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Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Centriolar satellites are small, microscopically visible granules that cluster around centrosomes. These structures, which contain numerous proteins directly involved in centrosome maintenance, ciliogenesis, and neurogenesis, have traditionally been viewed as vehicles for protein trafficking towards the centrosome. However, the recent identification of several new centriolar satellite components suggests that this model offers only an incomplete picture of their cellular functions. While the mechanisms controlling centriolar satellite status and function are not yet understood in detail, emerging evidence points to these structures as important hubs for dynamic, multi-faceted regulation in response to a variety of cues. In this review, we summarize the current knowledge of the roles of centriolar satellites in regulating centrosome functions, ciliogenesis, and neurogenesis. We also highlight newly discovered regulatory mechanisms targeting centriolar satellites and their functional status, and we discuss how defects in centriolar satellite components are intimately linked to a wide spectrum of human diseases.

Hepatic angiosarcomas are rare mesenchymal tumors with few case series and reports describing their imaging findings in the last two decades. The computed tomography and magnetic resonance imaging findings are variable and may appear like hemangioma at one end of the spectrum and hepatoma at the other end. The use of hepatocyte specific contrast on magnetic resonance imaging may also be insufficient in making a reliable imaging diagnosis. These tumors can easily mimic hypervascular liver cancer, atypical hemangioma or metastases. This case report highlights the imaging features of this entity and also underlines that, this tumor should always be considered as a differential diagnosis for hypervascular tumors in cirrhotic patients. The current imaging paradigms for its diagnosis are also discussed.

The comparison of DNA methylation patterns across cancer types (pan-cancer methylome analyses) has revealed distinct subgroups of tumors that share similar methylation patterns. Integration of these data with the wealth of information derived from cancer genome profiling studies performed by large international consortia has provided novel insights into the cellular aberrations that contribute to cancer development. There is evidence that genetic mutations in epigenetic regulators (such as DNMT3, IDH1/2 or H3.3) mediate or contribute to these patterns, although a unifying molecular mechanism underlying the global alterations of DNA methylation has largely been elusive. Knowledge gained from pan-cancer methylome analyses will aid the development of diagnostic and prognostic biomarkers, improve patient stratification and the discovery of novel druggable targets for therapy, and will generate hypotheses for innovative clinical trial designs based on methylation subgroups rather than on cancer subtypes. In this review, we discuss recent advances in the global profiling of tumor genomes for aberrant DNA methylation and the integration of these data with cancer genome profiling data, highlight potential mechanisms leading to different methylation subgroups, and show how this information can be used in basic research and for translational applications. A remaining challenge is to experimentally prove the functional link between observed pan-cancer methylation patterns, the associated genetic aberrations, and their relevance for the development of cancer.

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP(C)) into a pathological isoform termed PrP(Sc). In wild-type mice, PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP(Sc) typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrP(Sc) accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrP(C) anchoring to the plasma membrane correlated not only with the type of PrP(Sc) deposition but also with unique biochemical pathways associated with pathogenesis.

Saitohin gene found within the tau gene is thought to play a role in the pathogenesis of neurodegenerative diseases. The rs62063857 polymorphism originally found in the saitohin gene seems to be the responsible SNP in this event. This polymorphism is studied mostly in patients with Alzheimer's disease. Data on Parkinson's disease are scarce. Therefore, we examined the rs62063857 polymorphism in 583 Parkinson's disease patients (347 male and 236 female) and 396 healthy controls (238 male and 158 female) by a polymerase chain reaction and restriction fragment length polymorphism method to see whether it was associated with Parkinson's disease from the City of Istanbul, Turkey. The G allele frequency was 22 % in overall controls and 16 % in Parkinson's disease patients. In this study, the saitohin rs62063857 polymorphism was associated with Parkinson's disease (χ2 = 16.765; P = 0.000). Individuals with the AA genotype showed 1.7-fold increased risk for Parkinson's disease (χ2 = 16.680; P = 0.000), whereas individuals with the AG genotype revealed protection against Parkinson's disease (χ2 = 14.554; P = 0.000). After the stratification analysis according to gender, both male and female PD patients showed association with the alleles and genotypes of the rs62063857 polymorphism of the saitohin gene (χ2 = 9.476, P = 0.009; χ2 = 7.593, P = 0.022, respectively). When the Parkinson's patients were divided into two groups with regard to onset of the disease, both groups showed association with the disease. The Parkinson's patients with disease onset below 65 years of age showed 1.8-fold increased risk for the disease. The Parkinson's patients with disease onset over 65 showed more robust association with a 2.051-fold increased risk for the disease. Consequently, the rs62063857 polymorphism of the saitohin gene is a genetic risk factor for Parkinson's disease. Hence, this polymorphism may play a role in the etiology of Parkinson's disease.

ABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a β-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes.

Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as increased levels of SCFA's can epigenetically modulate cell function further supporting their role as environmental contributors to ASD.

NLRP3 is a key component of caspase-activating macromolecular protein complexes called inflammasomes. It has been found that DHX33 is a cytosolic dsRNA sensor for the NLRP3 inflammasome, which induces caspase-1-dependent production of IL-1β and IL-18 upon activation. However, how the cytosolic dsRNAs induce the interaction between DHX33 and the NLRP3 inflammasome remains unknown. In this study, we report that TRIM33, a member of the tripartite motif (TRIM) family, can bind DHX33 directly and induce DHX33 ubiquitination via the lysine 218 upon dsRNA stimulation. Knocking down of TRIM33 abolished the dsRNA-induced NLRP3 inflammasome activation in both THP-1-derived macrophages and human monocyte-derived macrophages. The ubiquitination of DHX33 by TRIM33 is lysine 63 specific and is required for the formation of the DHX33-NLRP3 inflammasome complex.

Crystal deposition in the cervical spine around the odontoid process may lead to acute neck pain. This rare condition is called crowned dens syndrome and should be considered in the differential diagnosis of a possible etiology for fever, headache and cervical pain of unknown origin. The syndrome is often overlooked, thus leading to misdiagnosis, invasive and useless investigations (lumbar puncture, biopsy), inappropriate treatment (steroids, antibiotics, antiviral drugs) and prolonged hospitalization. This can be prevented by imaging, based on a cervical CT scan that allows an accurate diagnosis. The disease has a good prognosis and symptoms usually subside within a few weeks. We describe a patient with crowned dens syndrome which manifested with clinical (acute occipital headache) and radiographic (calcium deposits in the alar ligament) features. Our patient recovered in four days with symptomatic therapy.

<h4>Background</h4>Previous studies have found interrelationships between the serotonin system and alcohol self-administration. The goal of this work was to directly observe in vivo effects of chronic ethanol self-administration on serotonin 5-HT1A receptor binding with [(18)F]mefway PET neuroimaging in rhesus monkeys. Subjects were first imaged alcohol-naïve and again during chronic ethanol self-administration to quantify changes in 5-HT1A receptor binding.<h4>Methods</h4>Fourteen rhesus monkey subjects (10.7-12.8 years) underwent baseline [(18)F]mefway PET scans prior to alcohol exposure. Subjects then drank gradually increasing ethanol doses over four months as an induction period, immediately followed by at least nine months ad libidum ethanol access. A post [(18)F]mefway PET scan was acquired during the final three months of ad libidum ethanol self-administration. 5-HT1A receptor binding was assayed with binding potential (BPND) using the cerebellum as a reference region. Changes in 5-HT1A binding during chronic ethanol self-administration were examined. Relationships of binding metrics with daily ethanol self-administration were also assessed.<h4>Results</h4>Widespread increases in 5-HT1A binding were observed during chronic ethanol self-administration, independent of the amount of ethanol consumed. A positive correlation between 5-HT1A binding in the raphe nuclei and average daily ethanol self-administration was also observed, indicating that baseline 5-HT1A binding in this region predicted drinking levels.<h4>Conclusions</h4>The increase in 5-HT1A binding levels during chronic ethanol self-administration demonstrates an important modulation of the serotonin system due to chronic alcohol exposure. Furthermore, the correlation between 5-HT1A binding in the raphe nuclei and daily ethanol self-administration indicates a relationship between the serotonin system and alcohol self-administration.

Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the genes encoding subunits of the phagocytic NADPH oxidase system. Patients can present with severe, recurrent infections and noninfectious conditions. Among the latter, inflammatory manifestations are predominant, especially granulomas and colitis. In this article, we systematically review the possible mechanisms of hyperinflammation in this rare primary immunodeficiency condition and their correlations with clinical aspects.

The devastating impact of congenital heart defects has made mechanisms of vertebrate heart and vascular development an active area of study. Because myocyte death is a common feature of acquired cardiovascular diseases and the adult heart does not regenerate, the need exists to understand whether features of the developing heart and vasculature-which are more plastic-can be exploited therapeutically in the disease setting. We know that a core network of transcription factors governs commitment to the cardiovascular lineage, and recent studies using genetic loss-of-function approaches and unbiased genomic studies have revealed the role for various chromatin modulatory events. We reason that chromatin structure itself is a causal feature that influences transcriptome complexity along a developmental continuum, and the purpose of this article is to highlight the areas in which 'omics technologies have the potential to reveal new principles of phenotypic plasticity in development. We review the major mechanisms of chromatin structural regulation, highlighting what is known about their actions to control cardiovascular differentiation. We discuss emergent mechanisms of regulation that have been identified on the basis of genomic and proteomic studies of cardiac nuclei and identify current challenges to an integrated understanding of chromatin structure and cardiovascular phenotype.

Endosomal entrapment is one of the main barriers that must be overcome for efficient gene expression along with cell internalization, DNA release, and nuclear import. Introducing pH-sensitive ionizable groups into the polycationic polymers to increase gene transfer efficiency has proven to be a useful method; however, a comparative study of introducing equal numbers of ionizable groups in both polymer and monomer forms, has not been reported. In this study, we prepared two types of histidine-grafted poly(L-lysine) (PLL), a stacking form of poly(L-histidine) (PLL-g-PHis) and a mono-L-histidine (PLL-g-mHis) with the same number of imidazole groups. These two types of histidine-grafted PLL, PLL-g-PHis and PLL-g-mHis, showed profound differences in hemolytic activity, cellular uptake, internalization, and transfection efficiency. Cy3-labeled PLL-g-PHis showed strong fluorescence in the nucleus after internalization, and high hemolytic activity upon pH changes was also observed from PLL-g-PHis. The arrangement of imidazole groups from PHis also provided higher gene expression than mHis due to its ability to escape the endosome. mHis or PHis grafting reduced the cytotoxicity of PLL and changed the rate of cellular uptake by changing the quantity of free ε-amines available for gene condensation. The subcellular localization of PLL-g-PHis/pDNA measured by YOYO1-pDNA intensity was highest inside the nucleus, while the lysotracker, which stains the acidic compartments was lowest among these polymers. Thus, the polymeric histidine arrangement demonstrate the ability to escape the endosome and trigger rapid release of polyplexes into the cytosol, resulting in a greater amount of pDNA available for translocation to the nucleus and enhanced gene expression.

The syndrome of cachexia, i.e., involuntary weight loss in patients with underlying diseases, sarcopenia, i.e., loss of muscle mass due to aging, and general muscle atrophy from disuse and/or prolonged bed rest have received more attention over the last decades. All lead to a higher morbidity and mortality in patients, and therefore, they represent a major socio-economic burden for the society today. This mini-review looks at recent developments in basic research that are relevant to the loss of skeletal muscle. It aims to cover the most significant publication of last 3 years on the causes and effects of muscle wasting, new targets for therapy development, and potential biomarkers for assessing skeletal muscle mass. The targets include the following: (1) E-3 ligases TRIM32, SOCS1, and SOCS3 by involving the elongin BC ubiquitin-ligase, Cbl-b, culling 7, Fbxo40, MG53 (TRIM72), and the mitochondrial Mul1; (2) the kinase MST1; and (3) the G-protein Gαi2. D(3)-creatine has the potential to be used as a novel biomarker that allows to monitor actual change in skeletal muscle mass over time. In conclusion, significant development efforts are being made by academic groups as well as numerous pharmaceutical companies to identify new target and biomarker muscles, as muscle wasting represents a great medical need, but no therapies have been approved in the last decades.

We previously demonstrated that the deletion of phospholipase C-related catalytically inactive protein-1/2 (PRIP-1/2) enhances the desensitization of GABAA receptors (GABAARs), while it facilitates their resensitization at the offset of GABA puff, causing a hump-like tail current (tail-I) in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex. In the present study, we investigated whether inhibitory synaptic transmission in L3 PCs in the barrel cortex is altered in the PRIP-1/2 double-knockout (PRIP-DKO) mice, and if so, how the interaction between excitation and inhibition is subsequently modified. PRIP-1/2 deletion resulted in the prolongation of the decay phase of inhibitory postsynaptic currents/potentials (IPSCs/IPSPs) in L3 PCs evoked by stimulation of L3, leaving the overall features of miniature IPSCs unchanged. An optical imaging revealed that the spatiotemporal profile of a horizontal excitation spread across columns in L2/3 caused by L4 stimulation in the barrel cortex was more restricted in PRIP-DKO mice compared to the wild type, while those obtained in the presence of bicuculline were almost identical between the two genotypes. These findings suggest that PRIP-1/2 deletion enhances the lateral inhibition by prolonging inhibitory synaptic actions to limit the intercolumnar integration in the barrel cortex. Considering the present findings together with our previous study including a mathematical simulation, the prolongation of inhibitory synaptic actions is likely to result from an enhancement of desensitization followed by an enhanced resensitization in GABAARs.

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca(2+)-activated Cl(-) channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca(2+) and voltage-dependent Cl(-) currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

<h4>Objectives</h4>Ventricular septal defect (VSD), one of the most common types of congenital heart disease (CHD), results from a combination of environmental and genetic factors. Recent studies demonstrated that microRNAs (miRNAs) are involved in development of CHD. This study was to characterize the expression of miRNAs that might be involved in the development or reflect the consequences of VSD.<h4>Methods</h4>MiRNA microarray analysis and reverse transcription-polymerase chain reaction (RT-PCR) were employed to determine the miRNA expression profile from 3 patients with VSD and 3 VSD-free controls. 3 target gene databases were employed to predict the target genes of differentially expressed miRNAs. miRNAs that were generally consensus across the three databases were selected and then independently validated using real time PCR in plasma samples from 20 VSD patients and 15 VSD-free controls. Target genes of validated 8 miRNAs were predicted using bioinformatic methods.<h4>Results</h4>36 differentially expressed miRNAs were found in the patients with VSD and the VSD-free controls. Compared with VSD-free controls, expression of 15 miRNAs were up-regulated and 21 miRNAs were downregulated in the VSD group. 15 miRNAs were selected based on database analysis results and expression levels of 8 miRNAs were validated. The results of the real time PCR were consistent with those of the microarray analysis. Gene ontology analysis indicated that the top target genes were mainly related to cardiac right ventricle morphogenesis. NOTCH1, HAND1, ZFPM2, and GATA3 were predicted as targets of hsa-let-7e-5p, hsa-miR-222-3p and hsa-miR-433.<h4>Conclusion</h4>We report for the first time the circulating miRNA profile for patients with VSD and showed that 7 miRNAs were downregulated and 1 upregulated when matched to VSD-free controls. Analysis revealed target genes involved in cardiac development were probably regulated by these miRNAs.

Vortioxetine (Brintellix(®), 1-[2-(2,4-dimethylphenyl-sulfanyl)-phenyl]-piperazine) is a multimodal antidepressant targeting the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT3, 5-HT7 receptors and the serotonin (5-HT) transporter (5-HTT). Vortioxetine administration induces antidepressant- and anxiolytic-like effects, and can enhance cognitive performance in rodents. Several clinical trials have reported the efficiency and a satisfactory tolerability of vortioxetine treatment in depressed patients. Remarkably, vortioxetine has a specific positive impact on cognitive symptoms in depressed patients. Overall, vortioxetine is an efficacious antidepressant drug for the treatment of patients with a major depressive episode and has a unique mechanism of action offering a new therapeutic option.

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by a CAG expansion mutation in HTT, the gene encoding huntingtin. Evidence from both human genotype-phenotype relationships and mouse model systems suggests that the mutation acts by dysregulating some normal activity of huntingtin. Recent work in the mouse has revealed a role for huntingtin in epigenetic regulation during development. Here, we examine the role of the Drosophila huntingtin ortholog (dhtt) in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, we found that dhtt acts as a suppressor of position-effect variegation (PEV), suggesting that it influences chromatin organization. We demonstrate that dhtt affects heterochromatin spreading in a PEV model by modulating histone H3K9 methylation levels at the heterochromatin-euchromatin boundary. To gain mechanistic insights into how dhtt influences chromatin function, we conducted a candidate genetic screen using RNAi lines targeting known PEV modifier genes. We found that dhtt modifies phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases (HDMs) and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the HDM dLsd1, in both the ovary and wing, and we demonstrate that dhtt appears to act as a facilitator of dLsd1 function in regulating global histone H3K4 methylation levels. These findings suggest that a fundamental aspect of huntingtin function in heterochromatin/euchromatin organization is evolutionarily conserved across phyla.

Silica (SiO2) nanoparticles (NPs) have found extensive applications in industrial manufacturing, biomedical and biotechnological fields. Therefore, the increasing exposure to such ultrafine particles requires studies to characterize their potential cytotoxic effects in order to provide exhaustive information to assess the impact of nanomaterials on human health. The understanding of the biological processes involved in the development and maintenance of a variety of pathologies is improved by genome-wide approaches, and in this context, gene set analysis has emerged as a fundamental tool for the interpretation of the results. In this work we show how the use of a combination of gene-by-gene and gene set analyses can enhance the interpretation of results of in vitro treatment of A549 cells with Ludox® colloidal amorphous silica nanoparticles. By gene-by-gene and gene set analyses, we evidenced a specific cell response in relation to NPs size and elapsed time after treatment, with the smaller NPs (SM30) having higher impact on inflammatory and apoptosis processes than the bigger ones. Apoptotic process appeared to be activated by the up-regulation of the initiator genes TNFa and IL1b and by ATM. Moreover, our analyses evidenced that cell treatment with LudoxÒ silica nanoparticles activated the matrix metalloproteinase genes MMP1, MMP10 and MMP9. The information derived from this study can be informative about the cytotoxicity of Ludox® and other similar colloidal amorphous silica NPs prepared by solution processes.

The green anole, Anolis carolinensis (ACA), is the model reptile for a vast array of biological disciplines. It was the first nonavian reptile to have its genome fully sequenced. During the genome project, the XX/XY system of sex chromosomes homologous to chicken chromosome 15 (GGA15) was revealed, and 106 X-linked genes were identified. We selected 38 genes located on eight scaffolds in ACA and having orthologs located on GGA15, then tested their linkage to ACA X chromosome by using comparative quantitative fluorescent real-time polymerase chain reaction applied to male and female genomic DNA. All tested genes appeared to be X-specific and not present on the Y chromosome. Assuming that all genes located on these scaffolds should be localized to the ACA X chromosome, we more than doubled the number of known X-linked genes in ACA, from 106 to 250. While demonstrating that the gene content of chromosome X in ACA and GGA15 is largely conserved, we nevertheless showed that numerous interchromosomal rearrangements had occurred since the splitting of the chicken and anole evolutionary lineages. The presence of many ACA X-specific genes localized to distinct contigs indicates that the ACA Y chromosome should be highly degenerated, having lost a large amount of its original gene content during evolution. The identification of novel genes linked to the X chromosome and absent on the Y chromosome in the model lizard species contributes to ongoing research as to the evolution of sex determination in reptiles and provides important information for future comparative and functional genomics.

Ras-driven cancer cells upregulate basal autophagy that degrades and recycles intracellular proteins and organelles. Autophagy-mediated proteome degradation provides free amino acids to support metabolism and macromolecular synthesis, which confers a survival advantage in starvation and promotes tumorigenesis. While the degradation of isolated protein substrates by autophagy has been implicated in controlling cellular function, the extent and specificity by which autophagy remodels the cellular proteome and the underlying functional consequences were unknown. Here we compared the global proteome of autophagy-functional and -deficient Ras-driven cancer cells, finding that autophagy affects the majority of the proteome yet is highly selective. While levels of vesicle trafficking proteins important for autophagy are preserved during starvation-induced autophagy, deleterious inflammatory response pathway components are eliminated even under basal conditions, preventing cytokine-induced paracrine cell death. This reveals the global, functional impact of autophagy-mediated proteome remodeling on cell survival and identifies critical autophagy substrates that mediate this process.

Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.

The striatum, a major component of the basal ganglia, performs multiple functions including control of movement, reward, and addiction. Dysfunction and death of striatal neurons are the main causes for the motor disorders associated with Huntington's disease (HD). Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is among factors that promote survival and proper function of this neuronal population. Here, we review recent studies showing that BDNF determines the size of the striatum by supporting survival of the immature striatal neurons at their origin, promotes maturation of striatal neurons, and facilitates establishment of striatal connections during brain development. We also examine the role of BDNF in maintaining proper function of the striatum during adulthood, summarize the mechanisms that lead to a deficiency in BDNF signaling and subsequently striatal degeneration in HD, and highlight a potential role of BDNF as a therapeutic target for HD treatment.

Iron is the most abundant metal in the human body and mainly works as a cofactor for proteins such as hemoglobin and various enzymes. No independent life forms on earth can survive without iron. However, excess iron is intimately associated with carcinogenesis by increasing oxidative stress via its catalytic activity to generate hydroxyl radicals. Biomolecules with redox-active sulfhydryl function(s) (thiol compounds) are necessary for the maintenance of mildly reductive cellular environments to counteract oxidative stress, and for the execution of redox reactions for metabolism and detoxification. Involvement of glutathione S-transferase and thioredoxin has long attracted the attention of cancer researchers. Here, I update recent findings on the involvement of iron and thiol compounds during carcinogenesis and in cancer cells. It is now recognized that the cystine/glutamate transporter (antiporter) is intimately associated with ferroptosis, an iron-dependent, non-apoptotic form of cell death, observed in cancer cells, and also with cancer stem cells; the former with transporter blockage but the latter with its stabilization. Excess iron in the presence of oxygen appears the most common known mutagen. Ironically, the persistent activation of antioxidant systems via genetic alterations in Nrf2 and Keap1 also contributes to carcinogenesis. Therefore, it is difficult to conclude the role of iron and thiol compounds as friends or foes, which depends on the quantity/distribution and induction/flexibility, respectively. Avoiding further mutation would be the most helpful strategy for cancer prevention, and myriad of efforts are being made to sort out the weaknesses of cancer cells.

The availability of ultra-high throughput DNA and RNA sequencing technologies in recent years has led to the identification of numerous novel transcripts, whose functions are unknown as yet. Evidence is accumulating that many of these molecules are deregulated in diseases, including prostate cancer, and potentially represent novel targets for diagnosis and therapy. In particular, functional genomic analysis of microRNA (miRNA) and long noncoding RNA (lncRNA) in cancer is likely to contribute insights into tumor development. Here, we compile recent efforts to catalog differential expression of miRNA and lncRNA in prostate cancer and to understand RNA function in tumor progression. We further highlight technologies for molecular characterization of noncoding RNAs and provide an overview of current activities to exploit them for the diagnosis and therapy of this complex tumor.

<h4>Background</h4>Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.<h4>Results</h4>Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167).<h4>Conclusion</h4>Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

<h4>Background</h4>Sertoli-Leydig cell tumor (SLCT) is a rare sex-cord tumor that usually occurs unilaterally and accounts for < 0.5% of all ovarian tumors. SLCT is uncommon in post-menopausal women, with the average age of diagnosis being 25 years.<h4>Case</h4>We present a case of a 63-year-old post-menopausal woman presenting with progressive hirsutism, and male-pattern baldness. Unusual nail changes were also observed.<h4>Methods</h4>Hormonal profile of the patient revealed increased testosterone and estradiol levels, and a 3.5 cm left ovarian mass. The patient was evaluated and was not found to be anemic or iron-deficient. Intraoperative frozen section assessment during laparoscopic exploration revealed SLCT, which was confirmed subsequently by histopathological and immunohistochemical (IHC) examination. Nail bed tissues were collected from normal females and evaluated by IHC for the presence of androgen receptors (AR).<h4>Results</h4>The patient had an excellent postoperative course and all her testosterone-related manifestations were reversed within one year of surgery. Following surgery, the patient's unique nail abnormalities also resolved gradually. The IHC evaluation also confirmed the presence of AR in nail bed tissues of females.<h4>Conclusion</h4>SLCT, albeit rare, should be considered in post-menopausal women presenting with virilization and elevated androgen levels. Unusual nail signs may develop in response to increased androgen levels in these patients.

Molecular docking studies were performed on 18 17β-carboxamide steroids in order to select compounds with potential local anti-inflammatory activity. These derivatives are amides of cortienic acids (obtained from hydrocortisone, prednisolone, and methylprednisolone) with methyl or ethyl esters of six amino acids. Interactions with the glucocorticoid receptor (GR), binding energies and ligand efficiency values of these compounds were compared with dexamethasone and cortienic acid obtained from prednisolone (inactive metabolite). On the basis of molecular docking studies, seven compounds were selected and their binding affinities for the GR were predicted by use of the exponential model created in this study. Subsequently, selected compounds were synthesized in good yields by use of modified N,N'-dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) coupling procedure. Finally, the local anti-inflammatory activity of the synthesized compounds was examined by use of the croton oil-induced ear edema test. In vivo evaluation of systemic side effects as well as in silico prediction of metabolism were performed on the derivative with the best local anti-inflammatory activity. The combination of molecular docking studies and the exponential model for the GR binding affinity prediction could be used as an in silico tool for the rational design of novel 17β-carboxamide steroids with potentially better biological profile than dexamethasone.

Only a small proportion of genetic variation in serum ferritin has been explained by variant genetic studies, and genome-wide association study (GWAS) for serum ferritin has not been investigated widely in Chinese population. We aimed at exploring the novel genetic susceptibility to serum ferritin, and performed this two stage GWAS in a healthy Chinese population of 3,495 men aged 20-69 y, including 1,999 unrelated subjects in the first stage and 1,496 independent individuals in the second stage. Serum ferritin was measured with electrochemiluminescence immunoassay, and DNA samples were collected for genotyping. A total of 1,940,243 SNPs were tested by using multivariate linear regression analysis. After adjusting for population stratification, age and BMI, the rs5742933 located in the 5'UTR region of PMS1 gene on chromosome 2 was the most significantly associated with ferritin concentrations (P-combined  = 2.329×10(-10)) (β  =  -0.11, 95% CI: -0.14, -0.07). Moreover, this marker was about 200 kb away from the candidate gene SLC40A1 which is responsible for iron export. PMS1 gene was the novel genetic susceptibility to serum ferritin in Chinese males and its relation to SLC40A1 needs further study.

The research on colorectal cancer (CRC) biology has been leading the oncology field since the early 1990s. The search for genetic alterations has allowed the identification of the main tumour suppressors or oncogenes. Recent work obtained in CRC has unexpectedly proposed the existence of novel category of tumour suppressors, the so-called 'dependence receptors'. These transmembrane receptors behave as Dr Jekyll and Mr Hyde with two opposite sides: they induce a positive signalling (survival, proliferation, differentiation) in presence of their ligand, but are not inactive in the absence of their ligand and rather trigger apoptosis when unbound. This trait confers them a conditional tumour suppressor activity: they eliminate cells that grow abnormally in an environment offering a limited quantity of ligand. This review will describe how receptors such as deleted in colorectal carcinoma (DCC), uncoordinated 5 (UNC5), rearranged during transfection (RET) or TrkC constrain CRC progression and how this dependence receptor paradigm may open up therapeutical perspectives.

