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<h4>Unlabelled</h4>Incidence rates of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) have increased in the United States. Metabolic syndrome is recognized as a risk factor for HCC and a postulated one for ICC. The magnitude of risk, however, has not been investigated on a population level in the United States. We therefore examined the association between metabolic syndrome and the development of these cancers. All persons diagnosed with HCC and ICC between 1993 and 2005 were identified in the Surveillance, Epidemiology, and End Results (SEER)-Medicare database. For comparison, a 5% sample of individuals residing in the same regions as the SEER registries of the cases was selected. The prevalence of metabolic syndrome as defined by the U.S. National Cholesterol Education Program Adult Treatment Panel III criteria, and other risk factors for HCC (hepatitis B virus, hepatitis C virus, alcoholic liver disease, liver cirrhosis, biliary cirrhosis, hemochromatosis, Wilson's disease) and ICC (biliary cirrhosis, cholangitis, cholelithiasis, choledochal cysts, hepatitis B virus, hepatitis C virus, alcoholic liver disease, cirrhosis, inflammatory bowel disease) were compared among persons who developed cancer and those who did not. Logistic regression was used to calculate odds ratios and 95% confidence intervals. The inclusion criteria were met by 3649 HCC cases, 743 ICC cases, and 195,953 comparison persons. Metabolic syndrome was significantly more common among persons who developed HCC (37.1%) and ICC (29.7%) than the comparison group (17.1%, P<0.0001). In adjusted multiple logistic regression analyses, metabolic syndrome remained significantly associated with increased risk of HCC (odds ratio=2.13; 95% confidence interval=1.96-2.31, P<0.0001) and ICC (odds ratio=1.56; 95% confidence interval=1.32-1.83, P<0.0001).<h4>Conclusion</h4>Metabolic syndrome is a significant risk factor for development of HCC and ICC in the general U.S. population.

Nanocarriers that combine multiple properties in an all-in-one system hold great promise for drug delivery. The absence of technology to assemble highly functionalized devices has, however, hindered progress in nanomedicine. To address this deficiency, we have chemically synthesized poly(ethylene oxide)-β-poly(ε-caprolactone) (PEO-b-PCL) block polymers modified at the apolar PCL terminus with thioctic acid and at the polar PEO terminus with an acylhydrazide, amine, or azide moiety. The resulting block polymers were employed to prepare nanoparticles that have a gold core, an apolar polyester layer for drug loading, a polar PEO corona to provide biocompatibility, and three different types of surface reactive groups for surface functionalization. The acylhydrazide, amine, or azide moieties of the resulting nanoparticles could be reacted with high efficiencies with modules having a ketone, isocyanate, or active ester and alkyne function, respectively. To demonstrate proof of principle of the potential of multisurface functionalization, we prepared nanoparticles that have various combinations of an oligo-arginine peptide to facilitate cellular uptake, a histidine-rich peptide to escape from lysosomes, and an Alexa Fluor 488 tag for imaging purposes. It has been shown that uptake and subcellular localization of the nanoparticles can be controlled by multisurface modification. It is to be expected that the modular synthetic methodology provides unique opportunities to establish optimal configurations of nanocarriers for disease-specific drug delivery.

Inflammatory bowel diseases (IBDs) are complex disorders caused by a combination of environmental, microbial, and genetic factors. Genome-wide association studies in humans have successfully identified multiple genes and loci associated with disease susceptibility, but the mechanisms by which these loci interact with each other and/or with environmental factors (i.e., intestinal microbiota) to cause disease are poorly understood. Helicobacter hepaticus-induced intestinal inflammation in mice is an ideal model system for elucidating the genetic basis of IBD susceptibility in a bacterially induced system, as there are significant differences in H. hepaticus-induced disease susceptibility among inbred mouse strains. Infected A/J mice develop acute overexpression of proinflammatory cytokines followed 2-3 months later by chronic cecal inflammation, whereas infected C57BL/6 mice fail to develop cecal inflammation or increased cytokine expression. The goal of this project was to use quantitative trait locus (QTL) mapping to evaluate genetic factors that contribute to the differential disease susceptibility between these two mouse strains. Using acute cecal IL-12/23p40 expression as a biomarker for disease susceptibility, QTL analysis of H. hepaticus-infected F(2) mice revealed involvement of multiple loci. The loci with the strongest association were located on Chromosome 3 and Chromosome 17, with logarithm of odds (LOD) scores of 6.89 and 3.09, respectively. Cecal expression of IL-12/23p40 in H. hepaticus-infected C57BL/6J-Chr3(A/J)/NaJ chromosome substitution mice had an intermediate phenotype, significantly higher than in resistant C57BL/6 but lower than in susceptible A/J mice, confirming the importance of this locus to the immune response to H. hepaticus infection.

<h4>Objective</h4>Systemic lupus erythematosus is a clinically heterogeneous autoimmune disease. A number of genetic loci that increase lupus susceptibility have been established. This study examines if these genetic loci also contribute to the clinical heterogeneity in lupus.<h4>Materials and methods</h4>4001 European-derived, 1547 Hispanic, 1590 African-American and 1191 Asian lupus patients were genotyped for 16 confirmed lupus susceptibility loci. Ancestry informative markers were genotyped to calculate and adjust for admixture. The association between the risk allele in each locus was determined and compared in patients with and without the various clinical manifestations included in the ACR criteria.<h4>Results</h4>Renal disorder was significantly correlated with the lupus risk allele in ITGAM (p=5.0 × 10(-6), OR 1.25, 95% CI 1.12 to 1.35) and in TNFSF4 (p=0.0013, OR 1.14, 95% CI 1.07 to 1.25). Other significant findings include the association between risk alleles in FCGR2A and malar rash (p=0.0031, OR 1.11, 95% CI 1.17 to 1.33), ITGAM and discoid rash (p=0.0020, OR 1.20, 95% CI 1.06 to 1.33), STAT4 and protection from oral ulcers (p=0.0027, OR 0.89, 95% CI 0.83 to 0.96) and IL21 and haematological disorder (p=0.0027, OR 1.13, 95% CI 1.04 to 1.22). All these associations are significant with a false discovery rate of <0.05 and pass the significance threshold using Bonferroni correction for multiple testing.<h4>Conclusion</h4>Signifi cant associations were found between clinical manifestations and the FCGR2A, ITGAM, STAT4, TNSF4 and IL21 genes. The findings suggest that genetic profiling might be a useful tool to predict disease manifestations in lupus patients in the future.

Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

Tau is a neuronal-specific microtubule-associated protein that plays an important role in establishing neuronal polarity and maintaining the axonal cytoskeleton. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism (FTDP-17), the early onset dementia observed in Down syndrome (DS; trisomy 21) and the dementia component of myotonic dystrophy type 1 (DM1). Splicing misregulation of adult-specific exon 10, which codes for a microtubule binding domain, results in expression of abnormal ratios of tau isoforms, leading to FTDP-17. Positions 3 to 19 of the intron downstream of exon 10 define a hotspot of splicing regulation: the region diverges between humans and rodents, and point mutations within it result in tauopathies. In this study, we investigated three regulators of exon 10 splicing: serine/arginine-rich protein SRp75 and heterogeneous nuclear ribonucleoproteins hnRNPG and hnRNPE2. SRp75 and hnRNPG inhibit splicing of exon 10 whereas hnRNPE2 activates it. Using co-transfections, co-immunoprecipitations and RNAi we discovered that SRp75 binds to the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region; and that hnRNPG and hnRNPE2 interact with SRp75. Thus, increased exon 10 inclusion in FTDP mutants may arise from weakened SRp75 binding. This work provides insights into the splicing regulation of the tau gene and into possible strategies for correcting the imbalance in tauopathies caused by changes in the ratio of exon 10.

DNA cleavage by the Type III Restriction-Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision.

For the past five years, genome-wide association studies (GWAS) have identified hundreds of common variants associated with human diseases and traits, including high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels. Approximately 95 loci associated with lipid levels have been identified primarily among populations of European ancestry. The Population Architecture using Genomics and Epidemiology (PAGE) study was established in 2008 to characterize GWAS-identified variants in diverse population-based studies. We genotyped 49 GWAS-identified SNPs associated with one or more lipid traits in at least two PAGE studies and across six racial/ethnic groups. We performed a meta-analysis testing for SNP associations with fasting HDL-C, LDL-C, and ln(TG) levels in self-identified European American (~20,000), African American (~9,000), American Indian (~6,000), Mexican American/Hispanic (~2,500), Japanese/East Asian (~690), and Pacific Islander/Native Hawaiian (~175) adults, regardless of lipid-lowering medication use. We replicated 55 of 60 (92%) SNP associations tested in European Americans at p<0.05. Despite sufficient power, we were unable to replicate ABCA1 rs4149268 and rs1883025, CETP rs1864163, and TTC39B rs471364 previously associated with HDL-C and MAFB rs6102059 previously associated with LDL-C. Based on significance (p<0.05) and consistent direction of effect, a majority of replicated genotype-phentoype associations for HDL-C, LDL-C, and ln(TG) in European Americans generalized to African Americans (48%, 61%, and 57%), American Indians (45%, 64%, and 77%), and Mexican Americans/Hispanics (57%, 56%, and 86%). Overall, 16 associations generalized across all three populations. For the associations that did not generalize, differences in effect sizes, allele frequencies, and linkage disequilibrium offer clues to the next generation of association studies for these traits.

