Context: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by a mutation in the G6PD gene on Xq28. Herein, we describe a Korean boy with G6PD deficiency resulting from a novel mutation in G6PD. A 20-month-old boy with hemolytic anemia was referred for molecular diagnosis. He had no relevant family history. The G6PD activity was severely decreased at 0.2 U/g Hb (severe deficiency). Direct sequencing analyses on the G6PD gene revealed that he was hemizygous for a novel missense variant, c.1187C>G (p.Pro396Arg), in exon 10 of G6PD. Family study involving his parents revealed the de novo occurrence of the mutation. This is the first report of genetically confirmed G6PD deficiency in Korea.


Context: Hutchinson-Gilford progeria syndrome (HGPS) is an autosomal-dominant genetic disease that leads to accelerated aging and often premature death caused by cardiovascular complications. Till now clinical management of HGPS has largely relied on the treatment of manifestations and on the prevention of secondary complications, cure for the disease has not yet been established. Addressing this need cannot only benefit progeria patients but may also provide insights into intervention design for combating physiological aging. By using the systematic review approach, this article revisits the overall progress in the development of strategies for HGPS treatment over the last ten years, from 2010 to 2019. In total, 1,906 articles have been retrieved, of which 56 studies have been included for further analysis. Based on the articles analyzed, the trends in the use of different HGPS models, along with the prevalence, efficiency, and limitations of different reported treatment strategies, have been examined. Emerging strategies for preclinical studies, and possible targets for intervention development, have also been presented as avenues for future research.


Context: Lung cancer is the leading cause of cancer-related death in the world. Prior to the era of targeted therapy, platinum-based doublet chemotherapy was the first-line therapy of choice for patients with metastatic non-small-cell lung cancer (NSCLC). The availability of agents that target epidermal growth factor receptor (EGFR)-tyrosine kinase, as well as inhibitors against anaplastic lymphoma kinase (ALK) gene rearrangement or ROS-1 gene rearrangement product, has provided promising clinical benefits in specific subpopulations of NSCLC. At present, only first-generation EGFR-tyrosine kinase inhibitors (TKIs) (erlotinib and gefitinib) are available for clinical use. Second-generation irreversible EGFR-TKIs, such as afatinib, are still in clinical trials. In current clinical practice, EGFR-TKI is the first-line treatment of choice for metastatic NSCLC patients with tumor EGFR mutation or as salvage therapy in NSCLC patients who received systemic chemotherapy previously. Platinum-based doublet chemotherapy continues to be the standard of care for those treatment-naïve patients with EGFR wild -type tumor or unknown EGFR status. Even though all investigators agree with the use of EGFR-TKI as the first-line treatment in tumor EGFR-mutated patients, only 10-30% of NSCLC patients have mutated EGFR, and there was no obvious survival difference when EGFR-TKIs were used in a second-line setting versus a first-line treatment in EGFR-mutated patients. Thus, the molecular complexity of lung cancer emphasizes the need for optimizing treatment by seeking a more personalized approach to care, including searching for driver oncogenes, managing the emergence of resistance and overcoming that resistance, and optimizing the sequence of treatment. Numerous other novel targeted agents are now in clinical development, including new agents targeting novel pathways and those that may have the potential to overcome the limitations or resistance associated with currently available EGFR-TKIs. In this report, we review the clinical data of EGFR-TKIs as molecular-targeted therapies in NSCLC.


Context: Cellular inhibitor of apoptosis proteins (cIAPs) have emerged as important anti-cell death mediators, particularly in cancer. Although they are known to be expressed in immune tissue, their specific immune function remains unclear. We observed that degradation of cIAPs with SMAC mimetic (SM) results in death of primary bone-marrow-derived macrophages. SM-induced death of macrophages occurred by programmed necrosis (necroptosis), which was dependent on TNF receptor expression. Consistent with necroptosis, SM-induced death of macrophages was abrogated by inhibition of receptor interacting protein 1 (Rip1) kinase signaling or by receptor interacting protein 3 (Rip3) knockdown. SM-induced necroptosis was also dependent on inhibition of SM-induced apoptosis due to the expression of the endogenous caspase inhibitor, xIAP. We found that cIAPs limit Rip3, and to a lesser extent Rip1, expression via post-transcriptional mechanisms, leading to inhibition of the Rip1-Rip3 death complex (necrosome). Reduced cIAP activity in vivo, via SM treatment or specific knockout of either cIAP, resulted in elevated macrophage cell death and compromised control of an intracellular bacterium, Listeria monocytogenes. These results show that cIAPs have an important role in limiting programmed necrosis of macrophages, which facilitates effective control of a pathogen.


Context: The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish.


Context: Ozanimod (RPC1063) is a specific and potent small molecule modulator of the sphingosine 1-phosphate receptor 1 (S1PR1) and receptor 5 (S1PR5), which has shown therapeutic benefit in clinical trials of relapsing multiple sclerosis and ulcerative colitis. Ozanimod and its active metabolite, RP-101075, exhibit a similar specificity profile at the S1P receptor family in vitro and pharmacodynamic profile in vivo. The NZBWF1 mouse model was used in therapeutic dosing mode to assess the potential benefit of ozanimod and RP-101075 in an established animal model of systemic lupus erythematosus. Compared with vehicle-treated animals, ozanimod and RP-101075 reduced proteinuria over the duration of the study and serum blood urea nitrogen at termination. Additionally, ozanimod and RP-101075 reduced kidney disease in a dose-dependent manner, as measured by histological assessment of mesangial expansion, endo- and exo-capillary proliferation, interstitial infiltrates and fibrosis, glomerular deposits, and tubular atrophy. Further exploration into gene expression changes in the kidney demonstrate that RP-101075 also significantly reduced expression of fibrotic and immune-related genes in the kidneys. Of note, RP-101075 lowered the number of plasmacytoid dendritic cells, a major source of interferon alpha in lupus patients, and reduced all B and T cell subsets in the spleen. Given the efficacy demonstrated by ozanimod and its metabolite RP-101075 in the NZBWF1 preclinical animal model, ozanimod may warrant clinical evaluation as a potential treatment for systemic lupus erythematosus.


Context: Rupture of an abdominal aortic aneurysm is readily diagnosed when the triad of abdominal or back pain, shock and a pulsatile abdominal mass are present. Clinical diagnosis can be difficult, however, when patients present with chronic pain and an aneurysm which is not readily palpable. In these patients with confusing abdominal symptoms, CT scan provides a rapid, noninvasive diagnosis. The acute leaking AAA has been documented, but only rarely. This report identifies two patients who ruptured an aortic aneurysm several "months" prior to operation--a "contained chronic" rupture.


Context: A retrospective study including more than 350,000 women, about 1400 of whom had developed meningioma, showed that the risk of meningioma was about twice as high in users of postmenopausal hormone replacement therapy as in non-users. Hormone replacement therapy should be discontinued if meningioma is diagnosed.


Context: Obesity is a significant contemporary health concern that carries wide-ranging implications for society, as well as for individual health and well-being. In particular, the neuropsychological sequelae of obesity carry wide ranging functional implications. While research in this area is growing, our knowledge of obesity-related cognitive dysfunction and brain alterations has not yet been synthesized. The present review integrates the recent literature regarding patterns of obesity-related cognitive dysfunction and brain alterations and also indicates potential mechanisms for these neuropathological changes. The review culminates in a preliminary model of obesity-related cognitive dysfunction and suggestions for future research, including the potential reversibility of these changes with weight-loss.


Context: Wernicke's encephalopathy is an acute neurolopsychiatric syndrome caused by thiamine deficiency, and classically presents with the triad of opthalmopathy, ataxia and altered mentality. Both prolonged total parenteral nutrition and reduced oral intake can induce Wernicke's encephalopathy during hematopoietic stem cell transplantation (HSCT). Although early treatment is important for recovery from Wernicke's encephalopathy, the vague symptoms and characteristics hinder early diagnosis. Furthermore, Wernicke's encephalopathy is not infrequent and can develop at any age during HSCT. Herein, we present two young patients developing Wernicke's encephalopathy during HSCT.


Context: In the past three decades, intravesical instillation of Mycobacterium bovis bacille Calmette-Guérin (BCG) has been used for treating bladder cancer and it still remains at the forefront of immunotherapy for cancer patients. Although BCG-based therapy is the most effective intravesical therapy for this kind of tumor and represents the only agent known to reduce progression into muscle invasive bladder cancer, BCG is ineffective in approximately 30-40 % of cases and disease recurs in up to 50 % of patients. Since that BCG is considered an effective vehicle for delivery of antigens due to its unique characteristics, the genetic manipulation of these mycobacteria has been appealing in the search for less toxic and more potent therapeutic agents for bladder cancer immunotherapy. Herein, we discuss current advances in recombinant BCG construction, research, concerns, and future directions to promote the development of this promising immunotherapeutic approach for bladder cancer.


