Gene Literature Dashboard

To identify susceptibility loci for meningioma, we conducted a genome-wide association study of 859 affected individuals (cases) and 704 controls with validation in two independent sample sets totaling 774 cases and 1,764 controls. We identified a new susceptibility locus for meningioma at 10p12.31 (MLLT10, rs11012732, odds ratio = 1.46, P(combined) = 1.88 × 10(-14)). This finding advances our understanding of the genetic basis of meningioma development.

<h4>Background</h4>Angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed.<h4>Methodology/principal findings</h4>Primary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H(2)O(2), but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation.<h4>Conclusion/significance</h4>Skeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair.

<h4>Objective</h4>This study aims to investigate the regulation of expression of Cartilage oligomeric matrix protein (COMP), which is predominately expressed by chondrocytes and functions to organize the extracellular matrix. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia and multiple epiphyseal dysplasia. The mechanism controlling COMP expression during chondrocyte differentiation is still poorly understood.<h4>Design</h4>Primary human bone marrow-derived stem cells were induced to differentiate into chondrocyte by pellet cultures. We then compared the temporal expression of COMP with the well-characterized cartilage-specific Type II collagen (Col2a1), and their response to transforming growth factor (TGF)β and Sox trio (Sox5, 6, and 9) stimulation.<h4>Results</h4>COMP and Col2a1 expression are differentially regulated by three distinct mechanisms. First, upregulation of COMP mRNA precedes Col2a1 by several days during chondrogenesis. Second, COMP expression is independent of high cell density but requires TGF-β1. Induction of COMP mRNA by TGF-β1 is detected within 2h in the absence of protein synthesis and is blocked by specific inhibitors of the TGFβ signaling pathway; and therefore, COMP is a primary TFGβ-response gene. Lastly, while Col2a1 expression is intimately controlled by the Sox trio, overexpression of Sox trio fails to activate the COMP promoter.<h4>Conclusion</h4>COMP and Col2a1 expression are regulated differently during chondrogenesis. COMP is a primary response gene of TGFβ and its fast induction during chondrogenesis suggests that COMP is suitable for rapidly accessing the chondrogenic potential of stem cells.

Striatal-enriched protein tyrosine phosphatase (STEP) has recently been implicated in several neuropsychiatric disorders with significant cognitive impairments, including Alzheimer's disease, schizophrenia, and fragile X syndrome. A model has emerged by which STEP normally opposes the development of synaptic strengthening and that disruption in STEP activity leads to aberrant synaptic function. We review the mechanisms by which STEP contributes to the etiology of these and other neuropsychiatric disorders. These findings suggest that disruptions in STEP activity may be a common mechanism for cognitive impairments in diverse illnesses.

Oxidative stress is commonly associated with a number of liver diseases and is thought to play a role in the pathogenesis of chronic hepatitis C, alcoholic liver disease, non-alcoholic steatohepatitis (NASH), haemochromatosis and Wilson's disease. Antioxidant therapy has thus been considered to have the possibility of beneficial effects in the management of these liver diseases. Despite this promise, antioxidants have produced mixed results in a number of clinical trials of efficacy. This review summarizes the results of clinical trials of antioxidants as sole or adjuvant therapy of chronic hepatitis C, alcoholic liver disease and non-alcoholic steatohepatitis (NASH). Overall, the most promising results to date are for vitamin E therapy of NASH but some encouraging results have been obtained with antioxidant therapy of acute alcoholic hepatitis as well. Despite evidence for small reductions of serum alanine aminotransferase, there is as yet no convincing evidence that antioxidant therapy itself is beneficial to patients with chronic hepatitis C. Problems such as small sample size, short follow up duration, inadequate endpoints, failure to demonstrate tissue delivery and antioxidant efficacy, and heterogeneous nature of the 'antioxidant' compounds used have complicated interpretation of results of the clinical studies. These limitations and their implications for future trial design are discussed.

<h4>Background</h4>In Huntington disease, the accurate determination of age-at-onset is critical to identify modifiers and therapies that aim to delay it.<h4>Methods</h4>Retrospective data from the European Huntington's Disease Network's REGISTRY and PREDICT-HD, a longitudinal study in prodromal huntingtin gene expansion mutation carriers. Data (age, gender, CAG repeat length, parent affected, and Unified Huntington's Disease Rating Scale motor score, total functional capacity) from at least three visits in 423 REGISTRY and 124 PREDICT-HD participants were included. Data based extrapolations of individual age-at-onset using generalized linear mixed models based on individual slopes of motor score or total functional capacity, and predictions using the Langbehn, or Ranen formula, were compared with clinicians' estimates.<h4>Results</h4>Concordance was best for the observed age-at-onset in PREDICT-HD and the calculated onset using the PREDICT-HD UHDRS longitudinal motor scores. This was superior to the REGISTRY data. For total functional capacity, the investigator's estimate was 4 years before the data derived age-at-onset. The concordance of predictions of probability of age-at-onset is better with the observed age-at-onset in the PREDICT-HD data (difference in 25%tile -5 to 10 years) than the REGISTRY data (±20 years).<h4>Conclusions</h4>Estimating or predicting age-at-onset in Huntington disease may be inaccurate. It can be useful to 1) add in the manifest population motor score regression derived age-at-onset as additional motor onset and 2) add total functional capacity regression derived age-at-onset for the onset of functional impact of Huntington disease when patients are in mid- to late-stage.

<h4>Background</h4>Interspecific hybridization may lead to the introgression of genes and genomes across species barriers and contribute to a reticulate evolutionary pattern and thus taxonomic uncertainties. Since several previous studies have demonstrated that introgressive hybridization has occurred among some species within Lepus, therefore it is possible that introgressive hybridization events also occur among Chinese Lepus species and contribute to the current taxonomic confusion.<h4>Results</h4>Data from four mtDNA genes, from 116 individuals, and one nuclear gene, from 119 individuals, provides the first evidence of frequent introgression events via historical and recent interspecific hybridizations among six Chinese Lepus species. Remarkably, the mtDNA of L. mandshuricus was completely replaced by mtDNA from L. timidus and L. sinensis. Analysis of the nuclear DNA sequence revealed a high proportion of heterozygous genotypes containing alleles from two divergent clades and that several haplotypes were shared among species, suggesting repeated and recent introgression. Furthermore, results from the present analyses suggest that Chinese hares belong to eight species.<h4>Conclusion</h4>This study provides a framework for understanding the patterns of speciation and the taxonomy of this clade. The existence of morphological intermediates and atypical mitochondrial gene genealogies resulting from frequent hybridization events likely contribute to the current taxonomic confusion of Chinese hares. The present study also demonstrated that nuclear gene sequence could offer a powerful complementary data set with mtDNA in tracing a complete evolutionary history of recently diverged species.

Metabolic syndrome is defined as a cluster of multiple risk factors, including central obesity, dyslipidemia, hypertension and impaired glucose tolerance, that increase cardiovascular disease morbidity and mortality. Genetic factors are important in the development of metabolic syndrome, as are environmental factors. However, the genetic background of metabolic syndrome is not yet fully clarified. There is evidence that obesity and obesity-related phenotypes are associated with variations in several genes, including NEGR1, SEC16B, TMEM18, ETV5, GNPDA2, BDNF, MTCH2, SH2B1, FTO, MAF, MC4R, KCTD15, SCG3, MTMR9, TFAP2B, MSRA, LYPLAL1, GCKR and FADS1. To investigate the relationship between metabolic syndrome and variations in these genes in the Japanese population, we genotyped 33 single-nucleotide polymorphisms (SNPs) in 19 genes from 1096 patients with metabolic syndrome and 581 control individuals who had no risk factors for metabolic syndrome. Four SNPs in the FTO gene were significantly related to metabolic syndrome: rs9939609 (P=0.00013), rs8050136 (P=0.00011), rs1558902 (P=6.6 × 10(-5)) and rs1421085 (P=7.4 × 10(-5)). rs3764220 in the SCG3 gene (P=0.0010) and rs2293855 in the MTMR9 gene (P=0.0015) were also significantly associated with metabolic syndrome. SNPs in the FTO, SCG3 and MTMR9 genes had no SNP × SNP epistatic effects on metabolic syndrome. Our data suggest that genetic variations in the FTO, SCG3 and MTMR9 genes independently influence the risk of metabolic syndrome.

Visceral fat accumulation has an important role in increasing morbidity and mortality rate by increasing the risk of developing several metabolic disorders, such as type 2 diabetes, dyslipidemia and hypertension. New genetic loci that contribute to the development of obesity have been identified by genome-wide association studies in Caucasian populations. We genotyped 1279 Japanese subjects (556 men and 723 women), who underwent computed tomography (CT) for measuring visceral fat area (VFA) and subcutaneous fat area (SFA), for the following single-nucleotide polymorphisms (SNPs): NEGR1 rs2815752, SEC16B rs10913469, TMEM18 rs6548238, ETV5 rs7647305, GNPDA2 rs10938397, BDNF rs6265 and rs925946, MTCH2 rs10838738, SH2B1 rs7498665, MAF rs1424233, and KCTD15 rs29941 and rs11084753. In the additive model, none of the SNPs were significantly associated with body mass index (BMI). The SH2B1 rs7498665 risk allele was found to be significantly associated with VFA (P=0.00047) but not with BMI or SFA. When the analysis was performed in men and women separately, no significant associations with VFA were observed (P=0.0099 in men and P=0.022 in women). None of the other SNPs were significantly associated with SFA. Our results suggest that there is a VFA-specific genetic factor and that a polymorphism in the SH2B1 gene influences the risk of visceral fat accumulation.

Prediction of the responses to neoadjuvant chemotherapy (NACT) can improve the treatment of patients with advanced breast cancer. Genes and proteins predictive of chemoresistance have been extensively studied in breast cancer tissues. However, noninvasive serum biomarkers capable of such prediction have been rarely exploited. Here, we performed profiling of N-glycosylated proteins in serum from fifteen advanced breast cancer patients (ten patients sensitive to and five patients resistant to NACT) to discover serum biomarkers of chemoresistance using a label-free liquid chromatography-tandem MS method. By performing a series of statistical analyses of the proteomic data, we selected thirteen biomarker candidates and tested their differential serum levels by Western blotting in 13 independent samples (eight patients sensitive to and five patients resistant to NACT). Among the candidates, we then selected the final set of six potential serum biomarkers (AHSG, APOB, C3, C9, CP, and ORM1) whose differential expression was confirmed in the independent samples. Finally, we demonstrated that a multivariate classification model using the six proteins could predict responses to NACT and further predict relapse-free survival of patients. In summary, global N-glycoproteome profile in serum revealed a protein pattern predictive of the responses to NACT, which can be further validated in large clinical studies.

<h4>Rationale</h4>Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3β1, α6β1, and α6β4) in nonendothelial cells.<h4>Objective</h4>We tested whether integrins are netrin-4 receptors in the endothelium.<h4>Methods and results</h4>The α6β1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or β1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or β1 inhibition. Finally, netrin-4 and α6β1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature.<h4>Conclusions</h4>The α6β1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.

Iron overload is associated with significant morbidity and mortality yet is easily treated. The objective of this study was to create a tool that could be easily adapted to clinical practice that indicates the likelihood of a patient having undetected iron overload. We used the National Health and Nutrition Examination Survey (NHANES) 1999-2002 for US adults aged 20 years and older to build a model (unweighted n=8,779). We chose potential variables for inclusion that could be gathered by self-report or measured without laboratory data and were suggested by past literature on hemochromatosis and iron overload. We computed logistic regressions to create the scores by initially evaluating the variables' relationship with elevated ferritin and elevated transferrin saturation and then using odds ratios to correspond to scores. The resulting score on the IRon Overload ScreeNing Tool (IRON) was then validated with data on 13,844 adults in the NHANES III, 1988-94. Predictors in the final tool were age, gender, previous diagnoses of liver condition, osteoporosis or thyroid disease. The IRON score yielded an area under the curve (AUC) in the NHANES 1999-02 of 0.720 and an AUC of 0.685 in the NHANES III validation sample. The IRON score is a tool to assist in identification of patients with iron overload that has several qualities that make it attractive for use in clinical practice with an undifferentiated patient population including brevity, easily collected information and predictive ability comparable to other tools that help in directing screening.

Esophageal adenocarcinoma (EAC) has become a major concern in Western countries due to rapid rises in incidence coupled with very poor survival rates. One of the key risk factors for the development of this cancer is the presence of Barrett's esophagus (BE), which is believed to form in response to repeated gastro-esophageal reflux. In this study we performed comparative, genome-wide expression profiling (using Illumina whole-genome Beadarrays) on total RNA extracted from esophageal biopsy tissues from individuals with EAC, BE (in the absence of EAC) and those with normal squamous epithelium. We combined these data with publically accessible raw data from three similar studies to investigate key gene and ontology differences between these three tissue states. The results support the deduction that BE is a tissue with enhanced glycoprotein synthesis machinery (DPP4, ATP2A3, AGR2) designed to provide strong mucosal defenses aimed at resisting gastro-esophageal reflux. EAC exhibits the enhanced extracellular matrix remodeling (collagens, IGFBP7, PLAU) effects expected in an aggressive form of cancer, as well as evidence of reduced expression of genes associated with mucosal (MUC6, CA2, TFF1) and xenobiotic (AKR1C2, AKR1B10) defenses. When our results are compared to previous whole-genome expression profiling studies keratin, mucin, annexin and trefoil factor gene groups are the most frequently represented differentially expressed gene families. Eleven genes identified here are also represented in at least 3 other profiling studies. We used these genes to discriminate between squamous epithelium, BE and EAC within the two largest cohorts using a support vector machine leave one out cross validation (LOOCV) analysis. While this method was satisfactory for discriminating squamous epithelium and BE, it demonstrates the need for more detailed investigations into profiling changes between BE and EAC.

Childhood maltreatment represents a significant risk factor for psychopathology. Recent research has begun to examine both the functional and structural neurobiological correlates of adverse care-giving experiences, including maltreatment, and how these might impact on a child's psychological and emotional development. The relationship between such experiences and risk for psychopathology has been shown to vary as a function of genetic factors. In this review we begin by providing a brief overview of neuroendocrine findings, which indicate an association between maltreatment and atypical development of the hypothalamic-pituitary-adrenal axis stress response, which may predispose to psychiatric vulnerability in adulthood. We then selectively review the magnetic resonance imaging (MRI) studies that have investigated possible structural and functional brain differences in children and adults who have experienced childhood maltreatment. Differences in the corpus callosum identified by structural MRI have now been reliably reported in children who have experienced abuse, while differences in the hippocampus have been reported in adults with childhood histories of maltreatment. In addition, there is preliminary evidence from functional MRI studies of adults who have experienced childhood maltreatment of amygdala hyperactivity and atypical activation of frontal regions. These functional differences can be partly understood in the context of the information biases observed in event-related potential and behavioral studies of physically abused children. Finally we consider research that has indicated that the effect of environmental adversity may be moderated by genotype, reviewing pertinent studies pointing to gene by environment interactions. We conclude by exploring the extent to which the growing evidence base in relation to neurobiological and genetic research may be relevant to clinical practice and intervention.

Like the other more well-characterized post-translational modifications (phosphorylation, methylation, acetylation, acylation, etc.), the attachment of the 76 amino acid ubiquitin (Ub) protein to substrates has been shown to govern countless cellular processes. As obligate intracellular parasites, viruses have evolved the capability to commandeer many host processes in order to maximize their own survival, whether it be to increase viral production or to ensure the long-term survival of latently infected host cells. The first evidence that viruses could usurp the Ub system came from the DNA tumor viruses and Adenoviruses, each of which use Ub to dysregulate the host cell cycle (Scheffner et al., 1990; Querido et al., 2001). Today, the list of viruses that utilize Ub includes members from almost every viral class, encompassing both RNA and DNA viruses. Among these, there are examples of Ub usage at every stage of the viral life cycle, involving both ubiquitination and de-ubiquitination. In addition to viruses that merely modify the host Ub system, many of the large DNA viruses encode their own Ub modifying machinery. In this review, we highlight the latest discoveries regarding the myriad ways that viruses utilize Ub to their advantage.

Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Friedreich's ataxia are the most common human neurodegenerative diseases pathologically characterized by a progressive and specific loss of certain neuronal populations. The exact mechanisms of neuronal cell death in these diseases are unclear, although some forms of the diseases are inherited and genes causing these diseases have been identified. Currently there are no effective clinical therapies for many of these diseases. The recently acquired ability to reprogram human adult somatic cells to induced pluripotent stem cells (iPSCs) in culture may provide a powerful tool for in vitro neurodegenerative disease modeling and an unlimited source for cell replacement therapy. In the present review, we summarize recent progress on iPSC generation and differentiation into neuronal cell types and discuss the potential application for in vitro disease mechanism study and in vivo cell replacement therapy.

The huntingtin (htt) mutation causes a polyglutamine expansion in the N-terminal region of protein. Mutant N-htt proteolytic fragments aggregate and cause cell death in Huntington's disease (HD). The normal huntingtin also can be cleaved by calpain and produce N-terminal htt fragments following ischemic injury, but the fate of cleaved fragment in dead neurons in the brain are unclear. To determine the localization of huntingtin following proteolysis, we examined htt expression after transient ischemic injury. Huntingtin immunoreactivity in mixed cultures of neuronal and astrocytes-derived clonal cells showed alteration of immunoreactivity from neurons into astrocytes. In the brain, both focal and global ischemia induced reactive astrocytes that were co-immunoreactive for huntingtin with elevated GFAP expression. The immunoreactive huntingtin was 55kDa calpain-cleaved N-terminal fragment, which appeared initially in the process, and extended into the cytoplasm of astrocytes. The results showed, after ischemic injury, huntingtin accumulated in astrocytes indicating that astrocytes may play a role in uptake of cleaved N-htt fragments.

The blood-brain barrier (BBB) is a multicellular vascular structure separating blood from the brain parenchyma that is composed of endothelial cells with tight intercellular junctions, surrounded by a basal lamina, astrocytes, and pericytes. Previous studies have generated detailed databases of the microvessel transcriptome; however, less information is available on the BBB at the protein level. In this study, we specifically focused on characterization of the membrane fraction of cells within the BBB to generate a more complete understanding of membrane transporters, tight junction proteins, and associated extracellular matrix proteins that are functional hallmarks of the BBB. We used Multidimensional Protein Identification Technology to identify a total of 1,143 proteins in mouse brain microvessels, of which 53% were determined to be membrane associated. Analyses of specific classes of BBB-associated proteins in the context of recent transcriptome reports provide a unique database to assess the relative contribution of genes at the level of both RNA and protein in the maintenance of normal BBB integrity.

Leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL) is a rare, autosomal recessive disorder caused by mutations in the gene encoding a mitochondrial aspartyl-tRNA synthetase, DARS2. The disease is characterized by progressive spastic ataxia and magnetic resonance imaging (MRI) shows a highly characteristic leukoencephalopathy with multiple long tract involvement. We describe the clinical and radiological features of two new cases of LBSL and report a novel pathogenic mutation in the DARS2 gene. Both patients had typical clinical and radiological findings, although no elevated lactate was found. The severity of MRI changes did not correlate with clinical course and severity suggesting that, although of highly specific diagnostic value, MRI does not necessarily reflect clinical activity and should not be used to assess disease severity or prognosis in LBSL.

Mutant transcripts containing expanded CUG repeats in the untranslated region are a pathogenic factor in myotonic dystrophy type 1 (DM1). The mutant RNA sequesters the muscleblind-like 1 (MBNL1) splicing factor and causes misregulation of the alternative splicing of multiple genes that are linked to clinical symptoms of the disease. In this study, we show that either long untranslated CAG repeat RNA or short synthetic CAG repeats induce splicing aberrations typical of DM1. Alternative splicing defects are also caused by translated CAG repeats in normal cells transfected with a mutant ATXN3 gene construct and in cells derived from spinocerebellar ataxia type 3 and Huntington's disease patients. Splicing misregulation is unlikely to be caused by traces of antisense transcripts with CUG repeats, and the possible trigger of this misregulation may be sequestration of the MBNL1 protein with nuclear RNA inclusions containing expanded CAG repeat transcripts. We propose that alternative splicing misregulation by mutant CAG repeats may contribute to the pathological features of polyglutamine disorders.

A general procedure, based on a new activated alkyne linker, for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition ('click' reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into 'clickable' azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 10(3) M(-1)s(-1) in the absence of heparin and 1.2 × 10(5) M(-1)s(-1) in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.

Cyclic AMP (cAMP) and calcium are ubiquitous, interdependent second messengers that regulate a wide range of cellular processes. During development of neuronal networks they are critical for the first step of circuit formation, transducing signals required for axon pathfinding. Surprisingly, the spatial and temporal cAMP and calcium codes used by axon guidance molecules are unknown. Here, we identify characteristics of cAMP and calcium transients generated in growth cones during Netrin-1-dependent axon guidance. In filopodia, Netrin-1-dependent Deleted in Colorectal Cancer (DCC) receptor activation induces a transient increase in cAMP that causes a brief increase in calcium transient frequency. In contrast, activation of DCC in growth cone centers leads to a transient calcium-dependent cAMP increase and a sustained increase in frequency of calcium transients. We show that filopodial cAMP transients regulate spinal axon guidance in vitro and commissural axon pathfinding in vivo. These growth cone codes provide a basis for selective activation of specific downstream effectors.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

<h4>Background</h4>Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator and autoantigen that is overexpressed in tumors, including prostate cancer (PCa). This oncoprotein promotes resistance to cell death induced by oxidative stress and chemotherapy by mechanisms that remain unclear. To get insights into these mechanisms we identified candidate target stress genes of LEDGF/p75 using pathway-specific gene expression profiling in PCa cells.<h4>Methods</h4>A "Human oxidative stress and antioxidant defense" qPCR array was used to identify genes exhibiting significant expression changes in response to knockdown or overexpression of LEDGF/p75 in PC-3 cells. Validation of array results was performed by additional qPCR and immunoblotting.<h4>Results</h4>Cytoglobin (CYGB), Phosphoinositide-binding protein PIP3-E/IPCEF-1, superoxidase dismutase 3 (SOD3), thyroid peroxidase (TPO), and albumin (ALB) exhibited significant transcript down- and up-regulation in response to LEDGF/p75 knockdown and overexpression, respectively. CYGB gene was selected for further validation based on its emerging role as a stress oncoprotein in human malignancies. In light of previous reports indicating that LEDGF/p75 regulates peroxiredoxin 6 (PRDX6), and that PRDXs exhibit differential expression in PCa, we also examined the relationship between these proteins in PCa cells. Our validation data revealed that changes in LEDGF/p75 transcript and protein expression in PCa cells closely paralleled those of CYGB, but not those of the PRDXs.<h4>Conclusions</h4>Our study identifies CYGB and other genes as stress genes potentially regulated by LEDGF/p75 in PCa cells, and provides a rationale for investigating their role in PCa and in promoting resistance to chemotherapy- and oxidative stress-induced cell death.

<h4>Objective</h4>To determine if selected pro-inflammatory and anti-inflammatory cytokines and/or mediators of inflammation reported to be related to the development of cerebral palsy (CP) predict neurodevelopmental outcome in extremely low birth weight infants.<h4>Study design</h4>Infants with birth weights ≤1000 g (n = 1067) had blood samples collected at birth and on days 3 ± 1, 7 ± 1, 14 ± 3, and 21 ± 3 to examine the association between cytokines and neurodevelopmental outcomes. The analyses were focused on 5 cytokines (interleukin [IL] 1β; IL-8; tumor necrosis factor-α; regulated upon activation, normal T-cell expressed, and secreted (RANTES); and IL-2) reported to be most predictive of CP in term and late preterm infants.<h4>Results</h4>IL-8 was higher on days 0-4 and subsequently in infants who developed CP compared with infants who did not develop CP in both unadjusted and adjusted analyses. Other cytokines (IL-12, IL-17, tumor necrosis factor-β, soluble IL rα, macrophage inflammatory protein 1β) were found to be altered on days 0-4 in infants who developed CP.<h4>Conclusions</h4>CP in former preterm infants may, in part, have a late perinatal and/or early neonatal inflammatory origin.

Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT) expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and genetic factors on fear-related behavior and the underlying neurophysiological basis is missing. Therefore, in this study we used the 5-HTT-deficient mouse model for studying emotional dysregulation to evaluate consequences of genotype specific disruption of 5-HTT function and repeated social defeat for fear-related behaviors and corresponding neurophysiological activities in the lateral amygdala (LA) and infralimbic region of the medial prefrontal cortex (mPFC) in male 5-HTT wild-type (+/+), homo- (-/-) and heterozygous (+/-) mice. Naive males and experienced losers (generated in a resident-intruder paradigm) of all three genotypes, unilaterally equipped with recording electrodes in LA and mPFC, underwent a Pavlovian fear conditioning. Fear memory and extinction of conditioned fear was examined while recording neuronal activity simultaneously with fear-related behavior. Compared to naive 5-HTT+/+ and +/- mice, 5-HTT-/- mice showed impaired recall of extinction. In addition, 5-HTT-/- and +/- experienced losers showed delayed extinction learning and impaired recall of extinction. Impaired behavioral responses were accompanied by increased theta synchronization between the LA and mPFC during extinction learning in 5-HTT-/- and +/- losers. Furthermore, impaired extinction recall was accompanied with increased theta synchronization in 5-HTT-/- naive and in 5-HTT-/- and +/- loser mice. In conclusion, extinction learning and memory of conditioned fear can be modulated by both the 5-HTT gene activity and social experiences in adulthood, accompanied by corresponding alterations of the theta activity in the amygdala-prefrontal cortex network.

Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.

