Microwave Studio 2013, as shown in [Fig. 1b](#f1){ref-type="fig"}. Here, the ECSs are sums of absorption cross sections and scattering cross sections. In [Fig. 1b](#f1){ref-type="fig"}, a peak can be clearly observed at 0.73 THz. To gain more insights into the resonance, we give the simulated electric field distributions (*E*~*z*~) in the *x-y* plane 5 μm above the metallic structure at 0.73 THz, as shown in [Fig. 1c](#f1){ref-type="fig"}. The electric fields distribution matches well with the previously reported spoof LSPs in the microwave frequency[@b23]. Meanwhile, we plot in [Fig. 1d](#f1){ref-type="fig"} the magnitude of the electric fields and electric field lines of this mode in a cross section *y* = 0. We can see that the arrows go from left of the structure to right. Clearly, this mode profiles correspond to the electric-dipole mode, indicating that the proposed structure supports spoof LSPs resonance at terahertz frequency. In [Fig. 2(a)](#f2){ref-type="fig"}, we show the calculated ECS spectra as a function of the outer radius *R*. When the radius *R* of the particle increases from 29 to 37 μm, the resonance frequency experiences an obvious redshift from 0.94 to 0.577 THz. To reveal the reason for the redshift in the proposed spoof LSP resonator, we first investigate the nature of spoof SPPs, since the spoof LSP resonator arises from the spoof SPP strip and the work frequency of the spoof SPPs has many influences on the resonance frequency of the relevant spoof LSPs. Textured surfaces can support spoof SPs due to the cavity-waveguide mode inside the groove, which is analogous to the penetration of fields into the metal. Either in straight or spiral groove, the frequency of existing SPs depends mainly on the depth of grooves. Here, although grooves in the proposed structure are spiral, it has been proved that the spiral grooves behave like the straight grooves approximatively[@b23]. The mentioned spoof SPP structure is illustrated in the inset of [Fig. 2(b)](#f2){ref-type="fig"}, whose parameters are set as *s* = 6.5 μm, *p* = 10 μm, and *a* = 5 μm. The depths of the straight grooves *h* are equal to the lengths of spiral curves in spoof LSP resonators, which are 45.7 μm, 53.8 μm, 62.5 μm, 71.8 μm, and 81.7 μm according to *R* = 29 μm, 31 μm, 33 μm, 35 μm, and 37 μm. The thicknesses of the metal and substrate are 0.2 μm and 20 μm, respectively. The calculated dispersion relations of spoof SPPs are presented in [Fig. 2(b)](#f2){ref-type="fig"}, from which we obtain that the deeper grooves correspond to the lower asymptotic frequencies. Actually the spoof LSP is a standing wave on the spoof LSP loop. Meanwhile because deeper grooves have lower frequencies for the same wavenumber, as shown in [Fig. 2(b)](#f2){ref-type="fig"}, hence the spoof LSP resonators composed of deeper grooves have lower resonance frequencies. Here, the deeper groove means longer spiral curve in the particle, which represents that the particle has bigger radius. Therefore, the resonance frequencies get smaller as the radiuses become bigger, as plotted in [Fig. 2(a)](#f2){ref-type="fig"}. In addition, we note that the radius *R* is much less than the resonant wavelength in the present design, making it a deep subwavelength structure. To gain an intuitional insight into their relationship, [Fig. 3](#f3){ref-type="fig"} further gives the resonance wavelengths *λ*~*res*~ as a function of the outer radius *R*, in which we see that the resonance wavelengths increase from about 319 to 520 μm as *R* increases from 29 to 37 μm. It can be calculated that a 27% change in size (from *R* = 29 μm to *R* = 37 μm) results in a 201 μm (or 63%) increase in resonance wavelength, demonstrating that *λ*~*res*~ can be tuned in large range by simply varying the radius *R*. Most importantly, by calculating the equivalent electrical length of the case when *R* equals 29 and 37 μm, we found that the bigger the radius R, the more the deep subwavelength effect is. This can be attributed to the large electrical length formed by the spiral grooves of particle, which effectively reduce the resonant frequency. Moreover, small change in radius *R* will result in great variation in the effective length of spiral grooves. The resonance frequency is not only highly dependent on the size of the meta-atom, but also depends on arrangement due to the electromagnetic interactions between neighbor particles. To evaluate these interactions, we simulate the transmission spectra using periodic boundaries. [Figure 4(a)](#f4){ref-type="fig"} shows the results for square arrays of meta-atoms with lattice constant *p* = 80--180 μm. As array spacing decreases, the resonance frequency first blue shifts for *p* larger than 80 μm and then redshifts for smaller distances. To analyze the unit coupling effects, we introduce a simple semi-analytical model which can help understand this trend qualitatively[@b26][@b27]. In this model, the retarded dipole sum *S* can reveal the variation of the spectra with arranged period. It was demonstrated that the real parts of *S* imply the resonant frequency shifts. The positive value corresponds to red shift of *λ*~*max*~, while the negative value represents blue shift. This sum *S* is given by where *d*~*ij*~ is the distance between the *i*th and *j*th particles, *θ*~*ij*~ is the angle between the vector from units *i* to *j* and the polarization vector. From [Fig. 4(a)](#f4){ref-type="fig"}, we find that the resonant frequencies are approximately 0.75 THz (400 μm). Then we calculate the values of *S* as the period *p* changes from 180 to 100 μm for *λ *= 400 μm. The results are presented in [Table 1](#t1){ref-type="table"}. [Table 1](#t1){ref-type="table"} demonstrates that the real part of *S* is negative when *p* changes from 180 to 100 μm, while is positive for *p* = 80 μm. The real part becomes less negative as the period *p* decreases and becomes positive until *p* = 80 μm. In other words, blue shifts occur for *p* = 180 to 100 μm and red shifts for *p* = 80 μm, which have good agreement with the trend in [Fig. 4(a)](#f4){ref-type="fig"}. Actually, the expression for *S* can be divided into two parts, one of which has 1/*d* component, representing the radiative dipole interaction, and the other of which has 1/*d*^*3*^ component, showing the static dipole coupling. Hence we find that for big lattice constant the radiative dipole interaction is dominant, whereas the static dipole coupling plays a more important role as the period *p* decreases. And the resonance blue shifts (negative value in real parts of *S*) are the result of the radiative dipole interaction for bigger separations, whereas red shifts due to the static dipole coupling for smaller lattice spacing. To experimentally verify the performance of the proposed spoof LSPs arrays, we fabricate samples, whose unit cells are distributed on a two-dimensional surface with lattice constants *p* from 80 to 180 μm. Each sample occupies an area of 15 × 15 mm, which is wide enough to cover the focused THz beam in experiments. [Figure 4(c)](#f4){ref-type="fig"} illustrate the sample with *p* = 180 μm and his zoomed image taken by an optical microscope (VHX-5000, Keyence Company). The measured transmission spectra are plotted in [Fig. 4(b)](#f4){ref-type="fig"}. In the measured spectra, the resonance wavelength first blue shifts and then red shifts, the switch in behavior occurring at 80 μm, which match with the trend in [Fig. 4(a)](#f4){ref-type="fig"}. Both calculation and experiment demonstrate that meta-atom arrangement and separation play an important role in resonance frequency. So far, magnetic resonance is not observed in the proposed structure under the normal incidence. Then we proceed to calculate the extinction cross sections (ECSs) of a single meta-atom under grazing incidence, aiming to excite magnetic resonance, shown in [Fig. 5(a)](#f5){ref-type="fig"}. The ECSs spectrum shows two distinct peaks, and this phenomenon closely resembles that discovered at microwave frequencies in previous published works[@b23][@b25]. The lower frequency at 0.73 THz corresponds to the electric resonance, which is same to the frequency of the resonance peak under the normal incidence. Then we plot the magnetic field distribution (*H*~*z*~) of the higher peak at 1.06 THz in [Fig. 5(b)](#f5){ref-type="fig"}. The magnetic field distribution is similar to a magnetic dipole. To confirm this mode, the magnitude of the magnetic fields and magnetic field lines in transverse plane *y* = 0 are simulated, shown in [Fig. 5(c)](#f5){ref-type="fig"}. The *H*-field lines is circulating around the disk, and thus the mode was termed as magnetic LSPs. We have demonstrated that for spoof LSPs resonance, normal incidence only excite the electric LSPs mode directly while the magnetic LSPs mode needs to be generated by grazing incidence. By this feature, magnetic LSPs mode is able to serve as EIT dark element. Generally, EIT spectral response is attributed to the destructive interference between bright and dark mode resonances, which corresponds to broad resonance and sharp resonance, respectively. Therefore, a metallic wire with length 90 μm and linewidth 5 μm is used to generate bright mode, as displayed in the left inset of [Fig. 6(a)](#f6){ref-type="fig"}. These wires are distributed on a polyimide substrate with lattice constant *p* = 180 μm. The simulated transmittance of the wires array under y-polarized (along the wire) normally incident terahertz wave are given in [Fig. 6(a)](#f6){ref-type="fig"} (black curve). The wires array show a low quality factor and broad resonance. To visualize this resonance mode, [Fig. 6(c)](#f6){ref-type="fig"} plots the simulated *E*~*z*~ near-field distribution at 1.07 THz, which shows a typical dipolar resonance. In this case, the metallic wires are strongly coupled to the normal incident wave, which enable them to be bright mode. Next, we place the straight wires close to the spoof LSPs resonators with distance of 5 μm, as shown in the right inset of [Fig. 6(a)](#f6){ref-type="fig"}. In this case, the normal terahertz waves can drive the wires directly while the spoof magnetic LSPs resonators have no response. However, the near field coupling between them excites the magnetic resonance in the spoof LSPs resonator. The destructive interference between the dipole and magnetic LSPs resonances results in a transparency window at 1.07 THz in the simulated transmission spectra, as illustrated in [Fig. 6(a)](#f6){ref-type="fig"} (the red line). To visually verify the strong EIT effect arising from destructive interference, the simulated *E*~*z*~ near-field distribution of the unit cell is shown in [Fig. 6(d)](#f6){ref-type="fig"}, in which strong spoof magnetic LSPs resonance can be clearly observed[@b23][@b25]. Comparing with [Fig. 6(c)](#f6){ref-type="fig"}, the dipole resonance in [Fig. 6(d)](#f6){ref-type="fig"} is very weak due the destructive interference effect. In order to experimentally verify the EIT behavior, a sample combining both wires and LSPs resonators is fabricated, as shown in the inset of [Fig. 6(b)](#f6){ref-type="fig"}. The experimental results of transmissions presented in [Fig. 6(b)](#f6){ref-type="fig"}, which are in good agreement with the simulation results. However, one may notice that the quality factor of the measured spectra is lower than simulation, which can be also observed in [Fig. 4(b)](#f4){ref-type="fig"}. This discrepancy is most likely due to surface roughness and the higher loss of metallic structure in the fabricated sample. Conclusion ========== In summary, we have numerically and experimentally demonstrated that spoof electric and magnetic LSPs can be realized at terahertz by metamaterial. The dimension of the meta-atoms is much smaller than resonance wavelength, making the proposed structure to be deep subwavelength scale. Both dimensions and arrangements can affect the resonance frequency. It is found that a sharp transparent transmission window can be achieved by placing the spoof LSPs resonator close to a metallic cut wire, between which destructive interference occurs. This work not only offers a way to obtain spoof LSPs resonance at terahertz frequency, but also make use of it to exhibit a EIT effect. Such plasmonic metamaterials may find interesting applications in plasmonic devices at terahertz frequency. Methods ======= Manufacturing method -------------------- The detailed fabrication processes are described as follows. First, a 20 μm thick polyimide is coated to a silicon wafer as the substrate. Then, it was cured on hotplate at 80, 120, 180 and 250 °C for 5, 5, 5 and 20 minutes, respectively. A standard photolithography was then conducted before depositing a Ti/Au layer (10/180 nm) with electron beam evaporation, after which a lift-off process helped form the final metallic pattern. Finally, we peeled off the sample from the silicon wafer by immersing it in HF solvent for 30 minutes. Simulation method ----------------- All numerical simulations are performed by the commercial software, CST Microwave Studio 2013. The relevant substrate is polyimide, whose permittivity and loss tangent are 3 and 0.03, respectively. The metal is chosen as gold, which is selected in the material library in CST. For ECS calculation, a plane-wave excitation and a broadband far-field monitor are applied to record absorption cross sections and scattering cross sections. The transmissions are simulated by the CST transient solver, in which the periodic boundary conditions are used. The calculation of dispersion relation is based on the CST eigenmode solver, in which the eigen frequencies of a unite cell is analyzed. Experimental setup and measurement ---------------------------------- The experimental setup is a commercial Terahertz Spectroscopic Systems (TAS7400SP). The samples are placed on a platform, as shown in [Fig. 7](#f7){ref-type="fig"}. All the experiments were measured in nitrogen environment (humidity smaller than 1%) under 25 °C. All spectra are normalized to the transmission without sample. Additional Information ====================== **How to cite this article**: Liao, Z. *et al*. Electromagnetically induced transparency metamaterial based on spoof localized surface plasmons at terahertz frequencies. *Sci. Rep*. **6**, 27596; doi: 10.1038/srep27596 (2016). This work was supported by the National Science Foundation of China (61302018, 61401089, 61571117, 61501112, 61501117, and 61138001), the 111 Project (111-2-05), the National Instrumentation Program (2013YQ200647), the Natural Science Foundation of Jiangsu Province (BK20130854), the Scientific Research Foundation of Graduate School at Southeast University (YBJJ1436), the Program for Postgraduate Research Innovation in University of Jiangsu Province (3204004910), and the Natural Science Foundation of Jiangsu Province under BK20150020. **Author Contributions** Z.L. and T.J.C. designed the samples and performed the simulations. Z.L., S.L., H.F.M., C.L., B.J. and T.J.C. conducted the experiment. Z.L., H.F.M. and T.J.C. wrote the manuscript. ![(**a**) Schematic diagram of the ultrathin metallic spiral structure. (**b**) The calculated ECS spectra at normal incidence. (**c**) The simulated *E*~*z*~ near-field pattern at the resonance frequency. (**d**) The magnitude of the electric fields at the resonance frequency, in which the arrows show the electric field lines.](srep27596-f1){#f1} ![(**a**) The simulated ECS spectra with respect to different outer radius. (**b**) The dispersion relations of spoof SPPs with different depth. The inset shows the schematic picture of spoof SPPs strip.](srep27596-f2){#f2} ![The resonant wavelengths of metamaterial particle as a function of the radius *R*.](srep27596-f3){#f3} ![(**a**) The simulated amplitude transmission spectra for the two-dimensional array with lattice constants *p* from 80 to 180 μm. (**b**) The measured amplitude transmission spectra for the two-dimensional array with lattice constants *p* from 80 to 180 μm. (**c**) The photo of sample and microscopic image of the fabricated structure.](srep27596-f4){#f4} ![(**a**) The calculated ECSs spectra at grazing incidence. (**b**) The simulation result of *H*~*z*~ near-magnetic-field distribution corresponding to the magnetic resonance. (**c**) The magnitude of the magnetic fields at the resonance frequency. The arrows show the magnetic field lines.](srep27596-f5){#f5} ![(**a**) The simulated amplitude transmission spectra of the sole-wire (the black line), and the EIT metamaterial (the red line). The insets present the structure of the simulated unit cell. (**b**) The measured amplitude transmission spectra of the sole-wire (the black line), and the EIT metamaterial sample (the red line). The insets present the microscopy images of the fabricated sample. The simulated electric field (*E*~*z*~) distributions of (**c**) the sole-wire and (**d**) the EIT metamaterial at 1.07 THz.](srep27596-f6){#f6} ![Photograph of the experimental setup used to measure the transmission of samples.](srep27596-f7){#f7} ###### The dipole sum *S* and its two components for λ = 400 μm. p(μm) S S(1/d^3^) S(1/d) ------- ---------------- --------------- --------------- 80 0.52 + 6.04i 3 + 3.61i −2.48 + 2.43i 100 −2.71 + 7.8i 2.21 + 4.29i −4.92 + 3.54i 120 −4.49 + 6.86i 1.23 + 4.8i −5.72 + 2.06i 140 −8.27 + 7.19i 0.15 + 5.14i −8.43 + 2.05i 160 −10.58 + 4.85i −1.04 + 5.25i −9.54-0.4i 180 −14.06 + 2.91i −2.3 + 5.1i −11.76-2.2i

{ "pile_set_name": "PubMed Central" }

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Glioblastomas (GBMs) are one of the most aggressive and deadly forms of human cancer. GBM treatment usually consists of surgical resection followed by radiotherapy combined with the alkylating agent temozolomide (TMZ)[@b1]. Although this therapeutic approach slightly improves the survival rate of GBM patients, a large fraction of these patients suffer from tumour recurrence[@b1]. Accumulating evidence suggests that tumour relapse may be driven by a component of heterogeneous tumour cells that retain stem cell-like properties, called "cancer stem cells" (CSCs). The potent tumourigenic capacity of glioma CSCs (GSCs), coupled with evidence of radio- and chemo-resistance, suggests that a stem cell-orientated therapy may represent an innovative strategy to reduce tumour recurrence and improve GBM prognosis[@b2]. Two main strategies are currently exploited to eradicate the heterogeneous population of GBM and GSCs: (a) chemotherapeutic regimens that specifically drive GSCs into cell death, and (b) driving GSCs into differentiation, thereby depleting the tumour reservoir. The latter strategy appears the most promising, considering that differentiated cells are in general more sensitive to chemotherapeutic agents with respect to CSCs[@b3]. Studies on human GBM samples have uncovered that the deregulation of signal transduction pathways is one of the most prominent[@b4][@b5]. The disruption of signal transduction in GBM occurs through over-expression or a gain-of-function mutation of tyrosine-kinase receptors[@b6][@b7], thus leading, among other events, to constitutive activation of Ras/extracellular signal-regulated kinase (ERK), AKT/mammalian target of rapamycin (mTOR). As a result, AKT is elevated in the majority of examined GBMs[@b8][@b9] with the subsequent amplification of pro-survival signals and blockage of oncosuppressor controls. The inactivation of the oncosuppressor protein p53 is certainly one of the main phenomena that allow GBM cells to escape cell cycle checkpoints. In particular, the intracellular levels of p53 are maintained low due to an excessive stimulation (mediated by AKT constitutive activation[@b10]) of the ubiquitin-ligase murine double minute 2 homologue (MDM2), the predominant natural endogenous inhibitor of the protein p53[@b11][@b12]. In addition to accelerating p53 degradation, MDM2 prevents p53 binding to DNA, blocking its transcriptional activity. As GBM cells typically express p53 with a wild-type amino acid sequence, the re-activation of p53 functionality can be restored through the inhibition of the oncogenic block exerted by the AKT/mTOR pathway, which causes an excessive stimulation of MDM2. In this respect, while agents inhibiting either the AKT/mTOR pathway[@b13][@b14][@b15] or the MDM2/p53 interaction[@b16][@b17][@b18] have provided some survival benefit in GBM, the effects of a co-therapy have not been deeply investigated to date, either in GBMs or in their stem cells. In acute myeloid leukaemia, the PI3K/mTOR inhibitor PI-103 acts synergistically with the MDM2 inhibitor nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion[@b19], supporting the aforementioned mechanistic rationale. In our previous work, a series of 2-oxindole derivatives (OXIDs) have been described[@b20] and demonstrated to act as inhibitors of the AKT/mTOR pathway. Herein, we identified FC85 as a new ligand, useful in establishing the preclinical *proof of concept* for the AKT/mTOR pathway, and whose activity could be amplified by co-treatment with an MDM2 inhibitor. The mechanism of action of FC85 was examined alone or in combination with an already characterized inhibitor of MDM2, ISA27[@b18], both in GBM cells and in their derived GSCs. In parallel experiments, the oral mTOR inhibitor everolimus[@b21][@b22] and the MDM2 inhibitor nutlin-3[@b17][@b18]were also used as reference compounds. Globally, our findings demonstrated that AKT/mTOR inhibitors actively enhance downstream p53 signalling and that a combination strategy aimed at inhibiting the PI3K/AKT/mTOR pathway and activating p53 signalling is potentially effective in GBMs and in GSCs ([Fig. 1a](#f1){ref-type="fig"}). Results ======= Design and Synthesis -------------------- Over recent years, new compounds with an indole/oxindole core have been widely investigated as agents able to target the activity of the serine/threonine kinases PDK1 and/or AKT[@b23]. Recently, we synthesized new OXIDs compounds by the combination of a tetrahydroisoquinoline nucleus with the 2-oxindole nucleus throughout a methylenamido moiety, and anchoring the 3-position of oxindole core to different heterocycles ([Fig. 1b and c](#f1){ref-type="fig"}). The new OXIDs[@b24] induced cell cycle arrest and inhibited AKT phosphorylation in non-small cell lung cancer cells (which overexpress the PI3K/AKT/mTOR pathway and show resistance to EGFR inhibitors), suggesting that the OXID nucleus can be used as central core to develop inhibitors of the PI3K/AKT/mTOR pathway. Specifically, we afforded the replacement of the amido moiety of OXIDs ([Fig. 1b and c](#f1){ref-type="fig"}) with its bioisosteric amidosulfonyl group. Sulfonamide is a well-known pharmacofore notorious as key element to confer anticancer properties, among others[@b25][@b26]. FC85 was obtained as depicted in [Figure 1b](#f1){ref-type="fig"}. Briefly, the 5-amino-2-oxindole **1** reacted with p-toluenesulfonyl chloride to give 4-methyl-N-(2-oxoindolin-5-yl)benzenesulfonamide **2**. The subsequent Knoevenagel condensation of **2** with the 1H-imidazole-5-carboxaldehyde afforded the target compound as a Z-isomer. In co-treatment experiments, we used the small-molecule MDM2 inhibitor with a spirooxoindolepyrrolidine core structure, ISA27, which was synthesized as previously described[@b27]. Effects of FC85 on the AKT/mTOR signalling pathway in U87MG cells ----------------------------------------------------------------- As a representative GBM cell line, we used U87MG cells, which is an appropriate model to study the interaction between the AKT/mTOR and MDM2-p53 pathways because of the following characteristics: i) U87MG cells maintain a wild type status of p53, and ii) U87MG cells are deficient for the tumour suppressor phosphatase and tensin homologue (PTEN), a negative regulator of the PI3K/AKT pathway; moreover, PTEN deficiency leads to MDM2 nuclear accumulation, thus inhibiting p53 functions[@b28]. To assess whether FC85 could effectively inhibit the mTOR/AKT pathway in U87MG cells, the effects of the new compound on AKT and mTOR activity were examined. FC85, tested at 100 nM, 1 µM and 10 µM, significantly inhibited AKT phosphorylation at Ser473 ([Suppl. Fig. 1a](#s1){ref-type="supplementary-material"}), as reported for the already published OXIDs[@b24]. The new compound reduced also mTOR constitutive phosphorylation at Ser2448 ([Suppl. Fig. 1b](#s1){ref-type="supplementary-material"}). These findings demonstrated that FC85 was able to target the AKT/mTOR signalling pathway in U87MG cells. Effects of FC85 on U87MG cell viability --------------------------------------- To examine the effects of FC85 on GBM cell growth/survival, U87MG cells were incubated with different concentrations of the compound for 24 h this new compound showed a dose-dependent inhibitory effect on the growth/survival of U87MG cells, with an IC~50~ value of 468.9±48.9 nM ([Fig. 2a](#f2){ref-type="fig"}). Trypan blue exclusion experiments confirmed a concentration-dependent decrease of live cells and an increase of dead cells ([Fig. 2b](#f2){ref-type="fig"}). To assess whether U87MG cells could resume proliferation, cells were treated for 96 h with the compound at 10 µM. At the end of the treatment period, the culture medium was replaced with fresh medium not containing the drug. As depicted in [Figure 2c](#f2){ref-type="fig"}, the number of viable cells did not significantly increase at either 1 or 3 days of wash-out with respect to control samples, suggesting the inability of cells to recover normal growth. To determine compound toxicity in non-tumour cells, we examined the effects of FC85 on the viability of normal Human Umbilical Vein Endothelial Cells (HUVECs). As shown in [Figure 2d](#f2){ref-type="fig"}, the viability of HUVECs was significantly decreased after a 48 h incubation with FC85 used at 1 µM and 10 µM. However, the effects on cell viability were not strictly concentration dependent, and the percentages of cell viability reduction were significantly lower with respect to those observed in U87MG cells, suggesting that the anti-proliferative effect elicited by FC85 was directed preferentially towards tumour cells. Effect of a combined therapy with an inhibitor of MDM2/p53 complex on U87MG cell viability ------------------------------------------------------------------------------------------ Because the inhibition of PI3K/AKT/mTOR signalling can augment p53-mediated apoptosis[@b29], AKT/mTOR may synergize with inhibitors of the MDM2/p53 complex to promote anti-tumoural activity in GBM cells that retain p53 function. To this end, we investigated the effects on U87MG cell viability of combining doses of FC85 with the already characterized MDM2 inhibitor, ISA27[@b18]. The theoretical additive IC~50,add~ values for the two drugs were calculated for two fixed ratios (1:2, 1:5), and the experimental IC~50,mix~values were determined for the same fixed-ratio combinations in the viability assay ([Fig. 3a](#f3){ref-type="fig"}). Statistical analysis of the data from isobolographic analysis revealed synergistic interactions between FC85 and ISA27 for the two examined fixed-ratio combinations ([Suppl. Table S1](#s1){ref-type="supplementary-material"}; [Fig. 3a](#f3){ref-type="fig"}). Similar experiments were performed using the oral mTOR inhibitor everolimus, currently in clinical use for several type of solid tumours[@b21][@b22], including GBMs (NCT00823459, NCT00831324, NCT00613132, NCT00553150, NCT00387400, NCT00085566, NCT00805961) and the MDM2 inhibitor nutlin-3, whose oral formulation has completed the first early phase clinical trials for both solid cancers and haematological malignancies[@b30][@b31]*.* Everolimus and nutlin-3 alone inhibited U87MG cell proliferation, yielding an IC~50~ of 50.0±2.8 nM and 10.3±1.1 µM, respectively ([Suppl. Fig. 2a](#s1){ref-type="supplementary-material"}). When combined, the two reference agents showed a synergic reduction of U87MG cell viability for both the examined fixed-ratio combinations ([Suppl. Table S2](#s1){ref-type="supplementary-material"}; [Suppl. Fig. 2b](#s1){ref-type="supplementary-material"}). Globally, these results confirmed that a combined therapy with an inhibitor of MDM2/p53 complex and an inhibitor of the AKT/mTOR pathway could be a useful anti-proliferative strategy in GBM cells. We then examined the effects of cell treatment with FC85, alone or in combination with ISA27, on the reactivation of p53 pathway, assessing accumulation of p53 protein and transcription induction of p53 target genes. The incubation of GBM cells with FC85 alone for 6 h led to a slight but significant increase in p53 protein levels ([Fig. 3b and c](#f3){ref-type="fig"}). As expected, challenging cells with ISA27 caused a higher accumulation of p53. When cells were treated with FC85 and ISA27, a significant enhancement of p53 protein accumulation with respect to cells treated with FC85 or ISA27 alone was observed ([Fig. 3b and c](#f3){ref-type="fig"}). In line with these data, short-term (6 h) treatment of U87MG cells with FC85 or ISA27 led to a significant increase in the mRNA levels of certain p53 target genes ([Fig. 3d](#f3){ref-type="fig"}): p21, a cell cycle inhibitor; PUMA, a gene product required for p53-controlled intrinsic apoptosis pathway; and MDM2, physiological inhibitor of p53, and its main transcriptional target. These results suggested that p53 stabilization in FC85-treated GBM cells led to an increase in MDM2, PUMA and p21 mRNA levels in a manner that was consistent with the activation of the p53 pathway. Moreover, a significant enhancement of the transcription of p53 target genes was observed in FC85-ISA27 co-treated cells with respect to single agent-treated cells ([Fig. 3d](#f3){ref-type="fig"}). In parallel experiments, neither nutlin-3 (10 µM) nor everolimus (50 nM) alone induced a significant accumulation of p53 protein in short-term (6 h) treatment ([Suppl. Fig. 3a and b](#s1){ref-type="supplementary-material"}). These data are consistent with those previously reported for nutlin-3[@b18]. When combined, the reference agents produced a significant p53 accumulation after 6 h of treatment ([Suppl. Fig. 3a and b](#s1){ref-type="supplementary-material"}). Real time RT-PCR experiments showed that nutlin-3 did not induce any significant increase in p53 target gene products after 6 h of treatment ([Suppl. Fig. 3c](#s1){ref-type="supplementary-material"}), according to published data[@b18]. Similar results were obtained with everolimus ([Suppl. Fig. 3c](#s1){ref-type="supplementary-material"}). When the two drugs were used together, a significant increase in MDM2 mRNA was noticed ([Suppl. Fig. 3c](#s1){ref-type="supplementary-material"}). These data are consistent with those obtained in western blotting analysis and suggest that the combined therapy could reactivate p53 function with increased efficacy compared with single therapy. To examine whether the p53 function was effectively reactivated after a prolonged exposure time, U87MG cells were incubated with everolimus and/or nutlin-3 for 24 h. Both agents caused a significant induction of MDM2 and p21 mRNA levels ([Suppl. Fig. 3d](#s1){ref-type="supplementary-material"}). Moreover, a synergistic and significant induction of PUMA mRNA level was noticed when the two drugs were combined ([Suppl. Fig. 3d](#s1){ref-type="supplementary-material"}). Globally, these results demonstrate that nutlin-3 and everolimus caused a slower re-activation kinetics of p53 function with respect to FC85 and ISA27. Effect of FC85, ISA27 and of their combined treatment on the induction of apoptosis and cell cycle block -------------------------------------------------------------------------------------------------------- Challenging U87MG cells with FC85 (500 nM) induced a significant phosphatidylserine externalization, both in the absence (early apoptosis) or presence of 7-amino-actinomysin binding to DNA (late apoptosis/death; [Fig. 4a and b](#f4){ref-type="fig"}). These data are consistent with the observed increase of PUMA mRNA level ([Fig. 3d](#f3){ref-type="fig"}). Similar results were observed with the MDM2 inhibitor ISA27 ([Fig. 4a and b](#f4){ref-type="fig"}). When the two drugs were combined together, a significant enhancement in the percentage of cells in late apoptosis/death was detected, with respect to cells treated with ISA27 or FC85 alone ([Fig. 4a and b](#f4){ref-type="fig"}). These findings suggest that the combined therapy may accelerate apoptosis induction in U87MG cells. In contrast, everolimus or nutlin-3 did not induced a significant level of apoptosis in U87MG cells within 24 h of treatment ([Suppl. Fig. 4](#s1){ref-type="supplementary-material"}), consistent with previously published data[@b17][@b18][@b32][@b33]. However, the co-treatment induced a slight but significant induction of apoptosis ([Suppl. Fig. 4](#s1){ref-type="supplementary-material"}), suggesting that everolimus could accelerate the induction of apoptosis elicited by nutlin-3 in more prolonged treatment[@b17][@b18]. These data are consistent with those observed in real-time RT-PCR experiments: after 24 h of incubation, the levels of PUMA mRNA, a p53-target gene product mainly involved in cellular apoptosis, were indeed significantly higher only when nutlin-3 and everolimus were used in combination. Cell cycle analysis demonstrated that challenging U87MG cells with FC85, as well as with ISA27, for 24 h arrested cell-cycle progression in the G0/G1-phase ([Fig. 4c and d](#f4){ref-type="fig"}). U87MG incubation with both FC85 and ISA27 was able to induce a significantly higher blockage in the G0-G1 phase compared with single-compound treated cells ([Fig. 4c and d](#f4){ref-type="fig"}). These results suggest that cell cycle arrest may have been a contributing factor in the observed increased sensitivity of GBM cells to the combined therapy. Effects of FC85, ISA27 and of their combined treatment on GSC viability ----------------------------------------------------------------------- A hyper activation of the AKT/mTOR pathway has been reported to play a pivotal role in GBM proliferation as well as in the self-renewal and propagation of GSCs. On this basis, we evaluated the effects of FC85 on GSCs, isolated from U87MG cells as previously reported[@b34]. As depicted in [Figure 5a](#f5){ref-type="fig"}, when GSCs were incubated with this new compound (1 µM or 10 µM), a significant inhibition of cell proliferation occurred starting from four days of treatment. Similar results were obtained using ISA27 ([Fig. 5b](#f5){ref-type="fig"}). A dose-response curve of FC85 and ISA27 was then performed after seven days of treatment: the two compounds were able to induce a concentration-dependent inhibition of GSC proliferation, with IC~50~ values of 104±12 nM and 540±68 nM, respectively ([Fig. 5c](#f5){ref-type="fig"}). In parallel experiments, nutlin-3 reduced GSC proliferation starting from four days of incubation, yielding an IC~50~ value of 18.5±2.1 µM after seven days of incubation ([Suppl. Fig. 5b and c](#s1){ref-type="supplementary-material"}). Everolimus exhibited a high ability in reducing GCS proliferation starting from 24 h of cell treatment and showing an IC~50~ value of 15.0±1.2 nM ([Suppl. Fig. 5a and c](#s1){ref-type="supplementary-material"}). The effects of a combined administration of FC85 and ISA27 were then investigated in GSCs. Isobolographic analysis revealed a marked synergic effect on the reduction of GSC proliferation, when the two compounds were used together ([Suppl. Table S3](#s1){ref-type="supplementary-material"}; [Fig. 5d](#f5){ref-type="fig"}), suggesting that a combined therapy with an inhibitor of the MDM2/p53 complex and an inhibitor of the AKT/mTOR pathway can be useful in GSC-orientated therapy. Similar synergistic interactions were confirmed also using everolimus and nutlin-3 ([Suppl. Fig. 5d](#s1){ref-type="supplementary-material"}; [Suppl. Table S4](#s1){ref-type="supplementary-material"}). Next, we assessed whether FC85 and/or ISA27-treated cells could resume proliferation after drug removal. After GSC challenging with FC85 or ISA27, and a wash-out period of seven and fourteen days, the percentages of proliferating/viable cells did not significantly increase, suggesting an overall inability to recover normal growth ([Fig. 5e](#f5){ref-type="fig"}). These effects were particularly evident when the two compounds were used simultaneously ([Fig. 5e](#f5){ref-type="fig"}). Effects of FC85, ISA27 and of their combined treatment on the induction of GSC apoptosis ---------------------------------------------------------------------------------------- We then investigated whether the reduction of GSC proliferation elicited by the two compounds was associated with apoptosis. After seven days of GSC treatment, both FC85 and ISA27 induced a significant phosphatidylserine externalization, both in the absence (early apoptosis) or in the presence of 7-amino-actinomysin 11 binding to DNA (late apoptosis/death; [Fig. 6a and b](#f6){ref-type="fig"}). The simultaneous incubation of GSCs with the two compounds induced both early and late apoptosis; the percentage of late apoptosis-death was significantly higher with respect to those observed with the two compounds alone ([Fig. 6a and b](#f6){ref-type="fig"}). Of note, whereas nutlin-3 alone did not show any significant effects on GSC apoptosis, everolimus induced slight but significant early apoptosis after seven days of treatment ([Suppl. Fig. 6](#s1){ref-type="supplementary-material"}). When used together, the two reference drugs caused a strong induction of early apoptosis, with significantly higher effects with respect to those obtained in single agent-treated GSCs ([Suppl. Fig. 6](#s1){ref-type="supplementary-material"}). These results confirmed that the chosen combined therapy could accelerate the induction of GSC apoptosis. Effects of FC85, ISA27 and of their combined treatment on GSC morphology and differentiation -------------------------------------------------------------------------------------------- The effects of FC85 and ISA27 on GSC morphology were evaluated by quantifying the area occupied by the cells in culture plates as well as the outgrowth of cellular processes. When GSCs were incubated with FC85 for seven days, an almost complete reduction in the area occupied by the floating spheres was noticed, and the cells showed prominent outgrowth of processes ([Fig. 7a-c](#f7){ref-type="fig"}), as previously reported as a response to other AKT/mTOR inhibitors[@b35][@b36]. Similar results were obtained with the MDM2/p53 inhibitor ([Fig. 7a-c](#f7){ref-type="fig"}), suggesting that the reactivation of the p53 pathway could also induce GSC differentiation. In both cases, the morphology of differentiated cells appeared to be heterogeneous, suggesting that FC85 and ISA27 could promote cell differentiation towards more than one phenotype. When FC85 and ISA27 were combined, a marked reduction in the size of neurospheres was evidenced, with few cells presenting small cellular processes ([Fig. 7a-c](#f7){ref-type="fig"}). To investigate the mechanisms through which FC85 and ISA27 affect GSC morphology, we then assessed the levels of stem and differentiation markers upon stimulation with the two compounds. In FC85-treated GSCs, a significant decrease in the stemness markers, CD133 and Nestin, was noticed ([Fig. 6c](#f6){ref-type="fig"}) accompanied by a significant increase of MAP and GFAP content, demonstrating that FC85 was able to promote GSC differentiation towards a neuronal and a glial phenotype. Similar results were obtained with ISA27 ([Fig. 6c](#f6){ref-type="fig"}). Surprisingly, despite the fact that no significant cellular processes had been noticed in the morphological analysis, when cells were treated with the two drugs, a significant decrease in the stemness markers was evidenced, together with a marked increase in the MAP, GFAP and Olig2 content ([Fig. 6c](#f6){ref-type="fig"}). Moreover, the enhancement of the neuronal and of the astrocyte markers were significantly higher in FC85+ISA27-treated cells with respect to that observed in single agent-treated cells ([Fig. 6c](#f6){ref-type="fig"}). Comparing the morphological/apoptosis analysis and RT-PCR data indicated that combined ISA27 and FC85 not only induced the apoptosis of GSCs but also promoted the differentiation of these cells. Effects of FC85, ISA27 and of their combined treatment on the kinetics of AKT and ERK1/2 phosphorylation -------------------------------------------------------------------------------------------------------- The putative intracellular signalling pathways underlying the effects elicited by the MDM2/p53 and the AKT/mTOR inhibitors were then investigated. Different signalling pathways have been demonstrated to play a pivotal role in GSC proliferation and differentiation[@b37]; among these, several evidence suggested that a crosstalk between the PI3K/AKT/mTOR and MEK/ERK pathways in the maintenance of self-renewal and tumourigenicity of GSCs[@b38]. To study in depth the mechanism underlying the observed effects, the kinetics of AKT and ERK1/2 inhibition were investigated. GSC incubation with FC85 and/or ISA27 did not alter total ERK or AKT levels ([Suppl. Fig. 7](#s1){ref-type="supplementary-material"}). Both FC85 and ISA27 induced a fast and significant inhibition of AKT phosphorylation, which persisted up to 60 min ([Fig. 8a](#f8){ref-type="fig"}). Challenging cells with both compounds prolonged p-AKT inhibition up to 120 min, with significantly higher percentages of inhibition with respect to single agent-treated cells ([Fig. 8a](#f8){ref-type="fig"}). Both ligands also affected p-ERK1/2, although showing a transient blockage (up to 30 min of GSC incubation, [Fig. 8b](#f8){ref-type="fig"}). In contrast, FC85+ISA27 produced a significant and sustained inhibition of ERK1/2 activity (up to 120 min); as in the case of p-AKT, the percentages of inhibition were significantly higher with respect to single agent-treated cells at 60 and 120 min of GSC treatment ([Fig. 8b](#f8){ref-type="fig"}). These results are in line with the additive/synergic effect on GSC differentiation elicited by the co-treatment FC85+ISA27, and support the previous demonstration that the dual and sustained inhibition of PI3K/AKT/mTOR and MEK/ERK signalling induced differentiation and inhibited the tumourigenicity of GSCs[@b38]. Discussion ========== In this study, we report the interactions of PI3K/AKT/mTOR and MDM2/p53 pathways in GBM cells, following the simultaneous inhibition of AKT/mTOR signalling and re-activation of the p53 pathway. First, we showed that a combined therapy utilizing the novel mTOR/AKT inhibitor FC85 and the MDM2/p53 inhibitor ISA27 produced a synergistic effect on the inhibition of GBM cell viability as well as on the reactivation of p53 pathway. Most importantly, similar synergistic effects were shown in GBM-derived CSCs, where the simultaneous use of the two compounds induced a strong differentiation of GSCs, as well as apoptosis. A synergistic inhibition of cell viability of both GBM and its derived CSCs was evidenced also using the mTOR inhibitor everolimus and the MDM2 inhibitor nutlin-3 as reference compounds. These findings suggest that a combination strategy aimed at inhibiting AKT/mTOR signalling and re-activating p53 signalling may be potentially effective in GBMs and GSCs. The multi-targeted strategy has assumed great importance in GBM therapy, based on the evidence that single-agent therapy is often not sufficient to control this tumour[@b39][@b40][@b41]. Because the hyper-activation of the PI3K/AKT/mTOR pathway and the inactivation of wild-type p53 by MDM2 over-expression are frequent molecular events in highly proliferative tumours, and the aforementioned pathways are directly related[@b29], a combined therapy with an inhibitor of AKT/mTOR pathway and a compound able to inhibit the MDM2/p53 interaction may represent a valuable approach in GBM. In this respect, a cell-based screen has demonstrated the efficacy of such combined therapy in several cancer cells[@b42], thus providing a framework for the rational design of new clinical trials. We found that FC85 inhibited mTOR and AKT constitutive activity in U87MG cells; as a result, the new compound decreased cell viability, induced cell cycle block, and triggered apoptosis. Moreover, the inhibition of cell viability was long-lasting and directed preferentially towards tumour cells, as demonstrated by the significantly lower effects observed in HUVECs. Real time RT-PCR and western blotting analyses demonstrated that FC85 alone led to the accumulation of p53 protein levels and to an increase of the mRNA levels of p53-target genes, demonstrating a reactivation of the p53 pathway. Challenging cells with FC85 and the MDM2/p53 inhibitor ISA27 caused an amplification response on the reactivation of the p53 signalling pathway. As a result, such combined therapy produced synergistic effects on the inhibition of cell viability and enhanced the induction of p53-mediated apoptosis and cell cycle block with respect to those elicited by the use of a single agent. A synergistic inhibition of GBM cell viability was confirmed using the mTOR inhibitor everolimus and the MDM2 inhibitor nutlin-3. However, slight differences were noted. When used alone, everolimus and nutlin-3 caused slower reactivation kinetics of p53 function compared with FC85 and ISA27, displaying a significant induction of p53 target genes only after 24 h of cell treatment, accordingly to data previously reported for the MDM2 inhibitor[@b18]. Moreover, neither everolimus nor nutlin-3 alone induced apoptosis of U87MG cells within 24 h of treatment, in line with literature findings, reporting a non-apoptotic mechanism for everolimus[@b32][@b33] and a tardive induction of apoptosis for nutlin-3[@b17][@b18][@b41]. Interestingly, the co-treatment induced a slight but significant induction of apoptosis, suggesting that everolimus could accelerate the induction of apoptosis elicited by nutlin-3 in prolonged treatment[@b17][@b18]. The different reactivation kinetics of p53 function and induction of apoptosis elicited by the two MDM2 inhibitors could be attributed to the more rapid accumulation of p53 protein levels caused by ISA27 with respect to nutlin-3[@b18]. With regard to everolimus and FC85, it is important to mention the intricacy of the AKT/mTOR pathway. mTOR protein kinase interacts with multiple proteins via its two distinct multiprotein complexes, mTORC1 and mTORC2[@b43][@b44], which have been shown to have distinct signalling in GBM[@b45]. Rapamycin and rapalogs, including everolimus, suppress mTOR activity via an allosteric mechanism and have been found to be incomplete inhibitors of mTORC1[@b46][@b47]. On this basis, and considering the lack of information on the detailed mechanism of AKT/mTOR inhibition elicited by FC85, further investigations are needed to obtain insight into the signalling pathways that are the basis of such differences. We next characterized our compounds, alone or in combination, in GSCs isolated from U87MG cells. First, we demonstrated that both FC85 and ISA27 induced a time- and concentration-dependent inhibition of GSC proliferation and triggered apoptosis. In addition, in GSCs, the combined therapy produced synergisticc effects, accelerating the induction of apoptosis and enhancing the inhibition of GSC viability. Such synergistic interactions were shown also using the reference compounds nutlin-3 and everolimus, thus confirming that a combined therapy with an inhibitor of MDM2/p53 complex and an inhibitor of the AKT/mTOR pathway is useful in anti-proliferative strategy in GSCs. To investigate the mechanism underlying these effects, the morphology and stemness/differentiation of GSCs upon treatment with the compounds were evaluated. Morphological and RT-PCR analyses demonstrated that FC85 and ISA27 alone significantly decreased the stemness of GBM-derived cells, and promoted their differentiation toward a neuronal-glial phenotype. Globally, these effects elicited by FC85 and ISA27 are in line with the differentiating/pro-apoptotic properties previously reported for AKT/mTOR inhibitors in GSCs[@b36][@b48], as well as with the abilities of p53 to induce differentiation and to suppress self-renewal in human embryonic stem cells[@b49]and in breast CSCs[@b50], respectively. When cells were concomitantly treated with the two drugs, a significant decrease in the stemness markers was evidenced, together with a marked increase in neuronal and glial markers, demonstrating that combining ISA27 and FC85 not only induced apoptosis of GSCs but also induced the differentiation of these cells. Then, the signalling pathways most likely implicated in the aforementioned effects were investigated. It has been demonstrated that the AKT/mTOR signaling pathway is critical for the maintenance of the properties of glioma CSCs[@b36][@b48]; on the other hand, in the same cells the MEK-ERK signalling is required for MDM2 expression, which prevents p53 activation and subsequent suppression of O(6)-methyl-guanine DNA methyltransferase expression, thus contributing to TMZ resistance[@b51]. In this study, we showed that FC85 and ISA27 alone inhibited AKT and ERK1/2 phosphorylation up to 30 or 60 min, respectively. Challenging cells with both compounds prolonged such inhibitions up to 120 min, with significant higher percentages of inhibition with respect to single agent-treated cells. These results are in line with the additive/synergic effect on GSC differentiation elicited by the co-treatment FC85+ISA27, and support the previous demonstrations that the dual inhibition of PI3K/AKT/mTOR and MEK/ERK signalling induced the differentiation of and inhibited the tumourigenic potential of GSCs[@b38]. Globally, our findings suggest that FC85 actively enhances downstream p53 signalling and that a combination strategy aimed at inhibiting PI3K/AKT/mTOR signalling and activating p53 signalling is potentially effective in GBM and in its CSC subpopulation, where TP53 mutations are rare, and the downstream p53 signalling is intact. The promising anti-tumour effects, confirmed by the combined use of everolimus and nutlin-3, sustain the strength of the present pharmacological approach, and encourage the extension of preclinical investigations that, above all, focus on molecular mechanisms at the basis of such cellular responses. Methods ======= Chemical synthesis ------------------ Melting points were determined on a Kofler apparatus and are uncorrected. Chemical shift (δ) is reported as part per million downfield from tetramethylsilane and referenced from solvent references.^1^H NMR and ^13^C NMR spectra of all compounds were obtained with a Varian Gemini 200 spectrometer (Labexchange, Burladingen, Germany) operating at 200 MHz, in a \~2% solution of DMSO-*d~6~*. The \>95% purity of tested compounds was confirmed by combustion analysis. Analytical TLC was performed on Merck 0.2-mm precoated silica gel aluminium (60 F~254~) sheets t hat were visualized under a UV lamp. Evaporation was performed *in vacuo* (rotating evaporator). Sodium sulfate was used as the drying agent. Commercially available chemicals were purchased from Sigma-Aldrich. The 5-amino-1,3-dihydro-2*H*-indol-2-one **(1)** was prepared according to reported procedures[@b20]. 4-methyl-*N*-(2-oxoindolin-5-yl)benzenesulfonamide (2) ------------------------------------------------------ To a stirred solution of 5-amino-2-oxindole **1** (148 mg, 1 mmol) in water (10 mL was added TsCl (228.8 mg, 1.2 mmol) at room temperature, and stirring was continued until the reaction was complete. Then, the solution was evaporated to dryness. The crude residue was diluted with MeOH, and the solid product was filtered off from the mixture. The collected solid was washed with diethyl ether and air-dried. (109 mg, 0.36 mmol, 36% yield): mp 210--212°C.[@b1]H NMR: δ 2.37 (s, 3H, Me); 3.44 (s, 2H, CH~2~); 6.65 (d, 1H, *J* = 8.2 Hz, Ar); 6.84 (d, 1H, *J* = 8.2 Hz, Ar); 6.95 (s, 1H, Ar); 7.32 (d, 1H, *J* = 8.1 Hz, Ar); 7.58 (d, 1H, *J* = 8.1 Hz, Ar); 9.89 (br s, 1H); 10.30 (br s, 1H) ppm. ^13^C NMR: δ 170.06, 141.81, 137.46, 135.73, 133.61, 128.85, 128.11, 127.25, 121.99, 117.19, 110.54, 36.42, 21.87 ppm. Anal. Calcd. for C~15~H~14~N~2~O~3~S: C, 59.59; H, 4.67; N, 9.27. Found: C, 59.38; H, 4.46; N, 9.03. (3*Z*)-N-(3-((1H-imidazol-5yl)-methylene)-2-oxoindolin-5-yl)-4-methylbenzenesulfonamide (FC85) ---------------------------------------------------------------------------------------------- To a solution of 4-methyl-*N*-(2-oxoindolin-5-yl)benzenesulfonamide (**2**) (118 mg, 0.39 mmol) in ethanol (7 mL) was added the 1*H*-imidazole-5-carboxaldehyde (41.32 mg, 0.43 mmol) and a catalytic amount of piperidine. The resulting solution was stirred and refluxed for 12 h; then, the solution was evaporated to dryness. The crude residue was purified by crystallization from EtOH, affording **FC85** as Z-isomer (79 mg, 0.21 mmol, 53% yield): mp 219--217°C. ^1^H NMR: δ 2.31 (s, 3H, Me); 6.70--6.82 (m, 2H, Ar); 7.32 (d, 1H, *J* = 8.1 Hz, Ar); 7.40 (s, 1H, Ar); 7.59 (d, 1H, J = 8.1 Hz, Ar); 7.73 (s, 1H, Ar); 7.77 (s, 1H, Ar); 8.02 (s, 1H, H-vinyl); 9.94 (br s, 1H); 10.95 (br s, 1H) ppm. ^13^C NMR: δ 168.90, 139.05, 136.74, 135.45, 134.95, 131.88, 131.40, 130.72, 128.46, 127.59, 127.44, 126.68, 121.33, 117.95, 111.01, 110.12, 21.30 ppm. Anal. Calcd. for C~19~H~16~N~4~O~3~S: C, 59.99; H, 4.24; N, 14.73. Found: C, 60.12; H, 4.46; N, 14.87. GBM cell culture and GSC isolation ---------------------------------- The U87MG cell line was obtained from the National Institute for Cancer Research of Genoa (Italy) and cultured as described[@b18]. To isolate GSCs, approximately 2.0 × 10^6^ cells were suspended in 1 mL of a defined serum-free Neural Stem Cell (NSC) medium[@b34]. After 3--4 days of culture, the neurospheres were collected, suspended in NSC medium, dissociated into single cells, and plated for the assays. For the long-term treatment of cells, NSC or complete medium containing drugs was replaced every two to three days. Cell proliferation/viability assays of GBM cells and CSCs --------------------------------------------------------- The human U87MG cells or GSCs were seeded at a density of 3 × 10^3^ cells/well. After 24 h, the cells were treated for one to 7 days with fresh growth medium containing different concentrations of FC85, ISA27, everolimus or nutlin-3. Following the treatment period, cell viability was determined using the MTS assay according to manufacturer\'s instruction. The absorbance of formazan at 490 nM was measured in a colorimetric assay with an automated plate reader (Victor Wallac 2, Perkin Elmer). For wash-out experiments, U87MG cells or GSCs were treated with FC85 and/or ISA27 for 96 h (U87MG cells) or seven days (GSCs). At the end of treatments, medium containing drugs was replaced by fresh medium, and cells were allowed to grow for the indicated days (one or three days in the case of U87MG, seven or fourteen days in the case of GSCs). At the end of treatments, cell viability was measured by the MTS assay. The results were calculated by subtracting the mean background from the values obtained from each evaluation and were expressed as the percentage of the control (untreated cells). Sigmoid dose-response curve was generated, from which the IC~50~ values were derived. The effects of compound treatment on U87MG cell viability were also evaluated using the trypan blue exclusion assay. Cells were treated with different concentrations (100 nM, 1 µM or 10 µM) of FC85. Following the treatment period, cells were collected and centrifuged at 300 × g for 5 minutes. The harvested cells were mixed with an equal volume of 0.4% trypan blue dye, and the blue (dead cells) and white (living cells) cells in each well were manually counted. The number of live cells for each condition was reported as the percentage of living cells relative to that in the control sample. Isobolar analysis ----------------- A graphical assessment of synergy with regard to growth inhibition was performed using isobolographic analysis[@b52]. In the present study, the dose of ISA27 or nutlin-3 required to give a 50% effect[@b18] was plotted on the abscissa, and the iso-effective dose of FC85 or everolimus was plotted on the ordinate. The theoretical additive effect of the two drugs is represented by the straight line connecting the two points. If the experimentally determined data points and their confidence interval fall on this line, the drug effects are additive (no interaction). If the points lie below this line, there is superadditivity (synergy), and if the points lie above this line, there is subadditivity (antagonism). To determine whether the interaction between the two drugs was synergistic, additive or antagonistic, the theoretical additive IC~50,add~was estimated from the dose-response curves of each drug administered individually. The interaction index, denoted by γ, is an assessment of the degree of synergism or antagonism. The index is defined by the isobolar relationship as follows[@b53][@b54]: γ = a/A + b/B where A and B are the doses of drug A (alone) and B (alone) that give the specified effect, and (a,b) are the combination doses that produce the same effect. p53 stabilization analysis in U87MG cells ----------------------------------------- The western blot analysis for the evaluation of p53 protein levels was performed as previously described[@b18]. In brief, U87MG cells were treated with DMSO (control) or with 500 nM FC85 and 2.5 µM ISA27, alone or in combination. Alternatively, cells were incubated with 50 nM everolimus and/or 10 µM nutlin-3, alone or in combination. Cells were then lysed for 60 min at 4°C, and equal amount of the cell extracts (40 μg of proteins) were diluted in Laemmli solution and resolved by SDS-PAGE[@b41]. RNA extraction and Real Time PCR analysis in U87MG cells and in CSCs -------------------------------------------------------------------- U87MG cells or GSCs were treated with DMSO (control), 500 nM FC85 and/or 2.5 µM ISA27 for 6 h or seven days, respectively. In some experiments, U87MG cells were incubated with 50 nM everolimus and/or 10 µM nutlin-3 for 6 h or 24 h. At the end of treatment, the cells were collected, and total RNA was extracted using RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\'s instructions. cDNA synthesis was performed with 500 ng of RNA using the i-Script cDNA synthesis kit (BioRad, Hercules, USA) following the manufacturer\'s instructions. RT-PCR reactions consisted of 25 µL of Fluocycle® II SYBR® (Euroclone, Milan, Italy), 1.5 µL of both 10 µM forward and reverse primers, 3 µL of cDNA, and 19 µL of H~2~O. All reactions were performed for 40 cycles using the following temperature profiles: 98 °C for 30 seconds (initial denaturation); T °C (see [Suppl. Table S5](#s1){ref-type="supplementary-material"}) for 30 seconds (annealing); and 72°C for 3 seconds (extension)[@b34]. Cell cycle analysis in U87MG cells ---------------------------------- The measurement of the percentage of cells in the different cell phases was performed using the Muse™ Cell Analyser, Merck KGaA, Darmstadt, Germany). Briefly, U87MG cells were treated for 24 h with DMSO or 500 nM FC85 and 2.5 µM ISA27, alone or in combination. Adherent cells were collected and centrifuged at 300 × g for 5 minutes. The pellet was washed with PBS and suspended in 100 µl of PBS; finally, the cells were slowly added to 1 ml of ice cold 70% ethanol and maintained o/n at −20°C. Then, a cell suspension aliquot (containing at least 2 × 10^5^ cells) was centrifuged at 300 × g for 5 minutes, washed once with PBS and suspended in the fluorescent reagent (Muse™ Cell Cycle reagent)[@b41]. Annexin V and 7-AAD staining in U87MG cells and in GSCs ------------------------------------------------------- Dual staining with Annexin V conjugated to fluorescein-isothiocyanate (FITC) and 7-amino-actinomysin (7-AAD) was performed using the commercially available kit (Muse Annexin V and Dead Cell Kit; Merck KGaA, Darmstadt, Germany). U87MG cells or GSCs were treated with DMSO (control), FC85 and/or ISA27 for 6 h or seven days, respectively. In some experiments, U87MG cells were incubated with 50 nM everolimus and/or 10 µM nutlin-3 for 24 h. At the end of the treatment periods, the percentages of living, apoptotic and dead cells were acquired and analysed by Muse™ Cell Analyser, as previously described[@b41]. Quantification of the occupied area and the cellular processes of neurospheres ------------------------------------------------------------------------------ GSCs were plated in complete growth medium (*day 0*) and treated for seven or fourteen days with 500 nM FC85 and/or 2.5 µM ISA27. At the end of the treatment period, the drug-containing media were replaced with fresh NSC medium, and the GSCs were allowed to grow for another 7 or 14 days. Images of the neurospheres were captured at days 0, 7, 14 and 21. Three different wells were analysed for each condition, and 15 images of each well were captured[@b34][@b55]. The response of the cultures to the various treatments was quantified by measuring the area occupied by neurospheres that had formed, using the ImageJ program (version 1.41; Bethesda, MD, USA). The cellular processes extending from the six to eight differentiating neurospheres per condition in three independent experiments were evaluated. ERK and AKT phosphorylation assays ---------------------------------- U87MG cells were cultured in 96-well microplates (5.000 cells/well) and treated for 2 hours with different FC85 concentrations (100 nM, 1 µM and 10 µM). In kinetic experiments, GSCs were treated for different times with 500 nM FC85 and/or 2.5 µM ISA27. At the end of the treatment period, the GSCs were centrifuged at 500 × g for 3 minutes; cells were washed twice using fresh saline and rapidly fixed with 4% (for adherent U87MG cells) or 8% (for suspension GSCs) formaldehyde to preserve the activation of specific protein modification. The levels of total and phosphorylated AKT and ERK1/2 were determined using specific primary antibodies. The subsequent incubation with a secondary HRP-conjugated antibody and the developing solution allowed for the colorimetric quantification of the levels of total and phosphorylated proteins. The relative number of cells in each well was then determined using the crystal violet assay. The results were calculated by subtracting the mean background value from the values obtained under each test condition: values were normalized to the number of cells in each well and are expressed as the percentages of the control (untreated cells) values. Statistical analyses -------------------- The nonlinear multipurpose curve-fitting program Graph-Pad Prism (GraphPad Software Inc., San Diego, CA) was used for data analysis and graphic presentations. All data are presented as the mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni\'s corrected t-test for post-hoc pair-wise comparisons. P\<0.05 was considered statistically significant. Author Contributions ==================== S.D., E.Z. and E.D.P. performed most of the biological work. S.S., G.N. and P.C. synthesized the compounds. B.C. and S.R. conceived the idea and conducted the design. C.M. coordinated the project. S.D., B.C. and S.R. wrote the main manuscript text. L.M. and E.N. provided important help in the significance of the results and in writing article discussion section. All authors reviewed the manuscript. Supplementary Material {#s1} ====================== ###### Supplementary Information Supplementary Information This work was supported by the FIRB, Bando Futuro in Ricerca 2010 (Grant RBFR10ZJQT). ![(a) Diagram of AKT/mTOR and MDM2/p53 signalling crosstalk in GBM and in GSCs.\ AKT/mTOR deactivation decreases MDM2 and p53 phosphorylation and increases stable p53, which triggers its downstream targets. Simultaneously, p53 may increase PTEN to suppress AKT activation further. FC85 inhibited both AKT (Ser473) and mTOR (Ser2448) phosphorylation. ISA27 dissociated the MDM2-p53 complex, thus re-activating p53. The combined therapy with FC85+ISA27 more efficiently re-activated the p53 pathway, producing a synergic effect on the inhibition of GBM cell viability; most importantly, the simultaneous inhibition of AKT/mTOR and of the MDM2-p53 complex led to a synergic effect in triggering cellular differentiation/apoptosis of GSC subpopulation. (**b**) The design of FC85 starting from the general structure of OXIDs. (**c**) The synthetic procedure for the preparation of FC85.](srep09956-f1){#f1} ![Effect of FC85 on U87MG and HUVEC cell viability.\ **(a)** U87MG cells were treated in complete medium with the indicated FC85 concentrations for 24 h. At the end of treatment, cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. (**b**) U87MG cells were treated as in (a); live and dead cells were then estimated using the trypan blue exclusion test. The data are expressed as the cell number per well and are the mean values ± SEM of two independent experiments, each performed in triplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \*\*\* p\<0.001 vs. control live cells; \#\#p\<0.01, \#\#\# p\<0.001 vs. control dead cells. (**c**) U87MG cells were treated with 10 µM FC85 for 24 h or 96 h, and then, the medium was replaced with drug-free fresh medium for another 24 h or 72 h. At the end of the treatment period, cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \*\*\* p\<0.001 vs. control. **(d)** U87MG (black bars) or HUVEC (white bars) cells were treated with different FC85 concentrations for 48 h. Cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \*\*\* p\<0.001 vs. respective control; \#p\<0.05, \#\#p\<0.01 vs. U87MG cells.](srep09956-f2){#f2} ![Synergistic effect of FC85 and ISA27 on the survival/growth of U87MG cells and on the reactivation of p53 pathway.\ (**a**) Isobologram 2-D showing the interactions between FC85 and ISA27 in MTS viability tests performed in U87MG cells treated for 24 h with FC85 and/or ISA27. The IC~50~values for FC85 and ISA27 are shown on the X- and Y-axis, respectively. The open points (○) on the additivity line depict the theoretical IC~50,add~values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. The solid points (●) depict the experimental IC~50,mix~values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. (**b**) U87MG cells were treated for 6 h with DMSO (control), 500 nM FC85 or 2.5 µM ISA27, alone or in combination. Lysates were subjected to western blot analysis using an antibody to p53. One representative Western blot is presented (b) for each cell treatment. β-actin was used as the loading control. The bar graph (c) shows the quantitative analysis of the Western blots, performed using ImageJ. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, set to 100%, and are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \*p0.05, \*\*\*p\<0.001 vs. control; \#\# p\<0.01 vs. FC85 alone; §§ p\<0.01 vs. ISA27 alone. Full-length blots are presented in the [Supplementary Information](#s1){ref-type="supplementary-material"} section titled ''Full-length blots relative to the cropped images showed in the main Figures". (**d**) U87MG cells were treated as in b. The relative mRNA quantification of p53 target genes (PUMA, p21 and MDM2) was performed by real-time RT-PCR as describe in the Methods section. The data are the mean values ± SEM of three different experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \*\*\* p\<0.001 vs. control; \#\# p\<0.01, \#\#\# p\<0.001 vs. FC85 alone; §§ p\<0.01, §§§ p\<0.001 vs. ISA27 alone.](srep09956-f3){#f3} ![The effect of FC85 and/or ISA27 on U87MG cell apoptosis and cell cycle.\ (**a**, **b)** U87MG cells were treated for 24 h with DMSO (control), or 500 nM FC85 or 2.5 µM ISA27, alone or in combination. At the end of the treatment period, the cells were collected, and the level of phosphatidylserine externalisation was evaluated using the Annexin V-staining protocol, as described in the Methods section. b) The data are expressed as the percentage of apoptotic cells (the data for the early-stage apoptotic cells shown in white, and the data for the late-stage apoptotic/necrotic cells shown in grey) versus the total number of cells. The data shown represent the mean ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \*\*\* p\<0.001 vs. control; \#\#\# p\<0.001 vs. FC85 alone; §§§ p\<0.001 vs. ISA27 alone. (**c, d**) U87MG cells were treated as in a. At the end of the treatment periods, the cell cycle was analysed as described in the Methods section. Representative cell cycle histograms of untreated and treated cells are shown (c). The data are expressed as the percentage of cells in the different phases (G0/G1, G2 or S) versus the total cell number and are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \*\* p\<0.01 vs. control in the respective cellular phase; \# p\<0.05, \#\# p\<0.01 vs. FC85 alone; § p\<0.05, §§ p\<0.01 vs. ISA27 alone.](srep09956-f4){#f4} ![Effect of FC85 and/or ISA27 on GSC proliferation/viability.\ U87MG-derived GSCs were incubated with the indicated concentrations of FC85 (**a**) or ISA27 (**b**) for 24 h, four days or seven days. At the end of the treatment periods, cell viability was measured using the MTS assay. (**c**) U87MG-derived GSCs were incubated for 7 days with increasing concentrations of FC85 or ISA27, and cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \*\* p\<0.01, \*\*\* p\<0.001 vs. control. (**d**) Isobologram 2-D showing the interactions between FC85 and ISA27 in MTS viability tests performed in U87MG-derived CSCs treated for seven days with FC85 and/or ISA27. The IC~50~ values for FC85 and ISA27 are shown on the X- and Y-axis, respectively. The open points (○) on the additivity line depict the theoretical IC~50,add~values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. The solid points (●) depict the experimental IC~50,mix~ values for total dose expressed as the proportion of FC85 and ISA27 that produced a 50% effect. (**e**) U87MG-derived GSCs were incubated with FC85 and/or ISA27 for seven days; then, the medium was replaced with drug-free fresh medium for other seven or fourteen days. At the end of the treatment period, cell viability was measured using the MTS assay. The data are expressed as a percentage with respect to that of untreated cells (control), which was set to 100%, and are the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \*\*p\<0.01, \*\*\* p\<0.001 vs. control.](srep09956-f5){#f5} ![The effect of FC85 and/or ISA27 on GSC apoptosis/differentiation.\ (**a**, **b**) GSCs were treated for 7 days with NSC medium containing DMSO (control), or 500 nM FC85 or 2.5 µM ISA27, alone or in combination. At the end of treatments, the cells were collected and the degree of phosphatidylserine externalisation was evaluated using the Annexin V protocol, as described in the Method section. b) The data were expressed as the percentage of apoptotic cells (early-apoptotic in white, late-apoptotic/necrotic in grey) relative to the total number of cells. The data are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \*\*\* p\<0.001 vs. control; \#\#\# p\<0.001 vs. FC85 alone; §§§ p\<0.001 vs. ISA27 alone. (**c**) GSCs were treated as in a; total RNA was extracted, and the relative mRNA quantification of the stem cell markers CD133 and nestin, the neuronal marker MAP, the astrocyte marker GFAP, and of the oligodendrocyte marker Olig2 was performed by RT-PCR. The data are expressed as the fold change vs. the levels of the control and are the mean values ± SEM of three different experiments. The significance of the differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \*\* p \<0.01, \*\*\* p\<0.001 vs. control; \#\#\# p\<0.001 vs. FC85 alone; §§ p\<0.01, §§§ p\<0.001 vs. ISA27 alone.](srep09956-f6){#f6} ![Effect of FC85 and/or ISA27 on sphere-derived cell morphology.\ GSCs were treated for seven or fourteen days with complete NSC medium containing DMSO (control), or 500 nM FC85, or 2.5 µM ISA27, alone or in combination. (**a**) Representative cell micrographs after seven and fourteen days of treatment are shown. The area of the culture plates occupied by the spheres (**b**) and the length of cellular processes (**c**) were scored after seven and fourteen days of treatment. The counts represent the mean values ± SEM of two independent experiments. The significance of differences was determined with a one-way ANOVA with Bonferroni post-test: \* p\<0.05, \*\*\* p\<0.001 vs. control; \# p\<0.05 vs. FC85 alone; § p\<0.05 vs. ISA27 alone.](srep09956-f7){#f7} ![Kinetics of FC85 and/or ISA27 inhibition of AKT and ERK1/2 phosphorylation.\ U87MG-derived CSCs were treated for the indicated times (0--120 min) with NSC medium containing DMSO (control), or 500 nM FC85, or 2.5 µM ISA27, alone or in combination. At the end of the treatment periods, the levels of AKT (**a**) and ERK 1/2 (**b**) phosphorylation were evaluated using ELISA kits, as described in the Methods section. The data are expressed as the percentage of phosphorylated AKT or ERK1/2 relative to those of untreated cells (control), which were set at 100%, and are the mean values ± SEM of three independent experiments performed in triplicate. The significance of differences was performed using one-way ANOVA with Bonferroni post-test: \* p\< 0.05, \*\* p\< 0.01, \*\*\* p\<0.001 vs. control; \# p\<0.05, \#\#p\< 0.01 vs. FC85 alone; § p\<0.05, §§ p\<0.01, §§§ p\<0.001 vs. ISA27 alone.](srep09956-f8){#f8}

{ "pile_set_name": "PubMed Central" }

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22,244

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"extraction", "Real", "Time", "PCR", "analysis", "cells", "CSCs", "cells", "GSCs", "treated", "DMSO", "control", "nM", "µM", "h", "seven", "days", "respectively", "experiments", "cells", "incubated", "nM", "everolimus", "µM", "h", "end", "treatment", "cells", "collected", "total", "RNA", "extracted", "using", "Mini", "Kit", "Qiagen", "Hilden", "Germany", "according", "instructions", "cDNA", "synthesis", "performed", "ng", "RNA", "using", "cDNA", "synthesis", "kit", "BioRad", "Hercules", "USA", "following", "instructions", "reactions", "consisted", "µL", "II", "Euroclone", "Milan", "Italy", "µL", "µM", "forward", "reverse", "primers", "µL", "cDNA", "µL", "reactions", "performed", "cycles", "using", "following", "temperature", "profiles", "seconds", "initial", "denaturation", "see", "Suppl", "Table", "seconds", "annealing", "seconds", "extension", "Cell", "cycle", "analysis", "cells", "measurement", "percentage", "cells", "different", "cell", "phases", "performed", "using", "Cell", "Analyser", "Merck", "KGaA", "Darmstadt", "Germany", "Briefly", "cells", "treated", "h", "DMSO", "nM", "µM", "alone", "combination", "Adherent", "cells", "collected", "centrifuged", "g", "minutes", "pellet", "washed", "PBS", "suspended", "µl", "PBS", "finally", "cells", "slowly", "added", "ml", "ice", "cold", "ethanol", "maintained", "cell", "suspension", "aliquot", "containing", "least", "cells", "centrifuged", "g", "minutes", "washed", "PBS", "suspended", "fluorescent", "reagent", "Cell", "Cycle", "reagent", "Annexin", "V", "staining", "cells", "GSCs", "Dual", "staining", "Annexin", "V", "conjugated", "FITC", "performed", "using", "commercially", "available", "kit", "Muse", "Annexin", "V", "Dead", "Cell", "Kit", "Merck", "KGaA", "Darmstadt", "Germany", "cells", "GSCs", "treated", "DMSO", "control", "h", "seven", "days", "respectively", "experiments", "cells", "incubated", "nM", "everolimus", "µM", "end", "treatment", "periods", "percentages", "living", "apoptotic", "dead", "cells", "acquired", "analysed", "Cell", "Analyser", "previously", "described", "Quantification", "occupied", "area", "cellular", "processes", "neurospheres", "GSCs", "plated", "complete", "growth", "medium", "day", "treated", "seven", "fourteen", "days", "nM", "µM", "end", "treatment", "period", "media", "replaced", "fresh", "NSC", "medium", "GSCs", "allowed", "grow", "another", "days", "Images", "neurospheres", "captured", "days", "Three", "different", "wells", "analysed", "condition", "images", "well", "captured", "response", "cultures", "various", "treatments", "quantified", "measuring", "area", "occupied", "neurospheres", "formed", "using", "ImageJ", "program", "version", "Bethesda", "MD", "USA", "cellular", "processes", "extending", "six", "eight", "differentiating", "neurospheres", "per", "condition", "three", "independent", "experiments", "evaluated", "ERK", "AKT", "phosphorylation", "assays", "cells", "cultured", "microplates", "treated", "hours", "different", "concentrations", "nM", "µM", "µM", "kinetic", "experiments", "GSCs", "treated", "different", "times", "nM", "µM", "end", "treatment", "period", "GSCs", "centrifuged", "g", "minutes", "cells", "washed", "twice", "using", "fresh", "saline", "rapidly", "fixed", "adherent", "cells", "suspension", "GSCs", "formaldehyde", "preserve", "activation", "specific", "protein", "modification", "levels", "total", "phosphorylated", "AKT", "determined", "using", "specific", "primary", "antibodies", "subsequent", "incubation", "secondary", "antibody", "developing", "solution", "allowed", "colorimetric", "quantification", "levels", "total", "phosphorylated", "proteins", "relative", "number", "cells", "well", "determined", "using", "crystal", "violet", "assay", "results", "calculated", "subtracting", "mean", "background", "value", "values", "obtained", "test", "condition", "values", "normalized", "number", "cells", "well", "expressed", "percentages", "control", "untreated", "cells", "values", "Statistical", "analyses", "nonlinear", "multipurpose", "program", "Prism", "GraphPad", "Software", "San", "Diego", "CA", "used", "data", "analysis", "graphic", "presentations", "data", "presented", "mean", "SEM", "Statistical", "analysis", "performed", "analysis", "variance", "ANOVA", "corrected", "comparisons", "considered", "statistically", "significant", "Author", "Contributions", "performed", "biological", "work", "synthesized", "compounds", "conceived", "idea", "conducted", "design", "coordinated", "project", "wrote", "main", "manuscript", "text", "provided", "important", "help", "significance", "results", "writing", "article", "discussion", "section", "authors", "reviewed", "manuscript", "Supplementary", "Material", "Supplementary", "Information", "Supplementary", "Information", "work", "supported", "FIRB", "Bando", "Futuro", "Ricerca", "Grant", "Diagram", "signalling", "crosstalk", "GBM", "deactivation", "decreases", "phosphorylation", "increases", "stable", "triggers", "downstream", "targets", "Simultaneously", "may", "increase", "PTEN", "suppress", "AKT", "activation", "inhibited", "AKT", "mTOR", "phosphorylation", "dissociated", "complex", "thus", "combined", "therapy", "efficiently", "pathway", "producing", "synergic", "effect", "inhibition", "GBM", "cell", "viability", "importantly", "simultaneous", "inhibition", "complex", "led", "synergic", "effect", "triggering", "cellular", "GSC", "subpopulation", "b", "design", "starting", "general", "structure", "OXIDs", "c", "synthetic", "procedure", "preparation", "Effect", "HUVEC", "cell", "cells", "treated", "complete", "medium", "indicated", "concentrations", "end", "treatment", "cell", "viability", "measured", "using", "MTS", "assay", "data", "expressed", "percentage", "respect", "untreated", "cells", "control", "set", "mean", "values", "SEM", "three", "independent", "experiments", "performed", "duplicate", "b", "cells", "treated", "live", "dead", "cells", "estimated", "using", "trypan", "blue", "exclusion", "test", "data", "expressed", "cell", "number", "per", "well", "mean", "values", "SEM", "two", "independent", "experiments", "performed", "triplicate", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "live", "cells", "control", "dead", "cells", "c", "cells", "treated", "µM", "h", "h", "medium", "replaced", "fresh", "medium", "another", "h", "end", "treatment", "period", "cell", "viability", "measured", "using", "MTS", "assay", "data", "expressed", "percentage", "respect", "untreated", "cells", "control", "set", "mean", "values", "SEM", "three", "independent", "experiments", "performed", "duplicate", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "black", "bars", "HUVEC", "white", "bars", "cells", "treated", "different", "concentrations", "Cell", "viability", "measured", "using", "MTS", "assay", "data", "expressed", "percentage", "respect", "untreated", "cells", "control", "set", "mean", "values", "SEM", "three", "independent", "experiments", "performed", "duplicate", "significance", "differences", "determined", "ANOVA", "Bonferroni", "respective", "control", "cells", "Synergistic", "effect", "cells", "reactivation", "Isobologram", "showing", "interactions", "MTS", "viability", "tests", "performed", "cells", "treated", "h", "shown", "respectively", "open", "points", "additivity", "line", "depict", "theoretical", "total", "dose", "expressed", "proportion", "produced", "effect", "solid", "points", "depict", "experimental", "total", "dose", "expressed", "proportion", "produced", "effect", "b", "cells", "treated", "h", "DMSO", "control", "nM", "µM", "alone", "combination", "Lysates", "subjected", "western", "blot", "analysis", "using", "antibody", "One", "representative", "Western", "blot", "presented", "b", "cell", "treatment", "used", "loading", "control", "bar", "graph", "c", "shows", "quantitative", "analysis", "Western", "blots", "performed", "using", "ImageJ", "data", "expressed", "percentage", "optical", "density", "immunoreactive", "band", "relative", "control", "set", "mean", "values", "SEM", "three", "different", "experiments", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "alone", "alone", "blots", "presented", "Supplementary", "Information", "section", "titled", "blots", "relative", "cropped", "images", "showed", "main", "Figures", "cells", "treated", "b", "relative", "mRNA", "quantification", "target", "genes", "PUMA", "performed", "describe", "Methods", "section", "data", "mean", "values", "SEM", "three", "different", "experiments", "performed", "duplicate", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "alone", "alone", "effect", "cell", "apoptosis", "cell", "b", "cells", "treated", "h", "DMSO", "control", "nM", "µM", "alone", "combination", "end", "treatment", "period", "cells", "collected", "level", "phosphatidylserine", "externalisation", "evaluated", "using", "Annexin", "protocol", "described", "Methods", "section", "b", "data", "expressed", "percentage", "apoptotic", "cells", "data", "apoptotic", "cells", "shown", "white", "data", "cells", "shown", "grey", "versus", "total", "number", "cells", "data", "shown", "represent", "mean", "SEM", "three", "different", "experiments", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "alone", "alone", "c", "cells", "treated", "end", "treatment", "periods", "cell", "cycle", "analysed", "described", "Methods", "section", "Representative", "cell", "cycle", "histograms", "untreated", "treated", "cells", "shown", "c", "data", "expressed", "percentage", "cells", "different", "phases", "versus", "total", "cell", "number", "mean", "values", "SEM", "three", "different", "experiments", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "respective", "cellular", "phase", "alone", "alone", "Effect", "GSC", "GSCs", "incubated", "indicated", "concentrations", "b", "h", "four", "days", "seven", "days", "end", "treatment", "periods", "cell", "viability", "measured", "using", "MTS", "assay", "c", "GSCs", "incubated", "days", "increasing", "concentrations", "cell", "viability", "measured", "using", "MTS", "assay", "data", "expressed", "percentage", "respect", "untreated", "cells", "control", "set", "mean", "values", "SEM", "three", "independent", "experiments", "performed", "duplicate", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "Isobologram", "showing", "interactions", "MTS", "viability", "tests", "performed", "CSCs", "treated", "seven", "days", "values", "shown", "respectively", "open", "points", "additivity", "line", "depict", "theoretical", "total", "dose", "expressed", "proportion", "produced", "effect", "solid", "points", "depict", "experimental", "values", "total", "dose", "expressed", "proportion", "produced", "effect", "e", "GSCs", "incubated", "seven", "days", "medium", "replaced", "fresh", "medium", "seven", "fourteen", "days", "end", "treatment", "period", "cell", "viability", "measured", "using", "MTS", "assay", "data", "expressed", "percentage", "respect", "untreated", "cells", "control", "set", "mean", "values", "SEM", "three", "independent", "experiments", "performed", "duplicate", "significance", "differences", "determined", "ANOVA", "Bonferroni", "vs", "control", "effect", "GSC", "b", "GSCs", "treated", "days", "NSC", "medium", "containing", "DMSO", "control", "nM", "µM", "alone", "combination", "end", "treatments", "cells", "collected", "degree", "phosphatidylserine", "externalisation", "evaluated", "using", "Annexin", "V", "protocol", "described", "Method", "section", "b", "data", "expressed", "percentage", "apoptotic", "cells", "white", "grey", "relative", "total", "number", "cells", "data", "mean", "values", "SEM", "three", "different", "experiments", "significance", "differences", "determined", "ANOVA", "Bonferroni", "control", "alone", "alone", "c", "GSCs", "treated", "total", "RNA", "extracted", 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"inhibition", "AKT", "CSCs", "treated", "indicated", "times", "min", "NSC", "medium", "containing", "DMSO", "control", "nM", "µM", "alone", "combination", "end", "treatment", "periods", "levels", "AKT", "ERK", "b", "phosphorylation", "evaluated", "using", "ELISA", "kits", "described", "Methods", "section", "data", "expressed", "percentage", "phosphorylated", "AKT", "relative", "untreated", "cells", "control", "set", "mean", "values", "SEM", "three", "independent", "experiments", "performed", "triplicate", "significance", "differences", "performed", "using", "ANOVA", "Bonferroni", "control", "alone", "alone" ]

Introduction {#sec1-1} ============ Placebo seems indispensable in modern clinical investigations. A placebo is a sham medical intervention that can produce a placebo effect. Common placebos are usually inert tablets or sham surgery based on false information.\[[@ref1]\] Since the publication of Henry K. Beecher\'s in 1955, the phenomenon has been considered to be clinically important.\[[@ref2]\] Placebo effects were shown to be genuine psychobiological events attributed to the overall therapeutic context.\[[@ref3][@ref4]\] From a psychological point of view, many mechanisms might contribute to placebo effects, including expectations, conditioning, learning, motivation, memory, reward and anxiety reduction, etc.\[[@ref5][@ref6]\] Owing to the placebo effect, it is sometimes difficult to evaluate new treatments. Apparent benefits of a new drug may derive from the placebo effect but not from the drug per se. Therefore, modern clinical trials control for this effect by using a placebo, in which the subjects are blinded as to whether they receive a drug treatment or a placebo. In this way, placebo-controlled trials might provide information about the real effectiveness of a drug. The view of placebo effect was notably challenged when a systematic review of clinical trials in 2001 concluded that there was no evidence of clinically important effects, except perhaps in the treatment of pain and other continuous subjective outcomes.\[[@ref7]\] Since evidence-based medicine (EBM) is increasingly emphasized, placebo is still widely used in clinical trials other than in the treatment of pain and continuous subjective outcomes. Currently, international journals as well as clinical researchers are inclined to avoid or even ignore the ethical aspects of placebo. However, accumulating ethical concern has arisen from the worldwide use of placebo in randomized control trials (RCTs), because the investigators in a certain trial may render its participants without early and optimal treatment. Where for compelling and scientifically sound methodological reasons the use of placebo is necessary to determine the efficacy or safety of an intervention and the patients who receive placebo or no treatment will not be subject to any risk of serious or irreversible harm. Extreme care must be taken to avoid abuse of this option. (Extracted from the Declaration of Helsinki)\[[@ref8]\] A recent study, the European Cooperative Acute Stroke Study III (ECASS III) reported by Bluhmki *et al* and by Hacke *et al* supported the use of alteplase three hours after the onset of stroke symptoms, while together with other studies, ECASS III raised some concerns about thrombolytic studies.\[[@ref9][@ref10]\] Despite repeated emphases of early treatment on acute ischemia, the control group in ECASS III failed to receive any treatment except the placebo. This is contradictory to the common sense "Time is brain".\[[@ref11]\] The lack of evidence-based therapies should not justify the exclusion of empirical use of anti-platelet agents, or others. In this regard, it is almost always the case in China, since traditional Chinese medicine has been a routine for patients suffering from cerebrovascular ischemia.\[[@ref12]\] Another example of clinical trial that may raise ethical concerns is the one conducted by Kappos, *et al*, about oral fingolimod in treating relapsing multiple sclerosis (MS). This placebo-controlled trial revealed that fingolimod improved the relapse rate, the risk of disability progression, and end points on MRI.\[[@ref13]\] However, the longer the trial lasts, the longer the patients in the placebo group must go without treatment. In this context, the well-acknowledged and widely used therapeutic approaches should be considered first and foremost as controls, when RCTs are designed. Although placebos may be helpful in treating MS, the better-acknowledged ways such as interferon-beta and glatiramer acetate can potently alleviate the sufferings of patients with MS\[[@ref14]\] and can readily serve as positive controls in that study. As a clinician, I feel suffocated when confronted with such ethically problematic studies. Where is the Hippocratic Oath? And where is Declaration of Helsinki. I hate the unreasonable use of placebos but cannot do anything. The sanctification of placebo is partly due to the repeated emphasis of RCTs as the gold standard of clinical trials. Thus for the time being, the ending of this sanctification seems incoercible. Dichotomy seems necessary in evaluating the role of placebo in scientific research; the need for placebo-controlled design cannot be denied at certain stage of drug development, whereas for larger phase IV studies, better alternatives should be considered. I thus would like to propose some solutions to such an ethical conundrum. Ethically, the clinicians are always expected to provide their patients with the best choices of treatment other than placebo, and the best available treatment as a positive control other than placebos is preferable to the patients. In neurological field, for example, aspirin is widely accepted as a control in trials with regard to prevention or treatment of cerebral ischemic disorders,\[[@ref15]\] because its prophylactic as well as therapeutic effects on cardiovascular events are well established.\[[@ref16][@ref17]\] Although the control group is a must in RCTs, an alternative way such as a delayed-start approach,\[[@ref18]\] can be utilized to avoid ethical issues that may arise. Delayed-start studies are designed primarily to test the disease-modifying effect of a drug, whereas they can reduce the ethical concerns that patients in the placebo group must go without treatment until the end of the study. The control subjects enrolled in the aforementioned thrombolytic study should at least start to accept conventional medication therapies immediately after the time-window for thrombolytic studies. As an old Chinese saying goes, doctors ought to be parentally considerate. Similarly, clinicians should always bear in mind that no treatment equals killing for their patients. Clinicians/researchers need to understand the implications of offering no treatment in certain situations before enrolling experimental subjects. More importantly, a thorough explanation about the aforementioned possible implications should be performed by the clinicians. The placebo effect is produced on the premise that the subjects believe the effectiveness of placebo. This is different from the practice in RCTs in which the subjects are informed of the possibility of accepting only a sham medical intervention. Informed consent for a study is usually required to be considered ethical, including the disclosure that some patients are to receive placebos only. In this case, the subjects do not know whether they might be getting a real treatment or a sham one, and thus might suspect the efficacy of the medical interventions even before the trials.\[[@ref19]\] As a result, placebos do not work as strongly in clinical trials, as in basic research. This may explain the insignificant clinically important effects as summarized by the said review.\[[@ref7]\] What\'s worse, the deception involved in the use of placebos creates conflicts between the Hippocratic Oath and the honesty of the doctor--patient relationship.\[[@ref20][@ref21]\] The purpose of the clinical trials should be explained in detail in the process of obtaining informed consent from the participants, while it is not always the case, especially in low-income countries. For patients in low-income countries, the participation in clinical trials is intriguing simply because poor patients have the chance of getting free treatment, or they can get rewards for attending the research. This may lead to selection bias as well as ethical debates because the poor participants have no better choice than attending the trials. The honest relationship between doctors and patients is further worsened because of the notion of the patients that they are utilized. In summary, although the ethics of placebos have been debated frequently in history,\[[@ref21]\] even in the revision process of the Declaration of Helsinki, placebos have given rise to ethical debates much more often than ever before. As the pilgrimage of placebo is still on the way, refinement of controls in RCTs is worth paying close attention to. This study was supported by the China Scholarship Council. **Source of Support:** Nil **Conflict of Interest:** None declared.

{ "pile_set_name": "PubMed Central" }

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22,245

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"routine", "patients", "suffering", "cerebrovascular", "Another", "example", "clinical", "trial", "may", "raise", "ethical", "concerns", "one", "conducted", "Kappos", "et", "al", "oral", "fingolimod", "treating", "relapsing", "multiple", "sclerosis", "MS", "trial", "revealed", "fingolimod", "improved", "relapse", "rate", "risk", "disability", "progression", "end", "points", "However", "longer", "trial", "lasts", "longer", "patients", "placebo", "group", "must", "go", "without", "treatment", "context", "widely", "used", "therapeutic", "approaches", "considered", "first", "foremost", "controls", "RCTs", "designed", "Although", "placebos", "may", "helpful", "treating", "MS", "ways", "glatiramer", "acetate", "potently", "alleviate", "sufferings", "patients", "readily", "serve", "positive", "controls", "study", "clinician", "feel", "suffocated", "confronted", "ethically", "problematic", "studies", "Hippocratic", "Oath", "Declaration", "Helsinki", "hate", "unreasonable", "use", "placebos", "anything", "sanctification", "placebo", "partly", "due", "repeated", "emphasis", "RCTs", "gold", "standard", "clinical", "trials", "Thus", "time", "ending", "sanctification", "seems", "incoercible", "Dichotomy", "seems", "necessary", "evaluating", "role", "placebo", "scientific", "research", "need", "design", "denied", "certain", "stage", "drug", "development", "whereas", "larger", "phase", "IV", "studies", "better", "alternatives", "considered", "thus", "would", "like", "propose", "solutions", "ethical", "conundrum", "Ethically", "clinicians", "always", "expected", "provide", "patients", "best", "choices", "treatment", "placebo", "best", "available", "treatment", "positive", "control", "placebos", "preferable", "patients", "neurological", "field", "example", "aspirin", "widely", "accepted", "control", "trials", "regard", "prevention", "treatment", "cerebral", "ischemic", "disorders", "prophylactic", "well", "therapeutic", "effects", "cardiovascular", "events", "well", "Although", "control", "group", "must", "RCTs", "alternative", "way", "approach", "utilized", "avoid", "ethical", "issues", "may", "arise", "studies", "designed", "primarily", "test", "effect", "drug", "whereas", "reduce", "ethical", "concerns", "patients", "placebo", "group", "must", "go", "without", "treatment", "end", "study", "control", "subjects", "enrolled", "aforementioned", "thrombolytic", "study", "least", "start", "accept", "conventional", "medication", "therapies", "immediately", "thrombolytic", "studies", "old", "Chinese", "saying", "goes", "doctors", "ought", "parentally", "considerate", "Similarly", "clinicians", "always", "bear", "mind", "treatment", "equals", "killing", "patients", "need", "understand", "implications", "offering", "treatment", "certain", "situations", "enrolling", "experimental", "subjects", "importantly", "thorough", "explanation", "aforementioned", "possible", "implications", "performed", "clinicians", "placebo", "effect", "produced", "premise", "subjects", 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"clinical", "trials", "intriguing", "simply", "poor", "patients", "chance", "getting", "free", "treatment", "get", "rewards", "attending", "research", "may", "lead", "selection", "bias", "well", "ethical", "debates", "poor", "participants", "better", "choice", "attending", "trials", "honest", "relationship", "doctors", "patients", "worsened", "notion", "patients", "utilized", "summary", "although", "ethics", "placebos", "debated", "frequently", "history", "even", "revision", "process", "Declaration", "Helsinki", "placebos", "given", "rise", "ethical", "debates", "much", "often", "ever", "pilgrimage", "placebo", "still", "way", "refinement", "controls", "RCTs", "worth", "paying", "close", "attention", "study", "supported", "China", "Scholarship", "Council", "Source", "Support", "Nil", "Conflict", "Interest", "None", "declared" ]