Interpreting Genome-Wide Association Studies (GWAS) at a gene level is an important step towards understanding the molecular processes that lead to disease. In order to incorporate prior biological knowledge such as pathways and protein interactions in the analysis of GWAS data it is necessary to derive one measure of association for each gene. We compare three different methods to obtain gene-wide test statistics from Single Nucleotide Polymorphism (SNP) based association data: choosing the test statistic from the most significant SNP; the mean test statistics of all SNPs; and the mean of the top quartile of all test statistics. We demonstrate that the gene-wide test statistics can be controlled for the number of SNPs within each gene and show that all three methods perform considerably better than expected by chance at identifying genes with confirmed associations. By applying each method to GWAS data for Crohn's Disease and Type 1 Diabetes we identified new potential disease genes.

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF-1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins.

This study evaluated the cellular localization of cyclic AMP-responsive element binding protein-binding protein (CBP) expression in pig retinas during postnatal development. Immunohistochemistry and Western blot analysis were performed on retinal tissue from 2-day-old, 5-week-old, and 6-month-old pigs. Western blot analysis detected the expression of CBP in the retinas of 2-day-old piglets and showed that it was significantly decreased in the retinas of 5-week-old and 6-month-old pigs. Immunohistochemically, CBP was intensely immunostained in protein kinase C alpha (PKCα)-positive-bipolar cells, glutamine synthetase-positive Müller cells, and in ganglion cells in 2-day-old piglets. CBP was detected weakly in the inner plexiform, outer nuclear, and rod and cone layers. CBP immunoreactivity in the ganglion cell layer was decreased in the retinas of 5-week-old and 6-month-old pigs, while clear CBP expression detected in the neurite of PKCα-positive bipolar cells in the inner nuclear layer. In addition, CBP immunoreactivity in Müller cells and glial fibrillary acidic protein-positive glial processes was particularly noteworthy in pig retinas, but not in rat retinas. The results indicate that CBP is expressed differentially in the retinal neurons and glial cells according to growth and animal species, and may play an important role in homeostasis in Müller cells, neurite extention in bipolar cells, and signal transduction in photoreceptor cells in the porcine retina.

The major defining pathological hallmarks of Alzheimer's disease (AD) are the accumulations of Aβ in senile plaques and hyperphosphorylated tau in neurofibrillary tangles and neuropil threads. Recent studies indicate that rather than these insoluble lesions, the soluble Aβ oligomers and hyperphosphorylated tau are the toxic agents of AD pathology. Such pathological protein species are accompanied by cytoskeletal changes, mitochondrial dysfunction, Ca(2+) dysregulation, and oxidative stress. In this review, we discuss how the binding of Aβ to various integrins, defects in downstream focal adhesion signaling, and activation of cofilin can impact mitochondrial dysfunction, cytoskeletal changes, and tau pathology induced by Aβ oligomers. Such pathological consequences can also feedback to further activate cofilin to promote cofilin pathology. We also suggest that the mechanism of Aβ generation by the endocytosis of APP is mechanistically linked with perturbations in integrin-based focal adhesion signaling, as APP, LRP, and β-integrins are physically associated with each other.

Misfolding and abnormal aggregation of proteins in the brain are implicated in the pathogenesis of various neurodegenerative diseases including Alzheimer's, Parkinson's, and the polyglutamine (polyQ) diseases. In the polyQ diseases, an abnormally expanded polyQ stretch triggers misfolding and aggregation of the disease-causing proteins, eventually resulting in neurodegeneration. In this paper, we introduce our therapeutic strategy against the polyQ diseases using polyQ binding peptide 1 (QBP1), a peptide that we identified by phage display screening. We showed that QBP1 specifically binds to the expanded polyQ stretch and inhibits its misfolding and aggregation, resulting in suppression of neurodegeneration in cell culture and animal models of the polyQ diseases. We further demonstrated the potential of protein transduction domains (PTDs) for in vivo delivery of QBP1. We hope that in the near future, chemical analogues of aggregation inhibitor peptides including QBP1 will be developed against protein misfolding-associated neurodegenerative diseases.

The trimeric RNA polymerase complex (3P, for PA-PB1-PB2) of influenza A virus (IAV) is an important viral determinant of pathogenicity and host range restriction. Specific interactions of the polymerase complex with host proteins may be determining factors in both of these characteristics and play important roles in the viral life cycle. To investigate this question, we performed a comprehensive proteomic analysis of human host proteins associated with the polymerase of the well-characterized H5N1 Vietnam/1203/04 isolate. We identified over 400 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), of which over 300 were found to bind to the PA subunit alone. The most intriguing and novel finding was the large number of mitochondrial proteins (∼20%) that associated with the PA subunit. These proteins mediate molecular transport across the mitochondrial membrane or regulate membrane potential and may in concert with the identified mitochondrion-associated apoptosis inducing factor (AIFM1) have roles in the induction of apoptosis upon association with PA. Additionally, we identified host factors that associated with the PA-PB1 (68 proteins) and/or the 3P complex (34 proteins) including proteins that have roles in innate antiviral signaling (e.g., ZAPS or HaxI) or are cellular RNA polymerase accessory factors (e.g., polymerase I transcript release factor [PTRF] or Supt5H). IAV strain-specific host factor binding to the polymerase was not observed in our analysis. Overall, this study has shed light into the complex contributions of the IAV polymerase to host cell pathogenicity and allows for direct investigations into the biological significance of these newly described interactions.

<h4>Objective</h4>It has been suggested that the high prevalence of subnormal free testosterone concentrations, along with low or inappropriately normal gonadotropins in men with type 2 diabetes, may be the result of an increase in plasma estradiol concentrations secondary to an increase in aromatase activity in the adipose tissue that leads to the suppression of the hypothalamo-hypophyseal-gonadal axis.<h4>Research design and methods</h4>To investigate this hypothesis, plasma estradiol, testosterone, leutinizing hormone, follicle-stimulating hormone, and sex hormone-binding globulin (SHBG) concentrations were measured in fasting blood samples of 240 men with type 2 diabetes. Free estradiol concentrations were either calculated (n = 198) using total estradiol and SHBG measured by immunoassay or directly measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and equilibrium dialysis (n = 102).<h4>Results</h4>The calculated free estradiol concentration in men with subnormal free testosterone concentrations was lower than that in men with normal free testosterone concentrations (median 0.047 vs. 0.063 ng/dL, P < 0.001). Directly measured (LC-MS/MS) free estradiol concentrations were also lower in men with subnormal free testosterone concentrations (median 0.025 vs. 0.045 ng/dL, P = 0.008). Free estradiol concentrations were directly related to free testosterone but not to BMI or age.<h4>Conclusions</h4>These data show that the suppression of the hypothalamo-hypophyseal-gonadal axis in patients with subnormal free testosterone concentrations and type 2 diabetes is not associated with increased estradiol concentrations. The pathogenesis of subnormal free testosterone concentrations in type 2 diabetes needs to be investigated further.

Anopheles gambiae, the primary African vector of malaria parasites, exhibits numerous rhythmic behaviors including flight activity, swarming, mating, host seeking, egg laying, and sugar feeding. However, little work has been performed to elucidate the molecular basis for these daily rhythms. To study how gene expression is regulated globally by diel and circadian mechanisms, we have undertaken a DNA microarray analysis of An. gambiae under light/dark cycle (LD) and constant dark (DD) conditions. Adult mated, non-blood-fed female mosquitoes were collected every 4 h for 48 h, and samples were processed with DNA microarrays. Using a cosine wave-fitting algorithm, we identified 1,293 and 600 rhythmic genes with a period length of 20-28 h in the head and body, respectively, under LD conditions, representing 9.7 and 4.5% of the An. gambiae gene set. A majority of these genes was specific to heads or bodies. Examination of mosquitoes under DD conditions revealed that rhythmic programming of the transcriptome is dependent on an interaction between the endogenous clock and extrinsic regulation by the LD cycle. A subset of genes, including the canonical clock components, was expressed rhythmically under both environmental conditions. A majority of genes had peak expression clustered around the day/night transitions, anticipating dawn and dusk. Genes cover diverse biological processes such as transcription/translation, metabolism, detoxification, olfaction, vision, cuticle regulation, and immunity, and include rate-limiting steps in the pathways. This study highlights the fundamental roles that both the circadian clock and light play in the physiology of this important insect vector and suggests targets for intervention.

A catalogue of molecular aberrations that cause ovarian cancer is critical for developing and deploying therapies that will improve patients' lives. The Cancer Genome Atlas project has analysed messenger RNA expression, microRNA expression, promoter methylation and DNA copy number in 489 high-grade serous ovarian adenocarcinomas and the DNA sequences of exons from coding genes in 316 of these tumours. Here we report that high-grade serous ovarian cancer is characterized by TP53 mutations in almost all tumours (96%); low prevalence but statistically recurrent somatic mutations in nine further genes including NF1, BRCA1, BRCA2, RB1 and CDK12; 113 significant focal DNA copy number aberrations; and promoter methylation events involving 168 genes. Analyses delineated four ovarian cancer transcriptional subtypes, three microRNA subtypes, four promoter methylation subtypes and a transcriptional signature associated with survival duration, and shed new light on the impact that tumours with BRCA1/2 (BRCA1 or BRCA2) and CCNE1 aberrations have on survival. Pathway analyses suggested that homologous recombination is defective in about half of the tumours analysed, and that NOTCH and FOXM1 signalling are involved in serous ovarian cancer pathophysiology.