Context: Since the discovery of sensitizing EGFR mutations as a predictive marker of sensitivity to EGFR tyrosine kinase inhibitors (TKIs), the field of targeted therapy in non-small cell lung cancer (NSCLC) has been revolutionized. Patients harbouring these sensitizing mutations treated with EGFR TKI have derived significant clinical outcome when compared with standard platinum based chemotherapy doublets. However disease progression invariably occurs at a median of about 9-13 months from initiation treatment, if acquired resistance commonly due to the development of EGFR T790M mutation. A novel class of "third generation" EGFR TKIs have been developed that is sensitising and T790M mutant-specific whilst sparing WT EGFR, representing a significant breakthrough in the treatment in NSCLC patients with acquired resistance harboring these genotypes. Early phase clinical data suggest the third generation EGFR TKIs such as osimertinib, rociletinib, and HM61713 are highly efficacious and well tolerated. Another promising class of EGFR TKI such as AZD3759 has been designed to penetrate blood brain barrier to treat brain metastases and leptomeningeal disease and has showed promising responses in patients with brain metastases. Acquired resistance to third generation EGFR TKIs has been reported including EGFR C797S. Given its non-invasive nature, plasma ctDNA is being explored as a possible approach to detect T790M mutation and to also inform on novel molecular mechansims of tertiary resistance to third generation EGFR TKIs. An understanding of the mechanisms of acquired resistance to the third-generation EGFR TKIs will greatly aid in the development of the next generation of EGFR TKIs.


Context: The three cerebral cavernous malformations (CCMs) genes namely CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10 have been identified for which mutations cause cerebral cavernous malformations. However, the protein products of these genes involved in forming CCM signaling, are still poorly understood imposing an urgent need to understand these genes and their signaling processes in details. So far involvement of CCM3/PDCD10 in the cavernous angioma has been characterized from biochemical and biophysical analyses. However, there is no comprehensive study illustrating the phylogenetic history and comprehensive genetic variants of CCM3/PDCD10. Herein, we explored the phylogenetic history and genetic variants of CCM3/PDCD10 gene. Synteny analyses revealed that CCM3/PDCD10 gene shared same genomic loci from Drosophila to human and the gene structure of CCM3/PDCD10 is conserved from human to Branchiostoma floridae for about 500 MYs with some changes in sea urchin and in insects. The conserved CCM3/PDCD10 is characterized by presence of indels in the N-terminal dimerization domain. We identified 951 CCM3/PDCD10 variants by analysis of 1092 human genomes with top three variation classes belongs to 84% SNPs, 6.9% insertions and 6.2% deletions. We identified 22 missense mutations in the human CCM3/PDCD10 protein and out of which three mutations are deleterious. We also identified four stop-codon gaining mutations at the positions E34*, E68*, E97* and E140*, respectively. This study is the first comprehensive analysis of the CCM3/PDCD10 gene based on phylogenetic origin and genetic variants. This study corroborates that the evolution of CCM proteins with tubular organization evolvements by endothelial cells.


Context: Salivary cortisol is commonly used as a clinical biomarker of endocrine status and also as a marker of psychosocial stress. Annexin-A1 (AnxA1) is an anti-inflammatory protein whose expression is modulated by glucocorticoids. Our principal objectives were to (i) detect the presence of and (ii) measure AnxA1 protein in whole human saliva and to (iii) investigate whether salivary cortisol and AnxA1 are correlated in healthy humans. A total of 37 healthy participants (male and female) were used in the study. Saliva was collected using salivette tubes. Salivary cortisol and AnxA1 protein were sampled at between 3 and 6 time points over 24h and measured for cortisol and AnxA1 protein using specific ELISA's. The presence of salivary AnxA1 protein was confirmed by Western blotting. AnxA1 protein is detectable in whole human saliva, as detected by Western blot analysis and ELISA. A diurnal rhythm was evident in both salivary cortisol (P<0.01) and AnxA1 (P<0.01) and was defined as a significant difference in time 0 (waking) samples compared to 'bed' (2300 h) samples. AnxA1 protein did not exhibit an awakening response (P>0.05), whereas salivary cortisol was significantly elevated between time 0 and 30 min post waking (P<0.001). AnxA1 protein correlates positively with salivary cortisol, indicating that cortisol is most likely a regulator of AnxA1 in human saliva.


Context: Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients. To compare signaling effects of peginesatide to recombinant human erythropoietin (rHuEPO), dose-dependent effects on protein phosphorylation and gene expression were evaluated using phosphoproteomics, quantitative signal transduction analyses, and gene profiling. After stimulation with peginesatide or rHuEPO, cell lysates were prepared from UT-7/EPO cells. Liquid chromatography-tandem mass spectrometry and MesoScale arrays were used to quantify phosphorylation events. Transcriptional changes were analyzed using microarrays and quantitative reverse transcription polymerase chain reaction. Peginesatide and rHuEPO were found to regulate the tyrosine phosphorylation of an essentially equivalent set of protein substrates, and modulate the expression of a similar set of target genes. Consistent with their roles in stimulating erythropoiesis, peginesatide and rHuEPO regulate similar cellular pathways.


Context: Arginine is a conditionally essential amino acid. To elucidate the influence of l-arginine on the activation of endogenous antioxidant defence, male Wistar rats were orally administered daily with l-arginine at different levels of 25, 50, 100 mg/100 g body weight. After 7 and 14 days feeding, the antioxidative capacities and glutathione (GSH) contents in the plasma and in the liver were uniformly enhanced with the increasing consumption of l-arginine, whereas the oxidative stress was effectively suppressed by l-arginine treatment. After 14 days feeding, the mRNA levels and protein expressions of Keap1 and Cul3 were gradually reduced by increasing l-arginine intake, resulting that the nuclear factor Nrf2 was activated. Upon activation of Nrf2, the expressions of antioxidant responsive element (ARE)-dependent genes and proteins (GCLC, GCLM, GS, GR, GST, GPx, CAT, SOD, NQO1, HO-1) were up-regulated by l-arginine feeding, indicating an upward trend in antioxidant capacity uniformly with the increasing consumption of l-arginine. The present study demonstrates that the supplementation of l-arginine stimulates GSH synthesis and activates Nrf2 pathway, leading to the up-regulation of ARE-driven antioxidant expressions via Nrf2-Keap1 pathway. Results suggest the availability of l-arginine is a critical factor to suppress oxidative stress and induce an endogenous antioxidant response.


Context: We report the results of long-term (6-year) treatment of Nelson's syndrome with the long-acting dopamine agonist, cabergoline, in a 55-year-old woman. The disease presented 26 years after bilateral adrenalectomy and radiation treatment for Cushing's disease, followed by glucocorticoid and mineralocorticoid replacement therapy. Signs of Nelson's syndrome included skin and mucosal hyperpigmentation accompanied by elevated plasma levels of adrenocorticotropic hormone (ACTH) (984 pmol/l; normal, 2.0-11.5 pmol/l). Magnetic resonance imaging of the pituitary demonstrated sellar enlargement with a 15 mm macroadenoma. The patient was initially treated with bromocriptine (10 mg/d) which had no effect on either ACTH level or tumor mass. Because of visual loss, transsphenoidal surgery was performed, with partial excision of the adenoma and chiasmal decompression, followed by radiosurgery. However, ACTH levels improved only temporarily, and then increased to previous levels. Therefore, cabergoline treatment (1.5 mg/week) was initiated. ACTH levels decreased dramatically from 1050 to 132 pmol/l, accompanied by clinical improvement. Repeated imaging studies demonstrated a stable residual pituitary tumor. This case demonstrates that long-term cabergoline treatment may be efficient in patients with Nelson's syndrome.


Context: Upon exposure to various environmental stresses such as arsenite, hypoxia, and heat shock, cells inhibit their translation and apoptosis and then repair stress-induced alterations, such as DNA damage and the accumulation of misfolded proteins. These types of stresses induce the formation of cytoplasmic RNA granules called stress granules (SGs). SGs are storage sites for the many mRNAs released from disassembled polysomes under these stress conditions and are essential for the selective translation of stress-inducible genes. Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is a component of SGs that initiates the assembly of SGs by forming a multimer. In this study, we examined the role of G3BP2, a close relative of G3BP1, in SG formation. Although single knockdown of either G3BP1 or G3BP2 in 293T cells partially reduced the number of SG-positive cells induced by arsenite, the knockdowns of both genes significantly reduced the number. G3BP2 formed a homo-multimer and a hetero-multimer with G3BP1. Moreover, like G3BP1, the overexpression of G3BP2 induced SGs even without stress stimuli. Collectively, these results suggest that both G3BP1 and G3BP2 play a role in the formation of SGs in various human cells and thereby recovery from these cellular stresses.


Context: The eukaryotic transcription factor TFIIS enhances elongation and nascent transcript cleavage activities of RNA polymerase II in a stalled elongation complex. By site-directed mutagenesis, we have demonstrated that invariant residues Asp-261 and Glu-262 of the nucleic acid-binding TFIIS Zn ribbon are critical for stimulation of both elongation and RNA cleavage activities of RNA polymerase II. Substitution of either of these residues inactivates both TFIIS functions, suggesting a related role in both activities. These acidic residues may participate in phosphoryl transfer reactions by a two-metal-ion mechanism in a manner analogous to Klenow fragment. The RNA polymerase II itself may contain a Zn ribbon, in as much as the polymerase's 15-kDa subunit contains a sequence that aligns well with the TFIIS Zn ribbon sequence, including a similarly placed pair of acidic residues.