The idea to lower mutant huntingtin is especially appealing in Huntington's disease (HD). It is autosomal dominant, so that expression of the mutant allele causes the disease. Advances in RNA and gene regulation provide foundations for the huntingtin gene (both normal and mutant alleles) and possibly the mutant allele only. There is much preclinical animal work to support the concept of gene and RNA silencing, but, to date, no clinical studies have been attempted in HD. Preventing expression of mutant huntingtin protein is at the cusp for a human trial. Antisense oligonucleotides delivered to patients with amyotrophic lateral sclerosis have been well tolerated; small RNAs administered to rodent and nonhuman primate brain knocked down huntingtin messenger RNA (mRNA); short-hairpin complementary DNA of microRNAs can be expressed in adeno-associated virus to provide long-term silencing of huntingtin mRNA and protein. We expect that these approaches will be ready for clinical studies in the near future, once safety has been validated. Our understanding of gene editing-changing the huntingtin gene itself-is rapidly progressing. Harnessing our knowledge of transcription and translation should push scientific creativity to new and exciting advances that overcome the lethality of the mutant gene in HD.

In genetically-modified Lmx1b(f/f/p) mice, selective deletion of LMX1B in Pet-1 expressing cells leads to failure of embryonic development of serotonin (5-HT) neurons. As adults, these mice have a decreased hypercapnic ventilatory response and abnormal thermoregulation. This mouse model has been valuable in defining the normal role of 5-HT neurons, but it is possible that developmental compensation reduces the severity of observed deficits. Here we studied mice genetically modified to express diphtheria toxin receptors (DTR) on Pet-1 expressing neurons (Pet-1-Cre/floxed DTR or Pet1/DTR mice). These mice developed with a normal complement of 5-HT neurons. As adults, systemic treatment with 2-35μg of diphtheria toxin (DT) reduced the number of tryptophan hydroxylase-immunoreactive (TpOH-ir) neurons in the raphe nuclei and ventrolateral medulla by 80%. There were no effects of DT on minute ventilation (VE) or the ventilatory response to hypercapnia or hypoxia. At an ambient temperature (TA) of 24°C, all Pet1/DTR mice dropped their body temperature (TB) below 35°C after DT treatment, but the latency was shorter in males than females (3.0±0.37 vs. 4.57±0.29days, respectively; p<0.001). One week after DT treatment, mice were challenged by dropping TA from 37°C to 24°C, which caused TB to decrease more in males than in females (29.7±0.31°C vs. 33.0±1.3°C, p<0.01). We conclude that the 20% of 5-HT neurons that remain after DT treatment in Pet1/DTR mice are sufficient to maintain normal baseline breathing and a normal response to CO2, while those affected include some essential for thermoregulation, in males more than females. In comparison to models with deficient embryonic development of 5-HT neurons, acute deletion of 5-HT neurons in adults leads to a greater defect in thermoregulation, suggesting that significant developmental compensation can occur.

A relationship has been shown between preeclampsia (PE) and intrauterine growth restriction (IUGR) and oxidative stress (OS). Since such pregnancies experience OS, we aimed to detect the distribution pattern and expression levels of a transcription factor, Nuclear factor erythroid 2-related factor-2 (Nrf2) that has a role in the regulation of antioxidant enzymes, and peroxiredoxin 6 (Prdx6) an antioxidant enzyme, in human healthy, IUGR, PE and in groups of rat healthy and IUGR placentas using immunohistochemistry and Western blotting. Both Nrf2 and Prdx6 immunoreactivities were weaker in human and rat IUGR group placentas compared to human and rat control group placentas, respectively. Nrf2 and Prdx6 were immunostained in labyrinth trophoblasts, decidua, giant, glycogen and fetal vessel endothelial cells in rat control and IUGR group placentas. Nrf2 and Prdx6 immunoreactivities were seen in the decidua, syncytiotrophoblasts, villous stromal cells, and vascular endothelium in human control, IUGR and PE group placentas. Results of Nrf2 and Prdx6 Western blotting applied for rat and human placentas were compatible with the results of Nrf2 and Prdx6 immunohistochemical observations with regard to rat and human placentas. Down-regulation of Nrf2 and Prdx6 proteins in human and rat IUGR group placentas may have led to the formation of OS which may have impaired proliferation and invasion of cytotrophoblasts.

Limb synovial joints are intricate structures composed of articular cartilage, synovial membranes, ligaments and an articular capsule. Together, these tissues give each joint its unique shape, organization and biomechanical function. Articular cartilage itself is rather complex and organized in distinct zones, including the superficial zone that produces lubricants and contains stem/progenitor cells. For many years there has been great interest in deciphering the mechanisms by which the joints form and come to acquire such unique structural features and diversity. Decades ago, classic embryologists discovered that the first overt sign of joint formation at each prescribed limb site was the appearance of a dense and compact population of mesenchymal cells collectively called the interzone. Work carried out since then by several groups has provided evidence that the interzone cells actively participate in joint tissue formation over developmental time. This minireview provides a succinct but comprehensive description of the many important recent advances in this field of research. These include studies using various conditional reporter mice to genetically trace and track the origin, fate and possible function of joint progenitor cells; studies on the involvement and roles in signaling pathways and transcription factors in joint cell determination and functioning; and studies using advanced methods of gene expression analyses to uncover novel genetic determinants of joint formation and diversity. The overall advances are impressive, and the findings are not only of obvious interest and importance but also have major implications in the conception of future translational medicine tools to repair and regenerate defective, overused or aging joints.

17Alpha-ethinylestradiol (EE2), used for birth control in humans, is a potent estrogen that is found in wastewater at low concentrations (ng/l). EE2 has the ability to interfere with the endocrine system of fish, affecting reproduction which can result in population level effects. The objective of this study was to determine if dietary exposure to EE2 would alter gene expression patterns and key pathways in the liver and ovary and whether these could be associated with reproductive endpoints in female largemouth bass during egg development. Female LMB received 70ng EE2/g feed (administered at 1% of body weight) for 60 days. EE2 dietary exposure significantly reduced plasma vitellogenin concentrations by 70%. Hepatosomatic and gonadosomatic indices were also decreased with EE2 feeding by 38.5% and 40%, respectively. Transcriptomic profiling revealed that there were more changes in steady state mRNA levels in the liver compared to the ovary. Genes associated with reproduction were differentially expressed, such as vitellogenin in the liver and aromatase in the gonad. In addition, a set of genes related with oxidative stress (e.g. glutathione reductase and glutathione peroxidase) were identified as altered in the liver and genes associated with the immune system (e.g. complement component 1, and macrophage-inducible C-type lectin) were altered in the gonad. In a follow-up study with 0.2ng EE2/g feed for 60 days, similar phenotypic and gene expression changes were observed that support these findings with the higher concentrations. This study provides new insights into how dietary exposure to EE2 interferes with endocrine signaling pathways in female LMB during a critical period of reproductive oogenesis.

Periodontal repair is a complex process in which regeneration of alveolar bone is a vital component. The aim of this study was to develop a biodegradable scaffold with good biocompatibility and osteoinductive ability. Two types of composite fibrous scaffolds were produced by electrospinning, ie, type I collagen/poly(ε-caprolactone) (COL/PCL) and type I collagen/poly(ε-caprolactone)/nanoscale hydroxyapatite (COL/PCL/nHA) with an average fiber diameter of about 377 nm. After a simulated body fluid (SBF) immersion test, the COL/PCL/nHA-SBF scaffold developed a rough surface because of the calcium phosphate deposited on the fibers, suggesting that the presence of nHA promoted the mineralization potential of the scaffold. Energy dispersive X-ray spectroscopy clearly showed the calcium and phosphorus content in the COL/PCL/nHA and COL/PCL/nHA-SBF scaffolds, confirming the findings of nHA and calcium phosphate precipitation on scanning electron micrographs. Water contact analysis revealed that nHA could improve the hydrophilic nature of the COL/PCL/nHA-SBF scaffold. The morphology of periodontal ligament cells cultured on COL/PCL-SBF and COL/PCL/nHA-SBF was evaluated by scanning electron microscopy. The results showed that cells adhered to either type of scaffold and were slightly spindle-shaped in the beginning, then extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has good biocompatibility and osteoinductive ability as a result of the characteristics of nHA, so could be innovatively applied to periodontal tissue engineering as a potential scaffold.

Serotonin type 7 receptors (5-HT7) are expressed in several brain areas, regulate brain development, synaptic transmission and plasticity, and therefore are involved in various brain functions such as learning and memory. A number of studies suggest that 5-HT7 receptors could be potential pharmacotherapeutic target for cognitive disorders. Several abnormalities of serotonergic system have been described in patients with autism spectrum disorder (ASD), including abnormal activity of 5-HT transporter, altered blood and brain 5-HT levels, reduced 5-HT synthesis and altered expression of 5-HT receptors in the brain. A specific role for 5-HT7 receptors in ASD has not yet been demonstrated but some evidence implicates their possible involvement. We have recently shown that 5-HT7 receptor activation rescues hippocampal synaptic plasticity in a mouse model of Fragile X Syndrome, a monogenic cause of autism. Several other studies have shown that 5-HT7 receptors modulate behavioral flexibility, exploratory behavior, mood disorders and epilepsy, which include core and co-morbid symptoms of ASD. These findings further suggest an involvement of 5-HT7 receptors in ASD. Here, we review the physiological roles of 5-HT7 receptors and their implications in Fragile X Syndrome and other ASD.

Iron is an essential mineral needed for normal physiologic processes. While its function in oxygen transport and other important physiologic processes is well known, less is understood about its role in inflammatory diseases such as atherosclerosis. Existing paradigms suggest iron as a driver of atherosclerosis through its actions as a pro-oxidant capable of causing lipid oxidation and tissue damage. Recently we and others have identified hemoglobin (Hb) derived iron as an important factor in determining macrophage differentiation and function in areas of intraplaque hemorrhage within human atherosclerosis. Hb associated macrophages, M(Hb), are distinct from traditional macrophage foam cells because they do not contain large amounts of lipid or inflammatory cytokines, are characterized by high levels of expression of mannose receptor (CD206) and CD163 in addition to producing anti-inflammatory cytokines such as IL-10. Despite the well-known role of iron as an catalyst capable of producing lipid peroxidation through generation of reactive oxygen species (ROS) such as hydroxyl radical, we and others have shown that macrophages in areas of intraplaque hemorrhage demonstrate reduced intracellular iron and ROS which triggers production of anti-inflammatory cytokines as well as genes involved in cholesterol efflux. These data suggest that manipulation of macrophage iron itself may be a promising pharmacologic target for atherosclerosis prevention through its effects on macrophage inflammation and lipid metabolism. In this review we will summarize the current understanding of iron as it relates to plaque inflammation and discuss how further exploration of this subject may lead to new therapies for atherosclerosis.

Cell-free nucleic acids (CFNA) have been reported by several authors in blood, stool, and urine of patients with colorectal cancer (CRC). These genetic biomarkers can be an indication of neoplastic colorectal epithelial cells, and can thus potentially be used as noninvasive tests for the detection of the disease in CRC patients and monitor their staging, without the need to use heavier and invasive tools. In a number of test-trials, these genetic tests have shown the advantage of non-invasiveness, making them well accepted by most of the patients, without major side effects. They have also shown a promising sensitivity and specificity in the detection of malignant and premalignant neoplasms. Moreover, costs for performing such tests are very low. Several studies reported and confirmed the proof of the principle for these genetic tests for screening, diagnosis, and prognosis; the main challenge of translating this approach from research to clinical laboratory is the validation from large and long-term randomized trials to prove sustainable high sensitivity and specificity. In this paper, we present a review on the noninvasive genetics biomarkers for CRC detection described in the literature and the challenges that can be encountered for validation processes.

Systems biology has gained a tremendous amount of interest in the last few years. This is partly due to the realization that traditional approaches focusing only on a few molecules at a time cannot describe the impact of aberrant or modulated molecular environments across a whole system. Furthermore, a hypothesis-driven study aims to prove or disprove its postulations, whereas a hypothesis-free systems approach can yield an unbiased and novel testable hypothesis as an end-result. This latter approach foregoes assumptions which predict how a biological system should react to an altered microenvironment within a cellular context, across a tissue or impacting on distant organs. Additionally, re-use of existing data by systematic data mining and re-stratification, one of the cornerstones of integrative systems biology, is also gaining attention. While tremendous efforts using a systems methodology have already yielded excellent results, it is apparent that a lack of suitable analytic tools and purpose-built databases poses a major bottleneck in applying a systematic workflow. This review addresses the current approaches used in systems analysis and obstacles often encountered in large-scale data analysis and integration which tend to go unnoticed, but have a direct impact on the final outcome of a systems approach. Its wide applicability, ranging from basic research, disease descriptors, pharmacological studies, to personalized medicine, makes this emerging approach well suited to address biological and medical questions where conventional methods are not ideal.

<h4>Background</h4>Hepatocellular carcinoma (HCC) is a heterogeneous disease with high mortality rate. Recent genomic studies have identified TP53, AXIN1, and CTNNB1 as the most frequently mutated genes. Lower frequency mutations have been reported in ARID1A, ARID2 and JAK1. In addition, hepatitis B virus (HBV) integrations into the human genome have been associated with HCC.<h4>Results</h4>Here, we deep-sequence 42 HCC patients with a combination of whole genome, exome and transcriptome sequencing to identify the mutational landscape of HCC using a reasonably large discovery cohort. We find frequent mutations in TP53, CTNNB1 and AXIN1, and rare but likely functional mutations in BAP1 and IDH1. Besides frequent hepatitis B virus integrations at TERT, we identify translocations at the boundaries of TERT. A novel deletion is identified in CTNNB1 in a region that is heavily mutated in multiple cancers. We also find multiple high-allelic frequency mutations in the extracellular matrix protein LAMA2. Lower expression levels of LAMA2 correlate with a proliferative signature, and predict poor survival and higher chance of cancer recurrence in HCC patients, suggesting an important role of the extracellular matrix and cell adhesion in tumor progression of a subgroup of HCC patients.<h4>Conclusions</h4>The heterogeneous disease of HCC features diverse modes of genomic alteration. In addition to common point mutations, structural variations and methylation changes, there are several virus-associated changes, including gene disruption or activation, formation of chimeric viral-human transcripts, and DNA copy number changes. Such a multitude of genomic events likely contributes to the heterogeneous nature of HCC.

Cholesterol-conjugated 5-fluorouracil prodrugs were designed to be carried in vivo via low density lipoproteins (LDL) and subsequently undergo LDL-receptor-mediated internalisation into cancer cells. In vivo anti-cancer evaluation was performed using 5-fluorouracil-cholesterol conjugate in a mouse model. The obtained prodrugs were more potent than 5-fluorouracil control drug at the same 5-fluorouracil content (3 mg·kg-1).

Cancer cells contain multiple genetic and epigenetic changes. The relative specificity of many epigenetic changes for neoplastic cells has allowed the identification of diagnostic, prognostic and predictive biomarkers for a number of solid tumors and hematological malignancies. Moreover, epigenetically-acting drugs are already in routine use for cancer and numerous additional agents are in clinical trials. Here, we review recent progress in the development and application of epigenetic strategies for the diagnosis, risk stratification and treatment of cancer.

Neurotrophins play a crucial role in mediating neuronal survival and synaptic plasticity. A lack of trophic factor support in the peripheral nervous system (PNS) is associated with a transcription-dependent programmed cell death process in developing sympathetic neurons. While most of the attention has been on events culminating in cell death in the PNS, the earliest events that occur after trophic factor withdrawal in the central nervous system (CNS) have not been investigated. In the CNS, brain-derived neurotrophic factor (BDNF) is widely expressed and is released in an activity-dependent manner to shape the structure and function of neuronal populations. Reduced neurotrophic factor support has been proposed as a mechanism to account for changes in synaptic plasticity during neurodevelopment to aging and neurodegenerative disorders. To this end, we performed transcriptional profiling in cultured rat hippocampal neurons. We used a TrkB ligand scavenger (TrkB-FC ) to sequester endogenous neurotrophic factor activity from hippocampal neurons in culture. Using a high-density microarray platform, we identified a significant decrease in genes that are associated with vesicular trafficking and synaptic function, as well as selective increases in MAP kinase phosphatases. A comparison of these changes with recent studies of Alzheimer's disease and cognitive impairment in postmortem brain tissue revealed striking similarities in gene expression changes for genes involved in synaptic function. These changes are relevant to a wide number of conditions in which levels of BDNF are compromised.

A flexible synthesis of dibenzo[e,g]isoindol-1-ones has been developed. Dibenzo[e,g]isoindol-1-ones represent simplified benzenoid analogues of biological indolo[2,3-a]pyrrolo[3,4-c]carbazol-5-ones (indolocarbazoles), compounds that have demonstrated a wide range of biological activity. The synthesis of the title compounds involved tetramic acid sulfonates. Different aryl groups were introduced at C4 of the heterocyclic ring via Suzuki-Miyaura cross-coupling reactions. Finally, mild Scholl-type oxidative cyclizations mediated by phenyliodine(III) bis(trifluoroacetate) (PIFA) converted some of the latter compounds into the corresponding dibenzo[e,g]isoindol-1-ones. A systematic study of the oxidative cyclization revealed the following reactivity trend: 3,4-dimethoxyphenyl ≫ 3-methoxyphenyl > 3,4,5-trimethoxyphenyl > 4-methoxyphenyl ≈ phenyl. Overall, the oxidative cyclization required at least two methoxy groups distributed in the aromatic rings, at least one of which had to be located para to the site of the cyclization.

Polycations are explored as carriers to deliver therapeutic nucleic acids. Polycations are conventionally pharmacological inert with the sole function of delivering therapeutic cargo. This study reports synthesis of a self-immolative polycation (DSS-BEN) based on a polyamine analogue drug N(1),N(11)-bisethylnorspermine (BENSpm). The polycation was designed to function dually as a gene delivery carrier and a prodrug targeting dysregulated polyamine metabolism in cancer. Using a combination of NMR and HPLC, we confirm that the self-immolative polycation undergoes intracellular degradation into the parent drug BENSpm. The released BENSpm depletes cellular levels of spermidine and spermine and upregulates polyamine catabolic enzymes spermine/spermidine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO). The synthesized polycations form polyplexes with DNA and facilitate efficient transfection. Taking advantage of the ability of BENSpm to sensitize cancer cells to TNFα-induced apoptosis, we show that DSS-BEN enhances the cell killing activity of TNFα gene therapy. The reported findings validate DSS-BEN as a dual-function delivery system that can deliver a therapeutic gene and improve the outcome of gene therapy as a result of the intracellular degradation of DSS-BEN to BENSpm and the subsequent beneficial effect of BENSpm on dysregulated polyamine metabolism in cancer.

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1β, IL-6, TGF-β, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-β) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.

Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.

The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

The known genetic cause of Huntington's disease (HD) has fueled considerable progress in understanding its pathobiology and the development of therapeutic approaches aimed at correcting specific changes linked to the causative mutation. Among the most promising is reducing expression of mutant huntingtin protein (mHTT) with RNA interference or antisense oligonucleotides; human trials are now being planned. Zinc-finger transcriptional repression is another innovative method to reduce mHTT expression. Modulation of mHTT phosphorylation, chaperone upregulation, and autophagy enhancement represent attempts to alter cellular homeostasis to favor removal of mHTT. Inhibition of histone deacetylases (HDACs) remains of interest; recent work affirms HDAC4 as a target but questions the assumed centrality of its catalytic activity in HD. Phosphodiesterase inhibition, aimed at restoring synaptic function, has progressed rapidly to human trials. Deranged cellular signaling provides several tractable targets, but specificity and complexity are challenges. Restoring neurotrophic support in HD remains a key potential therapeutic approach. with several approaches being pursued, including brain-derived neurotrophic factor (BDNF) mimesis through tyrosine receptor kinase B (TrkB) agonism and monoclonal antibodies. An increasing understanding of the role of glial cells in HD has led to several new therapeutic avenues, including kynurenine monooxygenase inhibition, immunomodulation by laquinimod, CB2 agonism, and others. The complex metabolic derangements in HD remain under study, but no clear therapeutic strategy has yet emerged. We conclude that many exciting therapeutics are progressing through the development pipeline, and combining a better understanding of HD biology in human patients, with concerted medicinal chemistry efforts, will be crucial for bringing about an era of effective therapies.

Vibrio vulnificus causes rapidly progressing septicemia with an extremely high mortality rate (≥50%), even with aggressive antibiotic treatment. The bacteria secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins, which are involved in the pathogenesis of Gram-negative Vibrio species. Recently, we reported that immunization with the C-terminal region of V. vulnificus RtxA1/MARTXVv, RtxA1-C, elicits a protective immune response against V. vulnificus through a poorly defined mechanism. In this study, we generated a panel of new monoclonal antibodies (MAbs) against V. vulnificus RtxA1-C and investigated their protective efficacies and mechanisms in a mouse model of infection. Prophylactic administration of seven MAbs strongly protected mice against lethal V. vulnificus infection (more than 90% survival). Moreover, three of these MAbs (21RA, 24RA, and 47RA) demonstrated marked efficacy as postexposure therapy. Notably, 21RA was therapeutically effective against lethal V. vulnificus infection by a variety of routes. Using Fab fragments and a neutropenic mouse model, we showed that 21RA and 24RA mediate protection from V. vulnificus infection through an Fc-independent and/or neutrophil-independent pathway. In contrast, 47RA-mediated protection was dependent on its Fc region and was reduced to 50% in neutropenic mice compared with 21RA-mediated and 24RA-mediated protection. Bacteriological study indicated that 21RA appears to enhance the clearance of V. vulnificus from the blood. Overall, these studies suggest that humoral immunity controls V. vulnificus infection through at least two different mechanisms. Furthermore, our panel of MAbs could provide attractive candidates for the further development of immunoprophylaxis/therapeutics and other therapies against V. vulnificus that target the MARTX toxin.

Ischemia/reperfusion (IR) injury has been associated with several retinal pathologies, and a few genes/gene products have been linked to IR injury. However, the big picture of temporal changes, regarding the affected gene networks, pathways, and processes remains to be determined. The purpose of the present study was to investigate initial, intermediate, and later stages to characterize the etiology of IR injury in terms of the pathways affected over time. Analyses indicated that at the initial stage, 0-hour reperfusion following the ischemic period, the ischemia-associated genes were related to changes in metabolism. In contrast, at the 24-hour time point, the signature events in reperfusion injury include enhanced inflammatory and immune responses as well as cell death indicating that this would be a critical period for the development of any interventional therapeutic strategies. Genes in the signal transduction pathways, particularly transmitter receptors, are downregulated at this time. Activation of the complement system pathway clearly plays an important role in the later stages of reperfusion injury. Together, these results demonstrate that the etiology of injury related to IR is characterized by the appearance of specific patterns of gene expression at any given time point during retinal IR injury. These results indicate that evaluation of treatment strategies with respect to time is very critical.

Nitrile oxide 1,3 dipolar cycloaddition is a simple and powerful coupling methodology. However, the self-dimerization of nitrile oxides has prevented the widespread use of this strategy for macromolecular coupling. By combining an in situ nitrile oxide generation with a highly reactive activated dipolarophile, we have overcome these obstacles and present a metal-free macromolecular coupling strategy for the modular synthesis of several ABA triblock copolymers. Nitrile oxides were generated in situ from chloroxime terminated poly(dimethylsiloxane) B-blocks and coupled with several distinct hydrophilic (poly(2-methyloxazoline) and poly(ethylene glycol)), and poly(N-isopropylacrylamide) or hydrophobic (poly(L-lactide) A-blocks terminated in activated dipolarophiles in a rapid fashion with high yield. This methodology overcomes many drawbacks of previously reported metal-free methods due to its rapid kinetics, versatility, scalability, and ease of introduction of necessary functionality. Nitrile oxide cycloaddition should find use as an attractive macromolecular coupling strategy for the synthesis of biocompatible polymeric nanostructures.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin protein (htt), leading to motor dysfunction, cognitive decline, psychiatric alterations, and death. The metabotropic glutamate receptor 5 (mGluR5) has been implicated in HD and we have recently demonstrated that mGluR5 positive allosteric modulators (PAMs) are neuroprotective in vitro. In the present study we demonstrate that the mGluR5 PAM, CDPPB, is a potent neuroprotective drug, in vitro and in vivo, capable of delaying HD-related symptoms. The HD mouse model, BACHD, exhibits many HD features, including neuronal cell loss, htt aggregates, motor incoordination and memory impairment. However, chronic treatment of BACHD mice with CDPPB 1.5 mg/kg s.c. for 18 weeks increased the activation of cell signaling pathways important for neuronal survival, including increased AKT and ERK1/2 phosphorylation and augmented the BDNF mRNA expression. CDPPB chronic treatment was also able to prevent the neuronal cell loss that takes place in the striatum of BACHD mice and decrease htt aggregate formation. Moreover, CDPPB chronic treatment was efficient to partially ameliorate motor incoordination and to rescue the memory deficit exhibited by BACHD mice. Importantly, no toxic effects or stereotypical behavior were observed upon CDPPB chronic treatment. Thus, CDPPB is a potential drug to treat HD, preventing neuronal cell loss and htt aggregate formation and delaying HD symptoms.

Bmi1 gene overexpression is found in various human tumors and has been shown as a potential target for gene treatment. However, siRNA-based treatments targeting Bmi1 gene have been restricted to limited delivery, low bioavailability and hence relatively reduced efficacy. To overcome these barriers, we developed a folate receptor targeted co-delivery system folate-doxorubicin/Bmi1 siRNA liposome (FA-DOX/siRNA-L). The FA-DOX/siRNA-L was prepared through electrostatic interaction between folate doxorubicin liposome (FA-DOX-L) and Bmi1 siRNA. In vitro and in vivo studies showed that FA-DOX/siRNA-L inhibited tumor growth by combinatory role of Bmi1 siRNA and doxorubicin (DOX). Co-delivery of Bmi1 siRNA and DOX by FA-DOX/siRNA-L showed significantly higher efficacy than sole delivery of either DOX or Bmi1 siRNA. Real-time PCR and western blot analysis showed that FA-DOX/siRNA-L silenced the expression of Bmi1 gene. In addition, higher accumulation of the siRNA and DOX in tumor cells indicated that folate ligand displayed tumor targeting effect. These results suggest that Bmi1 is an effective therapeutic target for siRNA based cancer treatment that can be further improved by co-delivery of DOX through targeted liposomes.

The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure-activity relationships, and previous salvinorin A-KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

Amyotrophic lateral sclerosis (ALS) is a rare degenerative condition of the motor neurons. Over 10% of ALS cases are linked to monogenic mutations, with the remainder thought to be due to other risk factors, including environmental factors, genetic polymorphisms, and possibly gene-environmental interactions. We examined the association between ALS and an intermediate CAG repeat expansion in the ATXN2 gene using a meta-analytic approach. Observational studies were searched with relevant disease and gene terms from MEDLINE, EMBASE, and PsycINFO from January 2010 through to January 2014. All identified articles were screened using disease terms, gene terms, population information, and CAG repeat information according to PRISMA guidelines. The final list of 17 articles was further evaluated based on the study location, time period, and authors to exclude multiple usage of the same study populations: 13 relevant articles were retained for this study. The range 30-33 CAG repeats in the ATXN2 gene was most strongly associated with ALS. The meta-analysis revealed that the presence of an intermediate CAG repeat (30-33) in the ATXN2 gene was associated with an increased risk of ALS [odds ratio (OR) = 4.44, 95%CI: 2.91-6.76)] in Caucasian ALS patients. There was no significant difference in the association of this CAG intermediate repeat expansion in the ATXN2 gene between familial ALS cases (OR = 3.59, 1.58-8.17) and sporadic ALS cases (OR = 3.16, 1.88-5.32). These results indicate that the presence of intermediate CAG repeat expansion in the ATXN2 gene is a specific genetic risk factor for ALS, unlike monogenic mutations with an autosomal dominant transmission mode, which cause a more severe phenotype of ALS, with a higher prevalence in familial ALS.