Mechanisms underlying stress-induced depression and antidepressant drug action were shown to involve alterations in serotonergic (5-HT) neurotransmission and expression of genes coding for proteins associated with neurotrophic signaling pathways and cell-survival in the hippocampus and cortex. Expression of these genes in the brainstem containing 5-HT neurons may also be related to vulnerability or resilience to stress-related psychopathology. Here we investigated 5-HT markers and expression of genes for Brain-Derived Neurotrophic Factor (BDNF) and apoptotic proteins in the brainstem in relation to swim stress-induced behavioral despair. We found that anti-apoptotic Bcl-xL gene is sensitive to stress during the course of fluoxetine administration. Responsiveness of this gene to stress appeared concomitantly with an antidepressant-like effect of fluoxetine in the forced swim test. Bcl-xL transcript levels showed negative correlations with duration of immobility in the test and 5-HT turnover in the brainstem. In contrast, BDNF and pro-apoptotic protein Bax mRNA levels were unchanged by either fluoxetine or stress, suggesting specificity of Bcl-xL gene responses to these treatments. We also found that the levels of mRNAs for tryptophan hydroxylase-2 (TPH2) and 5-HT transporter (5-HTT) were significantly down-regulated following prolonged treatment with fluoxetine, but were not affected by stress. Unlike TPH2 and 5-HTT, 5-HT1A receptor mRNA levels were not altered by fluoxetine but significantly increased in response to swim stress. These data show that long-term fluoxetine treatment leads to changes in 5-HT and Bcl-xL responses to stress associated with antidepressant-like effects of the drug. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Expression of the glycosylphosphatidylinositol-anchored membrane protein CD24 correlates with a poor prognosis for many human cancers, and in experimental tumors can promote metastasis. However, the mechanism by which CD24 contributes to tumor progression remains unclear. Here we report that in MTLy breast cancer cells CD24 interacts with and augments the kinase activity of c-src, a protein strongly implicated in promoting invasion and metastasis. This occurs within and is dependent upon intact lipid rafts. CD24-augmented c-src kinase activity increased formation of focal adhesion complexes, accelerated phosphorylation of FAK and paxillin and consequently enhanced integrin-mediated adhesion. Loss and gain of function approaches showed that c-src activity is necessary and sufficient to mediate the effects of CD24 on integrin-dependent adhesion and cell spreading, as well as on invasion. Together these results indicate that c-src is a CD24-activated mediator that promotes integrin-mediated adhesion and invasion, and suggest a mechanism by which CD24 might contribute to tumor progression through stimulating the activity of c-src or another member of the Src family.

Given the continued high incidence of HIV infection in the United States among racial/ethnic minority young men who have sex with men (YMSM), and an appreciation that antiretroviral therapy (ART) can provide personal and public health benefits, attention is needed to enhance the detection of HIV-infected youth and engage them in medical care and support services that encourage sustained HIV treatment and suppression of viremia. Poor retention in clinical care has been associated with higher mortality, an increase in HIV RNA, and decreased CD4 cell count. The goal of the current study was to evaluate the health care utilization and health outcomes of HIV-infected racial/ethnic minority YMSM enrolled in an outreach, linkage, and retention study funded by the Health Resources and Services Administration (HRSA) HIV/AIDS Bureau (HAB). We hypothesized that among racial/ethnic minority YMSM, baseline CD4 counts and usage of ART are influenced by age, race, drug and alcohol use, and mental health symptoms. Overall, 155 subjects had at least a baseline CD4 count recorded at study entry. There was a low rate of ART use in this population with only one-half of the cohort with CD4 counts ≤ 350 cells/mm(3) being prescribed ART to treat their infection. However, of those youth who were started on ART, the majority (74%) did achieve undetectable viral loads (< 400 copies). Given the continued increase in cases of HIV infection among racial/ethnic minority YMSM, efforts to increase both the provision of ART and support services that encourage adherence in this population are warranted.

<h4>Background</h4>Suicidal behaviour is known to aggregate in families. Patients with psychiatric disorders are at higher risk for suicide attempts (SA), however protective and risk genetic variants for suicide appear to be independent of underlying psychiatric disorders. Here we investigate genetic variants in genes important for neurobiological pathways linked to suicidal behaviour and/or associated endophenotypes, for association with SA among patients with co-existing psychiatric illness. Selected gene-gene and gene-environment interactions were also tested.<h4>Methods</h4>DNA was obtained from bloods of 159 patients (76 suicide attempters and 83 non-attempters), who were profiled for DSM-IV Axis I psychiatric diagnosis. Twenty-eight single nucleotide polymorphisms (SNPs) from 18 candidate genes (COMT, 5-HT2A, 5-HT1A, 5-HTR1B, TPH1, MAO-A, TPH2, DBH, CNR1, BDNF, ABCG1, GABRA5, GABRG2, GABRB2, SLC1A2, SLC1A3, NTRK2, CRHR1) were genotyped. Genotyping was performed by KBioscience. Tests of association between genetic variants and SA were conducted using Chi squared and Armitage Trend tests. Binary logistical regression analyses were performed to evaluate the contribution of individual genetic variants to the prediction of SA, and to examine SNPs for potential gene-gene and gene-environment interactions.<h4>Results</h4>Our analysis identified 4 SNPs (rs4755404, rs2269272, rs6296 and rs1659400), which showed evidence of association with SA compared to a non-attempter control group. We provide evidence of a 3-locus gene-gene interaction, and a putative gene-environment interaction, whereby genetic variation at the NTRK2 locus may moderate the risk associated with history of childhood abuse.<h4>Conclusion</h4>Preliminary findings suggest that allelic variability in SLC1A2/3, 5-HTR1B and NTRK2 may be relevant to the underlying diathesis for suicidal acts.

<h4>Background</h4>Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes.<h4>Methods</h4>A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays.<h4>Results</h4>Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype.<h4>Conclusion</h4>Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

DNA methylation may mediate persistent changes in gene function following chronic stress. To examine this hypothesis, we evaluated African American subjects matched by age and sex, and stratified into four groups by post-traumatic stress disorder (PTSD) diagnosis and history of child abuse. Total Life Stress (TLS) was also assessed in all subjects. We evaluated DNA extracted from peripheral blood using the HumanMethylation27 BeadChip and analyzed both global and site-specific methylation. Methylation levels were examined for association with PTSD, child abuse history, and TLS using a linear mixed model adjusted for age, sex, and chip effects. Global methylation was increased in subjects with PTSD. CpG sites in five genes (TPR, CLEC9A, APC5, ANXA2, and TLR8) were differentially methylated in subjects with PTSD. Additionally, a CpG site in NPFFR2 was associated with TLS after adjustment for multiple testing. Notably, many of these genes have been previously associated with inflammation. Given these results and reports of immune dysregulation associated with trauma history, we compared plasma cytokine levels in these subjects and found IL4, IL2, and TNFα levels associated with PTSD, child abuse, and TLS. Together, these results suggest that psychosocial stress may alter global and gene-specific DNA methylation patterns potentially associated with peripheral immune dysregulation. Our results suggest the need for further research on the role of DNA methylation in stress-related illnesses.

Genomewide association meta-analysis studies have identified > 100 independent genetic loci associated with blood cell indices, including volume and count of platelets and erythrocytes. Although several of these loci encode known regulators of hematopoiesis, the mechanism by which most sequence variants exert their effect on blood cell formation remains elusive. An example is the Rho guanine nucleotide exchange factor, ARHGEF3, which was previously implicated by genomewide association meta-analysis studies in bone cell biology. Here, we report on the unexpected role of ARHGEF3 in regulation of iron uptake and erythroid cell maturation. Although early erythroid differentiation progressed normally, silencing of arhgef3 in Danio rerio resulted in microcytic and hypochromic anemia. This was rescued by intracellular supplementation of iron, showing that arhgef3-depleted erythroid cells are fully capable of hemoglobinization. Disruption of the arhgef3 target, RhoA, also produced severe anemia, which was, again, corrected by iron injection. Moreover, silencing of ARHGEF3 in erythromyeloblastoid cells K562 showed that the uptake of transferrin was severely impaired. Taken together, this is the first study to provide evidence for ARHGEF3 being a regulator of transferrin uptake in erythroid cells, through activation of RHOA.

BCP are considered the most promising biomaterials for bone reconstruction. This study aims at analyzing the outcomes of patients who received BCP as bone substitutes in orthopaedic surgeries. Sixty-six patients were categorized according to the etiology and morphology of the bone defects and received scores after clinical and radiographic evaluations. The final results corresponded to the combination of both parameters and varied from 5 (excellent result) to 2 or lower (poor result). Most of the patients who presented cavitary defects or bone losses due to prosthesis placement or revision, osteotomies, or arthrodesis showed good results, and some of them excellent results. However, patients with segmental defects equal or larger than 3 cm in length were classified as moderate results. This study established clinical parameters where the BCP alone can successfully support the osteogenic process and where the association with other tissue engineering strategies may be considered.

Pin1 specifically recognizes and catalyzes the cis-trans isomerization of phosphorylated-Ser/Thr-Pro bonds, which modulate the stability, localization, and function of numerous Pin1 targets involved in tumor progression. However, the role of Pin1 in cancer remains enigmatic as the gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under-expressed in kidney cancer, we have explored its role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under-expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may have a mild tumor suppressive role in ccRCC.

Hereditary etiologies of pancreatic and hepatobiliary cancers are increasingly recognized. An estimated >10% of pancreatic and increasing number of hepatobiliary cancers are hereditary. The cumulative risk of hereditary pancreatic cancer ranges from measurable but negligible in cystic fibrosis to a sobering 70% in cases of hereditary pancreatitis. Candidates for pancreatic cancer surveillance are those with a risk pancreatic cancer estimated to be >10-fold that of the normal population. Screening for pancreatic cancer in high-risk individuals is typically performed by endoscopic ultrasound and should begin at least 10 years prior to the age of the youngest affected relative. Disease states known to be associated with increased risk of hepatocellular cancer include hereditary hemochromatosis, autoimmune hepatitis, porphyria, and α1-antitrypsin deficiency, with relative risks as high as 36-fold. Although much less is known about hereditary bile-duct cancers, Muir-Torre syndrome and bile salt export pump deficiency are diseases whose association with hereditary carcinogenesis is under investigation.