Recent evidence suggests that exercise improves functional outcome in animal models of cerebral ischemia. Since netrin-1 and its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5B (Unc5B), act as important regulators in neural and vascular activities, we sought to determine whether netrin-1 and DCC and Unc5B are involved in the neuroprotective effects of exercise on rats with induced cerebral ischemia. A total of 108 rats were randomly distributed into three groups: sham-operated group (n = 12), middle cerebral artery occlusion (MCAO) group (n = 48), MCAO+treadmill exercise group (n = 48). Behavioral testing indicated that treadmill exercise could significantly improve neurologic deficits of rats with cerebral ischemia at day 14 and 28 after MCAO (n = 12, P<0.05 and P<0.01), but there was no significant difference at day 4 and 7. Quantitative reverse transcription polymerase chain reaction (qPCR) and Western blot analysis revealed that treadmill exercise enhanced netrin-1 and DCC expression, while it suppressed Unc5B expression in rat peri-ischemic brain area, especially at day 14 and 28 after MCAO (n = 4, P<0.05 or P<0.01). Immunofluorescence analysis showed that in the peri-ischemic area, netrin-1 was expressed in neuronal perikarya, DCC, however, was expressed in neural processes and peri-vascular astrocytes, while Unc5B was expressed mostly in neuronal perikarya and some processes. These results suggest that netrin-1 and its receptors DCC and Unc5B may engage in exercise-induced neural circuit remodeling in the peri-ischemic area, and exercise may promote survival of neurons in this area by regulating netrin-1-Unc5B signaling. Additionally, netrin-1 may also play a role in brain-blood barrier via DCC-immunoreactive peri-vascular astrocytes. In conclusion, we demonstrate that treadmill exercise has beneficial effects that may be attributed, at least in part, to the involvement of netrin-1 and its receptors DCC and Unc5B in the neuronal and vascular activities in brain-ischemic rats.

Aminoacyl p-nitroaniline (aminoacyl-pNA) and aminoacyl 7-amino-4-methylcoumarin (aminoacyl-AMC) are important synthons for the synthesis of chromogenic/fluorogenic protease substrates. A new efficient method was developed to synthesize aminoacyl-pNA and aminoacyl-AMC derivatives in excellent yields starting from either amino acids or their corresponding commercially available N-hydroxysuccinimide esters. The method involved the in situ formation of selenocarboxylate intermediate of protected amino acids and the subsequent non-nucleophilic amidation with an azide. Common protecting groups used in amino acid/peptide chemistry were all well-tolerated. The method was also successfully applied to the synthesis of a dipeptide conjugate, indicating that the methodology is applicable to the synthesis of chromogenic substrates containing short peptides. The method has general applicability to the synthesis of chromogenic and fluorogenic peptide substrates and represents a convenient and high-yield synthesis of N(α)-protected-aminoacyl-pNAs/AMCs, providing easy access to these important synthons for the construction of chromogenic/fluorogenic protease substrates through fragment condensation or stepwise elongation.

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.

Deferiprone was shown to reverse iron deposition in Friedreich's ataxia. This multi-center, unblinded, single-arm pilot study evaluated safety and efficacy of deferiprone for reducing cerebral iron accumulation in neurodegeneration with brain iron accumulation. Four patients with genetically-confirmed pantothenate kinase-associated neurodegeneration, and 2 with parkinsonism and focal dystonia, but inconclusive genetic tests, received 15 mg/kg deferiprone bid. Magnetic resonance imaging and neurological examinations were conducted at baseline, six and 12 months. Chelation treatment caused no apparent hematologic or neurological side effects. Magnetic resonance imaging revealed decreased iron accumulation in the globus pallidus of 2 patients (one with pantothenate kinase-associated neurodegeneration). Clinical rating scales and blinded video rating evaluations documented mild-to-moderate motor improvement in 3 patients (2 with pantothenate kinase-associated neurodegeneration). These results underline the safety and tolerability of deferiprone, and suggest that chelating treatment might be effective in improving neurological manifestations associated with iron accumulation. (Clinicaltrials.gov Identifier: NTC00907283).

Common sequence variants have recently joined rare structural polymorphisms as genetic factors with strong evidence for association with schizophrenia. Here we extend our previous genome-wide association study and meta-analysis (totalling 7 946 cases and 19 036 controls) by examining an expanded set of variants using an enlarged follow-up sample (up to 10 260 cases and 23 500 controls). In addition to previously reported alleles in the major histocompatibility complex region, near neurogranin (NRGN) and in an intron of transcription factor 4 (TCF4), we find two novel variants showing genome-wide significant association: rs2312147[C], upstream of vaccinia-related kinase 2 (VRK2) [odds ratio (OR) = 1.09, P = 1.9 × 10(-9)] and rs4309482[A], between coiled-coiled domain containing 68 (CCDC68) and TCF4, about 400 kb from the previously described risk allele, but not accounted for by its association (OR = 1.09, P = 7.8 × 10(-9)).

We have previously shown that copper supplementation extends the replicative life span of Saccharomyces cerevisiae when grown under conditions forcing cells to respire. We now show that copper's effect on life span is through Fet3p, a copper containing enzyme responsible for high affinity transport of iron into yeast cells. Life span extensions can also be obtained by supplementing the growth medium with 1mM ferric chloride. Extension by high iron levels is still dependent on the presence of Fet3p. Life span extension by iron or copper requires growth on media containing glycerol as the sole carbon source, which forces yeast to respire. Yeast grown on glucose containing media supplemented with iron show no extension of life span. The iron associated with cells grown in media supplemented with copper or iron is 1.4-1.8 times that of cells grown without copper or iron supplementation. As with copper supplementation, iron supplementation partially rescues the life span of superoxide dismutase mutants. Cells grown with copper supplementation display decreased production of superoxide as measured by dihydroethidium staining.

Heparan sulfate (HS) chains play crucial biological roles by binding to various signaling molecules including fibroblast growth factors (FGFs). Distinct sulfation patterns of HS chains are required for their binding to FGFs/FGF receptors (FGFRs). These sulfation patterns are putatively regulated by biosynthetic enzyme complexes, called GAGOSOMES, in the Golgi. While the structural requirements of HS-FGF interactions have been described previously, it is still unclear how the FGF-binding motif is assembled in vivo. In this study, we generated HS structures using biosynthetic enzymes in a sequential or concurrent manner to elucidate the potential mechanism by which the FGF1-binding HS motif is assembled. Our results indicate that the HS chains form ternary complexes with FGF1/FGFR when enzymes carry out modifications in a specific manner.

In attempt to discover novel aberrantly hypermethylated genes with putative tumor suppressor function in epithelial ovarian cancer (EOC), we applied expression profiling following pharmacologic inhibition of DNA methylation in EOC cell lines. Among the genes identified, one of particular interest was DOK1, or downstream of tyrosine kinase 1, previously recognized as a candidate tumor suppressor gene (TSG) for leukemia and other human malignancies. Using bisulfite sequencing, we determined that a 5'-non-coding DNA region (located at nt -1158 to -850, upstream of the DOK1 translation start codon) was extensively hypermethylated in primary serous EOC tumors compared with normal ovarian specimens; however, this hypermethylation was not associated with DOK1 suppression. On the contrary, DOK1 was found to be strongly overexpressed in serous EOC tumors as compared to normal tissue and importantly, DOK1 overexpression significantly correlated with improved progression-free survival (PFS) values of serous EOC patients. Ectopic modulation of DOK1 expression in EOC cells and consecutive functional analyses pointed toward association of DOK1 expression with increased EOC cell migration and proliferation, and better sensitivity to cisplatin treatment. Gene expression profiling and consecutive network and pathway analyses were also confirmative for DOK1 association with EOC cell migration and proliferation. These analyses were also indicative for DOK1 protective role in EOC tumorigenesis, linked to DOK1-mediated induction of some tumor suppressor factors and its suppression of pro-metastasis genes. Taken together, our findings are suggestive for a possible tumor suppressor role of DOK1 in EOC; however its implication in enhanced EOC cell migration and proliferation restrain us to conclude that DOK1 represents a true TSG in EOC. Further studies are needed to more completely elucidate the functional implications of DOK1 and other members of the DOK gene family in ovarian tumorigenesis.

Bipolar disorder (BD) is a severe mental disorder characterized by recurrent episodes of mania and depression. Serotonin transporter (HTT) is a target of antidepressants and is one of the strongest candidate molecules of mood disorder, however, genetic study showed equivocal results. Here, we performed promoter-wide DNA methylation analysis of lymphoblastoid cell lines (LCLs) derived from two pairs of monozygotic twins discordant for BD. To rule out the possible discordance of copy number variation (CNV) between twins, we performed CNV analysis and found the copy number profiles were nearly identical between the twin pairs except for immunoglobulin-related regions. Among the three genes we obtained as candidate regions showing distinct difference of DNA methylation between one of the two pairs, hypermethylation of SLC6A4, encoding HTT, in the bipolar twin was only confirmed by bisulfite sequencing. Then, promoter hypermethylation of SLC6A4 in LCLs of BD patients was confirmed in a case-control analysis. DNA methylation of SLC6A4 was significantly correlated with its mRNA expression level in individuals with the S/S genotype of HTTLPR, and mRNA expression level was lower in BD patients carrying the S/S genotype. Finally, DNA methylation of the same site was also higher in the postmortem brains of BD patients. This is the first study to report the role of epigenetic modification of SLC6A4 in BD using an unbiased approach, which provides an insight for its pathophysiology.

The heat shock response (HSR) is a highly conserved protective mechanism that enables cells to withstand diverse environmental stressors that disrupt protein homeostasis (proteostasis) and promote protein misfolding. It has been suggested that small-molecule drugs that elicit the HSR by activating the transcription factor heat shock factor 1 might help mitigate protein misfolding and aggregation in several devastating neurodegenerative disorders, including Huntington disease (HD). In this issue of the JCI, Labbadia et al. use a brain-penetrant Hsp90 inhibitor, HSP990, to induce the HSR in mouse models of HD. Unexpectedly, they observed that HSP990 confers only transient amelioration of a subset of HD-related phenotypes, because alterations in chromatin architecture impair the HSR upon disease progression. These findings suggest that synergistic combination therapies that simultaneously unleash the HSR and prevent its impairment are likely to be needed to restore proteostasis in HD.

Huntington disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. Previous studies have proposed that activation of the heat shock response (HSR) via the transcription factor heat shock factor 1 (HSF1) may be of therapeutic benefit. However, the effect of disease progression on the HSR and the therapeutic potential of this pathway are currently unknown. Here, we used a brain-penetrating HSP90 inhibitor and physiological, molecular, and behavioral readouts to demonstrate that pharmacological activation of HSF1 improves huntingtin aggregate load, motor performance, and other HD-related phenotypes in the R6/2 mouse model of HD. However, the beneficial effects of this treatment were transient and diminished with disease progression. Molecular analyses to understand the transient nature of these effects revealed altered chromatin architecture, reduced HSF1 binding, and impaired HSR accompanied disease progression in both the R6/2 transgenic and HdhQ150 knockin mouse models of HD. Taken together, our findings reveal that the HSR, a major inducible regulator of protein homeostasis and longevity, is disrupted in HD. Consequently, pharmacological induction of HSF1 as a therapeutic approach to HD is more complex than was previously anticipated.

Increased activity of SRC family kinases promotes tumor invasion and metastasis, and overexpression of the mitotic regulator Aurora kinase A (AURKA) drives tumor aneuploidy and chromosomal instability. These functions nominate SRC and AURKA as valuable therapeutic targets for cancer, and inhibitors for SRC and Aurora kinases are now being used in the clinic. In this study, we demonstrate potent synergy between multiple inhibitors of Aurora and SRC kinases in ovarian and colorectal cancer cell lines, but not in normal ovarian epithelial cell lines. Combination of Aurora and SRC inhibitors selectively killed cells that have undergone a preceding aberrant mitosis, and was associated with a postmitotic reattachment defect, and selective removal of aneuploid cell populations. Combined inhibition of Aurora kinase and SRC potentiated dasatinib-dependent loss of activated (Y(416)-phosphorylated) SRC. SRC and AURKA share a common interaction partner, NEDD9, which serves as a scaffolding protein with activities in cell attachment and mitotic control, suggesting SRC and AURKA might interact directly. In vitro, we observed physical interaction and mutual cross-phosphorylation between SRC and AURKA that enhanced SRC kinase activity. Together, these findings suggest that combination of SRC and Aurora-targeting inhibitors in the clinic may be a productive strategy.

<h4>Background</h4>Endocrine tissues play a fundamental role in maintaining homeostasis of plasma metabolites such as non-esterified fatty acids and glucose, the levels of which reflect the energy balance or the health status of animals. However, the relationship between the transcriptome of endocrine tissues and plasma metabolites has been poorly studied.<h4>Methods</h4>We determined the blood levels of 12 plasma metabolites in 27 pigs belonging to five breeds, each breed consisting of both females and males. The transcriptome of five endocrine tissues i.e. hypothalamus, adenohypophysis, thyroid gland, gonads and backfat tissues from 16 out of the 27 pigs was also determined. Sex and breed effects on the 12 plasma metabolites were investigated and associations between genes expressed in the five endocrine tissues and the 12 plasma metabolites measured were analyzed. A probeset was defined as a quantitative trait transcript (QTT) when its association with a particular metabolic trait achieved a nominal P value < 0.01.<h4>Results</h4>A larger than expected number of QTT was found for non-esterified fatty acids and alanine aminotransferase in at least two tissues. The associations were highly tissue-specific. The QTT within the tissues were divided into co-expression network modules enriched for genes in Kyoto Encyclopedia of Genes and Genomes or gene ontology categories that are related to the physiological functions of the corresponding tissues. We also explored a multi-tissue co-expression network using QTT for non-esterified fatty acids from the five tissues and found that a module, enriched in hypothalamus QTT, was positioned at the centre of the entire multi-tissue network.<h4>Conclusions</h4>These results emphasize the relationships between endocrine tissues and plasma metabolites in terms of gene expression. Highly tissue-specific association patterns suggest that candidate genes or gene pathways should be investigated in the context of specific tissues.

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

<h4>Background</h4>The D variant phenotypes are often categorized into weak D types and partial D types. Although the molecular basis underlying the partial D phenotype has been investigated in several races, data from Chinese populations are rare.<h4>Study design and methods</h4>We collected partial D samples from 1,274,540 blood donors, as well as from sporadic patients in the Chinese population, over a 4-year period. Samples with partial D phenotype were determined by commercial monoclonal anti-D panels and molecular methods. Blood samples with discrepant results of serologic and molecular methods were further investigated by polymerase chain reaction (PCR) with sequence-specific primers and nucleotide sequencing of RHD exons. The detection of antibodies was performed.<h4>Results</h4>A total of 44 samples with partial D phenotypes were confirmed. Molecular typing revealed five different known aberrant alleles as well as four new RHD alleles. As described previously, DVI represented the most frequent partial D type in China with a total of 36 samples. However, discrepant results were observed in four DVI samples with serotyping and genotyping (i.e., DVI category identified by D-screen test and grossly intact RHD gene identified by multiplex PCR). We also found four novel alleles, termed DFR-4, DCS-3, DCC, and DLX.<h4>Conclusion</h4>To date, this study presents the most comprehensive report on partial D in China. The distribution of partial D types in China was found to be complicated and polymorphic, whereas RhD genotyping of DVI-variant samples might give inaccurate results due to a relatively high incidence of RHD(1227G>A) in the Chinese population.

Cocaine dependence is associated with orbitofrontal cortex (OFC)-dependent cognitive inflexibility in both humans and laboratory animals. A critical question is whether cocaine self-administration affects pre-existing individual differences in cognitive flexibility. Serotonin transporter knockout (5-HTT(-/-) ) mice show improved cognitive flexibility in a visual reversal learning task, whereas 5-HTT(-/-) rats self-administer increased amounts of cocaine. Here we assessed: (1) whether 5-HTT(-/-) rats also show improved cognitive flexibility (next to mice); and (2) whether this is affected by cocaine self-administration, which is increased in these animals. Results confirmed that naïve 5-HTT(-/-) rats (n = 8) exhibit improved cognitive flexibility, as measured in a sucrose reinforced reversal learning task. A separate group of rats was subsequently trained to intravenously self-administer cocaine (0.5 mg/kg/infusion), and we observed that the 5-HTT(-/-) rats (n = 10) self-administered twice as much cocaine [632.7 mg/kg (±26.3)] compared with 5-HTT(+/+) rats (n = 6) [352.3 mg/kg (±62.0)] over 50 1-hour sessions. Five weeks into withdrawal the cocaine-exposed animals were tested in the sucrose-reinforced reversal learning paradigm. Interestingly, like the naïve 5-HTT(-/-) rats, the cocaine exposed 5-HTT(-/-) rats displayed improved cognitive flexibility. In conclusion, we show that improved reversal learning in 5-HTT(-/-) rats reflects a pre-existing trait that is preserved during cocaine-withdrawal. As 5-HTT(-/-) rodents model the low activity s-allele of the human serotonin transporter-linked polymorphic region, these findings may have heuristic value in the treatment of s-allele cocaine addicts.

<h4>Background</h4>MicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir.<h4>Methodology/principal findings</h4>We employed a proteomics technique called "stable isotope labelling by amino acids in cell culture" (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation.<h4>Conclusions/significance</h4>To the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins in other cell lines, we provided further evidence for the cell line specificity of microRNAs.

We investigated the activity of curcumin against Proteus mirabilis and the struvite crystallization in relation to urinary stones formation. In order to evaluate an activity of curcumin we performed an in vitro experiment of struvite growth from artificial urine. The crystallization process was induced by Proteus mirabilis to mimic the real urinary tract infection, which usually leads to urinary stone formation. The results demonstrate that curcumin exhibits the effect against Proteus mirabilis inhibiting the activity of urease-an enzyme produced by these microorganisms. Addition of curcumin increases the induction time and decreases the efficiency of growth of struvite compared with the absence of curcumin. Interestingly, the addition of curcumin does not affect the crystal morphology and habit. In conclusion, curcumin has demonstrated its significant potential to be further investigated for its use in the case of struvite crystallization induced for the growth by Proteus mirabilis in relation to urinary stone formation.

<h4>Purpose</h4>The purpose of this study was to evaluate by microarray the hypothesis that LH(BETA)T(AG) retinoblastoma tumors exhibit regional and temporal variations in gene expression.<h4>Methods</h4>LH(BETA)T(AG) mice aged 12, 16, and 20 weeks were euthanatized (n = 9). Specimens were taken from five tumor areas (apex, anterior lateral, center, base, and posterior lateral). Samples were hybridized to gene microarrays. The data were preprocessed and analyzed, and genes with a P < 0.01, according to the ANOVA models, and a log(2)-fold change >2.5 were considered to be differentially expressed. Differentially expressed genes were analyzed for overlap with known networks by using pathway analysis tools.<h4>Results</h4>There were significant temporal (P < 10(-8)) and regional differences in gene expression for LH(BETA)T(AG) retinoblastoma tumors. At P < 0.01 and log(2)-fold change >2.5, there were significant changes in gene expression of 190 genes apically, 84 genes anterolaterally, 126 genes posteriorly, 56 genes centrally, and 134 genes at the base. Differentially expressed genes overlapped with known networks, with significant involvement in regulation of cellular proliferation and growth, response to oxygen levels and hypoxia, regulation of cellular processes, cellular signaling cascades, and angiogenesis.<h4>Conclusions</h4>There are significant temporal and regional variations in the LH(BETA)T(AG) retinoblastoma model. Differentially expressed genes overlap with key pathways that may play pivotal roles in murine retinoblastoma development. These findings suggest the mechanisms involved in tumor growth and progression in murine retinoblastoma tumors and identify pathways for analysis at a functional level, to determine significance in human retinoblastoma. Microarray analysis of LH(BETA)T(AG) retinal tumors showed significant regional and temporal variations in gene expression, including dysregulation of genes involved in hypoxic responses and angiogenesis.

<h4>Background</h4>Hepcidin is the main regulator of iron homeostasis: inappropriate production of hepcidin results in iron overload or iron deficiency and anaemia.<h4>Aims</h4>To study variation of serum hepcidin concentration in a normal population.<h4>Results</h4>Hepcidin showed age and sex dependent variations that correlated with ferritin but not with serum iron and transferrin saturation. The size of the study population was underpowered to find genome wide significant associations with hepcidin concentrations but it allowed to show that association with serum iron, transferrin saturation and erythrocyte traits of common DNA variants in HFE (rs1800562) and TMPRSS6 (rs855791) genes is not exclusively dependent on hepcidin values. When multiple interactions between environmental factors, the iron parameters and hepcidin were taken into account, the HFE variant, and to lesser extent the TMPRSS6 variant, were associated with ferritin and with hepcidin normalised to ferritin (the hepcidin/ferritin ratio).<h4>Conclusions</h4>The results suggest a mutual control of serum hepcidin and ferritin concentrations, a mechanism relevant to the pathophysiology of HFE haemochromatosis, and demonstrate that the HFE rs1800562 C282Y variant exerts a direct pleiotropic effect on the iron parameters, in part independent of hepcidin.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expansion of CAG repeats in the Htt gene. Examination of the post-mortem brains of HD patients shows the presence of diffuse nuclear htt immunoreactivity and intra-nuclear inclusions. The aim of this study was to produce a detailed characterization of the neuronal pathology in the R6/1 transgenic mouse model. The R6/1 carrier mice demonstrate intra-nuclear and extra-nuclear inclusions with the S830 htt antibody at 2-11 months of age. The distribution pattern of neuronal intra-nuclear inclusions (NIIs) was irregular in several brain regions including the striatum, cortex and hippocampus. A greater number of NIIs were found in the ventral striatum than in the dorsal striatum. In the globus pallidus, cerebellum and thalamus the pattern of inclusion formation was relatively consistent over time. At 4 and 6 months of age, the R6/1 mice showed increased glial fibrillary acid protein (GFAP) immunoreactivity in the cortex compared to their wildtype littermates, yet no difference was found in the striatum. Analysis by electron microscopy found that neurons from the R6/1 carriers contained a densely packed cytoplasm at 1.5 months of age, with some neurons displaying structural abnormalities including vacuolization and nuclear membrane folding. No NIIs were detected at this age, but by 7 months of age, NIIs were present with severe cellular vacuolization. The present study indicates that a decrease in striatal volume with cell loss is present in young (2 months) R6/1 mice, and the distribution of NIIs is robust and widespread, with considerably temporal and spatial variation in NII development between mice.

The spinocerebellar ataxias (SCAs) are dominantly inherited disorders that primarily affect coordination of motor function but also frequently involve other brain functions. The models described in this review address mechanisms of trinucleotide-repeat expansions, particularly those relating to polyglutamine expression in the mutant proteins. Modeling chronic late-onset human ataxias in mice is difficult because of their short life-span. While this potential hindrance has been partially overcome by using over-expression of the mutant gene, and/or worsening of the mutation by increasing the length of the trinucleotide repeat expansion, interpretation of results from such models and extrapolation to the human condition should be cautious. Nevertheless, genetically engineered murine models of these diseases have enhanced our understanding of the pathogenesis of many of these conditions. A common theme in many of the polyglutamine-repeat diseases is nuclear localization of mutant protein, with resultant effects on gene regulation. Conditional mutant models and transgenic knock-down therapy have demonstrated the potential for reversibility of disease when production of mutant protein is halted. Several other genetically engineered murine models of SCA also have begun to show utility in the identification and assessment of more classical drug-based therapeutic modalities.

Pioneer longitudinal axons grow long distances parallel to the floor plate and precisely maintain their positions using guidance molecules released from the floor plate. Two receptors, Robo1 and Robo2, are critical for longitudinal axon guidance by the Slit family of chemorepellents. Previous studies showed that Robo1(-/-);2(-/-) double mutant mouse embryos have disruptions in both ventral and dorsal longitudinal tracts. However, the role of each Robo isoform remained unclear, because Robo1 or 2 single mutants have mild or no errors. Here we utilized a more sensitive genetic strategy to reduce Robo levels for determining any separate functions of the Robo1 and 2 isoforms. We found that Robo1 is the predominant receptor for guiding axons in ventral tracts and prevents midline crossing. In contrast, Robo2 is the main receptor for directing axons within dorsal tracts. Robo2 also has a distinct function in repelling neuron cell bodies from the floor plate. Therefore, while Robo1 and 2 have some genetic overlap to cooperate in guiding longitudinal axons, each isoform has distinct functions in specific longitudinal axon populations.

Most nucleosides in solution typically exist in equilibrium between two major sugar pucker forms, N-type and S-type, but bridged nucleosides can be locked into one of these conformations depending on their specific structure. While many groups have researched these bridged nucleosides for the purpose of determining their binding affinity for antisense applications, we opted to look into the potential for biological activity within these conformationally-locked structures. A small library of 2',4'- and 3',4'-bridged nucleoside analogues was synthesized, including a novel 3',4'-carbocyclic bridged system. The synthesized compounds were tested for antibacterial, antitumor, and antiviral activities, leading to the identification of nucleosides possessing such biological activities. To the best of our knowledge, these biologically active compounds represent the first example of 2',4'-bridged nucleosides to demonstrate such properties. The most potent compound, nucleoside 33, exhibited significant antiviral activity against pseudoviruses SF162 (IC(50)=7.0 μM) and HxB2 (IC(50)=2.4 μM). These findings render bridged nucleosides as credible leads for drug discovery in the anti-HIV area of research.

<h4>Background</h4>Ovarian cancer is the most deadly gynecological cancer with a very poor prognosis. Xenograft mouse models have proven to be one very useful tool in testing candidate therapeutic agents and gene function in vivo. In this study we identify genes and gene networks important for the efficacy of a pre-clinical anti-tumor therapeutic, MT19c.<h4>Methods</h4>In order to understand how ovarian xenograft tumors may be growing and responding to anti-tumor therapeutics, we used genome-wide mRNA expression and DNA copy number measurements to identify key genes and pathways that may be critical for SKOV-3 xenograft tumor progression. We compared SKOV-3 xenografts treated with the ergocalciferol derived, MT19c, to untreated tumors collected at multiple time points. Cell viability assays were used to test the function of the PPARγ agonist, Rosiglitazone, on SKOV-3 cell growth.<h4>Results</h4>These data indicate that a number of known survival and growth pathways including Notch signaling and general apoptosis factors are differentially expressed in treated vs. untreated xenografts. As tumors grow, cell cycle and DNA replication genes show increased expression, consistent with faster growth. The steroid nuclear receptor, PPARγ, was significantly up-regulated in MT19c treated xenografts. Surprisingly, stimulation of PPARγ with Rosiglitazone reduced the efficacy of MT19c and cisplatin suggesting that PPARγ is regulating a survival pathway in SKOV-3 cells. To identify which genes may be important for tumor growth and treatment response, we observed that MT19c down-regulates some high copy number genes and stimulates expression of some low copy number genes suggesting that these genes are particularly important for SKOV-3 xenograft growth and survival.<h4>Conclusions</h4>We have characterized the time dependent responses of ovarian xenograft tumors to the vitamin D analog, MT19c. Our results suggest that PPARγ promotes survival for some ovarian tumor cells. We propose that a combination of regulated expression and copy number can identify genes that are likely important for chemotherapy response. Our findings suggest a new approach to identify candidate genes that are critical for anti-tumor therapy.

