Context: Most transcription factors (TFs) belong to protein families that share a common DNA binding domain and have very similar DNA binding preferences. However, many paralogous TFs (i.e. members of the same TF family) perform different regulatory functions and interact with different genomic regions in the cell. A potential mechanism for achieving this differential in vivo specificity is through interactions with protein co-factors. Computational tools for studying the genomic binding profiles of paralogous TFs and identifying their putative co-factors are currently lacking. Here, we present an interactive web implementation of COUGER, a classification-based framework for identifying protein co-factors that might provide specificity to paralogous TFs. COUGER takes as input two sets of genomic regions bound by paralogous TFs, and it identifies a small set of putative co-factors that best distinguish the two sets of sequences. To achieve this task, COUGER uses a classification approach, with features that reflect the DNA-binding specificities of the putative co-factors. The identified co-factors are presented in a user-friendly output page, together with information that allows the user to understand and to explore the contributions of individual co-factor features. COUGER can be run as a stand-alone tool or through a web interface: http://couger.oit.duke.edu.


Context: Vsr endonuclease, which initiates very short patch repair, has been hypothesized to regulate mutation in stationary-phase cells. Overexpression of Vsr does dramatically increase the stationary-phase reversion of a Lac- frameshift allele, but the absence of Vsr has no effect. Thus, at least in this case, Vsr has no regulatory role in stationary-phase mutation, and the effects of Vsr overproduction are likely to be artifactual.


Context: Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the locations of FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (sn(X2)) on FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost sn(X2) by this mechanism on a historical timescale. Finally, we characterize the original allele of the Bar gene (B(1)) that is carried on FM7, and validate the hypothesis that the origin and subsequent reversion of the B(1) duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila.


Context: HESR1 is a basic helix-loop-helix transcription factors regulated by the Notch signaling pathway in vertebrate and Drosophila embryos, and is related to the HES/Hairy/E (sp1) family. HESR1 is a downstream target of Notch in endothelial cells and could be an effector of Notch signaling in these cells. HESR1 is necessary for the induction of a tubular network and for continued maintenance of mature and quiescent blood vessels. To examine the role of HESR1 in retinal neovascularization, we transfected retinal vascular endothelial cells (HRCECs) with the HESR1 gene and studied its effects on the expression of angiogenic factors, on the proliferation and migration of endothelial cells, and on the formation of tube-like structures (TLSs). Overexpression of HESR1 downregulated VEGFR-2 expression, upregulated occludin expression, inhibited the migration and proliferation of HRCECs, and inhibited the formation of TLSs. Thus, HESR1 plays a key role in the finely tuned network of molecules involved in the regulation of retinal vascular homeostasis. HESR1 seems to inhibit the vessel-promoting effects of VEGF, shift endothelial cells from a proliferative state to a quiescent state, and restore normal vessel structures. Expression of the HESR1 gene in retinal vascular endothelial cells may protect retinal blood vessels and may be useful in the treatment of diseases involving damage to the retinal vasculature, including diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.


Context: Lu-AA21004, an oral, multimodal serotonergic agent, is currently under development by H Lundbeck and Takeda Pharmaceutical, for the potential treatment of depression and anxiety. Lu-AA21004 belongs to a novel chemical class of antidepressant agents, the bisarylsulfanyl amines, and possesses a novel pharmacological profile, with activity at serotonergic receptors 5-HT3, 5-HT7 and 5-HT1A, and also at the 5-HT transporter. Acute administration of Lu-AA21004 in rats inhibited the firing activity of serotonergic neurons of the dorsal raphe nucleus through 5-HT3 receptor blockade, with rapid recovery of firing activity upon cessation of treatment compared with an antidepressant of the SSRI class. Results from phase II clinical trials have reported improvement in depression and anxiety symptoms after 6 weeks of treatment. Lu-AA21004 was generally well tolerated, with adverse events related to sexual dysfunction occurring in a lower number of patients receiving Lu-AA21004 compared with venlafaxine. Phase III clinical trials with Lu-AA21004 in patients with major depressive disorder are underway and phase III trials in patients with generalized anxiety disorder have been completed. If initial outcomes from these clinical trials prove positive, Lu-AA21004 may pave the way for new multimodal therapies for the treatment of depression and anxiety.


Context: The lunar cycle seems to affect the incidence of intracranial aneurysm rupture, with the new moon being associated with an increased risk of aneurysmal SAH.


Context: Bath-PUVA therapy has been described as successful treatment for palmoplantar eczema. However, our own observations showed that patients with palmoplantar eczema of the dyshidrotic or hyperkeratotic type responded only partially to bath-PUVA therapy. In order to evaluate environmental influences possibly having an impact on the efficacy of this therapy, smokers and non-smokers suffering from palmoplantar eczema treated with bath-PUVA therapy were compared. A retrospective study was conducted involving 62 patients, 39 non-smokers and 23 smokers, with palmar and/or plantar eczema resistant to local corticosteroids. Bath-PUVA therapy was performed according to the European standard regimen for oral PUVA therapy. The total number of treatments and the cumulative UVA-dose were similar in smokers and non-smokers (smokers 24+/-17.7 (mean+/-SD) and 67.6+/-51.3 J/cm2 vs. non-smokers 25.7+/-16.3 and 68.5+/-49.3 J/cm2). In the group of non-smokers, 31% showed complete remission (CR; 100% clearance), 33% partial remission (PR; more than 50% clearance) and 36% no change after treatment (NC; less than 50% clearance). In contrast, the group of smokers showed only 13% CR and 22% PR, whereas 65% exhibited NC. The differences regarding complete or partial remission between the groups were statistically significant (Student t-test for paired samples; P<0.05). Regarding the different type of eczema, bath-PUVA proved to be more successful in the dyshidrotic type of eczema as compared to the hyperkeratotic type in non-smokers (P<0.05). In the group of smokers no CR was achieved in patients suffering from the dyshidrotic form of eczema. Smoking is likely to be a reason for the failure of bath-PUVA therapy in the treatment of chronic palmoplantar eczema, in particular regarding smokers with eczema of the dyshidrotic type where no complete remission was achieved.


Context: Teriflunomide is a new active drug which has recently been approved as a first-line treatment of relapsing forms of MS in the US, Australia, Argentina, and the European Union. It is characterized by a once-daily oral application and a well-established long-term safety profile. The main therapeutic effect is considered to be mediated via the inhibition of the de novo synthesis of pyrimidine in proliferating immune cells. Two phase III clinical trials (TEMSO, TOWER) tested teriflunomide in patients with relapsing forms of MS: efficacy was shown, with positive effects on relapse rates and disease progression for 14 mg/day. Overall, the safety profile in these studies was favorable. In patients treated with teriflunomide, the regular monitoring of blood cell counts and liver enzymes is required. Teriflunomide must not be used during pregnancy. In this article, we review recent phase II and phase III clinical trial data, and discuss the potential of teriflunomide for the treatment of relapsing forms of MS.


Context: Hetlioz(®) (tasimelteon) is the first approved treatment in the United States for Non-24-Hour Sleep-Wake Disorder (Non-24). We present here data on the in vitro binding affinity of tasimelteon for both human melatonin receptors MT1 and MT2, as well as the extended screen of other receptors and enzymes. Results indicate that tasimelteon is a potent Dual Melatonin Receptor Agonist (DMRA) with 2.1-4.4 times greater affinity for the MT2 receptor believed to mediate circadian rhythm phase-shifting (Ki = 0.0692 nM and Ki = 0.17 nM in NIH-3T3 and CHO-K1 cells, respectively), than for the MT1 receptor (Ki = 0.304 nM and Ki = 0.35 nM, respectively). Tasimelteon was also shown to have no appreciable affinity for more than 160 other pharmacologically relevant receptors and several enzymes.


Context: Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.


Context: TFIIB is essential for transcription initiation by RNA polymerase II. TFIIB also cross-links to terminator regions and is required for gene loops that juxtapose promoter-terminator elements in a transcription-dependent manner. The Saccharomyces cerevisiae sua7-1 mutation encodes an altered form of TFIIB (E62K) that is defective for both start site selection and gene looping. Here we report the isolation of an ssl2 mutant, encoding an altered form of TFIIH, as a suppressor of the cold-sensitive growth defect of the sua7-1 mutation. Ssl2 (Rad25) is orthologous to human XPB and is a member of the SF2 family of ATP-dependent DNA helicases. The ssl2 suppressor allele encodes an arginine replacement of the conserved histidine residue (H508R) located within the DEVH-containing helicase domain. In addition to suppressing the TFIIB E62K growth defect, Ssl2 H508R partially restores both normal start site selection and gene looping. Moreover, Ssl2, like TFIIB, associates with promoter and terminator regions, and the diminished association of TFIIB E62K with the PMA1 terminator is restored by the Ssl2 H508R suppressor. These results define a novel, functional interaction between TFIIB and Ssl2 that affects start site selection and gene looping.