The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been identified in two recurring chromosomal translocations, involving either MLL or MLLT10 (AF10). We developed a mouse model of CALM-AF10(+) leukemia to examine the hypothesis that disruption of endocytosis contributes to leukemogenesis. Exclusion of the C-terminal portion of CALM from the fusion protein, which is required for optimal binding to clathrin, resulted in the development of a myeloproliferative disease, whereas inclusion of this domain led to the development of acute myeloid leukemia and changes in gene expression of several cancer-related genes, notably Pim1 and Crebbp. Nonetheless, the development of leukemia could not be attributed directly to interference with endocytosis or consequential changes in proliferation and signaling. In leukemia cells, full-length CALM-AF10 localized to the nucleus with no consistent effect on growth factor endocyctosis, and suppressed histone H3 lysine 79 methylation regardless of the presence of clathrin. Using fluorescence resonance energy transfer analysis, we show that CALM-AF10 has a propensity to homo-oligomerize, raising the possibility that the function of endocytic proteins involved in chimeric fusions may be to provide dimerization properties, a recognized mechanism for unleashing oncogenic properties of chimeric transcription factors, rather than disrupting the internalization of growth factor receptors.

The ROP5 family is a closely related set of polymorphic pseudokinases that are critical to the ability of Toxoplasma to cause disease. Polymorphisms in ROP5 also make it a major determinant of strain-specific differences in virulence. ROP5 possesses all of the major kinase motifs required for catalysis except for a substitution at the catalytic Asp. We show that this substitution in the catalytic loop of ROP5 is part of a motif conserved in other pseudokinases of both Toxoplasma and human origin, and that this motif is required for the full activity in vivo of ROP5. This suggests evolutionary selection at this site for a biochemical function, rather than simple drift away from catalysis. We present the crystal structures of a virulent isoform of ROP5 both in its ATP-bound and -unbound states and have demonstrated that despite maintaining the canonical ATP-binding motifs, ROP5 binds ATP in a distorted conformation mediated by unusual magnesium coordination sites that would not be predicted from the primary sequence. In addition, we have mapped the polymorphisms spread throughout the primary sequence of ROP5 to two major surfaces, including the activation segment of ROP5. This suggests that the pseudoactive site of this class of pseudokinases may have evolved to use the canonical ATP-binding motifs for non-catalytic signaling through allostery.

Co-repressor histone deacetylase 9 (HDAC9) plays a key role in the development and differentiation of many types of cells, including regulatory T cells. However, the biological function of HDAC9 in T effector cells is unknown. Systemic autoimmune diseases like lupus, diabetes, and rheumatoid arthritis have dysfunctional effector T cells. To determine the role of HDAC9 in systemic autoimmunity, we created MRL/lpr mice with HDAC9 deficiency that have aberrant effector T cell function. HDAC9 deficiency led to decreased lympho-proliferation, inflammation, autoantibody production, and increased survival in MRL/lpr mice. HDAC9-deficient mice manifested Th2 polarization, decreased T effector follicular cells positive for inducible co-stimulator, and activated T cells in vivo compared with HDAC9-intact MRL/lpr mice. HDAC9 deficiency also resulted in increased GATA3 and roquin and decreased BCL6 gene expression. HDAC9 deficiency was associated with increased site-specific lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) globally that was localized to IL-4, roquin, and peroxisome proliferator-activated receptor-γ promoters with increased gene expression, respectively. In kidney and spleen, HDAC9 deficiency decreased inflammation and cytokine and chemokine production due to peroxisome proliferator-activated receptor γ overexpression. These findings suggest that HDAC9 acts as an epigenetic switch in effector T cell-mediated systemic autoimmunity.

Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.

The central nervous system normally functions at O(2) levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O(2)-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O(2) tensions compared to the cell culture standard of 20% O(2), to investigate their ability to sense and translate this O(2) information to transcriptional activity. Variance of ambient O(2) tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O(2) tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O(2) tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O(2) tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional 'programs' may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity.

<h4>Background</h4>Protein kinase CK2 is a highly conserved, ubiquitous protein serine/threonine kinase that phosphorylates many substrates and has a global role in numerous biological and pathological processes. Overexpression of the protein kinase CK2α subunit (CK2α) has been associated with the malignant transformation of several tissues, with not nearly as much focus on the role of CK2α in colorectal cancer (CRC). The aims of this study are to investigate the function and regulatory mechanism of CK2α in CRC development.<h4>Methods</h4>Expression levels of CK2α were analyzed in 144 patients (104 with CRC and 40 with colorectal adenoma) by immunohistochemistry. Proliferation, senescence, motility and invasion assays as well as immunofluorescence staining and western blots were performed to assess the effect of CK2α in CRC.<h4>Results</h4>The immunohistochemical expression of nuclear CK2α was stronger in tumor tissues than in adenomas and normal colorectal tissues. Suppression of CK2α by small-interfering RNA or the CK2α activity inhibitor emodin inhibited proliferation of CRC cells, caused G0/G1 phase arrest, induced cell senescence, elevated the expression of p53/p21 and decreased the expression of C-myc. We also found that knockdown of CK2α suppressed cell motility and invasion. Significantly, CK2α inhibition resulted in β-catenin transactivation, decreased the expression levels of vimentin and the transcription factors snail1 and smad2/3, and increased the expression of E-cadherin, suggesting that CK2α regulates the epithelial-mesenchymal transition (EMT) process in cancer cells.<h4>Conclusions</h4>Our results indicate that CK2α plays an essential role in the development of CRC, and inhibition of CK2α may serve as a promising therapeutic strategy for human CRC.

<h4>Purpose</h4>To globally characterize the stroma expression profile of superficial bladder transitional cell carcinoma and to discuss the cancer biology as well as biomarker discovery from stromal cells.<h4>Methods</h4>Laser capture microdissection was used to harvest purified bladder cancer stromal cells and normal stromal cells from 4 paired samples. Next, two-dimensional liquid chromatography-tandem mass spectrometry was used to identify the proteome expression profile. The differential proteins were further analyzed using bioinformatics tools and compared with the published literature.<h4>Results</h4>A total of 606/601 proteins commonly appeared in 4 paired samples. Six hundred and thirty-seven proteins were differentially expressed between cancer and normal stroma. Among them, 321/316 proteins specific expressed in cancer/normal stromal cells. Differential proteins were compared with the entire list of the international protein index (IPI). 36/49 GO terms exhibited as enriched and 25/14 exhibited as depleted in biological process; 39/40 GO terms exhibited as enriched and 9/4 exhibited as depleted in cellular component, respectively. Significantly altered pathways between cancer/normal stroma mainly include focal adhesion, EHEC, oxidative phosphorylation, glycolysis/gluconeogenesis, TCA cycle. Finally, descriptive statistics show the data that PI ≤ 8 have normal distribution and the proteins with extremes of PI and MW have the same probability to be a biomarker.<h4>Conclusions</h4>The present study identified the proteome expression profile of bladder cancer stromal cells and normal stromal cells. Furthermore, our results confirm proteins origin from stroma play important role in cancer biology and highlight a novel view of cancer and stress the importance of including factors related to the cancer stroma into biomarker discovery.

The 5-HT(1B) receptor has been implicated in disorders such as depression, anxiety and obsessive-compulsive disorder. In mice lacking the 5-HT(1B) receptor (5-HT(1B) knockout mice), important changes in physiology and behavior exist. In the absence of presynaptic 5-HT(1B) receptor inhibition, chronic SSRI treatment may differentially affect 5-HT(1A) receptor functionality. The present studies tested the hypothesis that chronically reducing 5-HT transporter (5-HTT) function with selective serotonin reuptake inhibitor (SSRI) treatment would accelerate 5-HT(1A) receptor desensitization in 5-HT(1B) knockout mice. Moreover, as 5-HT(1B) knockout mice have been found to display exaggerated autonomic and locomotor responses to environmental stressors, the effects of chronic SSRI treatment on the hyperreactive phenotype of 5-HT(1B) knockout mice were investigated. The stress-reducing effect of the 5-HT(1A) receptor agonist flesinoxan on increases in body temperature, heart rate and locomotor activity was similar in wild type and 5-HT(1B) knockout mice before and after chronic 21-day treatment with the SSRI fluvoxamine, indicating no apparent alteration of 5-HT(1A) receptor sensitivity in 5-HT(1B) knockout mice. Also, chronic SSRI treatment did not alter the increased stress reactivity to mild environmental stressors in 5-HT(1B) knockout mice. We demonstrate that no apparent differences in 5-HT(1A) receptor sensitivity occur between 5-HT(1B) knockout and wild type mice after chronic fluvoxamine treatment. Also, the hyperreactive phenotype of 5-HT(1B) knockout mice is unresponsive to chronic SSRI treatment. Taken together, these results indicate that constitutive absence of 5-HT(1B) receptors does not result in adaptive changes in 5-HT(1A) receptor functionality and that chronic SSRI treatment does not modify stress reactivity in 5-HT(1B) knockout mice.