<h4>Background</h4>The authors previously showed that the C→T polymorphism (rs6929846) of butyrophilin, subfamily 2, member A1 gene (BTN2A1) was significantly associated with myocardial infarction in Japanese individuals. Given that metabolic syndrome (MetS) is an important risk factor for myocardial infarction, the association of the rs6929846 of BTN2A1 with myocardial infarction might be attributable, at least in part, to its effect on susceptibility to MetS.<h4>Aim</h4>The aim of the present study was to examine the relation of the rs6929846 of BTN2A1 to MetS in East Asian populations.<h4>Methods</h4>The study population comprised 5210 Japanese or Korean individuals (3982 individuals with MetS, 1228 controls) from three independent subject panels. Japanese subject panels A and B comprised 1322 individuals with MetS and 654 controls, and 1909 individuals with MetS and 170 controls, respectively, whereas the Korean population samples comprised 751 individuals with MetS and 404 controls.<h4>Results</h4>Comparison of genotype distributions using the χ(2) test revealed that the genotype distributions and allele frequencies of rs6929846 were significantly (p<0.05) associated with MetS in Japanese subject panels A (T allele frequency: MetS, 0.091; controls, 0.054; p=6.1×10(-5)) and B (T allele frequency: MetS, 0.091; controls, 0.039; p=0013) but not in the Korean population samples (T allele frequency: MetS, 0.102; controls, 0.125; p=0.0997). Multivariable logistic regression analysis with adjustment for covariates revealed that the rs6929846 of BTN2A1 was significantly (p<0.017) associated with MetS in Japanese subject panel A (p=0.0055, OR 1.97) and in all individuals (p=0.0038, OR 1.38), with the T allele representing a risk factor for this condition.<h4>Conclusion</h4>BTN2A1 may be a susceptible gene for MetS in Japanese individuals.

Hepcidin has been established as a central regulator of iron metabolism. In most patients with chronic kidney disease (CKD), serum hepcidin levels are relatively high, favoring iron sequestration in several cell types and organs and thereby leading to iron-related complications. In the absence of overt inflammation, serum hepcidin has been found to be most closely associated with serum ferritin in healthy subjects and in CKD patients. Intestinal iron absorption is tightly regulated by both iron stores and hepcidin. The expression of the mammalian iron exporter, ferroportin (FPN), limits the growth of intracellular bacteria by depleting cytosolic iron. An upregulation of hepcidin could diminish FPN and favor bacterial growth. Of note, in patients with hyperferritinemia impaired hepcidin expression caused by a mutation in the hemochromatosis gene associates with an attenuation of atherosclerosis. Thus, hepcidin might accelerate atherosclerosis by preventing iron exit from macrophages or other cells in the arterial wall. High hepcidin levels have also been found to be linked to good erythropoiesis-stimulating agents (ESAs) response, in conjunction with the strong hepcidin-ferritin correlation. Finally, hepcidin may also play a significant role by itself in the pathogenesis of CKD complications associated with disturbed iron metabolism, i.e. unrelated to ESA hyporesponsiveness, such as bacterial infections and atherosclerosis.

<h4>Background</h4>Patients suffering from ulcerative colitis (UC) bear an increased risk for colorectal cancer. Due to the sparsity of colitis-associated cancer (CAC) and the long duration between UC initiation and overt carcinoma, elucidating mechanisms of inflammation-associated carcinogenesis in the gut is particularly challenging. Adequate murine models are thus highly desirable. For human CACs a high frequency of chromosomal instability (CIN) reflected by aneuploidy could be shown, exceeding that of sporadic carcinomas. The aim of this study was to analyze mouse models of CAC with regard to CIN. Additionally, protein expression of p53, beta-catenin and Ki67 was measured to further characterize murine tumor development in comparison to UC-associated carcinogenesis in men.<h4>Methods</h4>The AOM/DSS model (n = 23) and IL-10(-/-) mice (n = 8) were applied to monitor malignancy development via endoscopy and to analyze premalignant and malignant stages of CACs. CIN was assessed using DNA-image cytometry. Protein expression of p53, beta-catenin and Ki67 was evaluated by immunohistochemistry. The degree of inflammation was analyzed by histology and paralleled to local interferon-γ release.<h4>Results</h4>CIN was detected in 81.25% of all murine CACs induced by AOM/DSS, while all carcinomas that arose in IL-10(-/-) mice were chromosomally stable. Beta-catenin expression was strongly membranous in IL-10(-/-) mice, while 87.50% of AOM/DSS-induced tumors showed cytoplasmatic and/or nuclear translocation of beta-catenin. p53 expression was high in both models and Ki67 staining revealed higher proliferation of IL-10(-/-)-induced CACs.<h4>Conclusions</h4>AOM/DSS-colitis, but not IL-10(-/-) mice, could provide a powerful murine model to mechanistically investigate CIN in colitis-associated carcinogenesis.

The pathogenesis of hepatic fibrosis involves significant deposition of fibrilar collagen and other extracellular matrix proteins. It is a rather dynamic process of wound healing in response to a variety of persistent liver injury caused by factors such as ethanol intake, viral infection, drugs, toxins, cholestasis, and metabolic disorders. Liver fibrosis distorts the hepatic architecture, decreases the number of endothelial cell fenestrations and causes portal hypertension. Key events are the activation and transformation of quiescent hepatic stellate cells into myofibroblast-like cells with the subsequent up-regulation of proteins such as α-smooth muscle actin, interstitial collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and proteoglycans. Oxidative stress is a major contributing factor to the onset of liver fibrosis and it is typically associated with a decrease in the antioxidant defense. Currently, there is no effective therapy for advanced liver fibrosis. In its early stages, liver fibrosis is reversible upon cessation of the causative agent. In this review, we discuss some aspects on the etiology of liver fibrosis, the cells involved, the molecular pathogenesis, and the current therapeutic approaches.

Estrogen receptor-alpha (ERα) is a key factor in the development of breast cancer in humans. The expression and activity of ERα is regulated by a multitude of intracellular and extracellular signals. Here we show a cross-talk between β-catenin and ERα in human breast cancer cells. Knockdown of β-catenin by RNAi resulted in significant reduction of ERα mRNA and/or protein levels in MCF-7, T-47D, and BT-474 breast cancer cells and in significant reduction of estradiol-induced expression of the ERα target genes pS2 and GREB1. In addition β-catenin silencing resulted in significant decrease of growth of MCF-7 cells both in the absence and presence of estradiol. β-catenin and ERα could not be co-immunoprecipitated by ERα antibodies from lysates of E2-treated or untreated cells suggesting lack of direct physical interaction. It is concluded that β-catenin is a positive regulator of ERα mRNA and protein expression.

<h4>Context</h4>Obesity from childhood to adulthood is associated with adverse health later in life. Increased youth BMI is a risk factor for later obesity, but it is unknown whether identification of other risk factors, including recently discovered genetic markers, would help to identify children at risk of developing adult obesity.<h4>Objectives</h4>Our objective was to examine the childhood environmental and genetic predictors of adult obesity.<h4>Design, setting, and participants</h4>We followed 2119 individuals of the Cardiovascular Risk in Young Finns Study for up to 27 yr since baseline (1980, age 3-18 yr).<h4>Main outcome measure</h4>We evaluated adult obesity [body mass index (BMI) ≥ 30 kg/m(2)].<h4>Results</h4>The independent predictors (P < 0.05) of adult obesity included childhood BMI, C-reactive protein (CRP), family income (inverse), mother's BMI, and polymorphisms near genes TFAP2B, LRRN6C, and FLJ35579. A risk assessment based on childhood BMI, mother's BMI, and family income was superior in predicting obesity compared with the approach using data only on BMI (C-statistics 0.751 vs. 0.772, P = 0.0015). Inclusion of data on childhood CRP and novel genetic variants for BMI did not incrementally improve C-value (0.779, P = 0.16). A nonlaboratory risk score (childhood BMI, mother's BMI, and family income) predicted adult obesity in all age groups between 3-18 yr (P always <0.001).<h4>Conclusions</h4>Childhood BMI, CRP, family income (inversely), mother's BMI, and polymorphisms near genes FLJ35779, TFAP2B, and LRRN6C are independently related to adulthood obesity. However, because genetic risk markers and CRP only marginally improve the prediction, our results indicate that children at high risk of adult obesity can be identified using a simple non-laboratory-based risk assessment.

Extraocular muscle (EOM) myofibers do not fit the traditional fiber typing classifications normally used in noncranial skeletal muscle, in part, due to the complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and shortening velocity were determined for 220 fibers. Each fiber was examined for myosin heavy chain (MyHC) isoform composition by densitometric analysis of electrophoresis gels. Rectus muscle serial sections were examined for coexpression of eight MyHC isoforms. A continuum was seen in single myofiber shortening velocities as well as force generation, both in absolute force (g) and specific tension (kN/m(2)). Shortening velocity correlated with MyHCIIB, IIA, and I content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical shortening velocities expressed significantly different ratios of MyHC isoforms. The vast majority of myofibers in both the orbital and global layers expressed more than one MyHC isoform, with up to six isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus EOM myofibers represent a continuum in their histological and physiological characteristics. This continuum would facilitate fine motor control of eye position, speed, and direction of movement in all positions of gaze and with all types of eye movements-from slow vergence movements to fast saccades. To fully understand how the brain controls eye position and movements, it is critical that this significant EOM myofiber heterogeneity be integrated into hypotheses of oculomotor control.

The specific genes and genetic pathways associated with pancreatic ductal adenocarcinoma are still largely unknown partially due to the low resolution of the techniques applied so far to their study. Here we used high-density 500 K single nucleotide polymorphism (SNP)-arrays to define those chromosomal regions which most commonly harbour copy number (CN) alterations and loss of heterozygozity (LOH) in a series of 20 PDAC tumors and we correlated the corresponding genetic profiles with the most relevant clinical and histopathological features of the disease. Overall our results showed that primary PDAC frequently display (>70%) extensive gains of chromosomes 1q, 7q, 8q and 20q, together with losses of chromosomes 1p, 9p, 12q, 17p and 18q, such chromosomal regions harboring multiple cancer- and PDAC-associated genes. Interestingly, these alterations clustered into two distinct genetic profiles characterized by gains of the 2q14.2, 3q22.1, 5q32, 10q26.13, 10q26.3, 11q13.1, 11q13.3, 11q13.4, 16q24.1, 16q24.3, 22q13.1, 22q13.31 and 22q13.32 chromosomal regions (group 1; n = 9) versus gains at 1q21.1 and losses of the 1p36.11, 6q25.2, 9p22.1, 9p24.3, 17p13.3 and Xp22.33 chromosomal regions (group 2; n = 11). From the clinical and histopathological point of view, group 1 cases were associated with smaller and well/moderately-differentiated grade I/II PDAC tumors, whereas and group 2 PDAC displayed a larger size and they mainly consisted of poorly-differentiated grade III carcinomas. These findings confirm the cytogenetic complexity and heterozygozity of PDAC and provide evidence for the association between tumor cytogenetics and its histopathological features. In addition, we also show that the altered regions identified harbor multiple cancer associate genes that deserve further investigation to determine their relevance in the pathogenesis of PDAC.

<h4>Background</h4>Dendritic morphology largely determines patterns of synaptic connectivity and electrochemical properties of a neuron. Neurons display a myriad diversity of dendritic geometries which serve as a basis for functional classification. Several types of molecules have recently been identified which regulate dendrite morphology by acting at the levels of transcriptional regulation, direct interactions with the cytoskeleton and organelles, and cell surface interactions. Although there has been substantial progress in understanding the molecular mechanisms of dendrite morphogenesis, the specification of class-specific dendritic arbors remains largely unexplained. Furthermore, the presence of numerous regulators suggests that they must work in concert. However, presently, few genetic pathways regulating dendrite development have been defined.<h4>Methodology/principal findings</h4>The Drosophila gene turtle belongs to an evolutionarily conserved class of immunoglobulin superfamily members found in the nervous systems of diverse organisms. We demonstrate that Turtle is differentially expressed in Drosophila da neurons. Moreover, MARCM analyses reveal Turtle acts cell autonomously to exert class specific effects on dendritic growth and/or branching in da neuron subclasses. Using transgenic overexpression of different Turtle isoforms, we find context-dependent, isoform-specific effects on mediating dendritic branching in class II, III and IV da neurons. Finally, we demonstrate via chromatin immunoprecipitation, qPCR, and immunohistochemistry analyses that Turtle expression is positively regulated by the Cut homeodomain transcription factor and via genetic interaction studies that Turtle is downstream effector of Cut-mediated regulation of da neuron dendrite morphology.<h4>Conclusions/significance</h4>Our findings reveal that Turtle proteins differentially regulate the acquisition of class-specific dendrite morphologies. In addition, we have established a transcriptional regulatory interaction between Cut and Turtle, representing a novel pathway for mediating class specific dendrite development.

Posttranslational modifications (PTMs) modulate protein function in most eukaryotes and have a ubiquitous role in diverse range of cellular functions. Identification, characterization, and mapping of these modifications to specific amino acid residues on proteins are critical towards understanding their functional significance in a biological context. The interpretation of proteome data obtained from the high-throughput methods cannot be deciphered unambiguously without a priori knowledge of protein modifications. An in-depth understanding of protein PTMs is important not only for gaining a perception of a wide array of cellular functions but also towards developing drug therapies for many life-threatening diseases like cancer and neurodegenerative disorders. Many of the protein modifications like ubiquitination play a decisive role in various drug response(s) and eventually in disease prognosis. Thus, many commonly observed PTMs are routinely tracked as disease markers while many others are used as molecular targets for developing target-specific therapies. In this paper, we summarize some of the major, well-studied protein alterations and highlight their importance in various chronic diseases and normal development. In addition, other promising minor modifications such as SUMOylation, observed to impact cellular dynamics as well as disease pathology, are mentioned briefly.

Inhibition of bacterial transcription represents an effective and clinically validated anti-infective chemotherapeutic strategy. We describe the evolution of our approach to the streptolydigin class of antibiotics that target bacterial RNA polymerases (RNAPs). This effort resulted in the synthesis and biological evaluation of streptolydigin, streptolydiginone, streptolic acid, and a series of new streptolydigin-based agents. Subsequent biochemical evaluation of RNAP inhibition demonstrated that the presence of both streptolic acid and tetramic acid subunits was required for activity of this class of antibiotics. In addition, we identified 10,11-dihydrostreptolydigin as a new RNAP-targeting agent, which was assembled with high synthetic efficiency of 15 steps in the longest linear sequence. Dihydrostreptolydigin inhibited three representative bacterial RNAPs and displayed in vitro antibacterial activity against S. salivarius . The overall increase in synthetic efficiency combined with substantial antibacterial activity of this fully synthetic antibiotic demonstrates the power of organic synthesis in enabling design and comprehensive in vitro pharmacological evaluation of new chemical agents that target bacterial transcription.

To identify the ligands for σ(1) receptors that are potent and selective, analogues of prezamicol and trozamicol scaffolds of carbonyl-containing vesicular acetylcholine transporter (VAChT) inhibitors were explored. Of the 23 analogues synthesized and tested, 5 displayed very high affinity for σ(1) (K(i) = 0.48-4.05 nM) and high selectivity for σ(1) relative to σ(2) receptors (σ(1)/σ(2) selectivity of >749-fold). Four of the five compounds (14a, 14b, 14c, and 14e) showed very low affinity for VAChT (K(i) > 290 nM), and the fifth compound (14g) showed moderate affinity for VAChT (K(i) = 44.2 nM). The compound [1'-(4-fluorobenzyl)-3'-hydroxy[1,4']bipiperidinyl-4-yl]-(4-fluorophenyl)methanone (14a) displayed very high affinity and selectivity for σ(1) receptor (K(i) = 0.48 nM, σ(1)/σ(2) > 3600). All four of these most promising compounds (14a, 14b, 14c, and 14e) can be radiosynthesized with fluorine-18 or carbon-11, which will allow further evaluation of their properties as PET probes for imaging σ(1) receptor in vivo.

Recent attempts to understand the biological bases of depression vulnerability have revealed that both the short allele of the serotonin transporter-linked polymorphic region (5-HTTLPR) and activity in the amygdala are associated with depression. Other studies have reported amygdala hyperactivity associated with the 5-HTTLPR short allele, linking the genetic and neuroimaging lines of research and suggesting a mechanism whereby the short allele confers depression risk. However, fewer investigations have examined the associations among depression, 5-HTTLPR variability, and amygdala activation in a single study. The current study thus investigated whether 5-HTTLPR genotype modulates the association between depressive symptoms and amygdala activity among psychiatrically healthy adults. Regional cerebral blood flow was measured with perfusion fMRI during a task-free scan. We hypothesized differential associations between depressive symptoms and amygdala activity among individuals homozygous for the short allele and individuals homozygous for the long allele. Both whole brain analyses and region-of-interest analyses confirmed this prediction, revealing a significant negative association among the long allele group and a trend of positive association among the short allele group. These results complement existing reports of short allele related amygdala hyperactivity and suggest an additional neurobiological mechanism whereby the 5-HTTLPR is associated with psychiatric outcomes.

Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J cm(-2), twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and α-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, α-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage.

<h4>Background</h4>Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication.<h4>Methods</h4>A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression.<h4>Results</h4>Thirty known proteins were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens.<h4>Conclusions</h4>S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question.

<h4>Background</h4>Adipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance.AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer.<h4>Methods</h4>Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 μg/ml) and/or metformin (24 h, 1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation.<h4>Results</h4>Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01), while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1 receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA expression (p < 0.05).<h4>Conclusions</h4>Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin resistance, but also to prevent obesity-related breast cancer development and progression.

The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.

Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that promotes the transcription of cytoprotective genes in response to oxidative and electrophilic stresses. Most functions of Nrf2 were identified by studying biological models with Nrf2 deficiency, however, little is known about the effects of graded Nrf2 activation. In the present study, genomic gene expression profiles by microarray analysis were characterized with a "gene dose-response" model in livers of Nrf2-null mice, wild-type mice, Kelch-like ECH associating protein 1 (Keap1)-knockdown (Keap1-KD) mice with enhanced Nrf2 activation, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum hepatic Nrf2 activation. Hepatic nuclear Nrf2 protein, glutathione concentrations, and known Nrf2 target genes were increased in a dose-dependent manner. In total, 115 genes were identified to be constitutively induced and 80 genes suppressed with graded Nrf2 activation. Messenger RNA of genes encoding enzymes in the pentose phosphate pathway and enzyme were low with Nrf2 deficiency and high with Nrf2 activation, indicating that Nrf2 is important for NADPH production. NADPH is the major reducing resource to scavenge oxidative stress, including regenerating glutathione and thioredoxin and is also used for anabolic pathways including lipid synthesis. High performance liquid chromatography-ultraviolet absorbance analysis confirmed that hepatic NADPH concentration was lowest in Nrf2-null mice and highest in Keap1-HKO mice. In addition, genes involved in fatty acid synthesis and desaturation were downregulated with graded Nrf2 activation. In conclusion, the present study suggests that Nrf2 protects against environmental insults by promoting the generation of NADPH, which is preferentially consumed by aiding scavenging of oxidative stress rather than fatty acid synthesis and desaturation.

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10(-25)), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control.

<h4>Background</h4>Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches.<h4>Methodology/principal findings</h4>Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent.<h4>Conclusions/significance</h4>These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.

Most estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER(+) xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers.

<h4>Background</h4>Improving the treatment of renal cell carcinoma (RCC) will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7), also known as B-FABP and BLBP, which is expressed normally in radial glial cells of the developing central nervous system and cells of the mammary gland. Melanomas, glioblastomas, and several types of carcinomas, including RCC, overexpress FABP7. The abundant expression of FABP7 in primary RCCs compared to certain RCC-derived cell lines may allow the definition of the molecular components of FABP7's regulatory system.<h4>Results</h4>We determined FABP7 mRNA levels in six RCC cell lines. Two were highly expressed, whereas the other and the embryonic kidney cell line (HEK293) were weakly expressed FABP7 transcripts. Western blot analysis of the cell lines detected strong FABP7 expression only in one RCC cell line. Promoter activity in the RCC cell lines was 3- to 21-fold higher than that of HEK293. Deletion analysis demonstrated that three FABP7 promoter regions contributed to upregulated expression in RCC cell lines, but not in the HEK293 cell. Competition analysis of gel shifts indicated that OCT1, OCT6, and nuclear factor I (NFI) bound to the FABP7 promoter region. Supershift experiments indicated that BRN2 (POU3F2) and NFI bound to the FABP7 promoter region as well. There was an inverse correlation between FABP7 promoter activity and BRN2 mRNA expression. The FABP7-positive cell line's NFI-DNA complex migrated faster than in other cell lines. Levels of NFIA mRNA were higher in the HEK293 cell line than in any of the six RCC cell lines. In contrast, NFIC mRNA expression was lower in the HEK293 cell line than in the six RCC cell lines.<h4>Conclusions</h4>Three putative FABP7 promoter regions drive reporter gene expression in RCC cell lines, but not in the HEK293 cell line. BRN2 and NFI may be key factors regulating the expression of FABP7 in certain RCC-derived cell lines.

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.

Condensins play a central role in global chromatin organization. In bacteria, two families of condensins have been identified, the MukBEF and SMC-ScpAB complexes. Only one of the two complexes is usually found in a given species, giving rise to a paradigm that a single condensin organizes bacterial chromosomes. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly present in diverse bacteria. The proteins appear distantly related to MukBEF, have a similar operon organization and similar predicted secondary structures albeit with notably shorter coiled-coils. All three subunits of MksBEF exhibit significant sequence variation and can be divided into a series of overlapping subfamilies. MksBEF often coexists with the SMC-ScpAB, MukBEF and, sometimes, other MksBEFs. In Pseudomonas aeruginosa, both SMC and MksB contribute to faithful chromosome partitioning, with their inactivation leading to increased frequencies of anucleate cells. Moreover, MksBEF can complement anucleate cell formation in SMC-deficient cells. Purified PaMksB showed activities typical for condensins including ATP-modulated DNA binding and condensation. Notably, DNA binding by MksB is negatively regulated by ATP, which sets it apart from other known SMC proteins. Thus, several specialized condensins might be involved in organization of bacterial chromosomes.

Gestation is a complex process that involves different growth factors, cytokines and adhesion proteins related with embryo development, cellular differentiation and proliferation, embryo-endometrium interaction, angiogenesis, maternal-embryonic recognition and growth development of placenta and embryos. In this study, we examine pre-implantational (at 6 days of gestation) and gestational (at 12 days and total from ovulation to birth) losses in two rabbit lines selected by different criteria (post-weaning daily gain and litter size) and the pattern of a set of candidate transcripts, at 6 days of gestation, related with embryo development and implantation process, such as Oct-4, epidermal growth factor receptor 3 (erbB3), Transforming Growth Factor β2, Vascular Endothelial Growth Factor and Interferon γ and related with insulin-like growth factors signalling as insulin growth factors I and II and their receptors in rabbit blastocysts and endometrial tissue. Similar pre-implantational losses were obtained in both lines. However, the gestational losses of the line selected by post-weaning daily gain clearly mirrored an increase in losses by 50% at 12 days and at birth (22.4 vs 9.5 and 50.2 vs 25.4, respectively, between line selected by post-weaning daily gain and line selected by litter size). In blastocysts and endometrial tissue at 6 days of gestation qRT-PCR assays indicated that the mean insulin-like growth factor (IGF)-IIR mRNA expression was down-regulated in line selected by post-weaning daily gain. Dysregulation of the IGF-IIR could be potential reasons for induced gestational losses. We conclude that IGF-IIR gene expression in blastocyst and endometrial tissue at 6th day of gestation tends to decline in line selected by post-weaning daily gain. The functional significance related with gestational losses is uncertain.

Adenosine monophosphate-activated protein kinase (AMPK) is a major energy sensor that maintains cellular energy homeostasis. Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (Htt) gene. In this paper, we report that activation of the α1 isoform of AMPK (AMPK-α1) occurred in striatal neurons of humans and mice with HD. Overactivation of AMPK in the striatum caused brain atrophy, facilitated neuronal loss, and increased formation of Htt aggregates in a transgenic mouse model (R6/2) of HD. Such nuclear accumulation of AMPK-α1 was activity dependent. Prevention of nuclear translocation or inactivation of AMPK-α1 ameliorated cell death and down-regulation of Bcl2 caused by mutant Htt (mHtt). Conversely, enhanced expression of Bcl2 protected striatal cells from the toxicity evoked by mHtt and AMPK overactivation. These data demonstrate that aberrant activation of AMPK-α1 in the nuclei of striatal cells represents a new toxic pathway induced by mHtt.

β-thalassemia is a disease characterized by anemia and is associated with ineffective erythropoiesis and iron dysregulation resulting in iron overload. The peptide hormone hepcidin regulates iron metabolism, and insufficient hepcidin synthesis is responsible for iron overload in minimally transfused patients with this disease. Understanding the crosstalk between erythropoiesis and iron metabolism is an area of active investigation in which patients with and models of β-thalassemia have provided significant insight. The dependence of erythropoiesis on iron presupposes that iron demand for hemoglobin synthesis is involved in the regulation of iron metabolism. Major advances have been made in understanding iron availability for erythropoiesis and its dysregulation in β-thalassemia. In this review, we describe the clinical characteristics and current therapeutic standard in β-thalassemia, explore the definition of ineffective erythropoiesis, and discuss its role in hepcidin regulation. In preclinical experiments using interventions such as transferrin, hepcidin agonists, and JAK2 inhibitors, we provide evidence of potential new treatment alternatives that elucidate mechanisms by which expanded or ineffective erythropoiesis may regulate iron supply, distribution, and utilization in diseases such as β-thalassemia.