Context: Although interstitial lung disease accounts for the majority of deaths of patients with systemic sclerosis, treatment options for this manifestation of the disease are limited. Few high-quality, randomized, controlled trials exist for systemic sclerosis-related interstitial lung disease, and historically, studies have favored the use of cyclophosphamide. However, the benefit of cyclophosphamide for this disease is tempered by its complex adverse event profile. More recent studies have demonstrated the effectiveness of mycophenolate for systemic sclerosis-related interstitial lung disease, including Scleroderma Lung Study II. This review highlights the findings of this study, which was the first randomized controlled trial to compare cyclophosphamide with mycophenolate for the treatment of systemic sclerosis-related interstitial lung disease. The results reported in this trial suggest that there is no difference in treatment efficacy between mycophenolate and cyclophosphamide; however, mycophenolate appears to be safer and more tolerable than cyclophosphamide. In light of the ongoing advances in our understanding of the pathogenic mechanisms underlying interstitial lung disease in systemic sclerosis, this review also summarizes novel treatment approaches, presenting clinical and preclinical evidence for rituximab, tocilizumab, pirfenidone, and nintedanib, as well as hematopoietic stem cell transplantation and lung transplantation. This review further explores how reaching a consensus on appropriate study end points, as well as trial enrichment criteria, is central to improving our ability to judiciously evaluate the safety and efficacy of emerging experimental therapies for systemic sclerosis-related interstitial lung disease.


Context: Avanafil has a favorable phosphodiesterase-5 selectivity profile compared to that of marketed phosphodiesterase-5 inhibitors. Avanafil shows excellent in vitro and in vivo potency, and fast onset of action for penile erection. Cumulative data suggest that avanafil has a promising pharmacological profile for erectile dysfunction.


Context: The lantibiotic mersacidin has been previously reported to interfere with bacterial peptidoglycan biosynthesis, [Brötz, H., Bierbaum, G., Markus, A., Molitor, E. & Sahl, H.-G. (1995) Antimicrob. Agents Chemother. 39, 714-719]. Here, we focus on the target reaction and describe a mersacidin-induced accumulation of UDP-N-acetylmuramoyl-pentapeptide, indicating that inhibition of peptidoglycan synthesis occurs after the formation of cytoplasmic precursors. In vitro studies involving a wall-membrane particulate fraction of Bacillus megaterium KM demonstrated that mersacidin did not prevent the synthesis of lipid II [undecaprenyl-diphosphoryl-N-acetylmuramoyl-(pentapeptide)-N-ac ety lglucosamine] but specifically the subsequent conversion of this intermediate into polymeric nascent glycan strands by transglycosylation. Comparison with other inhibitors of transglycosylation shows that the effective concentration of mersacidin in vitro is in the range of that of the glycopeptide antibiotic vancomycin but 2-3 orders of magnitude higher than that of the competitive enzyme inhibitor moenomycin. The analogy to the glycopeptides may hint at an interaction of mersacidin with the peptidoglycan precursor rather than with the enzyme. Unlike vancomycin however, mersacidin inhibits peptidoglycan formation from UDP-N-acetylmuramoyl-tripeptide and is active against Enterococcus faecium expressing the vanA resistance gene cluster. This indicates that the molecular target site of mersacidin differs from that of vancomycin and that no cross-resistance exists between the two antibiotics.


Context: Classically, the maintenance and control of liver homeostasis are assigned to the metabolic activity of parenchymal cells. However, recent evidence highlights complex and tightly regulated interactions between hepatocytes and other intra-hepatic cells. Kupffer cells--the resident macrophages of the liver--are able to release a tremendous array of mediators upon inflammatory conditions, such as infection, and their role in innate immunity is well described in the literature. However, the impact of these Kupffer cell-derived mediators on liver homeostasis is unknown. In this study, we investigated the physiological involvement of Kupffer cells in the regulation of hepatic metabolism. It was first necessary to validate the use of a compound able to selectively deplete Kupffer cells. We confirmed that gadolinium chloride (GdCl3) injection to rats eliminated ED2-positive Kupffer cells and strongly decreased both their phagocytic and peroxidase activities. Moreover, we demonstrated that precision-cut liver slices (PCLS)--an original in vitro model allowing to maintain intact hepatic architecture and cellular heterogeneity--obtained from GdCl3-treated rats released lower amounts of inflammatory mediators. Therefore, we proposed to use GdCl3 prior to PCLS preparation in order to investigate the role of Kupffer cells in the control of hepatic metabolism. Among various metabolic functions of the liver, we focused, in particular, on paracetamol and lipid metabolism as example of drug and intermediary metabolism, respectively. Our results suggest that the presence of Kupffer cells in liver tissue can affect the viability of PCLS in culture and are involved in the regulation of paracetamol metabolism, in particular the glucuronidation pathway. Furthermore, inhibition of Kupffer cells leads to a metabolic shift of fatty acids towards their esterification (at least, in fasted rats) and accumulation in the liver tissue, supporting a key role of Kupffer cells in the regulation of intra-hepatic lipid metabolism. Results obtained from in vitro studies suggest that Kupffer cell-derived prostaglandin E2 might be involved in the higher capacity of lipid synthesis observed in PCLS obtained from GdCl3-treated rats. Final objectives of this work were devoted to highlight the impact of the diet on Kupffer cell activity, offering possibilities to modulate Kupffer cell functions by nutrients under various physio-pathological conditions, such as inflammation. Our results demonstrate that a supplementation of glycine, a simple amino acid, in the diet could influence lipid metabolism, namely in the liver. A direct relationship between those metabolic effects and Kupffer cell activity has not been demonstrated. Nevertheless, we have shown that the use of glycine in vitro offers the possibility to elucidate complex interactions between Kupffer cells and hepatocytes, by using PCLS in culture, and could constitute an alternative tool to inhibit in vitro Kupffer cell-derived mediators. Furthermore, dietary oligofructose, a fermentable and nondigestible carbohydrate known as prebiotic, was able to increase phagocytic activity of the liver and the production of Kupffer cell-derived mediators by PCLS. Our results indicate that improvement of Kupffer cell activities might be involved in the hepatoprotection against septic challenge observed after a diet enriched with oligofructose.


Context: Typical- and delayed-onset Friedreich's ataxia are different and Friedreich's ataxia is heterogeneous. Late-onset Friedreich's ataxia and very-late-onset Friedreich's ataxia appear to belong to the same clinical and molecular continuum and should be considered together as "delayed-onset Friedreich's ataxia." As the most frequently inherited ataxia, Friedreich's ataxia should be considered facing compatible pictures, including atypical phenotypes (spastic ataxia, retained reflexes, lack of dysarthria, and lack of extraneurological signs), delayed disease onset (even after 60 years of age), and/or slow disease progression.


Context: Pumilio is a member of the highly conserved PUF family of RNA-binding proteins that function as a developmental regulator in diverse animal species. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals and are found to be involved in sperm development, neuron development as well as human diseases such as neurodegeneration. Generation of animal models disrupting different parts of Pum protein could help to further dissect their physiological function. Here we described characterization and analysis of a mouse line possessing a gene trap mutation of the Pumilio1 (Pum1) gene. Mice homozygous for the mutation (Pum1(XE002)) cannot be recovered in the adult offspring, at birth or at different time points of embryonic development (E18, E14, E12). Careful analysis of preimplantation embryos showed that no homozygous blastocysts could be detected on day 3.5 of gestation. 96-hr in vitro culture of 1-cell embryos either by natural mating or in vitro fertilization between heterozygotes failed to uncover any homozygous blastocysts, suggesting an early loss of homozygous preimplantation embryos. The lack of Pum1 gene trap homozygotes suggests a role of Pum1 in very early embryonic development or fertilization. This novel animal model affecting the beginning of embryonic development could help to understand not only the genetic mechanism underlying preimplantation embryonic development but also the translational regulation in development and diseases.


Context: Immune infiltration of colorectal cancer (CRC) is closely associated with clinical outcome. However, previous work has not accounted for the diversity of functionally distinct cell types that make up the immune response. In this study, based on a deconvolution algorithm (known as CIBERSORT) and clinical annotated expression profiles, we comprehensively analyzed the tumor-infiltrating immune cells present in CRC for the first time. The fraction of 22 immune cells subpopulations was evaluated to determine the associations between each cell type and survival and response to chemotherapy. As a result, profiles of immune infiltration vary significantly between paired cancer and paracancerous tissue and the variation could characterize the individual differences. Of the cell subpopulations investigated, tumors lacking M1 macrophages or with an increased number of M2 macrophages, eosinophils, and neutrophils were associated with the poor prognosis. Unsupervised clustering analysis using immune cell proportions revealed five subgroups of tumors, largely defined by the balance between macrophages M1, M2, and NK resting cells, with distinct survival patterns, and associated with well-established molecular subtype. Collectively, our data suggest that subtle differences in the cellular composition of the immune infiltrate in CRC appear to exist, and these differences are likely to be important determinants of both prognosis and response to treatment.