Apart from its role in axon guidance, netrin-1 is also known to be pro-angiogenic. The aim of this study is to determine whether adeno-associated viral (AAV) mediated overexpression of netrin-1 improves post-stroke neurovascular structure and recovery of function. AAV-Netrin-1 or AAV-LacZ of 1×10(10) genome copies each was injected medial and posterior to ischemic lesion at one hour following reperfusion using the distal middle cerebral artery occlusion (MCAO) method. Quantitative RT-PCR revealed that the expression of netrin-1 transgene began as early as one day and increased dramatically about 3 weeks following vector injection. Western blot analysis and confocal microscopy suggested that both the endogenous and transduced netrin-1 were expressed in the neurons of the peri-infarct cortex after MCAO. AAV-mediated netrin-1 overexpression significantly increased vascular density in the peri-infarct cortex and promoted the migration of immature neurons into the peri-infarct white matter, but it did not significantly reduce infarct size. Netrin-1 overexpression also enhanced post-stroke locomotor activity, improved exploratory behavior, and reduced ischemia-induced motor asymmetry in forelimb usage. However, it had little effect on post-stroke spatial learning and memory. Our results suggest that AAV mediated netrin-1 overexpression improves peri-infarct vascular density and post stroke motor function.

Whether the variations in the hemochromatosis (HFE) gene increase Alzheimer's disease (AD) risk is still undetermined. We performed a meta-analysis in order to systematically summarize the possible association. Studies were identified by searching PUBMED, Web of Science and EMBASE databases complemented with screening the references of the retrieved studies. The association was measured using random-effect or fixed-effect odds ratio (OR) combined with 95% confidence intervals (CIs) according to the studies' heterogeneity. For C282Y polymorphism, we did not find any association using data from 22 studies including 4,365 cases and 8,652 controls. For H63D polymorphism, on the basis of 2,795 cases and 7,424 controls from 17 studies, we observed a significant association (allele contrast: OR = 0.902, 95% CI = 0.819-0.994, P = 0.037; minor-allele-dominant model: OR = 0.887, 95% CI = 0.790-0.996, P = 0.043). No publication bias was detected in this meta-analysis. The synthesis of available evidence supports mutant of HFE H63D polymorphism plays a protective role for AD risk.

Prostate-derived sterile 20-like kinases (PSKs) 1-α, 1-β, and 2 are members of the germinal-center kinase-like sterile 20 family of kinases. Previous work has shown that PSK 1-α binds and stabilizes microtubules whereas PSK2 destabilizes microtubules. Here, we have investigated the activation and autophosphorylation of endogenous PSKs and show that their catalytic activity increases as cells accumulate in G(2)/M and declines as cells exit mitosis. PSKs are stimulated in synchronous HeLa cells as they progress through mitosis, and these proteins are activated catalytically during each stage of mitosis. During prophase and metaphase activated PSKs are located in the cytoplasm and at the spindle poles, and during telophase and cytokinesis stimulated PSKs are present in trans-Golgi compartments. In addition, small interfering RNA (siRNA) knockdown of PSK1-α/β or PSK2 expression inhibits mitotic cell rounding as well as spindle positioning and centralization. These results show that PSK catalytic activity increases during mitosis and suggest that these proteins can contribute functionally to mitotic cell rounding and spindle centralization during cell division.

Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing genes in T1D, we performed an in silico "phenome-interactome analysis" on a genome-wide linkage scan dataset. This method prioritizes candidates according to their physical interactions at the protein level with other proteins involved in diabetes. A total of 11 genes were predicted to be likely disease genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated rat and human islets. Overexpression of HIP14 was associated with a decrease in IL-1β-induced NF-κB activity and protection against IL-1β-mediated apoptosis. Our study demonstrates that the current network biology approach is a valid method to identify genes of importance for T1D and may therefore embody the basis for more rational and targeted therapeutic approaches.

The use of animal models in medical research provides insights into molecular and cellular mechanisms of human disease, and helps identify and test novel therapeutic strategies. Drosophila melanogaster--the common fruit fly--is one of the most well-established model organisms, as its study can be performed more readily and with far less expense than for other model animal systems, such as mice, fish, or primates. In the case of fruit flies, standard assays are based on the analysis of longevity and basic locomotor functions. Here we present the iFly tracking system, which enables to increase the amount of quantitative information that can be extracted from these studies, and to reduce significantly the duration and costs associated with them. The iFly system uses a single camera to simultaneously track the trajectories of up to 20 individual flies with about 100 μm spatial and 33 ms temporal resolution. The statistical analysis of fly movements recorded with such accuracy makes it possible to perform a rapid and fully automated quantitative analysis of locomotor changes in response to a range of different stimuli. We anticipate that the iFly method will reduce very considerably the costs and the duration of the testing of genetic and pharmacological interventions in Drosophila models, including an earlier detection of behavioural changes and a large increase in throughput compared to current longevity and locomotor assays.

Parkinson's disease (PD), an adult neurodegenerative disorder, has been clinically linked to the lysosomal storage disorder Gaucher disease (GD), but the mechanistic connection is not known. Here, we show that functional loss of GD-linked glucocerebrosidase (GCase) in primary cultures or human iPS neurons compromises lysosomal protein degradation, causes accumulation of α-synuclein (α-syn), and results in neurotoxicity through aggregation-dependent mechanisms. Glucosylceramide (GlcCer), the GCase substrate, directly influenced amyloid formation of purified α-syn by stabilizing soluble oligomeric intermediates. We further demonstrate that α-syn inhibits the lysosomal activity of normal GCase in neurons and idiopathic PD brain, suggesting that GCase depletion contributes to the pathogenesis of sporadic synucleinopathies. These findings suggest that the bidirectional effect of α-syn and GCase forms a positive feedback loop that may lead to a self-propagating disease. Therefore, improved targeting of GCase to lysosomes may represent a specific therapeutic approach for PD and other synucleinopathies.

The iron-regulatory hormone, hepcidin, regulates systemic iron homeostasis by interacting with the iron export protein ferroportin (FPN1) to adjust iron absorption in enterocytes, iron recycling through reticuloendothelial macrophages, and iron release from storage in hepatocytes. We previously demonstrated that FPN1 was highly expressed in erythroblasts, a cell type that consumes most of the serum iron for use in hemoglobin synthesis. Herein, we have demonstrated that FPN1 localizes to the plasma membrane of erythroblasts, and hepcidin treatment leads to decreased expression of FPN1 and a subsequent increase in intracellular iron concentrations in both erythroblast cell lines and primary erythroblasts. Moreover, injection of exogenous hepcidin decreased FPN1 expression in BM erythroblasts in vivo, whereas iron depletion and associated hepcidin reduction led to increased FPN1 expression in erythroblasts. Taken together, hepcidin decreased FPN1 expression and increased intracellular iron availability of erythroblasts. We hypothesize that FPN1 expression in erythroblasts allows fine-tuning of systemic iron utilization to ensure that erythropoiesis is partially suppressed when nonerythropoietic tissues risk developing iron deficiency. Our results may explain why iron deficiency anemia is the most pronounced early manifestation of mammalian iron deficiency.

Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.

Many cancers display increased NF-κB activity, and NF-κB inhibition is known to diminish tumor development in multiple mouse models, supporting an important role of NF-κB in carcinogenesis. NF-κB activation in premalignant or cancer cells is believed to promote tumor development mainly by protecting these cells from apoptosis. However, it remains unclear to what extent NF-κB activation exhibits additional protumorigenic functions in premalignant cells that could be sufficient to induce spontaneous tumor development. Here we show that expression of constitutively active IκB kinase 2 (IKK2ca) in mouse intestinal epithelial cells (IECs) induced spontaneous tumors in aged mice and also strongly enhanced chemical- and Apc mutation-mediated carcinogenesis. IECs expressing IKK2ca displayed altered Wnt signaling and increased proliferation and elevated expression of genes encoding intestinal stem cell-associated factors including Ascl2, Olfm4, DLK1, and Bmi-1, indicating that increased IKK2/NF-κB activation synergized with Wnt signaling to drive intestinal tumorigenesis. Moreover, IECs expressing IKK2ca produced cytokines and chemokines that induced the recruitment of myeloid cells and activated stromal fibroblasts to become myofibroblasts, thus creating a tumor-promoting microenvironment. Taken together, our results show that constitutively increased activation of IKK2/NF-κB signaling in the intestinal epithelium is sufficient to induce the full spectrum of cell-intrinsic and stromal alterations required for intestinal tumorigenesis.

Dental fluorosis occurs as a result of excess fluoride ingestion during tooth formation. Enamel fluorosis and primary dentin fluorosis can only occur when teeth are forming, and therefore fluoride exposure (as it relates to dental fluorosis) occurs during childhood. In the permanent dentition, this would begin with the lower incisors, which complete mineralization at approximately 2-3 years of age, and end after mineralization of the third molars. The white opaque appearance of fluorosed enamel is caused by a hypomineralized enamel subsurface. With more severe dental fluorosis, pitting and a loss of the enamel surface occurs, leading to secondary staining (appearing as a brown color). Many of the changes caused by fluoride are related to cell/matrix interactions as the teeth are forming. At the early maturation stage, the relative quantity of amelogenin protein is increased in fluorosed enamel in a dose-related manner. This appears to result from a delay in the removal of amelogenins as the enamel matures. In vitro, when fluoride is incorporated into the mineral, more protein binds to the forming mineral, and protein removal by proteinases is delayed. This suggests that altered protein/mineral interactions are in part responsible for retention of amelogenins and the resultant hypomineralization that occurs in fluorosed enamel. Fluoride also appears to enhance mineral precipitation in forming teeth, resulting in hypermineralized bands of enamel, which are then followed by hypomineralized bands. Enhanced mineral precipitation with local increases in matrix acidity may affect maturation stage ameloblast modulation, potentially explaining the dose-related decrease in cycles of ameloblast modulation from ruffle-ended to smooth-ended cells that occur with fluoride exposure in rodents. Specific cellular effects of fluoride have been implicated, but more research is needed to determine which of these changes are relevant to the formation of fluorosed teeth. As further studies are done, we will better understand the mechanisms responsible for dental fluorosis.

