Gene Literature Dashboard

<h4>Background</h4>Colorectal carcinomas spread easily to nearby tissues around the colon or rectum, and display strong potential for invasion and metastasis. CSE1L, the chromosome segregation 1-like protein, is implicated in cancer progression and is located in both the cytoplasm and nuclei of tumor cells. We investigated the prognostic significance of cytoplasmic vs. nuclear CSE1L expression in colorectal cancer.<h4>Methods</h4>The invasion- and metastasis-stimulating activities of CSE1L were studied by in vitro invasion and animal experiments. CSE1L expression in colorectal cancer was assayed by immunohistochemistry, with tissue microarray consisting of 128 surgically resected specimens; and scored using a semiquantitative method. The correlations between CSE1L expression and clinicopathological parameters were analyzed.<h4>Results</h4>CSE1L overexpression was associated with increased invasiveness and metastasis of cancer cells. Non-neoplastic colorectal glands showed minimal CSE1L staining, whereas most colorectal carcinomas (99.2%, 127/128) were significantly positive for CSE1L staining. Cytoplasmic CSE1L was associated with cancer stage (P=0.003) and depth of tumor penetration (P=0.007). Cytoplasmic CSE1L expression also correlated with lymph node metastasis of the disease in Cox regression analysis<h4>Conclusions</h4>CSE1L regulates the invasiveness and metastasis of cancer cells, and immunohistochemical analysis of cytoplasmic CSE1L in colorectal tumors may provide a useful aid to prognosis.

Huntington disease is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine (polyQ) expansion (> 35Q) in the first exon (EX1) of huntingtin protein (Htt). mHtt protein is thought to adopt one or more toxic conformation(s) that are involved in pathogenic interactions in cells . However, the structure of mHtt is not known. Here, we present a near atomic resolution structure of mHtt36Q-EX1. To facilitate crystallization, three histidine residues (3H) were introduced within the Htt36Q stretch resulting in the sequence of Q 7HQHQHQ 27. The Htt36Q3H region adopts α-helix, loop, β-hairpin conformations. Furthermore, we observed interactions between the backbone of the Htt36Q3H β-strand with the aromatic residues mimicking putative-toxic interactions with other proteins. Our findings support previous predictions that the expanded mHtt-polyQ region adopts a β-sheet structure. Detailed structural information about mHtt improves our understanding of the pathogenic mechanisms in HD and other polyQ expansion disorders and may form the basis for rational design of small molecules that target toxic conformations of disease-causing proteins.

<h4>Objective</h4>Sepsis in older children and adults modifies immune system function. We compared serotype-specific antibody responses to heptavalent pneumococcal conjugate vaccine (PCV7) in very low birth weight infants (<1500 g,VLBWs) with and without blood stream infection (BSI) during their birth hospitalization.<h4>Study design</h4>Retrospective analysis of prospectively collected data for the Neonatal Research Network study of PCV7 responses among VLBWs. Infants received PCV7 at 2, 4 and 6 months after birth with blood drawn 4 to 6 weeks after third dose. Serotype antibodies were compared between infants with or without a history of BSI. Regression models were constructed with BW groups and other confounding factors identified in the primary study.<h4>Result</h4>In all, 244 infants completed the vaccine series and had serum antibody available; 82 had BSI. After adjustment, BSI was not associated with reduced odds of serum antibody 0.35 μg ml(-1).<h4>Conclusion</h4>BSI was not associated with reduced odds of World Health Organization-defined protective PCV7 responses in VLBWs.

The molecular target of the adipokine vaspin (visceral adipose tissue-derived serpin; serpinA12) and its mode of action are unknown. Here, we provide the vaspin crystal structure and identify human kallikrein 7 (hK7) as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro. We detect vaspin-hK7 complexes in human plasma and find co-expression of both proteins in murine pancreatic β-cells. We further demonstrate that hK7 cleaves human insulin in the A- and B-chain. Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion. By application of vaspin and generated inactive mutants, we find the significantly improved glucose tolerance in C57BL/6NTac and db/db mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspin-mediated changes in insulin sensitivity as determined by euglycemic-hyperinsulinemic clamp studies. Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice, supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation. In conclusion, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissue-derived serpin vaspin, and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesity-induced insulin resistance.

Mitochondrial dysfunction is both a contributing mechanism and complication of diabetes, and oxidative stress contributes to that dysfunction. Mitochondrial manganese-superoxide dismutase (MnSOD) is a metalloenzyme that provides antioxidant protection. We have previously shown in a mouse model of hereditary iron overload that cytosolic iron levels affected mitochondrial manganese availability, MnSOD activity, and insulin secretion. We therefore sought to determine the metallation status of MnSOD in wild-type mice and whether altering that status affected β-cell function. 129/SvEVTac mice given supplemental manganese exhibited a 73% increase in hepatic MnSOD activity and increased metallation of MnSOD. To determine whether manganese supplementation offered glucose homeostasis under a situation of β-cell stress, we challenged C57BL/6J mice, which are more susceptible to diet-induced diabetes, with a high-fat diet for 12 weeks. Manganese was supplemented or not for the final 8 weeks on that diet, after which we examined glucose tolerance and the function of isolated islets. Liver mitochondria from manganese-injected C57BL/6J mice had similar increases in MnSOD activity (81%) and metallation as were seen in 129/SvEVTac mice. The manganese-treated group fed high fat had improved glucose tolerance (24% decrease in fasting glucose and 41% decrease in area under the glucose curve), comparable with mice on normal chow and increased serum insulin levels. Isolated islets from the manganese-treated group exhibited improved insulin secretion, decreased lipid peroxidation, and improved mitochondrial function. In conclusion, MnSOD metallation and activity can be augmented with manganese supplementation in normal mice on normal chow, and manganese treatment can increase insulin secretion to improve glucose tolerance under conditions of dietary stress.

A combination of autozygosity mapping and exome sequencing identified a null mutation in SLC24A4 in a family with hypomineralized amelogenesis imperfect a (AI), a condition in which tooth enamel formation fails. SLC24A4 encodes a calcium transporter upregulated in ameloblasts during the maturation stage of amelogenesis. Screening of further AI families identified a missense mutation in the ion-binding site of SLC24A4 expected to severely diminish or abolish the ion transport function of the protein. Furthermore, examination of previously generated Slc24a4 null mice identified a severe defect in tooth enamel that reflects impaired amelogenesis. These findings support a key role for SLC24A4 in calcium transport during enamel formation.

Aim of this study was to investigate phosphatidylethanolamine binding protein 1 (PEBP1) in GIST and its relations with MEK1/2 activation and ETV1 by immunohistochemistry. Loss of PEBP1 was found in 22/161 (13.7%) GIST, was associated with clinical risk factors and with a trend towards shorter disease free survival, but not with pMEK1/2 and ETV1 expression. So downregulation of PEBP1 does not activate the Ras-Raf-1-MEK1/2-ERK1/2 pathway by phosphorylation of MEK1/2 and does not influence ETV1 expression in GIST. Loss of PEBP1 associates with clinical risk factors, but since no significant influence on survival was found, further studies are required.

Retrovirus is frequently used in the genetic modification of mammalian cells and the establishment of induced pluripotent stem cells (iPSCs) via cell reprogramming. Vector-induced genotoxicity could induce profound effect on the physiology and function of these stem cells and their differentiated progeny. We analyzed retrovirus-induced genotoxicity in somatic cell Jurkat and two iPSC lines. In Jurkat cells, retrovirus frequently activated host gene expression and gene activation was not dependent on the distance between the integration site and the transcription start site of the host gene. In contrast, retrovirus frequently down-regulated host gene expression in iPSCs, possibly due to the action of chromatin silencing that spreads from the provirus to the nearby host gene promoter. Our data raises the issue that some of the phenotypic variability observed among iPSC clones derived from the same parental cell line may be caused by retrovirus-induced gene expression changes rather than by the reprogramming process itself. It also underscores the importance of characterizing retrovirus integration and carrying out risk assessment of iPSCs before they can be applied in basic research and clinics.

Epidemiologic data suggest that maternal microbial infections may cause fetal neurodevelopmental disorders, potentially increasing susceptibility to heavy psychopathologies such as schizophrenia, schizophreniform disorder, autism, pervasive developmental disorders, bipolar disorders, psychosis, epilepsy, language and speech disorders, and cognitive impairment in adult offspring. However, the molecular pathomechanisms underlying such a relationship are not clear. Here we analyze the potential role of the maternal immune response to viral infection in determining fetal brain injuries that increase the risk of neurological disorders in the adult. We use influenza infection as a disease model and human axon guidance pathway, a key process in the formation of neural network during midgestation, as a potential fetal target of immune insults. Specifically, we examined influenza A H1N1 hemagglutinin (HA), an antigenic viral protein, for amino acid sequence similarity to a random library of 188 axon guidance proteins. We obtain the results that (1) contrary to any theoretical expectations, 45 viral pentapeptide matches are distributed throughout a subset of 36 guidance molecules; (2) in 24 guidance proteins, the peptide sharing with HA antigen involves already experimentally validated influenza HA epitopes; and (3) most of the axon guidance vs HA peptide overlap is conserved among influenza A viral strains and subsets. Taken together, our data indicate that immune cross-reactivity between influenza HA and axon guidance molecules is possible and may well represent a pathologic mechanism capable of determining neurodevelopmental disruption in the fetus.

During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN⁺ cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN⁺ blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV⁺ serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN⁺ DC that were isolated directly from HIV-1⁺ individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.

Using a phenome-wide association study (PheWAS) approach, we comprehensively tested genetic variants for association with phenotypes available for 70,061 study participants in the Population Architecture using Genomics and Epidemiology (PAGE) network. Our aim was to better characterize the genetic architecture of complex traits and identify novel pleiotropic relationships. This PheWAS drew on five population-based studies representing four major racial/ethnic groups (European Americans (EA), African Americans (AA), Hispanics/Mexican-Americans, and Asian/Pacific Islanders) in PAGE, each site with measurements for multiple traits, associated laboratory measures, and intermediate biomarkers. A total of 83 single nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) were genotyped across two or more PAGE study sites. Comprehensive tests of association, stratified by race/ethnicity, were performed, encompassing 4,706 phenotypes mapped to 105 phenotype-classes, and association results were compared across study sites. A total of 111 PheWAS results had significant associations for two or more PAGE study sites with consistent direction of effect with a significance threshold of p<0.01 for the same racial/ethnic group, SNP, and phenotype-class. Among results identified for SNPs previously associated with phenotypes such as lipid traits, type 2 diabetes, and body mass index, 52 replicated previously published genotype-phenotype associations, 26 represented phenotypes closely related to previously known genotype-phenotype associations, and 33 represented potentially novel genotype-phenotype associations with pleiotropic effects. The majority of the potentially novel results were for single PheWAS phenotype-classes, for example, for CDKN2A/B rs1333049 (previously associated with type 2 diabetes in EA) a PheWAS association was identified for hemoglobin levels in AA. Of note, however, GALNT2 rs2144300 (previously associated with high-density lipoprotein cholesterol levels in EA) had multiple potentially novel PheWAS associations, with hypertension related phenotypes in AA and with serum calcium levels and coronary artery disease phenotypes in EA. PheWAS identifies associations for hypothesis generation and exploration of the genetic architecture of complex traits.

Mitochondrial transcription, translation, and respiration require interactions between genes encoded in two distinct genomes, generating the potential for mutations in nuclear and mitochondrial genomes to interact epistatically and cause incompatibilities that decrease fitness. Mitochondrial-nuclear epistasis for fitness has been documented within and between populations and species of diverse taxa, but rarely has the genetic or mechanistic basis of these mitochondrial-nuclear interactions been elucidated, limiting our understanding of which genes harbor variants causing mitochondrial-nuclear disruption and of the pathways and processes that are impacted by mitochondrial-nuclear coevolution. Here we identify an amino acid polymorphism in the Drosophila melanogaster nuclear-encoded mitochondrial tyrosyl-tRNA synthetase that interacts epistatically with a polymorphism in the D. simulans mitochondrial-encoded tRNA(Tyr) to significantly delay development, compromise bristle formation, and decrease fecundity. The incompatible genotype specifically decreases the activities of oxidative phosphorylation complexes I, III, and IV that contain mitochondrial-encoded subunits. Combined with the identity of the interacting alleles, this pattern indicates that mitochondrial protein translation is affected by this interaction. Our findings suggest that interactions between mitochondrial tRNAs and their nuclear-encoded tRNA synthetases may be targets of compensatory molecular evolution. Human mitochondrial diseases are often genetically complex and variable in penetrance, and the mitochondrial-nuclear interaction we document provides a plausible mechanism to explain this complexity.

A unique synaptic activity-responsive element (SARE) sequence, composed of the consensus binding sites for SRF, MEF2 and CREB, is necessary for control of transcriptional upregulation of the Arc gene in response to synaptic activity. We hypothesize that this sequence is a broad mechanism that regulates gene expression in response to synaptic activation and during plasticity; and that analysis of SARE-containing genes could identify molecular mechanisms involved in brain disorders. To search for conserved SARE sequences in the mammalian genome, we used the SynoR in silico tool, and found the SARE cluster predominantly in the regulatory regions of genes expressed specifically in the nervous system; most were related to neural development and homeostatic maintenance. Two of these SARE sequences were tested in luciferase assays and proved to promote transcription in response to neuronal activation. Supporting the predictive capacity of our candidate list, up-regulation of several SARE containing genes in response to neuronal activity was validated using external data and also experimentally using primary cortical neurons and quantitative real time RT-PCR. The list of SARE-containing genes includes several linked to mental retardation and cognitive disorders, and is significantly enriched in genes that encode mRNA targeted by FMRP (fragile X mental retardation protein). Our study thus supports the idea that SARE sequences are relevant transcriptional regulatory elements that participate in plasticity. In addition, it offers a comprehensive view of how activity-responsive transcription factors coordinate their actions and increase the selectivity of their targets. Our data suggest that analysis of SARE-containing genes will reveal yet-undescribed pathways of synaptic plasticity and additional candidate genes disrupted in mental disease.

<h4>Background</h4>Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections.<h4>Methodology/principal finding</h4>In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6) that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization.<h4>Conclusion</h4>The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

<h4>Background</h4>Gonadotrophin-releasing hormone agonist (GnRHa) is commonly used to switch off (down regulate) the pituitary gland and thus suppress ovarian activity in women undergoing in vitro fertilisation (IVF). Other fertility drugs (gonadotrophins) are then used to stimulate ovulation in a controlled manner. Among the various types of pituitary down regulation protocols in use, the long protocol achieves the best clinical pregnancy rate. The long protocol requires GnRHa administration until suppression of ovarian activity occurs, within approximately 14 days. GnRHa can be used either as daily low-dose injections or through a single injection containing higher doses of the drug (depot). It is unclear which of these two forms of administration is best, and whether single depot administration may require higher doses of gonadotrophins.<h4>Objectives</h4>To compare the effectiveness and safety of a single depot dose of GHRHa versus daily GnRHa doses in women undergoing IVF.<h4>Search methods</h4>We searched the following databases: Cochrane Menstrual Disorders and Subfertility Group Trials Register (searched July 2012), Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2012, Issue 7), MEDLINE (1966 to July 2012), EMBASE (1980 to July 2012) and LILACS (1982 to July 2012). We also screened the reference lists of articles.<h4>Selection criteria</h4>We included RCTs comparing depot and daily administration of GnRHa for long protocols in IVF treatment cycles in couples with any cause of infertility, using various methods of ovarian stimulation. The primary review outcomes were live birth or ongoing pregnancy, clinical pregnancy and ovarian hyperstimulation syndrome (OHSS). Other outcomes included number of oocytes retrieved, miscarriage, multiple pregnancy, number of gonadotrophin (FSH) units used for ovarian stimulation, duration of gonadotrophin treatment, cost and patient convenience.<h4>Data collection and analysis</h4>Two review authors independently selected studies, extracted data and assessed study quality. For dichotomous outcomes, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) per woman randomised. Where appropriate, we pooled studies.<h4>Main results</h4>Sixteen studies were eligible for inclusion (n = 1811 participants), 12 (n = 1366 participants) of which were suitable for meta-analysis. No significant heterogeneity was detected.There were no significant differences between depot GnRHa and daily GnRHa in live birth/ongoing pregnancy rates (OR 0.95, 95% CI 0.70 to 1.31, seven studies, 873 women), but substantial differences could not be ruled out. Thus for a woman with a 24% chance of achieving a live birth or ongoing pregnancy using daily GnRHa injections, the corresponding chance using GnRHa depot would be between 18% and 29%.There was no significant difference between the groups in clinical pregnancy rate (OR 0.96, 95% CI 0.75 to 1.23, 11 studies, 1259 women). For a woman with a 30% chance of achieving clinical pregnancy using daily GnRHa injections, the corresponding chance using GnRHa depot would be between 25% and 35%.There was no significant difference between the groups in the rate of severe OHSS (OR 0.84, 95% CI 0.29 to 2.42, five studies, 570 women), but substantial differences could not be ruled out. For a woman with a 3% chance of severe OHSS using daily GnRHa injections, the corresponding risk using GnRHa depot would be between 1% and 6%.Compared to women using daily GnRHa, those on depot administration required significantly more gonadotrophin units for ovarian stimulation (standardised mean difference (SMD) 0.26, 95% CI 0.08 to 0.43, 11 studies, 1143 women) and a significantly longer duration of gonadotrophin use (mean difference (MD) 0.65, 95% CI 0.46 to 0.84, 10 studies, 1033 women).Study quality was unclear due to poor reporting. Only four studies reported live births as an outcome and only five described adequate methods for concealment of allocation.<h4>Authors' conclusions</h4>We found no evidence of a significant difference between depot and daily GnRHa use for pituitary down regulation in IVF cycles using the long protocol, but substantial differences could not be ruled out. Since depot GnRHa requires more gonadotrophins and a longer duration of use, it may increase the overall costs of IVF treatment.

<h4>Background</h4>Major stroke clinical trials have failed during the past decades. The failures suggest the presence of heterogeneity among stroke patients. Biomarkers refer to indicators found in the blood, other body fluids or tissues that predicts physiologic or disease states, increased disease risk, or pharmacologic responses to a therapeutic intervention. Stroke biomarkers could be used as a guiding tool for more effective personalized therapy.<h4>Main contents</h4>Three aspects of stroke biomarkers are explored in detail. First, the possible role of biomarkers in patients with stroke is discussed. Second, the limitations of conventional biomarkers (especially protein biomarkers) in the area of stroke research are presented with the reasons. Lastly, various types of biomarkers including traditional and novel genetic, microvesicle, and metabolomics-associated biomarkers are introduced with their advantages and disadvantages. We especially focus on the importance of comprehensive approaches using a variety of stroke biomarkers.<h4>Conclusion</h4>Although biomarkers are not recommended in practice guidelines for use in the diagnosis or treatment of stroke, many efforts have been made to overcome the limitations of biomarkers. The studies reviewed herein suggest that comprehensive analysis of different types of stroke biomarkers will improve the understanding of individual pathophysiologies and further promote the development of screening tools for of high-risk patients, and predicting models of stroke outcome and rational stroke therapy tailored to the characteristics of each case.

There is an ongoing effort to develop better methods for noninvasive detection and characterization of thrombi. Here we describe the synthesis and evaluation of three new fibrin-targeted positron emission tomography (PET) probes (FBP1, FBP2, FBP3). Three fibrin-specific peptides were conjugated as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-monoamides at the C- and N-termini and chelated with (64)CuCl2. Probes were prepared with a specific activity ranging from 10 to 130 μCi/nmol. Both the peptides and the probes exhibited nanomolar dissociation constants (Kd) for the soluble fibrin fragment DD(E), although the Cu-DOTA derivatization resulted in a 2-3 fold loss in affinity relative to the parent peptide. Biodistribution and imaging studies were performed in a rat model of carotid artery thrombosis. For FBP1 and FBP2 at 120 min post injection, the vessel containing the thrombus showed the highest concentration of radioactivity after the excretory organs, that is, the liver and kidneys. This was confirmed ex vivo by autoradiography, which showed >4-fold activity in the thrombus-containing artery compared to the contralateral artery. FBP3 showed much lower thrombus uptake, and the difference was traced to greater metabolism of this probe. Hybrid MR-PET imaging with FBP1 or FBP2 confirmed that these probes were effective for the detection of an arterial thrombus in this rat model. A thrombus was visible on PET images as a region of high activity that corresponded to a region of arterial occlusion identified by simultaneous MR angiography. FBP1 and FBP2 represent promising new probes for the molecular imaging of thrombi.

Deletions involving 17q21-q24 have been identified previously to result in two clinically recognizable contiguous gene deletion syndromes: 17q21.31 and 17q23.1-q23.2 microdeletion syndromes. Although deletions involving 17q22 have been reported in the literature, only four of the eight patients reported were identified by array-comparative genomic hybridization (array-CGH) or flourescent in situ hybridization. Here, we describe five new patients with 1.8-2.5-Mb microdeletions involving 17q22 identified by array-CGH. We also present one patient with a large karyotypically visible deletion involving 17q22, fine-mapped to ~8.2 Mb using array-CGH. We show that the commonly deleted region in our patients spans 0.24 Mb and two genes; NOG and C17ORF67. The function of C17ORF67 is not known, whereas Noggin, the product of NOG, is essential for correct joint development. In common with the 17q22 patients reported previously, the disease phenotype of our patients includes intellectual disability, attention deficit hyperactivity disorder, conductive hearing loss, visual impairment, low set ears, facial dysmorphology and limb anomalies. All patients displayed NOG-related bone and joint features, including symphalangism and facial dysmorphology. We conclude that these common clinical features indicate a novel clinically recognizable, 17q22 contiguous microdeletion syndrome.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western world and its incidence is increasing rapidly. NAFLD is a spectrum ranging from simple steatosis, which is relatively benign hepatically, to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis. Obesity, insulin resistance, type 2 diabetes mellitus, and dyslipidemia are the most important risk factors for NAFLD. Due to heavy enrichment with metabolic risk factors, individuals with NAFLD are at significantly higher risk for cardiovascular disease. Individuals with NAFLD have higher incidence of type 2 diabetes. The diagnosis of NAFLD requires imaging evidence of hepatic steatosis in the absence of competing etiologies including significant alcohol consumption. Liver biopsy remains the gold standard for diagnosing NASH and for determining prognosis. Weight loss remains a cornerstone of treatment. Weight loss of ~5% is believed to improve steatosis, whereas ~10% weight loss is necessary to improve steatohepatitis. A number of pharmacologic therapies have been investigated to treat NASH, and agents such as vitamin E and thiazolidinediones have shown promise in select patient subgroups.

During meiosis, evolutionarily conserved mechanisms regulate chromosome remodeling, leading to the formation of a tight bivalent structure. This bivalent, a linked pair of homologous chromosomes, is essential for proper chromosome segregation in meiosis. The formation of a tight bivalent involves chromosome condensation and restructuring around the crossover. The synaptonemal complex (SC), which mediates homologous chromosome association before crossover formation, disassembles concurrently with increased condensation during bivalent remodeling. Both chromosome condensation and SC disassembly are likely critical steps in acquiring functional bivalent structure. The mechanisms controlling SC disassembly, however, remain unclear. Here we identify akir-1 as a gene involved in key events of meiotic prophase I in Caenorhabditis elegans. AKIR-1 is a protein conserved among metazoans that lacks any previously known function in meiosis. We show that akir-1 mutants exhibit severe meiotic defects in late prophase I, including improper disassembly of the SC and aberrant chromosome condensation, independently of the condensin complexes. These late-prophase defects then lead to aberrant reconfiguring of the bivalent. The meiotic divisions are delayed in akir-1 mutants and are accompanied by lagging chromosomes. Our analysis therefore provides evidence for an important role of proper SC disassembly in configuring a functional bivalent structure.

Oxidative stress and endoplasmic reticulum (ER) stress are emerging as crucial events in the etiopathology of many neurodegenerative diseases. While the neuroprotective contributions of the dietary compound curcumin has been recognized, the molecular mechanisms underlying curcumin's neuroprotection under oxidative and ER stresses remains elusive. Herein, we show that curcumin protects HT22 from oxidative and ER stresses evoked by the hypoxia (1% O(2) or CoCl(2) treatment) by enhancing peroxiredoxin 6 (Prdx6) expression. Cells exposed to CoCl(2) displayed reduced expression of Prdx6 with higher reactive oxygen species (ROS) expression and activation of NF-κB with IκB phosphorylation. When NF-κB activity was blocked by using SN50, an inhibitor of NF-κB, or cells treated with curcumin, the repression of Prdx6 expression was restored, suggesting the involvement of NF-κB in modulating Prdx6 expression. These cells were enriched with an accumulation of ER stress proteins, C/EBP homologous protein (CHOP), GRP/78, and calreticulin, and had activated states of caspases 12, 9, and 3. Reinforced expression of Prdx6 in HT22 cells by curcumin reestablished survival signaling by reducing propagation of ROS and blunting ER stress signaling. Intriguingly, knockdown of Prdx6 by antisense revealed that loss of Prdx6 contributed to cell death by sustaining enhanced levels of ER stress-responsive proapoptotic proteins, which was due to elevated ROS production, suggesting that Prdx6 deficiency is a cause of initiation of ROS-mediated ER stress-induced apoptosis. We propose that using curcumin to reinforce the naturally occurring Prdx6 expression and attenuate ROS-based ER stress and NF-κB-mediated aberrant signaling improves cell survival and may provide an avenue to treat and/or postpone diseases associated with ROS or ER stress.

Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1(r)) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1(r) can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1(r) application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1(r) also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1(r) reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.

We report here that the acidic ribosomal protein P0 is a component of the membrane-associated Potato virus A (PVA) ribonucleoprotein complex. As a constituent of the ribosomal stalk, P0 functions in translation. Although the ribosomal stalk proteins P0, P1, P2, and P3 are all important for PVA infection, P0 appears to have a distinct role from those of the other stalk proteins in infection. Our results indicate that P0 also regulates viral RNA functions as an extraribosomal protein. We reported previously that PVA RNA can be targeted by VPg to a specific gene expression pathway that protects the viral RNA from degradation and facilitates its translation. Here, we show that P0 is essential for this activity of VPg, similar to eIF4E/eIF(iso)4E. We also demonstrate that VPg, P0, and eIF(iso)4E synergistically enhance viral translation. Interestingly, the positive effects of VPg and P0 on viral translation were negatively correlated with the cell-to-cell spread of infection, suggesting that these processes may compete for viral RNA.

Huntington's disease (HD) is an autosomal disease caused by a CAG repeat expansion in the huntingtin (HTT) gene. The resultant mutant HTT protein (mHTT) forms aggregates in various types of cells, including neurons and glial cells and preferentially affects brain function. We found that two HD mouse models (Hdh(150Q) and R6/2) were more susceptible than wild-type (WT) mice to lipopolysaccharide-evoked systemic inflammation and produced more proinflammatory cytokines in the brain. Such an enhanced inflammatory response in the brain was not observed in N171- 82Q mice that express mHTT only in neurons, but not in glial cells. Thus, HD glia might play an important role in chronic inflammation that accelerates disease progression in HD mice. Intriguingly, enhanced activation of nuclear factor (NF)-κB-p65 (p65), a transcriptional mediator of inflammatory responses, was observed in astrocytes of patients and mice with HD. Results obtained using primary R6/2 astrocytes suggest that these cells exhibited higher IκB kinase (IKK) activity that caused prolongation of NF-κB activation, thus upregulating proinflammatory factors during inflammation. R6/2 astrocytes also produced a more-damaging effect on primary R6/2 neurons than did WT astrocytes during inflammation. Blockage of IKK reduced the neuronal toxicity caused by R6/2 astrocytes and ameliorated several HD symptoms of R6/2 mice (e.g. decreased neuronal density, impaired motor coordination and poor cognitive function). Collectively, our results indicate that enhancement of the p65-mediated inflammatory response in astrocytes contributes to HD pathogenesis. Therapeutic interventions aimed at preventing neuronal inflammation may be an important strategy for treating HD.