Context: We conducted a phase II study of the combination of temozolomide and angiogenesis inhibitors for treating adult patients with newly diagnosed glioblastoma. Patients who had stable disease following standard radiation therapy received temozolomide for 5 days in 28-day cycles, in combination with daily thalidomide and celecoxib. Patients were treated until tumor progression or development of unacceptable toxicity. Four-month progression-free survival (PFS) from study enrollment was the primary end point, and overall survival (OS) was the secondary end point. In addition, we sought to correlate response with O(6)-methylguanine-DNA methyltransferase promoter methylation status and serum levels of angiogenic peptides. Fifty patients with glioblastoma were enrolled (18 women, 32 men). Median age was 54 years (range, 29-78) and median KPS score was 90 (range, 70-100). From study enrollment, median PFS was 5.9 months (95% confidence interval [CI]: 4.2-8.0) and 4-month PFS was 63% (95% CI: 46%-75%). Median OS was 12.6 months (95% CI: 8.5-16.4) and 1-year OS was 47%. Of the 47 patients evaluable for best response, none had a complete response, five (11%) had partial response, four (9%) had minor response, 22 (47%) had stable disease, and 16 (34%) had progressive disease. Analysis of serial serum samples obtained from 47 patients for four angiogenic peptides failed to show a significant correlation with response or survival for three of the peptides; higher vascular endothelial growth factor levels showed a trend toward correlation with decreased OS (p=0.07) and PFS (p=0.09). The addition of celecoxib and thalidomide to adjuvant temozolomide was well tolerated but did not meet the primary end point of improvement of 4-month PFS from study enrollment.


Context: Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified by microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high-affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU upregulated the most. Quantitative PCR analysis of pstB and phoU transcript levels in G. sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium-bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve because of the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.


Context: The introduction of cereals in human nutrition 10 000 years ago caused the occurrence of gluten induced diseases. This protein complex is involved in pathogenesis of wheat allergy, celiac disease, and gluten sensitivity. Wheat allergy and celiac disease are mediated by the system of adaptive immunity. Gluten sensitivity is a recently defined entity induced by innate immune mechanisms. These subjects present various intestinal and particularly extraintestinal symptoms. The differences between celiac disease and gluten intolerance include permeability of the intestinal mucosal barrier, histology of duodenal biopsy, and mucosal gene expression. The symptoms of gluten sensitivity may also have another genetic background of food intolerance independent of the HLADQ2, - DQ8 system and tissue transglutaminase (eg. in some psychiatric disorders). At present, there is no specific bio-marker of gluten sensitivity. The diagnosis is possible only by exclusion of other causes of symptoms and improvement on a glutenfree diet applied in a doubleblind placebo controlled manner with optional sequence of both stages to exclude the placebo effect due to nutritional intervention.


Context: In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.


Context: The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.


Context: Epigenetics refers to structural modifications to genes that do not change the nucleotide sequence itself but instead control and regulate gene expression. DNA methylation, histone modification, and RNA regulation are some of the mechanisms involved in epigenetic modification. Epigenetic changes are believed to be a result of changes in an organism's environment that result in fixed and permanent changes in most differentiated cells. Some environmental changes that have been linked to epigenetic changes include starvation, folic acid, and various chemical exposures. There are periods in an organism's life cycle in which the organism is particularly susceptible to epigenetic influences; these include fertilization, gametogenesis, and early embryo development. These are also windows of opportunity for interventions during the reproductive life cycle of women to improve maternal-child health. New data suggest that epigenetic influences might be involved in the regulation of fetal development and the pathophysiology of adult diseases such as cancer, diabetes, obesity, and neurodevelopmental disorders. Various epigenetic mechanisms may also be involved in the pathogenesis of preeclampsia and intrauterine growth restriction. Additionally, environmental exposures are being held responsible for causing epigenetic changes that lead to a disease process. Exposure to heavy metals, bioflavonoids, and endocrine disruptors, such as bisphenol A and phthalates, has been shown to affect the epigenetic memory of an organism. Their long-term effects are unclear at this point, but many ongoing studies are attempting to elucidate the pathophysiological effects of such gene-environment interactions.


Context: A commonly accepted model of Wnt/β-catenin signaling involves target gene activation by a complex of β-catenin with a T-cell factor (TCF) family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves β-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative in vivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process.


Context: Several lines of recent evidence support a role for chromatin in splicing regulation. Here, we show that splicing can also contribute to histone modification, which implies bidirectional communication between epigenetic mechanisms and RNA processing. Genome-wide analysis of histone methylation in human cell lines and mouse primary T cells reveals that intron-containing genes are preferentially marked with histone H3 Lys36 trimethylation (H3K36me3) relative to intronless genes. In intron-containing genes, H3K36me3 marking is proportional to transcriptional activity, whereas in intronless genes, H3K36me3 is always detected at much lower levels. Furthermore, splicing inhibition impairs recruitment of H3K36 methyltransferase HYPB (also known as Setd2) and reduces H3K36me3, whereas splicing activation has the opposite effect. Moreover, the increase of H3K36me3 correlates with the length of the first intron, consistent with the view that splicing enhances H3 methylation. We propose that splicing is mechanistically coupled to recruitment of HYPB/Setd2 to elongating RNA polymerase II.


Context: Adipokine leptin regulates neuroendocrine circuits that control energy expenditure, thermogenesis and weight loss. However, canonic regulators of leptin secretion, such as insulin and malonyl CoA, do not support these processes. We hypothesize that epiregulin (EREG), a growth factor that is secreted from fibroblasts under thermogenic and cachexia conditions, induces leptin secretion associated with energy dissipation. The effects of EREG on leptin secretion were studied ex vivo, in the intra-abdominal white adipose tissue (iAb WAT) explants, as well as in vivo, in WT mice with diet-induced obesity (DIO) and in ob/ob mice. These mice were pair fed a high-fat diet and treated with intraperitoneal injections of EREG. EREG increased leptin production and secretion in a dose-dependent manner in iAb fat explants via the EGFR/MAPK pathway. After 2 weeks, the plasma leptin concentration was increased by 215% in the EREG-treated group compared to the control DIO group. EREG-treated DIO mice had an increased metabolic rate and core temperature during the active dark cycle and displayed cold-induced thermogenesis. EREG treatment reduced iAb fat mass, the major site of leptin protein production and secretion, but did not reduce the mass of the other fat depots. In the iAb fat, expression of genes supporting mitochondrial oxidation and thermogenesis was increased in EREG-treated mice vs control DIO mice. All metabolic and gene regulation effects of EREG treatment were abolished in leptin-deficient ob/ob mice. Our data revealed a new role of EREG in induction of leptin secretion leading to the energy expenditure state. EREG could be a potential target protein to regulate hypo- and hyperleptinemia, underlying metabolic and immune diseases.


Context: We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C(2)-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91(phox) and p47(phox). C(2)-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47(phox) to the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C(2)-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.


Context: Nivolumab is a programmed death 1 (PD-1) inhibitor currently approved as second-line treatment for advanced renal cell carcinomas (RCC) after failure of standard antiangiogenic treatment. Motzer et al. have recently published in the New England Journal of Medicine the findings of CheckMate 214 trial, using nivolumab and ipilimumab, a cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor, versus sunitinib in previously untreated advanced RCC. The combination demonstrated a higher 18-month overall survival rate of 75% versus 60%, and a higher objective response rate of 42% versus 27%, for the combination in favor over sunitinib monotherapy. These results herald the rapidly changing role of immune checkpoint inhibitor therapy as first-line treatment for metastatic RCC.


Context: Aramchol, an oral stearoyl-coenzyme-A-desaturase-1 inhibitor, has been shown to reduce hepatic fat content in patients with primary nonalcoholic fatty liver disease (NAFLD); however, its effect in patients with human immunodeficiency virus (HIV)-associated NAFLD is unknown. The aramchol for HIV-associated NAFLD and lipodystrophy (ARRIVE) trial was a double-blind, randomized, investigator-initiated, placebo-controlled trial to test the efficacy of 12 weeks of treatment with aramchol versus placebo in HIV-associated NAFLD. Fifty patients with HIV-associated NAFLD, defined by magnetic resonance imaging (MRI)-proton density fat fraction (PDFF) ≥5%, were randomized to receive either aramchol 600 mg daily (n = 25) or placebo (n = 25) for 12 weeks. The primary endpoint was a change in hepatic fat as measured by MRI-PDFF in colocalized regions of interest. Secondary endpoints included changes in liver stiffness using magnetic resonance elastography (MRE) and vibration-controlled transient elastography (VCTE), and exploratory endpoints included changes in total-body fat and muscle depots on dual-energy X-ray absorptiometry (DXA), whole-body MRI, and cardiac MRI. The mean (± standard deviation) of age and body mass index were 48.2 ± 10.3 years and 30.7 ± 4.6 kg/m , respectively. There was no difference in the reduction in mean MRI-PDFF between the aramchol group at -1.3% (baseline MRI-PDFF 15.6% versus end-of-treatment MRI-PDFF 14.4%, P = 0.24) and the placebo group at -1.4% (baseline MRI-PDFF 13.3% versus end-of-treatment MRI-PDFF 11.9%, P = 0.26). There was no difference in the relative decline in mean MRI-PDFF between the aramchol and placebo groups (6.8% versus 1.1%, P = 0.68). There were no differences in MRE-derived and VCTE-derived liver stiffness and whole-body (fat and muscle) composition analysis by MRI or DXA. Compared to baseline, end-of-treatment aminotransferases were lower in the aramchol group but not in the placebo arm. There were no significant adverse events. Conclusion: Aramchol, over a 12-week period, did not reduce hepatic fat or change body fat and muscle composition by using MRI-based assessment in patients with HIV-associated NAFLD (clinicaltrials.gov ID:NCT02684591).