Context: The objective of this study was to assess the association between melanocortin-1 receptor (MC1R) variants and the severity of facial skin photoaging. The study population comprised 530 middle-aged French women. A trained dermatologist graded the severity of facial skin photoaging from photographs using a global scale. Logistic regressions were performed to assess the influence of MC1R polymorphisms on severe photoaging with adjustment for possible confounders (demographic and phenotypic data and sun exposure intensity). Among the fifteen MC1R variants identified, the nine most common were V60L, V92M, R151C, R160W, R163Q, R142H, D294H, D84E, and I155T. One hundred and eighty-five individuals (35%) were WT homozygotes, 261 (49%) had one common variant, 78 (15%) had two common variants, and six (1%) had at least one rare variant. After adjustment for possible confounders, the presence of two common variants was already a risk factor for severe photoaging (AOR (95% confidence interval): 2.33 (1.17-4.63)). This risk reached 5.61 (1.43-21.96) when two major diminished-function variants were present. Surprisingly, the minor variant, V92M, was associated with increased risk of photoaging (2.57 (1.23-5.35)). Our results suggest that genetic variations of MC1R are important determinants for severe photoaging.


Context: Silver staining is widely used to detect protein in polyacrylamide gels when high sensitivity is required. A simple and rapid protocol for silver staining of proteins following PAGE was developed in the present study. The number of steps was reduced compared to conventional protocol by combining fixing, rinsing, and soaking into a single impregnating step, thus achieving detection of proteins in 20 min. The present method is as sensitive as current protocols with the advantage of saving time and costs.


Context: Accurate reconstruction of the regulatory networks that control gene expression is one of the key current challenges in molecular biology. Although gene expression and chromatin state dynamics are ultimately encoded by constellations of binding sites recognized by regulators such as transcriptions factors (TFs) and microRNAs (miRNAs), our understanding of this regulatory code and its context-dependent read-out remains very limited. Given that there are thousands of potential regulators in mammals, it is not practical to use direct experimentation to identify which of these play a key role for a particular system of interest. We developed a methodology that models gene expression or chromatin modifications in terms of genome-wide predictions of regulatory sites and completely automated it into a web-based tool called ISMARA (Integrated System for Motif Activity Response Analysis). Given only gene expression or chromatin state data across a set of samples as input, ISMARA identifies the key TFs and miRNAs driving expression/chromatin changes and makes detailed predictions regarding their regulatory roles. These include predicted activities of the regulators across the samples, their genome-wide targets, enriched gene categories among the targets, and direct interactions between the regulators. Applying ISMARA to data sets from well-studied systems, we show that it consistently identifies known key regulators ab initio. We also present a number of novel predictions including regulatory interactions in innate immunity, a master regulator of mucociliary differentiation, TFs consistently disregulated in cancer, and TFs that mediate specific chromatin modifications.


Context: Blocking the action of negative muscle regulators through the activin type II receptors with bimagrumab treatment safely increased skeletal muscle mass but did not improve functional capacity in patients with COPD and low muscle mass. Clinical trial registered with www.clinicaltrials.gov (NCT01669174).


Context: Two secretases are involved in the generation of amyloid beta-peptide, the principal component of amyloid plaques in the brains of Alzheimer's disease patients. While beta-secretase is a classical aspartyl protease, gamma-secretase activity is associated with a high molecular weight complex. One of the complex components, which is critically required for gamma-secretase activity is nicastrin (NCT). Here we investigate the assembly of NCT into the gamma-secretase complex. NCT mutants either lacking the entire cytoplasmic tail, the cytoplasmic tail, and the transmembrane domain (TMD), or containing a set of heterologous TMDs were expressed in cells with strongly reduced levels of endogenous NCT. Maturation of exogenous NCT, gamma-secretase complex formation and proteolytic function was then investigated. This revealed that the cytoplasmic tail of NCT is dispensable for gamma-secretase complex assembly and function. In contrast, the authentic TMD of NCT is critically required for the interaction with gamma-secretase complex components and for formation of an active gamma-secretase complex. Neither soluble NCT lacking any membrane anchor nor NCT containing a heterologous TMD were inserted into the gamma-secretase complex. We identified the N-terminal region of the NCT TMD as a functionally important entity of NCT. These data thus demonstrate that NCT interacts with other gamma-secretase complex components via its TMD.


Context: Methylation at the 5' position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.


Context: To evaluate the use of general-purpose graphics processing units (GPGPUs) to improve the performance of MODFLOW, an unstructured preconditioned conjugate gradient (UPCG) solver has been developed. The UPCG solver uses a compressed sparse row storage scheme and includes Jacobi, zero fill-in incomplete, and modified-incomplete lower-upper (LU) factorization, and generalized least-squares polynomial preconditioners. The UPCG solver also includes options for sequential and parallel solution on the central processing unit (CPU) using OpenMP. For simulations utilizing the GPGPU, all basic linear algebra operations are performed on the GPGPU; memory copies between the central processing unit CPU and GPCPU occur prior to the first iteration of the UPCG solver and after satisfying head and flow criteria or exceeding a maximum number of iterations. The efficiency of the UPCG solver for GPGPU and CPU solutions is benchmarked using simulations of a synthetic, heterogeneous unconfined aquifer with tens of thousands to millions of active grid cells. Testing indicates GPGPU speedups on the order of 2 to 8, relative to the standard MODFLOW preconditioned conjugate gradient (PCG) solver, can be achieved when (1) memory copies between the CPU and GPGPU are optimized, (2) the percentage of time performing memory copies between the CPU and GPGPU is small relative to the calculation time, (3) high-performance GPGPU cards are utilized, and (4) CPU-GPGPU combinations are used to execute sequential operations that are difficult to parallelize. Furthermore, UPCG solver testing indicates GPGPU speedups exceed parallel CPU speedups achieved using OpenMP on multicore CPUs for preconditioners that can be easily parallelized.


Context: The low-density lipoprotein receptor (LDLr) plays a pivotal role in cholesterol homeostasis. Mutations in the LDLr gene (LDLR), which is located on chromosome 19, cause familial hypercholesterolemia (FH), an autosomal dominant disorder characterized by severe hypercholesterolemia associated with premature coronary atherosclerosis. To date almost 300 mutations have been identified in the LDLR gene. To facilitate the mutational analysis of the LDLR gene, and promote the analysis of the relationship between genotype and phenotype, a software package along with a computerized database (currently listing 210 entries) have been created.


Context: Abasic or AP sites generated by spontaneous DNA damage accumulate at a higher rate in actively transcribed regions of the genome in S. cerevisiae and are primarily repaired by base excision repair (BER) pathway. We have demonstrated that transcription-coupled nucleotide excision repair (NER) pathway can functionally replace BER to repair those AP sites located on the transcribed strand much like the strand specific repair of UV-induced pyrimidine dimers. Previous reports indicate that Rad26, a yeast homolog of transcription-repair coupling factor CSB, partly mediates strand-specific repair of UV-dimers as well as AP lesions. Here, we report that Def1, known to promote ubiquitination and degradation of stalled RNA polymerase complex, also directs NER to AP lesions on the transcribed strand of an actively transcribed gene but that its function is dependent on metabolic state of the yeast cells. We additionally show that Dst1, a homolog of mammalian transcription elongation factor TFIIS, interferes with NER-dependent repair of AP lesions while suppressing homologous recombination pathway. Overall, Def1 and Dst1 mediate very different outcomes in response to AP-induced transcription arrest.


Context: Optogenetics, the use of light to stimulate or inhibit neural circuits via viral transduction of protein channels, has emerged as a possible method of treating epilepsy. By introducing viral vectors carrying algal-derived cation or anion channels, known as opsins, neurons that initiate or propagate seizures may be silenced. To date, studies using this technique have been performed in animal models, and current efforts are moving toward more sophisticated nonhuman primate models. In this paper, the authors present a brief overview of the development of optogenetics and review recent studies investigating optogenetic modification of circuits involved in seizures. Further developments in the field are explored, with an emphasis on how optogenetics may influence future neurosurgical interventions.


Context: Shigellosis is a leading cause of diarrhea worldwide prompting vaccine development. The Shigella flexneri Invaplex 50 is a macromolecular complex containing IpaB, IpaC, and LPS, formulated from an aqueous extract of virulent Shigella delivered via nasal administration. Preclinical vaccine testing demonstrated safety, immunogenicity and efficacy. An open-label dose-escalating phase 1 study evaluated a 3-dose (2-week intervals) regimen via nasal pipette delivery. Thirty-two subjects were enrolled into one of four vaccine dose groups (10, 50, 240, or 480 microg). The vaccine was well tolerated with minor short-lived nasal symptoms without evidence of dose effect. Antibody-secreting cell (ASC) responses were elicited at doses > or =50 microg with the highest IgG ASC, Invaplex 50 (100%) and S. flexneri 2a LPS (71%), as well as, serologic responses (43%) occurring with the 240 microg dose. Fecal IgA responses, Invaplex 50 (38.5%) and LPS (30.8%), were observed at doses > or =240 microg. The Invaplex 50 nasal vaccine was safe with encouraging mucosal immune responses. Follow-on studies will optimize dose, delivery mechanism and assess efficacy in a S. flexneri 2a challenge study.


Context: Hepatocellular carcinoma, one of the most common virus-associated cancers, is the third most frequent cause of cancer-related death worldwide. By massively parallel sequencing of a primary hepatitis C virus-positive hepatocellular carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same individual, we identified more than 11,000 somatic substitutions of the tumor genome that showed predominance of T>C/A>G transition and a decrease of the T>C substitution on the transcribed strand, suggesting preferential DNA repair. Gene annotation enrichment analysis of 63 validated non-synonymous substitutions revealed enrichment of phosphoproteins. We further validated 22 chromosomal rearrangements, generating four fusion transcripts that had altered transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense substitution in a subpopulation of the tumor cells. This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor.