1. Introduction {#sec1} =============== 1.1. Anaerobic Methane-Oxidizing Archaea (ANME) {#sec1.1} ----------------------------------------------- Anaerobic methane-oxidizing archaea (ANME) perform anaerobic oxidation of methane (AOM) via reversal of the methanogenic pathway. ANME were first discovered in marine sediments where AOM was coupled to sulfate reduction (SR) ([Table 1](#tab1){ref-type="table"}, reaction (1)). Here, ANME formed metabolically interdependent consortia with sulfate-reducing bacteria (SRB) that belong to the Deltaproteobacteria \[[@B1]--[@B3]\]. Three distinct methanotrophic groups were identified: ANME-1 (subclusters a and b), ANME-2 (subclusters a, b, and c), and ANME-3. The ANME-1 cluster is related to Methanomicrobiales and Methanosarcinales but forms a separate cluster \[[@B2]\], ANME-2 are related to cultivated members of the Methanosarcinales \[[@B4]\], and ANME-3 are more related to*Methanococcoides* spp. \[[@B5]\] ([Figure 1](#fig1){ref-type="fig"}). The ANME clades are not monophyletic with each other and the phylogenetic distance between the subgroups is large, with 16S rRNA gene sequence similarity of only 75--92% \[[@B6]\]. Subclusters ANME-2a and ANME-2b form a coherent clade that is distinguished from ANME-2c \[[@B7]\] and are therefore often grouped together as ANME-2a/b ([Figure 1](#fig1){ref-type="fig"}). The wide phylogenetic distribution is reflected in the ecological niche adaptation of the different ANME clades. ANME clades involved in sulfate-dependent AOM (S-AOM) co-occur in many different marine environments, except for ANME-3 that was mainly found in mud volcanoes and in some seep sediments \[[@B6], [@B8], [@B9]\]. In marine sediments, a distinct zonation occurs where ANME-2a/b dominate upper layers and ANME-2c and/or ANME-1 abundance increases in deeper zones, indicating ecological niche separation \[[@B10]--[@B15]\]. ANME also form a versatile partnership with non-SRB such as beta-proteobacteria \[[@B16]\] and Verrucomicrobia \[[@B17]\]. ANME, and especially ANME-1, have also been observed without a (closely associated) bacterial partner \[[@B5], [@B12], [@B13], [@B18]--[@B22]\]. It was therefore suggested that ANME could perform AOM alone, using electron acceptors such as metal oxides, or perform other processes such as methanogenesis \[[@B23], [@B24]\]. Indications exist that AOM can be coupled to the reduction of different metal (oxides) ([Table 1](#tab1){ref-type="table"}, reactions (3)--(5)), but limited experimental evidence exists to date that ANME are responsible for this reaction (discussed in [Section 3.3](#sec3.3){ref-type="sec"}). Besides marine environments, ANME involved in S-AOM can be found in terrestrial \[[@B25], [@B26]\] and freshwater ecosystems \[[@B27]\]. A member of a fourth subcluster, "*Candidatus (Ca.)* Methanoperedens nitroreducens," was recently discovered to perform nitrate-dependent AOM (N-AOM) \[[@B28]\] ([Table 1](#tab1){ref-type="table"}, reaction (2)). This cluster was named "ANME-2d" \[[@B29]\] but later renamed to "GOM Arc I" \[[@B30]\] and "AOM-associated archaea (AAA)" \[[@B6]\]. Phylogenetic analysis shows that the ANME-2d cluster is monophyletic with "*Ca.* M. nitroreducens*"* and other AAA/GoM Arc I sequences, but distinct from other ANME-2 subclusters ([Figure 1](#fig1){ref-type="fig"}). ANME-2d were initially enriched in bioreactors inoculated with freshwater samples \[[@B28], [@B31]--[@B33]\]. As ANME-2d archaea were only recently recognized, their environmental preferences remain insufficiently studied. So far they have been found in freshwater canals \[[@B31]\], soils and rice paddy fields \[[@B34]--[@B36]\], lakes and rivers \[[@B35]\], and wastewater treatment plants \[[@B33]\]. In situ AOM activity of ANME-2d was determined recently for the first time \[[@B36]\]. More ANME phylotypes in different environments and possibly new archaeal clades involved in AOM may yet have to be discovered. For example, methyl coenzyme M reductase A genes (*mcrA*) from Bathyarchaeota (formerly known as Miscellaneous Crenarchaeota Group) and from the new archaeal phylum Verstraetearchaeota were recently found, indicating their involvement in methane metabolism \[[@B37], [@B38]\]. This review focusses on archaea performing AOM through the reversal of the methanogenesis pathway. We describe the reversibility of the central methanogenic pathway, including the key enzyme in methanogenesis and anaerobic methanotrophy (i.e., methyl coenzyme M reductase, Mcr). The possibility of methanogens to perform methane oxidation and of ANME to perform methanogenesis is also addressed. Lastly, the physiological adaptations of ANME to perform respiration using different electron acceptors during AOM are discussed. 1.2. ANME versus Methanogens: Domain Based (Meta)Genome Comparison {#sec1.2} ------------------------------------------------------------------ In order to find additional differences between archaeal methanotrophs and related methanogens that could validate and complement findings in the literature, we performed domain based (meta)genome comparison between selected metagenomes of ANME and genomes of methanogens, as done previously for bacterial genomes \[[@B39]\]. For archaeal methanotrophs, we used the metagenomes of ANME-1 \[[@B40], [@B41]\], ANME-2a \[[@B42]\], and ANME-2d \[[@B28], [@B43]\]. For methanogens, we used genomes of closely and distantly related species able to perform acetoclastic methanogenesis (A:*Methanosaeta concilii* GP6), methylotrophic methanogenesis (M-1:*Methanococcoides burtonii* DSM6242, M-2:*Methanolobus tindarius* DSM2278, and M-3:*Methanohalophilus mahii*DSM5219), hydrogenotrophic methanogenesis (H-1:*Methanospirillum hungatei* JF-1, H-2:*Methanobacterium formicicum* DSM3637, H-3:*Methanococcus maripaludis* C5, and H-4:*Methanoregula formicica* SMSP), and both acetoclastic and methylotrophic methanogenesis (AM:*Methanosarcina acetivorans*C2A). The genome of a sulfate-reducing archaeon that contained most enzymes for methanogenesis except for Mcr (S:*Archaeoglobus fulgidus* DSM 4304) was also included in the comparison. For each dataset the protein domains were obtained through InterProScan 5.17-56.0 using the TIGRFAM, ProDom, SMART, PROSITE, PfamA, PRINTS, SUPERFAMILY, COILS, and Gene3D domain databases. Results of the analysis are given in [Table 2](#tab2){ref-type="table"} and Table S1 of the Supplementary Material available online at <https://doi.org/10.1155/2017/1654237>. Since the ANME-1 metagenome assembled by Stokke et al. 2012 \[[@B40]\] contained many bacterial genes, we did not refer to this data for the domain based (meta)genome comparison but only used the ANME-1 metagenome described by Meyerdierks et al., 2010 \[[@B41]\]. We included both ANME-1 metagenomes to analyze the organization of genes for the formaldehyde-activating enzyme (Table S2) and the iron-only hydrogenase (Table S3). 2. Reversal of the Methanogenesis Pathway {#sec2} ========================================= 2.1. The Central Methanogenesis Pathway {#sec2.1} --------------------------------------- ANME are described to perform "reverse methanogenesis" \[[@B44]\] which implies the complete reversal of methanogenesis from H~2~ and CO~2~, that is, hydrogenotrophic methanogenesis (for kinetic and thermodynamic considerations, the reader is referred to \[[@B45]\]). During "forward" hydrogenotrophic methanogenesis, CO~2~ is reduced to CH~4~ with reducing equivalents derived from H~2~ ([Figure 2](#fig2){ref-type="fig"}). During methylotrophic methanogenesis, this pathway is already partly reversed. Methylotrophic methanogens utilize one-carbon compounds such as methylamines, methanol or methylated sulfur compounds (methanethiol, dimethyl sulfide) that are activated to methyl coenzyme M. About 75% of the methyl coenzyme M (CH~3~-CoM) is reduced to produce CH~4~ and about 25% of CH~3~-CoM is oxidized to CO~2~ using the methanogenesis pathway in reverse during methylotrophic growth. The oxidative part provides reducing equivalents that are needed for the generation of the proton motive force in the methanogenic respiratory chain and the reduction of CH~3~-CoM by methyl coenzyme M reductase (Mcr) \[[@B46]\] ([Figure 3](#fig3){ref-type="fig"}). In all methanogens, the Mcr reaction operates in the forward reaction and yields methane and the heterodisulfide of coenzyme B and coenzyme M (CoB-S-S-CoM):$$\begin{matrix} \left. {}{CH}_{3}\text{-}CoM + CoB\text{-}SH\longrightarrow{CH}_{4} + CoB\text{-}S\text{-}S\text{-}CoM; \right. \\ {\mspace{21210mu}\Delta G^{0} = - 30\, kJ\,{mol}^{- 1}\left\lbrack 52 \right\rbrack} \\ \end{matrix}$$The heterodisulfide is a central intermediate and acts as terminal electron acceptor in all methanogens. In hydrogenotrophic methanogens without cytochromes, it is the electron acceptor of the cytoplasmic electron-bifurcating CoB-S-S-CoM reductase (HdrABC) and F~420~-nonreducing hydrogenase (MvhADG) complex \[[@B47], [@B48]\] that is needed to provide reduced ferredoxin for the first step in methanogenesis; the reduction of CO~2~ to a formyl group. Within the methanogens with cytochromes, only a few members of the genus*Methanosarcina*are able to grow on H~2~/CO~2~. They use a ferredoxin-dependent hydrogenase (Ech) for ferredoxin reduction and an additional membrane bound methanophenazine-dependent hydrogenase (Vho) for H~2~ oxidation coupled to the reduction of the heterodisulfide by the membrane bound CoB-S-S-CoM reductase (HdrDE). F~420~ cycling can be accomplished using the F~420~-dependent hydrogenase (Frh) and F~420~H~2~: phenazine oxidoreductase (Fpo) complex \[[@B48]\] ([Figure 2](#fig2){ref-type="fig"}). For methanogens it is of crucial importance that Mcr operates in the forward reaction to yield methane and the heterodisulfide. If all reactions of the methanogenic pathway are reversed such as during AOM, the pathway requires the input of energy and produces electron donors (Figures [4](#fig4){ref-type="fig"} [](#fig5){ref-type="fig"}--[6](#fig6){ref-type="fig"}). Therefore, during AOM, an external electron acceptor is needed which makes the reaction thermodynamically favourable ([Table 1](#tab1){ref-type="table"}). The reverse reaction of Mcr is therefore an essential step in AOM and is discussed in [Section 2.2](#sec2.2){ref-type="sec"}. The respiratory chain that is needed for using different terminal electron acceptors will be discussed in [Section 3](#sec3){ref-type="sec"}. Evidence that ANME use the reverse methanogenesis pathway during AOM is derived from metagenomic and metatranscriptomic analyses. This showed that all genes for the (reverse) methanogenic pathway were present and expressed in ANME-2a \[[@B42]\] ([Figure 4](#fig4){ref-type="fig"}) and ANME-2d \[[@B28]\] ([Figure 5](#fig5){ref-type="fig"}). ANME-1 were consistently lacking the gene encoding N^5^,N^10^-methylene- tetrahydromethanopterin (H~4~MPT) reductase (Mer), which is an enzyme needed to oxidize methyl-H~4~MPT during methane oxidation \[[@B40], [@B41], [@B44], [@B49]\] ([Figure 6](#fig6){ref-type="fig"}, [Table 2](#tab2){ref-type="table"}). Possible explanations could be that (1) the*mer* gene is present but not yet detected, (2) ANME-1 used a bypass of this step, and (3) Mer was replaced by a structural analogue. The first possibility is highly unlikely. Although no closed genome of ANME-1 is publicly available to date, all ANME-1 metagenomes consistently only lack Mer and no other methanogenic genes. The second possibility was proposed previously where ANME-1 uses a bypass of Mer via the formation of methanol or methylamine \[[@B41]\], as was detected in deletion mutants of*Methanosarcina* \[[@B50], [@B51]\]. Here, CH~3~-CoM was presumably converted to methanol by a methyltransferase and subsequently to formaldehyde by a methanol dehydrogenase (Mdh). Then, formaldehyde would be converted to N^5^,N^10^ methylene-H~4~MPT using a fusion protein of the formaldehyde-activating enzyme (Fae) and a hexulose-6-phosphate synthase (Hps) \[[@B51]\] ([Figure 6](#fig6){ref-type="fig"}). Both Fae and Hps were found in the ANME-1 metagenome \[[@B41]\] and metaproteome \[[@B40], [@B49]\]. However, no genes coding for enzymes involved in methanol metabolism were detected in these ANME-1 datasets \[[@B40], [@B41], [@B49]\] ([Table 2](#tab2){ref-type="table"}), indicating that this alternative pathway probably does not occur. The presence of the Fae/Hps fusion protein in ANME-1 during AOM could also be explained by its involvement in ribose phosphate synthesis and not in AOM \[[@B52]\]. Indeed, the Fae gene domains of ANME-1 were located in between ribulose-phosphate binding barrel and ribosomal protein S5 domains (Table S2). The third possibility of a structural analogue is most likely since an analogue of N^5^,N^10^-methylene tetrahydrofolate (H~4~HF) reductase (MetF) was expressed by ANME-1 during AOM which could replace Mer \[[@B40]\] ([Figure 6](#fig6){ref-type="fig"}). 2.2. Methyl Coenzyme M Reductase (Mcr) {#sec2.2} -------------------------------------- The enzymatic reaction of a purified Mcr from ANME has not been measured to date. The key question is whether a methanogenic Mcr can explain the observed in situ AOM rates or if the Mcr of ANME is structurally altered. There are three main factors to be considered: the kinetic limitations as defined by enzyme properties (i.e., half-maximal activity at a specific *K* ~*M*~ value), the thermodynamic constraints of the enzymatic reaction, and the maximal or ambient rate of the enzymatic reaction. For the Mcr from*Methanothermobacter marburgensis*, kinetic parameters have been determined to illustrate the reversibility of reaction ([1](#EEq1){ref-type="disp-formula"}). In the methanogenic reaction, the purified Mcr isoform I \[[@B53]\] catalyzes the production of methane with a *V* ~max~ of 30 U mg^−1^ and a *K* ~*M*~ of 5 mM for CH~3~-CoM. The same (methanogenic) enzyme was able to oxidize methane to CH~3~-CoM with a rate of 0.0114 U mg^−1^ and a *K* ~*M*~ for methane of \~10 mM \[[@B54]\]. To answer if the observed AOM rates are in accordance with the measured methane oxidation rate for the purified Mcr enzyme from*M. marburgensis*, the Mcr activity during AOM is needed. Estimates for AOM rates in terms of activity (per cell dry mass) range between \<1 and 20 mmol day^−1^ and g cell dry mass^−1^ \[[@B54]--[@B64]\]. This equals an activity of 0.7 to \~14 nmol min and mg cell dry mass^−1^. About half of the cell dry mass is protein, so the activity for the ANME archaea would approximate 1.4 to 28 nmol min and mg protein^−1^. To estimate the activity per mg of Mcr, the proportion of Mcr to cellular protein is needed. It was reported that 7% of the protein of ANME microbial mats from the Black Sea \[[@B65], [@B66]\] and 10.4% of peptides from Hydrate Ridge mesocosms is Mcr \[[@B49]\]. As these were no pure cultures, the actual percentages of Mcr in ANME cells may be higher. Transcriptome data for ANME-2d \[[@B43]\] showed that about 19% of the total transcripts were derived from the*mcr* genes indicating (though not demonstrating) a high Mcr content in ANME-2d. Estimating that 10% of the cellular protein would be Mcr, the specific activity of the enzyme would be between 14 and 280 nmol min and mg Mcr protein^−1^, which is up to 25 times higher than the measured reverse reaction rate of the*M. marburgensis* enzyme (\~12 nmol min and mg Mcr^−1^ \[[@B54]\]). However, the reverse reaction rate of the*M. marburgensis* Mcr was determined under nonsaturating substrate conditions and was therefore not possibly representing the true maximum rate. Nevertheless, both reverse reaction rates are in the same order of magnitude, other than the forward reaction of 30,000--100,000 nmol min and mg Mcr^−1^. Thus, it seems that the Mcr in ANME may have similar catalytic properties as the methanogenic enzyme and that the high amount of Mcr per mg total cell biomass in ANME may in part compensates for the apparently relatively slow catalysis. Considering the thermodynamic constraints, the Gibbs free energy change of the Mcr forward reaction under standard conditions is around −30 kJ mol^−1^ \[[@B54]\]. Therefore, the reverse reaction is endergonic under standard conditions and will not proceed. However, high methane concentrations (10^5^ according to reaction ([1](#EEq1){ref-type="disp-formula"}) \[[@B67], [@B68]\]) may lead to a favourable change in the Gibbs free energy in the direction of AOM. High methane partial pressure prevails at many habitats where AOM has been detected. The solubility of methane at atmospheric pressure is only 1.3 mM \[[@B69]\]. Consequently, increased AOM rates were reported upon pressurizing samples of different geographical origin \[[@B57], [@B58], [@B70], [@B71]\]. The *K* ~*M*~ of Mcr of*M. marburgensis* for methane was determined at or above 10 mM and reported *K* ~*M*~ values of S-AOM varied from (at least) 1.1 mM \[[@B72]\], a few mM \[[@B55]\], to even 37 mM (equivalent to 3 MPa CH~4~) \[[@B56]\]. Thus, high pressure and therefore high concentrations of methane in the natural habitat accelerate the oxidation rate of methane by Mcr. Future research to accurately determine *K* ~*M*~ values and rates for Mcr at different methane partial pressures is however needed. This may seem difficult, but microbial activity measurements at in situ methane partial pressure were shown to be successful in the laboratory \[[@B73]\]. It was suggested that the Mcr reaction is the rate limiting step in reverse methanogenesis \[[@B54]\] which is in line with the above described challenges. Supporting these findings, there does not seem to be a major change in the amino acid structure that determines whether the backwards or the forward reaction of Mcr is favoured. Amino acid alignments \[[@B65]\] and the crystal structure of ANME-1 Mcr \[[@B74]\] indicated high overall similarity of the methanogenic and methanotrophic enzyme and unambiguously demonstrated that CoM-SH and CoB-SH are substrates of the methanotrophic enzyme. However, several posttranslational modifications of amino acids were different between methanogens and ANME archaea, and the cofactor F~430~ (the prosthetic group of Mcr) is modified in ANME-1, but not in ANME-2 or ANME-3 archaea \[[@B49], [@B61], [@B65], [@B66], [@B74], [@B75]\]. Furthermore, ANME-1 seems to lack the noncatalytic protein D domain of the*mcr*gene that is present in all other methanogens and methanotrophs but of which the function is unknown (IPR003901, [Table 2](#tab2){ref-type="table"}) \[[@B49]\]. A metabolically engineered*Methanosarcina acetivorans*was able to convert methane and CO~2~ to acetate with a plasmid containing Mcr derived from ANME-1 \[[@B76]\]. It is thus unclear if only thermodynamic constraints and the abundance of Mcr ensure AOM activity, or if also specific modifications can have an effect on the reverse activity of Mcr. 2.3. Methane Oxidation by Methanogens {#sec2.3} ------------------------------------- Pure cultures of methanogens were not able to oxidize methane under high methane and low hydrogen concentrations (reviewed in \[[@B77], [@B78]\]). Methanogens are only able to oxidize methane during net methane production \[[@B79]\]. Labeled methane addition (^13^C or ^14^C) to pure cultures of methanogens showed production of labeled CO~2~ during net methane production. This characteristic was confirmed with several pure cultures of methanogens \[[@B80]--[@B82]\]. The process was called "trace methane oxidation" (TMO), since the CO~2~ was formed in trace amounts relative to the methane produced \[[@B81]\]. It is not clear if TMO is due to the reported reversibility of individual enzymes \[[@B64]\], or if it is an active microbial process from which energy can be conserved. TMO was speculated to be an active metabolic process for three reasons: (1) the amount of methane oxidized varied between different species of methanogens grown on the same methanogenic substrate; (2) the amount of methane oxidized varied between different methanogenic substrates; and (3) TMO products varied between different methanogenic substrates \[[@B81], [@B82]\]. For instance, when grown on acetate,*Methanosarcina acetivorans* produced labeled acetate from labeled methane. When grown on carbon monoxide, it produced both labeled acetate and methyl sulfides from labeled methane \[[@B82]\]. During hydrogenotrophic and methylotrophic methanogenesis, TMO mainly produced CO~2~ from labeled methane \[[@B81]\]. However, in contrast with AOM, TMO rates never exceeded methanogenesis rates, even during long-term incubation with methane and sulfate \[[@B83]\]. It seems that methanogens are not able to conserve energy from TMO, even under thermodynamically favourable conditions. TMO occurs both in absence and presence of an external electron acceptor and only during net methanogenesis. It is therefore most likely caused by the reported back flux of individual enzymes of the methanogenic pathway \[[@B64]\]. TMO also occurred in granular sludge and in freshwater and terrestrial samples. These mixed communities showed higher TMO rates than pure cultures, reaching up to 90% of the methane produced \[[@B27], [@B83]--[@B85]\]. TMO should therefore be carefully considered in the experimental setup and interpretation of results when studying AOM in environmental samples, especially since TMO rates were, like AOM, stimulated by a high methane partial pressure \[[@B70], [@B84], [@B87]\]. Sulfate reduction was also stimulated by higher methane partial pressures \[[@B83]\]. Thus, a high methane partial pressure can have a stimulating effect on methane oxidation (either through AOM or TMO) and SR, which could be unrelated to S-AOM. Moreover, addition of iron sulfate (FeSO~4~) or manganese oxide (MnO~2~) also increased TMO rates \[[@B84]\]. Therefore, methane-dependent SR and sulfate- or metal-dependent methane oxidation are not necessarily indications for AOM in mixed cultures. In conclusion, when studying complex "black-box" communities, only net methane oxidation is proof for AOM activity. 2.4. Methane Production by ANME {#sec2.4} ------------------------------- The process of S-AOM is at the energetic limit for sustaining life, with estimates of Gibbs free energy yields between −18 and −35 kJ mol^−1^ \[[@B45], [@B77], [@B88]--[@B90]\] and doubling times between 1.1 and 7.5 months \[[@B63], [@B70], [@B71], [@B91], [@B92]\]. Since S-AOM operates close to its thermodynamic equilibrium, the reversibility of individual enzymes leads to measurable back flux, producing methane (3--7% of AOM) and sulfate (5.5--13% of SR) during S-AOM \[[@B64]\]. This "trace methane production" was observed in situ \[[@B11]\] and in sediment slurries, with methanogenesis around 10% \[[@B60], [@B93]\] or even as high as 50% of AOM \[[@B34]\]. When sulfate becomes depleted, Gibbs free energy yields become even lower (less negative) and the enzymatic back flux becomes even more apparent, up to 78% of net AOM \[[@B94]\]. Previous measurements of ^13^C depletion below the sulfate-methane transition zone (SMTZ) of marine sediments that were thought to be indicative for methanogenesis could therefore instead be attributed to the back flux of AOM \[[@B94]\]. The occurrence of ANME-1 without a bacterial partner in sediment layers where sulfate was depleted was previously interpreted as evidence that ANME-1 perform methanogenesis \[[@B24]\], but in light of the above it could also indicate AOM. There are indeed reports of AOM activity below the SMTZ in the methanogenic zone \[[@B95]--[@B98]\]. In contrast, AOM with other electron acceptors than sulfate operates far from the thermodynamic equilibrium with Gibbs free energy changes between −517.2 and −841.4 kJ mol methane^−1^ ([Table 1](#tab1){ref-type="table"}). Here, the reported anaerobic back flux \[[@B64]\] is expected to be less apparent. In laboratory incubations, researchers were not able to stimulate net methanogenesis through addition of methanogenic substrates to AOM performing sediments \[[@B60], [@B93]\]. In two cases, researchers were successful \[[@B23], [@B99]\]. In one of these cases, sediment-free long-term AOM enrichments that were dominated by ANME/SRB were incubated with methanogenic substrates. The resulting methanogenic activity most likely came from the enrichment of a minor population of methanogens (up to 7‰ of total archaeal gene tag sequences) that was present in the inoculum \[[@B99]\]. In the second study, methanogenic substrates were added to ANME-1 and ANME-2 dominated microbial mat samples and methanogenesis also occurred \[[@B23]\]. However, no information was provided for the total archaeal community composition, which makes it impossible to exclude methanogens as the responsible organisms. Genomic information of ANME also gives indication on potential methanogenic routes. Considering methylotrophic methanogenesis, no gene homologues catalyzing methyl transfer from methylated substrates to coenzyme M were detected in ANME ([Table 2](#tab2){ref-type="table"}) \[[@B40]--[@B43]\]. Acetoclastic methanogenesis needs either the AMP- and ADP-forming acetyl-coenzyme A synthetase (Acs and Acd, resp.) or proceeds via acetate kinase and phosphotransacetylase. In ANME-1, only the alpha subunit of a homologue of Acd was expressed during AOM \[[@B41]\], but in an ANME-1 proteome of active AOM biomass, no Acd was detected \[[@B40]\]. The Acd gene was detected in the single-aggregate genome and transcriptome of ANME-2a \[[@B42]\] and in ANME-2d \[[@B43]\]. However, gene domains for Acd are also present in methanogens unable to use acetate as substrate ([Table 2](#tab2){ref-type="table"}) and are probably used for lipid metabolism. In hydrogenotrophic methanogenesis, hydrogenases are used to replenish reduced coenzyme B and to recycle oxidized F~420~ (discussed in [Section 2.1](#sec2.1){ref-type="sec"}). Both the cytoplasmic Mvh complex and the membrane bound Vho were not present in ANME-2d \[[@B43]\] and not expressed in ANME-2a (which also lacked Ech and F~420~-dependent hydrogenase (Frh)) \[[@B42]\], making hydrogenotrophic methanogenesis unlikely. In ANME-1, both the cytoplasmic HdrABC and MvhD are present, as well as homologues of Frh and Ech, but these were lacking catalytic subunits \[[@B40], [@B41]\]. An iron hydrogenase was found in both ANME-1 metagenomes but not in any other methanotroph or methanogen \[[@B41]\] ([Table 2](#tab2){ref-type="table"}). This iron hydrogenase domain is part of a gene that is 70% identical to a \[FeFe\]-hydrogenase of*Dehalococcoides mccartyi*. \[FeFe\]-hydrogenases catalyze reversible H~2~ production and uptake, but these were presumed to have no key function in AOM \[[@B41]\]. However, the gene is part of a gene cluster of three genes containing a 51 kDa NADH: ubiquinone oxidoreductase subunit (Table S3), which could potentially form a complex that generates a proton motive force during hydrogen oxidation. Therefore, hydrogenotrophic methanogenesis by ANME-1 cannot be excluded yet. 3. Respiration during Anaerobic Oxidation of Methane {#sec3} ==================================================== For net AOM to occur, an external electron acceptor is needed which results in a favourable Gibbs free energy change ([Table 1](#tab1){ref-type="table"}). A variety of terminal electron acceptors have been discovered for AOM which will be discussed in Sections 3.1--3.3. 3.1. Sulfate-Dependent AOM {#sec3.1} -------------------------- During sulfate-dependent AOM, electrons are transferred from ANME to the sulfate-reducing bacterial partner. Previous work tried to uncover how electrons were transferred and most compounds that could act as interspecies electron carrier (IEC) were excluded to be involved in AOM, such as methanol, hydrogen, methanethiol, acetate, and carbon monoxide \[[@B55], [@B59], [@B60], [@B93], [@B100]\]. Indications that polysulfide could act as IEC were found, and ANME-2a archaea were thought to perform both AOM and sulfate reduction (SR) \[[@B101]\]. However, in marine seeps, hydrothermal vents, and other nondiffusion based sediments, AOM rates are high and ANME form close associations with SRB in dense aggregates \[[@B1], [@B5], [@B102], [@B103]\]. In these aggregates, the high AOM rates could not be explained by diffusion of an IEC, which made direct interspecies electron transfer (DIET) a more plausible mechanism \[[@B88], [@B104], [@B105]\]. Cellular activities were independent of aggregate type and distance between the syntrophic partners within the aggregate, which is best explained by DIET \[[@B106]\]. DIET is normally achieved using multiheme cytochrome c proteins (MHCs) and conductive pili (i.e., nanowires) which are mainly found in bacteria that donate electrons extracellularly, such as*Geobacter* and*Shewanella* species \[[@B107]--[@B113]\]. Indeed, ANME-2a from seep-sediment samples seem to transfer electrons directly using large MHCs \[[@B106]\], which were found in the metagenome of ANME-2a \[[@B106], [@B114]\]. ANME-1 and the associated bacterial partner also overexpressed genes for extracellular MHCs during AOM \[[@B115]\], which complements previous findings of transcription \[[@B41]\] and translation \[[@B40]\] of ANME-1 related MHC genes. Domain based (meta)genome analysis shows the high abundance of MHC domains in different ANME as compared to methanogens ([Table 2](#tab2){ref-type="table"}). A recently isolated bacterial partner of ANME-1 ("HotSeep-1") also produced cell-to-cell connections using pili-derived nanowires \[[@B115]\], which explain previously detected*Deltaproteobacteria*-related pili genes in an AOM sample dominated by ANME-1 \[[@B40]\]. How ANME use MHCs to donate electrons to the bacterial partner is not yet clear. For ANME-2a, the electrons probably flow from methanophenazine to membrane integrated di-heme cytochromes (cytochrome *b*/*b* ~6~) that transfer the electrons through the S-layer via MHC/S-layer fusion proteins to extracellular MHCs ([Figure 4](#fig4){ref-type="fig"}) (Figure 4 in \[[@B106]\]). Exosortases and archaea-specific archaeosortases are involved in export of cell surface proteins, such as the archaeal S-layer proteins. These transpeptidases recognize specific signal peptides for protein- sorting; that is, archaeosortase A recognizes the protein-sorting signal PGF-CTERM and archaeosortase C recognizes the PEF-CTERM signal \[[@B116]\]. Both ANME-2a and 2d show presence of di-heme cytochromes, archaeosortase A (IPR014522), archaeosortase C (IPR022504), and other exosortase gene domains ([Table 2](#tab2){ref-type="table"}). Moreover, some genes of both ANME-2a and 2d contained both MHC and PGF or PEF-CTERM domains. Lastly, some genes of both ANME-2a and 2d contained both MHC and S-layer domains \[[@B106]\], indicating that these could form the above-stated MHC/S-layer fusion proteins. ANME-1 do not seem to have di-heme cytochromes ([Table 2](#tab2){ref-type="table"}) \[[@B114]\]. PGF related domains (IPR026453 and IPR026371) were present in all ANME but PEF-CTERM (IPR017474) related domains were absent in ANME-1 ([Table 2](#tab2){ref-type="table"}). Moreover, ANME-1 lacked archaeosortase A (IPR014522) and archaeosortase C gene domains (IPR022504), as well as some other exosortases ([Table 2](#tab2){ref-type="table"}). A search in NCBI\'s conserved domains database (CCD, \[[@B117]\]) and the EMBL InterPro database \[[@B118]\] of amino acids sequences of all genes from ANME metagenomes that contain MHC domains showed that ANME-1 did not have any protein-sorting signal or S-layer domains within these genes. In fact, S-layer domains were completely absent in ANME-1 ([Table 2](#tab2){ref-type="table"}). These results imply that ANME-1 do not use di-heme cytochromes for electron transfer to MHCs and do not produce an S-layer ([Figure 6](#fig6){ref-type="fig"}). This implies that ANME-1 use a different mechanism for DIET and could explain the need for less MHCs by ANME-1 ([Table 2](#tab2){ref-type="table"}) and the observed pili-derived nanowires produced by the bacterial partner \[[@B115]\]. The genome of the bacterial partner of ANME-1 ("HotSeep-1") encoded 24*c*-type cytochromes of which 10 were similar to secreted MHCs of*Geobacter sulfurreducens*\[[@B119]\] which also uses pili for DIET \[[@B120]\]. In the case of ANME-2a, it is not clear if pili (i.e., nanowires) were formed during AOM. It was previously thought that electrically conductive pili seemed to be a prerequisite for current production and DIET \[[@B121], [@B122]\], even when syntrophs were closely associated \[[@B123]\] such as within ANME-2/SRB aggregates. However, conductive materials such as granular activated carbon were shown to be able to substitute pili in DIET \[[@B123]\]. Although in previous work conductive materials such as phenazines or humic acids did not seem to stimulate AOM rates \[[@B59]\], in a recent study AOM was decoupled from SR using artificial electron acceptors \[[@B124]\]. This indicates that conductive materials can indeed replace pili and that ANME-2a/b could possibly couple AOM to metal oxide reduction or any other suitable electron acceptor (discussed in [Section 3.3](#sec3.3){ref-type="sec"}). However, it needs to be proven if in ANME-2/SRB aggregates no pili are formed and if the mechanism of DIET is fundamentally different from ANME-1. As for the polysulfide shuttling theory \[[@B101]\], canonical genes for dissimilatory sulfate reduction such as adenylyltransferase (Sat), APS reductase (Apr), and dissimilatory sulfite reductase (Dsr) which are all present in the sulfate-reducing archeon*A. fulgidus* (Table S2) were not found in metagenomes of ANME-1 \[[@B41]\] and ANME-2a (Table S1). The enzymes Sat and Dsr were also not detected in ANME cells using fluorescent immunolabelling \[[@B125]\]. ANME-1 were previously found to encode most proteins for assimilatory sulfate reduction \[[@B41]\]. ANME-2d only harbor gene domains that encode Sat and assimilatory ATP sulfurylase, APS kinase, and sodium/sulfate symporters, which were not present in ANME-2a (Table S1). It is therefore clear that at least ANME-2a cannot donate electrons to sulfate but need to donate electrons to a sulfate-reducing partner. 3.2. Nitrate-Dependent AOM {#sec3.2} -------------------------- Unlike during S-AOM, ANME-2d that perform N-AOM do not need a bacterial partner but transfer electrons directly to a membrane bound nitrate reductase (Nar) \[[@B28], [@B43]\] ([Figure 5](#fig5){ref-type="fig"}). The ANME-2d genomes contain most MHCs found so far in archaea \[[@B106], [@B114]\] ([Table 2](#tab2){ref-type="table"}). Of the 87 proteins that contained a CxxCH binding motif, of which 43 seemed to be true*c*-type cytochromes \[[@B114]\], 23 CxxCH motif-encoding transcripts were expressed during N-AOM \[[@B43]\]. The function of most of these MHCs is not known, but they are likely involved in nitrate reduction since*c*-type cytochromes are capable of operating in the wide range of redox potentials that couple nitrate reduction (*E* ^0′^ (NO~3~ ^−^/NO~2~ ^−^) = +433 mV) and methane oxidation (*E* ^0′^ (CoM-S-S-CoB/CoM-SH+CoB-SH) = −143 mV) \[[@B43]\]. Nitrate as terminal electron acceptor in anaerobic respiration has been found in halophilic and thermophilic archaea \[[@B126]\]. The*nar* gene cluster of "*Ca.* M. nitroreducens" comprises several genes including the catalytic alpha (NarG, molybdopterin) and beta (NarH, iron sulfur cluster) subunit of nitrate reductase \[[@B28], [@B43]\]. The (halo)archaeal nitrate reductase complex was reported to be located at the extracellular side of the cytoplasmic membrane \[[@B127]\] and in most archaea associated with the cytoplasmic membrane via NarM \[[@B128]\]. The "*Ca.*M. nitroreducens" genome does not encode NarM but encodes a TAT signal peptide at the N-terminus of NarG for translocation across the cytoplasmic membrane \[[@B28], [@B43]\]. Interestingly, the NarG and NarH seem to have been acquired from the Proteobacteria via lateral gene transfer \[[@B28]\]. It is not yet clear at which point in the metabolism "*Ca.* M. nitroreducens" conserves energy. During reverse methanogenesis, N^5^-methyl-H~4~MPT:CoM methyltransferase (Mtr) dissipates the sodium ion potential across the cytoplasmic membrane so subsequent steps in N-AOM have to be coupled to the build-up of a proton or sodium motive force to make the overall process energetically favourable. The analysis of an environmental genome \[[@B43]\] indicated presence of several protein complexes involved in electron transport and energy conservation. Electrons that enter the respiratory chain could be transported by membrane-integral electron carriers (i.e., menaquinones) to a Rieske-cytochrome*b* complex that may use cytochrome*c* as electron acceptor. This in turn may be the electron donor for the unusual nitrate reductase complex. Energy conservation is thermodynamically and mechanistically feasible at the F~420~H~2~ dehydrogenase and the Rieske-cytochrome*b*complex ([Figure 5](#fig5){ref-type="fig"}) (Figure 2 in \[[@B43]\]). Further investigations are needed to determine whether nitrate reductase is also involved in energy conservation, but this working hypothesis is strengthened by the presence of cupredoxin, multicopper oxidase domains, and copper centers related to the periplasmic domain of cytochrome*c* oxidase subunit II (HCO II) in ANME-2d (Table S1). Both ANME-2d genomes discussed here are derived from bioreactors where "*Ca.* M. nitroreducens" formed syntrophic cultures with nitrite scavenging bacteria, either with*"Ca.*Kuenenia stuttgartiensis" (anammox bacteria) \[[@B28]\] or*"Ca.*Methylomirabilis oxyfera" (NC10 bacteria) \[[@B31], [@B43]\]. This indicated that ANME-2d could be dependent on those bacteria for nitrite removal. However, in addition to nitrite, "*Ca.* M. nitroreducens" may also produce ammonium during AOM by a pentaheme*c*-type nitrite reductase (NrfAH) encoded in the genome \[[@B43]\] ([Figure 5](#fig5){ref-type="fig"}). In fact, both ANME-2d genomes contain domains for NrfA (IPR003321) and NrfH (IPR017571) (Table S1) implying that both ANME-2d species are not necessarily dependent on a nitrite scavenger during AOM. 3.3. Metal-Dependent AOM {#sec3.3} ------------------------ Evidence for metal-dependent AOM was found in marine sediments \[[@B129]--[@B131]\]. In nonmarine environments, AOM was also hypothesized to be coupled to iron and/or manganese oxide reduction \[[@B26], [@B132]--[@B135]\] or even coupled to the reduction of humic acids \[[@B135], [@B136]\]. However, organisms responsible for metal-dependent AOM were not identified in these studies. It was speculated that JS1 bacteria, methanogenic archaea, and*Methanohalobium*/ANME-3 could be responsible for iron-dependent AOM \[[@B137]\]. Other researchers speculated that either ANME-1 or*Methanococcoides*/ANME-3 together with a bacterial partner were responsible for manganese-dependent AOM \[[@B138]\]. In another study where AOM was decoupled from SR, ANME were not detected, which leaves open the possibility that other archaeal clades besides ANME could perform metal-dependent AOM \[[@B130]\]. It was recently observed that cultures containing ANME-2a and ANME-2c could decouple AOM from SR in the presence of artificial electron acceptors, humic acids, and soluble iron \[[@B124]\], which confirmed previous findings of AOM not connected to SR in ANME dominated samples \[[@B139]\]. This suggests that ANME-2 could also use insoluble metal oxide minerals as electron acceptor during AOM. The MHCs of ANME-2a/b and ANME-2d are larger than those in*Shewanella* and*Geobacter* species \[[@B106]\], which are known to be capable of extracellular electron conduction. It was speculated that both ANME-2d and*Ferroglobus placidus*, of which the latter can perform solid iron reduction, can fold CxxCH motifs into extracellular conductive structures or pili \[[@B114]\]. Many of the MHCs of ANME-2d were not expressed when grown with nitrate (discussed in section 3.2), implying that these are not needed for nitrate reduction \[[@B43]\]. This strengthens the hypothesis that also ANME-2d could couple AOM to reduction of other extracellular electron acceptors than nitrate and even to insoluble metal oxides. Indeed, recent work showed that ANME-2d could be involved in AOM coupled to chromium(VI) reduction \[[@B140]\] ([Table 1](#tab1){ref-type="table"}, reaction (5)) and in AOM coupled to the reduction of soluble iron and insoluble ferrihydrite and birnessite minerals \[[@B141]\] ([Table 1](#tab1){ref-type="table"}, reactions (3) and (4)). ANME-2d could even possibly donate electrons to a bacterial partner: besides nitrate-rich environments, ANME-2d archaea have been found in wells of an aquifer where sulfate and methane concentrations overlap \[[@B142]\]. Moreover, ANME-2d was the only clade detected in sediments of a freshwater lake where S-AOM occurred \[[@B143]\]. Sulfate concentrations in these studies were low, but above 1 mM and thus higher than the lowest reported concentrations for S-AOM to occur \[[@B144], [@B145]\]. Lastly, sequences of ANME-2d were relatively more abundant in freshwater sediments fed with methane and sulfate than in sediments fed with only methane or only sulfate and no N-AOM activity was detected when fed with nitrate and methane \[[@B27]\]. These indications hold promise that direct experimental evidence for sulfate-dependent AOM by ANME-2d could be found in the future. The ANME-1 genome contains fewer MHC gene domains as compared to ANME-2a and ANME-2d and some other archaea, such as some methylotrophic methanogens ([Table 2](#tab2){ref-type="table"}) and some members of the Archaeoglobales \[[@B114]\]. The MHCs of ANME-1 also have a smaller heme count as compared to other ANME and some other archaea, with the largest being an octaheme cytochrome \[[@B106]\]. Each heme within a MHC has its own redox potential and therefore structurally different MHCs represent a large range of redox potentials that can be used for bioenergetic electron transfer (reviewed in \[[@B146]\]). For instance, metal oxide reduction in*Shewanella oneidensis* MR-1 is catalyzed by a chain of a tetraheme (CymA), two decaheme (MtrA and MtrB), and eventually extracellular decaheme cytochromes OmcA/MtrC that reduce the iron minerals \[[@B120], [@B147], [@B148]\]. In*Geobacter sulfurreducens*, iron mineral reduction seems to be catalyzed by the tetraheme cytochrome OmcE and hexaheme cytochrome OmcS transferring electrons from the outer membrane to type IV pili that transmit the electrons to iron minerals \[[@B120], [@B149], [@B150]\]. Since ANME-1 lack MHCs of the correct size and lack gene domains for pili production \[[@B115]\], they seem unable to reduce minerals via both mechanisms present in*Shewanella*and*Geobacter*. It can therefore be speculated that ANME-1 are less versatile in electron acceptor use and are not able to reduce solid metal oxides. However, DIET mechanisms are still not well understood and the true differences between MHCs of different ANME types need to be investigated using biochemical methods. This would allow uncovering the true capabilities concerning DIET. 3.4. Menaquinones and Methanophenazines {#sec3.4} --------------------------------------- ANME-2a encoded a protein with a domain specific for PhzF, which is an enzyme involved in phenazine biosynthesis in*Pseudomonas fluorescens* \[[@B151], [@B152]\]. The respective gene is not present in all methanophenazine containing methanogens (in our genome comparison only in*M. acetivorans* and*M. formicica*, [Table 2](#tab2){ref-type="table"}) so it is unclear whether this enzyme is really involved in methanophenazine biosynthesis. It is however likely that ANME-2a use methanophenazines in their respiratory chain since gene domains for menaquinone biosynthesis were absent ([Table 2](#tab2){ref-type="table"}). "*Ca*. M. nitroreducens" probably uses menaquinones as membrane-integral electron carrier since environmental genomes \[[@B28], [@B43]\] encoded the futalosine (mqn) biosynthesis pathway as reported for*Archaeoglobus fulgidus*\[[@B153]\] ([Table 2](#tab2){ref-type="table"}). Moreover, "*Ca*. M. nitroreducens" enrichment cultures showed absence of methanophenazines \[[@B43]\]. ANME-1 also contained gene domains for menaquinone biosynthesis via the futalosine (mqn) biosynthesis pathway ([Table 2](#tab2){ref-type="table"}). Indications for a quinone biosynthesis pathway in ANME-1 were previously found to be weak since only some of the Ubi homologues of the oxic ubiquinone biosynthesis pathway were detected \[[@B41]\], but the futalosine (mqn) biosynthesis pathway was overlooked in that particular analysis. Additionally, ANME-1 have Fqo homologues similar to*Archaeoglobus fulgidus*\[[@B44]\] and expressed the catalytic subunit FqoF \[[@B41]\]. However, since the phenazine biosynthesis domain PhzF was also present in the ANME-1 genomes ([Table 2](#tab2){ref-type="table"}) and Fpo and Fqo are homologues, we cannot conclude on genomic information alone which redox shuttle is used by ANME-1 during AOM. If ANME-1 would use menaquinones, this would have implications for subsequent electron transfer to MHCs since methanophenazine (*E* ^0^ = −165 mV) \[[@B154]\] and menaquinone (*E* ^0^ = −80 mV) \[[@B155]\] have different redox potentials. 3.5. Cell Adhesion {#sec3.5} ------------------ Some gene domains involved in cellular adhesion were more abundant in ANME than in methanogens ([Table 2](#tab2){ref-type="table"}), especially in ANME-2a and ANME-1 that are known to form syntrophic interactions for electron transfer during respiration. These domains include HYR (IPR003410) and CARDB domains (IPR011635) that both have a direct role in cellular adhesion \[[@B156]\] ([Table 2](#tab2){ref-type="table"}). Interestingly, also domains related to the cellulosome of*Clostridium* species, termed dockerin and cohesin, were highly abundant in both ANME-1 and ANME-2a as compared to ANME-2d and methanogens, together with many carbohydrate binding domains ([Table 2](#tab2){ref-type="table"}). In*Clostridium* species, dockerin and cohesin form an anchor to the bacterial cell wall that contains a scaffold with cellulose-degrading enzymes and a carbohydrate binding module that binds cellulose, altogether forming the cellulosome \[[@B157]\]. These domains have been found in all domains of life irrespective of cellulose utilization, but the function of such proteins outside of the cellulosome is not known \[[@B158]\]. Therefore, dockerin, cohesin, and carbohydrate binding domains in ANME could hypothetically form a construct that binds to carbohydrates and could possibly have a function in cell-to-cell contact or MHC adhesion, but this needs further investigation. 4. Future Challenges {#sec4} ==================== Advances in (meta)genomics, transcriptomics, and proteomics have produced the valuable metabolic blueprints of different ANME with hypotheses on how central metabolism, electron transport, and energy conservation may function. Future experiments are needed to biochemically demonstrate that these hypotheses are correct. The bottleneck for biochemical studies of ANME is the lack of pure cultures due to the slow and syntrophic growth. However, the recent milestone discovery of direct electron transfer provides opportunities to grow ANME, as stated previously, on electron accepting electrodes (i.e., anodes) \[[@B159]\]. In this way, pure or highly enriched cultures of different ANME without their respective syntrophic partner could be obtained and MHCs responsible for electric conduction could be biochemically characterized. It seems that ANME-1 are limited by the size and abundance of MHCs which could relate to differences in behavior on an anode. The partner bacteria of ANME-1 and ANME-2 could be investigated on an electron donating electrode (i.e., cathode), with specific focus on the ability to produce pili (i.e., nanowires). Also worth investigating is if besides ANME-2d, ANME-2a/b could also use insoluble electron acceptors and if ANME-2d could donate electrons to a bacterial partner. Another future challenge is to isolate and characterize Mcr from different ANME clades. It needs to be investigated if Mcr abundance in ANME cells compensates for the slow reverse activity or if modifications in the Mcr of ANME-1 also contribute to a better reverse activity of Mcr. Moreover, the effect of methane partial pressure on reaction rates and enzyme kinetics needs to be determined in situ. ANME-1 are potentially able to perform hydrogenotrophic methanogenesis due to the presence of a hydrogenase in the genome. Genetic indications of menaquinones as electron carrier in ANME-1 and the various cellulosome and cell adhesion related gene domains in all ANME are also topics that could be explored further. Methanogenic archaea are likely not able to perform AOM, but additional studies on TMO and more genetic modifications to stimulate AOM in methanogens could help in understanding the parameters needed for AOM to occur. Ultimately, physiological understanding of ANME will help to explain the observed ecological niche separation of different ANME clades and the occurrence of ANME without a bacterial partner. This would greatly enhance our knowledge of the methane cycle in anoxic environments. Supplementary Material {#supplementary-material-sec} ====================== ###### This supplementary material contains data on domain based (meta)genome comparison of selected metagenomes of methanotrophs and genomes of selected archaea that belongs to the article "Reverse methanogenesis, and respiration in methanotrophic archaea" by Peer H.A. Timmers, Cornelia U. Welte, jasper J. Koehorst, Caroline M. Plugge, Mike S. M. Jetten and Alfons J.M. Stams. The authors thank Stefanie Berger (RU, Nijmegen) for critical reading of the manuscript. This research is supported by the Soehngen Institute of Anaerobic Microbiology (SIAM) Gravitation grant (024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Organisation for Scientific Research (NWO). Mike S. M. Jetten was further supported by ERC AG 339880 Eco-MoM and Alfons J. M. Stams was supported by ERC AG 323009 Novel Anaerobes. Competing Interests =================== The authors declare that there is no conflict of interests regarding the publication of this paper. ![Phylogenetic tree of full length archaeal 16S rRNA sequences showing all methanotrophic clades so far described (grey) and other archaeal clades used in our domain based (meta)genome comparison (black). The tree was constructed with the ARB software package (version arb-6.0.1.rev12565) \[[@B160]\] using 2800 sequences from the SILVA SSURef NR 99 database (release 119.1) \[[@B161]\]. Trees were calculated by maximum likelihood analysis (RAxML, PHYML) and the ARB neighbor-joining method with terminal filtering and the Jukes-Cantor correction. Resulting trees were compared manually and a consensus tree was constructed. Sulfolobales as outgroup was removed after tree calculations. The scale bar represents the percentage of changes per nucleotide position.](ARCHAEA2017-1654237.001){#fig1} ![Hydrogenotrophic methanogenesis in cytochrome containing*Methanosarcina barkeri*. Black lines represent presence of conversions. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.002){#fig2} ![Methylotrophic methanogenesis in cytochrome containing*Methanosarcina barkeri*. Black lines represent presence of conversions and red lines indicate reversal of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.003){#fig3} ![Methanotrophic pathway during S-AOM by ANME-2a \[[@B42]\]. Red lines indicate reversal of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.004){#fig4} ![Methanotrophic pathway during N-AOM by "*Ca.*M. nitroreducens" MPEBLZ (ANME-2d) \[[@B43]\]. Red lines indicate reversal of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.005){#fig5} ![Methanotrophic pathway during S-AOM by ANME-1 \[[@B40], [@B41]\]. Red lines indicate reversal of the hydrogenotrophic methanogenic pathway, grey lines represent absence of conversions, and blue lines indicate a bypass of the hydrogenotrophic methanogenic pathway. See [Table 3](#tab3){ref-type="table"} for nomenclature.](ARCHAEA2017-1654237.006){#fig6} ###### Gibbs free energy changes under standard conditions (Δ*G* ^0^) for anaerobic methane oxidation coupled to different electron acceptors (possibly) performed by ANME. Reaction Gibbs free energy (Δ*G* ^0^, kJ mol^−1^) -------------------------------------------------------------------------------- ------------------------------------------ $1$ CH~4~ + SO~4~ ^2−^ → HCO~3~ ^−^ + HS^−^+ H~2~O −16.3 $2$ CH~4~ + 4 NO~3~ ^−^ → HCO~3~ ^−^ + 4 NO~2~ ^−^ + H~2~O + H^+^ −517.2 $3$ CH~4~ + 8 Fe(OH)~3~ + 16 H^+^ → CO~2~ + 8 Fe^2+^ + 22 H~2~O −571.2 $4$ CH~4~ + 4 MnO~2~ + 8 H^+^ → CO~2~ + 4 Mn^2+^ + 6 H~2~O −763.2 $5$ CH~4~ + 4/3 Cr~2~O~7~ ^2−^ + 32/3 H^+^ → 8/3 Cr^3+^ + CO~2~ + 22/3 H~2~O −841.4 ###### Domain based (meta)genome comparison of selected metagenomes of methanotrophs and selected genomes of other archaea. Domain abundance in every (meta)genome is indicated by numbers. S-AOM performing ANME include ANME-1-s \[[@B40]\], ANME-1-m \[[@B41]\], and ANME-2a \[[@B42]\]. N-AOM performing ANME include ANME-2d-h \[[@B28]\] and ANME-2d-a \[[@B43]\]. The acetoclastic (A) and methylotrophic (M) methanogens include *Methanosarcina acetivorans*C2A (AM), *Methanosaeta concilii* GP6 (A), *Methanococcoides burtonii* DSM6242 (M-1), *Methanolobus tindarius* DSM2278 (M-2), and *Methanohalophilus mahii*DSM5219 (M-3). Hydrogenotrophic methanogens (H) include *Methanospirillum hungatei* JF-1 (H-1), *Methanobacterium formicicum* DSM3637 (H-2), *Methanococcus maripaludis* C5 (H-3), and *Methanoregula formicica* SMSP (H-4). The sulfate-reducing archaeon (S) is *Archaeoglobus fulgidus* DSM 4304. InterPro ID ANME-1 S ANME-1 M ANME-2a ANME-2d H ANME-2d A AM A M-1 M-2 M-3 H-1 H-2 H-3 H-4 S ---------------------------------------------------------------------- ------------- ---------- ---------- --------- ----------- ----------- ---- ---- ----- ----- ----- ----- ----- ----- ----- ---- *Central pathway* Mcr, alpha subunit, N-terminal IPR003183 4 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, alpha subunit, N-terminal subdomain 1 IPR015811 2 3 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, alpha subunit, N-terminal subdomain 2 IPR015823 2 2 2 2 2 2 2 2 2 2 2 4 2 4 0 Mcr, alpha subunit, C-terminal IPR009047 3 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, alpha/beta subunit, C-terminal IPR008924 6 4 2 2 2 2 2 2 2 2 2 4 2 4 0 Mcr, alpha/beta subunit, domain 2, C-terminal IPR022681 9 6 3 3 3 3 3 3 3 3 3 6 3 6 0 Mcr, beta subunit IPR003179 1 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, beta subunit, C-terminal IPR022679 3 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, beta subunit, N-terminal IPR022680 4 2 1 1 1 1 1 1 1 1 1 2 1 2 0 Mcr, gamma subunit IPR003178 14 8 4 4 4 4 4 4 4 4 4 8 4 8 0 Mcr, protein C IPR007687 2 1 1 1 1 1 1 1 1 1 1 1 1 1 0 Mcr, protein C-like IPR026327 5 2 2 2 2 2 2 1 2 2 2 2 2 1 0 Mcr, protein D IPR003901 0 0 3 6 6 3 3 3 3 3 3 9 3 6 0 Mcr, ferredoxin-like fold IPR009024 12 6 3 3 3 3 3 3 3 3 3 6 3 6 0 5,10-methylenetetrahydromethanopterin reductase IPR019946 0 0 2 1 1 1 1 1 1 1 1 1 1 1 1 *Acetoclastic methanogenesis* CO dehydrogenase/acetyl-CoA synthase complex alpha subunit IPR004460 0 1 1 1 1 3 1 1 1 1 1 1 1 1 2 CO dehydrogenase/acetyl-CoA synthase complex beta subunit IPR004461 13 4 2 2 2 4 4 2 2 2 2 2 2 4 2 CO dehydrogenase/acetyl-CoA synthase delta subunit IPR004486 5 1 1 1 1 2 1 1 1 1 1 1 1 1 1 CO dehydrogenase/acetyl-CoA synthase delta subunit, TIM barrel IPR016041 11 2 5 3 2 4 2 2 3 3 3 2 2 5 2 CO dehydrogenase b subunit/acetyl-CoA synthase epsilon subunit IPR003704 3 2 2 2 2 4 2 2 2 2 2 2 2 2 4 *Methylotrophic methanogenesis* Methyltransferase MtaA/CmuA IPR006360 0 0 0 0 0 7 0 4 5 6 0 0 1 0 0 Methanol-cobalamin methyltransferase, B subunit IPR021079 0 0 0 0 0 3 0 2 2 2 0 0 0 0 0 Monomethylamine methyltransferase MtmB IPR008031 3 0 0 0 0 12 0 12 21 15 0 0 0 0 0 Trimethylamine methyltransferase IPR010426 11 0 0 0 0 4 0 5 2 2 0 0 0 0 0 Dimethylamine methyltransferase MtbB IPR012653 0 0 0 0 0 9 0 6 6 6 0 0 0 0 0 Trimethylamine methyltransferase, *Methanosarcina* IPR012740 0 0 0 0 0 2 0 1 1 1 0 0 0 0 0 Methylamine methyltransferase corrinoid protein reductive activase IPR026339 0 0 0 0 0 4 0 2 2 2 0 0 0 0 0 *C-type cytochromes* Di-haem cytochrome, transmembrane, nitrate reduction IPR016174 2 0 4 3 7 4 0 0 4 0 1 1 0 0 1 Doubled CXXCH motif IPR010177 33 0 3 2 3 0 0 0 0 0 0 0 0 0 0 Cytochrome c-like domain IPR009056 2 0 6 0 17 15 0 4 8 6 0 0 0 0 4 Class III cytochrome C (tetraheme cytochrome) IPR020942 2 0 0 3 3 0 0 0 0 0 0 0 0 0 0 Tetraheme cytochrome domain, flavocytochrome c3 (*Shewanella*) IPR012286 4 0 4 5 4 2 0 0 1 0 1 0 0 0 2 Octaheme c-type cytochrome IPR024673 1 0 2 0 0 0 0 0 2 2 0 0 0 0 1 Methanogenesis multiheme c-type cytochrome IPR027594 0 0 1 0 0 1 0 1 1 1 0 0 0 0 0 Multiheme cytochrome IPR011031 78 15 52 80 73 3 0 6 8 14 0 0 0 1 7 *S-layer domains* S-layer family duplication domain IPR006457 13 0 29 16 26 34 19 16 44 17 0 0 0 0 0 Sarcinarray family protein IPR026476 0 0 6 0 4 1 0 0 2 1 0 0 0 0 0 S-layer homology domain IPR001119 1 0 2 0 0 0 0 0 0 0 0 0 0 0 0 *Cell export and cell adhesion* HYR domain IPR003410 1 5 2 0 0 0 0 0 0 0 0 0 0 0 0 CARDB domain IPR011635 64 14 15 6 6 30 4 1 4 0 9 0 0 3 8 Collagen-binding surface protein Cna-like, B-type domain IPR008454 4 1 2 1 8 0 6 0 2 2 0 0 0 0 0 von Willebrand factor, type A IPR002035 55 28 17 32 14 37 23 17 42 9 28 8 0 27 7 VWA N-terminal IPR013608 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 Adhesion lipoprotein IPR006128 8 4 4 5 0 0 5 3 0 4 4 5 0 0 0 Adhesin B IPR006129 0 3 4 5 0 0 3 0 0 4 0 3 0 0 0 Invasin/intimin cell-adhesion fragments IPR008964 17 0 3 2 6 7 0 2 7 0 1 2 0 2 2 Putative cell wall binding repeat 2 IPR007253 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 Archaeosortase A IPR014522 2 0 1 2 2 1 1 1 1 1 1 0 0 2 1 Archaeosortase B IPR026430 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 Archaeosortase C IPR022504 0 0 1 2 1 0 0 1 1 0 0 0 0 0 0 Exosortase/archaeosortase domain IPR026392 5 0 2 5 3 1 1 2 2 2 1 0 1 2 1 Exosortase, EpsH IPR013426 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 Exosortase EpsH-related IPR019127 4 2 2 3 2 1 1 2 1 1 1 0 1 2 1 Archaeosortase family protein ArtE IPR026485 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 Cell wall hydrolase/autolysin, catalytic IPR002508 2 2 1 0 0 0 0 0 0 0 0 0 0 1 0 PEF-CTERM protein sorting domain IPR017474 0 0 3 4 1 0 0 10 19 0 0 0 0 0 0 PGF-pre-PGF domain IPR026453 1 1 18 9 8 24 0 7 21 11 0 0 0 1 1 PGF-CTERM archaeal protein-sorting signal IPR026371 9 4 6 2 1 2 0 2 1 2 1 0 0 1 3 LPXTG cell wall anchor domain IPR019931 3 0 1 0 0 0 1 1 0 0 0 0 0 0 0 VPXXXP-CTERM protein sorting domain IPR026428 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 *Cellulosome-related/Dockerin* Cellulosome anchoring protein, cohesin domain IPR002102 80 77 52 4 12 2 0 4 15 3 1 1 0 0 4 Dockerin domain IPR016134 149 112 44 4 6 5 17 2 4 0 9 0 0 0 2 Dockerin type I repeat IPR002105 1 0 5 0 0 0 0 0 0 0 0 0 0 0 1 *Carbohydrate-binding domains* Carbohydrate-binding domain IPR008965 50 42 39 2 6 2 0 3 13 2 3 0 1 0 2 Carbohydrate-binding-like fold IPR013784 14 5 3 10 19 0 0 0 0 1 3 0 0 6 1 Carbohydrate-binding, CenC-like IPR003305 1 0 7 0 17 0 0 0 0 0 0 0 0 0 0 Carbohydrate-binding/sugar hydrolysis domain IPR006633 27 22 5 2 2 27 3 7 8 8 0 5 2 1 8 Carbohydrate-binding domain, family 9 IPR010502 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 Bacteroidetes-associated carbohydrate-binding often N-terminal IPR024361 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Carbohydrate binding module, xylan-binding domain IPR031768 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 Galactose-binding domain-like IPR008979 12 1 17 6 39 4 4 2 1 3 0 0 0 0 0 *Menaquinones* 3-Demethylubiquinone-9 3-methyltransferase IPR028973 0 0 0 3 3 0 0 0 0 0 0 1 0 0 0 Succinate dehydrogenase/fumarate reductase, flavoprotein subunit IPR014006 0 0 1 2 1 0 0 0 0 1 0 0 0 0 1 UbiE/COQ5 methyltransferase IPR004033 3 0 0 2 2 1 0 1 0 0 0 0 0 0 0 UbiE/COQ5 methyltransferase, conserved site IPR023576 3 0 0 2 2 0 0 0 1 0 0 0 0 0 0 NrfD family IPR005614 3 0 0 4 3 0 0 0 0 0 0 0 0 0 3 Futalosine hydrolase IPR019963 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 Cyclic dehypoxanthine futalosine synthase IPR022431 1 1 0 1 1 0 0 0 0 0 0 0 0 0 1 Aminodeoxyfutalosine synthase IPR022432 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 Menaquinone synthesis (chorismate dehydratase & naphthoate synthase) IPR003773 8 6 0 2 2 0 0 0 0 0 0 0 0 0 2 FO synthase, subunit 2 IPR020050 3 3 1 3 3 2 1 2 1 2 2 2 2 2 3 *Phenazines* Phenazine biosynthesis PhzF protein IPR003719 1 1 4 0 0 2 0 0 0 0 0 0 0 2 0 ###### Nomenclature. -------------------------------- --------------------------------------------------------------------------------- *Central methanogenic pathway* Fmd Formylmethanofuran (CHO-MFR) dehydrogenase Ftr Formylmethanofuran-tetrahydromethanopterin (H~4~MPT) formyltransferase Mch N^5^,N^10^-methenyl-H~4~MPT cyclohydrolase Mtd F~420~H~2~-dependent methylene -H~4~MPT dehydrogenase Mer N^5^,N^10^-methylene-H~4~MPT reductase Mtr N^5^-methyl-H~4~MPT:coenzyme M (CoM) methyltransferase Mcr Methyl coenzyme M (CH~3~-CoM) reductase Mdh Methanol dehydrogenase Fae/Hps Fusion protein of formaldehyde activating enzyme/ hexulose-6-phosphate synthase MetF N^5^,N^10^-methylene tetrahydrofolate (H~4~HF) reductase analogue *Electron transport* Mvh F~420~-nonreducing hydrogenase Vho Methanophenazine-dependent hydrogenase Fpo F~420~H~2~:phenazine oxidoreductase Fqo F~420~H~2~:quinone oxidoreductase Hdr Coenzyme B-coenzyme M heterodisulfide (CoB-S-S-CoM) reductase Frh F~420~-dependent hydrogenase Ech Ferredoxin-dependent hydrogenase MePh/MePhH~2~ Methanophenazine MQ/MQH~2~ Menaquinone Cyt *b* Cytochrome *b* Cyt *c* Cytochrome *c* MHC Multiheme *c*-type cytochrome Rieske Rieske cytochrome *b* complex Orf7 Pseudoperiplasmic *b*-type cytochrome Nar Nitrate reductase Nap Periplasmic nitrate reductase Nrf Nitrite reductase -------------------------------- --------------------------------------------------------------------------------- [^1]: Academic Editor: Michael W. Friedrich

{ "pile_set_name": "PubMed Central" }

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"Nitrite", "reductase", "Academic", "Editor", "Michael", "Friedrich" ]

Background {#Sec1} ========== It is well recognized that patients with end stage renal disease (ESRD) requiring intermittent hemodialysis (HD) suffer excess cardiac morbidity and mortality \[[@CR1]\]. There are disproportinaltely higher rates of heart failure and cardiac arrhythmia \[[@CR2]\] in this population and cardiovascular disease remains the leading cause of mortaliy. In addition to the traditional cardiovascular risk factors, intra-dialytic hypotension (IDH) may be a key contributor to myocardial injury, and repeated injury during routine dialysis further precipitate progression of cardiovascular deterioration. For instance, previous investigations have found evidence of reduction in myocardial blood flow during dialysis, resulting in ischemia and myocardial stunning. Over time, such repeated injury can lead to ventricular remodeling that may further contribute to hemodynamic instability and dysregulation \[[@CR3], [@CR4]\]. Intra-dialytic hypotension is one of the most common complication of HD treatment, occurring in approximately 20--30% of HD sessions \[[@CR5]\]. Clinically, IDH often leads to premature cessation of dialysis sessions, or decreased fluid removal, both of which not only decrease dialysis efficiency and effect, but also leads to decreased quality of life. Furthermore, IDH has been associated with other medical complications such as vascular access thrombosis, and mesenteric ischemia \[[@CR6]\]. Current treatment strategies for IDH are aimed to address the rate of fluid shift through altering the ultrafiltration rate and dialysate electrolyte concentrations. Vasoconstrictive medications such as midodrine are commonly used with some success, however, effect tends to diminish over time and their safety has been called into question \[[@CR7]\]. Novel strategies are needed to further address this issue. One such approach to the management of IDH is mechanical augmentation via the application of lower limb compression stockings during dialysis \[[@CR8]--[@CR10]\]. These devices increase venous return and contributes to additional intravascular volume, preload, and in turn, cardiac output. In particular, the non-pneumatic anti-shock compression garments have been used since the early 1900s to manage shock \[[@CR11]\]. Benefit has been demonstrated in patients with hypovolemic shock, such as traumatic injury, abdominal aortic aneurysm rupture, and postpartum hemorrhage; and in distributive shock, such as in the setting of anaphylaxis \[[@CR11]\]. In addition to improving cardiac pre-load, when applied to the abdomen, the anti-shock garments may exert hormonal effects via sympathetic stimulation, and further improve blood pressure \[[@CR12]\]. In the dialysis population, the benefit of compression stockings is conflicting. Two studies have examined the benefit of pneumatic compression stockings: a randomized crossover study by Tai et al. did not find significant benefit \[[@CR13]\], while Alvares et al. \[[@CR14]\], also using a randomized crossover design, did find a reduction in IDH with the use of pneumatic compression in the first hour of dialysis. However, the use of non-pneumatic devices remains un-explored. In contrast to pneumatic compression stockings which provide thigh high intermittent compression, non-pneumatic anti-shock compression garments apply continuous pressure to not only the lower limbs but also upper thigh and abdomen. The ZOEX Non-Inflatable Anti-Shock Garment (NASG) is a specific type of anti-shock garment originally developed from NASA anti-G-force technology which received FDA device approval for management of haemorrhagic shock in 1991. They are a lightweight neoprene garment that is made up of six segments that close tightly with Velcro. This garment is worn on the lower extremities (legs) and applies 20--40 mmHg pressure to the lower body and abdomen. The utility of NASG to raise systemic blood pressure is already well established through work done on obstetrical haemorrhages in remote locations. Two independent groups working in Pakistan report a marked elevation in systolic blood pressure within minutes of application of NASG to patient suffering from obstetrical haemorrhage \[[@CR15], [@CR16]\]. Later prospective work by Miller et al. demonstrated a decrease in both blood loss, morbidity and mortality in patients with postpartum bleeding using NASG \[[@CR17]--[@CR19]\]. Furthermore, this work demonstrated that the use of NASG over extended periods is safe. Over a 36-h period of continuous use, no adverse events were reported in the study group \[[@CR17]--[@CR19]\]. The immediacy in which the Zoex NASG modulates hemodynamic variables combined with the lack of adverse effects even over extended use makes this device promising for use in the dialysis setting. The proven benefit in other settings, the relatively low cost, and the ease of application, make these devices a worthwhile option for further exploration. We therefore conducted this pilot study to examine the effects of non-pneumatic compression garments on individuals with varying degree of renal dysfunction. Through this small and focused trial. We aim 1) to assess the tolerability of these garments, 2) to examine the hemodynamic effects of these compression garments through continuous hemodynamic monitoring using the Finapres technology, and 3) to assess the feasibility for potential larger scale clinical trials. Methods {#Sec2} ======= Study design {#Sec3} ------------ In this prospective, interventional, pilot study, non-pneumatic anti-shock compression garments were applied to individuals with varying degrees of renal disease. Three groups of participants were analyzed: 1) Healthy participants with no known kidney disease which served as a control group, 2) participants with stage 4 or 5 chronic kidney disease (CKD) not receiving dialysis, and 3) individuals with end stage renal disease currently receiving intermittent hemodialysis. Participants {#Sec4} ------------ Five patients were recruited for each arm of the study. Healthy participants were recruited via poster board advertisement at London Health Science Centre (LHSC), Victoria Campus, London, Ontario, while CDK patients were recruited from the LHSC CKD clinic based at a community dialysis site. Dialysis patients were recruited from the Victoria Hospital dialysis unit population, also at LHSC. Inclusion and exclusion criteria {#Sec5} -------------------------------- All participants were required to be over the age of 18 and were of both sexes. Healthy participants had no clinical diagnosis of CKD. Non-dialysis CKD patients were required to have diagnosed stage 4 or 5 CKD but were not receiving and had never had any form of dialysis. Dialysis patients were required to be undergoing active intermittent hemodialysis at the time of the study protocol. Participants were excluded if they had New York Heart Association Class IV heart failure, absence of lower limbs or lower limb injury, or individuals with established peripheral vascular disease or clear symptoms of claudication. Additionally, patients who declined participation, were pregnant, or did not meet inclusion criteria were excluded. Compression garments {#Sec6} -------------------- ZOEX Non-Inflatable Anti-Shock Garment (NASG), supplied by ZOEX NIASC, Coloma, CA, USA were used for all participants of this study. They are a compression garment consisting of 6 segments that close tightly with Velcro that applies a continuous pressure of 20--40 mmHg to the lower limbs and abdomen. Finometer {#Sec7} --------- The use of the Finometer (Finapres Medical Systems, Arnhem, Netherlands) in dialysis patients has been described in detail elsewhere \[[@CR20]\] and was used to monitor a variety of hemodynamic parameters, including cardiac output and mean arterial pressure, during the study protocol. Study protocol {#Sec8} -------------- On the day of the study visit, participants were subjected to a basic history review and a baseline set of routine vitals were taken to calibrate the Finometer. Patients were weighed, and their current weight was compared against their ideal weight as an estimate of fluid balance (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Participants were laid supine on an examination table with the Finometer attached and without compression garments on. Baseline data for each participant was collected for a total of 15 min. Five minutes into the session a baseline echocardiogram was performed. Following this first interval, participants donned the compression garments and were again laid supine on an examination table. Data was again collected for 15 continuous minutes with an echocardiogram being performed 5 min after application of the compression garments. For dialysis patients, the study visit occurred on a day they would normally receive dialysis, but before their dialysis session. Regarding statistical analysis {#Sec9} ------------------------------ This pilot study was designed to characterize the hemodynamic of patients with varying degrees of renal dysfunction in the presence and absence of lower body compression. This study was not sufficiently powered to show an effect on cardiac output or mean arterial pressure with the application compression garments and as such a statistical analysis to that end was not performed. Descriptive and summary statistics were computed with Microsoft Excel 2018. Results {#Sec10} ======= Patients {#Sec11} -------- Baseline characteristics for study participants are presented in Table [1](#Tab1){ref-type="table"}. Significant heterogeneity was observed between individuals within groups, particularly with regards to their medications and comorbidities. Healthy participants were significantly younger than both CKD and dialysis participants and represent a cardiovascularly healthy optimal control. Table 1Baseline characteristics for study participants, each arm contained five patientsGroupDialysis (*n* = 5)CKD (*n* = 5)Healthy (*n* = 5)Average Age66.374.446.8Causes for CKD (n) Diabetes Mellitus41N/ABaseline SBP (mmHg)136.5155.8135.8Baseline DBP (mmHg)83.581.488.4Baseline HR (bmp)94.57372.6Medications (n) Beta-Blocker120 Calcium Channel Blocker130 Diuretic020 ACE inhibitor/ARB010 MRA010Cardiovascular Comorbidities (n) CAD100 Hypertension440 Atrial Fibrillation400Note: *CKD* Chronic kidney disease, *SBP* Systolic blood pressure, *DBP* Diastolic blood pressure, *HR* Heart rate, *ACE* Angiotensin converting enzyme, *ARB* Angiotensin receptor blocker, *MRA* Mineralocorticoid receptor antagonist, *CAD* Coronary artery disease Hemodynamic response {#Sec12} -------------------- Figure [1](#Fig1){ref-type="fig"} illustrates the continuous cardiac output of a representative individual from each group over the course of the study procedure. Mild fluctuations in cardiac output are seen throughout in each patient, however a stable baseline is readily apparent. No obvious or reproducible changes in cardiac output were observed over the 15-min interval in which the compression garments were worn, indicating that any hemodynamic effect caused by application of the garments was immediate and sustained. Similar scatter plots were generated for each study participant for several hemodynamic parameters. Individual responses to the compression garments were quite variable (Fig. [2](#Fig2){ref-type="fig"}, Table [2](#Tab2){ref-type="table"}) and no clear or significant trend was observed in any group with the exception of CKD participants who uniformly experienced an increase in mean arterial pressure when wearing the compression garments. Fig. 1Representative data series of cardiac output from individual patients of each cohort. Baseline characteristics in approximately 15-min intervals displayed in orange. Application of non-pneumatic compression stocks occurred subsequently, and hemodynamic response is shown in blue Fig. 2Hemodynamic response of patients with kidney disease to non-pneumatic compression stockings. The relative change in cardiac output, mean arterial pressure and global longitudinal strain after the application of non-pneumatic compression stockings in heathy individuals, CKD patients not on dialysis, and dialysis patients are displayed. Cardiac output and mean arterial pressure data were collected via Finometer while global longitudinal strain was obtained via echocardiography Table 2Hemodynamic response to non-pneumatic compression stockings. Hemodynamic parameters from healthy individuals (H), CKD patients (C) and participants receiving hemodialysis (D) are displayed. Data is related as a percent change from baseline (without compression garments)GroupHeart RateStroke VolumeCardiac OutputPeripheral ResistanceSystolic PressureMean Arterial PressureDiastolic PressureH1−0.9%−1.7%−2.7%5.8%1.0%1.3%2.7%H2−3.3%20.2%16.8%−13.1%9.6%6.1%2.7%H31.1%−1.5%2.5%4.7%1.5%0.4%0.5%H4−8.4%−3.2%−4.1%14.5%3.2%4.3%6.1%H54.2%−13.0%−9.4%21.8%2.8%5.3%8.1%Mean−1.5%0.1%0.6%6.7%3.6%3.5%4.0%St. Dev.4.8%12.0%10.0%13.0%3.0%3.0%3.0%C18.2%−10.6%−8.8%17.8%6.2%5.6%8.6%C2−0.5%−3.2%−3.0%11.6%3.5%5.4%4.9%C3−1.0%−19.8%−20.6%47.1%−2.3%5.5%11.4%C414.5%−16.5%−12.4%43.8%8.9%15.9%16.1%C54.4%16.0%21.3%−11.1%15.1%10.1%5.5%Mean5.1%−6.8%−4.7%21.8%6.3%8.5%9.3%St. Dev.6.5%14.2%15.9%24.1%6.4%4.6%4.6%D1−3.0%11.4%8.0%8.6%21.0%15.7%14.1%D2−3.4%3.0%1.7%10.4%11.5%13.1%9.0%D3−3.0%−7.8%−12.0%0.1%−16.9%−12.3%−9.9%D4−3.8%17.7%19.2%−19.2%4.6%3.4%2.7%D58.4%−15.7%−9.1%−12.5%−22.4%− 17.8%−15.9%Mean−0.9%1.7%1.5%−2.5%−0.4%0.4%0.0%St. Dev.5.3%13.6%12.7%13.0%18.6%15.0%12.6% Discussion {#Sec13} ========== In this pilot study we assessed the tolerability and hemodynamic response of a small group of patients with varying degrees of kidney disease to the application of non-pneumatic anti-shock compression garments. In general, the compression garments were well tolerated by all study participants, however, individuals with pre-existing peripheral vascular disease were noted to most likely decline participation in the trial. We performed continuous hemodynamic monitoring via the Finapres system and obtained multiple cardiac parameters. Interestingly, despite complete and robust data collection from each patient, no consistent trend was observed within or between groups of varying renal function. However, there seem to be greater degree of variability in the CKD and HD groups, as compared to the healthy group. The cause of the variability in response to compression garments is likely multifactorial. In this small scale study, there is significant heterogeneity in participant's characteristics. Underlying medical comorbidity, cardiovascular health, and hemodynamic modifying mediations are among some of the factors that have likely contributed to the response of each individual in our study. For instance, in the HD group, the experiments were conducted prior to dialysis sessions for ease of participant scheduling. As a result, these participants may be relatively hypervolemic, and augmentation of preload in this state may have diminished benefit, or even detrimental effect. Therefore, the lack of consistent response may simply reflect the variable fluid status in these individuals. Patient fluid balance as estimated by change from the patient's ideal weight (Additional file [1](#MOESM1){ref-type="media"}: Table S1) did not appear to be related to the hemodynamic response observed. Furthermore, the higher degree of variability between individuals was not unexpected as the increase in pre-load may be substantial, and different responses may reflect underlying cardiovascular fitness and right heart function. In comparison between groups, our qualitative observation of higher degrees of hemodynamic variability in the two groups with renal dysfunction may be at least partly explained by impaired autonomic regulation, and inability to respond to the increased pre-load in these populations. Another source of variability is the concurrent use of beta blockers or renin-aldosterone system blockade agents which may further impair this regulation system \[[@CR21]\], leading to additional sources of variability. In light of our data, large scale studies such as the study by Tai et al. may not observe a significant benefit statistically as the underlying variability due to patient heterogeneity may dilute the true effect. As such, study of generalized application of these compression stockings may not be the best approach. Our study highlights the need for further characterization of factors that would predict beneficial response. For instance, compression garments may be more suitable to individuals with certain known echocardiographic features, or application of these garments may be reserved on an as-needed basis, rather than universal application. However, in clinical setting, this may be difficult, as clinical variables such as intravascular volume status is difficult to determine accurately, especially since this variability naturally exist between individuals and within each individual across time. Therefore, such tailored but restricted approach may not be pragmatic in a clinical setting. Our study is limited in its small size, and descriptive in nature. However, our protocol reflected the pragmatic approach that would be applied in a large scaled study, and the smaller sample size allowed for mechanistic assessment of the intervention. Despite excellent tolerability, we found significant variation in hemodynamic response. This inconsistency calls into questions the likely utility of universal application of our compression garments to patients with renal dysfunction and informs that large scale study based on similar a protocol will likely result in statistically negative trial. Our results provide mechanistic insights into the use of these garments and poses questions for future research. Conclusions {#Sec14} =========== Non-pneumatic anti-shock compression garments inexpensive and readily available devices that are tolerable and appear feasible to utilize in the CKD and dialysis setting from a practical perspective. We have also highlighted the significant heterogeneity of hemodynamic responses to lower body compression in patients with renal dysfunction, which may explain previous studies' failure to show benefit with generalized application of these garments. Further study is warranted to refine patient selection and identify candidate who would be most likely to receive benefit from nonpneumatic compression garments and we remain optimistic for novel application methods of these devices. Supplementary information ========================= {#Sec15} **Additional file 1: Table S1.** Study participants fluid balance as estimated by change from ideal weight. CKD participants (C) and dialysis participants (D) were weighted on the day of study. This weight was compared to their ideal weight, obtained from their recent nephrology clinic notes. The difference between study weight and ideal weight is intended to provide an estimate to the fluid balance of the study participant on the day of study. ACE : Angiotensin converting enzyme ARB : Angiotensin receptor blocker CAD : Coronary artery disease CKD : Chronic kidney disease DBP : Diastolic blood pressure ESRD : End stage renal disease HD : Hemodialysis HR : Heart rate IDH : Intra-dialytic hypotension LHSC : London Health Science Centre MRA : Mineralocorticoid receptor antagonist NASG : Non-inflatable anti-shock garment SBP : Systolic blood pressure **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. R. Marinovich and Z. Li are co-first authors. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s12882-019-1680-8. Not applicable. RM and ZL with the literature review, organized the study, recruited participants, were responsible of executing the study protocol as well as data interpretation, analysis, manuscript preparation and submission. TT was involved with experimental design, execution of the study protocol and data collection and interpretation. KQ, and SW were involved with the literature review, experimental design and bringing the study through the approval process. CM is credited for the original research question and acted as supervising principal investigator for this study. He was involved the literature review, experimental design, data analysis and manuscript preparation. All authors have read and approved the final manuscript. Funding requirements were minimal for this study, as most materials used were already available to our research group. Purchase of the ZOEX NASG was supported by departmental funds. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. This trial was reviewed and approved by the University of Western Ontario Health Sciences Research Ethics Board prior to initiation of any study related procedures. Written, informed consent was obtained from all participants in this trial. All participants in this study have provided written, informed consent that their data will be made available for publication. The authors declare that they have no competing interests.

{ "pile_set_name": "PubMed Central" }

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Introduction {#s1} ============ Prior to the emergence of the highly pathogenic severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in 2003 [@ppat.1004166-Drosten1]--[@ppat.1004166-Peiris1] only two circulating human coronaviruses (HCoVs), HCoV-229E [@ppat.1004166-Hamre1] and HCoV-OC43 [@ppat.1004166-McIntosh1] causing relatively mild common cold-like respiratory tract infections, were known, and coronaviruses have not been regarded as significant threat for human health. Now, more than ten years later, the emergence of another highly pathogenic coronavirus of zoonotic origin, the Middle East respiratory syndrome coronavirus (MERS-CoV) [@ppat.1004166-Bermingham1]--[@ppat.1004166-Zaki1], boosted community awareness towards the pending need to develop effective therapeutic options to combat coronavirus infections. Coronaviruses are enveloped viruses and their positive strand RNA genome, the largest of all RNA viruses, encodes for as many as 16 non-structural proteins (nsps), 4 major structural proteins, and up to 8 accessory proteins (reviewed in [@ppat.1004166-Perlman1]). Many of these proteins provide essential, frequently enzymatic, functions during the viral life cycle and are therefore attractive targets for antiviral intervention. Antiviral strategies are mainly proposed for targeting coronavirus entry and essential enzymatic functions, such as coronavirus protease or RNA-dependent RNA polymerase (RdRp) activities. For example, the spike (S) protein mediates binding of different HCoVs to their specific cellular receptors [@ppat.1004166-Yeager1]--[@ppat.1004166-Raj1], an event associated with preferential virus tropism for either ciliated or non-ciliated cells of the airway epithelium [@ppat.1004166-Dijkman1]. The S protein also mediates fusion between lipids of the viral envelope and the host cell plasma membrane or membranes of endocytic vesicles to promote delivery of viral genomic RNA into the cytoplasm. Virus binding and cell entry events can be inhibited by antibodies directed against the S protein, antibodies or small molecules interfering with the virus receptors, or synthetic peptides derived from the fusion-triggering heptad repeat regions of the S protein (reviewed in [@ppat.1004166-Cinatl1]). Following virus entry, the coronavirus genome, a positive sense, capped and polyadenylated RNA strand, is directly translated resulting in the synthesis of coronavirus replicase gene-encoded nsps. Coronavirus nsps are translated as two large polyproteins harboring proteolytic enzymes, namely papain-like and chymotrypsin-like proteinases that extensively process coronavirus polyproteins to liberate up to 16 nsps (nsp 1--16) [@ppat.1004166-Perlman1], [@ppat.1004166-Gorbalenya1]--[@ppat.1004166-Ziebuhr2]. These proteolytic functions are considered essential for coronavirus replication and, consequently, a number of candidate drugs were reported to inhibit coronavirus polyprotein processing [@ppat.1004166-Anand1]--[@ppat.1004166-Anand2]. Likewise, the coronavirus RdRp activities, which reside in nsp8 [@ppat.1004166-Imbert1] and nsp12 [@ppat.1004166-Gorbalenya2], are considered essential for coronavirus replication and attractive targets for antiviral intervention. In addition to these classical drug targets, coronaviruses encode an array of RNA-processing enzymes representing additional candidate targets. These include a helicase activity linked to an NTPase activity in nsp13, a 3′-5′-exonuclease activity linked to a N7-methyltransferase activity in nsp14, an endonuclease activity in nsp15, and a 2′-O-methyltransferase activity in nsp16 (reviewed in [@ppat.1004166-Gorbalenya2]). Like all positive strand RNA viruses, coronaviruses synthesize viral RNA at organelle-like structures in order to compartmentalize this critical step of the viral life cycle to a specialized environment that is enriched in replicative viral and host-cell factors, and at the same time protected from antiviral host defense mechanisms [@ppat.1004166-Ahlquist1]--[@ppat.1004166-Overby1]. There is now a growing body of knowledge concerning the involvement, rearrangement and requirement of cellular membranes for RNA synthesis of a number of positive-strand RNA viruses, including coronaviruses [@ppat.1004166-denBoon1], [@ppat.1004166-Knoops1]--[@ppat.1004166-Welsch1]. Three coronaviral nsps, i.e., nsp3, nsp4, and nsp6 [@ppat.1004166-Perlman1], [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1] are thought to participate in formation of these sites for viral RNA synthesis. In particular, these proteins contain multiple trans-membrane domains that are thought to anchor the coronavirus replication complex through recruitment of intracellular membranes to form a reticulovesicular network (RVN) of modified, frequently paired, membranes that includes convoluted membranes [@ppat.1004166-Knoops1] and double membrane vesicles (DVM) [@ppat.1004166-Gosert1] interconnected via the outer membrane with the rough ER [@ppat.1004166-Knoops1]. Indeed, Angelini and colleagues [@ppat.1004166-Angelini1] have recently shown that co-expression of all three transmembrane domain-containing SARS-CoV nsps (nsp3, nsp4, and nsp6) is required to induce DMVs that are similar to those observed in SARS-CoV-infected cells. Such organelle-like compartments harboring membrane-bound replication complexes show remarkable parallels amongst a broad range of positive-strand RNA virus families, and are potentially evolutionary linked to similar mechanisms in the life cycle of double-strand (ds)RNA, reverse-transcribing, and cytoplasmic replicating DNA viruses [@ppat.1004166-Ahlquist1]. Coronavirus ER-derived DMVs are induced early after virus entry into the host cell cytoplasm [@ppat.1004166-Perlman1], [@ppat.1004166-Knoops1], [@ppat.1004166-Ulasli1], [@ppat.1004166-Gosert1]--[@ppat.1004166-Hagemeijer1], and display striking similarities to DMVs induced by hepatitis C virus [@ppat.1004166-RomeroBrey1]. The evolutionary conservation of engaging host cell-derived organelle-like membranous structures for virus RNA synthesis and genetic evidence that impairment of coronavirus DMV integrity is associated with severe reduction of virus replication [@ppat.1004166-Clementz1], [@ppat.1004166-Gadlage1] suggest that antiviral intervention by targeting membranes involved in virus replication represents an attractive, however yet largely unexplored approach. In this work, we describe a novel inhibitor of coronavirus replication that specifically interferes with membrane-bound coronaviral RNA synthesis. This novel mode-of-action is characterized by severe impairment of DMV formation that results in near-complete inhibition of RNA synthesis. Notably, the inhibitor displayed antiviral activity against a broad range of animal and human coronaviruses, including the recently emerging MERS-CoV. Results {#s2} ======= Identification of anti-HCoV-229E hit compound K22 {#s2a} ------------------------------------------------- To identify novel inhibitors of coronavirus infectivity we screened the ChemBioNet collection of 16671 compounds for antiviral activity against HCoV-229E. To this end, MRC-5 cells growing on 384-well plates were supplemented with a specific library compound (20 µM) and then inoculated with HCoV-229E. Compounds that reduced or abolished viral cytopathic effect were re-tested in 24-well plate format for more precise evaluation of their antiviral potential. This two-step screening procedure resulted in several hits including two structurally similar compounds referred to as K22 ([Figure 1A](#ppat-1004166-g001){ref-type="fig"}) and J15 ([Figure S1A](#ppat.1004166.s001){ref-type="supplementary-material"}). The former compound, K22, whose structural name is (*Z*)-N-(3-(4-(4-bromophenyl)-4-hydroxypiperidin-1-yl)-3-oxo-1-phenylprop-1-en-2-yl)benzamide was examined in detail. The compound was completely soluble in medium up to 50 µM. The concentration of K22 that inhibited the number of HCoV-229E plaques by 50% (IC~50~) was 0.7 µM ([Figure 1B](#ppat-1004166-g001){ref-type="fig"}). K22 did not reduce viability of MRC-5 cells by \>50% (CC~50~) at a concentration range of 0.032--500 µM ([Figure 1C](#ppat-1004166-g001){ref-type="fig"}). However this compound decreased proliferation of MRC-5 cells with a CC~50~ value of 110 µM ([Figure 1C](#ppat-1004166-g001){ref-type="fig"}). Hence, using the CC~50~ value determined in cell proliferation assay, the selective index for K22, i.e. the CC~50~/IC~50~ quotient, was 157. Compound J15, although showing anti-HCoV-229E activity similar to that of K22 exhibited a somewhat less favorable cytotoxicity profile in the cell viability assay ([Figure S1B](#ppat.1004166.s001){ref-type="supplementary-material"}). ![K22 structure, antiviral activity, and cytotoxicity.\ (**A**) K22 structure. (**B**) Anti-HCoV-229E activity of K22 in MRC-5 cells. K22 and HCoV-229E were added to MRC-5 cells, and the number of viral plaques developed after 48 h were assessed. Data shown are means (±SD) of duplicate determinations from three independent experiments. PFU, plaque forming unit. (**C**) Viability and proliferation of MRC-5 cells in the presence of K22. MRC-5 cells were incubated with K22 or DMSO solvent for 48 h at 37°C and the cell viability determined using tetrazolium-based reagent while cell proliferation was assayed by counting of cells. Data shown are means (±SD) of duplicate determinations from two independent experiments. (**D**) K22 affects the post-entry phase of viral life cycle. K22 (4 µM) or DMSO solvent were incubated with cells for a period of 2 h either before (−2 h), during (0 h) or after a 2 h period of cell inoculation with HCoV-229E, and the number of viral plaques developed after 48 h were assessed. Data shown are means of duplicate determinations from three independent experiments.\**P*\<0.05; *n* = 3. \*\*\**P*\<0.005; *n* = 3. (**E-F**) K22 exhibits potent antiviral activity when added up to 6 h after infection of cells. MRC-5 cells were inoculated with HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) added at specific time points relative to the end of inoculation period. The culture medium and cells were harvested after 24 h of incubation at 37°C, and the viral RNA (**E**) and infectivity (**F**) determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. EX, extracellular medium; CA, cell-associated sample.](ppat.1004166.g001){#ppat-1004166-g001} K22 inhibits HCoV-229E during the early, post entry phase of the viral life cycle {#s2b} --------------------------------------------------------------------------------- To assess which step of the HCoV-229E life cycle is affected by K22, a time-of-addition/removal experiment was performed. K22 (4 µM) was incubated with cells for a period of only two hours either prior to, during, or after infection with HCoV-229E. As shown in [Figure 1D, K](#ppat-1004166-g001){ref-type="fig"}22 treatment prior to infection resulted in only marginal reduction of virus infectivity thus excluding blockade of cellular receptor(s) for HCoV-229E as its mode-of-action. Simultaneous addition of K22 and virus resulted in ∼50% reduction of virus infectivity suggesting that the compound may interact with viral particles thus inactivating their binding or cell-entry activity. To clarify this possibility, the virus was incubated with ∼70 IC~50~ doses of K22 or DMSO solvent for 15 min at 37°C, followed by virus dilution and its titration at non-inhibitory compound concentrations. Similar titers were observed for the virus treated with K22 (7.2×10^5^/ml±8.9%) and DMSO (7.5×10^5^/ml±4.7%) (n = 2; two experiments), indicating that K22 exhibited no virus particle-inactivating activity. Thus, the ∼50% reduction in plaque number ([Figure 1D](#ppat-1004166-g001){ref-type="fig"}) observed by simultaneous addition of K22 and virus is likely due to cellular uptake of K22 and inhibitory activity of probably not yet metabolically processed compound during a very early step of virus replication rather than the drug binding to viral particles and interference with their penetration into cells. This idea is further corroborated by the most pronounced inhibition of HCoV-229E replication when K22 was added after infection ([Figure 1D](#ppat-1004166-g001){ref-type="fig"}). To more precisely determine the time window of efficient K22-mediated inhibition of HCoV-229E, K22 (10 µM) was added to infected cells at different time points post infection (p.i.), and intra- and extracellular viral RNA, and infectious particles were quantified at 24 hours p.i.. As shown in [Figures 1E-F, K](#ppat-1004166-g001){ref-type="fig"}22 addition within the first 6 hours p.i. resulted in near complete inhibition of viral RNA synthesis and ∼1000-fold reduction of produced infectious virus, suggesting that K22 inhibits most potently post virus entry during the early phase of the HCoV-229E life cycle. K22 resistant mutants contain substitutions in nsp6 {#s2c} --------------------------------------------------- To obtain further insight concerning the target of K22 inhibition we aimed to generate K22-resistant mutants and therefore subjected plaque purified HCoV-229E to 10--13 consecutive passages on MRC-5 cells in presence of increasing concentrations of K22 (2--16 µM). In two independent experiments we isolated and plaque purified several variants displaying moderate (∼2-fold) to strong (∼12-fold) K22 resistance (IC~50~ of 1.6--8.5 µM; [Table 1](#ppat-1004166-t001){ref-type="table"}). Whole genome sequencing analysis of wild type (wt) HCoV-229E, mock passaged virus, and K22 passaged virus revealed two amino acid substitutions within nsp 6 (H121L; M159V) that were associated with strong K22 resistance ([Table 1](#ppat-1004166-t001){ref-type="table"}). Sequence alignment and prediction of potential transmembrane regions of nsp6 homologs of HCoV-229E and other coronaviruses used in this study, revealed presence of 7 potential membrane-spanning domains ([Figure 2](#ppat-1004166-g002){ref-type="fig"}) 6 of which are proposed to be used as membrane anchors in other coronaviruses [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1], and that mutations conferring resistance to K22 are located in or near these regions ([Figure 3A](#ppat-1004166-g003){ref-type="fig"}). Subsequent generation of recombinant mutants, designated HCoV-229E^H121L^, HCoV-229E^M159V^, and HCoV-229E^H121L/M159V^, carrying the nsp6 mutations individually or combined by reverse genetics confirmed that these mutations confer resistance to K22 inhibition as revealed by plaque inhibition ([Table 1](#ppat-1004166-t001){ref-type="table"}) and the time-of-addition ([Figures 3B-C](#ppat-1004166-g003){ref-type="fig"}) assays. Thus, as expected from the previous experiment ([Figure 1E](#ppat-1004166-g001){ref-type="fig"}), K22 addition within the first 6 hours p.i. with the wt HCoV-229E resulted in near complete inhibition of viral RNA synthesis ([Figure 3C](#ppat-1004166-g003){ref-type="fig"}), an effect completely abrogated in the drug-resistant recombinant mutant viruses ([Figure 3B](#ppat-1004166-g003){ref-type="fig"}). Notably, although the amount of intracellular ([Figure 3D](#ppat-1004166-g003){ref-type="fig"}) and extracellular ([Figure 3E](#ppat-1004166-g003){ref-type="fig"}) viral RNA was comparable between K22-resistant mutants and parental wt HCoV-229E, production of infectious particles during infection with K22-resistant mutant viruses was greatly reduced (up to 34 fold at 48h p.i.) ([Figure 3F](#ppat-1004166-g003){ref-type="fig"}). This difference cannot be attributed to the presence of free viral RNA in preparations of extracellular virus, since the treatment of K22-resistant HCoV-229E^M159V^ mutant virus with ribonuclease A did not reduce the quantity of viral RNA ([Figure S2](#ppat.1004166.s002){ref-type="supplementary-material"}). This observation suggests that K22 resistance-conferring mutations in nsp6 are associated with a fitness cost (reduced specific infectivity). ![Alignment of coronavirus nsp6 sequences.\ Alignment of nsp6 sequences derived from coronaviruses used in this study was performed with Geneious Software (Biomatters Ltd, New Zealand). Coronavirus species and corresponding GenBank accession numbers are indicated. Membrane domains predicted by TMHMM Server v. 2.0 (<http://www.cbs.dtu.dk/services/TMHMM/>) are indicated by cyan shading while conserved amino acid residues are highlighted by black/grey shading. K22 resistance-conferring mutations in HCoV-229E nsp6, identified in this study, are depicted.](ppat.1004166.g002){#ppat-1004166-g002} ![Analysis of recombinant HCoV-229E nsp6 mutants.\ (**A**) Predicted topological structure of HCoV-229E nsp6 indicating the location of K22 resistance mutations. Concerning transmembrane domains VI and VII two proposed topologies are shown. (**B-C**) Recombinant nsp6 mutant viruses are resistant to K22. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L^, HCoV-229E^M159V^, HCoV-229E^H121L/M159V^ or wild-type HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) was added at specific time points relative to the end of inoculation period. The infectious cell culture medium and cells were harvested after 24 h of incubation at 37°C, and copy numbers of cell-associated (CA) or extracellular (EX) viral RNA was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. (**D-F**) Replication kinetics of recombinant nsp6 mutant viruses. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L^, HCoV-229E^M159V^, HCoV-229E^H121L/M159V^ or wild-type HCoV-229E at an moi of 0.05 for 1 h at 4°C. The infectious cell culture medium and cells were harvested at specific time points relative to the end of inoculation period, and copy numbers of cell-associated (CA; **D**) or extracellular (EX; **E**) viral RNA and infectivity (**F**) was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments.](ppat.1004166.g003){#ppat-1004166-g003} 10.1371/journal.ppat.1004166.t001 ###### Alterations detected in the K22 resistant variants of HCoV-229E. ![](ppat.1004166.t001){#ppat-1004166-t001-1} Alteration[a](#nt101){ref-type="table-fn"} ------------------------------------------------------- -------------------------------------------- ---------------------- ------------------------------------------ ---------- Initial[b](#nt102){ref-type="table-fn"} None None 0.7[c](#nt103){ref-type="table-fn"} KF293664 K22 passage 10 a10455t H121L (Nsp6) 9.8 (14)[d](#nt104){ref-type="table-fn"} KF293666 c19463t T281I (Nsp15) c26667t P328S (Nucleocapsid) A a10455t H121L (Nsp6) 8.2 (12) KF285470 B a10455t H121L (Nsp6) 8.2 (12) KF285471 D a10455t H121L (Nsp6) 7.6 (11) KF285472 G a10455t H121L (Nsp6) 6.9 (10) KF285473 K c19463t T281I (Nsp15) 1.6 (2) KF285481 c26667t P328S (Nucleocapsid) KF293662 L c19463t T281I (Nsp15) 2.2 (3) KF285482 c26667t P328S (Nucleocapsid) KF293663 K22 passage 13 - M[e](#nt105){ref-type="table-fn"} a10568g M159V (Nsp6) 6.7 (10) KF285474 a23130c N854T (Spike) KF285480 N a10568g M159V (Nsp6) 7.1 (10) KF285475 O a10568g M159V (Nsp6) 7.7 (11) KF285476 P a10568g M159V (Nsp6) 8.5 (12) KF285477 Q a10568g M159V (Nsp6) 7.7 (11) KF285478 R a10568g M159V (Nsp6) 6.8 (10) KF285479 HCoV-229E[f](#nt106){ref-type="table-fn"} 0.6 [g](#nt107){ref-type="table-fn"}HCoV-229E^H121L^ a10455t H121L (Nsp6) 7.2 (12) [g](#nt107){ref-type="table-fn"}HCoV-229E^M159V^ a10568g M159V (Nsp6) 6.3 (11) [g](#nt107){ref-type="table-fn"}HCoV229E^H121L/M159V^ a10455t H121L (Nsp6) 8.2 (14) a10568g M159V (Nsp6) Detected by comparison of the nucleotide sequences of HCoV-229E subjected to 10--13 passages in the presence of K22 including its plaque purified variants A-R with those of initial virus or mock-passaged virus (accession number KF293665). Plaque purified HCoV-229E that served as initial material for the virus passages. IC50 (µM). Fold resistance to K22 as related to initial virus is shown in parentheses. Virus preparation and its plaque purified variants M-R obtained in separate K22 selection experiment. The virus used for preparation of recombinant nsp6 mutants. K22 resistant recombinant viruses. K22 treatment results in loss of DMVs {#s2d} ------------------------------------- The observation that amino acid substitutions in nsp6 confer K22 resistance strongly suggests a mode-of-action based on interference with host cell membranes required for coronavirus replication. Nsp6 is expressed as a membrane-spanning integral component of the viral replication complex, and is, together with nsp3 and nsp4, implicated in anchoring the coronavirus replicase complex to DMVs or related membrane structures [@ppat.1004166-Perlman1], [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1], [@ppat.1004166-Angelini1], [@ppat.1004166-Hagemeijer1]. Indeed, there is genetic and experimental evidence concerning nsp4-mediated alterations of coronavirus DMVs [@ppat.1004166-Clementz1], [@ppat.1004166-Gadlage1], and that ectopic expression of nsp6 results in the formation of ER-derived vesicles [@ppat.1004166-Cottam1]. We therefore assessed if K22 may impact the formation of coronavirus-induced DMV by electron microscopy ([Figure 4](#ppat-1004166-g004){ref-type="fig"}). As expected, perinuclear DMV clusters as well as viral particles were readily detectable in wt HCoV-229E-infected cells ([Figure 4A](#ppat-1004166-g004){ref-type="fig"}). In sharp contrast, no DMV clusters or viral particles were detectable in wt HCoV-229E-infected and K22-treated (4 µM) cells ([Figure 4A](#ppat-1004166-g004){ref-type="fig"}). Since double-stranded (ds) RNA is indicative of coronavirus replication and has been shown to reside predominantly within the inner lumen of coronavirus-induced DMVs [@ppat.1004166-Knoops1] we also performed immunofluorescence analysis and stained HCoV-229E-infected cells for viral replicase complex (nsp8) and dsRNA. Strikingly, the characteristic perinuclear immunofluorescence staining pattern for viral replicase complexes and dsRNA visible in wt HCoV-229E-infected cells was completely absent under K22 treatment ([Figure 5](#ppat-1004166-g005){ref-type="fig"}), confirming the remarkable efficacy of K22-mediated inhibition of viral replication and supporting the notion that K22 blocks the formation of DMVs. In contrast to parental wt HCoV-229E and irrespectively whether K22 was applied, recombinant K22 escape mutants were still capable of inducing the formation of DMVs ([Figure 4B](#ppat-1004166-g004){ref-type="fig"}) and displayed the characteristic staining pattern for replicase complexes and dsRNA ([Figure 5](#ppat-1004166-g005){ref-type="fig"}). Likewise, compound J15 efficiently blocked replication ([Figure S1B](#ppat.1004166.s001){ref-type="supplementary-material"}) and DMV formation of wt HCoV-229E but not K22 resistant nsp6 recombinant HCoV-229E^M159V^ ([Figure S3](#ppat.1004166.s003){ref-type="supplementary-material"}) suggesting that J15 may have the same target and mode-of-action. Notably, in cells infected with K22 escape mutants the overall number of DMVs per cell was reduced (30.3±29.7 in HCoV-229E^M159V^ versus 65±50.1 in wt HCoV-229E infected cells; *P*\<0.05; *n* = 20), similar as previously described for mouse hepatitis virus (MHV) nsp4 mutants [@ppat.1004166-Clementz1], [@ppat.1004166-Gadlage1], while the number of intracellular viral particles that were often packed in tubular vesicle-like structures ([Figures 4A-B](#ppat-1004166-g004){ref-type="fig"}) was comparable to that of wt virus (471.8±212.6 in HCoV-229E^M159V^ versus 438.3±96.8 in wt virus infected cells; *n* = 10). We could also frequently detect DMVs displaying partially collapsed inner membranes in cells infected with K22 escape mutants (irrespectively whether or not K22 was applied; [Figure 4B](#ppat-1004166-g004){ref-type="fig"}), again similarly as reported for MHV nsp4 mutants [@ppat.1004166-Gadlage1], suggesting that nsp6, like nsp4, has a pivotal role in coronavirus DMV formation. Overall, these findings demonstrate that the antiviral activity of K22 (and that of the structurally similar compound J15) results in complete loss of DMVs. This efficient block in replication can be overcome by resistance mutations in nsp6, and DMVs induced by nsp6 mutant viruses are reduced in numbers and structurally impaired -- both findings concurring with the established function of nsp6 in DMV formation. ![K22 affects formation of double membrane vesicles (DMVs).\ MRC-5 cells growing on Melinex polyester film were infected with wild type HCoV-229E (WT) or with K22-resistant recombinant nsp6 mutant HCoV-229E^M159V^ (M159V) and incubated for 18 h at 37°C with or without K22. The cells were then fixed with glutaraldehyde and processed for electron microscopy without their scrapping or pelleting. (**A**) Electron micrographs of cells infected with WT virus show presence of perinuclear clusters of DMVs (arrow) and viral particles (arrowhead), and the lack of their production upon K22 treatment (4 µM). (**B**) Note presence of DMVs and viral particles in cells infected with K22-resistant nsp6 recombinant HCoV-229E^M159V^ (M159V) irrespective of the addition of K22. Each image shown was selected from a pool of over 30 images captured in three separate experiments.](ppat.1004166.g004){#ppat-1004166-g004} ![K22 affects formation of coronavirus replication complex in cells.\ MRC-5 cells were infected with wild type HCoV-229E (WT) and K22-resistant recombinants HCoV-229E^H121L^ (H121L), HCoV-229E^M159V^ (M159V), and HCoV-229E^H121L/M159V^ (H121L/M159V) and incubated for 18 h with or without the presence of K22. The cells were then fixed with 4% paraformaldehyde and immunostained for immunofluorescence analysis. Note the lack of detection of dsRNA and nsp8 upon K22 treatment (4 µM) of cells infected with WT but not recombinant viruses. Scale bar is 10 µM.](ppat.1004166.g005){#ppat-1004166-g005} K22 does not impact cellular autophagy {#s2e} -------------------------------------- Our data show that K22 targets a very early step in the HCoV-229E life cycle, and the appearance of resistance-conferring mutations in nsp6 suggests that K22 impairs DMV formation. We therefore assessed if K22 treatment may, independent of virus infection, impact autophagy, a cellular process displaying similarities to coronaviral DMV formation. To this end we first transfected Huh7 cells with a plasmid encoding LC3B-GFP in order to trace rapamycin-induced autophagsomes by life imaging. This analysis revealed that three to six hours after adding rapamycin to the culture medium green fluorescent autophagocytic vesicles become apparent, irrespectively if K22 (20 µM) was added or not (data not shown). We corroborated this result by immunofluorescence analysis of Huh7 cells that were stained for endogenous LC3B at six hours post addition of rapamycin. As shown in supplementary [Figure S4](#ppat.1004166.s004){ref-type="supplementary-material"} rapamycin-incuced autophagocytic vesicles were again readily detectable in the presence of K22 (20 µM), suggesting that K22 does not impact cellular autophagy. K22 inhibits a number of diverse coronaviruses {#s2f} ---------------------------------------------- Since K22 inhibits a crucial step in the HCoV-229E life cycle, we assessed the antiviral activity of K22 against a panel of diverse coronaviruses representing the major phylogenetic lineages of α-, β- and ???-coronaviruses. As shown in [Figure 6A-D](#ppat-1004166-g006){ref-type="fig"} and supplementary [Figure S5](#ppat.1004166.s005){ref-type="supplementary-material"}, K22 indeed displayed antiviral activity against recombinant MHV (strain A59 [@ppat.1004166-Coley1]) expressing *Gaussia* luciferase as marker for virus replication, recombinant type-I feline coronavirus (FCoV; strain Black [@ppat.1004166-Tekes1]) expressing *Renilla* luciferase as marker for virus replication, avian infectious bronchitis virus (IBV; strain Beaudette [@ppat.1004166-Casais1]), and SARS- CoV (strain Frankfurt-1 [@ppat.1004166-Thiel1]), suggesting that K22 targets a broad range of coronaviruses. Furthermore, there was no cytotoxicity detectable in cells of feline (FCWF cells), murine (L929 cells), and primate (Vero cells) origin in the K22 concentration range assessed, and analysis of K22 cytostatic activities in the cell proliferation assay revealed CC~50~ values ≥40 µM ([Table S1](#ppat.1004166.s007){ref-type="supplementary-material"}), i.e., the highest drug concentration used in antiviral assays. Notably, the efficacy of K22-mediated inhibition varied amongst different coronaviruses, however whether this is related, as in HCoV-229E, to nsp6 function would require generation and analysis of K22 resistant variants for all coronaviruses tested. In contrast, K22 exhibited little or no effect on replication of poliovirus ([Figure S6](#ppat.1004166.s006){ref-type="supplementary-material"}), a pathogen that like coronaviruses induces rearrangement of cellular membranes to assist RNA replication. ![K22 affects replication of diverse coronaviruses including MERS-CoV.\ (**A-D**) The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 during MHV-Gluc (**A**), FCoV-RL (**B**), SARS-CoV (**C**) and IBV (**D**) infection on representative continuous cell lines of murine (L-929 cells; **A**), feline (FCWF cells; **B**), or primate (Vero cells; **C-D**) origin. Data are shown as mean (±SD) of a representative experiment, from two independent experiments performed in triplicate. Toxicity values for Vero cells in panels C and D are derived from the same experiments. (**E-F**). The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 in HCoV-229E-ren (**E**) and MERS-CoV (**F**) infected differentiated human airway epithelial (HAE) cultures. Data are shown as mean (±SD) of three independent experiments performed in triplicate (log reduction), or mean (±SD) of a representative experiment, from two independent experiments performed in triplicate (cell viability). (**G-H**) Immunofluorescence analysis of HAE cultures infected with MERS-CoV in presence or absence of K22 in a representative overview (**G**, 20x; **H**, 40x) confocal Z-stack image. Stainings were performed using antibodies directed against (**G**) dsRNA (green), and DAPI (cell nucleus; blue), and (**H**) dsRNA, DAPI, β-tubulin (ciliated cells; white), and ZO1 (tight junctions, red). Scale bars are 50 (**G**) or 20 (**H**) µm.](ppat.1004166.g006){#ppat-1004166-g006} Inhibition of HCoV-229E and MERS-CoV in primary human airway epithelia cultures {#s2g} ------------------------------------------------------------------------------- Finally, we assessed the efficacy of K22 inhibition in the primary target cells of respiratory virus infection, the human airway epithelium. Fully differentiated primary human airway epithelia (HAE) cultures [@ppat.1004166-Dijkman1], [@ppat.1004166-Kindler1] derived from three different donors and grown under air-liquid interphase conditions were infected with a recombinant HCoV-229E expressing *Renilla* luciferase as marker for virus replication [@ppat.1004166-vandenWorm1], and with MERS-CoV [@ppat.1004166-Zaki1], [@ppat.1004166-Kindler1]. MERS-CoV was first described in 2012 and was isolated from a 60-year old man with acute pneumonia, renal failure and fatal outcome in Saudi Arabia [@ppat.1004166-Zaki1]. The virus is most likely of zoonotic origin [@ppat.1004166-vanBoheemen1], [@ppat.1004166-Reusken1] and by February 2014 the number of laboratory-confirmed cases of MERS-CoV infection reported to the World Health Organization exceeded 182, including more than 79 cases with fatal outcome. We have previously shown that MERS-CoV can readily replicate on primary HAE cells [@ppat.1004166-Kindler1] by infecting non-ciliated cells expressing the cellular receptor dipeptidyl peptidase 4 [@ppat.1004166-Raj1]. As shown in [Figure 6](#ppat-1004166-g006){ref-type="fig"}, HCoV-229E and MERS-CoV infections were inhibited by K22 treatment with remarkable efficacy, illustrated by reduction of viral replication by several orders of magnitude ([Figure 6E-F](#ppat-1004166-g006){ref-type="fig"}) and substantial reduction of dsRNA in MERS-CoV-infected primary HAE cultures ([Figure 6G-H](#ppat-1004166-g006){ref-type="fig"}). This result demonstrates that the broad anti-coronaviral activity of K22 makes this compound particularly promising for the development of efficacious treatment options for emerging coronaviruses, such as MERS-CoV. Discussion {#s3} ========== Here we describe the discovery of a novel class of inhibitor and propose a mode-of-action that targets membrane-bound viral replication. Like all positive strand RNA viruses, coronaviruses employ host cell membranes to assemble the viral replicase complex. This evolutionary conserved strategy provides a compartment for viral RNA synthesis that is enriched in replicative viral and host cell-derived proteins and believed to protect from antiviral host cell defense mechanisms. The remarkable efficacy of K22-mediated inhibition of coronavirus replication confirms that the employment of host cell membranes for viral RNA synthesis is a crucial step in the coronavirus life cycle, and importantly, demonstrates that this step is extremely vulnerable and also druggable for antiviral intervention. The observation that K22 resistance is mediated through mutations in nsp6 defines transmembrane domain-containing nsps implicated in anchoring viral replicase complexes to host cell-derived membranes, as novel targets for anti-coronaviral intervention. Moreover, we expect this mode-of-action to serve as a paradigm for the development of similar antiviral drugs to combat infections caused by many other positive strand RNA viruses. Notably, resistance conferring mutations in nsp6 emerged only after 10--13 consecutive passages of HCoV-229E under K22 selection, and we were so far not successful in obtaining K22-resistant MHV-A59 mutants (data not shown). This suggests that escape mutations in membrane domain-containing coronavirus nsps compatible with maintaining efficient RNA synthesis are limited. In addition, the nsp6 escape mutants we have obtained for HCoV-229E display a remarkable reduction of specific infectivity. Thus, although RNA synthesis appears to be unaffected and viral RNA detected in preparations of extracellular virus was ribonuclease insensitive implying its adequate package in viral particles, mutations in nsp6 seem to reduce virus fitness. Thus, it is conceivable that the nsp6 mutants may be functionally impaired during an early step in the viral life cycle. Since dsRNA is localized in DMVs and nsp6 escape mutants induced decreased number of DMVs that are structurally impaired, it is possible that the reduced specific infectivity of these viruses could be related to dsRNA-triggered innate immune responses. SARS-CoV nsp6 was recently found to contribute to the establishment of the virus-induced RVN by promoting vesicle formation in transfected cells [@ppat.1004166-Angelini1], and our observation that K22 resistant mutants generated decreased number of DMVs implies that specific alterations may adversely affect the vesicle-forming capability of nsp6. Nsp6 of HCoV-229E (this report), MHV, and SARS-CoV [@ppat.1004166-Baliji1], [@ppat.1004166-Oostra1] is predicted as a hexaspaning protein comprising a conserved C-terminal cytoplasmic tail. The latter domain may serve as a wedge-like amphipathic helix which upon insertion into the lipid membrane can trigger its bending due to induction of positive membrane curvature (reviewed in [@ppat.1004166-McMahon1]). The vesicle formation would also require a putative ion channel activity that depolarizes curved membranes thus facilitating their fusion and vesicle scission. The question as to whether nsp6 or other components of the coronavirus replicase complex exhibit such activities would require further investigation. Although our data reveal that the K22 escape mutations occur in nsp6, further binding experiments are required to clarify whether K22 targets nsp6 directly. We observed that K22 is most active in inhibiting replication of the tested α-coronaviruses (HCoV-229E, FCoV) and the γ-coronavirus IBV, whereas amongst β-coronaviruses K22 was highly active in inhibiting MERS-CoV, but only moderately against MHV or SARS-CoV ([Figure 6](#ppat-1004166-g006){ref-type="fig"}). It is conceivable that K22 may strong inhibit α-coronaviruses, since K22 has been identified by screening for anti-HCoV-229E activity. However, the limited nsp6 sequence similarity between coronaviruses ([Figure 2](#ppat-1004166-g002){ref-type="fig"}) does not allow predicting the strength of K22-mediated inhibition of replication based on nsp6 homology. We also like to address in future studies a question of how the moderately resistant virus variant L (containing mutations in nsp15 and nucleocapsid) can escape K22-mediated inhibition of replication. This variant, in contrast to these containing resistance mutations in nsp6, exhibited only moderate resistance to K22 (∼2-3-fold) and was not consistently selected in separate selection experiments. Although nsp15 and nucleocapsid protein have not yet been described as being directly involved in DMV formation, these proteins are components of the replicase complex that may somehow affect/modulate nsp6 functions, and compensatory mutations in these proteins may partially relieve K22 blockade of nsp6. An alternative possibility is that the actual K22 target may be a cellular protein or a process of recruitment of a cellular protein that participates in coronavirus-induced membrane rearrangements by interacting with nsp6. While we could not observe any detectable impact of K22 on the formation of autophagosomes, further studies are required to address if K22 may target similar vesicles, such as EDEMosomes [@ppat.1004166-Reggiori1]. Both possibilities are compatible with the observed phenotype of DMV impairment and the detection of resistance mutations at regions of HCoV-229E nsp6 that are structurally conserved while displaying only limited sequence similarity. It is thus conceivable that membrane domain-containing nsp3 and nsp4 may represent additional drug targets. Similar as described for the related arteriviruses, where co-expression of membrane-spanning nsp2 and nsp3 results in membrane alterations and DMV formation similar to those observed during arterivirus infection [@ppat.1004166-Snijder2], [@ppat.1004166-Posthuma1], co-expression of coronavirus nsp3, nsp4 and nsp6 is required to produce coronavirus-like membrane rearrangements including DMVs [@ppat.1004166-Angelini1]. Expression of nsp3, nsp4 or nsp6 alone or in combinations of two induces aberrant membrane rearrangements that only partially mimic membrane structures known from coronavirus infection [@ppat.1004166-Angelini1]. Thus, there is growing evidence that nsp3, nsp4, nsp6, and possibly ER membrane-resident host cell proteins [@ppat.1004166-Reggiori1], [@ppat.1004166-Bernasconi1], orchestrate critical events that lead to the development of suitable membrane structures facilitating coronavirus RNA synthesis. Since K22 apparently interferes with these processes, inhibitors like K22 and corresponding escape mutants will likely become valuable tools to further our understanding on the induction of membrane alterations and DMV formation that take place during the early phase of the coronavirus life cycle. For example, co-expression of nsp3, nsp4 and native or mutated nsp6 in the absence of virus replication, similar as described by Angelini and colleagues [@ppat.1004166-Angelini1], may help to clarify whether presence of K22 would affect formation of DMV by directly targeting nsp6 or cellular protein(s) required and recruited for DMV formation. We emphasize that the identification of K22 and its proposed mode-of-action is only the very first step towards an approved drug for therapeutic use in animals or humans. Specifically, we are currently focusing on the structure-activity relationship analysis of K22 analogs, with the aim to identify compounds with improved antiviral and cytotoxic profiles prior to their assessment *in vivo*. However, one important lesson of the past SARS-CoV and recent MERS-CoV outbreaks is that zoonotic transmission of coronaviruses into the human population can pose considerable threat to human health and that it is warranted to eventually invest significant efforts to developing efficacious and approved drugs to increase preparedness and combat coronavirus infections. The antiviral activity against a number of diverse coronaviruses makes K22 an ideal candidate for further development towards an efficacious "pan-coronavirus inhibitor". Broad anti-coronaviral activity has been proposed for inhibitors targeting highly conserved enzymatic functions, such as coronavirus proteinase activities [@ppat.1004166-Anand2], [@ppat.1004166-Ren1], or more recently, for compounds targeting host cell factors required for efficient replication, such as cyclophilins [@ppat.1004166-Pfefferle1], [@ppat.1004166-deWilde1]. The concept of targeting multiple key functions of viral replication led to the development of efficacious treatment regimens against HIV and hepatitis C virus by combining multiple antiviral drugs [@ppat.1004166-Deeks1], [@ppat.1004166-Delang1] and it is tempting to speculate that this concept will be applicable to combat coronavirus infections in the future. Moreover, with the identification of K22, we demonstrate that there are yet additional critical steps in the life cycle of positive strand RNA viruses to explore as targets for antiviral intervention. Materials and Methods {#s4} ===================== Ethics statement {#s4a} ---------------- Human bronchial epithelial cells were isolated from patients (\>18 years old) who underwent bronchoscopy and/or surgical lung resection in their diagnostic pathway for any pulmonary disease and that gave written informed consent. This was done in accordance with local regulation of the Kanton St. Gallen, Switzerland, as part of the St. Gallen Lung Biopsy Biobank (SGLBB) of the Kantonal Hospital, St. Gallen, which received approval by the ethics committee of the Kanton St. Gallen (EKSG 11/044, EKSG 11/103). Cells and viruses {#s4b} ----------------- Human embryonic lung diploid fibroblasts (MRC-5), African green monkey kidney cells (Vero), baby hamster kidney cells (BHK-21), felis catus whole fetus 4 cells (FCWF-4), were purchased from the American Type Culture Collection (ATCC), murine fibroblast cells (L929), African green monkey kidney cells (CV-1) were purchased from the European Collection of Cell Cultures. D980R cells were a kind gift from G. L. Smith, Imperial College, London, United Kingdom. African green monkey kidney (GMK AH1) cells were obtained from the Swedish Institute for Infectious Disease Control, Stockholm. Cells were grown in Eagle\'s minimum essential medium (EMEM) (MRC-5, CV-1, D980R, L929, BHK-21, GMK AH1 cells) or in Dulbecco\'s modified EMEM (DMEM) (FCWF-4, Vero cells), supplemented with 5--10% heat-inactivated fetal calf serum, (HI-FCS), 1% L-glutamine, penicillin (60 µg/ml) and streptomycin (100 µg/ml) (PEST). Isolation and cultivation of primary human bronchial epithelial cells to form pseudostratified/differentiated human airway epithelial (HAE) cultures was performed as described previously [@ppat.1004166-Dijkman1], [@ppat.1004166-Dijkman2]. Human CoV strain 229E [@ppat.1004166-Hamre1] (HCoV-229E) was obtained from ATCC (VR-740). HCoV-229E stocks were prepared from virus passages 6--8 in MRC-5 cells growing in EMEM supplemented with 2% HI-FCS, 1% L-glutamine, HEPES (10 mM) and PEST (EMEM-FP). In some experiments, the virus was concentrated by centrifugation of infectious culture fluid of MRC-5 cells over a 1.5 ml cushion of 20% sucrose for 2 h at 22000 rpm (SW28.1 rotor, Beckman). The pellet was covered with PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO), left overnight at 4°C, and then gently suspended by pipetting. The following viruses and their propagation were described previously: recombinant HCoV- 229E [@ppat.1004166-Thiel2], recombinant HCoV-229E-Ren expressing Renilla luciferase [@ppat.1004166-vandenWorm1], recombinant feline coronavirus (strain Black) expressing Renilla luciferase (recFCoV-RL) [@ppat.1004166-Tekes1], SARS-CoV strain Frankfurt-1 [@ppat.1004166-Thiel1], recombinant avian infectious bronchitis virus (IBV, strain Beaudette) [@ppat.1004166-Casais1], MERS-CoV [@ppat.1004166-Zaki1], [@ppat.1004166-Kindler1]. Recombinant MHV strain A59 expressing Gaussia luciferase (MHV-Gluc) was generated based on the previously described reverse genetics system [@ppat.1004166-Coley1], [@ppat.1004166-Eriksson1]. Briefly, the MHV-A59 accessory gene 4 was replaced by the gene encoding the codon-optimized Gaussia luciferase [@ppat.1004166-Tannous1] (hGLuc) using vaccinia-virus-mediated homologous recombination essentially as described for the generation of MHV-GP33-GFP [@ppat.1004166-Zust1]. The plasmid DNA used for recombination contained MHV-A59 nucleotides (nts) 27500--27967, the hGLuc Gaussia luciferase gene, and MHV-A59 nts 28265--28700. Recombinant HCoV-229E containing mutations conferring K22 resistance in nsp6 were generated based on the previously described reverse genetics system [@ppat.1004166-Thiel2], [@ppat.1004166-Eriksson1]. Briefly, vaccinia virus HCoV-inf1 (containing the full-length HCoV-229E cDNA) [@ppat.1004166-Thiel2] was used to recombine with a plasmid based on pGPT1 [@ppat.1004166-Hertzig1] where the *Escherichia coli* guanine phosphoribosyltransferase (GPT) gene was flanked by HCoV-229E nts 9398--10098 and 10930--11580. The resulting GPT-positive vaccinia virus was then used to recombine with plasmids containing the HCoV-229E nts 9398--11580 with modification of nucleotide 10455 (A to T; HCoV-229E^H121L^), or nt 10568 (A to G; HCoV-229E^M159V^), or both nts 10455 and 10568 (HCoV-229E^H121L/M159V^). The resulting vaccinia viruses were then used to rescue HCoV-229E^H121L^, HCoV-229E^M159V^, and HCoV-229E^H121L/M159V^ as described previously [@ppat.1004166-Thiel2], [@ppat.1004166-Eriksson1]. The identity of plasmid DNA and recombinant vaccinia viruses and recombinant coronaviruses was confirmed by sequencing. In some experiments poliovirus 1 strain Sabin (obtained from the Swedish Institute for Infectious Disease Control, Stockholm) was used. Reagents {#s4c} -------- The ChemBioNet diversity library of 16671 compounds was obtained from the Leibniz Institute for Molecular Pharmacology (Berlin, Germany). Library was provided in a 384 well plate format, each well containing 5 µl of a compound solubilized in DMSO to a final concentration of 10 mM. Hit compound K22 was purchased from ChemDiv (San Diego, CA; catalog number 4295--0370). The correct structure and purity of K22 (\>95%) was confirmed in our laboratory by NMR and LCMS analyses. Immunofluorescence analysis {#s4d} --------------------------- MRC-5 cells were infected at a multiplicity of infection (moi) of 0.05 with wtHCoV-229E and K22-resistant recombinants HCoV-229E^H121L^, HCoV-229E^M159V^, and HCoV-229E^H121L/M159V^ with or without the presence of K22 (4 µM). The cells were fixed at 18 h p.i. with 4% paraformaldehyde (PFA) and immunostained [@ppat.1004166-Dijkman3] using the mouse monoclonal anti-dsRNA (J2, English & Scientific Consulting Bt.) and rabbit anti-HCoV-229E nsp8 [@ppat.1004166-Ziebuhr3] (kindly provided by John Ziebuhr, University of Giessen, Germany) as primary antibodies for detection of double-stranded (ds) RNA and viral replication complexes. Donkey derived, Dylight 488 labeled, anti-mouse IgG (H+L) and Dylight 647 labeled, anti-rabbit IgG (H+L) (Jackson Immunoresearch) were applied as secondary antibodies. Cells were counterstained with DAPI (4\',6-diamidino-2-phenylindole; Invitrogen) to visualize nuclei. HAE cell cultures were inoculated with 40000 plaque forming units (PFU), with or without the presence of K22 (50 µM) and fixed with 4% PFA 48 h p.i. Staining was performed with the mouse monoclonal antibody directed against dsRNA (J2) and goat polyclonal anti-ZO1 (tight junctions; ab99462, Abcam) as primary antibodies. Dylight 488-labeled donkey anti-mouse IgG (H+L), Dylight 546-labeled donkey anti-goat IgG (H+L) (Jackson Immunoresearch) were applied as secondary antibodies, followed by two separate incubation steps with Alexa Fluor647-conjugated rabbit monoclonal anti-beta-Tubulin antibody (ciliated cells; 9F3, Cell Signal) and DAPI (Invitrogen). Images were acquired using EC-plan Neofluar 20x/50 M27 or EC Plan-Neofluar 40x/1.30 Oil DIC M27 objectives on a Zeiss LSM 710 confocal microscope. Image capture, analysis and processing were performed using the ZEN 2010 (Zeiss) and Imaris (Bitplane Scientific Software) software packages. Anti-coronavirus compound screening assay {#s4e} ----------------------------------------- The screening assay was performed as described previously for respiratory syncytial virus [@ppat.1004166-Lundin1]. Briefly, MRC-5 cells were seeded in 384 well plates (CLS-3701; Costar-Corning, NY, USA) to become ∼70--90% confluent after one day of culture. The growth medium was removed, and the cells supplemented consecutively with 25 µl of EMEM-FP medium, 1 µl volumes of library compounds at 1 mM concentration, and ∼350 PFU of HCoV-229E in 25 µl of EMEM-FP. The last two columns of the 384 well plate received either virus or EMEM-FP medium to serve as controls. The cells were observed under the microscope for their protection from the virus-induced cytopathic effect after 3 and 6 days of incubation at 37°C. Antiviral assays {#s4f} ---------------- Plaque reduction assay to determine the antiviral effect of K22 on HCoV-229E was done as follows. MRC-5 cells were seeded in 12-well plates to become nearly confluent after one day of culture. Serial fivefold dilution of K22 (0--100 µM) and 100 PFU of HCoV-229E virus in 0.5 ml of EMEM-FP medium were added to and incubated with cells for 3 h at 37°C, 5% CO2. Subsequently, the virus-compound mixtures were removed from cells, and 1.5 ml volumes of 1% methylcellulose (MC) solution in EMEM-FP medium supplemented with the same concentration of K22 were added. The plates with cells were further incubated at 37°C, 5% CO2 for 2--3 days, and then stained with 0.2% solution of crystal violet to visualize the viral plaques. Viral yield reduction assays were done to determine the antiviral effect of K22 on HCoV-229E-Ren, recFCoV-RL, MHV-Gluc, SARS-CoV, IBV, MERS-CoV, and poliovirus replication. Briefly, K22 or its DMSO solvent in medium was added at the indicated concentrations to nearly confluent monolayers of corresponding cell lines or to HAE cultures at the basolateral side and incubated for 4 h at 37°C, 5% CO2. The cells were then inoculated with recFCoV-RL (moi = 0.1 on FCWF-4 cells), MHV-Gluc (moi = 0.001 on L929 cells), SARS-CoV (moi = 0.001 on Vero cells), IBV (moi = 1 on Vero cells), HCoV-229E-Ren (4×10^3^ PFU on HAE cultures apically), MERS-CoV (4×10^3^ PFU on HAE cultures apically) or poliovirus (moi = 0.001 on GMK AH1 cells). After 2 h the viral inoculum was removed, cells were rinsed three times with PBS, and fresh medium containing the same concentrations of K22 or DMSO was added. Coronavirus replication was assessed from cell culture supernatant by determining titer as TCID50 (tissue culture infectious dose that will produce pathological change in 50% of cell cultures inoculated) for IBV or poliovirus at 48 h p.i., by determining the amount of viral genome RNA produced by qRT-PCR specific for SARS-CoV and MERS-CoV at 48 h p.i. as described previously [@ppat.1004166-Kindler1], or by determining the level of Renilla expression at 48 h p.i. (HCoV-229E-Ren) or 72 h p.i. (recFCoV-RL) using *Renilla* Luciferase Assay System (Promega, E2820), or Gaussia luciferase expression (MHV-Gluc) at 24 h p.i. using the BioLux *Gaussia* Luciferase Assay Kit (NEB,E3300), respectively. For the virucidal assay, 200 µl of HCoV-229E suspension (∼3×10^4^ PFU) in EMEM-FP medium was mixed with 50 µM K22 and incubated for 15 min at 37°C. In the control sample, virus was incubated with the DMSO solvent at a final concentration corresponding to that present in the test compound. Then, both mixtures were diluted serially tenfold in EMEM-FP medium and the residual virus infectivity determined by the viral plaque assay. Cell toxicity and proliferation assays {#s4g} -------------------------------------- The toxicity of K22 or its solvent (DMSO) for MRC-5 cells was evaluated using the tetrazolium-based CellTiter 96 AQueous One Solution cytotoxicity assay (Promega; G3580). The effect of K22 or its solvent on proliferation of MRC-5 cells was studied as follows. The cells were seeded in 48 well plates to become ∼50% confluent after one day of culture. The growth medium was removed, and cells incubated with specific concentrations of K22 or its solvent in EMEM-FP medium for 72 h at 37°C. The cells were then dissociated with trypsin/EDTA solution and counted. The effect of K22 or DMSO on viability of Vero, L929, and FCFW-4 cells was assessed using the CytoTox-Glo Cytotoxicity Assay kit (Promega, G9291) while the toxicity of test compound for differentiated HAE cultures was evaluated with CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, G7571). Time-of-addition assay {#s4h} ---------------------- MRC-5 cells growing in 12 well plates were precooled for 15 min at room temperature and for another 15 min at 4°C. The cells were rinsed once with 500 µl of cold EMEM-FP and inoculated with HCoV-229E at moi of 0.05. Following virus adsorption to cells for 45 min at 4°C, the cells were rinsed twice with 500 µl of cold EMEM-FP, and 990 µl of warm EMEM-FP medium was added. Subsequently 10 µl of 1 mM K22 was added at specific time points relative to the end of the virus adsorption period, and the infectious cell culture medium and cells harvested at the time point 24 h. The cell culture supernatant medium was clarified by centrifugation at 1000×*g* for 5 min while the pelleted cells were suspended in RNase-free water and stored at −80°C until quantification in RT-PCR assay. To study the effect of K22 on early virus-cell interaction the "time-of-addition" assay was modified as follows. MRC-5 cells were rinsed once with 1 ml of EMEM-FP and 500 µl of EMEM-FP supplemented with 4 µM K22 was added. The compound was incubated with cells for 2 h at 37°C either prior to, during or after a 2 h period of infection of cells with ∼100 PFU of 229E virus in 500 µl of EMEM-FP. The cells were washed once with 1 ml of EMEM-FP after each 2 h period of their incubation with compound and/or virus. Finally, the cells were overlaid with the MC solution, and after incubation for 2 days at 37°C stained with crystal violet to visualize the viral plaques. RT-PCR {#s4i} ------ The RT TaqMan PCR was carried out as described by Brittain-Long et al. [@ppat.1004166-BrittainLong1]. Briefly, the extraction of RNA was conducted in the Magnapure LC robot using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Mannheim, Germany), and amplification was performed using a TaqMan 7300 Real Time PCR system (Applied Biosystems, Foster City, CA), with a pair of forward 5′-CAGTCAAATGGGCTGATGCA-3′ and reverse 5′-AAAGGGCTATAAAGAGAATAAGGTATTCT-3′ primers as well as a probe 3′CCCTGACGACCACGTTGTGGTTCA 5′ specific for HCoV-229E genome fragment coding for nucleocapsid protein [@ppat.1004166-Gunson1]. The number of HCoV-229E RNA copies was determined by relating the detected cycle threshold values to a standard curve prepared based on five tenfold dilutions of the specific plasmid (pUC57) comprising a 94 bp insert from the nucleocapsid sequence of HCoV-229E. qRT-PCR assays to quantify SARS-CoV and MERS-CoV genomic RNA have been described previously [@ppat.1004166-Kindler1]. Preparation of drug-resistant variants of HCoV-229E and sequencing analysis {#s4j} --------------------------------------------------------------------------- A procedure described previously for respiratory syncytial virus [@ppat.1004166-Lundin1] was used. Briefly, plaque purified HCoV-229E was subjected to 10--13 consecutive passages in MRC-5 cells in the presence of increasing concentrations (2--16 µM) of K22. For control purposes, the same virus was also passaged in MRC-5 cells in the absence of inhibitor. The virus was then subjected to two rounds of plaque purification in the presence of inhibitor, and its relative drug-resistance tested using the viral plaque reduction assay. Genomic RNA of original, mock-passaged, and the K22-resistant virus from passage 10--13 was extracted from extracellular fluid of the 229E-infected MRC-5 cells using the QIAamp viral RNA purification kit (Qiagen). Overlapping DNA fragments covering the entire coding sequence were produced by reverse transcription PCR and subjected to nucleotide sequencing using the ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems). Nucleotide sequence analysis was performed using Sequencher 4.9 software (Gene Codes Corporation). HCoV-229E replication kinetics {#s4k} ------------------------------ MRC-5 cells growing in 12 well plates were precooled for 15 min at room temperature and for another 15 min at 4°C. The cells were rinsed once with 500 µl of cold EMEM-FP and inoculated with concentrated preparation (see the Cells and Viruses section) of HCoV-229E (moi = 0.05). Following virus adsorption to cells for 1 h at 4°C, the cells were rinsed thrice with 500 µl of cold EMEM-FP, and 500 µl of warm EMEM-FP medium was added. The supernatant fluid and infected cells were harvested at specific time points relative to the end of the virus adsorption period, and processed for determination of viral RNA and infectivity as described under the "time-of-addition" assay. Ribonuclease treatment of HCoV-229E {#s4l} ----------------------------------- The infectious culture medium comprising HCoV-229E or recombinant nsp6 mutant HCoV-229E M159V were clarified by centrifugation at 1000×g for 5 min, and then 100 µl volumes of the supernatant were supplemented with 2 µl (20 µg) of ribonuclease A (Thermo Fisher Scientific; EN0531) or its solvent. All samples were spiked with ∼7 µg of RNA purified from human respiratory syncytial virus (RSV) to serve as an internal control of ribonuclease activity. Following incubation of the virus-enzyme mixture for 30 min at 37°C, the coronaviral and RSV RNA were quantified by RT TaqMan PCR as described by Brittain-Long et al. [@ppat.1004166-BrittainLong1] while coronavirus infectivity was determined by plaque titration. Autophagy {#s4m} --------- To assess the time-frame where autophagy vesicle formation occurs we seeded Huh-7 cells (100.000 cells) on glass bottom 12-well cluster plates (MatTek). Forty-eight hours prior to stimulation cells were transfected with LC3B-GFP plasmid [@ppat.1004166-Kabeya1] using lipofectamine2000 (Invitrogen), according to manufactures protocol. Hereafter cells were exposed to 100 nM of rapamycin (Invivogen) alone or in presence of either 20 µM of K22 or an equal volume of DMSO for the duration of 18 hours at 37°C. Fluorescent and differential interference contrast (DIC) images were acquired with 30 minute interval using EC Plan Neo-fluar 40x/1.30 Oil DIC M27 objective on a Zeiss LSM 710 confocal microscope. Image capture, analysis and processing were performed using the ZEN 2010 (Zeiss). To determine whether K22 inhibits endogenous autophagy vesicle formation we stimulated Huh-7 cells (40.000 cells) with 100 nM of rapamycin alone or in presence of either 20 µM of K22 or an equal volume of DMSO for duration of six hours at 37°C. Unstimulated cells were used as mock control. Cells were fixed and immunostained as previously described [@ppat.1004166-Dijkman3]. Rabbit polyclonal anit-LC3B (L7543, Sigma Aldrich) was applied as primary antibody for the detection of autophagy vesicles. Goat derived, Cy3 labeled, anti-rabbit IgG (H+L; Jackson ImmunoResearch) was applied as secondary antibody. Thereafter cells were counterstained with DAPI (Invitrogen). Fluorescent images were acquired using a PLAPON 60xO/1.42 objective on an Olympus FV-1000 confocal microscope. Image capture, analysis and processing were performed using the Olympus Fluoview software. Electron microscopy {#s4n} ------------------- MRC-5 cells growing on a Melinex polyester film (Agar Scientific Ltd., Stansted, U.K.) in 24 well cluster plates were infected with HCoV-229E (moi = 0.04) in the presence of 10 µM of K22. After 18 h of infection at 37°C, the culture medium was removed, the cells rinsed twice with Eagle\'s medium, and a fresh Eagle\'s medium supplemented with 2.5% glutaraldehyde was added and incubated for 45 min at 37°C. The cells were washed twice with 0.05 M Tris-HCl buffer (pH 7.4) supplemented with 2 mM CaCl2, and further processed for electron microscopy as described [@ppat.1004166-Widehn1]. Experiments with recombinant nsp6 mutant viruses and original virus were carried out in a similar manner except that the cells were inoculated at a moi of ∼0.25 and incubated with or without the presence of 4 µM K22. Supporting Information {#s5} ====================== ###### **J15 structure, antiviral activity, and cytotoxicity.** (**A**) J15 structure. (**B**) Anti-HCoV-229E activity and cytotoxicity of J15 in MRC-5 cells. J15 and wild type (WT) HCoV-229E or nsp6 recombinant HCoV-229E^M159V^ (M159V) were added to MRC-5 cells, and the number of viral plaques developed after 48 h were assessed. For cytotoxicity assessment, MRC-5 cells were incubated with J15 for 48 h at 37°C and the cell viability determined using tetrazolium-based reagent. Data shown are means (±SD) of duplicate determinations from two independent experiments. PFU, plaque forming unit. (TIF) ###### Click here for additional data file. ###### **Ribonuclease treatment of HCoV-229E.** Infectious culture medium comprising wild type HCoV-229E or mutant nsp6 recombinant HCoV-229E^M159V^ (M159V) was spiked with RNA purified from human respiratory syncytial virus (RSV) and then incubated for 30 min at 37°C in the presence of ribonuclease A (RNase) or without this enzyme (mock). The number of copies of coronaviral RNA (A) or control RSV RNA (B) was determined by qPCR while titer of infectious coronavirus (C) by viral plaque assay. Data shown are means (±SD) of four determinations obtained in four independent experiments (qPCR) or duplicate determinations from two independent experiments (infectivity). PFU, plaque forming unit; n.d., not detectable; n.s., not significant. (TIF) ###### Click here for additional data file. ###### **J15 affects formation of double membrane vesicles (DMVs).** MRC-5 cells growing on Melinex polyester film were infected with wild type HCoV-229E (WT) or with K22-resistant recombinant nsp6 mutant HCoV-229E^M159V^ (M159V) and incubated for 18 h at 37°C with or without J15. The cells were then fixed with glutaraldehyde and processed for electron microscopy without their scrapping or pelleting. (**A**) Electron micrographs of cells infected with WT virus show presence of clusters of DMVs (arrow) and viral particles (arrowhead), and the lack of their production upon J15 treatment (4 µM). (**B**) Electron micrographs of MRC-5 cells infected with K22-resistant recombinant nsp6 mutant M159V showing presence of DMVs and viral particles irrespective of the addition of J15. (TIF) ###### Click here for additional data file. ###### **K22 does not inhibit autophagy vesicle formation.** To determine whether K22 inhibits autophagy vesicle formation Huh-7 cells were stimulated with rapamycin alone or in presence of either 20 µM of K22 or an equal volume of DMSO solvent for 6 h at 37°C. Unstimulated cells were used as mock control. Fixed cells were stained with Anti-LC3B (red) and DAPI (blue) to annotate autophagy vesicles and cell nucleus, respectively. (TIF) ###### Click here for additional data file. ###### **K22 affects replication of diverse coronaviruses including MERS-CoV.** (**A-D**) The antiviral activity (bars) and cell toxicity (data points above bars) of K22 (black bars) or DMSO solvent (white bars) during MHV-Gluc (**A**), FCoV-RL (**B**), SARS-CoV (**C**) and IBV (**D**) infection on representative continuous cell lines of murine (L-929 cells; **A**), feline (FCWF cells; **B**), or primate (Vero cells; **C-D**) origin. Data are shown as mean (±SD) of a representative experiment, from two independent experiments performed in triplicate. (**E-F**). The antiviral activity (bars) and cell toxicity (data points above bars) of K22 (black bars) or DMSO solvent (white bars) in HCoV-229E-ren (**E**) and MERS-CoV (**F**) infected differentiated human airway epithelial (HAE) cultures. Data are shown as mean (±SD) of three independent experiments performed in triplicate (viral yield), or mean (±SD) of a representative experiment, from two independent experiments performed in triplicate (cell viability). Ns, not significant (*P*\>0.05); \* *P*\<0.05; \*\* *P*\<0.01 (paired t-test). (TIF) ###### Click here for additional data file. ###### **K22 exhibits little or no activity against poliovirus 1.** GMK AH1 cells were pretreated with K22 (black bars) or DMSO solvent (white bars) for 4 h at 37°C and then infected with poliovirus 1 Sabin strain at a moi of 0.001. Following incubation of infected cells in the presence of K22 or DMSO for 48 h at 37°C, the titer of extracellular infectious virus in culture medium was determined. The results shown are means of duplicate determinations from two separate experiments. TCID~50~, tissue culture infectious dose. (TIF) ###### Click here for additional data file. ###### **Effect of K22 on proliferation and viability of cultured cells.** (DOCX) ###### Click here for additional data file. We are grateful to Dr. Regulo Rodriguez and Dr. Christoph Zeisel, Kantonal Hospital St.Gallen, Switzerland, for their support to obtain human lung tissue, to Sibylle Widehn, Department of Pathology, University of Gothenburg, Sweden, for help with electron microscopy, and to Dr. Alexandra Trkola, Dr. Silke Stertz and Dr. Jovan Pavlovic, Institute of Medical Virology, University of Zürich, Switzerland, for their generous help and availability of the BSL3+ facility. [^1]: JK is employed by a commercial company, Merck Animal Health. This does not alter our adherence to all PLOS Pathogens policies on sharing data and materials. [^2]: Conceived and designed the experiments: AL TB CH BA VT ET. Performed the experiments: AL RD EK HRJ JK DM. Analyzed the data: AL BA CH NK ET VT RD EK HRJ DM MAM CD MF. Contributed reagents/materials/analysis tools: NK. Wrote the paper: VT ET RD AL. [^3]: Current address: Avian Viral Diseases Programme, The Pirbright Institute, Compton Laboratory, Compton, United Kingdom