Context: Long non-coding RNAs (lncRNAs) are frequently dysregulated in a variety of human cancers. However, their biological roles in these cancers remain incompletely understood. In this study, we analyze the gene expression profiles of colon cancer tissues and identify a previously unannotated lncRNA, FLJ39051, that we term GSEC (G-quadruplex-forming sequence containing lncRNA), as a lncRNA that is upregulated in colorectal cancer. We further demonstrate that knockdown of GSEC results in the reduction of colon cancer cell motility. We also show that GSEC binds to the DEAH box polypeptide 36 (DHX36) RNA helicase via its G-quadruplex-forming sequence and inhibits DHX36 G-quadruplex unwinding activity. Moreover, knockdown of DHX36 restores the reduced migratory activity of colon cancer cells caused by GSEC knockdown. These results suggest that GSEC plays an important role in colon cancer cell migration by inhibiting the function of DHX36 via its G-quadruplex structure.


Context: Mucin is a glycoprotein that is the primary component of the mucus overlaying the epithelial tissues. Because mucin functions as a first line of the innate immune system, Pseudomonas aeruginosa appears to require interaction with mucin to establish infection in the host. However, the interactions between P. aeruginosa and mucin have been poorly understood. In this study, using in vivo expression technology (IVET), we attempted to identify mucin-inducible promoters that are likely to be involved in the establishment of P. aeruginosa infection. The IVET analysis revealed that the genes encoding glycosidases, sulfatases, and peptidases that are thought to be required for the utilization of mucin as a nutrient are present in 13 genes downstream of the identified promoters. Our results indicated that, among them, sdsA1 encoding a secreted sulfatase plays a central role in the degradation of mucin. It was then demonstrated that disruption of sdsA1 leads to a decreased release of sulfate from mucin and sulfated sugars. Furthermore, the sdsA1 mutant showed a reduction in the ability of mucin gel penetration and an attenuation of virulence in leukopenic mice compared with the wild-type strain. Collectively, these results suggest that SdsA1 plays an important role as a virulence factor of P. aeruginosa.


Context: McLeod syndrome is an X-linked multisystem disorder characterized by abnormalities in the neuromuscular and hematopoietic systems. We have assembled a cosmid contig of 360 kb that encompasses the McLeod gene locus. A 50 kb deletion was detected by screening DNA from patients with radiolabeled whole cosmids, and two transcription units were identified within this deletion. The mRNA expression pattern of one of them, designated as XK, correlates closely to the McLeod phenotype. XK encodes a novel protein with structural characteristics of prokaryotic and eukaryotic membrane transport proteins. Nucleotide sequence analysis of XK from two unrelated McLeod patients has identified point mutations at conserved splice donor and acceptor sites. These findings provide direct evidence that XK is responsible for McLeod syndrome.


Context: B cells that carry the complement receptor (CR+) were separated from B cells that lack the complement receptor (CR-) by velocity sedimentation or by passage through C-coated Sephadex columns. The kinetics of responses to bacterial lipopolysaccharide (LPS) in both B cell subpopulations were determined in three assay procedures: 1) incorporation of radioactive thymidine into DNA; 2) incorporation of radioactive leucine into immunoglobulin; 3) enumeration of cells forming polyclonal antibody to the 2,4,6-trinitrophenyl hapten. Although both subpopulations of B cells responded to LPS, they differed in the time course. CR- B cells responded with a delay of approximately 24 hr as compared with the response of CR+ B cells. The implications to the ontogenetic status of CR+ and CR- B subpopulations are discussed.


Context: Infantile hemangiomas (IH) are common benign tumors in infancy, affecting 5-10% of all infants and they can still cause disfigurement and serious complications depending on their location and size, which can be associated with ulcerations and haemorrhage. Since 2008, propranolol has become the first choice of therapy for complicated IH, compared to conventional approach with systemic corticosteroid therapy as first-line treatment and then interferon or vincristine as second- or third-line therapeutic agents. We report three cases of hemangioma, successfully treated with propranolol. Oral propranolol was given for a period of 6 months with monthly follow up. All cases showed dramatic response without any relapse after stopping the treatment. Propranolol is novel and safe medication for treatment of infantile hemangioma.


Context: The isotretinoin, a 13-cis-retinoic acid, has revolutionized the management of severe treatment-resistant acne and it has been widely used for a range of dermatological conditions, in 90% of the time in young women between 13 and 45 years of age. This agent has severe teratogenic effects, as serious craniofacial, cardiovascular, thymic and central nervous system malformations. The baseline population risk of malformations is 3-5%, but it increases to almost 30% in women exposed to isotretinoin during the first trimester of pregnancy. Generally, patients in treatment with isotretinoin avoid eventual pregnancy during assumption and, after its stopping, fertility and foetal development are normal once circulating isotretinoin levels return to normal. There are no known deleterious effects on male fertility and on long-term teratogenic effect of isotretinoin. In this report, we suppose the possibility to develop a foetal malformations after a long-term wash out from isotretinoin therapy. A 32 year-old healthy nullipara pregnant woman, with an uneventful past gynaecological history, was admitted in Hospital, with a severe depressive syndrome in a 18 weeks malformed pregnancy for thoraco-omphalopagus conjoined twins. She only assumed isotretinoin, at dose of 1 mg/kg a day, for a severe and scarring acne for 7 months. After 3 months of pharmacological wash out, patient become pregnant and manifested this severe malformation. Woman interrupted gestation, by labour induction.


Context: Idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN) syndrome is a disease characterized by multiple retinal macroaneurysms, neuroretinitis and peripheral capillary non-perfusion, leading to irreversible visual loss. It includes five stages and has previously been rarely reported. IRVAN syndrome is especially rare in Asia. In this report, we describe laser diagnosis and therapy in an Asian patient with IRVAN syndrome over two years of follow-up. We observed non-perfusion retina and dilated retinal capillaries in the contralateral eye. Photocoagulation is an effective therapy to control retinal macroaneurysms and nonperfusions and to prevent visual loss, particularly in the early stages of IRVAN syndrome (stages 2 & 3). To the best of our knowledge, this is the first long-term observation of photocoagulation in IRVAN syndrome. We discovered the early signs of such lesions, which may be beneficial for clinical diagnosis and therapy.


Context: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint inflammation, systemic inflammation, and immunological abnormalities. Because cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 play a major role in the development of RA, their targeting could constitute a reasonable novel therapeutic strategy for treating RA. Indeed, worldwide clinical trials of TNF inhibiting biologic disease modifying antirheumatic drugs (bDMARDs) including infliximab, adalimumab, golimumab, certolizumab pegol, and etanercept as well as the humanized anti-human IL-6 receptor antibody, tocilizumab, have demonstrated outstanding clinical efficacy and tolerable safety profiles, resulting in worldwide approval for using these bDMARDs to treat moderate to severe active RA in patients with an inadequate response to synthetic disease modifying antirheumatic drugs (sDMARDs). Although bDMARDs have elicited to a paradigm shift in the treatment of RA due to the prominent efficacy that had not been previously achieved by sDMARDs, a substantial percentage of patients failed primary or secondary responses to bDMARD therapy. Because RA is a heterogeneous disease in which TNF-α and IL-6 play overlapping but distinct pathological roles, further studies are required to determine the best use of TNF inhibitors and tocilizumab in individual RA patients.


Context: Fertilization is fundamental for sexual reproduction, yet its molecular mechanisms are poorly understood. We found that an oocyte-expressed Ly6/uPAR protein, which we call Bouncer, is a crucial fertilization factor in zebrafish. Membrane-bound Bouncer mediates sperm-egg binding and is thus essential for sperm entry into the egg. Remarkably, Bouncer not only is required for sperm-egg interaction but is also sufficient to allow cross-species fertilization between zebrafish and medaka, two fish species that diverged more than 200 million years ago. Our study thus identifies Bouncer as a key determinant of species-specific fertilization in fish. Bouncer's closest homolog in tetrapods, SPACA4, is restricted to the male germline in internally fertilizing vertebrates, which suggests that our findings in fish have relevance to human biology.


Context: Spinal epidural hematoma is a rare complication associated with pain control procedures such as facet block, acupuncture, epidural injection, etc. Although it is an uncommon cause of acute myelopathy, and it may require surgical evacuation. We report four patients with epidural hematoma developed after pain control procedures. Two procedures were facet joint blocks and the others were epidural blocks. Pain was the predominant initial symptom in these patients while two patients presented with post-procedural neurological deficits. Surgical evacuation of the hematoma was performed in two patients while in remaining two patients, surgery was initially recommended but not performed since symptoms were progressively improved. Three patients showed near complete recovery except for one patient who recovered with residual deficits. Although, spinal epidural hematoma is a rare condition, it can lead to serious complications like spinal cord compression. Therefore, it is important to be cautious while performing spinal pain control procedure to avoid such complications. Surgical treatment is an effective option to resolve the spinal epidural hematoma.


Context: Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer-promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here, we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By reanalyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that nonspecific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation.