<h4>Background</h4>The neurochemical serotonin (5-HT) is an important signaling molecule in the gastrointestinal motor and sensory functions. A key regulator of 5-HT levels is the transmembrane serotonin transporter (5-HTT; SLC6A4) that governs the reuptake of 5-HT. Recent studies have indicated 5-HTT expression may be regulated by epigenetic mechanisms. We investigated DNA methylation status of SLC6A4 gene in the gastric mucosa from functional dyspepsia (FD) because of their potential role in dyspeptic symptoms.<h4>Methods</h4>Endoscopic gastric biopsies were obtained from 78 subjects with no upper abdominal symptoms and 79 patients with FD. Bisulfite Pyrosequencing was carried out to determine the methylation status of promoter CpG islands (PCGIs), promoter non-CpG islands (PNCGIs) and gene body non-CpG islands (NPNCGIs) in the SLC6A4 gene. Gene expression was examined by real-time PCR.<h4>Results</h4>In overall, methylation level of PCGIs was significantly lower in FD compared to control subjects (p = 0.04). On the other hand, methylation level of NPNCGIs was significantly higher in FD compared to control subjects (p = 0.03). Lower methylation level in PNCGIs was highlighted in the patients with PDS (p = 0.01), while higher methylation level in NPNCGIs was more prominent in the patients with EPS (p = 0.017). Methylation levels of PCGIs and PNCGIs were inversely correlated, while methylation levels of NPNCGIs was positively correlated with SLC6A4 mRNA levels in FD patients.<h4>Conclusions</h4>Our data suggest that change in DNA methylation pattern of SLC6A4 in the gastric mucosa may have a role for developing FD. A role of epigenetics for developing FD needs to be further evaluated.

<h4>Background</h4>Netrin-1 has been reported to promote retinal neovascularization in oxygen-induced retinopathy (OIR). However, netrin-1 receptors, which may mediate netrin-1 action during retinal neovascularization, have not been characterized. In this study, we investigated netrin-1 receptor subtype expression and associated changes in the retinas of mice with OIR.<h4>Methods</h4>C57BL/6J mice were exposed to 75±2% oxygen for 5 days and then returned to normal air to induce retinal neovascularization. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to examine the expression of netrin-1 receptor subtypes in the mouse retinas. Double staining of netrin-1 receptor subtypes and isolectin B4 was used to determine the location of the netrin-1 receptor subtypes in the retinas. Inhibition of retinal neovascularization was achieved by UNC5B shRNA plasmid intravitreal injection. Retinal neovascularization was examined by fluorescein angiography and quantification of preretinal neovascular nuclei in retinal sections.<h4>Results</h4>RT-PCR results showed that, except for UNC5A, netrin-1 receptor subtypes UNC5B, UNC5C, UNC5D, DCC, neogenin, and A2b were all expressed in the retinas of OIR mice 17 days after birth. Western blots showed that only UNC5B expression was significantly increased on that day, and immunofluorescence results showed that only UNC5B and neogenin were expressed in retinal vessels. Treatment of OIR mice with the UNC5B shRNA plasmid dramatically reduced neovascular tufts and neovascular outgrowth into the inner limiting membrane.<h4>Conclusions</h4>UNC5B may promote retinal neovascularization in OIR mice.

Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that allows them to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H2S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfur concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulfide product, PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a (13)C3-labeled phosphine sulfide, PS1, as the internal standard. The specificity and efficiency of the method were proven by model reactions. It was also applied to the measurement of sulfane sulfurs in mouse tissues including brain, kidney, lung, liver, heart, spleen, and blood.

The 3-dimensional spatial organization of eukaryotic genomes is important for regulation of gene expression as well as DNA damage repair. It has been proposed that one basic biophysical property of all nuclei is that interphase chromatin must be kept in a condensed prestressed state in order to prevent entropic pressure of the DNA polymer from expanding and disrupting the nuclear envelope. Although many factors can contribute to specific organizational states to compact chromatin, the mechanisms through which such interphase chromatin compaction is maintained are not clearly understood. Condensin proteins are known to exert compaction forces on chromosomes in anticipation of mitosis, but it is not known whether condensins also function to maintain interphase prestressed chromatin states. Here we show that RNAi depletion of the N-CAP-H2, N-CAP-D3 and SMC2 subunits of human condensin II leads to dramatic disruption of nuclear architecture and nuclear size. This is consistent with the idea that condensin mediated chromatin compaction contributes significantly to the prestressed condensed state of the interphase nucleus, and when such compaction forces are disrupted nuclear size and shape change due to chromatin expansion.

Unraveling the pathophysiology and testing candidate therapeutics in neurodegenerative disorders is, necessarily, highly dependent on model systems. Because Huntington's disease (HD) is caused by a single (expanded CAG tract) mutation in the huntingtin (HTT) gene, a richness of model systems, particularly mice, have been engineered to both dissect disease mechanisms and test potential therapeutics. Even so, as with other neurodegenerative diseases, very little success has been achieved in translating HD mouse model drug testing results to the clinic. Because of the considerable costs-human, opportunity, and financial-there is a pressing need to improve the use of existing HD models and also to develop models in higher species beyond rodent, such as sheep, minipig, and nonhuman primate, to bridge the translational gap from preclinical to clinical testing of candidate therapeutics.

Omics profiling significantly expanded the molecular landscape describing clinical phenotypes. Association analysis resulted in first diagnostic and prognostic biomarker signatures entering clinical utility. However, utilizing Omics for deepening our understanding of disease pathophysiology, and further including specific interference with drug mechanism of action on a molecular process level still sees limited added value in the clinical setting. We exemplify a computational workflow for expanding from statistics-based association analysis toward deriving molecular pathway and process models for characterizing phenotypes and drug mechanism of action. Interference analysis on the molecular model level allows identification of predictive biomarker candidates for testing drug response. We discuss this strategy on diabetic nephropathy (DN), a complex clinical phenotype triggered by diabetes and presenting with renal as well as cardiovascular endpoints. A molecular pathway map indicates involvement of multiple molecular mechanisms, and selected biomarker candidates reported as associated with disease progression are identified for specific molecular processes. Selective interference of drug mechanism of action and disease-associated processes is identified for drug classes in clinical use, in turn providing precision medicine hypotheses utilizing predictive biomarkers.

<h4>Background</h4>PCDH8 is a novel tumor suppressor gene, and frequently inactivated by promoter methylation in human cancers. However, there is little information regarding PCDH8 methylation in non-muscle invasive bladder cancer (NMIBC). The aim of this study was to investigate the methylation status of PCDH8 in NMIBC and its clinical significance.<h4>Methods</h4>The methylation status of PCDH8 in 233 NMIBC tissues and 43 normal bladder epithelial tissues was examined by methylation-specific PCR (MSP), and then analyzed the correlations between PCDH8 methylation and clinicopatholocial features. Subsequently, Kaplan-Meier survival analysis and Multivariate Cox proportional hazard model analysis was used to investigate the correlation between PCDH8 methylation and prognosis of patients with NMIBC.<h4>Results</h4>PCDH8 methylation occurred frequently in NMIBC tissues than those in normal bladder epithelial tissues. In addition, PCDH8 methylation significantly correlated with advanced stage, high grade, larger tumor size, tumor recurrence and progression in NMIBC. Kaplan-Meier survival analysis revealed that patients with PCDH8 methylated have shorter recurrence-free survival, progression-free survival and five-year overall survival than patients with PCDH8 unmethylated. Multivariate analysis suggested that PCDH8 methylation was an independent prognostic biomarker for recurrence-free survival, progression-free survival and five-year overall survival simultaneously.<h4>Conclusions</h4>PCDH8 methylation may be associated with tumor progression and poor prognosis in NMIBC and may be used as a potential biomarker to predict the prognosis of patients with NMIBC.

<h4>Background</h4>Salmonella Typhi is a human-restricted pathogen, which causes typhoid fever and remains a global health problem in the developing countries. Although previously reported host expression datasets had identified putative biomarkers and therapeutic targets of typhoid fever, the underlying molecular mechanism of pathogenesis remains incompletely understood.<h4>Methods</h4>We used five gene expression datasets of human peripheral blood from patients suffering from S. Typhi or other bacteremic infections or non-infectious disease like leukemia. The expression datasets were merged into human protein interaction network (PIN) and the expression correlation between the hubs and their interacting proteins was measured by calculating Pearson Correlation Coefficient (PCC) values. The differences in the average PCC for each hub between the disease states and their respective controls were calculated for studied datasets. The individual hubs and their interactors with expression, PCC and average PCC values were treated as dynamic subnetworks. The hubs that showed unique trends of alterations specific to S. Typhi infection were identified.<h4>Results</h4>We identified S. Typhi infection-specific dynamic subnetworks of the host, which involve 81 hubs and 1343 interactions. The major enriched GO biological process terms in the identified subnetworks were regulation of apoptosis and biological adhesions, while the enriched pathways include cytokine signalling in the immune system and downstream TCR signalling. The dynamic nature of the hubs CCR1, IRS2 and PRKCA with their interactors was studied in detail. The difference in the dynamics of the subnetworks specific to S. Typhi infection suggests a potential molecular model of typhoid fever.<h4>Conclusions</h4>Hubs and their interactors of the S. Typhi infection-specific dynamic subnetworks carrying distinct PCC values compared with the non-typhoid and other disease conditions reveal new insight into the pathogenesis of S. Typhi.

Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2(kd/kd)) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2(+/+)) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2(kd/kd) mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2(kd/kd) neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2(kd/kd) neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.

Long non-coding RNAs are a new class of non-coding RNAs that are at the crosshairs in many human diseases such as cancers, cardiovascular disorders, inflammatory and autoimmune disease like Inflammatory Bowel Disease (IBD) and Type 1 Diabetes (T1D). Nearly 90% of the phenotype-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) lie outside of the protein coding regions, and map to the non-coding intervals. However, the relationship between phenotype-associated loci and the non-coding regions including the long non-coding RNAs (lncRNAs) is poorly understood. Here, we systemically identified all annotated IBD and T1D loci-associated lncRNAs, and mapped nominally significant GWAS/ImmunoChip SNPs for IBD and T1D within these lncRNAs. Additionally, we identified tissue-specific cis-eQTLs, and strong linkage disequilibrium (LD) signals associated with these SNPs. We explored sequence and structure based attributes of these lncRNAs, and also predicted the structural effects of mapped SNPs within them. We also identified lncRNAs in IBD and T1D that are under recent positive selection. Our analysis identified putative lncRNA secondary structure-disruptive SNPs within and in close proximity (+/-5 kb flanking regions) of IBD and T1D loci-associated candidate genes, suggesting that these RNA conformation-altering polymorphisms might be associated with diseased-phenotype. Disruption of lncRNA secondary structure due to presence of GWAS SNPs provides valuable information that could be potentially useful for future structure-function studies on lncRNAs.

HIV sensory neuropathy and distal neuropathic pain (DNP) are common, disabling complications associated with combination antiretroviral therapy (cART). We previously associated iron-regulatory genetic polymorphisms with a reduced risk of HIV sensory neuropathy during more neurotoxic types of cART. We here evaluated the impact of polymorphisms in 19 iron-regulatory genes on DNP in 560 HIV-infected subjects from a prospective, observational study, who underwent neurological examinations to ascertain peripheral neuropathy and structured interviews to ascertain DNP. Genotype-DNP associations were explored by logistic regression and permutation-based analytical methods. Among 559 evaluable subjects, 331 (59%) developed HIV-SN, and 168 (30%) reported DNP. Fifteen polymorphisms in 8 genes (p<0.05) and 5 variants in 4 genes (p<0.01) were nominally associated with DNP: polymorphisms in TF, TFRC, BMP6, ACO1, SLC11A2, and FXN conferred reduced risk (adjusted odds ratios [ORs] ranging from 0.2 to 0.7, all p<0.05); other variants in TF, CP, ACO1, BMP6, and B2M conferred increased risk (ORs ranging from 1.3 to 3.1, all p<0.05). Risks associated with some variants were statistically significant either in black or white subgroups but were consistent in direction. ACO1 rs2026739 remained significantly associated with DNP in whites (permutation p<0.0001) after correction for multiple tests. Several of the same iron-regulatory-gene polymorphisms, including ACO1 rs2026739, were also associated with severity of DNP (all p<0.05). Common polymorphisms in iron-management genes are associated with DNP and with DNP severity in HIV-infected persons receiving cART. Consistent risk estimates across population subgroups and persistence of the ACO1 rs2026739 association after adjustment for multiple testing suggest that genetic variation in iron-regulation and transport modulates susceptibility to DNP.

SOX17 is a key transcriptional regulator that can act by regulating other transcription factors including HNF1β and FOXA2, which are known to regulate postnatal β cell function. Given this, we investigated the role of SOX17 in the developing and postnatal pancreas and found a novel role for SOX17 in regulating insulin secretion. Deletion of the Sox17 gene in the pancreas (Sox17-paLOF) had no observable impact on pancreas development. However, Sox17-paLOF mice had higher islet proinsulin protein content, abnormal trafficking of proinsulin, and dilated secretory organelles suggesting that Sox17-paLOF adult mice are prediabetic. Consistant with this, Sox17-paLOF mice were more susceptible to aged-related and high fat diet-induced hyperglycemia and diabetes. Overexpression of Sox17 in mature β cells using Ins2-rtTA driver mice resulted in precocious secretion of proinsulin. Transcriptionally, SOX17 appears to broadly regulate secretory networks since a 24-hour pulse of SOX17 expression resulted in global transcriptional changes in factors that regulate hormone transport and secretion. Lastly, transient SOX17 overexpression was able to reverse the insulin secretory defects observed in MODY4 animals and restored euglycemia. Together, these data demonstrate a critical new role for SOX17 in regulating insulin trafficking and secretion and that modulation of Sox17-regulated pathways might be used therapeutically to improve cell function in the context of diabetes.

No abstract available.

In Huntington's disease, iron accumulation in basal ganglia accompanies neuronal loss. However, if iron content changes with disease progression and how it relates to gray matter atrophy is not clear yet. We explored iron content in basal ganglia and cortex and its relationship with gray matter volume in 77 mutation carriers [19 presymptomatic, 8 with soft symptoms (SS), and 50 early-stage patients) and 73 matched-controls by T2*relaxometry and T1-weighted imaging on a 3T scanner. The ANCOVA model showed that iron accumulates in the caudate in presymptomatic subjects (P = 0.004) and remains relatively stable along disease stages in this nucleus; while increases in putamen and globus pallidus (P < 0.05). Volume instead decreases in basal ganglia, starting from the caudate (P < 0.0001) and extending to the putamen and globus pallidus (P ≤ 0.001). The longer the disease duration and the higher the CAG repeats, the higher the iron accumulation and the smaller the volume. In the cortex, iron decreases in parieto-occipital areas in SS (P < 0.027); extending to premotor and parieto-temporo-occipital areas in patients (P < 0.003); while volume declines in frontoparietal and temporal areas in presymptomatic (P < 0.023) and SS (P < 0.045), and extends throughout the cortex, with the exception of anterior frontal regions, in patients (P < 0.023). There is an inverse correlation between volume and iron levels in putamen, globus pallidus and the anterior cingulate; and a direct correlation in cortical structures (SMA-sensoriomotor and temporo-occipital). Iron homeostasis is affected in the disease; however, there appear to be differences in the role played by iron in basal ganglia and in cortex.

Clinical trials of histone deacetylase (HDAC) inhibitors as antitumor therapy have been conducted for gastric cancer. Expression of SIRT1, a class III HDAC, is related to poor prognosis in some malignancies. We investigated the correlation between SIRT1 expression and progression and prognosis of gastric cancers comparing with molecules linked to SIRT1 in order to better predict the efficacy of HDAC inhibitors in treating this disease. We evaluated SIRT1 expression by western blot in 51 cases and SIRT1, DBC1, acetylated H4K16 (H4K16Ac), acetylated H3K9 (H3K9Ac), and p53 by immunohistochemistry (IHC) in 557 cases of gastric cancer. Western blotting showed that SIRT1 high expression related with statistics to advanced tumor progression, positive lymphatic invasion, positive venous invasion, and advanced stage but not to poor prognosis. IHC revealed that SIRT1 high expression correlated with worse clinico-pathological prognostic factors as same as in western blotting and related poor prognosis both by univariate and multivariate analyses. By the contrast, DBC1 and H4K16Ac were related to favorable prognostic factors and linked to favorable prognosis by univariate analysis but not by multivariate analysis. H3K16Ac correlated only favorable prognostic factors. Results of p53 were very similar to those of SIRT1. We found that SIRT1 high expression closely correlates with progression and prognosis in gastric cancer patients. And it was also indicated that SIRT1 acts as an oncogene by the results of DBC1, H4K16Ac, and H3K9Ac and might be a target molecule of HDAC inhibitor treatment for gastric cancer patients.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy characterized by clinical and genetic heterogeneity. Although more than 30 loci harboring CMT-causing mutations have been identified, many other genes still remain to be discovered for many affected individuals. For two consanguineous families with CMT (axonal and mixed phenotypes), a parametric linkage analysis using genome-wide SNP chip identified a 4.3 Mb region on 12q24 showing a maximum multipoint LOD score of 4.23. Subsequent whole-genome sequencing study in one of the probands, followed by mutation screening in the two families, revealed a disease-specific 5 bp deletion (c.247-10_247-6delCACTC) in a splicing element (pyrimidine tract) of intron 2 adjacent to the third exon of cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1), which is a component of mitochondrial respiratory complex IV (cytochrome c oxidase [COX]), within the autozygous linkage region. Functional analysis showed that expression of COX6A1 in peripheral white blood cells from the affected individuals and COX activity in their EB-virus-transformed lymphoblastoid cell lines were significantly reduced. In addition, Cox6a1-null mice showed significantly reduced COX activity and neurogenic muscular atrophy leading to a difficulty in walking. Those data indicated that COX6A1 mutation causes the autosomal-recessive axonal or mixed CMT.

The formation of cartilage is restricted to the core of the limb bud mesenchyme by ectodermal Wnts, which can irreversibly silence expression of the prochondrogenic transcription factor Sox9. In contrast, fibroblast growth factor (FGF) signals from the apical ectodermal ridge maintain the competence of chondrogenic precursors to undergo chondrogenesis once these cells go out of the range of ectodermal Wnt signals. We have found that Wnt signals induce both a repressive chromatin mark (H3K27me3) and DNA methylation over the Sox9 promoter and that Wnt-induced irreversible silencing of the Sox9 gene requires DNA methylation of this locus, which is specifically countered by FGF signals. FGF blocks the recruitment of the de novo DNA methyltransferase, DNMT3A, to the Sox9 promoter by inducing the interaction and phosphorylation of DNMT3A by ERK1/ERK2 and thereby controls whether expression of Sox9 is either irreversibly or reversibly silenced by Wnt signals in limb bud mesenchymal cells.

<h4>Background</h4>We report a case of a neonate with proximal spinal muscular atrophy (SMA) type 1 (also known as Werdnig-Hoffmann disease or severe infantile acute SMA) associated with a Blake's pouch cyst; a malformation that is currently classified within the spectrum of Dandy-Walker complex. The association of the two conditions has not been previously reported in the English literature. A comprehensive review of the pertinent literature is presented.<h4>Case description</h4>A male neonate was noted to have paucity of movement of the four limbs with difficulty of breathing and poor feeding soon after birth. Respiratory distress with tachypnea, necessitated endotracheal intubation and mechanical ventilation. Pregnancy was uneventful except for decreased fetal movements reported by the mother during the third trimester. Neurological examination revealed generalized hypotonia with decreased muscle power of all limbs, nonelicitable deep tendon jerks, and occasional tongue fasciculations. Molecular genetic evaluation revealed a homozygous deletion of both exons 7 and 8 of the survival motor neuron 1 (SMN1) gene, and exon 5 of the neuronal apoptosis inhibitory protein (NAIP) gene on the long arm of chromosome 5 consistent with Werdnig-Hoffmann disease (SMA type 1). At the age of 5 months, a full anterior fontanelle and abnormal increase of the occipito-frontal circumference were noted. Computed tomographic (CT) scan and magnetic resonance imaging (MRI) of the brain revealed a tetraventricular hydrocephalus and features of Blake's pouch cyst of the fourth ventricle.<h4>Conclusions</h4>This case represents a previously unreported association of Blake's pouch cyst and SMA type 1.

The hepatitis B virus (HBV) carrier rate has decreased in Japan; however, the incidence of HBV infection among hepatocellular carcinoma (HCC) patients has not decreased accordingly. In this study, we aimed to assess the time trends of the clinical characteristics in HCC patients with chronic HBV infection. Between 2000 and 2012, we enrolled a total of 156 HCC patients with chronic HBV infection in our field survey. The HCC risk was evaluated using the HCC prediction score, which is constructed from the characteristics of age, presence of liver cirrhosis and serum levels of albumin, bilirubin and HBV DNA. Lifestyle factors and the presence of diabetes mellitus were also evaluated. The time trends of patient characteristics were analyzed using the Jonckheere-Terpstra proportion trend test. Among HCC patients with chronic HBV infection, the proportion of patients at high risk according to the HCC prediction score significantly decreased during the study period (P=0.0005). Similarly, the proportion of patients with liver cirrhosis, ≤3.5 g/dl serum albumin level, >4 log copies/ml serum HBV DNA level and ≥60 g/day alcohol intake were also significantly decreased. The proportion of male and obese patients was not significantly altered, whereas the proportion of elderly (≥65 years) and diabetic patients tended to increase during the study period (P=0.0654 and P=0.0528, respectively). In this study, we analyzed the time trends of the clinical characteristics in HCC patients with chronic HBV infection and demonstrated that aging and diabetes mellitus may be involved in the hepatocarcinogenesis in patients with chronic HBV infection.

Although a number of tissue adhesives and sealants for surgical use are currently available, attaining a useful balance in high strength, high compliance, and low swelling has proven difficult. Recent studies have demonstrated that a four-arm poly(propylene oxide)-poly(ethylene oxide) block copolymer, Tetronic, can be chemically modified to form a hydrogel tissue adhesive (Cho et al., Acta Biomater 2012;8:2223-2232; Barrett et al., Adv Health Mater 2012;1-11; Balakrishnan, Evaluating mechanical performance of hydrogel-based adhesives for soft tissue applications. Clemson University, All Theses, Paper 1574: Tiger Prints; 2013). Building on the success of these studies, this study explored bifunctionalization of Tetronic with acrylates for chemical crosslinking of the hydrogel and N-hydroxysuccinimide (NHS) for reaction with tissue amines. The adhesive bond strengths of various uni and bifunctional Tetronic blends (T1107 ACR: T1107 ACR/NHS) determined by lap shear testing ranged between 8 and 74 kPa, with the 75:25 (T1107 ACR: T1107 ACR/NHS) blend displaying the highest value. These results indicated that addition of NHS led to improvement of tissue bond strength over acrylation alone. Furthermore, ex vivo pressure tests using the rat bladder demonstrated that the bifunctional Tetronic adhesive exhibited high compliance and maintained pressures under hundreds of filling and emptying cycles. Together, the results of this study provided evidence that the bifunctional Tetronic adhesive with a proper blend ratio may be used to achieve an accurate balance in bulk and tissue bond strengths, as well as the compliance and durability for soft tissue such as the bladder.

Electrochemical studies are reported on a series of peptides constrained into either a 310-helix (1-6) or β-strand (7-9) conformation, with variable numbers of electron rich alkene containing side chains. Peptides (1 and 2) and (7 and 8) are further constrained into these geometries with a suitable side chain tether introduced by ring closing metathesis (RCM). Peptides 1, 4 and 5, each containing a single alkene side chain reveal a direct link between backbone rigidity and electron transfer, in isolation from any effects due to the electronic properties of the electron rich side-chains. Further studies on the linear peptides 3-6 confirm the ability of the alkene to facilitate electron transfer through the peptide. A comparison of the electrochemical data for the unsaturated tethered peptides (1 and 7) and saturated tethered peptides (2 and 8) reveals an interplay between backbone rigidity and effects arising from the electron rich alkene side-chains on electron transfer. Theoretical calculations on β-strand models analogous to 7, 8 and 9 provide further insights into the relative roles of backbone rigidity and electron rich side-chains on intramolecular electron transfer. Furthermore, electron population analysis confirms the role of the alkene as a "stepping stone" for electron transfer. These findings provide a new approach for fine-tuning the electronic properties of peptides by controlling backbone rigidity, and through the inclusion of electron rich side-chains. This allows for manipulation of energy barriers and hence conductance in peptides, a crucial step in the design and fabrication of molecular-based electronic devices.

<h4>Background</h4>We have previously reported overexpression of the immunoregulatory protein B7-H3 in colorectal cancer and that nuclear expression predicted poor outcome in colon cancer patients. The present study was performed to examine the prognostic role of B7-H3 in an independent colorectal cancer cohort.<h4>Methods</h4>Using tissue microarrays from 731 colorectal cancer patients, tumour B7-H3 expression was assessed by immunohistochemistry. Associations with clinicopathological parameters and patient outcome were investigated.<h4>Results</h4>Nuclear expression of B7-H3 in cancer cells was present in 27% of the samples in the total study cohort, while cytoplasmic/membrane and stromal expression was seen in 86% and 77% of the samples, respectively. Nuclear B7-H3 had no prognostic relevance in the complete outcome cohort, neither in colon cancer patients. However, nuclear B7-H3 was significantly associated with reduced recurrence-free survival in TNM stage I colorectal cancer patients.<h4>Conclusions</h4>Overexpression of B7-H3 in colorectal cancer was confirmed, but in contrast to previous results, nuclear B7-H3 was not a strong prognostic biomarker in this cohort. The discrepancy might be related to the use of single-core tissue microarrays for detection of the heterogeneously expressed B7-H3, and the role of B7-H3 in colorectal cancer still needs further examination.

Many of the neurodegenerative diseases that afflict people in later life are associated with the formation of protein aggregates. These so-called "proteinopathies" include Alzheimer's disease (AD) and Huntington's disease (HD). The insulin/insulin-like growth factor signalling (IIS) pathway has been proposed to modulate such diseases in model organisms, as well as the general ageing process. In this pathway, insulin-like growth factor binds to insulin-like growth factor receptors, such as the insulin-like growth factor 1 receptor (IGF-1R). Heterozygous deletion of Igf-1r has been shown to lead to increased lifespan in mice. Reducing the activity of this pathway had benefits in a HD C. elegans model, and some of these may be attributed to the expected inhibition of mTOR activity resulting in an increase in autophagy, which would enhance mutant huntingtin clearance. Thus, we tested if heterozygous deletion of Igf-1r would lead to benefits in HD related phenotypes in the mouse. Surprisingly, reducing Igf-1r levels led to some beneficial effects in HD females, but also led to some detrimental effects in HD males. Interestingly, Igf-1r deficiency had no discernible effects on downstream mTOR signalling in HD mice. These results do not support a broad beneficial effect of diminishing the IIS pathway in HD pathology in a mammalian system.