{ "pile_set_name": "PubMed Central" }

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"ppatanand", "lsm", "efficaci", "cytoplasm", "environ", "ic", "dose", "predict", "free", "timeofaddit", "catalog", "consist", "essenti", "min", "observ", "zealand", "rdrp", "hcovinf", "reversetranscrib", "analys", "san", "β", "prolifer", "question", "lumen", "ppatwidehn", "occur", "emerg", "impact", "dilut", "hcov", "tight", "propag", "like", "variant", "largest", "strong", "analog", "phenotyp", "ppattann", "μmppatgppatg", "appear", "anim", "place", "pose", "cellderiv", "accessori", "ccic", "agar", "±", "tabl", "antigoat", "induc", "click", "nahpo", "coronaviru", "exhibit", "evalu", "larg", "autophagi", "membranebound", "ribonucleas", "resistanceconf", "trkola", "pfa", "one", "emem", "understand", "fusion", "data", "transcript", "christoph", "favor", "βtubulin", "gh", "last", "immunostain", "sppatsreftypesupplementarymateri", "treat", "parent", "particleinactiv", "pathogen", "supplementari", "comparison", "cycl", "ad", "extracellular", "ek", "stanst", "mm", "ppatbrittainlong", 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"therefore", "attractive", "targets", "antiviral", "intervention", "Antiviral", "strategies", "mainly", "proposed", "targeting", "coronavirus", "entry", "essential", "enzymatic", "functions", "coronavirus", "protease", "RNA", "polymerase", "RdRp", "activities", "example", "spike", "protein", "mediates", "binding", "different", "HCoVs", "specific", "cellular", "receptors", "event", "associated", "preferential", "virus", "tropism", "either", "ciliated", "cells", "airway", "epithelium", "protein", "also", "mediates", "fusion", "lipids", "viral", "envelope", "host", "cell", "plasma", "membrane", "membranes", "endocytic", "vesicles", "promote", "delivery", "viral", "genomic", "RNA", "cytoplasm", "Virus", "binding", "cell", "entry", "events", "inhibited", "antibodies", "directed", "protein", "antibodies", "small", "molecules", "interfering", "virus", "receptors", "synthetic", "peptides", "derived", "heptad", "repeat", "regions", "protein", "reviewed", "Following", "virus", "entry", "coronavirus", "genome", "positive", "sense", "capped", "polyadenylated", "RNA", "strand", "directly", "translated", "resulting", "synthesis", "coronavirus", "replicase", "nsps", "Coronavirus", "nsps", "translated", "two", "large", "polyproteins", "harboring", "proteolytic", "enzymes", "namely", "proteinases", "extensively", "process", "coronavirus", "polyproteins", "liberate", "nsps", "nsp", "proteolytic", "functions", "considered", "essential", "coronavirus", "replication", "consequently", "number", "candidate", "drugs", "reported", "inhibit", "coronavirus", "polyprotein", "processing", "Likewise", "coronavirus", "RdRp", "activities", "reside", "considered", "essential", "coronavirus", "replication", "attractive", "targets", "antiviral", "intervention", "addition", "classical", "drug", "targets", "coronaviruses", "encode", "array", "enzymes", "representing", "additional", "candidate", "targets", "include", "helicase", "activity", "linked", "NTPase", "activity", "activity", "linked", 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"double", "membrane", "vesicles", "DVM", "interconnected", "via", "outer", "membrane", "rough", "ER", "Indeed", "Angelini", "colleagues", "recently", "shown", "three", "transmembrane", "nsps", "required", "induce", "DMVs", "similar", "observed", "cells", "compartments", "harboring", "replication", "complexes", "show", "remarkable", "parallels", "amongst", "broad", "range", "RNA", "virus", "families", "potentially", "evolutionary", "linked", "similar", "mechanisms", "life", "cycle", "ds", "RNA", "cytoplasmic", "replicating", "DNA", "viruses", "Coronavirus", "DMVs", "induced", "early", "virus", "entry", "host", "cell", "cytoplasm", "display", "striking", "similarities", "DMVs", "induced", "hepatitis", "C", "virus", "evolutionary", "conservation", "engaging", "host", "membranous", "structures", "virus", "RNA", "synthesis", "genetic", "evidence", "impairment", "coronavirus", "DMV", "integrity", "associated", "severe", "reduction", "virus", "replication", "suggest", "antiviral", 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1. Introduction {#s0005} =============== The leishmaniases, caused by protozoan parasites of the genus *Leishmania* (Kinetoplastida: Trypanosomatidae), are worldwide vector-borne diseases transmitted by phlebotomine sand flies. *Leishmania* spp. infect a wide range of hosts including sylvatic and domestic animals. In humans, the leishmaniases are an enormous public health problem with a global prevalence of 12 million cases and a yearly incidence of 2 million cases ([@b0120]). Infection with *Leishmania* can cause a broad spectrum of clinical presentations ranging from asymptomatic to simple cutaneous or destructive mucocutaneous lesions, or severe visceral leishmaniasis (VL) that is fatal without effective chemotherapy. Historically, the population structure of *Leishmania* has been considered to be fundamentally clonal ([@b0110; @b0105]) but with some limited historical evidence of recombination, for example, between *Leishmania braziliensis* and *Leishmania panamensis/guyanensis*, from multilocus enzyme electrophoresis (MLEE) and random amplification of polymorphic DNA ([@b0015; @b0010]). Diversity observed within natural populations was thus generally attributed to the accumulation of mutations over time, with perhaps rare instances of genetic exchange. Nevertheless, evidence of genetic exchange between different *Leishmania* spp. in natural populations has been reported on several occasions ([@b0015; @b0040; @b0010; @b0080; @b0065; @b0100; @b0075]). However, at the intra-species level, the identification of hybrids among natural populations has been more difficult and more equivocal due to the limited discriminatory power of methods such as MLEE. A few recent studies characterising natural *Leishmania* populations using highly polymorphic microsatellite markers have suggested the presence of genetic exchange ([@b0020; @b0030]). In addition, [@b0090] described evidence of inbreeding in natural populations of both *Leishmania* (*Viannia*) *braziliensis* and *Leishmania* (*Viannia*) *guyanensis* based on linkage disequilibrium (LD), yet with a deficit of heterozygosity. The proposed reproductive strategy is therefore an alternating model of clonality in both the vertebrate and invertebrate hosts with non-obligatory sexual recombination in the sand fly. Whole genome sequencing of vector-isolated *Leishmania infantum* from southeastern Turkey supported the occurrence of hybridization and subsequent selfing; one of the parental genotypes was not identified but was likely to be from the *Leishmania donovani* complex ([@b0085]). The extant capacity of *Leishmania* spp. to undergo genetic exchange in the sand fly has been proven by generating experimental *Leishmania major* hybrids following co-passage of transgenic strains ([@b0005; @b0035]). Hybrids isolated from the sand fly gut had genotypes consistent with meiosis. Furthermore, experimental crosses of fluorescent transgenic *L. donovani* in the sand fly produced dual expression in single cells, consistent with intra-species genetic exchange ([@b0095]). Genetic exchange has potential implications for heterosis (hybrid vigour), the emergence and spread of virulent strains, resistance to chemotherapeutics, exploitation of different hosts and vectors, and adaptation to new ecological niches that may provide a selective advantage. For example, a startling observation by [@b0115] provided clear evidence that *L. infantum/major* hybrids possess enhanced transmission potential. *Leishmania infantum* is not normally able to infect the broad ranging *Phlebotomus papatasi* but, remarkably, the resultant hybrids were able to do so with potentially profound epidemiological consequences. *Leishmania braziliensis*/*Leishmania peruviana* hybrids have also been implicated as agents of destructive forms of mucocutaneous leishmaniasis ([@b0065]). The work described here was designed to accomplish three main objectives applied to a panel of 11 natural isolates of north Ethiopian origin: (i) to characterise four putative hybrids using a range of high resolution markers; (ii) to identify likely parental genotypes among seven north Ethiopian *L. donovani* representatives that did not have hybrid-like microsatellite genotypes; and (iii) to discount the possibility that hybrid-like microsatellite genotypes were the result of mixed infections. We applied a novel combination of multilocus sequence typing (MLST) targets and highly resolutive multilocus microsatellite typing (MLMT) markers ([@b0045; @b0030]) in conjunction with mitochondrial (maxicircle) sequencing and DNA content analysis. We demonstrate that four natural isolates are bona fide hybrids and not the result of mixed infection. The likely parental origins and epidemiological implications are discussed. 2. Materials and methods {#s0010} ======================== 2.1. Parasite strains and DNA isolation {#s0015} --------------------------------------- A panel of 11 north Ethiopian *L. donovani* isolates was available, originating from a population of Ethiopian isolates containing some putatively characterised as hybrids by [@b0030], participants were enroled according to written informed consent procedures and approved by the Institutional Review Boards of the Faculty of Medicine, Addis Ababa University, Addis Ababa. Full World Health Organization (WHO) isolate labels are shown in [Table 1](#t0005){ref-type="table"} and isolate characteristics are shown in [Supplementary Table S1](#s0075){ref-type="sec"}. Isolates that were available comprised four putative hybrids, DM19, DM62, DM295 and DM299. Two of the hybrids were isolated from an HIV-infected patient; DM62 and DM299 were isolated from the same HIV/VL co-infected patient during different episodes of the disease. Parent-like representatives were DM20, DM297 (designated hypothetical parent group A), and DM256, DM257, DM259, DM481, DM559 (designated hypothetical parent group B, [Fig. 1](#f0005){ref-type="fig"}, [Table 1](#t0005){ref-type="table"}). A total of 90 biological clones were generated, a maximum of 10 per isolate (DM19, eight clones; DM62, 10 clones; DM256, nine clones; DM257, nine clones; DM259, nine clones; DM295, nine clones; DM 297, nine clones; DM299, eight clones; DM481, nine clones; DM599, 10 clones). *Leishmania* were cultured in supplemented RPMI liquid medium at 28 °C, as previously described ([@b0055]). Isolates were then cloned on solid media as described by [@b0125]. Once single colonies became visible they were removed and inoculated into liquid culture medium. DNA was isolated from each clonal culture using DNA isolation kits (Promega, UK) following the manufacturer's instructions. 2.2. MLMT {#s0020} --------- Five microsatellite markers were selected ([Table 2](#t0010){ref-type="table"}) based on their ability to distinguish putative hybrids from parents ([@b0030]). Fluorescent-conjugated forward and non-conjugated reverse primers (Eurofins, Germany) were used for amplification. The amplification conditions and reaction cycles were performed as described by [@b0070] with slight modifications. After an initial denaturation step at 95 °C for 10 min, samples were processed through 35 cycles consisting of 95 °C for 30 s, annealing for 30 s at the temperature (*T~A~*) indicated in [Table 2](#t0010){ref-type="table"} and 72 °C for 1 min, followed by a terminal elongation step at 72 °C for 10 min. Each reaction was performed in a final volume of 20 μl consisting of 1× ThermoPol reaction buffer (New England Biolabs (NEB), UK), 1.5 mM MgCl~2~, 0.2 mM dNTPs, 10--25 pmol of each primer, 1 unit of *Taq* polymerase (NEB, UK) and 20--30 ng of genomic DNA. Allele sizes were estimated using an automated capillary sequencer (ABI3730, Applied Biosystems, UK) and were checked manually. 2.3. MLST {#s0025} --------- MLST markers were amplified from genomic DNA extracted from the isolates and biological clones. [Table 3](#t0015){ref-type="table"} summarises PCR amplification primers and annealing temperatures. Genetic markers consisted of four coding nuclear markers (glucose-6-phosphate isomerase, *GPI*; isocitrate dehydrogenase, *ICD* ; *Ch28; Ch36-1190*) and one non-coding nuclear marker (*Ch36-0350*). Targets were chosen following screening of a panel of markers known to exhibit sequence diversity within the *L. donovani* complex on the basis of GenBank sequences. Amplifications were performed with an initial denaturation step at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, with annealing and elongation steps for each marker as described herein, and a final elongation step at 72 °C for 10 min. The annealing temperatures for *GPI* and *ICD* were 57 °C and 61 °C, respectively, for 90 s and for *CH28-0190, CH36-1130 and CH36-0350* were 53 °C, 59 °C and 61 °C, respectively, for 60 s. Each reaction was performed in a 20 μl total volume containing: 20 ng of genomic DNA, 20 pmol of each primer, 2 mM dNTPs, 1.5 mM MgCl~2,~ and 5 U *Taq* (BIO-21086, Bioline, UK). PCR products were visualised on 1.5% agarose gels and the appropriate bands excised and purified using a QIAGEN Gel Extraction Kits (Qiagen, UK) or SureClean (Bioline). Bi-directional sequencing was performed, using internal primers where required ([Table 3](#t0015){ref-type="table"}), with Big Dye Terminator Cycle Sequencing V3.1 (Applied Biosystems) and an ABI PRISM 377 DNA Sequencer (Applied Biosystems) according to the manufacturer's protocols. Sequence data were assembled manually in BioEdit v7.0.9.0 (Ibis Biosciences, USA) and ambiguous peripheral regions of aligned sequences were discarded to produce unambiguous partial gene sequences for each isolate or clone. Chromatograms were examined visually in both directions for the presence of heterozygous bi-allelic single nucleotide polymorphisms (SNPs) at a single locus ([Fig. 2](#f0010){ref-type="fig"}). Re-sequencing was undertaken if results were ambiguous. SNP differences between isolates and clones were recorded and tabulated together with their associated positions ([Fig. 2](#f0010){ref-type="fig"}). 2.4. Mitochondrial maxicircle gene sequencing (mMLST) {#s0030} ----------------------------------------------------- The cytochrome b gene (*CYTb*) was amplified and sequenced using primer pairs LCBF: GGTGTAGGTTTTAGTTTAGG and LCBR2: CTACAATAAACAAATCATAATATACAATT ([@b0050]). Cycle conditions consisted of an initial denaturation at 94 °C for 30 s followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 68 °C for 5 min and a final extension step at 72 °C for 7 min. The amplified fragments were sequenced in both directions using the same set of primers and following the sequencing methods described above. 2.5. DNA content analysis {#s0035} ------------------------- Approximately 1 × 10^7^ cloned *L. donovani* parasites were harvested from mid-log phase liquid cultures. Parasites were pelleted by centrifugation at 3,000*g* (20 min), washed twice in ice-cold PBS and fixed with 100% methanol for 48 h at 4 °C. Two subsequent washes with ice-cold PBS were performed, after which cells were resuspended in PBS to a final density of 1 × 10^6^ cells/ml. Propidium iodide and RNAse A were added to a final concentration of 10 μg/ml and the mixture incubated for 45 min at 37 °C in the dark. Fluorescence was detected using a FACSCalibur flow cytometer, on channel FL2. For each sample a minimum of 10,000 events was counted. For each strain/clone, two independent assays were performed. Data analysis used FlowJo software (Tree Star Inc., Oregon, USA). Gates were created for G1-0 (2*n*) peaks and for G2-M (4*n*) peaks. Mean G1-0 values were taken to infer relative DNA content. Relative DNA content values were calculated as a ratio in comparison with an internal standard (*L. major,* Friedlin strain). For putative hybrids, the ratios relative to each putative parent (A or B) were also recorded. 3. Results {#s0040} ========== 3.1. MLMT hybrids, parental genotypes and exclusion of mixed infections {#s0045} ----------------------------------------------------------------------- Uncloned stocks from Ethiopia had identified a subset of putative hybrids possessing heterozygous profiles, with MLMT genotypes composed of alleles that were otherwise restricted to two distinct *L. donovani* populations in northern Ethiopia ([@b0030]). Heterozygous genotypes could have been the result of amplification of multiple alleles from multiclonal sources. We therefore prepared a maximum set of 10 biological clones for each of the 11 selected strains and conducted MLMT analyses. MLMT profiles across five loci are summarised in [Table 1](#t0005){ref-type="table"}. Overall in the four putative hybrids, MLMT analyses of clones showed high levels of heterozygosity that were not a result of multiclonality, in stark contrast with the high levels of homozygosity in putative parents (group A and group B). In more detail, the two non-hybrid (group A) strains and all of their derived clones were consistently homozygous across the five microsatellite markers. Similarly, the five non-hybrid (group B) strains and derived clones were generally homozygous across the five loci, with the exception of the heterozygous genotypes DM259 at locus Li41-56 and DM559 at loci Li41-56 and Li71-7. Three of the four hybrid strains (and their biological clones) were heterozygous across all five loci. The exception was DM19, which was heterozygous in three loci (Li46-67, Li22-35 and Li71-33) and homozygous at Li41-56 and Li71-7. Thus the heterozygous alleles in the five hybrid microsatellite loci matched with corresponding homozygous alleles among the parent-like isolates. However there were four examples where the origins of donor alleles in the putative hybrids could not be determined. As shown in [Table 1](#t0005){ref-type="table"}, these were: (i) the 103 bp allele at the heterozygous Li71-33 site in the hybrids was absent from both putative parental groups; (ii) for the heterozygous loci at Li41-56 (89/91), Li46-67 (70/72) and Li71-7 (90/92) in the hybrids one allele, 89, 72 or 92, respectively, could be found among both parental groups; and (iii) at locus (Li71-7), one isolate among parental group B was heterozygous (90/92). An exception was also seen at a single site for the MLST target *ICD* (see Section [3.2](#s0050){ref-type="sec"}). Taken alone, microsatellite data were less robust than MLST data (Section [3.2](#s0050){ref-type="sec"}) in identifying likely parental donors. 3.2. MLST comparisons reveal hybrids and putative parents {#s0050} --------------------------------------------------------- Although MLMT data detected consistent hybrid profiles, allele sharing across hypothetical non-hybrid groups (A and B) meant that inferring likely parental multilocus genotype donors was not possible. We therefore sought to analyse nucleotide sequences from several nuclear loci, reasoning that patterns of SNP marker inheritance would be more discriminatory. Gene identities (IDs) were confirmed by BLAST searches and submitted to TriTypDB 4.2 (<http://TriTrypDB.org>) to determine chromosomal locations. Locations for each of the nuclear gene fragments are shown in [Table 3](#t0015){ref-type="table"}. Two gene fragments (*Ch36-0350 and Ch36-1190*) were located on chromosome (Ch)36 (P:95153--94109 and P:429635--431277, respectively). The remaining genes were on independent chromosomes 10, 12 and 28. Nucleotide sequence data reported in this paper are available in the GenBank™ database under the Accession Nos. KJ907394- KJ907448. We characterised a total of 90 biological clones originating from both the putative hybrids (four isolates) and parent-like strains (seven isolates). The *GPI* locus was monomorphic and homozygous across all 11 strains and for the 90 biological clones; *GP*I was therefore not included in subsequent analysis. A summary of the distribution of SNPs and their relative positions in the remaining four loci is shown in [Fig. 1](#f0005){ref-type="fig"}. A total of 34 unambiguous SNP positions were detected across all loci. Locus *Ch28* was the most diverse fragment with 18 SNPs, followed by *Ch36-1190* with eight, *ICD* with five and *Ch36-0350* with three. Heterozygous sequence profiles were found only in the hybrids; 16 SNPs displayed bi-allelic heterozygous profiles in at least one isolate: locus *Ch28* with one such heterozygous site, *Ch36-1190* with seven, *ICD* with five and *Ch36-0350* with three. In contrast the putative parent-like isolates (A and B) were homozygous across all SNP positions. The high number of heterozygous sites seen in the hybrids, in comparison with the relative rarity of heterozygosity in the putative parents, is consistent with the patterns of heterozygosity and homozygosity seen in the MLMT data. Each of the four hybrid strains had a unique multilocus genetic profile. Interestingly, even hybrid isolates DM62 and DM299, which were isolated sequentially from the same patient, had distinct *Ch36-0350* and *ICD* sequences, with one and four SNP differences, respectively, suggesting either multiclonality in this patient or clonal diversification in vitro. Variable sites were then examined with respect to their presence/absence in strains and clones from the hybrid group ([Fig. 1](#f0005){ref-type="fig"}). Three classes of marker distribution were observed. In some cases SNP distribution in hybrid group strains was consistent with acquisition of a group A and a group B allele, for example, DM62, DM299 and DM295 had heterozygous sequences compatible with inheritance of an A-like and a B-like *Ch36* sequence. In other cases the hybrid group strains were homozygous for a group A-like or a group B-like sequence, for example, DM19 for the *Ch28* and *Ch36* loci and DM299 for the *ICD* locus. There were also several instances where the sequences in hybrid group strains were predominantly homozygous for an A-like or B-like sequence but with clear heterozygosity at the extreme 5′ and/or 3′ regions, e.g. DM62 and DM299 had *Ch28* sequences that were homozygous for A-like SNPs at all informative positions except the most 5′ site (position 273) for which it had both an A-like and B-like SNP. Finally, both heterozygous sites for *ICD* in DM62, DM295 and DM299 were not present in non-hybrid groups A and B and were not informative with respect to inheritance. Thus, although it is clear the MLST data overall support the hypothesis that the hybrid group strains are derived from a hybridization event between group A and group B, the distribution of specific markers suggests additional complexity that may be a result of several post-hybridization events including loss of heterozygosity due to gene conversion or subsequent rounds of hybridization (inbreeding), or that the diversity within groups A and B may not have been sampled adequately. We next examined the hybrid and parental strains in order to identify instances of strain-specific marker inheritance. There was only one intra-group SNP in non-hybrid group A (*Ch36-1190*, position 545, G/C); the 'G' allele was uniquely present in group A strain DM20 and three of the hybrid group strains, DM19, DM62 and DM299 ([Fig. 1](#f0005){ref-type="fig"}). This indicates that DM20 is a more similar candidate A parent of these hybrids than DM297. There was a greater degree of intra-group variation in non-hybrid group B. The group B-like *Ch28* and *ICD* alleles present in hybrid group strain DM295 were clearly most closely related to the group B strain DM481. Where informative markers were present for hybrid group strains DM62, DM299 and DM19, the sequences indicated higher identity with group B strains DM256, DM257 and DM259 than DM481 or DM559. The high number of SNP differences separating the different likely B-like allele donors for DM295 and DM62, DM299 and DM19 suggest two independent hybridization events, although a single event followed by significant subsequent diversification cannot be ruled out. It is also possible that DM19 stems from a third independent event, particularly given its distinct MLMT profile. In summary the MLST data showed a pattern of marked but not fixed heterozygosity in the hybrids in comparison with the rarity of heterozygosity in non-hybrid group strains, similar to that observed for the MLMT data. Taken alone, microsatellite data are less robust than MLST data in identifying likely parental contributors. But taken together, both the MLMT and MLST data indicate overwhelmingly that strains DM19, DM62, DM295 and DM299 are genetic hybrids. The distribution of SNP markers allowed some tentative parent-hybrid relationships to be identified and indicated the hybrid group strains have either three independent origins or, less likely, a single origin with extensive subsequent divergence. 3.3. Kinetoplast DNA (kDNA) maxicircle-encoded CYTb sequences support uniparental mitochondrial inheritance {#s0055} ----------------------------------------------------------------------------------------------------------- SNP markers were identified in a region of the *CYTb* gene to determine inheritance patterns of the mitochondrial maxicircles. Six SNPs were identified spanning a 748 bp fragment of the *CYT*b locus. Relative positions were p19 (G, T), p350 (A, T), p499 (G, A), p566 (A, C), p583 (A, G) and p649 (G, A). Two maxicircle genotypes were present among the isolates and clones studied here, with either TTACGA or GAGAAG SNP profiles across the six SNP sites. All hybrids and derived hybrid clones (a minimum of four clones per strain) had the type GAGAAG SNP profile, consistent with uniparental inheritance of the maxicircle DNA, in contrast to the biparental inheritance of the nuclear markers. There were no other combinations of maxicircle profiles and there was no heterozygosity. The GAGAAG maxicircle profiles found in the hybrid group strains were identical in both non-hybrid A strains (DM20, DM297) and in group B strains (DM559, DM481). Within group B, both mitochondrial genotypes were present. 3.4. Flow cytometry measurement of cellular DNA content {#s0060} ------------------------------------------------------- An example of the DNA content determinations on fixed parasites of hybrid and parents, stained with the fluorescent DNA binding dye propidium iodide, is shown in [Fig. 2](#f0010){ref-type="fig"}. A reference diploid (*2n*) strain was included in each run and duplicate runs were performed. All isolates produced clear G1-0 (*2n*) and G2-M (*4n*) fluorescence peaks. There was no significant difference in relative DNA contents between any of the hybrid or non-hybrid strains. The results are a strong indication of diploidy in all clones that were analysed. 4. Discussion {#s0065} ============= Here we provide evidence for bona fide hybrid strains and ancestral-like genotypes selected from a natural population of *L. donovani* causing VL in northern Ethiopia. Our evidence is based on high resolution MLMT and novel MLST targets applied to the nuclear genomes of 11 north Ethiopian *L. donovani* isolates and 90 clones derived from those. Importantly, the genotypes of all of the clones corresponded with the genotypes of the original isolates, and these results were therefore not confounded by artefactual analysis of isolate mixtures. By comparing genetic profiles of the north Ethiopian isolates and their derivative clones, four of the isolates were shown to be hybrids. The hybrids were heterozygous at both MLST and MLMT nuclear loci, with significant corresponding homozygosity present among putative parental strains of groups A and B. Breakpoints within individual alleles were not detected when applied to recombination algorithms (RDP V4), suggesting the inheritance of a discrete allele from each of the parents. Strikingly, the heterozygosity in the MLST data from hybrids almost precisely matched the homozygous SNPs at the same sites in the deduced pairs of parents. Results were mirrored in the MLMT analysis, although the technique is less robust and not suitable to fully resolve parents and donors using the five loci. However, together MLST analysis and MLMT spanning nine loci proved to be powerful and complementary techniques for identifying hybrids of *L. donovani*. As such, the novel combination of markers may be useful for screening whole populations for hybrids when sequencing is not feasible. Not all of the four *L. donovani* hybrid isolates were identical and there were varying degrees of SNP differences between them. Two hybrids (DM62 and DM299) had very similar genetic profiles, possibly originating from the same event. Remaining hybrids (DM295 and DM19) were more distinct. Our data show that DM62 and DM299 appear to possess a different parent (B) allele than DM295 and we consider this suggests more than one recombination event. Hybrid DM19 was an unusual outlier exhibiting reduced heterozygosity, relative to the other hybrids, yet still retaining a hybrid signal in one MLST locus and two MLMT loci. One interpretation of these data is another recombination event, although long-term divergence from hybrids (DM62 and DM299) or divergence via gene conversion cannot be entirely excluded. Recent whole genome sequencing of 11 *L. infantum* isolates from a focus of cutaneous leishmaniasis (CL) in southeastern Turkey ([@b0085]) suggested that they derived from a single cross of two diverse strains followed by recombination within the population. Although one of the parental types was not present, it was likely to be from the *L. donovani* complex. They concluded that recombination in *L. infantum* is probably rare in the field as judged by the length of parental sequence blocks, and the pattern of linkage between SNPs. Concerning our data, the allelic contributions from both parental types were present and appear relatively conserved, also suggesting recent genetic exchange events. However, further sampling would be required at the population level to resolve these processes and to assess more accurately the presence and frequency of endogamy which, at high frequencies, would be characterised by a trend towards homozygosity within a population ([@b0090]). Not surprisingly, the genotypes considered here to be parental did not correspond precisely with those of predicted parents; for example one of the SNPs at the heterozygous *ICD* locus is missing from the putative parents. There are a number of explanations for differences between the hybrids and putative parent-like isolates. Firstly, it is most likely that multiple other genotypes circulating naturally were not sampled and some of these may be even more similar to the predicted parents. Secondly, some gene conversion or mutation is inevitable with time since hybridization, restoring homozygosity or generating divergent genotypes at some loci. Differences between the hybrids could be also accounted for by repeated selfing, although the largely corresponding heterozygous SNPs and corresponding homozygous "donors" in MLST and MLMT outputs make this unlikely. We also investigated the *L. donovani* maxicircle kDNA inheritance patterns by sequencing the *CYTb* locus. Several SNPs in *CYTb* were identified. Identified SNPs formed two distinct mitochondrial marker combinations among the non-hybrid *L. donovani* isolates and clones. As expected, for haploid mitochondrial markers no heterozygosity was observed. We found that all four hybrid isolates and their derivative clones only inherited a single maxicircle DNA genotype. Furthermore, this was the mitochondrial type genotype of DM20, one of the most likely parent-like genotypes of all four hybrids. Thus, we find uniparental inheritance of mitochondrial DNA. This accords with mitochondrial inheritance of the experimental hybrids of *L. major*, in which 12 of 18 hybrids inherited the mitochondrial genotype of one parent and six inherited the genotype of the other parent ([@b0005]). We determined the DNA contents of the hybrids relative to the putative parents by fluorescence activated cell sorting (FACS analysis). The FACS analysis demonstrated that hybrids, non-hybrids and resultant clones were all diploid. In contrast, stable hybrid aneuploids have been described in *L. major* following extensive serial passage in vitro ([@b0005; @b0035]). Reversion to diploidy over longer time periods cannot be ruled out and has been observed with aneuploid genomes that are a product of parasexual processes. One hybrid tetraploid clone of *L. major*, stable in vitro and passaged through mice, resulted in the recovery of diploids ([@b0035]). Although there is striking synteny at the gene content level between different *Leishmania* genomes, there is extensive variation in some at the level of chromosomes ([@b0025]) and the characteristics of recombination in *L. donovani* may not be identical to that seen in *L. major.* On balance, the hybrids observed here among the north Ethiopian population of *L. donovani* appear compatible with products of meiosis in that: there are high levels of heterozygosity relative to the parents, with clearly identifiable donor SNPs; one homologue is present from each parent and recombinant genotypes are observed across multiple loci, consisting of nuclear and microsatellite markers; there is uniparental maxicircle inheritance, and a majority 2*n* ploidy. The routine generation of experimental hybrids would be needed to fully address the nature of recombination in *L. donovani* and whether hybridization follows the pattern of orthodox meiosis. It is interesting to note that two of the reported isolates (Dm62 and Dm299) were from a single HIV co-infected patient during different episodes of the disease in northern Ethiopia. Although the two hybrids were similar, SNP differences were apparent. The presence of multiple genotypes in a single severely immunocompromised patient may increase the likelihood of recombination between genotypes within the vertebrate host ([@b0080]). Alternatively, immunocompromised individuals may be more susceptible to infection with a wider range of genotypes from a sand fly vector in which genetic exchange has occurred, as is supported by the experimental proof of capacity for genetic exchange in the sand fly. Additionally, an immunocompromised status may enable less fit hybrids to persist when they would otherwise be cleared from immune competent individuals ([@b0060]). The full consequences of genetic exchange in *L. donovani* are as yet uncertain. For example, do the hybrids described here also differ in their ability to exploit different hosts and vectors or cause differential pathology in humans? The *L. donovani* hybrid and parental isolates characterised provide an excellent opportunity for follow up analysis and for further experiments on hybridization, the mechanisms of genetic exchange and the associated generation of phenotypic diversity. Appendix A. Supplementary data {#s0075} ============================== Supplementary data 1 We wish to thank Bethlehem Getachew (Leishmaniasis Research and Diagnostic Laboratory, School of Medicine, Addis Ababa University, Ethiopia) for her help with parasite cultivation. We also thank the Leishmaniasis Research and Treatment Centres in Gondar University and Arba Minch Hospital, Ethiopia. We specifically thank Dr Zewdu Hurissa and Dr Workagegnehu Hailu (Gondar University) and Dr. Teklu Weldegebreal (Arba Minch Hospital) for their help during field studies. This work was supported by the Wellcome Trust. ![Summary of single nucleotide polymorphism distributions across four multilocus sequence typing loci (Chromosome (*Ch*)*28*, *Ch36-0350*, *Ch36-1190* and isocitrate dehydrogenase (*ICD*)) in *Leishmania* clones. Data from the glucose-6-phosphate isomerase (*GPI*) locus is not shown (all sequences were identical). Isolate names are abbreviated with World Health Organization strain codes shown in [Table 1](#t0005){ref-type="table"}. DM19, DM62, DM295 and DM299 hybrids possess heterozygous single nucleotide polymorphisms. DM20 and DM297 (in colour) represent parent-like A donors and DM256, DM257, DM259, DM481 and DM559 (in colour) represent possible parent-like B donors. Full IUPAC codes for heterozygote single nucleotide polymorphisms are as follows: K(G,T); M(A,C); R(G,A), S(G,C); Y(T,C). Numbers indicate the nucleotide position within the loci. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)](gr1){#f0005} ![Flow cytometric analysis and sequencing electropherograms. **(**A) Flow cytometric analysis of relative DNA content in *Leishmania* clones. Data is plotted as a FL2 area histogram with gates were created for G1-0 (2*n*) peaks and for G2-M (4*n*) peaks and overlaid DNA histograms illustrating comparable (2*n*) DNA content in hybrid and parent-like isolates. (B) Electropherograms showing two variable single nucleotide polymorphisms present in homozygous non-hybrids (DM20, DM256) and the equivalent bi-allelic locus present in hybrid DM62 at the same locus.](gr2){#f0010} ###### Microsatellite profiles at five loci of hybrids and their corresponding putative parents for *Leishmania* isolates. Strain WHO code Marker ----------------------------------- ---------------------------------------------------- -------- ------- --------- --------- ------- Hypothetical parent A (*n *= 2) MHOM/ET/2007/DM20 89/89 72/72 78/78 99/99 92/92 MHOM/ET/2008/DM297 83/83 70/70 78/78 113/113 92/92    Hybrid isolates (*n *= 4) MHOM/ET/2007/DM19[a](#tblfn1){ref-type="table-fn"} 91/91 70/72 78/98 99/103 92/92 MHOM/ET/2007/DM62 89/91 70/72 78/98 99/103 90/92 MHOM/ET/2008/DM295 89/91 70/72 78/98 99/103 90/92 MHOM/ET/2008/DM299 89/91 70/72 78/98 99/103 90/92    Hypothetical parental B (*n *= 5) MHOM/ET/2008/DM256 91/91 72/72 98/98 117/117 92/92 MHOM/ET/2008/DM257 91/91 72/72 98/98 117/117 92/92 MHOM/ET/2008/DM259 83/89 72/72 98/98 117/117 92/92 MHOM/ET/2009/DM481 89/89 72/72 98/98 113/113 92/92 MHOM/ET/2009/DM559 83/89 72/72 98/98 113/113 90/92 WHO, World Health Organization. Hybrid DM19 is heterozygous at three loci unlike DM62, DM295 and DM299, which are heterozygous across all five loci, as is congruent with multilocus sequence typing (MLST) data ([Fig. 2](#f0010){ref-type="fig"}). ###### Properties of microsatellite markers used in this study. Marker *T~A~* (°C) Chromosome Forward (5′--3′) Reverse (5′--3) --------- ------------- ------------ ------------------------ ----------------------- Li22-35 52 1 CTTGATGTTCGGGTTAGCAAG ATGCACACCAAAAATCATGTG Li41-56 50 36 TTGCTTCATGATAACAACTTGG CCTGTTGGTGTGAGTTCGTG Li46-67 50 31 TCTTCTTTCGTTAGCTGAGTGC CTGTATCACCCATGAGGGGC Li71-7 50 30 GCTGCAGCAGATGAGAAGG GTGAGAAGGCAGGGATTCAA Li71-33 50 31 CTCCTTTCACACCCGCCTCT GAGAGAAGACGAGCCGAAGT *T~A~*, temperature of annealing. ###### Multilocus sequence typing targets for *Leishmania donovani*. ------------------------------------------------------------------------------------------------------------------------------------------------------------------- Locus Chromosome number No. of polymorphic sites/SNPs Primer sequences (5′--3′) Annealing temp (*T~A~* °C) bp -------------------------------------------- ------------------- ------------------------------- ------------------------------ ---------------------------- ------ GPI[a](#tblfn2){ref-type="table-fn"} 12 0 F:GACCGAGGCACTTGAAG\ 57 1150 R6: TGAATGAGCTGGTAGAATG ICD[a](#tblfn2){ref-type="table-fn"} 10 5 F:ATGTTCCGCCATGTTTCGG\ 61 1267 R:TTACGCGCTCATCGCCTT Ch28[a](#tblfn2){ref-type="table-fn"} 28 18 INTF:GTCGCAGTCCAACTCCCATA\ 53 1530 INTR:CGCATAGCAAAAGCCAAA Ch36-1190[a](#tblfn2){ref-type="table-fn"} 36 8 F:GCTTCTCGCTATTGCTCGTC\ 59 1640 R:ACTGGCAGGCACACATCAG\ INTFA: GCGGCTACCTCGCCCTCAGT\ INTFB:GTGAAGGACCAAGCTGCCTGG\ INTRA;TGTGAAGCACCAGCAGGACGG Ch36-0350[b](#tblfn3){ref-type="table-fn"} 36 3 F:ACTTGGTCTTGGTACGG\ 61 850 R:TGGAGGACGGAGAGACTTTG\ INTF:GTGAATGGAGGGCAGACG\ INTR:GTCGTGAAAAGCGAGAAGGT ------------------------------------------------------------------------------------------------------------------------------------------------------------------- SNP, single nucleotide polymorphism; *T~A~*, temperature of annealing; *GPI,* glucose-6-phosphate isomerase; *ICD,* isocitrate dehydrogenase; *Ch*, chromosome. Coding nuclear marker. Non-coding nuclear marker.

{ "pile_set_name": "PubMed Central" }

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"linkag", "friedlin", "glucosephosph", "found", "kdna", "centrifug", "extract", "sought", "′′", "health", "differ", "detail", "vitro", "arba", "intern", "consid", "vari", "provid", "no", "potenti", "sylvat", "stark", "nonconjug", "undergo", "assembl", "known", "homozygos", "trend", "statu", "inocul", "miss", "four", "less", "experiment", "power", "individu", "subsequ", "level", "cytochrom", "strain", "bind", "intragroup", "atgcacaccaaaaatcatgtg", "media", "serial", "overlaid", "vectorborn", "agaros", "either", "mgcl", "mutat", "nine", "abil", "howev", "immun", "posit", "exchang", "denatur", "presenc", "respect", "without", "combin", "basi", "hailu", "trust", "biosystem", "inc", "compos", "aneuploid", "mhometdm", "visualis", "eurofin", "five", "herein", "chemotherapeut", "stem", "medicin", "propidium", "protocol", "eight", "differenti", "condit", "v", "wider", "includ", "associ", "particularli", "cellsml", "recombin", "show", "among", "equivoc", "yet", "distinguish", "microsatellit", "histogram", "model", "genbank", "mlee", "chromosom", "revers", "ii", "hivvl", "implic", "per", "braziliensi", "snp", "adapt", "make", "guyanensi", "autom", "minch", "vectorisol", "resist", "check", "ch", "diagnost", "pmol", "kinetoplast", "phase", "infer", "liquid", "p", "direct", "discriminatori", "electrophoresi", "produc", "via", "methanol", "interest", "equival", "gpi", "patholog", "result", "strikingli", "mhometdmatblfnreftypetablefn", "account", "concentr", "properti", "express", "opportun", "north", "measur", "heterozygot", "incid", "genu", "ecolog", "follow", "inform", "accumul", "exclus", "stock", "predict", "articlegrf", "variabl", "correspond", "capac", "estim", "determin", "consequ", "leishmaniasi", "dual", "cytometri", "consist", "resolut", "ctacaataaacaaatcataatatacaatt", "manual", "nuclear", "min", "observ", "taken", "order", "gagagaagacgagccgaagt", "feasibl", "unusu", "fulli", "analys", "entir", "conjunct", "abl", "modif", "reveal", "target", "occur", "congruent", "emerg", "freftypefig", "evid", "peruviana", "set", "six", "accomplish", "nonobligatori", "homologu", "viscer", "donovani", "enzym", "slight", "record", "panel", "event", "broad", "meant", "strike", "cytometr", "although", "due", "channel", "strainspecif", "like", "intrcgcatagcaaaagccaaa", "marker", "sequenti", "rnase", "strong", "transmit", "univers", "similarli", "degre", "dntp", "restor", "phenotyp", "cannot", "sinc", "tetraploid", "biolog", "cultiv", "demonstr", "label", "appear", "ploidi", "star", "present", "anim", "fgaccgaggcacttgaag", "getachew", "icd", "standard", "nonhybrid", "ancestrallik", "uniqu", "gut", "clearli", "histor", "heterosi", "exploit", "group", "prepar", "lcbr", "predominantli", "precis", "address", "faculti", "abbrevi", "main", "domest", "thermopol", "sever", "tabl", "peak", "flow", "confirm", "lesion", "importantli", "bethlehem", "pellet", "minimum", "destruct", "gc", "blike", "cctgttggtgtgagttcgtg", "instanc", "vertebr", "coloni", "cytomet", "weldegebr", "public", "help", "characterist", "algorithm", "genom", "contribut", "exhibit", "cultur", "prove", "larg", "ttgcttcatgataacaacttgg", "mix", "million", "rule", "robust", "rttacgcgctcatcgcctt", "reader", "nucleotid", "repeat", "rpmi", "tgaatgagctggtagaatg", "partial", "least", "gel", "share", "one", "tcttctttcgttagctgagtgc", "seen", "leishmania", "likelihood", "′", "intfgtgaatggagggcagacg", "panamensisguyanensi", "°c", "firstli", "bona", "overal", "would", "dye", "data", "pair", "discuss", "ttacga", "mice", "describ", "summaris", "calcul", "furthermor", "episod", "ethiopian", "discret", "pattern", "occurr", "specif", "advantag", "duplic", "candid", "fli", "exampl", "conclud", "recoveri", "illustr", "intratgtgaagcaccagcaggacgg", "tent", "version", "rare", "parent", "kit", "maxicircleencod", "densiti", "except", "mac", "across", "supplementari", "sexual", "comparison", "visibl", "host", "addi", "still", "ctcctttcacacccgcctct", "neb", "reproduct", "cycl", "worldwid", "reason", "hypothet", "ad", "alik", "board", "seven", "mm", "homozyg", "fragment", "laboratori", "biolin", "sand", "sequenc", "nich", "plot", "enhanc", "deficit", "compet", "fluoresc", "genet", "propos", "excis", "parasit", "protozoan", "maximum", "interestingli", "paper", "requir", "repres", "profil", "routin", "web", "u", "independ", "chromatogram", "gm", "institut", "workagegnehu", "transgen", "relat", "size", "approxim", "phlebotomu", "object", "avail", "tritypdb", "relationship", "synteni", "wellcom", "dehydrogenas", "sgc", "rang", "see", "fig", "sureclean", "disequilibrium", "braziliensisleishmania", "close", "shown", "tree", "addit", "total", "μl", "high", "abi", "rtggaggacggagagactttg", "reaction", "activ", "limit", "note", "hivinfect", "concern", "approv", "subset", "perhap", "fatal", "examin", "resuspend", "output", "zewdu", "polymorph", "stain", "fl", "align", "instruct", "altern", "ld", "previous", "copassag", "sampl", "structur", "databas", "presenceabs", "meiosi", "count", "acquisit", "identif", "case", "materi", "deriv", "inherit", "haploid", "display", "mlmt", "capillari", "visual", "immunocompromis", "studi", "diploidi", "techniqu", "fit", "gate", "chbtblfnreftypetablefn", "gpiatblfnreftypetablefn", "isomeras", "harvest", "temp", "becam", "dm", "secondli", "dark", "ident", "three", "gtgagaaggcagggattcaa", "fluorescentconjug", "colour", "mitochondri", "highli", "manufactur", "usa", "iodid", "hybridlik", "product", "conserv", "spp", "research", "deduc", "find", "enorm", "pcr", "intfbgtgaaggaccaagctgcctgg", "content", "startl", "figur", "clinic", "restrict", "band", "submit", "rdp", "appar", "creat", "period", "conduct", "explan", "cttgatgttcgggttagcaag", "anoth", "method", "random", "step", "new", "form", "prism", "mlst", "resolv", "qiagen", "loss", "match", "procedur", "suitabl", "uk", "section", "distinct", "stabl", "diversif", "gondar", "ctgtatcacccatgaggggc", "enrol", "fac", "virul", "buffer", "elong", "noncod", "contrast", "ssreftypesec", "gctgcagcagatgagaagg", "b", "effect", "fundament", "contain", "gcggctacctcgccctcagt", "ggtgtaggttttagtttagg", "circul", "ababa", "isocitr", "monomorph", "within", "otherwis", "facscalibur", "problem", "hospit", "sourc", "span", "unclon", "peripher", "passag", "mean", "cross", "number", "introduct", "kinetoplastida", "tabul", "patient", "biolab", "ng", "centr", "appendix", "togeth", "multiclon", "persist", "extens", "ratio", "human", "infantummajor", "kj", "papatasi", "taq", "chemotherapi", "code", "variat", "next", "extrem", "parasexu", "complementari", "spread", "site", "iii", "nevertheless", "review", "accur", "need", "ethiopia", "expect", "enabl", "third", "facttggtcttggtacgg", "n", "oregon", "yearli", "medium", "possess", "bioscienc", "design", "singl", "suscept", "exclud", "indic", "flowjo", "ta", "c", "biparent", "httptritrypdborg", "maxicircl", "iupac", "alon", "natur", "fide", "field", "unlik", "possibl", "frequenc", "bp", "consent", "school", "locu", "resequenc", "simpl", "remark", "discount", "clear", "hypothesi", "confound", "search", "diseas", "agent", "investig", "r", "undertaken", "endogami", "analysi", "cytb", "complex", "icdatblfnreftypetablefn", "legend", "g", "strainclon", "asymptomat", "could", "genotyp", "lcbf", "sitessnp", "anneal", "balanc", "unit", "chosen", "primer", "trypanosomatida", "dna", "volum", "region", "icecold", "heterozygos", "unambigu", "increas", "softwar", "access", "biallel", "incub", "report", "similar", "×", "isol", "summari", "big", "treftypet", "hurissa", "area", "termin", "gagaag", "turkey", "clonal", "contributor", "clone", "attribut", "therefor", "adequ", "strategi", "northern", "detect", "amplifi" ]

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[ "Introduction", "leishmaniases", "caused", "protozoan", "parasites", "genus", "Leishmania", "Kinetoplastida", "Trypanosomatidae", "worldwide", "diseases", "transmitted", "phlebotomine", "sand", "flies", "Leishmania", "spp", "infect", "wide", "range", "hosts", "including", "sylvatic", "domestic", "animals", "humans", "leishmaniases", "enormous", "public", "health", "problem", "global", "prevalence", "million", "cases", "yearly", "incidence", "million", "cases", "Infection", "Leishmania", "cause", "broad", "spectrum", "clinical", "presentations", "ranging", "asymptomatic", "simple", "cutaneous", "destructive", "mucocutaneous", "lesions", "severe", "visceral", "leishmaniasis", "VL", "fatal", "without", "effective", "chemotherapy", "Historically", "population", "structure", "Leishmania", "considered", "fundamentally", "clonal", "limited", "historical", "evidence", "recombination", "example", "Leishmania", "braziliensis", "Leishmania", "multilocus", "enzyme", "electrophoresis", "MLEE", "random", "amplification", "polymorphic", "DNA", "Diversity", "observed", "within", "natural", "populations", "thus", "generally", "attributed", "accumulation", "mutations", "time", "perhaps", "rare", "instances", "genetic", "exchange", "Nevertheless", "evidence", "genetic", "exchange", "different", "Leishmania", "spp", "natural", "populations", "reported", "several", "occasions", "However", "level", "identification", "hybrids", "among", "natural", "populations", "difficult", "equivocal", "due", "limited", "discriminatory", "power", "methods", "MLEE", "recent", "studies", "characterising", "natural", "Leishmania", "populations", "using", "highly", "polymorphic", "microsatellite", "markers", "suggested", "presence", "genetic", "exchange", "addition", "described", "evidence", "inbreeding", "natural", "populations", "Leishmania", "Viannia", "braziliensis", "Leishmania", "Viannia", "guyanensis", "based", "linkage", "disequilibrium", "LD", "yet", "deficit", "heterozygosity", "proposed", "reproductive", "strategy", "therefore", "alternating", "model", "clonality", "vertebrate", "invertebrate", "hosts", "sexual", "recombination", "sand", "fly", "Whole", "genome", "sequencing", "Leishmania", "infantum", "southeastern", "Turkey", "supported", "occurrence", "hybridization", "subsequent", "selfing", "one", "parental", "genotypes", "identified", "likely", "Leishmania", "donovani", "complex", "extant", "capacity", "Leishmania", "spp", "undergo", "genetic", "exchange", "sand", "fly", "proven", "generating", "experimental", "Leishmania", "major", "hybrids", "following", "transgenic", "strains", "Hybrids", "isolated", "sand", "fly", "gut", "genotypes", "consistent", "meiosis", "Furthermore", "experimental", "crosses", "fluorescent", "transgenic", "donovani", "sand", "fly", "produced", "dual", "expression", "single", "cells", "consistent", "genetic", "exchange", "Genetic", "exchange", "potential", "implications", "heterosis", "hybrid", "vigour", "emergence", "spread", "virulent", "strains", "resistance", "chemotherapeutics", "exploitation", "different", "hosts", "vectors", "adaptation", "new", "ecological", "niches", "may", "provide", "selective", "advantage", "example", "startling", "observation", "provided", "clear", "evidence", "hybrids", "possess", "enhanced", "transmission", "potential", "Leishmania", "infantum", "normally", "able", "infect", "broad", "ranging", "Phlebotomus", "papatasi", "remarkably", "resultant", "hybrids", "able", "potentially", "profound", "epidemiological", "consequences", "Leishmania", "braziliensis", "Leishmania", "peruviana", "hybrids", "also", "implicated", "agents", "destructive", "forms", "mucocutaneous", "leishmaniasis", "work", "described", "designed", "accomplish", "three", "main", "objectives", "applied", "panel", "natural", "isolates", "north", "Ethiopian", "origin", "characterise", "four", "putative", "hybrids", "using", "range", "high", "resolution", "markers", "ii", "identify", "likely", "parental", "genotypes", "among", "seven", "north", "Ethiopian", "donovani", "representatives", "microsatellite", "genotypes", "iii", "discount", "possibility", "microsatellite", "genotypes", "result", "mixed", "infections", "applied", "novel", "combination", "multilocus", "sequence", "typing", "MLST", "targets", "highly", "resolutive", "multilocus", "microsatellite", "typing", "MLMT", "markers", "conjunction", "mitochondrial", "maxicircle", "sequencing", "DNA", "content", "analysis", "demonstrate", "four", "natural", "isolates", "bona", "fide", "hybrids", "result", "mixed", "infection", "likely", "parental", "origins", "epidemiological", "implications", "discussed", "Materials", "methods", "Parasite", "strains", "DNA", "isolation", "panel", "north", "Ethiopian", "donovani", "isolates", "available", "originating", "population", "Ethiopian", "isolates", "containing", "putatively", "characterised", "hybrids", "participants", "enroled", "according", "written", "informed", "consent", "procedures", "approved", "Institutional", "Review", "Boards", "Faculty", "Medicine", "Addis", "Ababa", "University", "Addis", "Ababa", "Full", "World", "Health", "Organization", "isolate", "labels", "shown", "Table", "table", "isolate", "characteristics", "shown", "Supplementary", "Table", "sec", "Isolates", "available", "comprised", "four", "putative", "hybrids", "Two", "hybrids", "isolated", "patient", "isolated", "patient", "different", "episodes", "disease", "representatives", "designated", "hypothetical", "parent", "group", "designated", "hypothetical", "parent", "group", "B", "Fig", "fig", "Table", "table", "total", "biological", "clones", "generated", "maximum", "per", "isolate", "eight", "clones", "clones", "nine", "clones", "nine", "clones", "nine", "clones", "nine", "clones", "DM", "nine", "clones", "eight", "clones", "nine", "clones", "clones", "Leishmania", "cultured", "supplemented", "RPMI", "liquid", "medium", "previously", "described", "Isolates", "cloned", "solid", "media", "described", "single", "colonies", "became", "visible", "removed", "inoculated", "liquid", "culture", "medium", "DNA", "isolated", "clonal", "culture", "using", "DNA", "isolation", "kits", "Promega", "UK", "following", "manufacturer", "instructions", "MLMT", "Five", "microsatellite", "markers", "selected", "Table", "table", "based", "ability", "distinguish", "putative", "hybrids", "parents", "forward", "reverse", "primers", "Eurofins", "Germany", "used", "amplification", "amplification", "conditions", "reaction", "cycles", "performed", "described", "slight", "modifications", "initial", "denaturation", "step", "min", "samples", "processed", "cycles", "consisting", "annealing", "temperature", "indicated", "Table", "table", "min", "followed", "terminal", "elongation", "step", "min", "reaction", "performed", "final", "volume", "μl", "consisting", "ThermoPol", "reaction", "buffer", "New", "England", "Biolabs", "NEB", "UK", "mM", "mM", "dNTPs", "pmol", "primer", "unit", "Taq", "polymerase", "NEB", "UK", "ng", "genomic", "DNA", "Allele", "sizes", "estimated", "using", "automated", "capillary", "sequencer", "Applied", "Biosystems", "UK", "checked", "manually", "MLST", "MLST", "markers", "amplified", "genomic", "DNA", "extracted", "isolates", "biological", "clones", "Table", "table", "summarises", "PCR", "amplification", "primers", "annealing", "temperatures", "Genetic", "markers", "consisted", "four", "coding", "nuclear", "markers", "isomerase", "GPI", "isocitrate", "dehydrogenase", "ICD", "one", "nuclear", "marker", "Targets", "chosen", "following", "screening", "panel", "markers", "known", "exhibit", "sequence", "diversity", "within", "donovani", "complex", "basis", "GenBank", "sequences", "Amplifications", "performed", "initial", "denaturation", "step", "min", "followed", "cycles", "annealing", "elongation", "steps", "marker", "described", "herein", "final", "elongation", "step", "min", "annealing", "temperatures", "GPI", "ICD", "respectively", "respectively", "reaction", 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"fig", "undertaken", "results", "ambiguous", "SNP", "differences", "isolates", "clones", "recorded", "tabulated", "together", "associated", "positions", "Fig", "fig", "Mitochondrial", "maxicircle", "gene", "sequencing", "mMLST", "cytochrome", "b", "gene", "CYTb", "amplified", "sequenced", "using", "primer", "pairs", "LCBF", "GGTGTAGGTTTTAGTTTAGG", "CTACAATAAACAAATCATAATATACAATT", "Cycle", "conditions", "consisted", "initial", "denaturation", "followed", "cycles", "denaturation", "annealing", "extension", "min", "final", "extension", "step", "min", "amplified", "fragments", "sequenced", "directions", "using", "set", "primers", "following", "sequencing", "methods", "described", "DNA", "content", "analysis", "Approximately", "cloned", "donovani", "parasites", "harvested", "phase", "liquid", "cultures", "Parasites", "pelleted", "centrifugation", "g", "min", "washed", "twice", "PBS", "fixed", "methanol", "h", "Two", "subsequent", "washes", "PBS", "performed", "cells", "resuspended", "PBS", 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"hybrids", "almost", "precisely", "matched", "homozygous", "SNPs", "sites", "deduced", "pairs", "parents", "Results", "mirrored", "MLMT", "analysis", "although", "technique", "less", "robust", "suitable", "fully", "resolve", "parents", "donors", "using", "five", "loci", "However", "together", "MLST", "analysis", "MLMT", "spanning", "nine", "loci", "proved", "powerful", "complementary", "techniques", "identifying", "hybrids", "donovani", "novel", "combination", "markers", "may", "useful", "screening", "whole", "populations", "hybrids", "sequencing", "feasible", "four", "donovani", "hybrid", "isolates", "identical", "varying", "degrees", "SNP", "differences", "Two", "hybrids", "similar", "genetic", "profiles", "possibly", "originating", "event", "Remaining", "hybrids", "distinct", "data", "show", "appear", "possess", "different", "parent", "B", "allele", "consider", "suggests", "one", "recombination", "event", "Hybrid", "unusual", "outlier", "exhibiting", "reduced", "heterozygosity", "relative", "hybrids", "yet", "still", "retaining", "hybrid", "signal", "one", "MLST", "locus", "two", "MLMT", "loci", "One", "interpretation", "data", "another", "recombination", "event", "although", "divergence", "hybrids", "divergence", "via", "gene", "conversion", "entirely", "excluded", "Recent", "whole", "genome", "sequencing", "infantum", "isolates", "focus", "cutaneous", "leishmaniasis", "CL", "southeastern", "Turkey", "suggested", "derived", "single", "cross", "two", "diverse", "strains", "followed", "recombination", "within", "population", "Although", "one", "parental", "types", "present", "likely", "donovani", "complex", "concluded", "recombination", "infantum", "probably", "rare", "field", "judged", "length", "parental", "sequence", "blocks", "pattern", "linkage", "SNPs", "Concerning", "data", "allelic", "contributions", "parental", "types", "present", "appear", "relatively", "conserved", "also", "suggesting", "recent", "genetic", "exchange", "events", "However", "sampling", "would", "required", "population", "level", "resolve", "processes", "assess", "accurately", "presence", "frequency", "endogamy", "high", "frequencies", "would", "characterised", "trend", "towards", "homozygosity", "within", "population", "surprisingly", "genotypes", "considered", "parental", "correspond", "precisely", "predicted", "parents", "example", "one", "SNPs", "heterozygous", "ICD", "locus", "missing", "putative", "parents", "number", "explanations", "differences", "hybrids", "putative", "isolates", "Firstly", "likely", "multiple", "genotypes", "circulating", "naturally", "sampled", "may", "even", "similar", "predicted", "parents", "Secondly", "gene", "conversion", "mutation", "inevitable", "time", "since", "hybridization", "restoring", "homozygosity", "generating", "divergent", "genotypes", "loci", "Differences", "hybrids", "could", "also", "accounted", "repeated", "selfing", "although", "largely", "corresponding", "heterozygous", "SNPs", "corresponding", "homozygous", "donors", "MLST", "MLMT", "outputs", "make", "unlikely", "also", "investigated", "donovani", "maxicircle", "kDNA", "inheritance", "patterns", "sequencing", "CYTb", "locus", "Several", "SNPs", "CYTb", "identified", "Identified", "SNPs", "formed", "two", "distinct", "mitochondrial", "marker", "combinations", "among", "donovani", "isolates", "clones", "expected", "haploid", "mitochondrial", "markers", "heterozygosity", "observed", "found", "four", "hybrid", "isolates", "derivative", "clones", "inherited", "single", "maxicircle", "DNA", "genotype", "Furthermore", "mitochondrial", "type", "genotype", "one", "likely", "genotypes", "four", "hybrids", "Thus", "find", "uniparental", "inheritance", "mitochondrial", "DNA", "accords", "mitochondrial", "inheritance", "experimental", "hybrids", "major", "hybrids", "inherited", "mitochondrial", "genotype", "one", "parent", "six", "inherited", "genotype", "parent", "determined", "DNA", "contents", "hybrids", "relative", "putative", 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"identifiable", "donor", "SNPs", "one", "homologue", "present", "parent", "recombinant", "genotypes", "observed", "across", "multiple", "loci", "consisting", "nuclear", "microsatellite", "markers", "uniparental", "maxicircle", "inheritance", "majority", "n", "ploidy", "routine", "generation", "experimental", "hybrids", "would", "needed", "fully", "address", "nature", "recombination", "donovani", "whether", "hybridization", "follows", "pattern", "orthodox", "meiosis", "interesting", "note", "two", "reported", "isolates", "single", "HIV", "patient", "different", "episodes", "disease", "northern", "Ethiopia", "Although", "two", "hybrids", "similar", "SNP", "differences", "apparent", "presence", "multiple", "genotypes", "single", "severely", "immunocompromised", "patient", "may", "increase", "likelihood", "recombination", "genotypes", "within", "vertebrate", "host", "Alternatively", "immunocompromised", "individuals", "may", "susceptible", "infection", "wider", "range", "genotypes", "sand", "fly", "vector", "genetic", "exchange", "occurred", "supported", "experimental", "proof", "capacity", "genetic", "exchange", "sand", "fly", "Additionally", "immunocompromised", "status", "may", "enable", "less", "fit", "hybrids", "persist", "would", "otherwise", "cleared", "immune", "competent", "individuals", "full", "consequences", "genetic", "exchange", "donovani", "yet", "uncertain", "example", "hybrids", "described", "also", "differ", "ability", "exploit", "different", "hosts", "vectors", "cause", "differential", "pathology", "humans", "donovani", "hybrid", "parental", "isolates", "characterised", "provide", "excellent", "opportunity", "follow", "analysis", "experiments", "hybridization", "mechanisms", "genetic", "exchange", "associated", "generation", "phenotypic", "diversity", "Appendix", "Supplementary", "data", "Supplementary", "data", "wish", "thank", "Bethlehem", "Getachew", "Leishmaniasis", "Research", "Diagnostic", "Laboratory", "School", "Medicine", "Addis", "Ababa", "University", "Ethiopia", "help", "parasite", "cultivation", "also", "thank", "Leishmaniasis", "Research", "Treatment", "Centres", "Gondar", "University", "Arba", "Minch", "Hospital", "Ethiopia", "specifically", "thank", "Dr", "Zewdu", "Hurissa", "Dr", "Workagegnehu", "Hailu", "Gondar", "University", "Teklu", "Weldegebreal", "Arba", "Minch", "Hospital", "help", "field", "studies", "work", "supported", "Wellcome", "Trust", "Summary", "single", "nucleotide", "polymorphism", "distributions", "across", "four", "multilocus", "sequence", "typing", "loci", "Chromosome", "Ch", "isocitrate", "dehydrogenase", "ICD", "Leishmania", "clones", "Data", "isomerase", "GPI", "locus", "shown", "sequences", "identical", "Isolate", "names", "abbreviated", "World", "Health", "Organization", "strain", "codes", "shown", "Table", "table", "hybrids", "possess", "heterozygous", "single", "nucleotide", "polymorphisms", "colour", "represent", "donors", "colour", "represent", "possible", "B", "donors", "Full", "IUPAC", "codes", "heterozygote", "single", "nucleotide", "polymorphisms", "follows", "K", "G", "C", "R", "G", "G", "C", "C", "Numbers", "indicate", "nucleotide", "position", "within", "loci", "interpretation", "references", "colour", "figure", "legend", "reader", "referred", "web", "version", "article", "Flow", "cytometric", "analysis", "sequencing", "electropherograms", "Flow", "cytometric", "analysis", "relative", "DNA", "content", "Leishmania", "clones", "Data", "plotted", "area", "histogram", "gates", "created", "n", "peaks", "n", "peaks", "overlaid", "DNA", "histograms", "illustrating", "comparable", "n", "DNA", "content", "hybrid", "isolates", "B", "Electropherograms", "showing", "two", "variable", "single", "nucleotide", "polymorphisms", "present", "homozygous", "equivalent", "locus", "present", "hybrid", "locus", "Microsatellite", "profiles", "five", "loci", "hybrids", "corresponding", "putative", "parents", "Leishmania", "isolates", "Strain", "code", "Marker", "Hypothetical", "parent", "n", "Hybrid", "isolates", "n", "Hypothetical", "parental", "B", "n", "World", "Health", "Organization", "Hybrid", "heterozygous", "three", "loci", "unlike", "heterozygous", "across", "five", "loci", "congruent", "multilocus", "sequence", "typing", "MLST", "data", "Fig", "fig", "Properties", "microsatellite", "markers", "used", "study", "Marker", "Chromosome", "Forward", "Reverse", "CTTGATGTTCGGGTTAGCAAG", "ATGCACACCAAAAATCATGTG", "TTGCTTCATGATAACAACTTGG", "CCTGTTGGTGTGAGTTCGTG", "TCTTCTTTCGTTAGCTGAGTGC", "CTGTATCACCCATGAGGGGC", "GCTGCAGCAGATGAGAAGG", "GTGAGAAGGCAGGGATTCAA", "CTCCTTTCACACCCGCCTCT", "GAGAGAAGACGAGCCGAAGT", "temperature", "annealing", "Multilocus", "sequence", "typing", "targets", "Leishmania", "donovani", "Locus", "Chromosome", "number", "polymorphic", "Primer", "sequences", "Annealing", "temp", "bp", "GPI", "F", "TGAATGAGCTGGTAGAATG", "ICD", "F", "R", "TTACGCGCTCATCGCCTT", "INTF", "INTR", "CGCATAGCAAAAGCCAAA", "F", "R", "INTFA", "INTFB", "INTRA", "TGTGAAGCACCAGCAGGACGG", "b", "F", "R", "INTF", "INTR", "GTCGTGAAAAGCGAGAAGGT", "SNP", "single", "nucleotide", "polymorphism", "temperature", "annealing", "GPI", "isomerase", "ICD", "isocitrate", "dehydrogenase", "Ch", "chromosome", "Coding", "nuclear", "marker", "nuclear", "marker" ]

T[he Biobanking and]{.smallcaps} Cohort Network (BCNet) was established by the International Agency for Research on Cancer (IARC) in 2013, as an opportunity for low- and middle-income countries (LMICs) to work together in a coordinated manner to jointly address many of the challenges in biobanking infrastructure, the acquisition and maintenance of high-quality samples and data, and governance and regulatory frameworks to guide in the sharing and reuse of resources for research. The BCNet currently has 34 LMIC member organizations from across the world and 13 partner organizations that have biobanking in their core activities. The BCNet initiative arose from the realization that despite improvements in developed countries, population cohorts and biobanking facilities are either underdeveloped or nonexistent in LMICs.^[@B1]^ In this context and in line with IARC\'s mission to contribute to worldwide cancer research, BCNet was set up as an opportunity for LMICs to work together in a coordinated and effective manner and jointly address the many challenges in biobanking infrastructure, including ethical, legal, and social issues.^[@B2],[@B3]^ In addition, BCNet facilitates the sharing of resources (e.g., expertise and protocols) and the development of joint projects, strengthening the competitiveness of LMIC biobanks in applying for international funding. IARC\'s Dr. Maimuna Mendy has expertly coordinated the BCNet with the help of a steering committee team focused on education and training, ethics and legal issues, and informatics (<http://bcnet.iarc.fr>). The BCNet steering committee has organized several training initiatives and resources; it has focused on the informatics necessary to support LMIC biobanking, in partnership with B3Africa; and has examined ethics and legal issues within and across LMIC biobanking, including sample and data sharing.^[@B4]^ The International Society for Biological and Environmental Repositories (ISBER) is one of the founding partner organizations of the BCNet and has taken a key role in the education and training portion of its outreach activities. ISBER entered into a partnership agreement with IARC-BCNet in 2015 to allow very low-cost access to the ISBER educational platform and the ISBER online biobanking training modules, Introduction to Biobanking, developed by the Canadian Tissue Resource Network. The purpose of the course is to give the viewer a general overview of the key issues in establishing, maintaining, and accessing a biobank. The education program includes nine online modules designed to provide "how-to" knowledge for researchers and biobankers. This course is currently available in the English and French languages. Now ISBER, in line with its mission to strengthen international collaboration^[@B5]^ and based on further recent evidence,^[@B6]^ has entered into a stronger partnership agreement with IARC BCNet. ISBER has made a firm commitment over the past few years to lower costs for resource-constrained biobankers and organizations, in addition to the partnership with IARC-BCNet. The new agreement between the two organizations welcomes all registered LMIC organizations that are members of BCNet into ISBER in a complimentary membership capacity. This new status provides BCNet members with full access to ISBER tools, including the ISBER forum discussion board, participation in working groups and committees, the ISBER best practices, science policy resources, and recorded conference sessions and webinars. In addition, BCNet members will be able to communicate with the ISBER membership through the provision of complimentary news features and the creation of a joint working group or task force for developing further educational material that would be LMICs specific and freely available to the members of both organizations. ISBER looks forward to this newly strengthened relationship to support our strategic commitment to education and harmonization of best practices in biobanking across the world, in all resource settings. [^1]: ISBER President (2017--2018).

{ "pile_set_name": "PubMed Central" }

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An outbreak of severe acute respiratory syndrome (SARS) began in Guangdong, China, on November 16, 2002. The first three SARS cases in Singapore were confirmed on March 6, 2003. By May 5, a total of 204 cases, including 27 deaths, had been confirmed. The last case was isolated on May 11, and by July 30, the end of the outbreak, 205 patients had recovered and 33 had died ([@R1]). Since SARS infection may come from ordinary contact with acquaintances, colleagues, or strangers, outbreaks can trigger anxiety and influence public perception of susceptibility, causing serious economic and social disruption. The need for health information and for crisis management by public health authorities is also high. We examine four areas of public reaction to the SARS outbreak in Singapore: preventive practices, perception of self-health, knowledge of SARS, and appraisal of SARS crisis management. Materials and Methods ===================== Sample ------ We interviewed a representative stratified random sample of 1,202 adults (≥21 years of age). To minimize personal contact during the outbreak, participants were interviewed by telephone instead of face-to-face. The residential telephone sampling covered 90% of households in Singapore. The response rate was 62.3%, and the sampling error ±3.5% ([Table 1](#T1){ref-type="table"}). We used Random Digit Dialing+1, a system commonly used in public health studies, to capture unlisted telephone numbers ([@R3]). ###### Demographic characteristics of study and total population Characteristics Study population Total population^a^ ------------------------------------------ --------------------------------------------- ------------------------------------------------------------- Ethnicity Chinese Malay Indian Other 900 (75.0) 172 (14.0) 82 (7.0) 47 (4.0) 2,505,400 (76.8) 453,600 (13.9) 257,800 (7.9) 46,400 (1.4) Age 21--29^b^ 30--39 40--49 50 and older 233 (19.0) 313 (26.0) 312 (26.0) 343 (28.0) 480,191 (20.4) 613,944 (26.1) 575,674 (24.5) 681,282 (29.0) Sex Male Female 599 (49.9) 602 (50.1) 1,630,293 (49.9) 1,632,916 (50.1) ^a^Ref. [@R2]. ^b^Total population figures refer to ages 20--29 years. Data Collection --------------- We modified and expanded a structured questionnaire provided by researchers from the Department of Community Medicine, University of Hong Kong (A.J. Hedley, T.H. Tan, G.M. Leung, B.H.Y. Chan, S.Y. Ho, L.M. Ho, unpub. data). The modified questionnaire ([Appendix](#SD1){ref-type="local-data"}) was translated into Mandarin, Malay, and English; interviews were conducted from May 5 to May 10, 2003. Factor analysis and logistic regression (SPSS for Windows \[Version 11.5\]) examined trends among four factors (SARS prevention, perception of self-health, knowledge of SARS, and perception of health authorities' crisis management). We also assessed how prevention measures correlated with other factors, including respondents' demographic characteristics. Preventive Measures ------------------- Eight questions focused on respondents' prevention practices in the 3 days before the interview. We constructed a composite index indicating the total number (from 0 to 8) of preventive measures taken. A dichotomous indicator of preventive behavior was calculated based on the mean number of precautions taken (4.68): "low" (≤5) versus "high" (≥6). Self-Health Perception ---------------------- Three sets of questions addressed respondents' perception of their own health. The first set covered nine physical health complaints. We created a composite index of symptoms by adding all instances of health complaints over the previous 2 weeks. This index was 0 to 7 in our study since no one reported having more than seven of the nine symptoms. The second set was a "frame of mind" index fashioned after B.A. Thyer's Clinical Anxiety Scale ([@R4]). Scores for positive items were 1 (not at all) to 4 (very much); negative item scores were reversed, so lower total scores indicated higher anxiety. The scale had an Alpha reliability coefficient of 0.8244. The third set addressed respondents' perceived susceptibility to SARS. Scores were 4 (very likely) to 0 (don't know). On the basis of the average score (1.5; standard deviation \[SD\] 1.01), we created a dichotomous variable to contrast respondents who believed they were susceptible to contracting SARS (scores 3 and 4) with those who did not (scores 0--2). Knowledge of SARS ----------------- Three questions tested SARS knowledge. Responses were scored 0 (incorrect) or 1 (correct); a composite index indicated the number of correct answers, from none correct (0) to all three correct ([@R3]). Appraisal of Crisis Management ------------------------------ Four sets of questions addressed respondents' appraisal of crisis management, but we discuss the three most relevant. The first set of five questions (Alpha reliability 0.8136) assessed opinions on information distribution. Scores were 1 (very negative) to 6 (very positive). On the basis of the mean score (4.83; SD 0.617), we calculated a dichotomous index: negative appraisal (scores ≤4.7) versus positive appraisal (scores ≥4.8). The second set of questions addressed openness of communication. Scores were 1 (very negative) to 6 (very positive). By using the sample's mean score (4.31; SD 1.25), this variable was dichotomized into "disagreement" (scores 1--3) and "agreement" (scores 4--6). The third set referred to the public's acceptance of quarantine regulations. The scores were dichotomized into "agreement" ([@R1]) versus "no agreement" and "don't know" ([@R2]). Results ======= Responses to the survey questions are summarized in [Table 2](#T2){ref-type="table"}. Variables were examined by using odds ratios (ORs) at 95% confidence intervals (CI). The statistically significant ORs are reported in [Table 3](#T3){ref-type="table"} with their respective level of significance from the Mantel-Haenszel common odds ratio estimates. ###### Variables used in analysis of public reaction and perspective of SARS crisis Variable \% Mean SD ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------- -------- -------- Symptoms (0--8) over past 2 weeks None One or more Main SARS-related symptoms Persistent high fever ≥38°C Cough Rapid breathing 77.6 22.4 1.0 9.0 1.0 0.3639 0.8286 Anxiety level High (1.0--2.2) Moderate (2.3--3.2) Low (3.3--4.0) 2.9 42.4 54.7 3.2307 .4819 Perceived likelihood of contracting SARS Very likely (4) Likely (3) Not very likely (2) Not likely at all (1) Don't know (0) 4.0 10.0 39.0 29.0 18.0 1.5304 1.014 Knowledge of SARS No knowledge (0 of 3 answers correct) (0) 1 of 3 answers correct (1) 2 of 3 answers correct (2) 3 of 3 answers correct (3) 11.7 25.0 42.5 20.7 1.7227 .9222 Appraisal of crisis management "Strongly agree" and "Agree" that information by health authorities is: Accurate Clear Sufficient Timely Trustworthy Population has chance to express personal views and concerns to the authorities, "strongly agree" or "agree." Agreeable to 10-day quarantine after nonclose contact with SARS-infected person and no symptoms Agree Don't agree Don't know 82.2 86.3 84.5 84.4 87.8 66.3 71.6 22.4 6.0 Years of formal education ≤10 years ≥11 years 57.1 42.9 10.07 3.9642 Practice of preventive measures Practicing each of eight measures "always" or "most of the time" during the past 3 days: Covered mouth with tissue when sneezing or coughing Covered mouth with bare hand when sneezing or coughing Washed hands after sneezing, coughing, or clearing nose Used soap or liquid hand-wash when washing hands Wore a mask Used serving utensils for shared food Took preventive measures when touching objects Washed hands after touching objects Preventive measures taken over past 3 days (score 0--8) Five or fewer of the eight measures Six or more of the eight measures 62.0 47.0 72.0 81.0 4.0 28.0 15.0 48.0 69.3 30.7 4.686 1.5286 ###### Practice of SARS preventive measures, 3 days before interviews^a^ Variable No. OR 95% CI ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------- ------------------------------------- ------------------------------------------------------------------------------------------- **Personal health evaluation** Symptoms in past 2 weeks None One or more Anxiety^b^ Moderate or high (score ≤3.25) Low anxiety (score \>3.25) Perceived likelihood of SARS Not likely Likely 932 269 544 657 1,034 167 1.012 0.960 0.861 1.140 1.031 0.833 0.947 to 1.082 0.766 to 1.203 0.757 to 0.978 1.031 to 1.283 0.979 to 1.085 0.621 to 1.118 **Knowledge of SARS** Two or fewer correct answers Three correct answers 952 249 1.012 0.954 0.950 to 1.079 0.753 to 1.079 **Appraisal of crisis management** Quality of official information Below average (negative) Above average (positive) Have chance to express opinion^c^ Disagree Agree Agreeable to quarantine when non-close contact with SARS-infected person and no symptoms Agree Do not agree or don't know 290 911 271 930 860 341 1.164 0.955 1.434 0.909 0.969 1.084 0.928 to 1.460 0.893 to 1.020 1.115 to 1.846 0.855 to 0.966 0.899 to 1.045 0.888 to 1.323 **Demographic characteristics** Years of formal education^d^ ≤10 \>10 Sex^c^ Male Female Age^c^ (y) \<35 ≥35 686 515 599 602 391 809 0.909 1.143 1.339 0.770 1.365 .872 0.821 to 1.006 0.985 to 1.325 1.166 to 1.539 0.689 to 0.861 1.123 to 1.658 0.806 to 0.943 ^a^SARS, severe acute respiratory syndrome; OR, odds ratio; CI, 95% confidence interval. ^b^Asymptotic significance (2-sided) ≤0.05. ^c^Asymptotic significance (2-sided) ≤0.001. ^d^Asymptotic significance (2-sided) ≤0.10. Recommended preventive measures were not practiced uniformly. The most practiced measures 3 days before the interview were using soap when washing hands (81%) and washing hands after sneezing, coughing, or clearing the nose (72%). The least practiced measure was wearing a mask over the mouth. A total of 4% wore masks, and most did so only when visiting a clinic or hospital or when the mask was part of a uniform (as in healthcare workers). The index of preventive measures indicates that most people (69.3%) took some preventive measures. Respondents' perception of their health was generally positive. A relatively low proportion (22.4%) of respondents reported having any of our nine physical health complaints over the previous 2 weeks, and fewer than 1% reported the three classic symptoms of SARS (fever ≥38°C, cough, rapid breathing). The mean number of health complaints reported in our sample was 0.369 (SD 0.828). The survey also showed low anxiety; only 2.9% of respondents reported high anxiety. The mean anxiety score was 3.23 (SD 0.48). Most respondents (68%) thought they were not very likely or not likely at all to contract SARS, and 18% were not sure of their likelihood. Those who thought they were likely to get the disease reported slightly more anxiety. Of the three aspects of health perception, only anxiety was associated with taking precautions (OR 0.861; 95% CI 0.757--0.978). In the high-anxiety group, 34% followed six or more of the eight preventive measures, in contrast to 28% of respondents who had low anxiety. Regarding knowledge of SARS, the sample correctly answered an average of 1.722 (SD 0.922) of 3 questions on SARS transmission. Approximately 63% answered two or more questions correctly; 11.7% did not answer any questions correctly. Respondents had a generally high opinion of authorities' crisis management. More than 80% thought official information was accurate, clear, sufficient, timely, and trustworthy, and 72% were prepared to accept a 10-day quarantine, even in the absence of SARS symptoms or close contact with a SARS patient. Of the three crisis management aspects, one had significant influence on preventive action: respondents' opinion of authorities' openness to communication. People who thought that authorities were open to communication were more inclined to practice six or more of the eight SARS preventive measures (OR 0.909; 95% CI 0.855 to 0.966) than those who thought they had no chance to express their concerns to the authorities (OR 1.434; 95% CI 1.115 to 1.846). Three demographic characteristics were associated with taking preventive measures against SARS: sex, age, and estimated years of formal education. Women were more inclined (OR 0.770; 95% CI 0.689 to 0.861) than men (OR 1.339; 95% CI 1.166 to 1.539) to take preventive measures; this finding is consistent with other studies on health behavior in Singapore ([@R5]*,*[@R6]). People ≥35 years of age were more inclined to take preventive measures (OR 0.872; 95% CI 0.806 to 0.943) than their younger counterparts (OR 1.365; 95% CI 1.123 to 1.658). The association with education disappeared when controlled for sex. Discussion ========== Information regarding the SARS outbreak was widely distributed by the media and government; while this information was essential to keep the public informed of the risks for infection and preventive measures, it also could increase anxiety. However, we found low levels of anxiety in Singapore, and few reported health complaints. Reporting health complaints was not associated with taking precautions against SARS, possibly because the nine symptoms of SARS covered in our questionnaire are associated with other common diseases in Singapore (e.g., dengue fever, the incidence of which was 86.2 per 100,000 in May 2003) and are not usually deemed serious. In fact, familiarity with symptoms was a key initial obstacle in preventing SARS spread in hospitals ([@R7]) and remains an impediment to raising community alertness. In our sample, anxiety appeared to motivate preventive behavior; those in the highest anxiety group took more precautions. However, anxiety was not associated with the perceived likelihood of contracting SARS. The low percentage of respondents who viewed SARS as a personal risk (14%, compared to 22% found in a similar survey in Toronto \[[@R8]\]) could be explained by the fact that healthcare workers were among the first SARS patients. By the time the interviews began, two physicians had died, and two hospitals had clusters of cases. Lay respondents (those with no contact with hospitals or healthcare workers) may have perceived SARS an occupational hazard. Distribution of SARS information and prevention advice in Singapore increased rapidly over the 2 months preceding the interviews. All types of media were used, including a public television channel, the "SARS Channel," established to give current and comprehensive information on world infection trends and Singapore's situation. The Ministry of Health provided SARS information on its Web site ([@R9]), taking advantage of the fact that, as of December 2001, Singapore had 1.9 million Internet subscribers (out of 3.3 million population) ([@R10]). Of respondents, 20.7% were able to correctly answer all three SARS questions, and these did not differ in the practice of preventive measures from those who had less SARS knowledge. The absence of a correlation between knowledge and behavior confirms that knowledge of a disease is not sufficient to trigger preventive action ([@R5],[@R6],[@R11]--[@R13]). Since SARS appeared unexpectedly, healthcare experts were uncertain how to control the epidemic. Consequently, assessing public opinion of authorities' crisis management in our survey was relevant to Singapore. Of the aspects we examined, only public opinion of authorities' openness to communication was correlated with taking preventive measures. The other two aspects (information dissemination and acceptance of quarantine regulations) did not affect preventive action, probably because of their very positive rating. The public's highly positive assessment of Singapore authorities' crisis management is distinctive. History shows that epidemics are politically perilous to governments as, among other things, they challenge their resolve, efficiency, and state of readiness ([@R14]). Political leaders of other SARS-affected Asian countries witnessed this principle directly. The SARS outbreak in Singapore appears to have worked in an opposite way: it corroborated the usefulness of public health and environmental regulations. In addition, this study's findings parallel the population's response to quarantine and other restrictive measures, confirming previous observations of a relatively high level of social discipline in the population ([@R15],[@R16]). Conclusion ========== Singapore was taken out of the official list of SARS-infected countries by the World Health Organization on May 30, 2003. The epidemic has left the crisis phase and entered a new phase, normalization and vigilance. As a new disease, SARS demands continuous scrutiny on all fronts, from the laboratory to the homes of the people. Supplementary Material ====================== ###### Appendix Questionnaire given to SARS survey respondents *Suggested citation for this article:* Quah SR, Lee H-P. Crisis prevention and management during SARS outbreak, Singapore. Emerg Infect Dis \[serial online\] Feb 2004 \[*date cited*\]. Available from: URL: <http://www.cdc.gov/ncidod/EID/vol10no2/03-0418.htm> We express our appreciation to the persons who consented to be interviewed for this study; John Wong, Director of the National University of Singapore--Office of Life Sciences, who facilitated the grant application; Calvin Fones, David Koh, and Paulin Straughan for their comments and suggestions on the questionnaire; two anonymous reviewers for their constructive feedback; and A.J. Hedley for sending us the questionnaire his research team applied in Hong Kong (April 2003) and suggesting that we apply it in Singapore. This study was supported by National University of Singapore research grant R-111-000-045-712. Dr. Quah is a professor of sociology at the National University of Singapore. Among her areas of interest are medical sociology and social policy. Dr. Lee is professor of public health at the National University of Singapore. Among his areas of interest are cancer epidemiology and community medicine.