Numerous studies have been done to understand genetic contributors to BMI, but only a limited number of studies have been done in nonwhite groups such as American Indians. A genome-wide association study (GWAS) for BMI was therefore performed in Pima Indians. BMI measurements from a longitudinal study of 1,120 Pima Indians and 454,194 single-nucleotide polymorphisms (SNPs) from the 1 million Affymetrix SNP panel were used (35% of SNPs were excluded due to minor allele frequency <0.05). Data included BMI measured at multiple examinations collected from 1965 to 2004, as well as the maximum BMI at one of these visits. General and within-family tests were performed using a maximum-likelihood based mixed model procedure. No SNP reached a genome-wide significance level (estimated at P < 4.94 × 10(-7)). For repeated measures analyses, the strongest associations for general and within-family tests mapped to two different regions on chromosome 6 (rs9342220 (P = 1.39 × 10(-6)) and rs7758764 (P = 2.51 × 10(-6)), respectively). For maximum BMI, the strongest association for the general tests mapped to chromosome 4 (rs17612333; P = 1.98 × 10(-6)) and to chromosome 3 (rs11127958; P = 1.53 × 10(-6)) for the within-family tests. Further analysis is important because only a few of these regions have been previously implicated in a GWAS and genetic susceptibility may differ by ethnicity.

Cardiac hypertrophy is a key structural feature of diabetic cardiomyopathy in the late stage of diabetes. Recent studies show that microRNAs (miRNAs) are involved in the pathogenesis of cardiac hypertrophy in diabetic mice, but more novel miRNAs remain to be investigated. In this study, diabetic cardiomyopathy, characterized by hypertrophy, was induced in mice by streptozotocin injection. Using microarray analysis of myocardial tissue, we were able to identify changes in expression in 19 miRNA, of which 16 miRNAs were further validated by real-time PCR and a total of 3212 targets mRNA were predicted. Further analysis showed that 31 GO functions and 16 KEGG pathways were enriched in the diabetic heart. Of these, MAPK signaling pathway was prominent. In vivo and in vitro studies have confirmed that three major subgroups of MAPK including ERK1/2, JNK, and p38, are specifically upregulated in cardiomyocyte hypertrophy during hyperglycemia. To further explore the potential involvement of miRNAs in the regulation of glucose-induced cardiomyocyte hypertrophy, neonatal rat cardiomyocytes were exposed to high glucose and transfected with miR-373 mimic. Overexpression of miR-373 decreased the cell size, and also reduced the level of its target gene MEF2C, and miR-373 expression was regulated by p38. Our data highlight an important role of miRNAs in diabetic cardiomyopathy, and implicate the reliability of bioinformatics analysis in shedding light on the mechanisms underlying diabetic cardiomyopathy.

<h4>Background</h4>Syndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression.<h4>Methodology/principal findings</h4>We utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration.<h4>Conclusions/significance</h4>Our results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression.

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k₂ = 21±1 s⁻¹) was much higher than the HNE deacylation step (k₃ = 0.57±0.05 s⁻¹). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k₁ 2.4-fold and reducing k₋₁ 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k₂ value, whereas the k₃ value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.

The treatment of chronic myelogenous leukemia (CML) was revolutionized by the development of imatinib mesylate, a small molecule inhibitor of several protein tyrosine kinases, including the ABL1 protein tyrosine kinase. The current second generation of FDA-approved ABL tyrosine kinase inhibitors, dasatinib and nilotinib, are more potent inhibitors of BCR-ABL1 kinase in vitro. Originally approved for the treatment of patients who were refractory to or intolerant of imatinib, dasatinib and nilotinib are now also FDA approved in the first-line setting. The choice of tyrosine kinase inhibitor (ie, standard or high dose imatinib, dasatinib, nilotinib) to use for initial therapy in chronic-phase CML (CML-CP) will not always be obvious. Therapy selection will depend on both clinical and molecular factors, which we will discuss in this review.

<h4>Background</h4>Cervical cancer continues to be an important worldwide health problem for women. Up to 35% of patients who are diagnosed with and appropriately treated for cervical cancer will recur and treatment results are poor for recurrent disease. Given these sobering statistics, development of novel therapies for cervical cancer remains a high priority. We evaluated the expression of Tissue Factor (TF) in cervical cancer and the potential of hI-con1, an antibody-like-molecule targeted against TF, as a novel form of immunotherapy against multiple primary cervical carcinoma cell lines with squamous- and adenocarcinoma histology.<h4>Methods</h4>Because TF is a transmembrane receptor for coagulation factor VII/VIIa (fVII), in this study we evaluated the in vitro expression of TF in cervical carcinoma cell lines by immunohistochemistry (IHC), real time-PCR (qRT-PCR) and flow cytometry. Sensitivity to hI-con1-dependent cell-mediated-cytotoxicity (IDCC) was evaluated in 5-hrs-51chromium-release-assays against cervical cancer cell lines in vitro.<h4>Results</h4>Cytoplasmic and/or membrane TF expression was observed in 8 out of 8 (100%) of the tumor tissues tested by IHC and in 100% (11 out of 11) of the cervical carcinoma cell lines tested by real-time-PCR and flow cytometry but not in normal cervical keratinocytes (p=0.0023 qRT-PCR; p=0.0042 flow cytometry). All primary cervical cancer cell lines tested overexpressing TF, regardless of their histology, were highly sensitive to IDCC (mean killing±SD, 56.2%±15.9%, range, 32.4%-76.9%, p<0.001), while negligible cytotoxicity was seen in the absence of hI-con1 or in the presence of rituximab-control-antibody. Low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (p=0.025) while human serum did not significantly decrease IDCC against cervical cancer cell lines (p=0.597).<h4>Conclusions</h4>TF is highly expressed in squamous and adenocarcinoma of the uterine cervix. hI-con1 induces strong cytotoxicity against primary cervical cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of cervical cancer refractory to standard treatment modalities.

<h4>Background</h4>One of the most consistent biological findings in autism is the elevated blood serotonin levels. Immune abnormalities, including autoimmunity with production of brain specific auto-antibodies, are also commonly observed in this disorder. Hyperserotonemia may be one of the contributing factors to autoimmunity in some patients with autism through the reduction of T-helper (Th) 1-type cytokines. We are the first to investigate the possible role of hyperserotonemia in the induction of autoimmunity, as indicated by serum anti-myelin-basic protein (anti-MBP) auto-antibodies, in autism.<h4>Methods</h4>Serum levels of serotonin and anti-MBP auto-antibodies were measured, by ELISA, in 50 autistic patients, aged between 5 and 12 years, and 30 healthy-matched children.<h4>Results</h4>Autistic children had significantly higher serum levels of serotonin and anti-MBP auto-antibodies than healthy children (P < 0.001 and P < 0.001, respectively). Increased serum levels of serotonin and anti-MBP auto-antibodies were found in 92% and 80%, respectively of autistic patients. Patients with severe autism had significantly higher serum serotonin levels than children with mild to moderate autism (P < 0.001). Serum serotonin levels had no significant correlations with serum levels of anti-MBP auto-antibodies in autistic patients (P = 0.39).<h4>Conclusions</h4>Hyperserotonemia may not be one of the contributing factors to the increased frequency of serum anti-MBP auto-antibodies in some autistic children. These data should be treated with caution until further investigations are performed. However, inclusion of serum serotonin levels as a correlate may be useful in other future immune studies in autism to help unravel the long-standing mystery of hyperserotonemia and its possible role in the pathophysiology of this disorder.

<h4>Background</h4>Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process.<h4>Methods</h4>Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model.<h4>Results</h4>TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1).<h4>Conclusions</h4>In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

Malignant peripheral nerve sheath tumor (MPNST) is a life-threatening complication of neurofibromatosis type 1 (NF1). NF1 is caused by mutation in the gene encoding neurofibromin, a negative regulator of Ras signaling. There are no effective pharmacologic therapies for MPNST. To identify new therapeutic approaches targeting this dangerous malignancy, we developed assays in NF1(+/+) and NF1(-/-) MPNST cell lines and in budding yeast lacking the NF1 homologue IRA2 (ira2Δ). Here, we describe UC1, a small molecule that targets NF1(-/-) cell lines and ira2Δ budding yeast. By using yeast genetics, we identified NAB3 as a high-copy suppressor of UC1 sensitivity. NAB3 encodes an RNA binding protein that associates with the C-terminal domain of RNA Pol II and plays a role in the termination of nonpolyadenylated RNA transcripts. Strains with deletion of IRA2 are sensitive to genetic inactivation of NAB3, suggesting an interaction between Ras signaling and Nab3-dependent transcript termination. This work identifies a lead compound and a possible target pathway for NF1-associated MPNST, and shows a novel model system approach to identify and validate target pathways for cancer cells in which NF1 loss drives tumor formation.

In Epstein-Barr virus (EBV) latency III (LTIII) infection, BHRF1 encodes three microRNAs (miRNAs). Herein we report that Drosha cleavage of LTIII BHRF1 RNA and cis-acting splicing effects inhibit splicing and inhibit BHRF1 RNA and protein expression. Evidence shown here supports the view that Drosha cleavage to generate mature miRNAs and cis-acting sequences that prevent mRNA maturation are independent processes that prevent LTIII BHRF1 expression in lymphoblastoid cell lines.