Context: p53 levels and activity are controlled in large part through regulated ubiquitination and subsequent destruction by the 26S proteasome. Monoubiquitination of p53 is mediated primarily by the RING-finger E3 ubiquitin ligase MDM2 and impacts p53 activity through modulation of p53 localization and transcription activities. Recently, several E4 ubiquitin ligases (E4s) have been identified which serve to extend these monoubiquitin chains. The ubiquitin ligase activity of these factors toward p53, and their contribution to p53 degradation, can be studied using a variety of in vitro and in vivo methods and reagents which will be described in this chapter. These methods include in vivo ubiquitination of p53 using HA-ubiquitin or his-ubiquitin; the in vitro E3 ubiquitin ligase assay, in which ubiquitin reaction components (URC) are incubated with a purified E3 or E4 ligase; a one-step E4 assay, in which URC are incubated with a substrate, E3, and E4; and a two-step E4 assay in which p53 is monoubiquitinated in an E3 reaction, and subsequently purified and incubated with an E4. Finally, we will describe an in vitro degradation assay in which ubiquitinated p53 is incubated with purified 26S proteasomes. Together, these assays can be used to provide insight into the biochemical nature of p53 ubiquitination and degradation.


Context: Hereditary hemochromatosis (HH) is a genetic disorder of iron metabolism that may lead to iron overload. Clinical penetrance is low, however those afflicted may develop cirrhosis, hepatocellular carcinoma, diabetes mellitus, and cardiomyopathy. Treatment of HH involves regular phlebotomy to reduce the systemic iron burden. In many countries-including the United States-numerous blood centers do not accept donated blood obtained from HH patients during therapeutic phlebotomy and there are inconsistent positions regarding this globally. This refusal of blood is borne out of a few concerns. First, there is a theoretical increase in the infectious risk of these blood products, particularly by siderophilic organisms such as Yersinia enterocolitica. Second, given the increased incidence of hepatitis C infection from nonvoluntary donors in the 1970s, there is a concern that blood units from HH donors may harbor additional risk given the nonvoluntary nature of their presentation. In this review, we examine the existing biological and clinical data concerning infectious risk and summarize clinical experience from centers allowing HH donors, and demonstrate that blood from HH patients is safe and should be allowed into the donor pool. We conclude that there is no convincing evidence to exclude this population from serving as blood donors. (Hepatology 2018;67:1150-1157).


Context: A 22 year-old-man with acute lymphoblastic leukaemia had received prophylactic cranial irradiation and intrathecal chemotherapy. Eighteen years later a cerebellar glioblastoma multiforme was diagnosed. The authors speculate about the possibility that this tumor may have been radiation and/or chemotherapy induced. Improvement in neuroimaging techniques, in particular magnetic resonance imaging, has helped characterize Central Nervous System abnormalities, namely secondary brain tumours.


Context: In cell lines established from Marek's disease tumors, several viral transcripts are expressed and among them the products of pp38/pp24 mRNA and 1.8 kb-mRNA have been suggested to be involved in viral oncogenicity. The long inverted repeats of Marek's Disease virus serotype 1 (MDV1) genome contain closely located transcriptional promoters for phosphorylated protein pp38/pp24 and 1.8 kb-mRNA. These promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. We have analyzed the transcription activity of these promoters in MDV1 (Md5 strain) infected CEF by transient expression of CAT reporter genes and found that the promoters were in fact active in infected cells and the promoter for 1.8 kb-mRNA was more active than the pp38/pp24 promoter. Deletion analysis of the short enhancer region revealed that the 30 bp region overlapping the enhancer elements for 1.8 kb-mRNA was important for promoter activity for pp38/pp24. The gel shift analysis revealed that nuclear factor(s) actually bound to the overlapping 30 bp region. In addition, the activity of these promoters in infected cells varied with MDV strains. These results suggest that pp38/pp24 and 1.8 kb-mRNA promoters share a common regulatory sequence but a viral or a cellular factor(s) induced by viral infection regulates the promoter by distinct mechanisms.


Context: Protein tyrosine kinases form a large family of signaling proteins implicated in both normal and malignant cell signaling. The aim of this study was to identify protein tyro-sine kinases that can transform hematopoietic cells to growth factor independent proliferation when constitutively activated by homodimerization. We used a modified retroviral insertion mutagenesis screen with a retroviral vector containing the homodimerization domain of ETV6 followed by an artificial splice donor site. Integration of this retroviral vector within a gene of the host genome would generate a fusion transcript containing the dimerization domain and part of the disrupted gene. Using this strategy with the IL3 dependent Ba/F3 cell line, we identified 8 different protein tyrosine kinases (Abl1, Fgfr1, Hck, Jak2, Lck, Mertk, Mst1r, Tnk1) that transformed the cells. These results characterize HCK, MERTK, MST1R and TNK1 as potential oncogenes and describe a method to identify gain-of-function fusion genes using a retroviral insertion screen.


Context: Facioscapulohumeral muscular dystrophy(FSHD) is an autosomal dominant neuromuscular disorder characterized by progressive weakness of the facial, shoulder and upper arm muscles. The major gene involved has been mapped to chromosome 4q35. There is the evidence for genetic heterogeneity. The FSHD- associated DNA rearrangements are due to deletions of integral copies of the 3.3 kb tandem repeated unit from the subtelomeric region on chromosome 4q35. A valuable molecular diagnostic test for FSHD has been created with the use of p13E-11 probe to detect the EcoR I/Bln I double digestion fragment which is usually smaller in FSHD patient than in normal indivdual. Since the FSHD gene has not been identified yet, the exact molecular pathogenesis of FSHD remains unclear. The hypothesis of position effect variegation has been postulated as the underlying genetic mechanism of FSHD. FRG1 (FSHD region gene 1) from human chromosome 4q35 is identified as a candidate gene for FSHD. A significant correlation between the size of rearrangements associated with FSHD and the clinical phenotype has been found. The various rearrangement fragment size may explain the wide range of clinical severity in FSHD.


Context: ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.


Context: Kearns Sayre Syndrome (KSS) is a rare mitochondrial disease characterized by a primary dysfunction of the mitochondrial respiratory chain. Cardiac involvement is a poor prognostic factor of KSS. Pregnancy and delivery in a KSS patient with cardiac involvement is uncommon, and strategies for the supervision and management of this group remain unclear. Herein, we report and discuss pregnancy and delivery complicated with acute cardiopulmonary failure in a woman with KSS.


Context: Although immunotherapies have been employed for many decades, immune checkpoint inhibitors have only recently entered the oncologic landscape. Avelumab is a fully human monoclonal antibody that blocks the interaction between PD-L1 on tumor cells and PD-1 on T cells, thereby inhibiting immunosuppression in the tumor microenvironment and reducing tumor growth. Most early clinical trials of avelumab as monotherapy and in combination regimens were part of the international JAVELIN clinical trial program, which included more than 7000 patients in more than 30 trials with at least 15 tumor types. Avelumab has been approved by the U.S. FDA for the treatment of metastatic Merkel cell carcinoma and metastatic urothelial carcinoma that has progressed during or following treatment with a platinum-based regimen. Its acceptable safety profile and ability to induce durable responses in otherwise deadly tumors provide the rationale for its use in other tumor types and in combination with other therapies.


Context: The transcriptional activity of an in vitro assembled human interferon-beta gene enhanceosome is highly synergistic. This synergy requires five distinct transcriptional activator proteins (ATF2/c-JUN, interferon regulatory factor 1, and p50/p65 of NF-kappaB), the high mobility group protein HMG I(Y), and the correct alignment of protein-binding sites on the face of the DNA double helix. Here, we investigate the mechanisms of enhanceosome-dependent transcriptional synergy during preinitiation complex assembly in vitro. We show that the stereospecific assembly of the enhanceosome is critical for the efficient recruitment of TFIIB into a template-committed TFIID-TFIIA-USA (upstream stimulatory activity complex) and for the subsequent recruitment of the RNA polymerase II holoenzyme complex. In addition, we provide evidence that recruitment of the holoenzyme by the enhanceosome is due, at least in part, to interactions between the enhanceosome and the transcriptional coactivator CREB, cAMP responsive element binding protein (CBP). These studies reveal a unique role of enhanceosomes in the cooperative assembly of the transcription machinery on the human interferon-beta promoter.


Context: The pleiomorphic adenoma gene 1 (PLAG1) gene is activated in a subset of pleomorphic adenomas of the salivary gland by gene fusion. Germ‑line mutation in cylindromatosis (CYLD), a tumor suppressor gene, causes familial cylindromatosis and Brook‑Spiegler syndrome. In the present study, aberrations in PLAG1 and CYLD were investigated in adenoid cystic carcinoma (ACC) of the salivary gland. Reverse‑transcription PCR and PCR direct sequencing were performed to detect gene fusion of PLAG1 and mutation of CYLD in 34 ACC tissues. No PLAG1 fusion was detected in ACC. However, silent mutation of CYLD was detected in 2 cases of ACC, but no missense mutation was detected in ACC. These results suggest that PLAG1 and CYLD do not play a role in ACC tumorigenesis.