{ "pile_set_name": "PubMed Central" }

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22,251

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1. Introduction {#sec1-ijms-18-00554} =============== Resveratrol is a stilbene-based, polyphenolic compound occurring naturally in nuts, berries, and the skin of grapes, and is present in many food items, particularly red wine \[[@B1-ijms-18-00554]\]. In recent years, the clinical effects of resveratrol have been studied intensively owing to a plethora of promising effects in cells and animals; among these are improvement in terms of the various consequences of obesity, e.g., impaired insulin sensitivity and low-grade inflammation \[[@B2-ijms-18-00554],[@B3-ijms-18-00554],[@B4-ijms-18-00554]\]. A landmark study demonstrated a shortened life span in diet-induced obese mice and a resveratrol-mediated improvement in longevity despite obesity \[[@B5-ijms-18-00554]\]. Owing to its purported salutary effects, resveratrol is already being sold as an over-the-counter dietary supplement. Unfortunately, translating the effects of resveratrol to a human setting has proven difficult. Some clinical studies have confirmed the pre-clinical results of resveratrol-mediated improved insulin sensitivity, diminished low-grade inflammation, reduced blood pressure, and reduced amounts of liver fat accumulation \[[@B6-ijms-18-00554],[@B7-ijms-18-00554]\]. In contrast, other studies fail to detect any positive physiological effects \[[@B8-ijms-18-00554],[@B9-ijms-18-00554]\]. These conflicting results may in part be attributable to differences in study designs, populations, and resveratrol formulations. Some studies have been performed in healthy individuals, and the ability of resveratrol to improve glucose handling in situations with normal glucose homoeostasis has been questioned \[[@B8-ijms-18-00554]\]. In contrast, it is feasible that the effect of a food ingredient is too weak to demonstrate any measurable consequences in full-blown type 2 diabetes. Thus, subjects suffering from modest degrees of insulin resistance are most likely optimal for the purpose of investigating the possible effects of resveratrol on insulin sensitivity, and probably most comparable to the original mice studies using diet-induced obese mice. Study length may also be of importance. Most studies include intervention periods of only a few weeks \[[@B6-ijms-18-00554],[@B9-ijms-18-00554]\]. Obviously, longer studies provide a better determination of the possible effects of resveratrol on chronic conditions such as insulin resistance and low-grade inflammation. Finally, the methods currently used in most studies on resveratrol are targeted towards whole-body effects. Insulin sensitivity, for instance, has been assessed via measurements of specific targets such as plasma insulin, glucose, and HbA1c \[[@B6-ijms-18-00554],[@B8-ijms-18-00554],[@B9-ijms-18-00554]\]. These techniques have their strengths, such as being very specific and applicable in daily clinics; however, the drawback is that findings will be limited to the pre-specified objectives. Likewise, when confining assessment to plasma samples, intracellular effects may be missed. Recently, the so-called metabolomics approach has been developed. Metabolomic analysis profiles small-molecule metabolites using either a targeted or non-targeted approach \[[@B10-ijms-18-00554]\]. Through systematic identification and quantification of metabolites in biological systems, e.g. tissues, organs, or fluids at a certain time point, metabolomics directly sample metabolic modulations. Targeted metabolomics allows for very precise measurement of a specific target or an isolated group of related metabolites, whereas non-targeted metabolomics assesses as wide a range of different metabolites as possible. This technique provides a simultaneous screening of multiple metabolic pathways and captures an instantaneous integrated snapshot of the entire physiology of an organism. Metabolomics has the advantage over other "omics" that it integrates changes in gene expression, protein levels, enzymatic activity, and post-translational changes. Previously, metabolomics made it possible to determine changes in the human plasma metabolome caused by various conditions and diseases like sleep restriction \[[@B11-ijms-18-00554],[@B12-ijms-18-00554]\], diabetes \[[@B13-ijms-18-00554]\], and bariatric surgery \[[@B14-ijms-18-00554]\]. Studies have demonstrated a weak correlation between plasma and muscle metabolite levels, indicating that plasma measurements are poor indicators of skeletal muscle metabolism \[[@B15-ijms-18-00554]\]. Consequently, metabolomic analysis requires tissue biopsies to allow a detailed examination of changes in the intracellular pathways within this specific tissue. Such metabolic profiling offers the possibility to identify biochemical signatures of cellular metabolism involved in different pathways like glucose, lipid, and protein handling. Massive research interest has gone into resveratrol as a potential protective compound in the management of obesity-associated complications, e.g., metabolic syndrome and low-grade inflammation. However, the particular pathways involved in potential salutary effects are by and large undetermined. Non-targeted metabolomic profiling may provide new insight into these matters. We have recently conducted a four-month randomized, placebo-controlled clinical trial describing the effects of resveratrol treatment in middle-aged males with metabolic syndrome and reported the findings on bone \[[@B16-ijms-18-00554]\], circulating steroids \[[@B17-ijms-18-00554]\], and glucose metabolism and inflammation \[[@B18-ijms-18-00554]\]. The aim of the present study is to provide a comprehensive metabolomic analysis of the changes caused by four months of high-dose resveratrol treatment in these middle-aged men with metabolic syndrome. As the effects of resveratrol in humans are incompletely characterized, we used a non-targeted metabolomics approach based on four different matrices: plasma, urine, adipose tissue, and skeletal muscle tissue from each study subject. In our study on circulating steroids and prostate size we established a dose-dependent decrease in plasma androgen precursors by resveratrol \[[@B17-ijms-18-00554]\]. In order to maximize the chances of detecting even subtle resveratrol-induced pathway changes, we therefore decided to employ the metabolomics analysis in the high-dose resveratrol (hRSV) (500 mg twice daily) group compared to the placebo group. 2. Results {#sec2-ijms-18-00554} ========== 2.1. Clinical Features {#sec2dot1-ijms-18-00554} ---------------------- The basic characteristics of the participants are outlined in [Table 1](#ijms-18-00554-t001){ref-type="table"}. Apart from a slight difference in age, the two groups were comparable at baseline and post-treatment. 2.2. Global Metabolic Profiling {#sec2dot2-ijms-18-00554} ------------------------------- Based on a combination of two Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) platforms and one Gas Chromatography--Mass Spectroscopy (GS-MS) platform, 405 metabolites in plasma, 282 metabolites in adipose tissue, 446 metabolites in skeletal muscle, and 604 metabolites in urine were identified. General platform methods, data analysis, and metabolite detection identification are described in detail in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"} and [Supplementary Tables S1--S4](#app1-ijms-18-00554){ref-type="app"}). A total of 88 identified metabolites were present in all four matrices, as illustrated in [Figure 1](#ijms-18-00554-f001){ref-type="fig"}. The relationship between significantly hRSV-changed metabolites in the four matrices is depicted in [Figure 2](#ijms-18-00554-f002){ref-type="fig"}. Adipose tissue and urine had the greatest overlap in terms of significantly changed metabolites. One metabolite, dehydroisoandrosterone sulfate (DHEA-S), was significantly changed in all four matrices. Random forest analysis (RF analysis) of adipose tissue, muscle tissue, urine, and plasma metabolic profiles resulted in 75%, 87%, 82%, and 89% accuracy in differentiating the hRSV and placebo groups, respectively, indicating that the differences in biochemical profiles between the two groups were quite pronounced. Random Forest classification results, methods, and data analysis are described in detail in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"} and [Supplementary Table S7](#app1-ijms-18-00554){ref-type="app"}). Among the 30 top-ranking metabolites resulting from the RF analysis, biochemicals involved in amino acid metabolism and lipid metabolism were particularly important with regards to the separation of the two groups. Many of the metabolites embraced in the RF-analysis are also included in the hRSV-altered metabolic pathways. Steroid hormones, especially, were well represented in the RF importance plots, in addition to being significantly altered by hRSV intervention. 2.3. Metabolic Profiling in Adipose Tissue {#sec2dot3-ijms-18-00554} ------------------------------------------ Of the 282 biochemicals identified in adipose, tissue 45 displayed a significant change (*p* ≤ 0.05) in response to hRSV treatment: 31 of these were elevated and 14 were reduced. Furthermore, for 28 compounds there was a trend towards a change (*p* \< 0.10), of which 24 compounds were increased and four compounds were reduced by hRSV treatment. The pathways that differed significantly between the hRSV group and controls in adipose tissue (*p* \< 0.05) are shown in [Figure 3](#ijms-18-00554-f003){ref-type="fig"}. Exploring the respective significantly changed pathways in detail reveals the specific hRSV-affected compounds. As illustrated in [Table 2](#ijms-18-00554-t002){ref-type="table"}, four out of 15 identified lipids are significantly elevated in the long-chain fatty acid pathway (myristate (14:0), myristoleate (14:1n5), palmitate (16:0), and palmitoleate (16:1n7)). These lipids depict the significant fold change between the hRSV group and the placebo group. In addition, examining the polyunsaturated fatty acid pathway demonstrated that six out of 13 identified lipids are significantly elevated in the hRSV group (stearidonate (18:4n3), docosapentaenoate (n3 DPA; 22:5n3), docosahexaenoate (DHA; 22:6n3) linoleate (18:2n6) adrenate (22:4n6), and mead acid (20:3n9)). Also of interest, the steroid hormone pathway reveals significant reductions in both of the identified steroids, dehydroisoandrosterone sulfate (DHEA-S) and 4-androsten-3β, 17β-diol disulfate. The most interesting trending changes (*p* \< 0.10) in adipose tissue pathways were displayed in the glycolysis, gluconeogenesis, and pyruvate metabolism pathway and the pentose phosphate pathway. As illustrated in [Table 2](#ijms-18-00554-t002){ref-type="table"}, trending elevation in three out of nine identified glycolytic pathway intermediates (glucose-6-phosphate (G6P), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, phosphoenolpyruvate (PEP)), and one in the pentose phosphate pathway intermediate was found (sedoheptulose-7-phosphate). 2.4. Metabolic Profiling in Plasma {#sec2dot4-ijms-18-00554} ---------------------------------- Of the 405 named compounds in plasma, 38 were statistically different between the two groups (*p ≤* 0.05); 13 of these were elevated and 25 were reduced. In addition, 11 compounds showed a trend towards a change by hRSV (*p* \< 0.10), of which eight were increased and three were reduced. The pathways that are significantly different (*p* \< 0.05) between the hRSV group and controls are shown in [Figure 4](#ijms-18-00554-f004){ref-type="fig"}. Particularly interesting changes in response to hRSV treatment were found in the steroid hormone pathway with consistent significant reductions in circulating levels of cholesterol-derived steroid hormones and sulfated steroid hormones. As illustrated in [Table 3](#ijms-18-00554-t003){ref-type="table"}, 13 out of 19 detected steroid hormones were significantly reduced. Also noteworthy, significant elevations in histidine and the related biochemical marker of muscle protein turnover 3-methylhistidine along with its acetylated derivative *N*-acetyl-3-methylhistidine were observed in plasma in response to hRSV treatment. In addition, subtle but consistent reductions in the dicarboxylic acids dodecanedioate, tetradecanedioate, sebacate, hexadecanedioate, and octadecanedioate were observed in the hRSV group. 2.5. Metabolic Profiling in Skeletal Muscle Tissue {#sec2dot5-ijms-18-00554} -------------------------------------------------- Of the 446 named biochemicals in skeletal muscle tissue, 24 changed significantly (*p* ≤ 0.05); 12 of these were elevated and 12 reduced by hRSV treatment. Additionally, 19 biochemicals showed a trend towards a change by hRSV (*p* \< 0.10), with 12 biochemicals increased and seven reduced. The pathways that are significantly different (*p* \< 0.05) between the hRSV group and controls are shown in [Figure 5](#ijms-18-00554-f005){ref-type="fig"}. The most striking change in muscle tissue was the effect of hRSV on the steroid hormone pathway, with four out of six compounds being significantly reduced, as illustrated in [Table 4](#ijms-18-00554-t004){ref-type="table"}. All four compounds comprised sulfated steroid metabolites (dehydroisoandrosterone sulfate (DHEA-S), epiandrosterone sulfate, androsterone sulfate, 4-androsten-3β, and 17β-diol disulfate 1). Regarding skeletal muscle lipid metabolism, our analysis revealed significant hRSV alterations in the short-chain fatty acids, medium-chain fatty acid, and long-chain fatty acid pathways. Generally, the hRSV group had lower levels of intracellular lipids. In addition, two out of 12 biochemicals in the polyunsaturated fatty acid pathway were increased (stearidonate (18:4n3) and linolenate \[α or γ; (18:3n3 or 6)\]). 2.6. Metabolic Profiling in Urine {#sec2dot6-ijms-18-00554} --------------------------------- Urine data were analyzed in both non-normalized and osmolality-normalized formats, and because normalization to osmolality values did not substantially affect results, non-normalized data were utilized for data interpretation. Of the 604 named biochemicals in urine, 43 changed significantly (*p* ≤ 0.05); 31 of these were elevated and 12 were reduced by hRSV treatment. Additionally, 27 biochemicals showed a trend towards a change by hRSV (*p* \< 0.10), with 22 biochemicals increased and five reduced. Pathways that demonstrated significantly modifications (*p* \< 0.05) in response to hRSV are shown in [Figure 6](#ijms-18-00554-f006){ref-type="fig"}. The most striking hRSV-induced changes in urine were observed in the steroid hormone pathway. Ten out of 28 named steroids were significantly changed, with the majority being elevated, as illustrated in [Table 5](#ijms-18-00554-t005){ref-type="table"}. Furthermore, several metabolites derived from aromatic amino acids that have a contribution from or are exclusively produced by the gut microbiota were altered in the urine. The tyrosine-derived metabolites tyramine, phenol sulfate and homovanillate (HVA), were changed in the hRSV group. Similarly, urinary levels of derivatives from the tryptophan metabolism (tryptamine and indolelactate), the phenyalanine metabolism (2-hydroxyphenylacetate), and the histidine metabolism (imidazole propionate) were significantly altered by hRSV treatment, as depicted in [Table 5](#ijms-18-00554-t005){ref-type="table"}. 3. Discussion {#sec3-ijms-18-00554} ============= The effects of resveratrol in human trials have proven to be very inconsistent. Most data represent standard biochemical analyses of selected parameters. The focus has been on inflammation \[[@B8-ijms-18-00554],[@B19-ijms-18-00554]\], glucose metabolism \[[@B9-ijms-18-00554],[@B20-ijms-18-00554]\], lipids \[[@B21-ijms-18-00554]\], liver function \[[@B22-ijms-18-00554]\], and steroids \[[@B17-ijms-18-00554]\], assessed by measurement in blood samples, thus representing a picture of whole body metabolism. The present paper describes an exhaustive metabolomic profile of blood, adipose tissue, skeletal muscle tissue, and urine from a comprehensive clinical trial of high-dose resveratrol-treated, middle-aged males with metabolic syndrome. This approach allows for a more elaborate investigation of subtle resveratrol effects on distinct intracellular pathways than what is possible by measurements confined to blood. In addition, this approach of unbiased measurements in various body compartments may help us to determine the cause of some of the effects we have previously detected. Recently, we reported that the androgen precursors in blood were significantly reduced after resveratrol treatment for four months \[[@B17-ijms-18-00554]\]. The presented metabolomic analysis was performed in the same study cohort, and by using this advanced technique we are able to verify the decrease in sulfated androgen precursors in the blood \[[@B17-ijms-18-00554]\]. In addition, we find significantly lower intracellular amounts of sulfated androgen precursors in skeletal muscle tissue as well as adipose tissue. Sulfation is a common modification to control levels of active steroid hormones at target sites by increasing solubility and renal excretion. The metabolomic profiling of urine revealed increased urinary excretion of the majority of the measured sulfated steroid hormones. In the original paper on reduced androgen precursors, we discussed whether the reduction was caused by decreased formation or increased excretion. The present results propose that at least part of the decrease in sulfated androgen precursors in blood and tissues is caused by an increase in urinary excretion of the sulfated steroids. The metabolomic analysis of lipids points to a striking accumulation of long-chain saturated, monounsaturated, and polyunsaturated (n3 and n6) free fatty acids in adipose tissue. However, in skeletal muscle two long-chain lipids were significantly changed; one was elevated (10-heptadecenoate (17:1n7)) whereas the other was significantly reduced (arachidate (20:0)). Deducing how resveratrol induces the increase in many of the long-chain polyunsaturated fatty acids (n3 and n6) in adipose tissue is complex. Usually these long-chain polyunsaturated fatty acids derive from increased intake of seafood or special plant oils. However, the two essential fatty acids alpha-linolenic acid (ALA; 18:3n3) and linoleic acid (LA; 18:2n6) can be metabolized into longer-chain polyunsaturated fatty acids through a series of desaturation and elongation processes \[[@B23-ijms-18-00554]\]. The literature describes a gender difference in the conversion of ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), with women having a much higher conversion rate than men \[[@B24-ijms-18-00554],[@B25-ijms-18-00554]\]. This difference depends upon sex steroids, and testosterone supplementation to female transsexuals reduced DHA concentration by 22% \[[@B26-ijms-18-00554]\]. Clearly, this indicates that the enzymes responsible for the desaturation and elongation conversion of ALA to DHA are negatively regulated by androgens. Based on this knowledge, we speculate that resveratrol might stimulate the conversion process of ALA and LA, resulting in an increase in long-chain polyunsaturated fatty acids through the robust reduction in androgen precursors. The health impacts of changes in levels of long-chain polyunsaturated fatty acids are still not fully determined. Long-chain polyunsaturated fatty acids have been associated with a positive effect on insulin signaling via the insulin receptor \[[@B27-ijms-18-00554]\], and even a minor increase of ALA content in adipose tissue of 1% has been associated with a 5 mmHg decrease in blood pressure \[[@B28-ijms-18-00554]\]. However, intervention studies providing ALA have yielded conflicting results on blood pressure, cholesterol levels, and triglyceride levels, as reviewed by Baker et al. \[[@B23-ijms-18-00554]\]. Also, despite the increase in these fatty acids in adipose tissue after resveratrol treatment, no clinically relevant improvements in blood pressure or insulin sensitivity were observed in our study population \[[@B18-ijms-18-00554]\]. In the adipose tissue we also detected a significant increase in intracellular glycerol and free fatty acids in the resveratrol-treated subjects. In support of this notion, previously published cell culture studies revealed a resveratrol-mediated enhancement of adrenergic stimulated lipolysis in fat cells \[[@B29-ijms-18-00554],[@B30-ijms-18-00554]\]. Moreover, resveratrol treatment caused consistent reductions in several dicarboxylic fatty acids in plasma. These lipids can be produced from peroxisomal-mediated degradation of very long chain fatty acids (\>22 carbons) and subsequently be metabolized via beta-oxidation. Collectively, a decrease in the dicarboxylic fatty acids in plasma could be a result of increased beta-oxidation in adipose tissue. Regarding glucose metabolism, we found trends toward elevation in glycolytic intermediates and the pentose phosphate pathway (PPP) intermediates (*p* \< 0.10) in adipose tissue in response to resveratrol. Stimulation of the PPP pathway is associated with increased insulin resistance, increased NADPH production, and thus, increased production of free fatty acids \[[@B31-ijms-18-00554]\]. Hence, our metabolomics findings may indicate increased glucose availability and utilization through glycolysis and the pentose phosphate pathway, owing to decreased insulin sensitivity in the males receiving resveratrol. Clinical parameters in the same study subjects support this assumption, as resveratrol increased fructosamine levels after four months of treatment, indicating poorer insulin sensitivity \[[@B18-ijms-18-00554]\]. In the resveratrol-treated group we demonstrated elevation in metabolites related to metabolism of the amino acid histidine. In particular, histidine was significantly increased in the blood and a trending elevation in the related biochemical marker of the muscle protein turnover 3-methylhistidine along with the acetylated derivative *N*-acetyl-3-methylhistidine was also observed. Increase in 3-methylhistidine, which is a post-translationally modified amino acid derived from the muscle contractile proteins actin and myosin, and *N*-acetyl-3-methylhistidine indicates increased muscle turnover in men treated with resveratrol. This finding ties in well with the study by Olesen et al. \[[@B32-ijms-18-00554]\], in which resveratrol blunted the positive effects of exercise training. In contrast, Scribbans et al. found that resveratrol in addition to exercise had positive effects on muscle metabolism with an increase in mitochondrial capacity \[[@B33-ijms-18-00554]\], while others found no effect in humans \[[@B34-ijms-18-00554]\]. Most rodent studies have found positive effects of resveratrol on muscle function \[[@B5-ijms-18-00554],[@B35-ijms-18-00554],[@B36-ijms-18-00554]\]. Clearly, more clinical studies are needed to determine the exact effects of resveratrol on skeletal muscle function. Finally, we found urinary changes in several tyrosine-derived, tryptophan-derived, phenylalanine-derived, and histidine-derived metabolites. Tryptophan, histidine, and phenylalanine are classified as essential amino acids, which cannot be synthesized de novo by the body. However, gut bacteria may contribute to their production and degradation \[[@B37-ijms-18-00554]\]. Fermentation of tyrosine and tryptophan by colonic bacteria is found to produce phenols and indoles, which are excreted in the urine \[[@B38-ijms-18-00554]\]. Also, imidazole propionate is known to be excreted in the urine and direct degradation of urocanate to imidazole propionate by the intestinal flora was demonstrated in an animal study \[[@B39-ijms-18-00554]\]. Changes in microbiota-related urinary metabolites are proposed to be of relevance to human health. For instance hippurate, phenylacetylglutamine, 4-cresylsulfate, and 4-hydroxyphenylpropionate have been related to body weight, blood pressure, and metabolic syndrome \[[@B40-ijms-18-00554]\], while urinary imiadazole propionate, which we found to be elevated by resveratrol, has been related to intestinal dysfunction \[[@B39-ijms-18-00554]\]. Therefore, the changes in urinary amino acids and derivatives revealed by the metabolomic analysis may imply a resveratrol-induced modulation of the gut flora in men with metabolic syndrome. This is in accordance with studies in rodents where resveratrol has been shown to modulate the gut microbiota directly \[[@B41-ijms-18-00554],[@B42-ijms-18-00554],[@B43-ijms-18-00554]\]. The strengths of the present clinical trial are a carefully selected, strong study design and the advanced metabolomics approach. A high dosage of resveratrol in a relatively long-term study was chosen. Participants were selected because they had metabolic syndrome, a condition considered a prime target for resveratrol. Furthermore, the advanced metabolomics technique was applied in an unbiased approach to four different matrices. However, a limitation of our study is the lack of liver tissue samples. The liver is a highly metabolic organ and a number of experimental studies have provided evidence of the hepatic benefits of resveratrol treatment \[[@B44-ijms-18-00554]\]. Metabolomic profiling of liver tissue from the same study subjects would help complete the metabolic puzzle of resveratrol-induced biochemical effects. Also, metabolomic analysis on feces would have provided insight into possible beneficial effects on the gut microbiota. Finally, it must be kept in mind that the present study used a very high dose of resveratrol (1000 mg), which is impossible to reach from natural food items. Resveratrol has a very low bioavailability owing to a rapid metabolism to glucuronide and sulfate conjugates in the liver. However, it is possible that ingestion of food items rich in resveratrol and other polyphenols might inhibit the metabolism or increase the absorption of resveratrol, resulting in a better bioavailability from food items than from purified resveratrol products \[[@B45-ijms-18-00554],[@B46-ijms-18-00554]\]. 4. Materials and Methods {#sec4-ijms-18-00554} ======================== 4.1. Study Design {#sec4dot1-ijms-18-00554} ----------------- Data in the present study comprise a subgroup from our original study describing the effects of resveratrol on bone \[[@B16-ijms-18-00554]\]. As we only obtained tissue samples from 24 subjects in the placebo group and 21 in the hRSV group, the sample size is lower compared to the original paper. The protocol was approved by the Regional Committee on Health Research Ethics (M-20110111) (24 May 2011) and the Danish Data Protection Agency, and the study was conducted in agreement with the Declaration of Helsinki II. Participants were given oral and written information about the purpose and nature of all procedures before informed consent was obtained. The protocol was registered at clinicaltrials.gov (NCT01412645). The study was a randomized, placebo-controlled, double-blinded, single-center study. Male test subjects with metabolic syndrome (MetS) were randomized to treatment for four months with tablets containing a placebo, low-dose resveratrol (75 mg twice daily), or high-dose resveratrol (500 mg twice daily). The study comprised 66 test subjects. Inclusion criteria were: Male gender, age between 30 and 60 years, and MetS. MetS was defined according to the International Diabetes Federation \[[@B47-ijms-18-00554]\] as central obesity (Waist circumference ≥94 cm and/or BMI \> 30 kg/m^2^) plus any two of the following: raised triglycerides (≥1.7 mmol/L), reduced high-density lipoprotein (HDL) (≤1.03 mmol/L), raised blood pressure (systolic ≥130 mmHg or diastolic ≥85 mmHg), raised fasting plasma glucose (≥5.6 mmol/L), or drug treatment for the individual features. The trial was performed under conditions of weight stability, unchanged diet, unchanged dietary supplements, and strict compliance with the study drug. Metabolomics results from the high-dose resveratrol (1000 mg daily) and the placebo groups after four months' treatment are reported in the present study. 4.2. Samples {#sec4dot2-ijms-18-00554} ------------ Blood and urine samples were collected between 7:30 and 11:00 a.m. after an overnight fast. Biopsy of the abdominal subcutaneous adipose tissue depot was obtained using a liposuction cannula and skeletal muscle biopsy was harvested from the musculus vastus lateralis using a Bergström cannula. All procedures were performed under sterile conditions. Before both biopsies were performed, the area was anesthetized using 5 to 10 mL lidocaine. 4.3. Metabolomic Analysis {#sec4dot3-ijms-18-00554} ------------------------- Urine, blood, adipose tissue, and skeletal muscle tissue samples were shipped to Metabolon, Inc.^®^, (Durham, NC, USA) on dry ice. Following receipt, samples were stored at −80 °C until analyzed. A recovery standard was added prior to the first step in preparation for quality control (QC). To remove protein, dissociate small molecules bound to protein or trapped in the protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for two minutes followed by centrifugation. The resulting extracts were divided into five fractions: one for analysis by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) with positive ion mode electrospray ionization, one for analysis by UPLC-MS/MS with negative ion mode electrospray ionization, one for LC polar platform, one for analysis by Gas Chromatography-Mass Spectroscopy (GC-MS), and one sample was reserved for backup. Raw data were extracted, peak-identified, and processed using Metabolon's hardware and software. More than 3300 commercially available purified standard compounds were used for the identification of compounds in the samples. General platform methods and metabolite detection identification are described in detail in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"}, [Tables S8 and S9](#app1-ijms-18-00554){ref-type="app"}, [Figures S1 and S2](#app1-ijms-18-00554){ref-type="app"}, and [Supplementary Tables S1--S4](#app1-ijms-18-00554){ref-type="app"}). 4.4. Statistical Analysis {#sec4dot4-ijms-18-00554} ------------------------- Baseline comparisons of the study population were evaluated by unpaired Student's *t*-test after being checked for normality and equal variance using SPSS Statistics version 20 software. Results are expressed as mean ± SEM. The level of significance was 0.05. The pathway enrichment value (PEV) used in [Figure 3](#ijms-18-00554-f003){ref-type="fig"}, [Figure 4](#ijms-18-00554-f004){ref-type="fig"}, [Figure 5](#ijms-18-00554-f005){ref-type="fig"} and [Figure 6](#ijms-18-00554-f006){ref-type="fig"} is calculated based on the following equation: A PEV greater than one indicates that the pathway holds more hRSV-regulated compounds relative to the study overall, suggesting that the pathway may be a target of interest in the intervention effects. In the metabolomic analysis Welch's two-sample *t*-test was used to identify biochemicals that differed significantly between treatment groups, following log transformation and, if necessary, imputation of missing values with the minimum observed value for each compound. *p*-Values ≤ 0.05 were considered statistically significant and values approaching significance (*p* \< 0.10) were reported as well. An estimate of the false discovery rate (*q*-value method) was calculated to take into account the multiple comparisons that normally occur in metabolomics-based studies. Metabolomics statistical methods are described in details in the [Supplementary Materials](#app1-ijms-18-00554){ref-type="app"} (see [Supplementary Appendix](#app1-ijms-18-00554){ref-type="app"} and [Table S10](#app1-ijms-18-00554){ref-type="app"}). 5. Conclusions {#sec5-ijms-18-00554} ============== Our comprehensive metabolomic analysis revealed small but robust changes in response to high-dose resveratrol treatment. The main finding is that resveratrol lowered sulfated androgen precursors in blood, adipose tissue, and muscle tissue and concurrently increased the content in urine, indicating increased urinary excretion of these sulfated steroids. Furthermore, the content of long-chain polyunsaturated fatty acids in adipose tissue was increased, probably by increased conversion of ALA and LA mediated through reduced androgen precursors. Several metabolites derived from aromatic amino acids associated with the gut microbiota changed, suggesting that resveratrol may affect either the composition or the metabolism of the gut flora. Considering the increasingly recognized role of the gut microbiota in human health and disease, this effect on gut flora activity may represent a potential and import mode of action of resveratrol. Lastly, our results indicate that non-targeted metabolomics is able to broaden our understanding of the many intra-cellular processes regulated by resveratrol treatment. Yet, our study also demonstrates that it is difficult to conclude solely from a single matric, since only a minor overlap between the four matrices we examined was found. Based on this, we suggest that metabolomics are performed in the tissue of interest. Future clinical studies on resveratrol should focus on the steroid metabolism pathway and the direct impact on human gut microbiota, preferably including liver tissue and feces samples. We wish to thank Lenette Pedersen and Pia Hornbek for their laboratory assistance. The study was supported by the Rasmus Riisfort Foundation, the Ejnar Danielsens Foundation, and the AP Møller Maersk Foundation. The study is part of the research program LIRMOI Research Center ([www.LIRMOI.com](www.LIRMOI.com)), which is supported by the Danish Council for Strategic Research (Grant 10-093499). Supplementary materials can be found at [www.mdpi.com/1422-0067/18/3/554/s1](www.mdpi.com/1422-0067/18/3/554/s1). ###### Click here for additional data file. Anne Sofie Korsholm took part in formulating the study design, analyzed the data, and wrote the manuscript with feedback from the co-authors. Marie Juul Ornstrup and Thomas Nordstrøm Kjær performed the experiment and took part in formulating the study design, drafting the manuscript, and revising it critically. Steen Bønløkke Pedersen conceived and designed the experiment, took part in analyzing the data, drafting the manuscript, and revising it critically, and contributed to important intellectual content of the manuscript. The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. hRSV High-dose resveratrol RF Random forest analysis PEV Pathway enrichment value DHA Docosahexaenoate ALA α-linolenic acid LA Linoleic acid MetS Metabolic syndrome ![Venn diagram of all detected metabolites in the four different matrices (for metabolite details see [Supplementary Table S5](#app1-ijms-18-00554){ref-type="app"}).](ijms-18-00554-g001){#ijms-18-00554-f001} ![Venn diagram of significantly hRSV-altered metabolites detected in the four different matrices (for metabolite details see [Supplementary Table S6](#app1-ijms-18-00554){ref-type="app"}).](ijms-18-00554-g002){#ijms-18-00554-f002} ![Resveratrol-mediated changes in adipose tissue, expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites); hRSV: high-dose resveratrol; Ctrl: Placebo group.](ijms-18-00554-g003){#ijms-18-00554-f003} ![Resveratrol-mediated changes in plasma expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites).](ijms-18-00554-g004){#ijms-18-00554-f004} ![Resveratrol-mediated changes in skeletal muscle tissue expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites); hRSV: high-dose resveratrol; Ctrl: Placebo group.](ijms-18-00554-g005){#ijms-18-00554-f005} ![Resveratrol-mediated changes in urine expressed as Pathway enrichment values (PEV). Values are based on the significant resveratrol-regulated compounds relative to all detected compounds in the pathway. PEV = (number of significant metabolites in pathway/total number of detected metabolites in pathway)/(total number of significant metabolites/total number of detected metabolites); hRSV: high-dose resveratrol; Ctrl: Placebo group.](ijms-18-00554-g006){#ijms-18-00554-f006} ijms-18-00554-t001_Table 1 ###### Clinical features at baseline and post-treatment. Characteristic Placebo (*n* = 24) hRSV (*n* = 21) *p*-Value -------------------------------- -------------------- ----------------- ------------ ------------ ------------- Age, years 47.8 ± 1.3 51.9 ± 1.3 *p* \< 0.05 Body mass index, kg/m^2^ 34.1 ± 0.8 33.4 ± 0.2 33.8 ± 0.7 33.7 ± 0.2 NS HOMA-IR 4.36 ± 0.5 4.19 ± 0.3 3.87 ± 0.5 4.50 ± 0.3 NS Systolic blood pressure, mmHg 150 ± 3 142 ± 3 146 ± 2 140 ± 3 NS Diastolic blood pressure, mmHg 91.3 ± 2.1 86.0 ± 1.3 89.3 ± 1.7 87.8 ± 1.4 NS ^1^ Data are expressed as mean ± SEM. NS = no significant difference. Comparisons between groups were evaluated by unpaired Student's *t*-test. The level of significance was 0.05. ijms-18-00554-t002_Table 2 ###### Summary of resveratrol-induced changes in adipose tissue. Identified metabolites in the long-chain fatty acid, polyunsaturated fatty acid (n3 and n6), steroids, glycolysis, gluconeogenesis, and pyruvate metabolism, and pentose phosphate pathways in adipose tissue. Red: Significant elevation (*p* ≤ 0.05). Orange: Trending elevation (*p* \< 0.10); Green: Significant reduction (*p* ≤ 0.05). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul} Ctrl *p*-Value *q*-Value ------------------------------------------------------------------------------------------------ -------------------------------------------------------------- ------------------------------ ------------------ ----------- ----------- **Lipid** Long-Chain Fatty Acid Myristate (14:0) 1.10 0.003 0.111 Myristoleate (14:1n5) 1.67 0.017 0.182 Pentadecanoate (15:0) 1.01 0.977 0.816 Palmitate (16:0) 1.10 0.033 0.240 Palmitoleate (16:1n7) 1.41 0.003 0.109 Margarate (17:0) 0.98 0.500 0.672 10-heptadecenoate (17:1n7) 1.12 0.086 0.309 Stearate (18:0) 1.02 0.610 0.731 Oleate (18:1n9) 1.17 0.109 0.328 *cis*-vaccenate (18:1n7) 1.17 0.304 0.538 Nonadecanoate (19:0) 1.00 0.866 0.794 10-nonadecenoate (19:1n9) 1.08 0.569 0.703 Arachidate (20:0) 0.94 0.472 0.660 Eicosenoate (20:1n9 or 11) 1.12 0.335 0.566 Erucate (22:1n9) 0.96 0.657 0.734 Polyunsaturated Fatty Acid (n3 and n6) Stearidonate (18:4n3) 1.50 0.045 0.240 Eicosapentaenoate (EPA; 20:5n3) 1.25 0.093 0.311 Docosapentaenoate (n3 DPA; 22:5n3) 1.31 0.012 0.177 Docosahexaenoate (DHA; 22:6n3) 1.38 0.042 0.240 Linoleate (18:2n6) 1.24 0.021 0.203 Linolenate \[α or γ; (18:3n3 or 6)\] 1.21 0.060 0.253 Dihomo-linolenate (20:3n3 or n6) 1.19 0.065 0.261 Arachidonate (20:4n6) 1.17 0.107 0.328 Adrenate (22:4n6) 1.69 0.010 0.177 Docosapentaenoate (n6 DPA; 22:5n6) 1.63 0.056 0.253 Docosadienoate (22:2n6) 1.10 0.275 0.498 Dihomo-linoleate (20:2n6) 1.11 0.216 0.443 Mead acid (20:3n9) 1.33 0.045 0.240 Steroid Dehydroisoandrosterone sulfate (DHEA-S) 0.40 0.013 0.177 4-androsten-3β,17β-diol disulfate (1) 0.37 0.009 0.177 **Carbohydrate** Glycolysis, Gluconeogenesis, and pyruvate metabolism pathway 1,5-anhydroglucitol (1,5-AG) 0.86 0.235 0.464 Glucose 0.98 0.594 0.722 Glucose-6-phosphate (G6P) 1.23 0.059 0.253 Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate, myo-inositol 1,4 or 1,3-Diphosphate 1.16 0.159 0.394 Dihydroxyacetone phosphate (DHAP) 1.30 0.080 0.303 3-phosphoglycerate 1.75 0.057 0.253 Phosphoenolpyruvate (PEP) 1.39 0.128 0.357 Lactate 1.04 0.396 0.599 Glycerate 1.08 0.771 0.777 Pentose phosphate pathway Sedoheptulose-7-phosphate 1.27 0.051 0.253 ijms-18-00554-t003_Table 3 ###### Summary of resveratrol-induced changes in plasma. Identified metabolites in the steroid, fatty acid dicarboxylate, and histidine metabolism pathways in plasma. Red: Significant elevation (*p* ≤ 0.05). Orange: Trending elevation (*p* \< 0.10); Green: Significant reduction (*p* ≤ 0.05). Light green: Trending reduction (*p* \< 0.10). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul} Ctrl *p*-Value *q*-Value ------------------------------------------------------ ---------------------- ---------------------- ------------------ ----------- ----------- **Lipid** Steroid Pregnenolone sulfate 0.63 0.001 0.018 21-hydroxypregnenolone disulfate 0.41 \<0.001 \<0.001 5α-pregnan-3β,20α-diol disulfate 0.42 \<0.001 \<0.001 5α-pregnan-3α,20β-diol disulfate 1 0.93 0.140 0.762 Pregnen-diol disulfate 0.53 0.001 0.015 Pregn steroid monosulfate 0.72 0.046 0.515 Pregnanediol-3-glucuronide 0.92 0.644 0.927 Cortisol 0.92 0.745 0.941 Cortisone 0.93 0.285 0.824 Dehydroisoandrosterone sulfate (DHEA-S) 0.57 \<0.001 0.013 Epiandrosterone sulfate 0.42 \<0.001 0.004 Androsterone sulfate 0.40 \<0.001 0.004 4-androsten-3β,17β-diol disulfate 1 0.51 \<0.001 0.016 4-androsten-3β,17β-diol disulfate 2 0.49 \<0.001 \<0.001 5α-androstan-3β,17α-diol disulfate 0.37 \<0.001 0.001 5α-androstan-3α,17β-diol disulfate 0.71 0.071 0.659 5α-androstan-3β,17β-diol disulfate 0.37 \<0.001 0.002 Andro steroid monosulfate 2 0.31 \<0.001 \<0.001 Estrone 3-sulfate 0.87 0.132 0.762 Fatty Acid, Dicarboxylate 2-hydroxyglutarate 0.98 0.660 0.934 Sebacate (decanedioate) 0.73 0.101 0.762 Dodecanedioate 0.61 \<0.001 0.001 Tetradecanedioate 0.75 0.021 0.278 Hexadecanedioate 0.85 0.206 0.762 Octadecanedioate 0.88 0.255 0.811 3-carboxy-4-methyl-5-propyl-2-furanpropanoate (CMPF) 0.95 0.955 0.964 **Amino Acid** Histidine Metabolism Histidine 1.16 0.008 0.147 3-methylhistidine 1.93 0.056 0.570 *N*-acetyl-3-methylhistidine 1.23 0.084 0.721 *trans*-urocanate 1.38 0.128 0.762 ijms-18-00554-t004_Table 4 ###### Summary of resveratrol-induced changes in skeletal muscle tissue. Identified metabolites in the steroid, short-chain fatty acid, medium-chain fatty acid, long-chain fatty acid, and polyunsaturated fatty acid (n3 and n6) pathways in skeletal muscle. Red: Significant elevation (*p* ≤ 0.05). Orange: Trending elevation (*p* \< 0.10); Green: Significant reduction (*p* ≤ 0.05). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul} Ctrl *p*-Value *q*-Value ----------------------------------------- ----------------------- ------------------ ------------------ ----------- ----------- **Lipid** Steroid Cortisol 1.40 0.211 0.962 Cortisone 1.14 0.778 0.987 Dehydroisoandrosterone sulfate (DHEA-S) 0.48 \<0.001 0.025 Epiandrosterone sulfate 0.29 \<0.001 0.001 Androsterone sulfate 0.24 \<0.001 0.001 4-androsten-3β,17β-diol disulfate (1) 0.46 0.002 0.125 Short-Chain Fatty Acid Valerate 0.86 \<0.001 0.026 Medium-Chain Fatty Acid Caproate (6:0) 1.09 0.442 0.962 Heptanoate (7:0) 0.82 \<0.001 0.032 Caprylate (8:0) 1.08 0.619 0.962 Undecanoate (11:0) 0.89 0.002 0.125 Long-Chain Fatty Acid Myristoleate (14:1n5) 1.78 0.252 0.962 Palmitate (16:0) 0.98 0.449 0.962 Margarate (17:0) 0.95 0.268 0.962 10-heptadecenoate (17:1n7) 1.09 0.029 0.655 Stearate (18:0) 0.97 0.209 0.962 Oleate (18:1n9) 1.01 0.994 0.996 *cis*-vaccenate (18:1n7) 1.02 0.904 0.995 10-nonadecenoate (19:1n9) 0.97 0.882 0.995 Arachidate (20:0) 0.84 0.003 0.168 Eicosenoate (20:1n9 or 11) 1.01 0.958 0.996 Erucate (22:1n9) 0.88 0.193 0.962 Polyunsaturated Fatty Acid (n3 and n6) Stearidonate (18:4n3) 1.45 0.015 0.474 Eicosapentaenoate (EPA; 20:5n3) 0.98 0.554 0.962 Docosapentaenoate (n3 DPA; 22:5n3) 1.04 0.511 0.962 Docosahexaenoate (DHA; 22:6n3) 1.07 0.344 0.962 Linoleate (18:2n6) 1.02 0.714 0.980 Linolenate \[α or γ; (18:3n3 or 6)\] 1.16 0.065 0.962 Dihomo-linolenate (20:3n3 or n6) 1.02 0.873 0.995 Arachidonate (20:4n6) 1.00 0.990 0.996 Docosapentaenoate (n6 DPA; 22:5n6) 0.99 0.994 0.996 Docosadienoate (22:2n6) 0.93 0.680 0.962 Dihomo-linoleate (20:2n6) 0.83 0.287 0.962 Mead acid (20:3n9) 0.93 0.619 0.962 ijms-18-00554-t005_Table 5 ###### Summary of resveratrol-induced changes in urine. Identified metabolites in the steroid, tryptophan metabolism, phenylalanine and tyrosine metabolism, and histidine metabolism pathways in urine. Red: Significant elevation (*p* ≤ 0.05). Orange: Trending elevation (*p* \< 0.10); Green: Significant reduction (*p* ≤ 0.05). Superpathway Sub Pathway Biochemical Name [hRSV]{.ul}Ctrl *p*-Value *q*-Value ------------------------------------------------------ ----------------------- ---------------------------------- ----------------- ----------- ----------- **Lipid** Steroid 21-hydroxypregnenolone disulfate 0.50 0.012 0.313 Pregnen-diol disulfate 0.79 0.421 0.861 Pregn steroid monosulfate 2.10 0.008 0.281 Pregnanediol-3-glucuronide 0.87 0.910 0.938 Cortisol 1.29 0.734 0.915 Cortisol glucuronide 1.36 0.600 0.893 Cortisone 1.13 0.757 0.915 Tetrahydrocortisone 1.54 0.270 0.762 Dehydroisoandrosterone sulfate (DHEA-S) 3.95 \<0.001 0.001 16a-hydroxy DHEA 3-sulfate 1.04 0.201 0.735 Epiandrosterone 0.87 0.967 0.938 Epiandrosterone sulfate 1.43 0.036 0.575 Androsterone sulfate 1.22 0.171 0.724 4-androsten-3β,17β-diol monosulfate (1) 3.75 \<0.001 0.001 4-androsten-3β,17β-diol monosulfate (2) 3.50 0.003 0.149 4-androsten-3α,17α-diol monosulfate (2) 6.78 \<0.001 0.001 4-androsten-3β,17β-diol disulfate (1) 0.72 0.804 0.920 4-androsten-3β,17β-diol disulfate (2) 0.89 0.736 0.915 5α-androstan-3β,17α-diol disulfate 0.20 0.002 0.127 5α-androstan-3β,17β-diol disulfate 0.68 0.414 0.861 Andro steroid monosulfate (1) 0.45 0.004 0.170 Testosterone sulfate 3.74 0.009 0.281 11-ketoetiocholanolone glucuronide 0.98 0.793 0.920 Etiocholanolone glucuronide 0.85 0.766 0.915 17α-hydroxypregnanolone glucuronide 1.10 0.216 0.747 5β-pregnan-3α,21-diol-11,20-dione 21-Glucosiduronate 1.51 0.604 0.893 11-ketoetiocholanolone sulfate 1.23 0.957 0.938 Dehydroepiandrosterone glucuronide 0.83 0.585 0.881 **Amino Acid** Tryptophan Metabolism Tryptophan 0.99 0.908 0.938 *N*-acetyltryptophan 1.37 0.194 0.726 Tryptamine 0.37 \<0.001 0.010 Indolelactate 1.56 0.050 0.613 Indoleacetate 0.58 0.201 0.735 3-indoxyl sulfate 1.39 0.124 0.724 Kynurenine 1.29 0.637 0.905 Kynurenate 1.17 0.325 0.793 Anthranilate 1.16 0.446 0.861 3-hydroxykynurenine 1.43 0.480 0.874 3-hydroxyanthranilate 1.59 0.057 0.638 Xanthurenate 1.23 0.231 0.748 Picolinate 1.16 0.180 0.724 5-hydroxytryptophan 1.11 0.383 0.848 5-hydroxyindoleacetate 1.02 0.563 0.881 Serotonin (5HT) 1.03 0.782 0.920 Indoleacetylglutamine 0.64 0.824 0.920 Tryptophan betaine 1.14 0.976 0.940 Indole-3-carboxylic acid 0.93 0.696 0.914 *C*-glycosyltryptophan 1.03 0.693 0.914 *N*-acetylkynurenine (2) 2.14 0.047 0.613 Phenylalanine and Tyrosine Metabolism Phenylalanine 1.11 0.588 0.881 *N*-acetylphenylalanine 1.31 0.185 0.726 Phenyllactate (PLA) 1.86 0.148 0.724 4-hydroxyphenylacetate 1.02 0.915 0.938 3-hydroxyphenylacetate 1.52 0.096 0.724 Phenylacetylglycine 1.13 0.801 0.920 Phenylacetylglutamine 0.97 0.720 0.914 Tyrosine 1.06 0.693 0.914 *N*-acetyltyrosine 1.47 0.051 0.613 4-hydroxycinnamate 1.83 0.172 0.724 Tyramine 0.60 0.001 0.075 m-tyramine 0.99 0.923 0.938 4-hydroxyphenylpyruvate 2.23 0.012 0.313 3-(4-hydroxyphenyl)lactate 1.33 0.280 0.762 Phenol sulfate 1.74 0.016 0.365 p-cresol sulfate 0.87 0.465 0.874 o-cresol sulfate 1.41 0.301 0.782 Dopamine 0.37 0.998 0.947 Vanillylmandelate (VMA) 1.08 0.392 0.858 3-methoxytyrosine 1.08 0.967 0.938 3-methoxytyramine 1.07 0.226 0.748 3-methoxytyramine sulfate 0.96 0.301 0.782 3,4-dihydroxyphenylacetate 1.32 0.107 0.724 Homovanillate (HVA) 1.33 0.040 0.583 Homovanillate sulfate 0.98 0.565 0.881 Gentisate 1.37 0.125 0.724 Phenylpropionylglycine 0.95 0.946 0.938 3-\[3-(sulfooxy)phenyl\]propanoic acid 0.66 0.602 0.893 3-(3-hydroxyphenyl)propionate 1.01 0.903 0.938 5-hydroxymethyl-2-furoic acid 0.77 0.442 0.861 2-hydroxyphenylacetate 1.31 0.075 0.721 Phenylacetylcarnitine 0.82 0.542 0.881 Dopamine sulfate (1) 0.48 0.963 0.938 Dopamine sulfate (2) 0.46 0.893 0.938 p-cresol-glucuronide 0.95 0.704 0.914 Tyramine *O*-sulfate 0.69 0.202 0.735 Vanillic alcohol sulfate 0.91 0.271 0.762 4-hydroxycinnamate sulfate 0.47 0.240 0.748 3,4-dihydroxyphenylacetate sulfate 0.69 0.548 0.881 Histidine Metabolism Histidine 1.10 0.402 0.861 *N*-acetylhistidine 1.12 0.152 0.724 1-methylhistidine 1.09 0.712 0.914 3-methylhistidine 2.02 0.080 0.724 *N*-acetyl-3-methylhistidine 2.10 0.037 0.576 *N*-acetyl-1-methylhistidine 1.07 0.482 0.874 Hydantoin-5-propionic acid 1.01 0.954 0.938 *trans*-urocanate 1.14 0.280 0.762 *cis*-urocanate 1.11 0.632 0.905 Formiminoglutamate 1.03 0.984 0.943 Imidazole propionate 1.67 0.010 0.290 Imidazole lactate 1.31 0.280 0.762 1-methylimidazoleacetate 1.13 0.424 0.861 4-imidazoleacetate 1.16 0.703 0.914 *N*-acetylhistamine 0.80 0.554 0.881

{ "pile_set_name": "PubMed Central" }

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"phenylacetylglutamin", "tyrosin", "receptor", "futur", "classifi", "sappijmsreftypeapp", "pcresolglucuronid", "owe", "shorten", "vastu", "shown", "homovanil", "arachidon", "specul", "vanillylmandel", "addit", "phenyalanin", "total", "high", "longchain", "subcutan", "metabolit", "activ", "limit", "picolin", "microbiotarel", "approv", "diastol", "middleag", "pronounc", "examin", "elev", "glycolyt", "fructosamin", "dpa", "mass", "surgeri", "qualiti", "octadecanedio", "statist", "mtyramin", "nut", "cortison", "took", "previous", "lipoprotein", "amino", "sampl", "plant", "møller", "prespecifi", "femal", "exercis", "identif", "superpathway", "resveratrolregul", "materi", "deriv", "regist", "rf", "obvious", "display", "nacetylhistamin", "depict", "longev", "gsm", "council", "estron", "bariatr", "conceiv", "stearat", "concurr", "nacetylmethylhistidin", "adipos", "studi", "techniqu", "cresylsulf", "explor", "harvest", "character", "three", "indoleacet", "glycolysi", "diet", "integr", "seri", "sappijmsreftypeappijmsgijmsf", "mitochondri", "highli", "usa", "ship", "αpregnanβαdiol", "tablet", "phenylacetylglycin", "hdl", "product", "metabolon", "fail", "isobar", "shortchain", "usual", "research", "palmitol", "hydroxyphenylacet", "microbiota", "deduc", "stilbenebas", "prefer", "find", "sebac", "α", "content", "figur", "clinic", "nontarget", "adrenerg", "serotonin", "restrict", "dhap", "indol", "backup", "period", "excret", "rapid", "conduct", "confin", "rodent", "part", "systol", "androgen", "method", "random", "affect", "step", "intellectu", "palmit", "new", "phenylalaninederiv", "helsinki", "methylhistidin", "recogn", "verifi", "procedur", "distinct", "discoveri", "wine", "approach", "variou", "subject", "contrast", "elong", "ap", "correl", "phenylacetylcarnitin", "hydroxyphenylpyruv", "effect", "lower", "omic", "pictur", "skin", "ga", "resveratrolinduc", "inc®", "wwwmdpicomswwwmdpicom", "metabolitestot", "fraction", "αandrostanβαdiol", "lenett", "disulf", "contain", "smallmolecul", "ht", "pedersen", "sem", "decis", "circul", "literatur", "ag", "docosapentaeno", "matric", "within", "anthranil", "overthecount", "hydroxyphenyllact", "rich", "notion", "tryptophanderiv", "hexadecanedio", "dietinduc", "span", "decid", "function", "danish", "decanedio", "hold", "mean", "number", "degrad", "introduct", "impli", "≥", "androstenββdiol", "andro", "cortisol", "rate", "liposuct", "other", "poor", "mediat", "purport", "fluid", "cm", "human", "advanc", "inconsist", "molecul", "docosahexaeno", "scribban", "modest", "male", "strateg", "seafood", "diabet", "depend", "placebocontrol", "hormon", "chain", "ppp", "socal", "flora", "overlap", "composit", "site", "et", "sofi", "promis", "review", "need", "captur", "preclin", "dicarboxyl", "care", "n", "matter", "sponsor", "stearidon", "propion", "intermedi", "satur", "design", "ala", "feder", "singl", "develop", "indic", "hydroxyindoleacet", "sulfat", "paramet", "format", "natur", "hydroxymethylfuro", "age", "bønløkke", "hbac", "possibl", "shake", "mmoll", "consent", "manuscript", "might", "plasma", "hrsvinduc", "hippur", "insight", "ann", "acetyl", "turnov", "daili", "diseas", "transform", "methoxytyramin", "investig", "etiocholanolon", "sub", "precursor", "dehydroisoandrosteron", "analysi", "complex", "cholesterolderiv", "reserv", "could", "solubl", "nct", "nacetylphenylalanin", "simultan", "apart", "chromatographymass", "greatest", "along", "raw", "action", "green", "fourmonth", "liver", "glycerol", "made", "varianc", "especi", "chosen", "yield", "region", "sold", "men", "phosphat", "neg", "lowdos", "ornstrup", "mode", "urocan", "lipolysi", "mg", "ion", "prime", "singlecent", "osmol", "polar", "increas", "softwar", "carbohydr", "improv", "revis", "report", "diagram", "oleat", "offer", "point", "isol", "≤", "summari", "clinicaltrialsgov", "area", "spectroscopi", "materialsappijmsreftypeapp", "hrsvul", "lc", "snapshot", "plu", "attribut", "depot", "therefor", "dehydroepiandrosteron", "heptadeceno", "detect" ]

22,252

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"accumulation", "contrast", "studies", "fail", "detect", "positive", "physiological", "effects", "conflicting", "results", "may", "part", "attributable", "differences", "study", "designs", "populations", "resveratrol", "formulations", "studies", "performed", "healthy", "individuals", "ability", "resveratrol", "improve", "glucose", "handling", "situations", "normal", "glucose", "homoeostasis", "questioned", "contrast", "feasible", "effect", "food", "ingredient", "weak", "demonstrate", "measurable", "consequences", "type", "diabetes", "Thus", "subjects", "suffering", "modest", "degrees", "insulin", "resistance", "likely", "optimal", "purpose", "investigating", "possible", "effects", "resveratrol", "insulin", "sensitivity", "probably", "comparable", "original", "mice", "studies", "using", "obese", "mice", "Study", "length", "may", "also", "importance", "studies", "include", "intervention", "periods", "weeks", "Obviously", "longer", "studies", "provide", "better", "determination", "possible", "effects", "resveratrol", "chronic", "conditions", "insulin", "resistance", "inflammation", "Finally", "methods", "currently", "used", "studies", "resveratrol", "targeted", "towards", "effects", "Insulin", "sensitivity", "instance", "assessed", "via", "measurements", "specific", "targets", "plasma", "insulin", "glucose", "techniques", "strengths", "specific", "applicable", "daily", "clinics", "however", "drawback", "findings", "limited", "objectives", "Likewise", "confining", "assessment", "plasma", "samples", "intracellular", "effects", "may", "missed", "Recently", "metabolomics", "approach", "developed", "Metabolomic", "analysis", "profiles", "metabolites", "using", "either", "targeted", "approach", "systematic", "identification", "quantification", "metabolites", "biological", "systems", "tissues", "organs", "fluids", "certain", "time", "point", "metabolomics", "directly", "sample", "metabolic", "modulations", "Targeted", "metabolomics", "allows", "precise", "measurement", "specific", "target", "isolated", "group", "related", "metabolites", "whereas", "metabolomics", "assesses", "wide", "range", "different", "metabolites", "possible", "technique", "provides", "simultaneous", "screening", "multiple", "metabolic", "pathways", "captures", "instantaneous", "integrated", "snapshot", "entire", "physiology", "organism", "Metabolomics", "advantage", "omics", "integrates", "changes", "gene", "expression", "protein", "levels", "enzymatic", "activity", "changes", "Previously", "metabolomics", "made", "possible", "determine", "changes", "human", "plasma", "metabolome", "caused", "various", "conditions", "diseases", "like", "sleep", "restriction", "diabetes", "bariatric", "surgery", "Studies", "demonstrated", "weak", "correlation", "plasma", "muscle", "metabolite", "levels", "indicating", "plasma", "measurements", "poor", "indicators", "skeletal", "muscle", "metabolism", "Consequently", "metabolomic", "analysis", "requires", "tissue", "biopsies", "allow", "detailed", "examination", "changes", "intracellular", "pathways", "within", "specific", "tissue", "metabolic", "profiling", "offers", "possibility", "identify", "biochemical", "signatures", "cellular", "metabolism", "involved", "different", "pathways", "like", "glucose", "lipid", "protein", "handling", "Massive", "research", "interest", "gone", "resveratrol", "potential", "protective", "compound", "management", "complications", "metabolic", "syndrome", "inflammation", "However", "particular", "pathways", "involved", "potential", "salutary", "effects", "large", "undetermined", "metabolomic", "profiling", "may", "provide", "new", "insight", "matters", "recently", "conducted", "randomized", "clinical", "trial", "describing", "effects", "resveratrol", "treatment", "males", "metabolic", "syndrome", "reported", "findings", "bone", "circulating", "steroids", "glucose", "metabolism", "inflammation", "aim", "present", "study", "provide", "comprehensive", "metabolomic", "analysis", "changes", "caused", "four", "months", "resveratrol", "treatment", "men", "metabolic", "syndrome", "effects", "resveratrol", "humans", "incompletely", "characterized", "used", "metabolomics", "approach", "based", "four", "different", "matrices", "plasma", "urine", "adipose", "tissue", "skeletal", "muscle", "tissue", "study", "subject", "study", "circulating", "steroids", "prostate", "size", "established", "decrease", "plasma", "androgen", "precursors", "resveratrol", "order", "maximize", "chances", "detecting", "even", "subtle", "pathway", "changes", "therefore", "decided", "employ", "metabolomics", "analysis", "resveratrol", "hRSV", "mg", "twice", "daily", "group", "compared", "placebo", "group", "Results", "Clinical", "Features", "basic", "characteristics", "participants", "outlined", "Table", "table", "Apart", "slight", "difference", "age", "two", "groups", "comparable", "baseline", "Global", "Metabolic", "Profiling", "Based", "combination", "two", "Ultrahigh", "Performance", "Liquid", "Mass", "Spectroscopy", "platforms", "one", "Gas", "Chromatography", "Mass", "Spectroscopy", "platform", "metabolites", "plasma", "metabolites", "adipose", "tissue", "metabolites", "skeletal", "muscle", "metabolites", "urine", "identified", "General", "platform", "methods", "data", "analysis", "metabolite", "detection", "identification", "described", "detail", "Supplementary", "Materials", "app", "see", "Supplementary", "Appendix", "app", "Supplementary", "Tables", "app", "total", "identified", "metabolites", "present", "four", "matrices", "illustrated", "Figure", "fig", "relationship", "significantly", "metabolites", "four", "matrices", "depicted", "Figure", "fig", "Adipose", "tissue", "urine", "greatest", "overlap", "terms", "significantly", "changed", "metabolites", "One", "metabolite", "dehydroisoandrosterone", "sulfate", "significantly", "changed", "four", "matrices", "Random", "forest", "analysis", "RF", "analysis", "adipose", "tissue", "muscle", "tissue", "urine", "plasma", "metabolic", "profiles", "resulted", "accuracy", "differentiating", "hRSV", "placebo", "groups", "respectively", "indicating", "differences", "biochemical", "profiles", "two", "groups", "quite", "pronounced", "Random", "Forest", "classification", "results", "methods", "data", "analysis", "described", "detail", "Supplementary", "Materials", "app", "see", "Supplementary", "Appendix", "app", "Supplementary", "Table", "app", "Among", "metabolites", "resulting", "RF", "analysis", "biochemicals", "involved", "amino", "acid", "metabolism", "lipid", "metabolism", "particularly", "important", "regards", "separation", "two", "groups", "Many", "metabolites", "embraced", "also", "included", "metabolic", "pathways", "Steroid", "hormones", "especially", "well", "represented", "RF", "importance", "plots", "addition", "significantly", "altered", "hRSV", "intervention", "Metabolic", "Profiling", "Adipose", "Tissue", "biochemicals", "identified", "adipose", "tissue", "displayed", "significant", "change", "p", "response", "hRSV", "treatment", "elevated", "reduced", "Furthermore", "compounds", "trend", "towards", "change", "p", "compounds", "increased", "four", "compounds", "reduced", "hRSV", "treatment", "pathways", "differed", "significantly", "hRSV", "group", "controls", "adipose", "tissue", "p", "shown", "Figure", "fig", "Exploring", "respective", "significantly", "changed", "pathways", "detail", "reveals", "specific", "compounds", "illustrated", "Table", "table", "four", "identified", "lipids", "significantly", "elevated", "fatty", "acid", "pathway", "myristate", "myristoleate", "palmitate", "palmitoleate", "lipids", "depict", "significant", "fold", "change", "hRSV", "group", "placebo", "group", "addition", "examining", "polyunsaturated", "fatty", "acid", "pathway", "demonstrated", "six", "identified", "lipids", "significantly", "elevated", "hRSV", "group", "stearidonate", "docosapentaenoate", "DPA", "docosahexaenoate", "DHA", "linoleate", "adrenate", "mead", "acid", "Also", "interest", "steroid", "hormone", "pathway", "reveals", "significant", "reductions", "identified", "steroids", "dehydroisoandrosterone", "sulfate", "disulfate", "interesting", "trending", "changes", "p", "adipose", "tissue", "pathways", "displayed", "glycolysis", "gluconeogenesis", "pyruvate", "metabolism", "pathway", "pentose", "phosphate", "pathway", "illustrated", "Table", "table", "trending", "elevation", "three", "nine", "identified", "glycolytic", "pathway", "intermediates", "dihydroxyacetone", "phosphate", "DHAP", "phosphoenolpyruvate", "PEP", "one", "pentose", "phosphate", "pathway", "intermediate", "found", "Metabolic", "Profiling", "Plasma", "named", "compounds", "plasma", "statistically", "different", "two", "groups", "p", "elevated", "reduced", "addition", "compounds", "showed", "trend", "towards", "change", "hRSV", "p", "eight", "increased", "three", "reduced", "pathways", "significantly", "different", "p", "hRSV", "group", "controls", "shown", 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"biochemicals", "showed", "trend", "towards", "change", "hRSV", "p", "biochemicals", "increased", "seven", "reduced", "pathways", "significantly", "different", "p", "hRSV", "group", "controls", "shown", "Figure", "fig", "striking", "change", "muscle", "tissue", "effect", "hRSV", "steroid", "hormone", "pathway", "four", "six", "compounds", "significantly", "reduced", "illustrated", "Table", "table", "four", "compounds", "comprised", "sulfated", "steroid", "metabolites", "dehydroisoandrosterone", "sulfate", "epiandrosterone", "sulfate", "androsterone", "sulfate", "disulfate", "Regarding", "skeletal", "muscle", "lipid", "metabolism", "analysis", "revealed", "significant", "hRSV", "alterations", "fatty", "acids", "fatty", "acid", "fatty", "acid", "pathways", "Generally", "hRSV", "group", "lower", "levels", "intracellular", "lipids", "addition", "two", "biochemicals", "polyunsaturated", "fatty", "acid", "pathway", "increased", "stearidonate", "linolenate", "α", "γ", "Metabolic", 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"Similarly", "urinary", "levels", "derivatives", "tryptophan", "metabolism", "tryptamine", "indolelactate", "phenyalanine", "metabolism", "histidine", "metabolism", "imidazole", "propionate", "significantly", "altered", "hRSV", "treatment", "depicted", "Table", "table", "Discussion", "effects", "resveratrol", "human", "trials", "proven", "inconsistent", "data", "represent", "standard", "biochemical", "analyses", "selected", "parameters", "focus", "inflammation", "glucose", "metabolism", "lipids", "liver", "function", "steroids", "assessed", "measurement", "blood", "samples", "thus", "representing", "picture", "whole", "body", "metabolism", "present", "paper", "describes", "exhaustive", "metabolomic", "profile", "blood", "adipose", "tissue", "skeletal", "muscle", "tissue", "urine", "comprehensive", "clinical", "trial", "males", "metabolic", "syndrome", "approach", "allows", "elaborate", "investigation", "subtle", "resveratrol", "effects", "distinct", "intracellular", "pathways", "possible", "measurements", "confined", "blood", "addition", "approach", "unbiased", "measurements", "various", "body", "compartments", "may", "help", "us", "determine", "cause", "effects", "previously", "detected", "Recently", "reported", "androgen", "precursors", "blood", "significantly", "reduced", "resveratrol", "treatment", "four", "months", "presented", "metabolomic", "analysis", "performed", "study", "cohort", "using", "advanced", "technique", "able", "verify", "decrease", "sulfated", "androgen", "precursors", "blood", "addition", "find", "significantly", "lower", "intracellular", "amounts", "sulfated", "androgen", "precursors", "skeletal", "muscle", "tissue", "well", "adipose", "tissue", "Sulfation", "common", "modification", "control", "levels", "active", "steroid", "hormones", "target", "sites", "increasing", "solubility", "renal", "excretion", "metabolomic", "profiling", "urine", "revealed", "increased", "urinary", "excretion", "majority", "measured", "sulfated", "steroid", 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"linoleic", "acid", "LA", "metabolized", "polyunsaturated", "fatty", "acids", "series", "desaturation", "elongation", "processes", "literature", "describes", "gender", "difference", "conversion", "ALA", "eicosapentaenoic", "acid", "EPA", "docosahexaenoic", "acid", "DHA", "women", "much", "higher", "conversion", "rate", "men", "difference", "depends", "upon", "sex", "steroids", "testosterone", "supplementation", "female", "transsexuals", "reduced", "DHA", "concentration", "Clearly", "indicates", "enzymes", "responsible", "desaturation", "elongation", "conversion", "ALA", "DHA", "negatively", "regulated", "androgens", "Based", "knowledge", "speculate", "resveratrol", "might", "stimulate", "conversion", "process", "ALA", "LA", "resulting", "increase", "polyunsaturated", "fatty", "acids", "robust", "reduction", "androgen", "precursors", "health", "impacts", "changes", "levels", "polyunsaturated", "fatty", "acids", "still", "fully", "determined", "polyunsaturated", "fatty", "acids", 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"sterile", "conditions", "biopsies", "performed", "area", "anesthetized", "using", "mL", "lidocaine", "Metabolomic", "Analysis", "Urine", "blood", "adipose", "tissue", "skeletal", "muscle", "tissue", "samples", "shipped", "Metabolon", "Durham", "NC", "USA", "dry", "ice", "Following", "receipt", "samples", "stored", "analyzed", "recovery", "standard", "added", "prior", "first", "step", "preparation", "quality", "control", "QC", "remove", "protein", "dissociate", "small", "molecules", "bound", "protein", "trapped", "protein", "matrix", "recover", "chemically", "diverse", "metabolites", "proteins", "precipitated", "methanol", "vigorous", "shaking", "two", "minutes", "followed", "centrifugation", "resulting", "extracts", "divided", "five", "fractions", "one", "analysis", "Ultrahigh", "Performance", "Liquid", "Mass", "Spectroscopy", "positive", "ion", "mode", "electrospray", "ionization", "one", "analysis", "negative", "ion", "mode", "electrospray", "ionization", "one", "LC", "polar", 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"main", "finding", "resveratrol", "lowered", "sulfated", "androgen", "precursors", "blood", "adipose", "tissue", "muscle", "tissue", "concurrently", "increased", "content", "urine", "indicating", "increased", "urinary", "excretion", "sulfated", "steroids", "Furthermore", "content", "polyunsaturated", "fatty", "acids", "adipose", "tissue", "increased", "probably", "increased", "conversion", "ALA", "LA", "mediated", "reduced", "androgen", "precursors", "Several", "metabolites", "derived", "aromatic", "amino", "acids", "associated", "gut", "microbiota", "changed", "suggesting", "resveratrol", "may", "affect", "either", "composition", "metabolism", "gut", "flora", "Considering", "increasingly", "recognized", "role", "gut", "microbiota", "human", "health", "disease", "effect", "gut", "flora", "activity", "may", "represent", "potential", "import", "mode", "action", "resveratrol", "Lastly", "results", "indicate", "metabolomics", "able", "broaden", "understanding", "many", "processes", 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"Adrenate", "Docosapentaenoate", "DPA", "Docosadienoate", "Mead", "acid", "Steroid", "Dehydroisoandrosterone", "sulfate", "disulfate", "Carbohydrate", "Glycolysis", "Gluconeogenesis", "pyruvate", "metabolism", "pathway", "Glucose", "Isobar", "fructose", "glucose", "Dihydroxyacetone", "phosphate", "DHAP", "Phosphoenolpyruvate", "PEP", "Lactate", "Glycerate", "Pentose", "phosphate", "pathway", "Summary", "changes", "plasma", "Identified", "metabolites", "steroid", "fatty", "acid", "dicarboxylate", "histidine", "metabolism", "pathways", "plasma", "Red", "Significant", "elevation", "p", "Orange", "Trending", "elevation", "p", "Green", "Significant", "reduction", "p", "Light", "green", "Trending", "reduction", "p", "Superpathway", "Sub", "Pathway", "Biochemical", "Name", "hRSV", "Ctrl", "p", "q", "Lipid", "Steroid", "Pregnenolone", "sulfate", "disulfate", "disulfate", "disulfate", "disulfate", "Pregn", "steroid", "monosulfate", "Cortisol", "Cortisone", "Dehydroisoandrosterone", "sulfate", 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"Acid", "Caproate", "Heptanoate", "Caprylate", "Undecanoate", "Fatty", "Acid", "Myristoleate", "Palmitate", "Margarate", "Stearate", "Oleate", "cis", "Arachidate", "Eicosenoate", "Erucate", "Polyunsaturated", "Fatty", "Acid", "Stearidonate", "Eicosapentaenoate", "EPA", "Docosapentaenoate", "DPA", "Docosahexaenoate", "DHA", "Linoleate", "Linolenate", "α", "γ", "Arachidonate", "Docosapentaenoate", "DPA", "Docosadienoate", "Mead", "acid", "Summary", "changes", "urine", "Identified", "metabolites", "steroid", "tryptophan", "metabolism", "phenylalanine", "tyrosine", "metabolism", "histidine", "metabolism", "pathways", "urine", "Red", "Significant", "elevation", "p", "Orange", "Trending", "elevation", "p", "Green", "Significant", "reduction", "p", "Superpathway", "Sub", "Pathway", "Biochemical", "Name", "hRSV", "Ctrl", "p", "q", "Lipid", "Steroid", "disulfate", "disulfate", "Pregn", "steroid", "monosulfate", "Cortisol", "Cortisol", "glucuronide", "Cortisone", "Tetrahydrocortisone", 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Introduction ============ Systemic anticancer therapy (SACT) ---------------------------------- There are many different treatment options for lung cancer, and these are influenced by type, extent and progression of disease. These treatments can be categorised into surgery, radiotherapy and systemic anticancer therapy (SACT), combinations of which are also possible. While surgery and radiotherapy tend to be of use in local, early-stage disease, SACT is largely used as first-line treatment in advanced stage IIIb-IV non-small-cell lung cancer (NSCLC), with extensive nodal involvement and metastases, to 'improve survival, disease control and quality of life' and is the preferred treatment for both limited and extensive small-cell lung cancer (SCLC) \[[@ref1]\]. It is planned in cycles, between which the patient is reviewed for response to therapy. SACT can be divided into three categories, based on mechanism of action in treating cancer. The most extensive group, chemotherapy, involves the use of cytotoxic drugs to directly destroy tumour cells. It has multiple applications in NSCLC, due to the various intentions of use across most stages of lung cancer. As an adjunct or in combination with radiotherapy, it can be considered pre-operatively in early-disease (stage I--II) patients suitable for surgery; adjuvant chemotherapy aiming to kill cancer cells following radiotherapy is an option in stage II--III disease \[[@ref2]\]. There are multiple classes of chemotherapy drug available, with a variety of intracellular targets such as DNA and microtubules. Biological therapies, the second group, are anticancer agents that prevent the spread of cancer by 'interfering with specific molecules involved in tumour growth and progression' \[[@ref3]\], as opposed to killing tumour cells directly. These targeted therapies have further developed as a result of advancements in tumour analysis for protein mutations that cause uncontrolled proliferation, such as those in epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) \[[@ref4]\]. The third mechanism of SACT is immunotherapy, where monoclonal antibodies recruit the immune system to recognise and attack malignant cells \[[@ref5]\]. [Table 1](#table1){ref-type="table"} details the SACT agents recommended for use in lung cancer. Application of SACT involves the use of combination therapies. The most recent statistics of the commonest regimens for lung cancer are shown in [Figure 1](#figure1){ref-type="fig"}. Supportive care medicines are often prescribed as part of the SACT regimen to prevent side effects from occurring. These include various antiemetics such as aprepitant, domperidone, and ondansetron, to combat nausea and vomiting, a common adverse effect of most anticancer agents \[[@ref6]\]. Notably, some have higher potential for nausea than others, particularly cisplatin, and different regimens thus require varying doses of antiemetic \[[@ref17]\]. Corticosteroids, such as dexamethasone, also counter nausea but additionally reduce the incidence and severity of skin rash, another frequent adverse reaction \[[@ref6]\]. Other medicines include folic acid and vitamin B12 for the antifolate drug pemetrexed \[[@ref18], [@ref19]\] and hydration guidelines for the nephrotoxic platinum-based compounds \[[@ref20]\], encompassing combinations of oral furosemide, intravenous saline, and magnesium or potassium salt solutions \[[@ref21], [@ref22]\]. Drug--drug interactions ======================= As discussed, cancer patients for whom SACT is indicated take a multitude of medicines, either within a complex regimen or due to a range of supportive therapies \[[@ref23]\]. This increases the likelihood of drug--drug interactions (DDIs), where concurrent administration of two drugs allows one to influence the activity of another \[[@ref24]\]. Moreover, interlinking these factors, older patients are the typical demographic of cancer and thus are more likely to be taking multiple regular medicines due to increased comorbidities \[[@ref25]--[@ref27]\]. DDIs, including those involving SACT medicines, can be beneficial and lead to a synergistic (augmented) effect of both drugs, a concept utilised in combination anticancer therapy \[[@ref28]\]. However, there is potential for negative outcomes; due to the large number and difference in mechanisms of action between anticancer agents, there are a multitude of adverse DDIs involving chemotherapy drugs \[[@ref28], [@ref29]\]. Importantly, several SACT agents, including cisplatin, crizotinib, and EGFR tyrosine kinase inhibitors (TKIs), are CYP450 substrates, meaning they can influence hepatic metabolism of many drugs \[[@ref29], [@ref30]\]. Other possible DDIs include platinum-based compounds with nephrotoxic agents such as NSAIDs, a synergistic interaction causing impaired renal function \[[@ref31]\], and EGFR-TKIs with antacids, which raise stomach pH and thus prevent absorption of the anticancer agent \[[@ref32]\]. The impact of DDIs directly on healthcare is poorly characterised. However, the broader group of adverse drug reactions (ADRs) place a significant strain on patients and hospitals, accounting for 6.5% of all hospital admissions, with a total cost per year of over £500,000 to the NHS \[[@ref33], [@ref34]\]. The proportion of this attributed to DDIs is debatable; some suggest an increased risk of readmission related to DDIs \[[@ref35]\], particularly in the elderly \[[@ref36]\], while other reviews propose the opposite \[[@ref37], [@ref38]\]. Nevertheless, as the potential for DDIs in SACT patients is high, methods to increase awareness are crucial to minimise risk of adverse events with which they are associated \[[@ref39], [@ref40]\]. Medicines reconciliation ------------------------ With regard to safe medicine prescribing, it is important that healthcare professionals (HCPs) are conscientious in maintaining the efficacy of medicines, in order for patients to get the best out of their treatment \[[@ref27], [@ref41]\]. This concept is known as medicines optimisation and comprises four guiding principles to improve patient outcome, as outlined by the Royal Pharmaceutical Society \[[@ref42]\]: aiming to understand the patient's experience; evidence-based choice of medicines; ensuring medicines use is as safe as possible; and making medicines optimisation part of routine practice. A major aspect of medicines optimisation that contributes to these aims is medicines reconciliation (Med-Rec), the process of ensuring the medicines a patient is taking are correctly documented \[[@ref43]\]. This involves 'creating and maintaining the most accurate list possible' of the patient's medicines, and then 'comparing... with the current list in use, recognising any discrepancies, and documenting any changes, thereby resulting in a complete list of medicines, accurately communicated' \[[@ref27], [@ref44]\], as defined by the Institute of Healthcare Improvement (IHI). These tasks can be summarised into three elements to form 'reliable' reconciliation: verification of the list of current medicines; validation (a review of the current medicines by a trained and competent HCP, noting whether to continue or alter any doses); and clarification, where the current list is compared with the prescribed 'medication order' \[[@ref41], [@ref44]\]. Previous studies highlight the potential for problems in healthcare without a formal Med-Rec procedure; unintentional discrepancies were found in 70% of medicines prescribed on admission (covering 60% of patients) in a large systematic review by Garfield \[[@ref41], [@ref45]\]. Actively implementing the process is also found to be beneficial; it decreases the rate of 'medication errors' by 70% and ADRs by 15% \[[@ref44], [@ref46], [@ref47]\] in one hospital setting, while another trial found that it reduced potential ADRs by 80% \[[@ref48]\]. This suggests Med-Rec is an important part of preventing harm to patients. Rationale for study ------------------- As clarified, there is a considerable risk of DDIs occurring between anticancer agents, plus various supportive care medicines as part of SACT regimens, and other medicines being taken by patients. The complexity of regimens in lung cancer specifically, utilising drugs with various mechanisms, adds to the potential risk of harm. It was therefore of interest to characterise the severity of these potential DDIs (PDDIs), and review whether established processes of Med-Rec, or simply documentation of PDDIs, could have a role in preventing harm. Numerous studies report aspects of these separately: DDIs involving general chemotherapy have been identified retrospectively in several studies \[[@ref49]--[@ref55]\]; while outcomes of pharmacy-led intervention with Med-Rec \[[@ref39], [@ref56]--[@ref62]\] have also been analysed. However, combining severity of PDDIs and improvement of patient safety has not been carried out previously. Aims, objectives and standards ============================== Aims ---- The aim is to evaluate the potential for DDIs between medicines in SACT regimens and other medicines taken by lung cancer patients treated at Chelsea and Westminster Hospital (CWH). Objectives ---------- 1. To identify and characterise PDDIs present between SACT medicines (comprising anticancer agents and supportive care medicines) and other medicines taken by the patients. 2. To assess the process of Med-Rec and evaluate documentation of PDDIs by prescribing HCPs (clinical audit). Standards for clinical audit ---------------------------- 1. 100% of consultations with a prescribing HCP since the patient started their current SACT regimen include a documented Med-Rec. 2. 100% of PDDIs between SACT medicines and other medicines had been acknowledged and documented by a prescribing HCP. Methodology =========== Study design, inclusion, and exclusion criteria ----------------------------------------------- This was a single-centre, retrospective case series study. Patients were selected for inclusion if diagnosed with lung cancer and undergoing a SACT regimen as of 31st March 2016 under the care of the Oncology team (led by Professor Mark Bower and Dr Tom Newsom-Davis) at CWH. Data collection --------------- For each patient, every instance of documented patient contact at CWH during their SACT regimen was compiled from the patient records. The chosen start point was the last consultation with a prescribing HCP before starting the current SACT regimen in which a full medicines history was taken. For patients on a maintenance SACT regimen, data were recorded from the last consultation in which a full medicines history was elicited before starting the SACT regimen. The endpoint was the cut-off date of 31st March 2016. Data recorded for each consultation include documentation of medicines being taken and changes to the medicine profile as a result of the consultation. Identifying PDDIs ----------------- A 'drug chart' overviewing how the medicine profile changed for each patient during their SACT regimen was then created from the data collection spreadsheet ([Appendix 1](#app1){ref-type="app"}). PDDIs were identified using three primary sources: the British National Formulary (BNF); Summary of Product Characteristics (SPC) at <www.medicines.org.uk>; and the London Cancer Alliance (LCA) protocols for each SACT regimen. Only PDDIs involving a SACT medicine---either the anticancer agent, or any prescribed supportive care medicine---were noted, and not between any two non-SACT medicines taken concurrently. Component drugs within a preparation were studied individually. Assessment of Med-Rec and documentation of PDDIs ------------------------------------------------ The same patient consultation records were analysed for documentation both of Med-Rec being performed and of any DDIs by prescribing HCPs. Definitions ----------- 'PDDI' (potential drug--drug interaction) is defined as a possible DDI between two medicines (as reported in the BNF, SPC, or LCA protocols) that may have occurred when the patient was taking both concurrently. 'Anticipated DDI' is defined as a possible DDI that was identified but did not occur due to intervention before both medicines were being taken simultaneously. There is therefore no possibility of an adverse event due to this DDI occurring. A DDI is 'identified' if noted by the author during retrospective analysis of the collected data; 'acknowledged' or 'documented' DDIs are those identified and written down during a consultation by a prescribing HCP. Results ======= Twenty-three patients met the criteria for inclusion in this study. SACT regimens being followed, listed by route of administration, are shown in [Table 2](#table2){ref-type="table"}. Identified PDDIs ---------------- A total of 88 instances of PDDIs involving SACT medicines across 21 patients were identified. This total includes anticipated DDIs (n = 13). [Figure 2](#figure2){ref-type="fig"} presents the SACT medicines with at least one identified PDDI. In order to present these data qualitatively, PDDIs were grouped based on effect of interaction and mechanism of the interacting medicine. These 30 distinct DDIs are summarised in [Table 3](#table3){ref-type="table"}. The most common identified DDI was that of dexamethasone + antihypertensives (12.5%), followed by: platinum-based compounds + nephrotoxic drugs (8.0%); crizotinib + UGT substrates, dexamethasone + antidiabetics; pemetrexed + nephrotoxic drugs (each 6.8%). The remaining PDDIs had five or fewer instances. In terms of severity, there were six 'potentially serious' DDIs, equating to 8.0% of the total number. Medicines reconciliation and documentation of PDDIs --------------------------------------------------- Standard 1: 100% of consultations with a prescribing HCP since the patient started their current SACT regimen include a documented Med-Rec. Outcome 1 (standard not met): 3.0% of consultations with a prescribing HCP since the patient started their current SACT regimen include a documented Med-Rec. A total of 480 instances of documented patient contact were recorded across all 23 patients. Forty-eight of these were excluded from further analysis: eight were phone calls, three were radiology scan appointments, and 37 were consultations with non-prescribing HCPs. Of the remaining 432 instances, 13 (3.0%, across 7 patients) included a documented Med-Rec. Additionally, 95.7% of patients had at least one full medicines history elicited by a prescribing HCP. This translates to a full medicines history being taken in 11.8% of consultations. Standard 2: 100% of identified PDDIs between SACT medicines and other medicines had been acknowledged and documented by a prescribing HCP. Outcome 2 (standard not met): 15.9% of identified PDDIs between SACT medicines and other medicines had been acknowledged and documented by a prescribing HCP. Of the 88 instances of PDDIs, 14 (15.9%, across 8 patients) were acknowledged and documented. These are summarised in [Table 4](#table4){ref-type="table"}. Comparison of severity and probability of documentation was performed using Fisher's exact test. Instances of PDDI with an unknown severity (*n* = 30 across both documented and non-documented DDIs) were omitted. Using a standard alpha-level of 0.05, the null hypothesis can be rejected (d.f.=1, two-tailed *p* = 0.019), indicating that potentially serious DDIs were significantly more likely to be documented. Documented PDDIs were also analysed in relation to Med-Rec processes. None of the 14 PDDIs were acknowledged by a prescribing HCP in the same consultation as a documented Med-Rec. Two (14.3%) were elicited after a full medicines history was taken in the same consultation. Thirteen of the 14 documented PDDIs can be considered anticipated DDIs, where intervention took place before the interaction could occur. The remaining one instance, gefitinib + warfarin, was acknowledged and documented after both medicines were taken concurrently. Discussion ========== Identified PDDIs ---------------- The wide-ranging nature of this study means there are several different aspects suitable for analysis. Firstly, the variety of SACT regimens---10 in total---confirm the multitude of treatment options for late-stage lung cancer patients and compare reasonably well with national statistics \[[@ref16]\]. Regarding the interactions, nearly all patients (91.3%) had at least one identified PDDI, highlighting the importance of characterising them well. Of particular significance were the PDDIs involving supportive care medicines, which accounted for 55% of the total. Most previous studies looking at DDIs involving chemotherapy only consider the anticancer agent \[[@ref50], [@ref51], [@ref54]\], although van Leeuwen did report supportive care medicines to be involved in 86% of all identified PDDIs for the cohort studied \[[@ref55]\], further supporting their clinical relevance in DDIs. The major SACT medicine of note here was dexamethasone, which comprised 39% of the total number. Moreover, two further PDDIs were identified involving dexamethasone prescribed separately from the SACT regimen. DDIs with corticosteroids have been reported in the literature---van Leeuwen's retrospective study found that dexamethasone was the major supportive care medicine involved \[[@ref55]\], and Lam considered it a 'cancer-related drug' with a variety of mechanisms of interaction \[[@ref49]\]. Additionally, a quarter of PDDIs involving dexamethasone were classified as potentially serious. This raises the question of its safety, particularly as it was a prescribed component of all intravenous combination SACT regimens for these patients ([Appendix 2](#app2){ref-type="app"}). However, regimens are carefully designed in terms of dose, timing, and frequency to minimise harm, with low-dose dexamethasone being taken for only three days for every cycle in these LCA-approved regimens. Nevertheless, caution should be exercised considering the wide range of drugs with which it potentially interacts, especially antihypertensives, which was the single-most common PDDI. Indeed, the high frequency reflects the high prevalence of hypertension in the population, particularly the elderly \[[@ref63]\] and therefore suggests it is an important comorbidity to consider. The next most commonly involved SACT medicine were the platinum-based compounds. This meant that cisplatin and carboplatin were the anticancer agents with the most PDDIs, a finding corroborated by Mouzon in a retrospective study \[[@ref50]\] and a review by Scripture \[[@ref29]\]. All PDDIs for these were linked with nephrotoxicity, which was also the potential outcome in the interactions involving pemetrexed, another nephrotoxic anticancer agent. Indeed, combining the PDDIs for all three makes kidney damage the most common potential outcome (20.4%). This is particularly dangerous because patients tend to be older and are more likely to have existing renal impairment \[[@ref64]\], putting them at greater risk. Other notable interactions are the remaining potentially serious PDDIs. Although only six of the 30 were classified in this way, each with low frequencies and thus constituting only 8.0% of the total PDDIs, possible outcomes such as increased risk of bleeding and adrenal suppression mean they should be actively avoided. It is also important to note that 10 PDDIs (totalling 27 instances) did not have a classified severity because they were identified from sources other than the BNF, and so any number could also be potentially serious DDIs, limiting the validity of the results. Medicines reconciliation and documentation of PDDIs --------------------------------------------------- The extent of identified interactions, with each patient on average having four PDDIs involving SACT medicines, highlights the necessity of safe prescribing and maintaining patient safety. Clinical audit was an effective method to measure this, identifying results as below expected. For the first standard, a remarkably low value of 3.0% of consultations including a documented Med-Rec implies there is almost a complete lack of the process occurring. Indeed, this was the case: the 13 documented Med-Recs were only performed when a patient was discharged from CWH, and never in clinic. UK and US guidelines focus primarily on Med-Rec for inpatients, with guidance stating it should be performed for any 'transfer of care', either on admission to hospital, during transfer between wards or on discharge \[[@ref27]\]. There is no formally established procedure for patients in ambulatory care at CWH, including this cohort of lung cancer patients attending regular Oncology clinics. This implies there is scope for implementation of Med-Rec processes for outpatients. IHI suggest collecting a full medicines history and then reviewing if there have been any changes during the consultation, after which a list can be kept 'on file' and verified for each subsequent appointment \[[@ref65]\]. Several intervention studies have assessed the advantages of reminders for patients to bring in their regular medicines to clinic followed by correction of the medicines list by patients themselves \[[@ref66]--[@ref68]\], all with encouraging results. A further justification to perform Med-Rec for these patients is that attending clinic could be seen as a 'temporary' transfer of care, during which the processes of Med-Rec should be carried out. These shortcomings are also reflected in the second standard regarding PDDI documentation. This again fell well below 100%, suggesting prescribing HCPs do not regularly review the safety of medicines being taken by the patient. Moreover, some were acknowledged simultaneously, meaning the 14 documented PDDIs were across eight consultations only. This below-standard practice could be due to the assumption made by HCPs that reviewing medicines comes under the remit of Pharmacy; involving pharmacists in a multidisciplinary team to ensure accurate medicines lists could improve the documentation of PDDIs and thus prevention of interactions, as seen in Lopez-Martin's single-centre study in Oncology outpatients \[[@ref61]\]. While increasing documentation of PDDIs is clearly key, there were some interesting patterns in the acknowledgement of certain categories. The results revealed the potentially serious interactions were significantly more likely to be documented than non-serious PDDIs. There are also three classes of interacting medicine that constitute the majority (71.4%) of the documented interactions: anticoagulants, PPIs, and antiretrovirals. This suggests that there may be awareness about certain types of DDI involving specific drugs, without the need for research. Finally, combining the aspects of the study, it appears that performing Med-Rec does not determine whether PDDIs are documented, which was reasonably assessed by the occurrence of both processes in the same consultation. However, this does not take into account the fact that Med-Rec may not always be carried out in a consultation, even though face-to-face Med-Rec is recommended in local guidelines \[[@ref41]\]; it could feasibly be done by comparing to the current medicines list without patient input. This means that there is possible underreporting of Med-Rec, potentially skewing results. Despite this, even just 'verification' correlates poorly (14.3%) with documentation of PDDIs, suggesting that the majority of acknowledged PDDIs were independent of the taking of a full medicines history in that consultation. Thus, it is beneficial to look at some of the many techniques already established to improve identification of potential interactions, such as the pharmacist-led PINCER \[[@ref69]\], the screening tools for elderly patients STOPP/START \[[@ref70]\], and My Medicines Passport, a patient-oriented booklet for recording medicines launched in CWH \[[@ref71]\]. Conclusion ========== In summary, this study provides a broad review of the common and potentially serious DDIs between SACT medicines and other medicines taken in a cohort of lung cancer patients. It also confirms the lack of established procedure for Med-Rec in these patients and highlights the scope for improvement in identifying more potential interactions, particularly as cancer patients are at risk of polypharmacy. However, the multidisciplinary care required in oncology, including general practice, must be taken into account; thus, implementing these processes in a hospital setting would not eliminate all risk of drug-related harm, but rather, contribute to the bigger picture of ensuring safe medicine prescribing across the whole of the patient journey. Ethical considerations ====================== Ethics approval is not required for this work as it is part of a service evaluation and improvement project. An ethics waiver was given by Chelsea and Westminster Hospital NHS Foundation Trust Research and Development lead and NRES for CLAHRC NWL Medicines Optimisation projects. ![](can-11-764fig3){#figure3} ---------------------------------------------------------------------------------------------------------------------------- SACT regimen Route of regimen Supportive care medicines used Route of supportive care medicine -------------------------------- ---------------------- -------------------------------- ----------------------------------- Pemetrexed\ Intravenous Folic acid Oral (maintenance) Ondansetron Intravenous and oral Vitamin B12 (hydroxocobalamin) Intramuscular Pemetrexed + cisplatin Intravenous Aprepitant Oral Dexamethasone Domperidone Folic acid Magnesium sulphate Intravenous Ondansetron Intravenous and oral Vitamin B12 (hydroxocobalamin) Intramuscular Gemcitabine + carboplatin Intravenous Domperidone Oral Dexamethasone Intravenous and oral Ondansetron Nivolumab Intravenous None N/A Etoposide +\ Intravenous Dexamethasone Intravenous carboplatin Ondansetron Pemetrexed + carboplatin Intravenous Aprepitant Oral Dexamethasone Domperidone Folic acid Magnesium sulphate Intravenous Ondansetron Intravenous and oral Vitamin B12 (hydroxocobalamin) Intramuscular Gefitinib Oral None N/A Erlotinib Oral None N/A Crizotinib Oral None N/A Carboplatin + vinorelbine Intravenous and oral Aprepitant Oral Dexamethasone Intravenous and oral Ondansetron Magnesium sulphate Intravenous Potassium chloride Docetaxel +\ Intravenous and oral Domperidone Oral nintedanib Dexamethasone Intravenous and oral Ondansetron ---------------------------------------------------------------------------------------------------------------------------- ![Most common SACT regimens for lung cancer between January and December 2014. Key: GEMCARBO=gemcitabine + carboplatin. Taken from \[[@ref16]\].](can-11-764fig1){#figure1} ![Frequency of PDDIs (total n = 88) for each of the 13 SACT medicines that had at least one identified PDDI.](can-11-764fig2){#figure2} ###### Summary of anticancer agents used in lung cancer with indications, class and mechanism of action. Taken and adapted with kind permission from \[[@ref6]\], contributions from \[[@ref1], [@ref2], [@ref4], [@ref7]--[@ref15]\]. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Anticancer agent Indication(s) in lung cancer Class of anticancer agent Main mechanism -------------------------------------------- ------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------- Cyclophosphamide Extensive SCLC Nitrogen mustard Intrastrand cross linking of DNA Carboplatin, cisplatin Stage I--II NSCLC (adjuvant) Platinum-based compound Stage IIIb--IV NSCLC (palliative) All-stage SCLC (palliative) Pemetrexed Stage IIIb--IV non-squamous NSCLC\ Folate antagonist Blocking the synthesis of DNA and/or RNA (first line) Gemcitabine Stage IIIb--IV squamous NSCLC (first-line) Pyrimidine pathway antimetabolite Doxorubicin Extensive SCLC Anthracycline Multiple effects on DNA/RNA synthesis and topoisomerase action Docetaxel, paclitaxel Stage IIIb--IV NSCLC (first line) Taxane Microtubule assembly; prevents spindle formation Vincristine Extensive SCLC Vinca alkaloid Vinorelbine Stage IIIb--IV squamous NSCLC (first line) Topotecan Relapsed SCLC Campothecin Inhibition of topoisomerase Etoposide All-stage SCLC (palliative) Other plant derivative Afatinib, erlotinib, gefitinib Stage IV--NSCLC + EGFR mutation\ Epidermal growth factor receptor\ Inhibition of kinases involved in growth factor receptor transduction (first line) (EGFR)-tyrosine kinase inhibitor (TKI) Stage IV NSCLC (second line if refractory) Crizotinib Stage IIIb--IV NSCLC + ALK translocation (second line) Anaplastic lymphoma kinase (ALK) inhibitor Nintedanib Stage IIIb--IV non-squamous NSCLC (second line, if progressive disease after first line) Vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-3) and platelet-derived growth factor receptor (PDGFRα,β) inhibitor Nivolumab Stage IV squamous and non-squamous NSCLC Anti-programmed cell death-1\ Recruitment of T cells (PD-1) monoclonal antibody -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ###### SACT regimens of all patients (n = 23). SACT regimen Frequency (%) ------------------------------------- --------------- **Intravenous** **14 (60.9)** -- Pemetrexed (maintenance) -- 7 (30.4) -- Post-pemetrexed + cisplatin -- 6 (26.1) -- Post-gemcitabine + carboplatin -- 1 (4.3) -- Nivolumab -- 3 (13.0) -- Gemcitabine + carboplatin -- 2 (8.7) -- Etoposide + carboplatin -- 1 (4.3) -- Pemetrexed + carboplatin -- 1 (4.3) **Oral** **7 (30.4)** -- Gefitinib -- 4 (17.4) -- Erlotinib -- 2 (8.7) -- Crizotinib -- 1 (4.3) **Combined (intravenous and oral)** **2 (8.7)** -- Carboplatin + vinorelbine -- 1 (4.3) -- Docetaxel + nintedanib -- 1 (4.3) ###### Summary of the identified PDDIs (*n*=30). Key: UGT=UDP-glucuronosyltransferase; ACEIs=angiotensin-converting enzyme (ACE) inhibitors; CCBs=calcium channel blockers; ARBs=angiotensin-II receptor blockers; PPIs=proton pump inhibitors. SACT medicine Interacting (class of) drug Possible outcome (effect of interaction) Severity of interaction n ---------------------------------------------------- -------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------- ------------------------- ---- Aprepitant Ritonavir Aprepitant toxicity (increased exposure) Non-serious 1 Warfarin Reduced anticoagulation (reduced exposure to warfarin) Non-serious 1 Crizotinib UGT substrates (amoxicillin, colecalciferol, diazepam, levomepromazine, metoclo-pramide, mirtazapine) Various (increased exposure to UGT substrates) Unknown 6 Dexamethasone Poor crizotinib efficacy (reduced exposure) Unknown 1 Dexamethasone Antihypertensives (ACEIs, CCBs, ARBs, beta-blockers, nitrates) Raised blood pressure (antagonised effect of antihypertensives) Non-serious 11 Antidiabetics (metformin, gliclazide, linagliptin) Raised blood glucose (antagonised effect of antidiabetics) Non-serious 6 Diuretics (furosemide, bendroflumethiazide) Hypokalaemia and associated signs and symptoms Non-serious 5 Aspirin Gastrointestinal bleeding and ulceration (reduced exposure to salicylate) Non-serious 5 Calcium carbonate Hypocalcaemia and associated signs and symptoms (reduced exposure to calcium salts) Non-serious 4 Coumarins Enhanced (high-dose corticosteroids) or reduced anticoagulation (increased or reduced exposure to coumarins) Potentially serious 1 Phenindione Enhanced or reduced anticoagulation (increased or reduced exposure to phenindione) Non-serious 1 Ritonavir Adrenal suppression (increased exposure to corticosteroids) Potentially serious 1 Docetaxel CYP3A inhibitors (paracetamol, PPIs) Docetaxel toxicity (increased exposure) Unknown 2 Clarithromycin Myelosuppression; docetaxel toxicity (increased exposure) Potentially serious 1 Domperidone Opioid analgesics (codeine, morphine) Gastroparesis (antagonised gastrointestinal effects of domperidone) Non-serious 5 Clarithromycin Domperidone toxicity (increased exposure to domperidone); ventricular arrhythmias Potentially serious 2 Tiotropium Gastroparesis (antagonised gastrointestinal effects of domperidone) Non-serious 1 Erlotinib PPIs/H2 antagonists/antacids (lansoprazole, ranitidine, sodium bicarbonate) Poor efficacy (reduced exposure to erlotinib) Unknown 3 Statins Myopathy Unknown 1 Folic acid Sodium bicarbonate Folate deficiency and associated signs and symptoms (reduced exposure to folic acid) Non-serious 2 Gefitinib CYP3A4 inhibitors (diclofenac, clindamycin) Gefitinib toxicity (increased exposure) Unknown 2 PPIs (lansoprazole, omeprazole) Poor gefitinib efficacy (reduced exposure) Unknown 2 CYP3A4 inducers (nevirapine, flucloxacillin) Poor gefitinib efficacy (reduced exposure) Unknown 2 Warfarin Enhanced anticoagulation (increased exposure to warfarin) Potentially serious 1 Nivolumab Dexamethasone Systemic immunosuppression Unknown 1 Ondansetron Sertraline Enhanced serotonergic effects Non-serious 1 Pemetrexed Nephrotoxic drugs (ibuprofen, ACEIs, sulphamethoxazole) Nephrotoxicity; pemetrexed toxicity (increased exposure) Non-serious 6 Platinum-based compounds (cisplatin, carboplatin) Nephrotoxic drugs (aspirin, ibuprofen, ACEIs, sulphamethoxazole) Nephrotoxicity Unknown 7 Diuretics (bendroflumethiazide, furosemide) Nephrotoxicity; ototoxicity Non-serious 5 Potassium chloride Irbesartan Hyperkalaemia and associated signs and symptoms Potentially serious 1 ###### Summary of acknowledged and documented instances of PDDIs (*n* = 14). ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Patient \# SACT medicine Interacting medicine Effect of interaction Severity of interaction --------------- --------------- -------------------------------------------------------------------------------------- ---------------------------------------------- ------------------------- 1 Aprepitant Ritonavir Increased exposure to aprepitant Non-serious Dexamethasone Ritonavir Increased exposure to corticosteroids, causing increased risk of adrenal suppression Potentially serious 2 Nivolumab Dexamethasone Increased risk of systemic immunosuppression Unknown 3 Aprepitant Warfarin Reduced anticoagulant effect of warfarin Non-serious Dexamethasone Coumarins Enhanced (high-dose corticosteroids) or\ Potentially serious reduced anticoagulant effect of coumarins Dexamethasone Phenindione Enhanced or reduced anticoagulant effect of phenindione Non-serious 4 Gefitinib Warfarin Enhanced anticoagulant effect of warfarin Potentially serious 5 Gefitinib Lansoprazole Reduced exposure to gefitinib Unknown 6 Gefitinib Flucloxacillin Reduced exposure to gefitinib Unknown 7 Erlotinib Lansoprazole Reduced exposure to erlotinib Unknown Erlotinib Statin Increased risk of myopathy Unknown 8 Gefitinib Nevirapine Reduced exposure to gefitinib Unknown Gefitinib Omeprazole Reduced exposure to gefitinib Unknown Gefitinib Diclofenac Increased exposure to gefitinib Unknown -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

{ "pile_set_name": "PubMed Central" }

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"immunotherapi", "tumour", "product", "antidiabet", "start", "journey", "irbesartan", "research", "crizotinib", "fell", "medicineeith", "mouzon", "prefer", "vitamin", "find", "polypharmaci", "valid", "encourag", "cancer", "extent", "figur", "clinic", "crucial", "antagonistsantacid", "rash", "creat", "domperidon", "correctli", "danger", "palli", "levomepromazin", "twotail", "anoth", "part", "method", "earlystag", "elimin", "primari", "attend", "form", "campothecin", "corrobor", "often", "nsclc", "adjuv", "carboplatin", "verifi", "cyclophosphamid", "suitabl", "procedur", "uk", "transduct", "distinct", "hydrat", "never", "doxorubicin", "variou", "interf", "correl", "b", "salt", "best", "effect", "squamou", "pictur", "skin", "pd", "underreport", "unintent", "call", "none", "proport", "averag", "outpati", "allstag", "within", "overview", "hospit", "consult", "problem", "encompass", "scope", "sourc", "counter", "elicit", "function", "poorli", "mean", "number", "cross", "introduct", "impli", 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"bendroflumethiazid", "statin", "lowdos", "analges", "nonsact", "metoclopramid", "cancerrel", "increas", "awar", "maintain", "improv", "clarif", "dnarna", "egfrtki", "report", "singlecentr", "aspect", "ppish", "point", "januari", "clarithromycin", "summari", "myelosuppress", "progress", "guidanc", "societi", "seriou", "advers", "attribut", "previou", "plu", "intent", "therefor", "widerang", "patientori", "suppress", "verif", "rather" ]

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"system", "recognise", "attack", "malignant", "cells", "Table", "table", "details", "SACT", "agents", "recommended", "use", "lung", "cancer", "Application", "SACT", "involves", "use", "combination", "therapies", "recent", "statistics", "commonest", "regimens", "lung", "cancer", "shown", "Figure", "fig", "Supportive", "care", "medicines", "often", "prescribed", "part", "SACT", "regimen", "prevent", "side", "effects", "occurring", "include", "various", "antiemetics", "aprepitant", "domperidone", "ondansetron", "combat", "nausea", "vomiting", "common", "adverse", "effect", "anticancer", "agents", "Notably", "higher", "potential", "nausea", "others", "particularly", "cisplatin", "different", "regimens", "thus", "require", "varying", "doses", "antiemetic", "Corticosteroids", "dexamethasone", "also", "counter", "nausea", "additionally", "reduce", "incidence", "severity", "skin", "rash", "another", "frequent", "adverse", "reaction", "medicines", "include", "folic", "acid", "vitamin", 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"negative", "outcomes", "due", "large", "number", "difference", "mechanisms", "action", "anticancer", "agents", "multitude", "adverse", "DDIs", "involving", "chemotherapy", "drugs", "Importantly", "several", "SACT", "agents", "including", "cisplatin", "crizotinib", "EGFR", "tyrosine", "kinase", "inhibitors", "TKIs", "substrates", "meaning", "influence", "hepatic", "metabolism", "many", "drugs", "possible", "DDIs", "include", "compounds", "nephrotoxic", "agents", "NSAIDs", "synergistic", "interaction", "causing", "impaired", "renal", "function", "antacids", "raise", "stomach", "pH", "thus", "prevent", "absorption", "anticancer", "agent", "impact", "DDIs", "directly", "healthcare", "poorly", "characterised", "However", "broader", "group", "adverse", "drug", "reactions", "ADRs", "place", "significant", "strain", "patients", "hospitals", "accounting", "hospital", "admissions", "total", "cost", "per", "year", "NHS", "proportion", "attributed", "DDIs", "debatable", "suggest", "increased", 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All relevant data are within the paper and its Supporting Information files. Introduction {#sec005} ============ Buruli ulcer (BU) is a Neglected Tropical Disease for which the World Health Organization (WHO) stressed the need to improve treatment \[[@pntd.0004076.ref001]\]. BU remains endemic in areas in West Africa, especially in Benin and Ghana \[[@pntd.0004076.ref002],[@pntd.0004076.ref003]\]. In Benin, prevalence rates between 5.4 and 60.7 per 10.000 inhabitants have been reported each year \[[@pntd.0004076.ref004]\] while in Ghana, prevalence rates have fluctuated between 2.0 and 15.0 per 10.000 inhabitants \[[@pntd.0004076.ref005]\]. BU destroys skin, subcutaneous fat, and sometimes bone \[[@pntd.0004076.ref006]\]. Patients typically present with non-ulcerated lesions; papules, nodules, plaques or edema, or undermined ulcers \[[@pntd.0004076.ref006],[@pntd.0004076.ref007]\]. Treatment entails antibiotics complemented with surgery if needed, together with dressing changes of which the frequency depends on the wound, and physiotherapy \[[@pntd.0004076.ref008]\]. Specialized treatment centers deliver the necessary care while antimicrobial treatment and dressings may also be delegated to local health centers \[[@pntd.0004076.ref009]\]. Irrespective of therapy (antibiotics or surgery), 47% of patients are left with functional limitations after healing \[[@pntd.0004076.ref010]\]. Although BU is described as relatively painless \[[@pntd.0004076.ref011]--[@pntd.0004076.ref013]\] clinical observations reveal that at least some patients experience considerable pain during their treatment---especially during wound care and physiotherapy. This is supported by two studies, i.e., one in Japanese patients \[[@pntd.0004076.ref014]\], and one in Ghanaian patients \[[@pntd.0004076.ref015]\]. Ghanaian BU patients sometimes complained of pain at the lesion site just before and during ulceration. This suggests a recovered sensation---perhaps due to a decrease in mycolactone concentrations \[[@pntd.0004076.ref016]\] in tissues at advanced disease stages, however this suggestion warrants verification by clinical studies. Clinical studies on pain and pain treatment among BU patients are lacking. The recognition, management and treatment of pain is a challenge worldwide, but limited resources in low- and middle-income countries further increase the risk of under treatment of pain \[[@pntd.0004076.ref017],[@pntd.0004076.ref018]\] Health care personnel (HCP) treating BU in Ghana and Benin stressed the need to include pain management \[[@pntd.0004076.ref019]\]. The WHO recommends integrating pain treatment as part of general treatment of patients globally \[[@pntd.0004076.ref018]\]. Pain management should follow a practice guideline, which is currently not available for BU treatment, including at least an analgesic ladder, i.e. the 'pain ladder', initially developed by the WHO originally for cancer pain control \[[@pntd.0004076.ref020],[@pntd.0004076.ref021]\], but now used for pain relief among patients with wound pain \[[@pntd.0004076.ref022]\]. The ladder consists of three steps of drugs with increasing analgesic effects. Previous studies showed that perceptions and beliefs of health professionals are important in pain treatment \[[@pntd.0004076.ref023]\]. In addition, barriers related to the availability of- and access to analgesics, a lack of education and information among HCP, and legal barriers such as regulatory restrictions on opioids exists, preventing effective pediatric pain treatment in sub-Saharan Africa \[[@pntd.0004076.ref024]\]. Furthermore, language barriers, fear of addiction to opioids, and cultural differences between patients and health professionals, influenced pain management in Central Africa \[[@pntd.0004076.ref025]\]. Firstly, this study investigates current pain practices in BU in Ghana and Benin, including the prescription of pain medication, and the use of the WHO pain ladder by the HCP. Secondly, this study aims to explore factors that might influence the success of a possible future guideline implementation. Methods {#sec006} ======= Design and participants {#sec007} ----------------------- A mixed methods approach was used, consisting of information on prescribed pain medication, retrieved from medical records from Ghana and Benin, and semi-structured interviews. Data collection was performed in the same period in both countries. Two out of the four BU treatment centers in Benin, and two out of the four BU treatment centers in Ghana were selected for the study. Information from the medical records of patients treated between 2008 and 2012 was collected between November 2012 and August 2013. In the two treatment centers in Ghana, medical records of all PCR confirmed BU patients admitted and treated in the selected time period, were included (n = 69). In the two treatment centers in Benin, a larger number of records of PCR confirmed BU cases was found in the selected time period, thus a systematic selection (every *8* ^th^ medical record) was performed in both centers to arrive at 40 records per center. Thus, in Ghana 69 medical records of patients were included, and in Benin 80 medical records of patients leading to a total of 149 patients. Eligible HCP were selected by purposive sampling based on their involvement in BU wound care, their profession---we selected medical doctors, nurses, physiotherapists and local health workers, to ensure heterogeneity of the interviewees-, and the ability to speak English. In total 13 HCP were eligible and approached. Interviews were conducted between August 2012 and May 2013. Data collection {#sec008} --------------- The interviews were held in private settings in the hospital or health center in Ghana, and were conducted by one of the authors (MA, JDZ or SL). Interviews were tape recorded and lasted between 60 and 90 min. Materials {#sec009} --------- Data on general characteristics and diagnostic tests were collected from the records. The type of pain medication and its indication were retrieved as well. An interview topic guide was developed, covering HCPs perceptions on current pain assessment and treatment. Specific topics included were; current practice, current prescription of pain medication, HCPs preferences for prescribing pain medication, satisfaction with current practice, wishes for improvements, perceptions on pain, pain assessment, and the acceptability of showing pain. Questions were derived from previously published work \[[@pntd.0004076.ref024]\]. Probes were used where necessary. Face and content validity of the interview guide were assessed by the study team, ensuring that questions adequately covered the objectives and were relevant. Data analysis {#sec010} ------------- Data on prescribing behavior and patient characteristics from the records were analyzed using Statistical Package for Social Science version 20. Descriptive analysis was performed on age and sex of patients whose records were included, including the type of lesion, indication for pain medication, types of pain medication prescribed during hospitalization and the types of pain medication prescribed in line with the WHO pain ladder. The total number of times that pain medication was prescribed for each step of the WHO pain ladder was counted. Interviews were transcribed verbatim by three different researchers (MA, JDZ and SL). A qualitative content analysis was performed using open coding and axial coding. To ensure reliability, interviews were analyzed consecutively by two researchers (MA and JDZ). These two researchers developed an initial codebook independently using one interview, and taking into account the interview questions. Both researchers participated in weekly meetings to extensively discuss the open coding analysis until consensus was reached, after which codes were merged into one codebook. The codebook was adapted throughout the analysis, based on new codes emerging from the data. A third and fourth researcher (AVR, YS) were consulted in case of disagreement, and ensured an acceptable coding of the data, ordering of the codes, and selection of themes. Qualitative and quantitative data were combined according to parallel data analysis \[[@pntd.0004076.ref026]\]. Ethics {#sec011} ------ Ethical clearance was obtained from the Medical Ethical Review Committee of the Kwame Nkrumah University of Science and Technology; School of Medical sciences, Komfo Anokye teaching hospital in Ghana (ref: CHRPE/AP/230/12). Data of medical records was handled in line with Good Clinical Practice. Written informed consent was obtained from all HCP. Before the interview started, voluntary participation, confidentiality, the aim, the topic and type of questions, the rights to withhold certain information, and to withdraw during the interview, were explained. Written informed consent was obtained from all HCP. In order to ensure anonymity and confidentiality, a number was assigned to each interview. Results {#sec012} ======= Prescribed pain medication {#sec013} -------------------------- ### Patient characteristics from medical records {#sec014} In total, 149 medical records were reviewed (69 from Ghana, 80 from Benin), of which 14 were incomplete, resulting in 135 records included in the analysis. In 113 (84%) of the 135 included records, pain medication was prescribed at least once during hospitalization. The median weeks of hospitalization was 17 (IQR: 10--23). The median age of the patients receiving pain medication was 14 years (IQR: 8--28), and 65 (58%) was male ([Table 1](#pntd.0004076.t001){ref-type="table"}). In 64% of the medical records, ulcers were reported, 15% plaque, 10% edema and 3% nodules (8% missing). 10.1371/journal.pntd.0004076.t001 ###### Patient characteristics (n = 113). ![](pntd.0004076.t001){#pntd.0004076.t001g} Total ---------------------------------------------------- -------------- ------------- Sex (male) 65 (58%) Age (Median, IQR) 14 (8--28) Type of ulcer Ulcer 64% Plaque 15% Edema 10% Nodule 3% Not reported 8% Duration of hospitalization in weeks (Median, IQR) 17 (10--23) ### Type of pain medication (different generics) prescribed during hospitalization {#sec015} In most records (43.4%; n = 49), two types of pain medication were prescribed, while in 35.4% of records (n = 40), one type of pain medication was prescribed. In 13.3% of the records (n = 15), three types were prescribed and in 7.9% of the records (n = 9), four or five types of medication were prescribed. ### Pain medication according to WHO ladder {#sec016} The total number of times pain medication was prescribed was 482. Step 1 medication (i.e. simple analgesics) was mostly prescribed (n = 430), for several indications. Pain medication is mostly prescribed after a surgical intervention (n = 165; 34%) ([Table 2](#pntd.0004076.t002){ref-type="table"}), however, often, no indication was noted in the records (n = 233; 48%). Step 2 and 3 medications (weak and strong opioids, respectively) were rarely prescribed (n = 52) ([Table 2](#pntd.0004076.t002){ref-type="table"}). Predominantly, pain medication is prescribed after an intervention, and less often for background pain. 10.1371/journal.pntd.0004076.t002 ###### Pain medication prescribed along different indications according to WHO ladder (n = 482). ![](pntd.0004076.t002){#pntd.0004076.t002g} Indication Step 1[\*](#t002fn001){ref-type="table-fn"} Step 2[\*\*](#t002fn002){ref-type="table-fn"} Step 3[\*\*\*](#t002fn003){ref-type="table-fn"} ------------------------------------------------------- --------------------------------------------- ----------------------------------------------- ------------------------------------------------- Post-surgical pain 151 12 2 (before) wound dressing 5 5 0 Other interventions (e.g. removal of necrotic tissue) 14 3 0 Infected wound/wound related pain 12 2 0 Generalized body pain 18 1 0 Not related to pain (e.g. fever, diarrhea) 22 2 0 Missing indication 208‡ 24 1 **Total** **430** **49** **3** \*Paracetamol/acetaminophen, ibuprofen, acetylsalicylic, diclofenac and diclofenac sodium. \*\* paracetamol/codeine, tramadol and dihydrocodeine. \*\*\* pethidin ‡ of the missing indications 136 times paracetamol/acetaminophen was prescribed Current pain assessment and treatment-results from the semi-structured interviews- {#sec017} ---------------------------------------------------------------------------------- 11/13 eligible HCP agreed to participate in the semi structured interview; nurses (4), medical doctors (2), BU coordinators (2), physiotherapist (1), health worker (1) and pharmacy technologist (1). Two participants refused because of a lack of time. Three factors in current pain assessment and treatment were important, i.e., perceptions on pain, pain assessment and pain treatment, including the use of the WHO pain ladder. Perceptions on pain in BU {#sec018} ------------------------- Pain is described as '*an uncomfortable feeling*' or '*an alarm*'. In early stages of the disease, BU is painless, however, in later stages patients start to feel pain: '*So when they start to heal*, *they start to feel pains'*. Perceived causes of pain were; wound pus, ulceration of the lesion, inflammation, nerve exposure, secondary infection, a graft at the donor site, proximity to bone or joint. An increase in pain is mentioned after wound treatment. > *'Yes*, *normally*, *patients with BU*, *actually don't have pain*. *Unless there is a secondary bacterial infection*. *There is involvement of the nerves and involvement of the bone tissue'*. Pain threshold is described by the HCP as '*The extent to which a person can handle pain*', or as '*How patients react to pain'*. According to the participating HCP, patients differ in their pain threshold and pain tolerance. Factors related to differences in pain tolerance are; previous experiences, gender differences, age of the patient, and size of the ulcer. Expression of pain and its assessment {#sec019} ------------------------------------- Nurses and a physiotherapist reported not to be trained in assessing mild pain, neither do they ask patients about mild pain. They attributed this to their culture; patients are able to handle mild pain. > '*We \[HCP\] are not trained to recognize the mild ones quickly*, *it is a cultural problem*, *we know people take the mild ones*, *so I don't ask*, *suggest pain to you \[patient\]*.*'* Instead of focusing on whether patients report pain, professionals pay attention to facial expression, body language, wound characteristics, individual characteristics, medical background, and patients' behavior. > *'But you can see from the face whether it is painful or not*. *So we look for signs*, *because otherwise*, *we are going to get wrong feedback*.' > *'Assuming you have a lesion*, *and I touch it*, *and you don't complain*, *that means you are ok*, *there is no pain*. *But if I touch it \[the lesion\] and you pull your hand*, *I mean the little thing I do*, *hurts to your hand*, *and you are preventing me*. *The moment you start and the patient is starting crying and screaming and all these things*, *you can easily know that now*, *the patient has a severe pain*.*'* Suppressing pain expression is ascribed to cultural factors; which is especially common among adult males, patients from the northern part of the country, patients from rural areas, and patients with a lower educational level. This cultural tendency hampers a proper pain assessment. > *'Location wise*, *you see people from the northern part of the country*, *they are very good adapted at taking pain*. *I would rather classify them as the affluent compared to the less affluent or the region poor or urban and the village*. *If it is in the city*, *well-educated parents know that they shouldn't be treated this way*, *so they get better treatment when you have more enlightened parents*. *People try to contain pain*. *It is a country wide a cultural thing*, *we are brought up to be able to contain an amount of pain*, *so you are a cry baby if you express too much pain*.*'* On the other hand, the majority of the HCP mentioned that patients exaggerate pain. This paradox could imply that---since HCP are part of the Ghanaian culture in which suppression of pain expression is commonly seen---they believe that patients who express pain overtly, are exaggerating. > *If it is true pain*, *or fake pain*, *we don't know*, *you measure*, *you look at the patient*.*'* > *'You rate it for the patient*, *but not what the patient tells you*. *Because psychologically*, *the patient would tell you 'oh*, *it is very painful'*. *Meanwhile*, *it is not like that*.*'* Pain treatment {#sec020} -------------- Important factors in current pain treatment include the financial constrains that patients often have to pay for pain medication. Moreover, HCP report a fear of side effects of pain medication, a fluctuation in availability of pain medication, and a shortage of time among medical doctors. > *'We shouldn't over rush in giving pain killers*, *because there are side-effects attached to the drugs'*. Furthermore, HCP report a discrepancy between their own as compared to their patients' expectations regarding pain relief. While professionals expect patients to endure pain to some extent, patients expect to be relieved from pain during hospital admission, according to the HCP. > *'They \[patients\] believe as soon as they're in the hospital*, *everything must be stopped*. *As soon as they're operated they should have no pain at all*, *even not the wound pain*. *I always tell them that operation does not mean that everything is healed*, *that pain stopped*, *the wound is there*, *so it pains'*. Besides, non-pharmacological factors in the current pain treatment include the different coping strategies used by the HCP to help the patient to handle pain. Examples are: counseling (providing information, reassurance, showing empathy) and giving advice. > *'Explaining to patient*, *oh no*, *this is just a little thing that I would just only have to give you*, *but it will minimize the pain that you are going through*, *because you fell from the bike*. *So your pain will minimize*.*'* Furthermore, while the HCP were not explicitly familiar with the WHO ladder, they reported to use the basic principles of the tool. > '*It is about*, *if someone is in pain then you start with paracetamol and then if someone has more severe pain you can give paracetamol and another pain killer which is more effective and then in the end you can give something like pethidin*.*'* Discussion {#sec021} ========== This study aimed to explore current pain practice in BU in Ghana and Benin. For most BU patients, pain medication was prescribed, and pain management mainly focused on severe pain. Professionals perceived later stages of BU as painful, and reported an increase in pain after wound treatment, and after a skin graft at the donor site. HCP reported a suppressed pain expression as well as exaggeration in patients, and differences in expectations between professionals and patients on what is an acceptable pain level without medication. Mainly WHO step 1 pain ladder medication was prescribed, while strong opioids were hardly prescribed. Explanations provided by the HCP on the mild prescriptions were; fear of side effects of strong opioids, fluctuation of availability of pain medication, and the shortage of time among medical doctors. These findings are in line with literature mentioning resistance among HCP to use morphine \[[@pntd.0004076.ref027]\]. Alternative explanations are that HCP did not ask whether patients experienced pain, or a lack of attention for mild pain. At the same time, the prescribed pain medication is typically for mild pain, according to the WHO pain ladder indicating that severe pain is treated with mild pain medication. It appeared that the indication for pain medication was often not documented in the medical records. Effective pain relief ultimately depends on accurate pain assessment, and the nature, severity, location and duration of pain should be assessed and documented to understand the possible etiology and to effectively treat pain \[[@pntd.0004076.ref028]\]. Both the finding that HCP believed that patients exaggerated their pain, and the expectation that patients should endure pain, might be influenced by sociocultural factors, since pain expression varies across cultures worldwide \[[@pntd.0004076.ref028]\]. In different African ethnic groups, stoicism is a valued response to pain \[[@pntd.0004076.ref029],[@pntd.0004076.ref029],[@pntd.0004076.ref030]\]. Despite the cultural factors, differences in pain expression can be due to individual differences \[[@pntd.0004076.ref031]\]. An important implication of our finding is that it complicates pain assessment for professionals. Patients and professionals differed in their expectation on acceptable pain levels without medication. HCP noticed that patients expected to be pain free during hospitalization, a statement that should be confirmed in studies using a patients' perspective. If these results can be confirmed, patients' expectations could be addressed during the preoperative pain assessment by collaboratively setting goals for pain control \[[@pntd.0004076.ref032]\], or during the intake for hospitalization. Furthermore, if documentation of pain will be integrated in daily practice, pain can be monitored, which is essential for adequate treatment. This study had several limitations. For the part on the medical records, an information bias might have occurred due to the retrospective design of the study. In a small proportion of medical records, no information on pain medication was found, and in case pain medication was prescribed, the indication was often missing. In addition, by using medical records, only the prescribing behavior of HCP was studied, while the actual intake remained unknown. A limitation of the interviews was the possible bias introduced by the role of non-native interviewers. Although the interviewers spent time in the different BU settings, cultural differences remained. This might have influenced the interviews. To conclude, these findings, together with a study on wound care-related pain in BU \[[@pntd.0004076.ref015]\], suggest that there is room for improvement to arrive at adequate pain treatment. Several factors could be taken into account when developing a pain guideline, such as the current practice on prescribing pain medication, the discrepancy between HCP and patients about pain relief, the views on pain expression and suppression, recognition and treatment of mild pain, and the lack of recorded indications. Furthermore, the findings that HCP tried to help the patient to cope with pain by providing information, reassurance, showing empathy, giving advice, as well as the finding that HCP were aware of the basic principles of the WHO ladder are useful in the development phase of such a protocol. We would like to thank the health care personnel from Benin and Ghana who participated in the interviews, as well as the personnel who assisted us with collecting the medical records of former BU patients. In addition, we would like to thank the board members of the participating hospitals for facilitating the study. [^1]: The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: JdZ MA YTB GS ROP TSvdW AVR YS. Performed the experiments: JdZ MA SL. Analyzed the data: JdZ MA YTB BM MP AVR YS. Wrote the paper: JdZ MA YTB GS ROP TSvdW SL BM MP AVR YS.