Context: Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models.


Context: The "hallmarks" of pancreatic ductal adenocarcinoma (PDAC) include proliferative, invasive, and metastatic tumor cells and an associated dense desmoplasia comprised of fibroblasts, pancreatic stellate cells, extracellular matrix, and immune cells. The oncogenically activated pancreatic epithelium and its associated stroma are obligatorily interdependent, with the resulting inflammatory and immunosuppressive microenvironment contributing greatly to the evolution and maintenance of PDAC. The peculiar pancreas-specific tumor phenotype is a consequence of oncogenes hacking the resident pancreas regenerative program, a tissue-specific repair mechanism regulated by discrete super enhancer networks. Defined as genomic regions containing clusters of multiple enhancers, super enhancers play pivotal roles in cell/tissue specification, identity, and maintenance. Hence, interfering with such super enhancer-driven repair networks should exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue. Novel drugs that directly or indirectly inhibit processes regulating epigenetic status and integrity, including those driven by histone deacetylases, histone methyltransferase and hydroxylases, DNA methyltransferases, various metabolic enzymes, and bromodomain and extraterminal motif proteins, have shown the feasibility of disrupting super enhancer-dependent transcription in treating multiple tumor types, including PDAC. The idea that pancreatic adenocarcinomas rely on embedded super enhancer transcriptional mechanisms suggests a vulnerability that can be potentially targeted as novel therapies for this intractable disease.


Context: Wide hybridization is a one of the important techniques in plant breeding. Oat (Avena sativa L.) and pearl millet (Pennisetum glaucum L.) belong to different subfamilies of Poaceae. In generally, such distant relative species show uniparental chromosome elimination after successful fertilization. However, all seven pearl millet chromosomes are retained beside the genome of oat during embryogenesis. Hybrid seedlings develop, but show necrosis after light irradiation. Here, a detailed protocol for wide hybridization between oat and pearl millet is described.


Context: Gastric cancer (GC) is the second leading cause of cancer-associated mortality worldwide. In advanced and metastatic GC, conventional chemotherapy results in limited efficacy and the average survival rate is currently approximately 10 months. Dysregulated activation of numerous genes, including zinc finger, DHHC-type containing 14; caspase-associated recruitment domain-containing protein; and Ras association domain family member 10, have been implicated in GC. The tumor suppressor function of lactotransferrin (LTF) has been reported in a variety of tumors, including GC, nasopharyngeal carcinoma (NPC) and prostate cancer. However, the mechanism of the tumor suppressor function of LTF in GC remains unclear. In the present study, the expression levels of LTF in patient GC tissue samples were investigated using reverse transcription-quantitative polymerase chain reaction, and it was demonstrated that the LTF mRNA expression level in GC tissue samples was reduced by ~20-fold compared with the adjacent non-cancerous tissues (t=4.56, P<0.01). A similar trend in LTF protein expression was observed by western blot analysis. Furthermore, the present study demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway intermediates p38, -Jun N-terminal kinase (JNK) and -Jun were highly expressed in GC tissue samples, and indicated that LTF downregulation may be associated with the dysregulation of the MAPK signaling pathway in GC tissues. In addition, the present study indicated that LTF overexpression reduced the expression of p38, JNK2 and -Jun in the GC cell line, SGC7901. The present study demonstrates that LTF expression is downregulated in GC tissues and that LTF may serve an important role in the dysregulation of the MAPK signaling pathway.


Context: The animal gastrointestinal tract houses a large microbial community, the gut microbiota, that confers many benefits to its host, such as protection from pathogens and provision of essential metabolites. Metagenomic approaches have defined the chicken fecal microbiota in other studies, but here, we wished to assess the correlation between the metagenome and the bacterial proteome in order to better understand the healthy chicken gut microbiota. Here, we performed high-throughput sequencing of 16S rRNA gene amplicons and metaproteomics analysis of fecal samples to determine microbial gut composition and protein expression. 16 rRNA gene sequencing analysis identified Clostridiales, Bacteroidaceae, and Lactobacillaceae species as the most abundant species in the gut. For metaproteomics analysis, peptides were generated by using the Fasp method and subsequently fractionated by strong anion exchanges. Metaproteomics analysis identified 3,673 proteins. Among the most frequently identified proteins, 380 proteins belonged to Lactobacillus spp., 155 belonged to Clostridium spp., and 66 belonged to Streptococcus spp. The most frequently identified proteins were heat shock chaperones, including 349 GroEL proteins, from many bacterial species, whereas the most abundant enzymes were pyruvate kinases, as judged by the number of peptides identified per protein (spectral counting). Gene ontology and KEGG pathway analyses revealed the functions and locations of the identified proteins. The findings of both metaproteomics and 16S rRNA sequencing analyses are discussed.


Context: alpha-neurotoxins from elapid snake venoms and alpha-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an alpha-neurotoxin from the false sea snake Laticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller alpha-conotoxins (from the marine snails Conus magus and Conus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake alpha-neurotoxins and their homologues. Six proteins unrelated to erabutoxin b were compared to the alpha-conotoxins to show that the extent of the erabutoxin b/alpha-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely alpha-cobratoxin from Naja naja siamensis and cytotoxin VII4 from Naja mossambica mossambica, were also analyzed. The extent of the matching with the alpha-conotoxins decreased in the series erabutoxin b greater than alpha-cobratoxin greater than cytotoxin VII4, and this also relates the order of similarity to the pharmacological properties of the alpha-conotoxins. The alpha-conotoxin-like area of the snake alpha-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake alpha-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.


Context: Background Hydroxychloroquine (HCQ), a 4-aminoquinolone antimalarial, is regarded as the oral therapy of choice for cutaneous and systemic lupus erythematosus (SLE). It is also licensed for rheumatoid arthritis (RA). Studies of HCQ-treated patients with SLE or RA have demonstrated a positive correlation between whole-blood HCQ levels and clinical response. Such studies have involved measuring whole-blood concentrations at any given time point after HCQ ingestion assuming that steady-state concentrations would undergo limited fluctuation over a daily interval because HCQ has a long half-life. This approach might not sufficiently take into account the potential intra-patient variation in HCQ blood levels that can occur over a 24-hour period. Such variation, if significant, could affect the credibility of any concentration-response relationship provided from these previous studies. Objectives The objectives of this report are to: (a) investigate the intra-patient variation in HCQ whole-blood levels and (b) suggest an optimum time for sampling patients for future studies. Methods Six patients were recruited with cutaneous lupus erythematosus who had each been on HCQ 200 mg twice daily for at least six months, so that they were at steady-state. Each patient was fasted overnight and had standardized meals and dosing schedule. Whole blood was sampled at seven time points over 24 hours. Whole-blood HCQ levels were measured with high-performance liquid chromatography using gradient elution, fluorimetric detection and chloroquine as an internal standard. The assay had a mean inter- and intra-day coefficient of variation of 10% and 5% respectively and a limit of detection of 5ng/ml. Results HCQ levels appeared to follow a biphasic pattern over the sampling period. Maximum levels were noted a median of four hours (range 2-6) after ingestion. Median intra-patient variation between trough and peak levels, 'Cmax' ((peak - trough)/trough × 100%), was 27% (range 8-150%). Conclusions This study demonstrated that whole-blood HCQ levels vary 27% (median, range 8-150%) within an individual over a 12-hour period. Drug levels might differ between individuals because of multiple factors, including variable adherence to medication. Measuring HCQ levels for assessment of drug adherence could be valuable in the 'real-world' clinical setting. This could be assessed by taking a blood sample at any time following HCQ ingestion. If patients were found to have very low or undetectable levels of HCQ, non-adherence to HCQ should be suspected.


Context: Istradefylline (ISD) is a new drug developed for the treatment of Parkinson's disease (PD). It is an adenosine receptor A antagonists that will represent an important option for patients with advanced PD where it has been demonstrated efficacy in decreasing daily OFF time and is well tolerated. ISD has been marketed in Japan since May 2013. Areas covered: The objective of this review is to summarize evidences emerged from clinical studies that have demonstrated the efficacy of ISD in advanced parkinsonian patients. It will then focus on the potential role in treating non-motor symptoms (NMS) and cognitive decline, which heavily affect quality of life for PD patients. Its putative role as neuroprotective agent will also be discussed. Expert opinion: ISD might represent an alternative option for patients with advanced PD. The reduction of OFF time highlighted in pivotal trials is comparable to that obtained with different levodopa adjunct therapies. The low profile of side effects makes ISD a more suitable drug for advanced patients whose illness is complicated by depression or cognitive impairment. Future studies are warranted to investigate the possible effects of this drug to delay the occurrence of dyskinesia and to impact significantly on NMS.


Context: Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1Δ fkh2Δ post-mitotic survival is epistatic to that observed in apc5(CA) mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5(CA) fkh1Δ fkh2Δ, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism under certain conditions, likely through combined regulation of stress response, genomic stability, and cell cycle regulation.


Context: The Grg gene encodes a 197-amino-acid protein homologous to the amino-terminal domain of the product of the groucho gene of the Drosophila Enhancer of split complex. We describe here the genomic organization of the mouse Grg gene. It spans approximately 7 kb on chromosome 10 and consists of seven exons. The 3' region of the Grg gene contains two functional polyadenylation sites that give rise to two transcripts that are differentially expressed among adult mouse tissues. The promoter region is very GC rich and lacks TATA box and "initiator" sequences. Primer extension analysis and ribonuclease protection assays show that Grg has a major transcription start site situated down-stream of putative binding motifs for the transcription factors Sp1, E2A, and PuF.