Context: The components of sweat provide an array of potential biomarkers for health and disease. Sweat chloride is of interest as a biomarker for cystic fibrosis, electrolyte metabolism disorders, electrolyte balance, and electrolyte loss during exercise. Developing wearable sensors for biomarkers in sweat is a major technological challenge. Potentiometric sensors provide a relatively simple technology for on-body sweat chloride measurement, however, equilibration between reference and test solutions has limited the time over which accurate measurements can be made. Here, we report on a wearable potentiometric chloride sweat sensor. We performed parametric studies to show how the salt bridge geometry determines equilibration between the reference and test solutions. From these results, we show a sweat chloride sensor can be designed to provide accurate measurements over extended times. We then performed on-body tests on healthy subjects while exercising to establish the feasibility of using this technology as a wearable device.


Context: DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through its influence on gene expression. DNA methyltransferase 1 (Dnmt1) is the enzyme responsible for maintaining the methylation marks through cell division. However, the de novo methyltransferases, Dnmt3a and Dnmt3b, can also contribute to the maintenance of the methylation pattern. Manipulation of these enzymes, especially Dnmt1, provides a means to alter DNA methylation levels. Manipulation of the DNA methylation pattern of somatic cells will allow a better understanding of the different molecular process associated with chromatin structure and gene expression. Different approaches to artificially manipulate the expression of Dnmt1 in somatic cells include the addition of 5-azacytidine, culture of cells for an extended period of time, and the use of small interfering RNA technologies.


Context: Pheochromocytoma is a rare catecholamine secreting neuroendocrine tumor with an estimated annual incidence of one to four per million and prevalence among hypertensive patients of 0.1 to 0.6%. The symptoms and signs of pheochromocytoma include the classic triad of episodic headache, increased sweating, and palpitations. These are as a result of an uncontrolled release of catecholamines. There exist only a small number of reports of pheochromocytoma simulating hypertrophic obstructive cardiomyopathy, few reports of pheochromocytoma-induced ischemic stroke and only two reported cases with pheochromocytoma-induced arterial thrombosis. We present a case of multiple, rare clinical complications of pheochromocytoma occurring in the same patient and the review of literature of these complications.


Context: We report a case of recurrent Zieve's syndrome, consisting of jaundice, hemolytic anemia and hyperlipoproteinemia, initiated by alcohol abuse. The crucial feature of this syndrome, which allows differentiation with common acute alcoholic hepatitis, is the presence of hemolysis. There is no specific treatment, and restraining from alcohol consumption remains the basic prophylactic measure.


Context: In multiple sclerosis (MS), B cell-depleting therapy using monoclonal anti-CD20 Abs, including rituximab (RTX) and ocrelizumab, effectively reduces disease activity. Based on indirect evidence, it is generally believed that elimination of the Ag-presenting capabilities and Ag nonspecific immune functions of B cells underlie the therapeutic efficacy. However, a small subset of T lymphocytes (T cells) was shown to also express CD20, but controversy prevails surrounding the true existence of this T cell subpopulation. Using single-cell imaging flow cytometry and expression profiling of sorted lymphocyte subsets, we unequivocally demonstrate the existence of CD3(+)CD20(dim) T cells. We show that in MS patients, increased levels of CD3(+)CD20(dim) T cells are effectively depleted by RTX. The pathological relevance of this T cell subset in MS remains to be determined. However, given their potential proinflammatory functionality, depletion of CD20-expressing T cells may also contribute to the therapeutic effect of RTX and other mAbs targeting CD20.


Context: The cohesin protein complex holds sister chromatids together to ensure proper chromosome segregation upon cell division and also regulates gene transcription. Partial loss of the Nipped-B protein that loads cohesin onto chromosomes, or the Pds5 protein required for sister chromatid cohesion, alters gene expression and organism development, without affecting chromosome segregation. Knowing if a reduced Nipped-B or Pds5 dosage changes how much cohesin binds chromosomes, or the stability with which it binds, is critical information for understanding how cohesin regulates transcription. We addressed this question by in vivo fluorescence recovery after photobleaching (FRAP) with Drosophila salivary glands. Cohesin, Nipped-B, and Pds5 all bind chromosomes in both weak and stable modes, with residence half-lives of some 20 seconds and 6 min, respectively. Reducing the Nipped-B dosage decreases the amount of stable cohesin without affecting its chromosomal residence time, and reducing the Pds5 dosage increases the amount of stable cohesin. This argues that Nipped-B and Pds5 regulate transcription by controlling how much cohesin binds DNA in the stable mode, and not binding affinity. We also found that Nipped-B, Pds5, and the Wapl protein that interacts with Pds5 all play unique roles in cohesin chromosome binding.


Context: Genetic disease transmission has been identified in a significant proportion of patients with dilated cardiomyopathy (DCM). Variable clinical characteristics and patterns of inheritance, as well as recent molecular genetic data, indicate the existence of several genes causing the disease. Several distinct subtypes of familial DCM have been identified. Autosomal dominant DCM is the most frequent form (56% of our cases), and several candidate disease loci have been identified by linkage analysis. Three disease genes are presently known: the cardiac actin gene, the desmin gene, and the lamin A/C gene. This latter gene has recently been found to be responsible for both the autosomal dominant form of DCM with subclinical skeletal muscle disease (7.7% of cases) and the familial form with conduction defects (2.6% of cases) or the autosomal dominant variant of Emery-Dreifuss muscular dystrophy. The autosomal recessive form of DCM accounts for 16% of cases and is characterized by a worse prognosis. An X-linked form of DCM (10% of cases) manifests in the adult population and is due to mutations in the dystrophin gene. In the rare infantile form of DCM, mutations in the G4.5 gene have been identified. Finally, some of the rare unclassifiable forms (7.7% of cases) may be due to mitochondrial DNA mutations. Clinical and experimental evidence based on animal models suggest that, in a large number of cases, DCMs are diseases of the cytoskeleton. However, other causes, such as alterations in regulatory elements and in signaling molecules, are possible. Moreover, other genes called modifier genes can influence the severity, penetrance, and expression of the disease, and they will be a main objective of future investigations. Familial DCM is frequent, cannot be predicted on a clinical or morphological basis and requires family screening for identification. The advances in the genetics of familial DCM can allow improved diagnosis, prevention and genetic counseling, and represent the basis for the development of new therapies.


Context: Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans.


Context: The limited number of progenitor stem cells in umbilical cord blood (UCB) enforces the optimization and strict control of all the procedures involved in its therapeutic use--ie, collection, processing, cryopreservation, thawing, and transportation--to ensure graft potency at transplantation. For this reason, international UCB standards recommend storage of a cell sample attached to the UCB unit as a quantitative and functional control of the unit selected for transplantation. To validate the use of the sample attached to the UCB unit as a quality-control tool for the final product, UCB units (n = 20) stored in liquid nitrogen with the Bioarchive system were analyzed. The UCB units and their attached segments were thawed, and the number and viability of total nucleated cells, mononucleated cells, CD45 + cells, and CD34+ cells were determined, as were colony-forming cell counts. There was no significant difference between UCB units and segments for any of the parameters assessed. Additionally, the linear correlation coefficient (R2) in these paired samples was 0.85 and 0.78 for CD34+ cells and colony-forming cells, respectively. In conclusion, the cell sample in the tube segment physically linked to the transplant UCB bag predicts the total cell content and functionality of the unit and may serve as a source for final quality control of the UCB unit before transplantation.


Context: 5-HT1A autoreceptor stimulation can act to attenuate supraphysiological swings in extracellular dopamine levels following long-term levodopa treatment and may be useful in the treatment and prevention of the motor complications. The purpose of this study was to investigate cellular and behavioral effects of 5-HT1A receptor agonist 8-OH-DPAT in a rat model of levodopa-induced motor complications. Two sets of experiments were performed. First, animals were treated with levodopa (50 mg/kg with benserazide 12.5 mg/kg, twice daily), intraperitoneally (i.p.) for 22 days. On day 23, animals received either 8-OH-DPAT (1 mg/kg, i.p.) or 8-OH-DPAT plus WAY-100635 (0.1 mg/kg, i.p) or vehicle with each levodopa dose. In the second set, animals were treated either with levodopa (50 mg/kg, i.p.) plus 8-OH-DPAT (1 mg/kg, i.p.) or levodopa (50 mg/kg, i.p.) plus vehicle, administered twice daily for 22 consecutive days. Our study showed that 8-OH-DPAT plus levodopa both prolonged the duration of the motor response and reduced peak turning. 8-OH-DPAT plus levodopa also decreased the frequency of failures to levodopa. Co-administration of WAY-100635, a 5-HT1A receptor antagonist, with 8-OH-DPAT eliminated the effect of 8-OH-DPAT on motor complications indicating that the observed 8-OH-DPAT responses were probably mediated at the 5-HT1A autoreceptor. Moreover, 8-OH-DPAT plus levodopa significantly reduced hyperphosphorylation of GluR1 at serine 845, which was closely associated with levodopa-induced motor complications.


Context: Chronic Myeloid Leukemia (CML) is myeloproliferative neoplasm characterized by Philadelphia chromosome which is a balanced translocation between chromosome 9 and 22 in 90% of cases. However, variant cytogenetic still happens in 5-10 % of cases, the importance of which is controversial as well as its response to therapy, prognosis and progression to acute leukemias. Here we report a male patient with CML and variant cytogenetic who responded to low dose of Dasatinib (50 mg daily).