{ "pile_set_name": "PubMed Central" }

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Introduction {#section1-1524838017697312} ============ Timely access to supportive and therapeutic resources for child sexual abuse (CSA) survivors can mitigate risk to the health and mental health well-being of children, youth, and adults. Identifying and understanding factors that promote or inhibit CSA disclosures have the potential to facilitate earlier disclosures, assist survivors to receive services without delay, and potentially prevent further sexual victimization. Increased knowledge on both the factors and the processes involved in CSA disclosures is timely when research continues to show high rates of delayed disclosures ([@bibr8-1524838017697312]; [@bibr9-1524838017697312]; [@bibr11-1524838017697312]; [@bibr17-1524838017697312]; [@bibr20-1524838017697312]; [@bibr25-1524838017697312]; [@bibr31-1524838017697312]; [@bibr42-1524838017697312]). Incidence studies in the United States and Canada report decreasing CSA rates ([@bibr13-1524838017697312]; [@bibr14-1524838017697312]; [@bibr49-1524838017697312], [@bibr50-1524838017697312]), while at the same time global trends from systematic reviews and meta-analyses have found concerning rates of CSA, with averages of 18--20% for females and of 8--10% for males ([@bibr37-1524838017697312]). The highest rates found for girls is in Australia (21.5%) and for boys in Africa (19.3%), with the lowest rates for both girls (11.3%) and boys (4.1%) reported in Asia ([@bibr46-1524838017697312]). These findings point to the incongruence between the low number of official reports of CSA to authorities and the high rates reported in prevalence studies. For example, a meta-analysis conducted by [@bibr46-1524838017697312]) combining estimations of CSA in 217 studies published between 1980 and 2008 revealed rates of CSA to be more than 30 times greater in studies relying on self-reports (127 in 1,000) than in official report inquiries, such as those based on data from child protection services and the police (4 in 1,000) ([@bibr23-1524838017697312]; [@bibr45-1524838017697312]). In other words, while 1 out of 8 people retrospectively report having experienced CSA, official incidence estimates indicate only 1 per 250 children. In a survey of Swiss child services, [@bibr35-1524838017697312]) further found 2.68 cases per 1,000 of CSA disclosures, while in a recent comprehensive review [@bibr31-1524838017697312]) details the high prevalence of delayed, partial, and nondisclosures in childhood indicating a persistent trend toward withholding CSA disclosure. It is our view that incidence statistics are likely an underestimation of CSA disclosures, and this drives the rationale for the current review. Given the persistence of delayed disclosures with research showing a large number of survivors only disclosing in adulthood ([@bibr8-1524838017697312]; [@bibr11-1524838017697312]; [@bibr21-1524838017697312]; [@bibr31-1524838017697312]; [@bibr42-1524838017697312]), these issues should be a concern for practitioners, policy makers, and the general public ([@bibr31-1524838017697312]). The longer disclosures are delayed, the longer individuals potentially live with serious negative effects and mental health problems such as depression, anxiety, trauma disorders, and addictions, without receiving necessary treatment. This also increases the likelihood of more victims falling prey to undetected offenders. Learning more about CSA disclosure factors and processes to help advance our knowledge base may help professionals to facilitate earlier disclosures. Previous literature reviews examining factors influencing CSA disclosure have served the field well but are no longer current. Important contributions on CSA disclosures include [@bibr36-1524838017697312] original review covering the literature largely from the premillennium era, followed by [@bibr30-1524838017697312] subsequent review, which may not have captured publications affected by "lag to print" delays so common in peer-reviewed journals. These reviews are now dated and therefore do not take into account the plethora of research that has been accumulated over the past 15 years. Other recent reviews exist but with distinct contributions on the *dialogical relational processes* of disclosure ([@bibr39-1524838017697312]), CSA disclosures in *adulthood* ([@bibr48-1524838017697312]), and delayed disclosures in *childhood* ([@bibr31-1524838017697312]). This literature review differs by focusing on CSA disclosures in children, youth, and adults from childhood and into adulthood---over the life course. Method {#section2-1524838017697312} ====== [@bibr27-1524838017697312] systematic review framework was utilized to establish what has been investigated in CSA disclosure research, through various mixed methods, to highlight the most convincing findings that should be considered for future research, practice, and program planning. This review centered on the question: *What is the state of CSA disclosure research and what can be learned to apply to future research and practice*? By way of clarification, the term systematic refers to a methodologically sound strategy for searching literature on studies for *knowledge construction*, in this case the CSA disclosure literature, rather than *intervention* studies. The years spanned for searching the literature were 2000--2016, building on previous reviews without a great deal of overlap. Retrieval of relevant research was done by searching international electronic databases: PsycINFO, PsycARTICLES, Educational Resources Information Center, Canadian Research Index, International Bibliography of the Social Sciences, Published International Literature on Traumatic Stress, Sociological Abstracts, Social Service Abstracts, and Applied Social Science Index and Abstracts. This review searched peer-reviewed studies. A search of the gray literature (unpublished literature such as internal agency documents, government reports, etc.) was beyond the scope of this review because unpublished studies are not subjected to a peer-review process. Keyword search terms used were child sexual abuse, childhood sexual abuse, disclosure, and telling. A search of the 9 databases produced 322 peer-reviewed articles. Selected search terms yielded 200 English publications, 1 French study, and 1 Portuguese review. The search was further refined by excluding studies focusing on forensic investigations, as these studies constitute a specialized legal focus on interview approaches and techniques. As well, papers that focused exclusively on rates and responses to CSA disclosure were excluded, as these are substantial areas unto themselves, exceeding the aims of the review question. Review articles were also excluded. Once the exclusion criteria were applied, the search results yielded 33 articles. These studies were subjected to a thematic analysis as described by [@bibr5-1524838017697312]). This entailed (1) multiple readings by the three authors; (2) identifying patterns across studies by coding and charting specific features; (3) examining disclosure definitions used, sample characteristics, and measures utilized; and (4) major findings were extrapolated. Reading of the articles was initially conducted by the authors to identify general trends in a first level of analyses and then subsequently to identify themes through a deeper second-level analyses. A table of studies was generated and was continuously revised as the selection of studies was refined (see [Table 1](#table1-1524838017697312){ref-type="table"}). ###### Child Sexual Abuse (CSA) Disclosure Studies: 2000--2016. ![](10.1177_1524838017697312-table1) Study Purpose Design Sample Findings Summary ------------------------------ --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- [@bibr16-1524838017697312]) To explore disclosure processes for male victims of CSA Phenomenological methodology used to interview male CSA survivors. The Long Interview Method (LIM) guided data collection and analyses. 17 men ranged in age from 19 to 67---average age 47. Purposive sampling strategy was used The majority of the men in the study waited until adulthood to disclose their abuse, with negative stereotypes contributing to their delayed disclosures. Negative stereotypes contributed to delayed disclosure with trying to forget. Breaking isolation was cited as a motivator to disclosure along with the aid of various forms of media on disclosure. Important contextual issues such as negative stereotyping of males, sexuality, and victims were noted. Social media was seen as a facilitator of disclosures All participants had disclosed and received services before participating in the study. Member checking could not be done with the participants to check themes. Small but sufficient size for a qualitative inquiry. Otherwise, high level of rigor in establishing trustworthiness of the data and analysis. Retrospective study could imply recall issues [@bibr6-1524838017697312] To explore the meaning African American women make of their traumatic experiences with CSA and how they disclosed across the life course Collective case study design with using narrative tradition (storyboard) for data collection and analysis. Qualitative interviewing 17 African American women in mid-life between 40 and 63 who experienced intrafamilial CSA. Purposive, snowballing strategy CSA onset was largely between the ages 5 and 9. No one ever talked to them about sex, so they didn't have language to disclose. Barriers: fear of family breakdown and removal, not wanting to tarnish the family's name, and fear of retribution by family members if they disclosed. Pattern of stifled and dismissed disclosures identified over the life course. All 17 participants identified spirituality as a primary source of strength throughout the life course One of few studies to focus exclusively on African American women. Small but sufficient size for a qualitative inquiry. Important cultural and contextual issues were brought forward. Retrospective study that may have been affected by recall issues. Use of a life-course perspective as a theoretical lens for understanding CSA in the middle to later years of life that should be considered in further investigations [@bibr8-1524838017697312]) To provide a mapping of factors that prevent CSA disclosures through an ecological lens from a sample of CSA adult survivors. Qualitative design using LIM. 67 male and female CSA adult survivors (76% identified as female and 24% as male). Age ranges from 19 to 69 years (*M* = 44.9). Purposive sampling strategy Three broad categories were identified as barriers to CSA disclosure: Barriers from within-internalized victim blaming, mechanisms to protect oneself, and immature development at time of abuse; barriers in relation to others---violence and dysfunction in the family, power dynamics, awareness of the impact of telling, and fragile social network; barriers in relation to the social world labeling, taboo of sexuality, lack of services available, and culture or time period. Half of the participants had not disclosed their CSA experiences before the age of 19. Retrospective aspect of the study could imply recall issues. All participants had disclosed and received counseling at some before participating in the study. High level of rigor in establishing trustworthiness of the data and analysis [@bibr29-1524838017697312] Study objectives investigated the factors that facilitate CSA disclosures Adult male child sexual offenders were interviewed to examine predictors of victim disclosure. Semistructured interviews based on the QID questionnaire. 369 adult males who had been convicted of a sexual offense against a child aged between 1 and 17 years old. Majority were White, uneducated, almost half unemployed before their arrest Disclosure increased with the age of the victim: if penetration had occurred, if the victim was related to the offender, if the victim was not living with the offender at the time of the abuse, or if the victim resisted during the offense. Male victims and victims from dysfunctional backgrounds were less likely to disclose Offender generated data through self-reports could be subject to cognitive distortions---minimization or exaggerations. Perspectives of offenders on vulnerability of victims in relation to disclosure could be important information to inform interventions [@bibr32-1524838017697312]) To investigate the feasibility of using child assessments as data sources of informal CSA disclosure. To assess if these reports provide substantive data on disclosures File reports of children seen for assessment in a child sexual abuse unit in a children's hospital were reviewed Content analysis was completed on 39 files (32 females and 7 males) based on a coding framework. Parents were asked to consent to have their child's file reviewed for the study. Victims assessed were 12--18 years of age Majority of children told their mothers (43%) and peers (33%) first. Three major themes were identified as influencing the disclosure process: (1) feeling distressed, (2) opportunity to tell, and (3) fears for self. Additional themes of being believed, shame/self-blame, and peer influence were also identified The sample size is small but will contribute to a large multisite study in Ireland. Serves as an important exploratory pilot bringing forward disclosure themes for consideration [@bibr10-1524838017697312]) This study aimed to explore how the relationship between the perpetrator and the victim, especially whether these relations are intrafamilial or extrafamilial, impact CSA disclosure File reports of children seen for assessment in a child sexual abuse unit in a children's hospital were reviewed 220 minor victims---78.2% female victims, 41.8% aged between 14 and 18 (most prevalent age range), and 48.2% were abused by a family member Disclosure processes were more complex when it concerned sexual abuse committed by intrafamilial perpetrator: 60% of the victims reveal the facts several years after, and most often to individuals outside the family (78.6% of the disclosures done at school); on the contrary, extrafamilial disclosures take place more spontaneously and quickly: 80% of the victims reveal the facts a few days after, most often to their mother or peers The relationship with the perpetrator has a significant impact on both timing and recipient of disclosure, with intrafamilial abuses less likely to be disclosed promptly and within the family system [@bibr12-1524838017697312] Study focus was on identification of barriers to CSA disclosure with male survivors Using qualitative content analysis, researchers conducted a secondary analysis of online survey data, the 2010 Health and Well-Being Survey, that included men with self-reported CSA histories with an open-ended item on disclosure barriers 460 men with CSA histories completed an anonymous, Internet-based survey. Recruited from survivors' organizations. Age range of 18--84 years. Two thirds of respondents reported clergy-related abuse. Majority of respondents were White Vast majority of participants (94.6%) were sexually abused by another male. Duration of sexual abuse broke down into: 30.2% less than 6 months, 32.3% 6 months to 3 years, and 34.3% more than 3 years. Ten years old was average age of CSA onset. Ten categories of barriers were classified into three domains: (1) sociopolitical: masculinity, limited resources; (2) interpersonal: mistrust of others, fear of being labeled "gay," safety and protection issues, past responses; and (3) personal: internal emotions, seeing the experience as sexual abuse, and sexual orientation. At time of the study, this was the largest qualitative data set to have been analyzed with an explicit focus on adult male survivors' perceptions of barriers to CSA disclosure. Because the sample was limited in terms of the low percentage of racial minorities (9.3%), disclosure differences based on race or ethnicity were not discerned. The majority of abuse reported was by clergy which might present a unique set of barriers to disclosure [@bibr11-1524838017697312] Study purpose was to describe male CSA disclosure processes using a life span approach examining differences based on age. Also, to explore relationships between disclosure attributes and men's mental health Cross-sectional survey design. Eligible participants were screened and completed an anonymous, Internet-based survey during 2010. Measures used: General Mental Health Distress Scale and General Assessment of Individual Needs. Questions related to CSA disclosure and supports were included Purposive sampling of 487 men from three national organizations devoted to raising awareness of CSA among men. Age range: 19--84 years. Mean age for onset of CSA was 10.3 years Older age and being abused by a family member were both related to delays in disclosure. Most participants who told someone during childhood did not receive emotionally supportive or protective responses and the helpfulness of responses across the life span was mixed. Delays in telling were significant periods of time (over 20 years). Approximately one half of the participants first told about the sexual abuse to a spouse/partner (27%) or a mental health professional (20%); 42% of participants reported that their most helpful discussion was with a mental health professional. However, unhelpful responses caused most mental distress. Clinical recommendations included more of a life-course perspective be adopted, understanding impact of unhelpful responses and the importance of expanding networks for male survivors Purposive sampling of men from awareness raising organizations may have attracted particular participants who had already disclosed and received help. Participants needed to have access to Internet which would have eliminated men in lower SES groups and required proficiency in English which would eliminate certain cultural groups. However, the sampling strategy gained access to a predominantly hidden population. Important clinical recommendations are made with an emphasis on a life-course focus [@bibr33-1524838017697312] Qualitative study asked the central research question: "How do children tell?" Objective was to develop theory of how children tell of their CSA disclosure experiences. Parents were interviewed. Grounded theory method study. Interviews were conducted. Line-by-line open and axial coding was conducted on verbatim transcripts Sample of 22 young people; 16 girls and 6 boys; age range: 8--18 years; 22 interviewed in total between the ages of 8 and 18. Mixed sample of some enduring intrafamilial CSA, some extrafamilial CSA, and two endured both forms A theoretical model was developed that conceptualizes the process of CSA disclosure as one of containing the secret: (1) the active withholding of the secret on the part of the child; (2) the experience of a "pressure cooker effect" reflecting a conflict between the wish to tell and the wish to keep the secret; and (3) the confiding itself which often occurs in the context of a trusted relationship. These were derived from eleven categories that were developed through open and axial coding Modest but sufficient sample for an exploratory qualitative inquiry. High level of trustworthiness rigor. A subsample of randomly selected transcripts was independently coded. Very young children and young adults were not captured in this sample. Transferability of findings can only be made to the age range sampled in the context of Ireland [@bibr41-1524838017697312] To investigate the process of CSA disclosure with adolescents from the general population who had experienced CSA. How many disclosed, who did they disclose to, and what were their motives for disclosing Data collection was through face-to-face qualitative interviews. Standardized questions and measures were administered on family situation, sociodemographic data, sexual victimization, general, and mental health. Sexual Assault Module of the Juvenile Victimization Questionnaire was used Convenience sample of 26 sexually victimized adolescents. 23 girls and 3 boys. Age range: 15--18 years. Online advertisements and flyers were used to recruit youth from community and counseling services Less than one third of participants immediately disclosed CSA to another person. In most cases, recipients of both immediate and delayed disclosure were to peers. More than one third of participants had never disclosed the abuse to a parent. Participants reported reluctance to disclose to parents so as not to burden them. Earlier disclosures were related to extrafamilial CSA, single occurrence CSA, age of victim at abuse onset, and parents who were living together. Higher levels of reported guilt and shame were related to delayed disclosures. Peers were viewed by this sample as more reliable confidants Two thirds of the sample did not disclose right away. Strengthening parent--child relationships may be one of the most important ways to increase disclosure to parents. Disclosure to peers has been found a common trend in other research and bears more examination [@bibr21-1524838017697312] Aim of this study was to develop a fuller understanding of CSA disclosures Narrative inquiry methodology. Face-to-face in-depth interviews were conducted with participants. Data were analyzed using Rosenthal and Fischer--[@bibr55-1524838017697312] method. Purposive sampling was employed. Sample consisted of 22 participants aged 25--70 years; 13 women and 9 men. Participants were sexually abused at 15 years or under with someone over the age of 18. Only 5 out of 22 participants told anyone about their early sexual experiences as children. Fear, shame, and self-blame were the main inhibitors to disclosure. These factors are further detailed through subthemes. Telling as a child and as an adult was further expanded upon using [@bibr1-1524838017697312]) framework verifying behavioral indirect attempts to tell and purposeful disclosure as categories. Thematic analysis supported that CSA disclosure should be conceptualized and viewed as a complex and lifelong process Delayed disclosure was common in this qualitative sample. Most participants did not make a selective disclosure until adulthood. These findings support [@bibr1-1524838017697312]) model of disclosure but also highlights the importance of life stage. Modest but sufficient sample size for a qualitative inquiry. Well-designed study with detailed analysis for transferability of findings [@bibr40-1524838017697312]) This study aimed to: (1) add direct inquiry about the process of a child's CSA disclosure; (2) determine if children will discuss process that led them to tell; and (3) describe factors that children identify that led them to tell about or caused them to delay CSA disclosure Study sought to find out if process issues of disclosure could be identified in the context of forensic interviews. Forensic interviewers were asked to incorporate questions about "telling" into an existing forensic interview protocol. Interview content related to the children's reasons for telling or waiting was extracted, transcribed, and analyzed using grounded theory method of analysis 191 interviews of CSA victims aged 3--18 over a 1-year period were used for the study. Inclusion criteria included children who made a statement about CSA prior to referral, reasons for telling or waiting to tell, and those who spoke English. Participants were children who were interviewed at a child sexual abuse clinic. 74% were female and 51% were Caucasian Reasons the children identified for telling were classified into three domains: **(**1) disclosure as a result of internal stimuli (e.g., the child had nightmares); (2) disclosure facilitated by outside influences (e.g., the child was questioned); and (3) disclosure due to direct evidence of abuse (e.g., the child's abuse was witnessed). The barriers to disclosure identified fell into five groups**: (**1) threats made by the perpetrator (e.g., the child was told she or he would get in trouble if she or he told), (2) fears (e.g., the child was afraid something bad would happen if she or he told), (3) lack of opportunity (e.g., the child felt the opportunity to disclose never presented), (4) lack of understanding (e.g., the child failed to recognize abusive behavior as unacceptable), and (5) relationship with the perpetrator (e.g., the child thought the perpetrator was a friend) An innovative study to try to assess if formal investigative interviews can facilitate disclosures of CSA. Data were based on a large number of interviews. Detailed analysis produced detailed findings supporting other study findings on CSA disclosure [@bibr3-1524838017697312]) The study aimed to identify factors impeding or promoting CSA disclosures. Overarching research question: What individual, interpersonal, environmental, and contextual influences impede or promote CSA disclosures. A qualitative phenomenological design, LIM, was used to interview adult CSA survivors about their disclosure experiences to provide retrospective accounts of CSA disclosure and meaning-making of these experiences. Thematic analysis was done through a social--ecological lens. Purposive sampling was employed. Snowball sampling was also used to recruit more male survivors. 40 adult survivors of CSA were interviewed: 36% men and 64% women. Age range of 18--65 with a mean age of 40.1 years. Average age of abuse onset was 5.3 years old. 36% of the sample was non-White. Diverse socioeconomic backgrounds Themes fell into four domains: (1) individual and developmental factors, developmental factors as to whether they comprehended what was happening, personality traits also had some bearing on their ability to tell, and anticipating not being believed; (2) disclosure inhibited by family characteristics such as rigidly fixed gender roles with dominating fathers, chaos and aggression, other forms of child abuse, domestic violence, dysfunctional communication, and social isolation; (3) neighborhood and community context, that is, lack of interest from neighbors and teachers not pursuing troubling behavior; and (4) cultural and societal attitudes, media messages and societal attitudes, feeling unheard as kids, gender socialization for males, and cultural attitudes influencing parent's reactions. Purposeful disclosure is higher than reported in other studies because of the sampling attempts to purposefully locate disclosers The study presents a comprehensive social--ecological analysis to CSA disclosure highlighting the multifaceted influences. Of note, 42% had disclosed the abuse during childhood; 26% had not disclosed because they had repressed the memory, or the abuse had occurred in preschool years and they had difficulty with recall. The remainder had attempted some form of disclosure in indirect ways during childhood. A retrospective approach that could be affected by recall issues [@bibr15-1524838017697312] This examination of CSA disclosure explored the ways culture affects processes of CSA disclosure and reporting, both in the United States and internationally Using published literature with clinical data, this article conducted an analysis to provide a culturally competent framework for CSA disclosure questioning Data consisted of published literature on disclosure and culture that was triangulated with clinical case material Cultural and structural factors affecting CSA disclosure are identified in in-depth detail. Recommendations made include (1) disclosure interviewing should be tailored to the child's cultural context, (2) questioning should also take into consideration age and gender factors, and (3) culture stands as an important factor in all cases in which children are considering disclosing or being asked to disclose, and not solely in cases in which children are from noticeable minority groups. Presents a comprehensive interview framework integrating cultural considerations One of the few works that adds knowledge to culturally contextual disclosure interviewing. Unique combination of literature findings with clinical material. Anecdotal accounts may preclude transferability of findings. Overall adds to an impoverished area of CSA disclosure information [@bibr51-1524838017697312] This study explored disclosure strategies with a national sample of youth focusing on (1) What are the hidden experiences of abuse among Canadian youth? (2) What impact does participation in abuse prevention programs have on youth to express their abuse experiences? (3) What disclosure barriers do youth face? (4) What are young people's disclosure patterns? and (5) Who do they tell? Forms were completed by youth following participation in abuse prevention programming by the Canadian Red Cross (RespectED). A series of focus groups and observations of the workshops were used to help contextualize the findings. Evaluation forms were analyzed from two violence prevention programs: (1) It's not your fault and (2) What's love got to do with It? Examination of results from a national sample of 1,621 evaluation forms where youth anonymously disclosed abuse experiences. Respondent's ages: 13 and under (27%), 14--15 (37%), 16--17 (25%), 18 and older (4%), and unknown (7%) Youth who have been abused or witnesses to abuse employ five disclosure strategies: using self-harming behaviors to signal the abuse to others; not talking at all about the abuse to prevent intrusive interventions by others; seeking help from peers; seeking help from informal adult supports; and seeking help from mandated service providers (social workers and police). Results suggest disclosure is an interactive process, with expectations regarding consequences to disclosure. Patterns of incrementally sharing abuse experiences are shaped by young people's interactions with peers, educators, and caregivers. About three-quarters of females previously disclosed; significantly less males disclosed This study highlights that disclosure is an interactive ongoing process. Findings lend support to studies that have identified similarly interactive models of disclosure such as those detailed by [@bibr1-1524838017697312]) and [@bibr44-1524838017697312]). This mixed sample of youth who experienced different forms of abuse and violence exposure were participants---not limited to CSA survivors [@bibr52-1524838017697312] This study explored abuse disclosure strategies with a national sample of Canadian youth who participated in violence prevention programming. One of the goals of the study was to document not previously identified experiences of abuse and youth attitudes toward disclosure of abuse experiences Exploratory design with a nonrepresentative samples. Qualitative analysis of 1,099 evaluation forms completed following Red Cross RespectED violence prevention programming delivered between 2000 and 2003. Forms of anonymous abuse disclosures by youth participants of neglect, emotional, physical, and sexual abuse. Twenty-seven interviews and focus groups were also done to understand contextual issues and engage youth and program facilitators in the interpretation of findings. A coding structure was developed for analysis to synthesize themes across data sources Purposeful sample of 1,099 evaluation forms completed following Red Cross RespectED violence prevention programming delivered between 2000 and 2003 Findings suggest high rates of hidden abuse, with less than one quarter of youth reporting a disclosure. 244 of the 1,099 youth who disclosed abuse on their evaluation forms identified specific individuals they told about their abuse. Disclosure patterns vary with boys, youth aged 14--15, victims of physical abuse, and those abused by a family member being most likely to disclose to professionals or the police. One third of disclosures were directed toward professionals and the least, 5% percent each, were directed toward friends, parents, and others. Participants were most likely to disclose sexual abuse to parents/family, professionals, and the police/courts, with fewer choosing friends. Out of all 1,099 participants, 225 males and 779 females indicated that they had been abused. Out of those, 43 males and 180 females indicated that they had disclosed the abuse. Of those who had disclosed, only a portion of males and females specified who they had disclosed the abuse to ("While 1,099 evaluations with disclosure statements were analyzed, only 22% made mention of people to whom disclosures occurred.") More females specified who they disclosed to compare to males. The data show perceptions among youth of negative consequences following disclosure Innovative design of this study provides insight into young people's perceptions of disclosure experiences. High level of rigor with trustworthiness of the data analysis ensured through use of youth focus groups, interviews, and observational data. The study results are somewhat limited in the thickness of the descriptions it can offer because most of the data are survey based. Regional differences may not have been picked up. Scope of the study is broad and approach is creative [@bibr38-1524838017697312]) This study aimed to investigate disclosure rates and disclosure patterns and examine predictors of nondisclosure in a sample of male and female adolescents with self-reported experiences of sexual abuse Participants completed 65-item questionnaire that included questions about background, consensual sex, sexual abuse experiences (noncontact, contact or penetrating abuse, including peer abuse), disclosure of CSA, own sexual abusive behavior, sexual attitudes, and experiences with pornography and sexual exploitation. The questionnaire included 6 modified items from the SCL-90 and 9 of 25 items from the Parental Bonding Instrument. The data for girls and boys were analyzed separately The sample consisted of 4,339 high school students in Sweden (2,324 girls and 2,015 boys). The mean age of the participants was 18.15 years. This study used a subsample of 1,962 participants who reported CSA and who answered disclosure questions Of the sample, 1,505 girls (65%) and 457 boys (23%) reported CSA. The disclosure rate was 81% (girls) and 69% (boys). Girls and boys disclosed most often to a friend of their own age. Few had disclosed to professionals, and even fewer had reported to the authorities. There were higher rates of disclosure to a professional with more severe abuse (contact abuse with or without penetration) for girls, but lower rates for boys The more severe the sexual abuse was, the less likely both girls and boys had talked to their mother, father, or a sibling. Girls were less likely to disclose if they had experienced contact sexual abuse with or without penetration, less frequent abuse, abuse by a family member, or if they had perceived their parents as less caring and less overprotective and highly overprotective. Boys were less likely to disclose if a family member abused them, they were studying a vocational program (vs. an academic program), lived with both parents or had perceived their parents as less caring and not overprotective. Adolescents who reported CSA perceived their mental health as poorer compared to adolescents without CSA. Nondisclosers reported more symptoms on the Mental Health Scale than those who had disclosed This study highlighted that sexual abuse is largely hidden from adult society, especially from professionals and the legal system. However, time lapsed to disclosure was not reported. Since friends appeared to be the main recipients of sexual abuse disclosures, practice implications of this could be to find ways to give young people better information and guidance about how to support a sexually abused peer. A qualitative component to the study would have provided a broader understanding of disclosure processes. Study limitations include a significant amount of boys who did not complete the questions regarding disclosure on; the timing of disclosures (whether they were delayed or not) was not measured; possibility of recall bias with retrospective studies based on self-reports; and youth participants may not have understood all the questions [@bibr43-1524838017697312] Study focused on disclosure challenges for male survivors of CSA to understand three issues: (1) To whom and in what contexts have they disclosed these experiences? (2) What do they have to say about their disclosure experiences? and (3) What are their perceptions of positive and negative aspects of their disclosure, including incentives and barriers? Male survivors of CSA were interviewed about their disclosure experiences. Analytic techniques included grounded theory method of analysis for coding and development of conceptually clustered matrices. Participants completed two in-depth, semi-structured interviews, lasting between 2 and 3 hr each taking place approximately a week apart The sample consisted of 16 male survivors of childhood sexual abuse; 11 Caucasian, 2 African American, 1 Puerto Rican, 1 part Native American, 1 African Cuban; age range of 24--61 years; 9 identified themselves as heterosexual, 5 as homosexual, and 2 as bisexual Barriers to disclosure were found to be operant in three interrelated domains: (1) personal (e.g., lack of cognitive awareness, intentional avoidance, emotional readiness, and shame); (2) relational (e.g., fears about negative repercussions, isolation); and (3) sociocultural (e.g., lack of acceptance for men to experience or acknowledge victimization). Only 1 of the 16 men in this sample disclosed the full extent of his sexual abuse experiences while he was still a child. The other men reported that they had not disclosed, although some reported attempts to tell that were indirect or incomplete. Several other men disclosed certain experiences or elements of their abuse, but concealed others. By the time of the study, many of these men had disclosed their past experiences in a variety of relationships, including those with family members, partners, therapists, and infrequently friends. Several had only limited discussions of their sexual abuse Since the vast majority of men in the sample had not disclosed in childhood, they may have been predisposed to identifying barriers to disclosure more readily. Retrospective accounts are subject to recall issues. Investigators made significant efforts to gather a diverse sample. High level of rigor was executed in the dependability of the data and iterative process of the interpretation of findings was conducted [@bibr20-1524838017697312] The goal of the present study was to examine how child victims of extrafamilial sexual abuse disclosed the abuse experience Alleged victims of sexual abuse and their parents were interviewed. Children were interviewed using the NICHD Investigative Interview Protocol by experienced youth investigators. Information on disclosure processes was obtained in the first formal interview, before any police investigation or child welfare intervention Thirty alleged victims of CSA; 18 boys and 12 girls. Child sample was 7- to 12-year-olds with an average age of 9.2 years. Twenty mothers and 10 fathers were also interviewed for a total of 30 parent interviews. A content analysis was conducted on child and parent interviews Disclosure categories were identified as follows: (1) delayed 53% of the children delayed disclosure for between 1 week and 2 years; (2) recipient of disclosure: 47% of children first disclosed to siblings or friends, 43% first disclosed to their parents, and 10% first disclosed to another adult. 57% of the children spontaneously disclosed abuse, but 43% disclosed only after they were prompted. 50% of the children reported feeling afraid or ashamed of their parents' responses. Parents' reactions: supportive (37%) and unsupportive (63%). There was a strong correlation between predicted and actual parental reactions suggesting children anticipated their parents' likely reactions accurately. Disclosure processes varied depending on the children's ages (e.g., younger children disclosed to parents), severity and frequency of abuse, parents' expected reactions, suspects' identities, and strategies used to foster secrecy Innovative design to gather disclosure data from young children. Focus is on extrafamilial CSA which may differ than disclosure patterns of intrafamilial CSA. Two thirds of the parents registered unsupportive responses which is high [@bibr4-1524838017697312]) The objectives of the current study were to identify a broad range of factors, including family dynamics that contribute to or hinder a child's ability to disclose CSA. A qualitative phenomenological design---LIM---was used to elicit disclosure experiences; facilitators and barriers; and relevant circumstances. Interviews were transcribed verbatim. Line-by-line open coding was conducted to capture family-level factors. Axial and selective coding facilitated identification of themes Purposive sampling was employed to recruit 20 adult survivors between the ages of 18 and 65 who were sexually abused by a family member. Average age of participants was 40.1 years; 60% of participants were female and 40% male. Average age of onset of abuse was 6.7 years. Mixed clinical and nonclinical sample. The majority had received treatment for CSA at some point in their lives Four major themes emerged suggesting that CSA disclosure can be significantly compromised when certain family conditions exist: (1) rigidly fixed, gender roles based on a patriarchy-based family structure; (2) presence of family violence; (3) closed, indirect family communication patterns; and (4) social isolation of the family as a whole, or specific members, played a part in CSA victims feeling they had no one safe to tell. Family systems formulations through a feminist lens are important in understanding children and families at risk of disclosure barriers Over half the participants had not disclosed the abuse during childhood. Of the nondisclosing participants, six did not disclose because they had repressed or forgotten the memory. Almost one third withheld disclosure intentionally. More data are needed on early disclosures to garner more information on facilitators of disclosure. Retrospective approach implies recall issues. High level of trustworthiness of the data and interpretations were achieved through credibility, dependability, and confirmability through direct quotes [@bibr2-1524838017697312]) The study purpose was to qualitatively explore dynamics that impede or promote disclosure by examining a range of factors including gender as a dynamic---how disclosures of females and males are similar and different, and in what ways gender affects CSA disclosure Survivors of CSA were interviewed about their disclosure experiences using LIM. Analysis of 30 participant narratives was used for theme development regarding impact of gender on disclosure. Interviews were transcribed verbatim for open, axial, and selective coding. Categories and subcategories were collapsed and refining into theme areas Purposive sampling of women and men, along with those who disclosed during the abuse and those who did not. 19 females and 11 males; 18--65 (mean 40.1) years who were sexually abused by a family member or a trusted adult. Average age of abuse onset was 5.3 years, 36% were nonwhite, and 58% had not disclosed during childhood Three themes emerged for men that inhibited or precipitated disclosure for reasons related to gender: (1) fear of being viewed as homosexual; (2) profound feelings of stigmatization or isolation because of the belief that boys are rarely victimized; and (3) fear of becoming an abuser, which acted as a precipitant for disclosure. Two predominant themes with female participants related to difficulties disclosing: (1) they felt more conflicted about who was responsible for the abuse and (2) they more strongly anticipated being blamed and/or not believed One in a dearth of studies that conduct gender analysis. Comparative analysis draws out important practice implications. Retrospective design of the study which implies possible recall issues. High level of trustworthiness of the data and interpretations were achieved through credibility, dependability, and confirmability through direct quotes [@bibr7-1524838017697312]). Study examined patterns of disclosure in a large representative sample of South African CSA victims. Two study objectives to: (1) examine how and when CSA victims disclose their abuse and (2) Identify factors associated with different patterns of disclosure File reviews of all social work and medical case files for CSA victims seen at the crisis center where all cases of CSA reported to the North Durban policing area were referred during the period of January 2001 to December 2003 1,737 cases of CSA reported in the North Durban area of KwaZulu-Natal, South Africa, during January 2001 to December 2003. 1,614 girls and 123 boys; average age of victimized children was 9.9 years. 47% reports were made within 72 hr of the abuse, 31% from 72 hr to 1 month, and 22% more than a month after the abuse Content analysis identified two broad dimensions of disclosure: (1) agency: child-initiated disclosure versus detection by a third party and (2) temporal duration: an event versus a process. These disclosure dimensions defined four discrete categories of disclosure: (1) purposeful disclosure (30% of cases), (2) indirect disclosure (9% of cases), (3) eyewitness detection (18% of cases), and (4) accidental detection (43% of cases). Disclosure independently predicted by victim's age, nature of the victim--perpetrator relationship, offender's age, frequency of abuse, and reporting latency. Mean age of purposeful disclosures (10.67) was higher than the mean age of indirect disclosures (5.84). Explicit forms of disclosure were less likely when the offender was a family member. Shorter reporting latency was more likely with repeated abuse These results fit into [@bibr1-1524838017697312]) disclosure framework. Through data analysis two raters coded disclosure categories using author's disclosure framework, which proved to be both exhaustive and mutually exclusive with the percentage of interrater agreement at 98%. Generalizability of this study is limited to child clients receiving a crisis assessment referred through a police report [@bibr19-1524838017697312]). This study aimed to identify characteristics of suspected child abuse victims that are associated with disclosure and nondisclosure during formal investigations Large database of suspected cases of physical and sexual abuse investigated in Israel between 1998 and 2002 was analyzed. Interviews were also conducted using standardized NICHD Investigative Interview Protocol. Archival data were analyzed The sample was comprised of 26,446 of 3- to 14-year-old alleged victims of sexual and physical abuse interviewed in Israel in the 5-year period from 1998 to 2002. 140 experienced trained youth investigators conducted interviews Overall, 65% of the 26,446 children made allegations when interviewed. Rates of disclosure were greater for sexual abuse (71%) over physical abuse (61%). Children of all ages were less likely to disclose/allege abuse when a parent was the suspected perpetrator. Disclosure rates increased as children grew older: 50% with 3- to 6-year-olds, 67% of the 7- to 10-year-olds, and 74% of the 11- to 14-year-olds disclosed abuse when questioned Overall findings indicated that rates of disclosure varied systematically depending on the nature of the alleged offences, the relationship between alleged victims and suspected perpetrators, and the age of the suspected victims. Analyses only involved cases that had come to the attention of official agencies, making it difficult to determine how many of abuse take place without ever triggering any kind of official investigation [@bibr22-1524838017697312]) This study investigated the context in which children were able to report their child sexual abuse experiences; their views as to what made it difficult to talk about abuse; what helped them in the disclosing process; and their parent's perceptions of their disclosure processes Qualitative approach to data collection and analysis was used. Therapeutic interviews o the children and mostly their mothers were analyzed through a qualitative approach. Follow-up interviews were held 1 year later 20 families with a total of 22 children participated. All children had told about experiences that created concerns for care-givers about CSA. Children's ages ranged between 3 and 16 years (average age 7.5 years); 15 girls and 7 boys. Sexually abused by someone in the family or a close person to the family None of the children told of abuse immediately after it occurred. Children exposed to repetitive abuse kept this as a secret for up to several years; 17 told their mothers first, 3 first told a friend, 1 told their father, and 1 their uncle. Majority of remarks that led to the suspicion of CSA were made in situations where someone engaged the child in a dialogue about what was bothering them, resulting in a referral. The children felt it was difficult to find situations containing enough privacy and prompts that they could share their experiences. When the children did disclose they did it in situations where the topic of child sexual abuse was in some form addressed or activated, where someone recognized the child's cues and probed further. They also were sensitive to others reactions, and whether their disclosures would be misinterpreted. Several of the children perceived negative consequences as major factors contributing to delaying disclosure. They were primarily concerned about negative effects for the mother. The mothers said they were also sensitive to the children's feelings. If their children showed signs of distress and did not want to talk, the mothers would change the subject or not pursue the topic further Evidence for delayed disclosures. The results indicate that disclosure is a fundamentally dialogical process that becomes less difficult if children perceive that there is an opportunity to talk, a purpose for speaking and a connection has been established to what they are talking about. Strengthening parent--child relationships is an important practice implication [@bibr44-1524838017697312]) The purpose of this study was to understand the full process of CSA disclosure and how this unfolded for preadolescent and adolescent girls. Examined what facilitated and hindered disclosure and subsequent consequences Secondary analysis of qualitative focus group data. Original project consisted of four focus groups conducted within the context of ongoing therapy for girls who had experienced CSA. Secondary analysis consisted of written narrative summaries of each session grouping these conceptually, and examining their interconnectedness Sample consisted of 34 participants from four groups. Sessions analyzed were between 60 and 90 min long; audiotaped and later transcribed for content analysis Findings are reported in three major domains: (1) self-phase: where children come to understand victimization internally; (2) confidant selection-reaction phase: where they select a time, place, and person to tell and then whether that person's reaction was supportive or hostile; and (3) consequences phase: good and bad that continued to inform their ongoing strategies of telling. The actions and reactions of adults were significant and informed the girls' decisions. The consequences phase was further subdivided into four aspects: (1) gossiping and news networks, (2) changing relationships, (3) institutional responses and the afterlife of telling, and (4) insider and outsider communities This study provided a contextual examination of the entire disclosure process, closer to the point in time when the abuse and disclosure occurred. Small groups of preadolescent and adolescent girls who had survived sexual abuse also served as consultants and were encouraged to share their knowledge for the benefit of professional practitioners [@bibr1-1524838017697312]) The study sought to examine influences that inhibit or promote children's disclosure of CSA to address gaps in knowledge about how, when, and under what circumstances victims of CSA disclose The study employed LIM---a phenomenological design. Intensive interviewing that were 2 hr long on average generated data for a thematic analysis. The interview guide was developed to probe for individual, interpersonal, environmental, and cultural factors influencing CSA disclosure Using purposive sampling 24 adult survivors of intrafamilial abuse between ages of 18 and 65 (average age 41.2) were recruited from agencies and one university; 57% male and 43% female; average age of abuse onset was 6.5 years; 42% of the participants had disclosed the abuse during childhood; 58% disclosed as adults Through analysis of the interview new categories of disclosure were identified to add to existing types. Three previously identified were confirmed in these data: accidental, purposeful, and prompted/elicited accounted for 42% of disclosure patterns in the study sample. Over half the disclosure patterns described by the study sample did not fit these previously established categories. Three additional disclosure categories emerged: behavioral and indirect verbal attempts, disclosures intentionally withheld, and disclosures triggered by recovered memories This study expanded types of CSA disclosures to more fully understand how children and adults disclose. And under what circumstances. Asking people to recount events that occurred in childhood is susceptible to memory failure, especially when memories were forgotten, delayed, or repressed and later recovered. Distortion and revision of events are also potential problems in recall. High degree of trustworthiness of the data was achieved and quotes provided supported the categories [@bibr9-1524838017697312]) The main goals of this study were to understand impediments that prevent adolescents from disclosing CSA and seeking help from their social network and/or the services In-depth telephone (anonymous) interviews were conducted after informed consent was explained and obtained. Three investigators experienced in counseling CSA counseling conducted the interviews which were recorded with permission. Three researchers independently scored the interviews according to a coding framework The sample was comprised of 36 young people who experienced sexual abuse in adolescence; 35 females and 1 male; aged 12--17. Some of the sample experienced sexual violence in a dating relationship The main impediments to disclose to a family member were: fear of not being believed, shame, and fear of causing trouble to the family. The main impediments for not seeking services were: unaware of appropriate services, wish to keep the secret, lack of awareness of being abused, mistrust of adults and professionals, and fear of the consequences of disclosing sexual abuse. When they did disclose to professionals, teens received very limited support as many professionals were not trained on sexual abuse and could not offer appropriate interventions to victims This study represented the findings of a mixed sample of survivors of child sexual abuse and intimate partner violence. The study was conducted in Italy and it is not clear what sexual abuse response training is available. There may have been a selection bias as the most dissatisfied survivors responded to the research call [@bibr25-1524838017697312] Study purpose was to explore how abuse traits, openness, reactions to CSA disclosure, and social support were related. Differences based on severity of abuse, timing and outcomes of disclosure, social support, and predicting factors of positive and negative reactions were probed Adult women reporting CSA by someone close were interviewed using semi-structured guides together with questionnaires. Data on victimization and current social support were retrieved through the questionnaires, and data on disclosure and reactions were gathered through the interviews with participants 122 adult women between 20 and 60 years old (average age of 41 years) reporting exposure to child sexual abuse by someone close before the age of 18 and had told someone about at least one abuse event. 90% were Swedish subjects. Purposive sampling strategy was used Abuse characteristics: abuse by multiple perpetrators was more common than by a single perpetrator. Age of onset was often before age of 7, with an average duration of 7 years. Severely abused women had talked to more of their social network, especially to professionals. Disclosures: 32% disclosed during childhood (before the age of 18) with an average of 21 years delay. Women who had disclosed in childhood reported more instances of physical abuse, multiple perpetrators, use of violence, and were more likely to have confronted a perpetrator, and had received a negative first reaction. Factors significantly predicting delay were younger age at first event and no use of violence. Disclosure outcomes: of the 26 women who told in childhood during a period with ongoing abuse, 15 women were continuously abused after disclosure 68% delayed disclosure until adulthood. At the time of the study, it was one of the first studies to focus on the interplay between social support networks and disclosure of child sexual abuse. The study results are somewhat limited by an overrepresentation of severely abused women. Retrospective study and self-report of information could imply recall issues and thus limits the accuracy of the information obtained on abuse and disclosure characteristics. Cross-sectional design does not allow for definite conclusions of cause and effect on the relationships found [@bibr28-1524838017697312]) The purpose of this study was to identify factors that influence the disclosures made by female survivors of USE in childhood and adolescence. The predictors of both the timing of disclosure and the recipient of the disclosure were investigated Data were gathered from a subsample of female adolescents that participated in the NSA, which consisted of structured phone interviews. USEs reported in the NSA were assessed using a modified version of the Incident Classification Interview. They were then asked a series of questions about each episode of unwanted sexual contact including event characteristics and perpetrator characteristics A subsample of 263 adolescent females between 12 and 17 years old, mean age of 15.2 years old, who reported at least one experience of unwanted sexual contact in the NSA. Participant characteristics, USE characteristics, and family contextual attributes were explored Children under the age of 7 were at a higher risk for delayed disclosures. Participants whose USE occurred between the ages of 7 and 13 were most likely to tell an adult. Adolescents (14--17) were more likely to tell only peers than children aged 7--10 years. Children under 11 were more likely to tell an adult, but were at risk for delaying disclosure beyond a month. Children aged 11--13 tended to disclose within a month. Closer relationship to the perpetrator or a family member was associated with delayed disclosure. Immediate disclosure was more likely with stranger perpetration. Fear for one's life during and penetration were associated with disclosure to adults. Family factors linked to disclosure were (1) drug abusing household member, which made survivors more likely to disclose more promptly and (2) never living with both parents was associated with nondisclosure This study examined factors including disclosures of USEs in childhood and adolescence in a nationally representative sample of female adolescents who participated in the NSA. Surveys for investigations of victimization experiences may be biased due to underreporting. Adolescents who refused to report or discuss an USE may represent a source of systematic bias and would make the results generalizable only to adolescents who are willing to disclose USE via survey. Although data may be retrospective, recall bias may have been minimized in this study since participants were adolescents, and so the time lag between the USE and the interview were presumably shorter than a study of adult participants recalling CSA experiences [@bibr17-1524838017697312]) The purpose of this study was to investigate variables associated with delay of disclosure of CSA and test a model for factors that influence how quickly children disclose sexual abuse Case file reviews of data obtained from prosecution files, as well as from structured interviews with the children's caretaker and observations of child interviews. Trained graduate students and one victim advocate completed the Sexual Assault Profile questionnaire for child characteristics, the abuse and their disclosure. Children's perception of responsibility and fear of negative consequences were probed. Correlational analyses were conducted with path analyses to test the hypothesized causal relations among variables Sample consisted of 218 children referred to prosecutors' offices for alleged CSA. All children in the sample had disclosed their abuse in some manner. Children ranged in age from 2 to 16 years at the beginning of abuse; 3--16 years at the end of the abuse, and 4--16 years at the time of the initial police report; 77% female, 70% Caucasian, 17% Hispanic, and 11% African American. Predominantly middle to low SES. Approximately 47% intrafamilial abuse 64% disclosed within a month and 29% within 6 months. Five variables for the model were tested. (1) age: children who were older took longer to disclose and older children feared more negative consequences to others than younger children; (2) type of abuse: victims of intrafamilial families took longer to disclose---victims of intrafamilial abuse feared greater negative consequences to others compared to victims of extrafamilial abuse; (3) fear of negative consequences: children who feared negative consequences of disclosure took longer to disclose, children who believed that their disclosure would bring harm to others took longer to disclose, fear of negative consequences to the self or the perpetrator was unrelated to time of disclosure, and girls more than boys feared negative consequences to others; (4) Perceived responsibility: children who felt greater responsibility for the abuse took longer to disclose and older children felt more responsibility for the abuse; and (5) gender was not significantly correlated with time to disclosure This study represents a higher rate of disclosers within a month. These cases had been reported to authorities and were in process of prosecution which may explain higher rate of early disclosures. Legal sample with higher rate of extrafamilial abuse (52%) may also account for earlier disclosures. Model suggests that older children, victims of intrafamilial abuse; felt greater responsibility for the abuse, and perceiving negative consequences to disclosing took longer to disclose. Well-designed study with high level of rigor. Produced a viable model of disclosure for further investigations. However, researchers were not able to interview children directly [@bibr42-1524838017697312]) The study focus was to gather data from a large sample of women about the length of time women who were raped before age 18 delayed disclosure who they disclosed to, and variables that predicted disclosure within 1 month Structured telephone interviews that lasted approximately 35 min were used to collect data using a computer-assisted telephone interview system. All telephone interviews were conducted with each question on a computer screen. The survey consisted of several measures designed to elicit demographic information, psychiatric symptoms, substance use, and victimization history. The present study reports on data from the demographic and child rape victimization questions Two probability samples. Wave 1 was a random sample of 2,009 respondents selected from stratified samples of defined jurisdictions. Random digital dialing was used to solicit households for listed and unlisted telephone numbers. Second random sample of 2,000 women between the ages of 18 and 34 was selected. Both Wave 1 and Wave 2 data were weighted to conform to the 1989 Census statistics 288 (9%) reported experiencing at least one event that met the study's definition of childhood rape. The average age at the time of the first rape was 10.9 years. Of the 288 women who reported a child rape, 28% stated that they had never told anyone about this sexual assault until specifically queried by the interviewer for this study. 58% did not disclose for over 1 year and up to 5 years post-rape. 27% disclosed within a month. Among women who disclosed prior to their NWS interview close friends were the most common person to whom victims made disclosures, followed by mothers and other immediate family members. Fewer than 10% of victims reported making their initial disclosure to social workers or law enforcement personnel. Only 12% of child rape victims stated that their assaults were reported to authorities at some point The time frame of this survey may have had contextual implications. The majority of child rapes reported by this sample occurred prior to the large-scale child assault prevention education programs that were begun in the 1980s that teach children that assaults (including CSA) are wrong and should be disclosed to responsible adults. This information may have influenced (and may currently be influencing) young women's disclosure patterns. For Wave 1, comparison of these data with the population parameters obtained from the U.S. Census Bureau indicated that the sample closely matched the demographic attributes of the population of U.S. women *Note*. SCL-90 = Symptom Check List-90; SES = socioeconomic status; LIM = long interview method; CSA = child sexual abuse; NICHD = National Institute of Child Health and Human Development; USE = unwanted sexual experiences; NSA = National Survey of Adolescents; NWS = National Women's Study; QIDS = Questionnaire informattsé sur les délinquants sexeuls. Key Findings {#section3-1524838017697312} ------------ First-level analysis of the studies identified key study characteristics. Trends emerged around definitions of CSA disclosure, study designs, and sampling issues. First, in regard to definitions, the term "telling" is most frequently used in place of the term disclosure. In the absence of standardized questionnaires or disclosure instruments, telling emerges as a practical term more readily understood by study participants. Several examples of this usage were found in the research questions, interview guides, and surveys examined: "How and when do people decide to tell others about their early sexual experiences with adults?" ([@bibr21-1524838017697312], p. 161); "Some men take many years to tell someone that they were sexually abused. Please describe why it may be difficult for men to tell about/discuss the sexual abuse" ([@bibr12-1524838017697312], p. 462). "Participants were asked a series of open-ended questions to elicit a narrative regarding their experiences of telling..." ([@bibr33-1524838017697312], p. 1160). "Who was the first person you told?" ([@bibr40-1524838017697312], p. 346). There was sound consistency between studies, defining disclosure in multifaceted ways with uniform use of categories of prompted, purposeful, withheld, accidental, direct, and indirect. However, defining the period of time that would delineate a disclosure as delayed varied widely across studies, wherein some studies viewed 1 week or 1 month as a delayed disclosure (i.e., [@bibr20-1524838017697312]; [@bibr28-1524838017697312]; [@bibr41-1524838017697312]). Other studies simply reported average years of delay sometimes as long as from 20 to 46 years ([@bibr11-1524838017697312]; [@bibr25-1524838017697312]; [@bibr42-1524838017697312]). Second, the number of qualitative studies has increased significantly over the last 15 years. This rise is in response to a previous dearth of qualitative studies. Based on [@bibr24-1524838017697312]) observation that disclosure factors and outcomes had been well documented through quantitative methods; in a widely read editorial, he recommended "Qualitative studies which are able to track the individual experiences of children and their perception of the influences upon them which led to their disclosure of information are needed to complement..." (p. 270). Third, although a few studies strived to obtain representative samples in quantitative investigations ([@bibr19-1524838017697312]; [@bibr28-1524838017697312]; [@bibr42-1524838017697312]), sampling was for the most part convenience based, relying on voluntary participation in surveys and consent-based participation in file reviews ([@bibr7-1524838017697312]; [@bibr38-1524838017697312]; [@bibr41-1524838017697312]; [@bibr51-1524838017697312]). Therefore, generalizability of findings is understandably limited. The qualitative studies used purposive sampling as is deemed appropriate for transferability of findings to similar populations. Some of those samples contained unique characteristics, since they were sought through counseling centers or sexual advocacy groups. These would be considered clinical samples producing results based on disclosures that may have been delayed or problematic. This might presumably produce data skewed toward barriers and bring forward less information on disclosure facilitators. Through an in-depth, second-level analysis, this review identified five distinct themes and subthemes beyond the general trends as noted earlier.**Theme 1**: Disclosure is viewed as an ongoing process as opposed to a discrete event---iterative and interactive in nature. A subtheme was identified regarding disclosure as being facilitated within a dialogical and relational context is being more clearly delineated.**Theme 2**: Contemporary disclosure models reflect a social--ecological, person-in-environment perspective to understand the complex interplay of individual, familial, contextual, and cultural factors involved in CSA disclosure. Subthemes include new categories of disclosure and a growing focus on previously missing cultural and contextual factors.**Theme 3**: Age and gender are strong predictors for delaying disclosure or withholding disclosure with trends showing fewer disclosures by younger children and boys. One subtheme emerged that intrafamilial abuse/family-like relationship of perpetrator has a bearing on disclosure delays or withholding.**Theme 4**: There is a lack of a cohesive life-course perspective. One subtheme includes the lack of data within the 18- to 24-year-old emerging adult population.**Theme 5**: Significantly more information is available on barriers than on facilitators of CSA disclosure. Subthemes of shame, self-blame, and fear are uniformly identified as disclosure deterrents. ### Disclosure as an ongoing process: Iterative and interactive in nature {#section4-1524838017697312} Disclosure is now generally accepted as a complex and lifelong process, with current trends showing that CSA disclosures are too often delayed until adulthood ([@bibr8-1524838017697312]; [@bibr11-1524838017697312]; [@bibr21-1524838017697312]). Knowledge building about CSA disclosure has moved in the direction of understanding this as an iterative and interactive process rather than a discrete, one-time event. Since the new millennium, disclosure is being viewed as a dynamic, rather than static, process and described "not as a single event but rather a carefully measured process" ([@bibr2-1524838017697312], p. 455). The catalyst for this view originates from [@bibr47-1524838017697312]) who initially conceptualized CSA disclosures as process based, although this notion was not fully explored until several years later. Examinations of [@bibr47-1524838017697312]) groundbreaking proposition of the CSA accommodation (CSAA) model produced varying results as to whether his five stages of secrecy, helplessness, entrapment and accommodation, delayed, conflicted, and unconvincing disclosures, and retraction or recantation, hold validity (for a review, see [@bibr30-1524838017697312]). However, the idea of disclosure as a process has been carried over into contemporary thinking. Recently, [@bibr33-1524838017697312]) detailed a process model of disclosure wherein they describe an interaction of internal factors with external motivators which they liken to a "pressure cooker" effect, preceded by a period of containment of the secret. Moreover, this and other studies strongly suggest disclosures are more likely to occur within a dialogical context---activated by discussions of abuse or prevention forums providing information about sexual abuse (Hershkowitz et al., [@bibr19-1524838017697312]; [@bibr22-1524838017697312]; [@bibr51-1524838017697312]). The term dialogical simply means to participate in dialogue. Key dialogical vehicles identified in these studies were providing sexual abuse information through prevention programs, being asked about sexual abuse, and being prompted to tell ([@bibr33-1524838017697312]; [@bibr51-1524838017697312]). ### Contemporary models of CSA disclosure reflect a social-ecological perspective {#section5-1524838017697312} Knowledge on CSA disclosure has been steadily advancing toward a holistic understanding of the complex interplay of individual, familial, contextual, and cultural factors ([@bibr4-1524838017697312]; [@bibr6-1524838017697312]; [@bibr15-1524838017697312]). Where at one time factors examined and identified were predominantly of intrapersonal factors of child victims, knowledge construction has shifted to fuller social--ecological, person-in-environment explanations ([@bibr3-1524838017697312]; [@bibr8-1524838017697312]; [@bibr12-1524838017697312]; [@bibr21-1524838017697312]; [@bibr52-1524838017697312]). Social--ecological explanations open up more opportunities to intervene in facilitating earlier disclosures. [@bibr3-1524838017697312]) proposes an ecological mapping of what individual, interpersonal, environmental, and contextual influences impede or promote CSA disclosures based on analysis of in-depth interview data of 40 adult survivors. Subsequently, based on a sample of 67 adult survivors, [@bibr8-1524838017697312]) identified three broad categories, closely aligned with an ecological framework that impede CSA disclosure: (1) barriers from within, (2) barriers in relation to others, and (3) barriers in relation to the social world which can be aligned to intrapersonal, interpersonal, and contextual factors. A summary of knowledge building using a social--ecological framework follows. Knowledge gained in the intrapersonal domain includes expanded conceptualization of disclosure by building on previous categories of accidental, purposeful, and prompted disclosure to also include behavioral and indirect attempts to tell, intentionally withheld disclosure, and triggered and recovered memories ([@bibr1-1524838017697312]). Categories of indirect behavioral disclosure patterns have been further verified in follow-up research by [@bibr21-1524838017697312]), and through an extensive file review that used [@bibr1-1524838017697312]) disclosure framework to analyze their data ([@bibr7-1524838017697312]) for verification. Interpersonal factors have also emerged in regard to certain family characteristics as disclosure barriers. Families with rigidly fixed gender roles, patriarchal attitudes, power imbalances, other forms of child abuse and domestic violence, chaotic family structure, dysfunctional communication, and social isolation have been found to suppress disclosure ([@bibr4-1524838017697312]; [@bibr8-1524838017697312]; [@bibr15-1524838017697312]). In addition, relationship with perpetrator is a factor whereby research indicates that disclosure is made more difficult when the perpetrator is a family member or close to the family ([@bibr10-1524838017697312];[@bibr11-1524838017697312]; [@bibr17-1524838017697312]; [@bibr19-1524838017697312]; [@bibr38-1524838017697312]; [@bibr41-1524838017697312]). This is especially a barrier when the perpetrator lives with the victim ([@bibr29-1524838017697312]). In terms of environmental factors, one study revealed that neighborhood/community conditions can hinder disclosure when there is lack of school involvement in providing a supportive environment, such as in following up on troubling student behavior ([@bibr3-1524838017697312]). Additionally, a child victim's anticipation of a negative response to disclosure, especially that they may not be believed by others outside their family such as neighbors or other community members, has shown to deter disclosure ([@bibr8-1524838017697312]). Cultural factors influencing CSA disclosure have been studied to a much lesser degree. Despite this, a few important studies examining critical sociocultural factors now exist for better understanding CSA disclosure within a cultural context ([@bibr6-1524838017697312]; [@bibr15-1524838017697312]). Among these important contributions, [@bibr6-1524838017697312] research has delineated CSA disclosure processes as "shaped by relational, racial, socio-cultural, historical, and developmental factors" (p. 182). In a unique study using culturally focused research literature as data triangulated with clinical case material, culturally based belief systems in many cultures have been found to foster family climates that can silence children from disclosing abuse ([@bibr15-1524838017697312]). Taboos about sexuality, patriarchal attitudes, and devaluation of women are among some of the cultural barriers that inhibit disclosure ([@bibr15-1524838017697312]). Clearly, disclosure conceptualizations are being integrated into a social--ecological model of individual and developmental factors, family dynamics, neighborhood, and community context as well as cultural and societal attitudes toward better understanding disclosure barriers and facilitators ([@bibr3-1524838017697312]), although more data are needed on cultural and contextual factors. ### Age and gender as predictors of disclosure {#section6-1524838017697312} #### Age {#section7-1524838017697312} Age is consistently found to be an influential factor in CSA disclosure, making the life stage of the victim/survivor a critical consideration. Studies draw distinctions in age-groups falling into either under or over 18 years of age. Eighteen years of age was the common age cutoff point that investigators chose in order to distinguish child/youth populations from adult samples. Sixteen of the studies drew on samples of children and youth, while the other 15 studies sampled adults over the age of 18, and a further two studies used mixed age-groups (refer to [Table 1](#table1-1524838017697312){ref-type="table"}). Among the child and youth samples, the age ranges spanned from preschool to late adolescence (3--17 years of age), with varying methodological approaches implemented across age cohorts. For younger cohorts, file reviews and secondary data analyses of CSA reports were typically undertaken. Adolescents were most often given surveys. Sometimes children and youth were interviewed as part of administering a survey or as a follow-up ([@bibr9-1524838017697312]; [@bibr19-1524838017697312]; [@bibr52-1524838017697312]). In the majority of child and adolescent samples, sexual abuse concerns were already flagged to investigative authorities. However, the work of [@bibr51-1524838017697312], [@bibr52-1524838017697312]) is one exception, whereby their survey elicited new disclosures. Adult studies typically had a mean age between 40 and 50 years. Interviews were the main data collection method with a few exceptions using survey designs (i.e., [@bibr11-1524838017697312]; [@bibr28-1524838017697312]; [@bibr42-1524838017697312]) and case file reviews (i.e., [@bibr7-1524838017697312]; [@bibr17-1524838017697312]). Results show a clear trend toward increased likelihood of disclosure in older youth, and findings from adult samples showing a preponderance of disclosures in adulthood, with the large majority of participants of adults reporting never having had a sexual abuse complaint filed with investigative authorities as a child or an adolescent (i.e., [@bibr21-1524838017697312]; [@bibr16-1524838017697312]; [@bibr43-1524838017697312]; [@bibr52-1524838017697312]). With children and youth under the ages of 18 distinct patterns emerged. First, accidental detection, rather than purposeful disclosure, is more likely to occur with younger children. For example, in one large-scale study of over 1,737 file reviews, over half of the CSA-related cases were identified through accidental and eyewitness detection (61%), while less than one third were purposeful disclosures initiated by the child victim ([@bibr7-1524838017697312]). A second pattern which emerged is that rates of disclosure increase with age, especially into adulthood, which is supported by persistent findings of high rates of delayed disclosure reported later in the life course by adult survivors ([@bibr7-1524838017697312]; [@bibr8-1524838017697312] ; [@bibr11-1524838017697312]; [@bibr25-1524838017697312]; [@bibr28-1524838017697312]; [@bibr29-1524838017697312]; [@bibr43-1524838017697312]). While gender and relationship with the perpetrator are considerable factors in CSA disclosure, age is consistently a stronger predictor of disclosure (or nondisclosure) ([@bibr19-1524838017697312]; [@bibr29-1524838017697312]). Third, younger children who disclose are more likely to do so in an interview situation or other environment that provides prompts or questions about sexual abuse ([@bibr19-1524838017697312]; [@bibr34-1524838017697312]; [@bibr40-1524838017697312]), but this trend can also be seen in older youth ([@bibr51-1524838017697312], [@bibr52-1524838017697312]). #### Gender {#section8-1524838017697312} A number of studies have recently focused on CSA disclosures with male victims, since males have been an understudied population ([@bibr2-1524838017697312]; [@bibr11-1524838017697312]; [@bibr12-1524838017697312]; [@bibr16-1524838017697312]). Most investigations that sampled both sexes show females outweighing male participants. Although women are at double the risk of being subjected to CSA, the ratio of women to men in most disclosure studies has not been representative. This finding may be indicative of male victims more likely delaying disclosing their CSA experiences, leaving male disclosure in child and youth samples underrepresented ([@bibr18-1524838017697312]; [@bibr52-1524838017697312]). [@bibr12-1524838017697312]) have been developing gender-specific modeling of disclosure examining male disclosures. Their proposed model groups male disclosures into barrier categories as determined by individual factors, interpersonal issues, and factors that are sociopolitical in nature. These authors suggest that predominant gender norms around masculinity reinforce the tendency for male victims of CSA to blame themselves for the abuse, resulting in no disclosure. Male participants in a subsequent study also relayed that gender norms and stereotypes contributed to them concealing the abuse because they were abused by a woman ([@bibr16-1524838017697312]). In the one study that compared male and female disclosures, investigator found that men's fears of being viewed as homosexual; profound feelings of stigmatization or isolation because of the belief that boys are rarely victimized; and fear of becoming an abuser acted as disclosure barriers. Whereas females felt more conflicted about who was responsible for the abuse and more strongly anticipated being blamed and not believed ([@bibr2-1524838017697312]). ### Lack of a life-course perspective {#section9-1524838017697312} Given that the study of CSA disclosure draws on age-groups ranging from samples of very young children to retrospective studies of adult survivors, with significant developmental considerations, this area of study lacks an intentional cohesive life-course perspective. Most data are derived from either cross-sectional or retrospective designs, with few longitudinal studies. There are a series of sound, yet disconnected, studies focusing on specific age-groups of children and adolescents, along with adult retrospective studies. Thus, the available knowledge base does not allow for a cohesive picture of CSA disclosure processes and pathways over the life course to emerge. The life-course perspective has long been recommended as a critical lens for the study of child abuse ([@bibr54-1524838017697312]; [@bibr53-1524838017697312]). For example, a life-course perspective has been utilized to understand the immediate- and long-term effects of CSA on the developing child victim ([@bibr54-1524838017697312]). Further, a life-course perspective is important in terms of examining age of onset of CSA to explain the differential effects of sexual victimization and developmental impacts in terms of understanding their ability to disclose---effects that need to be understood within a developmental context, especially for designing appropriate interventions for disclosure at critical transitions from early childhood through to adolescence and into adulthood. In addition, important "turning points" in life may facilitate disclosures. For example, entry into adulthood given that delayed disclosure occurs more often in adulthood. [@bibr1-1524838017697312], [@bibr2-1524838017697312]) found that being in a committed relationship or the birth of children acted as facilitators for some survivors to disclose, especially to their spouses. These significant life events, as contributing to disclosures, bear further examination. ### Summary of barriers and facilitators {#section10-1524838017697312} Research over the past 15 years continues to uncover barriers to CSA disclosure at a higher frequency than that of facilitators. As stated previously, this might be the result of sampling methods whereby participants who volunteer for disclosure research may have had more negative disclosure experiences, especially since many report delays in disclosure. The following section outlines the major trends in both barriers and facilitators (see [Table 2](#table2-1524838017697312){ref-type="table"}). ###### Factors Influencing Child Sexual Abuse Disclosures. ![](10.1177_1524838017697312-table2) Barriers Facilitators ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Age: The younger the child victim, the less likely they will purposefully disclose. Age: Disclosures increase with age, especially in adulthood. Gender: Males may be less likely to disclose in childhood/adolescence, fear of being seen as homosexual and as a victim, females experience more self-blame and anticipation of being blamed and/or not believed Gender: Slight trend toward females who are older (adolescent) to disclose before adulthood Relationship to perpetrator: If the perpetrator is a family member or in a family-like role, disclosure is less likely to happen Relationship to perpetrator: If the perpetrator is not living with the victim, disclosure rates increase Internal: Shame, self-blame, and fear are psychological barriers. In addition, fear of negative consequences on the family and for self-safety inhibits disclosure Dialogical context: Opportunities to disclose through discussion, therapeutic relationship, information sessions on sexuality, and sexual abuse prevention programs Family relations: Families with a patriarchal structure, rigidly fixed gender roles, dysfunctional communication, other forms of abuse (i.e., domestic violence), and isolation inhibit disclosure Family relations: Supportive parent--child relationship. Involvement of others: Eyewitnesses coming forward and reporting; detection through community members, professionals Environmental and cultural context: Lack of discussion about sexuality; passive acceptance that unwanted sexual experiences are inevitable; not wanting to bring shame to the family by admitting sexual abuse; lack of involvement from neighbors, school personnel; and stigma perpetuated by societal perceptions Environmental and cultural context: Promotion of open discussion of sexuality; community member involvement #### Barriers {#section11-1524838017697312} Age and gender were found to contribute to barriers as covered in Theme 3. Disclosures generally increase with age as children gain more developmental capacity, understanding of sexual abuse as victimization, and increased independence. Males are somewhat less likely to disclose, but this is often in interaction with other factors in the environment such as societal attitudes that promote hypermasculinity as desirable, attitudes that perpetuate negative views of boys and men who are victims, and homophobic attitudes ([@bibr3-1524838017697312]; [@bibr12-1524838017697312]; [@bibr16-1524838017697312]). Victims of intrafamilial abuse when the offender is a parent, caregiver, significant family member, or someone in a family-like role are less likely to disclose immediately or at all in childhood/adolescence because of obvious power differentials and dependency needs ([@bibr7-1524838017697312]; [@bibr10-1524838017697312]; [@bibr19-1524838017697312]; [@bibr28-1524838017697312]; [@bibr29-1524838017697312]; [@bibr36-1524838017697312]; [@bibr40-1524838017697312]). Further, the perpetrator residing with their victim(s) increases the likelihood of no disclosure ([@bibr29-1524838017697312]). Internalized victim-blaming, mechanisms to protect oneself (such as minimizing the impact of the abuse), and developmental immaturity at the onset of abuse constituted internal barriers. Further, shame, self-blame, and fear have been identified as significant factors deterring disclosure ([@bibr8-1524838017697312]; [@bibr9-1524838017697312]; [@bibr17-1524838017697312]; [@bibr21-1524838017697312]; [@bibr28-1524838017697312]; [@bibr32-1524838017697312]; [@bibr34-1524838017697312]). However, aspects of shame, self-blame and fear, and have not been fully explored in research. Since these are strong predictors of disclosure suppression, they bear further examination in future research to understand more fully how they operate in disclosure processes. In terms of interpersonal and environmental factors, family dynamics can play a part in deterring disclosure. As previously mentioned, families characterized by rigidly defined gender roles, patriarchal attitudes that perpetuate power imbalances between men and women, parents and children, presence of other forms of child abuse and/or domestic violence, chaotic family structure, dysfunctional communication, and social isolation have been found to suppress disclosure ([@bibr4-1524838017697312]; [@bibr8-1524838017697312]; [@bibr15-1524838017697312]). In regard to broader environmental factors, disclosure can be hindered when involved and supportive community members are not available, or not trained in sensitive responses, or when child victims anticipate not being believed by neighbors and other people outside the family ([@bibr3-1524838017697312]; [@bibr8-1524838017697312]). Further, barriers in relation to the social world were identified as stigmatization, the negative labeling of sexual abuse victims, and taboos surrounding sexuality and talking about sex as driven by cultural norms ([@bibr8-1524838017697312]; [@bibr15-1524838017697312]). Identification of cultural barriers is important recent contribution to understanding disclosure processes---and in particular to the obstacles. Findings related to cultural barriers included themes of children's voices not being heard leading to silencing, the normalization of the sexualization and objectification of girls and women, and the perpetuation of hypermasculinity in men---all acting as barriers in terms of stigma to disclosure ([@bibr2-1524838017697312], [@bibr3-1524838017697312]; [@bibr12-1524838017697312]). [@bibr6-1524838017697312] similarly found that lack of discussions about sex, young age at the onset of sexual abuse, therefore not having the language to express what was happening to them, and preserving the family good name by not talking about abuse in the family were also barriers to disclosure. Finally, it may be the case that more barriers continue to be identified over facilitators of CSA disclosure perhaps because of the methods employed in studies---particularly those drawing on adult populations who delayed disclosure. These samples may not be representative of the overall population of CSA victims, since they may have had more negative disclosure experiences, consequently more readily identifying barriers. On the other hand, these findings may speak to the actual imbalance between facilitating factors and barriers for disclosure, the latter carrying more weight in the victims/survivors' experiences, thus, explaining the high rates of disclosures delayed until adulthood. #### Facilitators {#section12-1524838017697312} Although fewer disclosure facilitators are identified in this review, very important facilitators were nonetheless uncovered---ones that should be noted for professionals in this field of practice. Internal factors that facilitate disclosures include symptoms that become unbearable, getting older with increased developmental efficacy, and realizing that an offence was committed ([@bibr8-1524838017697312]; [@bibr9-1524838017697312]; [@bibr11-1524838017697312]; [@bibr20-1524838017697312]; [@bibr34-1524838017697312]; [@bibr40-1524838017697312]). Circumstantial facilitators are those where the child discloses because there has been evidence provided, eye-witnessing has occurred, and a report has been made. Environmental factors include settings that provide opportunities such as counseling, interviews, information sessions and educational forums/workshops, and prevention programs for children and youth to disclose. To elaborate, dialogical contexts about CSA for children and youth can provide opportunities for discussion that may facilitate disclosures ([@bibr22-1524838017697312]). The research shows creating open dialogue in relationship contexts, to offset the power and influence of the perpetrator, can facilitate earlier disclosure. Among disclosure facilitators is being asked about abuse and given the opportunity to "tell" ([@bibr34-1524838017697312]); workshops on abuse and sexual abuse, in particular, can facilitate disclosures ([@bibr52-1524838017697312]); and using culturally sensitive probes and questions ([@bibr15-1524838017697312]). In [@bibr16-1524838017697312] study, positive disclosure experiences were described by participants as those where they felt that they had been listened to, were safe, were believed, and were not judged by the person they disclosed to. Further, family members and friends (peers) of the child victim can act as key supports to creating an open relational context and fostering positive responses ([@bibr22-1524838017697312]; [@bibr38-1524838017697312]; [@bibr41-1524838017697312]; [@bibr52-1524838017697312]). In particular, as children grow older, they are more likely to disclose to a peer, as shown in a number of studies, and this is an important reality for counselors and educators to be aware of ([@bibr10-1524838017697312]; [@bibr28-1524838017697312]; [@bibr41-1524838017697312]; [@bibr52-1524838017697312]). Discussion {#section13-1524838017697312} ========== Through examination of 33 studies published since the year 2000, this review identified five distinct themes regarding CSA disclosure: (1) Disclosure is best viewed as an iterative, interactive process rather than a discrete event done within a relational context; (2) contemporary models reflect a social--ecological, person-in-environment framework for understanding the complex interplay of individual, familial, contextual, and cultural factors involved in CSA disclosure; (3) age and gender are significant disclosure factors; (4) there is a lack of a life-course perspective; and (5) barriers to disclosure continue to outweigh facilitators. Based on these themes, a number of conclusions are drawn from this review. First, disclosure as a process is emphasized throughout contemporary research. Advances have been made in understanding these complex processes. However, the disclosure process over time---for example, how the first detection of CSA or attempts to disclose in childhood impact later disclosures---are not well understood. This is the result of the absence of a cohesive life-course perspective in investigations, although age consistently surfaces as significantly influencing CSA disclosure. Using a life-course perspective through the use of longitudinal studies is recommended. The use of varied methodological designs, depending on the developmental stage of the victims/survivors, influences the data generated and subsequent findings. For example, most studies on children and youth are based on file reviews of cases that have been brought to the attention of authorities, or surveys, with only a few studies using interviewing of younger children. Therefore, there is less information available on process issues with children and youth. In contrast, research on adult populations largely favors the use of qualitative interview methods for retrospective inquiry producing important process findings. In addition, investigations have not yet captured the disclosure experiences of adults in the "emerging adult" stage given that adult studies have failed to recognized that the age range of 18--24, which is now considered a developmental phase defined by neurobiological developmental uniqueness. As well, late adulthood has not been given attention as shown by the absence of participants representing this age-group in current research (70+). With a swelling geriatric population in North America, issues of historic CSA can be expected to surface and, with that, new disclosures. This trend is also anticipated due to attitudinal shifts that have presumably occurred over the last two generations about revealing such traumas and changing views about discussing sexual victimization. Interview guides used in a number of studies intentionally probed for facilitators, producing notable findings. For example, one such finding focuses on the importance of creating a contextually supportive environment to promote disclosure across the life course. These include developing therapeutic relational contexts for disclosure by providing information about sexuality, sexual abuse, prevention programming, and by asking directly. Disclosures to professionals are positive outcomes of how therapeutic contexts work; however, for forensic purposes prompting such disclosures would be viewed as problematic in legal settings, seriously compromising testimonies for trial proceedings. This is one example that speaks to the structural barriers victims and survivors run up against time and time again. Facilitators that show evidence to promote disclosure in one domain (therapeutic) are seen to work against CSA survivors in another domain---such as legal settings when perpetrators face prosecution. Defense attorneys will use this as evidence that the disclosure was prompted, and therefore the disclosure is potentially seen as not credible. Broadcasting of the frequency of acquitted cases or rulings in favor of the perpetrator through media outlets, often sensationalized, become a further compounding barrier. Given the review findings, we conclude that barriers and facilitators to CSA disclosures are nuanced and clearly embedded within intrapersonal, interpersonal, environmental, contextual, and cultural domains---often interlocked in complex ways. Limitations {#section14-1524838017697312} ----------- Although comprehensive in nature with its life-course coverage, this review may be limited by its qualitative, thematic focus rather than providing an evaluative, quantitative accounting of CSA disclosures. However, because of the recent focus on disclosure processes, the authors chose a suitably compatible approach---qualitative in nature. As well, a traditional checklist approach in rating the studies was not employed for interrater reliability, since two of the authors hold expertise in CSA disclosure research and are well versed with the literature. This expertise, and through closely following a systematic review framework ([@bibr27-1524838017697312]), assures that a thorough adjudication of the research literature was completed. Implications for Research and Practice {#section15-1524838017697312} -------------------------------------- These review findings have implications that can be useful in guiding future research and practice:Solid strides are being made in the use of a social--ecological framework to underpin investigations in the CSA disclosure investigations. Research efforts and practice considerations should continue in this vein. Investigating environmental factors and contextual and cultural forces is understudied, necessitating more research in these areas to more fully fill out understanding of CSA disclosure from a social--ecological perspective.There is good evidence that CSA disclosures are more likely to occur in a dialogical context---formal helping relationships but as well as other relationships such as peers and trusted adults. Providing information and education on topics of sexuality in general, and sexual abuse specifically, can help children and youth to disclose. Raising awareness and prevention programs can promote disclosures of sexual violence committed against children and youth.Goals of therapeutically supported disclosures (i.e., through therapy) may need to take precedence over forensic approaches, if well-being of child victims and adult survivors is to be made paramount. Legal processes may act to facilitate disclosures but can also act as barriers because of the negative outcomes experienced in the court process.Practitioners need to keep in mind that the legal system is lagging far behind in knowledge uptake of recent evidence on CSA disclosures so that victims and survivors continue to be systemically and structurally disadvantaged in legal proceedings.Health-care practitioners (i.e., child abuse pediatricians, family practice doctors, clinical nurse specialists, and public health nurses) should be made aware of the evidence in the CSA disclosure literature to create environments for facilitating therapeutic disclosures.Given that age is a stable predictor of disclosure of CSA, more studies are needed that make use of a life-course perspective. More longitudinal studies are needed to better identify trends over different life stages.The emerging young adult as a developmental age group needs specific investigation. Neuroscience research has established that ages 18--24 is a distinct developmental phase. Late adulthood is another life stage that deserves to be researched.Gender needs to be more fully investigated in relation to impact on disclosure. Awareness that boys and girls have unique challenges and barriers in disclosing CSA should be paramount for practitioners.Intervention planning should take note that disclosures increase when perpetrators no longer reside with victims, and this finding should be heeded by policy and law makers.Shame, self-blame, and fear are intrapersonal factors that persistently emerge as barriers to CSA disclosures and warrant more research to understand how to redress these barriers for earlier disclosures. Conclusion {#section16-1524838017697312} ========== There are still a substantial number of children and youth who are subjected to sexual abuse, despite preventative efforts. Just as concerning is the fact that many victims continue to suffer in silence as evidenced by the high numbers of delayed disclosure. These hidden cases should not be overlooked, and these victims should not be forgotten. Despite significant progress in bringing the issue of CSA to the forefront, improving facilitation of disclosure and increasing positive influences on disclosure processes are still critical in order to protect current and future generations of children and youth from the grave effects of sexual violence. Further, the focus should not be simply on strengthening and shoring up intrapersonal resources of victims to disclose but rather to change environmental conditions to create a more supportive and safer context for CSA victims and survivors to disclose. **Declaration of Conflicting Interests:** The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. **Funding:** The author(s) received no financial support for the research, authorship, and/or publication of this article. **Ramona Alaggia**, MSW, PhD, is an associate professor in social work and the Factor-Inwentash Chair in Children's Mental Health at the University of Toronto. Her teaching and research focuses on gender and violence, sexual abuse disclosures, domestic violence exposure, and resilience processes. **Delphine Collin-Vézina**, PhD, is an associate professor for School of Social Work, McGill University and director for Centre for Research on Children and Families. She holds the Nicolas Steinmetz and Gilles Julien Chair in Social Pediatrics in Community and the Canada Research Chair (Tier II) in Child Welfare. Her work focuses on research and clinical topics related to child maltreatment, child sexual abuse, and trauma. **Rusan Lateef**, MSW, is a social worker employed in the criminal justice system with adult male offenders in Ontario, Canada. She specializes in the intersection of health and mental health, child sexual abuse disclosures, and she is a researcher on the "Make Resilience Matter" project examining childhood exposure to domestic violence with Dr. Alaggia at the Factor-Inwentash Faculty of Social Work, University of Toronto.