<h4>Background</h4>The invasive nature of liver biopsy makes the histopathological diagnosis of non-alcoholic fatty liver disease (NAFLD) difficult and its diagnostic performance unsatisfactory. The present study aimed to identify a serum microRNA (miRNA) expression profile that could serve as a novel diagnostic biomarker for NAFLD.<h4>Methods</h4>Serum miRNA expression was investigated using three cohorts comprising 465 participants (healthy controls and NAFLD patients) recruited between August 2010 and June 2013. miRNA expression was initially screened by Illumina sequencing using serum samples pooled from 20 patients and 20 controls. Quantitative reverse transcriptase polymerase chain reaction assay was then used to evaluate the expression of selected miRNAs. A logistic regression model was constructed using a training cohort (n = 242) and validated using another cohort (n = 183). The area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy.<h4>Results</h4>We identified an miRNA panel (hsa-miR-122-5p, hsa-miR-1290, hsa-miR-27b-3p, and hsa-miR-192-5p) with a high diagnostic accuracy for NAFLD. The satisfactory diagnostic performance of the miRNA panel remained regardless of the NAFLD activity score (NAS) status. There was significant difference between the AUC values of the miRNA panel and those of ALT (AUC = 0.786, 95% CI = 0.717-0.855; P = 0.142) and FIB-4 (AUC = 0.795, 95% CI = 0.730-0.860; sensitivity = 69.9%, specificity = 83.7%.<h4>Conclusion</h4>We identified a serum microRNA panel with considerable clinical value in NAFLD diagnosis. The results indicate that the miRNA panel is a more sensitive and specific biomarker for NAFLD than ALT and FIB-4.

Knowledge of the cognitive manifestation of Huntington's disease has burgeoned over the past two decades. Many studies from independent datasets have shown that cognitive impairment is evident before motor diagnosis, and annual cognitive decline is a robust marker of disease progression. Additionally, cognition is a critical concern to patients and families and is associated with meaningful outcomes, including functional capacity, driving, loss of accustomed work, and quality of life. In the past few years, Huntington's disease animal models of cognition have increased, preparing for preclinical experimental therapeutics with cognitive endpoints. A longitudinal analysis of cognitive variables was conducted with 559 gene-positive cases and 233 controls showing no signs of motor abnormalities over approximately a 3-year period. Results show statistically significant differences in rate of annual change for some cognitive variables, such that the cases group had worsening performance over time. These findings show that cognitive deterioration can be seen in persons with the Huntington's disease gene expansion with no overt motor signs or symptoms, suggesting that cognitive onset of Huntington's disease may precede motor.

Tau abnormalities play a central role in several neurodegenerative diseases, collectively known as tauopathies. In the present study, we examined whether mutant huntingtin (mHtt), which causes Huntington's disease (HD), modifies Tau phosphorylation and subcellular localization using cell and mouse HD models. Initially, we used novel bimolecular fluorescence complementation assays in live cells to evaluate Tau interactions with either wild type (25QHtt) or mutant huntingtin (103QHtt). While 25QHtt and Tau interacted at the level of the microtubule network, 103QHtt and Tau interacted and formed 'ring-like' inclusions localized in the vicinity of the microtubular organizing center (MTOC). Fluorescence recovery after photobleaching experiments also indicated that, whereas homomeric 103QHtt/103QHtt pairs rapidly re-entered into inclusions, heteromeric 103QHtt/Tau pairs remained excluded from the 'ring-like' inclusions. Interestingly, in vitro Tau relocalization was associated to Tau hyperphosphorylation. Consistent with this observation, we found strong Tau hyperphosphorylation in brain samples from two different mouse models of HD, R6/2 and 140CAG knock-in. This was associated with a significant reduction in the levels of Tau phosphatases (PP1, PP2A and PP2B), with no apparent involvement of major Tau kinases. Thus, the present study strongly suggests that expression of mHtt leads to Tau hyperphosphorylation, relocalization and sequestration through direct protein-protein interactions in inclusion-like compartments in the vicinity of the MTOC. Likewise, our data also suggest that Tau alterations may also contribute to HD pathogenesis.

Olanzapine is widely prescribed for treating schizophrenia and other mental disorders, although it leads to severe body weight gain/obesity. Chronic co-treatment with betahistine has been found to significantly decrease olanzapine-induced weight gain; however, it is not clear whether this co-treatment affects the therapeutic effects of olanzapine. This study investigated the effects of chronic treatment of olanzapine and/or betahistine on the binding density of the serotonergic 5-HT2A (5-HT2AR) and 5-HT2C (5-HT2CR) receptors, 5-HT transporter (5-HTT), and dopaminergic D₂ receptors (D₂R) in the brain regions involved in antipsychotic efficacy, including the prefrontal cortex (PFC), cingulate cortex (Cg), nucleus accumbens (NAc), and caudate putamen (CPu). Rats were treated with olanzapine (1 mg/kg, t.i.d.) or vehicle for 3.5 weeks, and then olanzapine treatment was withdrawn for 19 days. From week 6, the two groups were divided into 4 groups (n=6) for 5 weeks' treatment: (1) olanzapine-only (1 mg/kg, t.i.d.), (2) betahistine-only (9.6 mg/kg, t.i.d.), (3) olanzapine and betahistine co-treatment (O+B), and (4) vehicle. Compared to the control, the olanzapine-only treatment significantly decreased the bindings of 5-HT2AR, 5-HT2CR, and 5-HTT in the PFC, Cg, and NAc. Similar changes were observed in the rats receiving the O+B co-treatment. The olanzapine-only treatment significantly increased the D₂R binding in the Cg, NAc, and CPu, while the betahistine-only treatment reduced D₂R binding. The co-treatment of betahistine reversed the D₂R bindings in the NAc and CPu that were increased by olanzapine. Therefore, chronic O+B co-treatment has similar effects on serotonin transmission as the olanzapine-only treatment, but reverses the D₂R that is up-regulated by chronic olanzapine treatment. The co-treatment maintains the therapeutic effects of olanzapine but decreases/prevents the excess weight gain.

Polymeric prodrug micelles for delivery of acyclovir (ACV) were synthesized. First, ACV was used directly to initiate ring-opening polymerization of ɛ-caprolactone to form ACV-polycaprolactone (ACV-PCL). Through conjugation of hydrophobic ACV-PCL with hydrophilic methoxy poly(ethylene glycol) (MPEG) or chitosan, polymeric micelles for drug delivery were formed. (1)H NMR, FTIR, and gel permeation chromatography were employed to show successful conjugation of MPEG or chitosan to hydrophobic ACV-PCL. Through dynamic light scattering, zeta potential analysis, transmission electron microscopy, and critical micelle concentration (CMC), the synthesized ACV-tagged polymeric micelles were characterized. It was found that the average size of the polymeric micelles was under 200nm and the CMCs of ACV-PCL-MPEG and ACV-PCL-chitosan were 2.0mgL(-1) and 6.6mgL(-1), respectively. The drug release kinetics of ACV was investigated and cytotoxicity assay demonstrates that ACV-tagged polymeric micelles were non-toxic.

Proteome analysis of the urine has shown that urine contains disease-specific information for a variety of urogenital system disorders, including prostate cancer (PCa). The aim of this study was to determine the protein components of urine from PCa patients. Urine from 8 patients with clinically and histologically confirmed PCa was analyzed by conventional 2D PAGE. The MS identification of the most prominent 125 spots from the urine map revealed 45 distinct proteins. According to Gene Ontology, the identified proteins are involved in a variety of biological processes, majority of them are secreted (71%), and half of them are enzymes or transporters. Comparison with the normal urine proteome revealed 11 proteins distinctive for PCa. Using Ingenuity Pathways Analysis, we have found 3 proteins (E3 ubiquitin-protein ligase rififylin, tumor protein D52, and thymidine phosphorylase) associated with cellular growth and proliferation (p = 8.35 × 10(-4) - 3.41 × 10(-2)). The top network of functional associations between 11 proteins was Cell Death and Survival, Cell-To-Cell Signaling and Interaction, and System Development and Function (p = 10(-30)). In summary, we have created an initial proteomic map of PCa patient's urine. The results from this study provide some leads to understand the molecular bases of prostate cancer.

Obesity, diabetes and insulin resistance are marked risk factors that promote the development of type I endometrial cancer. Previous studies have demonstrated that insulin-like growth factor 1 (IGF-1) and IGF-2 promote cell proliferation in endometrial cancer cells, while metformin reverses this effect and inhibits cell proliferation. However, the effects of metformin on the regulation of the IGF signaling pathway are unclear. The aim of this study was to investigate the regulation of IGF signaling by metformin in endometrial cancer cells, and to determine the effects of metformin combined with IGF-1 receptor (IGF-1R) inhibitor on cell proliferation and apoptosis. Cell proliferation was assessed following exposure of Ishikawa and HEC-1B endometrial cancer cell lines to metformin and/or the IGF-1R inhibitor, PPP. Apoptosis was assessed by TdT-mediated dUTP nick end labeling assay. Metformin was observed to downregulate IGF-1R and upregulate IGF binding protein-1 (IGFBP-1) mRNA and protein expression, while compound C, an adenosine monophosphate protein kinase inhibitor, reversed this effect. Metformin administered with PPP inhibited endometrial cancer cell proliferation to a greater degree than treatment with either agent alone. At high concentrations (1 or 2 mM), metformin induced apoptosis in endometrial cancer cells. Metformin combined with IGF-1R axis inhibitors may act synergistically to kill tumor cells, as metformin was shown to delay and prevent IGF-1R feedback. In conclusion, this study supported the results of animal studies and subclinical studies, demonstrating the feasibility of metformin combined with IGF-1R axis inhibitors in the treatment of endometrial cancer.

<h4>Introduction</h4>National Health and Nutrition Examination Survey (NHANES III), 1988-1994, first time reported a significant, positive association in iron storage and heart disease risk. Thereafter several researchers have found an association between iron overload, serum ferritin (SF) and MI. No such Indian study was available in the literature and so we decided to find out the relation of lipid profile and Serum Ferritin with myocardial infarction (MI).<h4>Materials and methods</h4>Fifty indian patients of AMI (study group) and fifty indian healthy volunteers (control group) were included for the present study. Lipid profile including TC, HDL-c, LDL-c, VLDL-c & TG and SF levels were estimated in all subjects.<h4>Observations and results</h4>Mean ± SD of TC level was 250.64 ± 25.61, of HDL-c was 36.52 ± 2.86, of LDL-c was 165.69 ± 26.80, of VLDL-c was 42.35 ± 8.53 and of TG was 211.83 ± 42.65 in study group while these values were 174.46±47.68, 43.2±12.52, 98.37±41.13, 32.88±21.45 and 164.42±107.29 respectively in control group. All the parameters were found not only raised in patients of acute myocardial infarction (AMI) but were also statistically significant when compared with control group (p=<0.01). Mean ± SD of SF levels was 268.43±30.17 ng/ml in study group and 110.96±56.5 ng/ml in control group; this level was found not only raised in patients of AMI but were also statistically significant when compared with control group (p=<0.01).<h4>Conclusion</h4>TC, LDL-c, VLDL-c, TG and SF levels were raised in patients of AMI and found to be statistically significant; while HDL-c levels were reduced in such patients and is also statistically significant. It can be concluded that there exists an association in lipid profile and SF with AMI therefore dyslipidemia and raised SF levels are the features of AMI.

<h4>Background</h4>As early and appropriate care of severe septic patients is associated with better outcome, understanding of the very first events in the disease process is needed. Pan-genomic analyses offer an interesting opportunity to study global genomic response within the very first hours after sepsis. The objective of this study was to investigate the systemic genomic response in severe intensive care unit (ICU) patients and determine whether patterns of gene expression could be associated with clinical severity evaluated by the severity score.<h4>Methods</h4>Twenty-eight ICU patients were enrolled at the onset of septic shock. Blood samples were collected within 30 min and 24 and 48 h after shock and genomic response was evaluated using microarrays. The genome-wide expression pattern of blood leukocytes was sequentially compared to healthy volunteers and after stratification based on Simplified Acute Physiology Score II (SAPSII) score to identify potential mechanisms of dysregulation.<h4>Results</h4>Septic shock induces a global reprogramming of the whole leukocyte transcriptome affecting multiple functions and pathways (>71% of the whole genome was modified). Most altered pathways were not significantly different between SAPSII-high and SAPSII-low groups of patients. However, the magnitude and the duration of these alterations were different between these two groups. Importantly, we observed that the more severe patients did not exhibit the strongest modulation. This indicates that some regulation mechanisms leading to recovery seem to take place at the early stage.<h4>Conclusions</h4>In conclusion, both pro- and anti-inflammatory processes, measured at the transcriptomic level, are induced within the very first hours after septic shock. Interestingly, the more severe patients did not exhibit the strongest modulation. This highlights that not only the responses mechanisms by themselves but mainly their early and appropriate regulation are crucial for patient recovery. This reinforces the idea that an immediate and tailored aggressive care of patients, aimed at restoring an appropriately regulated immune response, may have a beneficial impact on the outcome.

A variety of cyclodextrin-based molecular structures, with substitutions of either primary or secondary faces of the natural oligosaccharide macrocycles of α-, β-, or γ-cyclodextrins, have been designed towards innovative applications of self-assembled cyclodextrin nanomaterials. Amphiphilic cyclodextrins have been obtained by chemical or enzymatic modifications of their macrocycles using phospholipidyl, peptidolipidyl, cholesteryl, and oligo(ethylene oxide) anchors as well as variable numbers of grafted hydrophobic hydrocarbon or fluorinated chains. These novel compounds may self-assemble in an aqueous medium into different types of supramolecular nanoassemblies (vesicles, micelles, nanorods, nanospheres, and other kinds of nanoparticles and liquid crystalline structures). This review discusses the supramolecular nanoarchitectures, which can be formed by amphiphilic cyclodextrin derivatives in mixtures with other molecules (phospholipids, surfactants, and olygonucleotides). Biomedical applications are foreseen for nanoencapsulation of drug molecules in the hydrophobic interchain volumes and nanocavities of the amphiphilic cyclodextrins (serving as drug carriers or pharmaceutical excipients), anticancer phototherapy, gene delivery, as well as for protection of instable active ingredients through inclusion complexation in nanostructured media.

Exposure to nicotine during pregnancy via maternal cigarette smoking is associated with visual deficits in children. This is possibly due to the activation of nicotinic acetylcholine receptors (nAChRs) in the occipital cortex, which are important in the development of visual mapping. Using a baboon model, we explored the effects of prenatal nicotine on parameters in the primary and associated visual cortices. Pregnant baboons were infused with nicotine (0.5 mg/h, intravenous) or saline from 86 days gestation. At 161 days gestation, fetal brains were collected (n = 5 per group) and the occipital lobe assessed for nAChRs and markers of the serotonergic and catecholaminergic systems using tissue autoradiography and/or high-performance liquid chromatography. Neuronal nAChRs and serotonergic markers were expressed in a region- and subunit-dependent manner. Prenatal nicotine exposure was associated with increased binding for (3) H-epibatidine sensitive nAChRs in the primary visual cortex [Brodmann areas (BA) 17] and BA 18, but not BA 19, of the associative visual cortex (P < 0.05). Markers of the serotonergic or catecholaminergic systems were not significantly altered. Thus, prenatal nicotine exposure is associated with alterations in the cholinergic system in the occipital lobe, which may aid in the explanation of the appearance of visual deficits in children from mothers who smoke during pregnancy.

<h4>Background</h4>Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.<h4>Results</h4>Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMR) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients.<h4>Conclusions</h4>These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.

Chronic alcohol consumption changes gene expression, likely causing persistent remodeling of synaptic structures via altered translation of mRNAs within synaptic compartments of the cell. We profiled the transcriptome from synaptoneurosomes (SNs) and paired total homogenates (THs) from mouse amygdala following chronic voluntary alcohol consumption. In SN, both the number of alcohol-responsive mRNAs and the magnitude of fold-change were greater than in THs, including many GABA-related mRNAs upregulated in SNs. Furthermore, SN gene co-expression analysis revealed a highly connected network, demonstrating coordinated patterns of gene expression and highlighting alcohol-responsive biological pathways, such as long-term potentiation, long-term depression, glutamate signaling, RNA processing and upregulation of alcohol-responsive genes within neuroimmune modules. Alterations in these pathways have also been observed in the amygdala of human alcoholics. SNs offer an ideal model for detecting intricate networks of coordinated synaptic gene expression and may provide a unique system for investigating therapeutic targets for the treatment of alcoholism.

Patients with HBV-acute-on-chronic liver failure (HBV-ACLF) have high mortality and frequently require liver transplantation; few reliable prognostic markers are available. As a class of functional lipids, sphingolipids are extensively involved in the process of HBV infection. However, their role in chronic HBV infection remains unknown. The aim of this study was to determine the serum sphingolipid profile in a population of patients with chronic HBV infection, paying special attention to exploring novel prognostic markers in HBV-ACLF. High performance liquid chromatography tandem mass spectrometry was used to examine the levels of 41 sphingolipids in 156 serum samples prospectively collected from two independent cohorts. The training and validation cohorts comprised 20 and 28 healthy controls (CTRL), 29 and 23 patients with chronic hepatitis B (CHB), and 30 and 26 patients with HBV-ACLF, respectively. Biometric analysis was used to evaluate the association between sphingolipid levels and disease stages. Multivariate analysis revealed difference of sphingolipid profiles between CHB and HBV-ACLF was more drastic than that between CTRL and CHB, which indicated that serum sphingolipid levels were more likely to associate with the progression HBV-ACLF rather than CHB. Furthermore, a 3-month mortality evaluation of HBV-ACLF patients showed that dhCer(d18 : 0/24 : 0) was significantly higher in survivors than in non-survivors (including deceased patients and those undergoing liver transplantation, p < 0.05), and showed a prognostic performance similar to that of the MELD score. The serum sphingolipid composition varies between CTRL and chronic HBV infection patients. In addition, dhCer(d18 : 0/24 : 0) may be a useful prognostic indicator for the early prediction of HBV-ACLF.

Enterobacterial H-NS-like proteins and Pseudomonas MvaT-like proteins share low homology at the amino acid sequence level, but both can function as xenogeneic silencers and are included in the H-NS family of proteins. H-NS family members have dimerization/oligomerization and DNA-binding domains connected by a flexible linker and form large nucleoprotein complexes using both domains. Pmr, an MvaT-like protein encoded on the IncP-7 carbazole-degradative plasmid pCAR1, is a key regulator of an interaction between pCAR1 and its host Pseudomonas putida KT2440. KT2440 has two transcribed genes that encode the MvaT-like proteins TurA and TurB. Our previous transcriptome analyses suggested that the functions of Pmr, TurA and TurB are non-equivalent, although the detailed underlying mechanisms remain unclear. In this study, we focused on the protein-protein interactions of Pmr, and assessed the homo-oligomerization capacity of various substituted and truncated Pmr derivatives by protein-protein cross-linking analysis. Six of the seven residues identified as important for homo-oligomerization in Pmr were located near the N-terminus, and the putative flexible linker or the region near that was not involved in homo-oligomerization, suggesting that Pmr homo-oligomerization is different from that of enterobacterial H-NS and that the functional mechanism differs between H-NS-like and MvaT-like proteins. In addition, we assessed homo- and hetero-oligomerization of Pmr by surface plasmon resonance analysis and found that the coupling ratio of TurB-Pmr oligomers is smaller than that of Pmr-Pmr or TurA-Pmr oligomers. These results raised the possibility that composition of the hetero-oligomers of Pmr, TurA, and TurB could explain why the different gene sets were affected by either pmr, turA, or turB disruption in our previous studies.

Cognitive bias, the altered information processing resulting from the background emotional state of an individual, has been suggested as a promising new indicator of animal emotion. Comparable to anxious or depressed humans, animals in a putatively negative emotional state are more likely to judge an ambiguous stimulus as if it predicts a negative event, than those in positive states. The present study aimed to establish a cognitive bias test for mice based on a spatial judgment task and to apply it in a pilot study to serotonin transporter (5-HTT) knockout mice, a well-established mouse model for the study of anxiety- and depression-related behavior. In a first step, we validated that our setup can assess different expectations about the outcome of an ambiguous stimulus: mice having learned to expect something positive within a maze differed significantly in their behavior towards an unfamiliar location than animals having learned to expect something negative. In a second step, the use of spatial location as a discriminatory stimulus was confirmed by showing that mice interpret an ambiguous stimulus depending on its spatial location, with a position exactly midway between a positive and a negative reference point provoking the highest level of ambiguity. Finally, the anxiety- and depression-like phenotype of the 5-HTT knockout mouse model manifested--comparable to human conditions--in a trend for a negatively distorted interpretation of ambiguous information, albeit this effect was not statistically significant. The results suggest that the present cognitive bias test provides a useful basis to study the emotional state in mice, which may not only increase the translational value of animal models in the study of human affective disorders, but which is also a central objective of animal welfare research.

<h4>Context</h4>Macrosomic infants are at increased risk for adverse metabolic outcomes. Improving prediction of large-for-gestational-age (LGA) birth may help prevent these outcomes.<h4>Objective</h4>This study sought to determine whether genes associated with obesity-related traits in adults are associated with newborn size, and whether a genetic risk score (GRS) predicts LGA birth.<h4>Setting and design</h4>Single nucleotide polymorphisms (SNPs) in 40 regions associated with adult obesity-related traits were tested for association with newborn size. GRS's for birth weight and sum of skinfolds (SSF) specific to ancestry were calculated using the most highly associated SNP for each ancestry in genomic regions with one or more SNPs associated with birth weight and/or SSF in at least one ancestry group or meta-analyses.<h4>Participants</h4>Newborns from the Hyperglycemia Adverse Pregnancy Outcomes Study were studied (942 Afro-Caribbean, 1294 Northern European, 573 Mexican-American, and 1182 Thai).<h4>Outcome measures</h4>Birth weight >90th percentile (LGA) and newborn SSF >90th percentile were primary outcomes.<h4>Results</h4>After adjustment for ancestry, sex, gestational age at delivery, parity, maternal genotype, maternal smoking/alcohol intake, age, body mass index, height, blood pressure and glucose, 25 and 23 SNPs were associated (P < .001) with birth weight and newborn SSF, respectively. The GRS was highly associated with both phenotypes as continuous variables across all ancestries (P ≤ 1.6 × 10(-19)) and improved prediction of birth weight and SSF >90th percentile when added to a baseline model incorporating the covariates listed above.<h4>Conclusions</h4>A GRS comprised of SNPs associated with adult obesity-related traits may provide an approach for predicting LGA birth and newborn adiposity beyond established risk factors.

<h4>Background</h4>We previously demonstrated that in the premanifest stage of Huntington's disease (preHD), a reduced functional connectivity exists compared to healthy controls. In the current study, we look at possible changes in functional connectivity occurring longitudinally over a period of 3 years, with the aim of assessing the potential usefulness of this technique as a biomarker for disease progression in preHD.<h4>Methods</h4>Twenty-two preHD and 17 healthy control subjects completed resting state functional magnetic resonance imaging (fMRI) scans in two visits with 3 years in between. Differences in resting state connectivity were examined for eight networks of interest using FSL with three different analysis types: a dual regression method, region of interest approach, and an independent component analysis. To evaluate a possible combined effect of gray matter volume change and the change in blood oxygenation level dependent signal, the analysis was performed with and without voxel-wise correction for gray matter volume. To evaluate possible correlations between functional connectivity change and the predicted time to disease onset, the preHD group was classed as preHD-A if ≥10.9 years and preHD-B if <10.9 years from predicted disease onset. Possible correlations between burden of pathology score and functional connectivity change in preHD were also assessed. Finally, longitudinal change in whole brain and striatal volumetric measures was assessed in the studied cohort.<h4>Results</h4>Longitudinal analysis of the resting state-fMRI (RS-fMRI) data revealed no differences in the degree of connectivity change between the groups over a period of 3 years, though a significantly higher rate of striatal atrophy was found in the preHD group compared to controls in the same period.<h4>Discussion</h4>Based on the results found in this study, the provisional conclusion is that RS-fMRI lacks sensitivity in detecting changes in functional connectivity in HD gene carriers prior to disease manifestation over a 3-year follow-up period.

Strong evidence links the 5-HTTLPR genotype to the modulation of amygdala reactivity during fear conditioning, which is considered to convey the increased vulnerability for anxiety disorders in s-allele carriers. In addition to amygdala reactivity, the 5-HTTLPR has been shown to be related to alterations in structural and effective connectivity. The aim of this study was to investigate the effects of 5-HTTLPR genotype on amygdala reactivity and effective connectivity during fear conditioning, as well as structural connectivity [as measured by diffusion tensor imaging (DTI)]. To integrate different classification strategies, we used the bi-allelic (s-allele vs l/l-allele group) as well as the tri-allelic (low-functioning vs high-functioning) classification approach. S-allele carriers showed exaggerated amygdala reactivity and elevated amygdala-insula coupling during fear conditioning (CS + > CS-) compared with the l/l-allele group. In addition, DTI analysis showed increased fractional anisotropy values in s-allele carriers within the uncinate fasciculus. Using the tri-allelic classification approach, increased amygdala reactivity and amygdala insula coupling were observed in the low-functioning compared with the high-functioning group. No significant differences between the two groups were found in structural connectivity. The present results add to the current debate on the influence of the 5-HTTLPR on brain functioning. These differences between s-allele and l/l-allele carriers may contribute to altered vulnerability for psychiatric disorders.

<h4>Background & aims</h4>Nonalcoholic fatty liver disease (NAFLD) is a risk factor for hepatocellular carcinoma (HCC). However, no systemic studies from the United States have examined temporal trends, HCC surveillance practices, and outcomes of NAFLD-related HCC.<h4>Methods</h4>We identified a national cohort of 1500 patients who developed HCC from 2005 through 2010 from Veterans Administration (VA) hospitals. We reviewed patients' full VA medical records; NAFLD was diagnosed based on histologic evidence for, or the presence of, the metabolic syndrome in the absence of hepatitis C virus (HCV) infection, hepatitis B, or alcoholic liver disease. We compared annual prevalence values for the main risk factors (NAFLD, alcohol abuse, and HCV), as well a HCC surveillance and outcomes, among HCC patients.<h4>Results</h4>NAFLD was the underlying risk factor for HCC in 120 patients (8.0%); the annual proportion of NAFLD-related HCC remained relatively stable (7.5%-12.0%). In contrast, the proportion of HCC cases associated with HCV increased from 61.0% in 2005 (95% confidence interval, 53.1%-68.9%) to 74.9% in 2010 (95% confidence interval, 69.0%-80.7%). The proportion of HCC cases associated with only alcohol abuse decreased from 21.9% in 2005 to 15.7% in 2010, and the annual proportion of HCC cases associated with hepatitis B remained relatively stable (1.4%-3.5%). A significantly lower proportion of patients with NAFLD-related HCC had cirrhosis (58.3%) compared with patients with alcohol- or HCV-related HCC (72.4% and 85.6%, respectively; P < .05). A significantly higher percentage of patients with NAFLD-related HCC did not receive HCC surveillance in the 3 years before their HCC diagnosis, compared with patients with alcohol- or HCV-associated HCC. A lower proportion of patients with NAFLD-related HCC received HCC-specific treatment (61.5%) than patients with HCV-related HCC (77.5%; P < .01). However, the 1-year survival rate did not differ among patients with HCC related to different risk factors.<h4>Conclusions</h4>NAFLD is the third most common risk factor for HCC in the VA population. The proportion of NAFLD-related HCC was relatively stable from 2005 through 2010. Although patients with NAFLD-related HCC received less HCC surveillance and treatment, a similar proportion survive for 1 year, compared with patients with alcohol-related or HCV-related HCC.