Context: Selective estrogen receptor modulators (SERMs) bind to estrogen receptor (ER) and develop tissue-selective actions as estrogen agonists or antagonists. As such, SERMs have been developed to exert estrogen-like beneficial effects against some disorders including osteoporosis, while reducing estrogen-related risks, including breast cancer. Prevention of vertebral fractures by a SERM, raloxifene (RLX), in osteoporotic postmenopausal women has been well established. RLX does not increase or decrease cardiovascular events, overall mortality, cardiovascular mortality or the overall number of strokes, but there appears to be a small increase in stroke mortality. Both RLX and tamoxifen similarly reduce the risk of ER-positive invasive breast cancer. At the same time, RLX treatment is associated with 36% fewer uterine cancer incidence and 29% less thromboembolic events. Keeping these results in mind, it is our responsibility to critically evaluate and decide timing and length of treatment, as well as subjects with benefits or risks for the treatment of osteoporosis by SERMs.


Context: TNFalpha plays a pivotal role not only in the inflammatory process but also in the normal response against pathogens and therefore, interfering with this cytokine may increase the risk of infection. TNFalpha antagonists are commonly used in daily clinical practice for the treatment of inflammatory rheumatic diseases including rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and juvenile idiopathic arthritis since the beginning of 2000. The spectrum of pathogens giving infectious disease in patients under anti-TNFalpha therapies ranges from common bacteria to more opportunistic organisms such as Mycobacterium tuberculosis. The infections which were described with TNFalpha inhibitors may have a benign course or may be a serious, life threatening disease, and may be localized or disseminated. These TNFalpha inhibitors related infections were described in the randomized clinical trials, and were then declared to post-marketing surveillance systems and special registries. Tuberculosis (TB) is the most frequent opportunistic infection which has been reported with TNFalpha antagonists and the highest risk appears to be associated with infliximab, and at a lesser extent with etanercept. Currently available data and recent patents on the risk of TB with adalimumab are not sufficient to conclude, but TB cases were also reported with this agent. The description of TB infections with TNFalpha inhibitors led to the establishment of new guidelines for screening patients at high risk of developing TB. These data highlight the importance of post-marketing surveillance and special registries for accurately evaluating the safety profile and particularly the infectious risk of this very effective class of drug in inflammatory rheumatic diseases.


Context: How long does prior SARS-CoV-2 infection provide protection against SARS-CoV-2 reinfection? Among >22 million individuals tested February 2020 through April 2021, the relative risk of reinfection among those with prior infection was 87% lower than the risk of infection among individuals without prior infection. This protection was durable for up to a year. Factors associated with increased likelihood of reinfection included older age (85+ years), comorbid immunologic conditions, and living in congregate care settings; healthcare workers had lower risk. Prior SARS-CoV-2 infection provides a durable, high relative degree of protection against reinfection.


Context: MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.


Context: Safinamide (Xadago™) is an oral α-aminoamide derivative marketed for the treatment of Parkinson's disease (PD). The drug has both dopaminergic properties, namely highly selective and reversible inhibition of monoamine oxidase B, and nondopamimetic properties, namely selective sodium channel blockade and calcium channel modulation, with consequent inhibition of excessive glutamate release. In 2014, safinamide was approved in the European Economic Area, as "an add-on therapy to stable dose levodopa, alone or in combination with other PD therapies in mid- to late-stage-fluctuating PD patients." In addition, evidence has been provided for safinamide in the treatment of motor symptoms in early PD patients. This article summarizes the pharmacological properties, development program, clinical indications for PD treatment, stratified according to several disease's stages and the safety profile of safinamide. A meta-analysis of the most frequent adverse events among Phase III trials has been also performed.


Context: Since the onset of the COVID-19 pandemic, there has been an advisory for regular and thorough cleaning of hands besides other measures such as social distancing and self-isolation. The rationale for the same is to prevent the transfer of the virus from hands that have come in contact with fomites. While both alcohol-based hand rubs (ABHR) or washing with soap and water are claimed to have been effective, hand sanitizers have gained more popularity due to the ease of use. The increased frequency of ABHR use and the aerosols generated pose a potential threat to the skin and exposed mucosal surfaces, especially that of the eye due to the proximity of use. The adverse effects of alcohol in these sanitizers can be manifold. An allergic or inflammatory response can occur depending on the predisposing or preexisting conditions. This article describes the risks, underlying mechanisms, and preventive measures for sanitizer aerosol-driven ocular surface disease.


Context: Gene looping juxtaposes the promoter and terminator regions of RNA polymerase II-transcribed genes in yeast and mammalian cells. Here we report an activator-dependent interaction of transcription initiation and termination factors during gene looping in budding yeast. Chromatin analysis revealed that MET16, INO1, and GAL1p-BUD3 are in a stable looped configuration during activated transcription. Looping was nearly abolished in the absence of transcription activators Met28, Ino2, and Gal4 of MET16, INO1, and GAL1p-BUD3 genes, respectively. The activator-independent increase in transcription was not accompanied by loop formation, thereby suggesting an essential role for activators in gene looping. The activators did not facilitate loop formation directly because they did not exhibit an interaction with the 3' end of the genes. Instead, activators physically interacted with the general transcription factor TFIIB when the genes were activated and in a looped configuration. TFIIB cross-linked to both the promoter and the terminator regions during the transcriptionally activated state of a gene. The presence of TFIIB on the terminator was dependent on the Rna15 component of CF1 3' end processing complex. Coimmunoprecipitation revealed a physical interaction of Rna15 with TFIIB. We propose that the activators facilitate gene looping through their interaction with TFIIB during transcriptional activation of genes.


Context: The causative mutations for familial Mediterranean fever (FMF) are located in the MEFV gene, which encodes pyrin. Pyrin modulates the susceptibility to apoptosis via its PYD domain, but how the mutated versions of pyrin affect apoptotic processes are poorly understood. Spontaneous and induced rates of systemic neutrophil apoptosis as well as the levels of proteins involved in apoptosis were investigated ex vivo in patients with FMF using flow cytometry and RT-qPCR. The freshly collected neutrophils from the patients in FMF remission displayed a significantly larger number of cells spontaneously entering apoptosis compared to control (6.27 ± 2.14 vs. 1.69 ± 0.18%). This elevated ratio was retained after 24 h incubation of neutrophils in the growth medium (32.4 ± 7.41 vs. 7.65 ± 1.32%). Correspondingly, the mRNA level for caspase-3 was also significantly increased under these conditions. In response to the inducing agents, the neutrophils from FMF patients also displayed significantly elevated apoptotic rates compared to control. The elevated rates, however, can be largely explained by the higher basal ratio of apoptotic cells in the former group. Monitoring of several proteins involved in apoptosis has not revealed any conventional mechanisms contributing to the enhanced apoptotic rate of neutrophils in FMF. Although the exact molecular mechanisms of accelerated neutrophil apoptosis in FMF remain unknown, it may provide a protection against excessive inflammation and tissue damage due to a massive infiltration of neutrophils in the acute period of the disease.


Context: The polyneuropathy of juvenile Greyhound show dogs shows clinical similarities to the genetically heterogeneous Charcot-Marie-Tooth (CMT) disease in humans. The pedigrees containing affected dogs suggest monogenic autosomal recessive inheritance and all affected dogs trace back to a single male. Here, we studied the neuropathology of this disease and identified a candidate causative mutation. Peripheral nerve biopsies from affected dogs were examined using semi-thin histology, nerve fibre teasing and electron microscopy. A severe chronic progressive mixed polyneuropathy was observed. Seven affected and 17 related control dogs were genotyped on the 50k canine SNP chip. This allowed us to localize the causative mutation to a 19.5 Mb interval on chromosome 13 by homozygosity mapping. The NDRG1 gene is located within this interval and NDRG1 mutations have been shown to cause hereditary motor and sensory neuropathy-Lom in humans (CMT4D). Therefore, we considered NDRG1 a positional and functional candidate gene and performed mutation analysis in affected and control Greyhounds. A 10 bp deletion in canine NDRG1 exon 15 (c.1080_1089delTCGCCTGGAC) was perfectly associated with the polyneuropathy phenotype of Greyhound show dogs. The deletion causes a frame shift (p.Arg361SerfsX60) which alters several amino acids before a stop codon is encountered. A reduced level of NDRG1 transcript could be detected by RT-PCR. Western blot analysis demonstrated an absence of NDRG1 protein in peripheral nerve biopsy of an affected Greyhound. We thus have identified a candidate causative mutation for polyneuropathy in Greyhounds and identified the first genetically characterized canine CMT model which offers an opportunity to gain further insights into the pathobiology and therapy of human NDRG1 associated CMT disease. Selection against this mutation can now be used to eliminate polyneuropathy from Greyhound show dogs.