{ "pile_set_name": "PubMed Central" }

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"bias", "throughout", "evalu", "larg", "volunt", "toronto", "brought", "overlook", "one", "vers", "seen", "understand", "prosecut", "victim", "data", "transcript", "assault", "plethora", "favor", "last", "offend", "episod", "discret", "sensit", "conclud", "rare", "parent", "preserv", "stress", "comparison", "thorough", "reason", "month", "caucasian", "south", "fear", "rational", "circumst", "le", "mind", "teen", "latenc", "obviou", "earlier", "compet", "exagger", "rise", "unconvinc", "bear", "tri", "qualit", "sometim", "chang", "coverag", "selfsafeti", "prompt", "unawar", "cite", "size", "practic", "special", "immatur", "profici", "therapi", "proposit", "rang", "older", "quantit", "say", "cue", "sign", "close", "perspectivether", "asia", "laps", "graduat", "thought", "typic", "stand", "subthem", "lend", "femal", "identif", "second", "belief", "referr", "forget", "expand", "ident", "swedish", "délinquant", "integr", "feminist", "thirti", "vast", "accommod", "percent", "research", "explicit", "fell", "content", "portugues", "extent", "clinic", "neglect", "neurosci", "nuanc", "stifl", "conduct", "causal", "affect", "educ", "random", "threat", "recogn", "verbatim", "often", "match", "distinct", "never", "predictor", "contrast", "promot", "lifecours", "entail", "childhood", "effect", "proceedingshealthcar", "mcgill", "pictur", "neighborhood", "contain", "underreport", "counselor", "csaa", "none", "matric", "administ", "hospit", "garner", "notion", "interconnected", "sourc", "wait", "deter", "beyond", "doubl", "polici", "mandat", "centr", "experienc", "suffici", "noncontact", "et", "len", "captur", "twentyseven", "subdivid", "expect", "news", "bother", "third", "shame", "matter", "elig", "challeng", "actual", "design", "plan", "suscept", "indic", "ethnic", "field", "offens", "possibl", "consent", "might", "remark", "csarel", "analysi", "attract", "could", "adolesc", "made", "yield", "oper", "factorsthem", "indepth", "abstract", "theoret", "contextform", "break", "access", "shameselfblam", "revis", "predispos", "therapist", "america", "issu", "isol", "previou", "therefor", "suppress", "creativ" ]

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"point", "incongruence", "low", "number", "official", "reports", "CSA", "authorities", "high", "rates", "reported", "prevalence", "studies", "example", "conducted", "combining", "estimations", "CSA", "studies", "published", "revealed", "rates", "CSA", "times", "greater", "studies", "relying", "official", "report", "inquiries", "based", "data", "child", "protection", "services", "police", "words", "people", "retrospectively", "report", "experienced", "CSA", "official", "incidence", "estimates", "indicate", "per", "children", "survey", "Swiss", "child", "services", "found", "cases", "per", "CSA", "disclosures", "recent", "comprehensive", "review", "details", "high", "prevalence", "delayed", "partial", "nondisclosures", "childhood", "indicating", "persistent", "trend", "toward", "withholding", "CSA", "disclosure", "view", "incidence", "statistics", "likely", "underestimation", "CSA", "disclosures", "drives", "rationale", "current", "review", "Given", "persistence", "delayed", "disclosures", "research", "showing", "large", "number", "survivors", "disclosing", "adulthood", "issues", "concern", "practitioners", "policy", "makers", "general", "public", "longer", "disclosures", "delayed", "longer", "individuals", "potentially", "live", "serious", "negative", "effects", "mental", "health", "problems", "depression", "anxiety", "trauma", "disorders", "addictions", "without", "receiving", "necessary", "treatment", "also", "increases", "likelihood", "victims", "falling", "prey", "undetected", "offenders", "Learning", "CSA", "disclosure", "factors", "processes", "help", "advance", "knowledge", "base", "may", "help", "professionals", "facilitate", "earlier", "disclosures", "Previous", "literature", "reviews", "examining", "factors", "influencing", "CSA", "disclosure", "served", "field", "well", "longer", "current", "Important", "contributions", "CSA", "disclosures", "include", "original", "review", "covering", "literature", "largely", "premillennium", "era", "followed", "subsequent", "review", "may", "captured", "publications", "affected", "lag", "print", "delays", "common", "journals", "reviews", "dated", "therefore", "take", "account", "plethora", "research", "accumulated", "past", "years", "recent", "reviews", "exist", "distinct", "contributions", "dialogical", "relational", "processes", "disclosure", "CSA", "disclosures", "adulthood", "delayed", "disclosures", "childhood", "literature", "review", "differs", "focusing", "CSA", "disclosures", "children", "youth", "adults", "childhood", "adulthood", "life", "course", "Method", "systematic", "review", "framework", "utilized", "establish", "investigated", "CSA", "disclosure", "research", "various", "mixed", "methods", "highlight", "convincing", "findings", "considered", "future", "research", "practice", "program", "planning", "review", "centered", "question", "state", "CSA", "disclosure", "research", "learned", "apply", "future", "research", "practice", "way", "clarification", "term", "systematic", "refers", "methodologically", "sound", "strategy", "searching", "literature", "studies", "knowledge", "construction", "case", "CSA", "disclosure", "literature", "rather", "intervention", "studies", "years", "spanned", "searching", "literature", "building", "previous", "reviews", "without", "great", "deal", "overlap", "Retrieval", "relevant", "research", "done", "searching", "international", "electronic", "databases", "PsycINFO", "PsycARTICLES", "Educational", "Resources", "Information", "Center", "Canadian", "Research", "Index", "International", "Bibliography", "Social", "Sciences", "Published", "International", "Literature", "Traumatic", "Stress", "Sociological", "Abstracts", "Social", "Service", "Abstracts", "Applied", "Social", "Science", "Index", "Abstracts", "review", "searched", "studies", "search", "gray", "literature", "unpublished", "literature", "internal", "agency", "documents", "government", "reports", "etc", "beyond", "scope", "review", "unpublished", "studies", "subjected", "process", "Keyword", "search", "terms", "used", "child", "sexual", "abuse", "childhood", "sexual", "abuse", "disclosure", "telling", "search", "databases", "produced", "articles", "Selected", "search", "terms", "yielded", "English", "publications", "French", "study", "Portuguese", "review", "search", "refined", "excluding", "studies", "focusing", "forensic", "investigations", "studies", "constitute", "specialized", "legal", "focus", "interview", "approaches", "techniques", "well", "papers", "focused", "exclusively", "rates", "responses", "CSA", "disclosure", "excluded", "substantial", "areas", "unto", "exceeding", "aims", "review", "question", "Review", "articles", "also", "excluded", "exclusion", "criteria", "applied", "search", "results", "yielded", "articles", "studies", "subjected", "thematic", "analysis", "described", "entailed", "multiple", "readings", "three", "authors", "identifying", "patterns", "across", "studies", "coding", "charting", "specific", "features", "examining", "disclosure", "definitions", "used", "sample", "characteristics", "measures", "utilized", "major", "findings", "extrapolated", "Reading", "articles", "initially", "conducted", "authors", "identify", "general", "trends", "first", "level", "analyses", "subsequently", "identify", "themes", "deeper", "analyses", "table", "studies", "generated", "continuously", "revised", "selection", "studies", "refined", "see", "Table", "table", "Child", "Sexual", "Abuse", "CSA", "Disclosure", "Studies", "Study", "Purpose", "Design", "Sample", "Findings", "Summary", "explore", "disclosure", "processes", "male", "victims", "CSA", "Phenomenological", "methodology", "used", "interview", "male", "CSA", "survivors", "Long", "Interview", "Method", "LIM", "guided", "data", "collection", "analyses", "men", "ranged", "age", "age", "Purposive", "sampling", "strategy", "used", "majority", "men", "study", "waited", "adulthood", "disclose", "abuse", "negative", "stereotypes", "contributing", "delayed", "disclosures", "Negative", "stereotypes", "contributed", "delayed", "disclosure", "trying", "forget", "Breaking", "isolation", "cited", "motivator", "disclosure", "along", "aid", "various", "forms", "media", "disclosure", "Important", "contextual", "issues", "negative", "stereotyping", "males", "sexuality", "victims", "noted", "Social", "media", "seen", "facilitator", "disclosures", "participants", "disclosed", "received", "services", "participating", "study", "Member", "checking", "could", "done", "participants", "check", "themes", "Small", "sufficient", "size", "qualitative", "inquiry", "Otherwise", "high", "level", "rigor", "establishing", "trustworthiness", "data", "analysis", "Retrospective", "study", "could", "imply", "recall", "issues", "explore", "meaning", "African", "American", "women", "make", "traumatic", "experiences", "CSA", "disclosed", "across", "life", "course", "Collective", "case", "study", "design", "using", "narrative", "tradition", "storyboard", "data", "collection", "analysis", "Qualitative", "interviewing", "African", "American", "women", "experienced", "intrafamilial", "CSA", "Purposive", "snowballing", "strategy", "CSA", "onset", "largely", "ages", "one", "ever", "talked", "sex", "language", "disclose", "Barriers", "fear", "family", "breakdown", "removal", "wanting", "tarnish", "family", "name", "fear", "retribution", "family", "members", "disclosed", "Pattern", "stifled", "dismissed", "disclosures", "identified", "life", "course", "participants", "identified", "spirituality", "primary", "source", "strength", "throughout", "life", "course", "One", "studies", "focus", "exclusively", "African", "American", "women", "Small", "sufficient", "size", "qualitative", "inquiry", "Important", "cultural", "contextual", "issues", "brought", "forward", "Retrospective", "study", "may", "affected", "recall", "issues", "Use", "perspective", "theoretical", "lens", "understanding", "CSA", "middle", "later", "years", "life", "considered", "investigations", "provide", "mapping", "factors", "prevent", "CSA", "disclosures", "ecological", "lens", "sample", "CSA", "adult", "survivors", "Qualitative", "design", "using", "LIM", "male", "female", "CSA", "adult", "survivors", "identified", "female", "male", "Age", "ranges", "years", "Purposive", "sampling", "strategy", "Three", "broad", "categories", "identified", "barriers", "CSA", "disclosure", "Barriers", "victim", "blaming", "mechanisms", "protect", "oneself", "immature", "development", "time", "abuse", "barriers", "relation", "others", "dysfunction", "family", "power", "dynamics", "awareness", "impact", "telling", "fragile", "social", "network", "barriers", "relation", "social", "world", "labeling", "taboo", "sexuality", "lack", "services", "available", "culture", "time", "period", "Half", "participants", "disclosed", "CSA", "experiences", "age", "Retrospective", "aspect", "study", "could", "imply", "recall", "issues", "participants", "disclosed", "received", "counseling", 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"conclusions", "drawn", "review", "First", "disclosure", "process", "emphasized", "throughout", "contemporary", "research", "Advances", "made", "understanding", "complex", "processes", "However", "disclosure", "process", "time", "example", "first", "detection", "CSA", "attempts", "disclose", "childhood", "impact", "later", "disclosures", "well", "understood", "result", "absence", "cohesive", "perspective", "investigations", "although", "age", "consistently", "surfaces", "significantly", "influencing", "CSA", "disclosure", "Using", "perspective", "use", "longitudinal", "studies", "recommended", "use", "varied", "methodological", "designs", "depending", "developmental", "stage", "influences", "data", "generated", "subsequent", "findings", "example", "studies", "children", "youth", "based", "file", "reviews", "cases", "brought", "attention", "authorities", "surveys", "studies", "using", "interviewing", "younger", "children", "Therefore", "less", "information", "available", "process", "issues", "children", "youth", "contrast", "research", "adult", "populations", "largely", "favors", "use", "qualitative", "interview", "methods", "retrospective", "inquiry", "producing", "important", "process", "findings", "addition", "investigations", "yet", "captured", "disclosure", "experiences", "adults", "emerging", "adult", "stage", "given", "adult", "studies", "failed", "recognized", "age", "range", "considered", "developmental", "phase", "defined", "neurobiological", "developmental", "uniqueness", "well", "late", "adulthood", "given", "attention", "shown", "absence", "participants", "representing", "current", "research", "swelling", "geriatric", "population", "North", "America", "issues", "historic", "CSA", "expected", "surface", "new", "disclosures", "trend", "also", "anticipated", "due", "attitudinal", "shifts", "presumably", "occurred", "last", "two", "generations", "revealing", "traumas", "changing", "views", "discussing", "sexual", "victimization", "Interview", "guides", "used", "number", "studies", "intentionally", "probed", "facilitators", "producing", "notable", "findings", "example", "one", "finding", "focuses", "importance", "creating", "contextually", "supportive", "environment", "promote", "disclosure", "across", "life", "course", "include", "developing", "therapeutic", "relational", "contexts", "disclosure", "providing", "information", "sexuality", "sexual", "abuse", "prevention", "programming", "asking", "directly", "Disclosures", "professionals", "positive", "outcomes", "therapeutic", "contexts", "work", "however", "forensic", "purposes", "prompting", "disclosures", "would", "viewed", "problematic", "legal", "settings", "seriously", "compromising", "testimonies", "trial", "proceedings", "one", "example", "speaks", "structural", "barriers", "victims", "survivors", "run", "time", "time", "Facilitators", "show", "evidence", "promote", "disclosure", "one", "domain", "therapeutic", "seen", "work", "CSA", "survivors", "another", "domain", "legal", "settings", "perpetrators", "face", "prosecution", "Defense", "attorneys", "use", "evidence", "disclosure", "prompted", "therefore", "disclosure", "potentially", "seen", "credible", "Broadcasting", "frequency", "acquitted", "cases", "rulings", "favor", "perpetrator", "media", "outlets", "often", "sensationalized", "become", "compounding", "barrier", "Given", "review", "findings", "conclude", "barriers", "facilitators", "CSA", "disclosures", "nuanced", "clearly", "embedded", "within", "intrapersonal", "interpersonal", "environmental", "contextual", "cultural", "domains", "interlocked", "complex", "ways", "Limitations", "Although", "comprehensive", "nature", "coverage", "review", "may", "limited", "qualitative", "thematic", "focus", "rather", "providing", "evaluative", "quantitative", "accounting", "CSA", "disclosures", "However", "recent", "focus", "disclosure", "processes", "authors", "chose", "suitably", "compatible", "approach", "nature", "well", "traditional", "checklist", "approach", "rating", "studies", "employed", "interrater", "reliability", "since", "two", "authors", "hold", "expertise", "CSA", "disclosure", "research", "well", "versed", "literature", "expertise", "closely", "following", "systematic", "review", "framework", "assures", "thorough", "adjudication", "research", "literature", "completed", "Implications", "Research", "Practice", "review", "findings", "implications", "useful", "guiding", "future", "research", "practice", "Solid", "strides", "made", "use", "social", "ecological", "framework", "underpin", "investigations", "CSA", "disclosure", "investigations", "Research", "efforts", "practice", "considerations", "continue", "vein", "Investigating", "environmental", "factors", "contextual", "cultural", "forces", "understudied", "necessitating", "research", "areas", "fully", "fill", "understanding", "CSA", "disclosure", "social", "ecological", "good", "evidence", "CSA", "disclosures", "likely", "occur", "dialogical", "context", "helping", "relationships", "well", "relationships", "peers", "trusted", "adults", "Providing", "information", "education", "topics", "sexuality", "general", "sexual", "abuse", "specifically", "help", "children", "youth", "disclose", "Raising", "awareness", "prevention", "programs", "promote", "disclosures", "sexual", "violence", "committed", "children", "therapeutically", "supported", "disclosures", "therapy", "may", "need", "take", "precedence", "forensic", "approaches", "child", "victims", "adult", "survivors", "made", "paramount", "Legal", "processes", "may", "act", "facilitate", "disclosures", "also", "act", "barriers", "negative", "outcomes", "experienced", "court", "need", "keep", "mind", "legal", "system", "lagging", "far", "behind", "knowledge", "uptake", "recent", "evidence", "CSA", "disclosures", "victims", "survivors", "continue", "systemically", "structurally", "disadvantaged", "legal", "practitioners", "child", "abuse", "pediatricians", "family", "practice", "doctors", "clinical", "nurse", "specialists", "public", "health", "nurses", "made", "aware", "evidence", "CSA", "disclosure", "literature", "create", "environments", "facilitating", "therapeutic", "age", "stable", "predictor", "disclosure", "CSA", "studies", "needed", "make", "use", "perspective", "longitudinal", "studies", "needed", "better", "identify", "trends", "different", "life", "emerging", "young", "adult", "developmental", "age", "group", "needs", "specific", "investigation", "Neuroscience", "research", "established", "ages", "distinct", "developmental", "phase", "Late", "adulthood", "another", "life", "stage", "deserves", "needs", "fully", "investigated", "relation", "impact", "disclosure", "Awareness", "boys", "girls", "unique", "challenges", "barriers", "disclosing", "CSA", "paramount", "planning", "take", "note", "disclosures", "increase", "perpetrators", "longer", "reside", "victims", "finding", "heeded", "policy", "law", "fear", "intrapersonal", "factors", "persistently", "emerge", "barriers", "CSA", "disclosures", "warrant", "research", "understand", "redress", "barriers", "earlier", "disclosures", "Conclusion", "still", "substantial", "number", "children", "youth", "subjected", "sexual", "abuse", "despite", "preventative", "efforts", "concerning", "fact", "many", "victims", "continue", "suffer", "silence", "evidenced", "high", "numbers", "delayed", "disclosure", "hidden", "cases", "overlooked", "victims", "forgotten", "Despite", "significant", "progress", "bringing", "issue", "CSA", "forefront", "improving", "facilitation", "disclosure", "increasing", "positive", "influences", "disclosure", "processes", "still", "critical", "order", "protect", "current", "future", "generations", "children", "youth", "grave", "effects", "sexual", "violence", "focus", "simply", "strengthening", "shoring", "intrapersonal", "resources", "victims", "disclose", "rather", "change", "environmental", "conditions", "create", "supportive", "safer", "context", "CSA", "victims", 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"maltreatment", "child", "sexual", "abuse", "trauma", "Rusan", "Lateef", "MSW", "social", "worker", "employed", "criminal", "justice", "system", "adult", "male", "offenders", "Ontario", "Canada", "specializes", "intersection", "health", "mental", "health", "child", "sexual", "abuse", "disclosures", "researcher", "Make", "Resilience", "Matter", "project", "examining", "childhood", "exposure", "domestic", "violence", "Alaggia", "Faculty", "Social", "Work", "University", "Toronto" ]

Background {#Sec1} ========== *Taenia solium* and *Taenia saginata*, also referred to as the pork and beef tapeworm, respectively, are food-borne parasites of global importance. In 2014, *T. solium*, for which humans act as final hosts after consumption of undercooked pork, was ranked by an international panel of experts as the food-borne parasite of greatest global concern, affecting millions of individuals every year and causing a substantial economic impact \[[@CR1]\]. In 2016, during a similar exercise carried out by a group of experts at the European level, *T. solium* was ranked tenth among 27 parasites as the number of human cysticercosis cases is not as high when Europe is considered as a whole. If individual regions were taken into account, the parasite was ranked higher in eastern Europe than in other parts of Europe \[[@CR2]\]. Human cysticercosis is a condition that may arise when humans ingest eggs of *T. solium*. When the parasite migrates to the central nervous system, the neurological condition is referred to as neurocysticercosis (NCC), which may lead to a number of potentially debilitating neurological manifestations including epilepsy and severe progressive headache \[[@CR3]\]. NCC is a major global public health concern. Recently, *T. solium* was identified as the number one food-borne parasite contributing to the global burden of disease leading to approximately 28,000 deaths per year and around 2.8 million disability-adjusted life years (DALYs) \[[@CR4]\]. *Taenia saginata* ranked lower in the multicriteria-based ranking, as symptoms of taeniosis are mild or even absent and generally the disease is not of substantial public health concern \[[@CR1], [@CR2]\]. Nevertheless, the parasite can pose major obstacles for trade and causes a substantial financial burden due to carcass condemnation, freezing and devaluation \[[@CR5]--[@CR8]\]. *Taenia solium* is highly endemic in pork-consuming poor communities of Asia, Africa and Latin America, while *T. saginata* is distributed worldwide. Little is known about the situation of *T. solium* in eastern Europe, and the suspicion of on-going transmission persists \[[@CR9]\]. For *T. saginata* a recent review on the epidemiology of bovine cysticercosis revealed the presence of the parasite in a large number of countries in western Europe; however, for eastern Europe data were scarce and of poor quality \[[@CR10]\]. In eastern Europe, various socioeconomic and political developments such as the collapse of the Soviet Union and the dissolution of the Socialict Federal Republic of Yugoslavia in 1991 following the wars, led to political and demographic changes. As a result a number of sectors, including the veterinary and public health sectors, were negatively affected. Large numbers of veterinary control officers were replaced by a cheaper working force leading to deteriorated veterinary control systems \[[@CR11]\]. The change to small-scale farms with reduced rearing standards and biosecurity and to small abattoirs with insufficient meat inspection and reduced government and veterinary oversight possibly contributed to an increased prevalence of zoonotic parasites such as *T. solium*, *T. saginata*, *Trichinella* spp. and *Toxoplasma gondii* \[[@CR11]\]. Backyard slaughtering without meat inspection has also become popular, possibly leading to the perpetuation of zoonotic diseases in parts of the region \[[@CR12]\]. Meat inspection is obligatory at slaughterhouses in the European Union (EU), according to European Regulation 854/2004 \[[@CR13]\]. Eastern Europe consists of "old" and "new" EU Members States as well as states which are still in the pre-accession phase as candidate EU countries. Therefore, adaptation of the national legislation and/or development of implementing regulations which integrate the main principles and features of the EU legislation and institutional structures towards more modern approaches to achieve better surveillance are ongoing \[[@CR11], [@CR14]\]. In the past decade, the number of diagnosed autochthonous NCC cases was increasing in eastern Europe \[[@CR15]--[@CR17]\], leading to the suspicion of on-going *T. solium* transmission occurring in the area; however, according to experts the numbers have gone down in the last few years. Changes in eating habits might further contribute to the current transmission patterns of the parasite, while imported *T. solium* tapeworm carriers and increased migration and travels further contribute to the risk \[[@CR18]\]. More recently, increasingly, European integration and introduction of regulatory legislation have influenced the region and, e.g. made it mandatory to have modern sanitation \[[@CR11]\]. Both *T. solium* and *T. saginata* have been considered endemic in eastern Europe. Results reported from the region in the past should however be evaluated critically for their representativeness and for the laboratory procedures applied \[[@CR14]\]. In the World Health Organization *T. solium* endemicity map, updated in 2015, uncertainty on the epidemiological situation in eastern Europe persists \[[@CR19]\]. In order to advance our knowledge on the occurrence of these zoonotic parasites and assess the potential needs for surveillance, we performed a systematic review on the epidemiology of taeniosis/cysticercosis in eastern Europe. This review is part of a series of two systematic reviews: the first one covered western Europe \[[@CR10]\] and the current one covers eastern Europe. Methods {#Sec2} ======= We conducted a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines \[[@CR20]\] (Additional file [1](#MOESM1){ref-type="media"}). To search for information on the epidemiology of *T. saginata* and *T. solium* in eastern Europe we used international databases and local, unpublished sources. Epidemiology was defined as the occurrence, prevalence, incidence and geographical distribution of human/porcine/bovine cysticercosis and taeniosis. Eastern Europe was defined based on regional proximity and on gross domestic product/gross national income and included 22 countries: Albania, Belarus, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Estonia, Former Yugoslav Republic of Macedonia, Greece, Hungary, Kosovo, Latvia, Lithuania, Malta, Moldova, Montenegro, Poland, Romania, Serbia, Slovakia and Ukraine \[[@CR10]\]. International databases {#Sec3} ----------------------- The online international bibliographical databases PubMed, ISI Web of Knowledge, CABDirect, OAIster, and OpenGrey were searched for all published data on taeniosis and (neuro)cysticercosis using the following search phrase: (cysticerc\* OR cisticerc\* OR neurocysticerc\* OR neurocisticerc\* OR \"C. bovis\" OR \"C. cellulosae\" OR taenia\* OR tenia\* OR saginata OR solium OR taeniosis OR teniosis OR ténia OR taeniid OR cysticerque) AND (Albania OR Belarus OR Bosnia OR Herzegovina OR Bulgaria OR Croatia OR Cyprus OR Czech Republic OR Estonia OR Former Yugoslav Republic of Macedonia OR Greece OR Hungary OR Kosovo OR Latvia OR Lithuania OR Malta OR Moldova OR Montenegro OR Poland OR Romania OR Serbia OR Slovakia OR Ukraine). The databases were searched for papers published from January 1st 1990 up to January 31st 2017. No language restriction was applied. Papers were excluded when fulfilling one or more of the following criteria: (i) studies concerning a different parasite than *T. saginata* and/or *T. solium*; (ii) studies reporting data from outside the specified countries; (iii) studies reporting results out of the scope of the review questions; (iv) duplicates. Papers were first screened for eligibility based upon title and abstract, and in the case of doubt, the full paper was assessed. For each eligible document, a narrative synthesis was made, which were then further digested into a qualitative review. Local sources {#Sec4} ------------- To identify additional, locally published and unpublished data sources, we distributed country sheets (Additional file [2](#MOESM2){ref-type="media"}) to members of the European Network on Taeniosis/Cysticercosis (CYSTINET) COST Action TD1302 and other non-member experts, asking to specify relevant national journals or epidemiological bulletins, MSc/PhD dissertations, national institutes, or registries, and to translate relevant search terms. Due to ethical constraints, unpublished hospital or laboratory data were only presented at an aggregated level. In addition, we searched for information in the proceedings of the CYSTINET (European Network for taeniosis/cysticercosis, COST Action TD1302) and European Network for Foodborne Parasites (COST Action FA1408, Euro-FBP) meetings. Finally, we explored the reference lists of recent topic-specific reviews \[[@CR15]--[@CR17]\] to find additional eligible papers. We applied the same inclusion and exclusion criteria to these additional sources and developed narrative syntheses for all eligible documents. Data collection and analyses {#Sec5} ---------------------------- The data collection was performed by three independent reviewers (CT, BD and SS). For data analysis, we followed the methodology described in \[[@CR10]\]. Briefly, cases reported as case reports providing information on individual characteristics of the patient were defined as individual cases and when no individual information was given, these cases were defined as aggregated. Tables summarizing individual cases included year of diagnosis, age, gender, nationality, and reported risk factors, while for aggregated cases or prevalence, the tables included country, level of data collection, timeframe, number of cases (or prevalence or incidence), *Taenia* species, risk factors (e.g. immigration/travel history) if available, and reference (i.e. authors and publication year). The following definitions were applied for the description of risk factors: endemic region (Asia, Africa and South and Central America, including Caribbean islands); immigrant from endemic region (any person born in or native from endemic region, or reported to have moved from endemic region); travelled/stayed in endemic region (having travelled, stayed, or resided in endemic region); no history of travels to endemic areas or immigration (autochthonous) (no history of travel/immigration reported). Cases in which the existence of duplicates was probable (e.g. cases included in two retrospective studies on the same area/hospital, covering overlapping time periods, cases diagnosed in the same hospital in the same time-frame but reported in different sources etc.) were included only once, by selecting the one that was published first. Descriptive analyses and maps were performed using the software environment for statistical computing R 3.5.0 and graphs were made in both R and Microsoft Excel \[[@CR21]\]. Results {#Sec6} ======= Search results {#Sec7} -------------- The initial search resulted in 1179 peer reviewed papers identified though international databases. After the screening process, 69 relevant references were identified and included in the review (Additional file [3](#MOESM3){ref-type="media"}: Table S1). In addition to this, researchers of 18 out of 22 eastern European countries were contacted from which additional data (92 relevant sources) from local and unpublished sources were obtained (Additional file [4](#MOESM4){ref-type="media"}: Table S2). Contacts of researchers working within the field from Belarus, Kosovo, Malta and Ukraine were not available; hence local, unpublished data for these countries are missing. The flow diagram of the search strategy steps is presented in Fig. [1](#Fig1){ref-type="fig"}. The countries for which data on *T. solium* or *T. saginata* in humans and/or animals were found are presented in Fig. [2](#Fig2){ref-type="fig"}.Fig. 1Flow diagram of the search strategy stepsFig. 2Summary of identified data on taeniosis and cysticercosis (in humans and animals) in eastern Europe (1990--2017): taeniosis (**a**); human cysticercosis (**b**); bovine cysticercosis (**c**); and porcine cysticercosis (**d**) Taeniosis {#Sec8} --------- In total, 58 unique sources were identified providing information on taeniosis in eastern Europe. Individual (8 records) and/or aggregated cases/prevalence (50 records) of taeniosis were reported in 14 out of 22 eastern European countries (Fig. [2](#Fig2){ref-type="fig"}). No cases of taeniosis were reported in Cyprus, and for 7 countries (Belarus, Bosnia and Herzegovina, Kosovo, Malta, Moldova, Montenegro and Ukraine), no reports on taeniosis could be retrieved for the years between 1990 and 2017. Poland was the country recording most data on taeniosis. Taeniosis case reports {#Sec9} ---------------------- In total only 17 individual cases reported in case reports from Croatia, Greece, Lithuania and Serbia, were available (Additional file [5](#MOESM5){ref-type="media"}: Table S3). Out of the 17 cases, 13 were reported as *Taenia* spp., three reported as confirmed and one suspected *T. saginata* using microscopy. No case reports describing *T. solium* taeniosis cases were identified through the search of published and other unpublished grey literature. Moreover none of all the case reports reported possible risk factors or how the parasite infection was acquired. Aggregated taeniosis cases {#Sec10} -------------------------- Data on aggregated taeniosis cases were obtained from scientific publications, authorities' reports, national registries, epidemiological bulletins, and from hospitals/laboratories (Additional file [5](#MOESM5){ref-type="media"}: Table S4). Aggregated taeniosis cases were reported in 14 eastern European countries and covered different years. Data were obtained from publications ranging from the 1990 up to 2017 (Fig. [3](#Fig3){ref-type="fig"}). The largest proportion of cases was reported as *Taenia* spp. and at species level the majority of cases were reported as *T. saginata*. *Taenia solium* taeniosis cases were a rare finding, reported only in Albania, Estonia, Latvia and Poland. No indication was given on how species identification was performed. For Latvia, the two suspected cases probably originated from abroad (probably Russia) (Deksne, personal communication, 2016). No additional information on the source of infection, potential risk factors or nationality was given for the other reported cases.Fig. 3Taeniosis cases reported in scientific publications, authority reports, epidemiologial bulletins, and laboratories in eastern Europe (published/reported between 1990--2017). Arrow pointing at the number, larger than 300 taeniosis cases. *Abbreviation*: FYROM, Former Yugoslav Republic of Macedonia The annual number of cases reported for each country varied, with Poland and Romania reporting the highest annual number of aggregated cases. In particular for Poland, a large number of data was available as epidemiological reviews published quarterly in the journal of the National Institute of Public Health - National Institute of Hygiene and the Polish Society of Epidemiology and Infectious Diseases. Taeniosis incidence data {#Sec11} ------------------------ Incidence data were available for seven counties (Bulgaria, Estonia, Lithuania, Poland, Romania, Serbia and Slovakia) and ranged from 0/100,000 people for Bulgaria in the year 2000 to 8.5/100,000 people in Poland in 1991 (Fig. [4](#Fig4){ref-type="fig"}).Fig. 4Taeniosis incidence data reported between the years 1990--2017 in authorities' reports, epidemiological bulletins and national registries in eastern Europe. *Abbreviations*: BG, Bulgaria (1998 and 2000); EE, Estonia (1990--1999); PL, Poland (1991--2009); RO, Romania (2007--2014); RS, Serbia (1997--2005); SK, Slovakia (1990--2014) Taeniosis prevalence data {#Sec12} ------------------------- In total, five epidemiological studies reporting taeniosis prevalence data published between 1990**--**2017 were identified (Additional file [5](#MOESM5){ref-type="media"}: Table S5). Within eastern Europe the prevalence of taeniosis ranged between 0--4.9%, with the highest prevalence reported in a study conducted in children of the Roma settlements of Košice and Prešov regions in Slovakia \[[@CR22]\]. A study carried out in Estonia reported a seroprevalence of *T. solium* of 0.7% (95% confidence interval, CI: 0.3--1.4%) using a commercial NovaLisa IgG enzyme immunoassay ELISA \[NovaTec Immunodiagnostica GmbH, Dietzenbach, Germany (Sp \> 95%, Se 94%)\], and 0.0% (95% CI: 0--0.3%) using the ELISA and a Western Blot IgG (LDBIO DIAGNOSTICS, Lyon, France) in series \[[@CR23]\], while the other four studies used microscopy as a diagnostic tool. Human cysticercosis {#Sec13} ------------------- Relevant information on human cysticercosis was obtained from 63 sources. In total, 40 reports reported individual, and 23 reports reported aggregated cysticercosis cases. Information on human cysticercosis cases was available for 15 out of 22 eastern European countries (Fig. [2](#Fig2){ref-type="fig"}), while no data could be retrieved from Albania, Belarus, Cyprus, Malta, Moldova, Montenegro and Ukraine in the form of either published or unpublished sources. Human cysticercosis case reports {#Sec14} -------------------------------- In total, 58 individual cases of cysticercosis were identified in authorities' reports, from hospital/laboratories or published case reports (Table [1](#Tab1){ref-type="table"}). Out of 58 cases, 45 were diagnosed in 7 eastern European countries, while 13 cases (22%) were originally from eastern Europe but were diagnosed abroad. The average age was 49 years and 49% of the cases were male, 42% female and for 9% the sex was not known (Additional file [5](#MOESM5){ref-type="media"}: Table S6). Of all the individual case reports, 17% were reportedly suspected to be autochthonous cases, in 14% the infection was considered aquired in endemic countries while travelling or living abroad, whereas for the majority of cases (69%) the place of infection was unknown or not recorded. For Lithuania, the cases were possibly connected with travels to Argentina and South Africa (S. Petkevičius, personal communication, 2015).Table 1Number of human cysticercosis cases per country and most likely place of infection (Data from published and unpublished sources between 1990 and 2017)CountryMost likely place of infectionTotal no. of casesEastern EuropeEndemic countryUnknownCroatia1010Czech Republic6410Greece33612Hungary22Latvia112Lithuania55Serbia156Cyprus11Czechoslovakia22Former Yugoslav Republic of Macedonia33Bosnia and Herzegovina55 Aggregated human cysticercosis cases {#Sec15} ------------------------------------ The number of aggregated human cysticercosis cases varied largely across countries, with over 300 cases (most of them reported as autochthonous) reported from Romania. Figure [5](#Fig5){ref-type="fig"} shows the number of human cysticercosis cases identified in eastern Europe in documents published from 1990 to 2017. Information on risk factors and place where the disease might have been acquired were absent for most cases (Additional file [5](#MOESM5){ref-type="media"}: Table S7).Fig. 5Number of identified human cysticercosis cases in eastern Europe - data sources published and unpublished between 1990--2017. Arrow pointing at the number, larger than 300 human cycsticercosis cases. *Abbreviation*: FYROM, Former Yugoslav Republic of Macedonia Only two serological studies using ELISA and/or western blot techniques were identified. One of the studies was in Croatia, where a prevalence of 1.5% was recorded among people with epilepsy \[[@CR24]\]. The other study was in Estonia, where a western-blot-confirmed *T. solium* cysticercosis prevalence (commercial ELISA and commercial western blot in series) was of 0.0% in samples from the general Estonian population, children, veterinarians and hunters \[[@CR23]\]. Porcine cysticercosis {#Sec16} --------------------- Relevant information on porcine cysticercosis was obtained from 53 sources, which reported the occurrence of the infection in 8 countries. In Poland the causative agent was specified as being "*cysticercus cellulosae*" (name used for *T. solium* cysticercus) in two publications (Additional file [5](#MOESM5){ref-type="media"}: Table S8)*.* The highest number of porcine cysticercosis cases (4487 cases) was recorded in Poland in 2003; however, in the report species were not specified and porcine cysticercosis could refer to "*cysticercus cellulosae*" or cysticerci of other species like "*cysticercus tenuicollis*" (name used for *Taenia hydatigena* cysticercus). Of the porcine cysticercosis cases reported, a molecular method was reportedly used only in Estonia, and *T. solium* could not be confirmed \[[@CR25]\]. No cases of porcine cysticercosis in pigs were reported from Bosnia and Herzegovina, Croatia, Cyprus, Czech Republic, Former Yugoslav Republic of Macedonia and Hungary, and no information was found for Albania, Belarus, Greece, Kosovo, Malta and Ukraine. Based on routine meat inspection, reported prevalence ranged from 0% to 0.18% in Bulgaria \[[@CR9]\] and varied within and between countries. A single serological study was identified: in Romania, an ELISA-based prevalence of active porcine cysticercosis was reported to be 6.4% \[[@CR26]\]. In Latvia, according to the Food and Veterinary Service, the main slaughtering monitoring authority, in the last 10 years no suspicious cysts have been found in pigs (Food and Veterinary Service Latvia, personal communication, 2015). Bovine cysticercosis {#Sec17} -------------------- Relevant information on bovine cysticercosis was obtained from 41 sources reporting data for 15 out of 22 countries (Additional file [5](#MOESM5){ref-type="media"}: Table S9). Only for Cyprus, no cases were recorded since 1990. For Albania, Hungary, Kosovo, Latvia, Malta and Montenegro, no data were retrieved. Based on routine meat inspection, reported prevalence ranged from 0.0% up to 1.7% and varied within and between countries (Fig. [6](#Fig6){ref-type="fig"}). No studies were found applying more sensitive methods to detect bovine cysticercosis. For Poland in particular, a large amount of data were available, in which the highest annual number of bovine cysticercosis cases (4718 cases) in the country was reported in 1994. The number of reported cases has decreased consistently over the years.Fig. 6Prevalence of bovine cysticercosis based on routine meat inspection detected in eastern Europe (1990--2017). *Abbreviations*: CZ, Czech Republic; EE, Estonia; HR, Croatia; MD, Moldova; MK, Former Yugoslav Republic of Macedonia; PL, Poland; RO, Romania; RS, Serbia; SK, Slovakia; UA, Ukraine Discussion {#Sec18} ========== Published and unpublished data sources were searched, and contacts with researchers of eastern European countries were established to obtain all possible information on the epidemiology of *T. solium* and *T. saginata* in eastern Europe. Individual case reports for taeniosis were only retrieved from four countries, while reports on aggregated cases were available for nearly all the eastern European countries. At a species level, most data were reported as *Taenia* spp. or *T. saginata*, leading to uncertainty regarding the true disease epidemiology. In a few countries, *T. solium* was reported as aggregated taeniosis cases. Given the risk of having *T. solium* carriers it is essential to improve on differential diagnosis and reporting. A similar scenario was also observed in western Europe, suggesting that diagnosis and case management is not performed adequately \[[@CR10]\]. Moreover, the laboratory procedures and the reporting seem unstandardized and often only a small part of the population is examined; therefore, the data from most countries are incomplete \[[@CR14]\]. Hence, the reported prevalence of human taeniosis from *T. saginata* and *T. solium* should be evaluated critically. Also, the origin of infection and risk factors were not reported, leading to continued uncertainty on the real endemic status of eastern Europe. Human cysticercosis reports of individual and aggregated cases were available from nearly one third of the 22 eastern European countries, while for western Europe, information was available from all 18 countries \[[@CR10]\]. Romania and Serbia reported the largest number of diagnosed human cysticercosis cases, highlighting both the reporting and possibly also the epidemiological differences among eastern European countries. Observing the difference in reported cases at a hospital level between countries, either an underestimation or an overestimation might be suspected for eastern Europe. However, several factors such as lack of knowledge, diagnostic capacity and compensation for the farmers, unwillingness or inability to report and logistic difficulties may contribute to the vicious cycle of underreporting and underdiagnosis \[[@CR27], [@CR28]\]. Of all cases, 14% were suspected to have travelled abroad, 17% to be autochthonous cases, while for the majority (69%) it was not specified. This lack of additional information does not allow us to unravel the question of an existing parasite transmission in eastern Europe. The results show that even if the assumption that the infection can be acquired abroad would be correct, then the same happens in Europe as frequently. The automatic assumption that brief visits abroad automatically outrule looking for answers locally, can lead to a barrier for dealing with the actual epidemiological situation. Nearly one quarter of the cases were diagnosed abroad, possibly indicating that either the local health system is not equipped to detect these cases, or that people are actively seeking medical attention elsewhere. For two thirds of the human cases the origin of infection was not given, further contributing to the persisting knowledge and underreporting gap. However, it should be noted that a lowering trend is registered. For instance in Serbia, no new cases were recorded after 2010. Cases of porcine cysticercosis were reported in a number of countries, and as for the human cases, the number of cases and reports per country varied significantly. Notably, in Poland over 4000 cases of porcine cysticercosis were recorded by the veterinary inspection services in 2003; however, no information on the *Taenia* spp. involved was given and the possibility of (some of) the cases being *T. hydatigena* cannot be excluded. Also, for most other porcine cysticercosis cases reported in eastern Europe, species identification was never performed, nor molecularly confirmed. Only in one study from Estonia, molecular techniques were applied to confirm a false positive case \[[@CR25]\]. Lack of differential diagnosis highlights the presence of poor meat inspection, due to the possible lack of: qualified meat inspectors, sensitive and specific diagnostic tools, awareness, funding and adequate mandatory reporting systems. Moreover the trend towards smaller-scale pig farming, with outdoor access and home slaughtering (without meat inspection) increases the risk of infection and therefore the need for monitoring. Bovine cysticercosis was reported in over two thirds of the countries in the region. The highest number of cases was reported in Poland, where 4718 bovine cysticercosis cases were recorded in 1994. General meat inspection has a very low sensitivity, hence slaughterhouse figures, even when well-recorded will always underestimate the prevalence \[[@CR29]\]. False positives, e.g. eosinophilic myositis as a consequence of sarcocystis infection or abscessess, can also occur although they usually contribute only a small (negligible) proportion of bovine cysticercosis records. Better, cost-effective and more sensitive diagnostic tools should be developed, and data recording systems should be improved to assure that data from individual slaughterhouses are centralised. Increased availability of data will further allow estimating the overall economic impact of *T. saginata* which has been shown to be significant in Belgium (858 €/heavily infected carcass and 586 €/lightly infected carcass) \[[@CR7]\], but less so in Catalonia \[northeastern Spain (1140 €/heavily infected carcass and 509 €/lightly infected carcass)\] \[[@CR8]\]. Without reliable data from the surveillance the default assumption should be that the parasite is present, not absent. To overcome case uncertainty and to be able to have more accurate data that will improve our knowledge on the epidemiological situation of *T. saginata* and *T. solium*, the possibility to molecularly confirm *T. solium* findings should be available. Recently, the laboratories across Europe that are running tests for the larval and adult stages of the parasites have been summarised and mapped, facilitating the process when suspected lesions are detected \[[@CR27]\]. The early species-level identification of the tapeworm and subsequent adapted management are crucial to avoid not only human-to-human transmission, but also human-to-pig/cattle transmission. Finally, for nearly all the countries, at least some data and research on the topic could be retrieved and both published and unpublished data sources were available. Particularly the unpublished sources were of added value and contributed to an improved picture of the epidemiological situation in eastern Europe, highlighting the importance of data access and collaborations through networks such as CYSTINET. Nevertheless, for Belarus, Kosovo, Malta and Ukraine a contact could not be established and no information could be retrieved leaving information gaps. In the absence of data the presence of *T. saginata* and *T. solium* in these countries cannot be excluded. For western Europe information and a contact was established with all counties of the region, and the amount of published and unpublished literature was more than double that of eastern Europe, which helped to provide a better and more complete overview of the epidemiological situation \[[@CR10]\]. The two zoonotic parasites would merit more attention and research in eastern Europe. Conclusions {#Sec19} =========== There is indication that taeniosis and cysticercosis are present across eastern Europe but information on the occurrence of *T. solium* and *T. saginata* across the region remains fragmented. For a number of countries, no information was available and for some, establishing a contact with experts within the field proved challenging. Species identification is not performed in most countries and the findings are not confirmed using molecular methods (especially for taeniosis and porcine cysticercosis), making the endemicity status in the region still unclear. Attention should be paid to: (i) suspected autochthonous human cysticercosis cases to rule out ongoing transmission; (ii) notification of taeniosis and human cysticercosis as these should be implemented to get better data and a clearer overview of the epidemiological situation; (iii) surveillance and reporting systems in animals; and (iv) methods for confirming findings. Additional files ================ {#Sec20} Additional file 1:PRISMA checklist. (PDF 68 kb) Additional file 2:Country sheets template. (PDF 81 kb) Additional file 3:**Table S1.** List of references included in the review retrieved through online international databases. (XLSX 24 kb) Additional file 4:**Table S2.** List of references included in the review made available through local sources. (XLSX 71 kb) Additional file 5:**Table S3.** Individual taeniosis cases identified in case reports in eastern Europe available from 1990 to 2017. **Table S4.** Aggregated taeniosis cases reported in documents (authorities' reports, epidemiological bulletins, national registries and publications) identified in eastern Europe available from 1990 to 2017. **Table S5.** Taeniosis prevalence data reported in epidemiological studies published between 1990--2017. **Table S6.** Individual human cysticercosis cases identified in case reports in eastern Europe (1990--2017). **Table S7.** Aggregated human cysticercosis cases identified in case reports and publications in eastern Europe (1990--2017). **Table S8.** Porcine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990--2017). **Table S9.** Bovine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990--2017). (DOCX 207 kb) CYSTINET : European Network on Taeniosis/Cysticercosis EFSA : European Food Safety Authority GDP/GNI : gross domestic product/gross national income ICD : International Classification of Diseases NCC : neurocysticercosis OIE : World Organization for Animal Health/Office International des Epizooties This work is a collaboration within the framework of CYSTINET, the European network on taeniasis/cysticercosis, COST ACTION TD1302. We acknowledge Dr G. Petrusevska and Dr I. Kuzmanovski, for the unpublished data about NCC in Former Yugoslav Republic of Macedonia, and Prim. Dr E. Miloskoska Blagoeska and Dr G. Kuzmanovska, Former Yugoslav Republic of Macedonia, for their valuable input for the conduction of this work. We also acknowledge Professor Olga Dulović, Clinic for Infectious and Tropical Diseases, Clinical Center of Serbia, Belgrade, Serbia, and School of Medicine, University of Belgrade, Belgrade, Serbia, for the assistance in providing medical records for Serbia. Funding {#FPar1} ======= This work is a collaboration within the framework of CYSTINET, the European network on taeniasis/cysticercosis, COST ACTION TD1302. Availability of data and materials {#FPar2} ================================== All references found eligible in our literature review are included in the supplemental materials (Additional files [3](#MOESM3){ref-type="media"} and [4](#MOESM4){ref-type="media"}). CT, BD and SS conducted the systematic review of the literature and extracted data. CT and BD analysed data and drafted the first version of the manuscript. AA, BD, CT, MLG, SS and VD contributed to the design of the study, interpretation of data and writing of the first draft. The remain co-authors (ZW, AK, AC, ASW, AA, BB, BL, CMC, CV, DA, GD, IB, IK, JK, JS, BK, MJP, MV, MP, MS, MK, OD, PJ, DSJ, VS, ZD, SG and PD) contributed to data collection and writing of the paper. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== This work is a collaboration within the framework of CYSTINET, the European network on taeniasis/cysticercosis, COST ACTION TD1302. Consent for publication {#FPar4} ======================= Not applicable. Competing interests {#FPar5} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

{ "pile_set_name": "PubMed Central" }

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22,256

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Sir, In recent years, we have seen an increasing use of noninvasive ventilation (NIV) in the context of acute respiratory insufficiency (acute respiratory failure). However, adherence to treatment is not perfect; partly because of patient\'s complaints relating to high-pressure levels. This led to envisaging if the use of lower ventilatory pressures could lead to the same clinical results of the higher pressures, on patients with the restrictive pulmonary disease. We have read with great interest study by Kadowaki *et al*. published in your journal, entitled "Low-intensity noninvasive ventilation: Lower pressure, more exacerbations of chronic respiratory failure," presenting a retrospective study where they investigated the effects of lower NIV pressure on chronic respiratory failure. It concluded that patients can develop more exacerbations, recommending the use of higher initial support pressure levels.\[[@ref1]\] It is an interesting and original article to read, but we would like to comment some parts of it: First, we do not agree with the nondiscrimination of hypercapnic respiratory insufficiency, being defined only by the value of PaCO~2~. There is not any reference to the value of oxygen (or pH) and we think that a patient with severe -- mild hypoxemia and hypercapnia are surely different from a patient with the only hypercapnia, and this may alter the results because these two subtypes are associated with different outcomes\[[@ref2]\]Second, according to the authors, its protocol to start with lower pressure levels (LPLs). We understand that the aim of this work was to study this issue and we also accept this strategy may be an attempt to improve the patients\' adherence; however, we do not agree to be standard starting with low inspiratory positive airway pressure (IPAP). Most recent guidelines report that even patients with restrictive disease should start with initial IPAP 15--20 cmH~2~O\[[@ref3]\]Finally, all patients had heated humidifier and its benefits are well known, although it is not unanimous its routine use.\[[@ref3]\] There are, however, data in literature showing that advantages are more prominent with higher IPAP pressure level.\[[@ref4]\] It is also widely known that use of these devices cause adverse effects such as the risk of infection or poor compliance. Could this have affected patients with LPLs and led to worse outcomes? Financial support and sponsorship {#sec2-1} ================================= Nil. Conflicts of interest {#sec2-2} ===================== There are no conflicts of interest.

{ "pile_set_name": "PubMed Central" }

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22,257

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Background ========== Chronic inflammatory demyelinating polyneuropathy (CIDP) is an uncommon manifestation of systemic lupus erythematosus (SLE). To our knowledge, few cases of CIDP and SLE have been previously reported in the literature \[[@b1-amjcaserep-18-980]\]. CIDP is a debilitating clinical condition that is characterized by symmetrical polyneuropathy with histologic findings of demyelination and occasionally remyelination, and is believed to be an acquired autoimmune disorder that targets myelin \[[@b1-amjcaserep-18-980]\]. CIDP continues to progress, or may relapse, for more than eight weeks, a clinical finding that differentiates it from acute inflammatory demyelinating polyneuropathy which is a monophasic sub-acute illness that reaches its nadir within three to four weeks. CIDP is characterized by muscular weakness with or without sensory loss in the extremities and can have a chronic progressive course with remission and repeated relapses \[[@b2-amjcaserep-18-980]\]. The diagnosis of CIDP is more likely when the patient has a predominance of sensory symptoms over motor symptoms. Although the cause of CIDP is unknown, there is evidence to support an autoimmune etiology with multiple immunological triggers \[[@b3-amjcaserep-18-980],[@b4-amjcaserep-18-980]\]. Both the cellular and humoral components of the immune system appear to be involved in the pathogenesis of CIDP and its variants \[[@b3-amjcaserep-18-980],[@b4-amjcaserep-18-980]\]. An estimated 10--20% of SLE patients show peripheral nervous system involvement and patients present with sensorimotor polyneuropathies, with less common syndromes including mononeuritis multiplex or asymmetric polyneuropathy and acute or chronic demyelinating polyneuropathy \[[@b5-amjcaserep-18-980]\]. Multiple factors, including early diagnosis of CIDP and presence of multiple antibodies associated with SLE, predict a good response to intravenous immunoglobulin (IVIG). Case Report =========== A 40-year-old African American woman with a past medical history of SLE, diagnosed at the age of 40 years, and treated with hydroxychloroquine, presented with a three-month history of slowly progressive tingling sensation and weakness in both her lower and upper extremities, and difficulty in walking. She initially presented with fatigue, fever, myalgia and arthralgia at the time of her diagnosis of SLE diagnosis. She had no known complications of SLE and no significant past medical history at the time of presentation. Review of here systems showed worsening fatigue, myalgia, headache, and numbness and some weakness of upper and lower extremities. The sensory and motor symptoms progressed in an ascending fashion resulting in impaired balance without bowel or bladder involvement. On her current admission to hospital, the differential diagnosis of her symptoms was broad and included idiopathic inflammatory myopathy, CIDP, subacute combined degeneration of spinal cord, cervical myelopathy, SLE neuropathy, thyroid myopathy, amyotrophic lateral sclerosis, multiple sclerosis, Eaton-Lambert syndrome, and paraneoplastic syndrome associated with an unknown primary malignancy. Physical examination showed a grading for motor strength of 4/5, decreased temperature of the limb extremities, reduced pinprick and vibration sense and absent reflexes of both the upper and lower extremities, and an unsteady gait. Her sensory symptoms were more prominent when compared to her motor weakness. Her positive and negative autoimmune panel, determined by indirect immunofluorescence, is listed in [Table 1](#t1-amjcaserep-18-980){ref-type="table"}. Other laboratory investigations showed a normal complete metabolic panel, cerebrospinal fluid analysis, thyroid profile, brain magnetic resonance imaging (MRI), an erythrocyte sedimentation rate (ESR) of 75 mm/hr, low C4 complement, leukopenia, and anemia. No paraproteins were identified on serum electrophoresis. Electromyography (EMG) showed axonal demyelinating polyradiculoneuropathy, abnormal peroneal distal latency with very low amplitude and disappearance of F waves consistent with CIDP. The patient was treated with intravenous immunoglobulin (IVIG) 2 gm/kg daily for five days and prednisone 60 mg daily for a total of seven days, while continuing hydroxychloroquine. Following treatment with IVIG and prednisone, the patient showed marked clinical improvement and regained her ability to walk with minor assistance. She was discharged home on a tapering dose of prednisone dose with follow-up in the rheumatology clinic. Discussion ========== Neurological and psychiatric symptoms are reported to occur in between 10--80% of patients with the diagnosis of systemic lupus erythematosus (SLE) during their illness \[[@b6-amjcaserep-18-980],[@b7-amjcaserep-18-980]\]. The range of neurological manifestations of SLE includes central nervous system encephalitis, hemiparesis, brain infarcts, seizures, schizophrenia, atypical trigeminal neuralgia, acute demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy (CIDP) \[[@b8-amjcaserep-18-980]\]. In 1948 Sedgwick and Von Hagen first described the clinical manifestation of a CIDP-like illness in patients with SLE ([@b9-amjcaserep-18-980]\]. Retchland and coworkers stated that CIDP can have an unusual presentation and can precede systemic symptoms of SLE \[[@b10-amjcaserep-18-980]\]. About 50% of the patients with a concomitant diagnosis of SLE and CIDP can achieve a good clinical response to IVIG, and the remainder may have a minimal response. Certain characteristics, including early CIDP diagnosis, the involvement of all four extremities, hyporeflexia or areflexia, slowed peripheral motor nerve conduction velocity, SLE involvement of critical internal organs, and the presence of multiple auto-antibodies associated with SLE, are of a good response to IVIG \[[@b1-amjcaserep-18-980]\]. Patients with SLE and CIDP may present with recurrent episodes of Guillain-Barré syndrome-like symptoms, mononeuritis multiplex, or symmetric polyradiculopathy, over a period of weeks to months. The proposed pathological mechanisms for the clinical symptoms of CIDP in SLE patients include abnormalities in blood vessels that supply the epineurium, resulting in nerve fiber loss, inflammation that involves the interstitial and causes separation of nerve fibers, and reduction in the amount of myelin \[[@b11-amjcaserep-18-980],[@b12-amjcaserep-18-980]\]. CIDP is now believed to be an autoimmune mediated process involving cellular and humoral response that involve T-cells, activated macrophages, cytokines, anti-myelin antibodies, and costimulatory inflammatory molecules, resulting in loss of myelin and axonal loss \[[@b11-amjcaserep-18-980],[@b12-amjcaserep-18-980]\]. The neuropathy of CIDP is usually polyradicular, symmetrical, and involves both proximal and distal muscles. \[[@b13-amjcaserep-18-980]\]. Electromyography (EMG) testing in CIDP may help to determine the extent of sensory and motor deficits and categorize the demyelinating process (prolonged terminal latency, slowing of nerve conduction velocity, dispersion and conduction block) and axonal form of neuropathy (marginal slowing of nerve conduction, small compound muscle or sensory action potential, and denervation on EMG) \[[@b14-amjcaserep-18-980]\]. Hereditary forms of demyelination are suggested by features of uniform demyelination, whereas variable demyelination of nerve segments of the same nerve favor acquired demyelination \[[@b14-amjcaserep-18-980]\]. Demyelination requires slowing of conduction velocities, according to the criteria by American Academy of Neurology (AAN) \[[@b15-amjcaserep-18-980]\]. Most CIDP patients do not meet these AAN criteria, as conduction slowing can be absent when not enough fibers are affected when demyelination is proximal and not affected by distal stimulation, or when there is severe secondary axonal degeneration \[[@b16-amjcaserep-18-980]\]. A study by Haq and colleagues showed that there was only 42% sensitivity of the electrodiagnostic criteria proposed by the AAN in CIDP patients who had sural nerve biopsies \[[@b17-amjcaserep-18-980]\]. The presence of anticardiolipin antibodies in CIDP may also reflect myelin damage \[[@b18-amjcaserep-18-980]\]. For patients with sensorimotor neuropathy, there is a good response to steroid pulse or IVIG treatment, despite age, concurrent illness, and positive anticardiolipin antibodies \[[@b18-amjcaserep-18-980]\]. According to Sindern and colleagues, CIDP is also reported to occur episodically in other autoimmune diseases, including myasthenia gravis, acute glomerulonephritis, Hashimoto's thyroiditis, and multiple sclerosis \[[@b19-amjcaserep-18-980]\]. Very rare associations have been reported with diseases that include hemophagocytic lymphohistiocytosis, which has an estimated prevalence of 0.9--4.6% in patients with SLE \[[@b20-amjcaserep-18-980]\]. CIDP can occur before, after, or simultaneously with the onset of SLE. As shown in this case report, early identification of peripheral limb weakness, areflexia or hyporeflexia, and slowed nerve conduction is crucial before treatment is commenced with steroids, IVIG, or plasmapheresis, all of which may be life-saving. Apart from treatment with steroids and IVIG, other immunosuppressive agents including azathioprine, mycophenolate, rituximab and cyclophosphamide may be considered \[[@b21-amjcaserep-18-980],[@b22-amjcaserep-18-980]\]. Inflammatory neuropathies respond to therapy with glucocorticoids in moderate to higher doses, such as prednisone 30--60 mg/day, but not all patients improve on this treatment. Therapy with glucocorticoids, IVIG, or plasmapheresis may be indicated in CIDP. Evidence of axonal damage on EMG, or vasculitis on nerve biopsy, are indications for initiating more potent immunosuppressive therapy, such as cyclophosphamide. In 2005, Vina and colleagues suggested that more aggressive treatment should be used when more severe systemic manifestation of SLE were in order to suppress the immune-mediated processes involved in the pathogenesis of CIDP \[[@b1-amjcaserep-18-980]\]. Conclusions =========== CIDP is an acquired autoimmune peripheral neuropathy that can be an unusual association seen in patients with SLE. As this case report has shown, early clinical diagnosis of CIDP, supported by serological autoantibody profiles associated with SLE, can predict a good response to steroids. Most patients with CIDP are treated successfully with steroids if the diagnosis is made early. IVIG, plasmapheresis, or immunosuppressive therapy should be considered if there is no response to steroids. **Conflict of interest** None. ###### Panel of autoimmune serology serological markers tested. **Serum markers and antibodies** **Patient result** **Normal range** ---------------------------------- -------------------- ------------------- ANA Positive Negative ANA titer 1: 1280 \<1: 80 ANA pattern Speckled C4 complement 14.3 mg/dl 18.0--55.0 mg/dl C3 complement 90.5 mg/dl 79.0--152.0 mg/dl Anti-ds DNA antibodies Positive Negative Anti-ds DNA antibodies titer 1: 44 IU \<1: 25 IU Anti SSA antibodies Positive Negative Anti SSA antibodies titer \>1: 25 IU \<1: 20 IU Anti SM antibodies Positive Negative Anti SM antibodies titer \>1: 25 IU \<1: 20 IU Anti SSB antibodies Negative Negative Anti RNP antibodies Negative Negative Anti SCL 70 Negative Negative Anti-histone antibody Negative Negative ANA -- antinuclear antibodies; SSA, SSB -- Sjögren syndrome A and B; SM -- smooth muscle; RNP -- ribonucleoprotein; SCL -- scleroderma. [^1]: Authors' Contribution: [^2]: Study Design [^3]: Data Collection [^4]: Statistical Analysis [^5]: Data Interpretation [^6]: Manuscript Preparation [^7]: Literature Search [^8]: Funds Collection [^9]: **Conflict of interest:** None declared

{ "pile_set_name": "PubMed Central" }

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22,258

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"Collection", "Conflict", "interest", "None", "declared" ]

Introduction {#s1} ============ Wild-type streptavidin is a tetrameric protein with four identical subunits. Each subunit has a biotin binding pocket and can bind biotin tightly with a dissociation constant (K~d~) around 10^−14^ M [@pone.0035203-Green1]. This binding is considered to be irreversible and has been applied in a wide range of biomedical and biotechnological applications [@pone.0035203-Wilchek1], [@pone.0035203-Laitinen1]. However, the tight biotin binding also makes streptavidin not suitable for affinity purification of biotinylated molecules. It would be ideal to develop engineered streptavidin muteins with reversible biotin binding capability. These engineered muteins can be applied to purify biotinylated molecules, develop automated high-throughput protein purification systems, reusable biosensor chips and bioreactors, study protein-protein interactions and design strippable probing agents (e.g. engineered muteins conjugated to horseradish peroxidase) for blot reprobing. To understand the strategies applied in engineering streptavidin with reversible biotin binding ability, it is vital to understand the structural features of streptavidin, its biotin binding pocket and the subunit interfaces. Each streptavidin subunit contains eight antiparallel strands that form a β-barrel structure [@pone.0035203-Weber1], [@pone.0035203-Hendrickson1]. Two of these subunits (A and B as well as C and D in [Fig. 1b](#pone-0035203-g001){ref-type="fig"}) have extensive interfacial interactions to form a relatively stable dimer. Two dimers then assemble into a tetramer via a weaker interface. Although each subunit can bind a biotin molecule, each of the four complete biotin pockets in the tetramer relies on the donation of Trp-120 [@pone.0035203-Sano1], [@pone.0035203-Freitag1], [@pone.0035203-Chilkoti1] located in loop~7--8~ from the neighboring subunit (e.g. subunit A needs Trp-120 from subunit D and vice versa, [Figure 1b](#pone-0035203-g001){ref-type="fig"}). Exceptionally tight biotin binding in streptavidin is mainly contributed by three sets of interactions [@pone.0035203-Stayton1]. The first set involves at least 6 residues (N23, S27, Y43, S88, T90 and D128) in the biotin binding pocket to form an extensive hydrogen bonding network with biotin. The second set involves strong hydrophobic interactions [@pone.0035203-Freitag1] between biotin and four tryptophan residues (79, 92, 108 and 120). Finally, residues in loop~3--4~ (S45, V47, G48, N49 and A50) play a critical role in binding and trapping biotin to the biotin binding pocket [@pone.0035203-Freitag2], [@pone.0035203-LeTrong1], [@pone.0035203-Chu1]. In the absence of biotin, loop~3--4~ has been shown to be flexible and is mainly in an open configuration. However, after biotin binding, loop~3--4~ becomes immobilized and is in a closed position ([Figure 1](#pone-0035203-g001){ref-type="fig"}, panels a and b) because of its interactions with biotin. Furthermore, biotin binding can also strengthen subunit interactions [@pone.0035203-Katz1], in particular, via interactions between biotin in one subunit and Trp-120 in loop~7--8~ from the neighboring subunit [@pone.0035203-Sano1]. In fact, the majority of the streptavidin-biotin complexes are in the tetrameric state in SDS-polyacrylamide gel even if the samples have been boiled before loading [@pone.0035203-Bayer1], [@pone.0035203-Waner1]. ![Biotin entry and exit paths in wild-type streptavidin (panels a and b) and models of the engineered mobile loop~7--8~ in streptavidin (panels c and d).\ Subunits A, B, C and D in wild-type streptavidin are colored in blue, red, yellow and green, respectively. Loop~3--4~ in subunit D is in the open conformation as shown in panel a to allow biotin binding. Trp-120 (blue) from subunit A (blue) is required to form a complete biotin binding pocket in subunit D (green). With biotin binding, loop~3--4~ is in the closed position as shown in panel b. To develop an engineered streptavidin (8-aa-loop-H127C) with reversible biotin binding, loop~7--8~ in wild-type streptavidin was replaced by an engineered mobile loop to function as a dynamic gateway for the exit of the bound biotin. The engineered mobile loop~7--8~ in subunit A is modeled in 10 different positions to illustrate its dynamic nature (panel c). The blue loop (loop~7--8~) in the upper positions will form a wall as part of the biotin binding pocket for the subunit D (green). At these stages, this gate is in the closed state. When loop~7--8~ is in the lower position (panels c and d), the gate is in the open state. With loop~3--4~ in subunit D in the closed conformation, biotin still can possibly escape from the biotin binding pocket at this stage (panel d).](pone.0035203.g001){#pone-0035203-g001} Two approaches have been taken to develop streptavidin muteins with reversible biotin binding ability. The first approach is to replace amino acid residues that are critical in hydrogen bonding to biotin. Although these changes can indeed lower the biotin binding affinities [@pone.0035203-Qureshi1], [@pone.0035203-Kopetzki1], many of the mutations also affect inter-subunit interactions in the streptavidin tetramer. Weakening inter-subunit interactions typically generates a heterogeneous population of streptavidin oligomers which leads to many practical problems. A second approach is to develop recombinant monomeric streptavidin [@pone.0035203-Wu1] which has lower biotin binding affinity. This approach exploits the fact that an individual streptavidin subunit lacks a complete biotin binding pocket since Trp-120 from a neighboring subunit forms a key part of the binding site ([Figure 1](#pone-0035203-g001){ref-type="fig"}, panels a and b). Replacing Trp-120 with alanine (W120A) results in a tetrameric streptavidin mutein with a K~d~ of 3×10^−9^ M for biotin [@pone.0035203-Kopetzki1]. To create monomeric streptavidin, charge repulsion and steric hindrance were introduced at the subunit interface [@pone.0035203-Wu1]. The resulting monomeric streptavidin has a biotin binding constant around 10^−7^ M. However, this mutein can be in the monomeric state only under certain conditions. First, the salt concentration has to be low to maximize electrostatic repulsion between streptavidin subunits. Second, the concentration of the monomers has to be low as exposure of the hydrophobic interface promotes non-specific aggregation. In this study, a new approach was designed to develop an engineered tetrameric streptavidin with reversible biotin binding capability and other desirable features. Results {#s2} ======= Rationale for the design of novel streptavidin muteins {#s2a} ------------------------------------------------------ Our novel approach relies on the effects of two loops (loop~3--4~ with residues 45--52 and loop~7--8~ with residues 114--121) on biotin binding ([Figure 1](#pone-0035203-g001){ref-type="fig"}). Loop~3--4~ forms the lid and Trp-120 from loop~7--8~ forms part of the wall of the biotin binding pocket. With various hydrogen bonding and hydrophobic interactions in the biotin binding pocket and the closure of the lid, biotin can hardly escape from the binding pocket. In this study, an attempt was made to create a dynamic "back door" formed by loop~7--8~ in streptavidin to allow biotin a second route to escape from the binding pocket when the "main door" primarily formed by loop~3--4~ is closed. This objective can potentially be achieved by several approaches. One is to develop a ΔW120 mutein. Since Trp-120 in loop~7--8~ is known to interact strongly with biotin [@pone.0035203-Sano1], [@pone.0035203-Freitag1], [@pone.0035203-Chilkoti1], the deletion of Trp-120 may allow the modified loop~7--8~ to become more flexible. A second approach is to create a mobile 8-amino-acid-loop (8-aa-loop) mutein in which loop~7--8~ is engineered to have a completely different sequence but retains the same length as the original loop in wild-type streptavidin ([Table 1](#pone-0035203-t001){ref-type="table"}). Asparagine and glycine were introduced at the center of the loop since they are known to introduce a turn in the loop structure [@pone.0035203-Wilmot1]. The DSS (aspartate, serine and serine) and SDG (serine, aspartate and glycine) sequences were introduced to form the left and right arms of the loop, respectively ([Table 1](#pone-0035203-t001){ref-type="table"}). These amino acids were selected because they have a high propensity for intrinsic disorder [@pone.0035203-Linding1]. This design minimizes interactions between loop~7--8~ and biotin, and was intended to allow the engineered loop to act as an unlocked mobile door swinging between the open and closed states even in the presence of biotin ([Figure 1](#pone-0035203-g001){ref-type="fig"}, panels c and d). To improve the chance of obtaining a mutein with its engineered "back door" open wide enough for the exit of the bound biotin, a third approach was applied to create a series of muteins (2-aa-loop, 4-aa-loop and 6-aa-loop muteins, [Table 1](#pone-0035203-t001){ref-type="table"}) with both the sequence of loop~7--8~ redesigned and the length of the loop shortened. Muteins with shorter loops were hypothesized to provide bigger openings around the biotin binding pocket which should facilitate the release of biotin. In a preliminary study, the 4-aa-loop mutein was immobilized on agarose matrix. Although this matrix could bind biotinylated proteins, streptavidin mutein was seen leaking from the column during washing and elution. Since the coupling condition allows on average one out of four subunits in the tetrameric streptavidin mutein to be coupled to the matrix to maximize the accessibility of the biotin binding sites, the leakage of streptavidin subunits suggests that changes in the loop~7--8~ structure might result in weakening of the inter-subunit interactions in the streptavidin mutein. To avoid this complication, the H127C mutation [@pone.0035203-Reznik1], [@pone.0035203-Chilkoti2] was introduced to two constructs (8-aa-loop mutein and ΔW120 mutein) to create 8-aa-loop-H127C mutein and ΔW120-H127C mutein, respectively. The H127C mutation has been reported [@pone.0035203-Reznik1], [@pone.0035203-Chilkoti2] to allow crosslink between subunits A and C (and also between subunits B and D) through the formation of a disulfide bond. These disulfide bonds can strengthen inter-subunit interactions. Since the matrix for the 4-aa-loop mutein had a subunit leakage problem, this mutein was not further characterized. The remaining four muteins (ΔW120-H127C, 2-aa-loop, 6-aa-loop and 8-aa-loop-H127C) were used for further analyses. 10.1371/journal.pone.0035203.t001 ###### Features of the loop~7--8~ muteins. ![](pone.0035203.t001){#pone-0035203-t001-1} Streptavidin Loop~7--8~ H127C ----------------- ----------------------------------------------- ------- Wild-type L T S G~113~ **T T E A N A W K** S~122~ T L V − ΔW120-H127C **T T E A N A K** \+ 8-aa-loop-H127C **D S S N G S D G** \+ 8-aa-loop **D S S N G S D G** − 6-aa-loop **D S N G S G** − 4-aa-loop **D N G G** − 2-aa-loop **N G** − The amino acid sequences in the natural and engineered loop~7--8~ loops are shown in bold. The sequences flanking the loop~7--8~ are shown in black. + or − indicates the presence or absence of H127C in the muteins. Production and purification of streptavidin muteins {#s2b} --------------------------------------------------- To avoid the formation of inclusion bodies and the need to refold the engineered streptavidin, all streptavidin muteins were produced in their soluble state from *B. subtilis* via secretion [@pone.0035203-Wu2]. The production yield of the muteins in a semi-defined medium [@pone.0035203-Wu3] was 40--60 mg/liter. Each mutein could be affinity purified in one step using the biotin-agarose matrix. The purification of the 8-aa-loop-H127C mutein showed typical results ([Figure 2a](#pone-0035203-g002){ref-type="fig"}). 8-aa-loop-H127C mutein was efficiently captured by the biotin-agarose matrix and could be eluted by buffer containing 4 mM biotin. The recovery was ∼90%. After dialysis to remove biotin from the pooled elution fractions, the purified mutein could rebind the biotin-agarose matrix and could be eluted again using biotin-containing buffer (data not shown). This result demonstrates the reversible biotin binding ability of the mutein. ![SDS-PAGE showing purification of 8-aa-loop-H127C mutein and its oligomeric state.\ In panel a, 8-aa-loop-H127C mutein was affinity purified from *B. subtilis* culture supernatant using biotin-agarose matrix. S represents the concentrated culture supernatant before purification. FT, W and E are the flow-through, wash and elution fractions, respectively. 5 µl from each fraction (800 µl per fraction, each fraction contained the same volume of sample) was reduced with β--mercaptoethanol and boiled before loading onto the gel. The arrowhead marks the position of the 8-aa-loop-H127C mutein. Panel b shows the tetrameric state of 8-aa-loop-H127C (MH127C). 8-aa-loop (M) and wild-type (wt) streptavidin were included as reference. B and U represent the boiled and unboiled samples respectively. ME represents β--mercaptoethanol (used at 0.7 M final concentration). M shows the position of the molecular weight markers (numbers on the left of both panels are expressed in kDa).](pone.0035203.g002){#pone-0035203-g002} Determination of kinetic parameters {#s2c} ----------------------------------- The kinetic parameters for biotin binding to various muteins were determined by surface plasmon resonance using the BIAcore biosensor with biotinylated IgG proteins as the ligand ([Table 2](#pone-0035203-t002){ref-type="table"}). ΔW120-H127C had a binding affinity for biotin (K~d~ = 8.1×10^−9^ M) that was comparable to that of the W120A mutein (K~d~∼3×10^−9^ M) [@pone.0035203-Kopetzki1]. Although muteins with smaller loops (2- and 6-aa-loop muteins) were expected to have lower biotin binding affinities, their binding affinities (∼1--3×10^−9^ M) were actually comparable to that of the W120A mutein. Streptavidin muteins with nano-molar binding affinity tend to bind biotinylated molecules too tightly for affinity chromatography purification, leading to the poor recovery of target proteins. The mutein with the lowest biotin binding affinity (1.9×10^−8^ M) in this study is the 8-aa-loop-H127C mutein. It was further characterized and its ability to act as an affinity agent for purifying biotinylated proteins was explored. 10.1371/journal.pone.0035203.t002 ###### Kinetic parameters of biotin binding in streptavidin muteins. ![](pone.0035203.t002){#pone-0035203-t002-2} Streptavidin mutein ΔW120-H127C 2-aa-loop 6-aa-loop 8-aa-loop-H127C --------------------------------------------- ------------- ------------- ------------- ----------------- k~a~ or k~on~ (M^−1^s^−1^) 4.96×10^4^ 6.3×10^4^ 2.43×10^4^ 2.21×10^4^ k~d~ or k~off~ (s^−1^) 4.0×10^−4^ 7.02×10^−5^ 7.59×10^−5^ 4.28×10^−4^ K~d~ (M) 8.1×10^−9^ 1.12×10^−9^ 3.13×10^−9^ 1.9×10^−8^ t~½~ (min)[\*](#nt103){ref-type="table-fn"} 28.9 165 152 27 Biotinylated IgG proteins were immobilized to the sensor chip, and purified muteins were injected to determine binding affinities. t~½~ is the half-life of biotin in the SAV-biotin complex. It is estimated based on the following equation: t~½~ (min) = 0.693/k~off~/60. Tetrameric state of 8-aa-loop-H127C mutein {#s2d} ------------------------------------------ Since the matrix with 4-aa-loop mutein immobilized showed leakage of streptavidin subunits during chromatography, this observation prompted the examination of the oligomeric state of the 8-aa-loop-H127C mutein. The migration pattern of the 8-aa-loop-H127C mutein resolved by SDS-PAGE was examined in the presence or absence of mercaptoethanol under boiled and non-boiled conditions ([Figure 2b](#pone-0035203-g002){ref-type="fig"}). 8-aa-loop mutein and wild-type streptavidin were included in the study as a comparison. Since the streptavidin inter-subunit interactions are weaker in the absence of biotin [@pone.0035203-Sano1], these analyses were performed in the absence of biotin. Under all conditions, 8-aa-loop mutein migrated like a monomer. By contrast, over 98% of wild-type streptavidin remained as tetramers if the sample was not boiled. This suggests that changes in the amino acid sequence of loop~7--8~ weaken inter-subunit interactions. A significant difference in the oligomeric states of 8-aa-loop and 8-aa-loop-H127C muteins was observed in the absence of reducing agent. Whereas 8-aa-loop mutein remained as monomers whether the sample was boiled or not, ∼80% of 8-aa-loop-H127C mutein remained in the dimeric form even after boiling. This indicates the successful formation of disulfide bonds between subunits. With the unboiled sample, most of 8-aa-loop-H127C mutein adopted the tetrameric state. Since this mutein exists mainly as tetramer even in the presence of detergent in SDS-PAGE, it should be predominantly in the tetramer state under non-denaturing conditions. Thus, the H127C mutation did strengthen inter-subunit interactions in 8-aa-loop-H127C mutein. Purification of biotinylated protein G and biotinylated IgG using 8-aa-loop-H127C mutein-agarose matrix {#s2e} ------------------------------------------------------------------------------------------------------- To explore the feasibility of using 8-aa-loop-H127C mutein for the affinity purification of biotinylated molecules, chemically biotinylated protein G and IgG were captured on the 8-aa-loop-H127C mutein-agarose matrix ([Figure 3](#pone-0035203-g003){ref-type="fig"}, panels a and b). After removal of nonspecifically bound proteins, the bound biotinylated proteins could be eluted from the column using 4 mM biotin. Absence of a 20-kDa streptavidin band in the flow-through, wash and elution fractions suggested that streptavidin subunit leakage was not a problem. Estimation of the amount of IgG molecules captured on the column and quantification of biotinylated IgG in the elution fractions indicated the recovery to be approximately 95%. To demonstrate binding specificity, HeLa cell extracts were applied to the column. Non-specific binding was not observed ([Figure 3c](#pone-0035203-g003){ref-type="fig"}). Non-biotinylated protein G and IgG also could not bind to the matrix (data not shown). When biotinylated IgG was mixed with the HeLa cell extract, biotinylated IgG could be affinity purified to homogeneity in one step ([Figure 3d](#pone-0035203-g003){ref-type="fig"}). ![Purification of biotinylated protein G and IgG using 8-aa-loop-H127C mutein-agarose matrix.\ Excess amounts of biotinylated proteins were loaded to the column. Panel a: Biotinylated protein G with an apparent molecular mass of 37 kDa is marked by arrowhead. Some biotinylated protein G molecules are in oligomeric state with higher molecular masses. Panel b: The 54- and 25-kDa protein bands are the biotinylated heavy (marked by closed arrowhead) and light chains (marked by gray arrowhead) of IgG, respectively. Panel c: HeLa cell extract was applied to the 8-aa-loop-H127C mutein-agarose matrix. Panel d: Biotinylated IgG mixed with HeLa cell extract. S: Sample before purification, FT, W, and E: Flow-through, wash and elution fractions. M: Molecular weight markers.](pone.0035203.g003){#pone-0035203-g003} To regenerate the column for repeated rounds of purification, the matrix was simply washed with 10 column volumes of binding buffer. Using biotinylated BSA as the target protein for purification, the 8-aa-loop-H127C mutein matrix saturated with excess amounts of biotinylated BSA could be regenerated in this manner to purify biotinylated BSA for 10 rounds with no observable loss in binding capacity (data not shown). Structural characterization of the 8-aa-loop-H127C mutein {#s2f} --------------------------------------------------------- To assess the conformation of the 8-aa-loop-H127C mutein, the protein was crystallized in the presence of biotin and its structure was solved using X-ray crystallography. Using data extending to 2.0 Å resolution, the structure clearly shows that the mutein crystallizes as a tetramer with a single subunit in the asymmetric unit ([Figure S1](#pone.0035203.s001){ref-type="supplementary-material"}). Biotin is bound in a manner indistinguishable from that of wild-type streptavidin, but there is very little electron density for loop~7--8~ (residues 114--121) and its neighboring residue 113 ([Figure 4](#pone-0035203-g004){ref-type="fig"}). The lack of electron density suggests that the modified sequence of loop~7--8~ confers a significant degree of dynamic disorder to the loop. The lack of interaction between loop~7--8~ and the biotin molecule bound to the adjacent subunit probably allows a higher level of mobility and dynamic motion in this loop in the mutein. ![Electron density map (2\|*F~o~*\|-\|*F~c~*\| coefficients, contoured at 1.1 sigma) contoured around the region expected to be occupied by residues 114--121 of loop~7--8~.\ The model for residues 110--112 and 122--124 fit the electron density quite well, but there is insufficient electron density to model residues 113--121. The structure of wild-type streptavidin (PDB code 1SWE) is superimposed and drawn in a thinner, gray line representation. Coefficients and phases were calculated using Refmac and the figure was prepared using PyMOL.](pone.0035203.g004){#pone-0035203-g004} Since inter-dimer interactions are critical for stabilizing the tetrameric structure and will likely affect the dynamics of the streptavidin tetramer, changes introduced to the 8-aa-loop-H127C mutein can possibly affect the inter-dimer arrangement. In fact, a significant alteration in the arrangement of subunits in the 8-aa-loop-H127C mutein was observed when compared with wild-type streptavidin. The orientation of the A/B dimer relative to the C/D dimer appears to be very similar in nearly all of the previously reported structures of biotin-bound streptavidin [@pone.0035203-Weber1], [@pone.0035203-Hendrickson1], partly because loop~7--8~ in one dimer interacts with a bound biotin molecule and nearby residues in the other dimer. Even when biotin is not present, Trp-120 and Leu-124 form mostly non-specific van der Waals contacts with Val-47 and Lys-121 from the opposing dimer, respectively. Thus, previously reported structures do not appear to have much difference in inter-dimer orientation or interactions. In this case, the loss of order in loop~7--8~ of 8-aa-loop-H127C mutein allows for a much larger rearrangement of the A/B dimer relative to the C/D dimer. When the A/B dimer of the mutein is superimposed onto the A/B dimer of wild-type streptavidin, the C/D dimer is rotated by 11° relative to the position of the C/D dimer in wild-type streptavidin ([Figure 5](#pone-0035203-g005){ref-type="fig"}). In contrast, the relative positions of the two dimers in other structures of biotin-bound streptavidin crystallized in different crystal forms differ by ∼1--3°. Significantly, an inter-dimer rotation of 5.4° was reported in the first paper comparing the structure of apo-streptavidin with biotin-bound streptavidin [@pone.0035203-Weber1]. The even larger inter-dimer rotation seen in the 8-aa-loop-H127C mutein supports the correlation of a disrupted inter-dimer interface with less order in loop~7--8~ and lower biotin-binding affinity. ![Comparison of quaternary structures of the 8aa-loop-H127C mutein and wild-type streptavidin (1SWE).\ Subunits A, B, C and D are colored blue, cyan, red and orange for both the mutein and wild-type structures. Biotin is drawn in space-filling representation and colored black. In all panels, only subunits A and B have been aligned. As a result, the cartoon diagrams and superimposed ribbon diagram at the far right show how the C/D dimer of the mutein is rotated by ∼10° relative to the C/D dimer of wild-type streptavidin when the A/B dimers of the mutein and wild-type streptavidin are superimposed. Loop~7--8~ (highlighted by the solid, black ovals in the front view) lies at the interface between dimer A/B and dimer C/D of wild-type streptavidin and is fixed in orientation in part by interactions between W120 and the biotin molecule bound to the opposing dimer. Loop~7--8~ is dynamically disordered in the mutein and hence is not shown in the cartoon model; the expected location of loop~7--8~ is denoted by the dashed, black ovals in the front view, and the location of the ends of the ordered parts of both loops (residues 112 and 122) are denoted by small circles. The side chains of Cys-127 from each subunit are drawn in space-filling representation, showing the formation of disulfide bonds between subunits A/C and B/D.](pone.0035203.g005){#pone-0035203-g005} An interesting feature also clearly seen in the crystal structure of the 8-aa-loop-H127C mutein is that a disulfide bond is formed between the Cys-127 residues of adjacent subunits at the A/B and C/D dimer interfaces ([Figure 6](#pone-0035203-g006){ref-type="fig"}). The Cys residue was introduced as a means of stabilizing the A/B and C/D dimers, based on the observed proximity of His-127 residues in adjacent subunits of the structure of wild-type streptavidin [@pone.0035203-Weber1], [@pone.0035203-Hendrickson1], [@pone.0035203-Reznik1], [@pone.0035203-Chilkoti2]. The formation of this disulfide bond in the crystallized mutein is consistent with the presence of disulfide-bonded protein in non-reducing SDS-PAGE analysis as described above ([Figure 2b](#pone-0035203-g002){ref-type="fig"}). ![Electron density map (2\|*F~o~*\|-\|*F~c~*\| coefficients, contoured at 1.1 sigma) contoured around the model of the disulfide bond formed by Cys-127 residues from adjacent subunits (A and C or B and D in the tetramer shown in [Figure 5](#pone-0035203-g005){ref-type="fig"}).\ Coefficients and phases were calculated using Refmac and the figure was prepared using PyMOL.](pone.0035203.g006){#pone-0035203-g006} Discussion {#s3} ========== An idealized streptavidin mutein for affinity chromatography applications should have the following desirable properties. First, it should be tetrameric and all four biotin binding sites are capable of binding biotin. In this state, the hydrophobic interface regions will not be exposed to the surface, thus minimizing non-specific hydrophobic interactions between the engineered streptavidin mutein and proteins in the crude sample to be analyzed. Second, intersubunit interactions should be strong. With one subunit of the tetramer immobilized, the other three subunits should stably associate with this covalently immobilized subunit so that no streptavidin subunits will be stripped off the column during the wash and elution steps. Third, the dissociation constant (K~d~) for biotin should be 10^−7^ to 10^−8^ M. Fourth, the off-rate (k~off~) for the bound biotin in the streptavidin-biotin complex is ideally around 10^−4^ sec^−1^ to allow the estimated half-life of the bound biotin to be around 10--30 minutes. With conditions 3 and 4 combined, the interaction would be both strong and specific enough to allow non-specifically bound molecules to be washed off the matrix without leakage of the specifically bound biotinylated molecules. At the same time, there should be efficient and quantitative elution of biotinylated molecules from the column. A fine balance between affinity towards biotinylated molecules and good recovery is essential for the ideal streptavidin affinity agent. Fifth, the engineered streptavidin muteins immobilized to the matrix should be stable enough to allow the matrix to be used for multiple rounds. Sixth, the engineered streptavidin should be produced with a reasonable production yield in a soluble and functional state without the requirement of refolding via inefficient and expensive denaturation and renaturation processes. The engineered 8-aa-loop-H127C mutein produced from *B. subtilis* via secretion meets all of the above requirements. Replacement of residues in loop~7--8~ of wild-type streptavidin allows loop~7--8~ to become flexible and changes the relative orientation of subunits in the tetrameric structure. These structural changes can lead to the lowering of the biotin binding affinity in 8-aa-loop-H127C mutein. Introduction of the H127C mutation is for the objective to stabilize the mutein in the tetrameric state. To confirm that loop replacement is the major factor contributing to the observed lower biotin binding affinity in the 8-aa-loop-H127C mutein, the kinetic parameters of the 8-aa-loop mutein (without the H127C mutation) for binding biotinylated proteins were also determined (data not shown). Its dissociation constant (3.96×10^−8^ M) was found to be comparable to that (1.9×10^−8^ M) of the 8-aa-loop-H127C mutein. In contrary, the streptavidin mutein carrying solely the H127C mutation did not show a significant decrease in biotin binding affinity since it could not be eluted off from the biotin-agarose matrix using a biotin containing buffer. The strength of biotin interactions with wild-type streptavidin and 8-aa-loop-H127C mutein was analyzed using the ligand energy inspector function in the Molegro molecular viewer program [@pone.0035203-Thomsen1] based on X-ray crystallographic data. The binding free energy is estimated by the MolDock scores as shown in [Table 3](#pone-0035203-t003){ref-type="table"}. The more negative values indicate stronger interactions. This analysis suggests that the lower biotin binding affinity in 8-aa-loop-H127C mutein is mainly contributed by both the absence of interactions (i.e. W120 and K121) and weaker interactions (D128, S45, N23 and L25) between biotin and residues in the biotin binding pocket. A previous study also suggests that D128, S45 and N23 play important roles in the biotin exit pathway [@pone.0035203-Freitag3]. Weakening the interactions between biotin and these residues is instrumental in enhancing biotin exit thereby increasing the biotin off rate (k~off~). 10.1371/journal.pone.0035203.t003 ###### Interaction of biotin with residues in the biotin binding pocket of subunit A. ![](pone.0035203.t003){#pone-0035203-t003-3} Protein 8-aa-loop-H127C Wild type SAV (2IZF) Difference (wild type -- mutein) ------------------------------ ----------------- ---------------------- ---------------------------------- MolDock score −125.6 −140.1 −14.4 **Trp 120 (from subunit D)** 0 −10.5 −10.5 **Lys 121 (from subunit D)** 0 −1.3 −1.3 **Asp128** −4.4 −7.7 −3.3 **Ser 45** −6.6 −8.0 −1.4 **Asn 23** −4.1 −5.2 −1.1 **Leu 25** −3.5 −4.3 −0.8 In addition to the interaction between biotin and specific residues in the biotin binding pocket of streptavidin, the strength of biotin binding appears to depend in part on the flexibility of loop~3--4~ and loop~7--8~. In both wild-type streptavidin [@pone.0035203-Freitag2] and the W120A mutein [@pone.0035203-Freitag1], both loops appear to be rigid in the presence of biotin. In contrast, the flexibility and mobility of loop~7--8~ in 8-aa-loop-H127C mutein creates a new exit path for the release of biotin from the biotin binding pocket even if loop~3--4~ is closed. This effect contributes to increases in the dissociation rate of the bound biotin. During the course of this study, a new streptavidin mutein called traptavidin was reported [@pone.0035203-Chivers1]. Biotin dissociates from traptavidin at least 10 times slower than from wild-type streptavidin and binds with 10 times higher affinity. The crystal structure of traptavidin [@pone.0035203-Chivers2] indicates that loop~3--4~ is well-ordered and adopts a closed conformation in both the presence and absence of biotin. By contrast, loop~3--4~ is well-ordered only when biotin is bound to wild-type streptavidin. Because the conformation of the biotin-bound complex in traptavidin is virtually identical to that seen in wild-type streptavidin, the increase in biotin binding affinity is clearly not due to additional contacts between biotin and traptavidin, as supported by the analysis of binding interactions with Molegro, which assigns similar MolDock scores for biotin binding to traptavidin and wild-type streptavidin (−139.3 and −140.1 respectively). The increased rigidity of loop~3--4~ thus accounts for the higher biotin binding affinity in traptavidin. Conversely, the increased mobility of the engineered loop~7--8~ in 8-aa-loop-H127 mutein may in part account for the observed lower biotin binding affinity. Most of the entire series of streptavidin muteins (ΔW120-H127C, 2- and 6-aa-loop muteins) have biotin binding affinities similar to that of the W120A mutein in the range of 10^−9^ M. Why the 2- and 6-aa-loop muteins do not have lower biotin binding affinities (K~d~\>10^−8^ M) is unclear at present, but the dynamics of loop~7--8~ are likely important. Structural and molecular dynamics studies of these muteins may help to explain relative binding affinities. Two matrices are commercially available to purify biotinylated molecules. One is the monomeric avidin matrix [@pone.0035203-Kohanski1]. Unfortunately, this approach has three drawbacks. First, the production cost is high as three avidin subunits are sacrificed for each avidin subunit immobilized to the matrix. Second, the large hydrophobic surface exposed in the generation of monomeric avidin typically introduces problematic non-specific interactions with unwanted proteins in the crude extract. Third, it is difficult to completely denature the tetrameric avidin because of the strong subunit interactions. Incomplete denaturation of avidin results in the presence of some tetrameric avidin in the matrix which can bind biotinylated molecules irreversibly. The second commercially available matrix contains the engineered streptavidin mutein [@pone.0035203-Kopetzki1]. The main concern with this matrix is the occasional observation of leakage of streptavidin subunits during protein loading, washing and elution. With the above-mentioned limitations, the engineered 8-aa-loop-H127C mutein has substantial advantages over the currently available matrices for affinity purification of biotinylated molecules. Many potential biotechnological applications of this mutein can be developed. In the post-genomic era with the discovery of many novel proteins which can be promising therapeutic targets, biomarkers for diseases, and agents with medical and biotechnological applications, the availability of a user-friendly high-throughput system to purify and immobilize these proteins for structural and functional studies is vital. However, protein purification requires reversibility in binding while immobilization requires ultra-tight interactions. Development of the 8-aa-loop-H127C mutein provides a solution to solve this dilemma. Biotinylated biomolecules can be affinity purified using the 8-aa-loop-H127C mutein matrix and immobilized to the wild-type streptavidin based protein chips. Furthermore, automated high-throughput purification platforms, reusable biosensor chips, protein arrays, bioreactors and magnetic beads can be developed using the 8-aa-loop-H127C mutein. With the reversible biotin binding capability, chemical conjugation or genetic fusion of 8-aa-loop-H127C muteins with other proteins such as alkaline phosphatase can allow blots to be reprobed without worrying masking other probing sites because of steric hindrance imposed by the bulky streptavidin conjugates. Although the 8-aa-loop-H127C mutein in the present format has many desirable features for biotechnological application, two areas need further improvement. First, it would be ideal if its production yield can be increased further (\>60 mg/liter). Since this mutein binds biotin reversibly, production of this mutein does not exert any toxic effect on its expression host by depleting biotin in both the cytoplasm and the culture medium. This is significantly different from the production of wild-type streptavidin which requires the production host to have the ability to produce sufficient quantities of biotin to sustain the cell growth [@pone.0035203-Wu3]. Many other expression systems from prokaryotes to eukaryotes can now be explored. Second, a hybrid tag containing six histidine residues and a single cysteine residue [@pone.0035203-Reznik2] can be fused to either end of the 8-aa-loop-H127C mutein. When this mutein is produced at industrial scale, a single-step purification using biotin-agarose column is usually not sufficient to purify the mutein to homogeneity. The added his-tag can offer another round of affinity purification of this mutein via a different mechanism. The added cysteine residue in the tag can be applied for orientation specific coupling of the mutein to the thiol-based coupling column matrices, biosensor chips or protein arrays. This approach not only improves the accessibility of the biotin binding sites of the immobilized mutein in the affinity matrix [@pone.0035203-Huang1] but also makes chemical coupling of this mutein to various matrices in a user friendly manner. Materials and Methods {#s4} ===================== Construction of expression plasmids for production of streptavidin muteins in *B. subtilis* {#s4a} ------------------------------------------------------------------------------------------- Plasmid pSSAV [@pone.0035203-Wu2] carrying a *B. subtilis* levansucrase signal peptide for secretion and a P43 promoter for transcription was used as the expression vector. This vector carries a synthetic gene for wild-type full-length streptavidin. To construct the expression vectors for the loop muteins, the gene encoding the wild-type streptavidin counterpart in pSSAV was replaced by the *Pst*I/*Bcl*I synthetic fragments encoding the loop muteins. *E. coli* pbluescript plasmids containing the synthetic gene fragments were ordered from Epoch Life Science, Inc. Texas, U.S.A. Each *E. coli* plasmid was digested with *Pst*I/*Bcl*I to release the synthetic gene fragment which would then be ligated to the *Pst*I/*Bcl*I cut pSSAV to generate the expression vectors. Production and affinity purification of streptavidin muteins {#s4b} ------------------------------------------------------------ *B. subtilis* WB800 [@pone.0035203-Wu4] cells carrying the expression vectors were cultivated in a semi-defined medium [@pone.0035203-Wu3] at 30°C for 9--12 hours. Culture supernatant was collected by centrifugation and streptavidin muteins were affinity purified using biotin-agarose as described previously [@pone.0035203-Wu1]. X-ray crystallographic studies {#s4c} ------------------------------ Crystals of 8-aa-loop-H127C mutein were obtained using the hanging-drop vapor diffusion technique by combining 2 µL protein (4 mg/mL, 10 mM Tris-Cl pH 7.5, 150 mM NaCl) with 2 µL reservoir solution (50% Tacsimate, pH 6.0 (Hampton Research), 10% (w/v) glycerol). Crystals grew to an approximate size of 0.2×0.2×0.1 mm after ∼2 weeks. They were suspended in a microfabricated loop (Mitegen) and flash-cooled under a nitrogen gas stream (∼100 K, Oxford Cryosystems). Data were measured using X-rays from a rotating anode generator (Rigaku RU-H3R), Multilayer optics (Osmic Blue) and Mar345 imaging plate detector (MarResearch). Diffraction data (200 images×0.5°; 10 minutes/image) were indexed and scaled using DENZO and Scalepack [@pone.0035203-Otwinowski1]. The space group was assigned by autoindexing, inspecting systematic absences and scaling. It was confirmed to be P4~2~2~1~2 by molecular replacement calculations and refinement. PHASER [@pone.0035203-Read1] was used for molecular replacement calculations, yielding a single very strong solution using a single subunit of wild-type streptavidin (1SWE) as the search model. Refinement calculations were performed using REFMAC (v. 5.5.0109) [@pone.0035203-Murshudov1], and model building was performed using COOT (v. 0.6.1) [@pone.0035203-Emsley1]. Geometric parameters were monitored using Procheck [@pone.0035203-Laskowski1] and Molprobity [@pone.0035203-Chen1]. Crystallographic statistics are reported in [Table 4](#pone-0035203-t004){ref-type="table"}. 10.1371/journal.pone.0035203.t004 ###### Crystallographic statistics. ![](pone.0035203.t004){#pone-0035203-t004-4} ----------------------------------------------------------------------------------- ----------------------- *Data collection* Space Group Mol/A.U. 1 Unit cell a, b, c (Å) 57.1, 57.1, 70.2 Resolution (Å)[a](#nt104){ref-type="table-fn"} 20.0−1.95 (2.02−1.95) R~sym~ [b](#nt105){ref-type="table-fn"} 0.063 (0.496) I/σI 28.4 (2.75) Completeness (%) 96.1 (88.0) Redundancy 6.7 (3.6) *Refinement* Resolution (Å) 19.39−2.00 Unique reflections 7585 R~work~ [c](#nt106){ref-type="table-fn"}/R~free~ [d](#nt107){ref-type="table-fn"} 0.189/0.232 No. of atoms 1003 Protein atoms 915 Biotin 16 Water 72 r.m.s.d. from ideal[e](#nt108){ref-type="table-fn"} geometry Bond lengths (Å) 0.009 Bond angles (°) 1.26 Ramachandran angles[f](#nt109){ref-type="table-fn"} %-favored 97.5% %-outliers 0% ----------------------------------------------------------------------------------- ----------------------- Values from the outermost resolution shell are given in parentheses. R~sym~ = Σ~i~\|I~i~−\<I\>\|/Σ~i~ I~i~ where I~i~ is the *i*th integrated intensity of a given reflection and \<I\> is the weighted mean of all measurements of I. R~work~ = Σ\|\|F~o~\|−\|F~c~\|\|/Σ\|F~o~\| for 95% of reflection data used in refinement. R~free~ = Σ\|\|F~o~\|−\|F~c~\|\|/Σ\|F~o~\| for 5% of reflection data excluded from refinement. Root-mean-square deviations from ideal geometry calculated by Procheck [@pone.0035203-Laskowski1]. Ramachandran plot analysis carried out using Molprobity [@pone.0035203-Chen1]. Analysis of interactions between biotin and streptavidin using Molegro viewer {#s4d} ----------------------------------------------------------------------------- The pdb files of the biotin complexes associated with 8-aa-loop-H127C mutein, wild-type streptavidin (2IZF) and traptavidin (2Y3F) [@pone.0035203-Chivers2] were used as the input files. Each of these files was imported to Molegro Molecular Viewer (Version 2.2.0) [@pone.0035203-Thomsen1]. Interaction energy analysis was performed using the ligand energy inspector module in the program. Before analysis, both the ligand and protein hydrogen bonding positions were optimized and the ligand was energy minimized using the action panel in the ligand energy inspector module. The protein-ligand interaction energy (E~inter~, sum of the steric interaction energy, hydrogen bonding energy, short- and long-range electrostatic interaction energies) was expressed in the form of the MolDock score [@pone.0035203-Thomsen1] in arbitrary units. A more negative value reflects a stronger interaction. Other methods {#s4e} ------------- Determination of kinetic parameters using BIAcore biosensor, preparation of streptavidin-agarose matrix and purification of biotinylated proteins were performed as previously described [@pone.0035203-Wu1], [@pone.0035203-Wu5]. The affinity column had a bed volume of 0.1 ml 8-aa-loop-H127C mutein coupled matrix. 200 µl sample containing 0.2 mg of biotinylated protein G or 0.44 mg biotinylated IgG from ThermoFisher was applied to the column. To purify biotinylated IgG from a mixture, HeLa cell extract containing 0.6 mg protein was mixed with 0.44 mg biotinylatyed IgG. This mixture was then applied to the affinity column for purification. The graphic drawings of the structure of the streptavidin-biotin complex shown in [Fig. 1](#pone-0035203-g001){ref-type="fig"} were generated by using the Yasara program [@pone.0035203-Krieger1], (YASARA Biosciences GmbH, Austria). To model the possible positions of loop~7--8~, the pdb file (1SWE) of the streptavidin-biotin complex was used as the input file. The amino acid residues (G113 and S122) at the base of loop~7--8~ were fixed. The possible conformations of loop were searched against the protein data bank using the loop modeling module in Yasara. Two modeled structures with loops in the upper and lower positions were selected as the input pdb files. These two input structures define the boundary of the loop movement. A pdb file containing 10 modelled structures generated from the Morph server [@pone.0035203-Krebs1] website was analyzed by Yasara to generate the animated structures shown in [Fig. 1c](#pone-0035203-g001){ref-type="fig"}. Supporting Information {#s5} ====================== ###### A complete tetramer of the 8aa-loop-H127C mutein is drawn, which each subunit drawn in a different color. The unit cell is drawn as a black box. The two-fold axes running through the origin are drawn according to standard crystallographic conventions. Three orthogonal views are drawn: each view is parallel to one of the three crystallographic axes. For each view, the two remaining axes orthogonal to axis being viewed down are labeled. (DOCX) ###### Click here for additional data file. We gratefully acknowledge access to crystal screening and data collection facilities at the Stanford Synchrotron Radiation Laboratory and the Canadian Light Source at various stages during the course of this project. **Competing Interests:**Eric Hommema and Greg T. Hermanson are employees of Thermo Fisher Scientific, Inc. Valerie J. O\'Sullivan and Mark Schofield were employees of Thermo Fisher Scientific, Inc. during the study. Mark Schofield is currently an employee of Pall Life Sciences. A US patent application to characterize the mutein has been made through Innovate Calgary, the technology transfer office of the University of Calgary. Thermo Fisher Scientific is interested in developing this product for marketing. This does not alter the authors\' adherence to all the PLoS ONE policies on sharing data and materials. **Funding:**The work was supported by Natural Sciences and Engineering Research Council of Canada, <http://www.nserc-crsng.gc.ca/Index_eng.asp>. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: VJO EH GTH MS IBN SCW KN SLW. Performed the experiments: VJO EH MS SCW CH IBN. Analyzed the data: VJO EH GTH MS IBN SCW CH KN SLW. Contributed reagents/materials/analysis tools: VJO EH MS GTH IBN KN SLW. Wrote the paper: VJO SCW IBN KN SLW. Manuscript editing: GTH MS. [^2]: Current address: Pall Life Sciences, Northborough, Massachusetts, United States of America

{ "pile_set_name": "PubMed Central" }

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22,259

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"complex", "shown", "Fig", "fig", "generated", "using", "Yasara", "program", "YASARA", "Biosciences", "GmbH", "Austria", "model", "possible", "positions", "pdb", "file", "complex", "used", "input", "file", "amino", "acid", "residues", "base", "fixed", "possible", "conformations", "loop", "searched", "protein", "data", "bank", "using", "loop", "modeling", "module", "Yasara", "Two", "modeled", "structures", "loops", "upper", "lower", "positions", "selected", "input", "pdb", "files", "two", "input", "structures", "define", "boundary", "loop", "movement", "pdb", "file", "containing", "modelled", "structures", "generated", "Morph", "server", "website", "analyzed", "Yasara", "generate", "animated", "structures", "shown", "Fig", "fig", "Supporting", "Information", "complete", "tetramer", "mutein", "drawn", "subunit", "drawn", "different", "color", "unit", "cell", "drawn", "black", "box", "axes", "running", "origin", "drawn", "according", "standard", "crystallographic", "conventions", "Three", 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INTRODUCTION ============ Spinal cord injury (SCI), which typically results from traffic accidents or long falls, is the most common cause of paralysis \[[@r1]\]. Two key mechanisms play roles in the pathology of SCI: a) primary injury resulting from bruising and compressive lesions, and b) secondary injuries, including inflammation, formation of glial scars, microvascular bleeding, and upregulation of apoptotic factors, all of which inhibit axonal regeneration \[[@r2], [@r3]\]. There are no effective therapies for patients with SCI, and novel treatments are urgently needed. Exosomes are nanosized extracellular vesicles 30--100 nm diameter which are formed as a result of inward budding in multiple cell types \[[@r4]\]. They were first discovered in sheep reticulocytes in 1983 \[[@r5]\]~,~ and the term "exosome" was coined by Johnstone in 1987 \[[@r6]\]. Exosomes may serve as biomarkers in many diseases due to the important role they play in intercellular communication and transport of proteins, mRNAs, and miRNAs into target cells \[[@r7], [@r8]\]. Exosomes can also have therapeutic effects in the nervous and respiratory systems \[[@r9]\]. Other studies demonstrated that exosomes derived from mesenchymal stem cells (MSCs) had therapeutic effects in liver, cardiovascular, and kidney diseases \[[@r10]--[@r12]\]. Furthermore, systemic administration of exosomes derived from MSCs promoted neurovascular reshaping and functional recovery after stroke in rats \[[@r13]\]. Exosomes can also reduce cognitive impairments after traumatic brain injury \[[@r14]\]. Recent research has demonstrated that exosomes containing large amounts of miRNA are pivotal for intercellular communication in the nervous system \[[@r15]\]. The miRNAs contained in MSC-derived exosomes also play important roles in intercellular signaling more generally \[[@r16]\]. Current knowledge indicates that the process by which miRNAs are loaded into exosomes is not random and differs in different cell types \[[@r17]\]. In addition, a recent study suggests that exosomes facilitate the transfer of miR-155 from smooth muscle cells to endothelial cells, which induces endothelial injury and promotes atherosclerosis \[[@r18]\]. Although neural stem cell-derived exosomes attenuate apoptosis and neuroinflammation after traumatic spinal cord injury \[[@r19]\], the mechanism underlying this effect remains unknown. Because insulin-like growth factor-1 (IGF-1) promotes regeneration after neural injury and proliferation in neural cells \[[@r20]--[@r22]\], we hypothesized that it may enhance the protective and regenerative effects of NSC-derived exosomes after SCI. The pathological effects of SCI are primarily a result of neuroinflammation, apoptosis, and oxidative stress injury \[[@r23]--[@r25]\]. We therefore established an H~2~O~2~-induced PC12 cell injury model to mimic SCI *in* *vitro* \[[@r26]\] and a rat SCI model using Allen's modified appliance *in* *vivo* \[[@r27], [@r28]\]. In this study, we examined the effects of IGF-1-induced (IGF-Exo) and normal exosomes (Nor-Exo) on neural inflammation, apoptosis, and regeneration after SCI and identified an miRNA-dependent mechanism responsible for those effects. RESULTS ======= Preparation and characterization of IGF-Exo ------------------------------------------- Neural stem cells were obtained from E15 fetal rat cerebral cortex and cultured in medium until neurospheres of similar sizes and shapes formed **(**Fig. 1A). These neurospheres were immunopositive for the NSC marker nestin (shown in red) ([Figure 1B](#f1){ref-type="fig"}). Rat NSCs collected from passage 3 were cultured in two kinds of culture medium: complete medium (DMEM/F12 medium supplemented with 20 ng/mL EGF, 10 ng/mL bFGF, 1× B27 supplement, 100 U/mL streptomycin, and 100 U/mL penicillin) or IGF-1 medium (100 ng/μL IGF-1 in complete medium). Exosomes were isolated from cell supernatants by ultracentrifugation. The shapes and sizes of both types of NSC-derived Exo were examined using transmission electron microscopy. While both the normal (Nor-Exo) and IGF-Exo had diameters of 30--300 nm, and the mean diameter of the IGF-Exo was slightly larger than that of the Nor-Exo ([Figure 1C](#f1){ref-type="fig"}). Western blot analysis indicated that levels of the exosomal markers CD9, CD63, and Alix were high in both Nor-Exo and IGF-Exo ([Figure 1D](#f1){ref-type="fig"}). Nanosight analysis of Exo size distributions revealed that the mean diameters of Nor-Exo and IGF-Exo were 101.1 ± 19.0 nm and 139.3 ± 34.0 nm, respectively ([Figure 1E](#f1){ref-type="fig"}). ![**Characteristics of neural stem cells (NSCs) and exosomes derived from NSCs.** (**A**) Morphology of neurospheres with typical shape examined by light microscopy. (**B**) Nestin immunofluorescence (red), a marker of NSCs, in neurospheres. (**C**) Exosome morphology examined by transmission electron microscopy. (**D**) Western blot analysis of exosome surface markers. (**E**) Particle size distribution of Nor-Exo and IGF-Exo by Nano Sight.](aging-11-102568-g001){#f1} IGF-Exo inhibits apoptosis and promotes regeneration in neural cells -------------------------------------------------------------------- We investigated the protective effect of IGF-Exo in PC12 cells treated with H~2~O~2~ to establish a cellular model of neural injury. CCK-8 assays showed that the 50% lethal dose of H~2~O~2~ was 200 μM for 24h in PC12 cells ([Supplementary Figure 1A](#SD2){ref-type="supplementary-material"}), and the optimal dose for IGF-1 in NSC culture was 200 ng/mL for 24h ([Supplementary Figure 1B](#SD2){ref-type="supplementary-material"}). After 24h of treatment with 200 μM H~2~O~2~, PC12 cells had damaged axons and decreased in number compared with the sham group. Additionally, cell viability was higher and axons were longer in IGF-Exo group PC12 cells than in the injury model group or Nor-Exo group (*P \<* 0.05) ([Figure 2A](#f2){ref-type="fig"}, [2B](#f2){ref-type="fig"}). In the TUNEL immunofluorescence assay, the ratio of TUNEL-positive cells in the IGF-Exo group was much higher than in the injury model group and slightly higher than in the Nor-Exo group (*P* \< 0.05) ([Figure 2C](#f2){ref-type="fig"}, [2D](#f2){ref-type="fig"}). To further explore the relationship between IFG-Exo and neural cell apoptosis, we measured Bax, Bcl-2, Beclin-1, and caspase-3 levels in the four PC12 cell groups. Compared with the sham group, Bax, Beclin-1, and caspase-3 expression were increased, while Bcl-2 expression decreased, in the injury group significantly increased (*P* \< 0.05). Bax, Beclin-1, and caspase-3 expression were also higher in the IGF-Exo group than in the injury group or the Nor-Exo (*P* \< 0.05) ([Figure 2E](#f2){ref-type="fig"}--[2I](#f2){ref-type="fig"}). ![**IGF-Exo inhibited H~2~O~2~-induced neural apoptosis in PC12 cells *invitro*.** (**A**) Morphology for each experimental group examined by light microscopy (control group: PC12 cells without treatment; H~2~O~2~ group: PC12 cells treated with H~2~O~2~ for 24h; Nor-Exo group: PC12 cells pretreated with Nor-Exo for 24h followed by H~2~O~2~ for 24h; IGF-Exo group: PC12 cells pretreated with IGF-Exo for 24h followed by H~2~O~2~ for 24h). (**B**) Cell viability in each experimental group; (**C**) TUNEL staining (red) indicating cell apoptosis in each experimental group; nuclear DAPI stain in blue. (**D**) TUNEL-positive cell numbers per field for each experimental group. (**E**) Western Blot analysis of apoptotic and anti-apoptotic proteins. (**F**--**I**) Relative expression of BAX, Bcl-2, Beclin-1 and Caspase-3. Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls *post hoc* test). \*\**P* \< 0.01 *vs*. control group; \#\#*P* \< 0.01 *vs*. H~2~O~2~ group.](aging-11-102568-g002){#f2} IGF-Exo reduces lesion size and promotes functional recovery after SCI ---------------------------------------------------------------------- To test the neuroprotective effect of IGF-Exo *in* *vivo*, we established a rat model of acute SCI using a modified Allen's weight drop apparatus ([Supplementary Figure 2](#SD2){ref-type="supplementary-material"}). Rats received systemic IGF-Exo, sham, PBS, or Nor-Exo injections via the tail vein ([Figure 3A](#f3){ref-type="fig"}). BBB locomotor scores were markedly lower in SCI group rats than in sham rats at 1, 3, 7, 14, and 28 days after SCI (*P \<* 0.01). Compared with the SCI group, BBB scores were higher in Nor-Exo and IGF-Exo rats at 7, 14, and 28 days after SCI (*P* \< 0.05). Moreover, the scores of IGF-Exo rats were higher than those of Nor-Exo rats at 14 and 28 days post-SCI (*P \<* 0.05) ([Supplementary Figure 3](#SD2){ref-type="supplementary-material"}). MRI and neuroelectrophysiological examinations were also used to evaluate whether IGF-Exo promoted recovery from SCI recovery. DTI indicated that reconnection of the neural fasciculus was increased in the IGF-Exo group compared to the Nor-Exo and SCI groups ([Figure 3B](#f3){ref-type="fig"}, [Supplementary Figures 4](#SD2){ref-type="supplementary-material"}, [5](#SD2){ref-type="supplementary-material"}). Neuroelectrophysiological examination revealed that MEP amplitudes were higher in the IGF-Exo group than in the Nor-Exo and SCI groups ([Figure 3C](#f3){ref-type="fig"}, [3D](#f3){ref-type="fig"}). In longitudinal sections of rat spinal cords collected for HE staining 28 days after SCI, injury areas (cavity) were smaller in the IGF-Exo group than in the SCI and Nor-Exo groups (*P \<* 0.05) ([Figure 3E](#f3){ref-type="fig"}, [3F](#f3){ref-type="fig"}). Immunostaining analysis of BrdU, a marker of new neurons, and NeuN, a general neural marker, revealed that BrdU and NeuN staining intensity were both lower in IGF-Exo group lesion areas than in the SCI and Nor-Exo groups at 28 days after SCI (*P \<* 0.05) ([Figure 3G](#f3){ref-type="fig"}). In a TUNEL assay performed 3 days after SCI, the TUNEL-positive cell (red) ratio was higher in the IGF-Exo group than in the injury model group and slightly higher than in the Nor-Exo group (*P* \< 0.05) ([Figure 3H](#f3){ref-type="fig"}, [3J](#f3){ref-type="fig"}). ![**IGF-Exo inhibited neural apoptosis and neuroinflammation after SCI *invivo*.** (**A**) Schematic of tail intravenous injections of Nor-Exo and IGF-Exo in SCI model rats. (**B**) DTIs constructed for the SCI, Nor-Exo, and IGF-Exo groups at 1 day and 28 days after surgery. (**C**) Neuroelectrophysiological examination results for each experimental group (control, SCI, Nor-Exo, and IGF-Exo) at 3 days and 28 days after surgery. (**D**) MEP amplitudes for each experimental group at 3 days and 28 days after surgery. (**E**) Hematoxylin-Eosin staining of sections containing SCI lesions in each experimental group at 3 days and 28 days after surgery. (**F**) Cavity space percentages for each experiment group at 3 days and 28 days after surgery. (**G**) BrdU and NeuN immunofluorescence indicative of neuron regeneration in each experimental group at 28 days after surgery. (**H**) TUNEL staining (green) indicative of apoptosis after SCI in each experimental group; DAPI in blue. (**J**) Numbers of TUNEL-positive cells per field for each experimental group. (**K**--**O**) Western Blot analysis of pro- and anti-apoptotic proteins (BAX, Bcl-2, Beclin-1, and Caspase-3). Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls *post* *hoc* test). \*\*P \< 0.01, *vs*. control group; \#\#P \< 0.01, *vs*. SCI group.](aging-11-102568-g003){#f3} Western blot analysis of Bax, Bcl-2, Beclin-1, and caspase-3 levels in the four groups of SCI rats were used to assess the anti-apoptosis effects of IGF-Exo *in* *vivo*. Compared with the sham group, Bax, Beclin-1, and caspase-3 expression increased, while Bcl-2 expression decreased, in the SCI group (*P* \< 0.05). However, Bax, Beclin-1, and caspase-3 expression was significantly higher in the IGF-Exo group than in the SCI and Nor-Exo groups (*P* \< 0.05) ([Figure 3K](#f3){ref-type="fig"}--[3O](#f3){ref-type="fig"}). Antiapoptotic miR-219a-2-3p is highly increased in IGF-Exo ---------------------------------------------------------- To illuminate the mechanism underling the neuroprotective effects of IGF-Exo, we characterized exosomal miRNA content though miRNA sequencing analysis. Six miRNAs were upregulated and 2 were downregulated in IGF-Exo compared to Nor-Exo ([Figure 4A](#f4){ref-type="fig"}--[4C](#f4){ref-type="fig"}; [Supplementary Figure 6](#SD2){ref-type="supplementary-material"}). qRT-PCR analysis was then performed to confirm that expression was higher for the 6 upregulated miRNAs identified in the sequencing analysis. All 6 miRNAs, namely rno-miR-138-5p (P \< 0.05), rno-miR-219a-2-3p (P \< 0.05), rno-miR-92b-3p (P \< 0.01), rno-miR-92b (P \< 0.05), rno-miR-25-5p (P \< 0.05), and rno-miR-674-3p (P \< 0.01), were significantly upregulated in IGF-Exo compared with Nor-Exo ([Figure 4D](#f4){ref-type="fig"}). ![**Screening and identification of upregulated miRNAs in Nor-Exo and IGF-Exo.** (**A**) Heat map showing the 6 miRNAs for which expression was upregulated ≥ 2-fold in IGF-Exo compared to Nor-Exo. (**B**) IGF-1-induced miRNA regulation network. (**C**) Among the 6 upregulated miRNAs, miR-219a-2-3p expression increased the most. (**D**) qRT-PCR comparison of rno-miR-219a-2-3p, 138-5p, 92b-3p, 92b, 25-5p, and 674-3p expression between Nor-Exo and IGF-Exo from 3 independent experiments. \*P \< 0.05, \*\*P \< 0.01 for IGF-Exo *vs*. Nor-Exo.](aging-11-102568-g004){#f4} Neuroprotective effects of IGF-Exo after SCI are associated with miR-219a-2-3p-dependent inhibition of YY1 ---------------------------------------------------------------------------------------------------------- We screened possible target genes of miR-219a-2-3p using bioinformatics methods (Target Scan v7.2) and identified YY1 was the potential target ([Figure 5A](#f5){ref-type="fig"}). We therefore constructed a YY1-3′UTR (3′-Untranslated Region) expression vector containing a luciferase gene and transfected it into PC12 cells. A conventional luciferase reporter assay showed that miR-219a-2-3p interacted with YY1 ([Figure 5A](#f5){ref-type="fig"}). Additionally, qRT-PCR showed that YY1 expression decreased when miR-219a-2-3p was upregulated ([Figure 5B](#f5){ref-type="fig"}). Furthermore, YY1 expression increased when miR-219a-2-3p was downregulated ([Figure 5C](#f5){ref-type="fig"}). Blockage of miR-219a-2-3p by Anti-miR-219a-2-3p (the miR-219a-2-3p inhibitor) inhibited IGF-Exo-mediated neuroprotective effects ([Figure 5D](#f5){ref-type="fig"}). CCK-8 assays, TUNEL staining, and Western blots were performed to confirm that miR-219a-2-3p protected against neural injury and inhibited resulting apoptosis ([Figure 5E](#f5){ref-type="fig"}--[5G](#f5){ref-type="fig"}). The results indicated that miR-219a-2-3p expression downregulated, while miR-219a-2-3p knockdown upregulated, the YY1/NF-kB-p65 axis in PC12 cells ([Figure 5H](#f5){ref-type="fig"}--[5J](#f5){ref-type="fig"}). ![**Inhibition of miR-219a-2-3p in exosomes reduced the protective effects of IGF-Exo.** (**A**) Luciferase activity was detected in PC12 cells co-transfected with pGL3 vector, pGL3-YY1, miR-219a-2-3p mimics, or miR-219a-2-3p mutant. (**B**) Relative YY1 expression after upregulation of miR-219a-2-3p measured by qRT-PCR. B) PC12 cells were transfected with miR-219a-2-3p mimics or miR-NC; relative YY1 expression was analyzed by qRT-PCR. (**C**) PC12 cells were transfected with Anti-miR-219a-2-3p (miR-219a-2-3p inhibitor) or Anti-NC (control); relative YY1 expression was analyzed by qRT-PCR. (**D**) Morphology in each experimental group examined by light microscopy (control group: PC12 cells without treatment; H~2~O~2~ group: PC12 cells treated with H~2~O~2~ for 24h; IGF-Exo group: PC12 cells pretreated with IGF-Exo for 24h followed by H~2~O~2~ for 24h); Anti-miR-219a-2-3p-IGF-Exo group: PC12 cells pretreated with miR-219a-2-3p inhibitor-transfected IGF-Exo for 24h followed by H~2~O~2~ for 24h). (**E**) Cell viability in each experimental group when miR-219a-2-3p was downregulated in IGF-Exo; (**F**) TUNEL staining (red) indicative of apoptosis in each experimental group; DAPI in blue. (**G**) Numbers of TUNEL-positive cells per field for each experiment group. (**H**--**J**) Western Blot analysis of YY1 and NF-kB-p65 (si-NC: siRNA blank vector; miR-NC: miRNA blank vector; si-YY1: YY1 siRNA; miR-219a-2-3p: miR-219a-2-3p mimics). Data are expressed as means ± SD (analysis of variance followed by Student-Newman-Keuls *post hoc* test). \*\**P* \< 0.01, *vs*. control group; \#\#*P* \< 0.01, *vs*. SCI group.](aging-11-102568-g005){#f5} DISCUSSION ========== Although basic research and clinical studies have attempted to identify more effective treatments for SCI patients, the prognosis remains very poor \[[@r29]\]. The pathological processes involved in SCI include neuroinflammation, apoptosis, and inhibition of neural regeneration \[[@r30], [@r31]\]. A previous study revealed that exosomes derived from MSC and NSC stem cells suppress inflammation and apoptosis after neural injury. However, the mechanisms underlying these effects remain unknown. Here, we used exosomes derived from NSCs exposed to IGF-1 to treat SCI for the first time and demonstrated that these IGF-Exo enhanced the neuroprotective effects of exosomes by inhibiting apoptosis and neuroinflammation in an miR-219a-2-3p-dependent manner. Several studies have demonstrated that stem cells (especially MSCs and NSCs) administered either systemically or via local transplantation are potential therapies for neural injury and nervous system diseases \[[@r32]\]. However, stem cell therapies face important challenges. Tumorigenesis due to chromosome instability and distal vascular occlusion resulting from the large size of stem cells can be fatal, and the survival rate of stem cells *in* *vivo* is relatively low \[[@r33], [@r34]\]. Fortunately, increasing evidence suggests that the biological effects of stem cells can mostly be attributed to molecules they secrete, including those secreted via exosomes \[[@r9], [@r35]\]. Recent studies indicated that exosomes derived from NSCs might aid in the discovery of treatments for central nervous system injury \[[@r36], [@r37]\], but the mechanisms underlying their protective effects are unknown, and NSCs produce relatively small quantities of exosomes. IGF-1 improves proliferation and metabolism in neural cells, and we confirmed this IGF-1-induced increase in NSC cell viability here in a CCK-8 assay. Exosomes play an important role in intercellular communication by transmitting bioactive RNAs and proteins, and exosomes derived from NSCs have therapeutic effects against ischemia and neurodegeneration \[[@r38], [@r39]\]. Because exosomes derived from NSCs can reduce apoptosis and neuroinflammation after SCI, we hypothesized that IGF-Exo could enhance these protective effects and promote regeneration after SCI. In this study, RNA sequencing of Nor-Exo and IGF-Exo identified 8 microRNAs that were differentially expressed between the two exosome types, and miR-219a-2-3p was determined to the most significant among them. The difference in miR-219a-2-3p expression between Nor-Exo and IGF-Exo was verified by qRT-PCR assay. Previous research indicated that miR-219a-2-3p is enriched in human white matter and oligodendrocytes, and may be a potential target in demyelinating disorders \[[@r40]\]. Moreover, bioinformatics screening revealed that miR-219a-2-3p interacts with the downstream gene YY1 \[[@r41]\]. Here, a luciferase assay confirmed that up-regulation of miR-219a-2-3p inhibited YY1 expression, while changes in YY1 expression had no effect on the expression of miR-219a-2-3p. However, YY1 expression is correlated with activity of the inflammatory NK-kB pathway, and up-regulation of YY1 enhanced inflammatory effects. Therefore, IGF-Exo likely inhibits the expression of YY1, and in turn NK-kB pathway activity, through the up-regulation of miR-219a-2-3p, thus inhibiting neuroinflammation and promoting neuroprotective effects. This mechanism was confirmed in a rescue experiment in which miR-219a-2-3p expression was inhibited by transfection of si-miR-219a-2-3p. Some important limitations of this study should be considered when interpreting the results and should be addressed in future studies. Firstly, additional *in* *vitro* studies are needed to fully characterize the mechanism underlying the neuroprotective effects of IGF-Exo, and their efficacy should be confirmed in clinical trials. Secondly, IGF-Exo might exert neuroprotective effects and inhibit the apoptosis and inflammation via the action of other miRNAs or pathways, and additional downstream genes of miR-219a-2-3p should be examined further. Finally, additional studies are needed to verify that the effects of miR-219a-2-3p on cell apoptosis and neural regeneration observed here are applicable in other *in* *vitro* cell models and under different conditions. In summary, this study demonstrated that exosomes derived from neural stem cells exposed to IGF-1, at least partly, suppress the nerve inflammation, inhibit apoptosis, and promote nerve regeneration via an miR-219a-2-3p-dependent mechanism after the spinal cord injury. This is the first microRNA mechanism to be identified that might explain the effects of NSC-derived exosomes in SCI and may inform the development of novel treatments for spinal cord injury. MATERIALS AND METHODS ===================== Animals ------- Adult female Sprague-Dawley rats (180--200g) and pregnant rats (15d) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal care and experimental protocols were approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force. All animals were housed in individual cages on a 12h light/dark cycle with ad-libitum access to food and water. Isolation and culture of rat embryonic NSCs ------------------------------------------- 15-day embryos were isolated from pregnant rats euthanized with 230 mg/kg sodium pentobarbital. Cerebral cortex tissues were then isolated from the embryos and placed in Hank's solution. After the meninges were removed, the cerebral cortex was washed twice with Hank's solution, cut into pieces, digested with accutase cell detachment solution at 37°C for 10 min, and dispersed by pipetting. The suspension was filtered with a 200-mesh screen and then centrifuged at 800 rpm for 5 min; NSCs were obtained from the pelleted material. The cells were then re-suspended in complete medium (DMEM/F12 medium supplemented with 20 ng/ml EGF, 10 ng/ml bFGF, 1 × B27 supplement, 100 U/mL streptomycin, and 100 U/mL penicillin). After cell density was adjusted to 1 × 10^5^/mL, cells were seeded into culture flasks and incubated at 37°C with 5% CO~2~. After 3-5 days, cells were passaged. All NSCs used in this study were from passage 3. Isolation and purification of NSC-Exo ------------------------------------- Exosomes were isolated from NSC supernatant as previously described \[[@r42]\]. The medium was then collected and centrifuged at 300 × g for 10 min, followed by centrifugation at 2000 × g for 20 min at 4°C. After centrifugation, the supernatant was filtered using a 0.22 μm filter to remove dead cells and large cellular debris. Small cellular debris were then pelleted by centrifugation at 10,000 × g for 30 minutes, and the supernatants were recentrifuged at 100,000 × g for 70 minutes. Finally, pelleted exosomes were resuspended in 100 μL PBS solution in the ultracentrifuge tubes. Exosomes were quantitated by measuring total protein concentration using a bicinchoninic acid assay (BCA; Thermo Fisher). Identification of NSC-Exo ------------------------- The morphology of NSC-derived exosomes (NSC-Exo) was assessed by transmission electron microscopy (TEM, Hitachi HT7700, Tokyo, Japan). Nanosizer^TM^ technology (Malvern Instruments, Malvern, UK) was used to analyze the diameter distribution of NSC-Exo. Western blotting was used to examine the specific surface markers encapsulated in the exosomes, including CD9, CD63, and Alix. *In vitro* experiment design ---------------------------- In the first *in vitro* experiment, PC12 cells were divided among the following four groups to verify the protective effect of IGF-Exo: control group (untreated PC12 cells), H~2~O~2~ group (PC12 cells treated with H~2~O~2~ for 24h), Nor**-**Exo group (PC12 cells pretreated with Nor-Exo for 24h followed by H~2~O~2~ for 24h), and the IGF**-**Exo group (PC12 cells pretreated with IGF-Exo for 24h followed by H~2~O~2~ for 24h). In the second *in vitro* experiment, PC12 cells were divided among the following four groups to verify the effects of miR-219a-2-3p: control group (untreated PC12 cells), H~2~O~2~ group (PC12 cells treated with H~2~O~2~ for 24h), NC**-**IGF**-**Exo group (PC12 cells pretreated with NC-IGF-Exo derived from IGF-1-induced NSCs transfected with miR inhibitor negative control (Anti-NC) for 24h followed by H~2~O~2~ for 24h), and Anti**-**miR**-**219a**-**2**-**3p**-**IGF**-**Exo group (PC12 cells pretreated with IGF-Exo derived from IGF-1-induced NSCs transfected with miR-219a-2-3p inhibitor (Anti- miR-219a-2-3p) for 24h followed by H~2~O~2~ for 24h). *In vivo* experiment design --------------------------- Adult female Sprague-Dawley rats were divided among the following four groups (n=8 per group): control group (spinal cord segment T10 was only exposed in a sham operation), SCI group (acute SCI via modified Allen's weight drop apparatus, 0.5 mL PBS systemic administration through tail vein injection after SCI), Nor**-**Exo group (100 μg of precipitated normal exosome protein in 0.5 mL of PBS was administered via tail vein injection after SCI), and IGF**-**Exo group (100 μg of precipitated IGF-1-induced exosome protein in 0.5 mL of PBS was administered via tail vein injection after SCI). Cell viability assay -------------------- The viability of PC12 cells and primary neurons was evaluated with a CCK-8 assay (Dojindo, Kumamoto, Japan) to examine the effects of NSC-Exo on neural cell proliferation. The cells were rinsed three times with 1 × PBS after 0, 24, 48, and 72 h of incubation. 10 μL of CCK-8 solution diluted in 90 μL of fresh culture medium was then added followed by incubated for another 2 h at 37°C. Quantitative real-time RT-PCR ----------------------------- Total RNA was isolated from cells and tissues using Trizol reagent (TransGen Biotech, Beijing) according to the manufacturer's instructions. For RNase R treatment, 2 mg total RNA was incubated for 15 min at 37°C with or without 3 U/mg RNase R (Epicentre Technologies, Madison, WI, USA). qRT-PCR was performed using SYBR Green (TransGen Biotech) according to the manufacturer\'s instructions ([Table 1](#t1){ref-type="table"}) and an ABI 7500 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). ###### Primers of miRNA used for qPCR in this study. -------------- ------------------------------------------- **Primer** **Sequence(5′to3′)** miR-138-5p Forward: ACACTCCAGCTGGGAGCtGGtGttGTGAATC Reverse: TGGTGTCGTGGAGTCG miR-219-2-3p Forward: ACACTCCAGCTGGGAGAATTGTGGCTGGAC Reverse: TGGTGTCGTGGAGTCG miR-25 Forward: ACACTCCAGCTGGGAGGCGGAGACACGGGC Reverse: TGGTGTCGTGGAGTCG miR-674-3p Forward: ACACTCCAGCTGGGCACAGCtCCCATCTCA Reverse: TGGTGTCGTGGAGTCG miR-92b Forward: ACACTCCAGCTGGGAGGGACGGGACGCGGTGC Reverse: TGGTGTCGTGGAGTCG miR-92b-3p Forward: ACACTCCAGCTGGGTATTGCACtCGTCCCG Reverse: TGGTGTCGTGGAGTCG YY1 Forward: AGTGGGAACAGAAGCAGGTG Reverse: GAGGTCAATGCCAGGTATCC -------------- ------------------------------------------- Transfection ------------ Rat miR-219a-2-3p mimics, anti-miR-219a-2-3p (miR-219a-2-3p inhibitor), si-YY1 (si-YY1), miR mimics negative control (miR-NC), miR inhibitor negative control (miR-NC), and si-YY1 negative control (si-NC) were purchased from GenePharma (Shanghai, China). Sequences are listed in [Table 2](#t2){ref-type="table"}. ###### miRNA mimics, inhibitors, and siRNAs used in this study. ---------------------------------------------- ------------------------ **Name** **Sequence(5′to3′)** miR-219a-2-3p mimics AGAAUUGUGGCUGGACAUCUGU miR-219a-2-3p inhibitor (Anti-miR-219a-2-3p) ACAGAUGUCCAGCCACAAUUCU siRNA-YY1(si-YY1) GGUAAUAAGAAGUGGGAACTT miR mimics negative control (miR-NC) UUGUACUACACAAAAGUACUG miR inhibitor negative control (Anti-NC) CAGUACUUUUGUGUAGUACAA si-YY1 negative control(si-NC) UUCUCCGAACGUGUCACGUTT ---------------------------------------------- ------------------------ Cell apoptosis assays --------------------- PC12 cells were incubated with or without NSC-Exo (100 μg/mL) after treatment with H~2~O~2~ (200 μM) for 24 h. A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL; Roche, Basel, Switzerland) was used to detect DNA strand breaks according to the manufacturer's instructions. Images were captured using a fluorescence microscope (Leica, Solms, Germany). Apoptotic neuronal cells were determined by counting total numbers of TUNEL- and DAPI-stained cells. Western blot analysis --------------------- Protein lysates were extracted from cells and 5 mm blocks of injured spinal cord tissue encompassing the lesion collected on days 3 and 28 after SCI by incubating with RIPA lysis and extraction buffer (KeyGEN Biotechnology, China). Protein concentration was determined using BCA. Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (EMD Millipore Corp., USA), incubated overnight at 4°C with primary antibodies, and blocked with bovine serum albumin (5%, v/v). The primary antibodies included Bax (1:2000, rabbit IgG; Abcam, USA), Bcl-2 (1:1000, rabbit IgG; Abcam, USA), Beclin-1 (1:1000, rabbit IgG; Abcam, USA), caspase-3 (1:1000, rabbit IgG; Abcam, USA), GAPDH (1:1000, Abcam, USA), YY1 (1:1000, mouse IgG1; Abcam, USA), and p65 (1:1000, rabbit IgG; Abcam, USA). The membranes were then incubated with secondary antibody (1:2000, Proteintech, USA) for 120 min at room temperature. Protein bands were visualized using enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology, Nanjing, China), and band density was analyzed semi-quantitatively using Image J software. Immunofluorescence staining --------------------------- For immunofluorescence staining, 1.0 x 10^5^ NSCs were cultured in 0.5 mL media unless otherwise specified. Cultured cells were fixed with 4% (wt/vol) paraformaldehyde for 30 min at room temperature, washed with 0.1 M Tris-buffered solution (pH 7.5, TBS), blocked with 10% (vol/vol) normal goat serum in TBS containing 0.3% (vol/vol) Triton X-100 at room temperature for 60 min, and incubated with primary antibodies at 4°C overnight. The primary antibodies were rabbit anti-Nestin (1:1000), rabbit anti-BrdU (1:2000), and donkey anti-NeuN (1:1000). The cells were washed with TBS and incubated with Alexa Fluor 488- and 555-conjugated secondary antibodies (1:1000, Proteintech) at room temperature for 60 min. To visualize nuclei, the cells were counterstained with 2 μg/mL 4\',6-diamidino-2-phenylindole (DAPI). Finally, the cells were mounted with 80% (vol/vol) glycerol, visualized under a fluorescent microscope (IX81, Olympus Corp., Tokyo, Japan), and image data were processed using MetaMorph (Molecular Devices, Sunnyvale, CA, USA) for quantification. Numbers of positive cells were counted in three to five fandom fields per well for three separate wells in each individual experiment. Behavioral tests ---------------- Hindlimb motor function assessments were performed 1, 3, 7, 14, and 28 days after SCI using the Basso Beattie Bresnahan (BBB) locomotor rating scale and slanting board test. The BBB score ranged from 0 (complete hindlimb paralysis) to 21 (normal locomotion). Scores were assigned by assessing the motor capacity of the experimental rats, including the movement of the hindlimbs, weight-bearing, and coordination of forelimb and hindlimb movements. The rats were placed in an open field of wooden models for 4 min and scored by 2 researchers using blinded methods. The slanting board test was performed according to the Rivlin method and involved placing a rat on a rectangular wooden oblique board covered with a rubber pad; the oblique plate was rotated to measure the angle of the inclined plate. When the longitudinal axis of the rat's body was parallel to the longitudinal axis of the inclined plate, the rat was tilted toward the side of the tilted plate to raise it in 5° increments. The maximum angle at which a rat could maintain its position for 5 s was measured. Measurements were performed five times for each animal, and the average values were analyzed. Statistical analysis -------------------- The data are presented as means ± standard deviation (SD) and were analyzed using one-way analysis of variance and Tukey's *post* *hoc* multiple comparison tests. All experimental data were analyzed using SPSS 17.0. *P-*values \< 0.05 were considered statistically significant. Additional experimental materials and methods details, including electrophysiological nerve and MRI assessments, are provided in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. Supplementary Material ====================== **AUTHOR CONTRIBUTIONS:** Ke Ma and Huiyou Xu performed statistical analysis and wrote the manuscript. Jian Zhang and Fei Zhao were involved in manuscript preparation. Renjie Wang and Xuyi Chen were responsible for study design and data collection. Haiqian Liang, Hongtao Sun, Ping Li, Sai Zhang, and Xuyi Chen were responsible for research funding. All authors approved the final version of the manuscript. **CONFLICTS OF INTEREST:** None of the authors declare any conflicts of interest. **FUNDING:** This study was support by the National Key Research and Development Plan of China (No. 2016YFC1101500), the National Natural Science Foundation of China (No. 11672332), the Key Science and Technology Support Foundation of Tianjin City (No. 17YFZCSY00620), the Major Project of Tianjin Science and Technology Military and Civilian Integration (No. 18ZXJMTG00260), and the Project of Tianjin Rescue Medicine Clinical Center (No. 15ZXLCSY00040). [^1]: Equal contribution