Cell invasion through basement membrane (BM) occurs in many physiological and pathological contexts. MIG-10, the Caenorhabditis elegans Lamellipodin (Lpd), regulates diverse biological processes. Its function and regulation in cell invasive behavior remain unclear. Using anchor cell (AC) invasion in C. elegans as an in vivo invasion model, we have previously found that mig-10's activity is largely outside of UNC-6 (netrin) signaling, a chemical cue directing AC invasion. We have shown that MIG-10 is a target of the transcription factor FOS-1A and facilitates BM breaching. Combining genetics and imaging analyses, we report that MIG-10 synergizes with UNC-6 to promote AC attachment to the BM, revealing a functional role for MIG-10 in stabilizing AC-BM adhesion. MIG-10 is also required for F-actin accumulation in the absence of UNC-6. Further, we identify mig-10 as a transcriptional target negatively regulated by EGL-43A (C. elegans Evi-1 proto-oncogene), a transcription factor positively controlled by FOS-1A. The revelation of this negative regulation unmasks an incoherent feedforward circuit existing among fos-1, egl-43 and mig-10. Moreover, our study suggests the functional importance of the negative regulation on mig-10 expression by showing that excessive MIG-10 impairs AC invasion. Thus, we provide new insight into MIG-10's function and its complex transcriptional regulation during cell invasive behavior.

Huntington׳s disease is a neurodegenerative disorder, attributable to an expanded trinucleotide repeat in the coding region of the human HTT gene, which encodes the protein huntingtin. These mutations lead to huntingtin fragment inclusions in the striatum of the brain. However, the exact function of normal huntingtin and the defect causing the disease remain obscure. Because there are indications that huntingtin plays a role in Ca(2+) homeostasis, we studied the deletion mutant of the HTT ortholog in the model developmental system Dictyostelium discoideum, in which Ca(2+) plays a role in receptor-regulated behavior related to the aggregation process that leads to multicellular morphogenesis. The D. discoideum htt(-)-mutant failed to undergo both K(+)-facilitated chemotaxis in spatial gradients of the major chemoattractant cAMP, and chemotaxis up a spatial gradient of Ca(2+), but behaved normally in Ca(2+)-facilitated cAMP chemotaxis and Ca(2+)-dependent flow-directed motility. This was the same phenotypic profile of the null mutant of Nhel, a monovalent cation/H(+)exchanger. The htt(-)-mutant also failed to orient correctly during natural aggregation, as was the case for the Nhel mutant. Moreover, in a K(+)-based buffer the normal localization of actin was similarly defective in both htt(-) and nhe1(-) cells in a K(+)-based buffer, and the normal localization of Nhe1 was disrupted in the htt(-) mutant. These observations demonstrate that Htt and Nhel play roles in the same specific cation-facilitated behaviors and that Nhel localization is directly or indirectly regulated by Htt. Similar cation-dependent behaviors and a similar relationship between Htt and Nhe1 have not been reported for mammalian neurons and deserves investigation, especially as it may relate to Huntington׳s disease.

Nitric oxide (NO) synthase 2 (NOS2), a major inflammatory protein, modulates disease progression via NO in a number of pathologies, including cancer. The role of NOS2-derived NO is not only flux-dependent, which is higher in mouse vs human cells, but also varies based on spatial and temporal distribution both within tumor cells and in the tumor microenvironment. NO donors have been utilized to mimic NO flux conditions and to investigate the effects of varied NO concentrations. As a wide range of effects mediated by NO and other nitrogen oxides such as nitroxyl (HNO) have been elucidated, multiple NO- and HNO-releasing compounds have been developed as potential therapeutics, including as tumor modulators. One of the challenges is to determine differences in biomarker expression from extracellular vs intracellular generation of NO or HNO. Taking advantage of new NO and HNO releasing agents, we have characterized the gene expression profile of estrogen receptor-negative human breast cancer (MDA-MB-231) cells following exposure to aspirin, the NO donor DEA/NO, the HNO donor IPA/NO andtheir intracellularly-activated prodrug conjugates DEA/NO-aspirin and IPA/NO-aspirin. Comparison of the gene expression profiles demonstrated that several genes were uniquely expressed with respect to NO or HNO, such as miR-21, HSP70, cystathionine γ-lyase and IL24. These findings provide insight into targets and pathways that could be therapeutically exploited by the redox related species NO and HNO.

Viral infection triggers the activation of antiviral innate immune responses in mammalian cells. Viral RNA in the cytoplasm activates signaling pathways that result in the production of interferons (IFNs) and IFN-stimulated genes. Some viral infections have been shown to induce cytoplasmic granular aggregates similar to the dynamic ribonucleoprotein aggregates termed stress granules (SGs), suggesting that these viruses may utilize this stress response for their own benefit. By contrast, some viruses actively inhibit SG formation, suggesting an antiviral function for these structures. We review here the relationship between different viral infections and SG formation. We examine the evidence for antiviral functions for SGs and highlight important areas of inquiry towards understanding cellular stress responses to viral infection.

Biomolecules have been widely investigated as potential therapeutics for various diseases. However their use is limited due to rapid degradation and poor cellular uptake in vitro and in vivo. To address this issue, we synthesized a new nano-carrier system comprising of cholic acid-polyethylenimine (CA-PEI) copolymer micelles, via carbodiimide-mediated coupling for the efficient delivery of small interfering ribonucleic acid (siRNA) and bovine serum albumin (BSA) as model protein. The mean particle size of siRNA- or BSA-loaded CA-PEI micelles ranged from 100-150 nm, with zeta potentials of +3-+11 mV, respectively. Atomic force, transmission electron and field emission scanning electron microscopy demonstrated that the micelles exhibited excellent spherical morphology. No significant morphology or size changes were observed in the CA-PEI micelles after siRNA and BSA loading. CA-PEI micelles exhibited sustained release profile, the effective diffusion coefficients were successfully estimated using a mathematically-derived cylindrical diffusion model and the release data of siRNA and BSA closely fitted into this model. High siRNA and BSA binding and loading efficiencies (95% and 70%, respectively) were observed for CA-PEI micelles. Stability studies demonstrated that siRNA and BSA integrity was maintained after loading and release. The CA-PEI micelles were non cytotoxic to V79 and DLD-1 cells, as shown by alamarBlue and LIVE/DEAD cell viability assays. RT-PCR study revealed that siRNA-loaded CA-PEI micelles suppressed the mRNA for ABCB1 gene. These results revealed the promising potential of CA-PEI micelles as a stable, safe, and versatile nano-carrier for siRNA and the model protein delivery.

Spontaneous immune tolerance in mouse liver transplantation has always been a hotspot in transplantation-immune research. Recent studies revealed that regulatory T cells (Tregs), hepatic satellite cells and Kupffer cells play a potential role in spontaneous immune tolerance, however the precise mechanism of spontaneous immune tolerance is still undefined. By using Microarray Chips, we investigated different immune regulatory factors to decipher critical mechanisms of spontaneous tolerance after mouse liver transplantation. Allogeneic (C57BL/6-C3H) and syngeneic (C3H-C3H) liver transplantation were performed by 6-8 weeks old male C57BL/6 and C3H mice. Graft samples (N = 4 each group) were collected from 8 weeks post-operation mice. 11 differentially expressed miRNAs in allogeneic grafts (Allografts) vs. syngeneic grafts (Syngrafts) were identified using Agilent Mouse miRNA Chips. It was revealed that 185 genes were modified by the 11 miRNAs, furthermore, within the 185 target genes, 11 of them were tightly correlated with immune regulation after Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Genbank data cross-comparison. Verified by real-time PCR and western blot, our results indicated that mRNA expression levels of IL-6 and TAB2 were respectively down regulated following miR-142-3p and miR-155 augment. In addition, increased miR-152 just silenced mRNA of CaMK II and down-regulated translation of CaMK II in tolerated liver grafts, which may play a critical role in immune regulation and spontaneous tolerance induction of mouse liver transplantation.

The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows it to bind many drugs and drug leads, possibly causing undesired drug-drug interactions. Therefore, it is crucial to evaluate whether chemicals or drugs bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. On the basis of our previously characterized 4 (BODIPY FL vinblastine), a high-affinity PXR probe, we developed 20 (BODIPY FL vindoline) and showed that it is a novel and potent PXR fluorescent probe with Kd of 256 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay with PXR. By using 20 (BODIPY FL vindoline) in the PXR TR-FRET assay, we obtained a more than 7-fold signal-to-background ratio and high signal stability (signal was stable for at least 120 min, and Z'-factor > 0.85 from 30 to 240 min). The assay can tolerate DMSO up to 2%. This assay has been used to evaluate a panel of PXR ligands for their PXR-binding affinities. The performance of 20 (BODIPY FL vindoline) in the PXR TR-FRET assay makes it an ideal PXR fluorescent probe, and the newly developed PXR TR-FRET assay with 20 (BODIPY FL vindoline) as a fluorescent probe is suitable for high-throughput screening to identify PXR-binding ligands.

The death-associated protein kinase 1 (DAPK1) is an important regulator of cell death and autophagy. Recently, we have identified that ATF6, an endoplasmic reticulum-resident transcription factor, in association with the transcription factor CEBP-β, regulates the gamma interferon (IFN-γ)-induced expression of Dapk1 (P. Gade et al., Proc. Natl. Acad. Sci. U. S. A. 109:10316-10321, 2012, doi.org/10.1073/pnas.1119273109). IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β were essential for the formation of a novel transcriptional complex that regulates DAPK1. Here, we report that IFN-γ activates the ASK1-MKK3/MKK6-p38 mitogen-activated protein kinase (MAPK) pathway for controlling the activity of ATF6. The terminal enzyme in this pathway, p38 MAPK, phosphorylates a critical threonine residue in ATF6 upstream of its DNA binding domain. ATF6 mutants defective for p38 MAPK phosphorylation fail to undergo proteolytic processing in the Golgi apparatus and drive IFN-γ-induced gene expression and autophagy. We also show that mice lacking Ask1 are highly susceptible to lethal bacterial infection owing to defective autophagy. Together, these results identify a novel host defense pathway controlled by IFN-γ signaling.

Short tandem repeats are among the most polymorphic loci in the human genome. These loci play a role in the etiology of a range of genetic diseases and have been frequently utilized in forensics, population genetics, and genetic genealogy. Despite this plethora of applications, little is known about the variation of most STRs in the human population. Here, we report the largest-scale analysis of human STR variation to date. We collected information for nearly 700,000 STR loci across more than 1000 individuals in Phase 1 of the 1000 Genomes Project. Extensive quality controls show that reliable allelic spectra can be obtained for close to 90% of the STR loci in the genome. We utilize this call set to analyze determinants of STR variation, assess the human reference genome's representation of STR alleles, find STR loci with common loss-of-function alleles, and obtain initial estimates of the linkage disequilibrium between STRs and common SNPs. Overall, these analyses further elucidate the scale of genetic variation beyond classical point mutations.

We characterized 30 community-associated extended-spectrum-β-lactamase-producing Escherichia coli isolates collected from five hospitals in the United States. Nineteen sequence types were identified. All sequence type 131 (ST131) isolates had the fimH30 allele. IncFII-FIA-FIB was the most common replicon type among the blaCTX-M-carrying plasmids, followed by IncFII-FIA and IncA/C. Restriction analysis of the IncFII-FIA-FIB and IncFII-FIA plasmids yielded related profiles for plasmids originating from different hospitals. The plasmids containing blaCTX-M or blaSHV were stably maintained after serial passages.

Non-coding RNAs (ncRNAs) play various roles during central nervous system development. MicroRNAs (miRNAs) are a class of ncRNAs that exert their function together with argonaute proteins by post-transcriptional gene silencing of messenger RNAs (mRNAs). Several studies provide evidence for alterations in miRNA expression in patients with neurodegenerative diseases. Among these is huntington's disease (HD), a dominantly inherited fatal disorder characterized by deregulation of neuronal-specific mRNAs as well as miRNAs. Recently, next-generation sequencing (NGS) miRNA profiles from human HD and neurologically normal control brain tissues were reported. Five consistently upregulated miRNAs affect the expression of genes involved in neuronal differentiation, neurite outgrowth, cell death and survival. We re-analyzed the NGS data publicly available in array express and detected nineteen additional differentially expressed miRNAs. Subsequently, we connected these miRNAs to genes implicated in HD development and network analysis pointed to miRNA-mediated downregulation of twenty-two genes with roles in the pathogenesis as well as treatment of the disease. In silico prediction and reporter systems prove that levels of BDNF, a central node in the miRNA-mRNA regulatory network, can be post-transcriptionally controlled by upregulated miR-10b-5p and miR-30a-5p. Reduced BDNF expression is associated with neuronal dysfunction and death in HD. Moreover, the 3'UTR of CREB1 harbors a predicted binding site for these two miRNAs. CREB1 is similarly downregulated in HD and overexpression decreased susceptibility to 3-nitropropionic-induced toxicity in a cell model. In contradiction to these observations, it is presumed that miR-10b-5p upregulation in HD exerts a neuroprotective role in response to the mutation in the huntingtin gene. Therefore, the function of miR-10b-5p and especially its effect on BDNF expression in HD requires further academic research.

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders, and 'a disease of synapses' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies, little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare, multiply affected, large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and, furthermore, dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders.

<h4>Background</h4>Cytomegalovirus (CMV) infection is the most important cause of congenital viral infection in developed countries. In utero transmission occurs at higher rates in seronegative women during primary infection, especially those in contact with young children in day-care centers (DCC). Nevertheless data on variability of CMV excretion among children in French DCCs are lacking, and are important for public health planning.<h4>Objectives</h4>Our main objective was to assess the feasibility of a salivary sample in DCCs in order to study CMV excretion among toddlers. Our secondary aims were to assess prevalence of CMV excretion in children attending Hospital Emergency Unit (EU) in comparison with various types of DCCs and to validate the analytical chain for collected specimens.<h4>Study design</h4>Excretion of CMV in saliva was quantified using a real-time PCR assay in children aged from 3 months to 6 years old in EU and in DCC, with gB, gH and gN genotypes determined in infected children. Salivary sampling was performed using small sponges placed into a DNA conservation medium. Socio cultural and medical information were collected from attending parents.<h4>Results</h4>A total of 625 children were included, with 256 from six DCCs and 369 from one EU. In DCCs, the acceptability of the procedure was 87.3% (95%CI 78.5-96.2) amongst parents and children, and in the EU, acceptability was higher at 97.6% (95%CI 95.5-98.9). CMV shedding overall prevalence was 21.7% (95%CI 17.6-26.2), with CMV shedding prevalence in DCCs of 51.9% (95%CI 22.8-81.1).<h4>Conclusion</h4>We validated the feasibility and acceptability of measuring CMV shedding in the saliva of French toddlers. The discrepancy between CMV infection rates in day care centers and in the general population (as sampled in the EU) indicates the need for a further study to determine risk factors and shedding levels in the DCC population.

There is a controversial relationship between HLA-A2 and Alzheimer's disease (AD). It has been suggested a modifier effect on the risk that depends on genetic loadings. Thus, the aims of this study were to evaluate this relationship and to reveal genes associated with both concepts the HLA-A gene and AD. Consequently, we did first a classical systematic review and a meta-analysis of case-control studies. Next, by means of an in silico approach, we used experimental knowledge of protein-protein interactions to evaluate the top ranked genes shared by both concepts, previously found through text mining. The meta-analysis did not show a significant pooled OR (1.11, 95% CI: 0.98 to 1.24 in Caucasians), in spite of the fact that four of the included studies had a significant OR > 1 and none of them a significant OR < 1. In contrast, the in silico approach retrieved nonrandomly shared genes by both concepts (P = 0.02), which additionally encode truly interacting proteins. The network of proteins encoded by APP, ICAM-1, ITGB2, ITGAL, SELP, SELL, IL2, IL1B, CD4, and CD8A linked immune to neurodegenerative processes and highlighted the potential roles in AD pathogenesis of endothelial regulation, infectious diseases, specific antigen presentation, and HLA-A2 in maintaining synapses.

<h4>Background</h4>MicroRNAs (miRNAs) have been identified as important posttranscriptional regulators involved in various biological and pathological processes of cells, but their association with tumor chemoresistance has not been fully understood.<h4>Methods</h4>We detected miR-27a expression in two lung adenocarcinoma cell lines, A549 and A549/CDDP, and then investigated the effects of miR-27a on the metastasis and the chemosensitivity of cancer cells, using both gain- and loss-of-function studies. The correlation between miR-27a level and chemoresistance was further investigated in clinical lung adenocarcinoma specimens.<h4>Results</h4>miR-27a was significantly up-regulated in cisplatin-resistant lung adenocarcinoma A549/CDDP cells compared with parental A549 cells. miR-27a regulates epithelial-mesenchymal transition (EMT) and cisplatin resistance in vitro and modulates response of lung adenocarcinoma cells to cisplatin in vivo. Further studies identified Raf Kinase Inhibitory Protein (RKIP) as a direct and functional target of miR-27a. Small interfering RNA-mediated RKIP knockdown revealed similar effects as that of ectopic miR-27a expression, while overexpression of RKIP attenuated the function of miR-27a in lung adenocarcinoma cells. Increased miR-27a expression was also detected in tumor tissues sampled from lung adenocarcinoma patients treated with cisplatin-based chemotherapy and was proved to be correlated with low expression of RKIP, decreased sensitivity to cisplatin, and poor prognosis.<h4>Conclusion</h4>Our results suggest that up-regulation of miR-27a could suppress RKIP expression and in turn contribute to chemoresistance of lung adenocarcinoma cells to cisplatin.

Dopaminergic (DA) neuron-like cells obtained through direct reprogramming of primary human fibroblasts offer exciting opportunities for treatment of Parkinson's disease. A significant obstacle is the low efficiency of conversion during the reprogramming process. Here, we demonstrate that the suppression of p53 significantly enhances the efficiency of transcription factor-mediated conversion of human fibroblasts into functional dopaminergic neurons. In particular, blocking p53 activity using a dominant-negative p53 (p53-DN) in IMR90 cells increases the conversion efficiency by 5-20 fold. The induced DA neuron-like cells exhibit dopamine neuron-specific gene expression, significant dopamine uptake and production capacities, and enables symptomatic relief in a rat Parkinson's disease model. Taken together, our findings suggest that p53 is a critical barrier in direct reprogramming of fibroblast into dopaminergic neurons.

Therapy-resistant cancer cells are a major problem in cancer research. Recent studies suggest that the epithelial-mesenchymal transition (EMT) is a key mechanism in therapy resistance. Yet, the expressions of EMT markers, EMT core regulators, and a stem cell marker of BMI1 during chemotherapy have been poorly analyzed in clinical breast cancer specimens. In the present study, we investigated the roles of RhoC under chemotherapy to follow up on earlier findings demonstrating the involvement of RhoC in prostate cancer resistance to endocrine therapy. Immunohistochemically, E-cadherin expression was significantly lower in human breast cancer specimens analyzed after chemotherapy than specimens biopsied before chemotherapy. Significant upregulation of fibronectin, a mesenchymal EMT marker, was found in post-chemotherapy analysis. A study of the EMT core regulators of SNAIL1, SNAIL2, TWIST1, and a well-known stem cell marker of BMI1 revealed no post-chemotherapy upregulation of these molecules. In contrast, RhoC expression was significantly upregulated in post-chemotherapy breast cancer specimens. MCF-7 cells stably transfected with the constitutive active (CA) RhoC plasmid manifested a reduced level of E-cadherin at the peripheries and disorganization of actin fibers, with no accompanying upregulation of SNAIL1, SNAIL2, TWIST1, or BMI1 in Western blots. Exposure of etoposide on MCF-7 cells showed RhoC upregulation together with reduced membranous expression of E-cadherin and disorganization of actin fibers. In MTT assay, however, the CA-RhoC-expressing MCF-7 cells failed to show chemotherapy resistance under etoposide treatment. Taken in sum, RhoC may contribute to an EMT-like process in human breast cancer during chemotherapy.

When cells die by necrosis in vivo they stimulate an inflammatory response. It is thought that this response is triggered when the injured cells expose proinflammatory molecules, collectively referred to as damage associated molecular patterns (DAMPs), which are recognized by cells or soluble molecules of the innate or adaptive immune system. Several putative DAMPs and/or their receptors have been identified, but whether and how much they participate in responses in vivo is incompletely understood, and they have not previously been compared side-by-side in the same models. This study focuses on evaluating the contribution of multiple mechanisms that have been proposed to or potentially could participate in cell death-induced inflammation: The third component of complement (C3), ATP (and its receptor P2X7), antibodies, the C-type lectin receptor Mincle (Clec4e), and protease-activated receptor 2 (PAR2). We investigate the role of these factors in cell death-induced inflammation to dead cells in the peritoneum and acetaminophen-induced liver damage. We find that mice deficient in antibody, C3 or PAR2 have impaired inflammatory responses to dying cells. In contrast there was no reduction in inflammation to cell death in the peritoneum or liver of mice that genetically lack Mincle, the P2X7 receptor or that were treated with apyrase to deplete ATP. These results indicate that antibody, complement and PAR2 contribute to cell death-induced inflammation but that Mincle and ATP- P2X7 receptor are not required for this response in at least 2 different in vivo models.

Panic disorder (PD) is a severe anxiety disorder that is characterized by recurrent panic attacks (PA), which can be unexpected (uPA, i.e., no clear identifiable trigger) or expected (ePA). Panic typically involves an abrupt feeling of catastrophic fear or distress accompanied by physiological symptoms such as palpitations, racing heart, thermal sensations, and sweating. Recurrent uPA and ePA can also lead to agoraphobia, where subjects with PD avoid situations that were associated with PA. Here we will review recent developments in our understanding of PD, which includes discussions on: symptoms and signs associated with uPA and ePAs; Diagnosis of PD and the new DSM-V; biological etiology such as heritability and gene×environment and gene×hormonal development interactions; comparisons between laboratory and naturally occurring uPAs and ePAs; neurochemical systems that are associated with clinical PAs (e.g. gene associations; targets for triggering or treating PAs), adaptive fear and panic response concepts in the context of new NIH RDoc approach; and finally strengths and weaknesses of translational animal models of adaptive and pathological panic states.

Adenosine A1 receptor (A1R) stimulation exerts beneficial effects in response to various insults to the brain and, although it was found neuroprotective in a lesional model of Huntington's disease (HD), the features of this receptor in genetic models of HD have never been explored. In the present study we characterized the expression, affinity and functional effects of A1Rs in R6/2 mice (the most widely used transgenic model of HD) and in a cellular model of HD. Binding studies revealed that the density of A1Rs was significantly reduced in the cortex and the striatum of R6/2 mice compared to age-matched wild-type (WT), while receptor affinity was unchanged. The selective A1R agonist cyclopentyladenosine (CPA, 300nM) was significantly more effective in reducing synaptic transmission in corticostriatal slices from symptomatic R6/2 than in age-matched WT mice. Such an effect was due to a stronger inhibition of glutamate release from the pre-synaptic terminal. The different functional activities of A1Rs in HD mice were associated also to a different intracellular signaling pathway involved in the synaptic effect of CPA. In fact, while the PKA pathway was involved in both genotypes, p38 MAPK inhibitor SB203580 partially prevented synaptic effects of CPA in R6/2, but not in WT, mice; moreover, CPA differently modulated the phosphorylation status of p38 in the two genotypes. In vitro studies confirmed a different behavior of A1Rs in HD: CPA (100 nM for 5h) modulated cell viability in STHdh(Q111/Q111) (mhttHD cells), without affecting the viability of STHdh(Q7/Q7) (wthtt cells). This effect was prevented by the application of SB203580. Our results demonstrate that in the presence of the HD mutation A1Rs undergo profound changes in terms of expression, pharmacology and functional activity. These changes have to be taken in due account when considering A1Rs as a potential therapeutic target for this disease.

Huntington's disease (HD) is caused by an expanded polyglutamine repeat in huntingtin protein that disrupts synaptic function in specific neuronal populations and results in characteristic motor, cognitive and affective deficits. Histopathological hallmarks observed in both HD patients and genetic mouse models include the reduced expression of synaptic proteins, reduced medium spiny neuron (MSN) dendritic spine density and decreased frequency of spontaneous excitatory post-synaptic currents (sEPSCs). Early down-regulation of cannabinoid CB1 receptor expression on MSN (CB1(MSN)) is thought to participate in HD pathogenesis. Here we present a cell-specific genetic rescue of CB1(MSN) in R6/2 mice and report that treatment prevents the reduction of excitatory synaptic markers in the striatum (synaptophysin, vGLUT1 and vGLUT2), of dendritic spine density on MSNs and of MSN sEPSCs, but does not prevent motor impairment. We conclude that loss of excitatory striatal synapses in HD mice is controlled by CB1(MSN) and can be uncoupled from the motor phenotype.

Faced with the need to find new drugs for all kinds of diseases, science sees that Nature offers numerous classes of compounds showing an impressively high biological potential. Among those are the cyclodepsipeptides, hybrid structures composed of amino and hydroxy acids. In the past decades numerous cyclodepsipeptides have been isolated and their potential as drugs has been studied extensively. For several cyclodepsipeptides total syntheses both in solution and on solid-phase have been established, allowing the production of combinatorial libraries. In addition, the biosynthesis of specific cyclodepsipeptides has been elucidated and used for the chemoenzymatic preparation of nonnatural analogues. This review summarizes the recent literature on cyclic tetra- to decadepsipeptides, composed exclusively of α-amino- and α-hydroxy acids.

The details of the total synthesis of viridicatumtoxin B (1) are described. Initial synthetic strategies toward this intriguing tetracycline antibiotic resulted in the development of key alkylation and Lewis acid-mediated spirocyclization reactions to form the hindered EF spirojunction, as well as Michael-Dieckmann reactions to set the A and C rings. The use of an aromatic A-ring substrate, however, was found to be unsuitable for the introduction of the requisite hydroxyl groups at carbons 4a and 12a. Applying these previous tactics, we developed stepwise approaches to oxidize carbons 12a and 4a based on enol- and enolate-based oxidations, respectively, the latter of which was accomplished after systematic investigations that revealed critical reactivity patterns. The herein described synthetic strategy resulted in the total synthesis of viridicatumtoxin B (1), which, in turn, formed the basis for the revision of its originally assigned structure. The developed chemistry facilitated the synthesis of a series of viridicatumtoxin analogues, which were evaluated against Gram-positive and Gram-negative bacterial strains, including drug-resistant pathogens, revealing the first structure-activity relationships within this structural type.