Protein phosphorylation is deeply involved in the pathological mechanism of various neurodegenerative disorders. However, in human pathological samples, phosphorylation can be modified during preservation by postmortem factors such as time and temperature. Postmortem changes may also differ among proteins. Unfortunately, there is no comprehensive database that could support the analysis of protein phosphorylation in human brain samples from the standpoint of postmortem changes. As a first step toward addressing the issue, we performed phosphoproteome analysis with brain tissue dissected from mouse bodies preserved under different conditions. Quantitative whole proteome mass analysis showed surprisingly diverse postmortem changes in phosphoproteins that were dependent on temperature, time and protein species. Twelve hrs postmortem was a critical time point for preservation at room temperature. At 4°C, after the body was cooled down, most phosphoproteins were stable for 72 hrs. At either temperature, increase greater than 2-fold was exceptional during this interval. We found several standard proteins by which we can calculate the postmortem time at room temperature. The information obtained in this study will be indispensable for evaluating experimental data with human as well as mouse brain samples.

Ozone is known to add across and cleave carbon-carbon double bonds. Ozonolysis is widely used for the preparation of pharmaceuticals, for bleaching substances and for killing microorganisms in air and water sources. Some polysaccharides and oligosaccharides, such as those prepared using chemical or enzymatic β-elimination, contain a site of unsaturation. We examined ozonolysis of low-molecular-weight heparins (LMWHs), enoxaparin and logiparin, and heparosan oligo- and polysaccharides for the removal of the nonreducing terminal unsaturated uronate residue. 1D (1)H NMR showed that these ozone-treated polysaccharides retained the same structure as the starting polysaccharide, except that the C4-C5 double bond in the nonreducing end unsaturated uronate had been removed. The anticoagulant activity of the resulting product from enoxaparin and logiparin was comparable to that of the starting material. These results demonstrate that ozonolysis is an important tool for the removal of unsaturated uronate residues from LMWHs and heparosan without modification of the core polysaccharide structure or diminution of anticoagulant activity. This reaction also has potential applications in the chemoenzymatic synthesis of bioengineered heparin from Escherichia coli-derived K5 heparosan.

Transfusion-dependent myelodysplastic (MDS) patients are prone to iron overload. We evaluated 43 transfused MDS patients with T2* magnetic resonance imaging scans. 81% had liver and 16·8% cardiac iron overload. Liver R2* (1000/T2*), but not cardiac R2*, was correlated with number of units transfused (r=0·72, P<0·0001) and ferritin (r=0·53, P<0·0001). The area under the curve of a time-ferritin plot was found to be much greater in patients with cardiac iron loading (median 53·7x10(5) Megaunits vs. 12·2x10(5) Megaunits, P=0·002). HFE, HFE2, HAMP or SLC40A1 genotypes were not predictors of iron overload in these patients.

<h4>Introduction</h4>Protein profiles of endoscopically collected pancreatic juice from normal, chronic pancreatitis patients and pancreatic cancer patients were compared to identify diagnostic biomarkers of pancreatic cancer.<h4>Methods</h4>Secretin was injected intravenously and pancreatic juice was collected via selective cannulation of the pancreatic duct during endoscopic retrograde cholangiopancreatography. Pancreatic juices consisting of three pooled samples for normal control, chronic pancreatitis, and pancreatic cancer patients were compared using two-dimensional gel electrophoresis, and the proteins were subsequently identified using MALDI-TOF-MS.<h4>Results</h4>Thirty-five protein spots were up-regulated twofold in pancreatic cancer compared with the levels in the normal controls, and 85 protein spots were present in pancreatic cancer samples but not in normal controls. After excluding spots that were also expressed in chronic pancreatitis, 26 protein spots that were up-regulated or only expressed in pancreatic cancer samples were identified. Among the identified proteins, we confirmed the expressions of BIG2, PRDX6, and REG1α in pancreatic cancer tissue using immunohistochemistry. ELISA showed that the serum level of REG1α was significantly higher in patients with pancreatic cancer than it was in the normal controls (P = 0.023). With the best cut-off value, the sensitivity and specificity of REG1α to differentiate normal and pancreatic cancer were 82.6 and 81.8%, compared with 69.6 and 100% for CA19-9.<h4>Conclusions</h4>We have shown that pancreatic juice is a good source of pancreatic cancer tumor markers. Further studies are needed to determine the clinical implications of REG1α and other markers.

To better understand the genomic organization and evolution of Sox genes in vertebrates, we cytogenetically mapped Sox2 and Sox14 genes in cichlid fishes and performed comparative analyses of their orthologs in several vertebrate species. The genomic regions neighboring Sox2 and Sox14 have been conserved during vertebrate diversification. Although cichlids seem to have undergone high rates of genomic rearrangements, Sox2 and Sox14 are linked in the same chromosome in the Etroplinae Etroplus maculatus that represents the sister group of all remaining cichlids. However, these genes are located on different chromosomes in several species of the sister group Pseudocrenilabrinae. Similarly, the ancestral synteny of Sox2 and Sox14 has been maintained in several vertebrates, but this synteny has been broken independently in all major groups as a consequence of karyotype rearrangements that took place during the vertebrate evolution.

<h4>Background</h4>Huntington's disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The expanded CAG repeats are translated into polyglutamine (polyQ), causing aberrant functions as well as aggregate formation of mutant Htt. Effective treatments for HD are yet to be developed.<h4>Methodology/principal findings</h4>Here, we report a novel dual-function compound, N(6)-(4-hydroxybenzyl)adenine riboside (designated T1-11) which activates the A(2A)R and a major adenosine transporter (ENT1). T1-11 was originally isolated from a Chinese medicinal herb. Molecular modeling analyses showed that T1-11 binds to the adenosine pockets of the A(2A)R and ENT1. Introduction of T1-11 into the striatum significantly enhanced the level of striatal adenosine as determined by a microdialysis technique, demonstrating that T1-11 inhibited adenosine uptake in vivo. A single intraperitoneal injection of T1-11 in wildtype mice, but not in A(2A)R knockout mice, increased cAMP level in the brain. Thus, T1-11 enters the brain and elevates cAMP via activation of the A(2A)R in vivo. Most importantly, addition of T1-11 (0.05 mg/ml) to the drinking water of a transgenic mouse model of HD (R6/2) ameliorated the progressive deterioration in motor coordination, reduced the formation of striatal Htt aggregates, elevated proteasome activity, and increased the level of an important neurotrophic factor (brain derived neurotrophic factor) in the brain. These results demonstrate the therapeutic potential of T1-11 for treating HD.<h4>Conclusions/significance</h4>The dual functions of T1-11 enable T1-11 to effectively activate the adenosinergic system and subsequently delay the progression of HD. This is a novel therapeutic strategy for HD. Similar dual-function drugs aimed at a particular neurotransmitter system as proposed herein may be applicable to other neurotransmitter systems (e.g., the dopamine receptor/dopamine transporter and the serotonin receptor/serotonin transporter) and may facilitate the development of new drugs for other neurodegenerative diseases.

Rationally designed asymmetrical alkylacyl phosphatidylcholines (APC) have been synthesized and evaluated as helper lipids for non-viral gene delivery. A long aliphatic chain (C22-C24) was introduced at the 1-position of glycerol backbone, a branched lipid chain (C18) at the 2-position, and a phosphocholine head group at the 3-position. The fusogenicity of APC depends on the length and degree of saturation of the alkyl chain. Cationic lipids were formulated with APC as either lipoplexes or nanolipoparticles, and evaluated for their stability, transfection efficiency, and cytotoxicity. APC mediated high in vitro transfection efficiency, and had low cytotoxicity. Small nanolipoparticles (less than 100 nm) can be obtained with APC by applying as low as 0.1% PEG-lipid. Our study extends the type of helper lipids that are suitable for gene transfer and points the way to improve non-viral nucleic acid delivery system other than the traditional cationic lipids optimization.

The development of in vitro three-dimensional cell culture matrices offers physiologically relevant alternatives to traditional culture on plastic surfaces. However methods to analyze cell subpopulations therein are poor. Here we present a simple and inexpensive method to analyze cell subpopulations in mixed-cell colonies using standard immunohistochemical (IHC) techniques. Briefly, Matrigel™ blocks are sandwiched between two layers of HistoGel™, hardened by rapid cooling then processed for routine fixation, paraffin embedding, and IHC. We demonstrate the assay using mono- and co-cultured normal human breast, human breast cancer, and transformed mouse stromal cells along with hormone treated breast cancer cells. Judicious selection of specific antibodies allows different cell types within heterotypic colonies to be identified. A brief pulse of bromodeoxyuridine in living colonies allows proliferation of cell subpopulations to be quantified. This simple assay is useful for multiple cell types, species, and conditions.

Hostility is a multidimensional personality trait with changing expression over the life course. We performed a genome-wide association study (GWAS) of the components of hostility in a population-based sample of Finnish men and women for whom a total of 2.5 million single-nucleotide polymorphisms (SNPs) were available through direct or in silico genotyping. Hostility dimensions (anger, cynicism and paranoia) were assessed at four time points over a 15-year interval (age range 15-30 years at phase 1 and 30-45 years at phase 4) in 982-1780 participants depending on the hostility measure. Few promising areas from chromosome 14 at 99 cM (top SNPs rs3783337, rs7158754, rs3783332, rs2181102, rs7159195, rs11160570, rs941898, P values <3.9 × 10(-8) with nearest gene Enah/Vasp-like (EVL)) were found suggestively to be related to paranoia and from chromosome 7 at 86 cM (top SNPs rs802047, rs802028, rs802030, rs802026, rs802036, rs802025, rs802024, rs802032, rs802049, rs802051, P values <6.9 × 10(-7) with nearest gene CROT (carnitine O-octanoyltransferase)) to cynicism, respectively. Some shared suggestive genetic influence for both paranoia and cynicism was also found from chromosome 17 at 2.8 cM (SNPs rs12936442, rs894664, rs6502671, rs7216028) and chromosome 22 at 43 cM (SNPs rs7510759, rs7510924, rs7290560), with nearest genes RAP1 GTPase activating protein 2 (RAP1GAP2) and KIAA1644, respectively. These suggestive associations did not replicate across all measurement times, which warrants further study on these SNPs in other populations.