<h4>Background</h4>Neurulation requires precise, spatio-temporal expression of numerous genes and coordinated interaction of signal transduction and gene regulatory networks, disruption of which may contribute to the etiology of neural tube defects (NTDs). MicroRNAs (miRNAs) are key modulators of cell and tissue differentiation. To define potential roles of miRNAs in development of the murine neural tube (NT), miRNA microarray analysis was conducted to establish expression profiles, and identify miRNA target genes and functional gene networks.<h4>Methods</h4>The miRNA expression profiles in murine embryonic NTs derived from gestational days 8.5, 9.0, and 9.5 were defined and compared utilizing miRXplore microarrays from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. Gene expression changes were verified by TaqMan quantitative Real-Time PCR. The clValid R package and the UPGMA (hierarchical) clustering method were utilized for cluster analysis of the microarray data. Functional associations among selected miRNAs were examined via Ingenuity Pathway Analysis.<h4>Results</h4>The miRXplore chips enabled examination of 609 murine miRNAs. Expression of approximately 12% of these was detected in murine embryonic NTs. Clustering analysis revealed several developmentally regulated expression clusters among these expressed genes. Target analysis of differentially expressed miRNAs enabled identification of numerous target genes associated with cellular processes essential for normal NT development. Utilization of Ingenuity Pathway Analysis revealed interactive biologic networks which connected differentially expressed miRNAs with their target genes, and highlighted functional relationships.<h4>Conclusions</h4>The present study defined unique gene expression signatures of a range of miRNAs in the developing NT during the critical period of NT morphogenesis. Analysis of miRNA target genes and gene interaction pathways revealed that specific miRNAs might direct expression of numerous genes encoding proteins, which have been shown to be indispensable for normal neurulation. This study is the first to identify miRNA expression profiles and their potential regulatory networks in the developing mammalian NT.

Transposable elements (TEs) are highly abundant in the genome and capable of mobility, two properties that make them particularly prone to transfer horizontally between organisms. Although the impact of horizontal transfer (HT) of TEs is well recognized in prokaryotes, the frequency of this phenomenon and its contribution to genome evolution in eukaryotes remain poorly appreciated. Here, we provide evidence that a DNA transposon called SPIN has colonized the genome of 17 species of reptiles representing nearly every major lineage of squamates, including 14 families of lizards, snakes, and amphisbaenians. Slot blot analyses indicate that SPIN has amplified to high copy numbers in most of these species, ranging from 2,000-28,000 copies per haploid genome. In contrast, we could not detect the presence of SPIN in any of the turtles (seven species from seven families) and crocodiles (four species) examined. Genetic distances between SPIN sequences from species belonging to different squamate families are consistently very low (average = 0.1), considering the deep evolutionary divergence of the families investigated (most are >100 My diverged). Furthermore, these distances fall below interfamilial distances calculated for two genes known to have evolved under strong functional constraint in vertebrates (RAG1, average = 0.24 and C-mos, average = 0.27). These data, combined with phylogenetic analyses, indicate that the widespread distribution of SPIN among squamates is the result of at least 13 independent events of HTs. Molecular dating and paleobiogeographical data suggest that these transfers took place during the last 50 My on at least three different continents (North America, South America and, Africa). Together, these results triple the number of known SPIN transfer events among tetrapods, provide evidence for a previously hypothesized transoceanic movement of SPIN transposons during the Cenozoic, and further underscore the role of HT in the evolution of vertebrate genomes.

Cell invasion through basement membrane is a specialized cellular behavior critical for many developmental processes and leukocyte trafficking. Invasive cellular behavior is also inappropriately co-opted during cancer progression. Acquisition of an invasive phenotype is accompanied by changes in gene expression that are thought to coordinate the steps of invasion. The transcription factors responsible for these changes in gene expression, however, are largely unknown. C. elegans anchor cell (AC) invasion is a genetically tractable in vivo model of invasion through basement membrane. AC invasion requires the conserved transcription factor FOS-1A, but other transcription factors are thought to act in parallel to FOS-1A to control invasion. Here we identify the transcription factor HLH-2, the C. elegans ortholog of Drosophila Daughterless and vertebrate E proteins, as a regulator of AC invasion. Reduction of HLH-2 function by RNAi or with a hypomorphic allele causes defects in AC invasion. Genetic analysis indicates that HLH-2 has functions outside of the FOS-1A pathway. Using expression analysis, we identify three genes that are transcriptionally regulated by HLH-2: the protocadherin cdh-3, and two genes encoding secreted extracellular matrix proteins, mig-6/papilin and him-4/hemicentin. Further, we show that reduction of HLH-2 function causes defects in polarization of F-actin to the invasive cell membrane, a process required for the AC to generate protrusions that breach the basement membrane. This work identifies HLH-2 as a regulator of the invasive phenotype in the AC, adding to our understanding of the transcriptional networks that control cell invasion.

In rodent models and in humans the impact of gestational diets on the offspring's phenotype was shown experimentally and epidemiologically. The underlying programming of fetal development was shown to be associated with an increased risk of degenerative diseases in adulthood, including the metabolic syndrome. There are clues that diet-dependent modifications of the metabolism during fetal life can persist until adulthood. This leads to the hypothesis that the offspring's transcriptomes show short-term and long-term changes depending on the maternal diet. To this end pregnant German landrace gilts were fed either a high protein diet (HP, 30% CP) or an adequate protein diet (AP, 12% CP) throughout pregnancy. Hepatic transcriptome profiles of the offspring were analyzed at prenatal (94 dpc) and postnatal stages (1, 28, 188 dpn). Depending on the gestational dietary exposure, mRNA expression levels of genes related to energy metabolism, N-metabolism, growth factor signaling pathways, lipid metabolism, nucleic acid metabolism and stress/immune response were affected either in a short-term or in a long-term manner. Gene expression profiles at fetal stage 94 dpc were almost unchanged between the diets. The gestational HP diet affected the hepatic expression profiles at prenatal and postnatal stages. The effects encompassed a modulation of the genome in terms of an altered responsiveness of energy and nutrient sensing pathways. Differential expression of genes related to energy production and nutrient utilization contribute to the maintenance of development and growth performance within physiological norms, however the modulation of these pathways may be accompanied by a predisposition for metabolic disturbances up to adult stages.

The multisubunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from Saccharomyces cerevisiae cells circular minichromosomes linked to condensin. We find that either linearization of minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin's structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide the arms with the structural rigidity necessary for their segregation.

<h4>Background</h4>Histone variants alter the composition of nucleosomes and play crucial roles in transcription, chromosome segregation, DNA repair, and sperm compaction. Modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates, but the urochordate, Oikopleura dioica, a member of the sister group to vertebrates, exhibits profound modification of this ancestral architecture.<h4>Results</h4>We show that a histone complement of 47 gene loci encodes 31 histone variants, grouped in distinct sets of developmental expression profiles throughout the life cycle. A particularly diverse array of 15 male-specific histone variants was uncovered, including a testes-specific H4t, the first metazoan H4 sequence variant reported. Universal histone variants H3.3, CenH3, and H2A.Z are present but O. dioica lacks homologs of macroH2A and H2AX. The genome encodes many H2A and H2B variants and the repertoire of H2A.Z isoforms is expanded through alternative splicing, incrementally regulating the number of acetylatable lysine residues in the functionally important N-terminal "charge patch". Mass spectrometry identified 40 acetylation, methylation and ubiquitylation posttranslational modifications (PTMs) and showed that hallmark PTMs of "active" and "repressive" chromatin were present in O. dioica. No obvious reduction in silent heterochromatic marks was observed despite high gene density in this extraordinarily compacted chordate genome.<h4>Conclusions</h4>These results show that histone gene complements and their organization differ considerably even over modest phylogenetic distances. Substantial innovation among all core and linker histone variants has evolved in concert with adaptation of specific life history traits in this rapidly evolving chordate lineage.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) and pulmonary arterial smooth muscle cells (PASMCs) are implicated in human genetic disorders. Here, we generated genome-wide maps of Smad1/5 binding sites in ECs and PASMCs. Smad1/5 preferentially bound to the region outside the promoter of known genes, and the binding was associated with target gene upregulation. Cell-selective Smad1/5 binding patterns appear to be determined mostly by cell-specific differences in baseline chromatin accessibility patterns. We identified, for the first time, a Smad1/5 binding motif in mammals, and termed GC-rich Smad binding element (GC-SBE). Several sequences in the identified GC-SBE motif had relatively weak affinity for Smad binding, and were enriched in cell type-specific Smad1/5 binding regions. We also found that both GC-SBE and the canonical SBE affect binding affinity for the Smad complex. Furthermore, we characterized EC-specific Smad1/5 target genes and found that several Notch signaling pathway-related genes were induced by BMP in ECs. Among them, a Notch ligand, JAG1 was regulated directly by Smad1/5, transactivating Notch signaling in the neighboring cells. These results provide insights into the molecular mechanism of BMP signaling and the pathogenesis of vascular lesions of certain genetic disorders, including hereditary hemorrhagic telangiectasia.

Huntington's disease (HD) is characterized by numerous alterations within the corticostriatal circuitry. The striatum is innervated by a dense array of dopaminergic (DA) terminals and these DA synapses are critical to the proper execution of motor functions. As motor disturbances are prevalent in HD we examined DA neurotransmission in the striatum in transgenic (tg) murine models of HD. We used in vivo microdialysis to compare extracellular concentrations of striatal DA in both a fragment (R6/2) model, which displays a rapid and severe phenotype, and a full-length (YAC128) model that expresses a more progressive phenotype. Extracellular striatal DA concentrations were significantly reduced in R6/2 mice and decreased concomitantly with age-dependent increasing motor impairments on the rotarod task (7, 9, and 11 weeks). In a sample of 11-week-old R6/2 mice, we also measured tissue concentrations of striatal DA and found that total levels of DA were significantly depleted. However, the loss of total DA content (<50%) was insufficient to account for the full extent of DA depletion in the extracellular fluid (ECF; ∼75%). We also observed a significant reduction in extracellular DA concentrations in the striatum of 7-month-old YAC128 mice. In a separate set of experiments, we applied d-amphetamine (AMPH; 10 μm) locally into the striatum to stimulate the release of intracellular DA into the ECF. The AMPH-induced increase in extracellular DA levels was significantly blunted in 9-week-old R6/2 mice. There also was a decrease in AMPH-stimulated DA efflux in 7-month-old YAC128 mice in comparison to WT controls, although the effect was milder. In the same cohort of 7-month-old YAC128 mice we observed a significant reduction in the total locomotor activity in response to systemic AMPH (2 mg/kg). Our data demonstrate that extracellular DA release is attenuated in both a fragment and full-length tg mouse model of HD and support the concept of DA involvement in aspects of the syndrome.

To probe the space at the floor of the orthosteric ligand binding site in the dopamine D(1) receptor, four methylated analogues of dihydrexidine (DHX) were synthesized with substitutions at the 7 and 8 positions. The 8α-axial, 8β-equatorial, and 7α-equatorial were synthesized by photochemical cyclization of appropriately substituted N-benzoyl enamines, and the 7β-axial analogue was prepared by an intramolecular Henry reaction. All of the methylated analogues displayed losses in affinity when compared to DHX (20 nM): 8β-Me(ax)-DHX (270 nM), 8α-Me(eq)-DHX (920 nM), 7β-Me(eq)-DHX (6540 nM), and 7α-Me(ax)-DHX (>10000 nM). Molecular modeling studies suggest that although the disruption of an aromatic interaction between Phe203(5.47) and Phe288(6.51) is the cause for the 14-fold loss in affinity associated with 8β-axial substitution, unfavorable steric interactions with Ser107(3.36) result in the more dramatic decreases in binding affinity suffered by the rest of the analogues.

Wiring of vascular and neural networks requires precise guidance of growing blood vessels and axons, respectively, to reach their targets during development. Both of the processes share common molecular signaling pathways. Transient receptor potential canonical (TRPC) channels are calcium-permeable cation channels and gated via receptor- or store-operated mechanisms. Recent studies have revealed the requirement of TRPC channels in mediating guidance cue-induced calcium influx and their essential roles in regulating axon navigation and angiogenesis. Dissecting TRPC functions in these physiological processes may provide therapeutic implications for suppressing pathological angiogenesis and improving nerve regeneration.

<h4>Background</h4>Understanding animal development and physiology at a molecular-biological level has been advanced by the ability to determine at high resolution the repertoire of mRNA molecules by whole transcriptome resequencing. This includes the ability to detect and quantify rare abundance transcripts and isoform-specific mRNA variants produced from a gene.The sex hierarchy consists of a pre-mRNA splicing cascade that directs the production of sex-specific transcription factors that specify nearly all sexual dimorphism. We have used deep RNA sequencing to gain insight into how the Drosophila sex hierarchy generates somatic sex differences, by examining gene and transcript isoform expression differences between the sexes in adult head tissues.<h4>Results</h4>Here we find 1,381 genes that differ in overall expression levels and 1,370 isoform-specific transcripts that differ between males and females. Additionally, we find 512 genes not regulated downstream of transformer that are significantly more highly expressed in males than females. These 512 genes are enriched on the × chromosome and reside adjacent to dosage compensation complex entry sites, which taken together suggests that their residence on the × chromosome might be sufficient to confer male-biased expression. There are no transcription unit structural features, from a set of features, that are robustly significantly different in the genes with significant sex differences in the ratio of isoform-specific transcripts, as compared to random isoform-specific transcripts, suggesting that there is no single molecular mechanism that generates isoform-specific transcript differences between the sexes, even though the sex hierarchy is known to include three pre-mRNA splicing factors.<h4>Conclusions</h4>We identify thousands of genes that show sex-specific differences in overall gene expression levels, and identify hundreds of additional genes that have differences in the abundance of isoform-specific transcripts. No transcription unit structural feature was robustly enriched in the sex-differentially expressed transcript isoforms. Additionally, we found that many genes with male-biased expression were enriched on the × chromosome and reside adjacent to dosage compensation entry sites, suggesting that differences in sex chromosome composition contributes to dimorphism in gene expression. Taken together, this study provides new insight into the molecular underpinnings of sexual differentiation.

Posttranscriptional status of messenger RNAs (mRNA) can be affected by many factors, most of which are RNA-binding proteins (RBP) that either bind mRNA in a nonspecific manner or through specific motifs, usually located in the 3' untranslated regions. RBPs can also be recruited by small noncoding RNAs (sncRNA), which have been shown to be involved in posttranscriptional regulations and transposon repression (eg, microRNAs or P-element-induced wimpy testis-interacting RNA) as components of the sncRNA effector complex. Non-sncRNA-binding RBPs have much more diverse effects on their target mRNAs. Some can cause degradation of their target transcripts and/or repression of translation, whereas others can stabilize and/or activate translation. The splicing and exportation of transcripts from the nucleus to the cytoplasm are often mediated by sequence-specific RBPs. The mechanisms by which RBPs regulate mRNA transcripts involve manipulating the 3' poly(A) tail, targeting the transcript to polysomes or to other ribonuclear protein particles, recruiting regulatory proteins, or competing with other RBPs. Here, we briefly review the known mechanisms of posttranscriptional regulation mediated by RBPs, with an emphasis on how these mechanisms might control spermatogenesis in general.

RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.

The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10(-12), OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10(-6), OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10(-7), OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10(-61), OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10(-76), OR = 8.84), and in NOTCH4 with ACA P = 8.84×10(-21), OR = 0.55) and ATA (P = 1.14×10(-8), OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc.

Chicken body weight is an economically important trait and great genetic progress has been accomplished in genetic selective for body weight. To identify genes and chromosome regions associated with body weight, we performed a genome-wide association study using the chicken 60 k SNP panel in a chicken F2 resource population derived from the cross between Silky Fowl and White Plymouth Rock. A total of 26 SNP effects involving 9 different SNP markers reached 5% Bonferroni genome-wide significance. A chicken chromosome 4 (GGA4) region approximately 8.6 Mb in length (71.6-80.2 Mb) had a large number of significant SNP effects for late growth during weeks 7-12. The LIM domain-binding factor 2 (LDB2) gene in this region had the strongest association with body weight for weeks 7-12 and with average daily gain for weeks 6-12. This GGA4 region was previously reported to contain body weight QTL. GGA1 and GGA18 had three SNP effects on body weight with genome-wide significance. Some of the SNP effects with the significance of "suggestive linkage" overlapped with previously reported results.

<h4>Background</h4>The molecular phenotype of Huntington's disease (HD) is known to comprise highly reproducible changes in gene expression involving striatal signaling genes. Here we test whether individual changes in striatal gene expression are capable of mitigating HD-related neurotoxicity.<h4>Methodology/principal findings</h4>We used protein-encoding and shRNA-expressing lentiviral vectors to evaluate the effects of RGS2, RASD2, STEP and NNAT downregulation in HD. Of these four genes, only RGS2 and RASD2 modified mutant htt fragment toxicity in cultured rat primary striatal neurons. In both cases, disease modulation was in the opposite of the predicted direction: whereas decreased expression of RGS2 and RASD2 was associated with the HD condition, restoring expression enhanced degeneration of striatal cells. Conversely, silencing of RGS2 or RASD2 enhanced disease-related changes in gene expression and resulted in significant neuroprotection. These results indicate that RGS2 and RASD2 downregulation comprises a compensatory response that allows neurons to better tolerate huntingtin toxicity. Assessment of the possible mechanism of RGS2-mediated neuroprotection showed that RGS2 downregulation enhanced ERK activation. These results establish a novel link between the inhibition of RGS2 and neuroprotective modulation of ERK activity.<h4>Conclusions</h4>Our findings both identify RGS2 downregulation as a novel compensatory response in HD neurons and suggest that RGS2 inhibition might be considered as an innovative target for neuroprotective drug development.

<h4>Purpose</h4>To investigate whether implantation of a glaucoma shunt device leads to inappropriate accumulation of plasma derived proteins in the aqueous humor.<h4>Methods</h4>Aqueous humor samples were collected from 11 patients (study group) with a glaucoma shunt device undergoing either cataract surgery or a corneal transplant and 11 patients (control) with senile cataract undergoing routine cataract extraction. Of the study group, 9 had an Ahmed valve implant and 2 eyes had a Baerveldt implant. Tryptic digests of the mixture of proteins in aqueous humor (AH) were analyzed using Liquid Chromatography/Mass Spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and compared quantitatively using a label-free platform (IdentiQuantXL™).<h4>Results</h4>We identified 135 proteins in the albumin-depleted fraction in both the study and control group AH. Using stringent criteria, 13 proteins were detected at a significantly higher level compared to controls. These proteins are known to play a role in oxidative stress, apoptosis, inflammation and/or immunity and include gelsolin (p=0.00005), plasminogen (p=0.00009), angiotensinogen (p=0.0001), apolipoprotein A-II (p=0.0002), beta-2-microglobulin (p=0.0002), dickkopf-3 (DKK-3; p=0.0002), pigment epithelium-derived factor (p=0.0002), RIG-like 7-1 (p=0.0002), afamin (p=0.0003), fibronectin 1 (FN1; p=0.0003), apolipoprotein A-I (p=0.0004), activated complement C4 protein (C4a; p=0.0004) and prothrombin (p=0.0004). Many of the identified proteins were novel proteins that have not been associated with glaucoma in prior studies. All but C4a (complement C4 is a plasma protein but not in an activated form) are known plasma proteins and the elevated levels of these proteins in the aqueous humor suggests a breach in the blood-aqueous barrier with passive influx into the anterior chamber of the eye.<h4>Conclusions</h4>The presence of these proteins in the aqueous humor suggests that glaucoma shunt device causes either a breach in blood-aqueous barrier or chronic trauma, increasing influx of oxidative, apoptotic and inflammatory proteins that could potentially cause corneal endothelial damage.

Rab GTPases are well-recognized targets in human disease, although are underexplored therapeutically. Elucidation of how mutant or dysregulated Rab GTPases and accessory proteins contribute to organ specific and systemic disease remains an area of intensive study and an essential foundation for effective drug targeting. Mutation of Rab GTPases or associated regulatory proteins causes numerous human genetic diseases. Cancer, neurodegeneration and diabetes represent examples of acquired human diseases resulting from the up- or downregulation or aberrant function of Rab GTPases. The broad range of physiologic processes and organ systems affected by altered Rab GTPase activity is based on pivotal roles in responding to cell signaling and metabolic demand through the coordinated regulation of membrane trafficking. The Rab-regulated processes of cargo sorting, cytoskeletal translocation of vesicles and appropriate fusion with the target membranes control cell metabolism, viability, growth and differentiation. In this review, we focus on Rab GTPase roles in endocytosis to illustrate normal function and the consequences of dysregulation resulting in human disease. Selected examples are designed to illustrate how defects in Rab GTPase cascades alter endocytic trafficking that underlie neurologic, lipid storage, and metabolic bone disorders as well as cancer. Perspectives on potential therapeutic modulation of GTPase activity through small molecule interventions are provided.

<h4>Objectives</h4>Neonatal hemochromatosis is a very bad prognosis disease; liver transplantation was the only way to avoid an unfavourable evolution. Nowadays, hypothesis of an alloimmune mechanism for this disease has purposed to administrate high doses of immunoglobulins.<h4>Patients and methods</h4>In this study, we report four cases of women whose previous child had neonatal hemochromatosis and who received such a treatment during the next pregnancy from 18 weeks to the term.<h4>Results</h4>This treatment allowed to lead their pregnancy to success. At birth, all four neonates were alive. Two of them presented transitory biologic symptoms of liver deficiency. All had a favourable evolution later.<h4>Discussion and conclusion</h4>Maternal treatment with high doses of immunoglobulins during pregnancy seems to improve dramatically the prognosis of neonatal hemochromatosis as it has been already reported. It could also apply to other diseases, which proceed from the same mechanism.

<h4>Background & aims</h4>HFE and transferrin receptor 2 (TFR2) are each necessary for the normal relationship between body iron status and liver hepcidin expression. In murine Hfe and Tfr2 knockout models of hereditary hemochromatosis (HH), signal transduction to hepcidin via the bone morphogenetic protein 6 (Bmp6)/Smad1,5,8 pathway is attenuated. We examined the effect of dietary iron on regulation of hepcidin expression via the Bmp6/Smad1,5,8 pathway using mice with targeted disruption of Tfr2, Hfe, or both genes.<h4>Methods</h4>Hepatic iron concentrations and messenger RNA expression of Bmp6 and hepcidin were compared with wild-type mice in each of the HH models on standard or iron-loading diets. Liver phospho-Smad (P-Smad)1,5,8 and Id1 messenger RNA levels were measured as markers of Bmp/Smad signaling.<h4>Results</h4>Whereas Bmp6 expression was increased, liver hepcidin and Id1 expression were decreased in each of the HH models compared with wild-type mice. Each of the HH models also showed attenuated P-Smad1,5,8 levels relative to liver iron status. Mice with combined Hfe/Tfr2 disruption were most affected. Dietary iron loading increased hepcidin and Id1 expression in each of the HH models. Compared with wild-type mice, HH mice demonstrated attenuated (Hfe knockout) or no increases in P-Smad1,5,8 levels in response to dietary iron loading.<h4>Conclusions</h4>These observations show that Tfr2 and Hfe are each required for normal signaling of iron status to hepcidin via the Bmp6/Smad1,5,8 pathway. Mice with combined loss of Hfe and Tfr2 up-regulate hepcidin in response to dietary iron loading without increases in liver Bmp6 messenger RNA or steady-state P-Smad1,5,8 levels.

<h4>Background and methods</h4>In two birth cohort studies with genetic, sensitive parenting, and attachment data of more than 1,000 infants in total, we tested main and interaction effects of candidate genes involved in the dopamine, serotonin, and oxytocin systems (DRD4, DRD2, COMT, 5-HTT, OXTR) on attachment security and disorganization. Parenting was assessed using observational rating scales for parental sensitivity (Ainsworth, Bell, & Stayton, 1974), and infant attachment was assessed with the Strange Situation Procedure.<h4>Results</h4>We found no consistent additive genetic associations for attachment security and attachment disorganization. However, specific tests revealed evidence for a codominant risk model for COMT Val158Met, consistent across both samples. Children with the Val/Met genotype showed higher disorganization scores (combined effect size d = .22, CI = .10-.34, p < .001). Gene-by-environment interaction effects were not replicable across the two samples.<h4>Conclusions</h4>This unexpected finding might be explained by a broader range of plasticity in heterozygotes, which may increase susceptibility to environmental influences or to dysregulation of emotional arousal. This study is unique in combining the two largest attachment cohorts with molecular genetic and observed rearing environment data to date.

5-Hydroxytryptamine (5-HT or serotonin) is an important neurotransmitter for a number of brain functions and widely distributed throughout the brain. Physiological and pharmacological relationship between 5-HT1A receptors and serotonin transporter (5-HTT) in the regulation of 5-HT neurotransmission has now been documented. A relationship between 5-HT1A receptors and 5-HTT is also suggested by the pathophysiology of depression and the mechanism of action of antidepressants. We have scanned 42 healthy adults with both [11C] WAY-100635 and [11C] DASB to investigate the anatomical co-distribution of multiple serotonergic markers. We hypothesized that lower 5-HTT densities in the dorsal raphe nucleus (DRN) and limbic regions will be accompanied by lower 5-HT1A receptor density in the same regions, contributing to the 5-HT1A receptor desensitization. In addition, variations in DRN 5-HT1A receptor density can theoretically influence the density and/or function of other serotonin receptor subtypes and the 5-HTT consequent to changes in serotonergic tone. In a comparatively large sample of volunteers, we have shown that the relationship between 5-HT1A and 5-HTT PET indices was complex. We were unable to demonstrate robust, intra-regional relationships between 5-HT1A and 5-HTT densities. Inter-regionally, DRN 5-HT1A receptors were related to cortical (temporal and frontal regions) and paralimbic (insula), but not limbic 5-HTT. This latter finding may reflect differences in 5-HT tone between individuals, and highlights probable substrates sensitive to variations in DRN 5-HT function.

Prenatal cannabis exposure can complicate in utero development of the nervous system. Cannabis impacts the formation and functions of neuronal circuitries by targeting cannabinoid receptors. Endocannabinoid signaling emerges as a signaling cassette that orchestrates neuronal differentiation programs through the precisely timed interaction of endocannabinoid ligands with their cognate cannabinoid receptors. By indiscriminately prolonging the 'switched-on' period of cannabinoid receptors, cannabis can hijack endocannabinoid signals to evoke molecular rearrangements, leading to the erroneous wiring of neuronal networks. Here, we formulate a hierarchical network design necessary and sufficient to describe the molecular underpinnings of cannabis-induced neural growth defects. We integrate signalosome components, deduced from genome- and proteome-wide arrays and candidate analyses, to propose a mechanistic hypothesis of how cannabis-induced ectopic cannabinoid receptor activity overrides physiological neurodevelopmental endocannabinoid signals, affecting the timely formation of synapses.