Context: Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT), and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we define for the first time the composition of the human cytoplasmic dynein-2 complex. We show that the proteins encoded by the ciliopathy genes WDR34 and WDR60 are bona fide dynein-2 intermediate chains and are both required for dynein-2 function. In addition, we identify TCTEX1D2 as a unique dynein-2 light chain that is itself required for cilia function. We define several subunits common to both dynein-1 and dynein-2, including TCTEX-1 (also known as DYNLT1) and TCTEX-3 (also known as DYNLT3), roadblock-1 (also known as DYNLRB1) and roadblock-2 (also known as DYNLRB2), and LC8-1 and LC8-2 light chains (DYNLL1 and DYNLL2, respectively). We also find that NudCD3 associates with dynein-2 as it does with dynein-1. By contrast, the common dynein-1 regulators dynactin, LIS1 (also known as PAFAH1B1) and BICD2 are not found in association with dynein-2. These data explain why mutations in either WDR34 or WDR60 cause disease, as well as identifying TCTEX1D2 as a candidate ciliopathy gene.


Context: The sarcoplasmic reticulum (SR) plays a critical role in excitation-contraction coupling by regulating the cytoplasmic calcium concentration of striated muscle. The histidine-rich calcium-binding protein (HRCBP) is expressed in the junctional SR, the site of calcium release from the SR. HRCBP is expressed exclusively in muscle tissues and binds calcium with low affinity and high capacity. In addition, HRCBP interacts with triadin, a protein associated with the ryanodine receptor and thought to be involved in calcium release. Its calcium binding properties, localization to the SR, and interaction with triadin suggest that HRCBP is involved in calcium handling by the SR. To determine the function of HRCBP in vivo, we inactivated HRC, the gene encoding HRCBP, in mice. HRC knockout mice exhibited impaired weight gain beginning at 11 months of age, which was marked by reduced skeletal muscle and fat mass, and triadin protein expression was upregulated in the heart of HRC knockout mice. In addition, HRC null mice displayed a significantly exaggerated response to the induction of cardiac hypertrophy by isoproterenol compared to their wild-type littermates. The exaggerated response of HRC knockout mice to the induction of cardiac hypertrophy is consistent with a regulatory role for HRCBP in calcium handling in vivo and suggests that mutations in HRC, in combination with other genetic or environmental factors, might contribute to pathological hypertrophy and heart failure.


Context: Specific outcomes upon activation of the c-Jun N-terminal kinase (JNK) pathway critically depend on the intensity and duration of signal transmission. Dual-specificity phosphatases (DUSPs) play a very important role in these events by modulating the extent of JNK phosphorylation and activation and thus regulating cellular responses to stress. M3/6 (DUSP8) is one of the dual-specificity protein phosphatases with distinct specificity towards JNK. It has been shown that M3/6 itself is phosphorylated by JNK upon stimulation with arsenite, but the role of this phosphorylation has not been investigated. In this study, we mapped JNK-induced phosphorylation sites on M3/6 using mass spectrometry. Phosphorylated residues Ser 515, Thr 518 and Ser 520 were identified and site-directed mutagenesis was employed to investigate their role. Upon arsenite stimulation, M3/6 mutated at these sites exhibited decreased phosphorylation compared to the wild-type protein. No difference was observed in terms of the enzyme's in vitro phosphatase activity, its substrate specificity towards JNK isoforms, its interactions with JNK and the scaffold family of JNK-interacting proteins (JIPs), its stability or its subcellular localization. Interestingly, expression of M3/6 phosphorylation mutants delayed the time-course of JNK phosphorylation and activation by arsenite. We propose that phosphorylation of the M3/6 phosphatase by JNK in response to stress stimuli results in attenuation of phosphatase activity and acceleration of JNK activation.


Context: Predictive modeling with electronic health record (EHR) data is anticipated to drive personalized medicine and improve healthcare quality. Constructing predictive statistical models typically requires extraction of curated predictor variables from normalized EHR data, a labor-intensive process that discards the vast majority of information in each patient's record. We propose a representation of patients' entire raw EHR records based on the Fast Healthcare Interoperability Resources (FHIR) format. We demonstrate that deep learning methods using this representation are capable of accurately predicting multiple medical events from multiple centers without site-specific data harmonization. We validated our approach using de-identified EHR data from two US academic medical centers with 216,221 adult patients hospitalized for at least 24 h. In the sequential format we propose, this volume of EHR data unrolled into a total of 46,864,534,945 data points, including clinical notes. Deep learning models achieved high accuracy for tasks such as predicting: in-hospital mortality (area under the receiver operator curve [AUROC] across sites 0.93-0.94), 30-day unplanned readmission (AUROC 0.75-0.76), prolonged length of stay (AUROC 0.85-0.86), and all of a patient's final discharge diagnoses (frequency-weighted AUROC 0.90). These models outperformed traditional, clinically-used predictive models in all cases. We believe that this approach can be used to create accurate and scalable predictions for a variety of clinical scenarios. In a case study of a particular prediction, we demonstrate that neural networks can be used to identify relevant information from the patient's chart.


Context: The theory of positive deviance holds that in any group, some people are more effective than others even when they have the same resources at hand. The object is to identify those people, see what they're doing differently and share it with the larger group. The Plexus Institute and the Robert Wood Johnson Foundation recently collaborated on a six-hospital positive-deviance study that reduced methicillin-resistant Staphylococcus aureus (MRSA) infection rates by 70 percent. What these hospitals learned may help others cut their MRSA rates.


Context: The balanced growth of a cell requires the integration of major systems such as DNA replication, membrane biosynthesis, and ribosome formation. An example of such integration is evident from our recent finding that, in Saccharomyces cerevisiae, any failure in the secretory pathway leads to severe repression of transcription of both rRNA and ribosomal protein genes. We have attempted to determine the regulatory circuit(s) that connects the secretory pathway with the transcription of ribosomal genes. Experiments show that repression does not occur through the circuit that responds to misfolded proteins in the endoplasmic reticulum, nor does it occur through circuits known to regulate ribosome synthesis, e.g. the stringent response, or the cAMP pathway. Rather, it appears to depend on a stress response at the plasma membrane that is transduced through protein kinase C (PKC). Deletion of PKC1 relieves the repression of both ribosomal protein and rRNA genes that occurs in response to a defect in the secretory pathway. We propose that failure of the secretory pathway prevents the synthesis of new plasma membrane. As protein synthesis continues, stress develops in the plasma membrane. This stress is monitored by Pkc1p, which initiates a signal transduction pathway that leads to repression of transcription of the rRNA and ribosomal protein genes. The importance of the transcription of the 137 ribosomal protein genes to the economy of the cell is apparent from the existence of at least three distinct pathways that can effect the repression of this set of genes.


Context: Retinitis pigmentosa (RP) is a disease that primarily affects the peripheral retina and ultimately causes visual impairment. X-chromosomal forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We show that the novel splice donor site (SDS) mutation c.1245+3A>T in intron 10 of RPGR cosegregates with RP in a five-generation Caucasian family. The mutation causes in-frame skipping of exon 10 from RPGR transcripts in patient-derived primary fibroblasts. To correct the splice defect, we developed a gene therapeutic approach using mutation-adapted U1 small nuclear RNA (U1). U1 is required for SDS recognition of pre-mRNAs and initiates the splice process. The mutation described herein interferes with the recognition of the SDS by U1. To overcome the deleterious effects of the mutation, we generated four U1 isoforms with increasing complementarity to the SDS. Lentiviral particles were used to transduce patient-derived fibroblasts with these U1 variants. Full complementarity of U1 corrects the splice defect partially and increases recognition of the mutant SDS. The therapeutic effect is U1-concentration dependent as we show for endogenously expressed RPGR transcripts in patient-derived cells. U1-based gene therapeutic approaches constitute promising technologies to treat SDS mutations in inherited diseases including X-linked RP.


Context: The c-Myc oncoprotein is overexpressed in many tumors and is essential for maintaining the proliferation of transformed cells. To function as a transcription factor, c-Myc must dimerize with Max via the basic helix-loop-helix leucine zipper protein (bHLH-ZIP) domains in each protein. The small molecule 7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (10074-G5) binds to and distorts the bHLH-ZIP domain of c-Myc, thereby inhibiting c-Myc/Max heterodimer formation and inhibiting its transcriptional activity. We report in vitro cytotoxicity and in vivo efficacy, pharmacodynamics, pharmacokinetics, and metabolism of 10074-G5 in human xenograft-bearing mice. In vitro, 10074-G5 inhibited the growth of Daudi Burkitt's lymphoma cells and disrupted c-Myc/Max dimerization. 10074-G5 had no effect on the growth of Daudi xenografts in C.B-17 SCID mice that were treated with 20 mg/kg 10074-G5 intravenously for 5 consecutive days. Inhibition of c-Myc/Max dimerization in Daudi xenografts was not seen 2 or 24 h after treatment. Concentrations of 10074-G5 in various matrices were determined by high-performance liquid chromatography-UV, and metabolites of 10074-G5 were identified by liquid chromatography/tandem mass spectrometry. The plasma half-life of 10074-G5 in mice treated with 20 mg/kg i.v. was 37 min, and peak plasma concentration was 58 μM, which was 10-fold higher than peak tumor concentration. The lack of antitumor activity probably was caused by the rapid metabolism of 10074-G5 to inactive metabolites, resulting in tumor concentrations of 10074-G5 insufficient to inhibit c-Myc/Max dimerization. Our identification of 10074-G5 metabolites in mice will help design new, more metabolically stable small-molecule inhibitors of c-Myc.