Glutaminase is a multifunctional enzyme encoded by gene Gls involved in energy metabolism, ammonia trafficking and regeneration of neurotransmitter glutamate. To address the proteomic basis for the neurophenotypes of glutaminase-deficient mice, brain proteins from late gestation wild type, Gls+/- and Gls-/- male mice were subjected to two-dimensional gel electrophoresis, with subsequent identification by mass spectrometry using nano-LC-ESI-MS/MS. Protein spots that showed differential genotypic variation were quantified by immunoblotting. Differentially expressed proteins unambiguously identified by MS/MS included neurocalcin delta, retinol binding protein-1, reticulocalbin-3, cytoskeleton proteins fascin and tropomyosin alpha-4-chain, dihydropyrimidinase-related protein-5, apolipoprotein IV and proteins from protein metabolism proteasome subunits alpha type 2, type 7, heterogeneous nuclear ribonucleoprotein C1/C2 and H, voltage-gated anion-selective channel proteins 1 and 2, ATP synthase subunit β and transitional endoplasmic reticulum ATPase. An interaction network determined by Ingenuity Pathway Analysis revealed a link between glutaminase and calcium, Akt and retinol signaling, cytoskeletal elements, ATPases, ion channels, protein synthesis and the proteasome system, intermediary, nucleic acid and lipid metabolism, huntingtin, guidance cues, transforming growth factor beta-1 and hepatocyte nuclear factor 4-alpha. The network identified involves (a) cellular assembly and organization and (b) cell signaling and cell cycle, suggesting that Gls is crucial for neuronal maturation.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder caused by polyglutamine repeat expansions in Ataxin-1. Recent evidence supports a role for microRNAs (miRNAs) deregulation in SCA1 pathogenesis. However, the extent to which miRNAs may modulate the onset, progression or severity of SCA1 remains largely unknown. In this study, we used a mouse model of SCA1 to determine if miRNAs are misregulated in pre- and post-symptomatic SCA1 cerebellum. We found a significant alteration in the steady-state levels of numerous miRNAs prior to and following phenotypic onset. In addition, we provide evidence that increased miR-150 levels in SCA1 Purkinje neurons may modulate disease pathogenesis by targeting the expression of Rgs8 and Vegfa.

Genomic copy number alterations are common in cancer. Finding the genes causally implicated in oncogenesis is challenging because the gain or loss of a chromosomal region may affect a few key driver genes and many passengers. Integrative analyses have opened new vistas for addressing this issue. One approach is to identify genes with frequent copy number alterations and corresponding changes in expression. Several methods also analyse effects of transcriptional changes on known pathways. Here, we propose a method that analyses in-cis correlated genes for evidence of in-trans association to biological processes, with no bias towards processes of a particular type or function. The method aims to identify cis-regulated genes for which the expression correlation to other genes provides further evidence of a network-perturbing role in cancer. The proposed unsupervised approach involves a sequence of statistical tests to systematically narrow down the list of relevant genes, based on integrative analysis of copy number and gene expression data. A novel adjustment method handles confounding effects of co-occurring copy number aberrations, potentially a large source of false positives in such studies. Applying the method to whole-genome copy number and expression data from 100 primary breast carcinomas, 6373 genes were identified as commonly aberrant, 578 were highly in-cis correlated, and 56 were in addition associated in-trans to biological processes. Among these in-trans process associated and cis-correlated (iPAC) genes, 28% have previously been reported as breast cancer associated, and 64% as cancer associated. By combining statistical evidence from three separate subanalyses that focus respectively on copy number, gene expression and the combination of the two, the proposed method identifies several known and novel cancer driver candidates. Validation in an independent data set supports the conclusion that the method identifies genes implicated in cancer.

<h4>Objective</h4>Premature ejaculation (PE) has been reported as the most common male sexual dysfunction with global prevalence rates estimated at approximately 30%. The neurobiogenesis of ejaculation is very complex and involves the serotoninergic (5-hydroxytryptamine, 5-HT) system. Recently, genetic polymorphisms located on SLC6A4 gene codifying for 5-HT transporter (5-HTT), the major regulator of serotonic neurotransmission, have been linked with the pathogenesis and risk of PE. Apparently studies of this type of polymorphism in PE have show conflicting results.<h4>Methods</h4>A meta-analysis was performed that are available in relation with 5-HTT gene-linked promoter region (5-HTTLPR) polymorphism and the risk of lifelong PE (LPE) in men to clarify this relationship. We searched Pubmed and Embase (last search updated on Aug 2012) using 'premature ejaculation', 'polymorphism or variant', 'genotype', 'ejaculatory function', and 'rapid ejaculation' as keywords and reference lists of studies corresponded to the inclusion criteria for meta-analysis. These studies involved the total number of 481 LPE men and 466 health control men subjects. Odds ratio (OR) and 95% confidence intervals (CIs) were used to evaluate this relationship.<h4>Results</h4>In the overall analysis, significant associations between LPE risk and 5-HTTLPR polymorphism were found (L-allele vs. S-allele OR = 0.86, 95% CI = 0.79-0.95, P = 0.002; LL vs. SS: OR = 0.80, 95% CI = 0.68-0.95, P = 0.009; LS vs. SS: OR = 0.85, 95% CI = 0.76-0.97, P = 0.012 and LL+LS vs. SS: OR = 0.88, 95% CI = 0.81-0.95, P = 0.002). Moreover, in subgroup analysis based on ethnicity, similar significant associations were detected. The Egger's test did not reveal presence of a publication bias.<h4>Conclusions</h4>Our investigations demonstrate that 5-HTTLPR (L>S) polymorphism might protect men against LPE risk. Further studies based on larger sample size and gene-environment interactions should be conducted the role of 5-HTTLPR polymorphism and LPE risk.

Classic genetic studies implicated several genes including immune response genes in the risk of developing type 1 diabetes in humans. However, recent evidence including discordant diabetes incidence among monozygotic twins suggested a role for epigenetics in disease manifestation. NOD mice spontaneously develop type 1 diabetes like humans and serve as an excellent model system to study the mechanisms of type 1 diabetes as well as the efficacy of maneuvers to manipulate the disease. Using this preclinical model, we have recently demonstrated that pharmacological inhibition of histone deacetylases can lead to histone hyperacetylation, selective up-regulation of interferon-γ and its transactivator Tbx21/Tbet, and amelioration of autoimmune diabetes. In the current study, we show that chromatin remodeling can render splenocytes incapable of transferring diabetes into immunodeficient NOD.scid mice. To elucidate the underlying mechanisms of drug-mediated protection against type 1 diabetes, we performed global gene expression profiling of splenocytes using high throughput microarray technology. This unbiased transcriptome analysis unraveled the exaggerated expression of a novel set of closely related inflammatory genes in splenocytes of acutely diabetic mice and their repression in mice cured of diabetes by chromatin remodeling. Analysis of gene expression by qRT-PCR using RNA derived from spleens and pancreata of cured mice validated the suppression of most of these genes, indicating an inverse correlation between the high levels of these inflammatory genes and protection against diabetes in NOD mice. In addition, higher-level expression of genes involved in insulin sensitivity, erythropoiesis, hemangioblast generation, and cellular redox control was evident in spleens of cured mice, indicating their possible contribution to protection against type 1 diabetes. Taken together, these results are consistent with the involvement of epistatic mechanisms in the manifestation of autoimmune diabetes and further indicate the utility of chromatin remodeling in curing this complex autoimmune disorder.

<h4>Background</h4>Vibrio vulnificus is an opportunistic human pathogen that is widely distributed in estuarine environments and is capable of causing necrotizing fasciitis and sepsis. In Japan, based on epidemiological research, the incidences of V. vulnificus were concentrated in Kyusyu, mainly in coastal areas of the Ariake Sea. To examine the virulence potential, various genotyping methods have recently been developed. This study aimed to investigate the distribution of virulence markers among V. vulnificus isolates of clinical and environmental origin in three coastal areas with different infection incidences and to determine whether these isolates have the siderophore encoding gene viuB.<h4>Methodology/principal findings</h4>We examined the distribution of genotypes of the 16S ribosomal ribonucleic acid (rRNA) gene, vvhA, vcg, and capsular polysaccharide (CPS), and the presence of viuB in 156 isolates collected from patients and environmental samples in Japan. The environmental samples were collected from three coastal areas: the Ariake Sea, Ise & Mikawa Bay, and Karatsu Bay. The results showed disparity in the ratios of genotypes depending on the sample origins. V. vulnificus isolates obtained from patients were classified into the clinical type for all genotypes. In the environmental isolates, the ratios of the clinical type for genotypes of the 16S rRNA gene, vvhA, and vcg were in the order of the Ariake Sea>Ise & Mikawa Bay>Karatsu Bay. Meanwhile, CPS analysis showed no significant difference. Most isolates possessed viuB.<h4>Conclusions</h4>Many V. vulnificus belonging to the clinical type existed in the Ariake Sea. Three coastal areas with different infection incidences showed distinct ratios of genotypes. This may indicate that the distribution of clinical isolates correlates with the incidence of V. vulnificus infection.

<h4>Aims/hypothesis</h4>Genome-wide association studies have identified over 50 new genetic loci for type 2 diabetes (T2D). Several studies conclude that higher dietary heme iron intake increases the risk of T2D. Therefore we assessed whether the relation between genetic loci and T2D is modified by dietary heme iron intake.<h4>Methods</h4>We used Affymetrix Genome-Wide Human 6.0 array data [681,770 single nucleotide polymorphisms (SNPs)] and dietary information collected in the Health Professionals Follow-up Study (n = 725 cases; n = 1,273 controls) and the Nurses' Health Study (n = 1,081 cases; n = 1,692 controls). We assessed whether genome-wide SNPs or iron metabolism SNPs interacted with dietary heme iron intake in relation to T2D, testing for associations in each cohort separately and then meta-analyzing to pool the results. Finally, we created 1,000 synthetic pathways matched to an iron metabolism pathway on number of genes, and number of SNPs in each gene. We compared the iron metabolic pathway SNPs with these synthetic SNP assemblies in their relation to T2D to assess if the pathway as a whole interacts with dietary heme iron intake.<h4>Results</h4>Using a genomic approach, we found no significant gene-environment interactions with dietary heme iron intake in relation to T2D at a Bonferroni corrected genome-wide significance level of 7.33 ×10(-) (8) (top SNP in pooled analysis: intergenic rs10980508; p = 1.03 × 10(-) (6)). Furthermore, no SNP in the iron metabolic pathway significantly interacted with dietary heme iron intake at a Bonferroni corrected significance level of 2.10 × 10(-) (4) (top SNP in pooled analysis: rs1805313; p = 1.14 × 10(-) (3)). Finally, neither the main genetic effects (pooled empirical p by SNP = 0.41), nor gene - dietary heme-iron interactions (pooled empirical p-value for the interactions = 0.72) were significant for the iron metabolic pathway as a whole.<h4>Conclusions</h4>We found no significant interactions between dietary heme iron intake and common SNPs in relation to T2D.

<h4>Purpose</h4>All-trans retinoic acid (RA)-incorporated nanoparticles were prepared using deoxycholic acid-conjugated dextran (DexDA). Anticancer activity of RA-incorporated DexDA nanoparticles were tested in vitro and in vivo.<h4>Methods</h4>RA-incorporated nanoparticles were prepared by dialysis. Antiproliferative and anti-invasive potential of RA-incorporated nanoparticles were studied using CT26 colorectal carcinoma cells.<h4>Results</h4>RA-incorporated nanoparticles have small particle sizes of around 70-300 nm and spherical shapes. The higher drug-feeding ratio and higher substitution degree of deoxycholic acid in the conjugates resulted in higher drug contents, lower loading efficiency, and larger particle size. RA release rate became slower at higher drug contents and higher substitution degree of deoxycholic acid in the DexDA conjugates. The antiproliferation activity, anti-invasive activity, and matrix metalloproteinase 2 expression of RA-incorporated nanoparticles against CT26 cells in vitro was similar to RA. However, RA-incorporated nanoparticles had superior antimetastatic activity in an animal pulmonary metastatic model of CT26 cells compared to RA itself.<h4>Conclusion</h4>RA-incorporated nanoparticles showed similar anticancer activity in vitro and superior antimetastatic activity in vivo in a pulmonary metastatic model of CT26 cells. We suggest that RA-incorporated nanoparticles are promising vehicles for efficient delivery of RA.

<h4>Unlabelled</h4>Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid.<h4>Conclusion</h4>Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host.

Histone deacetylase (HDAC) inhibitors have been demonstrated to be beneficial in animal models of neurodegenerative diseases. Such results were mainly associated with the epigenetic modulation caused by HDACs, especially those from class I, via chromatin deacetylation. However, other mechanisms may contribute to the neuroprotective effect of HDAC inhibitors, since each HDAC may present distinct specific functions within the neurodegenerative cascades. Such an example is HDAC6 for which the role in neurodegeneration has been partially elucidated so far. The strategy to be adopted in promising therapeutics targeting HDAC6 is still controversial. Specific inhibitors exert neuroprotection by increasing the acetylation levels of α-tubulin with subsequent improvement of the axonal transport, which is usually impaired in neurodegenerative disorders. On the other hand, an induction of HDAC6 would theoretically contribute to the degradation of protein aggregates which characterize various neurodegenerative disorders, including Alzheimer's, Parkinson's and Hutington's diseases. This review describes the specific role of HDAC6 compared to the other HDACs in the context of neurodegeneration, by collecting in silico, in vitro and in vivo results regarding the inhibition and/or knockdown of HDAC6 and other HDACs. Moreover, structure, function, subcellular localization, as well as the level of HDAC6 expression within brain regions are reviewed and compared to the other HDAC isoforms. In various neurodegenerative diseases, the mechanisms underlying HDAC6 interaction with other proteins seem to be a promising approach in understanding the modulation of HDAC6 activity.

<h4>Background</h4>Individual skeletal elements of the vertebrate limbs arise through a segmentation process introducing joints in specific locations. However, the molecular pathways controlling joint formation and subsequent joint maintenance are largely unknown. In this study, we focused on SOX11, and its contribution to the regulation of GDF5, a secreted signal necessary for proper joint formation and postnatal joint homeostasis.<h4>Results</h4>Sox11 is initially expressed broadly in the murine cartilage condensations at early stages of skeletal development, but its expression is specifically increased in the forming joint interzone as is forms. SOX11 overexpression can directly activate GDF5 expression both in vitro and in micromass cell cultures prepared from chick limb buds. Conserved SOX family binding sites are present in the 5' UTR region of the GDF5 gene and we show SOX11 can specifically bind to one of them. While misexpression of Sox11 in developing chick limbs through RCAS virus infection does not induce Gdf5 expression in ectopic locations, it does enhance its expression. To explore the roles of Sox11 in joint homeostasis, we analyzed adult knee joints in an osteoarthritis mouse model where the medial meniscus and the medial collateral ligament were removed. We also analyzed knee joints from human subjects who underwent total knee replacement surgery. We find that SOX11 is mainly expressed in the weight-bearing areas of knee joints, and its expression is decreased in degraded cartilage during progression of knee osteoarthritis in both mice and humans.<h4>Conclusions</h4>This work implicates SOX11 as a potential regulator of GDF5 expression in joint maintenance and suggests a possible role in the pathogenesis of osteoarthritis.

<h4>Unlabelled</h4>Antidepressants are associated with bone loss and fractures in older adults. We treated depressed older adults with an antidepressant and examined its effects on bone turnover by comparing blood samples before and after treatment. Bone resorption increased after antidepressant treatment, which may increase fracture risk.<h4>Introduction</h4>Antidepressants have been associated with increased bone loss and fractures in older adults in observational studies, but the mechanism is unclear. We examined the effects of a serotonin-norepinephrine reuptake inhibitor, venlafaxine, on biomarkers of bone turnover in a prospective treatment study of late-life depression.<h4>Methods</h4>Seventy-six individuals aged 60 years and older with current major depressive disorder received a 12-week course of venlafaxine XR 150-300 mg daily. We measured serum C-terminal cross-linking telopeptide of type I collagen (β-CTX) and N-terminal propeptide of type I procollagen (P1NP), measures of bone resorption and formation, respectively, before and after treatment. We then analyzed the change in β-CTX and P1NP within each participant. Venlafaxine levels were measured at the end of the study. We assessed depression severity at baseline and remission status after treatment.<h4>Results</h4>After 12 weeks of venlafaxine, β-CTX increased significantly, whereas P1NP did not significantly change. The increase in β-CTX was significant only in participants whose depression did not remit (increase by 10 % in non-remitters vs. 4 % in remitters). Change in β-CTX was not correlated with serum levels of venlafaxine or norvenlafaxine.<h4>Conclusion</h4>Our findings suggest that the primary effect of serotonergic antidepressants is to increase bone resorption. However, such an increase in bone resorption seemed to depend on whether or not participants' depression remitted. Our results are in agreement with prior observational studies reporting increased bone loss in older adults taking serotonergic antidepressants. These negative effects on bone homeostasis could potentially contribute to increased fracture risk in older adults.

It has long been known that there is a genetic component to obesity, and that characterizing this underlying factor would likely offer the possibility of better intervention in the future. Monogenic obesity has proved to be relatively straightforward, with a combination of linkage analysis and mouse models facilitating the identification of multiple genes. In contrast, genome-wide association studies have successfully revealed a variety of genetic loci associated with the more common form of obesity, allowing for very strong consensus on the underlying genetic architecture of the phenotype for the first time. Although a number of significant findings have been made, it appears that very little of the apparent heritability of body mass index has actually been explained to date. New approaches for data analyses and advances in technology will be required to uncover the elusive missing heritability, and to aid in the identification of the key causative genetic underpinnings of obesity.

<h4>Background</h4>The identification of signatures of natural selection has long been used as an approach to understanding the unique features of any given species. Genes within segmental duplications are overlooked in most studies of selection due to the limitations of draft nonhuman genome assemblies and to the methodological reliance on accurate gene trees, which are difficult to obtain for duplicated genes.<h4>Results</h4>In this work, we detected exons with an accumulation of high-quality nucleotide differences between the human assembly and shotgun sequencing reads from single human and macaque individuals. Comparing the observed rates of nucleotide differences between coding exons and their flanking intronic sequences with a likelihood-ratio test, we identified 74 exons with evidence for rapid coding sequence evolution during the evolution of humans and Old World monkeys. Fifty-five percent of rapidly evolving exons were either partially or totally duplicated, which is a significant enrichment of the 6% rate observed across all human coding exons.<h4>Conclusions</h4>Our results provide a more comprehensive view of the action of selection upon segmental duplications, which are the most complex regions of our genomes. In light of these findings, we suggest that segmental duplications could be subjected to rapid evolution more frequently than previously thought.

Mood disorders such as major depressive disorder and bipolar disorder--and their consequent effects on the individual and society--are among the most disabling and costly of all medical illnesses. Although a number of antidepressant treatments are available in clinical practice, many patients still undergo multiple and lengthy medication trials before experiencing relief of symptoms. Therefore a tremendous need exists to improve current treatment options and to facilitate more rapid, successful treatment in patients suffering from the deleterious neurobiological effects of ongoing depression. Toward that end, ongoing research is exploring the identification of biomarkers that might be involved in prevention, diagnosis, treatment response, severity, or prognosis of depression. Biomarkers evaluating treatment response will be the focus of this review, given the importance of providing relief to patients in a more expedient and systematic manner. A novel approach to developing such biomarkers of response would incorporate interventions with a rapid onset of action--such as sleep deprivation or intravenous drugs (e.g., ketamine or scopolamine). This alternative translational model for new treatments in psychiatry would facilitate shorter studies, improve feasibility, and increase higher compound throughput testing for these devastating disorders.

Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease.

Melioidosis (Burkholderia pseudomallei infection) is a common cause of community-acquired sepsis in Northeast Thailand and northern Australia. B. pseudomallei is a soil saprophyte endemic to Southeast Asia and northern Australia. The clinical presentation of melioidosis may mimic tuberculosis (both cause chronic suppurative lesions unresponsive to conventional antibiotics and both commonly affect the lungs). The two diseases have overlapping risk profiles (e.g., diabetes, corticosteroid use), and both B. pseudomallei and Mycobacterium tuberculosis are intracellular pathogens. There are however important differences: the majority of melioidosis cases are acute, not chronic, and present with severe sepsis and a mortality rate that approaches 50% despite appropriate antimicrobial therapy. By contrast, tuberculosis is characteristically a chronic illness with mortality <2% with appropriate antimicrobial chemotherapy. We examined the gene expression profiles of total peripheral leukocytes in two cohorts of patients, one with acute melioidosis (30 patients and 30 controls) and another with tuberculosis (20 patients and 24 controls). Interferon-mediated responses dominate the host response to both infections, and both type 1 and type 2 interferon responses are important. An 86-gene signature previously thought to be specific for tuberculosis is also found in melioidosis. We conclude that the host responses to melioidosis and to tuberculosis are similar: both are dominated by interferon-signalling pathways and this similarity means gene expression signatures from whole blood do not distinguish between these two diseases.

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

Arsenic trioxide loaded biocompatible PHB-PVA(1) nanoparticles (<100 nm in size) with folate functionalized surface were synthesized using poly-[(R)-3-hydroxybutyric acid] (PHB) produced by Bacillus firmus NII 0830. Folate functionalization was carried using dicyclohexyl carbodiimide (DCC) as a catalyst and 10-bromodecanol as a linker to conjugate glutamic acid terminal of folate with the hydroxylate groups present on the surface of PHBA-PVA(2) nanotrojans. The effect of fabrication parameters on shape, size distribution and PDI of the PHB nanoparticles were also investigated. It was observed that increase in sonication time and polyvinyl alcohol (PVA) concentration greatly reduced the size of nanoparticles. The drug release studies on arsenic trioxide incorporated PHB-PVA nanoparticles were conducted at physiological pH and temperature. FOL-PHBA-PVA(3) nanoparticles showed greater extent of cytotoxicity towards murine fibrosarcoma L929 cells than PHBA-PVA nanoparticles alone without conjugated folate, indicating the significance of folate as ligand for specific targeting of FR+ cancer cells.

Colorectal cancer is a leading cause of cancer mortality in both developed and developing countries. Transforming basic research results into clinical practice is one of the key tasks of translational research, which will greatly improve the diagnosis and treatments of colorectal cancer. In this paper, a translational research platform for colorectal cancer, named crcTRP, is introduced. crcTRP serves the colorectal cancer translational research by providing various types of biomedical information related with colorectal cancer to the community. The information, including clinical data, epidemiology data, individual omics data, and public omics data, was collected through a multisource biomedical information collection solution and then integrated in a clinic-omics database, which was constructed with EAV-ER model for flexibility and efficiency. A preliminary exploration of conducting translational research on crcTRP was implemented and worked out a set of clinic-genomic relations, linking clinical data with genomic data. These relations have also been applied to crcTRP to make it more conductive for cancer translational research.

Implants that simultaneously function as an osteoconductive matrix and as a device for local drug or growth factor delivery could provide an attractive system for bone regeneration. In our previous work, we prepared hollow hydroxyapatite (abbreviated HA) microspheres with a high surface area and mesoporous shell wall and studied the release of a model protein, bovine serum albumin (BSA), from the microspheres into phosphate-buffered saline (PBS). The present work is an extension of our previous work to study the release of BSA from similar HA microspheres into a biocompatible hydrogel, poly(ethylene glycol) (PEG). BSA-loaded HA microspheres were placed in a PEG solution which was rapidly gelled using ultraviolet radiation. The BSA release rate into the PEG hydrogel, measured using a spectrophotometric method, was slower than into PBS, and it was dependent on the initial BSA loading and on the microstructure of the microsphere shell wall. A total of 35-40% of the BSA initially loaded into the microspheres was released into PEG over ~14 days. The results indicate that these hollow HA microspheres have promising potential as an osteoconductive device for local drug or growth factor delivery in bone regeneration and in the treatment of bone diseases.

The association of tobacco smoke with decreased cell motility and wound healing is well documented; however, the cellular mechanisms and specific toxic tobacco constituents responsible for this effect are not well understood. Tobacco-specific N-nitrosamines (TSNAs) are among the most important classes of carcinogens found in tobacco products. The TSNA N'-nitrosonornicotine (NNN) is present at relatively high levels in tobacco and its smoke, as well as second- and third-hand smoke. To investigate the cellular pathways that are perturbed upon NNN exposure, we employed a quantitative proteomic approach, utilizing stable isotope labeling by amino acids in cell culture and mass spectrometry, to assess the NNN-induced alteration of protein expression in GM00637 human skin fibroblast cells. With this approach, we were able to quantify 2599 proteins, 191 of which displayed significantly changed expression following NNN exposure. One of the main findings from our proteomic analysis was the down-regulation of six different subunits of myosin, particularly nonmuscle myosin II heavy chain, isoforms A, B, and C. In addition, we found the altered expression of several extracellular matrix proteins and proteins involved in cellular adhesion. Together, our quantitative proteomic results suggested that NNN exposure may interfere with fibroblast motility. An in vitro scratch wound assay result supported that NNN exposure reduced the ability of dermal fibroblast to migrate into the scratched area. The results from the present study offer novel insights into the cellular mechanisms of NNN toxicity and identify NNN as a specific tobacco constituent that contributes to decreased fibroblast migration.

<h4>Purpose</h4>This phase II study evaluated bortezomib-based secondary induction and stem cell mobilization in 38 transplant-eligible patients with myeloma who had an incomplete and stalled response to, or had relapsed after, previous immunomodulatory drug-based induction.<h4>Experimental design</h4>Patients received up to six 21-day cycles of bortezomib plus dexamethasone, with added liposomal doxorubicin for patients not achieving partial response or better by cycle 2 or very good partial response or better (≥VGPR) by cycle 4 (DoVeD), followed by bortezomib, high-dose cyclophosphamide, and filgrastim mobilization. Gene expression/signaling pathway analyses were conducted in purified CD34+ cells after bortezomib-based mobilization and compared against patients who received only filgrastim ± cyclophosphamide. Plasma samples were similarly analyzed for quantification of associated protein markers.<h4>Results</h4>The response rate to DoVeD relative to the pre-DoVeD baseline was 61%, including 39% ≥ VGPR. Deeper responses were achieved in 10 of 27 patients who received bortezomib-based mobilization; postmobilization response rate was 96%, including 48% ≥ VGPR, relative to the pre-DoVeD baseline. Median CD34+ cell yield was 23.2 × 10(6) cells/kg (median of 1 apheresis session). After a median follow-up of 46.6 months, median progression-free survival was 47.1 months from DoVeD initiation; 5-year overall survival rate was 76.4%. Grade ≥ 3 adverse events included thrombocytopenia (13%), hand-foot syndrome (11%), peripheral neuropathy (8%), and neutropenia (5%). Bortezomib-based mobilization was associated with modulated expression of genes involved in stem cell migration.<h4>Conclusion</h4>Bortezomib-based secondary induction and mobilization could represent an alternative strategy for elimination of tumor burden in immunomodulatory drug-resistant patients that does not impact stem cell yield.