Context: In a cohort of pregnant women with mostly sufficient levels of serum 25(OH)D, vitamin D deficiency was not associated with GDM. Further studies are warranted with larger cohorts, especially in populations with lower levels of vitamin D.


Context: We have analyzed the interference of antitumoral drugs acting through the inhibition of DNA topoisomerase II on the human HeLa cell metabolism. Different compounds characterized by a diverse mechanism of action have been used, namely m-amsacrine, an intercalative drug, etoposide, which does not intercalate DNA, and suramin, which exerts its effect through an unknown mechanism. In HeLa cells treated with increasing doses of these drugs, we have examined cell viability and DNA synthesis capacity, and we have evaluated topoisomerase II activity. Cellular morphology and DNA integrity have been studied in order to characterize the mechanism of cell death. The results we have obtained clearly indicate that topoisomerase II poisons induce cell death by apoptosis. These observations suggest a role of the inhibition of topoisomerase II activity in the apoptotic program.


Context: MLN4924 is a first-in-class cancer drug that inhibits the Nedd8-activating enzyme (NAE). Herein, we report that MLN4924 inhibits Vpx/Vpr-induced SAMHD1 degradation by inhibiting the neddylation of E3 ubiquitin ligase and blocks macaque simian immunodeficiency virus (SIVmac) replication in myeloid cells. SAMHD1 is required for MLN4924-mediated SIVmac inhibition. Our findings indicate the potential efficacy of inhibiting neddylation as an antiretroviral strategy and identify the readily available anticancer drug MLN4924 as a candidate agent for that purpose.


Context: Saccharomyces cerevisiae RecQ helicase (Sgs1) combines with DNA topoisomerase III (Top3) and RecQ-mediated genome instability 1 (Rmi1) to form an evolutionarily conserved complex that is required for processing homologous recombination intermediates and restarting collapsed replication forks. It was previously reported that Rmi1 contributes to sister chromatid cohesion; however, the underlying molecular mechanism has been unclear. In the present study, Rmi1 was found to be enriched at the region close to an early-firing replication origin when replication forks were arrested near their origins in the presence of hydroxyurea. Genetic analyses revealed that Rmi1 promoted sister chromatid cohesion in a process that was distinct from both the cohesion establishment pathway involving Ctf4, Csm3, and Chl1 and the pathway involving the acetylation of Smc3. On the other hand, Rmi1 seemed to function in a pathway involving the Ctf18-RFC complex and Mrc1, which were previously predicted to regulate leading-strand DNA replication.


Context: Management of hypertension in diabetes is critical for reduction of cardiovascular mortality and morbidity. While blood pressure (BP) control has improved over the past two decades, the control rate is still well below 50% in the general population of patients with type 2 diabetes mellitus (T2DM). A new class of oral glucose-lowering agents has recently been approved; the sodium-glucose co-transporter 2 (SGLT2) inhibitors, which act by eliminating large amounts of glucose in the urine. Two agents, dapagliflozin and canagliflozin, are currently approved in the United States and Europe, and empagliflozin and ipragliflozin have reported Phase 3 trials. In addition to glucose lowering, SGLT2 inhibitors are associated with weight loss and act as osmotic diuretics, resulting in a lowering of BP. While not approved for BP-lowering, they may potentially aid BP goal achievement in people within 7-10 mm Hg of goal. It should be noted that the currently approved agents have side effects that include an increased incidence of genital infections, predominantly in women. The approved SGLT2 inhibitors have limited use based on kidney function and should be used only in those with an estimated glomerular filtration rate (eGFR) > 60 mL/min/1.73 m2 for dapagliflozin and ≥45 mL/min/1.73 m2 for canagliflozin. Cardiovascular outcome trials are ongoing with these agents and will be completed within the next 4-5 years.


Context: Capture and representation of scientific knowledge in a structured format are essential to improve the understanding of biological mechanisms involved in complex diseases. Biological knowledge and knowledge about standardized terminologies are difficult to capture from literature in a usable form. A semi-automated knowledge extraction workflow is presented that was developed to allow users to extract causal and correlative relationships from scientific literature and to transcribe them into the computable and human readable Biological Expression Language (BEL). The workflow combines state-of-the-art linguistic tools for recognition of various entities and extraction of knowledge from literature sources. Unlike most other approaches, the workflow outputs the results to a curation interface for manual curation and converts them into BEL documents that can be compiled to form biological networks. We developed a new semi-automated knowledge extraction workflow that was designed to capture and organize scientific knowledge and reduce the required curation skills and effort for this task. The workflow was used to build a network that represents the cellular and molecular mechanisms implicated in atherosclerotic plaque destabilization in an apolipoprotein-E-deficient (ApoE(-/-)) mouse model. The network was generated using knowledge extracted from the primary literature. The resultant atherosclerotic plaque destabilization network contains 304 nodes and 743 edges supported by 33 PubMed referenced articles. A comparison between the semi-automated and conventional curation processes showed similar results, but significantly reduced curation effort for the semi-automated process. Creating structured knowledge from unstructured text is an important step for the mechanistic interpretation and reusability of knowledge. Our new semi-automated knowledge extraction workflow reduced the curation skills and effort required to capture and organize scientific knowledge. The atherosclerotic plaque destabilization network that was generated is a causal network model for vascular disease demonstrating the usefulness of the workflow for knowledge extraction and construction of mechanistically meaningful biological networks.


Context: Tuberous sclerosis complex (TSC) and severe cortical dysplasia (CD), or CD type II according to Palmini classification, share histopathologic similarities, specifically the presence of cytomegalic neurons and balloon cells. In this study we examined the morphologic and electrophysiologic properties of cells in cortical tissue samples from pediatric patients with TSC and CD type II who underwent surgery for pharmacoresistant epilepsy. Normal-appearing pyramidal neurons from TSC and CD type II cases had similar passive membrane properties. However, the frequency of excitatory postsynaptic currents (EPSCs) was higher in neurons from TSC compared to severe CD cases, particularly the frequency of medium- and large-amplitude synaptic events. In addition, EPSCs rise and decay times were slower in normal cells from TSC compared to severe CD cases. Balloon cells were found more frequently in TSC cases, whereas cytomegalic pyramidal neurons occurred more often in CD type II cases. Both cell types were similar morphologically and electrophysiologically in TSC and severe CD. These results suggest that even though the histopathology in TSC and severe CD is similar, there are subtle differences in spontaneous synaptic activity and topographic distribution of abnormal cells. These differences may contribute to variable mechanisms of epileptogenesis in patients with TSC compared with CD type II.


Context: Neurexins and neuroligins are two distinct families of single-pass transmembrane proteins localized at pre- and postsynapses, respectively. They trans-synaptically interact with each other and induce synapse formation and maturation. Common variants and rare mutations, including copy number variations, short deletions, and single or small nucleotide changes in neurexin and neuroligin genes have been linked to the neurodevelopmental disorders, such as autism spectrum disorders (ASDs). In this review, we summarize the structure and basic synaptic function of neurexins and neuroligins, followed by behaviors and synaptic phenotypes of knock-in and knock-out mouse of these family genes. From the studies of these mice, it turns out that the effects of neurexins and neuroligins are amazingly neural circuit dependent, even within the same brain region. In addition, neurexins and neuroligins are commonly involved in the endocannabinoid signaling. These finding may provide not only insight into understanding the pathophysiology, but also the concept for strategy of therapeutic intervention for ASDs.


Context: The North Star Ambulatory Assessment measures motor performance in ambulatory boys with Duchenne muscular dystrophy, a hereditary and degenerative muscle disorder. To use the North Star Ambulatory Assessment in Brazilian boys, we performed the cross-cultural adaptation to the Portuguese language spoken in Brazil and evaluated the reliability and validity of the instrument. Cross-cultural adaptation included: independent translations, synthesis, committee review, pre-testing in 12 boys, back-translation and comparison with the original instrument. Thirty-five boys with Duchenne muscular dystrophy and 38 healthy age-matched controls were recruited for further analyses. Reliability was assessed by internal consistency and reproducibility. Validity studies included face, content, construct and known-groups analyses. Cross-cultural adaptation resulted in an adequate instrument. Reliability studies demonstrated high internal consistency (Cronbach's alpha = 0.935) and adequate intra and inter-rater reproducibility (intraclass correlation coefficient = 0.988 and 0.962). Validation analyses indicated satisfactory content, face and convergent construct validities, with positive correlations with the Motor Function Measure total score (r = 0.863) and the 6-minute walk test (r = 0.433). The known group validity was demonstrated by higher scores in younger boys with Duchenne muscular dystrophy (p = 0.005). North Star Ambulatory Assessment in Brazilian Portuguese is a reliable and valid instrument to measure functional capacity in boys with Duchenne muscular dystrophy.