{ "pile_set_name": "PubMed Central" }

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"sybr", "epicentr", "challeng", "design", "develop", "plan", "indic", "microvascular", "mirbp", "c", "agaauuguggcuggacaucugu", "sulfat", "dead", "regener", "locomot", "format", "field", "′untransl", "natur", "side", "possibl", "neun", "thermo", "hoinduc", "jfreftypefig", "bp", "rivlin", "manuscript", "vivo", "might", "diseas", "dfreftypefig", "pipet", "r", "investig", "mimic", "analysi", "g", "could", "embryo", "intercellular", "bca", "axon", "nanj", "action", "green", "motor", "vital", "liver", "glycerol", "varianc", "especi", "region", "apoptot", "primer", "scar", "neg", "dna", "oper", "acut", "behavior", "mg", "sham", "break", "obliqu", "rpm", "tb", "injuri", "increas", "access", "improv", "mirap", "softwar", "incub", "report", "maintain", "similar", "shape", "×", "genepharma", "isol", "inject", "downstream", "tukey", "exo", "summari", "treftypet", "area", "polyacrylamid", "termin", "bcl", "hoc", "previou", "attribut", "therefor", "suppress", "yy′utr", "cognit", "sightaginggf" ]

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1. Case Report {#sec1} ============== A 48-year-old male finished Triple-Ironman distance in 41 hours and 18 minutes (11.4 km swimming, 540 km cycling, and 126 km running). At the time of the examinations, he had been practising his current diet of raw vegan diet for 6 years. Prior to this, the vegan athlete had been living as a vegan for 3 years and as a vegetarian for the previous 13 years. All last competitions were performed only based on a raw diet. At the time of both examinations, the vegan athlete was 48 years of age and 1.80 metres in height. In the sporting season he was 79.7 kg in weight, with a body fat index of 12.9%; in the off-season he weighed 80.3 kg with a body fat index of 16.3%. Clinical examination showed a regular heart rhythm at 60 beats/min. Blood pressure was 115/70. The heart sounds were normal. Prior to the spiroergometry, echocardiography was performed based on ASA criteria (1). For comparison purposes, we refer to the values for 10 Ironman triathletes of similar age living on a mixed diet. The results of the spiroergometry are shown in [Table 1](#tab1){ref-type="table"}, those of echocardiography are in [Table 2](#tab2){ref-type="table"}, and blood analysis findings are presented in [Table 3](#tab3){ref-type="table"}. The athletes of the control group were aged 47.4 ± 5.2, weighed 76.2 ± 8.9 kg (with 13.4 ± 2.0% body fat), and were 1.816 metres ±6.6 cm in height. In the active phase the vegan athlete was training on average 18 hours per week, consisting of 2 hours of swimming, 11 hours of cycling, and 5 hours running. This involved covering distances of 5 km (swimming), 330 km (cycling) and 50 km (running). The athletes of the control group were training for a total of 15.9 ± 2.1 hours weekly, involving 2.5 ± 0.7 hours and 5.6 ± 1.5 km swimming, 8.6 ± 1.5 hours and 215.5 ± 53.0 km cycling, and 4.9 ± 0.7 hours and 55.2 ± 6.5 km running. In terms of performance diagnostics, the vegan athlete showed comparable VO~2~max, VO~2~ at VAT, and %VO~2~max at VAT values as compared with the control group (statements regarding significant differences are not possible). VO~2~ and %VO~2~max at RCP were somewhat higher for the vegan athlete. The maximum ergometric performance is higher for the vegan athlete in absolute terms but not relative to the body weight. At RCP the ergometric performance of the vegan athlete is somewhat higher than that of the control group. The vegan athlete had lower pulse rates at rest, at VAT, at RCP, and at the endurance limit. In comparison with the off-season, in the active season the vegan athlete had a higher maximum ergometric performance, VO~2~, and %VO~2~max, as well as cardiac frequency at RCP. In morphological terms, the vegan athlete showed a greater left ventricular end diastolic diameter, with consecutively higher end diastolic volume and stroke volume. The systolic and diastolic functions seem to be similar for the vegan athlete and the control group. Besides a mild thrombopenia, slightly higher CK-NAC levels in the active season and a slight drop of free testosterone (in both examinations) all remaining values were within normal ranges. 2. Discussion {#sec2} ============= It is well known that a vegetarian or vegan diet, when sensibly managed, can make a contribution to the prevention and therapy of illnesses in all phases of life \[[@B1]\]. More and more people are thus adopting a vegetarian (2) or vegan (2) lifestyle. It is equally understandable that such people may want to take part in endurance sports. It follows that the effects of vegan diet on sporting performance are a matter of general interest. This case report is set out to investigate an ultra endurance triathlete, who had been living on a vegetarian diet since 22 years and a purely vegan diet for 9 years, with reference to his ability to perform, cardiac status, and any symptoms of deficiency. It was also a matter of interest for the study to ascertain whether his performance parameters were different from those of mixed diet triathletes. Even competitive athletes can adopt a diet of this kind with a view to the health benefits, without any risk of suffering from dietary deficiencies \[[@B2]--[@B4]\]. With its disciplines of swimming, cycling, and running, the triathlon is a very popular type of endurance sport. As well as competitions involving classic sprint distances (0.5, 20 and 5 km), short distances (1--1.5, 40, and 10 km), and medium distances (ca. 2, 80--90, and 21.2 km), it also offers competitions involving ultra distances: Ironman (3, 8, 180, and 42.2 km) and multiple (2× until 10×) Ironman. As is the case with other types of endurance sport, triathletes undergo various physical adaptations in the course of training. These include an increased cardiac output under stress and increased arteriovenous oxygen extraction with consecutive rises in the maximum oxygen intake (VO~2~max) \[[@B5]\]. In echocardiographic terms, triathletes show physical changes similar to those of cyclists, resembling a mixed state of static/dynamic stress and involving a significant increase in the left ventricular muscle mass and internal left ventricular diameter \[[@B6]\]. In terms of spiroergometry, successful triathletes not only show an increased oxygen intake, but they also demonstrate a capacity for continuous high performance over long periods, as a result of effective internal economy, high fractional extraction of VO~2~max at the given stress level (%VO~2~max), and a high anaerobic threshold \[[@B7]\]. Good indicators of the limit of endurance are the ventilatory anaerobic threshold (VAT) and the respiratory compensation point (RCP) \[[@B7], [@B8]\]. It is not known whether or to what extent triathletes living on a vegan or vegetarian diet differ from triathletes living on a mixed diet. It is known that nonsporting vegans show better metabolic parameters than mixed diet athletes (9). To the best of our knowledge, no data are to be found in the extant literature on the spiroergometric, echocardiographic, or haematological parameters of ultra endurance athletes living on a vegan diet. As a result, this is a ground-breaking case report involving the first ever presentation of the characteristic parameters of a vegan ultra endurance athlete. In view of the equivalence in terms of age and other similar anthropometric features, the data for the vegan athlete are well comparable with those of the control group. On the other hand, we are dealing here with an individual case, so the meaningfulness of the results, in the nature of the case, remains limited. Both the vegan athlete and the control group trained for a comparable number of hours. The vegan athlete\'s cycling training, on the other hand, was more intense and more prolonged. Comparison of the competition results would be a way of checking this, but in view of the fact that the athletes\' preferred competition distances vary from one another, this is not possible. Here, however, we should draw attention to the considerably different age of the athletes. Millet et al. \[[@B8]\] report on athletes of 24.8 ± 2.6 years of age. In comparison with the off-season, in the active season the RCP and the maximum ergometric performance rose moderately, whereas the VAT and VO~2~max remained at a constant level. This study demonstrated a significant rise in the RCP and maximum ergometric performance (with VAT and VO~2~max remaining constant), based on comparison between the pre-competitive and competitive seasons. The echocardiographic parameters of the vegan athlete are different from those of the control group. Essentially the vegan athlete has a greater LVEDD value, with higher EDV and SV, which however do not exceed standard and normal values \[[@B10]\]. Overall the echocardiographic values of the sporting season and the off-season do not differ in any significant degree. Laboratory parameters showed no pathological values. A slight thrombopenia in the sporting season can be explained by the reduced thrombocyte activation and adhesion caused by training. In itself this is not necessarily a pathological manifestation. In conjunction with a threshold value for vitamin B12 and standard iron, ferritin, and folic acid status, the standard haemoglobin, haematocrit, MCH, MCV, and MCHC readings exclude the possibility of manifest or latent anaemia. Raised CK-NAC levels are not unusual with active athletes. Mougious et al. have therefore written about the necessity of separate CK reference values for active sportspersons. Reduced testosterone values in the blood have also been described. An influence on the bone density or the muscle mass, however, cannot be demonstrated. None of the usual laboratory parameters gives grounds for suspecting the presence of any detriment to health caused by sport or by diet. Quite on the contrary, the vegan athlete even shows ideal cholesterol values. 3. Critical Remarks {#sec3} =================== The case report of a vegan triathlete can only provide indications that a vegan diet is at least not detrimental to health, even in connection with a long-distance triathlon, and that a vegan diet results in similar performance as compared with a mixed diet. These conclusions cannot be extrapolated to short distances and Olympic distances, where an explosive performance---above all, a performance in the lactic acid zone---is frequently necessary. Taking place for the most part in the aerobic range, long-distance races may work more in favour of vegan diet than races that approach the anaerobic threshold. In view of the shortage of suitable and willing test subjects, it will not be easy to arrange a group comparison of vegan and traditional triathletes for study purposes. A case study like the present one can supply indications, but the meaningfulness of its findings remains subject to limit. This case report seems to suggest that an ultra triathlete living on a vegan diet has a similar profile, physiologically and in performance terms, to that of triathletes of similar age living on a conventional diet. The cardiac adaptations are likewise similar in both cases. The higher laboratory parameters do not permit conclusions with regard to dietary deficiency or any detriment to health. A well-planned vegan diet can apparently also be adopted by ultra endurance athletes without any risk to health necessarily being incurred. To what extent a vegan diet offers benefits or disadvantages for the performance capacity/health of a triathlete would be a matter for investigation in a future study, though in view of the highly individual training regimes of triathletes, this would be difficult to arrange. Conflict of Interests ===================== There is no data relating to financial interests and relationships (patents, fees, support by companies, etc.). ###### Spiroergometric parameters of the vegan athlete and the control group (values given as a mean value ± standard deviation). Vegan Off-S. Vegan season Control group (*n* = 10) ----------------------------- --------- -------------- -------------- -------------------------- VO~2~ max L/min 4.8 4.7 4.3 ± 0.6 mL∗min^−1^∗kg^−1^ 60.0 60.0 56.9 ± 7.5 VO~2~ VAT L/min 3.4 3.3 3.3 ± 0.6 mL∗min^−1^∗kg^−1^ 43 42 43.7 ± 7.4 \% VO~2~ max 71 70 76.1 ± 9.3 VO~2~ RCP L/min 3.9 4.4 3.6 ± 0.6 mL∗min^−1^∗kg^−1^ 48 55 48.2 ± 8.3 \% VO~2~ max 80.6 92 84.4 ± 6.9 HR At rest 55 49 58.6 ± 2.9 At VAT 127 123 147.7 ± 11.4 At RCP 141 147 157.0 ± 13.1 Maximal load 163 159 176.7 ± 11.0 W At VAT 280 280 262 ± 42.9 At RCP 340 370 286 ± 56.2 Maximal load 370 400 337 ± 53.8 Maximal load/kg body weight 4.61 5.02 4.46 ± 0.73 HF: heart rate/min, Off-S: off-season, season: active season, vegan: vegan athlete, VO~2~ max: maximum oxygen intake, VO~2~ VAT: oxygen intake at ventilatory anaerobic threshold, VO~2~ RCP: oxygen intake at respiratory compensation point, and W: ergometric performance. ###### Echocardiographic parameters. Vegan athlete, off-season Vegan athlete, active season Control group (*n* = 10) -------------- ------ --------------------------- ------------------------------ -------------------------- Aortic root cm 3.7 3.4 3.1 ± 0.4 Left atrium cm 3.0 3.0 2.6 ± 0.3 Left atrium ml 36 30 29.6 ± 8.6 LVEDD cm 5.0 5.4 4.64 ± 0.4 IVSD cm 1.2 1.1 1.2 ± 0.1 PWTD cm 1.2 1.0 1.2 ± 0.1 LVMM g 211.3 206.3 203.0 ± 35.3 LVMM g/m² 105.68 103.67 103.1 ± 14.5 EDV mL 119 140 100.2 ± 16.7 ESV mL 48 58 39 ± 6.1 SV mL 72 82 61.2 ± 11.2 EF \% 60 59 61.0 ± 2.1 SF \% 32 31 35.5 ± 9.4 MV E/A ratio 1.45 1.56 1.39 ± 0.20 EDV: end diastolic volume, EF: ejection fraction, ESV: end systolic volume, IVSD: intraventricular septum in diastole, LVEDD: left ventricular end diastolic diameter, LVMM: left ventricular muscle mass, MV E/A ratio: mitral valve E/A ratio, PWTD: posterior wall thickness in diastole, SF: shortening fraction, and SV: stroke volume. ###### Results of blood investigation of the vegan ultra triathlete. Analysis Off-season Active season ------------------------------------------- ------------ --------------- Leukocytes (1000 s/uL) 4.6 4.7 Erythrocytes (Mill·uL) 5.1 4.9 Haematocrit (%) 43 42 Haemoglobin (g/dL) 14.9 14.1 MCV (fl) 83 87 MCH (pg) 29.0 29.1 MCHC (g/dL) 35.1 33.3 Thrombocytes (1000 s/uL) 171 138 Alkaline phosphatase (U/L) 68 70 Glutamyl transpeptidase 8 15 Glutamate oxaloacetate transaminase (U/L) 36 40 Glutamate pyruvate transaminase (U/L) 32 42 Lactate dehydrogenase (U/L) 199 238 Amylase (U/L) 63 61 Lipase (U/L) 35 36 CK-NAC (U/L) 155 209 Protein (g/dL) 6.6 6.7 Cholesterol (mg/dL) 134 113 HDL (mg/dL) 47 51 LDL (mg/dL) 74 50 Triacylglyceride (mg/dL) 148 84 TSH basal (mU/L) 1.23 0.73 Iron (*µ*g/dL) 95.0 82.0 Free testosterone (pg/mL) 5.5 5.7 Vitamin B12 (pg/mL) 329 347 Ferritin (ng/mL) 83.7 82.6 Folic acid (ng/mL) 16.8 14.3 [^1]: Academic Editors: G. Minardi and J. Peteiro

{ "pile_set_name": "PubMed Central" }

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Introduction {#sec1-1} ============ Rubella is usually a mild self-limiting illness in children, presenting with fever and rash; however, in pregnant women, infection during the first 16 weeks of pregnancy can result in miscarriage, fetal death, or an infant born with congenital birth defects known as congenital rubella syndrome (CRS).\[[@ref1][@ref2][@ref3][@ref4]\] Rubella is of significant public health importance due to these teratogenic effects. Up to 60% of rubella cases may not present with a rash, many cases are not detected or reported. Seroprevalence surveys have documented widespread circulation of the rubella virus in all parts of the world.\[[@ref4]\] The presence of rubella-specific IgG in an unvaccinated population is a long-term marker of previous rubella infection and immunity status, and the antibodies persist life long, protecting the individual from further infections.\[[@ref1][@ref2]\] A study conducted in Tamil Nadu (South India) among unvaccinated girls aged 10--16 shows the presence of protective antibodies in 86.5%.\[[@ref1]\] A similar study in North India (mean age of 10.7 years) reported that 90% have protective antibodies.\[[@ref5]\] It was estimated that in India, about 50% of children acquire rubella antibodies by the age of 5 years and 80--90% become immune by 15 years by naturally acquired rubella.\[[@ref1][@ref5]\] All these studies were conducted before the widespread use of rubella vaccine in private sectors of India. Childhood exposure renders immunity to majority of women; however, periodic epidemics still occur among children and spread to involve a small portion of susceptible adult women, leading to epidemics of CRS.\[[@ref6]\] Several sero-epidemiological surveys from other countries have reported that a substantial number of women reach childbearing age without acquiring natural immunity to rubella, making them more susceptible to infection.\[[@ref4][@ref7][@ref8]\] Due to the high incidence of subclinical infections during childhood, eliciting a reliable history is difficult making seroprevalence studies using representative sampling from different age groups superior in determining the age groups for vaccination.\[[@ref4][@ref9]\] Indian newborns consist of 20% of annual birth cohorts of the world. Regardless, no country-wide estimates of CRS burden and susceptibility to rubella infection are available due to lack of a national surveillance and registry for rubella.\[[@ref2][@ref3][@ref10]\] There are no systematic reviews or nation-wide studies assessing the susceptibility of Indian population to rubella infection in general or specific to children or adolescent girls.\[[@ref10]\] The WHO evaluation studies validate the inclusion of childhood rubella vaccination as cost-effective in countries having measles vaccine coverage of more than 80%. Countries which can sustain the above coverage level can introduce rubella vaccination in their UIP program for the desired effects.\[[@ref3][@ref5]\] The latest data show that 140 of the 194 WHO member countries have incorporated rubella vaccine in combination with mumps and measles at the age of 12--18 months.\[[@ref11]\] Rubella and CRS control was the goal established in the South-East Asian region as an initial step toward elimination and many developed countries have been successful in doing so.\[[@ref11]\] Rubella vaccine is not yet included in the National childhood vaccination program (UIP) in India and it is given as an optional vaccine by private providers. This results in low coverage and thereby reduced herd immunity since only a small proportion of children from families who can afford the vaccine gets vaccinated. This unregulated use of vaccine may rise the "effective reproduction rate 'R' and ultimately lead to 'age shifting' and paradoxical increase of CRS in future."\[[@ref2][@ref3][@ref12]\] The state of Kerala, with best health indicators in the country, has been facing periodic epidemics of rubella among adult population in recent years. The Social Justice Department, with the help of Health and Education Department, in 2014, introduced the monovalent rubella vaccine among school girls but faced resistance from the community due to less perceived risks and resulted in low coverage (\<25%). Age-specific seroprevalence among girls, which the WHO considers to be a sensitive tool for risk assessment of CRS, has never been done here.\[[@ref3][@ref12]\] This will also help in understanding the degree of impact of private sector/optional vaccine. Rubella IgG estimation in children will provide data for the necessary modification in the immunization strategy. Although rubella and CRS are a public health problem, no studies were done from the state on this subject. With the aim to understand the age-specific susceptibility of acquiring rubella infections and future risk of CRS, estimation of rubella IgG antibodies among girls in the age group of 13--15 was conducted. Participants and Methods {#sec1-2} ======================== The study was conducted at a randomly selected rural area named Mavoor Panchayath of Kozhikode District, Kerala, among adolescent girls. Being the only high school in the area, with 100% school enrollment and zero dropouts, all the adolescent girls from the area were admitted in the selected school, hence representing the population of girls in the community. With an expected rubella-specific IgG prevalence of 80% and for precision of ± 5, the minimum sample size required was estimated using Epi Info software 7 World health organization (WHO) as 250.\[[@ref1]\] The protocol of the study was approved by the Institutional Ethics Committee. The study was conducted in Mavoor Government high school, among students of 8^th^ and 9^th^ standard (ages 13--15) with the permission of the school authorities and the parent--teacher association (PTA). All the girls studying in 8--9^th^ standards in the selected school (*n* = 280), whose parents have given written informed consent and accent by the girls, were enrolled as participants and they were selected consecutively according to the roll number in the school registers till we got the required sample size of 250. Data and blood sample collection was done by conducting screening camps in the school on fixed days from 10 am to 2 pm. Those who were absent on the fixed day, their data were collected during the subsequent visits. A standardized questionnaire was used by the interviewer to collect the demographic, morbidity, and immunization data (specifically measles-mumps-rubella \[MMR\]). Some of the variables used include history of exanthematous fever and past and recent hospitalizations. The variables include history of any exanthematous fever, recently or past and hospitalization. Medical examination was conducted to detect any disease such as fever/exanthema/lymph node enlargement and anomaly-cataract, deafness, and congenital heart disease to rule out rubella or features of CRS. From every participant, 5 ml venous blood was collected from the antecubital vein under aseptic precautions using vacutainers with gel separators. Serum was separated and brought to the microbiology department at the medical college in vaccine carriers maintained at a temperature of 4°C on the same day and stored at −20°C till further analysis was made. Rubella-specific IgG antibody was measured by quantitative enzyme-linked immunosorbent assay using BEIA Rubella IgG Quant Technogenetics SRL Italy kit, with a relative sensitivity and specificity of 100%. The tests were done using standard procedures by a laboratory technician with adequate experience under the supervision of a microbiologist (author). The results are expressed in IU/ml. As per the product manual (BEIA Rubella IgG Quant Ed. 01-09-2006), IgG titer of \>15 IU is considered positive, 8--15 as equivocal, and \<8 IU as negative. About 10% of the samples were retested at Kasturba Medical College, Manipal Laboratory, a central laboratory accredited by the National Accreditation Board for Testing Laboratories of the Department of Science and Technology, Government of India, and the validity and consistency were assured. The collected data were processed in an excel data sheet with codes, with personal anonymity, and statistical analysis was performed using SSPS program. Chi-square test was applied to find out significant difference and Fisher\'s exact test wherever applicable. Results {#sec1-3} ======= Data and blood sample collection was done from 250 girls. The age ranged from 13 to 15 with a mean age of 14.2 ± 0.7 years. IgG estimation was done on 224 samples (90%), and due to sample lysis, the rest was discarded. Out of which 3 girls had received MMR vaccine who were also excluded from the analysis, so 221 samples were finally included for the analysis. Age wise distribution of girls were 24.5%, 51%, 24.5% respectively in 13, 14 and 15 years. The mean IgG titer was 151.93 ± 128.78 IU, and as per the criteria, 68.3% were positive, 28.5% negative, and 3.2% equivocal \[[Table 1](#T1){ref-type="table"}\]. The class-wise details of IgG values are given in [Table 2](#T2){ref-type="table"}. There is no association between the titer values and the age of the participants (Pearson\'s correlation: 0.043, *P* = 0.53). ###### Prevalence of IgG antibody titer among girls ![](JFMPC-5-658-g001) ###### Class wise distribution of IgG antibody titer among girls ![](JFMPC-5-658-g002) The childhood immunization details were collected from parents by crosschecking immunization cards and it was found that 61% had given Bacillus Calmette--Guérin, 57% had given three doses of OPV and DPT, and 54% had given measles vaccine. Twenty-four girls had a history of mumps. None of the participants showed features of rubella (fever, rashes, and cervical nodes), congenital anomalies, or features of CRS on clinical examination. The susceptibility to rubella may considerably vary with socioeconomic strata. As a proxy of this, the educational status of both parents was collected, this revealed a 100% literacy rate. Nearly 87% of fathers and 85% of mothers have completed \>10 years of schooling. There was no variation of IgG status between the strata (*P* = 0.2, 0.4) in both parents meaning that rubella is uniformly transmitting among all income groups \[Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}\]. ![Relation of IgG tire values with father\'s education status\*. \*10th standard, Plus two, Degree, Postgraduate](JFMPC-5-658-g003){#F1} ![Relation of IgG tire values with mother\'s education status\*. \*10th standard, Plus two, Degree, Postgraduate](JFMPC-5-658-g004){#F2} Discussion {#sec1-4} ========== Documentation of rubella infection has been difficult due to the challenges in diagnosing the disease due to its subclinical and atypical presentations. Hence, serological surveys play a precise role in defining infectious disease epidemiology, especially in the case of rubella, in determining its potential health impact, and also to help policymakers to decide the vaccination policies to contain the consequences of the disease. In our study, even though girls were not immunized against rubella, 68.3% of the girls demonstrated a protective level of IgG by naturally acquired childhood infection. Since most of them were subclinical or with mild symptoms, their parents could not recall the past infections. The IgG prevalence reported here was less than reported earlier from Tamil Nadu (86.5%) Maharashtra (76%), and New Delhi (90%).\[[@ref1][@ref5][@ref6]\] This may be due to the improved socioeconomic status and higher standard of living in the state. A similar rate of prevalence (67.3%) was reported among girls aged 11--18 from Jammu and Kashmir.\[[@ref13]\] These figures throw light on the widespread reach of rubella among the children of the state, thereby rendering them lifelong immunity.\[[@ref4][@ref12]\] The IgG prevalence among girls did not differ significantly by religion, educational status, or family income, which is consistent with the reports from a hospital-based study previously conducted in the state.\[[@ref14]\] It is seen that more than 30% of the cohort was susceptible to rubella. It is noteworthy that the majority of girls get protection until they attain adolescence; however, not all were protected and therefore some girls may remain susceptible to infection during adulthood and pregnancy. The WHO reports that even when the susceptibility levels in women are below 10%, there is a chance of CRS in the future.\[[@ref15]\] With the buildup of such susceptible cohorts, pregnant women can acquire rubella infection from younger children, leading to epidemics among adult population. Studies from different states of the country on acquired rubella infection among pregnant women during pregnancy period reported 3%, 6.7%, and 8.3% prevalence from Kerala, Andhra Pradesh, and New Delhi, respectively, as evidenced by IgM estimation.\[[@ref14][@ref16]\] With an average annual incidence of 5 lakh deliveries, we could expect about 15,000 infections from Kerala among pregnant women and proportional CRS. Since rubella vaccine was not included in the childhood vaccination program in India only three of our subjects have gave history of previous rubella vaccination. They received it from private providers as an optional vaccine with out-of-pocket expenditure. Currently, about 15--25% of the child population are getting rubella vaccine (RA 27/3) combined with mumps and measles (MMR). Reviews have shown that RA 27/3 vaccine has a duration of protection ranging from 10 to 21 years in children with a seropositivity rate of 95%.\[[@ref9]\] As per the district level household surveys (DLHS I, II. III), the immunization coverage for the Kerala state is 84%, 78.6%, and 83%, with few districts below the state average, and at country average, it is only 65%.\[[@ref17]\] Hence, with the current status of subpopulation immunization leading to "age shifting," oscillating herd immunity, the probability of paradoxical rubella outbreaks among adult population and CRS, as occurred in Greece, cannot be ruled out in India.\[[@ref3][@ref11][@ref12][@ref18]\] This study shows that rubella virus infection is prevalent in Kerala in all socioeconomic strata, and more than 30% girls reach childbearing age without acquiring natural immunity against the disease. Studies conducted across India suggest similar baseline information on the susceptibility profile of women of childbearing age.\[[@ref19]\] Although rubella vaccine is safe and effective, clear policy regarding rubella immunization of children either at 15 months or young girls at 9--12 years has not been outlined in India. For controlling CRS, the "indirect strategy" is to immunize children and reduce transmission of rubella, and the "direct strategy" is to immunize adolescents to prevent rubella infection and CRS.\[[@ref18][@ref19][@ref20]\] The first may pose a risk to adults and the second cannot prevent rubella transmission as described, so both are needed in a combined, coordinated form. Consider a scenario, in which combination MMR vaccine has been introduced in the UIP of India. The state with expected average immunization coverage of 80% and vaccine efficacy of 95% will produce 24% birth cohort without immunity to rubella in each year. Considering the birth rate of 15 per thousand, within a 10--15 year period, it will produce a large number of rubella-susceptible youth population in the state, later leading to CRS. In other states where the immunization coverage is poor, the extrapolation of the data predicts a worse situation. As supported by the WHO, Gavi, Kerala, India, may introduce MMR vaccine in UIP for children in the near future. Many authors have warned that *Rubella vaccination: must not be business as usual*. The childhood immunization programs against rubella without specifically addressing the countries epidemiology will not achieve the desired results.\[[@ref19][@ref20][@ref21]\] To prevent these negative impacts, efforts should be taken to maintain the "herd immunity threshold" at a level above 83% among the adult population without regional variation within the country.\[[@ref3][@ref20][@ref21]\] Therefore, the best results can be achieved only by a combined immunization policy as adopted by Denmark, Sweden, most of the European countries, and the USA, where the first dose is offered as MMR vaccine at 15--18 months of age and the second dose as MMR vaccine at 6--12 years, or only rubella vaccine exclusively to girls at 12--14 years of age is best for India too to eliminate CRS in the near future along with surveillance of rubella.\[[@ref5][@ref18]\] Conclusion {#sec1-5} ========== Substantial numbers of women reach childbearing age without immunity against rubella and thus are at a risk of passing the infection to their fetuses, who can then develop subsequent congenital defects leading to CRS. An immunization policy recommending rubella-containing vaccine is highly desirable to prevent rubella and CRS. Financial support and sponsorship {#sec2-1} --------------------------------- This study was financially supported by the State Board of Medical Research, Government of Kerala. Conflicts of interest {#sec2-2} --------------------- There are no conflicts of interest. This study was funded by the State Board of Medical Research (SBMR) -- Directorate of Medical Education, Government of Kerala. The authors express thanks to the staff and PTA students of Mavoor, Government high school. Thanks to Dr. Thomas Bina, Head of the Department, Community Medicine, for the help, and Dr. Rema Devi, Former Head of the Department of Microbiology, for providing laboratory support for the study.

{ "pile_set_name": "PubMed Central" }

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Figure 1Cells lacking some nucleoporins die on FOA because they derepress a telomeric URA3 gene.Telomere association with nuclear pores is critical not only for transcriptional silencing but also for efficient repair of double-stranded breaks in the subtelomeric region of budding yeast chromosomes, according to Therizols et al. (page 189). As in many organisms, yeast telomeres localize to the nuclear periphery. To determine whether nuclear pore proteins are involved in telomere tethering, Therizols et al. looked for telomere localization in cells lacking functional Nup84 complexes, which are essential components of the pore. They found that the telomeres no longer associated with the nuclear periphery in these mutants. As might be expected from previous work on transcriptional silencing, transgenes located in the subtelomeric region were no longer silent in Nup84 complex mutants, indicating that localization of the telomere to the nuclear periphery was functionally important. Surprisingly, when double-stranded breaks were introduced into subtelomeric sites in the mutants, DNA repair efficiency dropped significantly relative to wild-type cells. The efficiency of break repair in central regions of chromosomes did not differ between wild-type and mutant cells. The DNA silencing and repair phenotypes were separated in cells mutant for Esc1p, a protein located at the nuclear periphery but which is not directly involved in the pore. In this case, telomere localization and DNA repair were disrupted, but silencing remained intact. The researchers conclude that anchoring telomeres to the nuclear pore is important for efficient DNA double-stranded break repair in the subtelomeric regions, though it is not yet clear why this is true. Because silencing remained intact in the Esc1p mutants but repair was disrupted, it appears that chromatin structure itself is not the problem. One possibility is that clusters of repair proteins may be concentrated near groups of tethered telomeres, thereby facilitating rapid repairs.

{ "pile_set_name": "PubMed Central" }

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All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Small non-coding RNAs (sRNAs) of 20\~30 nucleotides (nt) have gained significant attention in recent years as they are widely involved in various biological processes such as the embryonic, neuronal, muscle, and germline development \[[@pone.0159487.ref001]--[@pone.0159487.ref003]\]. QDE-2-interacting small RNAs (qiRNAs) of typically 20\~21 nt are a new class of sRNAs. qiRNAs are induced by DNA damage \[[@pone.0159487.ref004]\] and mediate gene silencing in the DNA damage response (DDR) pathway in *Neurospora crassa* by inhibiting protein translation \[[@pone.0159487.ref004]\]. Although qiRNAs were first discovered in 2009, only little is known about their biogenesis and functionality. It has been demonstrated that the biogenesis of qiRNAs requires DNA-damage-induced aberrant RNAs (aRNAs) as precursors \[[@pone.0159487.ref004]\]. Moreover, RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3, as well as dicers have been previously shown to be involved in the production of qiRNAs \[[@pone.0159487.ref004]\]. After DNA damage, aRNAs are highly induced and then specifically recognized by RNA-dependent polymerases to produce double-stranded RNAs (dsRNAs). Subsequently, dsRNAs are converted to qiRNAs by dicers \[[@pone.0159487.ref004], [@pone.0159487.ref005]\]. However, the detailed mechanism for the generation of qiRNAs is largely unknown and requires elucidation in further experimental studies. Previously, novel qiRNAs in *Neurospora crassa* \[[@pone.0159487.ref004]\] or rice were identified almost exclusively by immunoprecipitation followed by sequencing. However, this conventional approach for the identification of qiRNAs is time-consuming, labor-intensive, and inefficient and even holds the risk of not detecting lowly expressed or issue-specific qiRNAs. Computational methods can overcome these experimental hurdles and extract general information from known qiRNAs to predict novel qiRNAs for further experimental manipulation \[[@pone.0159487.ref006]\]. However, as far as we know, no qiRNA prediction tool has been implemented so far. Therefore, the development of efficient computational approaches for qiRNA prediction is urgently needed and intriguing. Common sequence or structure conservation-based approaches in microRNAs (miRNAs) prediction cannot be directly adopted for qiRNA prediction as no evidence has been reported for a high evolutionary conservation of the sequence and structure of qiRNAs across species. Unlike Piwi-interacting RNAs (piRNAs), the density of plot of qiRNAs does not show a striking clustering characteristic \[[@pone.0159487.ref004]\]. Thus, no clustering characteristic can be used for qiRNA prediction. However, it has been demonstrated that qiRNAs exhibit strong position-specific preferences for uracil (U) at the first nucleotide of the 5' end and for adenine (A) at the first nucleotide of the 3' end \[[@pone.0159487.ref004]\]. In bioinformatics, position-specific nucleotide preferences are usually employed for sRNAs prediction. For example, ping-pong-dependent piRNAs show some position-specific nucleotide preferences, such as for thymine (T) at the first position (1T) and for A at the 10th position (10A) \[[@pone.0159487.ref006]\]. To capture and leverage these sequence features for piRNA prediction, Betel *et al*. constructed a 21 × 4 feature vector based on a 21-base window around the 5' end (plus 10 nt upstream and 10 nt downstream) and trained a support vector machine (SVM) model for piRNA prediction \[[@pone.0159487.ref007]\]. In 2011, Zhang *et al*. used a simple *k*-mer scheme to construct 1364 dimension feature vectors for describing the candidate piRNA sequences and then constructed the software piRNApredictor for piRNA prediction based on an improved Fisher linear algorithm \[[@pone.0159487.ref006]\]. Moreover, position-specific scoring matrix (PSSM) is a commonly used representation of sequence motifs, which has been widely applied to the prediction of significant biological signals such as DNA binding sites \[[@pone.0159487.ref008]\], RNA binding sites \[[@pone.0159487.ref009]\], promoters \[[@pone.0159487.ref010]\], protein secondary structure prediction \[[@pone.0159487.ref011]\], etc. In this work, we developed an ingenious method for the extraction of features based on position probability matrices (PPM) of biosequences and then constructed a novel qiRNApredictor (qiRNA predictor) software package with 80 features. Firstly, the experimentally verified qiRNAs were collected manually from the scientific literatures and then used for training the Random forest (RF) model using the randomForest R package. Subsequently, the performance and robustness of qiRNApredictor were extensively evaluated by *n*-fold cross-validations (CV) as well as the leave-one-out cross-validation (LOO-CV). Upon LOO-CV, qiRNApredictor exhibits a promising sensitivity of 93.55% and a specificity of 71.61%, which can be used for the prediction of qiRNAs. Materials and Methods {#sec002} ===================== Data preparation {#sec003} ---------------- Herein, only qiRNA sequences in *Neurospora crassa* were considered (no qiRNA in rice was sequenced in the work by Chen *et al*. \[[@pone.0159487.ref005]\]). Lee *et al*. identified 184 individual qiRNA sequences in *Neurospora crassa* \[[@pone.0159487.ref004]\]. However, 23 qiRNA sequences were directly discarded as they included undefined bases. We further eliminated six individual qiRNA sequences as they were identical to other qiRNA sequences. Finally, 155 experimentally verified qiRNAs were collected as positive samples to train a RF model. Three negative datasets were built. For the convenience of discussion, we called them the "Random", the "sRNA-segment", and "milRNA" negative datasets, according to the ways we collected them or the data origin. The "Random" negative dataset was randomly extracted from NONCODE database. As previously described \[[@pone.0159487.ref006]\], non-coding RNA sequences obtained from the NONCODE database (version 3.0) \[[@pone.0159487.ref012]\] were fragmented into non-overlapping segments. For each of these non-qiRNA segments, we shuffled it 10000 times to destroy any potentially functional structures \[[@pone.0159487.ref006]\]. Then, the same amount of negative samples were randomly selected from the segments under the constraint condition that the length distribution of the selected segments was identical with that of positive qiRNAs. The "sRNA-segement" negative dataset was collected from Rfam database. The sequences of sRNAs of *Neurospora crassa* were retrieved from Rfam database (release 12.0) \[[@pone.0159487.ref013]\]. Because the lengths of sRNAs are much longer than that of qiRNAs, we fragmented the sequences of sRNAs into non-overlapping segments under the constraint that the length distribution of sRNA segments was similar to that of positive qiRNAs. Then, the same amount of sRNA segments were randomly selected and regarded as the analogue of the degraded fragments of sRNAs. miRNA-like small RNAs (milRNA) are about 19\~25 nt and have a strong preference (51.08%) for U at the 5' end. They were firstly discovered in *Neurospora crassa* and called milRNAs due to their similarities with miRNAs \[[@pone.0159487.ref014]\]. Furthermore, 325 milRNAs were manually collected from the scientific literatures to construct the "milRNA" negative dataset. Since only 25 milRNAs were obtained from *Neurospora crassa*, we also collected the putative milRNAs from other fungi species, including *Trichoderma reesei* \[[@pone.0159487.ref015]\], *Sclerotinia sclerotiorum* \[[@pone.0159487.ref016]\], *Metarhizium anisopliae* \[[@pone.0159487.ref017]\], *Zymoseptoria tritici* \[[@pone.0159487.ref018]\], *Fusarium oxysporum* \[[@pone.0159487.ref019]\], *Fusarium graminearum* \[[@pone.0159487.ref020]\], *Antrodia cinnamomea* \[[@pone.0159487.ref021]\], *Aspergillus flavus* \[[@pone.0159487.ref022]\], *Penicillium chrysogenum* \[[@pone.0159487.ref023]\], and *Penicillium marneffei* \[[@pone.0159487.ref024]\]. Finally, these three negative datasets were incorporated with the positive dataset respectively to construct the "Random", "sRNA-segment" and "milRNA" training datasets. Feature extraction {#sec004} ------------------ The extraction of an appropriate set of features for training a prediction model is one of the vital, yet most challenging issues in machine learning-based prediction approaches. To capture the position-specific preference of nucleotides, the occurrence probabilities of each nucleotide at the first ten positions (1 through 10) and the last ten positions (-1 to -10) of both positive and negative samples were calculated to create four PPMs ([Fig 1](#pone.0159487.g001){ref-type="fig"}). It should be noted that qiRNA sequences of less than 20 nt exhibit some overlaps of the first and last ten positions. The score of nucleotide *i* at position *j* can be calculated according to the formula $$\text{S}\left( {\mathbf{i},\mathbf{j}} \right) = \text{log}_{2}\left( \frac{\mathbf{P}_{ij}}{\mathbf{N}_{ij}} \right),\text{ }\mathbf{i} \in \left\lbrack {\text{A},\text{C},\text{G},\text{U}} \right\rbrack,\text{ }\mathbf{j} \in \left\lbrack {- 10,\cdots, - 1;\, 1,\cdots,10} \right\rbrack$$ where ***P***~*ij*~ and ***N***~*ij*~ are the probability of nucleotide *i* at position *j* in PPMs produced by positive and negative samples, respectively ([Fig 1](#pone.0159487.g001){ref-type="fig"}). The score gives an indication how much the position-specific preference of positive samples differs from that of negative samples. For the cases with ***P***~*ij*~ = ***N***~*ij*~, the score equals zero, which means this feature cannot offer any information for the prediction. However, when ***P***~*ij*~ or ***N***~*ij*~ is zero, it doesn't mean this feature has great significance. Instead, it may be simply because of the limited sample size. To circumvent the infinite value caused by inadequate sampling, we assigned a zero score for the cases that ***P***~*ij*~ or ***N***~*ij*~ is zero. In total, 80 scoring values were directly regarded as features to train a RF model. Then *F*-score was used to measure the discriminatory power of each feature above \[[@pone.0159487.ref025]\]. The *F*-score of the *i*th feature is defined as: $$F\left( i \right) \equiv \frac{\left( {{\overline{x}}_{i}^{( + )} - {\overline{x}}_{i}} \right)^{2} + \left( {{\overline{x}}_{i}^{( - )} - {\overline{x}}_{i}} \right)^{2}}{\frac{1}{n_{+} - 1}{\sum\limits_{k = 1}^{n_{+}}\left( {x_{k,i}^{( + )} - {\overline{x}}_{i}^{( + )}} \right)^{2}} + \frac{1}{n_{-} - 1}{\sum\limits_{k = 1}^{n_{-}}\left( {x_{k,i}^{( - )} - {\overline{x}}_{i}^{( - )}} \right)^{2}}}$$ where *n*~+~ and *n*~−~ are the numbers of positive and negative samples, respectively; ${\overline{x}}_{i}$, ${\overline{x}}_{i}^{( + )}$, and ${\overline{x}}_{i}^{( - )}$ are the average of the *i*th feature of total, positive, and negative samples, respectively. $x_{k,i}^{( + )}$ and $x_{k,i}^{( - )}$ are the *i*th feature of the *k*th positive and negative sample, respectively. Larger *F*-scores indicate better discrimination \[[@pone.0159487.ref025]\]. ![The flowchart of qiRNApredictor.](pone.0159487.g001){#pone.0159487.g001} Random forest {#sec005} ------------- RF is an ensemble learning classifier consisting of a multitude of tree-structured classifiers \[[@pone.0159487.ref026]\]. RF makes use of two powerful machine-learning methods---the bagging and random feature selection in the tree induction \[[@pone.0159487.ref027]\]. In the bagging algorithm, each tree is trained by using a bootstrap sample of the training data, and the prediction results of the ensemble are aggregated by majority vote or averaging rule to give the final prediction results \[[@pone.0159487.ref027]\]. Instead of using all features, only a random subset of features was used here to split at each node when growing a single tree. A type of CV together with the training step using out-of bag (OOB) samples was used to assess the prediction performance of RF. More specifically, a particular bootstrap sample was adopted to grow each tree during the process of training. Since bootstrapping sampling is sampling with replacement, some of the samples were ignored, while others were reused. The ignored samples constitute the OOB sample. On average, 1−*e*^−1^ ≅ 2/3 of the training samples was used for growing the tree leaving *e*^−1^ ≅ 1/3 as OOB, which have not been used for tree construction. Therefore, these samples could be used to evaluate the prediction performance \[[@pone.0159487.ref026], [@pone.0159487.ref027]\]. The RF algorithm was implemented by the randomForest R package. Performance evaluation {#sec006} ---------------------- As previously described \[[@pone.0159487.ref028]\], among the predicted positive results obtained by qiRNApredictor, the real positives are called true positives (*TP*), while the other positive results are called false positives (*FP*). Among the predicted negative results obtained by qiRNApredictor, real negatives are called true negatives (*TN*), while the other negative results are called false negatives (*FN*). The performance is evaluated based on four measurements of specificity (*Sp*), sensitivity (*Sn*), accuracy (*Ac*), and Matthew's correlation coefficient (*MCC*). These indexes are defined as $$Sn = \frac{TP}{TP + FN},\, Sp = \frac{TN}{TN + FP},\, Ac = \frac{TP + TN}{TP + FP + TN + FN}$$ and $$MCC = \frac{\left( {TP \times TN} \right) - \left( {FN \times FP} \right)}{\sqrt{\left( {TP + FN} \right) \times \left( {TN + FP} \right) \times \left( {TP + FP} \right) \times \left( {TN + FN} \right)}}$$ In this study, we performed 4-, 6-, 8-, and 10-fold CVs as well as the LOO-CV. Receiver Operating Characteristic (ROC) curves were plotted for performance visualization. Results {#sec007} ======= A novel algorithm for qiRNA prediction {#sec008} -------------------------------------- In this work, we collected experimentally identified qiRNAs from the scientific literatures. After eliminating redundancy in sequences and directly removing sequences that include undefined bases, a dataset of 155 experimentally verified qiRNA sequences was obtained ([S1 Table](#pone.0159487.s002){ref-type="supplementary-material"}). As previously described, a negative dataset containing 155 samples was constructed. Finally, a balanced database containing 155 positive and negative samples was used for model construction. qiRNAs are very short of approximately 20\~21 nt in length ([Fig 2](#pone.0159487.g002){ref-type="fig"}), which renders it very difficult to predict qiRNAs with high accuracy. However, the first and last nucleotide of qiRNAs have a high preference for U and A, respectively ([Fig 3](#pone.0159487.g003){ref-type="fig"}). Therefore, we attempted to predict qiRNAs with the position-specific preferences of nucleotides in qiRNA sequences. To this goal, PPMs were firstly constructed for characterizing position-specific properties of qiRNAs. Based on the PPMs, 80 log-likelihood scores were calculated as features for training prediction model (see details in [Materials and Methods](#sec002){ref-type="sec"}). Based on the training datasets, we used the *F*-score \[[@pone.0159487.ref025]\] to rank 80 features. As expected, nucleotides at the first and last position, such as 1U, 1A, 1C, or -1A, exhibit high *F*-scores ([Fig 4](#pone.0159487.g004){ref-type="fig"}). This is consistent with the position-specific preferences of nucleotides in qiRNA sequences, which demonstrates that we have succeeded in capturing these characteristics in qiRNA sequences. ![The length distribution of qiRNAs in *Neurospora crassa*.](pone.0159487.g002){#pone.0159487.g002} ![Position-specific nucleotide preferences of qiRNAs in *Neurospora crassa* for (A) the first 15 nt substring from 5' end to 3' end and (B) the last 15 nt substring from 5' end to 3' end.\ The insets are Frequency Plot of qiRNA sequences. The sequence logo analysis was implemented by WebLogo \[[@pone.0159487.ref029]\].](pone.0159487.g003){#pone.0159487.g003} ![The *F*-scores of PPM features.\ Larger *F*-scores indicate better discrimination.](pone.0159487.g004){#pone.0159487.g004} To evaluate the performance and robustness of the qiRNApredictor, LOO-CV and 4-, 6-, 8-, and 10-fold CVs were performed. The LOO-CV results based on "Random" training dataset show that our method predicts at 93.55% sensitivity, 71.61% specificity, 82.58% accuracy and 0.6679 *MCC* value ([Table 1](#pone.0159487.t001){ref-type="table"}). The results of the 4-, 6-, 8-, and 10-fold CVs are also close to those of the LOO-CV. From the ROC curves ([Fig 5A](#pone.0159487.g005){ref-type="fig"}), AUC (area under ROC curves) values were calculated as 0.8779 (LOO-CV), 0.8772 (4-fold CV), 0.8752 (6-fold CV), 0.8761 (8-fold CV), and 0.8765 (10-fold CV), respectively ([Fig 5A](#pone.0159487.g005){ref-type="fig"}). As no qiRNA prediction tool existed to date, the performance of our qiRNA prediction tool could not be compared to any existing tool. To further test the ability of qiRNApredictor in distinguishing qiRNAs among other sRNAs, we also run it through "sRNA-segement" and "milRNA" datasets. milRNA is 19\~25 nt in length with a strong preference (51.08%) for U at the 5' end, which is somewhat similar to qiRNA. As shown in [Fig 5B](#pone.0159487.g005){ref-type="fig"} & [Table 1](#pone.0159487.t001){ref-type="table"}, the results demonstrate that the qiRNApredictor is able to identify qiRNAs among other sRNAs with quite significant AUC, *MCC*, sensitivity and specificity. Taken together, qiRNApredictor is a promising tool for the prediction of candidate qiRNAs. 10.1371/journal.pone.0159487.t001 ###### The performances of qiRNApredictor on each training dataset by the LOO CV. ![](pone.0159487.t001){#pone.0159487.t001g} Dataset *Ac* (%) *Sn* (%) *Sp* (%) *MCC* -------------------------------------- ---------- ---------- ---------- -------- **"Random" training dataset** 82.58 93.55 71.61 0.6679 **"sRNA-segement" training dataset** 80.97 86.45 75.48 0.6231 **"milRNA" training dataset** 79.17 70.32 83.38 0.5303 ![The prediction performance of qiRNApredictor.\ (A) The LOO-CV as well as 4-, 6-, 8-, and 10-fold CVs based on "Random" training dataset were calculated. The ROC curves and AUCs were also drawn and analyzed. To increase the specificity, we recommended a stringent cut-off value 0.667 for experimental investigation. The specificity of qiRNApredictor with the cut-off value 0.667 is 92.26%, while the sensitivity is 54.84%. (B) The ROC curves and AUCs were drawn and analyzed for the "Random", "sRNA-segment", and "milRNA" training datasets, respectively, by LOO-CV.](pone.0159487.g005){#pone.0159487.g005} Comparison to k-mer feature classes {#sec009} ----------------------------------- In bioinformatics, *k*-mers usually refer to *k*-gram or *k*-tuples of DNA or protein sequences and can be used to find certain regions within biosequences or be employed as *k*-mer statistics for giving discrete probability distributions of possible *k*-mer combinations \[[@pone.0159487.ref006]\]. *K*-mers can be used to distinguish qiRNA from non-qiRNA based on differences of string usages between the different sequence classes. Here, 1--5 nt strings were used to characterize positive or negative sequences by a vector consisting of the frequencies of *k*-mer strings. We first adjusted the *k* parameter in *k*-mer feature classes to obtain a better performance based on the same training dataset. With increasing *k* values, the AUC values exhibit first a rapid increase, which slows down in the following ([Fig 6A](#pone.0159487.g006){ref-type="fig"}). Although the increase of features may result in over-fitting, the best performance (AUC = 0.7790) of *k*-mer feature classes is lower than that of PPM features classes (AUC = 0.8779) ([Fig 6B](#pone.0159487.g006){ref-type="fig"}). This demonstrates that PPM is more suitable for qiRNA prediction. Moreover, we combined PPM and *k*-mer feature classes for the prediction of qiRNAs. However, the prediction performance of the combined feature classes exhibits to be very close to that of single PPM feature classes ([Fig 6B](#pone.0159487.g006){ref-type="fig"}). ![Comparison to *k*-mer feature classes.\ (A) Effect of the *k* parameter in *k*-mer feature classes on the prediction performance by 5-fold CV; (B) The ROC curves for RFs trained with different feature classes.](pone.0159487.g006){#pone.0159487.g006} Discussion {#sec010} ========== As a new regulatory factor for mediating gene silencing in the DNA damage response, qiRNAs have increasingly attracted considerable interest and investigative efforts. The identification of qiRNAs is fundamental for dissecting regulatory functions and molecular mechanisms. Compared to other expensive and time-consuming experimental methods, the computational prediction of qiRNAs is a conveniently rapid method of getting useful information for subsequent experimental verification. However, no qiRNA prediction tool existed to date. Therefore, developing a novel approach for qiRNA prediction is required and very intriguing. In this work, we designed a novel software named qiRNApredictor based on PPM features and RF algorithm. The performance and robustness of qiRNApredictor were extensively evaluated by n-fold CVs as well as LOO-CV, which gave very promising results. Here, the window size refers to the length of the region of interest at both ends of the sequences. To evaluate the effect of window size on the prediction performance, the window size was adjusted, and the AUC value was calculated based on the results of 5-fold CV. The AUC values first rapidly increase and then slow down with an increasing window size ([Fig 7](#pone.0159487.g007){ref-type="fig"}). There is a qiRNA sequence of only 15 nt, which was simply ignored when testing the window of 16 nt. Interestingly, the combination of two adjacent bases at both ends of sequences obtained a high AUC value of 0.8357. This either demonstrates that the characteristic signal at both ends of the qiRNA sequences is very significant, or it suggests that non-qiRNAs are not very similar to genuine qiRNAs. However, the approach of non-qiRNAs construction is standard according to previous studies \[[@pone.0159487.ref006]\]. ![Effect of the window size in PPM feature classes on the prediction performance by 5-fold CV.](pone.0159487.g007){#pone.0159487.g007} The performance of a classifier system would be improved by selecting the most discriminative set of features due to reducing the complexity of the classifier system. In contrast, the simple combination of PPM and *k*-mer feature classes does not improve the performance of the classifier system. This suggests that the feature set exhibits redundancies, which leaves room for optimization of the feature set used in qiRNApredictor in the subsequent work. Furthermore, the prediction performance of qiRNApredictor would be improved by finding the secondary structure features of qiRNA precursors. Taken together, our studies provide a novel and promising approach for qiRNA prediction and will facilitate further functional studies of qiRNAs. Supporting Information {#sec011} ====================== ###### The compressed files in ZIP format of the local package of qiRNApredictor. (ZIP) ###### Click here for additional data file. ###### The dataset of 155 experimentally verified qiRNAs in *Neurospora crassa* obtained from the work by Lee *at al* \[[@pone.0159487.ref004]\]. (XLS) ###### Click here for additional data file. The authors thank Dr. Ya Jia (CCNU) and Dr. Anbang Li (CCNU) for their helpful suggestions on the project implementation. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: YY HD QL. Performed the experiments: YY HD. Analyzed the data: YY HD QL MY. Contributed reagents/materials/analysis tools: YY HD QL WC RG CM MY. Wrote the paper: YY HD.

{ "pile_set_name": "PubMed Central" }

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By analyzing SNP array data on "trios" samples including premalignant atypical adenomatous hyperplasias (AAH) of the lung, matched normal and lung adenocarcinoma (LUAD) tissues from 16 patients, Sivakumar et al. \[[@bb0005]\] in this issue investigated the allelic imbalance (AI) events in the premalignant lesions of lung adenocarcinoma. With a modified statistic method to infer the AI events sensitively, they identified AI events in more than 50% of AAH tissues, indicating the importance of chromosomal aberrations and genome instability in the development of AAH. In addition, they identified a number of chromosomal aberrations harboring tumor suppressors and oncogenes, such as 17p loss affecting well-known *TP53*. It was worth to note that the same group previously reported the somatic mutations and RNA-Seq data from the same samples \[[@bb0010]\]. Thus, they presented a comprehensive molecular landscape of the evolution process from AAH to LUAD, which is important for understanding the etiology of LUAD. First of all, the premalignant data provided evidence that chromosomal aberrations initiated at the early stage of LUAD and emerged even earlier than many driver mutations. Both somatic mutations and copy number aberrations were the hallmarks of cancer. The TCGA study revealed a heavy burden of somatic mutations and copy number alterations in LUAD and most of them are "passengers" that have no effect on the neoplastic process \[[@bb0015]\]. Thus, it is difficult to identify which somatic alterations are drivers that promote a selective growth advantage. Previous studies suggested that genes carried "driver" alterations had a higher alteration rate than other genes and proposed statistical methods to identify driver genes \[[@bb0020]\]. By using the methods, TCGA studies reported that *TP53* was a commonly mutated (46%) gene in LUAD \[[@bb0025]\]. In addition, somatic alterations usually affect oncogenes in the receptor tyrosine kinase (RTK)/RAS/RAF pathways in LUAD and tumor suppressor genes including *STK11*, *KEAP1*, *NF1*, *RB1,* and *CDKN2A* \[[@bb0025]\]. They also observed copy number amplified regions (3q26, 5p15, 7p11, 8q24, 11q13, 12q13, 12q15, 14q13) and deleted regions (17p13, 9p21, 9p23) in LUAD \[[@bb0025]\]. However, it is difficult to decipher the causal relationship between somatic mutations and copy number alterations in the cancer genomics data. With the evidence from AAH, we can differentiate the initial driver alterations of LUAD like 17p loss involving *TP53*. Furthermore, in this issue Sivakumar et al. \[[@bb0005]\] reported an important association between AI burden and smoking behavior in AAH. Interestingly, a similar trend was observed in their matched LUAD samples, but the difference was not significant, and at the same time, a higher AI burden was seen in *EGFR* mutated non-smokers. The results suggested that the underlying mechanisms of AI were quite different in smokers and non-smokers: AI served as a founding event in smoking patients and initiated the development of LUAD, while it was only a bypass product of genome instability in non-smoker patients and therefore occurred only in LUAD tissues. In the phylogenetic tree construction, Sivakumar et al. \[[@bb0005]\] found that 6 of 7 patients with evidence of shared AI between matched AAH and LUAD were smokers, which further supported the conclusion above. Thus, the AI events in smokers and non-smokers should not be considered equally in the following studies. However, the sample size involved in this analysis is relatively small and more well-designed epidemiological studies with larger sample size are warranted to further illuminate the differences. The molecular landscapes of premalignant tissues could also be used to identify potential biomarkers to predict the progress from the prelesions to cancers. Teiseira et al. recently profiled the genomic, transcriptomic, and epigenomic landscape of the prelesion of lung squamous cancer \[[@bb0030]\]. With longitudinally monitoring information, they successfully generated a predictive model to identify which lesions will progress with remarkable accuracy, which offered a new strategy for the early detection of lung cancer. Lastly, the public available multi-omics data from "trios" LUAD patients are a valuable resource for the following studies of LUAD. For instance, we recently conducted whole-genome sequencing and RNA-Seq on 90 lung cancer and observed an association between infiltrating B cells and *EGFR* mutations in tumor tissues of LUAD \[[@bb0035]\]. We performed a re-analysis of the RNA-Seq data from the previous Sivakumar et al.\'s paper \[[@bb0010]\] and found that the elevation of infiltrating B cells and inflammatory microenvironments preceded the emergence of *EGFR* mutations \[[@bb0035]\]. However, there are still great challenges to investigate the evolution process from AAH to LUAD. Intratumor heterogeneity of lung cancer was one of them. A recent study conducted whole-exome sequencing on the multi-regions of non-small-cell lung cancer (NSCLC) \[[@bb0040]\]. They observed widespread intratumor heterogeneity for somatic copy-number alterations as well as mutations. A number of known driver alterations occurred in the sub-clones of LUAD. To clearly infer the clonal evolution and trace the origin of these alterations, a multi-region sampling of AAH are necessary for future studies. In addition, the low cell fraction of somatic alterations in AAH also limited the genomic studies and the subsequent evolution analysis. The authors overcome the challenge by jointly modeling the allelic imbalance statistics, but the technology advance may provide new insights into the era. Single-cell RNA-Seq has recently used to decipher the microenvironments and subclones of lung cancers \[[@bb0045]\] and additional methods have been developed to estimate the copy number alterations and copy-neutral loss-of-heterozygosity events in single-cell RNA-Seq data \[[@bb0050]\], which might be used in the future studies of AAH to solve the problems of cell heterogeneity. Conflicts of interest {#s0005} ===================== The authors declare no conflicts of interest.

{ "pile_set_name": "PubMed Central" }

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All relevant data are within the manuscript. The measurement data are available from the figshare database (doi: [10.6084/m9.figshare.8088167](https://doi.org/10.6084/m9.figshare.8088167)). Introduction {#sec001} ============ The chance of being struck by lightning increases when humans are outdoors in a thunderstorm. They are especially exposed to lightning strikes directly in the head in open elevated areas \[[@pone.0223133.ref001]--[@pone.0223133.ref003]\]. A direct lightning strike can cause physical damages like burns on the skin, fractures in the skull, or haemorrhage in the brain due to the heat and explosive effects, as described in several pathological diagnosis reports and other literature \[[@pone.0223133.ref004]--[@pone.0223133.ref010]\]. These effects are caused by currents up to 200 kA and voltages of 1 MV and higher in discharges of a few milliseconds \[[@pone.0223133.ref011]--[@pone.0223133.ref013]\]. Overall, the mortality of lightning injury is only about 10% \[[@pone.0223133.ref014],[@pone.0223133.ref015]\] but increases to about 30% when people are struck by lightning outdoors \[[@pone.0223133.ref001],[@pone.0223133.ref002]\]. There are five known mechanisms of lightning injury: direct strikes accounting for 3--5% of the cases, side flashes for 30--35%, contact injury 3--5%, upward streamer 10--15% and ground current 50--55% \[[@pone.0223133.ref003],[@pone.0223133.ref015]\]. A possible mechanism responsible for the relatively large chances of surviving a lightning strike despite the mentioned medical issues could be the formation of a flashover outside the body. In a flashover, most of the current flows outside the body and only a few Amperes flow inside the body \[[@pone.0223133.ref012],[@pone.0223133.ref013]\], as estimated in a theoretical study by Uman \[[@pone.0223133.ref012]\]. This reduced current inside the body during a flashover might be small enough to not be lethal. This might be especially true for the head owing to the assumed additional protective effect of the skull caused by its low conductivity. The head is one of the most important parts of the human body and is most exposed during a direct strike or side flash. However, there are no reports in the literature on measured current distributions inside the head before and during a flashover in a direct lightning strike. Consequently, we aim to establish an experimental setup to measure current distributions for the main compartments of the human head using a physical head phantom. With these measurements, we want to establish normative values that can serve as a basis for later experimental investigations on the possibilities of lightning protection equipment for humans according to the standard DIN EN 1149 \[[@pone.0223133.ref016]\]. To achieve this aim, we have designed human head phantoms comprising scalp, skull, and intracranial compartments, mimicking the geometry, relative permittivity, and electric conductivity of real biological human tissue. In contrary to the homogeneous models used previously to investigate the flashover mechanism \[[@pone.0223133.ref017]\] the nonhomogeneous head phantoms allow to quantify the current distribution in the three compartments. Moreover, the head phantoms allow a qualitative analyses of visible damages per compartment. Consequently, we will be able to experimentally quantify the additional protective effect of the skull caused by its low conductivity. In addition to the currents flowing in the three head compartments, we measured the flashover current with an electrode outside and underneath the head phantom. With these measurements, we strive to obtain an experimental validation of the theoretical values proposed by Uman \[[@pone.0223133.ref012]\]. Materials and methods {#sec002} ===================== Overview {#sec003} -------- We applied pulses to the head phantoms using three different pulse generators with different properties. A Marx generator delivered up to 450 kV and relatively low currents of up to 2 kA with an 1.2/50 μs voltage pulse; a combination wave generator delivered up to 12 kV and 6 kA with low noise (due to the compact design) and 1.2/50 μs voltage pulse or 8/20 μs current pulse; a 10/350 μs current pulse generator delivered up to 12 kV and 42 kA. The various generators emulated different aspects of a lightning flash. For example, the standardized positive first lightning current is defined by front time 10 μs, the maximum amplitude and time to half-value on tail 350 μs, identified as 10/350 μs according to the standard IEC 62305 \[[@pone.0223133.ref018]\]. The equivalent frequency is 25 kHz for the front of this pulse. [Fig 1](#pone.0223133.g001){ref-type="fig"} illustrates the calculation of the equivalent frequency. After the peak, the frequency content decreases to 1 kHz and lower \[[@pone.0223133.ref011]\]. ![Calculation of the equivalent front frequency of a lightning pulse current.\ The front time (T~1~) of the 10/350 *μ*s lightning current is 10 *μ*s. The equivalent frequency (f~eq~) is based on a sine wave and the corresponding equivalent period (T~eq~).](pone.0223133.g001){#pone.0223133.g001} Creation of head phantom {#sec004} ------------------------ We created three-compartment head phantoms based on the computer tomography data of an individual human head similar to Tidswell et al. \[[@pone.0223133.ref019]\] and Sperandio et al. \[[@pone.0223133.ref020]\]. We segmented the inner and outer boundaries of the skull and the outer skin boundary from the CT data \[[@pone.0223133.ref021]\] using Seg3D (SCI Institute, Salt Lake City, UT, United States) and designed forms resampling these boundaries as negative moulds for the head phantom compartments in SolidWorks® (Dassault Systèmes, Vélizy-Villacoublay, France). We produced the moulds of the compartments through a fused deposition modelling process. The moulds are made of the polymer Lignin (GreenTec, Extrudr®, Lauterach, Austria). The casting process of the compartments started with the innermost compartment, the intracranial volume (brain), continued to the central compartment, the neurocranium (skull), and ended with the outermost compartment the scalp; in each case, the preceding compartment provided the base for the next compartment. These head phantoms resampled the geometry of the head and the dielectric properties of biological tissue. We used 2% agarose (Agarose Broad Range, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) in deionized water as the basic phantom material. The dielectric properties of the compartments were set by doping the basic phantom material with different fractions of sodium chloride (NaCl), graphite (Graphite d50, Algin-Chemie e. K., Neustadt-Glewe, Germany), or carbon black (carbon fibre powder, EMV Vega, Recklinghausen, Germany). We used the mixtures (shown in [Table 1](#pone.0223133.t001){ref-type="table"}) for creating head phantoms, in accordance to literature values for the dielectric properties. The dielectric properties were analysed with a Hewlett Packard 4284A 20 Hz to 1 MHz precision LCR meter (HP Inc., Palo Alto, CA, United States) in a two-plate method. 10.1371/journal.pone.0223133.t001 ###### Dielectric properties of the compartments of the two types of head phantoms in the range of 20 Hz to 1 MHz. ![](pone.0223133.t001){#pone.0223133.t001g} ---------------------------------------------------------------------------------------------------------------- Phantom compartment Mixture Electric conductivity in S/m\ Relative permittivity\ 20 Hz--1 MHz 20 Hz--1 MHz -------------------------- ---------------------------- ------------------------------- ------------------------ **Head phantom Type I** **Brain Type I** 2 % agarose, 0.17 % NaCl 0.12--0.38 72.5·10^6^--930 **Skull Type I** 2 % agarose, 0.01 % NaCl,\ 0.024--0.063 9·10^6^--196 4 % graphite **Scalp Type I** 2 % agarose, 0.17 % NaCl,\ 0.054--0.63 43·10^6^--1290 4 % carbon black **Head phantom Type II** **Brain Type II** 2 % agarose, 0.17 % NaCl 0.12--0.38 72.5·10^6^--930 **Skull Type II** 2 % agarose, 0.01 % NaCl 0.015--0.048 8.1·10^6^--155 **Scalp Type II** 2 % agarose, 0.17 % NaCl 0.12--0.38 72.5·10^6^--930 ---------------------------------------------------------------------------------------------------------------- Two types of phantoms were created, with (Type I) and without carbon additives (Type II). Type I phantoms had the advantage of better approximating the dielectric properties found in the literature \[[@pone.0223133.ref022]--[@pone.0223133.ref025]\]. The phantoms without carbon additives were transparent, which allowed us to later add dyes for the visualization of micro damages and perforations caused by electric current pulses. For dyeing, all transparent head phantoms were placed completely in an emulsion of 1% ironhexacyanoferrate and deionized water for two days after the tests. [Table 1](#pone.0223133.t001){ref-type="table"} shows the dielectric properties for both types of head phantoms. [Fig 2A](#pone.0223133.g002){ref-type="fig"} shows the scalp from head phantom Type I and [Fig 2B](#pone.0223133.g002){ref-type="fig"} depicts the structure of head phantoms in a CAD model. The scalp compartment appears black due to the carbon additives. The maximum diameter of each phantom is 200 mm and the maximum height is 165 mm. Furthermore, we created a third type of head phantom (head phantom Type III) which was built up like head phantom Type I but with the addition of four silver/silver-chloride microelectrodes (diameter: 1 mm, length: 2.5 mm, BMP1 from MedCat GmbH, Munich, Germany) in the brain compartment. Thus, it was possible to measure voltages inside head phantom Type III during an applied pulse. The microelectrodes were plugged in holders, which were produced in a fused deposition modelling process and were made of Lignin. Each holder had a diameter of 3 mm. Two holders had a length of 50 mm and the other two had a length of 100 mm. We named the anterior microelectrode ME1, the left microelectrode ME2, the posterior microelectrode ME3, and the right positioned microelectrode ME4 ([Fig 2C](#pone.0223133.g002){ref-type="fig"} and [Fig 2D](#pone.0223133.g002){ref-type="fig"}). ![Head phantom geometry and electrode setup of microelectrodes.\ (A) Formed scalp in a lateral view of a head phantom with carbon additives (head phantom Type I). (B) Sagittal cut through the head phantom. The intracranial volume (brain) is coloured blue, the neurocranium (skull) yellow, and the scalp red. (C) Double cut of the head phantom Type III with the microelectrodes ME1 and ME2 and their holders. (D) Double cut of the head phantom Type III with the microelectrodes ME3 and ME4 and their holders. The inset in D shows a photograph of a microelectrode (grey) with a physical cut through a holder (white) and the wire insulation (yellow).](pone.0223133.g002){#pone.0223133.g002} We created an electrode setup with four separate brass electrodes to detect the current distribution across the compartments of the head phantom. These electrodes were grounded in all the experiments. The geometry of each electrode was adapted to the base area of the compartments to minimize the current path from adjacent compartments. When the head phantom was placed on the electrode setup, the innermost electrode (E1) contacted the brain, the second electrode (E2) the skull and the third electrode (E3) the scalp. The fourth electrode (E4) concentrically surrounded the base of the phantom in a distance of 5 mm (E4: inner diameter 205 mm, outer diameter 215 mm). The chosen dimensions of electrode E4 and the distance to E3 allowed to determine the current distribution in the flashover separately since the ends of the flashovers hit only electrode E4. Flashovers are discharges that start from an impact point on the surface of the head phantom and propagate to the electrode ring E4. These events were detected by a single lens reflex (SLR) camera (EOS 5D Mark II, Canon Inc., Tokyo, Japan). [Fig 3](#pone.0223133.g003){ref-type="fig"} shows the electrode setup and its dimensions. ![Electrode setup.\ (A) Schematic of electrode setup with electrodes innermost E1 to outermost E4. (B) Dimensions of the electrode setup in a top view. Each electrode E1--E4 has a thickness of 10 mm.](pone.0223133.g003){#pone.0223133.g003} Experiments with Marx generator {#sec005} ------------------------------- We used a Marx generator to replicate the high-voltage effects on the human head during a lightning strike. The Marx generator (MG, HIGHVOLT Prueftechnik Dresden GmbH, Germany, properties of MG: Energy 6 kJ, max. charging voltage 125 kV per stage) produces a standardized 1.2/50 μs voltage pulse and can reach a total load voltage of 450 kV \[[@pone.0223133.ref026],[@pone.0223133.ref027]\]. An MG can reach a high voltage at the output with one pulse, making it possible to ionize the air over long distances. The discharge is more like a real lightning strike discharge, but the current level of the generated pulse is much lower. In the first test series, one head phantom Type I was placed on the electrode setup E1--E4 underneath the output from the MG. The distance between the high voltage point electrode (output) and the highest point of the head phantom (vertex) was 150 mm. We applied 15 single positive voltage pulses to the head phantom, each pulse with a total load voltage from the MG of 450 kV. An SLR camera documented the expansion of the lightning discharge. In a second test series, we placed a head phantom Type II on the electrode setup (E1--E4) underneath the output of the MG in the same distance of 150 mm. Again, 15 single voltage pulses were applied, each pulse with a total load voltage of 450 kV. The head phantom Type II was dyed before slicing, as explained in Section: Creation of head phantom. We examined and sliced both head phantoms after the test series. For slicing, we used a strained wire with a diameter of 0.5 mm. The slicing always started with a sagittal cut through one of the observed perforations on the surface of the head phantoms. Measurements with combination wave generator {#sec006} -------------------------------------------- A head phantom Type III was tested with an in-house combination wave generator (CWG) to investigate the electrical behaviour of the head phantom and measure the potential distribution in the brain compartment. The used CWG had the following parameters: Energy 350 J, V~max~ 12 kV 1.2/50 μs, I~max~ 6 kA 8/20 μs \[[@pone.0223133.ref026]\]. The head phantom was placed on the electrode setup (E4 was decoupled since no flashovers were expected) and a brass electrode (diameter: 25 mm, height: 24.9 mm) was galvanically coupled to the top of the head phantom, near the vertex. This brass electrode was necessary for applying the pulse to the head phantom, because the voltage of the CWG is not high enough to break through larger air gaps like the Marx generator. Further, the influence of an air gap was avoided in the tests to get the electrical behaviour of only the head phantom. For the measurements, two Pearson current monitors Model 101 and one Pearson current monitor Model 110 (Pearson Electronics Inc., Palo Alto, CA, United States) detected the currents of E1 to E3, while one voltage probe PHV4002-3 (PMK GmbH, Kassel, Germany) detected the voltage drop over the head phantom. Four voltage probes PHV 1000 (PMK GmbH, Kassel, Germany) recorded the potentials of each microelectrode in the brain compartment. All signals were measured simultaneously by an eight-channel oscilloscope DLM4000 (Yokogawa Electric Corp., Musashino, Japan). We applied 10 positive voltage pulses with an amplitude of 5 kV to the head phantom. The measurement setup is shown in [Fig 4](#pone.0223133.g004){ref-type="fig"}. After that, we applied 10 positive voltage pulses with an amplitude of 12 kV to the head phantom. In this test series, the four voltage probes PHV 1000 were disconnected due to their limited admissible voltage of 6 kV. Possible discharges were documented with the SLR camera. ![Measurement setup for the experiments with the combination wave generator (CWG).\ (A) Interconnected measuring equipment with the combination wave generator, head phantom, and oscilloscope. E1 to E3 are the grounded electrodes, each with a current monitor. The four microelectrodes (ME1 to ME4) in the brain compartment were connected by four voltage probes (only two of four probes are symbolized in the schematic). The dashed lines symbolize the measurement wires to the oscilloscope. (B) Photograph of the head phantom Type III placed at the electrode setup and connected via the stimulation electrode to the CWG. A mirror shows the backside of the head phantom Type III.](pone.0223133.g004){#pone.0223133.g004} Measurements with 10/350 μs current pulse generator {#sec007} --------------------------------------------------- We tested four head phantoms on a 10/350 μs current pulse generator to emulate the high-current effects of a direct lightning strike to the head \[[@pone.0223133.ref018]\]. A current pulse generator reaches a higher current level, different from that obtained by combination wave generator or Marx generator. Therefore, a more realistic lightning current level could be applied to the tested head phantoms. Distances between the output of the generator and the head phantoms were restricted due to the lower voltage. Therefore, the experiments replicated the impact point of a lightning discharge on the head without possible upward connecting lightning leaders. The generator (IP176/12S of HIGHVOLT Prueftechnik Dresden GmbH, Dresden, Germany) uses 12 switchable single capacitors (called HV-CAP) IS-Phr 12 (VISHAY Electronic GmbH, Selb, Germany). Each HV-CAP has a capacitance of 175 μF and can be loaded with a voltage of up to 12 kV, which results in an energy of 12.6 kJ per charged capacitor. Each head phantom was placed with the electrode setup in the examination chamber of the current pulse generator. The distance from the power output electrode of the current pulse generator to the surface of the head phantom was 55 mm. Therefore, it was necessary to bridge the distance with a copper ignition wire with a diameter of 0.1 mm, which ended about 4 mm above the surface of the head phantom at the vertex. We used three current monitors with a transmission ratio of 1:1000 (Model 1423 Pearson Electronics Inc., Palo Alto, CA, United States) and two current monitors with a transmission ratio of 1:100 (Model 101) to capture the currents in each compartment of the head phantom. The voltage probe (PHV4002-3) detected the voltage drop from the output of the current pulse generator to the ground. All signals were simultaneous recorded from parallel triggered oscilloscopes 44MXI-A and HDO6054 (Teledyne LeCroy, CA, USA). [Fig 5](#pone.0223133.g005){ref-type="fig"} shows the measurement setup for the tests with the 10/350 μs current pulse generator. ![Measurement setup on the 10/350 μs current pulse generator.\ (A) Schematics of the setup. E1 to E4 are the grounded electrodes. They are connected to the current monitors. The dashed lines indicate the measurement wires to the oscilloscope. (B) Photograph of the head phantom Type II placed in the examination chamber of the current pulse generator.](pone.0223133.g005){#pone.0223133.g005} We performed different measurement series with this setup on the current pulse generator by changing the number of HV-CAPs to vary the resulting current. Four head phantoms were examined and sliced after the measurements in order to investigate the optically visible damages. In addition, the head phantom Type II was dyed with ironhexacyanoferrate to highlight perforations and damages. - Test series I: The series started with 12 HV-CAPs @ 12 kV. The number of the HV-CAPs was reduced by three after three applied pulses to the phantom Type 1. At three used HV-CAPs, the number was reduced to one HV-CAP. The charging voltage was always constant. In the last step, the charging voltage was reduced (one HV-CAP @ 6 kV). The used HV-CAP numbers were: 12 @ 12 kV, 9 @ 12 kV, 6 @ 12 kV, 3 @ 12 kV, 1 @ 12 kV, and 1 @ 6 kV. This test series was performed to determine the electrical behaviour and current distribution of the head phantom at different current levels while the positive 10/350 μs pulse. - Test series II: Repeat of test series I with a newly casted head phantom Type I. - Test series III: A single pulse with 12 HV-CAPs and a charging voltage of 12 kV was applied to one head phantom Type II. This test was performed to find potential damages and perforations at the transparent head phantom Type II which are not visible with the head phantom Type I. - Test series IV: The series was performed with a head phantom Type I similar to I and II, but the sequence for the number of used HV-CAPs was pseudo-randomized, starting with six HV-CAPs @ 12 kV. Results {#sec008} ======= Results of experiments on Marx generator {#sec009} ---------------------------------------- We observed discharges on the surface of the head phantom Type I and head phantom Type II for each 450kV voltage pulse applied from the Marx generator, as shown in [Fig 6A and 6B](#pone.0223133.g006){ref-type="fig"}. It can be seen that the discharge channel hit the head phantoms and branched in discharges. The spilt began either at the surface of the head or in the air gap. After the tests, we examined the head phantoms. The head phantom Type I showed a dried ellipsoidal area around the vertex with a length of 120 mm in the anterior--posterior direction and 100 mm in the lateral direction. Also, we found punctured regions with diameters of 2--5 mm. No further particularities were observed in the brain or skull compartment after the slicing of the head phantom Type I. Several perforations occurred in the vicinity of the optically detected impact points at the head phantom Type II. The four largest ones had a diameter of 3--6 mm (marked with X [Fig 6C](#pone.0223133.g006){ref-type="fig"}). We sliced the head phantom Type II in a sagittal cut through one impact point, as marked in [Fig 6C](#pone.0223133.g006){ref-type="fig"}, and found a dyed channel, which started at the impact point and ended after a length of approx. 50 mm in the brain compartment (highlighted with blue colour in [Fig 6D](#pone.0223133.g006){ref-type="fig"}). This perforation was the only one penetrating the brain compartment---all other observed penetrations ended in the skull compartment. There was no systematic difference in the results from head phantom Type I and head phantom Type II. In a previous study, we measured currents of about 2 kA in a simplified head phantom \[[@pone.0223133.ref028]\] originating from the same pulses of the Marx generator. For the present study, we estimated similar currents in the head phantom Type I and Type II. ![Results of the experiments with the Marx generator.\ (A) Head phantom Type I and a discharge over the surface of the scalp. A mirror behind the head phantom Type I shows possible discharges on the side facing away from the SLR camera. (B) A discharge over the surface of the scalp from the head phantom Type II. (C) Top view on the scalp compartment from the head phantom Type II after 15 applied single voltage pulses. We marked the biggest perforations with X. The cut line for the first slice is marked black. (D) Sagittal cut along the black line in C, the arrows in C show the view direction. We found a tubular perforation, highlighted in blue colour.](pone.0223133.g006){#pone.0223133.g006} Results of measurements on combination wave generator {#sec010} ----------------------------------------------------- No flashover or discharges on the surface of the head phantom Type III were optically detected in the experiments with the CWG and an applied 1.2/50 μs voltage pulse of 5 kV. [Fig 7A](#pone.0223133.g007){ref-type="fig"} shows the average recorded time courses of the voltage and the currents of E1 to E3. The mean values at 2.4 μs are additionally indicated in [Fig 7A](#pone.0223133.g007){ref-type="fig"}. We calculated the relative current distribution of each compartment with the values at 2.4 μs (maximum measured voltage). The brain was exposed to 11.2%, the skull to 8.6% and the scalp to 80.2% of the current. An average voltage of 530 V was detected at the first microelectrode ME1 at 2.4 μs. Further, the voltage reached 1250 V at ME2, 520 V at ME3, and 1220 V at ME4 ([Fig 7B](#pone.0223133.g007){ref-type="fig"}). We calculated the electric field strength (based on the recorded electric potential at 2.4 μs and the distance of the microelectrode to the ground) at these four points: ME1: 10.6 kV/m at ME1; ME2: 12.5 kV/m; ME3: 10.4 kV/m; ME4: 12.2 kV/m. ![Results of measurements on the combination wave generator with an applied voltage of 5 kV.\ (A) Average time courses of recorded voltage and measured currents at the electrodes E1 to E3. (B) Time courses of the recorded voltages for the four microelectrodes (ME) in the brain compartment.](pone.0223133.g007){#pone.0223133.g007} We optically detected multiple discharges at the surface of the scalp compartment with an applied voltage of 12 kV ([Fig 8A](#pone.0223133.g008){ref-type="fig"}). The discharges started from the attached stimulation electrode. [Fig 8B](#pone.0223133.g008){ref-type="fig"} depicts the average recorded time courses of the voltage at the generator output and currents from the electrodes E1 to E3 with labelled mean values at 2.4 μs (maximum measured voltage). The relative current distribution at 2.4 μs was: brain compartment 13.1%, skull compartment 6.1% and scalp compartment 80.8%. ![Results of measurements on the combination wave generator with an applied voltage of 12 kV.\ (A) Registered discharges at the surface of the scalp compartment. The mirror in the background shows the backside of the head phantom. (B) Average time courses of recorded voltage at the generator output and currents from E1 to E3.](pone.0223133.g008){#pone.0223133.g008} Results of measurements on 10/350 μs current pulse generator {#sec011} ------------------------------------------------------------ In each test series, we optically detected a flashover across the head phantoms in the measurements with the 10/350 μs current pulse generator. In almost all tests, the discharge sat on near the highest point of the head phantoms (vertex) and went over the scalp to E4. [Fig 9](#pone.0223133.g009){ref-type="fig"} depicts four different morphology examples. [Fig 9A and 9B](#pone.0223133.g009){ref-type="fig"} show exemplary discharges of the test series I and III with 12 HV-CAPs @ 12 kV with a head phantom Type I and the head phantom Type II, respectively. In both cases, the discharge channel appeared structured in the vicinity of the scalp. However, the discharge channel was surrounded by a corona. The discharge and the resulting flashover were less intensive and the corona around the channel was smaller with one HV-CAP @ 6 kV in contrast to 12 HV-CAPs @ 12 kV. An example of a discharge with one HV-CAP @ 6 kV is shown in [Fig 9C](#pone.0223133.g009){ref-type="fig"}. [Fig 9D](#pone.0223133.g009){ref-type="fig"} depicts the last applied current pulse of the test series IV (after three times six HV-CAPS @ 12 kV and one time 12 HV-CAPS @ 12 kV). The main discharge over the scalp and the discharge channel are difficult to identify. Discharges next to the scalp appeared especially on the right side of the head phantom Type I. The discharges busted the phantom material at several points. ![Detected flashovers across head phantoms for the tests with the 10/350 μs current pulse generator.\ (A) An example of the test series I with 12 HV-CAPs @ 12 kV and with a head phantom Type I. (B) An example of test series III with 12 HV-CAPs @ 12 kV and with head phantom Type II. (C) An exemplary discharge of the test series I with one HV-CAP @ 6 kV and with head phantom Type I. (D) An exemplary discharge of the test series IV at 12 HV-CAPs @ 12 kV with head phantom Type I. The head phantom Type I was destroyed at this discharge.](pone.0223133.g009){#pone.0223133.g009} All recorded voltages and currents showed characteristic time courses. The voltage increased up to the load voltage in about 1 μs; simultaneously, the current increased through the head phantom. Then the voltage decreased rapidly to a value of about 1.5 kV in approximately 50 μs. At the same time, the current in E4 increased and showed an aperiodic pulse behaviour. [Fig 10A and 10B](#pone.0223133.g010){ref-type="fig"} present the average time courses of the test series I and II at 12 HV-CAPs @ 12 kV with the most important key values. It can be seen that simultaneous to the first rise of the voltage, the current in E1, E2, E3 and the total current also increase. The time point t~start~ indicates the beginning of the increasing voltage and current. The discharge process started over the head phantom at this time point. After the increase of the current in E1 and E2 to the maximum, these currents broke down rapidly, along with the voltage and the current in E3 (Case 1). We defined this point as the moment of the beginning of the evolvement of the complete flashover across the head phantom and labelled it t~0~, determined by the highest decrease speed with the first derivation of the voltage. After the rapid collapse of the current in E1 and E2 close to zero ampere, the current in E4 increased from zero ampere to a high amplitude (in [Fig 10A and 10B](#pone.0223133.g010){ref-type="fig"}, up to 41.8 kA or 41.3 kA). Furthermore, we found a second possible case (Case 2) for the current time course in E3. The current in E3 broke in first, increased again, and reached a multiple higher peak, for example from 930 A at t~0~ to 5.7 kA after further 14.4 μs. This case is shown as an example in [Fig 10B](#pone.0223133.g010){ref-type="fig"}. [Fig 10C](#pone.0223133.g010){ref-type="fig"} depicts the time courses from the test series I and II at one HV-CAP @ 6 kV. The duration to t~0~ increases in comparison to the time courses in [Fig 10A and 10B](#pone.0223133.g010){ref-type="fig"}, but the time course shows some similarity in shape. The applied pulses ended about 2.6 ms after t~0~. As a further result, we extracted the time points t~start~ for each test for the test series I and II. The distribution is shown in [Fig 10D](#pone.0223133.g010){ref-type="fig"}. The time of starting of the flashover (t~start~) for the head phantom was in median between 40 μs and 45 μs at an energy level between 151.2 kJ and 75.6 kJ. The time increased from the level of 37.8 kJ at 53.6 μs over 12.6 kJ at 69.5μs to 6.3 kJ at 257 μs. The time t~start~ differed most at 6.3 kJ between 219 μs and 694 μs. ![Time courses from test series I and II with the 10/350 μs current pulse generator.\ (A) Time courses for 12 HV-CAPs @ 12 kV for an example Case 1 at test series I and II. (B) Time courses for 12 HV-CAPs @ 12 kV for an example Case 2 at test series I and II. (C) Time courses for one HV-CAP @ 6 kV. (D) Violin plots of t~start~ for each tested capacitor stage (applied energy). The time t~start~ was defined as latency at which we registered a first appearance of voltages or currents different from zero. The time to start of the flashover was called t~0~. M: mean, Mdn: median, SD: standard deviation.](pone.0223133.g010){#pone.0223133.g010} [Table 2](#pone.0223133.t002){ref-type="table"} provides the calculated relative current distribution for the compartments at t~0~ and at the latency of the maximum of the total current after the fully formed flashover (t~maxtot~), based on the values (test series I and II) of the currents from each tested capacitor stage. The skin compartment was most exposed to the applied current at t~0~ for each used HV-CAP stage, followed by the brain compartment and the skull compartment. After a flashover, the highest currents were detected at t~maxtot~ in electrode E4 for each capacitor stage; we found fractions of current between 98.2% and 99.5% in E4. The next highest part flowed through the scalp compartment. Brain and skull compartments had a smaller exposition, each with a current below 7 A. For the brain and skull compartments, the relative current distribution increased with a lower energy level at t~maxtot~. 10.1371/journal.pone.0223133.t002 ###### Relative current distribution for the test series I and II at the time points before (t~0~) and after a fully formed flashover (t~maxtot~). ![](pone.0223133.t002){#pone.0223133.t002g} -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- HV-CAPS\ Total energy in kJ Applied max. current @ t~0~ in kA Applied max. current @ t~maxtot~ in kA Rel. current distribution for flashover in % Rel. current distribution for scalp in % Rel. current distribution for skull in % Rel. current distribution for brain in % charged up to 12 kV --------------------- -------------------- ----------------------------------- ---------------------------------------- ---------------------------------------------- ------------------------------------------ ------------------------------------------ ------------------------------------------ ----- -------- ------ -------- **12** 151.2 1.00 41.5 n/a 99.5 85.8 0.45 5.1 \<0.02 9.1 \<0.02 **9** 113.4 0.95 31.8 n/a 99.5 87.2 0.32 4.6 \<0.02 8.2 \<0.02 **6** 75.6 1.40 21.5 n/a 99.5 90.4 0.38 3.5 \<0.02 6 \<0.02 **3** 37.8 0.77 10.9 n/a 98.2 83.5 1.7 5.9 0.05 10.6 0.06 **1** 12.6 0.97 3.7 n/a 98.9 87.2 0.8 4.3 0.08 8.5 0.2 **1 (6 kV)** 6.3 0.48 1.55 n/a 99.2 90.5 0.3 3.1 0.2 6.4 0.3 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- The time courses of the currents in test series III were also aperiodic, as in test series I and II for case 2, but we found a second increase in the current in E3, probably caused by an incomplete flashover. The currents in E1, E2, and E3 decreased and the current increased up to approximately 2.5 kA in E4 after the breakdown of the voltage. After that, we registered an increase of the voltage, an increase of the currents at E1--E3, and a collapse to 0 A in E4 (named collapse in [Fig 11A](#pone.0223133.g011){ref-type="fig"}). After another 5 μs, the voltage broke down again as well as the currents in E1 to E3. The current in E4 increased and followed the typical aperiodic time course. The measurement of the time courses from test series IV with 12 HV-CAPs @ 12 kV are shown in [Fig 11B](#pone.0223133.g011){ref-type="fig"}. The time courses were aperiodic too, but the time to the event points were longer than the measured durations from other test series. We could not record the event t~start~. The moment t~maxtot~ was reached after 131 μs after t~0~. Further, the time course showed waves between t~0~ and t~maxtot~. ![Time courses from test series III and IV with the 10/350 μs current pulse generator.\ (A) Shows the time courses for the test series III, the only one test series with the head phantom Type II and 12 HV-CAPs @ 12 kV. (B) Depicts the recorded time courses for the test series IV at the second repeat with 12 HV-CAPs @ 12 kV.](pone.0223133.g011){#pone.0223133.g011} The maximum current steepness is a parameter to define and compare lightning current pulses. We calculated the maximum current steepness of the measured total currents with the 30%--90%-rule of amplitude. We used the 30%--90%-rule because the current amplitude at 10% was not measurable \[[@pone.0223133.ref018]\]. The maximum current steepness showed a reduction from about 1855 A/μs at an applied energy of 151.2 kJ down to 64 A/μs at 6.3 kJ. Compared to the ideal 10/350 μs current pulses (assumed double exponential function), the measured current pulses had a less current steepness with a factor between 0.95 and 2.08, as shown in [Table 3](#pone.0223133.t003){ref-type="table"}. The difference of current steepness between the measured current pulses and the ideal 10/350 μs current pulses is due to impedances of the head phantom and the discharge channel. The 10/350 μs current pulse generator is not an ideal constant current source; additional impedances extend the pulse form. 10.1371/journal.pone.0223133.t003 ###### Current steepness of measured total currents in test series I and II and comparison with ideal 10/350 μs current pulses. ![](pone.0223133.t003){#pone.0223133.t003g} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ HV-CAPS\ Total energy in kJ Current steepness in A/μs of measured total current pulses Current steepness in A/μs of ideal 10/350 μs current pulses Factor between measured and ideal current pulses charged up to 12 kV --------------------- -------------------- ------------------------------------------------------------ ------------------------------------------------------------- -------------------------------------------------- **12** 151.2 1855 3557 1.92 **9** 113.4 1501 2726 1.82 **6** 75.6 1260 1843 1.46 **3** 37.8 655 934 1.43 **1** 12.6 334 317 0.95 **1 (6 kV)** 6.3 64 133 2.08 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ We examined and sliced the head phantoms after the test series, with the following results. - Test series I and II: We found similar damages on both head phantoms Type I from test series I and II, which are described for one phantom in the following: 10 impact points were found on the top surface of the scalp ([Fig 12A](#pone.0223133.g012){ref-type="fig"}), ranging from 5 mm to 13 mm in diameter---they are marked with Y. Several smaller perforations are marked with Z in [Fig 12A](#pone.0223133.g012){ref-type="fig"}. One path (outgoing from an impact point) was found in the right parietal occipital region, with a length of about 45 mm (marked with W in [Fig 12A](#pone.0223133.g012){ref-type="fig"}). We sliced the head phantoms with a sagittal cut through an impact point. In the sagittal cut of one head phantom Type I ([Fig 12B](#pone.0223133.g012){ref-type="fig"}), we noticed damages at the upper part of the skull near the impacts in the form of drying and melting, marked as V. The brain compartment seemed to be unaffected ([Fig 12B](#pone.0223133.g012){ref-type="fig"}). - Test series III: This head phantom Type II was dyed with ironhexacyanoferrate to highlight the damages. We found an ellipsoidal impact point on the scalp near the vertex, with an approx. length of 7 mm from anterior to posterior and 10 mm from right to left. Three obvious burn paths were found to be outgoing from the main impact point. The first path had a length of 110 mm to posterior direction, the second path a length of 95 mm to right parietal, and the third path a length of 80 mm to right parietal occipital. Furthermore, we detected perforations in the upper part of the skull near the impact point. We sliced the head phantom Type II with a sagittal cut through the impact point. One perforation had a length of 10 mm down to the skull, marked with L in [Fig 12C](#pone.0223133.g012){ref-type="fig"}. The impact point is marked with O in [Fig 12C](#pone.0223133.g012){ref-type="fig"}. We found melting and blistering points around the impact point (marked with P [Fig 12C](#pone.0223133.g012){ref-type="fig"}). No optical damages were found in the brain compartment. - Test series VI: The head phantom Type I ([Fig 12D](#pone.0223133.g012){ref-type="fig"}) was destroyed after three pulses applied with six HV-CAPs and two pulses applied with 12 HV-CAPs (in the second applied pulse). The scalp was blown up and the skull was broken on the right side of the head phantom. We found a melted area in the brain compartment under the cracked skull with the dimensions of 120 mm anterior to posterior and 95 mm inferior to superior. ![Examined head phantoms after the tests with the 10/350 μs current pulse generator.\ (A) Damaged and dried areas at the top of the scalp compartment for the head phantom Type I after the test series I. The impact points are marked with Y smaller perforations with Z and a current path with W. The black line marks the cut line. The arrows indicate the view direction for the cut. (B) Slice of the head phantom Type I indicated in A. Burned and perforated areas were magnified in the inset (marked with V). (C) Sagittal sliced head phantom Type II from test series III. A perforation was marked with L. Inset shows magnified area around the impact point O and P mark blistering in the scalp compartment. (D) The destroyed head phantom Type I after the test series IV. The scalp and skull compartments were blown open, and the brain compartment was damaged and melted.](pone.0223133.g012){#pone.0223133.g012} Discussion {#sec012} ========== Spatial current distribution in the head phantom {#sec013} ------------------------------------------------ We obtained the relative current distribution in the compartments of the created head phantoms and in discharge channels during various applied voltage and current pulses. In case of no flashover, the scalp compartment was always most exposed to the current (80--90%) in all experiments. The brain compartment was exposed to 6--13% of the current. The skull compartment experienced the smallest amount of current---about 3--6%. In case of flashover formation, the relative current distribution changed and 98--99% of the current flowed in the discharge channel due to its higher electrical conductivity compared to the head phantom. The remaining 1--2% of the current again predominantly flowed in the scalp, followed by the brain and skull compartments. We observed this distribution pattern (most current in the scalp, followed by brain and skull compartments) across all experiments, independent of the applied voltage and current pulse forms. To the best of our knowledge, this is the first time that a study has reported such current distributions that establish normative values to be used in later experimental investigations on the possibilities of lightning protection equipment for humans. Voltage and current time courses {#sec014} -------------------------------- The time courses of the recorded voltages and currents show distinct differences for the cases without and with flashover. For the cases without flashover, we found the expected rise and fall of both voltage and current time courses according to the applied 1.2/50 μs pulse. For cases with flashover, we found three time intervals: a short rising time of the voltage lasting approximately 1 μs after t~start~ marked the initial interval indicating the connecting leader phase \[[@pone.0223133.ref012]\]. Thereafter, the time interval until t~0~, the initial return stroke phase \[[@pone.0223133.ref012]\], is characterized by increasing current in and a constant voltage across the head phantom. We measured a time of approximately 30--700 μs for the second interval. The third interval starts with t~0~ and ends after 2.6 ms and is mostly dominated by the high pulse current; this interval is the denominated surface flashover phase \[[@pone.0223133.ref012],[@pone.0223133.ref013]\]. Contrary to the theoretical predictions by Uman \[[@pone.0223133.ref012]\], our experiments show an almost constant voltage across the head phantom and a nonlinearly increasing current in the head phantom for the first and second time intervals. While Uman \[[@pone.0223133.ref012]\] used an impressed pulse voltage for the first and second time intervals (t~start~ to t~0~), in our experiments, the 10/350 μs current pulse generator provided an impressed constant voltage during the first and second time intervals. In one experiment, the head phantom was destroyed by the impressed pulse. In this specific case, in the third time interval, we found time courses for voltages and currents that exhibited a step-like and spiky behaviour ([Fig 11B](#pone.0223133.g011){ref-type="fig"}). In this interval, the voltage and current irregularly increased and decreased, possibly indicating a change in the electrical conductivity. We assume that these changes in the electrical conductivity might be caused by thermal ionization and plasma processes directly in the head phantom \[[@pone.0223133.ref029]\]. Flashover {#sec015} --------- The flashover is a phenomenon that occurs in technical setups and during lightning accidents with humans \[[@pone.0223133.ref012],[@pone.0223133.ref013],[@pone.0223133.ref029]\]. In our experiments, the flashover took approximately 30--700 μs to form. Differently, a time of 30--150 ns characterizes flashovers for lightning accidents \[[@pone.0223133.ref013]\] and a few 100 ns for technical setups \[[@pone.0223133.ref029],[@pone.0223133.ref030]\]. The time to form a flashover mainly depends on the voltage amplitude and the pulse form. The observed difference in time were likely caused by the voltage differences: our experiments operated with 12 kV while a lightning strike has about 1 MV \[[@pone.0223133.ref011],[@pone.0223133.ref013]\]. Bazelian and Raizer characterize times up to 200 μs for fully formed discharge channels along water surfaces \[[@pone.0223133.ref031]\]. This implies that the time to form a flashover increases with a lower impedance such as biological tissues. The time to form a flashover increased in median from 69.5 μs at 1 CAP at 12 kV to 257 μs for 1 CAP at 6 kV ([Fig 10D](#pone.0223133.g010){ref-type="fig"}). This behaviour is expected for such impulse voltage-time characteristics \[[@pone.0223133.ref029],[@pone.0223133.ref030]\]. Implications for lightning protection {#sec016} ------------------------------------- Our experiments confirmed that most of the current (98--99%) flowed outside of the human head in the discharge channel after a fully formed flashover \[[@pone.0223133.ref012],[@pone.0223133.ref013]\]. Consequently, the faster a flashover is formed over the head, the higher are the chances of survival. Further, the current flowing in the brain compartment is typically a factor of 20 units less in amplitude compared to the current flowing in the scalp. This difference is caused by the protective property of the skull due to its low electrical conductivity and relative permittivity compared to the scalp and brain compartments \[[@pone.0223133.ref022]\]. Electroporation can cause cell damage and death \[[@pone.0223133.ref032]--[@pone.0223133.ref035]\]. An electric field strength of 6 kV/m and above can cause reversible or irreversible electroporation in biological cells \[[@pone.0223133.ref032]--[@pone.0223133.ref035]\]. An electric field strength of about 100--600 kV/m close to the ground are typical values during lightning \[[@pone.0223133.ref011]\]. Our experiments show an electric field strength of about 10.4--12.5 kV/m in the brain compartment during an applied 1.2/50 μs voltage pulse with an amplitude of 5 kV. These values indicate that electroporation can occur even for the lower current and voltage amplitudes in our experiments. Thus, neurological damages during a lightning strike caused by electroporation are likely to occur \[[@pone.0223133.ref006],[@pone.0223133.ref035]\]. We applied high voltage (Marx generator) and high current pulses (10/350 μs current pulse generator) to different head phantoms and optically analysed the damages to the head phantoms. In all setups, we found similar damages, e.g. perforations and burns on the scalp compartment or dried areas throughout the scalp compartment down to the skull. These observations are similar to autopsy reports after lightning accidents \[[@pone.0223133.ref003],[@pone.0223133.ref008],[@pone.0223133.ref015]\]. According to the DIN EN 1149, a personal lightning protection equipment, for example in the form of a coat with a hood consisting of modacrylics (flame retardant synthetic copolymer), can reduce the current amplitudes in the head compartments due to the material's electrostatic discharge behaviour \[[@pone.0223133.ref016]\]. Consequently, this lightning protection equipment can prevent burns and perforation as well. Experimental setup and limitations {#sec017} ---------------------------------- Physical head phantoms allow us to gain insight into head internal field and current distributions. They can provide explanations for observed lightning phenomena and can help to develop novel hypotheses and experiments. However, physical head phantoms are simplifications. Our head phantoms replicate the three main head compartments in terms of their geometric and dielectric properties, but neglect the highly complex interior structures of the human head, like the sulcus/gyrus structure, the anisotropic conductivity of the white matter, according Haueisen et al. \[[@pone.0223133.ref036]\], or hair on the scalp. Future research should include more detailed head phantoms. Another limitation was the measurement setup for the current distribution. The spatial and temporal resolutions of our measurement equipment (oscilloscopes, current monitors, voltage probes) were limited as well. Due to these limitations, we were unable to measure the initial connecting leader phase, in which a negative current typically flows at the start of the discharge. At present, voltage and current generators cannot provide a high voltage and a high current simultaneously similar to a lightning discharge. For this reason, we use three different generators to replicate the voltage and current effects on the head phantoms. We compare the generators in the following paragraph. - The Marx generator (MG) generates a 1.2/50 μs high voltage of up to 450 kV. The high voltage ionizes the air gap over a larger distance between the high voltage point electrode and the head phantom, mimicking the air distance between the cloud and the ground during a lightning discharge \[[@pone.0223133.ref027]\]. The energy and the resulting current (about 2 kA) values are low in comparison to natural lightning. Further, no flashovers are possible due to the low energy and the impedance of the head phantom. The used MG is only suitable for visually investigating the high voltage effects which manifested as perforations on the head phantoms. - The combination wave generator (CWG) produces a 1.2/50 μs voltage up to 12 kV (open-circuit) or an 8/20 μs current up to 6 kA (short-circuit), depending on the impedance of the test object according to IEC 61000--4--5 \[[@pone.0223133.ref026]\]. The voltage amplitude was not high enough to ionize the air over larger distances. For this reason, a stimulation electrode galvanically contacted the scalp compartment in our experiments. The low noise and low interference setup enabled a voltage and current measurement with a high signal noise ratio and a high reproducibility in our experiments. This setup also allowed the measurement of voltages at electrodes integrated in the brain compartment (ME1--ME4). Another advantage of the CWG is the similarity of its voltage pulse form to a lightning voltage pulse form. - The 10/350 μs current pulse generator can be charged up to 12 kV according to IEC 62305 \[[@pone.0223133.ref018]\]. The maximum current in the experiments was about 42 kA. Due to the low voltage, an air gap of only 4 mm between the output of the generator and the test object was possible. We used an ignition wire to increase this distance to 55 mm. Consequently, no connecting upward leader was formed. The used current amplitude and current steepness corresponded to real lightning currents. For the estimation of the current distribution in the head phantom, including the flashover, the 10/350 μs current pulse generator was best suited for comparison to real lightning currents. Conclusion and outlook {#sec018} ====================== We created head phantoms made of agarose to replicate the scalp, the neurocranium, and the intracranial volume of a human head. These head phantoms were tested on pulse generators, which modelled lightning components to investigate the physical and biological effects during a lightning strike. We found: 1. The scalp compartment is most exposed to the current during a lightning strike, with a fraction of approximately three quarters. 2. A fully formed flashover rapidly reduces the current in the brain compartment and the current exposition is reduced in interior structures the faster a flashover is formed. This mechanism can explain why people can survive a lightning strike. 3. The skull has protective properties due to its low electrical conductivity and relative permittivity. 4. The electric field strength in our experiments reaches a level where electroporation is possible. The mechanism of electroporation may explain neurological effects and damages after a lightning strike. We established normative values in our experiments for the human head to lightning discharges without any protective equipment. In the next steps, we will compare the found values with protective equipment applied to the head phantom. Further, we will improve our head phantoms to be more realistic models (e.g. add hair on scalp compartment). [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: ‡ These authors also contributed equally to this work.