Hepatitis C virus (HCV) is the etiological agent accounting for chronic liver disease in approximately 2-3% of the population worldwide. HCV infection often leads to liver fibrosis and cirrhosis, various metabolic alterations including steatosis, insulin and interferon resistance or iron overload, and development of hepatocellular carcinoma or non-Hodgkin lymphoma. Multiple molecular mechanisms that trigger the emergence and development of each of these pathogenic processes have been identified so far. One of these involves marked induction of a reactive oxygen species (ROS) in infected cells leading to oxidative stress. To date, markers of oxidative stress were observed both in chronic hepatitis C patients and in various in vitro systems, including replicons or stable cell lines expressing viral proteins. The search for ROS sources in HCV-infected cells revealed several mechanisms of ROS production and thus a number of cellular proteins have become targets for future studies. Furthermore, during last several years it has been shown that HCV modifies antioxidant defense mechanisms. The aim of this review is to summarize the present state of art in the field and to try to predict directions for future studies.

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.

Autoreactive B cells are one of the key immune cells that have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). In addition to the production of harmful auto-antibodies (auto-Abs), B cells prime autoreactive T cells as antigen-presenting cells and secrete a wide range of pro-inflammatory cytokines that have both autocrine and paracrine effects. Agents that modulate B cells may therefore be of potential therapeutic value. Current strategies include targeting B-cell surface antigens, cytokines that promote B-cell growth and functions, and B- and T-cell interactions. In this article, we review the role of B cells in SLE in animal and human studies, and we examine previous reports that support B-cell modulation as a promising strategy for the treatment of this condition. In addition, we present an update on the clinical trials that have evaluated the therapeutic efficacy and safety of agents that antagonize CD20, CD22 and B-lymphocyte stimulator (BLyS) in human SLE. While the results of many of these studies remain inconclusive, belimumab, a human monoclonal antibody against BLyS, has shown promise and has recently been approved by the US Food and Drug Administration as an indicated therapy for patients with mild to moderate SLE. Undoubtedly, advances in B-cell immunology will continue to lead us to a better understanding of SLE pathogenesis and the development of novel specific therapies that target B cells.

Korean red ginseng (KRG) possesses neuroprotective activity. However, the potential neuroprotective value of KRG for the striatal toxicity is largely unknown. We investigated whether KRG extract (KRGE) could have a neuroprotective effect in a 3-nitropropionic acid- (3-NP) induced (i.p.) Huntington's disease (HD) model. KRGE (50, 100, and 250 mg/kg/day, p.o.) was administrated 10 days before 3-NP injection (pre-administration), from the same time with 3-NP injection (co-administration), or from the peak point of neurological impairment by 3-NP injection (post-administration). Pre-administration of KRGE produced the greatest neuroprotective effect in this model. Pre-administration of KRGE significantly decreased 3-NP-induced neurological impairment, lethality, lesion area, and neuronal loss in the 3-NP-injected striatum. KRGE attenuated microglial activation and phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signal pathway. KRGE also reduced the level of mRNA expression of tumor necrosis factor-alpha, interleukin- (IL-) 1β, IL-6, inducible nitric oxide synthase, and OX-42. Interestingly, the intrathecal administration of SB203580 (a p38 inhibitor) or PD98059 (an inhibitor of MAPK Kinase, MEK) increased the survival rate in the 3-NP-induced HD model. Pre-administration of KRGE may effectively inhibit 3-NP-induced striatal toxicity via the inhibition of the phosphorylation of MAPKs and NF-κB pathways, indicating its therapeutic potential for suppressing Huntington's-like symptoms.

Research on the molecular mechanisms involved in Huntington's disease, a monogenic disorder with a complex phenotype including motor, behaviour, and cognitive impairments, is advancing at a rapid path. Knowledge on several of the multimodal pathways has now lead to the establishment of rational strategies to prepare trials of several compounds in affected people. Furthermore, improved understanding of the phenotype and on ways of assessing it, as well as the process of developing biomarkers, allows setting the frame for such studies. In this brief review, the present status of some of these aspects is examined.

The Drosophila fat body is a liver- and adipose-like tissue that stores fat and serves as a detoxifying and immune responsive organ. We have previously shown that a high sugar diet leads to elevated hemolymph glucose and systemic insulin resistance in developing larvae and adults. Here, we used stable isotope tracer feeding to demonstrate that rearing larvae on high sugar diets impaired the synthesis of esterified fatty acids from dietary glucose. Fat body lipid profiling revealed changes in both carbon chain length and degree of unsaturation of fatty acid substituents, particularly in stored triglycerides. We tested the role of the fat body in larval tolerance of caloric excess. Our experiments demonstrated that lipogenesis was necessary for animals to tolerate high sugar feeding as tissue-specific loss of orthologs of carbohydrate response element-binding protein or stearoyl-CoA desaturase 1 resulted in lethality on high sugar diets. By contrast, increasing the fat content of the fat body by knockdown of king-tubby was associated with reduced hyperglycemia and improved growth and tolerance of high sugar diets. Our work supports a critical role for the fat body and the Drosophila carbohydrate response element-binding protein ortholog in metabolic homeostasis in Drosophila.

Protein misfolding and aggregation are the hallmark of a number of diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and the prion diseases. In all cases, a naturally-occurring protein misfolds and forms aggregates that are thought to disrupt cell function through a wide range of mechanisms that are yet to be fully unraveled. Fourier transform infrared (FTIR) spectroscopy is a technique that is sensitive to the secondary structure of proteins and has been widely used to investigate the process of misfolding and aggregate formation. This review focuses on how FTIR spectroscopy and spectroscopic microscopy are being used to evaluate the structural changes in disease-related proteins both in vitro and directly within cells and tissues. Finally, ongoing technological advances will be presented that are enabling time-resolved FTIR imaging of protein aggregation directly within living cells, which can provide insight into the structural intermediates, time scale, and mechanisms of cell toxicity associated with aggregate formation. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Genomic variation is a trend observed in various human diseases including cancer. Genetic studies have set out to understand how and why these variations result in cancer, why some populations are pre-disposed to the disease, and also how genetics affect drug responses. The melanoma incidence has been increasing at an alarming rate worldwide. The burden posed by melanoma has made it a necessity to understand the fundamental signaling pathways involved in this deadly disease. Signaling cascades such as mitogen-activated protein kinase and PI3K/AKT have been shown to be crucial in the regulation of processes that are commonly dysregulated during cancer development such as aberrant proliferation, loss of cell cycle control, impaired apoptosis, and altered drug metabolism. Understanding how these and other oncogenic pathways are regulated has been integral in our challenge to develop potent anti-melanoma drugs. With advances in technology and especially in next generation sequencing, we have been able to explore melanoma genomes and exomes leading to the identification of previously unknown genes with functions in melanomagenesis such as GRIN2A and PREX2. The therapeutic potential of these novel candidate genes is actively being pursued with some presenting as druggable targets while others serve as indicators of therapeutic responses. In addition, the analysis of the mutational signatures of melanoma tumors continues to cement the causative role of UV exposure in melanoma pathogenesis. It has become distinctly clear that melanomas from sun-exposed skin areas have distinct mutational signatures including C to T transitions indicative of UV-induced damage. It is thus necessary to continue spreading awareness on how to decrease the risk factors of developing the disease while at the same time working for a cure. Given the large amount of information gained from these sequencing studies, it is likely that in the future, treatment of melanoma will follow a highly personalized route that takes into account the differential mutational signatures of each individual's cancer.

G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β(2)-adrenergic receptor (β(2)AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β(2)AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β(2)AR pull-down, 242 proteins in the inverse agonist-activated β(2)AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β(2)AR-interacting proteins isolated was confirmed by Western blot; three known β(2)AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β(2)AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β(2)AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.

Cadherin cell adhesion molecules play crucial roles in vertebrate development including the development of the retina. Most studies have focused on examining functions of classic cadherins (e.g. N-cadherin) in retinal development. There is little information on the function of protocadherins in the development of the vertebrate visual system. We previously showed that protocadherin-17 mRNA was expressed in developing zebrafish retina during critical stages of the retinal development. To gain insight into protocadherin-17 function in the formation of the retina, we analyzed eye development and differentiation of retinal cells in zebrafish embryos injected with protocadherin-17 specific antisense morpholino oligonucleotides (MOs). Protocadherin-17 knockdown embryos (pcdh17 morphants) had significantly reduced eyes due mainly to decreased cell proliferation. Differentiation of several retinal cell types (e.g. retinal ganglion cells) was also disrupted in the pcdh17 morphants. Phenotypic rescue was achieved by injection of protocadherin-17 mRNA. Injection of a vivo-protocadherin-17 MO into one eye of embryonic zebrafish resulted in similar eye defects. Our results suggest that protocadherin-17 plays an important role in the normal formation of the zebrafish retina.

Changes in DNA methylation, whether hypo- or hypermethylation, have been shown to be associated with the progression of colorectal cancer. Methylation changes substantially in the progression from normal mucosa to adenoma and to carcinoma. This phenomenon has not been studied extensively and studies have been restricted to individual CpG islands, rather than taking a whole-genome approach. We aimed to study genome-wide methylation changes in colorectal cancer. We obtained 10 fresh-frozen normal tissue-cancer sample pairs, and five fresh-frozen adenoma samples. These were run on the lllumina HumanMethylation27 whole-genome methylation analysis system. Differential methylation between normal tissue, adenoma and carcinoma was analysed using Bayesian regression modelling, gene set enrichment analysis (GSEA) and hierarchical clustering (HC). The highest-rated individual gene for differential methylation in carcinomas versus normal tissue and adenomas versus normal tissue was GRASP (padjusted  = 1.59 × 10(-5) , BF = 12.62, padjusted  = 1.68 × 10(-6) , BF = 14.53). The highest-rated gene when comparing carcinomas versus adenomas was ATM (padjusted  = 2.0 × 10(-4) , BF = 10.17). Hierarchical clustering demonstrated poor clustering by the CIMP criteria for methylation. GSEA demonstrated methylation changes in the Netrin-DCC and SLIT-ROBO pathways. Widespread changes in DNA methylation are seen in the transition from adenoma to carcinoma. The finding that GRASP, which encodes the general receptor for phosphoinositide 1-associated scaffold protein, was differentially methylated in colorectal cancer is interesting. This may be a potential biomarker for colorectal cancer.

CREB3 proteins comprise a set of ER-localized bZip transcription factors defined by the presence of a transmembrane domain. They are regulated by inter-compartmental transport, Golgi cleavage and nuclear transport where they promote appropriate transcriptional responses. Although CREB3 proteins play key roles in differentiation, inflammation and metabolism, a general framework relating their defining features to these diverse activities is lacking. We identify unique features of CREB3 organization including the ATB domain, which we show it is essential for transcriptional activity. This domain is absent in all other human bZip factors, but conserved in Drosophila CREBA, which controls secretory pathway genes (SPGs). Furthermore, each of the five human CREB3 factors was capable of activating SPGs in Drosophila, dependent upon the ATB domain. Expression of the CREB3 protein, CREB-H, in 293 cells, upregulated genes involved in secretory capacity, extracellular matrix formation and lipid metabolism and increased secretion of specific cargos. In liver cells, which normally express CREB-H, the active form specifically induced secretion of apolipoproteins, including ApoA-IV, ApoAI, consistent with data implicating CREB-H in metabolic homeostasis. Based on these data and other recent studies, we propose a general role for the CREB3 family in regulating secretory capacity, with particular relevance to specialized cargos.

Cytochromes P450 (CYPs) are potential enzymes responsible for hydroxylation of many xenobiotics and endogenous chemicals in living organisms. It has been found that 4-monochlorobiphenyl (PCB3), mainly an airborne pollutant, can be metabolized to hydroxylated transformation products (OH-PCB3s) in whole poplars. However, the enzymes involved in the hydroxylation of PCB3 in whole poplars have not been identified. Therefore, two CYP suicide inhibitors, 1-aminobenzotriazole (ABT) and 17-octadecynoic acid (ODYA), were selected to probe the hydroxylation reaction of PCB3 in whole poplars in this work. Poplars (Populus deltoides × nigra, DN34) were exposed to PCB3 with or without inhibitor for 11 days. Results showed both ABT and ODYA can decrease the concentrations and yields of five OH-PCB3s in different poplar parts via the inhibition of CYPs. Furthermore, both ABT and ODYA demonstrated a dose-dependent relationship to the formation of OH-PCB3s in whole poplars. The higher the inhibitor concentrations, the lower the total yields of OH-PCB3s. For ABT spiked-additions, the total mass yield of five OH-PCB3s was inhibited by a factor of 1.6 times at an ABT concentration of 2.5 mg L(-1), 4.0 times at 12.5 mg L(-1), and 7.0 times at 25 mg L(-1). For the inhibitor ODYA, the total mass of five OH-PCB3s was reduced by 2.1 times compared to the control at an ODYA concentration of 2.5 mg L(-1). All results pointed to the conclusion that CYP enzymes were the agents which metabolized PCB3 to OH-PCB3s in whole poplars because suicide CYP inhibitors ABT and ODYA both led to sharp decreases of OH-PCB3s formation in whole poplars. A dose-response curve for each of the suicide inhibitors was developed.

Multiple genetic loci have been associated with body mass index (BMI) and obesity. The aim of this study was to investigate the effects of established adult BMI and childhood obesity loci in a Greek adolescent cohort. For this purpose, 34 variants were selected for investigation in 707 (55.9% females) adolescents of Greek origin aged 13.42 ± 0.88 years. Cumulative effects of variants were assessed by calculating a genetic risk score (GRS-34) for each subject. Variants at the FTO, TMEM18, FAIM2, RBJ, ZNF608 and QPCTL loci yielded nominal evidence for association with BMI and/or overweight risk (p < 0.05). Variants at TFAP2B and NEGR1 loci showed nominal association (p < 0.05) with BMI and/or overweight risk in males and females respectively. Even though we did not detect any genome-wide significant associations, 27 out of 34 variants yielded directionally consistent effects with those reported by large-scale meta-analyses (binomial sign p = 0.0008). The GRS-34 was associated with both BMI (beta = 0.17 kg/m(2) /allele; p < 0.001) and overweight risk (OR = 1.09/allele; 95% CI: 1.04-1.16; p = 0.001). In conclusion, we replicate associations of established BMI and childhood obesity variants in a Greek adolescent cohort and confirm directionally consistent effects for most of them.

<h4>Background</h4>In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established.<h4>Results</h4>Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks.<h4>Conclusion</h4>mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway.

Cyclodepsipeptide natural products often display intriguing biological activities that along with their complex molecular scaffolds, makes them interesting targets for chemical synthesis. Although cyclodepsipeptides feature highly diverse chemical structures, their synthesis is often associated with similar synthetic challenges such as the establishment of a suitable macrocyclization methodology. This review therefore compiles case studies of synthetic approaches to different bioactive cyclodepsipeptide natural products, thereby illustrating obstacles of cyclodepsipeptide synthesis as well as their overcomings.

The liver, pancreas, and heart are particularly susceptible to iron-related disorders. These tissues take up plasma iron from transferrin or non-transferrin-bound iron, which appears during iron overload. Here, we assessed the effect of iron status on the levels of the transmembrane transporters, ZRT/IRT-like protein 14 and divalent metal-ion transporter-1, which have both been implicated in transferrin- and non-transferrin-bound iron uptake. Weanling male rats (n=6/group) were fed an iron-deficient, iron-adequate, or iron-overloaded diet for 3 weeks. ZRT/IRT-like protein 14, divalent metal-ion transporter-1 protein and mRNA levels in liver, pancreas, and heart were determined by using immunoblotting and quantitative reverse transcriptase polymerase chain reaction analysis. Confocal immunofluorescence microscopy was used to localize ZRT/IRT-like protein 14 in the liver and pancreas. ZRT/IRT-like protein 14 and divalent metal-ion transporter-1 protein levels were also determined in hypotransferrinemic mice with genetic iron overload. Hepatic ZRT/IRT-like protein 14 levels were found to be 100% higher in iron-loaded rats than in iron-adequate controls. By contrast, hepatic divalent metal-ion transporter-1 protein levels were 70% lower in iron-overloaded animals and nearly 3-fold higher in iron-deficient ones. In the pancreas, ZRT/IRT-like protein 14 levels were 50% higher in iron-overloaded rats, and in the heart, divalent metal-ion transporter-1 protein levels were 4-fold higher in iron-deficient animals. At the mRNA level, ZRT/IRT-like protein 14 expression did not vary with iron status, whereas divalent metal-ion transporter-1 expression was found to be elevated in iron-deficient livers. Immunofluorescence staining localized ZRT/IRT-like protein 14 to the basolateral membrane of hepatocytes and to acinar cells of the pancreas. Hepatic ZRT/IRT-like protein 14, but not divalent metal-ion transporter-1, protein levels were elevated in iron-loaded hypotransferrinemic mice. In conclusion, ZRT/IRT-like protein 14 protein levels are up-regulated in iron-loaded rat liver and pancreas and in hypotransferrinemic mouse liver. Divalent metal-ion transporter-1 protein levels are down-regulated in iron-loaded rat liver, and up-regulated in iron-deficient liver and heart. Our results provide insight into the potential contributions of these transporters to tissue iron uptake during iron deficiency and overload.

Huntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA)(Gln-CUG) that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNA(Gln-CUG). The concentration of tRNA(Gln-CUG) also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels.

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (Nrg1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan low density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.

Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/-) mouse embryonic fibroblasts (MEFs). DHCR24(-/-) MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/-) MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/-) MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(V)β(3) integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/-) MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

<h4>Background</h4>Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease.<h4>Methodology/principal findings</h4>To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+) T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++) algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain.<h4>Conclusions/significance</h4>This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.

Typhoid fever is a major cause of death worldwide with a major part of the disease burden in developing regions such as the Indian sub-continent. Bangladesh is part of this highly endemic region, yet little is known about the spatial and temporal distribution of the disease at a regional scale. This research used a Geographic Information System to explore, spatially and temporally, the prevalence of typhoid in Dhaka Metropolitan Area (DMA) of Bangladesh over the period 2005-9. This paper provides the first study of the spatio-temporal epidemiology of typhoid for this region. The aims of the study were: (i) to analyse the epidemiology of cases from 2005 to 2009; (ii) to identify spatial patterns of infection based on two spatial hypotheses; and (iii) to determine the hydro-climatological factors associated with typhoid prevalence. Case occurrences data were collected from 11 major hospitals in DMA, geocoded to census tract level, and used in a spatio-temporal analysis with a range of demographic, environmental and meteorological variables. Analyses revealed distinct seasonality as well as age and gender differences, with males and very young children being disproportionately infected. The male-female ratio of typhoid cases was found to be 1.36, and the median age of the cases was 14 years. Typhoid incidence was higher in male population than female (χ(2) = 5.88, p<0.05). The age-specific incidence rate was highest for the 0-4 years age group (277 cases), followed by the 60+ years age group (51 cases), then there were 45 cases for 15-17 years, 37 cases for 18-34 years, 34 cases for 35-39 years and 11 cases for 10-14 years per 100,000 people. Monsoon months had the highest disease occurrences (44.62%) followed by the pre-monsoon (30.54%) and post-monsoon (24.85%) season. The Student's t test revealed that there is no significant difference on the occurrence of typhoid between urban and rural environments (p>0.05). A statistically significant inverse association was found between typhoid incidence and distance to major waterbodies. Spatial pattern analysis showed that there was a significant clustering of typhoid distribution in the study area. Moran's I was highest (0.879; p<0.01) in 2008 and lowest (0.075; p<0.05) in 2009. Incidence rates were found to form three large, multi-centred, spatial clusters with no significant difference between urban and rural rates. Temporally, typhoid incidence was seen to increase with temperature, rainfall and river level at time lags ranging from three to five weeks. For example, for a 0.1 metre rise in river levels, the number of typhoid cases increased by 4.6% (95% CI: 2.4-2.8) above the threshold of 4.0 metres (95% CI: 2.4-4.3). On the other hand, with a 1 °C rise in temperature, the number of typhoid cases could increase by 14.2% (95% CI: 4.4-25.0).

<h4>Background</h4>Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD).<h4>Methodology/principal findings</h4>Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.<h4>Conclusion/significance</h4>Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

Microsatellite stable (MSS), CpG island methylator phenotype (CIMP)-negative colorectal tumors, the most prevalent molecular subtype of colorectal cancer, are associated with extensive copy number alteration (CNA) events and aneuploidy. We report on the identification of characteristic recurrent CNA (with frequency >25%) events and associated gene expression profiles for a total of 40 paired tumor and adjacent normal colon tissues using genome-wide microarrays. We observed recurrent CNAs, namely gains at 1q, 7p, 7q, 8p12-11, 8q, 12p13, 13q, 20p, 20q, Xp, and Xq and losses at 1p36, 1p31, 1p21, 4p15-12, 4q12-35, 5q21-22, 6q26, 8p, 14q, 15q11-12, 17p, 18p, 18q, 21q21-22, and 22q. Within these genomic regions we identified 356 genes with significant differential expression (P < 0.0001 and ±1.5-fold change) in the tumor compared to adjacent normal tissue. Gene ontology and pathway analyses indicated that many of these genes were involved in functional mechanisms that regulate cell cycle, cell death, and metabolism. An amplicon present in >70% of the tumor samples at 20q11-20q13 contained several cancer-related genes (AHCY, POFUT1, RPN2, TH1L, and PRPF6) that were upregulated and demonstrated a significant linear correlation (P < 0.05) for gene dosage and gene expression. Copy number loss at 8p, a CNA associated with adenocarcinoma and poor prognosis, was observed in >50% of the tumor samples and demonstrated a significant linear correlation for gene dosage and gene expression for two potential tumor suppressor genes, MTUS1 (8p22) and PPP2CB (8p12). The results from our integration analysis illustrate the complex relationship between genomic alterations and gene expression in colon cancer.

<h4>Background</h4>Inherited Copy Number Variants (CNVs) can modulate the expression levels of individual genes. However, little is known about how CNVs alter biological pathways and how this varies across different populations. To trace potential evolutionary changes of well-described biological pathways, we jointly queried the genomes and the transcriptomes of a collection of individuals with Caucasian, Asian or Yoruban descent combining high-resolution array and sequencing data.<h4>Results</h4>We implemented an enrichment analysis of pathways accounting for CNVs and genes sizes and detected significant enrichment not only in signal transduction and extracellular biological processes, but also in metabolism pathways. Upon the estimation of CNV population differentiation (CNVs with different polymorphism frequencies across populations), we evaluated that 22% of the pathways contain at least one gene that is proximal to a CNV (CNV-gene pair) that shows significant population differentiation. The majority of these CNV-gene pairs belong to signal transduction pathways and 6% of the CNV-gene pairs show statistical association between the copy number states and the transcript levels.<h4>Conclusions</h4>The analysis suggested possible examples of positive selection within individual populations including NF-kB, MAPK signaling pathways, and Alu/L1 retrotransposition factors. Altogether, our results suggest that constitutional CNVs may modulate subtle pathway changes through specific pathway enzymes, which may become fixed in some populations.

<h4>Background</h4>Select retrotransposons in the long terminal repeat (LTR) class exhibit interindividual variation in DNA methylation that is altered by developmental environmental exposures. Yet, neither the full extent of variability at these "metastable epialleles," nor the phylogenetic relationship underlying variable elements is well understood. The murine metastable epialleles, Avy and CabpIAP, result from independent insertions of an intracisternal A particle (IAP) mobile element, and exhibit remarkably similar sequence identity (98.5%).<h4>Results</h4>Utilizing the C57BL/6 genome we identified 10802 IAP LTRs overall and a subset of 1388 in a family that includes Avy and CabpIAP. Phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. To characterize interindividual variation across clades, liver DNA from 17 isogenic mice was subjected to combined bisulfite and restriction analysis (CoBRA) for 21 separate LTR transposons (7 per clade). The lowest and highest mean methylation values were 59% and 88% respectively, while methylation levels at individual LTRs varied widely, ranging from 9% to 34%. The clade with the most conserved elements had significantly higher mean methylation across LTRs than either of the two diverged clades (p = 0.040 and p = 0.017). Within each mouse, average methylation across all LTRs was not significantly different (71%-74%, p > 0.99).<h4>Conclusions</h4>Combined phylogenetic and DNA methylation analysis allows for the identification of novel regions of variable methylation. This approach increases the number of known metastable epialleles in the mouse, which can serve as biomarkers for environmental modifications to the epigenome.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease involving progressive motor, cognitive and behavioural decline, leading to death approximately 20 years after motor onset. The disease is characterised pathologically by an early and progressive striatal neuronal cell loss and atrophy, which has provided the rationale for first clinical trials of neural repair using fetal striatal cell transplantation. Between 2000 and 2003, the 'NEST-UK' consortium carried out bilateral striatal transplants of human fetal striatal tissue in five HD patients. This paper describes the long-term follow up over a 3-10-year postoperative period of the patients, grafted and non-grafted, recruited to this cohort using the 'Core assessment program for intracerebral transplantations-HD' assessment protocol. No significant differences were found over time between the patients, grafted and non-grafted, on any subscore of the Unified Huntington's Disease Rating Scale, nor on the Mini Mental State Examination. There was a trend towards a slowing of progression on some timed motor tasks in four of the five patients with transplants, but overall, the trial showed no significant benefit of striatal allografts in comparison with a reference cohort of patients without grafts. Importantly, no significant adverse or placebo effects were seen. Notably, the raclopride positron emission tomography (PET) signal in individuals with transplants, indicated that there was no obvious surviving striatal graft tissue. This study concludes that fetal striatal allografting in HD is safe. While no sustained functional benefit was seen, we conclude that this may relate to the small amount of tissue that was grafted in this safety study compared with other reports of more successful transplants in patients with HD.

A sizable fraction of patients experiences adverse drug events or lack of drug efficacy. A part of this variability in drug response can be explained by genetic differences between patients. However, pharmacogenomic data as well as computational clinical decision support systems for interpreting such data are still unavailable in most healthcare settings. We address this problem by introducing the medicine safety code (MSC), which captures compressed pharmacogenomic data in a two-dimensional barcode that can be carried in a patient's wallet. We successfully encoded data about 385 genetic polymorphisms in MSC and were able to decode and interpret MSC quickly with common mobile devices. The MSC could make individual pharmacogenomic data and decision support available in a wide variety of healthcare settings without the set up of large-scale infrastructures or centralized databases.

Previous proteomics studies have partially unraveled the complexity of endothelial protein secretion but have not investigated glycosylation, a key modification of secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol-12-myristate-13 acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. In addition to 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by zwitterionic hydrophilic interaction liquid chromatography resins and were either treated with PNGase F and H2(18)O or directly analyzed using a recently developed workflow combining higher-energy C-trap dissociation (HCD) with electron-transfer dissociation (ETD) for a hybrid linear ion trap-orbitrap mass spectrometer. After deglycosylation with PNGase F in the presence of H2(18)O, 123 unique peptides displayed (18)O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptide analysis via HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted, and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD. The former detected predominantly high-intensity, multiply charged glycopeptides, whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction. This study represents the most comprehensive characterization of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status of complex biological samples.

<h4>Background and aim</h4>GBC is a lethal and multifaceted disease. Deleted in colorectal carcinoma (DCC) is a well known tumor suppressor gene. Recently a small genome-wide association study (GWAS) identified DCC to be significantly associated with gallbladder cancer (GBC) susceptibility in a Japanese population sample. However, the study sample size was small and lacked independent replication. Therefore, the present study was carried out to replicate the association of two GWAS identified DCC SNPs (A>Grs4078288, C>Trs7504990) and two other SNPs (C>Grs2229080 and A>Grs714) previously associated with various cancers.<h4>Methodology</h4>The study was accomplished in 406 GBC cases and 260 healthy control samples from North India. Genotyping was carried out by PCR-RFLP and Taqman genotyping assays. Statistical analysis was performed by using SPSS ver16 and functional prediction of these variants was carried out using Bioinformatics tools (FAST-SNP, F-SNP).<h4>Result</h4>We did not observe association with GWAS-identified SNPs of DCC but other SNPs showed significant association. In addition, a DCC haplotype Grs2229080-Ars4078288-Crs7504990-Ars714 conferred high risk of GBC in India. The haplotype associated risk was independent of gallstone, sex or tobacco usages which are well-known modifiers of GBC risk. Further analysis suggested DCC A>Grs714 as a major risk conferring SNP in the Indian population.<h4>Conclusion</h4>This study re-affirms the role of plausible tumor suppressor DCC variants, in gallbladder carcinogenesis and the risk haplotype may be explored as a useful marker for GBC susceptibility.