RKIP is proposed as a new metastasis suppressor. Our recent study showed that RKIP inhibits malignant phenotypes of gastric cancer cells. However, the underlying mechanism of RKIP function in gastric cancer is unclear. This study aimed to investigate the correlation of RKIP, STAT3 and cyclin D1 expression in the tumorigenesis of gastric cancer. RKIP, STAT3 and cyclin D1 proteins were detected by immunohistochemistry in tissues of gastric ulcer (n = 27), gastric adenomatous polyp (n = 7), intestinal metaplasia (n = 26), dysplasia (n = 40), gastric carcinoma (n = 169) and metastatic lymph node (n = 36). RKIP, STAT3 and cyclin D1 mRNA levels were analyzed by RT-PCR in SGC7901 cells. We found that RKIP protein expression was significantly decreased in advanced gastric cancer and metastatic lymph node tissues while cyclin D1 and STAT3 protein expression was markedly increased in severe dysplasia, gastric cancer and metastatic lymph node tissue (P < 0.01). RKIP expression in gastric cancer was negatively correlated with the invasion, TNM stage and lymphoid node metastasis (P < 0.01), while cyclin D1 and STAT3 expression was positively correlated with histological differentiation and lymphoid node metastasis (P < 0.01). RKIP protein level was negatively correlated with cyclin D1 and STAT3 protein level, while cyclin D1 protein level was positively correlated with STAT3 protein level in gastric cancer samples. Moreover, reconstitution of RKIP in SGC7901 gastric cancer cells led to reduced cyclin D1 and STAT3 mRNA levels. In conclusion, these data suggest that RKIP inhibits gastric cancer metastasis via the downregulation of its downstream target genes STAT3 and cyclin D1.

Most solid tumors contain cancer-associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF-like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF-specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n = 145) but not in the stroma of normal ovaries (n = 4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n = 8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.

<h4>Introduction</h4>Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin (htt) protein, which underlies the loss of striatal and cortical neurons. Glutamate has been implicated in a number of neurodegenerative diseases, and several studies suggest that the metabotropic glutamate receptor 5 (mGluR5) may represent a target for the treatment of HD.<h4>Areas covered</h4>The main goal of this review is to discuss the current data in the literature regarding the role of mGluR5 in HD and evaluate the potential of mGluR5 as a therapeutic target for the treatment of HD. mGluR5 is highly expressed in the brain regions affected in HD and is involved in movement control. Moreover, mGluR5 interacts with htt and mutated htt profoundly affects mGluR5 signaling. However, mGluR5 stimulation can activate both neuroprotective and neurotoxic signaling pathways, depending on the context of activation.<h4>Expert opinion</h4>Although the data published so far strongly indicate that mGluR5 plays a major role in HD-associated neurodegeneration, htt aggregation and motor symptoms, it is not clear whether mGluR5 stimulation can diminish or intensify neuronal cell loss and HD progression. Thus, future experiments will be necessary to further investigate the outcome of drugs acting on mGluR5 for the treatment of neurodegenerative diseases.

Paclitaxel is one of the most effective chemotherapy drugs for advanced cervical cancer. However, acquired resistance of paclitaxel represents a major barrier to successful anticancer treatment. In this study, paclitaxel-resistant HeLa sublines (HeLa-R cell lines) were established by continuous exposure and increased autophagy level was observed in HeLa-R cells. 3-Methyladenine or ATG7 siRNA, autophagy inhibitors, could restore sensitivity of HeLa-R cells to paclitaxel compared with parental HeLa cells. To determine the underlying molecular mechanism, differentially expressed proteins between HeLa and HeLa-R cells were identified by two-dimensional gel electrophoresis coupled with electrospray ionization quadrupole time-of-flight MS/MS. We found glycolysis-associated proteins were upregulated in HeLa-R cell lines. Inhibition of glycolysis by 2-deoxy-D-glucose or koningic acid could decrease autophagy and enhance sensitivity of HeLa-R cells to paclitaxel. Moreover, glycolysis could activate HIF1-α. Downregulation of HIF1-α by specific siRNA could decrease autophagy and resensitize HeLa-R cells to paclitaxel. Taken together, a possible Warburg effect activated HIF1-α-mediated signaling-induced autophagic pathway is proposed, which may provide new insight into paclitaxel chemoresistance.

Understanding how human neonates respond to infection remains incomplete. Here, a system-level investigation of neonatal systemic responses to infection shows a surprisingly strong but unbalanced homeostatic immune response; developing an elevated set-point of myeloid regulatory signalling and sugar-lipid metabolism with concomitant inhibition of lymphoid responses. Innate immune-negative feedback opposes innate immune activation while suppression of T-cell co-stimulation is coincident with selective upregulation of CD85 co-inhibitory pathways. By deriving modules of co-expressed RNAs, we identify a limited set of networks associated with bacterial infection that exhibit high levels of inter-patient variability. Whereas, by integrating immune and metabolic pathways, we infer a patient-invariant 52-gene-classifier that predicts bacterial infection with high accuracy using a new independent patient population. This is further shown to have predictive value in identifying infection in suspected cases with blood culture-negative tests. Our results lay the foundation for future translation of host pathways in advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis.

We have developed a dual-functional nanocarrier composed of a hydrophilic polyethylene glycol (PEG) and a hydrophobic farnesylthiosalicylate (FTS, a nontoxic Ras antagonist), which is effective in delivery of hydrophobic anticancer drug, paclitaxel (PTX). To facilitate the retention of the therapeutic activity of the carrier, FTS was coupled to PEG via a reduction-sensitive disulfide linkage (PEG5k-S-S-FTS2). PEG5k-S-S-FTS2 conjugate formed uniform micelles with very small size (∼30 nm) and the hydrophobic drug PTX could be readily incorporated into the micelles. Interestingly, inclusion of a disulfide linkage into the PEG5k-FTS2 micellar system resulted in a 4-fold decrease in the critical micelle concentration (CMC). In addition, the PTX loading capacity and colloidal stability of PTX-loaded micelles were improved. HPLC-MS showed that parent FTS could be more effectively released from PEG5k-S-S-FTS2 conjugate in tumor cells/tissues compared to PEG5k-FTS2 conjugate in vitro and in vivo. PEG5k-S-S-FTS2 exhibited a higher level of cytotoxicity toward tumor cells than PEG5k-FTS2 without a disulfide linkage. Furthermore, PTX-loaded PEG5k-S-S-FTS2 micelles were more effective in inhibiting the proliferation of cultured tumor cells compared to Taxol and PTX loaded in PEG5k-FTS2 micelles. More importantly, PTX-loaded PEG5k-S-S-FTS2 micelles demonstrated superior antitumor activity compared to Taxol and PTX formulated in PEG5k-FTS2 micelles in an aggressive murine breast cancer model (4T1.2).

A common metaphor for describing development is a rugged "epigenetic landscape" where cell fates are represented as attracting valleys resulting from a complex regulatory network. Here, we introduce a framework for explicitly constructing epigenetic landscapes that combines genomic data with techniques from spin-glass physics. Each cell fate is a dynamic attractor, yet cells can change fate in response to external signals. Our model suggests that partially reprogrammed cells are a natural consequence of high-dimensional landscapes, and predicts that partially reprogrammed cells should be hybrids that co-express genes from multiple cell fates. We verify this prediction by reanalyzing existing datasets. Our model reproduces known reprogramming protocols and identifies candidate transcription factors for reprogramming to novel cell fates, suggesting epigenetic landscapes are a powerful paradigm for understanding cellular identity.

We recently discovered a secreted and diffusible midline cue called MADD-4 (an ADAMTSL) that guides migrations along the dorsoventral axis of the nematode Caenorhabditis elegans. We showed that the transmembrane receptor, UNC-40 (DCC), whose canonical ligand is the UNC-6 (netrin) guidance cue, is required for extension towards MADD-4. Here, we demonstrate that MADD-4 interacts with an EVA-1/UNC-40 co-receptor complex to attract cell extensions. EVA-1 is a conserved transmembrane protein with predicted galactose-binding lectin domains. EVA-1 functions in the same pathway as MADD-4, physically interacts with both MADD-4 and UNC-40, and enhances UNC-40's sensitivity to the MADD-4 cue. This enhancement is especially important in the presence of UNC-6. In EVA-1's absence, UNC-6 interferes with UNC-40's responsiveness to MADD-4; in UNC-6's absence, UNC-40's responsiveness to MADD-4 is less dependent on EVA-1. By enabling UNC-40 to respond to MADD-4 in the presence of UNC-6, EVA-1 may increase the precision by which UNC-40-directed processes can reach their MADD-4-expressing targets within a field of the UNC-6 guidance cue.

<h4>Background</h4>The hepatitis C virus (HCV) genotype-specific impacts on the host metabolic alterations remained inconclusive.<h4>Methods</h4>A prospective study including 229 (118 genotype 1 (G1) and 111 G2) consecutive chronic HCV patients who had completed a course of anti-HCV treatment and underwent pre- and 24 weeks post-treatment surveys of metabolic profiles was conducted. Patients were stratified according to the therapeutic response, viral genotype and baseline insulin resistance (IR: homeostasis model assessments of IR (HOMA-IR) ≥ 2.5). Paired t-tests were used to compare the pre- and post-treatment variables.<h4>Results</h4>Significant post-therapeutic increases in cholesterol, triglyceride, HDL, LDL, apolipoprotein A1 and apolipoprotein B were observed in patients with sustained virological response (SVR) but not in those without. Among those with SVR, post-therapeutic increases in HDL (p<0.001) and apolipoprotein A1 (p = 0.012) were only found in G2, whereas increased triglyceride/HDL (p = 0.01) ratios were only found in G1 patients. When stratified by baseline IR among those with SVR, a significant increase in post-treatment HDL (p = 0.019) and apolipoprotein A1 (p = 0.012) but a decrease in HOMA-IR (p = 0.04), C-peptide (p = 0.019) and hemoglobin A1c (p = 0.047) were found in patients with baseline IR; a significant increase in HOMA-IR (p = 0.002) was found in patients without baseline IR. The latter change was observed only in G1 (p = 0.01) but not G2 patients. Although the pre-treatment metabolic profiles of G1 and G2 patients were indifferent, G1 had higher post-treatment triglyceride/HDL ratios (p = 0.041) and triglyceride (p = 0.044) levels than G2 patients.<h4>Conclusions</h4>G2 benefit more than G1 patients from viral clearance in metabolic alterations, particularly in those without baseline IR.

Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.

Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.

Columnar cell hyperplasia (CCH) is the earliest histologically identifiable breast lesion linked to cancer progression and is characterized by increased proliferation, decreased apoptosis and elevated oestrogen receptor α (ERα) expression. The mechanisms underlying the initiation of these lesions have not been clarified but might involve early and fundamental changes in cancer progression. MiRNAs are key regulators of several biological processes, acting by influencing the post-transcriptional regulation of numerous targets, thus making miRNAs potential candidates in cancer initiation. Here we have defined novel epithelial as well as stromal miRNA signatures from columnar cell hyperplasia lesions compared to normal terminal duct lobular units by using microdissection and miRNA microarrays. Let-7c were among the identified downregulated epithelial miRNAs and its functions were delineated in unique CCH derived cells and breast cancer cell line MCF-7 suggesting anti-proliferative traits potentially due to effects on Myb and ERα. MiR-132 was upregulated in the stroma surrounding CCH compared to stoma surrounding normal terminal duct lobular units (TDLUs), and overexpression of miR-132 in immortalized fibroblasts and in fibroblasts co-cultured with epithelial CCH cells caused substantial expression changes of genes involved in metabolism, DNA damage and cell motility. The miRNA signatures identified in CCH indicate early changes in the epithelial and stromal compartment of CCH and could represent early key alterations in breast cancer progression that potentially could be targeted in novel prevention or treatment schedules.

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.

<h4>Purpose</h4>Mutations in the cilia-centrosomal protein of centrosomal protein of 290 kDa (CEP290) result in severe ciliopathies, including autosomal recessive early onset childhood blindness disorder Leber congenital amaurosis (LCA). The Cep290(rd16) (retinal degeneration 16) mouse model of CEP290-LCA exhibits accumulation of CEP290-interacting protein Raf-1 kinase inhibitory protein (RKIP) prior to onset of retinal degeneration (by postnatal day P14). We hypothesized that reducing RKIP levels in the Cep290(rd16) mouse will delay or improve retinal phenotype.<h4>Methods</h4>We generated double mutant mice by combining the Cep290(rd16) and Rkip(ko) alleles (Cep290(rd16):Rkip(+/ko) and Cep290(rd16):Rkip(ko/ko)). Retinal function was assessed by ERG and retinal morphology and protein trafficking were assessed by histology, transmission electron microscopy (TEM), and immunofluorescence analysis. Cell death was examined by apoptosis.<h4>Results</h4>Prior to testing our hypothesis, we examined ERG and retinal morphology of Rkip(ko/ko) mice and did not find any detectable differences compared with wild-type mice. The Cep290(rd16):Rkip(+/ko) mice exhibited similar retinopathy as Cep290(rd16); however, Cep290(rd16): Rkip(ko/ko) double knockout mice demonstrated a substantial improvement (>9-fold) in photoreceptor function and structure at P18 as of Cep290(rd16) mice. We consistently detected transient preservation of photoreceptors at P18 and polarized trafficking of opsins to sensory cilia in the double mutant mice; however, retinal degeneration ensued by P30.<h4>Conclusions</h4>Our studies implicate CEP290-RKIP pathway in CEP290-retinal degeneration and suggest that targeting RKIP levels can delay photoreceptor degeneration, assisting in extending the time-window for treating such rapidly progressing blindness disorder.

With the continued extension of lifespan, aging and age-related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons' DNA. When the accumulation of DNA damage exceeds a neuron's capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in the nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.

The function of neural circuits depends on the precise connectivity between populations of neurons. Increasing evidence indicates that disruptions in excitatory or inhibitory synapse formation or function lead to excitation/inhibition (E/I) imbalances and contribute to neurodevelopmental and psychiatric disorders. Leucine-rich repeat (LRR)-containing surface proteins have emerged as key organizers of excitatory and inhibitory synapses. Distinct LRR proteins are expressed in different cell types and interact with key pre- and postsynaptic proteins. These protein interaction networks allow LRR proteins to coordinate pre- and postsynaptic elements during synapse formation and differentiation, pathway-specific synapse development, and synaptic plasticity. LRR proteins, therefore, play a critical role in organizing synaptic connections into functional neural circuits, and their dysfunction may contribute to neuropsychiatric disorders.

Coreceptor CD4 and CD8αβ double-negative (DN) TCRαβ(+) intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαβ(+) intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαβ(+) lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is nonoverlapping with that of CD4(+) or CD8αβ(+) T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαβ(+) T cells form a third lineage of TCRαβ T lymphocytes expressing a variable TCR repertoire, which serve nonredundant immune functions.

CAG/CTG trinucleotide repeats are unstable, fragile sequences that strongly position nucleosomes, but little is known about chromatin modifications required to prevent genomic instability at these or other structure-forming sequences. We discovered that regulated histone H4 acetylation is required to maintain CAG repeat stability and promote gap-induced sister chromatid recombination. CAG expansions in the absence of H4 HATs NuA4 and Hat1 and HDACs Sir2, Hos2, and Hst1 depended on Rad52, Rad57, and Rad5 and were therefore arising through homology-mediated postreplication repair (PRR) events. H4K12 and H4K16 acetylation were required to prevent Rad5-dependent CAG repeat expansions, and H4K16 acetylation was enriched at CAG repeats during S phase. Genetic experiments placed the RSC chromatin remodeler in the same PRR pathway, and Rsc2 recruitment was coincident with H4K16 acetylation. Here we have utilized a repetitive DNA sequence that induces endogenous DNA damage to identify histone modifications that regulate recombination efficiency and fidelity during postreplication gap repair.

<h4>Purpose</h4>The purpose of this study was to evaluate the properties of a porous zirconia scaffold coated with bioactive materials and compare the in vitro cellular behavior of MC3T3-E1 preosteoblastic cells to titanium and zirconia disks and porous zirconia scaffolds.<h4>Materials and methods</h4>Titanium and zirconia disks were prepared. A porous zirconia scaffold was fabricated with an open cell polyurethane disk foam template. The porous zirconia scaffolds were coated with β-TCP, HA and a compound of β-TCP and HA (BCP). The characteristics of the specimens were evaluated using scanning electron microscopy (SEM), energy dispersive x-ray spectrometer (EDX), and x-ray diffractometry (XRD). The dissolution tests were analyzed by an inductively coupled plasma spectrometer (ICP). The osteogenic effect of MC3T3-E1 cells was assessed via cell counting and reverse transcriptase-polymerase chain reaction (RT-PCR).<h4>Results</h4>The EDX profiles showed the substrate of zirconia, which was surrounded by the Ca-P layer. In the dissolution test, dissolved Ca(2+) ions were observed in the following decreasing order; β-TCP > BCP > HA (P<.05). In the cellular experiments, the cell proliferation on titanium disks appeared significantly lower in comparison to the other groups after 5 days (P<.05). The zirconia scaffolds had greater values than the zirconia disks (P<.05). The mRNA level of osteocalcin was highest on the non-coated zirconia scaffolds after 7 days.<h4>Conclusion</h4>Zirconia had greater osteoblast cell activity than titanium. The interconnecting pores of the zirconia scaffolds showed enhanced proliferation and cell differentiation. The activity of osteoblast was more affected by microstructure than by coating materials.

Through synthetic lethality screening of isogenic cell lines with and without the oncogenic KRAS gene and through lead compound optimization, we recently developed a novel anticancer agent designated NSC-743380 (oncrasin-72) that has promising in vitro and in vivo anticancer activity in a subset of cancer cell lines, including KRAS-mutant cancer cells. However, NSC-743380 tends to form dimers, which dramatically reduces its anticancer activity. To improve the physicochemical properties of NSC-743380, we synthesized a prodrug of NSC-743380, designated oncrasin-266, by modifying NSC-743380 with cyclohexylacetic acid and evaluated its in vitro and in vivo properties. Oncrasin-266 spontaneously hydrolyzed in phosphate-buffered saline in a time-dependent manner and was more stable than NSC-743380 in powder or stock solutions. In vivo administration of oncrasin-266 in mice led to the release of NSC-743380 which improved the pharmacokinetics of NSC-743380. Tissue distribution analysis revealed that oncrasin-266 was deposited in liver, whereas released NSC-743380 was detected in liver, lung, kidney, and subcutaneous tumor. Oncrasin-266 was better tolerated in mice at a higher dose level treatment (150-300 mg/kg, ip) than the parent agent was, suggesting that the prodrug reduced the acute toxicity of the parent agent. Our results demonstrated that the prodrug strategy could improve the stability, pharmacokinetic properties, and safety of NSC-743380.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate transcriptional and posttranscriptional gene regulation of the cell. Experimental evidence shows that miRNAs have a direct role in different cellular processes, such as immune function, apoptosis, and tumorigenesis. In a viral infection context, miRNAs have been connected with the interplay between host and pathogen, occupying a major role in pathogenesis. While numerous viral miRNAs from DNA viruses have been identified, characterization of functional RNA virus-encoded miRNAs and their potential targets is still ongoing. Here, we used an in silico approach to analyze dengue Virus genome sequences. Pre-miRNAs were extracted through VMir software, and the identification of putative pre-miRNAs and mature miRNAs was accessed using Support Vector Machine web tools. The targets were scanned using miRanda software and functionally annotated using ClueGo. Via computational tools, eight putative miRNAs were found to hybridize with numerous targets of morphogenesis, differentiation, migration, and growth pathways that may play a major role in the interaction of the virus and its host. Future approaches will focus on experimental validation of their presence and target messenger RNA genes to further elucidate their biological functions in human and mosquito cells.

Prenatal diagnosis (PD) is recommended in pregnancies after a Preimplantation Genetic Diagnosis (PGD). However, conventional PD entails a risk of fetal loss which makes PGD patients reluctant to undergo obstetric invasive procedures. The presence of circulating fetal DNA in maternal blood allows performing a non-invasive prenatal diagnosis (NIPD) without risk for the pregnancy outcome. This work shows the introduction of NIPD for confirmation of PGD results in eight pregnancies. In those pregnancies referred to PGD for an X-linked disorder (six out of eight), fetal sex determination in maternal blood was performed to confirm fetal sex. One pregnancy referred to PGD for Marfan syndrome and one referred for Huntington disease (HD) were also analyzed. In seven out of eight cases, PGD results were confirmed by NIPD in maternal blood. No results were obtained in the HD pregnancy. NIPD in PGD pregnancies can be a reliable alternative for couples that after a long process feel reluctant to undergo PD due to the risk of pregnancy loss.

Massive parallel DNA sequencing combined with chromatin immunoprecipitation and a large variety of DNA/RNA-enrichment methodologies is at the origin of data resources of major importance. Indeed these resources, available for multiple genomes, represent the most comprehensive catalogue of (i) cell, development and signal transduction-specified patterns of binding sites for transcription factors ('cistromes') and for transcription and chromatin modifying machineries and (ii) the patterns of specific local post-translational modifications of histones and DNA ('epigenome') or of regulatory chromatin binding factors. In addition, (iii) the resources specifying chromatin structure alterations are emerging. Importantly, these types of "omics" datasets populate increasingly public repositories and provide highly valuable resources for the exploration of general principles of cell function in a multi-dimensional genome-transcriptome-epigenome-chromatin structure context. However, data mining is critically dependent on the data quality, an issue that, surprisingly, is still largely ignored by scientists and well-financed consortia, data repositories and scientific journals. So what determines the quality of ChIP-seq experiments and the datasets generated therefrom and what refrains scientists from associating quality criteria to their data? In this 'opinion' we trace the various parameters that influence the quality of this type of datasets, as well as the computational efforts that were made until now to qualify them. Moreover, we describe a universal quality control (QC) certification approach that provides a quality rating for ChIP-seq and enrichment-related assays. The corresponding QC tool and a regularly updated database, from which at present the quality parameters of more than 8000 datasets can be retrieved, are freely accessible at www.ngs-qc.org.

Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization.

<h4>Background</h4>The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia.<h4>Principal findings</h4>By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their "pharmacological target(s)" by uncompetitive mechanism without the requirement of phospholipids/Ca(2+). The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.<h4>Conclusion/significance</h4>In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed.

microRNAs (miRNAs) are important modulators of development. Owing to their ability to simultaneously silence hundreds of target genes, they have key roles in large-scale transcriptomic changes that occur during cell fate transitions. In somatic stem and progenitor cells--such as those involved in myogenesis, haematopoiesis, skin and neural development--miRNA function is carefully regulated to promote and stabilize cell fate choice. miRNAs are integrated within networks that form both positive and negative feedback loops. Their function is regulated at multiple levels, including transcription, biogenesis, stability, availability and/or number of target sites, as well as their cooperation with other miRNAs and RNA-binding proteins. Together, these regulatory mechanisms result in a refined molecular response that enables proper cellular differentiation and function.

Single nucleotide polymorphisms (SNPs) of factor V Leiden have been associated with osteonecrosis of the femoral head (ONFH) in Caucasians but remains controversial in Asians. We used an SNP microarray to screen 55 loci of factor V gene in patients with ONFH of Chinese. Significantly different candidate SNPs at 14 loci were analyzed in 146 patients and 116 healthy controls using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and gene sequencing. The factor V Leiden (rs6025) was not found in all participants. Six SNP loci (rs9332595, rs6020, rs9332647, rs3766110, rs10919186, and rs12040141) were confirmed with significant differences in patients but not in controls. The rs6020 G-to-A polymorphism was found in 88.9% of the patients. In addition, a high percentage (87.6%) of the patients had an abnormal coagulation profile that included hyperfibrinogen, elevated fibrinogen degradation products, elevated D-dimer, abnormal protein S, abnormal protein C, or a decrease in anti-thrombin III. Patients with the rs6020 G-to-A polymorphism (mutation) had a higher risk (odds ratio: 4.62; 95% confidence interval: 1.44-14.8) of having coagulation abnormalities than did those without the mutation (wild-type) (χ(2) p  =  0.006). Our findings suggested that the rs6020 polymorphism might be the genetic trait that accounts for the higher prevalence of ONFH in the Chinese population than in Westerners. Exposure to risk factors such as alcohol and steroids in patients with the rs6020 polymorphism causes coagulation abnormalities and, subsequently, thromboembolisms in the femoral head.

Calcineurin (CN) is a highly conserved Ca(2+)-calmodulin (CaM)-dependent phosphatase that senses Ca(2+) concentrations and transduces that information into cellular responses. Ca(2+) homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca(2+), thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase's activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.

The polyglutamine expansion within huntingtin is the causative factor in the pathogenesis of Huntington's disease (HD). Although the underlying mechanisms by which mutant huntingtin causes neuronal dysfunction and degeneration have not been fully elucidated, compelling evidence suggests that mitochondrial dysfunction and compromised energy metabolism are key players in HD pathogenesis. Longitudinal studies of HD subjects have shown reductions in glucose utilization before the disease clinical onset. Preferential striatal neurodegeneration, a hallmark of HD pathogenesis, also has been associated with interrupted energy metabolism. Data from genetic HD models indicate that mutant huntingtin disrupts mitochondrial bioenergetics and prevents adenosine triphosphate (ATP) generation, implying altered energy metabolism as an important component of HD pathogenesis. Here we revisit the evidence of abnormal energy metabolism in the central nervous system of HD patients, review our current understanding of the molecular mechanisms underlying abnormal metabolism induced by mutant huntingtin, and discuss the promising therapeutic development by halting abnormal metabolism in HD.

During the immune response, striking the right balance between positive and negative regulation is critical to effectively mount an anti-microbial defense while preventing detrimental effects from exacerbated immune activation. Intra-cellular immune signaling is tightly regulated by various post-translational modifications, which allow for this dynamic response. One of the post-translational modifiers critical for immune control is ubiquitin, which can be covalently conjugated to lysines in target molecules, thereby altering their functional properties. This is achieved in a process involving E3 ligases which determine ubiquitination target specificity. One of the most prominent E3 ligase families is that of the tripartite motif (TRIM) proteins, which counts over 70 members in humans. Over the last years, various studies have contributed to the notion that many members of this protein family are important immune regulators. Recent studies into the mechanisms by which some of the TRIMs regulate the innate immune system have uncovered important immune regulatory roles of both covalently attached, as well as unanchored poly-ubiquitin chains. This review highlights TRIM evolution, recent findings in TRIM-mediated immune regulation, and provides an outlook to current research hurdles and future directions.

Despite large gaps in our knowledge on the intracellular mechanism leading to cervical cancer, the pathways induced by oncogenic high-risk Human Papilloma Virus (HPV) and those finally causing cervical cancer are increasingly being unraveled. Assuming that precancerous tissue is recognized and lysed by the immune system-which is in many cases incomplete because of the counteraction by the HPV virus-we hypothesize that several intracellular factors, involved in induction and development of precancerous lesions and/or cervical cancer are being released into the cervicovaginal fluid (CVF). These factors can then be seen as markers for the precancerous state, and when they persist they are indicative for an increased risk for cervical carcinoma. In a previous study, we analyzed the proteomic profiles of six CVF samples from women with different stages of precancerous lesions and compared these with the CVF proteomes from healthy women. Here, we extend these observations by investigating these proteomes by Ingenuity Pathway Analysis (IPA). We show that proteins in CVF from precancerous women are clearly more involved in pathways that make up the 'hallmarks of cancer', as compared to CVF proteins from healthy persons. Moreover, after literature search, proteins classified by IPA in the 'cancer' category, were more correlated with cervical cancer when they originated from CVF from precancerous women. Many of these proteins formed a network with angiotensin II as central mediator. The search for 'network biomarkers', rather than single biomarkers, could drastically increase specificity, sensitivity and prognostic value of cervical cancer diagnosis, making use of an easy to handle fluid, the CVF.

No abstract available.