The Enhancer of Zeste 2 (EZH2) protein has been reported to stimulate cell growth in some cancers and is therefore considered to represent an interesting new target for therapeutic intervention. Here, we investigated a possible role of EZH2 for the growth control of colon cancer cells. RNA interference (RNAi)-mediated intracellular EZH2 depletion led to cell cycle arrest of colon carcinoma cells at the G1/S transition. This was associated with a reduction of cell numbers upon transient transfection of synthetic EZH2-targeting siRNAs and with inhibition of their colony formation capacity upon stable expression of vector-borne siRNAs. We furthermore tested whether EZH2 may repress the growth-inhibitory p27 gene, as reported for pancreatic cancer. However, expression analyses of colon cancer cell lines and colon cancer biopsies did not reveal a consistent correlation between EZH2 and p27 levels. Moreover, EZH2 depletion did not re-induce p27 expression in colon cancer cells, indicating that p27 repression by EZH2 may be cell- or tissue-specific. Whole genome transcriptome analyses identified cellular genes affected by EZH2 depletion in colon cancer cell lines. They included several cancer-associated genes linked to cellular proliferation or invasion, such as Dag1, MageD1, SDC1, Timp2, and Tob1. In conclusion, our results demonstrate that EZH2 depletion blocks the growth of colon cancer cells. These findings might provide benefits for the treatment of colon cancer.

In the brain, serotonin production is controlled by tryptophan hydroxylase 2 (TPH2), a genotype. Previous studies found that mutations on the TPH2 locus in humans were associated with depression and studies of mice and studies of rhesus macaques have shown that the TPH2 locus was involved with aggressive behavior. We previously reported a functional single nucleotide polymorphism (SNP) in the form of an amino acid substitution, Q468R, in the chimpanzee TPH2 gene coding region. In the present study we tested whether this SNP was associated with neuroticism in captive and wild-born chimpanzees living in Japan and Guinea, respectively. Even after correcting for multiple tests (Bonferroni p = 0.05/6 = 0.008), Q468R was significantly related to higher neuroticism (β = 0.372, p = 0.005). This study is the first to identify a genotype linked to a personality trait in chimpanzees. In light of the prior studies on humans, mice, and rhesus macaques, these findings suggest that the relationship between neuroticism and TPH2 has deep phylogenetic roots.

<h4>Objective</h4>A potentially useful tool for understanding the distribution and determinants of emotional dysregulation in children is a Child Behavior Checklist profile, comprising the Attention Problems, Anxious/Depressed, and Aggressive Behavior clinical subscales (CBCL-DP). The CBCL-DP indexes a heritable trait that increases susceptibility for later psychopathology, including severe mood problems and aggressive behavior. We have conducted a genome-wide association study of the CBCL-DP in children with attention-deficit/hyperactivity disorder (ADHD).<h4>Method</h4>Families were ascertained at Massachusetts General Hospital and University of California, Los Angeles. Genotyping was conducted with the Illumina Human1M or Human1M-Duo BeadChip platforms. Genome-wide association analyses were conducted with the MQFAM multivariate extension of PLINK.<h4>Results</h4>CBCL data were available for 341 ADHD offspring from 339 ADHD affected trio families from the UCLA (N = 128) and the MGH (N = 213) sites. We found no genome-wide statistically significant associations but identified several plausible candidate genes among findings at p < 5E-05: TMEM132D, LRRC7, SEMA3A, ALK, and STIP1.<h4>Conclusions</h4>We found suggestive evidence for developmentally expressed genes operant in hippocampal dependent memory and learning with the CBCL-DP.

Quercetin is a flavonoid natural product, that is, found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying all potential targets of quercetin. We describe an O7-biotinylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing quercetin binding proteins, which can then be identified by LC-MS/MS. BioQ was shown to inhibit heat induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor. BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin antibody. Analysis of the protein bands by trypsinization and LC-MS/MS led to the identification of heat shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quercetin. Most of the proteins identified have also been previously suggested to be potential anticancer targets, suggesting that quercetin's antitumor activity may be due to its ability to inhibit multiple target proteins.

Four guanidine derivatives of N4,N9-diacylated spermine have been designed, synthesized, and characterized. These guanidine-containing cationic lipids bound siRNA and formed nanoparticles. Two cationic lipids with C18 unsaturated chains, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in terms of GFP expression reduction compared to the other cationic lipids with shorter C12 (12:0) and very long C22 (22:1) chains. N1,N12-Diamidino-N4-linoleoyl-N9-oleoylspermine siRNA lipoplexes resulted in GFP reduction (26%) in the presence of serum, and cell viability (64%). These data are comparable to those obtained with TransIT TKO. Thus, cationic lipid guanidines based on N4,N9-diacylated spermines are good candidates for non-viral delivery of siRNA to HeLa cells using self-assembled lipoplexes.

<h4>Background</h4>Hypovitaminosis D has been recently recognized as a worldwide epidemic. Since vitamin D exerts significant metabolic activities, comprising free fatty acids (FFA) flux regulation from the periphery to the liver, its deficiency may promote fat deposition into the hepatocytes. Aim of our study was to test the hypothesis of a direct association between hypovitaminosis D and the presence of NAFLD in subjects with various degree of insulin-resistance and related metabolic disorders.<h4>Methods</h4>We studied 262 consecutive subjects referred to the Diabetes and Metabolic Diseases clinics for metabolic evaluation. NAFLD (non-alcoholic fatty liver disease) was diagnosed by upper abdomen ultrasonography, metabolic syndrome was identified according to the Third Report of National Cholesterol Education Program/Adult Treatment Panel (NCEP/ATPIII) modified criteria. Insulin-resistance was evaluated by means of HOMA-IR. Fatty-Liver-Index, a recently identified correlate of NAFLD, was also estimated. Serum 25(OH)vitamin D was measured by colorimetric method.<h4>Results</h4>Patients with NAFLD (n=162,61.8%) had reduced serum 25(OH) vitamin D levels compared to subjects without NAFLD (14.8±9.2 vs 20.5±9.7 ng/ml, p<0.001, OR 0.95, IC 95% 0.92-0.98). The relationship between NAFLD and reduced 25(OH)vitamin D levels was independent from age, sex, triglycerides, high density lipoproteins (HDL) and glycaemia (p<0.005) and Fatty Liver Index inversely correlated with low 25(OH) vitamin D regardless sex, age and HOMA-IR (p<0.007).<h4>Conclusions</h4>Low 25(OH)vitamin D levels are associated with the presence of NAFLD independently from metabolic syndrome, diabetes and insulin-resistance profile.

Pontocerebellar Hypoplasia (PCH) is group of very rare, inherited progressive neurodegenerative disorders with prenatal onset. Up to now seven different subtypes have been reported (PCH1-7). The incidence of each subtype is unknown. All subtypes share common characteristics, including hypoplasia/atrophy of cerebellum and pons, progressive microcephaly, and variable cerebral involvement. Patients have severe cognitive and motor handicaps and seizures are often reported. Treatment is only symptomatic and prognosis is poor, as most patients die during infancy or childhood. The genetic basis of different subtypes has been elucidated, which makes prenatal testing possible in families with mutations. Mutations in three tRNA splicing endonuclease subunit genes were found to be responsible for PCH2, PCH4 and PCH5. Mutations in the nuclear encoded mitochondrial arginyl- tRNA synthetase gene underlie PCH6. The tRNA splicing endonuclease, the mitochondrial arginyl- tRNA synthetase and the vaccinia related kinase1 are mutated in the minority of PCH1 cases. These genes are involved in essential processes in protein synthesis in general and tRNA processing in particular. In this review we describe the neuroradiological, neuropathological, clinical and genetic features of the different PCH subtypes and we report on in vitro and in vivo studies on the tRNA splicing endonuclease and mitochondrial arginyl-tRNA synthetase and discuss their relation to pontocerebellar hypoplasia.

Eukaryotic chromatin is a combination of DNA and histone proteins. It is established fact that epigenetic mechanisms are associated with DNA and histones. Initial studies emphasize on core histones association with DNA, however later studies prove the importance of linker histone H1 epigenetic. There are many types of linker histone H1 found in mammals. These subtypes are cell specific and their amount in different types of cells varies as the cell functions. Many types of post-translational modifications which occur on different residues in each subtype of linker histone H1 induce conformational changes and allow the different subtypes of linker histone H1 to interact with chromatin at different stages during cell cycle which results in the regulation of transcription and gene expression. Proposed O-glycosylation of linker histone H1 promotes condensation of chromatin while phosphorylation of linker histone H1 is known to activate transcription and gene regulation by decondensation of chromatin. Interplay between phosphorylation and O-β-GlcNAc modification on Ser and Thr residues in each subtype of linker histone H1 in Homo sapiens during cell cycle may result in diverse functional regulation of proteins. This in silico study describes the potential phosphorylation, o-glycosylation and their possible interplay sites on conserved Ser/Thr residues in various subtypes of linker histone H1 in Homo sapiens.

<h4>Objective</h4>Recent genome-wide association studies (GWAS) have identified multiple novel loci associated with adiposity in European-derived study populations. Limited study of these loci has been reported in African Americans. Here we examined the effects of these previously identified adiposity loci in African Americans.<h4>Methods</h4>A total of 46 representative single-nucleotide polymorphisms (SNPs) in 19 loci that were previously reported in GWAS in Europeans (including FTO and MC4R) were genotyped in 4992 subjects from six African-American cohorts. These SNPs were tested for association with body mass index (BMI) after adjustment for age, gender, disease status and population structure in each cohort. Meta-analysis was conducted to combine the results.<h4>Results</h4>Meta-analysis of 4992 subjects revealed seven SNPs near four loci, including NEGR1, TMEM18, SH2B1 /ATP2A1 and MC4R, showing significant association at 0.005<P<0.05, and had effect sizes between 0.04 and 0.06 s.d. units (or 0.30 to 0.44  g m(-2)) of BMI for each copy of the BMI-increasing allele. The most significantly associated SNPs (rs9424977, rs3101336 and rs2568958) are located in the NEGR1 gene (P=0.005, 0.020 and 0.019, respectively).<h4>Conclusion</h4>We replicated the association of variants at four loci in six African-American cohorts that demonstrated a consistent direction of association with previous studies of adiposity in Europeans. These loci are all highly expressed in the brain, consistent with an important role for central nervous system processes in weight regulation. However, further comprehensive examination of these regions may be necessary to fine map and elucidate for possible genetic differences between these two populations.

In Sjögren's syndrome, like in most other autoimmune diseases, the enigma leading to a pathogenic attack against self has not yet been solved. By definition, the disease must be mediated by specific immune reactions against endogenous tissues to qualify as an autoimmune disease. In Sjögren's syndrome the autoimmune response is directed against the exocrine glands, which, as histopathological hallmark of the disease, display persistent and progressive focal mononuclear cell infiltrates. Clinically, the disease in most patients is manifested by two severe symptoms: dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). A number of systemic features have also been described and the presence of autoantibodies against the ubiquitously expressed ribonucleoprotein particles Ro (Sjögren's-syndrome-related antigen A - SSA) and La (SSB) further underline the systemic nature of Sjögren's syndrome. The original explanatory concept for the pathogenesis of Sjögren's syndrome proposed a specific, self-perpetuating, immune mediated loss of acinar and ductal cells as the principal cause of salivary gland hypofunction. Although straightforward and plausible, the hypothesis, however, falls short of accommodating several Sjögren's syndrome-related phenomena and experimental findings. Consequently, researchers considered immune-mediated salivary gland dysfunction prior to glandular destruction and atrophy as potential molecular mechanisms underlying the symptoms of dryness in Sjögren's syndrome. Accordingly, apoptosis, fibrosis and atrophy of the salivary glands would represent consequences of salivary gland hypofunction. The emergence of advanced bio-analytical platforms further enabled the identification of potential biomarkers with the intent to improve Sjögren's syndrome diagnosis, promote the development of prognostic tools for Sjögren's syndrome and the long-term goal to identify possible processes for therapeutic treatment interventions. In addition, such approaches allowed us to glimpse at the apparent complexity of Sjögren's syndrome.

<h4>Background</h4>To identify in vivo new cardiac binding sites of serum response factor (SRF) in genes and to study the response of these genes to mild over-expression of SRF, we employed a cardiac-specific, transgenic mouse model, with mild over-expression of SRF (Mild-O SRF Tg).<h4>Methodology</h4>Microarray experiments were performed on hearts of Mild-O-SRF Tg at 6 months of age. We identified 207 genes that are important for cardiac function that were differentially expressed in vivo. Among them the promoter region of 192 genes had SRF binding motifs, the classic CArG or CArG-like (CArG-L) elements. Fifty-one of the 56 genes with classic SRF binding sites had not been previously reported. These SRF-modulated genes were grouped into 12 categories based on their function. It was observed that genes associated with cardiac energy metabolism shifted toward that of carbohydrate metabolism and away from that of fatty acid metabolism. The expression of genes that are involved in transcription and ion regulation were decreased, but expression of cytoskeletal genes was significantly increased. Using public databases of mouse models of hemodynamic stress (GEO database), we also found that similar altered expression of the SRF-modulated genes occurred in these hearts with cardiac ischemia or aortic constriction as well.<h4>Conclusion and significance</h4>SRF-modulated genes are actively regulated under various physiological and pathological conditions. We have discovered that a large number of cardiac genes have classic SRF binding sites and were significantly modulated in the Mild-O-SRF Tg mouse hearts. Hence, the mild elevation of SRF protein in the heart that is observed during typical adult aging may have a major impact on many SRF-modulated genes, thereby affecting cardiac structure and performance. The results from our study could help to enhance our understanding of SRF regulation of cellular processes in the aged heart.

Classical population genetics shows that varying permutations of genes and risk factors permit or disallow the effects of causative agents, depending on circumstance. For example, genes and environment determine whether a fox kills black or white rabbits on snow or black ash covered islands. Risk promoting effects are different on each island, but obscured by meta-analysis or GWAS data from both islands, unless partitioned by different contributory factors. In Alzheimer's disease, the foxes appear to be herpes, borrelia or chlamydial infection, hypercholesterolemia, hyperhomocysteinaemia, diabetes, cerebral hypoperfusion, oestrogen depletion, or vitamin A deficiency, all of which promote beta-amyloid deposition in animal models-without the aid of gene variants. All relate to risk factors and subsets of susceptibility genes, which condition their effects. All are less prevalent in convents, where nuns appear less susceptible to the ravages of ageing. Antagonism of the antimicrobial properties of beta-amyloid by Abeta autoantibodies in the ageing population, likely generated by antibodies raised to beta-amyloid/pathogen protein homologues, may play a role in this scenario. These agents are treatable by diet and drugs, vitamin supplementation, pathogen detection and elimination, and autoantibody removal, although again, the beneficial effects of individual treatments may be tempered by genes and environment.

A low p50 hemoglobin (Hb) (p50 indicates O(2) tension at which Hb is half-saturated)-based oxygen carrier conjugated to antioxidant enzymes via dicarboxymethylated poly(ethylene glycol) (PEG) linker may have the beneficial effect in protecting pancreatic beta cells from severe hypoxia at transplantation sites. In this study, the oxygen dissociation curves, Hill plots, Bohr Effect, and oxygen content of Hb conjugates were measured. The protective effect due to incubation of Hb-conjugates (Hb/PEG molar ratio 1:10) with pancreatic beta cells (RINm5F) against hypoxia (6%, 3%, and 1% oxygen) was evaluated by an MTT assay and confocal microscopy. Quantitatively, Hb conjugates with antioxidant enzymes offered statistically significant protection (p<0.01, increased viability ∼80%) from hypoxia compared to control cells in 1% oxygen environment. Confocal images also showed that the low p50 Hb conjugates with antioxidants protected RINm5F cells from hypoxia.

<h4>Background</h4>Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in E. coli had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. The objective of this study was to establish a procedure for expressing full-length DGAT1 in E. coli.<h4>Results</h4>An expression plasmid containing the open reading frame for tung tree (Vernicia fordii) DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in E. coli BL21(DE3). Immunoblotting showed that the recombinant DGAT1 (rDGAT1) was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The rDGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized rDGAT1 was partially purified by Ni-NTA affinity chromatography. PreScission protease digestion confirmed the identity of rDGAT1 and showed extensive precipitation following Ni-NTA affinity purification.<h4>Conclusions</h4>This study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system. The results suggest that recombinant DGAT1 is degraded extensively from the carboxyl terminus and associated with other proteins, lipids, and membranes.

Hepatitis C Virus (HCV) is a single stranded RNA virus which produces negative strand RNA as a replicative intermediate. We analyzed 75 RT-PCR studies that tested for negative strand HCV RNA in liver and other human tissues. 85% of the studies that investigated extrahepatic replication of HCV found one or more samples positive for replicative RNA. Studies using in situ hybridization, immunofluorescence, immunohistochemistry, and quasispecies analysis also demonstrated the presence of replicating HCV in various extrahepatic human tissues, and provide evidence that HCV replicates in macrophages, B cells, T cells, and other extrahepatic tissues. We also analyzed both short term and long term in vitro systems used to culture HCV. These systems vary in their purposes and methods, but long term culturing of HCV in B cells, T cells, and other cell types has been used to analyze replication. It is therefore now possible to study HIV-HCV co-infections and HCV replication in vitro.

<h4>Background</h4>Chronic iron overload (CIO) is associated with blood disorders such as thalassemias and hemochromatosis. A major prognostic indicator of survival in patients with CIO is iron-mediated cardiomyopathy characterized by contractile dysfunction and electrical disturbances, including slow heart rate (bradycardia) and heart block.<h4>Methods and results</h4>We used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced heart rate (≈20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting that bradycardia originates from altered intrinsic SAN pacemaker function. Indeed, spontaneous action potential frequencies in SAN myocytes with CIO were reduced in association with decreased L-type Ca(2+) current (I(Ca,L)) densities and positive (rightward) voltage shifts in I(Ca,L) activation. Pacemaker current (I(f)) was not affected by CIO. Because I(Ca,L) in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of Ca(V)1.3 channels (in addition to Ca(V)1.2 channels), our data suggest that elevated iron preferentially suppresses Ca(V)1.3 channel function. Consistent with this suggestion, CIO reduced Ca(V)1.3 mRNA levels by ≈40% in atrial tissue (containing SAN) and did not lower heart rate in Ca(V)1.3 knockout mice. CIO also induced PR-interval prolongation, heart block, and atrial fibrillation, conditions also seen in Ca(V)1.3 knockout mice.<h4>Conclusions</h4>Our results demonstrate that CIO selectively reduces Ca(V)1.3-mediated I(Ca,L), leading to bradycardia, slowing of electrical conduction, and atrial fibrillation as seen in patients with iron overload.

<h4>Purpose</h4>Colorectal cancer (CRC) develops as a result of a series of accumulated genomic changes that produce oncogene activation and tumor suppressor gene loss. These characteristics may classify CRC into subsets of distinct clinical behaviors.<h4>Patients and methods</h4>We studied two of these genomic defects-mismatch repair deficiency (MMR-D) and loss of heterozygosity at chromosomal location 18q (18qLOH)-in patients enrolled onto two phase III cooperative group trials for treatment of potentially curable colon cancer. These trials included prospective secondary analyses to determine the relationship between these markers and treatment outcome. A total of 1,852 patients were tested for MMR status and 955 (excluding patients with MMR-D tumors) for 18qLOH.<h4>Results</h4>Compared with stage III, more stage II tumors were MMR-D (21.3% v 14.4%; P < .001) and were intact at 18q (24.2% v 15.1%; P = .001). For the combined cohort, patients with MMR-D tumors had better 5-year disease-free survival (DFS; 0.76 v 0.67; P < .001) and overall survival (OS; 0.81 v 0.78; P = .029) than those with MMR intact (MMR-I) tumors. Among patients with MMR-I tumors, the status of 18q did not affect outcome, with 5-year values for patients with 18q intact versus 18qLOH tumors of 0.74 versus 0.65 (P = .18) for DFS and 0.81 versus 0.77 (P = .18) for OS.<h4>Conclusion</h4>We conclude that MMR-D tumor status, but not the presence of 18qLOH, has prognostic value for stages II and III colon cancer.

<h4>Background</h4>In spite of intensive efforts, understanding of the genetic aspects of familial prostate cancer (PC) remains largely incomplete. In a previous microsatellite-based linkage scan of 1,233 PC families, we identified suggestive evidence for linkage (i.e., LOD ≥ 1.86) at 5q12, 15q11, 17q21, 22q12, and two loci on 8p, with additional regions implicated in subsets of families defined by age at diagnosis, disease aggressiveness, or number of affected members.<h4>Methods</h4>In an attempt to replicate these findings and increase linkage resolution, we used the Illumina 6000 SNP linkage panel to perform a genome-wide linkage scan of an independent set of 762 multiplex PC families, collected by 11 International Consortium for Prostate Cancer Genetics (ICPCG) groups.<h4>Results</h4>Of the regions identified previously, modest evidence of replication was observed only on the short arm of chromosome 8, where HLOD scores of 1.63 and 3.60 were observed in the complete set of families and families with young average age at diagnosis, respectively. The most significant linkage signals found in the complete set of families were observed across a broad, 37 cM interval on 4q13-25, with LOD scores ranging from 2.02 to 2.62, increasing to 4.50 in families with older average age at diagnosis. In families with multiple cases presenting with more aggressive disease, LOD scores over 3.0 were observed at 8q24 in the vicinity of previously identified common PC risk variants, as well as MYC, an important gene in PC biology.<h4>Conclusions</h4>These results will be useful in prioritizing future susceptibility gene discovery efforts in this common cancer.

<h4>Background</h4>Leukoencephalopathy with brain stem and spinal cord involvement and brain lactate elevation (LBSL) was recently shown to be caused by mutations in the DARS2 gene, encoding a mitochondrial aspartyl-tRNA synthetase. So far, affected individuals were invariably compound heterozygous for two mutations in DARS2, and drug treatments have remained elusive.<h4>Methods</h4>Prospective 2-year follow-up of the natural history of the main presenting symptoms in a homozygous DARS2 mutation carrier, followed by a 60 day treatment with acetazolamide in two different doses and with two random treatment interruptions.<h4>Results</h4>The patient presented with exercise-induced paroxysmal gait ataxia and areflexia as an atypical phenotype associated with a novel homozygous DARS2 mutation. These features showed an excellent dose-dependent, sustained treatment response to a carbonic anhydrase inhibitor. Pathogenic mutations in episodic ataxia genes were excluded, thus making it highly unlikely that this phenotype was because of episodic ataxia as a second disorder besides LBSL.<h4>Conclusions</h4>This case demonstrates that DARS2 mutation homozygosity is not lethal, as suggested earlier, but compatible with a rather benign disease course. More importantly, it extends the phenotypic spectrum of LBSL and reveals that at least some DARS2-associated phenotypic features might be readily treatable. However, future observations of paroxsymal ataxia and, possibly, areflexia in other DARS2-mutated patients are warranted to further corroborate our finding that DARS2 mutations can lead to a paroxsymal ataxia phenotype.

<h4>Background</h4>Neurons and astrocytes are generated from common neural precursors, yet neurogenesis precedes astrocyte formation during embryogenesis. The mechanisms of neural development underlying suppression and de-suppression of differentiation-related genes for cell fate specifications are not well understood.<h4>Methodology/principal findings</h4>By using an in vitro system in which NTera-2 cells were induced to differentiate into an astrocyte-like lineage, we revealed a novel role for Sin3A in maintaining the suppression of GFAP in NTera-2 cells. Sin3A coupled with MeCP2 bound to the GFAP promoter and their occupancies were correlated with repression of GFAP transcription. The repression by Sin3A and MeCP2 may be an essential mechanism underlying the inhibition of cell differentiation. Upon commitment toward an astrocyte-like lineage, Sin3A- MeCP2 departed from the promoter and activated STAT3 simultaneously bound to the promoter and exon 1 of GFAP; meanwhile, olig2 was exported from nuclei to the cytoplasm. This suggested that a three-dimensional or higher-order structure was provoked by STAT3 binding between the promoter and proximal coding regions. STAT3 then recruited CBP/p300 to exon 1 and targeted the promoter for histone H3K9 and H3K14 acetylation. The CBP/p300-mediated histone modification further facilitates chromatin remodeling, thereby enhancing H3K4 trimethylation and recruitment of RNA polymerase II to activate GFAP gene transcription.<h4>Conclusions/significance</h4>These results provide evidence that exchange of repressor and activator complexes and epigenetic modifications are critical strategies for cellular differentiation and lineage-specific gene expression.

Dcc is the key receptor that mediates attractive responses of axonal growth cones to netrins, a family of axon guidance cues used throughout evolution. However, a Dcc homolog has not yet been identified in the chicken genome, raising the possibility that Dcc is not present in avians. Here we show that the closely related family member neogenin may functionally substitute for Dcc in the developing chicken spinal cord. The expression pattern of chicken neogenin in the developing spinal cord is a composite of the distribution patterns of both rodent Dcc and neogenin. Moreover, whereas the loss of mouse neogenin has no effect on the trajectory of commissural axons, removing chicken neogenin by RNA interference results in a phenotype similar to the functional inactivation of Dcc in mouse. Taken together, these data suggest that the chick neogenin is functionally equivalent to rodent Dcc.

Inhibition of dihydroorotate dehydrogenase (DHODH) for P. falciparum potentially represents a new treatment option for malaria, since DHODH catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and P. falciparum is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. We report herein the synthesis and structure-activity relationship of a series of 5-(2-methylbenzimidazol-1-yl)-N-alkylthiophene-2-carboxamides that are potent inhibitors against PfDHODH but do not inhibit the human enzyme. On the basis of efficacy observed in three mouse models of malaria, acceptable safety pharmacology risk assessment and safety toxicology profile in rodents, lack of potential drug-drug interactions, acceptable ADME/pharmacokinetic profile, and projected human dose, 5-(4-cyano-2-methyl-1H-benzo[d]imidazol-1-yl)-N-cyclopropylthiophene-2-carboxamide 2q was identified as a potential drug development candidate.