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington's disease. Here, we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (tryptophan zipper, disulfide, d-Pro-Gly, Coulombic attraction, l-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n=4 for a simple, unbroken Q23 sequence to approximate unitary n values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of solid-state NMR (13)C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn values remain quite low (~10(-)(10)) and there is no evidence for significant enhancement of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.

<h4>Background</h4>Understanding how genes are expressed specifically in particular tissues is a fundamental question in developmental biology. Many tissue-specific genes are involved in the pathogenesis of complex human diseases. However, experimental identification of tissue-specific genes is time consuming and difficult. The accurate predictions of tissue-specific gene targets could provide useful information for biomarker development and drug target identification.<h4>Results</h4>In this study, we have developed a machine learning approach for predicting the human tissue-specific genes using microarray expression data. The lists of known tissue-specific genes for different tissues were collected from UniProt database, and the expression data retrieved from the previously compiled dataset according to the lists were used for input vector encoding. Random Forests (RFs) and Support Vector Machines (SVMs) were used to construct accurate classifiers. The RF classifiers were found to outperform SVM models for tissue-specific gene prediction. The results suggest that the candidate genes for brain or liver specific expression can provide valuable information for further experimental studies. Our approach was also applied for identifying tissue-selective gene targets for different types of tissues.<h4>Conclusions</h4>A machine learning approach has been developed for accurately identifying the candidate genes for tissue specific/selective expression. The approach provides an efficient way to select some interesting genes for developing new biomedical markers and improve our knowledge of tissue-specific expression.

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.

<h4>Unlabelled</h4>Enterovirus 71 (EV71), a major causative agent of hand-foot-and-mouth disease (HFMD), causes outbreaks among children in the Asia-Pacific region. A vaccine is urgently needed. Based on successful pre-clinical work, phase I and II clinical trials of an inactivated EV71 vaccine, which included the participants of 288 and 660 respectively, have been conducted. In the present study, the immune response and the correlated modulation of gene expression in the peripheral blood mononuclear cells (PBMCs) of 30 infants (6 to 11 months) immunized with this vaccine or placebo and consented to join this study in the phase II clinical trial were analyzed. The results showed significantly greater neutralizing antibody and specific T cell responses in vaccine group after two inoculations on days 0 and 28. Additionally, more than 600 functional genes that were up- or down-regulated in PBMCs were identified by the microarray assay, and these genes included 68 genes associated with the immune response in vaccine group. These results emphasize the gene expression profile of the immune system in response to an inactivated EV71 vaccine in humans and confirmed that such an immune response was generated as the result of the positive mobilization of the immune system. Furthermore, the immune response was not accompanied by the development of a remarkable inflammatory response.<h4>Clinical trial registration</h4>NCT01391494 and NCT01512706.

<h4>Background</h4>With less than a 5% survival rate pancreatic adenocarcinoma (PDAC) is almost uniformly lethal. In order to make a significant impact on survival of patients with this malignancy, it is necessary to diagnose the disease early, when curative surgery is still possible. Detailed knowledge of the natural history of the disease and molecular events leading to its progression is therefore critical.<h4>Methods and findings</h4>We have analysed the precursor lesions, PanINs, from prophylactic pancreatectomy specimens of patients from four different kindreds with high risk of familial pancreatic cancer who were treated for histologically proven PanIN-2/3. Thus, the material was procured before pancreatic cancer has developed, rather than from PanINs in a tissue field that already contains cancer. Genome-wide transcriptional profiling using such unique specimens was performed. Bulk frozen sections displaying the most extensive but not microdissected PanIN-2/3 lesions were used in order to obtain the holistic view of both the precursor lesions and their microenvironment. A panel of 76 commonly dysregulated genes that underlie neoplastic progression from normal pancreas to PanINs and PDAC were identified. In addition to shared genes some differences between the PanINs of individual families as well as between the PanINs and PDACs were also seen. This was particularly pronounced in the stromal and immune responses.<h4>Conclusions</h4>Our comprehensive analysis of precursor lesions without the invasive component provides the definitive molecular proof that PanIN lesions beget cancer from a molecular standpoint. We demonstrate the need for accumulation of transcriptomic changes during the progression of PanIN to PDAC, both in the epithelium and in the surrounding stroma. An identified 76-gene signature of PDAC progression presents a rich candidate pool for the development of early diagnostic and/or surveillance markers as well as potential novel preventive/therapeutic targets for both familial and sporadic pancreatic adenocarcinoma.

Regulation of key proteins by microRNAs (miRNAs) is an emergent field in biomedicine. Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is a relevant molecule for cardiovascular diseases, since it is the target of oral anticoagulant drugs and plays a role in soft tissue calcification. The objective of this study was to determine the influence of miRNAs on the expression of VKORC1. Potential miRNAs targeting VKORC1 mRNA were searched by using online algorithms. Validation studies were carried out in HepG2 cells by using miRNA precursors; direct miRNA interaction was investigated with reporter assays. In silico studies identified two putative conserved binding sites for miR-133a and miR-137 on VKORC1 mRNA. Ex vivo studies showed that only miR-133a was expressed in liver; transfection of miRNA precursors of miR-133a in HepG2 cells reduced VKORC1 mRNA expression in a dose-dependent manner, as assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as well as protein expression. Reporter assays in HEK293T cells showed that miR-133a interacts with the 3'UTR of VKORC1. Additionally, miR-133a levels correlated inversely with VKORC1 mRNA levels in 23 liver samples from healthy subjects. In conclusion, miR-133a appears to have a direct regulatory effect on expression of VKORC1 in humans; this regulation may have potential importance for anticoagulant therapy or aortic calcification.

The proton chemical shift (CS) tensor is a sensitive probe of structure and hydrogen bonding. Highly accurate quantum-chemical protocols exist for computation of (1)H magnetic shieldings in the various contexts, making proton chemical shifts potentially a powerful predictor of structural and electronic properties. However, (1)H CS tensors are not yet widely used in protein structure calculation due to scarcity of experimental data. While isotropic proton shifts can be readily measured in proteins even in the solid state, determination of the (1)H chemical shift anisotropy (CSA) tensors remains challenging, particularly in molecules containing multiple proton sites. We present a method for site-resolved measurement of amide proton CSAs in fully protonated solids under magic angle spinning. The approach consists of three concomitant 3D experiments yielding spectra determined by either mainly (1)H CSA, mainly (1)H–(15)N dipolar, or combined (1)H CSA and (1)H–(15)N dipolar interactions. The anisotropic interactions are recoupled using RN-sequences of appropriate symmetry, such as R12(1)(4), and (15)N/(13)C isotropic CS dimensions are introduced via a short selective (1)H–(15)N cross-polarization step. Accurate (1)H chemical shift tensor parameters are extracted by simultaneous fit of the lineshapes recorded in the three spectra. An application of this method is presented for an 89-residue protein, U-(13)C,(15)N-CAP-Gly domain of dynactin. The CSA parameters determined from the triple fits correlate with the hydrogen-bonding distances, and the trends are in excellent agreement with the prior solution NMR results. This approach is generally suited for recording proton CSA parameters in various biological and organic systems, including protein assemblies and nucleic acids.

<h4>Introduction</h4>RhoB has been reported to exert positive and negative effects on cancer pathophysiology but an understanding of its role in breast cancer remains incomplete. Analysis of data from the Oncomine database showed a positive correlation between RhoB expression and positivity for both estrogen receptor alpha (ERα) and progesterone receptor (PR).<h4>Methods</h4>This finding was validated by our analysis of a tissue microarray constructed from a cohort of 113 patients and then investigated in human cell models.<h4>Results</h4>We found that RhoB expression in tissue was strongly correlated with ERα and PR expression and inversely correlated with tumor grade, tumor size and count of mitosis. In human breast cancer cell lines, RhoB attenuation was associated with reduced expression of both ERα and PR, whereas elevation of RhoB was found to be associated with ERα overexpression. Mechanistic investigations suggested that RhoB modulates ERα expression, controlling both its protein and mRNA levels, and that RhoB modulates PR expression by accentuating the recruitment of ERα and other major co-regulators to the promoter of PR gene. A major consequence of RhoB modulation was that RhoB differentially regulated the proliferation of breast cancer cell lines. Interestingly, we documented crosstalk between RhoB and ERα, with estrogen treatment leading to RhoB activation.<h4>Conclusion</h4>Taken together, our findings offer evidence that in human breast cancer RhoB acts as a positive function to promote expression of ERα and PR in a manner correlated with cell proliferation.

<h4>Background</h4>Children born small-for-gestational-age (SGA) are at increased risk of developing obesity and metabolic diseases later in life, a risk which is magnified if followed by accelerated postnatal growth. We investigated whether common gene variants associated with adult obesity were associated with increased postnatal growth, as measured by BMI z-score, in children born SGA and appropriate for gestational age (AGA) in the Auckland Birthweight Collaborative.<h4>Methods</h4>A total of 37 candidate SNPs were genotyped on 547 European children (228 SGA and 319 AGA). Repeated measures of BMI (z-score) were used for assessing obesity status, and results were corrected for multiple testing using the false discovery rate.<h4>Results</h4>SGA children had a lower BMI z-score than non-SGA children at assessment age 3.5, 7 and 11 years. We confirmed 27 variants within 14 obesity risk genes to be individually associated with increasing early childhood BMI, predominantly in those born AGA.<h4>Conclusions</h4>Genetic risk variants are less important in influencing early childhood BMI in those born SGA than in those born AGA, suggesting that non-genetic or environmental factors may be more important in influencing childhood BMI in those born SGA.

<h4>Background</h4>Airway inflammation is commonly assessed by sputum induction followed by a differential cell count (DCC) using light microscopy. This method is prone to intercounter variability and poor reproducibility. We aimed to develop a more objective method using flow cytometry (FCM).<h4>Methods</h4>Fifty-six sputum inductions were conducted in 41 adults (23 asthmatics). Sputum was processed, a cytospin prepared for DCC, and the remainder immunolabeled for FCM using CD45, CD14, and CD16-specific antibodies to distinguish major leukocyte populations. Aliquots of 15 samples were frozen at -80°C to assess the effects of cryostorage. DCC and FCM were compared, and viability of individual cell populations was determined by FCM.<h4>Results</h4>FCM and DCC, and fresh and frozen samples, were significantly correlated, R = 0.54-0.87; all P < 0.0001, and R = 0.57 to 1; P < 0.005, respectively. There was a significant neutrophil loss after cryostorage (from median 30.5-17.4% of total leukocytes; P < 0.0001). Cell viability was higher for lymphocytes compared to granulocytes or macrophages (P < 0.001). With the exception of the expected higher levels of eosinophils (P < 0.005), no significant difference in cell differentials or viability was observed between asthmatics and nonasthmatics using either DCC or FCM.<h4>Conclusions</h4>FCM is a suitable means of assessing leukocyte populations in induced sputum. Sample storage at -80°C prior to FCM is feasible, but may be detrimental to neutrophils, although good correlations were still observed between fresh and frozen samples. Large differences in viability were found between individual cell populations suggesting that viability dye use may be necessary.

The TrkC neurotrophin receptor belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is a classic tyrosine kinase receptor and therefore generally considered to be a proto-oncogene. We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Finally, we provide evidence that a mutation of TrkC detected in a sporadic cancer is a loss-of-proapoptotic function mutation. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor.

Huntington disease (HD) is a devastating, late-onset, inherited neurodegenerative disorder that manifests with personality changes, movement disorders, and cognitive decline. It is caused by a CAG repeat expansion in exon 1 of the HTT gene that translates to a polyglutamine tract in the huntingtin protein (HTT). The formation of HTT fragments has been implicated as an essential step in the molecular pathogenesis of HD and several proteases that cleave HTT have been identified. However, the importance of smaller N-terminal fragments has been highlighted by their presence in HD postmortem brains and by the fact that nuclear inclusions are only detected by antibodies to the N terminus of HTT. Despite an intense research effort, the precise length of these fragments and the mechanism by which they are generated remains unknown. Here we show that CAG repeat length-dependent aberrant splicing of exon 1 HTT results in a short polyadenylated mRNA that is translated into an exon 1 HTT protein. Given that mutant exon 1 HTT proteins have consistently been shown to be highly pathogenic in HD mouse models, the aberrant splicing of HTT mRNA provides a mechanistic basis for the molecular pathogenesis of HD. RNA-targeted therapeutic strategies designed to lower the levels of HTT are under development. Many of these approaches would not prevent the production of exon 1 HTT and should be reviewed in light of our findings.

The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by central nervous system (CNS) demyelination and axonal degeneration. Although the cause of MS is still unknown, it is widely accepted that novel drug targets need to focus on both decreasing inflammation and promoting CNS repair. In MS and experimental autoimmune encephalomyelitis, non-coding small microRNAs (miRNAs) are dysregulated in the immune system and CNS. Since individual miRNAs are able to down-regulate multiple targeted mRNA transcripts, even minor changes in miRNA expression may lead to significant alterations in gene expression. Herein, we review miRNA signatures reported in CNS tissue and immune cells of MS patients and consider how altered miRNA expression may influence MS pathology.

Pancreatogenic diabetes (PD) is a potentially fatal disease that occurs secondary to pancreatic disorders. The current anti-diabetic therapy for PD is fraught with adverse effects that can increase morbidity. Here we investigated the efficacy of novel peptide nanomedicine: pancreatic polypeptide (PP) in sterically stabilized micelles (SSM) for management of PD. PP exhibits significant anti-diabetic efficacy but its short plasma half-life curtails its therapeutic application. To prolong and improve activity of PP in vivo, we evaluated the delivery of PP in SSM. PP-SSM administered to rats with PD, significantly improved glucose tolerance, insulin sensitivity and hepatic glycogen content compared to peptide in buffer. The studies established the importance of micellar nanocarriers in protecting enzyme-labile peptides in vivo and delivering them to target site, thereby enhancing their therapeutic efficacy. In summary, this study demonstrated that PP-SSM is a promising novel anti-diabetic nanomedicine and therefore should be further developed for management of PD.<h4>From the clinical editor</h4>Pancreatic peptide was earlier demonstrated to address pancreatogenic diabetes, but its short half-life represented major difficulties in further development for therapeutic use. PP-SSM (pancreatic polypeptide in sterically stabilized micelles) is a promising novel anti-diabetic nanomedicine that enables prolonged half-life and increased bioactivity of PP, as shown in this novel study, paving the way toward clinical studies in the near future.

Meningiomas are central nervous system tumors that originate from the meningeal coverings of the brain and spinal cord. Most meningiomas are pathologically benign or atypical, but 3-5% display malignant features. Despite previous studies on benign and atypical meningiomas, the key molecular pathways involved in malignant transformation remain to be determined, as does the extent of epigenetic alteration in malignant meningiomas. In this study, we explored the landscape of DNA methylation in ten benign, five atypical and four malignant meningiomas. Compared to the benign tumors, the atypical and malignant meningiomas demonstrate increased global DNA hypomethylation. Clustering analysis readily separates malignant from atypical and benign tumors, implicating that DNA methylation patterns may serve as diagnostic biomarkers for malignancy. Genes with hypermethylated CpG islands in malignant meningiomas (such as HOXA6 and HOXA9) tend to coincide with the binding sites of polycomb repressive complexes (PRC) in early developmental stages. Most genes with hypermethylated CpG islands at promoters are suppressed in malignant and benign meningiomas, suggesting the switching of gene silencing machinery from PRC binding to DNA methylation in malignant meningiomas. One exception is the MAL2 gene that is highly expressed in benign group and silenced in malignant group, representing de novo gene silencing induced by DNA methylation. In summary, our results suggest that malignant meningiomas have distinct DNA methylation patterns compared to their benign and atypical counterparts, and that the differentially methylated genes may serve as diagnostic biomarkers or candidate causal genes for malignant transformation.

Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism.

Inhibitors of tumour necrosis factor (TNF) are among the most successful protein-based drugs (biologics) and have proven to be clinically efficacious at reducing inflammation associated with several autoimmune diseases. As a result, attention is focusing on the therapeutic potential of additional members of the TNF superfamily of structurally related cytokines. Many of these TNF-related cytokines or their cognate receptors are now in preclinical or clinical development as possible targets for modulating inflammatory diseases and cancer as well as other indications. This Review focuses on the biologics that are currently in clinical trials for immune-related diseases and other syndromes, discusses the successes and failures to date as well as the expanding therapeutic potential of modulating the activity of this superfamily of molecules.

Recent studies suggest that Peroxiredoxin 1 (Prdx1), in addition to its known H₂O₂-scavenging function, mediates cell signaling through redox-specific protein-protein interactions. Our data illustrate how Prdx1 specifically coordinates p38MAPK-induced signaling through regulating p38MAPKα phosphatases in an H₂O₂ dose-dependent manner. MAPK phosphatases (MKP-1 and/or MKP-5), which are known to dephosphorylate and deactivate the senescence-inducing MAPK p38α, belong to a group of redox-sensitive phosphatases (protein tyrosine phosphatases) characterized by a low pKa cysteine in their active sites. We found that Prdx1 bound to both MKP-1 and MKP-5, but dissociated from MKP-1 when the Prdx1 peroxidatic cysteine Cys52 was over-oxidized to sulfonic acid, which in turn resulted in MKP-1 oxidation-induced oligomerization and inactivity toward p38MAPKα. Conversely, over-oxidation of Prdx1-Cys52 was enhancing in the Prdx1:MKP-5 complex with increasing amounts of H₂O₂ concentrations and correlated with a protection from oxidation-induced oligomerization and inactivation of MKP-5 so that activation toward p38MAPK was maintained. Further examination of this Prdx1-specific mechanism in a model of reactive oxygen species-induced senescence of human breast epithelial cells revealed the specific activation of MKP-5, resulting in decreased p38MAPKα activity. Taken together, our data suggest that Prdx1 orchestrates redox signaling in an H₂O₂ dose-dependent manner through the oxidation status of its peroxidatic cysteine Cys52.

Aurora kinase A (AURKA) localizes to centrosomes and mitotic spindles where it mediates mitotic progression and chromosomal stability. Overexpression of AURKA is common in cancer, resulting in acquisition of alternate non-mitotic functions. In the current study, we identified a novel role for AURKA in regulating ovarian cancer cell dissemination and evaluated the efficacy of an AURKA-selective small molecule inhibitor, alisertib (MLN8237), as a single agent and combined with paclitaxel using an orthotopic xenograft model of epithelial ovarian cancer (EOC). Ovarian carcinoma cell lines were used to evaluate the effects of AURKA inhibition and overexpression on migration and adhesion. Pharmacological or RNA interference-mediated inhibition of AURKA significantly reduced ovarian carcinoma cell migration and adhesion and the activation-associated phosphorylation of the cytoskeletal regulatory protein SRC at tyrosine 416 (pSRC(Y416)). Conversely, enforced expression of AURKA resulted in increased migration, adhesion and activation of SRC in cultured cells. In vivo tumor growth and dissemination were inhibited by alisertib treatment as a single agent. Moreover, combination of alisertib with paclitaxel, an agent commonly used in treatment of EOC, resulted in more potent inhibition of tumor growth and dissemination compared with either drug alone. Taken together, these findings support a role for AURKA in EOC dissemination by regulating migration and adhesion. They also point to the potential utility of combining AURKA inhibitors with taxanes as a therapeutic strategy for the treatment of EOC patients.

Huntington's disease (HD) is a neuropsychiatric disorder characterized by choreiform movement of the limbs, cognitive disability, psychosis and dementia. It is invariably associated with an abnormally long CAG expansion within the IT15 gene on human chromosome 4. Although the mutant huntingtin protein is ubiquitously expressed in HD patients, cellular degeneration occurs predominantly in neurons within the corpus striatum and cerebral cortex. The Ras homolog Rhes is expressed very selectively in the precise brain areas affected by HD. Recent in vitro work suggests that Rhes may be a co-factor with mutant huntingtin in cell death. The objective of the present study was to examine whether the inhibition of Rhes would attenuate or delay the symptoms of HD in vivo. We used a transgenic mouse model of HD crossed with Rhes knockout mice to show that the behavioral symptoms of HD are regulated by Rhes. HD(+)/Rhes(-/-) mice showed significantly delayed expression of HD-like symptoms in this in vivo model. Drugs that block or inhibit the actions of Rhes may be useful as the first treatments for HD.

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin's DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.

Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

Meningiomas are the most common primary nervous system tumor. The tumor suppressor NF2 is disrupted in approximately half of all meningiomas, but the complete spectrum of genetic changes remains undefined. We performed whole-genome or whole-exome sequencing on 17 meningiomas and focused sequencing on an additional 48 tumors to identify and validate somatic genetic alterations. Most meningiomas had simple genomes, with fewer mutations, rearrangements and copy-number alterations than reported in other tumors in adults. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements, including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors and found alterations in epigenetic modifiers in an additional 8% of tumors. A subset of meningiomas lacking NF2 alterations harbored recurrent oncogenic mutations in AKT1 (p.Glu17Lys) and SMO (p.Trp535Leu) and exhibited immunohistochemical evidence of activation of these pathways. These mutations were present in therapeutically challenging tumors of the skull base and higher grade. These results begin to define the spectrum of genetic alterations in meningiomas and identify potential therapeutic targets.

Osterix (Osx) is a transcription factor required for osteoblast differentiation during intramembranous and endochondral ossification. Recently, several reports have described novel functions of Osx in chondrocyte differentiation. In an in vitro study, in which the effects of Osx gene silencing were examined in mouse chondrogenic ATDC5 cells, chondrocyte marker genes were found to be expressionally downregulated and chondrocyte differentiation reduced. On the other hand, in vivo studies based on chondrocyte-specific Osx knockouts demonstrated impaired endochondral bone formation with delayed chondrocyte differentiation and reduced cartilage matrix ossification. However, little is known about the mechanism or targets of Osx involved in the control of chondrocyte differentiation. Here, we attempted to high-density of Affymetrix GeneChip microarray to investigate global gene expression profile changes caused by Osx knockdown in ATDC5 chondrocytes. The mRNA expressions of 112 genes were significantly modified by Osx knockdown: 68 genes were upregulated and 44 genes downregulated. Functional categories of gene expression classified by gene ontology demonstrated that genes related to cell adhesion, development, and signal transduction were highly affected by Osx knockdown. The expressions of differential genes, such as Sfrp2, Sema3a, Nox4, Rgs4, Zfp521, Has2, Sox6, Scn2a1, Sirpa, and Thbs2, were validated by quantitative real-time PCR. This study shows that expression profiling can be used to identify genes that are transcriptionally modified following Osx knockdown and to reveal the molecular mechanism of chondrocyte differentiation regulated by Osx.

<h4>Background</h4>Streptococcus pneumoniae is one of the major bacterial pathogens colonizing nasopharynx, and often causes upper respiratory tract infections in children. We investigated the prevalence of S. pneumoniae in nasopharynx and adenoid core in 57 children aged 2-5 years who underwent adenoidectomy for recurrent pharyngotonsillitis, and we determined serotypes and antibiotic resistance patterns of the isolated pneumococci.<h4>Material/methods</h4>The nasopharyngeal specimens obtained before adenoidectomy and the adenoids after the surgery were cultured for pneumococci. All isolates were serotyped by means of Quellung reaction. Susceptibility to antibiotics was determined according to EUCAST recommendations.<h4>Results</h4>S. pneumoniae colonization was observed in 40 (70.2%) children. From 29 (50.9%) children S. pneumoniae was isolated both from nasopharynx and adenoid core; 2 or 3 different isolates were identified in 8 (14.0%) children. In 8 (14.0%) children pneumococci were obtained from adenoid core only and in 3 (5.3%) children from nasopharynx only. Among the isolates, 35.3% were susceptible to all tested antimicrobials and 45.1% had decreased susceptibility to penicillin. Multidrug resistance was present in 52.9% of the isolates. The most frequent was serotype 19F (25.5%). The prevalence of serotypes included in pneumococcal conjugate vaccines PCV10 and PCV13 was 51.0% and 62.7%, respectively.<h4>Conclusions</h4>The adenoids, like the nasopharynx, can be regarded as a reservoir of pneumococci, including multidrug resistant strains, especially in children with indication for adenoidectomy due to recurrent respiratory tract infections refractory to antibiotic therapy. Good vaccine coverage among the isolated pneumococci confirmed the validity of the routine immunization by PCVs in young children.

A new, mechanically stable silica microparticle with macrosized internal pores (1.6 μm particles with 100 nm pores) has been developed for chromatography. The particles are characterized by an extensive network of interconnected macropores with a high intraparticle void volume, as observed by transmission electron microscopy (TEM). They are synthesized by an aerosol assembly technique called ultrasonic spray pyrolysis (USP). The particles have a high surface area for a macroporous material, ∼200 m(2)/g, making them suitable for large biomolecular separations. To demonstrate their potential for bioseparations, they have been functionalized with lectins for affinity enrichment of glycoproteins. The material was derivatized with two lectins, Concanavalin A (Con A) and Aleuria aurantia lectin (AAL), and binding properties were tested with standard glycoproteins. The columns exhibited excellent binding capacities for microaffinity enrichment: Con A was able to bind 75 μg of a standard glycoprotein in a 50 × 1 mm column. Following initial tests, the lectin microcolumns were utilized for enrichment of glycoproteins from 1 μL volumes of blood serum samples, performed in triplicate for each lectin. The enriched serum fractions were subjected to side-by-side glycomic and glycoproteomic profiling analyses with mass spectrometry to show that the new particles offer excellent sensitivity for microscale analyses of precious biological sample materials. The unique combination of the macroporous architecture and small particle diameter suggests the material may have advantages for conventional modes of chromatographic separation of macromolecules in an ultra-high-pressure liquid chromatography (UHPLC) format.

<h4>Background</h4>ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis.<h4>Methods</h4>UCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time.<h4>Results</h4>UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations.<h4>Conclusion</h4>The results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

Glycosylation is an important and common form of posttranscriptional modification of proteins in cells. During the last decade, a vast array of biological functions has been ascribed to glycans because of a rapid evolution in glycomic technologies. Glycogenes that are highly expressed at the human ocular surface include families of glycosyltransferases, proteoglycans, and glycan degradation proteins, as well as mucins and carbohydrate-binding proteins, such as the galectins. On the apical glycocalyx, mucin O-glycans promote boundary lubrication, prevent bacterial adhesion and endocytic activity, and maintain epithelial barrier function through interactions with galectins. The emerging roles attributed to glycans are contributing to the appreciation of their biological capabilities at the ocular surface.

The Gene Ontology (GO) is the de facto standard for the functional description of gene products, providing a consistent, information-rich terminology applicable across species and information repositories. The UniProt Consortium uses both manual and automatic GO annotation approaches to curate UniProt Knowledgebase (UniProtKB) entries. The selection of a protein set prioritized for manual annotation has implications for the characteristics of the information provided to users working in a specific field or interested in particular pathways or processes. In this article, we describe an organelle-focused, manual curation initiative targeting proteins from the human peroxisome. We discuss the steps taken to define the peroxisome proteome and the challenges encountered in defining the boundaries of this protein set. We illustrate with the use of examples how GO annotations now capture cell and tissue type information and the advantages that such an annotation approach provides to users. Database URL: http://www.ebi.ac.uk/GOA/ and http://www.uniprot.org.