<h4>Background</h4>Good prognostic tools for predicting disease progression in early stage prostate cancer (PCa) are still missing. Detection of molecular subtypes, for instance by using microarray gene technology, can give new prognostic information which can assist personalized treatment planning. The detection of new subtypes with validation across additional and larger patient cohorts is important for bringing a potential prognostic tool into the clinic.<h4>Methods</h4>We used fresh frozen prostatectomy tissue of high molecular quality to further explore four molecular subtype signatures of PCa based on Gene Set Enrichment Analysis (GSEA) of 15 selected gene sets published in a previous study. For this analysis we used a statistical test of dependent correlations to compare reference signatures to signatures in new normal and PCa samples, and also explore signatures within and between sample subgroups in the new samples.<h4>Results</h4>An important finding was the consistent signatures observed for samples from the same patient independent of Gleason score. This proves that the signatures are robust and can surpass a normally high tumor heterogeneity within each patient. Our data did not distinguish between four different subtypes of PCa as previously published, but rather highlighted two groups of samples which could be related to good and poor prognosis based on survival data from the previous study.The poor prognosis group highlighted a set of samples characterized by enrichment of ESC, ERG-fusion and MYC + rich signatures in patients diagnosed with low Gleason score,. The other group consisted of PCa samples showing good prognosis as well as normal samples. Accounting for sample composition (the amount of benign structures such as stroma and epithelial cells in addition to the cancer component) was important to improve subtype assignments and should also be considered in future studies.<h4>Conclusion</h4>Our study validates a previous molecular subtyping of PCa in a new patient cohort, and identifies a subgroup of PCa samples highly interesting for detecting high risk PCa at an early stage. The importance of taking sample tissue composition into account when assigning subtype is emphasized.

SWI/SNF chromatin remodeling complexes regulate critical cellular processes, including cell-cycle control, programmed cell death, differentiation, genomic instability, and DNA repair. Inactivation of this class of chromatin remodeling complex has been associated with a variety of malignancies, including lung, ovarian, renal, liver, and pediatric cancers. In particular, approximately 10% of primary human lung non-small cell lung cancers (NSCLC) display attenuations in the BRG1 ATPase, a core factor in SWI/SNF complexes. To evaluate the role of BRG1 attenuation in NSCLC development, we examined the effect of BRG1 silencing in primary and established human NSCLC cells. BRG1 loss altered cellular morphology and increased tumorigenic potential. Gene expression analyses showed reduced expression of genes known to be associated with progression of human NSCLC. We demonstrated that BRG1 losses in NSCLC cells were associated with variations in chromatin structure, including differences in nucleosome positioning and occupancy surrounding transcriptional start sites of disease-relevant genes. Our results offer direct evidence that BRG1 attenuation contributes to NSCLC aggressiveness by altering nucleosome positioning at a wide range of genes, including key cancer-associated genes.

Intracranial aneurysms, also called cerebral aneurysms, are dilatations in the arteries that supply blood to the brain. Rupture of an intracranial aneurysm leads to a subarachnoid hemorrhage, which is fatal in about 50% of the cases. Intracranial aneurysms can be repaired surgically or endovascularly, or by combining these two treatment modalities. They are relatively common with an estimated prevalence of unruptured aneurysms of 2%-6% in the adult population, and are considered a complex disease with both genetic and environmental risk factors. Known risk factors include smoking, hypertension, increasing age, and positive family history for intracranial aneurysms. Identifying the molecular mechanisms underlying the pathogenesis of intracranial aneurysms is complex. Genome-wide approaches such as DNA linkage and genetic association studies, as well as microarray-based mRNA expression studies, provide unbiased approaches to identify genetic risk factors and dissecting the molecular pathobiology of intracranial aneurysms. The ultimate goal of these studies is to use the information in clinical practice to predict an individual's risk for developing an aneurysm or monitor its growth or rupture risk. Another important goal is to design new therapies based on the information on mechanisms of disease processes to prevent the development or halt the progression of intracranial aneurysms.

<h4>Purpose</h4>To determine genetic variants associated with severe retinopathy of prematurity (ROP) in a candidate gene cohort study of US preterm infants.<h4>Methods</h4>Preterm infants in the discovery cohort were enrolled through the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network, and those in the replication cohort were from the University of Iowa. All infants were phenotyped for ROP severity. Because of differences in the durations of enrollment between cohorts, severe ROP was defined as threshold disease in the discovery cohort and as threshold disease or type 1 ROP in the replication cohort. Whole genome amplified DNA from stored blood spot samples from the Neonatal Research Network biorepository was genotyped using an Illumina GoldenGate platform for candidate gene single nucleotide polymorphisms (SNPs) involving angiogenic, developmental, inflammatory, and oxidative pathways. Three analyses were performed to determine significant epidemiologic variables and SNPs associated with levels of ROP severity. Analyses controlled for multiple comparisons, ancestral eigenvalues, family relatedness, and significant epidemiologic variables. Single nucleotide polymorphisms significantly associated with ROP severity from the discovery cohort were analyzed in the replication cohort and in meta-analysis.<h4>Results</h4>Eight hundred seventeen infants in the discovery cohort and 543 in the replication cohort were analyzed. Severe ROP occurred in 126 infants in the discovery and in 14 in the replication cohort. In both cohorts, ventilation days and seizure occurrence were associated with severe ROP. After controlling for significant factors and multiple comparisons, two intronic SNPs in the gene BDNF (rs7934165 and rs2049046, P < 3.1 × 10(-5)) were associated with severe ROP in the discovery cohort and were not associated with severe ROP in the replication cohort. However, when the cohorts were analyzed together in an exploratory meta-analysis, rs7934165 increased in associated significance with severe ROP (P = 2.9 × 10(-7)).<h4>Conclusions</h4>Variants in BDNF encoding brain-derived neurotrophic factor were associated with severe ROP in a large candidate gene study of infants with threshold ROP.

In this study, we evaluated the association between bone mineral density (BMD) and 10 single-nucleotide polymorphisms (SNPs) within eight osteoporosis susceptibility genes that were previously identified in genome-wide association studies (GWASs). A total of 494 men and 493 postmenopausal women participating in the Chungju Metabolic Disease cohort study in Korea were included. The following 10 SNPs were genotyped: ZBTB40 rs6426749, MEF2C rs1366594, ESR1 rs2941740, TNFRSF11B rs3134070, TNFRSF11B rs2073617, SOX6 rs711785, LRP5 rs599083, TNFSF11 rs227438, TNFSF11 rs9594782, and FOXL1 rs10048146; and the association between these SNPs and bone metabolism-related markers was assessed. Two SNPs, TNFSF11 rs2277438 and FOXL1 rs1004816, were associated with lumbar spine BMD. TNFSF11 rs2277438 in men and SOX6 rs7117858 and FOXL1 rs10048146 in postmenopausal women were found to be associated with lumbar BMD. ZBTB40 rs6426749, MEF2C rs1366594, and LRP5 rs599083 showed significant associations with femur neck BMD. These three SNPs in men and MEF2C rs1366594 and ESR1 rs2941740 in postmenopausal women were associated with femur neck BMD. A significant association between MEF2C rs1366594 and serum calcium levels was observed in men. Serum phosphorus levels were related to SOX6 rs7117858. Serum PTH levels were significantly associated with TNFRSF11B rs3134070 in men, and SOX6 rs711858 in postmenopausal women. In conclusion, our study independently confirmed associations between several SNPs: ZBTB40, MEF2C, ESR1, SOX6, LRP5, TNFSF11, and FOXL1 and bone marrow density in the Korean population.

<h4>Background</h4>Obesity is associated with increased adipose tissue and glucose intolerance. High-fat diets (HFDs) are known to induce obesity and increase proinflammatory adipokines. The consumption of green tea may improve the health of obese individuals because it contains a potent antioxidant that has effects on body weight, energy expenditure and serum cholesterol concentrations.<h4>Methods</h4>We examined the effects of epigallocatechin-3-gallate (EGCG) (50 mg/kg body weight per day) or saline after 30 or 60 days of treatment. Mice were distributed into four groups: 1) NS: normolipidic diet receiving saline; 2) NE: normolipidic diet receiving EGCG; 3) HFS: high-fat diet receiving saline; 4) HFE: high-fat diet receiving EGCG.<h4>Results</h4>We observed that administration of a HFD plus EGCG treatment for 60 days reduced delta weight, the relative weights of the mesenteric adipose tissue (MES), retroperitonial adipose tissue (RET), epididymal adipose tissue (EPI), the sum of the adipose tissues (SAT), reduced triacylglycerol (TG) and improved both high-density lipoprotein (HDL) cholesterol levels and the adiponectin/STA ratio when compared with HFS.<h4>Conclusions</h4>Our results suggest that the chronic administration of EGCG (60 days) promoted a significant improvement in glucose tolerance, decreased adipose tissue deposits, weight mass, TG and HDL-C only when associated with high-fat diet treatment.

Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

Evidence suggests that advanced fibrosis, as determined by the noninvasive NAFLD fibrosis score (NFS), is a predictor of cardiovascular mortality in individuals with ultrasonography-diagnosed NAFLD. Whether the severity of histology (i.e., fibrosis stage) is associated with more pronounced cardiovascular organ damage is unsettled. In this study, we analyzed the clinical utility of NFS in assessing increased carotid intima-media thickness (cIMT), and left ventricular mass index (LVMI). In this cross-sectional study NFS, cIMT and LVMI were assessed in 400 individuals with ultrasonography-diagnosed steatosis. As compared with individuals at low probability of liver fibrosis, individuals both at high and at intermediate probability of fibrosis showed an unfavorable cardio-metabolic risk profile having significantly higher values of waist circumference, insulin resistance, high sensitivity C-reactive protein (hsCRP), fibrinogen, cIMT, and LVMI, and lower insulin-like growth factor-1 (IGF-1) levels. The differences in cIMT and LVMI remained significant after adjustment for smoking and metabolic syndrome. In a logistic regression model adjusted for age, gender, smoking, and diagnosis of metabolic syndrome, individuals at high probability of fibrosis had a 3.9-fold increased risk of vascular atherosclerosis, defined as cIMT>0.9 mm, (OR 3.95, 95% CI 1.12-13.87) as compared with individuals at low probability of fibrosis. Individuals at high probability of fibrosis had a 3.5-fold increased risk of left ventricular hypertrophy (LVH) (OR 3.55, 95% CI 1.22-10.34) as compared with individuals at low probability of fibrosis. In conclusion, advanced fibrosis, determined by noninvasive fibrosis markers, is associated with cardiovascular organ damage independent of other known factors.

Alpha-synuclein (α-Syn) aggregates are the main component of Lewy bodies, which are the characteristic pathological feature in Parkinson's disease (PD) brain. Evidence that α-Syn aggregation can be propagated between neurones has led to the suggestion that this mechanism is responsible for the stepwise progression of PD pathology. Decreasing α-Syn expression is predicted to attenuate this process and is thus an attractive approach to delay or halt PD progression. We have used α-Syn small interfering RNA (siRNA) to reduce total and aggregated α-Syn levels in mouse brains. To achieve widespread delivery of siRNAs to the brain we have peripherally injected modified exosomes expressing Ravies virus glycoprotein loaded with siRNA. Normal mice were analyzed 3 or 7 days after injection. To evaluate whether this approach can decrease α-Syn aggregates, we repeated the treatment using transgenic mice expressing the human phosphorylation-mimic S129D α-Syn, which exhibits aggregation. In normal mice we detected significantly reduced α-Syn messenger RNA (mRNA) and protein levels throughout the brain 3 and 7 days after treatment with RVG-exosomes loaded with siRNA to α-Syn. In S129D α-Syn transgenic mice we found a decreased α-Syn mRNA and protein levels throughout the brain 7 days after injection. This resulted in significant reductions in intraneuronal protein aggregates, including in dopaminergic neurones of the substantia nigra. This study highlights the therapeutic potential of RVG-exosome delivery of siRNA to delay and reverse brain α-Syn pathological conditions.

Initially, indoleamine-2,3-dioxygenase (IDO) has been introduced as a bactericidal effector mechanism and has been linked to T-cell immunosuppression and tolerance. In recent years, evidence has been accumulated that IDO also plays an important role during viral infections including HIV, influenza, and hepatitis B and C. Moreover, novel aspects about the role of IDO in bacterial infections and sepsis have been revealed. Here, we review these recent findings highlighting the central role of IDO and tryptophan metabolism in many major human infections. Moreover, we also shed light on issues concerning human-specific and mouse-specific host-pathogen interactions that need to be considered when studying the biology of IDO in the context of infections.

The C→T polymorphism (rs6929846) of the butyrophilin, subfamily 2, member A1 (<i>BTN2A1</i>) gene has been previously identified as a susceptibility locus for myocardial infarction by a genome-wide association study. As hypertension is a major risk factor for myocardial infarction, the association between the <i>BTN2A1</i> polymorphism, rs6929846, and myocardial infarction may be partly due to its effect on hypertension susceptibility. The aim of the present study was to examine the possible association of rs6929846 with hypertension. The study subjects comprised 5,959 community-dwelling individuals (2,183 subjects with hypertension and 3,776 controls) who were recruited to a population-based cohort study. The rs6929846 genotype was determined by a method that combined polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Comparisons between the genotype distributions (P=0.0090) and allele frequencies (P=0.0051) by the χ² test revealed that rs6929846 was significantly associated with hypertension. Multivariable logistic regression analysis with adjustment for age, gender, body mass index and smoking status revealed that rs6929846 was significantly associated with hypertension (P=0.0008; odds ratio, 1.29; dominant model), with the minor <i>T</i> allele representing a risk factor for this condition. Among all the individuals, systolic, diastolic and mean blood pressure was significantly higher in the combined group of individuals with the <i>CT</i> or <i>TT</i> genotypes compared to the <i>CC</i> genotype group. <i>BTN2A1</i> may thus be a susceptibility gene for hypertension. Therefore, determining the genotype for this polymorphism may provide genetic risk assessment information for hypertension.

F17 fimbriae are produced by pathogenic Escherichia coli involved in diarrhea and septicemia outbreaks in calves and lambs. These proteins result from the expression of four different clustered genes, namely f17A, f17D, f17C and f17G, encoding a pilin protein, a periplasmic protein, an anchor protein and an adhesin protein, respectively. Several variants of f17A and f17G genes have been reported and found genetically associated with typical virulence factors of bovine pathogenic E. coli strains. In this study, a new F17e-A variant, closely related to F17b-A, was identified from a collection of 58 E. coli isolates from diarrheic calves in Iran. While highly prevalent in Iranian F17-producing clinical isolates from calves, this variant was rare among E. coli from a French healthy adult bovine population, suggesting a possible association with virulence. The f17Ae gene was also found in the genome of the Shiga-like toxin variant Stx1d-producing bovine E. coli strain MHI813, and belonged to a gene cluster also encoding a new F17-G3 variant, which greatly differed from F17-G1 and F17-G2. This gene cluster was located on a pathogenicity island integrated in the tRNA pheV gene. The gene coding for a third new F17f-A variant corresponding to a combination of F17c-A and F17d-A was also identified on the pVir68 plasmid in the bovine pathogenic E. coli strain 6.0900. In conclusion, we identified three new F17-A and F17-G variants in cattle E. coli, which may also have significant impact on the development of new diagnostics and vaccination tools.

In the present study, we investigated whether Raf-1 kinase inhibitory protein (RKIP) is important for neural cell apoptosis induced by microwave exposure and explored the role of MEK/ERK/CREB pathway regulated by RKIP in the apoptosis. Differentiated PC12 cells were exposed to continuous microwave radiation at 2.856 GHz for 5 min with average power density of 30 mW/cm(2). RKIP sense and anti-sense recombinant plasmids were constructed and transfected into PC12 cells, respectively. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay were used to detect cell apoptosis. The results showed that RKIP was downregulated after microwave exposure while the MEK/ERK/CREB signaling pathway was activated excessively. Moreover, the ratio of Bcl-2/Bax decreased, activity of caspase-3 increased, and thus apoptotic DNA fragmentation increased. RKIP overexpression significantly inhibited the phosphorylation of MEK, ERK, and CREB, while RKIP downregulation had the reverse effect. Furthermore, U0126 was found to antagonize the changes caused by RKIP downregulation after exposure to radiation. In conclusion, RKIP plays an important role in the neural cell apoptosis induced by microwave radiation, and the regulation of cell apoptosis by RKIP is partly through the MEK/ERK/CREB pathway. This suggests that RKIP may act as a key regulator of neuronal damage caused by microwave radiation.

The ability of cells to maintain low levels of Ca(2+) under resting conditions and to create rapid and transient increases in Ca(2+) upon stimulation is a fundamental property of cellular Ca(2+) signaling mechanism. An increase of cytosolic Ca(2+) level in response to diverse stimuli is largely accounted for by the inositol 1,4,5-trisphosphate receptor (IP3R) present in the endoplasmic reticulum membranes of virtually all eukaryotic cells. Extensive information is currently available on the function of IP3Rs and their interaction with modulators. Very little, however, is known about their molecular architecture and therefore most critical issues surrounding gating of IP3R channels are still ambiguous, including the central question of how opening of the IP3R pore is initiated by IP3 and Ca(2+). Membrane proteins such as IP3R channels have proven to be exceptionally difficult targets for structural analysis due to their large size, their location in the membrane environment, and their dynamic nature. To date, a 3D structure of complete IP3R channel is determined by single-particle cryo-EM at intermediate resolution, and the best crystal structures of IP3R are limited to a soluble portion of the cytoplasmic region representing ∼15% of the entire channel protein. Together these efforts provide the important structural information for this class of ion channels and serve as the basis for further studies aiming at understanding of the IP3R function.

Inspired from occurrence of anti-inflammatory activity of 3-substituted coumarins and antiulcer activity of various 2-substituted benzimidazoles, novel compounds have been designed by coupling coumarin derivatives at 3-position directly or through amide linkage with benzimidazole nucleus at 2-position. The resultant compounds are expected to exhibit both anti-inflammatory and antioxidant activities along with less gastric toxicity profile. Two series of coumarin-benzimidazole derivatives (4a-e and 5a-e) were synthesized and evaluated for anti-inflammatory activity and antioxidant activity. Compounds 4c, 4d and 5a displayed good anti-inflammatory (45.45%, 46.75% and 42.85% inhibition, respectively, versus 54.54% inhibition by indomethacin) and antioxidant (IC50 of 19.7, 13.9 and 1.2 µmol/L, respectively, versus 23.4 µmol/L for butylatedhydroxytoluene) activities. Evaluation of ulcer index and in vivo biochemical estimations for oxidative stress revealed that compounds 4d and 5a remain safe on gastric mucosa and did not induce oxidative stress in tissues. Calculation of various molecular properties suggests the compounds to be sufficiently bioavailable.

Microglia have long been the focus of much attention due to their strong proliferative response (microgliosis) to essentially any kind of damage to the CNS. More recently, we reached the realization that these cells play specific roles in determining progression and outcomes of essentially all CNS disease. Thus, microglia has ceased to be viewed as an accessory to underlying pathologies and has now taken center stage as a therapeutic target. Here, we review how our understanding of microglia's involvement in promoting or limiting the pathogenesis of diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, multiple sclerosis, X-linked adrenoleukodystrophy (X-ALD) and lysosomal storage diseases (LSD) has changed over time. While strategies to suppress the deleterious and promote the virtuous functions of microglia will undoubtedly be forthcoming, replacement of these cells has already proven its usefulness in a clinical setting. Over the past few years, we have reached the realization that microglia have a developmental origin that is distinct from that of bone marrow-derived myelomonocytic cells. Nevertheless, microglia can be replaced, in specific situations, by the progeny of hematopoietic stem cells (HSCs), pointing to a strategy to engineer the CNS environment through the transplantation of modified HSCs. Thus, microglia replacement has been successfully exploited to deliver therapeutics to the CNS in human diseases such as X-ALD and LSD. With this outlook in mind, we will discuss the evidence existing so far for microglial involvement in the pathogenesis and the therapy of specific CNS disease.

MITF and pigmentation play important roles in both normal melanocyte and transformed melanoma cell biology. MITF is regulated by many pathways and it also regulates many targets, some of which are still being discovered and functionally validated. MITF is involved in a wide range of processes in melanocytes, including pigment synthesis and lineage survival. Pigmentation itself plays an important role as the interface between genetic and environmental factors that contribute to melanoma.

Cognitive abilities vary among people. About 40-50% of this variability is due to general intelligence (g), which reflects the positive correlation among individuals' scores on diverse cognitive ability tests. g is positively correlated with many life outcomes, such as education, occupational status and health, motivating the investigation of its underlying biology. In psychometric research, a distinction is made between general fluid intelligence (gF) - the ability to reason in novel situations - and general crystallized intelligence (gC) - the ability to apply acquired knowledge. This distinction is supported by developmental and cognitive neuroscience studies. Classical epidemiological studies and recent genome-wide association studies (GWASs) have established that these cognitive traits have a large genetic component. However, no robust genetic associations have been published thus far due largely to the known polygenic nature of these traits and insufficient sample sizes. Here, using two GWAS datasets, in which the polygenicity of gF and gC traits was previously confirmed, a gene- and pathway-based approach was undertaken with the aim of characterizing and differentiating their genetic architecture. Pathway analysis, using genes selected on the basis of relaxed criteria, revealed notable differences between these two traits. gF appeared to be characterized by genes affecting the quantity and quality of neurons and therefore neuronal efficiency, whereas long-term depression (LTD) seemed to underlie gC. Thus, this study supports the gF-gC distinction at the genetic level and identifies functional annotations and pathways worthy of further investigation.

Dosage compensation adjusts the expression levels of genes on one or both targeted sex chromosomes in heterogametic species. This process results in the normalized transcriptional output of important and essential gene families encoded on multiple chromosomes. The mechanisms of dosage compensation have been studied in many model organisms, including Drosophila melanogaster (fly), Caenorhabditis elegans (worm), and Mus musculus (mouse). Although the mechanisms of dosage compensations differ among these species, all of these processes rely on the initial discrimination of the X chromosome from autosomes. Recently, a new paradigm for how the X chromosome is targeted for regulation was identified in Drosophila. This mechanism involves a newly identified zinc finger protein, CLAMP. Here, we review important factors involved in dosage compensation across species with special focus on the fly. Understanding how the newly identified CLAMP protein is involved in X targeting in the fly could provide key insights into how the X chromosome is initially identified across species.

Inhibitory local circuit neurons (LCNs), often called interneurons, have vital roles in the development and function of cortical networks. Their inhibitory influences regulate both the excitability of cortical projection neurons on the level of individual cells, and the synchronous activity of projection neuron ensembles that appear to be a neural basis for major aspects of cognitive processing. Dysfunction of LCNs has been associated with neurological and psychiatric diseases, such as epilepsy, schizophrenia, and autism. Here we review progress in understanding LCN fate determination, their nonradial migration to the cortex, their maturation within the cortex, and the contribution of LCN dysfunction to neuropsychiatric disorders.

Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3-24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing.

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28°C as compared to 37°C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology.

<h4>Background</h4>Despite abundant evidence for pathogenicity of large copy number variants (CNVs) in neurodevelopmental disorders (NDDs), the individual significance of genome-wide rare CNVs <500 kb has not been well elucidated in a clinical context.<h4>Methods</h4>By high-resolution chromosomal microarray analysis, we investigated the clinical significance of all rare non-polymorphic exonic CNVs sizing 1-500 kb in a cohort of 714 patients with undiagnosed NDDs.<h4>Results</h4>We detected 96 rare CNVs <500 kb affecting coding regions, of which 58 (60.4%) were confirmed. 6 of 14 confirmed de novo, one of two homozygous and four heterozygous inherited CNVs affected the known microdeletion regions 17q21.31, 16p11.2 and 2p21 or OMIM morbid genes (CASK, CREBBP, PAFAH1B1, SATB2; AUTS2, NRXN3, GRM8). Two further de novo CNVs affecting single genes (MED13L, CTNND2) were instrumental in delineating novel recurrent conditions. For the first time, we here report exonic deletions of CTNND2 causing low normal IQ with learning difficulties with or without autism spectrum disorder. Additionally, we discovered a homozygous out-of-frame deletion of ACOT7 associated with features comparable to the published mouse model. In total, 24.1% of the confirmed small CNVs were categorised as pathogenic or likely pathogenic (median size 130 kb), 17.2% as likely benign, 3.4% represented incidental findings and 55.2% remained unclear.<h4>Conclusions</h4>These results verify the diagnostic relevance of genome-wide rare CNVs <500 kb, which were found pathogenic in ∼2% (14/714) of cases (1.1% de novo, 0.3% homozygous, 0.6% inherited) and highlight their inherent potential for discovery of new conditions.

The cell cycle genes homology region (CHR) has been identified as a DNA element with an important role in transcriptional regulation of late cell cycle genes. It has been shown that such genes are controlled by DREAM, MMB and FOXM1-MuvB and that these protein complexes can contact DNA via CHR sites. However, it has not been elucidated which sequence variations of the canonical CHR are functional and how frequent CHR-based regulation is utilized in mammalian genomes. Here, we define the spectrum of functional CHR elements. As the basis for a computational meta-analysis, we identify new CHR sequences and compile phylogenetic motif conservation as well as genome-wide protein-DNA binding and gene expression data. We identify CHR elements in most late cell cycle genes binding DREAM, MMB, or FOXM1-MuvB. In contrast, Myb- and forkhead-binding sites are underrepresented in both early and late cell cycle genes. Our findings support a general mechanism: sequential binding of DREAM, MMB and FOXM1-MuvB complexes to late cell cycle genes requires CHR elements. Taken together, we define the group of CHR-regulated genes in mammalian genomes and provide evidence that the CHR is the central promoter element in transcriptional regulation of late cell cycle genes by DREAM, MMB and FOXM1-MuvB.

A major barrier to the clinical use of erythrocytes generated in vitro from pluripotent stem cells or cord blood progenitors is failure of these erythrocytes to express adult hemoglobin. The key regulators of globin switching KLF1 and BCL11A are absent or at a lower level than in adult cells in K562 and erythroid cells differentiated in vitro from induced pluripotent stem cells and cord blood progenitors. Transfection or transduction of K562 and cord blood erythroid cells with either KLF1 or BCL11A-XL had little effect on β-globin expression. In contrast, transduction with both transcription factors stimulated β-globin expression. Similarly, increasing the level of BCL11A-XL in the induced pluripotent stem cell-derived erythroid cell line HiDEP-1, which has levels of endogenous KLF1 similar to adult cells but lacks BCL11A, resulted in levels of β-globin equivalent to that of adult erythroid cells. Interestingly, this increase in β-globin was coincident with a decrease in ε- and ζ-, but not γ-globin, implicating BCL11A in repression of embryonic globin expression. The data show that KLF1 and BCL11A-XL together are required, but sufficient to induce adult levels of β-globin in induced pluripotent stem cell and cord blood-derived erythroid cells that intrinsically express embryonic or fetal globin.