Context: Depressive disorders in women are not uncommon during the reproductive period. Identifying women at risk for the development of depression, early detection of depressive symptoms and an adequate treatment are important issues in preventing severe chronic depressive disorders. Do psychotherapeutic interventions not show any sufficient benefits, the use of antidepressants is indicated. In the meantime sufficient data are available concerning the use of anti-depressants during pregnancy to exclude any serious damage to the fetus or newborn. As a basic principle, a monotherapy should be favoured and the drug applicated in the lowest efficient dose. Due to the SSRI's side effect profile the choice of the antidepressant should be done in favour of serotonin reuptake inhibitors rather than tri- and tetracyclic antidepressants. For both substance groups there is no hint for any teratogenic potential. Benzodiazepine should be prescribed with caution. Negative long term effects to the newborn cannot be excluded. Based on few available data, indeed, there exists no reservation against the use of St. John's wort during pregnancy and breastfeeding. A profound ultrasound screening of the organs should be performed in case of lithium exposition, whereas newer studies showed no increase in risk for Ebstein anomaly. A multidisciplinary approach, including psychiatry, obstetrics and neonatology should be always aimed at in case of depressive disorders during pregnancy.


Context: Congenital craniofacial malformations vary widely in both expression and gravity. To understand congenital craniofacial malformations, knowledge of embryonic development is of essential importance. Craniosynostosis has its origin in the failure of suture development between 2 bone centres or in early closure of the suture by bone centre tissue fusion. Hereditary craniosynostosis phenotypes predominantly arise by autosomal dominant inheritance. So far, the majority of mutations have been found in fibroblast growth-factor receptor genes (FGFR-genes). Different phenotypes are not primarily created by disparities of the receptors, but particularly by tissue-specific expressions.


Context: Non-melanoma skin cancer (NMSC) comprises basal cell carcinoma (BCC) and squamous cell carcinoma, together with a host of rare tumours. NMSC is the commonest malignancy among Caucasians and its incidence continues to rise annually. Exposure to UV radiation initiates approximately 90% of NMSC, causing malignant transformation of keratinocytes and suppression of the inflammatory response. Risk factors include sun exposure and immunosuppression. There are several subtypes of BCC, although histological overlap is common. Surgery has traditionally been regarded as the 'gold-standard' treatment, offering excellent cure rates and cosmetic results. Other treatment modalities include physical destruction (radiotherapy, curettage and cautery, and cryotherapy), chemical destruction (photodynamic therapy and topical 5-flurouracil) and immunomodulatory therapy (topical imiquimod). The recent development of novel hedgehog pathway inhibitors for high-risk BCC (including oral vismodegib and sonidegib) may represent a paradigm shift towards medical management of NMSC.


Context: COVID-19 vaccines are effective and important to control the ongoing pandemic, but vaccine reactogenicity may contribute to poor uptake. Analgesics or antipyretic medications are often used to alleviate vaccine side effects, but their effect on immunogenicity remains uncertain. Few studies have assessed the effect of analgesics/antipyretics on vaccine immunogenicity and reactogenicity. Some studies revealed changes in certain immune response parameters post-vaccination when analgesics/antipyretics were used either prophylactically or therapeutically. Still, there is no evidence that these changes impact vaccine efficacy. Specific data on the impact of analgesic/antipyretic medications on immunogenicity of COVID-19 vaccines are limited. However, available data from clinical trials of licensed vaccines, along with recommendations from public health bodies around the world, should provide reassurance to both healthcare professionals and vaccine recipients that short-term use of analgesics/antipyretics at non-prescription doses is unlikely to affect vaccine-induced immunity.


Context: Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are important biological markers and regulators of cardiac function. The natriuretic peptide receptor A (NPRA), also known as NPR1 or guanylyl cyclase A, binds ANP and BNP to initiate transmembrane signal transduction by elevating the intracellular levels of cyclic guanosine monophosphate. However, the effects and mechanisms downstream of NPRA are largely unknown. The aim of the present study was to evaluate the changes in the global pattern of mRNA and circular RNA (circRNA) expression in NPRA and NPRA myocardium. Differentially expressed mRNA molecules were characterised using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis and were found to be primarily related to metabolic processes. Moreover, circRNA expression was also examined, and a possible competing endogenous RNA network consisting of circRNA, microRNA (miRNA), and mRNA molecules was constructed. The results of this study indicated that NPRA may play a role in cardiac metabolism, which could be mediated by circRNA through endogenous competition mechanisms. These findings may provide insight into future characterisation of various ceRNA network pathways.


Context: We present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs.


Context: The diagnosis of acute human immunodeficiency virus (HIV) infection (AHI) plays a unique role in preventing the spread of HIV and ending the epidemic. Acutely infected individuals are thought to contribute substantially to forward transmissions of HIV; however, diagnosing AHI in resource-limited settings has proven to be a challenge. While fourth generation antigen-antibody combination assays have been successful in high-resource settings, rapid point of care (POC) versions of these assays have yet to demonstrate high sensitivity to detect AHI. Newer RNA/DNA based POC technologies are being validated, but the challenge to understand the additional value of these devices depends on the quality of study evaluations, in particular choice of study designs and case mix of included populations. In this commentary, we aimed to review the quality of studies evaluating a new fourth generation rapid test for detecting AHI, to identify general methodological limitations and biases in diagnostic accuracy studies, and to recommend strategies for avoiding them in future evaluations. The new studies that were evaluated continued to report the same weaknesses and biases that were seen in previous evaluations of fourth generation rapid tests. We recommend that investigators design future studies carefully, keeping in mind how diagnostic performance may be influenced by prevalence, population, patient case mixes, and reference standards. Care must be taken to avoid biases specific to diagnostic accuracy studies (spectrum, verification, incorporation and reference standard biases). To improve on quality, reporting checklists and guidelines such as Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) and Standards for Reporting Diagnostic accuracy studies (STARD) should be reviewed prior to conducting studies.


Context: Thromboembolism in pregnancy and the puerperium and inherited or acquired thrombophilia are associated. Thrombophilia can be revealed by pregnancy. Thrombotic risk during pregnancy and the puerperium is higher in asymptomatic women with than without thrombophilia. Antithrombin deficiency, combined deficiencies and homozygous or double-heterozygotes factor V Leiden and factor II G 20210 A mutations are associated with a higher thrombotic risk than heterozygote mutations or protein S and C deficiencies, whereas hyperhomocysteinemia does not appear as a risk factor for maternal thromboembolic disease. Antiphospholipid syndrome with lupus anticoagulant is strongly associated with thrombotic risk in pregnancy and the puerperium. Further studies are required to assess the thrombotic risk in women with preeclampsia as well as early or late recurrent pregnancy loss.


Context: In archaeological studies, the isotopic enrichment values of carbon and nitrogen in bone collagen give a degree of information on dietary composition. The isotopic enrichments of individual amino acids from bone collagen and dietary protein have the potential to provide more precise information about the components of diet. A limited amount of work has been done on this, although the reliability of these studies is potentially limited by fractionation arising through hydrolysis of whole plant tissue (where reaction between amino acids and carbohydrates may occur) and, for certain amino acids, the use of derivatives (particularly trifluoroacetyl derivatives) for gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis. The present study takes the approach of extracting the protein components of plant tissues before hydrolysis and using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS), which does not require derivatisation, for measurement of the isotopic enrichment of the amino acids. The protocol developed offers a methodology for consistent measurement of the δ(13)C values of amino acids, allowing isotopic differences between the individual amino acids from different plant tissues to be identified. In particular, there are highly significant differences between leaf and seed protein amino acids (leaf minus grain) in the cases of threonine (-4.1‰), aspartic acid (+3.5‰) and serine (-3.2‰). In addition to its intended application in archaeology, the technique will be of value in the fields of plant sciences, nutrition and environmental food-web studies.


Context: Gene regulatory programs in different cell types are largely defined through cell-specific enhancers activity. The histone variant H2A.Z has been shown to play important roles in transcription mainly by controlling proximal promoters, but its effect on enhancer functions remains unclear. Here, we demonstrate by genome-wide approaches that H2A.Z is present at a subset of active enhancers bound by the estrogen receptor alpha (ERα). We also determine that H2A.Z does not influence the local nucleosome positioning around ERα enhancers using ChIP sequencing at nucleosomal resolution and unsupervised pattern discovery. We further highlight that H2A.Z-enriched enhancers are associated with chromatin accessibility, H3K122ac enrichment and hypomethylated DNA. Moreover, upon estrogen stimulation, the enhancers occupied by H2A.Z produce enhancer RNAs (eRNAs), and recruit RNA polymerase II as well as RAD21, a member of the cohesin complex involved in chromatin interactions between enhancers and promoters. Importantly, their recruitment and eRNAs production are abolished by H2A.Z depletion, thereby revealing a novel functional link between H2A.Z occupancy and enhancer activity. Taken together, our findings suggest that H2A.Z acts as an important player for enhancer functions by establishing and maintaining a chromatin environment required for RNA polymerase II recruitment, eRNAs transcription and enhancer-promoters interactions, all essential attributes of enhancer activity.


Context: Skin inflammation often evokes pruritus, which is the major subjective symptom in many inflammatory skin diseases such as atopic dermatitis and prurigo nodularis. Pruritus or itch is a specific sensation found only in the skin. Recent studies have stressed the pivotal role played by interleukin-31 (IL-31) in the sensation of pruritus. IL-31 is produced by various cells including T helper 2 cells, macrophages, dendritic cells and eosinophils. IL-31 signals via a heterodimeric receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor β. Recent clinical trials have shown that the anti-IL-31RA antibody nemolizumab can successfully decrease pruritus in patients with atopic dermatitis and prurigo nodularis. The IL-31 pathway and pruritic skin are highlighted in this review article.