<h4>Objective</h4>Preterm infants with intrauterine growth restriction are at increased risk of respiratory distress syndrome and bronchopulmonary dysplasia (BPD). A randomized clinical trial by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Neonatal Research Network demonstrated that vitamin A supplementation in extremely low-birth-weight (ELBW) preterm infants requiring early respiratory support decreased the risk of developing BPD.<h4>Study design</h4>A subgroup analysis of small-for-gestational-age (SGA) infants from the original NICHD trial was performed to test the hypothesis that in infants requiring early respiratory support, vitamin A supplementation decreases the relative risk of BPD or death in premature SGA infants to a greater extent than in gestational age-equivalent vitamin A-treated appropriate-for-gestational-age (AGA) infants.<h4>Results</h4>Although vitamin A supplementation significantly increased serum retinol concentrations in AGA ELBW infants (median [5th percentile, 95th percentile]: 16.3 [-7.0, 68.8] versus 2.4 [-13.9, 55.1]; p < 0.001), no increases were noted in SGA ELBW infants.<h4>Conclusions</h4>Given the limited power of this analysis due to a low number of SGA infants, these data did not provide evidence to support the hypothesis that vitamin A supplementation in preterm SGA infants requiring early respiratory support decreases the relative risk of BPD or death as compared with preterm AGA infants.

Infertility is a medical condition with an increasing impact in Western societies with causes linked to toxins, genetics, and aging (primarily delay of motherhood). Within the different pathologies that can lead to infertility, poor quality or reduced quantity of gametes plays an important role. Gamete donation and therefore demand on donated sperm and eggs in fertility clinics is increasing. It is hoped that a better understanding of the conditions related to poor gamete quality may allow scientists to design rational treatments. However, to date, relatively little is known about human germ cell development in large part due to the inaccessibility of human development to molecular genetic analysis. It is hoped that pluripotent human embryonic stem cells and induced pluripotent stem cells may provide an accessible in vitro model to study germline development; these cells are able to differentiate to cells of all three primary embryonic germ layers, as well as to germ cells in vitro. We review the state of the art in germline differentiation from pluripotent stem cells.

The traditional notion of chelation therapy is the administration of a chemical agent to remove metals from the body. But formation of a metal-chelate can have biological ramifications that are much broader than metal elimination. Exploring these other possibilities could lead to pharmacological interventions that alter the concentration, distribution, or reactivity of metals in targeted ways for therapeutic benefit. This review highlights recent examples that showcase four general strategies of using principles of metal chelation in medicinal contexts beyond the traditional notion of chelation therapy. These strategies include altering metal biodistribution, inhibiting specific metalloenzymes associated with disease, enhancing the reactivity of a metal complex to promote cytotoxicity, and conversely, passivating the reactivity of metals by site-activated chelation to prevent cytotoxicity.

Venous thrombosis is a major medical disorder caused by both genetic and environmental factors. Little is known about the genetic background of venous thrombosis in the Chinese population. A total of 1,304 individuals diagnosed with a first venous thrombosis and 1,334 age- and sex-matched healthy participants were enrolled in this study. Resequencing of THBD (encoding thrombomodulin) in 60 individuals with venous thrombosis and 60 controls and a functional assay showed that a common variant, c.-151G>T (rs16984852), in the 5' UTR significantly reduced the gene expression and could cause a predisposition to venous thrombosis. Therefore, this variant was genotyped in a case-control study, and results indicated that heterozygotes had a 2.80-fold (95% confidence interval = 1.88-4.29) increased risk of venous thrombosis. The THBD c.-151G>T variant was further investigated in a family analysis involving 176 first-degree relatives from 38 index families. First-degree relatives with this variant had a 3.42-fold increased risk of venous thrombosis, and their probability of remaining thrombosis-free was significantly lower than that of relatives without the variant. In addition, five rare mutations that might be deleterious were also identified in thrombophilic individuals by sequencing. This study is the largest genetic investigation of venous thrombosis in the Chinese population. Further study on genetics of thrombosis should focus on resequencing of THBD and other hemostasis genes in different populations.

The genetic and epigenetic events of hepato-carcinogenesis are relatively poorly understood. By analyzing genes from human hepatocellular carcinoma (HCC) with restriction landmark genomic scanning, several aberrantly methylated genes, including Delta-like 3 (DLL3), have been isolated. In this study, we investigated the function of DLL3 in hepatocarcinogenesis. Methylation of the DLL3 gene in HCC cell lines was investigated with methylation-specific PCR and expression of DLL3 mRNA in HCC cells was examined by RT-PCR. Reactivation of DLL3 expression by treatment with a demethylating agent was examined in methylation-silenced HuH2 cells. Human DLL3 cDNA was cloned and DLL3 function was examined by restoring DLL3 expression in HuH2 cells. The effects of DLL3 on cell growth were evaluated by colony formation assay. Induction of cell death by overexpression of DLL3 was examined by flow cytometric assay using Annexin V and PI. Apoptotic cells were detected by TUNEL staining and the amount of single-stranded DNA was measured by ELISA. As a result, the promoter region of the DLL3 gene was methylated in four of ten HCC cell lines. This aberrant methyl-ation correlated well with the suppression of RNA expression and a demethylating agent reactivated DLL3 expression in methylation-silenced HCC cells. Interestingly, the restoration of DLL3 in the methylation-silenced HuH2 cells led to growth suppression on colony formation assay. Flow cytometric assay with Annexin V and PI showed that this growth suppression by DLL3 expression is associated with the induction of apoptosis. Furthermore, these apoptotic effects were confirmed by TUNEL staining and measurement of single-stranded DNA. These results suggest that DLL3 was silenced by methylation in human HCC and that it negatively regulates the growth of HCC cells.

Genetic variants in intron 1 of the fat mass- and obesity-associated (FTO) gene have been consistently associated with body mass index (BMI) in Europeans. However, follow-up studies in African Americans (AA) have shown no support for some of the most consistently BMI-associated FTO index single nucleotide polymorphisms (SNPs). This is most likely explained by different race-specific linkage disequilibrium (LD) patterns and lower correlation overall in AA, which provides the opportunity to fine-map this region and narrow in on the functional variant. To comprehensively explore the 16q12.2/FTO locus and to search for second independent signals in the broader region, we fine-mapped a 646-kb region, encompassing the large FTO gene and the flanking gene RPGRIP1L by investigating a total of 3,756 variants (1,529 genotyped and 2,227 imputed variants) in 20,488 AAs across five studies. We observed associations between BMI and variants in the known FTO intron 1 locus: the SNP with the most significant p-value, rs56137030 (8.3 × 10(-6)) had not been highlighted in previous studies. While rs56137030was correlated at r(2)>0.5 with 103 SNPs in Europeans (including the GWAS index SNPs), this number was reduced to 28 SNPs in AA. Among rs56137030 and the 28 correlated SNPs, six were located within candidate intronic regulatory elements, including rs1421085, for which we predicted allele-specific binding affinity for the transcription factor CUX1, which has recently been implicated in the regulation of FTO. We did not find strong evidence for a second independent signal in the broader region. In summary, this large fine-mapping study in AA has substantially reduced the number of common alleles that are likely to be functional candidates of the known FTO locus. Importantly our study demonstrated that comprehensive fine-mapping in AA provides a powerful approach to narrow in on the functional candidate(s) underlying the initial GWAS findings in European populations.

The liver is one of the most sex-dimorphic organs in both oviparous and viviparous animals. In order to understand the molecular basis of the difference between male and female livers, high-throughput RNA-SAGE (serial analysis of gene expression) sequencing was performed for zebrafish livers of both sexes and their transcriptomes were compared. Both sexes had abundantly expressed genes involved in translation, coagulation and lipid metabolism, consistent with the general function of the liver. For sex-biased transcripts, from in addition to the high enrichment of vitellogenin transcripts in spawning female livers, which constituted nearly 80% of total mRNA, it is apparent that the female-biased genes were mostly involved in ribosome/translation, estrogen pathway, lipid transport, etc, while the male-biased genes were enriched for oxidation reduction, carbohydrate metabolism, coagulation, protein transport and localization, etc. Sexual dimorphism on xenobiotic metabolism and anti-oxidation was also noted and it is likely that retinol x receptor (RXR) and liver x receptor (LXR) play central roles in regulating the sexual differences of lipid and cholesterol metabolisms. Consistent with high ribosomal/translational activities in the female liver, female-biased genes were significantly regulated by two important transcription factors, Myc and Mycn. In contrast, Male livers showed activation of transcription factors Ppargc1b, Hnf4a, and Stat4, which regulate lipid and glucose metabolisms and various cellular activities. The transcriptomic responses to sex hormones, 17β-estradiol (E2) or 11-keto testosterone (KT11), were also investigated in both male and female livers and we found that female livers were relatively insensitive to sex hormone disturbance, while the male livers were readily affected. E2 feminized male liver by up-regulating female-biased transcripts and down-regulating male-biased transcripts. The information obtained in this study provides comprehensive insights into the sexual dimorphism of zebrafish liver transcriptome and will facilitate further development of the zebrafish as a human liver disease model.

<h4>Background</h4>The timing of associations between common genetic variants and changes in growth patterns over childhood may provide insight into the development of obesity in later life. To address this question, it is important to define appropriate statistical models to allow for the detection of genetic effects influencing longitudinal childhood growth.<h4>Methods and results</h4>Children from The Western Australian Pregnancy Cohort (Raine; n=1,506) Study were genotyped at 17 genetic loci shown to be associated with childhood obesity (FTO, MC4R, TMEM18, GNPDA2, KCTD15, NEGR1, BDNF, ETV5, SEC16B, LYPLAL1, TFAP2B, MTCH2, BCDIN3D, NRXN3, SH2B1, MRSA) and an obesity-risk-allele-score was calculated as the total number of 'risk alleles' possessed by each individual. To determine the statistical method that fits these data and has the ability to detect genetic differences in BMI growth profile, four methods were investigated: linear mixed effects model, linear mixed effects model with skew-t random errors, semi-parametric linear mixed models and a non-linear mixed effects model. Of the four methods, the semi-parametric linear mixed model method was the most efficient for modelling childhood growth to detect modest genetic effects in this cohort. Using this method, three of the 17 loci were significantly associated with BMI intercept or trajectory in females and four in males. Additionally, the obesity-risk-allele score was associated with increased average BMI (female: β=0.0049, P=0.0181; male: β=0.0071, P=0.0001) and rate of growth (female: β=0.0012, P=0.0006; male: β=0.0008, P=0.0068) throughout childhood.<h4>Conclusions</h4>Using statistical models appropriate to detect genetic variants, variations in adult obesity genes were associated with childhood growth. There were also differences between males and females. This study provides evidence of genetic effects that may identify individuals early in life that are more likely to rapidly increase their BMI through childhood, which provides some insight into the biology of childhood growth.

<h4>Background</h4>Mounting evidence indicates that obesity may be associated with the risk of colorectal cancer (CRC). To conduct a systematic review of prospective studies assessing the association of obesity with the risk of CRC using meta-analysis.<h4>Methodology/principal findings</h4>Relevant studies were identified by a search of MEDLINE and EMBASE databases before January 2012, with no restrictions. We also reviewed reference lists from retrieved articles. We included prospective studies that reported relative risk (RR) estimates with 95% confidence intervals (CIs) for the association between general obesity [measured using body mass index (BMI)] or central obesity [measured using waist circumference (WC)] and the risk of colorectal, colon, or rectal cancer. Approximately 9, 000, 000 participants from several countries were included in this analysis. 41 studies on general obesity and 13 studies on central obesity were included in the meta-analysis. The pooled RRs of CRC for the obese vs. normal category of BMI were 1.334 (95% CI, 1.253-1.420), and the highest vs. lowest category of WC were 1.455 (95% CI, 1.327-1.596). There was heterogeneity among studies of BMI (P<0.001) but not among studies of WC (P=0.323).<h4>Conclusions</h4>Both of general and central obesity were positively associated with the risk of CRC in this meta-analysis.

<h4>Background</h4>Whereas many causes and mechanisms of neurodegenerative diseases have been identified, very few therapeutic strategies have emerged in parallel. One possible explanation is that successful treatment strategy may require simultaneous targeting of more than one molecule of pathway. A new therapeutic approach to have emerged recently is the engagement of microRNAs (miRNAs), which affords the opportunity to target multiple cellular pathways simultaneously using a single sequence.<h4>Methodology/principal findings</h4>We identified miR-22 as a potentially neuroprotective miRNA based on its predicted regulation of several targets implicated in Huntington's disease (histone deacetylase 4 (HDAC4), REST corepresor 1 (Rcor1) and regulator of G-protein signaling 2 (Rgs2)) and its diminished expression in Huntington's and Alzheimer's disease brains. We then tested the hypothesis that increasing cellular levels of miRNA-22 would achieve neuroprotection in in vitro models of neurodegeneration. As predicted, overexpression of miR-22 inhibited neurodegeneration in primary striatal and cortical cultures exposed to a mutated human huntingtin fragment (Htt171-82Q). Overexpression of miR-22 also decreased neurodegeneration in primary neuronal cultures exposed to 3-nitropropionic acid (3-NP), a mitochondrial complex II/III inhibitor. In addition, miR-22 improved neuronal viability in an in vitro model of brain aging. The mechanisms underlying the effects of miR-22 included a reduction in caspase activation, consistent with miR-22's targeting the pro-apoptotic activities of mitogen-activated protein kinase 14/p38 (MAPK14/p38) and tumor protein p53-inducible nuclear protein 1 (Tp53inp1). Moreover, HD-specific effects comprised not only targeting HDAC4, Rcor1 and Rgs2 mRNAs, but also decreasing focal accumulation of mutant Htt-positive foci, which occurred via an unknown mechanism.<h4>Conclusions</h4>These data show that miR-22 has multipartite anti-neurodegenerative activities including the inhibition of apoptosis and the targeting of mRNAs implicated in the etiology of HD. These results motivate additional studies to evaluate the feasibility and therapeutic efficacy of manipulating miR-22 in vivo.

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

<h4>Background</h4>Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.<h4>Methodology/principal findings</h4>In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus.<h4>Conclusions/significance</h4>We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.

After entry into the cell, the early steps of the human immunodeficiency virus type 1 (HIV-1) replication cycle are mediated by two functionally distinct nucleoprotein complexes, the reverse transcription complex (RTC) and preintegration complex (PIC). These two unique viral complexes are responsible for the conversion of the single-stranded RNA genome into double-stranded DNA, transport of the DNA into the nucleus, and integration of the viral DNA into the host cell chromosome. Prior biochemical analyses suggest that these complexes are large and contain multiple undiscovered host cell factors. In this study, functional HIV-1 RTCs and PICs were partially purified by velocity gradient centrifugation and fractionation, concentrated, trypsin digested, and analyzed by LC-MS/MS. A total of seven parallel infected and control biological replicates were completed. Database searches were performed with Proteome Discoverer and a comparison of the HIV-1 samples to parallel uninfected control samples was used to identify unique cellular factors. The analysis produced a total data set of 11055 proteins. Several previously characterized HIV-1 factors were identified, including XRCC6, TFRC, and HSP70. The presence of XRCC6 was confirmed in infected fractions and shown to be associated with HIV-1 DNA by immunoprecipitation-PCR experiments. Overall, the analysis identified 94 proteins unique in the infected fractions and 121 proteins unique to the control fractions with ≥ 2 protein assignments. An additional 54 and 52 were classified as enriched in the infected and control samples, respectively, based on a 3-fold difference in total Proteome Discoverer probability score. The differential expression of several candidate proteins was validated by Western blot analysis. This study contributes additional novel candidate proteins to the growing published bioinformatic data sets of proteins that contribute to HIV-1 replication.

Impaired energy metabolism in neurons is integral to a range of neurodegenerative diseases, from Alzheimer's disease to stroke. To investigate the complex molecular changes underpinning cellular adaptation to metabolic stress, we have defined the proteomic response of the SH-SY5Y human neuroblastoma cell line after exposure to a metabolic challenge of oxygen glucose deprivation (OGD) in vitro. A total of 958 proteins across multiple subcellular compartments were detected and quantified by label-free liquid chromatography mass spectrometry. The levels of 130 proteins were significantly increased (P<0.01) after OGD and the levels of 63 proteins were significantly decreased (P<0.01) while expression of the majority of proteins (765) was not altered. Network analysis identified novel protein-protein interactomes involved with mitochondrial energy production, protein folding, and protein degradation, indicative of coherent and integrated proteomic responses to the metabolic challenge. Approximately one third (61) of the differentially expressed proteins was associated with the endoplasmic reticulum and mitochondria. Electron microscopic analysis of these subcellular structures showed morphologic changes consistent with the identified proteomic alterations. Our investigation of the global cellular response to a metabolic challenge clearly shows the considerable adaptive capacity of the proteome to a slowly evolving metabolic challenge.

<h4>Purpose</h4>Due to ethnic, genetic and environmental factors, the clinical and molecular characteristics of Turkish colorectal cancer (CRC) patients are different from those of Western populations. The aim of this study was to clarify the relevant alterations of gene expression associated with colorectal carcinogenesis in early-onset patients and to identify specific biomarkers that could provide novel therapeutic molecular targets in this population.<h4>Methods</h4>The expression profiles of 114 different genes were evaluated using mRNA PCR arrays in 39 tumors and 20 surgical margin tissue samples from 39 sporadic CRC patients diagnosed at less than 50 years of age.<h4>Results</h4>The expression levels of IMPDH2, CK20, MAP3K8 and EIF5A were strongly up-regulated in CRC tissues compared with normal colorectal tissues (p < 0.05). The highly significant expression ratios of CK20, MAP3K8 and EIF5A observed in the colorectal tumors of patients predicted recurrence (p < 0.05). The expression of IMPDH2, CK20, MAP3K8 and EIF5A was significantly higher in the tumors of patients with short median survival (log-rank p value < 0.05). Progression-free survival was also significantly increased in patients with low expression of the EIF5A gene compared with those who exhibited high expression of this gene (log-rank p value < 0.05).<h4>Conclusion</h4>We demonstrated that high CK20, MAP3K8 and EIF5A expression levels were significant prognostic factors for poor overall survival in CRC patients. Further studies and validations are required; these genes may provide novel therapeutic molecular targets for CRC treatment, as well as new directions for the development of anticancer drugs.

Hippocampal neurogenesis, important for memory and mood function, wanes greatly in old age. Studies in rat models have implied that this decrease is not due to loss of neural stem cells (NSCs) in the subgranular zone of the dentate gyrus (DG) but rather due to an increased quiescence of NSCs. Additional studies have suggested that changes in the microenvironment, particularly declines in the concentrations of neurotrophic factors, underlie this change. In this study, we compared the expression of 84 genes that are important for NSC proliferation and neurogenesis between the DG of young (4 months old) and aged (24 months old) Fischer 344 rats, using a quantitative real-time polymerase chain reaction array. Interestingly, the expression of a vast majority of genes that have been reported previously to positively or negatively regulate NSC proliferation was unaltered with aging. Furthermore, most genes important for cell cycle arrest, regulation of cell differentiation, growth factors and cytokine levels, synaptic functions, apoptosis, cell adhesion and cell signaling, and regulation of transcription displayed stable expression in the DG with aging. The exceptions included increased expression of genes important for NSC proliferation and neurogenesis (Stat3 and Shh), DNA damage response and NF-kappaB signaling (Cdk5rap3), neuromodulation (Adora1), and decreased expression of a gene important for neuronal differentiation (HeyL). Thus, age-related decrease in hippocampal neurogenesis is not associated with a decline in the expression of selected genes important for NSC proliferation and neurogenesis in the DG.

In biological environments, nanomaterials associate with proteins forming a protein corona (PC). The PC may alter the nanomaterial's pharmacokinetics and pharmacodynamics, thereby influencing toxicity. Using a label-free mass spectrometry-based proteomics approach, the composition of the PC is examined for a set of nanotubes (NTs) including unmodified and carboxylated single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), polyvinylpyrrolidone (PVP)-coated MWCNT (MWCNT-PVP), and nanoclay. NTs are incubated for 1 h in simulated cell culture conditions, then washed, resuspended in PBS, and assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for their associated PC. To determine those attributes that influence PC formation, the NTs are extensively characterized. NTs had negative zeta potentials in water (SWCNT-COOH < MWCNT-COOH < unmodified NTs) while carboxylation increases their hydrodynamic sizes. All NTs are also found to associate a common subset of proteins including albumin, titin, and apolipoproteins. SWCNT-COOH and MWCNT-COOH are found to bind the greatest number of proteins (181 and 133 respectively) compared to unmodified NTs (<100), suggesting covalent binding to protein amines. Modified NTs bind a number of unique proteins compared to unmodified NTs, implying hydrogen bonding and electrostatic interactions are involved in PC formation. PVP-coating of MWCNT did not influence PC composition, further reinforcing the possibility of hydrogen bonding and electrostatic interactions. No relationships are found between PC composition and corresponding isoelectric point, hydropathy, or aliphatic index, implying minimal roles of hydrophobic interaction and pi-stacking.

Increasing evidence suggests that high-frequency deep brain stimulation of the nucleus accumbens (NAcb-DBS) may represent a novel therapeutic strategy for individuals suffering from treatment-resistant depression, although the underlying mechanisms of action remain largely unknown. In this study, using a unique mouse model of enhanced depression- and anxiety-like behavior (HAB), we investigated behavioral and neurobiological effects of NAcb-DBS. HAB mice either underwent chronic treatment with one of three different selective serotonin reuptake inhibitors (SSRIs) or received NAcb-DBS for 1 h per day for 7 consecutive days. Animals were tested in established paradigms revealing depression- and anxiety-related behaviors. The enhanced depression-like behavior of HAB mice was not influenced by chronic SSRI treatment. In contrast, repeated, but not single, NAcb-DBS induced robust antidepressant and anxiolytic responses in HAB animals, while these behaviors remained unaffected in normal depression/anxiety animals (NAB), suggesting a preferential effect of NAcb-DBS on pathophysiologically deranged systems. NAcb-DBS caused a modulation of challenge-induced activity in various stress- and depression-related brain regions, including an increase in c-Fos expression in the dentate gyrus of the hippocampus and enhanced hippocampal neurogenesis in HABs. Taken together, these findings show that the normalization of the pathophysiologically enhanced, SSRI-insensitive depression-like behavior by repeated NAcb-DBS was associated with the reversal of reported aberrant brain activity and impaired adult neurogenesis in HAB mice, indicating that NAcb-DBS affects neuronal activity as well as plasticity in a defined, mood-associated network. Thus, HAB mice may represent a clinically relevant model for elucidating the neurobiological correlates of NAcb-DBS.

After replicating in epithelial cells, alphaherpesviruses such as pseudorabies virus (PRV) invade axons of peripheral nervous system neurons and undergo retrograde transport toward the distant cell bodies. Although several viral proteins engage molecular motors to facilitate transport, the initial steps and neuronal responses to infection are poorly understood. Using compartmented neuron cultures to physically separate axon infection from cell bodies, we found that PRV infection induces local protein synthesis in axons, including proteins involved in cytoskeletal remodeling, intracellular trafficking, signaling, and metabolism. This rapid translation of axonal mRNAs is required for efficient PRV retrograde transport and infection of cell bodies. Furthermore, induction of axonal damage, which also induces local protein synthesis, prior to infection reduces virion trafficking, suggesting that host damage signals and virus particles compete for retrograde transport. Thus, similar to axonal damage, virus infection induces local protein translation in axons, and viruses likely exploit this response for invasion.

Iron abnormalities within the brain are associated with several rare but severe neurodegenerative conditions. There is growing evidence that more common systemic iron loading disorders such as hemochromatosis can also have important effects on the brain. To identify features that are common across different forms of hemochromatosis, we used microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) to assess brain transcriptome profiles of transferrin receptor 2 mutant mice (Tfr2(mut)), a model of a rare type of hereditary hemochromatosis, relative to wildtype control mice. The results were compared with our previous findings in dietary iron-supplemented wildtype mice and Hfe(-/-) mice, a model of a common type of hereditary hemochromatosis. For transcripts showing significant changes relative to controls across all three models, there was perfect (100%) directional concordance (i.e. transcripts were increased in all models or decreased in all models). Comparison of the two models of hereditary hemochromatosis, which showed more pronounced changes than the dietary iron-supplemented mice, revealed numerous common molecular effects. Pathway analyses highlighted changes for genes relating to long-term depression (6.8-fold enrichment, p=5.4×10(-7)) and, to a lesser extent, long-term potentiation (3.7-fold enrichment, p=0.01), with generalized reductions in transcription of key genes from these pathways, which are involved in modulating synaptic strength and efficacy and are essential for memory and learning. The agreement across the models suggests the findings are robust and strengthens previous evidence that iron loading disorders affect the brain. Perturbations of brain phenomena such as long-term depression and long-term potentiation might partly explain neurologic symptoms reported for some hemochromatosis patients.

Alterations in serotonin (5-HT) neurochemistry have been implicated in the aetiology of all major neuropsychiatric disorders, ranging from schizophrenia to mood and anxiety-spectrum disorders. This review will focus on the multifaceted implications of 5-HT-ergic dysfunctions in the pathophysiology of aggressive and suicidal behaviours. After a brief overview of the anatomical distribution of the 5-HT-ergic system in the key brain areas that govern aggression and suicidal behaviours, the implication of 5-HT markers (5-HT receptors, transporter as well as synthetic and metabolic enzymes) in these conditions is discussed. In this regard, particular emphasis is placed on the integration of pharmacological and genetic evidence from animal studies with the findings of human experimental and genetic association studies. Traditional views postulated an inverse relationship between 5-HT and aggression and suicidal behaviours; however, ample evidence has shown that this perspective may be overly simplistic, and that such pathological manifestations may reflect alterations in 5-HT homoeostasis due to the interaction of genetic, environmental and gender-related factors, particularly during early critical developmental stages. The development of animal models that may capture the complexity of such interactions promises to afford a powerful tool to elucidate the pathophysiology of impulsive aggression and suicidability, and identify new effective therapies for these conditions.

The accessory sex gland (ASG) is an important component of the male reproductive system, which functions to enhance the fertility of spermatozoa during male reproduction. Certain proteins secreted by the ASG are known to bind to the spermatozoa membrane and affect its function. The ASG gene expression profile in Chinese mitten crab (Eriocheir sinensis) has not been extensively studied, and limited genetic research has been conducted on this species. The advent of high-throughput sequencing technologies enables the generation of genomic resources within a short period of time and at minimal cost. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset for the ASG of E. sinensis using Illumina sequencing technology. This analysis yielded a total of 33,221,284 sequencing reads, including 2.6 Gb of total nucleotides. Reads were assembled into 85,913 contigs (average 218 bp), or 58,567 scaffold sequences (average 292 bp), that identified 37,955 unigenes (average 385 bp). We assembled all unigenes and compared them with the published testis transcriptome from E. sinensis. In order to identify which genes may be involved in ASG function, as it pertains to modification of spermatozoa, we compared the ASG and testis transcriptome of E. sinensis. Our analysis identified specific genes with both higher and lower tissue expression levels in the two tissues, and the functions of these genes were analyzed to elucidate their potential roles during maturation of spermatozoa. Availability of detailed transcriptome data from ASG and testis in E. sinensis can assist our understanding of the molecular mechanisms involved with spermatozoa conservation, transport, maturation and capacitation and potentially acrosome activation.