Dendritic cells (DC) are critical for the initiation of immune responses; however, their role in priming IL-4-producing Th2 cells in vivo is not fully understood. We used a model of intradermal injection with fluorescent-labeled, nonviable larvae from the helminth parasite nonviable Nippostrongylus brasiliensis L3 larvae (Nb), a strong inducer of Th2 responses, together with IL-4-GFP reporter mice that enable a sensitive detection of IL-4 production to examine the contribution of DC to the priming of IL-4-producing CD4(+) T cells in vivo. We found that parasite material is taken up by two distinct DC populations in draining lymph nodes: a mostly CD11c(int)MHC class II (MHCII)(hi)CD11b(+)Ly6C(-) dermal DC population and a CD11c(hi)MHCII(int)CD11b(+)Ly6C(+) monocyte-derived DC population. After Nb treatment, these two DC populations appeared in the draining lymph nodes in comparable numbers and with similar kinetics; however, treatment with pertussis toxin blocked the migration of dermal DC and the priming of IL-4-producing T cells, but only partially affected monocyte-derived DC numbers. In line with this observation, transfer of OVA-loaded CD11c(int)MHCII(hi) DC from Nb-treated mice into naive hosts could sensitize OVA-specific CD4(+) T cells to IL-4 production, whereas transfer of CD11c(int)MHCII(hi) DC from naive mice, or CD11c(hi)MHCII(int) DC from Nb-treated or naive mice, induced CD4(+) T cell expansion but no IL-4 production. Phenotypic analysis of Nb-loaded CD11c(int)MHCII(hi) DC revealed expression of programmed death ligand 2, CD301b, IFN regulatory factor 4, and moderate upregulation of OX40 ligand. However, thymic stromal lymphopoietin and OX40 ligand were not required for Th2 priming. Thus, our data suggest that appropriate stimuli can induce DC to express the unique signals sufficient to direct CD4(+) T cells to Th2 differentiation.

The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, and Huntington's disease, leading to the hypothesis that proteasomal impairment is contributing to these diseases. So far, most research related to the UPS in neurodegenerative diseases has been focused on neurons, while glial cells have been largely disregarded in this respect. However, glial cells are essential for proper neuronal function and adopt a reactive phenotype in neurodegenerative diseases, thereby contributing to an inflammatory response. This process is called reactive gliosis, which in turn affects UPS function in glial cells. In many neurodegenerative diseases, mostly neurons show accumulation and aggregation of ubiquitinated proteins, suggesting that glial cells may be better equipped to maintain proper protein homeostasis. During an inflammatory reaction, the immunoproteasome is induced in glia, which may contribute to a more efficient degradation of disease-related proteins. Here we review the role of the UPS in glial cells in various neurodegenerative diseases, and we discuss how studying glial cell function might provide essential information in unraveling mechanisms of neurodegenerative diseases.

Rubella virus causes a relatively benign disease in most cases, although infection during pregnancy can result in serious birth defects. An effective vaccine has been available since the early 1970s and outbreaks typically do not occur among highly vaccinated (≥2 doses) populations. Nevertheless, considerable inter-individual variation in immune response to rubella immunization does exist, with single-dose seroconversion rates ~95 %. Understanding the mechanisms behind this variability may provide important insights into rubella immunity. In the current study, we examined associations between single nucleotide polymorphisms (SNPs) in selected cytokine, cytokine receptor, and innate/antiviral genes and immune responses following rubella vaccination in order to understand genetic influences on vaccine response. Our approach consisted of a discovery cohort of 887 subjects aged 11-22 at the time of enrollment and a replication cohort of 542 older adolescents and young adults (age 18-40). Our data indicate that SNPs near the butyrophilin genes (BTN3A3/BTN2A1) and cytokine receptors (IL10RB/IFNAR1) are associated with variations in IFNγ secretion and that multiple SNPs in the PVR gene, as well as SNPs located in the ADAR gene, exhibit significant associations with rubella virus-specific IL-6 secretion. This information may be useful, not only in furthering our understanding immune responses to rubella vaccine, but also in identifying key pathways for targeted adjuvant use to boost immunity in those with weak or absent immunity following vaccination.

Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV.

Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.

Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.

The objective of this study was to identify the aggregation pheromone of the melon thrips Thrips palmi, a major pest of vegetable and ornamental plants around the world. The species causes damage both through feeding activities and as a vector of tospoviruses, and is a threat to world trade and European horticulture. Improved methods of detecting and controlling this species are needed and the identification of an aggregation pheromone will contribute to this requirement. Bioassays with a Y-tube olfactometer showed that virgin female T. palmi were attracted to the odour of live males, but not to that of live females, and that mixed-age adults of both sexes were attracted to the odour of live males, indicating the presence of a male-produced aggregation pheromone. Examination of the headspace volatiles of adult male T. palmi revealed only one compound that was not found in adult females. It was identified by comparison of its mass spectrum and chromatographic details with those of similar compounds. This compound had a structure like that of the previously identified male-produced aggregation pheromone of the western flower thrips Frankliniella occidentalis. The compound was synthesised and tested in eggplant crops infested with T. palmi in Japan. Significantly greater numbers of both males and females were attracted to traps baited with the putative aggregation pheromone compared to unbaited traps. The aggregation pheromone of T. palmi is thus identified as (R)-lavandulyl 3-methyl-3-butenoate by spectroscopic, chromatographic and behavioural analysis.

Type 2 diabetes (T2D) is more prevalent in African Americans than in Europeans. However, little is known about the genetic risk in African Americans despite the recent identification of more than 70 T2D loci primarily by genome-wide association studies (GWAS) in individuals of European ancestry. In order to investigate the genetic architecture of T2D in African Americans, the MEta-analysis of type 2 DIabetes in African Americans (MEDIA) Consortium examined 17 GWAS on T2D comprising 8,284 cases and 15,543 controls in African Americans in stage 1 analysis. Single nucleotide polymorphisms (SNPs) association analysis was conducted in each study under the additive model after adjustment for age, sex, study site, and principal components. Meta-analysis of approximately 2.6 million genotyped and imputed SNPs in all studies was conducted using an inverse variance-weighted fixed effect model. Replications were performed to follow up 21 loci in up to 6,061 cases and 5,483 controls in African Americans, and 8,130 cases and 38,987 controls of European ancestry. We identified three known loci (TCF7L2, HMGA2 and KCNQ1) and two novel loci (HLA-B and INS-IGF2) at genome-wide significance (4.15 × 10(-94)<P<5 × 10(-8), odds ratio (OR)  = 1.09 to 1.36). Fine-mapping revealed that 88 of 158 previously identified T2D or glucose homeostasis loci demonstrated nominal to highly significant association (2.2 × 10(-23) < locus-wide P<0.05). These novel and previously identified loci yielded a sibling relative risk of 1.19, explaining 17.5% of the phenotypic variance of T2D on the liability scale in African Americans. Overall, this study identified two novel susceptibility loci for T2D in African Americans. A substantial number of previously reported loci are transferable to African Americans after accounting for linkage disequilibrium, enabling fine mapping of causal variants in trans-ethnic meta-analysis studies.

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity.

Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC.

Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily; dysregulated expression of NGAL has been observed in several benign and malignant diseases. In the present study, differentially expressed genes, in comparison with those of control cells, in the mRNA expression profile of EC109 esophageal squamous cell carcinoma (ESCC) cells following NGAL overexpression were analyzed by multiple bioinformatic tools for a comprehensive understanding. A total of 29 gene ontology (GO) terms associated with immune function, chromatin structure and gene transcription were identified among the differentially expressed genes (DEGs) in NGAL overexpressing cells. In addition to the detected GO categories, the results from the functional annotation chart revealed that the differentially expressed genes were also associated with 101 functional annotation category terms. A total of 59 subpathways associated locally with the differentially expressed genes were identified by subpathway analysis, a markedly greater total that detected by traditional pathway enrichment analysis only. Promoter analysis indicated that the potential transcription factors Snail, deltaEF1, Mycn, Arnt, MNB1A, PBF, E74A, Ubx, SPI1 and GATA2 were unique to the downregulated DEG promoters, while bZIP910, ZNF42 and SOX9 were unique for the upregulated DEG promoters. In conclusion, the understanding of the role of NGAL overexpression in ESCC has been improved through the present bioinformatic analysis.

Mercury (Hg) is neurotoxic, and children may be particularly susceptible to this effect. A current major challenge is identification of children who may be uniquely susceptible to Hg toxicity because of genetic predisposition. We examined the possibility that common genetic variants that are known to affect neurologic functions or Hg handling in adults would modify the adverse neurobehavioral effects of Hg exposure in children. Three hundred thirty subjects who participated as children in the recently completed Casa Pia Clinical Trial of Dental Amalgams in Children were genotyped for 27 variants of 13 genes that are reported to affect neurologic functions and/or Hg disposition in adults. Urinary Hg concentrations, reflecting Hg exposure from any source, served as the Hg exposure index. Regression modeling strategies were employed to evaluate potential associations between allelic status for individual genes or combinations of genes, Hg exposure, and neurobehavioral test outcomes assessed at baseline and for 7 subsequent years during the clinical trial. Among boys, significant modification of Hg effects on neurobehavioral outcomes over a broad range of neurologic domains was observed with variant genotypes for 4 of 13 genes evaluated. Modification of Hg effects on a more limited number of neurobehavioral outcomes was also observed for variants of another 8 genes. Cluster analyses suggested some genes interacting in common processes to affect Hg neurotoxicity. In contrast, significant modification of Hg effects on neurobehavioral functions among girls with the same genotypes was substantially more limited. These observations suggest increased susceptibility to the adverse neurobehavioral effects of Hg among children, particularly boys, with genetic variants that are relatively common to the general human population. These findings advance public health goals to identify factors underlying susceptibility to Hg toxicity and may contribute to strategies for preventing adverse health risks associated with Hg exposure.

Recent genomic analyses of pathologically defined tumor types identify "within-a-tissue" disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head and neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multiplatform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All data sets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies.

We investigated the effects of simulated weightlessness on cellular morphology, proliferation, cell cycle, and apoptosis of the human gastric carcinoma cell line SGC-7901 and the human gastric normal cell line HFE-145. A rotating clinostat was used to simulate weightlessness. The Image-Pro4.5 image analysis system was used for morphometric analysis. Proliferating cell nuclear antigen expression was examined by immunohistochemical staining. Changes in the cell cycle were examined using a cytometer. Apoptosis was measured using the terminal dUTP nick-end labeling (TUNEL) method. When subjected to simulated weightlessness, the cellular morphology of SGC-7901 cells was changed at 12, 24, 48, and 72 h, cell conversion from the G1 to S phase was blocked, proliferation was inhibited at 48 and 72 h, and the apoptosis index was increased at 72 h. The same changes were observed for HFE-145 cells at 12 h when subjected to simulated weightlessness, but no significant changes were found afterward compared with controls. SGC-7901 cells change their cellular morphology and biological characteristics during clinostat-simulated weightlessness at 72 h, but HFE-145 cells only change at 12 h and adapt to simulated weightlessness after that point.

Netrin-1 is a guidance cue that can trigger either attraction or repulsion effects on migrating axons of neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. Here we present the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC) receptor. We show that one netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, we demonstrate that UNC5A can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. We propose that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses.

Despite the development of potent RAF/mitogen-activated protein kinase (MAPK) pathway inhibitors, only a fraction of BRAF-mutant patients benefit from treatment with these drugs. Using a combined chemogenomics and chemoproteomics approach, we identify drug-induced RAS-RAF-MEK complex formation in a subset of BRAF-mutant cancer cells characterized by primary resistance to vemurafenib. In these cells, autocrine interleukin-6 (IL-6) secretion may contribute to the primary resistance phenotype via induction of JAK/STAT3 and MAPK signaling. In a subset of cell lines, combined IL-6/MAPK inhibition is able to overcome primary resistance to BRAF-targeted therapy. Overall, we show that the signaling plasticity exerted by primary resistant BRAF-mutant cells is achieved by their ability to mimic signaling features of oncogenic RAS, a strategy that we term "oncogene mimicry." This model may guide future strategies for overcoming primary resistance observed in these tumors.

Distinct midbrain dopamine (mDA) neuron subtypes are found in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), but it is mainly SNc neurons that degenerate in Parkinson's disease. Interest in how mDA neurons develop has been stimulated by the potential use of stem cells in therapy or disease modeling. However, very little is known about how specific dopaminergic subtypes are generated. Here, we show that the expression profiles of the transcription factors Sox6, Otx2, and Nolz1 define subpopulations of mDA neurons already at the neural progenitor cell stage. After cell-cycle exit, Sox6 selectively localizes to SNc neurons, while Otx2 and Nolz1 are expressed in a subset of VTA neurons. Importantly, Sox6 ablation leads to decreased expression of SNc markers and a corresponding increase in VTA markers, while Otx2 ablation has the opposite effect. Moreover, deletion of Sox6 affects striatal innervation and dopamine levels. We also find reduced Sox6 levels in Parkinson's disease patients. These findings identify Sox6 as a determinant of SNc neuron development and should facilitate the engineering of relevant mDA neurons for cell therapy and disease modeling.

Selective neuronal loss is a hallmark of neurodegenerative diseases, including Huntington's disease (HD). Although mutant huntingtin, the protein responsible for HD, is expressed ubiquitously, a subpopulation of neurons in the striatum is the first to succumb. In this review, we examine evidence that protein quality control pathways, including the ubiquitin proteasome system, autophagy, and chaperones, are significantly altered in striatal neurons. These alterations may increase the susceptibility of striatal neurons to mutant huntingtin-mediated toxicity. This novel view of HD pathogenesis has profound therapeutic implications: protein homeostasis pathways in the striatum may be valuable targets for treating HD and other misfolded protein disorders.

Cephalexin is a very commonly prescribed orally administered antibiotic which has many potential side effects. Amongst these cholestatic jaundice has been infrequently reported as an adverse reaction. We present a case of a 57-year-old male who exhibited features of cholestatic jaundice including elevated liver function tests (LFTs) after taking cephalexin and showed improvement on removal of the offending agent. During this time he was symptomatically treated with cholestyramine. Complete resolution of LFTs was seen in four weeks. Cephalexin induced cholestasis is rare and hence requires a high degree of clinical suspicion for prompt diagnosis and treatment.

Amphiphilic macromolecules (AMs) composed of sugar backbones modified with branched aliphatic chains and a poly(ethylene glycol) (PEG) tail can inhibit macrophage uptake of oxidized low-density lipoproteins (oxLDL), a major event underlying atherosclerosis development. Previous studies indicate that AM hydrophobic domains influence this bioactivity through interacting with macrophage scavenger receptors, which can contain basic and/or hydrophobic residues within their binding pockets. In this study, we compare two classes of AMs to investigate their ability to promote athero-protective potency via hydrogen-bonding or hydrophobic interactions with scavenger receptors. A series of ether-AMs, containing methoxy-terminated aliphatic arms capable of hydrogen-bonding, was synthesized. Compared to analogous AMs containing no ether moieties (alkyl-AMs), ether-AMs showed improved cytotoxicity profiles. Increasing AM hydrophobicity via incorporation of longer and/or alkyl-terminated hydrophobic chains yielded macromolecules with enhanced oxLDL uptake inhibition. These findings indicate that hydrophobic interactions and the length of AM aliphatic arms more significantly influence AM bioactivity than hydrogen-bonding.

<h4>Background</h4>The menopausal transition (MT) is a biological inevitability for all ageing women that can be associated with changes in mood, including depressive symptoms. There is tentative evidence that women who develop depression during the MT have greater risk of subsequent depressive episodes, as well as increased health morbidity and mortality. Thus, preventing depression during the MT could enhance both current and the future health and well-being of women. This study aims to test the efficacy of a client-centred health promotion intervention to decrease the 12-month incidence of clinically significant symptoms of depression among women undergoing the MT.<h4>Methods/design</h4>This randomised controlled trial will recruit 300 women undergoing the MT living in the Perth metropolitan area. They will be free of clinically significant symptoms of depression and of psychotic or bipolar disorders. Consenting participants will be stratified for the presence of subsyndromal symptoms of depression and then randomly assigned to the intervention or control group. The intervention will consist of eight telephone health promotion sessions that will provide training in problem solving and education about the MT, healthy ageing, depression and anxiety, and management of chronic health symptoms and problems. The primary outcome of interest is the onset of a major depressive episode according the DSM-IV-TR criteria during the 12-month follow-up or of clinically significant symptoms of depression, as established by a score of 15 or greater on the Patient Health Questionnaire (PHQ-9). Secondary outcomes of interest include changes in the severity of symptoms of depression and anxiety (Hospital Anxiety and Depression Scale, HADS), quality of life (Short Form Health Survey, SF-12), and lifestyle.<h4>Discussion</h4>Current evidence shows that depressive symptoms and disorders are leading causes of disability worldwide, and that they are relatively common during the MT. This study will use a multifaceted health promotion intervention with the aim of preventing depression in these women. If successful, the results of this trial will have implications for the management of women undergoing the MT.<h4>Trial registration</h4>Australian and New Zealand Clinical Trials Registry ACTRN12613000724774. Date registered: 1 July 2013.

<h4>Background</h4>Inflammation triggered by infection or injury is tightly controlled by glucocorticoid hormones which signal via a dedicated transcription factor, the Glucocorticoid Receptor (GR), to regulate hundreds of genes. However, the hierarchy of transcriptional responses to GR activation and the molecular basis of their oftentimes non-linear dynamics are not understood.<h4>Results</h4>We investigated early glucocorticoid-driven transcriptional events in macrophages, a cell type highly responsive to both pro- and anti-inflammatory stimuli. Using whole transcriptome analyses in resting and acutely lipopolysaccharide (LPS)-stimulated macrophages, we show that early GR target genes form dense networks with the majority of control nodes represented by transcription factors. The expression dynamics of several glucocorticoid-responsive genes are consistent with feed forward loops (FFL) and coincide with rapid GR recruitment. Notably, GR binding sites in genes encoding members of the KLF transcription factor family colocalize with KLF binding sites. Moreover, our gene expression, transcription factor binding and computational data are consistent with the existence of the GR-KLF9-KLF2 incoherent FFL. Analysis of LPS-downregulated genes revealed striking enrichment in multimerized Zn-fingers- and KRAB domain-containing proteins known to bind nucleic acids and repress transcription by propagating heterochromatin. This raises an intriguing possibility that an increase in chromatin accessibility in inflammatory macrophages results from broad downregulation of negative chromatin remodelers.<h4>Conclusions</h4>Pro- and anti-inflammatory stimuli alter the expression of a vast array of transcription factors and chromatin remodelers. By regulating multiple transcription factors, which propagate the initial hormonal signal, GR acts as a coordinating hub in anti-inflammatory responses. As several KLFs promote the anti-inflammatory program in macrophages, we propose that GR and KLFs functionally cooperate to curb inflammation.

Early life stress has been linked with poorer lifelong health outcomes across species, including modern and ancient humans. Epigenetic mechanisms, such as DNA methylation patterning of stress pathway genes in stress-responsive tissue, may play an important role in the long-term health effects of early stress across species. The relationships among early maternal care quality, DNA methylation patterns in a candidate stress pathway gene (serotonin transporter, 5-HTT) linked region in blood DNA, and adult health outcomes were examined in male and female rhesus macaques, excellent models of human health. Male (n = 12) and female (n = 32) infants were observed with their mothers for the first 12 weeks of life and 5-HTT linked DNA methylation was measured in blood at 12 weeks of age. Approximately 8 years later, health-related measures were collected for the 25 animals (6 male and 19 female) that were available for study. Health composite scores were generated using factor analysis of body condition, body weight, and diagnosis of diarrhea during the lifespan. Better quality maternal care predicted lower 5-HTT linked methylation at 12 weeks of age. Lower 5-HTT methylation, in turn, predicted better health composite scores in adulthood, including better body condition, greater body weight and absence of lifetime diarrhea. These data suggest that the epigenetic regulation of 5-HTT may be one biomarker of the link between early stress and lifetime health trajectories. Future studies will examine whether epigenetic signatures in modern and ancient human DNA lends insight into stress and health and natural selection in human evolutionary history.

Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.

The gene silencing transcription factor REST [repressor element 1 silencing transcription factor]/NRSF (neuron-restrictive silencer factor) actively represses a large array of coding and noncoding neuron-specific genes important to synaptic plasticity including miR-132. miR-132 is a neuron-specific microRNA and plays a pivotal role in synaptogenesis, synaptic plasticity and structural remodeling. However, a role for miR-132 in neuronal death is not, as yet, well-delineated. Here we show that ischemic insults promote REST binding and epigenetic remodeling at the miR-132 promoter and silencing of miR-132 expression in selectively vulnerable hippocampal CA1 neurons. REST occupancy was not altered at the miR-9 or miR-124a promoters despite the presence of repressor element 1 sites, indicating REST target specificity. Ischemia induced a substantial decrease in two marks of active gene transcription, dimethylation of lysine 4 on core histone 3 (H3K4me2) and acetylation of lysine 9 on H3 (H3K9ac) at the miR-132 promoter. RNAi-mediated depletion of REST in vivo blocked ischemia-induced loss of miR-132 in insulted hippocampal neurons, consistent with a causal relation between activation of REST and silencing of miR-132. Overexpression of miR-132 in primary cultures of hippocampal neurons or delivered directly into the CA1 of living rats by means of the lentiviral expression system prior to induction of ischemia afforded robust protection against ischemia-induced neuronal death. These findings document a previously unappreciated role for REST-dependent repression of miR-132 in the neuronal death associated with global ischemia and identify a novel therapeutic target for amelioration of the neurodegeneration and cognitive deficits associated with ischemic stroke.

Hydroxyapatite (HA) is considered to be a bioactive material that favorably influences the adhesion, growth, and osteogenic differentiation of osteoblasts. To optimize the cell response on the hydroxyapatite composite, it is desirable to assess the optimum concentration and also the optimum particle size. The aim of our study was to prepare composite materials made of polydimethylsiloxane, polyamide, and nano-sized (N) or micro-sized (M) HA, with an HA content of 0%, 2%, 5%, 10%, 15%, 20%, 25% (v/v) (referred to as N0-N25 or M0-M25), and to evaluate them in vitro in cultures with human osteoblast-like MG-63 cells. For clinical applications, fast osseointegration of the implant into the bone is essential. We observed the greatest initial cell adhesion on composites M10 and N5. Nano-sized HA supported cell growth, especially during the first 3 days of culture. On composites with micro-size HA (2%-15%), MG-63 cells reached the highest densities on day 7. Samples M20 and M25, however, were toxic for MG-63 cells, although these composites supported the production of osteocalcin in these cells. On N2, a higher concentration of osteopontin was found in MG-63 cells. For biomedical applications, the concentration range of 5%-15% (v/v) nano-size or micro-size HA seems to be optimum.

Serotonin (5-HT) has consistently been implicated in the pathophysiology of impulsive aggression. In the current study, we tested the hypothesis that 5-HT transporter (5-HTT) binding is reduced in the anterior cingulate cortex (ACC) in impulsive aggressive patients. Additionally, we characterized pathological personality dimensions, with a specific focus on callousness (i.e. emotional indifference, a facet of psychopathy). Callousness is putatively positively correlated with presynaptic 5-HT, and thus could potentially confound the hypothesized negative relation between 5-HTT levels and trait aggression. We determined 5-HTT binding with positron emission tomography and [(11)C]DASB in 29 patients with intermittent explosive disorder (IED-IR) and 30 controls. We assessed group differences in 5-HTT binding in the pregenual ACC, amygdala and subcortical regions and examined correlations between 5-HTT binding and clinical measures. There were no significant differences in 5-HTT binding between IED-IR patients and controls. Trait callousness exhibited a significant, positive correlation with ACC 5-HTT availability. Among IED-IR patients, a trend-level negative partial correlation was observed between trait aggression and ACC 5-HTT availability, while covarying for callousness and age. Exploratory analyses revealed a significant negative correlation between state aggression levels and 5-HTT availability in subcortical regions, namely striatum and thalamus. We did not confirm our hypothesis of lower ACC 5-HTT availability in impulsive aggressive patients, however, the positive correlation between callousness and ACC 5-HTT availability likely played a confounding role. Subtypes of aggression (e.g., reactive vs. proactive aggression), which are differentially associated with pathological personality dimensions such as callousness, may contribute to variability between 5-HT functioning and aggression.

Charcot spine is rare condition whose association with Parkinson's disease (PD) has not been reported yet. The authors reported the cases of two patients with PD who developed Charcot spine. Both patients presented with a history of back pain and bilateral radicular leg pain. They had complete clinical and radiological assessment. Lumbar spine was involved in both patients. Clinical features and response to treatment were described. In the first case, circumferential fusion and stabilization were performed on the dislocated vertebral levels. A solid and stable fusion of the spine was obtained with satisfactory clinical outcome. Surgical treatment has been recommended to the other patient. In both cases, no other neurological etiology was found to account for Charcot spine. In conclusion, Charcot spine is associated with several neurological affections but has not previously been reported in association with Parkinson's disease.

<h4>Background</h4>Decreased antithrombin III (ATIII) activity and large splenic vein diameter (SVD) are risk factors for portal vein thrombosis (PVT) after splenectomy in liver cirrhosis with portal hypertension. Antithrombin III concentrates can prevent PVT. This study was designed to stratify risks for PVT after splenectomy in cirrhotic patients and to develop prophylactic protocols for PVT.<h4>Study design</h4>In 53 patients (testing cohort), the cutoff level of preoperative ATIII activity (≤60%) was evaluated for administration of ATIII concentrates. Antithrombin III activity and SVD were re-evaluated as criteria for prophylaxis of PVT. In 57 patients (validation cohort), the risk stratification of PVT and prophylactic protocols were validated.<h4>Results</h4>In the testing cohort, 10 (19%) of 53 patients had PVT. Risk level of PVT was stratified and prophylactic protocols were developed. Patients at low risk (ATIII activity ≥70% and SVD <10 mm) were not treated; those at high risk (ATIII activity <70% or SVD ≥10 mm) received ATIII concentrates (1,500 U/day) for 3 days; and those at highest risk (SVD ≥15 mm) received ATIII concentrates for 3 days, followed by danaparoid sodium (2,500 U/day) for 14 days and warfarin. In the validation cohort, 0 of 14 low-risk and 2 of 32 high-risk patients had PVT. Although 8 of 11 patients at highest risk had temporary PVT, it disappeared within 3 months postoperatively. Finally, only 2 (3.5%) of 57 patients had PVT.<h4>Conclusions</h4>Risk stratification of PVT after splenectomy and prophylaxis with ATIII concentrates and danaparoid sodium dramatically reduced the incidence of PVT.

<h4>Background</h4>Aberrant methylation of protocadherin 17 (PCDH17) has been reported in several human cancers. However, the methylation status of PCDH17 in prostate cancer and its clinical significance remains unclear. The aim of this study was to investigate the methylation status of PCDH17 and its clinical significance in patients with prostate cancer after radical prostatectomy.<h4>Material/methods</h4>The methylation status of PCDH17 in 152 prostate cancer tissues and 51 non-tumoral prostate tissues was examined by methylation-specific PCR (MSP). Then the association between PCDH17 methylation and clinicopathologic parameters was analyzed. Kaplan-Meier survival analysis, log-rank test and multivariate Cox proportional hazard model analysis were used to analyze the correlation between PCDH17 methylation and prognosis of patients with prostate cancer.<h4>Results</h4>Our data demonstrated that PCDH17 methylation occurred frequently in prostate cancer. PCDH17 methylation was significantly associated with higher pathological Gleason score (P=0.0315), advanced pathological stage (P=0.0260), higher level of preoperative PSA (P=0.0354), positive angiolymphatic invasion (P=0.0461), positive lymph node metastasis (P=0.0362), and biochemical recurrence (BCR) (P=0.0018). In addition, PCDH17 methylation was an independent predictor of poor biochemical recurrence-free (BCR-free) survival and overall survival for patients with prostate cancer.<h4>Conclusions</h4>PCDH17 methylation is a frequent tumor-specific event in prostate cancer, and is significantly correlated with shorter BCR-free survival and overall survival of patients with prostate cancer after radical prostatectomy. PCDH17 methylation in tumor samples after radical prostatectomy may be used as an independent prognostic biomarker.

5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine.

<h4>Background</h4>In Huntington's disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice.<h4>Results</h4>To this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections.<h4>Conclusions</h4>These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.