{ "pile_set_name": "PubMed Central" }

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22,266

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Introduction {#s1} ============ Peroxisomes are defined as organelles encasing the metabolism of H~2~O~2~. This highly reactive molecule is generated by several oxidative reactions and degraded inside the organelle by abundant amounts of catalase. In addition, peroxisomes fulfill a number of important metabolic functions for eukaryotic cells requiring an active communication between the peroxisomal lumen and the cytosol or other organelles. Major functions include the β-oxidation of fatty acids, parts of the glyoxylate cycle and parts of the photorespiration (Hu et al., [@B38]; Waterham and Wanders, [@B91]). For β-oxidation fatty acids have to enter peroxisomes, and the resulting acetyl-CoA is distributed throughout the cells. Photorespiration in plants involves mitochondria, chloroplasts and peroxisomes and therefore requires extensive metabolite exchange. A key intermediate shared by the photorespiration and the glyoxylate cycle is glyoxylate, usually generated and metabolized inside peroxisomes. The glyoxylate cycle was originally considered a metabolic process localized to peroxisomes (Breidenbach and Beevers, [@B16]). This localization was rationalized as a means to increase the efficiency of the flux of intermediates. However, the finding that parts of the whole cycle are extra-peroxisomal in the yeast *Saccharomcyes cerevisiae* and in the plant *Arabidopsis thaliana* indicated, that a functional glyoxylate cycle requires the transfer of various metabolites across the peroxisomal membrane, too (Minard and McAlister-Henn, [@B51]; Courtois-Verniquet and Douce, [@B20]; Taylor et al., [@B75]; Kunze et al., [@B42]; Pracharoenwattana et al., [@B60]). The nature of the molecules crossing the peroxisomal membrane varies with the organism under study. The single membrane separating the peroxisomal matrix from the surrounding was shown to be permeable only for small molecules such as tri- and dicarboxylates and amino acids (Antonenkov et al., [@B4]). Specific transporters have not yet been identified but features compatible with pore-like structures were demonstrated (Verleur and Wanders, [@B87]; Antonenkov and Hiltunen, [@B2]). In this review we will address the questions how and where glyoxylate is generated and how metabolites of the two most prominent pathways partially localized to peroxisomes, the glyoxylate cycle and the photorespiration, are thought to cross the membrane on their way in and out of the peroxisomes. Peroxisomes and glyoxylate metabolism ===================================== Inside peroxisomes the accumulation of glyoxylate is prevented by conversion into glycine making use of a transaminase reaction or by condensation with acetyl-CoA into malate catalyzed by malate synthase (MLS), one of the key enzymes of the glyoxylate pathway (for reviews see Wanders and Waterham, [@B90]; Theodoulou and Eastmond, [@B76]). Glyoxylate can be produced from different precursor molecules (Figure [1A](#F1){ref-type="fig"}). Cleavage of isocitrate generates glyoxylate and succinate in the glyoxylate cycle (Figure [1B](#F1){ref-type="fig"}). The oxidation of glycolate to glyoxylate is catalyzed by glycolate oxidase being part of the photorespiratory process generating H~2~O~2~ within peroxisomes (Figure [1C](#F1){ref-type="fig"}). Another possible source of glyoxylate is the degradation of purines in those organisms metabolizing the intermediate uric acid (Figure [1D](#F1){ref-type="fig"}). Moreover, in mammals glyoxylate may represent a degradation product of hydroxyproline originally derived from collagen (Salido et al., [@B68]) or alternatively, may be the result of a conversion from glycine catalyzed by D-amino acid oxidase generating H~2~O~2~ (Ohide et al., [@B53]). ![**Glyoxylate generation and consumption. (A)** Glyoxylate is generated from different precursor molecules and converted into stable metabolites for further utilization. The proteins involved are ureidoglycolate hydrolase (UGH), isocitrate lyase (ICL), glycolate oxidase (GO), malate synthase (MLS), glutamine:glyoxylate aminotransferase (GGAT). **(B)** In the glyoxylate cycle two acetyl-CoA are condensed to succinate and 2 CoA (not shown). The proteins involved are citrate synthase (CIT), aconitase (ACO), isocitrate lyase (ICL), malate synthase (MLS), malate dehydrogenase (MDH). **(C)** The generation and consumption of glyoxylate in the photorespiratory process. The stoichiometry of the reaction sequence to obtain three molecules of 3-phospho-glycerate from two oxygenation products of ribulose-1,5-bisphosphate is indicated by the numbers below the molecules (1×, 2×). The proteins involved are ribulose-bisphosphate-carboxylase-oxygenase (RuBisCO), phosphoglycolate phosphatase (PGLP), glycolate oxidase (GO), glutamine:glyoxylate aminotransferase (GGAT), glycine-decarboxylase (GlyDC), serine-hydroxymethyltransferase (SHMT), serine:glyoxylate aminotransferase (SGAT), hydroxypyruvate reductase (HPR), glycerate kinase (GLYK). **(D)** The reactions in the purine degradation pathway leading to glyoxylate. The proteins involved are urate oxidase (UO), allantoin synthase (HIU-hydrolase + OHCU decarboxylase) (AS), allantoinase (alantoin amidohydrolase) (AL), allantoate amidohydrolase (AAH), ureidoglycine aminohydrolase (UGlyAH), ureidoglycolate amidohydrolase (UAH).](fphys-04-00204-g0001){#F1} The glyoxylate cycle allows the formation of 4-carbon units from 2-carbon units (Figure [1B](#F1){ref-type="fig"}). The resulting succinate serves to replenish the tricarboxylic acid (TCA) cycle representing the major collector and distributor of small carbon units. Alternatively, succinate or its follow-up product oxaloacetate serves as precursor for many biosynthetic processes. This biosynthetic pathway is absent in all animals except nematodes (Kondrashov et al., [@B41]). Acetyl-CoA fed into the glyoxylate cycle can be derived from different sources, such as β-oxidation of fatty acids, degradation of amino acids or in case of microbial organisms from external carbon sources such as ethanol or acetate. The glyoxylate cycle shares a series of three enzymatic activities with the TCA-cycle, namely malate dehydrogenase (MDH), citrate synthase (CIT), and aconitase (ACO) activity. The two unique activities, isocitrate lyase (ICL) and malate synthase (MLS) generate and consume the name-giving metabolite glyoxylate. The cleavage of isocitrate bypasses the decarboxylation reactions and the synthase reaction leads to the net-condensation of acetyl-CoA units. In many organisms these activities can be carried out by two or more isoenzymes with different localization signals and different expression patterns catalyzing the respective reactions. Photorespiration is required in all photosynthetic organisms and serves as carbon recovery system (Maurino and Peterhansel, [@B47]). Oxygenation of ribulose-1,5-bisphosphate by ribulose-bisphosphate-carboxylase-oxygenase (RuBisCO) competes with CO~2~ fixation upon high partial oxygen pressure leading to the formation of 2-phosphoglycolate and 3-phosphoglycerate in the chloroplast (Figure [1C](#F1){ref-type="fig"}). 3-phosphoglycerate is channeled into the Calvin cycle and 2-phosphoglycolate is recycled into 3-phosphoglycerate in a series of reactions involving peroxisomes and mitochondria. In peroxisomes glycolate is oxidized to glyoxylate generating H~2~O~2~, which is immediately decomposed by catalase. Glyoxylate is transaminated to glycine using either serine or glutamine as nitrogen source and further transported to the mitochondria. Here, two glycines are converted into one serine which is now returned into peroxisomes. The peroxisomal serine:glyoxylate aminotransferase and hydroxypyruvate reductase convert serine into glycerate which is transferred to chloroplasts, where it enters the Calvin cycle in the form of 3-phosphoglycerate. In summary, two glycolate molecules are consumed to produce glycerate and CO~2~ and the generation of two H~2~O~2~ is confined to peroxisomes. The degradation pathway of purines leads to uric acid, which in many organisms is further metabolized within peroxisomes to allantoine and allantoic acid (Figure [1D](#F1){ref-type="fig"}). In some animals the allantoicase activity leads to the cleavage of allantoic acid into urea and glyoxylate (Hayashi et al., [@B33]). In plants, some fungi and bacteria allantoic acid is further processed within the endoplasmic reticulum into ureidoglycine and ureidoglycolate, both giving rise to glyoxylate upon degradation (Werner and Witte, [@B92]). The glyoxylate derived from purine degradation is supposedly converted into glycine or condensed with acetyl-CoA catalyzed by a MLS activity providing the versatile metabolite malate. In essence, the heterocyclic core structure is converted into a nitrogen containing product and glyoxylate. The latter is recycled into biosynthetic processes in all organisms, the nitrogen containing product is either excreted (animals) or recycled (plants). Enzymes allocated to the glyoxylate cycle ========================================= Due to the fact that three of the five enzymatic activities contributing to the glyoxylate cycle are shared with the TCA cycle and additional enzymatic activities have been described an assignment of individual proteins to the glyoxylate cycle is not trivial. Some of the encoded proteins can be excluded due to an inappropriate expression pattern, but for some of the proteins only the phenotype of cells lacking the respective activity is a convincing argument for the function in the glyoxylate cycle. As the situation in the yeast *S. cerevisiae* has been analysed in detail previously (Kunze et al., [@B43]), we will only summarize the most important information and then concentrate on more recent results obtained in the thale cress *A. thaliana* as the plant model organism and in the human opportunistic fungus *Candida albicans*. The corresponding proteins are listed in Table [1](#T1){ref-type="table"}. ###### **Enzymatic activities required for the glyoxylate cycle**. **Enzyme** ***Saccharomyces cerevisiae*** ***Candida albicans*** ***Arabidopsis thaliana*** ------------------ -------------------------------- ------------------------ ---------------------------- ------ -------------- --------------- ------------- ----------- --------------- Isocitrate lyase **[ICL1]{.ul}** YER065C Cytosolic ICL1 CaO19.14134 Peroxisomal ICL At3g21720 Peroxisomal ICL2 YPR006C Mitochondrial Malate synthase **[MLS1]{.ul}** YNL117W Peroxisomal MLS1 CaO19.12296 Peroxisomal MLS At5g03860 Peroxisomal DAL7 YIR031C PTS1 Malate MDH1 YKL085W Mitochondrial MDH1 CaO19.12072 MITO pMDH1 At2g22780 Peroxisomal dehydrogenase **[MDH2]{.ul}** YOL126C Cytosolic MDH2 CaO19.7481 pMDH2/MDHG1 At5g09660 Peroxisomal MDH3 YDL078C Peroxisomal MDH3 CaO19.1278 PTS1 mDH3/MDHM1 At1g53240 Mitochondrial mDH4/MDHM2 At3g15020 Mitochondrial MDH5/MDHC1 At1g04410 MDH6/MDHC2 At5g43330 MDH7/MDHC3 AT5G56720 cMDH8/MDHP1 At3g47520 Chloroplast Citrate synthase CIT1 YNR001C Mitochondrial CIT1 CaO19.11871 Mitochondrial CSY1 At3g58740 PTS2 **[CIT2]{.ul}** YCR005C Peroxisomal Q59ZZ5 CSY2 At3g58750 Peroxisomal CIT3 YPR001W Mitochondrial CSY3 At2g42790 Peroxisomal CSY4 At2g44350 Mitochondrial CSY5 At3g60100 Mitochondrial Aconitase **[ACO1]{.ul}** YLR304C Mitochondrial ACO1 CaO19.13742 MITO ACO1 At4g35830 Mitochondrial ACO2 YJL200C Mitochondrial ACO2 CaOrf19.6632 MITO ACO2 At4g26970 Mitochondrial ACO3 At2g05710 Mitochondrial Proteins, corresponding genes, accession number, and targeting information are shown for enzymes carrying out activities required for the glyoxylate cycle in S. cerevisiae, C. albicans, and A. thaliana. PTS and MITO indicates predicted targeting information (general prediction: <http://wolfpsort.org/>, PTS1-predictor: <http://mendel.imp.ac.at/pts1/>, Mito-predictor: <http://ihg.gsf.de/ihg/mitoprot.html>). Cytosolic, peroxisomal, mitochondrial, and chloroplast indicates experimentally verified localization. Bold underlined are proteins experimentally proven to contribute to the glyoxylate cycle. In the genome of *S. cerevisiae* the key enzymes ICL and MLS are encoded by two genes each. Based on the high expression levels when cells grow on ethanol, acetate or fatty acids and the inability of cells lacking these proteins to grow on these carbon sources Icl1p and Mls1p have been attributed to the glyoxylate cycle (Fernandez et al., [@B26]; Hartig et al., [@B32]). The two others, Icl2p and Dal7p contribute to the mitochondrial propionate metabolism and the purine degradation, respectively (Hartig et al., [@B32]; Luttik et al., [@B46]). From the three genes coding for MDH, a mitochondrial (Mdh1p; McAlister-Henn and Thompson, [@B48]), a cytosolic (Mdh2p; Minard and McAlister-Henn, [@B51]), and a peroxisomal (Mdh3p; Steffan and McAlister-Henn, [@B72]) gene product are derived. Only the cytosolic and the mitochondrial variants are expressed when cells grow on C~2~-carbon sources (ethanol/acetate) and the cytosolic enzyme was required for growth under these conditions (Minard and McAlister-Henn, [@B51]; McCammon, [@B49]). This experimental evidence indicates that the cytosolic Mdh2p participates in the glyoxylate cycle. Similarly, among the three CIT proteins encoded within the yeast genome only Cit2p is considered to contribute to the glyoxylate cycle, because of its expression pattern on various carbon sources and because of the mitochondrial location of the other two enzymes Cit1p and Cit3p. Interestingly, Cit2p is essential for growth on ethanol or acetate only, when an alternative route probably involving one of the two mitochondrial activities is blocked (Van Roermund et al., [@B82]). Finally, one gene encoding ACO (ACO1; Gangloff et al., [@B28]) gives rise to the cytosolic and the mitochondrial activity (Regev-Rudzki et al., [@B62]) attributing Aco1p to the TCA- and the glyoxylate cycle, whereas Aco2p may be important for fermentation (Van den Berg et al., [@B81]). Even more complex is the situation in *A. thaliana*. Although only one gene codes for ICL (Eastmond et al., [@B22]) and one for MLS (Cornah et al., [@B19]), eight genes encode NAD^+^ dependent MDHs, five genes code for CITs and three genes code for ACOs (summarized in Table [1](#T1){ref-type="table"}). The localization, expression pattern and phenotype of the corresponding T-insertion mutants may help to discern the respective function of each individual protein. Nevertheless, the attribution of a function in the glyoxylate cycle appears difficult. Three of the five CITs, CYS1-3 harbor peroxisomal targeting information whereas CYS4 and CYS5 include mitochondrial leader peptides (Pracharoenwattana et al., [@B59]). Similarly, among the MDH proteins two are peroxisomal, two mitochondrial, one is observed in chloroplasts and three in the cytosol (Pracharoenwattana et al., [@B60]) \[The Arabidopsis Information Resource (TAIR), <http://www.arabidopsis.org/>, Huala et al., [@B39]\]. The expression pattern of enzymes contributing to the glyoxylate cycle is very characteristic, namely high expression after imbibition during seed germination followed by strong reduction upon postgerminative growth. Isoenzymes that are not expressed in the phase of germination can be excluded from a contribution to the glyoxylate cycle, such as CYS1 (Pracharoenwattana et al., [@B59]). During this developmental phase both, the β-oxidation of fatty acids and the glyoxylate cycle are equally needed and therefore the expression profiles of the corresponding genes are similar (Eastmond and Graham, [@B23]; Rylott et al., [@B67]). A detailed and precise cluster analysis of expression profiles during seedling development in soybean remained inconclusive since the expression pattern of the two key enzymes ICL and MLS did not fall into the same cluster and expression differences of the isoforms of CIT and MDH were not distinguishable (Gonzalez and Vodkin, [@B30]). An unambiguous attribution of a function in the glyoxylate cycle to a distinct protein could be based on the characteristics of cells deficient in the glyoxylate cycle function. *A. thaliana* seeds lacking ICL are able to germinate, but their seedling establishment is severely impaired in the absence of light or carbohydrates offered as alternative carbon source (Eastmond et al., [@B22]). In other words, gluconeogenesis is compromised but lipid respiration is still active in these mutants. *A. thaliana* seeds lacking MLS, the other unique enzyme of the glyoxylate cycle, display a similar defect in seedling establishment in the absence of light or carbohydrates. Regarding the compensation of the establishment defect by light the seeds respond differently. In the absence of MLS activity a lower light dose is required to promote seedling establishment than in the absence of ICL activity (Cornah et al., [@B19]). The latter phenotype led to the suggestion that in the absence of MLS glyoxylate, which is produced by ICL can enter gluconeogenesis by hijacking enzyme activities from the photorespiratory pathway. Phenotypes of plants lacking various isoforms of either CYS or MDH are less revealing to discern a specific function. During seedling establishment both peroxisomal MDHs (pMDH1 and pMDH2) and peroxisomal CITs (CYS2 and CYS3) are expressed. Seeds lacking either one of the peroxisomal activities present with disturbed fatty acid degradation (Pracharoenwattana et al., [@B59], [@B60]). Thus, a contribution of these proteins to the glyoxylate cycle cannot be delineated from the block in seedling establishment obtained in these mutant plants, because the lack of fatty acid degradation elicits a similar block in seedling establishment. Conversely, no noticeable defect in establishment was observed for seedlings from plants lacking mitochondrial MDH excluding an essential role of these MDHs in either β-oxidation of fatty acids or the glyoxylate cycle (Tomaz et al., [@B78]). When the metabolic distribution of exogenously added acetate within seedlings is used as indicator for the functionality of the glyoxylate cycle, no differences between seedlings lacking the peroxisomal MDH and wild type seedlings could be observed (Pracharoenwattana et al., [@B60]). However, this assay shows differences in the re-routing of acetate between seedlings from wild-type plants and plants, which are blocked in the glyoxylate cycle due to lack of ICL or MLS, although the differences appear small in the absence of MLS (Cornah et al., [@B19]). Experiments to discern the individual roles of the three cytosolic MDH were not yet carried out. Similarly, among the three ACO proteins a function in the glyoxylate cycle has not been assigned to anyone of them (Peyret et al., [@B56]; Arnaud et al., [@B8]). Altogether, *in A. thaliana* the assignment of individual ACOs, CITs, and MDHs to the glyoxylate cycle remains open. The situation in the yeast *C. albicans* is different. Each one of the enzymes ICL, MLS, and CIT is encoded by a single gene (Piekarska et al., [@B58]). In this organism the same enzyme Cit1p seems to contribute to the mitochondrial TCA-cycle and the glyoxylate cycle such as demonstrated for *Candida tropicalis* (Ueda et al., [@B80]). However, it cannot be excluded that a cytosolic form is derived from alternative translation as two variants are described (C4YLG7 and Q59ZZ5), but none of the described variants contains a peroxisomal targeting signal type 1 (PTS1; C-terminus KYIELVKNINKA). ACO activity is encoded by two genes, MDH activity by three genes (Jones et al., [@B40]). In each case one of the variants has a closer similarity to the respective glyoxylate cycle enzyme in *S. cerevisiae*, but experimental evidence for their role is missing. Subcellular localization of the enzymes of the glyoxylate cycle --------------------------------------------------------------- In *S. cerevisiae* the enzymes contributing to the glyoxylate cycle are distributed between the peroxisomal matrix and the cytosol. Icl1p, Mdh2p, and Aco1p were described in the cytosol (Minard and McAlister-Henn, [@B51]; Taylor et al., [@B75]; Regev-Rudzki et al., [@B62]), and Cit2p was found in the peroxisomal fraction (Lewin et al., [@B44]). Mls1p is either targeted to peroxisomes when cells utilize oleic acid or distributed across the cytosol when cells utilize ethanol (McCammon et al., [@B50]; Kunze et al., [@B42]). Upon growth on ethanol or acetate as sole carbon source with all other glyoxylate cycle enzymes in the cytosol the extra-peroxisomal fraction of Cit2p might suffice to generate four-carbon units through the glyoxylate cycle. In *A. thaliana* the key enzymes ICL and MLS are considered peroxisomal containing a C-terminal PTS1 \[EGTSLVVAKSRM for ICL and IVAHYPINVSRL for MLS, Arabidopsis subcellular database (SUBA), (Heazlewood et al., [@B34])\]. In contrast, ACOs lack peroxisomal targeting information and were allocated to the cytosol and the mitochondria (Arnaud et al., [@B8]). The extra-peroxisomal location of ACO activity was confirmed in castor bean and potato (Courtois-Verniquet and Douce, [@B20]). Which of the other gene products, namely of MDH and of CIT, are contributing to the glyoxylate cycle is unclear. Among the MDHs it can be assumed that one or more of the three cytosolic isoforms participates in the glyoxylate cycle as mutant plants lacking either the peroxisomal (Pracharoenwattana et al., [@B60]) or the mitochondrial (Tomaz et al., [@B78]) isoenzymes show no signs of a glyoxylate cycle defect. In *C. albicans* both key enzymes of the glyoxylate cycle, ICL and MLS were found in peroxisomes irrespective of the carbon source used (Piekarska et al., [@B58]), rendering this microorganism an alternative yeast model with key enzyme distribution similar to *A. thaliana*. However, the exclusive presence of one citrate synthase gene (CIT1) and the necessity of a peroxisomal and mitochondrial shuttle mechanism for acetyl-CoA for growth on oleic acid indicates that peroxisomal CIT is not available, but a cytosolic isoform might well be. The similarity of the MDH proteins to the homologs from *S. cerevisiae* suggests that the cytosolic Mdh2p contributes to the glyoxylate cycle as well. Finally, the cytosolic ACO Aco1p (Jones et al., [@B40]) might be supplemented by a minor peroxisomal subfraction that is caused by a weak PTS1 \[HGSALNFIKSKY, <http://mendel.imp.ac.at/pts1/>, Neuberger et al., [@B52]\]. Briefly summarized, the localization of proteins participating in the glyoxylate cycle on different sides of the peroxisomal membrane in all three model organisms requires the transport of intermediates across the lipid barrier. Transfer of metabolites ======================= Usually, the concentration of free intermediates occurring in metabolic pathways is relatively low, because a local accumulation of participating enzymes, e.g., within an organelle, or a physical interaction between successive enzymes allows a channeling of intermediates. In extreme cases, large protein complexes such as the fatty acid synthase transfer small molecules from one active center to the next. If a membrane separates consecutive steps an efficient metabolic pathway requires a direct shuttling of intermediates either through a transporter protein or through a proteinaceous channel linking enzymes on both sides of the membrane. Alternatively, a comparably high net concentration of intermediates might facilitate diffusion controlled transfer, which appears unlikely for highly reactive compounds such as glyoxylate. Permeability of the peroxisomal membrane ---------------------------------------- To seal certain reactions in a compartment and to restrict the generation of reactive molecules cells seem to spare no effort to translocate the corresponding proteins and metabolic precursors into distinct compartments. The localization of various oxidases to peroxisomes is a typical example for an energy spending activity preventing contamination of the cytosol by H~2~O~2~. In turn, the membrane delimiting a compartment thought to protect the cellular interior from detrimental effects by small, highly reactive molecules could be expected to be impermeable for such substances. However, early studies provided evidence for a permeability of the peroxisomal membrane for small solutes such as urate or amino acids and for density gradient material such as sucrose (De Duve and Baudhuin, [@B21]; Van Veldhoven et al., [@B86]). For cofactors and larger substrates required for peroxisomal enzymes such as NAD or acetyl-CoA the peroxisomal membrane was shown to act as barrier similar to the inner mitochondrial membrane (Van Roermund et al., [@B82]). Shuttle systems were proposed to functionally connect the peroxisomal lumen with the cytosol exchanging substrates and keeping the cofactors in the reduced or in the oxidized state as required (Elgersma et al., [@B24]; Van Roermund et al., [@B82]; Antonenkov and Hiltunen, [@B2]; Visser et al., [@B89]). A concept for the peroxisomal membrane permeability reconciling conflicting data was put forward by Hiltunen and co-workers (Antonenkov et al., [@B6],[@B7]). Pore-like structures permit the free exchange of small solutes (MW \< 300D) across an otherwise impermeable membrane inhibiting the transfer of molecules such as acetyl-CoA, ATP, or NAD. Accordingly, pore-forming activities were reported in peroxisomes isolated from plants, mammalian tissue and yeast (Reumann et al., [@B64]; Antonenkov et al., [@B5]; Grunau et al., [@B31]), but the molecular nature of the channel proteins remained largely unknown. In contrast, specific transporters for adenine nucleotides and NAD were identified corroborating the impermeability of the peroxisomal membrane for such bulky molecules (Palmieri et al., [@B55]; Bernhardt et al., [@B13]; for reviews see Antonenkov and Hiltunen, [@B3]; Hu et al., [@B38]). The localization of enzymatic activities of the glyoxylate cycle on different sides of the peroxisomal membrane requires an efficient transport of intermediates across the lipid barrier (Kunze et al., [@B43]). When acetyl-CoA is generated inside the peroxisomal matrix---usually via β-oxidation of fatty acids---it remains confined to peroxisomes probably because of its size (Van Roermund et al., [@B82]; Antonenkov and Hiltunen, [@B2]). CIT catalyzes the condensation of acetyl-CoA with oxaloacetate into citrate, which in turn is exported and serves as substrate for extra-peroxisomal ACO. The resulting isocitrate is imported into peroxisomes in those organisms in which the corresponding cleavage activity, ICL resides inside peroxisomes (*A. thaliana, C. albicans*). Thereby, succinate, the net product of the cycle is released within peroxisomes and requires an additional export mechanism. Conversely, glyoxylate is directly handed over to the second acetyl-CoA consuming enzyme, MLS, which is a peroxisomal constituent in all organisms. Some organisms such as *S. cerevisiae* do not harbor a peroxisomal ICL (Taylor et al., [@B75]), but instead generate the products of this reaction in the cytosol. The disadvantage that a small reactive molecule such as glyoxylate needs to be translocated across a membrane to reach MLS might be balanced by the advantage that succinate is already in the cytosol, which is one step closer to its usual final destination, mitochondria. The interesting observation that the relevant MDH activity for the glyoxylate cycle is exerted by cytosolic isoforms in the yeast *S. cerevisiae* and supposedly also in *A. thaliana* calls for an additional export of malate and the subsequent import of oxaloacetate to close the cycle (Minard and McAlister-Henn, [@B51]; Pracharoenwattana et al., [@B60]). All in all a series of transport steps is required to complete a full round of the glyoxylate cycle. If ICL is extra-peroxisomal, e.g., in the yeast *S. cerevisiae*, citrate and malate are exported, and glyoxylate and oxaloacetate are imported (Figure [2A](#F2){ref-type="fig"}). If ICL resides inside peroxisomes citrate, malate and succinate are exported, whereas isocitrate and oxaloacetate are imported (Figure [2B](#F2){ref-type="fig"}), a situation occurring in the plant *A. thaliana*. A similar flux of intermediates occurs in *C. albicans*, although citrate is generated in the cytosol and does not occur in peroxisomes (Figure [2C](#F2){ref-type="fig"}). ![**Metabolites of the glyoxylate cycle crossing the peroxisomal membrane.** The peroxisomal membrane facilitates the transfer of small metabolites. The transport of glyoxylate cycle intermediates and of C~2~-units is shown for *S. cerevisiae* **(A)**, *A. thaliana* **(B)**, and *C. albicans* **(C)**. Hypothetical pore-forming proteins permitting the export of intermediates are colored orange, hypothetical pore-forming proteins permitting the import of intermediates are colored green. Broken lines are drawn to close the glyoxyate cycle. Intermediates that cross the peroxisomal membrane are indicated bold. *Px*, peroxisomal side of the membrane, *Cyt*, cytosolic side of the membrane.](fphys-04-00204-g0002){#F2} The export of malate and import of oxaloacetate as part of the glyoxylate cycle mimics a redox-shuttle system consisting of a peroxisomal and a cytosolic MDH activity that mediates a net-export of reduction equivalents generated by the β-oxidation of fatty acids to the cytosol. Thus, peroxisomal malate generated by MLS might be fueled into the same path to the cytosol as malate generated by the re-oxidation of NADH (peroxisomal *Sc*Mdh3p or *At*pMDH1/pMDH2). Similarly, the transport of isocitrate might be part of a shuttle mechanism coupling the import of isocitrate and the export of α-oxo-glutarate with the reduction of NADP^+^ inside peroxisomes. Such a hypothetical net-exchange is based on a cytosolic isocitrate dehydrogenase (*Sc*Idp2p) and a peroxisomal isoform (*Sc*Idp3p) converting isocitrate into α-oxo-glutarate and CO~2~ and *vice versa*. The export of citrate is not restricted to the glyoxylate cycle but also presents a mode of export for C~2~-units that are generated as acetyl-CoA by fatty acid β-oxidation within peroxisomes. In this case the export of citrate is ultimately balanced by the import of oxaloacetate, the precursor for CIT. The export of citrate is considered the only export pathway for C~2~-units in plants since lack of the peroxisomal CITs, CYS2 and CYS3, blocks β-oxidation of fatty acids in *A. thaliana* (Pracharoenwattana et al., [@B59]), whereas in the yeast *S. cerevisiae* an additional export system for acetyl-units exists. For the latter, a peroxisomal form of carnitine acetyl-transferase (Cat2p; Elgersma et al., [@B24]) generates acetyl-carnitine that can be translocated across the membrane and rebuilt into acetyl-CoA either in the cytosol or within mitochondria employing either one of three carnitine acetyltransferases, Cat2p, Yat1p, or Yat2p (Swiegers et al., [@B74]). In *S. cerevisiae* the β-oxidation of fatty acids requires only one mode of export and thus the lack of either the peroxisomal CIT Cit2p or the carnitine acetyltransferase Cat2p localized to the peroxisomes and the mitochondria is tolerated for growth on oleic acid as carbon source, but upon deletion of both genes (ΔCIT2ΔCAT2) cells were not able to utilize oleate (Van Roermund et al., [@B84]). Moreover, under these conditions the cytosolic re-conversion of acetyl-carnitine into acetyl-CoA is also essential, as mutant cells lacking CIT2 and either one of the other carnitine acetyl-transferases (YAT1 or YAT2) were unable to consume oleic acid (Swiegers et al., [@B74]). In *C. albicans* a peroxisomal condensation of acetyl-CoA with oxaloacetate producing citrate is not possible due to the lack of a peroxisomal CIT. Therefore, the export of acetyl-units depends on the carnitine form and consequently, in the absence of the peroxisomal isoform of Cat2p cells were unable to grow on oleic acid (Strijbis et al., [@B73]). The transfer of the various small intermediates is compatible either with specific transporter proteins or with atypical permeability properties of the membrane. The mitochondrial and chloroplast membranes contain numerous transporter proteins specific for small organic compounds, but corresponding proteins were not identified in peroxisomes. Few examples may illustrate this. In mitochondria of *S. cerevisiae* a carnitine acylcarnitine carrier protein Crc1p and a citrate-oxoglutarate carrier were identified, but no peroxisomal paralog was discovered yet (Van Roermund et al., [@B84]; Castegna et al., [@B17]). Similarly, a succinate-fumarate transporter was identified in the inner mitochondrial membranes of *S. cerevisiae* and *A. thaliana* \[Sfc1p/Acr1p (Bojunga et al., [@B15]) and AtMSFC1 (Catoni et al., [@B18]), respectively\], but no homologous protein was identified to mediate plant peroxisomal succinate export. Furthermore, a glycolate/glycerate transporter required for photorespiration (PLGG1) has been identified in the chloroplast membrane (Pick et al., [@B57]), but no homologous proteins were found in plant peroxisomes. Despite overwhelming evidence for the free exchange of small solutes the molecular nature of pore-forming activities in plant and yeast peroxisomes remained unknown (Reumann et al., [@B63], [@B65]; Grunau et al., [@B31]). Moreover, the yeast peroxisomal membrane pores were shown to conduct solutes of the glyoxylate cycle (Antonenkov et al., [@B4]; Grunau et al., [@B31]). So far, only in mammalian peroxisomes a protein, Pxmp2, was identified that exhibits channel-forming capacities (Rokka et al., [@B66]). Provided that the transport of intermediates is essential for the metabolic activity, the lack of such pores should result in non-functional peroxisomes. Therefore, the corresponding genes should have turned up in various genetic screens searching for mutants with dysfunctional peroxisomes. However, more than one gene could encode redundant functions escaping the detection of these genes in screens. Likewise, such porins should be rather abundant constituents of the peroxisomal membrane and as such should have been identified in various proteomic approaches. The most abundant yeast peroxisomal membrane protein reported is Pex11p, but its localization at the outer surface renders a function in solute transport rather unlikely (Erdmann and Blobel, [@B25]; Van Roermund et al., [@B85]; Opalinski et al., [@B54]). Bipartite enzyme distribution of glyoxylate cycle enzymes: subcellular distribution follows generation of substrates ==================================================================================================================== The hypothesis that substrate availability was the driving force for changes in enzyme localization, has been proposed based on observations in the yeast *S. cerevisiae*. In this organism Mls1p localization differs between cells grown in medium containing ethanol as sole carbon source, when acetyl-CoA is generated primarily in the cytosol, and cells utilizing oleic acid generating acetyl-CoA within peroxisomes (Kunze et al., [@B42], [@B43]). In other organisms such as the plant *A. thaliana* ICL and MLS are peroxisomal. In this case an efficient coupling of the glyoxylate cycle to β-oxidation of fatty acids is essential, because both, energy and biomass production for germination and seedling outgrowth rests on the β-oxidation of fatty acid. The resulting acetyl-CoA is used to generate energy via the TCA-cycle and oxidative phosphorylation and to feed the glyoxylate cycle to cover the needs for biosynthetic processes. For the opportunistic fungus *C. albicans* carbohydrates and fatty acids are the prevalent carbon sources in its natural habitat, the mammalian gut. Thus, acetyl-CoA is expected to be obtained primarily inside mitochondria following glycolysis or inside peroxisomes via β-oxidation under such conditions. Even when these cells were grown in the presence of ethanol or acetate both key enzymes (ICL and MLS) are located in peroxisomes (Piekarska et al., [@B58]). Needless to mention, that neither *C. albicans* nor plants ever face ethanol or acetate as carbon source in their natural environment. This supports the hypothesis that the prevailing source of acetyl-CoA under natural conditions determines the localization of glyoxylate cycle key enzymes. For both model systems neither evolutionary pressure nor man-made selection forced the organism to change the intracellular location of parts of the glyoxylate cycle. In contrast, strains of baker\'s yeast, *S. cerevisiae*, have been selected for efficient growth in the presence of ethanol. Consequently, this organism most efficiently utilizes acetyl-CoA generated from this carbon source in the cytosol, which is apparently supported by the potential to relocate the complete glyoxylate cycle to the cytosol. The bipartite localization of enzymes provokes unusual intricacies for a straightforward metabolic pathway consisting of five enzymatic activities. Evolutionary optimization and pressure to increase efficiency demand a physiological advantage to compensate for this complexity. We suggest that the distribution of the glyoxylate cycle enzymes on different sides of the peroxisomal membrane might be due to the combination of (i) the unavoidable provision of its substrate, acetyl-CoA, inside peroxisomes by fatty acid β-oxidation (Kunze et al., [@B43]) and (ii) the incompatibility of some of its enzymes, namely ACO (Verniquet et al., [@B88]) and to a certain extent also ICL (Yanik and Donaldson, [@B93]), with the oxidative milieu within peroxisomes including high H~2~O~2~ concentrations. Thus, during evolution neither compartmentation of the complete pathway within peroxisomes nor the transfer of all enzymes into the cytosol appeared as feasible alternative. However, the tight coupling of peroxisomal acetyl-CoA generation and its fueling into the glyxoylate cycle that appears optimal for growth of *A. thaliana* and *C. albicans* in their natural habitats might restrain the incorporation of extra-peroxisomal acetyl-CoA into the glyoxylate cycle. When such organisms utilize the less physiological carbon source acetate as carbon source they satisfy all their energetic and biosynthetic needs from acetyl-CoA that is primarily generated in the cytosol. In this case the peroxisomal membrane could act as barrier that separates cytosolic acetyl-CoA from those peroxisomal enzymes of the glyoxylate cycle that utilize it, namely MLS in both organisms and CIT in *A. thaliana*. Under these conditions the mitochondrial energy production from cytosolic acetyl-CoA is not expected to be limited, but the biosynthetic capacity of the glyoxylate cycle is restricted by the transfer of acetyl-CoA into peroxisomes involving a specific import mechanism for this intermediate. Such a limitation is avoided in *S. cerevisiae*, where the glyoxylate cycle can be relocated to the cytosol. Interestingly, *A. thaliana* and *C. albicans* can grow under conditions when acetate is utilized as sole carbon source (Hooks et al., [@B36]; Zhou and Lorenz, [@B94]), but they are dependent on specific peroxisomal functions. In *A. thaliana* a genetic screen for mutants that are unable to utilize exogenous acetate identified two genes encoding peroxisomal proteins (Hooks et al., [@B36]). The peroxisomal transporter protein *COMATOSE* known to transport fatty acids (Hooks et al., [@B37]) and the intraperoxisomal short chain acyl-CoA/acetyl-CoA synthase (At3g16910, AAE7) (Turner et al., [@B79]; Shockey and Browse, [@B70]) are required for the integration of acetate into organic compounds (Allen et al., [@B1]). Interestingly, neither *C. albicans* nor *S. cerevisiae* cells require functional peroxisomes for the utilization of acetate, as cells harboring a deletion of a PEX-gene can grow on medium solely containing this carbon source (Piekarska et al., [@B58]). However, upon selective interruption of fatty acid β-oxidation by the ablation of the enzyme exerting the second step (Fox2p), *C. albicans* cells cannot utilize acetate any more, but *S. cerevisae* cells can do so (Hiltunen et al., [@B35]; Piekarska et al., [@B58]). This phentotype of *C. albicans* cells might be due to their inability to feed cytosolic acetyl-CoA into the glyoxylate cycle, because MLS is enclosed by the peroxisomal membrane. This notion is supported by the observation, that the ability to utilize acetate efficiently can be restored in these cells (ΔFOX2), when the compartmentation of peroxisomal enzymes is prevented by the deletion of the PTS1-receptor PEX5 (ΔFOX2ΔPEX5) (Piekarska et al., [@B58]). Thus, in organisms optimized for an efficient consumption of fatty acids the redirection of cytosolic acetyl-CoA units obtained from other sources such as acetate into peroxisomes is a critical step for its fueling into the glyoxylate cycle. Metabolic fluxes in related metabolic pathways ============================================== Photorespiration represents another example for a metabolic process in which glyoxylate is formed and intermediates traverse the peroxisomal membrane. Considering only the transport reactions between chloroplasts and peroxisomes two molecules of glycolate are transported into peroxisomes and one molecule glycerate is exported. The exchange with the mitochondria includes the export of two molecules of glycines and the import of one molecule serine (Figure [3](#F3){ref-type="fig"}). Already 40 years ago peroxisomes, mitochondria, and chloroplasts were often seen in close vicinity in the electron microscope (Frederick and Newcomb, [@B27]). A direct apposition of the peroxisomal membrane and the outer membranes of chloroplasts (Schumann et al., [@B69]) supports the hypothesis of direct exchange of metabolites thereby avoiding diffusion of intermediates into the cytosol. In leaves of *A. thaliana*, in which due to a mutation in a peroxisomal membrane protein the close apposition of chloroplasts and peroxisomes is lost, glyoxylate accumulates (Schumann et al., [@B69]). ![**Metabolite transport processes for the glyoxylate cycle and the photorespiratory process are considered to be similar.** In plant peroxisomes glyoxylate is a key metabolite of the glyoxylate cycle and the photorespiration, the latter involving mitochondria and chloroplasts. Hypothetical pore-forming proteins permitting the export are colored orange, hypothetical pore-forming proteins permitting the import are colored green. The broken lines with arrow heads indicate reactions of the glyoxylate cycle and the conversion of two molecules of glycine into serine inside mitochondria without further details. Reactions of the photorespiration process are encircled.](fphys-04-00204-g0003){#F3} Under normal conditions the glyoxylate cycle and the photorespiration do not overlap although they occur in the same organelle and share glyoxylate as key intermediate. This separation is due to the developmental program, which shows highest glyoxylate key enzyme activities during seed germination \[2--4 days postimbibition (Eastmond and Graham, [@B23])\], whereas the enzymes of the photorespiration are induced by light (Bertoni and Becker, [@B14]) upon cotyledon development. Upon this development the change of glyoxylate cycle containing peroxisomes ("glyoxysomes," see Pracharoenwattana and Smith, [@B61]) into photorespiratory active peroxisomes is considered prototypical for the exchange of enzymatic equipment of such organelles (Titus and Becker, [@B77]; Behrends et al., [@B12]) and is even accompanied by the highly surprising removal of ICL from "glyoxsomes" (Lingard et al., [@B45]). However, employing artificial experimental conditions by exposing outgrowing seedlings to light these developmental programs become blended, and so does the enzymatic equipment. This is demonstrated by the comparison of plants in which the glyoxylate cycle is interrupted either by ICL deficiency or MLS deficiency. The observed growth defect of these plants in the dark can be completely overcome by exposing seedlings lacking MLS to light, but seedlings lacking ICL can only partially recover (Cornah et al., [@B19]). The suggestion that the addition of photorespiratory equipment allows the re-direction of glyoxylate from MLS to serine/glutamine-glyoxylate aminotransferase is supported by the re-organization of the incorporation pattern of radioactively labeled acetyl-CoA into different water soluble intermediates. Thereby, wild type plants and plants lacking MLS display a more similar phenotype than plants lacking ICL. Model for the flux of intermediates across a membrane ===================================================== The localization of enzymes catalyzing successive metabolic reactions on opposite sides of the peroxisomal membrane and the permeability of this membrane for small molecules raises the question how fast small metabolites can cross the barrier membrane. Such a transfer of intermediates might be accomplished either (i) by an unspecific pore or (ii) by a series of specific transporter or facilitator proteins or (iii) by the direct coupling of specific enzymes on both sides of an unspecific pore forming a transmembrane metabolon. A simple diffusion controlled mechanism involving only a pore-like structure appears unlikely, because after reaching the other side of the membrane small metabolites could easily diffuse and the concentration might become (quite) low. This may happen, permitting the adjustment of concentrations between the cytosol and the peroxisomal matrix. However, such a model appears less likely for instable, highly reactive or even toxic intermediates such as glyoxylate. Alternatively, specific transporters or facilitators might close the gap between successive enzymatic reactions and guarantee specificity of transport, however these proteins have not been found so far. An interesting alternative might be a transmembrane metabolon comprised of transiently accumulating metabolic enzymes such as the glyoxylate cycle enzymes on both sides of an unspecific pore. We propose that in this extended form of the classical metabolon (Srere, [@B71]) pore-like channels participate in the assembly of supramolecular complexes, thereby linking proteins on both sides of the membrane enabling the swift transfer or exchange of metabolites. Such structural arrangement could enhance efficiency and ensure sufficient flux of intermediates across the peroxisomal membrane. Importantly, the transiently formed transmembrane metabolon would generate only local concentration peaks of intermediates at the entry sides of the pores. In our model metabolically active proteins transiently acquire a pore leading to a rapid transfer of the corresponding metabolites across a short distance. In this respect the observation seems interesting that polyethylene glycol (PEG), which stabilizes peroxisomal membrane integrity during the isolation procedure (Antonenkov et al., [@B6]) is also known as stabilizing agent for supramolecular protein complexes such as metabolons (Beeckmans and Kanarek, [@B11]; Barnes and Weitzman, [@B9]). Such a model would explain how a variety of substrates could cross the membrane without specific transporter molecules. The selectivity would be maintained via the transient association of proteins on both sides of the pore-like channels providing control and efficiency. According to our model in *S. cerevisiae* the loosely associated protein complex outside peroxisomes would consist of ACO, ICL, and MDH, on the inside of MLS and CIT, and the channel(s) would allow transferring citrate and malate from the matrix to the cytosol and/or glyoxylate and oxaloacetate from the cytosol to the peroxisomal matrix. When the peroxisomal Mdh3p has no access to the complex formed it is excluded from participation in the glyoxylate cycle. In plants, five metabolites have to cross the membrane. ACO and MDH acting outside peroxisomes require the export of citrate and malate and the import of isocitrate and oxaloacetate for further processing. The product succinate needs to be exported as well to make it available for the cellular metabolism. Since according to our model ICL would loosely associate with CIT and MLS it is reasonable to suggest that the simultaneous export of succinate using the same channel unit represents an additional drive for the cleavage reaction. Furthermore, the peroxisomal membrane would *per se* not represent a principal barrier for glyoxylate cycle intermediates or any other small metabolite, but controls the velocity of the continuous flux. In addition, this model could also explain how shuttles may function. A local concentration of enzymes on both sides of a membrane-spanning pore could facilitate the shuttling of reduction equivalents in \[isocitrate/α-oxo-glutarate (Van Roermund et al., [@B83])\] or out \[malate/oxaloacetate (Van Roermund et al., [@B82]), lactate/pyruvate (Baumgart et al., [@B10]), G3P/DHAP (Gee et al., [@B29])\] of the peroxisomal matrix (for review see Antonenkov and Hiltunen, [@B2]). The exchange of the reduced and oxidized intermediates would occur due to a local association of the corresponding enzymes, Mdh2p/Mdh3p or Idp2p/Idp3p, on each side of a channel. Conclusions and perspectives ============================ The peroxisomal matrix is surrounded by a single membrane that allows the enclosure of a variety of highly reactive and even toxic compounds. However, *in vitro* the membrane appears permeable to small molecules and neither a chemical nor an electrochemical gradient has been detected, which represents a major difference to the mitochondrial inner membrane or the thylakoid membrane. The biophysical properties of the peroxisomal membrane demands pore-like structures in addition to the few transmembrane proteins with transporter function for larger molecules. The frequent exchange of intermediates between peroxisomes and the intracellular environment requires directed transport processes with high selectivity, which appears incompatible with pore-like exchange modules. However, a model in which soluble enzymes accumulate in proximity to both ends of such pore-like structures forming a transmembrane metabolon would explain rapid and selective exchange based on a local increase in intermediate concentration. The observation that the enzymes of the glyoxylate cycle are distributed across both sides of the peroxisomal membrane would be prototypical for such transport processes, because an efficient transfer of small organic metabolites across the peroxisomal membrane is essential for this pathway. The high variability in the nature of small organic molecules transported across the peroxisomal membranes between *S. cerevisiae*, *C. albicans*, and *A. thaliana* may reflect the ease of adaptation processes whenever specific transporter proteins are not involved. Similar considerations are applicable to the photorespiration, although the direct exchange of metabolites with other organelles renders the latter apparently more complex. A transport system of such high versatility generates an organelle of high plasticity and allows rapid adjustments to environmental changes and to developmental programs generally considered an important feature of peroxisomes. Altogether, the peroxisomal membrane markedly differs from membranes of other metabolically active organelles, which might represent an intermediate step in the development of an organelle with novel properties. Thus, it can serve as model system to investigate and to understand transport processes across membranes with reduced complexity. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. [^1]: Edited by: Vladimir I. Titorenko, Concordia University, Canada [^2]: Reviewed by: J. Kalervo Hiltunen, University of Oulu, Finland; Vladimir I. Titorenko, Concordia University, Canada [^3]: This article was submitted to Frontiers in Integrative Physiology, a specialty of Frontiers in Physiology.

{ "pile_set_name": "PubMed Central" }

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"Finland", "Vladimir", "Titorenko", "Concordia", "University", "Canada", "article", "submitted", "Frontiers", "Integrative", "Physiology", "specialty", "Frontiers", "Physiology" ]

Methylamine (CH~3~NH~2~) is the simplest primary amine in chemistry. Its CH~3~ group is connected to the NH~2~ group by the C−N bond that exhibits a vibrational stretching motion. As for the two groups themselves, the CH~3~ group has an internal torsional motion while the NH~2~ group has an inversion motion, which makes the CH~3~NH~2~ a prototype in molecular physics for non-rigid molecules having two coupled large-amplitude internal motions. The torsion splits the rotational energy levels into a threefold pattern of *E* and *A* symmetry components with an *E*/*A* spin statistical weight ratio of 1/2, and inversion produces a further splitting into *s* and *a* doublets with spin weight ratio of 1/3. Thus, each vibration-rotation level of CH~3~NH~2~ has *Aa, As, Ea* and *Es* sublevels, resulting in a complex vibration-rotation-torsion-inversion energy structures and rich but highly crowded spectra with a wide range of relative line intensities. Therefore, the interesting microwave[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9][@b10], far-infrared[@b11][@b12][@b13][@b14][@b15][@b16][@b17], and infrared[@b18][@b19][@b20][@b21][@b22][@b23] spectra of CH~3~NH~2~ have been extensively studied for many years to explore the splittings and the symmetry species, leading to the valuable application in the detection of interstellar methylamine[@b24][@b25][@b26][@b27]. In contrast, despite the fact that it is of great practical interest related to optically pumped far-infrared laser lines and interstellar detection, spectroscopic study by the sub-Doppler technique on the stretching of the C−N bond that connects the CH~3~ and NH~2~ groups has remained rare until recently[@b28]. Study of the C−N stretching band of CH~3~NH~2~ has been of great spectroscopic, practical, and astrophysical interest for decades. The C−N stretching band of CH~3~NH~2~, earlier studied and reported sixty years ago at the relatively low resolution of 1 cm^−1^ and an accuracy of about 0.1 cm^−1^ by Gray and Lord[@b19], displays strong characteristic absorptions in the infrared region around 1044 cm^−1^. It is characterized by a parallel structure with vibrational *P, Q*, and *R* branches, which overlaps well with the CO~2~ laser bands. Thus, CH~3~NH~2~ has been an important member of the class of laser media employed to generate far-infrared laser lines by optically pumping with CO~2~ laser transitions. However, few of the CH~3~NH~2~ lines have so far been identified as to their quantum numbers due to the limited accurate experimental information available on this important band. It is only recently that high resolution spectroscopy of the C−N stretching band of CH~3~NH~2~ has been reported. In 2011, Lees *et al*.[@b21] and Gulaczyk *et al*.[@b22] investigated Doppler-limited Fourier-transform (FT) spectra of this band. Numerous transitions were assigned, perturbations from several resonances between the C−N stretching state and high-lying torsional substates of the ground state were analyzed, with both Fermi and Coriolis resonances being observed. Yet to date there are still many overlapped lines and unresolved asymmetry doublet lines in the FT spectrum for this band of CH~3~NH~2~ due to limit on spectral resolution imposed by the Doppler width. It is very desirable to carry out sub-Doppler observations on this band employing a spectrometer with very high resolution and accuracy in order to make confident and reliable measurements and line assignments. In 2010, we observed the precise Lamb-dip spectra of the C−N stretching band of CH~3~NH~2~ at a spectral resolution of 0.4 MHz and determined the transition frequencies with an accuracy of ±0.1 MHz[@b28], which is the first sub-Doppler observation in any spectral range of CH~3~NH~2~. However, the first Lamb-dip spectra of this band have been observed just for 43 saturation dips which are primarily in the C−N stretching *Q*-branch region and only in two sub-states. There is a clear need to observe many more Lamb-dip signals over a wider range of transitions in *P*-, *Q*-, and *R*-branch to obtain accurate experimental information and parameters for detailed spectroscopic analysis. These precise Lamb-dip measurements are important not only for disentangling overlapped features in the Doppler-limited spectra but also in providing a grid of standard reference frequencies for accurate calibration of the overall FT spectrum. Accordingly, in the present work, using frequency-tunable microwave sidebands of a much larger group of CO~2~ laser lines in our spectrometer[@b28], the Lamb-dip spectroscopy of the C−N stretching band of CH~3~NH~2~ has been systematically studied to cover a greatly expanded set of quantum states. We now report our extended experimental results. Results and Discussion ====================== A frequency-tunable infrared source for Lamb-dip observation ------------------------------------------------------------ Although the C−N stretching band center of CH~3~NH~2~ overlaps well with the CO~2~ laser bands, it is difficult to observe many absorption lines of CH~3~NH~2~ by using just a grating CO~2~ laser, because the available spectral coverage between the CO~2~ laser and CH~3~NH~2~ absorption lines is limited to the overlap in Doppler widths of only about 60--100 MHz for each laser line. This situation was recently improved greatly by the application of a frequency-tunable infrared source in a dual-mode sideband spectrometer to CH~3~NH~2~[@b28]. The schematic of the experimental setup used in the present work for the Lamb-dip spectroscopy of CH~3~NH~2~ is shown in [Fig. 1](#f1){ref-type="fig"}, and has been described in detail previously[@b28]. The tunable radiation is generated by adding microwave sidebands to the CO~2~ laser lines in a GaAs waveguide modulator. In the modulator, the added microwave radiation with a frequency *f*~MW~ produces a periodic variation of the refractive index of the GaAs crystal and a corresponding small phase change of *Δϕ* to the incident CO~2~ laser field. Therefore, the laser output from the modulator includes both the input field of the CO~2~ laser plus microwave-modulated field components. The latter are called microwave sidebands of the CO~2~ laser lines. In practice, the output from the modulator shows three spectral signals at *f*~L~ (frequency of CO~2~ laser carrier), *f*~L~ + *f*~MW~ (upper sideband), and *f*~L~−*f*~MW~ (lower sideband), respectively. These upper and lower microwave sidebands of CO~2~ laser lines provide a powerful radiation source for precision spectroscopy, operating at room-temperature with narrow linewidths and continuously tunable and precisely controlled frequencies in the 9--11 *μ*m region. Our infrared source has the three main features of 1) a tunability range about 23.6 GHz (±6.7 to ±18.5 GHz) for each CO~2~ laser line by sweeping the microwave frequency *f*~MW~; 2) a typical microwave-modulated CO~2~ laser power about 10 mW for either the upper or lower sideband; 3) accurate radiation frequency. In operation, the CO~2~ laser is stabilized to the center of a 4.3 *μ*m fluorescence Lamb-dip signal and has an estimated frequency uncertainty of 33 kHz[@b29]. High-resolution observation and precise measurement of the spectral Lamb-dips ----------------------------------------------------------------------------- A Lamb-dip signal has a much narrower spectral linewidth than that of the Doppler-broadened spectral line. The Lamb-dip spectroscopic technique can thus enable blended lines in the Doppler-limited spectrum to be fully resolved and the centers of these resolved absorption lines to be determined very precisely. [Figure 2](#f2){ref-type="fig"} shows a Lamb-dip spectrum for a blended line in the extremely congested *Q*-branch of the C−N stretching band center of CH~3~NH~2~ around 1044.5930 cm^−1^ in the FT spectrum. A 50 MHz scan of the lower microwave sideband of the 9*P*22 CO~2~ laser line was recorded at a pressure of 10 mTorr in 2nd derivative (2*f*) detection mode using a lock-in amplifier time constant of 30 ms. We can see that two spectral lines with an interval of just 30 MHz have been clearly resolved. This shows the usefulness of the high power and wide frequency-tunability of the CO~2~-microwave sidebands for resolving the overlapped features by Lamb-dip observations. Furthermore, in order to precisely measure the absolute transition frequencies for each of the individual line, we narrowed the microwave scanning range down to 3 MHz and swept the microwave sideband both upward and downward 5 times for each line with a frequency step-size of 10 kHz to record their saturation-dip 2*f* signals. [Figure 3](#f3){ref-type="fig"} displays the result for signal I of [Fig. 2](#f2){ref-type="fig"}, recorded at 14 mTorr pressure with a lock-in time constant of 30 ms. We then fitted the experimental trace (black) to determine the center transition frequency by a least-squares fit to the second derivative of a Gaussian profile[@b29] where *G*~0~ is a baseline constant, *G*~1~ is the integrated intensity, ν~0~ is the center frequency, and *Δ*ν~*pp*~ is the frequency separation between positive and negative peaks of the first-derivative *G*'(ν). The fitting trace is shown as a red solid curve in [Fig. 3](#f3){ref-type="fig"}. From this fit, the microwave frequency at line center was determined to be 12839.477 MHz, which yields an infrared transition frequency as 9*P*22--12839.477 MHz, giving 31 316 122.019 MHz with an accuracy of 0.1 MHz when the known frequency of the 9*P*22 CO~2~ laser line[@b30] is added. In the current study, we have measured more than 150 saturated absorption dips for spectral lines which belong to 27 C−N stretching substates. The assignments of these transitions, the measured transition frequencies, and the determined upper-state energy term values are presented in [Table 1](#t1){ref-type="table"}. The observed infrared transitions and assignments of CH~3~NH~2~ --------------------------------------------------------------- In [Table 1](#t1){ref-type="table"}, the transition notation of *P*/*Q*/*R*(*υ*~*t*~ *S*~*t−i*~ *K, J*) expresses the assigned quantum numbers for each of the identified spectral lines belonging to the *P*-, *Q*-, and *R*-branch, respectively. Here, *υ*~*t*~ is the torsional quantum number, *S*~*t-i*~ is the torsion-inversion symmetry (*A* or *E* for torsional species and *a* or *s* for inversion species), and *K* is the projection along the molecular near-symmetry *a*-axis of the rotational angular momentum *J*. For asymmetry *K*-doubling levels of *A* torsional symmetry, we add a superscript + or − to *K* to indicate the resolved doublet components. The C−N stretching fundamental is a parallel *a*-type band, the transition selection rules are Δ*υ*~t~ = 0, Δ*K* = 0, and Δ*J* = 0, ±1. Other researchers[@b22] use another common notation of the *G*~12~ or *D*~3h~ molecular symmetry group species *A, B, E*~1~, and *E*~2~, which corresponds here to *As, Aa, Ea*, and *Es*, respectively. The second and third columns list the determined transition frequencies in MHz and in wavenumbers, respectively, according to the specific CO~2~-microwave sideband used. For example, the Lamb-dip signals I and II in [Fig. 2](#f2){ref-type="fig"} observed with the lower microwave sideband of the 9*P*22 CO~2~ laser line are assigned as transition *Q*(0 *As* 8, 8) at a transition frequency of 1044.593391 cm^−1^ and as *Q*(0 *As* 5, 5) at 1044.592375 cm^−1^, respectively. The latter was first observed and reported in our previous work[@b28], and has been confirmed here. In [Table 1](#t1){ref-type="table"}, a letter U in the first column denotes a line that is still unassigned in the spectrum. As lines of *s* inversion symmetry are weak with only 1/3 the intensity of the corresponding *a* lines due to the relative spin statistical weights, our data are primarily for the *a* inversion species. For a substantial number of transitions, we could test our assignments and measurement accuracy via application of the Rydberg-Ritz combination principle to closed loops involving the observed lines. As an example for illustration, an energy-level diagram for the (*υ*~*t*~ *S*~*t−i*~ *K*) = (0 *Aa* 3) sub-band is shown in [Fig. 4](#f4){ref-type="fig"}. For each pair of transitions sharing a common upper-state level in [Fig. 4](#f4){ref-type="fig"}, we can form four closed loops involving the eight infrared transitions with labels *a* to *h* and the eight microwave transitions in the ground state. By using the predicted microwave frequencies[@b17] (uncertainty of 0.06 MHz) in Loops 1 and 2 and the observed microwave frequencies[@b17] (uncertainty of 0.06 MHz) in Loops 3 and 4, we calculate the loop closure defects (in MHz) as follows: Loop 1: δ~8~^+^ = a − c − ν~1~ − ν~2~ = (9*P*10 + 14334.992) − (9*P*36 − 15429.241) − 399010.809 − 354681.521 = − 0.14, Loop 2: δ~8~^−^ = b − d − ν~3~ − ν~4~ = (9*P*10 + 14339.825) − (9*P*36 − 15450.205) − 399027.383 − 354690.570 = 0.01, Loop 3: δ~13~^+^ = e − g − α~1~ − α~2~ = (9*P*2 + 13882.574) − (9*P*44 − 14507.345) − 620591.113 − 576287.579 = 0.01, Loop 4: δ~13~^−^ = f − h − β~1~ − β~2~ = (9*P*2 + 13914.907) − (9*P*44 − 14736.996) − 620746.167 − 576394.504 = − 0.01. Since each loop contains two infrared transitions, the fact that these loop defects are all so close to zero confirms the transition assignments in each of the loops and our experimental uncertainty of 0.1 MHz. Over 30 closed combination loops of transitions have been formed from the present and previous sub-Doppler observations. Line assignments shown with a letter L in [Table 1](#t1){ref-type="table"} are confirmed from these frequency combination closure relations. Such calculations of loop defects are very useful for providing secure assignments for the resolved *K*-doublet lines and for the component lines in blended features, especially for those spectral lines located around the crowded band center. We noticed that when term values for the ground state (kindly provided by N. Ohashi from his microwave and FIR analyses[@b7][@b14]) are used to calculate the related energy differences involved in the above mentioned loops, we found that the loop defects in MHz from Loop 1 to Loop 4 are −1.05, −2.25, −0.72, and −2.58, respectively. This indicates the estimated uncertainty of those term values to be about 1 MHz, consistent with the estimate from the microwave experimental study by Ilyushin *et al*.[@b9]. Comparison of the measured transition frequencies with those in the FT spectrum ------------------------------------------------------------------------------- The last two columns of [Table 1](#t1){ref-type="table"} show the frequency differences between the present Lamb-dip measurements and the data in the FT spectrum reported in Ref. [@b21] and Ref. [@b22], respectively. A blank space indicates either a new assignment or an unidentified U line. A histogram illustrating the distribution of these differences is given in [Fig. 5](#f5){ref-type="fig"}. The inset shows the histogram for the FT infrared data reported in Ref. [@b21]. We see that most of the deviations are less than several megahertz, but some of them are tens of megahertz. The mean value of the absolute deviation is about 5 MHz and 8 MHz for the FT data in Ref. [@b21] and Ref. [@b22], respectively, which reflects the frequency accuracy of the FT data estimated in these two works for infrared transitions in the C−N stretching band of CH~3~NH~2~. Power-series expansions of the energy term values in the excited state ---------------------------------------------------------------------- The fourth column of [Table 1](#t1){ref-type="table"} lists energy term values *W*(*υ*~*t*~ *S*~*t* − *i*~ *K, J*) for the upper levels of the corresponding transitions in the first column, obtained by adding our measured transition frequencies in wavenumbers to the Ohashi calculated ground-state energies, referenced to the (*υ*~*t*~ *S*~*t−i*~ *K, J*) = (0 *As* 0, 0) ground level as the energy zero. When two or three transitions were observed having a common upper level but giving independent values, the average of those term values was taken. In order to determine the *J*-independent origins of 27 C−N stretching substates, their term values were least-squares fitted to *J*(*J*+1) power-series expansions where *a*~0~ is the *J*-independent substate origin, *a*~1~ is an effective rotational constant, and *a*~2~ and *a*~3~ are effective centrifugal distortion constants. The obtained expansion coefficients *a*~0~, *a*~1~, *a*~2~, and *a*~3~ for 27 substates are shown in [Table 2](#t2){ref-type="table"}, in which the 1-*σ* standard deviations are in units of the last quoted digit. Asymmetry splittings and asymmetry-splitting constants of *A* symmetry levels in the excited state -------------------------------------------------------------------------------------------------- A rotational level with *A* symmetry of CH~3~NH~2~ may split due to the higher-order vibration-rotation interactions. For the resolved *K-*doublet lines, the transition selection rules are *K*^±^ ↔ *K*^±^ for Δ*J* = ±1 and *K*^±^ ↔ *K* ^∓^ for Δ*J* = 0. We have observed 57 Lamb-dip signals for resolved *K*-doublet lines resulting from the asymmetry splittings and precisely determined their transition frequencies. From the calculated level splittings in the ground state, asymmetry splittings for *Aa* levels with *K* = 1, 2, and 3 in the *υ*~*t*~ = 0 excited state have been determined and are shown in [Table 3](#t3){ref-type="table"}. The observed asymmetry splittings Δ*E*(*υ*~t~ *S*~t−i~ *K, J*) can be approximately represented by where *b*~0~ is the principal asymmetry splitting constant and other *b*~m~ with *m* ≥ 1 are higher-order corrections. The resulting series expansion coefficients from least-squares fits of the observed asymmetry splittings to [Eq. (3)](#eq3){ref-type="disp-formula"} for the three *K*-states are presented in [Table 3](#t3){ref-type="table"}. Conclusion ========== In this work for a molecule with the two strongly coupled large-amplitude internal motions of torsion and inversion, by using microwave sidebands of CO~2~ laser lines as frequency-tunable infrared sources in a sub-Doppler spectrometer, Lamb-dip spectroscopy of the C−N stretching band of CH~3~NH~2~ has been systematically studied. Many blended features and unresolved *K*-doublet lines involving wide variations in relative intensities in dense Doppler-limited spectra have been separated at high resolution. More than 50 *K*-doublet lines have been observed and the asymmetry-splitting constants for levels with *K* = 1, 2, and 3 in the excited state have been determined. Over 150 transitions have been assigned and identified into 27 substates and their transition frequencies have been precisely measured with an absolute accuracy of ±0.1 MHz. Energy term values for the upper levels of these assigned transitions have been determined and have been fitted to *J*(*J* + 1) power-series expansions for each substate to determine the *J*-independent C−N stretching substate origins and effective rotational constants. The Rydberg-Ritz combination principle was fully used in calculations of the defects for closed combination loops involving our observed transitions for confirming, revising and extending the transition assignments from previously reported results. Our experiment demonstrates the capabilities of the current experimental setup to precisely study the sub-Doppler infrared spectroscopies of isotopic species of CH~3~NH~2~ and of molecules with more than two internal motions. Our results constitute a high-accuracy database for frequency calibration in the 9--11 *μ*m region, provide more accurate spectral information for excited vibrational states to clearly map the rich and complex energy structures, to reveal the complex interaction mechanisms relevant to the C−N stretching of CH~3~NH~2~, to support further possible astronomical detections of interstellar CH~3~NH~2~, and to assign more energy levels and transition systems for optically pumped far-infrared laser emissions of CH~3~NH~2~. Methods ======= The tunable microwave radiation is generated by a microwave synthesizer and is amplified by a traveling-wave-tube amplifier to a power of about 20W before being fed to the modulator. Under this condition, the typical conversion efficiency into the sidebands from the incident laser beam is about 0.8%. In order to saturate the transitions of CH~3~NH~2~ at low pressures of several mTorr in this work, the full output radiation from the modulator is focused into our multi-reflection absorption cell with a total absorption path of 9.6 m in 16 transits. For observation of the saturation Lamb-dips by generating a counter-propagating beam inside the cell, a mirror M5 is placed at the exit of the cell window to reflect the radiation which has passed through the absorption cell back into the cell. This mirror is adjusted carefully so that the retro-reflected radiation nearly coincides with the incoming radiation but is slightly shifted and passes through a tunable Fabry--Pérot etalon filter for selecting only the desired sideband containing the CH~3~NH~2~ absorption signal. In such an optical arrangement, only those molecules moving at zero velocity parallel to the beam can absorb both the two counter-propagating laser radiations which have the same frequency, creating a saturation dip at the center of the absorption curve. This narrow dip is then detected by a liquid-N~2~-cooled HgCdTe detector as a Lamb-dip signal. In order to increase the probing sensitivity for the spectral signals, we use 1 kHz modulation of the sideband laser frequency for source modulation and demodulate the detected signal with a digital lock-in amplifier operating in the second-derivative (2*f*) detection mode to display the spectral lines. The signals are then sent to a computer for recording and analysis. A commercial CH~3~NH~2~ sample supplied by BOC Specialty Gases with a stated purity of 99.5% was used in the experiment without further purification. High attention should be taken for avoiding the possible confusion in the spectral analysis from NH~3~ impurity lines. Additional Information ====================== **How to cite this article**: Sun, Z.-D. *et al*. Lamb-dip spectroscopy of the C − N stretching band of methylamine by using frequency-tunable microwave sidebands of CO~2~ laser lines. *Sci. Rep.* **6**, 34270; doi: 10.1038/srep34270 (2016). We express our thanks to Prof. N. Ohashi for providing a list of calculated ground-state energies. This research was partially supported by the National Natural Science Foundation of China (Grants No. 11174186, 91536105, and 11374191) and the Tianshan Scholar Program. L.H.X and R.M.L acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada. **Author Contributions** Z.-D.S. contributed to the experimental setup, conducted measurements, analyzed the data, and wrote the manuscript. S.-D.Q. performed the data analyses. R.M.L. and L.-H.X. conducted the calculation and revised the manuscript. All authors contributed to the scientific discussions and approved the final manuscript. ![Schematic of the sub-Doppler spectrometer using microwave sidebands of CO~2~ laser lines as frequency-tunable infrared sources for the Lamb-dip spectroscopy of CH~3~NH~2~.\ M1--M5, mirrors; BS1, BS2, beam splitters; MW, microwave; TWT Amp., traveling wave tube amplifier; F.--P. etalon, Fabry--Pérot etalon filter; Detector1, InSb detector; Detector2, HgCdTe detector.](srep34270-f1){#f1} ![Observation of two Lamb-dip signals I and II resolved from an overlapped feature in the FT spectrum of CH~3~NH~2~.\ The spectrum was recorded in a scanning range of 50 MHz using the lower microwave sideband of the 9*P*22 CO~2~ laser line. The sample pressure was 20 mTorr and the lock-in time constant was 30 ms. Signals I and II are assigned as transitions *Q*(0 *As* 8, 8) and *Q*(0 *As* 5, 5), respectively. The observation of Signal II has previously been reported in Ref. [@b28] and has been confirmed here. This chart demonstrates features of the high power for observation of the saturated absorption spectra and the wide frequency-tunability of the microwave sidebands of CO~2~ laser lines.](srep34270-f2){#f2} ![Lamb-dip signal for the *Q*(0 *As* 8, 8) line of the C−N stretching band of CH~3~NH~2~ (signal I in [Fig. 2](#f2){ref-type="fig"}), scanned in a range of 3 MHz with a step-size of 10 kHz and recorded with the lock-in amplifier in 2*f* detection mode.\ The lock-in time constant was 30ms and the sample pressure of CH~3~NH~2~ was 14 mTorr. The solid curve (red) is a least-squares fit of a second-derivative Gaussian profile to the experimental trace (black). The microwave frequency at line center was determined from the fit to be 12839.477 MHz.](srep34270-f3){#f3} ![*K*−doublet energy-level diagram for the observed 8 infrared transitions accessing the (*υ*~*t*~ *S*~*t − i*~ *K*) = (0 *Aa* 3) substate of CH~3~NH~2~ in the Lamb-dip sub-Doppler spectrum.\ The measured infrared transition frequencies have been given in [Table 1](#t1){ref-type="table"}. The calculated frequencies (in MHz) for ground-state transitions in Ref. [@b17] are ν~1~ = 399010.809, ν~2~ = 354681.521, ν~3~ = 399027.383, ν~4~ = 354690.570. The measured transition frequencies (in MHz) in the ground states in Ref. [@b17] are α~1~ = 620591.113, α~2~ = 576287.579, β~1~ = 620746.167, β~2~ = 576394.504. The asymmetry splittings of the energy levels are exaggerated for clarity.](srep34270-f4){#f4} ![Histogram of the deviations (in MHz) for transition frequencies between our Lamb-dip measurements presented in [Table 1](#t1){ref-type="table"} and the FT infrared data reported in Ref. [@b22].\ The inset shows the histogram for the FT infrared data reported in Ref. [@b21].](srep34270-f5){#f5} ###### Observed infrared transitions and their upper-state term values for the C−N stretching band of CH~3~NH~2~. [a](#t1-fn1){ref-type="fn"}Transition *R*/*P*/*Q*(υ~t~ *S*~t−i~ *K, J*) [b](#t1-fn2){ref-type="fn"}CO~2~ laser line ± microwave req. (MHz) [c](#t1-fn3){ref-type="fn"}ν~obs~. (cm^−1^) Upper-state term value (cm^−1^) [d](#t1-fn4){ref-type="fn"}ν~FT1~ - ν~obs.~ (MHz) [e](#t1-fn5){ref-type="fn"}ν~FT2~ - ν~obs.~ (MHz) ------------------------------------------------------------------------- -------------------------------------------------------------------- --------------------------------------------- --------------------------------- --------------------------------------------------- --------------------------------------------------- *R*(0 *Ea* 4, 23) 9*R*16 − 11898.876 1075.590916 1527.054716 −29.3 −26.3 *R*(0 *Aa* 1^−^, 23) 9*R*16 − 12671.411 1075.565147 1489.380347 −15.5 −14.7 *R*(0 *Ea* 4, 20) 9*R*10 + 10911.802 1072.247745 1426.195845 −13.9 −15.4 *R*(0 Ea 7, 20) 9*R*10 + 10894.123 1072.247155 1515.100055 3.8 *R*(0 *Aa* 6, 20) 9*R*10 + 10689.436 1072.240327 1480.052327 1.3 −1.0 *R*(0 *Aa* 5, 20) 9*R*10 + 10553.581 1072.235796 1450.347096 −61.6 −45.4 *R*(0 *Aa* 3^+^, 20) 9*R*10 + 10507.877 1072.234271 1407.452171 −15.9 0.3 *R*(0 *Aa* 1^−^, 20) 9*R*10 + 10487.973 1072.233607 1388.031807 4.0 20.2 *R*(0 *Aa* 3^−^, 20) 9*R*10 + 10453.378 1072.232453 1407.567753 38.6 54.8 *R*(0 *Ea* −2, 20) 9*R*10 + 10305.241 1072.227512 1393.266612 −9.6 −6.7 *R*(0 *Aa* 2^−^, 18) 9*R*8 − 8791.434 1070.169057 1333.969857 −1.7 2.7 *R*(0 *Aa* 2^+^, 18) 9*R*8 − 9899.311 1070.132103 1334.975403 −1.8 −4.0 *R*(0 *Ea* −3, 18) 9*R*8 − 11187.383 1070.089137 1347.673137 9.4 6.9 *R*(0 *Ea* 3, 18) 9*R*8 − 11829.536 1070.067717 1347.269817 6.4 18.5 *R*(0 *Aa* 1^+^, 18) 9*R*8 − 15025.886 1069.961098 1322.810798 1.6 0.5 *R*(0 *Aa* 6, 18) 9*R*8 − 15611.927 1069.941550 1420.150150 −14.7 −25.5 *R*(0 *Aa* 3^−^, 18) 9*R*8 − 15931.388 1069.930894 1347.569494 −15.7 −18.4 *R*(0 *Ea* −2, 18) 9*R*8 − 16465.53 1069.913077 1333.443077 −3.5 0.3 *R*(0 *Ea* 2, 18) 9*R*8 − 16694.656 1069.905434 1334.541634 3.8 0.8 *R*(0 *Aa* 7, 18) 9*R*6 + 11061.409 1069.383062 1454.799162 6.0 −0.7 *R*(0 *Aa* 4^+^, 18) 9*R*6 + 10912.404 1069.378092 1365.464792 −6.9 −8.3 *R*(0 *Aa* 4^−^, 18) 9*R*6 + 10896.661 1069.377566 1365.462566 8.8 7.4 *R*(0 *Ea* −1, 17) 9*R*6 − 8350.046 1068.735565 1295.477665 16.7 *R*(0 *Aa* 4^−^, 16) 9*R*4 − 15504.517 1067.021935 1311.376435 8.6 4.1 *R*(0 *Aa* 7, 16) 9*R*4 − 16034.697 1067.004250 1400.722550 −3.2 −0.5 *R*(0 *Aa* 2^+^, 15) 9*R*2 + 15797.556 1066.564310 1255.648210 *R*(0 *Aa* 2^−^, 15) 9*R*2 + 15795.090 1066.564227 1255.115127 −0.7 2.1 *R*(0 *Ea* −5, 15) 9*R*2 + 13606.873 1066.491236 1311.601836 0.8 0.5 *R*(0 *Ea* −3, 15) 9*R*2 + 12983.316 1066.470437 1268.633737 1.1 0.6 *R*(0 *Aa* 8, 15) 9*R*2 + 11893.876 1066.434097 1416.913897 −25.6 *R*(0 *Ea* 3, 15) 9*R*2 + 11845.906 1066.432497 1268.214197 22.3 19.9 *R*(0 *Ea* 1, 15)L 9*R*2 + 10302.783 1066.381024 1248.412624 2.3 −0.2 *R*(0 *Aa* 6, 15) 9*R*2 + 10069.842 1066.373254 1341.234754 11.0 20.7 *R*(0 *Aa* 3^−^, 15) 9*R*2 + 9488.821 1066.353873 1268.554973 −31.2 −28.9 *R*(0 *Aa* 5, 15) 9*R*2 + 9277.964 1066.346840 1311.482840 −6.5 −4.5 *R*(0 *Ea* −2, 15) 9*R*2 + 8780.626 1066.330250 1254.592450 −24.5 −24.2 *R*(0 *Ea* −5, 12) 9*P*2 + 17768.953 1062.758673 1245.792673 −5.1 −7.1 *R*(0 *Ea* −3, 12)L 9*P*2 + 16567.900 1062.718611 1202.770811 4.3 −1.7 *R*(0 *Aa* 8, 12) 9*P*2 + 16467.650 1062.715267 1351.145967 2.9 5.2 *R*(0 *Ea* 1, 12) 9*P*2 + 15260.579 1062.675003 1182.112603 0.9 −0.3 *R*(0 *Aa* 1^−^, 12)L 9*P*2 + 14932.119 1062.664047 1181.933847 −15.4 −15.5 *R*(0 *Ea* 4, 12)L 9*P*2 + 14173.561 1062.638744 1221.484244 −2.7 −1.2 *R*(0 *Ea* 5, 12) 9*P*2 + 13932.828 1062.630714 1245.862714 −29.1 *R*(0 *Aa* 3^−^, 12)L 9*P*2 + 13914.907 1062.630116 1202.713316 −11.2 −13.7 *R*(0 *Aa* 3^+^, 12)L 9*P*2 + 13882.574 1062.629038 1202.706238 21.1 18.6 *R*(0 *Aa* 5, 12) 9*P*2 + 13705.152 1062.623120 1245.683420 −2.3 0.7 *R*(0 *Ea* −2, 12)L 9*P*2 + 13373.523 1062.612058 1188.822658 −6.7 −4.7 U 9*P*2 + 13332.331 1062.610684 *R*(0 *Ea* 2, 12) 9*P*2 + 12741.567 1062.590978 1189.173578 −9.7 −5.7 *R*(0 *Ea* −1, 12) 9*P*2 + 12730.848 1062.590620 1179.746320 1.0 5.1 *R*(0 *Aa* 1^+^, 12) 9*P*2 + 11318.384 1062.543506 1179.529006 −3.7 −0.2 *R*(0 *Aa* 4, 11) 9*P*4 + 8971.098 1060.869910 1201.743110 −4.7 −0.1 *R*(0 *Aa* 1^+^, 9)L 9*P*6 − 8477.145 1058.665947 1127.387047 −3.1 0.7 *R*(0 *Aa* 2^+^, 8) 9*P*8 + 9991.249 1057.633433 1122.155833 −4.7 −0.7 *R*(0 *Aa* 2^−^, 8) 9*P*8 + 9671.323 1057.622762 1122.096162 −3.2 −2.1 *R*(0 *Aa* 4, 8) 9*P*8 − 9088.952 1056.996986 1153.512786 −1.5 0.1 *R*(0 *Ea* 1, 7) 9*P*10 + 16357.707 1056.170702 1101.016902 −0.5 2.2 *R*(0 *Ea* −3, 7)L 9*P*10 + 16082.288 1056.161515 1122.270215 −1.5 −5.9 *R*(0 *Aa* 6^−^, 7) 9*P*10 + 15748.817 1056.150392 1195.035492 *R*(0 *Aa* 6^+^, 7) 9*P*10 + 15748.072 1056.150367 1195.035467 −14.7 −12.4 *R*(0 *Aa* 1^−^, 7) 9*P*10 + 15431.349 1056.139802 1100.808202 −6.8 −4.1 *R*(0 *Aa* 3^−^, 7)L 9*P*10 + 14339.825 1056.103393 1122.240693 −7.4 −7.5 *R*(0 *Aa* 3^+^, 7)L 9*P*10 + 14334.992 1056.103232 1122.240332 −2.6 −2.7 *R*(0 *Aa* 5^−^, 7) 9*P*10 + 14226.358 1056.099608 1165.243108 *R*(0 *Aa* 5^+^, 7) 9*P*10 + 14224.595 1056.099549 1165.243049 −10.6 −7.4 *R*(0 *Ea* 3, 7)L 9*P*10 + 14191.512 1056.098446 1121.821546 22.5 25.6 *R*(0 *Ea* −2, 7)L 9*P*10 + 13868.159 1056.087660 1108.348360 1.3 −0.9 *R*(0 *Ea* 2, 7)L 9*P*10 + 12586.227 1056.044899 1108.598499 1.7 −1.8 *R*(0 *Aa* 1^+^, 7)L 9*P*10 + 11458.187 1056.007272 1099.853072 −0.5 −0.1 *R*(0 *Ea* −1, 7)L 9*P*10 + 11298.615 1056.001949 1100.011549 2.6 4.6 *R*(0 *Aa* 4, 6) 9*P*12 + 12380.893 1054.336485 1128.671685 −5.4 −5.4 *R*(0 *Ea* 1, 5)L 9*P*12 − 14021.903 1053.455783 1078.900283 0.7 1.2 *R*(0 *Aa* 1^−^, 5)L 9*P*12 − 15401.199 1053.409774 1078.668574 −5.6 −1.9 *R*(0 *Aa* 3, 5) 9*P*12 − 16245.122 1053.381624 1100.293124 −1.4 −3.2 *R*(0 *Ea* 3, 5) 9*P*12 − 16712.009 1053.366050 1099.863150 11.3 10.2 *R*(0 *Ea* −2, 5) 9*P*12 − 16817.968 1053.362516 1086.392416 5.4 4.2 *R*(0 *Ea* 2, 5) 9*P*12 − 18065.890 1053.320890 1086.642890 −3.4 3.2 *Q*(0 *Aa* 3^+^, 4) 9*P*22 − 10210.545 1044.681083 1084.198083 2.3 *Q*(0 *As* 8, 8) 9*P*22 − 12839.477 1044.593391 1270.813991 −17.5 −19.6 *Q*(0 *Ea* −6, 6) 9*P*22 − 15577.846 1044.502049 1172.979549 −5.4 *Q*(0 *Ea* 9, 9) 9*P*22 − 16084.970 1044.485133 1330.001133 2.0 5.6 U 9*P*22 − 17775.882 1044.428730 U 9*P*24 + 17848.480 1043.758600 *Q*(0 *Ea* −8, 13) 9*P*24 + 17816.997 1043.757550 1351.457050 6.9 7.0 *Q*(0 *Ea* 9, 13) 9*P*24 + 17809.443 1043.757298 1397.236398 14.4 14.6 *Q*(0 *Aa* 11, 13) 9*P*24 + 16894.708 1043.726786 1505.045186 −2.3 −2.2 *Q*(0 *Aa* 8, 12) 9*P*24 + 16465.533 1043.712470 1332.143170 −0.3 -0.5 *Q*(0 *Aa* 7, 8) 9*P*24 + 16151.914 1043.702009 1229.649909 −5.1 −0.2 *Q*(0 *Aa* 6, 12) 9*P*24+14714.233 1043.654053 1256.44835 −0.1 1.0 *Q*(0 *Aa* 9, 12) 9*P*24 + 14041.047 1043.631598 1377.577598 −2.7 *Q*(0 *Aa* 7, 9) 9*P*24 + 11963.481 1043.562298 1242.813298 3.0 6.5 U 9*P*24 + 10809.657 1043.523810 *Q*(0 *Aa* 11, 14) 9*P*24 + 10233.275 1043.504584 1525.496884 −10.1 −8.6 *P*(0 *Aa* 1^+^, 6) 9*P*32 + 10145.824 1035.812044 1069.412640 0.8 0.3 *P*(0 *Ea* 1, 7)L 9*P*34 + 16971.825 1034.054118 1078.900320 2.5 5.0 *P*(0 *Aa* 1^−^, 7)L 9*P*34 + 15354.390 1034.000166 1078.668566 2.3 1.7 *P*(0 *Aa* 2^−^, 9) 9*P*36 − 9648.259 1031.155600 1108.931200 −0.8 0 *P*(0 *Aa* 1^+^, 9)L 9*P*36 − 10356.138 1031.131987 1099.853087 1.7 0.4 *P*(0 *Ea* −1, 9)L 9*P*36 − 10624.323 1031.123042 1100.011642 −1.8 −0.4 *P*(0 *Aa* 2^+^, 9) 9*P*36 − 10809.311 1031.116871 1108.969271 −0.8 0.9 *P*(0 *Ea* −3, 9) 9*P*36 − 13698.547 1031.020497 1122.270197 −5.8 −11.9 *P*(0 *As* 3, 9) 9*P*36 − 14350.507 1030.998750 1122.176450 −3.2 −1.7 *P*(0 *Ea* 4, 9) 9*P*36 − 14830.98 1030.982723 1141.037623 −9.9 −10.6 *P*(0 *Aa* 3^+^, 9) 9*P*36 − 15429.241 1030.962767 1122.240367 −19.0 −11.6 *P*(0 *Aa* 3^−^, 9) 9*P*36 − 15450.205 1030.962068 1122.240768 2.0 9.4 *P*(0 *Ea* 3, 9)L 9*P*36 − 15604.449 1030.956923 1121.821623 −0.3 −0.4 *P*(0 *Ea* −2, 9)L 9*P*36 − 16137.042 1030.939157 1108.348360 3.2 1.1 *P*(0 *Ea* 2, 9)L 9*P*36 − 17653.154 1030.888585 1108.598490 4.1 2.7 *P*(0 *Aa* 1^−^, 10) 9*P*38 − 9484.598 1029.125720 1114.089120 −2.0 1.3 *P*(0 *Aa* 4, 10) 9*P*38 − 16148.642 1028.903431 1153.512831 0.7 0 *P*(0 *Ea* −1, 11) 9*P*40 + 17092.541 1027.952318 1127.560120 1.4 4.1 *P*(0 *Aa* 1^+^, 11) 9*P*40 + 16989.214 1027.948871 1127.387171 −0.2 2.1 *P*(0 *Aa* 2^−^, 11) 9*P*40 + 15993.048 1027.915643 1136.722040 0.9 2.8 *P*(0 *Aa* 2^+^, 11) 9*P*40 + 13721.694 1027.839878 1136.811378 11.0 14.0 *P*(0 *Ea* 4, 11) 9*P*40 + 10281.031 1027.725110 1168.829210 7.0 4.5 *P*(0 *Aa* 5, 11) 9*P*40 + 9887.452 1027.711982 1193.033982 9.9 18.7 *P*(0 *Aa* 3^+^, 11) 9*P*40 + 9730.095 1027.706733 1150.039030 −15.0 −18.6 *P*(0 *Ea* −2, 11) 9*P*40 + 9187.589 1027.688637 1136.157337 2.3 3.7 *P*(0 *Ea* 1, 12) 9*P*42 + 15646.148 1025.819764 1145.257364 −2.1 0.1 *P*(0 *Aa* 1^−^, 12) 9*P*42 + 15032.225 1025.799286 1145.069086 0.2 −0.5 *P*(0 *Ea* −1, 13) 9*P*42 − 16840.998 1024.736110 1160.897510 −1.6 −1.1 *P*(0 *Aa* 1^+^, 13) 9*P*42 − 17569.495 1024.711810 1160.702410 −1.6 −1.1 *P*(0 *Ea* −3, 14)L 9*P*44 − 11971.706 1022.790042 1202.770842 *P*(0 *Aa* 2^+^, 14) 9*P*44 − 12189.339 1022.782782 1189.601080 −2.6 −4 *P*(0 *Aa* 6, 14) 9*P*44 − 12928.003 1022.758143 1275.454543 1.0 2.1 *P*(0 *Ea* 3, 14) 9*P*44 − 13533.332 1022.737952 1202.336550 −1.7 −0.8 *P*(0 *Ea* 4, 14)L 9*P*44 − 13960.687 1022.723697 1221.484200 −9.6 −9.5 *P*(0 *Ea* −2, 14)L 9*P*44 − 14254.030 1022.713912 1188.822610 7.9 26.0 *P*(0 *Aa* 3^+^, 14)L 9*P*44 − 14507.345 1022.705462 1202.706262 −2.3 −4.9 *P*(0 *Aa* 3^−^, 14)L 9*P*44 − 14736.996 1022.697802 1202.713402 0.7 −0.8 *P*(0 *Ea* −1, 15) 9*P*46 + 14919.509 1021.554573 1200.096773 −3.2 *P*(0 *Aa* 1^+^, 15) 9*P*46 + 11162.841 1021.429264 1199.799864 −5.3 −2.1 *P*(0 *Aa* 1^−^, 15) 9*P*46 − 10682.479 1020.700583 1202.570383 1.6 −0.1 *P*(0 *Aa* 4, 15) 9*P*46 − 16527.760 1020.505605 1241.210105 −3.9 −4.9 *P*(0 *Ea* −3, 16) 9*P*48 + 14709.120 1019.391336 1245.215836 0 −1.9 *P*(0 *Ea* 3, 16) 9*P*48 + 13389.423 1019.347316 1244.790816 −34.8 −28.5 *P*(0 *Ea* 7, 16) 9P48 + 13353.259 1019.346110 1352.910110 1.4 *P*(0 *Aa* 6, 16) 9*P*48 + 13345.498 1019.345851 1317.848051 9.2 15.4 *P*(0 *Ea* −2, 16) 9*P*48 + 12891.933 1019.330722 1231.214822 0.2 0.6 *P*(0 *Aa* 2^+^, 16) 9*P*48 + 12421.741 1019.315038 1232.157838 −1.4 −2.7 *P*(0 *Ea* 4, 16) 9*P*48 + 12335.332 1019.312155 1263.896255 −21.0 −24.5 *P*(0 *Ea* 5, 16) 9*P*48 + 12295.030 1019.310811 1288.263811 19.3 15.8 *P*(0 *Aa* 5, 16) 9*P*48 + 12229.499 1019.308625 1288.089125 −2.4 3.0 *P*(0 *Aa* 3^+^, 16) 9*P*48 + 11781.408 1019.293678 1245.128078 0.2 4.6 *P*(0 *Aa* 3^−^, 16) 9*P*48 + 11308.036 1019.277888 1245.144388 −0.7 0.2 *P*(0 *Ea* 2, 17) 9*P*50 + 17961.451 1017.320072 1255.207572 −1.3 0 *P*(0 *Ea* 1, 17)L 9*P*50 + 15685.814 1017.244164 1248.412664 1.3 0.8 *P*(0 *Aa* 1^−^, 17) 9*P*50 + 15357.100 1017.233200 1248.244800 −11.2 −7.4 *P*(0 *Aa* 4, 17) 9*P*50 + 9686.673 1017.044055 1286.526755 −24.8 −23.6 *P*(0 *Aa* 2^−^, 18) 9*P*50 − 17254.659 1016.145389 1279.946189 3.9 1.5 *P*(0 *Ea* −2, 19) 9*P*52 − 10876.861 1014.155075 1305.707275 2.5 5.5 *P*(0 *Aa* 6, 19) 9*P*52 − 12143.902 1014.112811 1392.386211 −0.1 −0.4 *P*(0 *Ea* −6, 19) 9*P*52 − 12921.993 1014.086857 1392.296957 −70.7 *P*(0 *Aa* 5, 19) 9*P*52 − 13002.243 1014.084180 1362.650680 9.5 15.1 *P*(0 *Aa* 3^−^, 19) 9*P*52 − 14888.525 1014.021260 1319.767360 −0.4 −0.6 *P*(0 *Aa* 2^+^, 19) 9*P*52 − 16384.082 1013.971374 1307.056674 1.0 2.5 ^a^Letter L indicates that the line assignment has been confirmed from a frequency combination loop; a letter U denotes an unassigned line. ^b^Observed transition frequencies in MHz are expressed by the frequency of the CO~2~ laser line and the determined microwave frequency from the fitting to the recorded Lamb-dip signal. ^c^Observed transition frequencies are converted into wavenumbers using a factor of 29979.2458 MHz/cm^−1^ and CO~2~ laser frequencies from Ref. [@b30]. ^d^Differences (in MHz) between the observed transition frequencies in the present work and values from the FT infrared wavenumbers reported in Ref. [@b21]. A blank space indicates either a new assignment or an unidentified U line to the data in Ref. 21. ^e^Differences (in MHz) between the observed transition frequencies in the present work and values from the FT infrared wavenumbers reported in Ref. [@b22]. A blank space indicates either a new assignment or an unidentified U line to the data in Ref. 22. ###### *J*(*J* + 1) power-series expansion coefficients (in cm^−1^) of upper-state term values in 27 substates of the C−N stretching band of CH~3~NH~2~. Substate (*υ*~*t*~ *S*~*t−i*~ *K*) *a*~0~ *a*~1~ *a*~2~ × 10^6^ *a*~3~ × 10^10^ ---------------------------------------- ------------------ --------------- ---------------- ----------------- (0 *Aa* 0)[\*](#t2-fn1){ref-type="fn"} 1044.912526(461) 0.732866(11) −10.72(8) 44.1(16) (0 *Aa* 1^+^) 1047.66065(167) 0.725175(4) −3.92(26) 27.3(4) (0 *Aa* 1^−^) 1047.662408(196) 0.738380(3) −3.34(1) −5.0(1) (0 *Aa* 2^+^) 1056.257642(711) 0.731710(12) 5.73(6) −29.0(9) (0 *Aa* 2^−^) 1056.254285(389) 0.731812(7) −2.63(3) 1.1(4) (0 *Aa* 3^+^) 1069.565927(15) 0.731614(0) −0.31(1) −5.5(0) (0 *Aa* 3^−^) 1069.566059(76) 0.731610(1) −0.28(1) 5.9(1) (0 *Aa* 4) 1087.76225(210) 0.730430(40) −1.69(20) −0.2(3) (0 *Aa* 5) 1112.580226(61) 0.731490(1) −0.84(0) −0.2(0) (0 *Aa* 6) 1142.383547(391) 0.731342(7) −1.01(3) 0.4(2) (0 *Aa* 7) 1176.989660(29) 0.7314660(0) −1.02(0) 0 (0 *Aa* 8) 1218.14342(465) 0.730775(22) (0 *Aa* 11) 1372.109149(0) 0.730418(0) (0 *Ea* 0)\* 1045.101654(472) 0.731802(12) −10.24(6) 34.2(9) (0 *Ea* 1) 1047.93850(858) 0.737074(237) 2.77(18) −107(37) (0 *Ea* 2) 1055.93689(927) 0.730844(209) 7.42(11) −34(17) (0 *Ea* 3) 1069.121205(380) 0.731947(11) 78(7) 1.4(0) (0 *Ea* 4) 1088.374933(240) 0.731462(4) -0.49(2) −9(2) (0 *Ea* 5) 1112.77336(123) 0.731406(29) −0.8(1) 0 (0 *Ea* 6) 1142.535800(365) 0.731324(6) (0 *Ea* 7) 1177.49078(396) 0.730786(131) (0 *Ea* 9) 1264.227504(0) 0.730818(0) (0 *Ea* −1) 1047.6819(828) 0.727080(889) −7(2) 0 (0 *Ea* -2) 1055.62915(546) 0.732554(97) −4.2(4) 10(5) (0 *Ea* −3) 1069.575107(628) 0.731907(12) −0.49(6) 8.0(1) (0 *Ea* −5) 1112.674698(42) 0.731554(1) −0.75(0) 0 (0 *Ea* −6) 1142.275112(0) 0.731058(0) (0 *Ea* −7) 1177.492590(0) 0.731449(0) (0 *Ea* −8) 1218.416547(0) 0.730994(0) ^\*^First reported in Ref. [@b28] and confirmed in this work. Energy of the level (*υ*~*t*~ *S*~*t−i*~ *K, J*) = (0 *As* 0, 0) in the ground state is taken as the zero. Errors in parentheses are one standard deviation in units of the last quoted digit. ###### Asymmetry splittings Δ*E*(*υ* ~t~ *S* ~*t−i*~ *K, J*) and asymmetry-splitting constants *b* ~m~ (in MHz) for the resolved *K*-doublet levels of *A* torsional symmetry of CH~3~NH~2~. *J* Δ*E*(*υ*~t~ *S*~*t−i*~ *K, J*) (MHz) [a](#t3-fn1){ref-type="fn"}Obs. − Calc. (MHz) *K* *b*~0~ *b*~1~ *b*~2~ *b*~3~ *b*~4~ *b*~5~ ----- -------------------------------------- ----------------------------------------------- ----- --------------- ---------------- --------------- ---------------- ---------------- ----------------- 7 28634.077 −0.087 1 496.599352 0.533819 −0.004838 12 72095.319 −1.861 15 83058.070 −6.763 8 1788.892 0.035 2 4.325212 −0.107269 9.44 × 10^−4^ −3.35 × 10^−6^ 4.10 × 10^−9^ 9 1141.340 0.068 11 2678.196 0.214 15 15981.426 1.268 18 30145.511 3.581 7 10.823 0 3 8.77 × 10^−4^ −2.91 × 10^−5^ 3.61 × 10^−7^ −2.01 × 10^−9^ 5.09 × 10^−12^ −4.72 × 10^−15^ 9 12.022 0 12 212.193 −0.001 14 214.052 −0.011 16 488.961 −0.078 20 3465.061 −1.431 ^a^Asymmetry splittings were calculated from the asymmetry-splitting constants in Table 3. The *K*-doublet levels are in the excited states with *K* = 1, 2, and 3 and in torsional state *υ*~t~ = 0 with the torsion-inversion symmetry *Aa*.

{ "pile_set_name": "PubMed Central" }

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22,268

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