Context: Monoclonal antibodies (mAbs) targeting calcitonin gene-related peptide (CGRP) signaling are being explored as prophylactic treatments for migraine. Erenumab (AMG 334) is the first potent, selective, and competitive human mAb antagonist of the CGRP receptor. We report the data from two phase I studies assessing the safety, pharmacokinetics (PK), and pharmacodynamics of single and multiple administrations of erenumab in healthy subjects and patients with migraine. The results indicate that the PK profile of erenumab is nonlinear from 1 mg to 70 mg and the linear portion of the clearance from 70 mg to 210 mg is consistent with other human immunoglobulin G2 antibodies. Single doses of erenumab resulted in >75% inhibition of capsaicin-induced dermal blood flow, with no apparent dose-dependency for erenumab ≥21 mg. Erenumab was generally well tolerated, with an acceptable safety profile, supporting further clinical development of erenumab for migraine prevention.


Context: The effects of natriuretic peptides on the proliferation and differentiation of osteoblast-like cells from rat calvariae were examined. Natriuretic peptides are physiological agonists that activate receptor guanylate cyclases, namely, natriuretic peptide receptor (NPR)-A and NPR-B. Exposure of cells to atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) resulted in large increases in the rate of intracellular production of guanosine 3',5'-cyclic monophosphate (cGMP). Moreover, CNP-like immunoreactivity was detected in the conditioned medium from osteoblast-like cells, while ANP was undetectable. In cells exposed to natriuretic peptides, a dose-dependent reduction in the rate of DNA synthesis was observed. Natriuretic peptides also stimulated the activity of alkaline phosphatase (ALPase) and the expression of mRNA for ALPase and osteocalcin and the mineralization of nodules by the cultured cells. These results could be reproduced by treating cells with 8-bromo-cGMP. Endothelin-1, whose physiological functions are the opposite of those of natriuretic peptides, decreased the ALPase activity and the mineralization of nodules. In the present study, natriuretic peptides were demonstrated to promote bone formation via the action of cGMP in a signal-transduction pathway mediated by specific receptors in osteoblast-like cells.


Context: Radiation enteritis is an old but emerging question induced by the application of radiation. However, no effective drugs for radiation enteritis in clinic. In this study, we found that thymoquinone (TQ) could mitigate intestinal damages induced by irradiation. After exposure to irradiation, TQ-treated improved the irradiated mice survival rate, ameliorated intestinal injury and increased the numbers of intestinal crypts. Furthermore, Lgr5 ISCs and their daughter cells, including Vil1 enterocytes, Ki67 cells and lysozyme Paneth cells, were all significantly increased with TQ treatment. In addition, P53, γH2AX, caspase8, caspase9 and caspase3 expression were all reduced by TQ. Our data showed that TQ modulated DNA damages and decreased the apoptosis in the small intestine. TQ might be used for radiation enteritis treatment.


Context: The spatial arrangement of interphase chromosomes in the nucleus is important for gene expression and genome function in animals and in plants. The recently developed Hi-C technology is an efficacious method to investigate genome packing. Here we present a detailed Hi-C map of the three-dimensional genome organization of the plant Arabidopsis thaliana. We find that local chromatin packing differs from the patterns seen in animals, with kilobasepair-sized segments that have much higher intrachromosome interaction rates than neighboring regions, representing a dominant local structural feature of genome conformation in A. thaliana. These regions, which appear as positive strips on two-dimensional representations of chromatin interaction, are enriched in epigenetic marks H3K27me3, H3.1, and H3.3. We also identify more than 400 insulator-like regions. Furthermore, although topologically associating domains (TADs), which are prominent in animals, are not an obvious feature of A. thaliana genome packing, we found more than 1000 regions that have properties of TAD boundaries, and a similar number of regions analogous to the interior of TADs. The insulator-like, TAD-boundary-like, and TAD-interior-like regions are each enriched for distinct epigenetic marks and are each correlated with different gene expression levels. We conclude that epigenetic modifications, gene density, and transcriptional activity combine to shape the local packing of the A. thaliana nuclear genome.


Context: Biallelic mutations in Fanconi anemia complementation group genes disrupt DNA repair and result in the complex Fanconi anemia phenotype. In addition, germ line mutations in the BRCA2/FANCD1 Fanconi anemia complementation group gene have also been implicated in predisposition to a number of cancers including pancreatic cancer. The recent identification of FANCC and FANCG mutations in resected pancreatic tumors selected for loss of heterozygosity on chromosome 9, some of which were present in the germ line DNA, suggests that inactivation of these and other Fanconi complementation group genes may contribute to pancreatic cancer. To further assess the relevance of FANCC and FANCG mutations to pancreatic cancer we conducted a mutation screen of these genes in DNA from blood of 421 sequentially collected pancreatic cancer cases diagnosed at the Mayo Clinic. Two truncating FANCC mutations but no truncating FANCG mutations were identified in young onset (<55 years) pancreatic cancer cases with no family history of pancreatic cancer. Both mutations were associated with loss of heterozygosity of the wild-type allele in corresponding pancreatic tumors. In addition, no truncating mutations were identified in germ line DNA from blood of 658 control individuals undergoing routine colonoscopy. Taken together these data support the assertion that inherited mutations in FANCC can predispose to pancreatic cancer.


Context: The developmental pathways involved in horn development are complex and still poorly understood. Here we report the description of a new dominant inherited syndrome in the bovine Charolais breed that we have named type 2 scurs. Clinical examination revealed that, despite a strong phenotypic variability, all affected individuals show both horn abnormalities similar to classical scurs phenotype and skull interfrontal suture synostosis. Based on a genome-wide linkage analysis using Illumina BovineSNP50 BeadChip genotyping data from 57 half-sib and full-sib progeny, this locus was mapped to a 1.7 Mb interval on bovine chromosome 4. Within this region, the TWIST1 gene encoding a transcription factor was considered as a strong candidate gene since its haploinsufficiency is responsible for the human Saethre-Chotzen syndrome, characterized by skull coronal suture synostosis. Sequencing of the TWIST1 gene identified a c.148_157dup (p.A56RfsX87) frame-shift mutation predicted to completely inactivate this gene. Genotyping 17 scurred and 20 horned founders of our pedigree as well as 48 unrelated horned controls revealed a perfect association between this mutation and the type 2 scurs phenotype. Subsequent genotyping of 32 individuals born from heterozygous parents showed that homozygous mutated progeny are completely absent, which is consistent with the embryonic lethality reported in Drosophila and mouse suffering from TWIST1 complete insufficiency. Finally, data from previous studies on model species and a fine description of type 2 scurs symptoms allowed us to propose different mechanisms to explain the features of this syndrome. In conclusion, this first report on the identification of a potential causal mutation affecting horn development in cattle offers a unique opportunity to better understand horn ontogenesis.


Context: miRNAs and DNA methylation are both critical regulators of gene expression. Aberration in miRNA expression or DNA methylation is a causal factor for numerous pathological conditions. DNA methylation can inhibit the transcription of miRNAs, just like coding genes, by methylating the CpG islands in the promoter regions of miRNAs. Conversely, certain miRNAs can directly target DNA methyltransferases and bring about their inhibition, thereby affecting the whole genome methylation pattern. Recently, methylation patterns have also been revealed in mRNA. Surprisingly, the two most commonly studied methylation states in mRNA (m6A and m5C) are found to be enriched in 3'-UTRs (untranslated regions), the target site for the majority of miRNAs. Whereas m5C is reported to stabilise mRNA, m6A has a destabilising effect on mRNA. However, the effect of mRNA methylation on its interaction with miRNAs is largely unexplored. The review highlights the complex interplay between microRNA and methylation at DNA and mRNA level.


Context: Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B. anthracis, showing that it lies closer to a newly described "C" branch than to either of two previously described "A" or "B" branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.


Context: Eosinophils are important proinflammatory cells that make a major contribution to the inflammation seen in allergic diseases including asthma. Interleukin-5 is central to eosinophil maturation, release from the bone marrow, and subsequent accumulation, activation, and persistence in the tissues. Reslizumab (Cinquil™) is a humanized monoclonal antibody with potent interleukin-5 neutralizing effects, which represents a potential treatment for poorly controlled eosinophilic asthma. This review will consider the current status of the clinical development of reslizumab for asthma and in other inflammatory diseases with a marked eosinophilic component.


Context: Charcot-Marie-Tooth disease type 2D (CMT2D) is an autosomal-dominant axonal peripheral neuropathy characterized by impaired motor and sensory function in the distal extremities. Mutations in the glycyl-tRNA synthetase (GARS) gene cause CMT2D. GARS is a member of the ubiquitously expressed aminoacyl-tRNA synthetase (ARS) family and is responsible for charging tRNA with glycine. To date, 13 GARS mutations have been identified in patients with CMT disease. While functional studies have revealed loss-of-function characteristics, only four GARS mutations have been rigorously studied. Here, we report the functional evaluation of nine CMT-associated GARS mutations in tRNA charging, yeast complementation, and subcellular localization assays. Our results demonstrate that impaired function is a common characteristic of CMT-associated GARS mutations. Additionally, one mutation previously associated with CMT disease (p.Ser581Leu) does not demonstrate impaired function, was identified in the general population, and failed to segregate with disease in two newly identified families with CMT disease. Thus, we propose that this variant is not a disease-causing mutation. Together, our data indicate that impaired function is a key component of GARS-mediated CMT disease and emphasize the need for careful genetic and functional evaluation before implicating a variant in disease onset.