Alternative splicing of pre-mRNA generates protein diversity. Dysfunction of splicing machinery and expression of specific transcripts has been linked to cancer progression and drug response. Exon microarray technology enables genome-wide quantification of expression levels of the majority of exons and facilitates the discovery of alternative splicing events. Analysis of exon array data is more challenging than the analysis of gene expression data and there is a need for reliable quantification of exons and alternatively spliced variants. We introduce a novel, computationally efficient methodology, Multiple Exon Array Preprocessing (MEAP), for exon array data pre-processing, analysis and visualization. We compared MEAP with existing pre-processing methods, and validation of six exons and two alternatively spliced variants with qPCR corroborated MEAP expression estimates. Analysis of exon array data from head and neck squamous cell carcinoma (HNSCC) cell lines revealed several transcripts associated with 11q13 amplification, which is related with decreased survival and metastasis in HNSCC patients. Our results demonstrate that MEAP produces reliable expression values at exon, alternatively spliced variant and gene levels, which allows generating novel experimentally testable predictions.

Positron emission tomography (PET) is a nuclear imaging technique, which allows in vivo estimations of important physiological parameters such as, glucose metabolism and neuroreceptor binding enabling greater understanding of the pathophysiology of Parkinson's disease (PD). The review will evaluate the role of PET in assessing both the dopaminergic (DA) and non-DA systems in relation to the pathophysiology of PD, differential diagnosis, progression of disease and pre-clinical disease. Medication side effects, genetic forms of PD, the non-motor symptoms of PD and alternative restorative approaches will also be discussed in relation to how PET imaging can enhance our understanding of these aspects of the disease. PET neuroimaging has to date, provided an excellent tool to assess the underlying mechanisms of the disease as well as evaluating the complications and management of PD and has the potential to be of great clinical value if the current limitations of costing and availability are resolved.

Head and neck cancer (HNC) is a heterogenous and complex entity including diverse anatomical sites and a variety of tumor types displaying unique characteristics and different etilogies. Both environmental and genetic factors play a role in the development of the disease, but the underlying mechanism is still far from clear. Previous studies suggest that alterations in the genes acting in cellular signal pathways may contribute to head and neck carcinogenesis. In cancer, DNA methylation patterns display specific aberrations even in the early and precancerous stages and may confer susceptibility to further genetic or epigenetic changes. Silencing of the genes by hypermethylation or induction of oncogenes by promoter hypomethylation are frequent mechanisms in different types of cancer and achieve increasing diagnostic and therapeutic importance since the changes are reversible. Therefore, methylation analysis may provide promising clinical applications, including the development of new biomarkers and prediction of the therapeutic response or prognosis. In this review, we aimed to analyze the available information indicating a role for the epigenetic changes in HNC.

Type 1 hereditary haemochromatosis (HH) is a common genetic disorder in Caucasoids resulting from mutations in the HFE gene. Routine diagnostic testing for type 1 HH involves genotyping for two of these described HFE mutations, C282Y and H63D. In some cases typing of a third mutation, S65C is also performed. Several techniques have been reported for HFE genotyping and these include polymerase chain reaction (PCR)-sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), PCR-sequence-specific oligonucleotide probe (SSOP), real-time PCR followed by melting curve analysis and TaqMan assay. The aim of this study was to develop an alternative method to both conventional PCR and real-time PCR/TaqMan assay to detect all three HFE mutations in a single assay using Luminex technology. DNA controls of known genotypes (n = 109) were used to evaluate this approach. These controls were selected to represent the three possible genotypes (wild type, mutant, heterozygous) for each mutation. Subsequently, blind DNA samples (n = 100) were used to validate this method. This new assay was then compared with current techniques (in-house PCR-SSP and TaqMan assay). Comparison of genotypes obtained with the Luminex method with those previously reported by both in-house PCR-SSP and TaqMan assay showed 100% concordance for both DNA controls and blind DNA samples and no discrepancies were observed. Allelic frequency for C282Y, H63D and S65C mutations were 22%, 16% and 2%, respectively. We report here a high-throughput, accurate and robust multiplex luminex bead assay for routine clinical testing of C282Y, H63D and S65C mutations in the HFE gene.

Amorphous Calcium Phosphate (ACP) is an essential mineral phase formed in mineralized tissues and the first commercial product as artificial hydroxyapatite. ACP is unique among all forms of calcium phosphates in that it lacks long-range, periodic atomic scale order of crystalline calcium phosphates. The X-ray diffraction pattern is broad and diffuse with a maximum at 25 degree 2 theta, and no other different features compared with well-crystallized hydroxyapatite. Under electron microscopy, its morphological form is shown as small spheroidal particles in the scale of tenths nanometer. In aqueous media, ACP is easily transformed into crystalline phases such as octacalcium phosphate and apatite due to the growing of microcrystalline. It has been demonstrated that ACP has better osteoconductivity and biodegradability than tricalcium phosphate and hydroxyapatite in vivo. Moreover, it can increase alkaline phosphatase activities of mesoblasts, enhance cell proliferation and promote cell adhesion. The unique role of ACP during the formation of mineralized tissues makes it a promising candidate material for tissue repair and regeneration. ACP may also be a potential remineralizing agent in dental applications. Recently developed ACP-filled bioactive composites are believed to be effective anti-demineralizing/remineralizing agents for the preservation and repair of tooth structures. This review provides an overview of the development, structure, chemical composition, morphological characterization, phase transformation and biomedical application of ACP in dentistry.

<h4>Background</h4>Apolipoprotein E (apoE), a component of plasma lipoproteins, increases septic mortality in a rodent model of sepsis, presumably by enhancing lipid antigen presentation to antigen-presenting cells via the low-density lipoprotein receptor (LDLR). Downstream, this culminates in natural killer T (NKT) cell activation and cytokine secretion. To determine whether apoE antagonism would protect against septic mortality in mice, apoE-LDLR binding was antagonized using heparin, which can inhibit apoE's LDLR-binding site.<h4>Methods</h4>C57BL/6 mice underwent cecal ligation and puncture (CLP) and heparin infusion. Serum partial thromboplastin time and alanine aminotransferase were measured at 24 hours, and survival was monitored for 7 days after CLP. LDLR+/+ and LDLR-/- fibroblasts were incubated with apoE and heparin to measure apoE internalization. Hepatic NKT cells and cytokine levels were quantified via fluorescence-activated cell sorting.<h4>Results</h4>Heparin decreased CLP-induced mortality by 50% versus saline-treated controls, independent of anticoagulation. LDLR+/+ fibroblasts displayed decreased uptake of apoE when treated concurrently with heparin for 12 hours. In septic mice, hepatic alanine aminotransferase levels, hepatic NKT cells, and plasma cytokine levels decreased after heparin treatment.<h4>Conclusions</h4>This study demonstrates that heparin protects against septic mortality independent of its anticoagulant effect. This protective effect is associated with the inhibition of apoE-LDLR binding, diminished NKT proliferation and cytokine production, and hepatic dysfunction. These findings indicate a potential clinical role for apoE antagonism in the treatment of sepsis.

Bifidobacterium longum DJO10A was previously demonstrated to produce a lantibiotic, but only during growth on agar media. To evaluate the feasibility of production of this lantibiotic in broth media, a transcription analysis of the lanA gene was undertaken. Comparative microarray analysis of broth and agar cultures of B. longum DJO10A revealed that the lantibiotic production, modification, transport/peptidase, and immunity genes were significantly upregulated in agar cultures, while the two-component regulatory genes were expressed equally under both conditions. This suggested that the signal transduction regulatory system should function in broth cultures. Real-time PCR and Northern hybridization confirmed that lanA gene expression was significantly repressed in broth cultures. A crude lantibiotic preparation from an agar-grown culture was obtained, and its antimicrobial spectrum analysis revealed a broad inhibition range. Addition of this extract to broth cultures of B. longum DJO10A induced lanA gene expression in a dose-dependent fashion. Subinoculation using >10% of an induced broth culture maintained lanA expression. The expression of lanA was log-phase specific, being significantly downregulated in stationary phase. Transcription start analysis of lanA revealed a 284-bp 5' untranslated region, which was proposed to be involved in repression of transcription, while an inverted repeat structure located at bp -75 relative to the transcription start was strategically located to likely function as a binding site for the two-component response regulator. Understanding the transcription regulation of this lanA gene is the first step toward enabling production of this novel and potentially interesting lantibiotic in broth cultures.

The aim of this study was to determine: (1) the frequency of VEGFA gene locus (6p12) amplification in colorectal cancers, (2) the effect of gene amplification on clinical outcome using two independent colorectal cancer patient cohorts and (3) the relationship between amplification and KRAS or BRAF gene mutation as well as with other RAS/MAPK signalling proteins. Single-punch (n=1280; cohort 1) and multiple-punch (n=195; cohort 2) tissue microarrays were used for dual-labelling fluorescence in situ hybridization (FISH). Amplification was defined as a ratio >2 times for 6p12/centromere 6 signals. Mutation analysis of KRAS (codons 12 and 13) and BRAF (codon V600E) and immunohistochemistry for p-MAPK3/MAPK1, PEBP1, HMMR, p-AKT, PLAU, PLAUR, TP53 and VEGFA were performed on cohort 1. In cohort 1, VEGFA amplification was found in 39/1280 (3%) cases and linked to higher pT stage (P=0.022), higher tumor grade (P=0.024) and vascular invasion (P=0.003). The 5-year disease-specific survival rates were 31% (95% CI 17-46) and 57% (95% CI 54-60) for amplified and nonamplified cases, respectively (P<0.001). Results were confirmed in cohort 2. In multivariable analysis, the relative risk for amplification was 2.09 (95% CI 1.4-3.1; P<0.001) and linked to more frequent BRAF mutation (P=0.015), overexpression of p-MAPK3/MAPK1 (P=0.012) and PLAU (P=0.048) and loss of metastasis suppressor protein PEBP1 (P=0.047). VEGFA gene locus amplification highlights a small but remarkably aggressive subgroup of colorectal cancers. Further studies are needed to elucidate the potential role of amplification as a prognostic or predictive biomarker in both metastatic and nonmetastatic patients.

Chromatin-modifying enzymes play a fundamental role in regulating chromatin structure so that DNA replication is spatially and temporally coordinated. For example, the lysine demethylase 4A/Jumonji domain-containing 2A (KDM4A/JMJD2A) is tightly regulated during the cell cycle. Overexpression of JMJD2A leads to altered replication timing and faster S phase progression. In this study, we demonstrate that degradation of JMJD2A is regulated by the proteasome. JMJD2A turnover is coordinated through the SKP1-Cul1-F-box ubiquitin ligase complex that contains cullin 1 and the F-box and leucine-rich repeat protein 4 (FbxL4). This complex interacted with JMJD2A. Ubiquitin overexpression restored turnover and blocked the JMJD2A-dependent faster S phase progression in a cullin 1-dependent manner. Furthermore, increased ubiquitin levels decreased JMJD2A occupancy and BrdU incorporation at target sites. This study highlights a finely tuned mechanism for regulating histone demethylase levels and emphasizes the need to tightly regulate chromatin modifiers so that the cell cycle occurs properly.

Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.

Depression is the most common psychiatric disorder in Huntington's disease (HD) patients. In the general population, women are more prone to develop depression and such susceptibility might be related to serotonergic dysregulation. There is yet to be a study of sexual dimorphism in the development and presentation of depression in HD patients. We investigated whether 8-week-old male and female R6/1 transgenic HD mice display depressive-like endophenotypes associated with serotonergic impairments. We also studied the behavioral effects of acute treatment with sertraline. We found that only female HD mice exhibited a decreased preference for saccharin as well as impaired emotionality-related behaviors when assessed on the novelty-suppressed feeding test (NSFT) and the forced-swimming test (FST). The exaggerated immobility time displayed by female HD in the FST was reduced by acute administration of sertraline. We also report an increased response to the 5-HT(1A) receptor agonist 8-OH-DPAT in inducing hypothermia and a decreased 5-HT(2A) receptor function in HD animals. While tissue levels of serotonin were reduced in both male and female HD mice, we found that serotonin concentration and hydroxylase-2 (TPH2) mRNA levels were higher in the hippocampus of males compared to female animals. Finally, the antidepressant-like effects of sertraline in the FST were blunted in male HD animals. This study reveals sex-specific depressive-related behaviors during an early stage of HD prior to any cognitive and motor deficits. Our data suggest a crucial role for disrupted serotonin signaling in mediating the sexually dimorphic depression-like phenotype in HD mice.

<h4>Objective</h4>Hyalinizing trabecular tumours (HTTs) are rare follicular-derived neoplasms that behave in an almost benign manner. HTT is frequently misdiagnosed as papillary carcinoma by fine needle aspiration (FNA) cytology or as papillary or medullary carcinoma on surgical resection.<h4>Methods</h4>The authors examined FNA material from seven cases of histologically verified HTT. Cytological findings were reviewed and correlated with ultrasonographic and histological features. In addition, MIB-1 and calcitonin immunostaining was performed on surgical specimens, and BRAF mutation analysis on three pre-operative FNA specimens and seven histology specimens.<h4>Results</h4>The original cytological diagnosis was either suspicious or positive for papillary carcinoma in all patients. The FNA-based differential diagnoses included HTT, papillary carcinoma or, less likely, medullary carcinoma in two patients. Aspirates showed oval to spindle-shaped cells with frequent intranuclear inclusions, isolated in loosely cohesive groups with a trabecular or syncytial pattern in a bloody background. Radiating arrangements of tumour cells surrounding hyaline stroma with serrated calcifications and a lack of papillary or sheet-like fragments may suggest HTT on FNA. Spherical calcified bodies and possible psammoma bodies were frequently found in three cases. Retrospectively, six of the seven cases showed membranous immunoreactivity for MIB-1, but none of the seven possessed the BRAF (V600E) mutation or showed calcitonin reactivity.<h4>Conclusions</h4>Although the recognition of HTT on FNA cytology is difficult, because of its morphological similarities to papillary and medullary carcinoma, its characteristic cytological features along with ultrasonographic findings may suggest the diagnosis preoperatively and avoid surgical over-treatment.

Huntington's disease (HD) is the most prevalent polyglutamine expansion disorder. HD is caused by an expansion of CAG triplet in the huntingtin (HTT) gene, associated with striatal and cortical neuronal loss. Central and peripheral metabolic abnormalities and altered insulin-like growth factor-1 (IGF-1) levels have been described in HD. Thus, we hypothesized that restoration of IGF-1-mediated signaling pathways could rescue R6/2 mice from metabolic stress and behavioral changes induced by polyglutamine expansion. We analyzed the in vivo effect of continuous peripheral IGF-1 administration on diabetic parameters, body weight and motor behavior in the hemizygous R6/2 mouse model of HD. We used 9 week-old and age-matched wild-type mice, subjected to continuously infused recombinant IGF-I or vehicle, for 14 days. IGF-1 treatment prevented the age-related decrease in body weight in R6/2 mice. Although blood glucose levels were higher in R6/2 mice, they did not reach a diabetic state. Even though, IGF-1 ameliorated poor glycemic control in HD mice. This seemed to be associated with a decrease in blood insulin levels in R6/2 mice, which was increased following IGF-1 infusion. Similarly, blood IGF-1 levels decreased during aging in both wild-type and R6/2 mice, being significantly improved upon its continuous infusion. Although no significant differences were found in motor function in R6/2-treated mice, IGF-1 treatment highly improved paw clasping scores. In summary, these results suggest that IGF-1 has a protective role against HD-associated impaired glucose tolerance, by enhancing blood insulin levels.

Iron is essential for human life, but toxic if present in excess. To avoid iron overload and maintain iron homeostasis, all cells are able to regulate their iron content through the post-transcriptional control of iron genes operated by the cytosolic iron regulatory proteins that interact with iron responsive elements on iron gene mRNA. At the systemic level, iron homeostasis is regulated by the liver peptide hepcidin. Disruption of these regulatory loops leads to genetic diseases characterized by iron deficiency (iron-refractory iron-deficiency anemia) or iron overload (hemochromatosis). Alterations of the same systems are also found in acquired disorders, such as iron-loading anemias characterized by ineffective erythropoiesis and anemia of chronic diseases (ACD) associated with common inflammatory conditions. In ACD, iron is present in the body, but maldistributed, being deficient for erythropoiesis, but sequestered in macrophages. Studies of the hepcidin regulation by iron and inflammatory cytokines are revealing new pathways that might become targets of new therapeutic intervention in iron disorders.

Hyperphosphorylated tau protein constitutes a significant portion of intracellular inclusions in some neurodegenerative diseases. In addition, mutations in tau protein cause familial forms of frontotemporal dementia (FTD), indicating that dysfunction of tau protein is responsible for neurodegeneration and dementia. P301S tau-transgenic (Tg) mouse expressing human mutant tau in neurons exhibits similar features of human tauopathies including neuronal degeneration and filament accumulation consisted of hyperphosphorylated tau protein. In the present study, we attempted to characterize protein expression profiles in P301S tau-Tg mouse by using two-dimensional differential in-gel electrophoresis (2D-DIGE) coupled by peptide mass fingerprinting (PMF). As a result, we identified four upregulated proteins; heat shock protein 27 (Hsp27), peroxiredoxin 6 (Prdx6), apolipoprotein E (ApoE), and latexin (LTXN), all of which may function as a neuroprotective mechanism against tau toxicity. In immunohistochemistry, these four proteins were increased invariably in astrocytes, and these astrocytes infiltrated the area in which there are numerous accumulations of hyperphosphorylated tau and neuronal loss. Therefore, these results may indicate that astrocytes provide a neuroprotective mechanism against tau toxicity.

Cancers of the colon and rectum, which rank among the most frequent human tumors, are currently treated by surgical resection in locally restricted tumor stages. However, disease recurrence and formation of local and distant metastasis frequently occur even in cases with successful curative resection of the primary tumor (R0). Recent technological advances in molecular diagnostic analysis have led to a wealth of knowledge about the changes in gene transcription in all stages of colorectal tumors. Differential gene expression, or transcriptome analysis, has been proposed by many groups to predict disease recurrence, clinical outcome, and also response to therapy, in addition to the well-established clinico-pathological factors. However, the clinical usability of gene expression profiling as a reliable and robust prognostic tool that allows evidence-based clinical decisions is currently under debate. In this review, we will discuss the most recent data on the prognostic significance and potential clinical application of genome wide expression analysis in colorectal cancer.

The promise of personalized medicine depends on the ability to integrate genetic sequencing information into disease risk assessment for individuals. As genomic sequencing technology enters the realm of clinical care, its scale necessitates answers to key social and behavioral research questions about the complexities of understanding, communicating, and ultimately using sequence information to improve health. Our study captured the motivations and expectations of research participants who consented to participate in a research protocol, ClinSeq, which offers to return a subset of the data generated through high-throughput sequencing. We present findings from an exploratory study of 322 participants, most of whom identified themselves as white, non-Hispanic, and coming from higher socio-economic groups. Participants aged 45-65 years answered open-ended questions about the reasons they consented to ClinSeq and about what they anticipated would come of genomic sequencing. Two main reasons for participating were as follows: a conviction to altruism in promoting research, and a desire to learn more about genetic factors that contribute to one's own health risk. Overall, participants expected genomic research to help improve understanding of disease causes and treatments. Our findings offer a first glimpse into the motivations and expectations of individuals seeking their own genomic information, and provide initial insights into the value these early adopters of technology place on information generated by high-throughput sequencing studies.

Iron homeostasis is crucial to many biological functions in nearly all organisms, with roles ranging from oxygen transport to immune function. Disruption of iron homeostasis may result in iron overload or iron deficiency. Iron deficiency may have severe consequences, including anemia or changes in immune or neurotransmitter systems. Here we report on the variability of phenotypic iron tissue loss and splenomegaly and the associated quantitative trait loci (QTLs), polymorphic areas in the mouse genome that may contain one or more genes that play a role in spleen iron concentration or spleen weight under each dietary treatment. Mice from 26 BXD/Ty recombinant inbred strains, including the parent C57BL/6 and DBA/2 strains, were randomly assigned to adequate iron or iron-deficient diets at weaning. After 120 days, splenomegaly was measured by spleen weight, and spleen iron was assessed using a modified spectrophotometry technique. QTL analyses and gene expression comparisons were then conducted using the WebQTL GeneNetwork. We observed wide, genetic-based variability in splenomegaly and spleen iron loss in BXD/Ty recombinant inbred strains fed an iron-deficient diet. Moreover, we identified several suggestive QTLs. Matching our QTLs with gene expression data from the spleen revealed candidate genes. Our work shows that individual differences in splenomegaly response to iron deficiency are influenced at least partly by genetic constitution. We propose mechanistic hypotheses by which splenomegaly may result from iron deficiency.

<h4>Background</h4>Viral epidemics or pandemics of acute respiratory infections like influenza or severe acute respiratory syndrome pose a global threat. Antiviral drugs and vaccinations may be insufficient to prevent their spread.<h4>Objectives</h4>To review the effectiveness of physical interventions to interrupt or reduce the spread of respiratory viruses.<h4>Search strategy</h4>We searched The Cochrane Library, the Cochrane Central Register of Controlled Trials (CENTRAL 2010, Issue 3), which includes the Acute Respiratory Infections Group's Specialised Register, MEDLINE (1966 to October 2010), OLDMEDLINE (1950 to 1965), EMBASE (1990 to October 2010), CINAHL (1982 to October 2010), LILACS (2008 to October 2010), Indian MEDLARS (2008 to October 2010) and IMSEAR (2008 to October 2010).<h4>Selection criteria</h4>In this update, two review authors independently applied the inclusion criteria to all identified and retrieved articles and extracted data. We scanned 3775 titles, excluded 3560 and retrieved full papers of 215 studies, to include 66 papers of 67 studies. We included physical interventions (screening at entry ports, isolation, quarantine, social distancing, barriers, personal protection, hand hygiene) to prevent respiratory virus transmission. We included randomised controlled trials (RCTs), cohorts, case-controls, before-after and time series studies.<h4>Data collection and analysis</h4>We used a standardised form to assess trial eligibility. We assessed RCTs by randomisation method, allocation generation, concealment, blinding and follow up. We assessed non-RCTs for potential confounders and classified them as low, medium and high risk of bias.<h4>Main results</h4>We included 67 studies including randomised controlled trials and observational studies with a mixed risk of bias. A total number of participants is not included as the total would be made up of a heterogenous set of observations (participant people, observations on participants and countries (object of some studies)). The risk of bias for five RCTs and most cluster-RCTs was high. Observational studies were of mixed quality. Only case-control data were sufficiently homogeneous to allow meta-analysis. The highest quality cluster-RCTs suggest respiratory virus spread can be prevented by hygienic measures, such as handwashing, especially around younger children. Benefit from reduced transmission from children to household members is broadly supported also in other study designs where the potential for confounding is greater. Nine case-control studies suggested implementing transmission barriers, isolation and hygienic measures are effective at containing respiratory virus epidemics. Surgical masks or N95 respirators were the most consistent and comprehensive supportive measures. N95 respirators were non-inferior to simple surgical masks but more expensive, uncomfortable and irritating to skin. Adding virucidals or antiseptics to normal handwashing to decrease respiratory disease transmission remains uncertain. Global measures, such as screening at entry ports, led to a non-significant marginal delay in spread. There was limited evidence that social distancing was effective, especially if related to the risk of exposure.<h4>Authors' conclusions</h4>Simple and low-cost interventions would be useful for reducing transmission of epidemic respiratory viruses. Routine long-term implementation of some measures assessed might be difficult without the threat of an epidemic.

No abstract available.

Addiction represents a complex interaction between the reward and stress neural circuits, with increasing drug use reflecting a shift from positive reinforcement to negative reinforcement mechanisms in sustaining drug dependence. Preclinical studies have indicated the involvement of regions within the extended amygdala as subserving this transition, especially under stressful conditions. In the addictive situation, the reward system serves to maintain habitual behaviors that are associated with the relief of negative affect, at the cost of attenuating the salience of other rewards. Therefore, addiction reflects the dysregulation between core reward systems, including the prefrontal cortex (PFC), ventral tegmental area (VTA), and nucleus accumbens (NAc), as well as the hypothalamic-pituitary-adrenal axis and extended amygdala of the stress system. Here, we consider the consequences of changes in neural function during or following addiction on parenting, an inherently rewarding process that may be disrupted by addiction. Specifically, we outline the preclinical and human studies that support the dysregulation of reward and stress systems by addiction and the contribution of these systems to parenting. Increasing evidence suggests an important role for the hypothalamus, PFC, VTA, and NAc in parenting, with these same regions being those dysregulated in addiction. Moreover, in addicted adults, we propose that parenting cues trigger stress reactivity rather than reward salience, and this may heighten negative affect states, eliciting both addictive behaviors and the potential for child neglect and abuse.

An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded 'fixed charge' sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS(3), and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S'-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of 'light' (S(CH(3))(2)) and 'heavy' (S(CD(3))(2)) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS(3) or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency.

High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2-4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence.

TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.

The use of tyrosine kinase inhibitors (TKIs) has become an integral component of cancer therapy. Imatinib mesylate, a breakpoint cluster region-Abelson BCR-ABL1 inhibitor, was the first TKI approved in cancer medicine and has served as a model for the development of similar agents for other cancers. An important drawback of TKI therapy is the development of resistance, frequently through the acquisition of mutations. Mutations at the gatekeeper residues of BCR-ABL1 (eg, the threonine-to-isoleucine mutation at codon 315) and other oncogenic kinases have proven highly resistant to currently available TKIs. Advances in the structural biology of oncogenic kinases have facilitated the rational development of TKIs that are active against gatekeeper mutations.

Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.

Capan-1 is a well-characterised BRCA2-deficient human cell line isolated from a liver metastasis of a pancreatic adenocarcinoma. Here we report a genome-wide assessment of structural variations and high-depth exome characterization of single nucleotide variants and small insertion/deletions in Capan-1. To identify potential somatic and tumour-associated variations in the absence of a matched-normal cell line, we devised a novel method based on the analysis of HapMap samples. We demonstrate that Capan-1 has one of the most rearranged genomes sequenced to date. Furthermore, small insertions and deletions are detected more frequently in the context of short sequence repeats than in other genomes. We also identify a number of novel mutations that may represent genetic changes that have contributed to tumour progression. These data provide insight into the genomic effects of loss of BRCA2 function.