Hepcidin, produced by the liver, is the master regulator of iron balance. Serum hepcidin is increased by high iron stores, blocks intestinal iron absorption, and impairs storage iron release. Conversely, iron deficiency lowers hepcidin levels and enhances intestinal iron absorption and the release of storage iron. As with ferritin, hepcidin is an acute phase reactant. Consequently, inflammation increases hepcidin and leads to impaired iron absorption, lowers serum iron and transferrin saturation, and contributes to the anemia of chronic kidney disease (CKD). We review the physiology of iron absorption, its relationship to hepcidin and the transmembrane iron transporter ferroportin, the role of hepcidin in CKD related anemia, and the possible diagnostic implications and limitations of using hepcidin as a marker of iron status.

A transgenic primate model for Huntington's Disease (HD) first reported by our group that (HD monkeys) carry the mutant Huntingtin (HTT) gene with expanded polyglutamine (CAG) repeats and, develop chorea, dystonia, and other involuntary motor deficiencies similar to HD [ 1 ]. More recently, we have found that longitudinal magnetic resonance imaging of the HD monkey brain revealed significant atrophy in regions associated with cognitive deficits symptomatic in HD patients, providing the first animal model which replicates clinical phenotypes of diagnosed humans. Here we report germline transmission of the pathogenic mutant HTT in HD monkey by the production of embryos and subsequent derivation of HD monkey embryonic stem cells (rHD-ESCs) using HD monkey sperm. rHD-ESCs inherit mutant HTT and green fluorescent protein (GFP) genes through the gametes of HD monkey. rHD-ESCs express mutant HTT and form intranuclear inclusion, a classical cellular feature of HD. Notably, mosaicism of the pathogenic polyQ region in the sperm as well as derived ESCs were also observed, consistent with intraindividual and intergenerational reports of mosaic CAG repeats [ 2 , 3 ]and CAG expansion in HD patients [ 4-7 ]. The confirmation of transgene inheritability and development of pathogenic HD phenotype in derived rHD-ESCs reported in this study is a milestone in the pursuit of a transgenic primate model with inherited mutant HTT for development of novel disease biomarkers and therapeutics.

Prescription opioids abuse and associated deaths are an emerging concern in the USA. Vaccination against prescription opioids may provide an alternative to pharmacotherapy. An oxycodone hapten containing a tetraglycine linker at the C6 position (6OXY(Gly)(4)OH) conjugated to keyhole limpet hemocyanin (KLH) has shown early proof-of-efficacy in rodents as a candidate immunogen (6OXY(Gly)(4)-KLH) for the treatment of oxycodone abuse. In this study, oxycodone-based and hydrocodone-based haptens were conjugated to KLH to generate immunogens that would recognize both oxycodone and hydrocodone. Vaccination with 6OXY(Gly)(4)-KLH increased drug binding in serum, reduced drug distribution to brain, and blunted analgesia for both oxycodone and hydrocodone. An analogous C6-linked hydrocodone vaccine blocked hydrocodone effects but less so than 6OXY(Gly)(4)-KLH. C8-Linked hydrocodone immunogens had only limited efficacy. Amide conjugation showed higher haptenation ratios and greater efficacy than thioether conjugation to maleimide activated KLH (mKLH). The 6OXY(Gly)(4)-KLH vaccine may be used for treatment of prescription opioid abuse.

<h4>Unlabelled</h4><h4>Background</h4>Iron deficiency anaemia and oral iron supplementation have been associated negatively with quality of life, and with adverse effects, respectively, in subjects with inflammatory bowel disease (IBD). Hence, the risk-benefit ratio of oral iron is not understood in this patient group. The present case-control study investigated whether dietary iron intake impacts on quality of life in IBD patients.<h4>Methods</h4>Quality of life, habitual dietary iron intakes and iron requirements were assessed in 29 patients with inactive or mildly active IBD as well as in 28 healthy control subjects.<h4>Results</h4>As expected, quality of life was worse in IBD patients as a whole in comparison to healthy controls according to EuroQol score and EuroQol VAS percentage (6.9 ± 1.6 vs 5.3 ± 0.6; p< 0.0001 and 77 ± 14% vs 88 ± 12%; p=0.004 respectively). For IBD subjects, 21/29 were iron deplete based upon serum iron responses to oral iron but, overall, were non-anaemic with mean haemoglobin of 13.3 ± 1.5 g/dL, and there was no difference in their quality of life compared to 8/29 iron replete subjects (Hb 14.0 ± 0.8 g/dL). Interestingly, total dietary iron intake was significantly negatively associated with quality of life in IBD patients, specifically for non-haem iron and, more specifically, for fortificant iron. Moreover, for total non-haem iron the negative association disappeared when fortificant iron values were subtracted. Finally, further sub-analysis indicated that the negative association between (fortificant) dietary iron intake and quality of life in IBD patients is driven by findings in patients with mildly active disease rather than in patients with quiescent disease.<h4>Conclusions</h4>Iron deficiency per se (i.e. without concomitant anaemia) does not appear to further affect quality of life in IBD patients with inactive or mildly active disease. However, in this preliminary study, dietary iron intake, particularly fortificant iron, appears to be significantly negatively associated with quality of life in patients with mildly active disease.

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25-40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression. They are characterized by specific maturation processes defined by canonical and non-canonical biogenic pathways. Analysis of ∼0.5 billion sequences from mouse data sets derived from different tissues, developmental stages and cell types, partly characterized by either ablation or mutation of the main proteins belonging to miRNA processor complexes, reveals 66 high-confidence new genomic loci coding for miRNAs that could be processed in a canonical or non-canonical manner. A proportion of the newly discovered miRNAs comprises mirtrons, for which we define a new sub-class. Notably, some of these newly discovered miRNAs are generated from untranslated and open reading frames of coding genes, and we experimentally validate these. We also show that many annotated miRNAs do not present miRNA-like features, as they are neither processed by known processing complexes nor loaded on AGO2; this indicates that the current miRNA miRBase database list should be refined and re-defined. Accordingly, a group of them map on ribosomal RNA molecules, whereas others cannot undergo genuine miRNA biogenesis. Notably, a group of annotated miRNAs are Dgcr8 independent and DICER dependent endogenous small interfering RNAs that derive from a unique hairpin formed from a short interspersed nuclear element.

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.

Atrogin-1/MAFbx is an ubiquitin E3 ligase that regulates myocardial structure and function through the ubiquitin-dependent protein modification. However, little is known about the effect of atrogin-1 activation on the gene expression changes in cardiomyocytes. Neonatal rat cardiomyocytes were infected with adenovirus atrogin-1 (Ad-atrogin-1) or GFP control (Ad-GFP) for 24 hours. The gene expression profiles were compared with microarray analysis. 314 genes were identified as differentially expressed by overexpression of atrogin-1, of which 222 were up-regulated and 92 were down-regulated. Atrogin-1 overexpression significantly modulated the expression of genes in 30 main functional categories, most genes clustered around the regulation of cell death, proliferation, inflammation, metabolism and cardiomyoctye structure and function. Moreover, overexpression of atrogin-1 significantly inhibited cardiomyocyte survival, hypertrophy and inflammation under basal condition or in response to lipopolysaccharide (LPS). In contrast, knockdown of atrogin-1 by siRNA had opposite effects. The mechanisms underlying these effects were associated with inhibition of MAPK (ERK1/2, JNK1/2 and p38) and NF-κB signaling pathways. In conclusion, the present microarray analysis reveals previously unappreciated atrogin-1 regulation of genes that could contribute to the effects of atrogin-1 on cardiomyocyte survival, hypertrophy and inflammation in response to endotoxin, and may provide novel insight into how atrogin-1 modulates the programming of cardiac muscle gene expression.

There has been significant interest in the bioactivity of the natural product psammaplin A, most recently as a potent and isoform selective HDAC inhibitor. Here we report our preliminary studies on thioester HDAC inhibitors derived from the active monomeric (thiol) form of psammaplin A, as a means to improve compound delivery into cells. We have discovered that such compounds exhibit both potent cytotoxicity and enzymatic inhibitory activity against recombinant HDAC1. The latter effect is surprising since previous SAR suggested that modification of the thiol functionality should detrimentally affect HDAC potency. We therefore also report our preliminary studies on the mechanism of action of this observed effect.

<h4>Background</h4>Diabetes mellitus (DM) is identified as a negative prognostic indicator in hepatocellular carcinoma (HCC), though the basis for this is unknown.<h4>Methods</h4>This is a retrospective analysis of a prospectively collected database of 191 HCC patients treated at the University of Rochester Medical Center (URMC) with orthotopic liver transplantation between 1998-2008. Clinical characteristics were compared between patients with and without DM prior to liver transplantation and logistic regression analyses were conducted to assess the effect of DM on clinical outcomes including vascular invasion.<h4>Results</h4>Eighty-four of 191 (44%) transplanted patients had DM at time of transplantation. An association of DM with invasive disease was found among transplanted HCC patients where histologically confirmed macrovascular invasion was found in 20.2% (17/84) of diabetics compared to 9.3% of non-diabetics (10/107) (p=0.032). This difference also remained significant when adjusting for tumor size, number of nodules, age, obesity and etiologic risk factors in multivariate logistic regression analysis (OR=3.2, p=0.025).<h4>Conclusions</h4>DM is associated with macrovascular invasion among a cohort of transplanted HCC patients.

<h4>Background</h4>Mycobacterium tuberculosis encodes 11 putative serine-threonine proteins Kinases (STPK) which regulates transcription, cell development and interaction with the host cells. From the 11 STPKs three kinases namely PknA, PknB and PknG have been related to the mycobacterial growth. From previous studies it has been observed that PknB is essential for mycobacterial growth and expressed during log phase of the growth and phosphorylates substrates involved in peptidoglycan biosynthesis. In recent years many high affinity inhibitors are reported for PknB. Previously implementation of data fusion has shown effective enrichment of active compounds in both structure and ligand based approaches .In this study we have used three types of data fusion ranking algorithms on the PknB dataset namely, sum rank, sum score and reciprocal rank. We have identified reciprocal rank algorithm is capable enough to select compounds earlier in a virtual screening process. We have also screened the Asinex database with reciprocal rank algorithm to identify possible inhibitors for PknB.<h4>Results</h4>In our work we have used both structure-based and ligand-based approaches for virtual screening, and have combined their results using a variety of data fusion methods. We found that data fusion increases the chance of actives being ranked highly. Specifically, we found that the ranking of Pharmacophore search, ROCS and Glide XP fused with a reciprocal ranking algorithm not only outperforms structure and ligand based approaches but also capable of ranking actives better than the other two data fusion methods using the BEDROC, robust initial enhancement (RIE) and AUC metrics. These fused results were used to identify 45 candidate compounds for further experimental validation.<h4>Conclusion</h4>We show that very different structure and ligand based methods for predicting drug-target interactions can be combined effectively using data fusion, outperforming any single method in ranking of actives. Such fused results show promise for a coherent selection of candidates for biological screening.

<h4>Background</h4>Little is known regarding the types of information African American and non-African American patients with chronic kidney disease (CKD) and their families need to inform renal replacement therapy (RRT) decisions.<h4>Methods</h4>In 20 structured group interviews, we elicited views of African American and non-African American patients with CKD and their families about factors that should be addressed in educational materials informing patients' RRT selection decisions. We asked participants to select factors from a list and obtained their open-ended feedback.<h4>Results</h4>Ten groups of patients (5 African American, 5 non-African American; total 68 individuals) and ten groups of family members (5 African American, 5 non-African American; total 62 individuals) participated. Patients and families had a range (none to extensive) of experiences with various RRTs. Patients identified morbidity or mortality, autonomy, treatment delivery, and symptoms as important factors to address. Family members identified similar factors but also cited the effects of RRT decisions on patients' psychological well-being and finances. Views of African American and non-African American participants were largely similar.<h4>Conclusions</h4>Educational resources addressing the influence of RRT selection on patients' morbidity and mortality, autonomy, treatment delivery, and symptoms could help patients and their families select RRT options closely aligned with their values. Including information about the influence of RRT selection on patients' personal relationships and finances could enhance resources' cultural relevance for African Americans.

<h4>Unlabelled</h4><h4>Background</h4>Hutchinson-Gilford progeria syndrome is a rare dominant human disease of genetic origin. The average life expectancy is about 20 years, patients' life quality is still very poor and no efficient therapy has yet been developed. It is caused by mutation of the LMNA gene, which results in accumulation in the nuclear membrane of a particular splicing form of Lamin-A called progerin. The mechanism by which progerin perturbs cellular homeostasis and leads to the symptoms is still under debate.Micro-RNAs are able to negatively regulate transcription by coupling with the 3' UnTranslated Region of messenger RNAs. Several Micro-RNAs recognize the same 3' UnTranslated Region and each Micro-RNA can recognize multiple 3' UnTranslated Regions of different messenger RNAs. When different messenger RNAs are co-regulated via a similar panel of micro-RNAs, these messengers are called Competing Endogenous RNAs, or ceRNAs.The 3' UnTranslated Region of the longest LMNA transcript was analysed looking for its ceRNAs. The aim of this study was to search for candidate genes and gene ontology functions possibly influenced by LMNA mutations that may exert a role in progeria development.<h4>Results</h4>11 miRNAs were isolated as potential LMNA regulators. By computational analysis, the miRNAs pointed to 17 putative LMNA ceRNAs. Gene ontology analysis of isolated ceRNAs showed an enrichment in RNA interference and control of cell cycle functions.<h4>Conclusion</h4>This study isolated novel genes and functions potentially involved in LMNA network of regulation that could be involved in laminopathies such as the Hutchinson-Gilford progeria syndrome.

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.

In this essay, we discuss new insights into the wide-ranging impacts of mammalian transposable elements (TE) on gene expression and function. Nearly half of each mammalian genome is comprised of these mobile, repetitive elements. While most TEs are ancient relics, certain classes can move from one chromosomal location to another even now. Indeed, striking recent data show that extensive transposition occurs not only in the germline over evolutionary time, but also in developing somatic tissues and particular human cancers. While occasional germline TE insertions may contribute to genetic variation, many other, similar TEs appear to have little or no impact on neighboring genes. However, the effects of somatic insertions on gene expression and function remain almost completely unknown. We present a conceptual framework to understand how the ages, allele frequencies, molecular structures, and especially the genomic context of mammalian TEs each can influence their various possible functional consequences.

Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.

<h4>Objective</h4>Serotonin transporter gene polymorphism (5-HTTLPR polymorphism) predicts the degree of structural and functional connectivity in the brain, and less consistently the degree of vulnerability for anxiety and depressive disorders. It is less known how 5-HTTLPR polymorphism influences on the coupling between brain and neuronal cardiovascular control. The present study demonstrates the impact of 5-HTTLPR polymorphism on the relations between heart rate (HR) corrected cardiac repolarization interval (QTc interval) and the brain 5-HTT binding.<h4>Material and methods</h4>Thirty healthy young adults (fifteen monozygotic twin pairs) (mean age 26±1.3 years, 16 females) were imagined with single-photon emission computed tomography (SPECT) using iodine-123 labeled 2β-carbomethoxy-3β-(4-iodophenyl) nortropane (nor-β-CIT). Continuous ECG recording was obtained from each participant at supine rest. Signal averaged QTc interval on continuous ECG was calculated and compared with the brain imaging results.<h4>Results</h4>In the two groups [l homozygotes (n = 16, 10 females), s carriers (n = 14, 8 female)] HR and the length of QTc interval were not influenced by 5-HTTLPR polymorphism. There were no significant relations between HR and 5-HTT binding in the brain. There were significant associations between QTc interval and nor-β-CIT binding in the brain in l homozygotes, but not in s carriers (correlations for QTc interval and nor-β-CIT binding of striatum, thalamus and right temporal region were -0.8--0.9, (p<0.0005), respectively).<h4>Conclusion</h4>The finding of longer QTc interval with less 5-HTT binding availability in major serotonergic binding sites in l homozygotes, but not in s carriers, implicate to differentiated control of QTc interval by 5-HTTLPR polymorphism.

<h4>Background</h4>The major reason for the poor prognosis of esophageal squamous cell carcinoma (ESCC) patients is lymph node (LN) metastases.<h4>Methodology/principal</h4>In the present study, gene expression profiling assay (GEP) was performed to identify the differences in gene expression profiles between primary ESCC tumors that were with LN metastases (N(+)) and those without LN metastases (N(-)).<h4>Conclusions/significance</h4>A total of 23 genes were identified as being significantly elevated, and 30 genes were sharply decreased in ESCC tumors that were N(+) compared with N- tumors. Among these genes, two transcripts of the short chain dehydrogenase/reductase family 9C, member 7 (SDR9C7) were observed 7 times more frequently in N(+) compared with N(-) tumors. Immunohistochemical staining showed that SDR9C7 expression closely correlated with metastasis, and would be a prognostic marker for ESCC patients. To investigate the role of SDR9C7 in the ESCC metastasis, repeated transwell assays were adopted to establish highly and non-invasive ESCC sublines, and western blot showed that SDR9C7 expression was markedly higher in highly invasive cells compared with non-invasive ones. Down-regulation of SDR9C7 dramatically inhibited the metastatic abilities in vitro and in vivo, and repressed the expression of MMP11 in highly invasive cells, indicating that SDR9C7 promotes ESCC metastasis partly through regulation of MMP11, and might be a potential prognostic and therapeutic marker for ESCC patients.

Systemic lupus erythematosus (SLE) is a serious prototype autoimmune disease characterized by chronic inflammation, auto-antibody production and multi-organ damage. Recent association studies have identified a long list of loci that were associated with SLE with relatively high statistical power. However, most of them only established the statistical associations of genetic markers and SLE at the DNA level without supporting evidence of functional relevance. Here, using publically available datasets, we performed integrative analyses (gene relationship across implicated loci analysis, differential gene expression analysis and functional annotation clustering analysis) and combined with expression quantitative trait loci (eQTLs) results to dissect functional mechanisms underlying the associations for SLE. We found that 14 SNPs, which were significantly associated with SLE in previous studies, have cis-regulation effects on four eQTL genes (HLA-DQA1, HLA-DQB1, HLA-DQB2, and IRF5) that were also differentially expressed in SLE-related cell groups. The functional evidence, taken together, suggested the functional mechanisms underlying the associations of 14 SNPs and SLE. The study may serve as an example of mining publically available datasets and results in validation of significant disease-association results. Utilization of public data resources for integrative analyses may provide novel insights into the molecular genetic mechanisms underlying human diseases.

<h4>Background</h4>The intestinal mucosa is the compartment that sustains the most severe injury in response to radiation and is therefore of primary interest. The use of whole gut extracts for analysis of gene expression may confound important changes in the mucosa. On the other hand, laser capture microdissection (LCM) is hampered by the unstable nature of RNA and by a more complicated collection process. This study assessed, in parallel samples from a validated radiation model, the indications for use of LCM for intestinal gene expression analysis.<h4>Methodology/principal findings</h4>RNA was extracted from mouse whole intestine and from mucosa by LCM at baseline and 4 h, 24 h, and 3.5 d after total body irradiation and subjected to microarray analysis. Among mucosal genes that were altered > = 2-fold, less than 7% were present in the whole gut at 4 and 24 h, and 25% at 3.5 d. As expected, pathway analysis of mucosal LCM samples showed that radiation activated the coagulation system, lymphocyte apoptosis, and tight junction signaling, and caused extensive up-regulation of cell cycle and DNA damage repair pathways. Using similar stringent criteria, regulation of these pathways, with exception of the p53 pathway, was undetectable in the whole gut. Radiation induced a dramatic increase of caspase14 and ectodysplasin A2 receptor (Eda2r), a TNFα receptor, in both types of samples.<h4>Conclusions/significance</h4>LCM-isolated mucosal specimens should be used to study cellular injury, cell cycle control, and DNA damage repair pathways. The remarkable increase of caspase14 and Eda2r suggests a novel role for these genes in regulating intestinal radiation injury. Comparative gene expression data from complex tissues should be interpreted with caution.

Regulatory region of milk protein alpha S1-casein (αS1-CN) gene was sequenced, characterized, and analyzed to detect variations among 13 Indian cattle (Bos indicus) breeds. Comparative analysis of 1,587 bp region comprising promoter (1,418 bp), exon-I (53 bp), and partial intron-I (116 bp) revealed 35 nucleotide substitutions (32 within promoter region, 1 in exon-I, and 2 in partial intron-I region) and 4 Indels. Within promoter, 15 variations at positions -1399 (A > G), -1288 (G > A), -1259 (T > C), -1158 (T > C), -1016 (A > T), -941 (T > G), -778 (C > T), -610 (G > A), -536 (A > G), -521 (A > G), -330 (A > C), -214 (A > G), -205 (A > T), -206 (C > A), and -175 (A > G) were located within the potential transcription factor binding sites (TFBSs), namely, NF-κE1/c-Myc, GATA-1, GATA-1/NF-E, Oct-1/POU3F2, MEF-2/YY1, GATA-1, AP-1, POU1F1a/GR, TMF, GAL4, YY1/Oct-1, HNF-1, GRalpha/AR, GRalpha/AR, and AP-1, respectively. Seventy-four percent (26/35) of the observed SNPs were novel to Indian cattle and 11 of these novel SNPs were located within one or more TFBSs. Collectively, these might influence the binding affinity towards their respective nuclear TFs thus modulating the level of transcripts in milk and affecting overall protein composition. The study provides information on several distinct variations across indicine and taurine αS1-CN regulatory domains.

IL-28B polymorphisms are predictors of response to therapy in adults infected with hepatitis C. We do not know whether they are markers of response to therapy in children and adolescents. The aim of this study was to determine whether single-nucleotide polymorphisms (SNPs) in the IL-28B gene could influence the probability of response to therapy compared with other known baseline prognostic factors and correlate with clinical findings in pediatric patients infected with hepatitis C virus (HCV) genotypes 1 or 4. We determined three SNPs of IL-28B (rs12979860, rs12980275, and rs8099917) in 82 patients with chronic HCV infection treated with pegylated interferon alpha and ribavirin (peg-IFNα/RBV). Treatment response and clinical data were analyzed. Overall, sustained virological response (SVR) was achieved by 45 % of patients infected with difficult-to-treat HCV genotypes 1 and 4. Except for IL-28B polymorphisms, there was no association of SVR with any other clinical data. IL-28B rs12979860 CC [odds ratio (OR), 6.81; p = 0.001] and rs8099917 TT (OR, 3.14; p = 0.013) genotypes were associated with higher SVR rates. IL-28B rs12980275 was not significantly associated with SVR (p = 0.058). Only the distribution between CC and CT-TT genotypes of rs12979860 significantly differentiated patients achieving early virological response (EVR) (OR, 10.0; p = 0.011). Children with the rs12979860 CC genotype had significantly higher baseline viral load compared with CT-TT patients (p = 0.010). In children and adolescents chronically infected with HCV genotypes 1 and 4, IL-28B rs12979860 and rs8099917 polymorphisms were the only predictors of response to peg-IFN/RBV.

Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGFβ3 mice) develop MPNSTs. To determine whether P(0)-GGFβ3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGFβ3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGFβ3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGFβ3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGFβ3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.

The diagnosis of hepatocellular carcinoma (HCC) became easier in relation to the improved radiological examinations; however, the neoplasm may occur under atypical presentations mimicking other benign or malignant processes. Multicystic HCC mimicking a liver abscess associated with septic-type fever and leukocytosis is rare, has a poor prognosis, and poses diagnostic and therapeutic dilemmas. We present the case of an 80-year-old patient, who presented with fever, leukocytosis, and large cystic masses involving right and left lobes of the liver initially considered abscesses and finally diagnosed as HCC after open drainage and liver biopsy. Although the patient died on the tenth postoperative day due to pulmonary oedema, the authors emphasize the high index of suspicion needed in the diagnosis of this unusual presentation of HCC.

In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥ 30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.

Cerebrospinal fluid (CSF) is a low protein content biological fluid with a dynamic range spanning at least 9 orders of magnitude in protein content and is in direct contact with the brain. A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the low volumes of CSF that are obtainable from mice. As a model system in which to test this approach, we utilized transgenic mice that overexpress the intermediate filament glial fibrillary acidic protein (GFAP). These mice are models for Alexander disease (AxD), a severe leukodystrophy in humans. From the CSF of control and transgenic mice we report the identification of 289 proteins, with relative quantification of 103 proteins. Biological and technical triplicates were performed to address animal variability as well as reproducibility in mass spectrometric analysis. Relative quantitation was performed using distributive normalized spectral abundance factor (dNSAF) spectral counting analysis. A panel of biomarker proteins with significant changes in the CSF of GFAP transgenic mice has been identified with validation from enzyme-linked immunosorbent assay (ELISA) and microarray data, demonstrating the utility of our methodology and providing interesting targets for future investigations on the molecular and pathological aspects of AxD.

The generation of chimeras, which is now a standard technology for producing gene modified mutant mice, was originally developed as a tool for developmental biology. However, the application of conventional single marker chimeric mice for developmental study was initially limited. This situation has been dramatically changed by development of multicolor chimeric mice using various kinds of fluorescent proteins. Now using our technology, up to ten different clones could be distinguished by their colors, which enable us to perform more accurate statistical analyses and lineage tracing experiments than by conventional methods. This method could be applied to visualize not only cell turnover of normal stem cells but also cancer development of live tissues in vivo. In the present review, we will discuss how these methods have been developed and what questions they are now answering by mainly focusing on intestinal stem cells and intestinal tumors.

Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34(+)) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34(+) progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository.

<h4>Background</h4>Although smoking behavior is known to affect body mass index (BMI), the potential for smoking to influence genetic associations with BMI is largely unexplored.<h4>Methods</h4>As part of the 'Population Architecture using Genomics and Epidemiology (PAGE)' Consortium, we investigated interaction between genetic risk factors associated with BMI and smoking for 10 single nucleotide polymorphisms (SNPs) previously identified in genome-wide association studies. We included 6 studies with a total of 56,466 subjects (16,750 African Americans (AA) and 39,716 European Americans (EA)). We assessed effect modification by testing an interaction term for each SNP and smoking (current vs. former/never) in the linear regression and by stratified analyses.<h4>Results</h4>We did not observe strong evidence for interactions and only observed two interactions with p-values <0.1: for rs6548238/TMEM18, the risk allele (C) was associated with BMI only among AA females who were former/never smokers (β = 0.018, p = 0.002), vs. current smokers (β = 0.001, p = 0.95, p(interaction) = 0.10). For rs9939609/FTO, the A allele was more strongly associated with BMI among current smoker EA females (β = 0.017, p = 3.5 x 10(-5)), vs. former/never smokers (β = 0.006, p = 0.05, p(interaction) = 0.08).<h4>Conclusions</h4>These analyses provide limited evidence that smoking status may modify genetic effects of previously identified genetic risk factors for BMI. Larger studies are needed to follow up our results.<h4>Clinical trial registration</h4>NCT00000611.

Significant advances have been made in understanding the genetic basis of systemic sclerosis (scleroderma) in recent years. Can these discoveries lead to individualized monitoring and treatment? Besides robustly replicated genetic susceptibility loci, several genes have been recently linked to various systemic sclerosis disease manifestations. Furthermore, inclusion of genetic studies in design and analysis of drug trials could lead to development of genetic biomarkers that predict treatment response. Future genetic studies in well-characterized systemic sclerosis cohorts paired with advanced analytic approaches can lead to development of genetic biomarkers for targeted diagnostic and therapeutic interventions in systemic sclerosis.