Context: Eukaryotic DNA replication is a complex process requiring the proper functioning of a multitude of proteins to create error-free daughter DNA strands and maintain genome integrity. Even though synthesis and joining of Okazaki fragments on the lagging strand involves only half the DNA in the nucleus, the complexity associated with processing these fragments is about twice that needed for leading strand synthesis. Flap endonuclease 1 (FEN1) is the central component of the Okazaki fragment maturation pathway. FEN1 cleaves flaps that are displaced by DNA polymerase delta (pol delta), to create a nick that is effectively joined by DNA ligase I. The Pif1 helicase and Dna2 helicase/nuclease contribute to the maturation process by elongating the flap displaced by pol delta. Though the reason for generating long flaps is still a matter of debate, genetic studies have shown that Dna2 and Pif1 are both important components of DNA replication. Our current knowledge of the exact enzymatic steps that govern Okazaki fragment maturation has heavily derived from reconstitution reactions in vitro, which have augmented genetic information, to yield current mechanistic models. In this review, we describe both the design of specific DNA substrates that simulate intermediates of fragment maturation and protocols for reconstituting partial and complete lagging strand replication.


Context: Preclinical studies have provided evidence that matrix metalloproteinases (MMPs), a family of zinc-containing proteolytic enzymes, facilitate tumor invasion, the establishment of metastases, and the promotion of tumor-related angiogenesis. Matrix metalloproteinase inhibitors (MMPIs) have been shown to inhibit tumor growth and dissemination in preclinical models. Not all lung cancers express the MMPs believed to be most important in promoting the neoplastic process, and there are conflicting reports regarding the prognostic significance of MMPs in lung cancer. However, it is possible that these observations are because of limitations in the procedures for measuring MMPs. Many investigators believe that MMPs are universally involved in tumor progression; this hypothesis was the basis for initiating seven phase III MMPI trials in lung cancer. Four studies were closed at completion of the predefined accrual goal, and three were closed early. There were no significant differences in survival in a non-small cell lung cancer prinomastat study, and in a small cell lung cancer marimastat trial. The results of the remaining five studies have not been reported. At this point it appears that MMPIs will probably not play a major role in the treatment of advanced lung cancer patients.


Context: Cancer cells can obtain their ability to invade and metastasise by undergoing epithelial-to-mesenchymal transition (EMT). Exploiting this mechanism of cellular plasticity, malignant cells can remodel their actin cytoskeleton and down-regulate proteins needed for cell-cell contacts. The mechanisms of cytoskeletal reorganisation resulting in mesenchymal morphology and increased invasive potential are poorly understood. Actin nucleating formins have been implicated as key players in EMT. Here, we analysed which formins are altered in squamous cell carcinoma related EMT. FHOD1, a poorly studied formin, appeared to be markedly upregulated upon EMT. In human tissues FHOD1 was primarily expressed in mesenchymal cells, with little expression in epithelia. However, specimens from oral squamous cell cancers demonstrated consistent FHOD1 upregulation in mesenchymally transformed cells at the invasive edge. This upregulation was confirmed in an oral squamous carcinoma model, where FHOD1 expression was markedly increased upon EMT in a PI3K signalling dependent manner. In the EMT cells FHOD1 contributed to the spindle-shaped morphology and mesenchymal F-actin organization. Furthermore, functional assays demonstrated that FHOD1 contributes to cell migration and invasion. Finally, FHOD1 depletion reduced the ability of EMT cancer cells to form invadopodia and to degrade extracellular matrix. Our results indicate that FHOD1 participates in cytoskeletal changes in EMT. In addition, we show that FHOD1 upregulation occurs during cancer cell EMT in vivo, which indicates that FHOD1 may contribute to tumour progression.


Context: Acupotomy showed promising results for some musculoskeletal disorders; however, additional high-quality evidence is required to make clinical recommendations regarding this procedure.


Context: Carnitine/organic cation transporter 2 (OCTN2) is localized at the basolateral membrane of epididymal epithelial cells, and mainly serves to reabsorb carnitine as an essential factor for sperm maturation; however, its functional features in epididymal epithelial cells have remained unclear. We isolated primary epididymal epithelial cells from rat epididymides and verified their phenotype by detecting the presence of cytokeratin-19 (CK-19, an epithelial cell marker) and the absence vimentin (an interstitial cell marker). We found that cultured epididymal epithelial cells isolated from rat epididymides expressed high levels of CK-19 but barely expressed vimentin. Gain-of-function assays, which included the CCK-8 assay and EdU flow cytometry assay, indicated that overexpression of OCTN2 significantly promoted epididymal epithelial cell growth and proliferation. Moreover, forced expression of OCTN2 inhibited the cell apoptosis process, and at the same time increased expression of the pro-apoptosis factor BAX, and decreased expression of the anti-apoptosis factors BCL-2 and Survivin. Furthermore, we also found that OCTN2 overexpression dramatically increased the levels of biomarkers associated with spermatogenesis, including azoospermia-like (DAZL), phosphoglycerate kinase 2 (PGK2), and protamine 2 (PRM2). These results demonstrate that OCTN2 plays a positive role in epididymal epithelial cells, and might be useful in the clinical treatment of male infertility by serving as a key regulatory factor.


Context: Epigenetic alterations are strongly associated with cancer development and drug resistance. The use of the DNA methylation inhibitor decitabine (Dacogen®) has been approved in the treatment of hematological malignancies, and its clinical effects on solid tumors have gained attention. Here, we present a review of the molecular regulation mechanisms, clinical experiences and biological evaluation for novel decitabine-based therapies in solid tumors. We also discuss the following questions: What is the best administration schedule of decitabine in solid tumors? Is there tumor type specificity for decitabine-based epigenetic therapy? What are the biological function and mechanism of decitabine in suppressing tumor development? Is there a correlation between DNA demethylation and clinical response? Importantly, low-dose decitabine and combined therapy show significant improvement in solid tumor treatment. However, the correlation studies are preliminary, and key biomarkers for prognosis need further investigation.


Context: Histone deacetylases (HDACs) influence diverse cellular processes and may contribute to tumor development and progression by multiple mechanisms. Class I HDACs are often overexpressed in cancers contributing to a genome-wide epigenetic state permitting increased proliferation, and diminished apoptosis and cell differentiation. Class IIA and IIB isoenzymes may likewise contribute to tumorigenesis as components of specific intranuclear repressor complexes or regulators of posttranslational protein modifications. As HDAC inhibitors may counteract these tumorigenic effects several of these compounds are currently tested in clinical trials. HDAC inhibitors are also considered for urothelial carcinoma, where novel therapeutic drugs are urgently required. However, only modest antineoplastic activity has been observed with isoenzyme-unspecific pan-HDAC inhibitors. Therefore, inhibition of specific HDAC isoenzymes might be more efficacious and tumor-specific. Here, we systematically review knowledge on the expression, function and suitability as therapeutic targets of the 11 classical HDACs in UC. Overall, the class I HDACs HDAC1 and HDAC2 are the most promising targets for antineoplastic treatment. In contrast, targeting HDAC8 and HDAC6 is likely to be of minor relevance in urothelial carcinoma. Class IIA HDACs like HDAC4 require further study, since their downregulation rather than upregulation could be involved in urothelial carcinoma pathogenesis.


Context: We present a case of Brooke-Spiegler syndrome with a germline deep intronic mutation in the CYLD gene leading to intronic exonization. Additionally, diverse somatic mutations were identified, namely loss of heterozygosity, a recurrent nonsense mutation, and a sequence mutation causing exon skipping. These somatic aberrations were identified in 4 different cylindromas that had been removed from the patient. Additionally, we microscopically studied a spiradenocylindroma that showed unusual histology, including foci of follicular differentiation. A deep intronic mutation resulting in exonization and a somatic sequence mutations causing exon skipping are hitherto unreported genetic mechanisms involving the CYLD gene in patients with Brooke-Spiegler syndrome.


Context: To clarify whether medical radiation exposure, especially from head computed tomography (CT), increases the risk of brain tumours in young patients in Japan, which ranks the second highest in the world in the number of paediatric CT examinations following the US. From 2011 to 2015, we performed a case-control study of 120 brain tumour patients and 360 appendicitis patients as controls. Reasons, the number of brain and head CT scans date were available from interviews. A cumulative radiation dose to the brain was calculated as a sum of doses received from head CT scans and from conventional X-rays and estimated using a reference table derived from a literature review of published studies. We performed conditional logistic regression to assess the risk of brain tumours from brain and head CT, and from conventional head X-ray procedures. The case group received on average 1.8 CTs to the brain area and 2.2 CTs to the whole head, with a mean estimated brain dose of 32 ±13 mGy. The odds ratio for developing a brain tumour from having a brain CT was 0.93 (95% confidence interval: 0.38-1.82). This was hardly altered when adjusting for parental educational history and for other diseases (history of neurological disease and attention-deficit disorder/attention-deficit hyperactivity disorder). Neither whole head CT nor cumulative brain dose to the brain increased the risk of glioma or of all brain tumours. Although this study conducted in Japan, where ranks second in the number of CT scans conducted in the world, did not show an increased risk of brain tumours related to CT scans, it should be taken with caution due to a case-control study with limited sample size.