Although increased oxidative stress has been associated with the impairment of proliferation and function of adult human muscle stem cells, proteins either involved in the stress response or damaged by oxidation have not been identified. A parallel proteomics approach was performed for analyzing the protein expression profile as well as proteins preferentially oxidized upon hydrogen peroxide-induced oxidative stress. Fifteen proteins involved in the oxidative stress response were identified. Among them, protein spots identified as peroxiredoxins 1 and 6, glyceraldehyde-3-phosphate dehydrogenase, and α-enolase were shifted to a more acidic isoelectric point upon oxidative stress, indicating posttranslational modifications. Oxidized proteins were evidenced by immunodetection of derivatized carbonyl groups followed by identification by mass spectrometry. The carbonylated proteins identified are mainly cytosolic and involved in carbohydrate metabolism, cellular assembly, cellular homeostasis, and protein synthesis and degradation. Pathway analysis revealed skeletal and muscular disorders, cell death, and cancer-related as the main molecular networks altered. Interestingly, these pathways were focused on two distinct proteins: p53 for altered protein expression and huntingtin for increased protein carbonylation. This study emphasizes the importance of performing analysis addressing different aspects of the cellular proteome to have a more accurate view of their changes upon stress.

Function of non-B DNA structures are poorly understood though several bioinformatics studies predict role of the G-quadruplex DNA structure in transcription. Earlier, using transcriptome profiling we found evidence of widespread G-quadruplex-mediated gene regulation. Herein, we asked whether potential G-quadruplex (PG4) motifs associate with transcription factors (TF). This was analyzed using 220 position weight matrices [designated as transcription factor binding sites (TFBS)], representing 187 unique TF, in >75,000 genes in human, chimpanzee, mouse and rat. Results show binding sites of nine TFs, including that of AP-2, SP1, MAZ and VDR, occurred significantly within 100 bases of the PG4 motif (P < 1.24E-10). PG4-TFBS combinations were conserved in 'orthologously' related promoters across all four organisms and were associated with >850 genes in each genome. Remarkably, seven of the nine TFs were zinc-finger binding proteins indicating a novel characteristic of PG4 motifs. To test these findings, transcriptome profiles from human cell lines treated with G-quadruplex-specific molecules were used; 66 genes were significantly differentially expressed across both cell-types, which also harbored conserved PG4 motifs along with one/more of the nine TFBS. In addition, genes regulated by PG4-TFBS combinations were found to be co-regulated in human tissues, further emphasizing the regulatory significance of the associations.

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4(+) Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4(+) T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4(+) Th cells upon activation, whereas the 'standard' proteasome is up-regulated in Tregs only upon activation.

Discrete and punctate nuclear RNA foci are characteristic molecular hallmarks of pathogenesis in myotonic dystrophy type 1 and type 2. Intranuclear RNA inclusions of distinct morphology have also been found in fragile X-associated tremor ataxia syndrome, Huntington's disease-like 2, spinocerebellar ataxias type 8, type 10 and type 31. These neurological diseases are associated with the presence of abnormally long simple repeat expansions in their respective genes whose expression leads to the formation of flawed transcripts with altered metabolisms. Expanded CUG, CCUG, CGG, CAG, AUUCU and UGGAA repeats are associated with the diseases and accumulate in nuclear foci, as demonstrated in variety of cells and tissues of human and model organisms. These repeat RNA foci differ in size, shape, cellular abundance and protein composition and their formation has a negative impact on cellular functions. This review summarizes the efforts of many laboratories over the past 15 years to characterize nuclear RNA foci that are recognized as important triggers in the mutant repeat RNA toxic gain-of-function mechanisms of pathogenesis in neurological disorders.

Pseudomonas aeruginosa possesses a type III secretion system (T3SS) to intoxicate host cells and evade innate immunity. This virulence-related machinery consists of a molecular syringe and needle assembled on the bacterial surface, which allows delivery of T3 effector proteins into infected cells. To accomplish a one-step effector translocation, a tip protein is required at the top end of the T3 needle structure. Strains lacking expression of the functional tip protein fail to intoxicate host cells. P. aeruginosa encodes a T3S that is highly homologous to the proteins encoded by Yersinia spp. The needle-tip proteins of Yersinia, LcrV, and P. aeruginosa, PcrV, share 37% identity and 65% similarity. Other known tip proteins are AcrV (Aeromonas), IpaD (Shigella), SipD (Salmonella), BipD (Burkholderia), EspA (EPEC, EHEC), Bsp22 (Bordetella), with additional proteins identified from various Gram-negative species, such as Vibrio and Bordetella. The tip proteins can serve as a protective antigen or may be critical for sensing host cells and evading innate immune responses. Recognition of the host microenvironment transcriptionally activates synthesis of T3SS components. The machinery appears to be mechanically controlled by the assemblage of specific junctions within the apparatus. These junctions include the tip and base of the T3 apparatus, the needle proteins and components within the bacterial cytoplasm. The tip proteins likely have chaperone functions for translocon proteins, allowing the proper assembly of translocation channels in the host membrane and completing vectorial delivery of effector proteins into the host cytoplasm. Multi-functional features of the needle-tip proteins appear to be intricately controlled. In this review, we highlight the functional aspects and complex controls of T3 needle-tip proteins with particular emphasis on PcrV and LcrV.

Multiple sclerosis (MS) affects the central nervous system leading to disability and is complicated by bone loss and fractures. Despite the acceptance of osteoporosis and fractures as two major public health problems, in people with MS the mechanisms have not been investigated adequately. Physicians and patients usually focus on the major cause of disability and neglect the multiple risk factors for osteoporosis and fractures in this specific population. This review updates the epidemiology and physiopathological mechanisms in MS.

Chronic liver injuries of different etiologies eventually lead to fibrosis, a scarring process associated with increased and altered deposition of extracellular matrix in the liver. Progression of fibrosis has a major worldwide clinical impact due to the high number of patients affected by chronic liver disease which can lead to severe complications, expensive treatment, a possible need for liver transplantation, and death. Liver fibrogenesis is characterized by activation of hepatic stellate cells and other extracellular matrix producing cells. Liver fibrosis may regress following specific therapeutic interventions. Other than removing agents causing chronic liver damage, no antifibrotic drug is currently available in clinical practice. The extent of liver fibrosis is variable between individuals, even after controlling for exogenous factors. Thus, host genetic factors are considered to play an important role in the process of liver scarring. Until recently it was believed that this process was irreversible. However, emerging experimental and clinical evidence is starting to show that even cirrhosis in its early stages is potentially reversible.

MYEOV and NEGR1 are novel candidate gene targets in neuroblastoma that were identified by chromosomal gain in 11q13 and loss in 1p31, respectively, through single nucleotide polymorphism array analysis. In the present study, to assess the involvement of MYEOV and NEGR1 in the pathogenesis of neuroblastoma, we analyzed their mutation status and/or expression profiles in a panel of 55 neuroblastoma samples, including 25 cell lines, followed by additional functional studies. No tumor-specific mutations of MYEOV or NEGR1 were identified in our case series. Expression of MYEOV was upregulated in 11 of 25 cell lines (44%) and in seven of 20 fresh tumors (35%). The siRNA-mediated knockdown of MYEOV in NB-19 cells, which exhibit high expression of MYEOV, resulted in a significant decrease in cell proliferation (P = 0.0027). Conversely, expression studies of NEGR1 revealed significantly lower expression of this gene in neuroblastomas at an advanced stage of the disease. Exogenous NEGR1 expression in neuroblastoma cells induced significant inhibition of cell growth (P = 0.019). The results of these studies provide supporting evidence for MYEOV and NEGR1 as gene targets of 11q13 gains and 1p31 deletions in a neuroblastoma subset. In addition, the findings suggest a possible prognostic value for NEGR1 in neuroblastoma.

APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca(2+) and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.

Many human neurological diseases are not currently curable and result in devastating neurologic sequelae. The increasing availability of induced pluripotent stem cells (iPSCs) derived from adult human somatic cells provides new prospects for cellreplacement strategies and disease-related basic research in a broad spectrum of human neurologic diseases. Patient-specific iPSC-based modeling of neurogenetic and neurodegenerative diseases is an emerging efficient tool for in vitro modeling to understand disease and to screen for genes and drugs that modify the disease process. With the exponential increase in iPSC research in recent years, human iPSCs have been successfully derived with different technologies and from various cell types. Although there remain a great deal to learn about patient-specific iPSC safety, the reprogramming mechanisms, better ways to direct a specific reprogramming, ideal cell source for cellular grafts, and the mechanisms by which transplanted stem cells lead to an enhanced functional recovery and structural reorganization, the discovery of the therapeutic potential of iPSCs offers new opportunities for the treatment of incurable neurologic diseases. However, iPSC-based therapeutic strategies need to be thoroughly evaluated in preclinical animal models of neurological diseases before they can be applied in a clinical setting.

<h4>Summary</h4>The impairment of osteoblast differentiation is one cause of the glucocorticoid-induced osteoporosis (GCOP). The quantitative proteomic analysis of the dexamethasone (DEX)-induced effects of osteoblast differentiation, proliferation, and apoptosis using stable-isotope labeling by amino acids in cell culture (SILAC) demonstrated drastic changes of some key proteins in MC3T3-E1 cells.<h4>Introduction</h4>The impairment of osteoblast differentiation is one of the main explanations of GCOP. SILAC enables accurate quantitative proteomic analysis of protein changes in cells to explore the underlying mechanism of GCOP.<h4>Methods</h4>Osteoprogenitor MC3T3-E1 cells were treated with or without 10(−6) M DEX for 7 days, and the differentiation ability, proliferation, and apoptosis of the cells were measured. The protein level changes were analyzed using SILAC and liquid chromatography-coupled tandem mass spectrometry.<h4>Results</h4>In this study, 10(−6) M DEX inhibited both osteoblast differentiation and proliferation but induced apoptosis in osteoprogenitor MC3T3-E1 cells on day 7. We found that 10(−6) M DEX increased the levels of tubulins (TUBA1A, TUBB2B, and TUBB5), IQGAP1, S100 proteins (S100A11, S100A6, S100A4, and S100A10), myosin proteins (MYH9 and MYH11), and apoptosis and stress proteins, while inhibited the protein levels of ATP synthases (ATP5O, ATP5H, ATP5A1, and ATP5F1), G3BP-1, and Ras-related proteins (Rab-1A, Rab-2A, and Rab-7) in MC3T3-E1 cells.<h4>Conclusions</h4>Several members of the ATP synthases, myosin proteins, small GTPase superfamily, and S100 proteins may participate in functional inhibition of osteoblast progenitor cells by GCs. Such protein expression changes may be of pathological significance in coping with GCOP.

Condensin, a major non-histone protein complex on chromosomes, is responsible for the formation of rod-shaped chromosome in mitosis. A heterodimer composed of SMC2 (structural maintenance of chromosomes) and SMC4 subunits constitutes the core part of condensin. Although extensive studies have been done in yeast, fruit fly and Xenopus to uncover the mechanisms and molecular nature of SMC proteins, little is known about the complex in mammalian cells. We have conducted a series of experiments to unveil the nature of condensin complex in human chromosome formation. The results show that overexpression of the C-terminal domain of SMC subunits disturbs chromosome condensation, leading to formation of swollen chromosomes, while knockdown of SMC subunits severely disturbs mitotic chromosome formation, resulting in chromatin bridges between daughter cells and multiple nuclei in single cells. The salt extraction assay indicates that a fraction of the condensin complex is bound to chromatin in interphase, but most of the condensin bind to chromatin at the onset of mitosis. Thus, disturbance in condensin function or expression affects chromosome condensation and influences mitotic progression.

<h4>Purpose</h4>To identify the changes in postnatal mouse conjunctival forniceal gene expression and their regulation by Klf4 during the eye-opening stage when the goblet cells first appear.<h4>Methods</h4>Laser microdissection (LMD) was used to collect conjunctival forniceal cells from postnatal (PN) day 9, PN14 and PN20 wild-type (WT), and PN14 Klf4-conditional null (Klf4CN) mice, in which goblet cells are absent, developing, present, and missing, respectively. Microarrays were used to compare gene expression among these groups. Expression of selected genes was validated by quantitative RT-PCR, and spatiotemporal expression was assessed by in situ hybridization.<h4>Results</h4>This study identified 668, 251, 1160, and 139 transcripts that were increased and 492, 377, 1419, and 57 transcripts that were decreased between PN9 and PN14, PN14 and PN20, PN9 and PN20, and PN14 WT and Klf4CN conjunctiva, respectively. Transcripts encoding transcription factors Spdef, FoxA1, and FoxA3 that regulate goblet cell development in other mucosal epithelia, and epithelium-specific Ets (ESE) transcription factor family members were increased during conjunctival development. Components of pathways related to the mesenchymal-epithelial transition, glycoprotein biosynthesis, mucosal immunity, signaling, and endocytic and neural regulation were increased during conjunctival development. Conjunctival Klf4 target genes differed significantly from the previously identified corneal Klf4 target genes, implying tissue-dependent regulatory targets for Klf4.<h4>Conclusions</h4>The changes in gene expression accompanying mouse conjunctival development were identified, and the role of Klf4 in this process was determined. This study provides new probes for examining conjunctival development and function and reveals that the gene regulatory network necessary for goblet cell development is conserved across different mucosal epithelia.

<h4>Objective</h4>The overexpression of interferon (IFN)-inducible genes is a prominent feature of systemic lupus erythematosus (SLE); it serves as a marker for active and more severe disease, and is also observed in other autoimmune and inflammatory conditions. This study was undertaken to investigate the genetic variations responsible for sustained activation of IFN-responsive genes in SLE.<h4>Methods</h4>We systematically evaluated association of SLE with a total of 1,754 IFN pathway-related genes, including IFN-inducible genes known to be differentially expressed in SLE patients and their direct regulators. We used a 3-stage study design in which 2 cohorts (total of 939 SLE cases and 3,398 controls) were analyzed independently and jointly for association with SLE, and the results were adjusted for the number of comparisons.<h4>Results</h4>A total of 15,166 single-nucleotide polymorphisms (SNPs) passed all quality control filters; 305 of these SNPs demonstrated replicated association with SLE in both cohorts. Nine variants were further genotyped for confirmation in an average of 1,316 independent SLE cases and 3,215 independent controls. Association with SLE was confirmed for several genes, including those for the transmembrane receptor CD44 (CD44 [rs507230]; P = 3.98 × 10⁻¹²), the cytokine pleiotrophin (PTN [rs919581]; P = 5.38 × 10⁻⁴), the heat-shock protein DnaJ (DNAJA1 [rs10971259]; P = 6.31 × 10⁻³), and the nuclear import protein karyopherin α1 (KPNA [rs6810306]; P = 4.91 × 10⁻²).<h4>Conclusion</h4>This study expands the number of candidate genes that have been shown to be associated with SLE and highlights potential of pathway-based approaches for gene discovery. Identification of the causal alleles will help elucidate the molecular mechanisms responsible for activation of the IFN system in SLE.

No abstract available.

This study was designed to investigate mechanisms of the protective effects of Salvia miltiorrhiza polysaccharide (SMPS) against lipopolysaccharide (LPS)-induced immunological liver injury (ILI) in Bacille Calmette-Guérin (BCG)-primed mice. Two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that three proteins are down-regulated and six proteins are up-regulated by SMPS. SMPS reduces the degree of liver injury by up-regulating the enzymes of the citric acid cycle, namely malate dehydrogenase (MDH) and 2-oxoglutarate dehydrogenase complex. LPS significantly increases nuclear factor kappa B (NF-κB) activation, inducible nitric oxide synthase (iNOS) expression and MDA level in BCG primed mice liver, whereas SMPS treatment protects against the immunological liver injury through inhibition of the NF-κB activation by up-regulation of PRDX6 and the subsequent attenuation of lipid peroxidation, iNOS expression and inflammation.

<h4>Unlabelled</h4>The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. Although liver iron content is independently positively correlated with hepatic Bmp6 messenger RNA (mRNA) expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice.<h4>Conclusion</h4>Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, whereas the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.

<h4>Purpose of review</h4>Several advances have been made to manipulate the rat genome in the last 2 years. This review aims to describe these advances in rat genetic manipulations, with an emphasis on their current status and their prospects and applications in the postgenomic era.<h4>Recent findings</h4>Authentic rat embryonic stem cells were derived in 2008 using the 2i/3i culture system. This led to the generation of the first gene knockout rats via embryonic stem cell-based gene targeting. The development of zinc-finger nucleases (ZFNs) provided an alternative approach that avoids the necessity of germline competent embryonic stem cells. Meanwhile, improvements have been made to the well established random mutagenesis mediated by transposons or N-ethyl-N-nitrosourea (ENU). The in-vitro rat spermatogonial stem cell (SSC) system has greatly optimized these phenotype-driven approaches for future applications.<h4>Summary</h4>The rat has long been a prime model organism in physiological, pharmacological and neurobehavioral studies. The recent advances of rat reverse genetic approaches, together with the classical ENU and transposon mutagenesis system, will contribute tremendously to the deciphering of gene functions and the creation of rat disease models.

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

Over 20 genetic loci with abnormal expansions of short tandem repeats have been associated with human hereditary neurological diseases. Of these, specific trinucleotide repeats located in non-coding and coding regions of individual genes implicated in these disorders are strongly overrepresented. Expansions of CTG, CGG and CAG repeats are linked to, respectively, myotonic dystrophy type 1 (DM1), fragile X-associated tremor/ataxia syndrome (FXTAS), as well as Huntington's disease (HD) and a number of spinocerebellar ataxias (SCAs). Expanded CAG repeats in translated exons trigger the most disorders for which a protein gain-of-function mechanism has been proposed to explain neurodegeneration by polyglutamine-rich (poly-Q) proteins. However, the results of last years showed that RNA composed of mutated CAG repeats can also be toxic and contribute to pathogenesis of polyglutamine disorders through an RNA-mediated gain-of-function mechanism. This mechanism has been best characterized in the non-coding repeat disorder DM1 and is also implicated in several other diseases, such as FXTAS, spinocerebellar ataxia type 8 (SCA8), Huntington's disease-like 2 (HDL2), as well as in myotonic dystrophy type 2 (DM2), spinocerebellar ataxia type 10 (SCA10) and type 31 (SCA31). In this review, we summarize recent findings that emphasize the participation of coding mutant CAG repeat RNA in the pathogenesis of polyglutamine disorders, and we discuss the basis of an RNA gain-of-function model in non-coding diseases such as DM1, FXTAS and SCA8.

<h4>Background</h4>Pegylated interferon, ribavirin, and telaprevir triple therapy is a new strategy expected to eradicate the hepatitis C virus (HCV) even in patients infected with difficult-to-treat genotype 1 strains, although adverse effects, such as anemia and rash, are frequent.<h4>Methods</h4>We assessed efficacy and predictive factors for sustained virological response (SVR) for triple therapy in 94 Japanese patients with HCV genotype 1. We included recently identified predictive factors, such as IL28B and ITPA polymorphism, and substitutions in the HCV core and NS5A proteins.<h4>Results</h4>Patients treated with triple therapy achieved comparatively high SVR rates (73%), especially among treatment-naive patients (80%). Of note, however, patients who experienced relapse during prior pegylated interferon plus ribavirin combination therapy were highly likely to achieve SVR while receiving triple therapy (93%); conversely, prior nonresponders were much less likely to respond to triple therapy (32%). In addition to prior treatment response, IL28B SNP genotype and rapid viral response were significant independent predictors for SVR. Patients with the anemia-susceptible ITPA SNP rs1127354 genotype typically required ribavirin dose reduction earlier than did patients with other genotypes.<h4>Conclusions</h4>Analysis of predictive factors identified IL28B SNP, rapid viral response, and transient response to previous therapy as significant independent predictors of SVR after triple therapy.

Intratumor heterogeneity is a major clinical problem because tumor cell subtypes display variable sensitivity to therapeutics and may play different roles in progression. We previously characterized 2 cell populations in human breast tumors with distinct properties: CD44+CD24- cells that have stem cell-like characteristics, and CD44-CD24+ cells that resemble more differentiated breast cancer cells. Here we identified 15 genes required for cell growth or proliferation in CD44+CD24- human breast cancer cells in a large-scale loss-of-function screen and found that inhibition of several of these (IL6, PTGIS, HAS1, CXCL3, and PFKFB3) reduced Stat3 activation. We found that the IL-6/JAK2/Stat3 pathway was preferentially active in CD44+CD24- breast cancer cells compared with other tumor cell types, and inhibition of JAK2 decreased their number and blocked growth of xenografts. Our results highlight the differences between distinct breast cancer cell types and identify targets such as JAK2 and Stat3 that may lead to more specific and effective breast cancer therapies.

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, also known as CD71), which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q, suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition, in 2 disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant antitumor activity, including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.

Asthma and allergy are common conditions with complex etiologies involving both genetic and environmental contributions. Recent genome-wide association studies (GWAS) and meta-analyses of GWAS have begun to shed light on both common and distinct pathways that contribute to asthma and allergic diseases. Associations with variation in genes encoding the epithelial cell-derived cytokines, interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), and the IL1RL1 gene encoding the IL-33 receptor, ST2, highlight the central roles for innate immune response pathways that promote the activation and differentiation of T-helper 2 cells in the pathogenesis of both asthma and allergic diseases. In contrast, variation at the 17q21 asthma locus, encoding the ORMDL3 and GSDML genes, is specifically associated with risk for childhood onset asthma. These and other genetic findings are providing a list of well-validated asthma and allergy susceptibility genes that are expanding our understanding of the common and unique biological pathways that are dysregulated in these related conditions. Ongoing studies will continue to broaden our understanding of asthma and allergy and unravel the mechanisms for the development of these complex traits.

<h4>Motivation</h4>Pinpointing genes that underlie human inherited diseases among candidate genes in susceptibility genetic regions is the primary step towards the understanding of pathogenesis of diseases. Although several probabilistic models have been proposed to prioritize candidate genes using phenotype similarities and protein-protein interactions, no combinatorial approaches have been proposed in the literature.<h4>Results</h4>We propose the first combinatorial approach for prioritizing candidate genes. We first construct a phenome-interactome network by integrating the given phenotype similarity profile, protein-protein interaction network and associations between diseases and genes. Then, we introduce a computational method called MAXIF to maximize the information flow in this network for uncovering genes that underlie diseases. We demonstrate the effectiveness of this method in prioritizing candidate genes through a series of cross-validation experiments, and we show the possibility of using this method to identify diseases with which a query gene may be associated. We demonstrate the competitive performance of our method through a comparison with two existing state-of-the-art methods, and we analyze the robustness of our method with respect to the parameters involved. As an example application, we apply our method to predict driver genes in 50 copy number aberration regions of melanoma. Our method is not only able to identify several driver genes that have been reported in the literature, it also shed some new biological insights on the understanding of the modular property and transcriptional regulation scheme of these driver genes.<h4>Contact</h4>ruijiang@tsinghua.edu.cn.

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 μm deep in the HeLa tumor.

Behaviour genetic research has shown that a given gene or gene pathway can influence categorically similar behaviours in different species. Questions about the conservation of gene function in behaviour are increasingly tractable. This is owing to the surge of DNA and 'omics data, bioinformatic tools, as well as advances in technologies for behavioural phenotyping. Here, we discuss how gene function, as a hierarchical biological phenomenon, can be used to examine behavioural homology across species. The question can be addressed independently using different levels of investigation including the DNA sequence, the gene's position in a genetic pathway, spatial-temporal tissue expression and neural circuitry. Selected examples from the literature are used to illustrate this point. We will also discuss how qualitative and quantitative comparisons of the behavioural phenotype, its function and the importance of environmental and social context should be used in cross-species comparisons. We conclude that (i) there are homologous behaviours, (ii) they are hard to define and (iii) neurogenetics and genomics investigations should help in this endeavour.

Conventional antineoplastic, novel immunosuppressive agents and antibiotics used in cancer treatment can directly affect the growth, development and virulence of Candida and Aspergillus species. Cytotoxic and cisplatin compounds have anti-Candida activity and may be synergistic with antifungal drugs; they also inhibit Candida and Aspergillus filamentation/conidation and effect increased virulence in vitro. Glucocorticoids enhance Candida adherence to epithelial cells, germination in serum and in vitro secretion of phospholipases and proteases, as well as growth of A. fumigatus. Calcineurin and target of rapamycin inhibitors perturb Candida and Aspergillus morphogenesis, stress responses and survival in serum, reduce azole tolerance in Candida, but yield conflicting in vivo data. Inhibition of candidal heat shock protein 90 and candidal-specific histone deacetylase represent feasible therapeutic approaches for candidiasis. Tyrosine kinase inhibitors inhibit fungal cell entry into epithelial cells and phagocytosis. Quinolone and other antibiotics may augment activity of azole and polyene agents. The correlation of in vitro effects with clinically meaningful in vivo systems is warranted. 

<h4>Background</h4>In the United States, the incidence of craniosynostosis (premature fusion of the sutures of the cranial vault) is one in 2000 to 3000 live births. The condition can cause increased intracranial pressure, severely altered head shape, and mental retardation. The authors have previously described a colony of rabbits with heritable coronal suture synostosis. This model has been instrumental in describing the postsurgical craniofacial growth associated with craniosynostosis. The molecular analysis of this model has been limited by the lack of molecular tools for use in rabbits. To understand the pathogenesis of craniosynostosis, the authors compared gene expression in perisutural tissues between wild-type and craniosynostotic rabbits using polymerase chain reaction suppression subtractive hybridization.<h4>Methods</h4>Suppression subtractive hybridization polymerase chain reaction was performed on RNA derived from pooled samples of calvariae from 10-day-old wild-type (n = 3) and craniosynostotic (n = 3) rabbits to obtain cDNA clones enriched in either wild-type tissues (underexpressed in craniosynostotic tissue) or craniosynostotic tissues (overexpressed in craniosynostotic compared with wild-type).<h4>Results</h4>Differential expression was identified for approximately 140 recovered cDNA clones up-regulated in craniosynostotic tissues and 130 recovered clones for wild-type tissues. Of these, four genes were confirmed by quantitative reverse-transcriptase polymerase chain reaction as being overexpressed in craniosynostotic sutural tissue: β-globin (HBB), osteopontin (SPP1), osteonectin (SPARC), and cathepsin K (CTSK). Two genes were confirmed to be underexpressed in the craniosynostotic samples: collagen 3, alpha 1 (COL3A1) and ring finger protein 12 (RNF12).<h4>Conclusion</h4>The differential expression of these gene products in our naturally occurring craniosynostotic model appears to be the result of differences in the normal bone formation/resorption pathway.