Increased level of endothelin-1 (ET-1), a potent vasoconstrictor, has been found in the cerebral spinal fluid (CSF) of patients with multi-infarction dementia, suggesting a possible role of ET-1 in cognitive deficit associated with stroke. Previously, we have reported that synthesis of ET-1 is induced in endothelial cells in hypoxic/ischemic conditions. Transgenic mice over-expressing endothelin-1 in endothelial cells (TET-1) developed systemic hypertension and showed more severe brain damage after transient ischemia. To further understand the significance of endothelial ET-1 in cognitive deficit, we subjected adult TET-1 mice to 30 min middle cerebral artery occlusion (MCAO) with 7 days reperfusion. At baseline, TET-1 mice showed similar locomotor activity, emotion and cognitive function compared to non-transgenic (NTg) mice. However, after 30 min MCAO and 7 days reperfusion, although the sensorimotor function measured by neurological scores was recovered in both genotypes, TET-1 mice showed increased anxiety-like behavior in the open field test and impaired spatial learning and reference memory in the Morris water maze. Parallel with these behavioral changes, TET-1 mice showed more severe brain damage with blood-brain-barrier breakdown (BBB), reactive astrogliosis, increased caspase-3, and increased peroxiredoxin 6 (Prx6) expressions around blood vessels in the ipsilateral hippocampus, compared to that of NTg mice, suggesting that ET-1 over-expression in the endothelial cells leads to more severe BBB breakdown and increased oxidative stress which may resulted in neuronal apoptosis and glial reactivity, which might contribute to the emotional changes and cognitive deficits after short-term ischemia with long-term reperfusion.

Embryonic development is characterized by dynamic changes in gene expression, yet the role of chromatin remodeling in these cellular transitions remains elusive. To address this question, we profiled the transcriptome and select chromatin modifications at defined stages during pancreatic endocrine differentiation of human embryonic stem cells. We identify removal of Polycomb group (PcG)-mediated repression on stage-specific genes as a key mechanism for the induction of developmental regulators. Furthermore, we discover that silencing of transitory genes during lineage progression associates with reinstatement of PcG-dependent repression. Significantly, in vivo- but not in vitro-differentiated endocrine cells exhibit close similarity to primary human islets in regard to transcriptome and chromatin structure. We further demonstrate that endocrine cells produced in vitro do not fully eliminate PcG-mediated repression on endocrine-specific genes, probably contributing to their malfunction. These studies reveal dynamic chromatin remodeling during developmental lineage progression and identify possible strategies for improving cell differentiation in culture.

<h4>Objective</h4>To identify lipodystrophy in prepubertal HIV-infected children using anthropometric parameters and body composition assessment.<h4>Methods</h4>Cross-sectional study including 40 prepubertal HIV-infected children of both genders seen at the Care Center of the Division of Pediatric Infectious Diseases - Universidade Federal de São Paulo, São Paulo city, Brazil, was carried out from August to December 2008. Age, clinical and immunological status, prophylaxis, transmission and highly active antiretroviral therapy were recorded. Body mass index z-score and height-for-age z-score were calculated to characterize the nutritional status. Circumferences were measured with flexible tape and skinfolds were assessed by an adipometry. Fat mass and lean mass were determined by dual-energy X-ray absorptiometry. Presence of clinical signs of lipodystrophy was assessed by a trained clinician. Data were analyzed using SPSS 12.0 software.<h4>Results</h4>The mean age and standard deviation were 9.8 (1.2) years, 50% were girls and 82.5% children from B and C categories. Clinical lipodystrophy and dislypidemia were present in 27.5% and 70%, respectively. The trunk to arm ratio and the limb to trunk ratio had positive association with lipodystrophy. Patients with lipodystrophy had short stature, higher triglycerides values and lower HDL-cholesterol.<h4>Conclusion</h4>The ratios obtained by skinfolds and dual-energy X-ray absorptiometry measurements can be considered as indicators of preclinical lipodystrophy. The cutoff points have not been determined yet; however, continuous assessment may be useful to identify early body composition changes.

The expression of the chicken delta-protocadherin (Pcdh) subfamily was investigated in the developing feather buds of the chicken. The expression profiles of the eight investigated Pcdhs in the cells and tissues of the feather buds differ from each other. Pcdh1, Pcdh7, Pcdh8 and Pcdh10 are differentially expressed in the epidermis of the feather bud. Expression of Pcdh1 and Pcdh10 is restricted to the periderm and Pcdh17 expression to the epidermis of interbud region. Pcdh19 is mostly expressed at the anterior side of epidermis as well as in the blood vessels of the feather buds. Furthermore, Pcdh9 and Pcdh18 both are regionally expressed in the dermis of the feather bud. These results suggest that Pcdhs may play a variety of roles during avian feather bud formation.

Alopecia areata (AA) is a common autoimmune disorder mostly presented as round patches of hair loss and subclassified into alopecia totalis/alopecia universalis (AT/AU) based on the area of alopecia. Although AA is relatively common, only 5% of AA patients progress to AT/AU, which affect the whole scalp and whole body respectively. To determine genetic determinants of this orphan disease, we undertook whole-exome sequencing of 6 samples from AU patients, and 26 variants in immune-related genes were selected as candidates. When an additional 14 AU samples were genotyped for these candidates, 6 of them remained at the level of significance in comparison with 155 Asian controls (p<1.92×10(-3)). Linkage disequilibrium was observed between some of the most significant SNPs, including rs41559420 of HLA-DRB5 (p<0.001, OR 44.57) and rs28362679 of BTNL2 (p<0.001, OR 30.21). While BTNL2 was reported as a general susceptibility gene of AA previously, HLA-DRB5 has not been implicated in AA. In addition, we found several genetic variants in novel genes (HLA-DMB, TLR1, and PMS2) and discovered an additional locus on HLA-A, a known susceptibility gene of AA. This study provides further evidence for the association of previously reported genes with AA and novel findings such as HLA-DRB5, which might represent a hidden culprit gene for AU.

Neural sexual differentiation begins during embryogenesis and continues after birth for a variable amount of time depending on the species and brain region. Because gonadal hormones were the first factors identified in neural sexual differentiation, their role in this process has eclipsed investigation of other factors. Here, we use a mouse with a spontaneous translocation that produces four different unique sets of sex chromosomes. Each genotype has one normal X-chromosome and a unique second sex chromosome creating the following genotypes: XY(*x) , XX, XY(*) , XX(Y) (*) . This Y(*) mouse line is used by several laboratories to study two human aneuploid conditions: Turner and Klinefelter syndromes. As sex chromosome number affects behavior and brain morphology, we surveyed brain gene expression at embryonic days 11.5 and 18.5 to isolate X-chromosome dose effects in the developing brain as possible mechanistic changes underlying the phenotypes. We compared gene expression differences between gonadal males and females as well as individuals with one vs. two X-chromosomes. We present data showing, in addition to genes reported to escape X-inactivation, a number of autosomal genes are differentially expressed between the sexes and in mice with different numbers of X-chromosomes. Based on our results, we can now identify the genes present in the region around the chromosomal break point that produces the Y(*) model. Our results also indicate an interaction between gonadal development and sex chromosome number that could further elucidate the role of sex chromosome genes and hormones in the sexual differentiation of behavior.

MicroRNA-137 (miR-137) expression has been reported to be decreased in astrocytic tumors in two expression profiling studies but its role in the pathogenesis of oligodendroglial tumors is still limited. In this study, we demonstrate that miR-137 expression is significantly downregulated in a cohort of 35 oligodendroglial tumors and nine glioma cell lines compared with normal brains. Lower miR-137 expression is associated with shorter progressive-free survival and overall survival. Restoration of miR-137 expression in an oligodendroglial cells TC620, and also glioblastoma cells of U87 and U373 significantly suppressed cell growth, anchorage-independent growth, as well as invasion. Demethylation and deacetylation treatments resulted in upregulation of miR-137 expression in TC620 cells. In silico analysis showed that CSE1 chromosome segregation 1-like (yeast) (CSE1L) is a potential target gene of miR-137. Luciferase reporter assay demonstrated that miR-137 negatively regulates CSE1L by interaction between miR-137 and complementary sequences in the 3' UTR of CSE1L. Immunohistochemistry revealed that CSE1L is upregulated in oligodendroglial tumors. Knockdown of CSE1L resulted in similar outcomes as overexpressing miR-137 in oligodendroglioma cells and glioblastoma cells. Overall, our data suggest that miR-137 regulates growth of glioma cells and targets CSE1L, providing further understanding in the tumorigenesis of gliomas.

<h4>Background</h4>Pica and restless legs syndrome (RLS) are associated with iron depletion and deficiency. The presence of pica and RLS was prospectively assessed in blood donors.<h4>Study design and methods</h4>During a 39-month period, 1236 donors deferred for fingerstick hemoglobin (Hb) level of less than 12.5 g/dL and 400 nondeferred "control" donors underwent health screening and laboratory testing (complete blood count, ferritin, iron, transferrin). Pica and RLS were assessed by direct questioning. Deferred donors and iron-deficient control donors were given 325 mg of ferrous sulfate daily for 60 days. Reassessments were performed and additional iron tablets dispensed at subsequent visits.<h4>Results</h4>Pica was reported in 11% of donors with iron depletion or deficiency, compared with 4% of iron-replete donors (p < 0.0001). Pagophagia (ice pica) was most common and often of extraordinary intensity. Female sex, younger age, and lower mean cell volume and transferrin saturation values were strongly associated with pica. Donors with pica given iron reported a marked reduction in the desire to consume the nonnutritive substance by Days 5 to 8 of therapy, with disappearance of symptoms by Days 10 to 14. RLS was reported in 16% of subjects with iron depletion or deficiency compared with 11% of iron-replete donors (p = 0.012). Iron replacement generally resulted in improvement of RLS symptoms; however, at least 4 to 6 weeks of iron therapy was necessary.<h4>Conclusion</h4>The presence of pica is associated with a high probability of iron depletion or deficiency in blood donors; however, RLS lacks a strong correlation in this population. Screening questions for pagophagia may be useful in the ascertainment of iron deficiency in donors and may identify those who would benefit from oral iron.

The role of angiogenesis factors in skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease (COPD) is unknown. The first objective of this study was to assess various pro- and antiangiogenic factor and receptor expressions in the vastus lateralis muscles of control subjects and COPD patients. Preliminary inquiries revealed that angiopoietin-2 (ANGPT2) is overexpressed in limb muscles of COPD patients. ANGPT2 promotes skeletal satellite cell survival and differentiation. Factors that are involved in regulating muscle ANGPT2 production are unknown. The second objective of this study was to evaluate how oxidants and proinflammatory cytokines influence muscle-derived ANGPT2 expression. Angiogenic gene expressions in human vastus lateralis biopsies were quantified with low-density real-time PCR arrays. ANGPT2 mRNA expressions in cultured skeletal myoblasts were quantified in response to proinflammatory cytokine and H2O2 exposure. Ten proangiogenesis genes, including ANGPT2, were significantly upregulated in the vastus lateralis muscles of COPD patients. ANGPT2 mRNA levels correlated negatively with forced expiratory volume in 1 s and positively with muscle wasting. Immunoblotting confirmed that ANGPT2 protein levels were significantly greater in muscles of COPD patients compared with control subjects. ANGPT2 expression was induced by interferon-γ and -β and by hydrogen peroxide, but not by tumor necrosis factor. We conclude that upregulation of ANGPT2 expression in vastus lateralis muscles of COPD patients is likely due to oxidative stress and represents a positive adaptive response aimed at facilitating myogenesis and angiogenesis.

Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions, and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA, and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, whereas epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors, and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by quantitative ChIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that human terminal erythropoiesis is surprisingly associated with hypomethylation of the genome.

<h4>Background</h4>The objective of this study was to discover molecular biomarkers associated with the recurrence of esophageal squamous cell carcinoma (ESCC).<h4>Methods</h4>The authors retrospectively analyzed the hypermethylation status of 11 genes using methylation-specific polymerase chain reaction (PCR) and the expression of epidermal growth factor receptor (EGFR), O-6 methylguanine-DNA methyltransferase (MGMT), tumor protein 53 (p53), and transforming growth factor β (TGFβ) using immunohistochemistry in 329 formalin-fixed, paraffin-embedded ESCCs.<h4>Results</h4>Recurrence was identified in 151 of 329 ESCCs (46%) at a median follow-up of 4.5 years. The recurrence was associated with hypermethylation of the genes cell adhesion molecule 1 (CADM1) (P = .003), deleted in colon carcinoma (DCC) (P = .04), or cyclin-dependent kinase inhibitor 2A (p14) (P = .02) in patients with stage I ESCC. Thirty-six of 37 Stage I ESCCs (97%) that had cohypermethylation of at least 2 of the 3 genes had hypermethylation of p14 plus either CADM1 or DCC or both CADM1 and DCC. The 5-year recurrence-free survival (RFS) rates were 93% in patients who had stage I disease without hypermethylation of the 3 genes and 56% in those who had cohypermethylation of p14 in combination with CADM1 and/or DCC. Patients who had stage I ESCC with cohypermethylation of p14 in combination with DCC and/or CADM1 had 7.13 times (95% confidence interval, 1.61-31.64 times; P = .009) poorer RFS compared with those who had no hypermethylation of the 3 genes after adjusting confounding factors. Hypermethylation of the other 8 genes and altered expression of 4 proteins were not associated with recurrence across pathologic stages.<h4>Conclusions</h4>The current results suggested that cohypermethylation of p14 in combination with DCC and/or CADM1 may be an independent prognostic factor for recurrence in patients with stage I ESCC.

Abnormalities in glycan biosynthesis have been conclusively linked to many diseases but the complexity of glycosylation has hindered the analysis of glycan data in order to identify glycoforms contributing to disease. To overcome this limitation, we developed a quantitative N-glycosylation model that interprets and integrates mass spectral and transcriptomic data by incorporating key glycosylation enzyme activities. Using the cancer progression model of androgen-dependent to androgen-independent Lymph Node Carcinoma of the Prostate (LNCaP) cells, the N-glycosylation model identified and quantified glycan structural details not typically derived from single-stage mass spectral or gene expression data. Differences between the cell types uncovered include increases in H(II) and Le(y) epitopes, corresponding to greater activity of α2-Fuc-transferase (FUT1) in the androgen-independent cells. The model further elucidated limitations in the two analytical platforms including a defect in the microarray for detecting the GnTV (MGAT5) enzyme. Our results demonstrate the potential of systems glycobiology tools for elucidating key glycan biomarkers and potential therapeutic targets. The integration of multiple data sets represents an important application of systems biology for understanding complex cellular processes.

Infection with Epstein-Barr virus (EBV) is highly prevalent worldwide, and it has been associated with infectious mononucleosis and severe diseases including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal lymphoma, and lymphoproliferative disorders. Although EBV has been the focus of extensive research, much still remains unknown concerning what makes some individuals more sensitive to infection and to adverse outcomes as a result of infection. Here we use an integrative genomics approach in order to localize genetic factors influencing levels of Epstein Barr virus (EBV) nuclear antigen-1 (EBNA-1) IgG antibodies, as a measure of history of infection with this pathogen, in large Mexican American families. Genome-wide evidence of both significant linkage and association was obtained on chromosome 6 in the human leukocyte antigen (HLA) region and replicated in an independent Mexican American sample of large families (minimum p-value in combined analysis of both datasets is 1.4×10(-15) for SNPs rs477515 and rs2516049). Conditional association analyses indicate the presence of at least two separate loci within MHC class II, and along with lymphocyte expression data suggest genes HLA-DRB1 and HLA-DQB1 as the best candidates. The association signals are specific to EBV and are not found with IgG antibodies to 12 other pathogens examined, and therefore do not simply reveal a general HLA effect. We investigated whether SNPs significantly associated with diseases in which EBV is known or suspected to play a role (namely nasopharyngeal lymphoma, Hodgkin lymphoma, systemic lupus erythematosus, and multiple sclerosis) also show evidence of associated with EBNA-1 antibody levels, finding an overlap only for the HLA locus, but none elsewhere in the genome. The significance of this work is that a major locus related to EBV infection has been identified, which may ultimately reveal the underlying mechanisms by which the immune system regulates infection with this pathogen.

The use of clinical features to define subtypes of a disorder may aid in gene identification for complex diseases. In particular, clinical subtypes of mania may distinguish phenotypic subgroups of bipolar subjects that may also differ genetically. To assess this possibility, we performed a genome-wide association study using genotype data from the Bipolar Genome Study (BiGS) and subjects that were categorized as having either irritable or elated mania during their most severe episode. A bipolar case-only analysis in the GAIN bipolar sample identified several genomic regions that differed between irritable and elated subjects, the most significant of which was for 33 SNPs on chromosome 13q31 (peak p = 2×10(-7)). This broad peak is in a relative gene desert over an unknown EST and between the SLITRK1 and SLITRK6 genes. Evidence for association to this region came predominantly from subjects in the sample that were originally collected as part of a family-based bipolar linkage study, rather than those collected as bipolar singletons. We then genotyped an additional sample of bipolar singleton cases and controls, and the analysis of irritable vs. elated mania in this new sample did not replicate our previous findings. However, this lack of replication is likely due to the presence of significant differences in terms of clinical co-morbity that were identified between these singleton bipolar cases and those that were selected from families segregating the disorder. Despite these clinical differences, analysis of the combined sample provided continued support for 13q31 and other regions from our initial analysis. Though genome-wide significance was not achieved, our results suggest that irritable mania results from a distinct set of genes, including a region on chromosome 13q31.

Noni (Morinda citrifolia) juice is a popular herbal dietary supplement globally used for preventive or therapeutic purposes in a variety of ailments, claiming to exhibit hepatoprotective properties as well. Herein we present the case of a 38-year-old woman who developed acute liver injury associated with noni juice consumption on a long-term (9 months) anticonvulsant therapy. Clinical presentation and liver biopsy were consistent with severe, predominantly hepatocellular type of injury. Both agents were stopped and corticosteroids were initiated. Five months later the patient had fully recovered. Although in the literature the hepatotoxicity of noni juice remains speculative, sporadic but emerging cases of noni juice-associated liver injury address the need to clarify and investigate potential harmful effects associated with this supplement.

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a toxic Huntingtin (HTT) protein. The use of short interfering RNAs (siRNAs) to silence the mutant protein is one of the most promising therapeutic strategies under investigation. The biggest caveat to siRNA-based approaches is the lack of efficient and nontoxic delivery vectors for siRNA delivery to the central nervous system. In this study, we investigated the potential of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as novel siRNA neuronal carriers. We show that CDs formed nanosize particles which were stable in artificial cerebrospinal fluid. Moreover, these complexes were able to reduce the expression of the HTT gene in rat striatal cells (ST14A-HTT120Q) and in human HD primary fibroblasts. Only limited toxicity was observed with CD·siRNA nanoparticles in any of the in vitro models used. Sustained knockdown effects were observed in the striatum of the R6/2 mouse model of HD after single direct injections of CD·siRNA nanoparticles. Repeated brain injections of CD·siRNA complexes resulted in selective alleviation of motor deficits in this mouse model. Together these data support the utility of modified β-CDs as efficient and safe siRNA delivery vectors for RNAi-based therapies for neuropsychiatric and neurodegenerative disorders.

In Huntington disease (HD), polyglutamine expansion in the huntingtin protein causes specific neuronal death. The consequences of the presence of mutant huntingtin in other tissues are less well understood. Here we propose that mutant huntingtin influences breast cancer progression. Indeed, we show that mammary tumours appear earlier in mouse breast cancer models expressing mutant huntingtin as compared to control mice expressing wild-type huntingtin. Tumours bearing mutant huntingtin have a modified gene expression pattern that reflects enhanced aggressiveness with the overexpression of genes favouring invasion and metastasis. In agreement, mutant huntingtin accelerates epithelial to mesenchymal transition and enhances cell motility and invasion. Also, lung metastasis is higher in HD conditions than in control mice. Finally, we report that in HD, the dynamin dependent endocytosis of the ErbB2/HER2 receptor tyrosine kinase is reduced. This leads to its accumulation and to subsequent increases in cell motility and proliferation. Our study may thus have important implications for both cancer and HD.

Bovine papillomaviruses (BPVs) are oncogenic DNA viruses, which mainly induce benign lesions of cutaneous and/or mucosal epithelia in cattle. Thirteen (BPV 1-13) different viral genotypes have been characterized so far. BPVs are usually species-specific but BPV 1/2 may also infect equids as well as buffaloes and bison and cause tumors in these species. BPV-induced benign lesions usually regress, however occasionally they develop into cancer particularly in the presence of environmental carcinogenic co-factors. The major transforming protein of BPV is E5, a very short hydrophobic, transmembrane protein with many oncogenic activities. E5 contributes to cell transformation through the activation of the cellular β receptor for the platelet-derived growth factor (PDGFβ-r), it also decreases cell surface expression of major histocompatibility complex class I (MHCI) causing viral escape from immunosurveillance, and plays a role in the inhibition of the intracellular communication by means of aberrant connexin expression. E7 is considered as a weak transforming gene, it synergies with E5 in cell transformation during cancer development. E7 expression correlates in vivo with the over-expression of β1-integrin, which plays a role in the regulation of keratinocytes proliferation and differentiation. Additionally, E7 is involved in cell-mediated immune responses leading to tumour rejection, in anoikis process by direct binding to p600, and in invasion process by upregulation of Matrix metalloproteinase1 (MMP-1) expression. Studies on the role of BPV E5 and E7 oncoproteins in naturally occurring tumours are of scientific value, as they may shed new light on the biological role of these two oncogenes in cell transformation.

The left and right sides of the nervous system communicate via commissural axons that cross the midline during development using evolutionarily conserved molecules. These guidance cues have been particularly well studied in the mammalian spinal cord, but it remains unclear whether these guidance mechanisms for commissural axons are similar in the developing forebrain, in particular for the corpus callosum, the largest and most important commissure for cortical function. Here, we show that Netrin1 initially attracts callosal pioneering axons derived from the cingulate cortex, but surprisingly is not attractive for the neocortical callosal axons that make up the bulk of the projection. Instead, we show that Netrin-deleted in colorectal cancer signaling acts in a fundamentally different manner, to prevent the Slit2-mediated repulsion of precrossing axons thereby allowing them to approach and cross the midline. These results provide the first evidence for how callosal axons integrate multiple guidance cues to navigate the midline.

High-mobility group box 1 protein (HMGB1), which mainly exists in the nucleus, has recently been shown to function as a sentinel molecule for viral nucleic acid sensing and an autophagy regulator in the cytoplasm. In this study, we studied the chaperone-like activity of HMGB1 and found that HMGB1 inhibited the chemically induced aggregation of insulin and lysozyme, as well as the heat-induced aggregation of citrate synthase. HMGB1 also restored the heat-induced suppression of cytoplasmic luciferase activity as a reporter protein in hamster lung fibroblast O23 cells with expression of HMGB1. Next, we demonstrated that HMGB1 inhibited the formation of aggregates and toxicity caused by expanded polyglutamine (polyQ), one of the main causes of Huntington disease. HMGB1 directly interacted with polyQ on immunofluorescence and coimmunoprecipitation assay, whereas the overexpression of HMGB1 or exogenous administration of recombinant HMGB1 protein remarkably reduced polyQ aggregates in SHSY5Y cells and hmgb1(-/-) mouse embryonic fibroblasts upon filter trap and immunofluorescence assay. Finally, overexpressed HMGB1 proteins in mouse embryonic primary striatal neurons also bound to polyQ and decreased the formation of polyQ aggregates. To this end, we have demonstrated that HMGB1 exhibits chaperone-like activity and a possible therapeutic candidate in polyQ disease.

Cohesin and condensin complexes are essential for defining the topology of chromosomes through the cell cycle. Here we look at the emerging role of these complexes in regulating chromatin structure and gene expression and reflect on how these activities could be linked with chromosome topology.

Hypoxia is one of the major stressors at high altitude. Exposure to hypobaric hypoxia induces several adverse consequences to the structural and functional integrity of brain. In an attempt to understand the proteome modulation, we used 2-DE coupled with MALDI-TOF/TOF for cortex and hippocampus exposed to short-term temporal (0, 3, 6, 12 and 24h) hypobaric hypoxia. This enabled us in the identification of 88 and 73 hypoxia responsive proteins in cortex and hippocampus respectively. We further compared the proteomes of both the regions and identified 37 common proteins along with 49 and 32 specific proteins for cortex and hippocampus respectively. We observed significant up-regulation of glycolytic enzymes like Gapdh, Pgam1, Eno1 and malate-aspartate shuttle enzymes Mdh1 and Got1in cortex as compared to hippocampus deciphering efficient use of energy producing substrates. This was coupled with concomitant increase in expression of antioxidant enzymes like Sod1, Sod2 and Pebp1 in cortex to neutralize the hypoxia-induced reactive oxygen species (ROS) generation. Our comparative proteomics studies demonstrate that efficient use of energy generating pathways in conjugation with abundance of antioxidant enzymes makes cortex less vulnerable to hypoxia than hippocampus.

Traumatic spinal cord injury (SCI) results in changes to the anatomical, neurochemical, and physiological properties of cells in the central and peripheral nervous system. Neurotrophins, acting by binding to their cognate Trk receptors on target cell membranes, contribute to modulation of anatomical, neurochemical, and physiological properties of neurons in sensorimotor circuits in both the intact and injured spinal cord. Neurotrophin signaling is associated with many post-SCI changes including maladaptive plasticity leading to pain and autonomic dysreflexia, but also therapeutic approaches such as training-induced locomotor improvement. Here we characterize expression of mRNA for neurotrophins and Trk receptors in lumbar dorsal root ganglia (DRG) and spinal cord after two different severities of mid-thoracic injury and at 6 and 12 weeks post-SCI. There was complex regulation that differed with tissue, injury severity, and survival time, including reversals of regulation between 6 and 12 weeks, and the data suggest that natural regulation of neurotrophins in the spinal cord may continue for months after birth. Our assessments determined that a coordination of gene expression emerged at the 12-week post-SCI time point and bioinformatic analyses address possible mechanisms. These data can inform studies meant to determine the role of the neurotrophin signaling system in post-SCI function and plasticity, and studies using this signaling system as a therapeutic approach.

Bipolar disorder (BPD) is a complex psychiatric trait with high heritability. Despite efforts through conducting genome-wide association (GWA) studies, the success of identifying susceptibility loci for BPD has been limited, which is partially attributed to the complex nature of its pathogenesis. Pathway-based analytic strategy is a powerful tool to explore joint effects of gene sets within specific biological pathways. Additionally, to incorporate other aspects of genomic data into pathway analysis may further enhance our understanding for the underlying mechanisms for BPD. Patterns of DNA methylation play important roles in regulating gene expression and function. A commonly observed phenomenon, allele-specific methylation (ASM) describes the associations between genetic variants and DNA methylation patterns. The present study aimed to identify biological pathways that are involve in the pathogenesis of BPD while incorporating brain specific ASM information in pathway analysis using two large-scale GWA datasets in Caucasian populations. A weighting scheme was adopted to take ASM information into consideration for each pathway. After multiple testing corrections, we identified 88 and 15 enriched pathways for their biological relevance for BPD in the Genetic Association Information Network (GAIN) and the Wellcome Trust Case Control Consortium dataset, respectively. Many of these pathways were significant only when applying the weighting scheme. Three ion channel related pathways were consistently identified in both datasets. Results in the GAIN dataset also suggest for the roles of extracellular matrix in brain for BPD. Findings from Gene Ontology (GO) analysis exhibited functional enrichment among genes of non-GO pathways in activity of gated channel, transporter, and neurotransmitter receptor. We demonstrated that integrating different data sources with pathway analysis provides an avenue to identify promising and novel biological pathways for exploring the underlying molecular mechanisms for bipolar disorder. Further basic research can be conducted to target the biological mechanisms for the identified genes and pathways.

The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.

To explore the molecular pathways underlying thiazolidinediones effects on pancreatic islets in conditions mimicking normo- and hyperglycemia, apoptosis rate and transcriptional response to Pioglitazone at both physiological and supraphysiological glucose concentrations were evaluated. Adult rat islets were cultured at physiological (5.6 mM) and supraphysiological (23 mM) glucose concentrations in presence of 10 μM Pioglitazone or vehicle. RNA expression profiling was evaluated with the PancChip 13k cDNA microarray after 24-h, and expression results for some selected genes were validated by qRT-PCR. The effects of Pioglitazone were investigated regarding apoptosis rate after 24-, 48- and 72-h. At 5.6 mM glucose, 101 genes were modulated by Pioglitazone, while 1,235 genes were affected at 23 mM glucose. Gene networks related to lipid metabolism were identified as altered by Pioglitazone at both glucose concentrations. At 23 mM glucose, cell cycle and cell death pathways were significantly regulated as well. At 5.6 mM glucose, Pioglitazone elicited a transient reduction in islets apoptosis rate while at 23 mM, Bcl2 expression was reduced and apoptosis rate was increased by Pioglitazone. Our data demonstrate that the effect of Pioglitazone on gene expression profile and apoptosis rate depends on the glucose concentration. The modulation of genes related to cell death and the increased apoptosis rate observed at supraphysiological glucose concentration raise concerns about Pioglitazone's direct effects in conditions of hyperglycemia and reinforce the necessity of additional studies designed to evaluate TZDs effects on the preservation of β-cell function in situations where glucotoxicity might be more relevant than lipotoxicity.

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.

The mood stabilizers lithium and valproic acid (VPA) are traditionally used to treat bipolar disorder (BD), a severe mental illness arising from complex interactions between genes and environment that drive deficits in cellular plasticity and resiliency. The therapeutic potential of these drugs in other central nervous system diseases is also gaining support. This article reviews the various mechanisms of action of lithium and VPA gleaned from cellular and animal models of neurologic, neurodegenerative, and neuropsychiatric disorders. Clinical evidence is included when available to provide a comprehensive perspective of the field and to acknowledge some of the limitations of these treatments. First, the review describes how action at these drugs' primary targets--glycogen synthase kinase-3 for lithium and histone deacetylases for VPA--induces the transcription and expression of neurotrophic, angiogenic, and neuroprotective proteins. Cell survival signaling cascades, oxidative stress pathways, and protein quality control mechanisms may further underlie lithium and VPA's beneficial actions. The ability of cotreatment to augment neuroprotection and enhance stem cell homing and migration is also discussed, as are microRNAs as new therapeutic targets. Finally, preclinical findings have shown that the neuroprotective benefits of these agents facilitate anti-inflammation, angiogenesis, neurogenesis, blood-brain barrier integrity, and disease-specific neuroprotection. These mechanisms can be compared with dysregulated disease mechanisms to suggest core cellular and molecular disturbances identifiable by specific risk biomarkers. Future clinical endeavors are warranted to determine the therapeutic potential of lithium and VPA across the spectrum of central nervous system diseases, with particular emphasis on a personalized medicine approach toward treating these disorders.

No abstract available.

Mutagenesis screens can establish mouse models of utility for the study of critical biological processes such as iron metabolism. Such screens can produce mutations in novel genes or establish novel alleles of known genes, both of which can be useful tools for study. In order to identify genes of relevance to hematologic as well as other phenotypes, we performed N-ethyl-N-nitrosourea mutagenesis in C57BL/6J mice. An anemic mouse was identified and a putative mutation was characterized by mapping, sequencing and in vitro activity analysis. The mouse strain was backcrossed for ten generations then phenotypically characterized with respect to a previously established null mouse strain. Potential modifying loci were identified by quantitative trait locus analysis. Mapping and sequencing in an anemic mouse termed hem8 identified an I286F substitution in Tmprss6, a serine protease essential for iron metabolism; this substitution impaired in vitro protease activity. After backcrossing to C57BL6/J for ten generations, the hem8(-/-) strain exhibited a phenotype similar in some but not all aspects to that of Tmprss6(-/-) mice. The hem8 and Tmprss6-null mutations were allelic. Both hem8(-/-) and Tmprss6(-/-) mice responded similarly to pharmacological modulators of bone morphogenetic protein signaling, a key regulator of iron metabolism. Quantitative trait locus analysis in the hem8 strain identified potential modifying loci on chromosomes 2, 4, 7 and 10. In conclusion, the hem8 mouse model carries a novel allele of Tmprss6. Potential uses for this strain in the study of iron metabolism are discussed.

Evidence is limited as to whether heritable risk of obesity varies throughout adulthood. Among >34,000 European Americans, aged 18-100 years, from multiple U.S. studies in the Population Architecture using Genomics and Epidemiology (PAGE) Consortium, we examined evidence for heterogeneity in the associations of five established obesity risk variants (near FTO, GNPDA2, MTCH2, TMEM18, and NEGR1) with BMI across four distinct epochs of adulthood: 1) young adulthood (ages 18-25 years), adulthood (ages 26-49 years), middle-age adulthood (ages 50-69 years), and older adulthood (ages ≥70 years); or 2) by menopausal status in women and stratification by age 50 years in men. Summary-effect estimates from each meta-analysis were compared for heterogeneity across the life epochs. We found heterogeneity in the association of the FTO (rs8050136) variant with BMI across the four adulthood epochs (P = 0.0006), with larger effects in young adults relative to older adults (β [SE] = 1.17 [0.45] vs. 0.09 [0.09] kg/m², respectively, per A allele) and smaller intermediate effects. We found no evidence for heterogeneity in the association of GNPDA2, MTCH2, TMEM18, and NEGR1 with BMI across adulthood. Genetic predisposition to obesity may have greater effects on body weight in young compared with older adulthood for FTO, suggesting changes by age, generation, or secular trends. Future research should compare and contrast our findings with results using longitudinal data.

We performed a genome-wide association study (GWAS) and a multistage meta-analysis of type 2 diabetes (T2D) in Punjabi Sikhs from India. Our discovery GWAS in 1,616 individuals (842 case subjects) was followed by in silico replication of the top 513 independent single nucleotide polymorphisms (SNPs) (P < 10⁻³) in Punjabi Sikhs (n = 2,819; 801 case subjects). We further replicated 66 SNPs (P < 10⁻⁴) through genotyping in a Punjabi Sikh sample (n = 2,894; 1,711 case subjects). On combined meta-analysis in Sikh populations (n = 7,329; 3,354 case subjects), we identified a novel locus in association with T2D at 13q12 represented by a directly genotyped intronic SNP (rs9552911, P = 1.82 × 10⁻⁸) in the SGCG gene. Next, we undertook in silico replication (stage 2b) of the top 513 signals (P < 10⁻³) in 29,157 non-Sikh South Asians (10,971 case subjects) and de novo genotyping of up to 31 top signals (P < 10⁻⁴) in 10,817 South Asians (5,157 case subjects) (stage 3b). In combined South Asian meta-analysis, we observed six suggestive associations (P < 10⁻⁵ to < 10⁻⁷), including SNPs at HMG1L1/CTCFL, PLXNA4, SCAP, and chr5p11. Further evaluation of 31 top SNPs in 33,707 East Asians (16,746 case subjects) (stage 3c) and 47,117 Europeans (8,130 case subjects) (stage 3d), and joint meta-analysis of 128,127 individuals (44,358 case subjects) from 27 multiethnic studies, did not reveal any additional loci nor was there any evidence of replication for the new variant. Our findings provide new evidence on the presence of a population-specific signal in relation to T2D, which may provide additional insights into T2D pathogenesis.

Blood pressure (BP) is a heritable determinant of risk for cardiovascular disease (CVD). To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP) and pulse pressure (PP), we genotyped ∼50 000 single-nucleotide polymorphisms (SNPs) that capture variation in ∼2100 candidate genes for cardiovascular phenotypes in 61 619 individuals of European ancestry from cohort studies in the USA and Europe. We identified novel associations between rs347591 and SBP (chromosome 3p25.3, in an intron of HRH1) and between rs2169137 and DBP (chromosome1q32.1 in an intron of MDM4) and between rs2014408 and SBP (chromosome 11p15 in an intron of SOX6), previously reported to be associated with MAP. We also confirmed 10 previously known loci associated with SBP, DBP, MAP or PP (ADRB1, ATP2B1, SH2B3/ATXN2, CSK, CYP17A1, FURIN, HFE, LSP1, MTHFR, SOX6) at array-wide significance (P < 2.4 × 10(-6)). We then replicated these associations in an independent set of 65 886 individuals of European ancestry. The findings from expression QTL (eQTL) analysis showed associations of SNPs in the MDM4 region with MDM4 expression. We did not find any evidence of association of the two novel SNPs in MDM4 and HRH1 with sequelae of high BP including coronary artery disease (CAD), left ventricular hypertrophy (LVH) or stroke. In summary, we identified two novel loci associated with BP and confirmed multiple previously reported associations. Our findings extend our understanding of genes involved in BP regulation, some of which may eventually provide new targets for therapeutic intervention.

Diverse life forms are driven by the evolution of gene regulatory programs including changes in regulator proteins and cis-regulatory elements. Alterations of cis-regulatory elements are likely to dominate the evolution of the gene regulatory networks, as they are subjected to smaller selective constraints compared with proteins and hence may evolve quickly to adapt the environment. Prior studies on cis-regulatory element evolution focus primarily on sequence substitutions of known transcription factor-binding motifs. However, evolutionary models for the dynamics of motif occurrence are relatively rare, and comprehensive characterization of the evolution of all possible motif sequences has not been pursued. In the present study, we propose an algorithm to estimate the strength of purifying selection of a motif sequence based on an evolutionary model capturing the birth and death of motif occurrences on promoters. We term this measure as the 'evolutionary retention coefficient', as it is related yet distinct from the canonical definition of selection coefficient in population genetics. Using this algorithm, we estimate and report the evolutionary retention coefficients of all possible 10-nucleotide sequences from the aligned promoter sequences of 27 748. orthologous gene families in 34 mammalian species. Intriguingly, the evolutionary retention coefficients of motifs are intimately associated with their functional relevance. Top-ranking motifs (sorted by evolutionary retention coefficients) are significantly enriched with transcription factor-binding sequences according to the curated knowledge from the TRANSFAC database and the ChIP-seq data generated from the ENCODE Consortium. Moreover, genes harbouring high-scoring motifs on their promoters retain significantly coherent expression profiles, and those genes are over-represented in the functional classes involved in gene regulation. The validation results reveal the dependencies between natural selection and functions of cis-regulatory elements and shed light on the evolution of gene regulatory networks.

High quality protocols facilitate proper conduct, reporting, and external review of clinical trials. However, the completeness of trial protocols is often inadequate. To help improve the content and quality of protocols, an international group of stakeholders developed the SPIRIT 2013 Statement (Standard Protocol Items: Recommendations for Interventional Trials). The SPIRIT Statement provides guidance in the form of a checklist of recommended items to include in a clinical trial protocol. This SPIRIT 2013 Explanation and Elaboration paper provides important information to promote full understanding of the checklist recommendations. For each checklist item, we provide a rationale and detailed description; a model example from an actual protocol; and relevant references supporting its importance. We strongly recommend that this explanatory paper be used in conjunction with the SPIRIT Statement. A website of resources is also available (www.spirit-statement.org). The SPIRIT 2013 Explanation and Elaboration paper, together with the Statement, should help with the drafting of trial protocols. Complete documentation of key trial elements can facilitate transparency and protocol review for the benefit of all stakeholders.

<h4>Purpose</h4>Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe(-/-) mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated.<h4>Methods</h4>Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4.<h4>Results</h4>Hfe(-/-) RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe(-/-) cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe(-/-) RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe(-/-) cells.<h4>Conclusions</h4>RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe(-/-) RPE cells as well as in Hjv(-/-) RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe(-/-) and Hjv(-/-) RPE cells.

In developing brains, neural progenitors exhibit cell cycle-dependent nuclear movement within the ventricular zone [interkinetic nuclear migration (INM)] and actively proliferate to produce daughter progenitors and/or neurons, whereas newly generated neurons exit from the cell cycle and begin pial surface-directed migration and maturation. Dysregulation of the balance between the proliferation and the cell cycle exit in neural progenitors is one of the major causes of microcephaly (small brain). Recent studies indicate that cell cycle machinery influences not only the proliferation but also INM in neural progenitors. Furthermore, several cell cycle-related proteins, including p27(kip1) , p57(kip2) , Cdk5, and Rb, regulate the migration of neurons in the postmitotic state, suggesting that the growth arrest confers dual functions on cell cycle regulators. Consistently, several types of microcephaly occur in conjunction with neuronal migration disorders, such as periventricular heterotopia and lissencephaly. However, cell cycle re-entry by disturbance of growth arrest in mature neurons is thought to trigger neuronal cell death in Alzheimer's disease. In this review, we introduce the cell cycle protein-mediated regulation of two types of nuclear movement, INM and neuronal migration, during cerebral cortical development, and discuss the roles of growth arrest in cortical development and neurological disorders.

<h4>Background</h4>Melatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis.<h4>Results</h4>In the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for "DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer".<h4>Conclusions</h4>These findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways.

The Atg1/ULK1 complex plays a central role in starvation-induced autophagy, integrating signals from upstream sensors such as MTOR and AMPK and transducing them to the downstream autophagy pathway. Much progress has been made in the last few years in understanding the mechanisms by which the complex is regulated through protein-protein interactions and post-translational modifications, providing insights into how the cell modulates autophagy, particularly in response to nutrient status. However, how the ULK1 complex transduces upstream signals to the downstream central autophagy pathway is still unclear. Although the protein kinase activity of ULK1 is required for its autophagic function, its protein substrate(s) responsible for autophagy activation has not been identified. Furthermore, examples of potential ULK1-independent autophagy have emerged, indicating that under certain specific contexts, the ULK1 complex might be dispensable for autophagy activation. This raises the question of how the autophagic machinery is activated independent of the ULK1 complex and what are the biological functions of such noncanonical autophagy pathways.

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian "output" genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of "core" clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

The first 17 amino acids of Huntington's disease (HD) protein, huntingtin, comprise an amphipathic alpha-helical domain that can target huntingtin to the endoplasmic reticulum (ER). N17 is phosphorylated at two serines, shown to be important for disease development in genetic mouse models, and shown to be modified by agents that reverse the disease phenotype in an HD mouse model. Here, we show that the hydrophobic face of N17 comprises a consensus CRM1/exportin-dependent nuclear export signal, and that this nuclear export activity can be affected by serine phospho-mimetic mutants. We define the precise residues that comprise this nuclear export sequence (NES) as well as the interaction of the NES, but not phospho-mimetic mutants, with the CRM1 nuclear export factor. We show that the nuclear localization of huntingtin depends upon the RanGTP/GDP gradient, and that N17 phosphorylation can also distinguish localization of endogenous huntingtin between the basal body and stalk of the primary cilium. We present a mechanism and multifunctional role for N17 in which phosphorylation of N17 not only releases huntingtin from the ER to allow nuclear entry, but also prevents nuclear export during a transient stress response event to increase the levels of nuclear huntingtin and to regulate huntingtin access to the primary cilium. Thus, N17 is a master localization signal of huntingtin that can mediate huntingtin localization between the cytoplasm, nucleus and primary cilium. This localization can be regulated by signaling, and is misregulated in HD.

The MHC fold is found in proteins that have a range of functions in the maintenance of an organism's health, from immune regulation to fat metabolism. Well adapted for antigen presentation, as seen for peptides in the classical MHC molecules and for lipids in CD1 molecules, the MHC fold has also been modified to perform Fc-receptor activity (e.g., FcRn) and for roles in host homeostasis (e.g., with HFE and ZAG). The more divergent MHC-like molecules, such as some of those that interact with the NKG2D receptor, represent the minimal MHC fold, doing away with the α3 domain and β2m while maintaining the α1/α2 platform domain for receptor engagement. Viruses have also co-opted the MHC fold for immune-evasive functions. The variations on the theme of a β-sheet topped by two semiparallel α-helices are discussed in this review, highlighting the fantastic adaptability of this fold for good and for bad.

During embryonic development of the mammalian cerebral cortex, postmitotic cortical neurons migrate radially from the ventricular zone to the cortical plate. Proper migration involves the correct orientation of migrating neurons and the transition from a multipolar to a mature bipolar morphology. Herein, we report that the 2 isoforms of Myosin-10 (Myo10) play distinct roles in the regulation of radial migration in the mouse cortex. We show that the full-length Myo10 (fMyo10) isoform is located in deeper layers of the cortex and is involved in establishing proper migration orientation. We also demonstrate that fMyo10-dependent orientation of radial migration is mediated at least in part by the netrin-1 receptor deleted in colorectal cancer. Moreover, we show that the headless Myo10 (hMyo10) isoform is required for the transition from multipolar to bipolar morphologies in the intermediate zone. Our study reveals divergent functions for the 2 Myo10 isoforms in controlling both the direction of migration and neuronal morphogenesis during radial cortical neuronal migration.

Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T) cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours), and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.

With the development of increasingly large and complex genomic and proteomic data sets, an enhancement in the complexity of available Venn diagram analytical programs is becoming increasingly important. Current freely available Venn diagram programs often fail to represent extra complexity among datasets, such as regulation pattern differences between different groups. Here we describe the development of VennPlex, a program that illustrates the often diverse numerical interactions among multiple, high-complexity datasets, using up to four data sets. VennPlex includes versatile output features, where grouped data points in specific regions can be easily exported into a spreadsheet. This program is able to facilitate the analysis of two to four gene sets and their corresponding expression values in a user-friendly manner. To demonstrate its unique experimental utility we applied VennPlex to a complex paradigm, i.e. a comparison of the effect of multiple oxygen tension environments (1-20% ambient oxygen) upon gene transcription of primary rat astrocytes. VennPlex accurately dissects complex data sets reliably into easily identifiable groups for straightforward analysis and data output. This program, which is an improvement over currently available Venn diagram programs, is able to rapidly extract important datasets that represent the variety of expression patterns available within the data sets, showing potential applications in fields like genomics, proteomics, and bioinformatics.

Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation.

A key feature of the innate antiviral immune response is a rapid nonspecific response to virus infection largely mediated by the induction and extracellular secretion of type I interferons (IFNs) that restrict virus replication. Cytoplasmic sensors such as RIG-I recognize viral RNA and trigger antiviral signaling pathways that upregulate IFN transcription. However, it remains largely unknown how antiviral signaling is negatively regulated to maintain homeostasis after the elimination of virus. In this report, we have identified the RING domain-containing protein RING finger 11 (RNF11) as a novel negative regulator of innate antiviral signaling. Overexpression of RNF11 downregulated IFN-β expression and enhanced viral replication whereas siRNA-mediated knockdown of RNF11 suppressed viral replication. RNF11 interacted with the noncanonical IKK kinases TBK1/IKKi and attenuated their Lys63-linked polyubiquitination by blocking interactions with the E3 ligase TRAF3. The inhibitory function of RNF11 was dependent on the ubiquitin-binding adaptor molecule TAX1BP1 which was required for RNF11 to target TBK1/IKKi. Collectively, these results indicate that RNF11 functions together with TAX1BP1 to restrict antiviral signaling and IFN-β production.

A novel bis-lipoyl derivative containing 8-hydroxyquinoline scaffold (LA-HQ-LA, 5) was synthesized as a new multifunctional drug candidate with antioxidant, chelant, and neuroprotective properties for the treatment of neurodegenerative diseases. We have investigated the potential effectiveness of LA-HQ-LA against the cytotoxicity induced by 6-OHDA and H2O2 on human neuroblastoma SH-SY5Y cell line. Our outcomes showed that LA-HQ-LA resulted in significant neuroprotective and antioxidant effects against H2O2- and 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cells, as assessed by MTT assay. In particular, it showed potent neuroprotective effects against 6-OHDA in RA/PMA differentiated cells at all the tested concentrations.

There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases-confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development.

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the 'cytochrome P450 genesis locus', where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.

<h4>Unlabelled</h4><h4>Background</h4>Different histone post-translational modifications (PTMs) fine-tune and integrate different cellular signaling pathways at the chromatin level. ADP-ribose modification of histones by cellular ADP-ribosyltransferases such as ARTD1 (PARP1) is one of the many elements of the histone code. All 5 histone proteins were described to be ADP-ribosylated in vitro and in vivo. However, the crosstalk between ADP-ribosylation and other modifications is little understood.<h4>Results</h4>In experiments with isolated histones, it was found that ADP-ribosylation of H3 by ARTD1 prevents H3 methylation by SET7/9. However, poly(ADP-ribosyl)ation (PARylation) of histone H3 surprisingly allowed subsequent methylation of H1 by SET7/9. Histone H1 was thus identified as a new target for SET7/9. The SET7/9 methylation sites in H1.4 were pinpointed to the last lysine residues of the six KAK motifs in the C-terminal domain (K121, K129, K159, K171, K177 and K192). Interestingly, H1 and the known SET7/9 target protein H3 competed with each other for SET7/9-dependent methylation.<h4>Conclusions</h4>The results presented here identify H1.4 as a novel SET7/9 target protein, and document an intricate crosstalk between H3 and H1 methylation and PARylation, thus implying substrate competition as a regulatory mechanism. Thereby, these results underline the role of ADP-ribosylation as an element of the histone code.

Molecular subclassification is rapidly informing the clinical management of medulloblastoma. However, the disease remains associated with poor outcomes and therapy-associated late effects, and the majority of patients are not characterized by a validated prognostic biomarker. Here, we investigated the potential of epigenetic DNA methylation for disease subclassification, particularly in formalin-fixed biopsies, and to identify biomarkers for improved therapeutic individualization. Tumor DNA methylation profiles were assessed, alongside molecular and clinical disease features, in 230 patients primarily from the SIOP-UKCCSG PNET3 clinical trial. We demonstrate by cross-validation in frozen training and formalin-fixed test sets that medulloblastoma comprises four robust DNA methylation subgroups (termed WNT, SHH, G3 and G4), highly related to their transcriptomic counterparts, and which display distinct molecular, clinical and pathological disease characteristics. WNT patients displayed an expected favorable prognosis, while outcomes for SHH, G3 and G4 were equivalent in our cohort. MXI1 and IL8 methylation were identified as novel independent high-risk biomarkers in cross-validated survival models of non-WNT patients, and were validated using non-array methods. Incorporation of MXI1 and IL8 into current survival models significantly improved the assignment of disease risk; 46 % of patients could be classified as 'favorable risk' (>90 % survival) compared to 13 % using current models, while the high-risk group was reduced from 30 to 16 %. DNA methylation profiling enables the robust subclassification of four disease subgroups in frozen and routinely collected/archival formalin-fixed biopsy material, and the incorporation of DNA methylation biomarkers can significantly improve disease-risk stratification. These findings have important implications for future risk-adapted clinical disease management.

<h4>Objective</h4>To examine whether preterm very low birth weight (VLBW) infants have an increased risk of late-onset sepsis (LOS) following early-onset sepsis (EOS).<h4>Study design</h4>Retrospective analysis of VLBW infants (401-1500 g) born September 1998 through December 2009 who survived >72 hours and were cared for within the National Institute of Child Health and Human Development Neonatal Research Network. Sepsis was defined by growth of bacteria or fungi in a blood culture obtained ≤ 72 hours of birth (EOS) or >72 hours (LOS) and antimicrobial therapy for ≥ 5 days or death <5 days while receiving therapy. Regression models were used to assess risk of death or LOS by 120 days and LOS by 120 days among survivors to discharge or 120 days, adjusting for gestational age and other covariates.<h4>Results</h4>Of 34,396 infants studied, 504 (1.5%) had EOS. After adjustment, risk of death or LOS by 120 days did not differ overall for infants with EOS compared with those without EOS [risk ratio (RR): 0.99 (0.89-1.09)] but was reduced in infants born at <25 weeks gestation [RR: 0.87 (0.76-0.99), P = .048]. Among survivors, no difference in LOS risk was found overall for infants with versus without EOS [RR: 0.88 (0.75-1.02)], but LOS risk was reduced in infants with birth weight 401-750 g who had EOS [RR: 0.80 (0.64-0.99), P = .047].<h4>Conclusions</h4>Risk of LOS after EOS was not increased in VLBW infants. Surprisingly, risk of LOS following EOS appeared to be reduced in the smallest, most premature infants, underscoring the need for age-specific analyses of immune function.

The mechanisms by which mesenchymal stromal cells (MSCs) induce immunomodulation are still poorly understood. In the current work, we show by a combination of polymerase chain reaction (PCR) array, flow cytometry, and multiplex cytokine data analysis that during the inhibition of an alloantigen-driven CD4+ T-cell response, MSCs induce a fraction of CD4+ T-cells to coexpress interferon-γ (IFNγ) and interleukin-10 (IL-10). This CD4+ IFNγ+ IL-10+ cell population shares properties with recently described T-cells originating from switched Th1 cells that start producing IL-10 and acquire a regulatory function. Here we report that IL-10-producing Th1 cells accumulated with time during T-cell stimulation in the presence of MSCs. Moreover, MSCs caused stimulated T-cells to downregulate the IFNγ receptor (IFNγR) without affecting IL-10 receptor expression. Further, the inhibitory effect of MSCs could be reversed by an anti-IFNγR-blocking antibody, indicating that IFNγ is one of the major players in MSC-induced T-cell suppression. Stimulated (and, to a lesser extent, resting) CD4+ T-cells treated with MSCs were able to inhibit the proliferation of autologous CD4+ T-cells, demonstrating their acquired regulatory properties. Altogether, our results suggest that the generation of IL-10-producing Th1 cells is one of the mechanisms by which MSCs can downmodulate an immune response.

The human 2-oxoglutarate (2OG) dependent oxygenases belong to a family of structurally related enzymes that play important roles in many biological processes. We report that competition-based NMR methods, using 2OG as a reporter ligand, can be used for quantitative and site-specific screening of ligand binding to 2OG oxygenases. The method was demonstrated using hypoxia inducible factor hydroxylases and histone demethylases, and K(D) values were determined for inhibitors that compete with 2OG at the metal center. This technique is also useful as a screening or validation tool for inhibitor discovery, as exemplified by work with protein-directed dynamic combinatorial chemistry.

The biological behavior of prostate cancer is uncertain, and therefore, search for molecular prognostic markers associated with disease progression seems to be essential. We performed microsatellite allelotyping of DNA isolated from primary prostate tumors biopsies (prostate adenocarcinoma, PCa). We evaluated the frequency of allelic imbalance (AI), including loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) as well as the association of these DNA alterations with clinicopathological variables. We assessed the significance of LOH/MSI alterations in selected imprinted and non-imprinted chromosomal regions (IR and NIR) in PCa. A total of 50 biopsies of prostate tumor (containing >75 % tumor cells) were histologically examined confirming prostate carcinoma. Microsatellite allelotyping using 16 microsatellite markers linked to the following chromosomal regions: 1p31.2, 3p21.3-25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1 was performed. The incidence of LOH/MSI alterations in prostate tumor cells was the highest for chromosomal regions 7q32.2 and 16q22.1 (31.25 and 26.60 %, respectively), followed by 1p31.2 and 3p21.3-25.3 (26.50 and 17.40 %, respectively). Statistically significant increase in LOH/MSI alterations has been observed for markers: D1S2137 (1p region; p = 0.00032), D9S974 (9p region; p = 0.0017), and D16S3025 (16q region; p = 0.0017). Statistically significant differences in frequency of LOH/MSI alterations in particular chromosomal regions have been found for 1p31.2, 7q32.2 and 16q22.1 (p = 0.027, p = 0.012 and p = 0.031, respectively). We documented statistically significant association between Fractional Allele Loss (FAL) index and advanced tumor stage (p < 0.05). We suggest that genomic instability of LOH/MSI type is a frequent event in prostate carcinogenesis and assessed as FAL index has clinical value for the molecular staging of prostate cancer in (TRUS)-guided prostate biopsy material.