Hyalinizing trabecular tumor (HTT) of the thyroid is a rare neoplasm that was first described by Carney in 1987. It is a tumor of follicular derivation with peculiar nuclear, architectural, histochemical, and immunohistochemical features. We report a case of HTT in a 69-year-old woman with a mutinodular goiter. Since the clinical and ultrasonographic features were nonspecific, fine needle aspiration biopsy (FNAB) of the left lobe-dominant node was performed that resulted in an indeterminate cytologic diagnosis (category THY-3). The patient underwent total thyroidectomy, with a histologic diagnosis of HTT. We discuss the clinical and diagnostic approach, including the role of FNAB, and the pathologic features of HTT with special reference to the possible differential diagnosis. Total thyroidectomy or hemithyroidectomy represent adequate treatments, while radioiodine ablation is not standard. Although rare cases of malignant HTT have been documented, this tumor should be considered a benign neoplasm or, at most, a neoplasm of extremely low malignant potential. As a consequence, once this diagnosis is rendered, clinical management should be conservative, which may include a precautionary annual follow-up in order to exclude the very rare possibility of recurrence, as exceptionally reported.

<h4>Background</h4>Modern dog breeds display traits that are either breed-specific or shared by a few breeds as a result of genetic bottlenecks during the breed creation process and artificial selection for breed standards. Selective sweeps in the genome result from strong selection and can be detected as a reduction or elimination of polymorphism in a given region of the genome.<h4>Results</h4>Extended regions of homozygosity, indicative of selective sweeps, were identified in a genome-wide scan dataset of 25 Boxers from the United Kingdom genotyped at ~20,000 single-nucleotide polymorphisms (SNPs). These regions were further examined in a second dataset of Boxers collected from a different geographical location and genotyped using higher density SNP arrays (~170,000 SNPs). A selective sweep previously associated with canine brachycephaly was detected on chromosome 1. A novel selective sweep of over 8 Mb was observed on chromosome 26 in Boxer and for a shorter region in English and French bulldogs. It was absent in 171 samples from eight other dog breeds and 7 Iberian wolf samples. A region of extended increased heterozygosity on chromosome 9 overlapped with a previously reported copy number variant (CNV) which was polymorphic in multiple dog breeds.<h4>Conclusion</h4>A selective sweep of more than 8 Mb on chromosome 26 was identified in the Boxer genome. This sweep is likely caused by strong artificial selection for a trait of interest and could have inadvertently led to undesired health implications for this breed. Furthermore, we provide supporting evidence for two previously described regions: a selective sweep on chromosome 1 associated with canine brachycephaly and a CNV on chromosome 9 polymorphic in multiple dog breeds.

<h4>Problem</h4>Regulatory T cells (Treg cells), a small subset of CD4(+) T cells maintaining tolerance by immunosuppression, are proposed contributors to the survival of the fetal semiallograft. We investigated Treg cells in paired decidual and peripheral blood (PB) samples from healthy women in early pregnancy and PB samples from non-pregnant women.<h4>Method of study</h4>Distribution, location, cytokine mRNA, and phenotype were assessed in CD4(+) CD25(+) Treg cells from paired samples using immunohistochemistry, immunofluorescence, flow cytometry, and real-time quantitative RT-PCR.<h4>Results</h4>The presence and in situ distribution of CD4(+) Foxp3(+) Treg cells in decidua are hereby demonstrated for the first time. Three Foxp3(+) cell populations, CD4(+) CD25(++) Foxp3(+), CD4(+) CD25(+) Foxp3(+), and CD4(+) CD25(-) Foxp3(+), were enriched locally in decidua. In contrast, no statistically significant difference in numbers of circulating Treg cells between pregnant and non-pregnant women was found. The Foxp3(+) cells expressed the surface molecules CD45RO, CTLA-4, CD103, Neuropilin-1, LAG-3, CD62L, and TGFβ1 mRNA consistent with Treg phenotype. The population of CD4(+) CD25(-) Foxp3(+) cells, not described in human decidua before, was enriched 10-fold compared with PB in paired samples. Their cytokine expression was often similar to Th3 profile, and the Foxp3 mRNA expression level in CD4(+) CD25(-) cells was stable and comparable to that of CD4(+) CD25(+) Treg cells implying that the majority of CD4(+) CD25(-) Foxp3(+) cells might be naïve Treg cells.<h4>Conclusion</h4>(i) There is a local enrichment of Treg cells in decidua (ii) The exclusive accumulation of decidual CD4(+) CD25(-) Foxp3(+) cells suggests an additional reservoir of Foxp3(+) naïve Treg cells that can be converted to 'classical' Treg cells in uterus.

Autophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of S-nitrosylation substrates, JNK1 and IKKβ. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2-Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKβ and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1-Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition.

Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here, we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N richness promotes assembly of benign self-templating amyloids; Q richness promotes formation of toxic nonamyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs.

We demonstrate that RING finger protein MSL2 in the MOF-MSL complex is a histone ubiquitin E3 ligase. MSL2, together with MSL1, has robust histone ubiquitylation activity that mainly targets nucleosomal H2B on lysine 34 (H2B K34ub), a site within a conserved basic patch on H2B tail. H2B K34ub by MSL1/2 directly regulates H3 K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells. The significance of MSL1/2-mediated histone H2B ubiquitylation is underscored by the facts that MSL1/2 activity is important for transcription activation at HOXA9 and MEIS1 loci and that this activity is evolutionarily conserved in the Drosophila dosage compensation complex. Altogether, these results indicate that the MOF-MSL complex possesses two distinct chromatin-modifying activities (i.e., H4 K16 acetylation and H2B K34 ubiquitylation) through MOF and MSL2 subunits. They also shed light on how an intricate network of chromatin-modifying enzymes functions coordinately in gene activation.

Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.

<h4>Study objectives</h4>To describe the rate, distribution and correlates of periodic limb movements in sleep (PLMS) in children with sickle cell disease (SCD).<h4>Design</h4>Prospective, cross-sectional.<h4>Setting</h4>Hospital-based sleep laboratory.<h4>Participants</h4>Sixty-four children aged 2-18 years with SCD, hemoglobin SS-type who had an overnight polysomnogram and a parent-completed Pediatric Sleep Questionnaire. Mean age was 8.4 years (SD 4.8); 50% were male.<h4>Interventions</h4>N/A.<h4>Measurements and results</h4>The mean PLMS index was 3.7 (6.6) and ranged from 0 to 31.8, with 23.4% of the sample having PLMS ≥ 5/h. Sleep efficiency was decreased (P = 0.03), and the total arousal index (P = 0.003) and PLMS arousal index (P < 0.001) were increased in children with PLMS ≥ 5/h compared to those with PLMS < 5/h. PLMS were most frequent in NREM stage 2 sleep and during the fourth hour of sleep. Inter-movement interval duration peaked at 25-30 s. "Growing pains worst in bed" or "restlessness of the legs", suggesting restless legs syndrome (RLS), were reported in 12.5% of the total sample and were more common in children with elevated PLMS. A PLMS score for identifying elevated PLMS in children, based on items from the Pediatric Sleep Questionnaire, did not significantly predict PLMS ≥ 5/h.<h4>Conclusions</h4>Elevated PLMS are common in children with SCD and are associated with sleep disruption and symptoms of RLS. Future research into the time structure of PLMS, their causes and consequences, and development of a disease-specific sleep disorders screening questionnaire, is needed in children with SCD.

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal ganglion (TG) and dorsal root ganglia (DRG) coexpress the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knock-out (DKO) mice. Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets, Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knock-out alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a-regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes.

Local β-actin synthesis in growth cones of developing axons plays an important role in growth cone steering; however, the mRNA binding proteins required for this process are unknown. Here we used Zbp1/Imp1(-/)(-) mice to test the hypothesis that zipcode binding protein 1 (ZBP1) is required for the regulation of β-actin mRNA transport and local translation underlying growth cone guidance. To address the biological function of ZBP1, we developed a novel in vitro turning assay with primary cortical neuron balls having axons >1 mm in length and demonstrate that growth cones of mammalian neurons exhibit protein synthesis-dependent attraction to either netrin-1 or brain-derived neurotrophic factor (BDNF). Interestingly, this attraction is lost in Zbp1-deficient neurons. Furthermore, BDNF-stimulated β-actin mRNA localization was attenuated in Zbp1-deficient neurons, which impaired enrichment of β-actin protein in the growth cone. Finally, using a photoconvertible translation reporter, we found that ZBP1 is necessary for netrin-1 stimulated local translation of β-actin mRNA in axonal growth cones. Together, these results suggest that netrin-1- and BDNF-induced growth cone attraction required ZBP1-mediated local translation of β-actin mRNA, and therefore ZBP1 regulates protein synthesis-dependent axon guidance. Thus, mRNA binding proteins regulating local translation can control spatiotemporal protein expression in response to guidance cues and directional cell motility.

<h4>Background</h4>It is suggested that targeted screening for hemochromatosis and iron overload may be worthwhile. The aim of this study was to examine uric acid as a potential indicator of the presence of iron overload.<h4>Methods</h4>We analyzed adults aged 20 and older in the National Health and Nutrition Examination Survey 1999 to 2002. We computed logistic regressions controlling for age, sex, race/ethnicity, liver or kidney condition, and alcohol use to see the relationship between combinations of uric acid and ferritin with the outcomes of elevated liver enzymes and proteinuria.<h4>Results</h4>In unadjusted analyses, 20.7% of individuals with high uric acid had high ferritin levels versus 8.8% of individuals with low uric acid levels (P<.001). Individuals with both elevated uric acid and elevated ferritin levels had significantly higher liver enzymes than individuals with either elevated uric acid or ferritin. With low uric acid and low ferritin as the reference category, individuals with high uric acid and high ferritin were significantly more likely to also have proteinuria (odds ratio, 2.66; 95% CI, 1.82-3.91).<h4>Conclusions</h4>Elevated levels of uric acid is associated with elevated ferritin levels and may serve as a risk stratification variable for presence of iron overload and hemochromatosis.

A computational model of blood coagulation is presented with particular emphasis on the regulatory effects of blood flow, spatial distribution of tissue factor (TF), and the importance of the thrombomodulin-activated protein C inhibitory pathway. We define an effective prothrombotic zone that extends well beyond the dimensions of injury. The size of this zone is dependent on the concentrations of all reactive species, the dimensions of TF expression, the densities of surface molecules, and the characteristics of the flow field. In the case of tandem sites of TF, the relationship between the magnitude of the effective prothrombotic zone and the interval distance between TF sites dictate the net response of the system. Multiple TF sites, which individually failed to activate the coagulation pathway, are shown to interact in an additive manner to yield a prothrombotic system. Furthermore, activation of the thrombomodulin-activated protein C pathway in the regions between sites of TF downregulate the thrombin response at subsequent TF sites. The implications of prothrombotic effects, which extend downstream beyond the discrete site of injury to interact with subsequent lesions are critical given the systemic nature of atherosclerotic disease.

Iron overload disease has a wide variety of genotypes. The genetic study of this disease confirms its hereditary nature and enables us to provide genetic counseling for first-degree relatives. We performed magnetic resonance imaging and liver biopsy in an asymptomatic patient with more than 1,000 µg/L of serum ferritin and studied the genes involved in this condition. The phenotype of iron overload is confirmed by a predominantly periportal pattern of iron deposits in the liver suggestive of genetic disease. In the case we present the molecular study revealed a double heterozygosity for the mutations c.187C>G (p.H63D) and c.840C>G (p.F280L) in the HFE and transferrin receptor 2 (TFR2) genes, respectively.

To promote functional recovery after CNS injuries, it is crucial to develop strategies that enhance both neuronal survival and regeneration. Here, we report that caspase-6 is upregulated in injured retinal ganglion cells and that its inhibition promotes both survival and regeneration in these adult CNS neurons. Treatment of rat retinal whole mounts with Z-VEID-FMK, a selective inhibitor of caspase-6, enhanced ganglion cell survival. Moreover, retinal explants treated with this drug extended neurites on myelin. We also show that caspase-6 inhibition resulted in improved ganglion cell survival and robust axonal regeneration following optic nerve injury in adult rats. The effects of Z-VEID-FMK were similar to other caspase inhibitory peptides including Z-LEHD-FMK and Z-VAD-FMK. In searching for downstream effectors for caspase-6, we identified caspase-8, whose expression pattern resembled that of caspase-6 in the injured eye. We then showed that caspase-8 is activated downstream of caspase-6 in the injured adult retina. Furthermore, we investigated the role of caspase-8 in RGC apoptosis and regenerative failure both in vitro and in vivo. We observed that caspase-8 inhibition by Z-IETD-FMK promoted survival and regeneration to an extent similar to that obtained with caspase-6 inhibition. Our results indicate that caspase-6 and caspase-8 are components of a cellular pathway that prevents neuronal survival and regeneration in the adult mammalian CNS.

In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of β-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC(50)'s were in the 50 μM range with no toxicity to the cells at concentrations up to 200 μM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.

Evaluation of: Avouac J, Fürnrohr BG, Tomcik M et al. Inactivation of the transcription factor STAT-4 prevents inflammation-driven fibrosis in animal models of systemic sclerosis. Arthritis Rheum. 63(3), 800-809 (2011). STAT4 has been identified as a genetic risk factor for the development of autoimmune diseases including systemic sclerosis. STAT4 regulates Th1 cell development and cell-mediated immunity, but it is not known how it may regulate the development of dermal fibrosis. Using the bleomycin-induced dermal fibrosis model, it has now been demonstrated that STAT4-deficient mice have reduced dermal fibrosis in part via STAT4-dependent alterations in T-cell proliferation and cytokine production. These data stress the importance of STAT4 in autoimmune diseases such as systemic sclerosis and provide an important direction for future research to improve our understanding of systemic sclerosis pathogenesis.

The sense of hearing depends on the faithful transmission of sound information from the ear to the brain by spiral ganglion (SG) neurons. However, how SG neurons develop the connections and properties that underlie auditory processing is largely unknown. We catalogued gene expression in mouse SG neurons from embryonic day 12, when SG neurons first extend projections, up until postnatal day 15, after the onset of hearing. For comparison, we also analyzed the closely related vestibular ganglion (VG). Gene ontology analysis confirmed enriched expression of genes associated with gene regulation and neurite outgrowth at early stages, with the SG and VG often expressing different members of the same gene family. At later stages, the neurons transcribe more genes related to mature function, and exhibit a dramatic increase in immune gene expression. Comparisons of the two populations revealed enhanced expression of TGFβ pathway components in SG neurons and established new markers that consistently distinguish auditory and vestibular neurons. Unexpectedly, we found that Gata3, a transcription factor commonly associated with auditory development, is also expressed in VG neurons at early stages. We therefore defined new cohorts of transcription factors and axon guidance molecules that are uniquely expressed in SG neurons and may drive auditory-specific aspects of their differentiation and wiring. We show that one of these molecules, the receptor guanylyl cyclase Npr2, is required for bifurcation of the SG central axon. Hence, our dataset provides a useful resource for uncovering the molecular basis of specific auditory circuit assembly events.

<h4>Aim</h4>To find out the association of common HFE mutations (viz., C282Y and H63D) with primary iron overload (PIL) in liver cirrhosis (CLD) patients of Indian origin.<h4>Methods</h4>Polymerase chain reaction-restriction fragment length polymorphism method was used for screening C282Y and H63D mutation in 496 CLD patients (hepatitis B virus associated cirrhosis (HBVc) = 74, hepatitis C virus associated cirrhosis (HCV) = 50, alcoholic cirrhosis with hepatitis (ALcW)  =  38, alcoholic cirrhosis without hepatitis (ALc) = 92, cryptogenic cirrhosis (CC) = 242) and 502 healthy controls. Transferrin saturation of >45 or serum ferritin of >300 ng/mL (males)/>200 ng/mL (females) with normal total exogenous iron intake was suggestive of PIL. Histological liver iron grading was done by Perl's Prussian blue stain.<h4>Results</h4>Of 496 patients, 13 (2.6; 9 CC, 2 ALc, 1 HBVc, 1 AlcW) had PIL. However, only two (15.3) of 13 patients (1 CC and 1 HBVc) were positive for H63D heterozygous mutation. All the subjects were found to be C282Y wild type, except a single case of double heterozygous (C282Y/H63D) who however, did not have PIL. Overall frequency of H63D allele in patients and controls was not significantly different (5.95 and 4.58 respectively, p = 0.17). A highly significant H63D allele frequency (p  <  0.005) was observed in HBVc (10.82) and ALcW (11.84) groups but they were not associated with PIL.<h4>Conclusion</h4>The frequency of PIL, and the HFE gene mutaion (C282Y) are both rare in Indian patients and explain why hemochromatosis is a rare cause of liver cirrhosis in India. A highly significant H63D allele frequency in HBV and alcohol-related cirrhosis suggest a possible predisposing role for liver fibrosis of this allele.

Alzheimer's disease (AD) pathogenesis is characterized by senile plaques in the brain and evidence of oxidative damage. Oxidative stress may precede plaque formation in AD; however, the link between oxidative damage and plaque formation remains unknown. Presenilins are transmembrane proteins in which mutations lead to accelerated plaque formation and early-onset familial Alzheimer's disease. Presenilins physically interact with two antioxidant enzymes thiol-specific antioxidant (TSA) and proliferation-associated gene (PAG) of the peroxiredoxin family. The functional consequences of these interactions are unclear. In the current study we expressed a presenilin transgene in Drosophila wing and sensory organ precursors of the fly. This caused phenotypes typical of Notch signaling loss-of-function mutations. We found that while expression of TSA or PAG alone produced no phenotype, co-expression of TSA and PAG with presenilin led to an enhanced Notch loss-of-function phenotype. This phenotype was more severe and more penetrant than that caused by the expression of Psn alone. In order to determine whether these phenotypes were indeed affecting Notch signaling, this experiment was performed in a genetic background carrying an activated Notch (Abruptex) allele. The phenotypes were almost completely rescued by this activated Notch allele. These results link peroxiredoxins with the in vivo function of Presenilin, which ultimately connects two key pathogenetic mechanisms in AD, namely, antioxidant activity and plaque formation, and raises the possibility of a role for peroxiredoxin family members in Alzheimer's pathogenesis.

<h4>Aim</h4>To investigate the role of laparoscopy in the surgical management of hepatocellular adenoma (HA).<h4>Methods</h4>We reviewed a prospectively collected database of consecutive patients undergoing laparoscopic liver resection for HA.<h4>Results</h4>Thirteen patients underwent fifteen pure laparoscopic liver resections for HA (male/female: 3/10; median age 42 years, range 22-72 years). Two patients with liver adenomatosis required two different laparoscopic operations for ruptured adenomas. Indications for surgery were: symptoms in 12 cases, need to rule out malignancy in 2 cases and preoperative diagnosis of large HA in one case. Symptoms were related to bleeding in 10 cases, sepsis due to liver abscess following embolization of HA in one case and mass effect in one case (shoulder tip pain). Five cases with ruptured bleeding adenoma required emergency admission and treatment with selective arterial embolization. Laparoscopic liver resection was then semi-electively performed. Eight patients (62%) required major hepatectomy [right hepatectomy (n = 5), left hepatectomy (n = 3)]. No conversion to open surgery occurred. The median operative time for pure laparoscopic procedures was 270 min (range 135-360 min). The median size of the excised lesions was 85 mm (range 25-180 mm). One patient with adenomatosis developed postoperative bleeding requiring embolization. Mortality was nil. The median hospital stay was 4 d (range 1-18 d) with a median high dependency unit stay of 1 d (range 0-7 d).<h4>Conclusion</h4>The laparoscopic approach represents a safe option for the management of HA in a semi-elective setting and when major hepatectomy is required.

Genome-wide association studies (GWAS) have identified several genetic variants associated with coronary heart disease (CHD), and variations in plasma lipoproteins and blood pressure (BP). Loci corresponding to CDKN2A/CDKN2B/ANRIL, MTHFD1L, CELSR2, PSRC1 and SORT1 genes have been associated with CHD, and TMEM57, DOCK7, CELSR2, APOB, ABCG5, HMGCR, TRIB1, FADS2/S3, LDLR, NCAN and TOMM40-APOE with total cholesterol. Similarly, CELSR2-PSRC1-SORT1, PCSK9, APOB, HMGCR, NCAN-CILP2-PBX4, LDLR, TOMM40-APOE, and APOC1-APOE are associated with variations in low-density lipoprotein cholesterol levels. Altogether, forty, forty three and twenty loci have been associated with high-density lipoprotein cholesterol, triglycerides and BP phenotypes, respectively. Some of these identified loci are common for all the traits, some do not map to functional genes, and some are located in genes that encode for proteins not previously known to be involved in the biological pathway of the trait. GWAS have been successful at identifying new and unexpected genetic loci common to diseases and traits, thus rapidly providing key novel insights into disease biology. Since genotype information is fixed, with minimum biological variability, it is useful in early life risk prediction. However, these variants explain only a small proportion of the observed variance of these traits. Therefore, the utility of genetic determinants in assessing risk at later stages of life has limited immediate clinical impact. The future application of genetic screening will be in identifying risk groups early in life to direct targeted preventive measures.

<h4>Aim</h4>To evaluate the relationship between hepatic fat infiltration and abdominal fat volume by using computed tomography (CT).<h4>Methods</h4>Three hundred and six patients who visited our obesity clinic between November 2007 and April 2008 underwent fat protocol CT scans. The age range of the patients was 19 to 79 years and the mean age was 49 years. The male to female ratio was 116:190. Liver and spleen attenuation measurements were taken with three regions of interests (ROIs) from the liver and two ROIs from the spleen. Hepatic attenuation indices (HAIs) were measured as follows: (1) hepatic parenchymal attenuation (CT(LP)); (2) liver to spleen attenuation ratio (LS ratio); and (3) difference between hepatic and splenic attenuation (LS(dif)). Abdominal fat volume was measured using a 3 mm slice CT scan starting at the level of the umbilicus and was automatically calculated by a workstation. Abdominal fat was classified into total fat (TF), visceral fat (VF), and subcutaneous fat (SF). We used a bivariate correlation method to assess the relationship between the three HAIs and TF, VF, and SF.<h4>Results</h4>There were significant negative correlations between CT(LP), LS ratio, and LS(dif) with TF, VF, and SF, respectively. The CT(LP) showed a strong negative correlation with TF and VF (r = -0.415 and -0.434, respectively, P < 0.001). The correlation between CT(LP) and SF was less significant (r = -0.313, P < 0.001).<h4>Conclusion</h4>Fatty infiltration of the liver was correlated with amount of abdominal fat and VF was more strongly associated with fatty liver than SF.

<h4>Background</h4>Iron overload is an important issue in the state of thalassemic patients due to the harmful effect of high concentration of iron deposited in different tissues in human body including endocrine glands. In the present work, an attempt is carried out to estimate the effect of iron overload in thalassemic patients on the function of endocrine glands through the estimation of their ability to secrete adequate amounts of certain hormones.<h4>Materials and methods</h4>Seventy eight male children with beta-thalassemia, in the age-group of 4-11 years, were enrolled for this research. These children were being treated with frequent transfusions and long-term iron chelation therapy. Thirty age and sex matched children without thalassemia constituted the control group. Ferritin and different hormones were estimated by ELISA technique.<h4>Results</h4>The results showed a mild reduction in the function of endocrine glands through the decrease in the level of some hormones. These changes due mainly to the hypoxia and precipitation of iron in certain glands and overlapping with the synthesis or secretion of the hormones.<h4>Conclusion</h4>There is a different hormonal disturbances in beta thalassemia patients. Reduction of total body iron store is an important goal of the treatment of thalassemia and measuring the hormones concentration is necessary for the follow up of the thalassemic patients especially during puberty.

The pathogenesis of both entities of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), is still complex and under investigation. The importance of the microbial flora in developing IBD is beyond debate. In the last few years, the focus has changed from adaptive towards innate immunity. Crohn's ileitis is associated with a deficiency of the antimicrobial shield, as shown by a reduced expression and secretion of the Paneth cell defensin HD5 and HD6, which is related to a Paneth cell differentiation defect mediated by a diminished expression of the Wnt transcription factor TCF4. In UC, the protective mucus layer, acting as a physical and chemical barrier between the gut epithelium and the luminal microbes, is thinner and in part denuded as compared to controls. This could be caused by a missing induction of the goblet cell differentiation factors Hath1 and KLF4 leading to immature goblet cells. This defective Paneth and goblet cell differentiation in Crohn's ileitis and UC may enable the luminal microbes to invade the mucosa and trigger the inflammation. The exact molecular mechanisms behind ileal CD and also UC must be further clarified, but these observations could give rise to new therapeutic strategies based on a stimulation of the protective innate immune system.

No abstract available.

<h4>Background/aims</h4>To identify potential new tumor markers in peripheral blood mononuclear cells for pancreatic adenocarcinoma screening.<h4>Methodology</h4>The total mRNA was extracted from the peripheral blood mononuclear cell samples of both pancreatic adenocarcinoma patients (n=5) and healthy donors (n=5). Having the RNA expression profiling of over 38500 genes via the Human Genome U133 Plus 2.0 Array performed in order to identify differentially expressed genes, the study underwent real-time Reverse Transcriptase PCR (RT-PCR) for validation.<h4>Results</h4>The mRNA expression profiles identified the Olfactomedin4 gene, which was significantly overexpressed (4.46-fold) versus the control group (p<0.01). Afterwards, this result was validated by quantitative RT-PCR, with the same expression trend (4.80-fold) as well as statistical significance (p<0.05).<h4>Conclusions</h4>Homo sapiens olfactomedin4 was revealed to be up-regulated as a cancer associated gene in peripheral blood mononuclear cells from pancreatic adenocarcinoma patients. Thus, Olfactomedin4, as well as its expression products, might promisingly prove to be the ideal screening tool and new tumor marker available in blood samples.