<h4>Aim</h4>To identify the key proteins involved in the nephrotoxicity induced by andrographolide sodium bisulfite (ASB).<h4>Methods</h4>Male ICR mice were intravenously administrated with ASB (1000 or 150 mg·kg⁻¹·d⁻¹) for 7 d. The level of malondialdehyde (MDA) and the specific activity of superoxide dismutase (SOD) in kidneys were measured. The renal homogenates were separated by two-dimensional electrophoresis, and the differential protein spots were identified using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry.<h4>Results</h4>The high dose (1000 mg/kg) of ASB significantly increased the MDA content, but decreased the SOD activity as compared to the control mice. The proteomic analysis revealed that 6 proteins were differentially expressed in the high-dose group. Two stress-responsive proteins, ie heat shock cognate 71 kDa protein (HSC70) and peroxiredoxin-6 (PRDX6), were regulated at the expression level. The remaining 4 proteins involving in cellular energy metabolism, including isoforms of methylmalonyl-coenzyme A mutase (MUT), nucleoside diphosphate-linked moiety X motif 19 (Nudix motif19), mitochondrial NADH dehydrogenase 1 alpha subcomplex subunit 10 (NDUFA10) and nucleoside diphosphate kinase B (NDK B), were modified at the post-translational levels.<h4>Conclusion</h4>Our findings suggest that the mitochondrion is the primary target of ASB and that ASB-induced nephrotoxicity results from oxidative stress mediated by superoxide produced by complex I.

<h4>Aims</h4>Hereditary hemochromatosis (HHC) is the most commonly identified autosomal recessive genetic disorder in the Caucasian population and HFE gene mutations are highly concentrated among European populations. This is the first study that screens for HHC-related gene mutations in a healthy Lebanese sample population.<h4>Methods</h4>Using the reverse hybridization Hemochromatosis StripAssay A from ViennaLab, the DNA extracted from a total of 116 healthy volunteers (59 males and 57 females) was analyzed, looking for 18 different mutations in the HFE, ferroportin, and transferrin genes.<h4>Results</h4>For the HFE gene, the C282Y mutation was not detected, but the H63D mutation was found with an overall carrier frequency of 25.8% (24.1% heterozygous and 1.7% homozygous). None of the mutations in the transferrin and ferroportin genes was identified.<h4>Conclusions</h4>The Hemochromatosis StripAssay A from ViennaLab provides an easy and reliable technique for simultaneous screening of the different HFE gene mutations. This first study in Lebanon represents a baseline report for further future studies in the field using this easy technique with a reasonable turnaround time for diagnosis. We also note that ferroportin and transferrin gene mutations have not been detected in this population sample and larger clinical studies will be needed to better estimate their prevalence.

Three-dimensional topology of DNA in the cell nucleus provides a level of transcription regulation beyond the sequence of the linear DNA. To study the relationship between the transcriptional activity and the spatial environment of a gene, we used allele-specific chromosome conformation capture-on-chip (4C) technology to produce high-resolution topology maps of the active and inactive X chromosomes in female cells. We found that loci on the active X form multiple long-range interactions, with spatial segregation of active and inactive chromatin. On the inactive X, silenced loci lack preferred interactions, suggesting a unique random organization inside the inactive territory. However, escapees, among which is Xist, are engaged in long-range contacts with each other, enabling identification of novel escapees. Deletion of Xist results in partial refolding of the inactive X into a conformation resembling the active X without affecting gene silencing or DNA methylation. Our data point to a role for Xist RNA in shaping the conformation of the inactive X chromosome at least partially independent of transcription.

Cortical γ-aminobutyric acid (GABA)ergic interneurons are characterized by extraordinary neurochemical and functional diversity. Although recent studies have uncovered some of the molecular components underlying interneuron development, including the cellular and molecular mechanisms guiding their migration to the cortex, the intracellular components involved are still unknown. Rac1, a member of the Rac subfamily of Rho-GTPases, has been implicated in various cellular processes such as cell cycle dynamics, axonogenesis, and migration. In this study, we have addressed the specific role of Rac1 in interneuron progenitors originating in the medial ganglionic eminence, via Cre/loxP technology. We show that ablation of Rac1 from Nkx2.1-positive progenitors, results in a migratory impairment. As a consequence, only half of GABAergic interneurons are found in the postnatal cortex. The rest remain aggregated in the ventral telencephalon and show morphological defects in their growing processes in vitro. Ablation of Rac1 from postmitotic progenitors does not result in similar defects, thus underlying a novel cell autonomous and stage-specific requirement for Rac1 activity, within proliferating progenitors of cortical interneurons. Rac1 is necessary for their transition from G1 to S phase, at least in part by regulating cyclin D levels and retinoblastoma protein phosphorylation.

Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.

<h4>Background</h4>The prevalence of obesity and related diseases has increased enormously in the last few decades, becoming a very important medical and social issue. Because of the increasing number of people who need weight loss therapies and the high costs associated with these, the search for reliable predictors of success for weight loss and weight maintenance treatments has become a priority.<h4>Objective</h4>A literature review was undertaken to identify possible predictors of outcome of weight loss and weight maintenance in patients treated with antiobesity drugs.<h4>Results</h4>For the majority of variables, published data are not sufficient to define their role on final outcomes. Among all considered factors, only early response to treatment appeared to be a reliable positive predictor, and diabetes a negative predictor of weight loss and maintenance.<h4>Conclusion</h4>To date, no definitive results have been obtained. Due to the great benefits of reliable predictors of outcome associated to currently available antiobesity drugs and those under development, identifying these predictors has to be supported and encouraged.

We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.

Sensitive and robust bioassays for glucocorticoids are very useful for the pharmaceutical industry, environmental scientists and veterinary control. Here, a recombinant yeast cell was constructed that expresses the human glucocorticoid receptor alpha and a green fluorescent reporter protein in response to glucocorticoids. Both the receptor construct and the reporter construct were stably integrated into the yeast genome. The correct and specific functioning of this yeast glucocorticoid bioassay was studied by exposures to cortisol and other related compounds and critically compared to a GR-CALUX bioassay based on a human bone cell. Although less sensitive, the new yeast glucocorticoid bioassay showed sensitivity towards all (gluco)corticoids tested, with the following order in relative potencies: budesonide >> corticosterone > dexamethasone > cortisol = betamethasone > prednisolone > aldosterone. Hormone representatives for other hormone nuclear receptors, like 17β-estradiol for the oestrogen receptor, 5α-dihydrotestosterone for the androgen receptor and progesterone for the progesterone receptor, showed no clear agonistic responses, whilst some polychlorinated biphenyls were clearly able to interfere with the GR activity.

Huntingtin (Htt) is a protein with a polyglutamine stretch in the N-terminus and expansion of the polyglutamine stretch causes Huntington's disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that post-translational modifications of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization, and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase inhibitors ameliorates HD-like symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1-612) in a cell culture model using mass spectrometry. Myc-tagged Htt23Q (1-612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDS-PAGE, digested and subjected to high performance liquid chromatography tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys-178, Lys-236, Lys-345 and two previously described sites Lys-9 and Lys-444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring MS assay was developed to compare quantitatively the Lys-178 acetylation level between wild-type Htt23Q and mutant Htt148Q (1-612). This report represents the first comprehensive mapping of lysine acetylation sites in N-terminal region of Htt.

NFE2L3 [Nuclear factor (erythroid-derived 2)-like 3] or NRF3, a member of the Cap'n'Collar (CNC) family, is a basic-region leucine zipper (bZIP) transcription factor that was first identified over 10 years ago. Contrary to its extensively studied homolog NFE2L2 (NRF2), the regulation and function of the NFE2L3 protein have not yet attracted as much attention. Nevertheless, several recent reports have now shed light on the possible roles of NFE2L3. Structural and biochemical studies revealed a series of domains and modifications that are critical for its cellular regulation. The control of the subcellular localization of NFE2L3 appears to be essential for understanding its role in various cellular processes. Importantly, newer studies provide fascinating insights linking NFE2L3 to differentiation, inflammation, and carcinogenesis. Here, we present an overview of the current level of knowledge of NFE2L3 transcription factor biology in humans and mice. From being the Cinderella of the CNC transcription factors for many years, NFE2L3 may now rapidly come into its own.

The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.

<h4>Background</h4>Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been implicated in tumor development and progression. The aim of this study was to investigate the role of TWEAK in colorectal cancer (CRC) progression.<h4>Methods</h4>To investigate the involvement of TWEAK in the progression of human CRC, normal, and tumor specimens from 174 patients were analyzed immunohistochemically for the expression of TWEAK. TWEAK recombinant protein treatment, transfection of expression plasmids, and small interfering RNA to knockdown TWEAK expression were performed to test invasive ability with a Boyden chamber. The mRNA expression profile in recombinant TWEAK treatment was compared to a control group by microarray analysis. To identify downstream effectors, Raf kinase inhibitor (RKIP) and its correlation with TWEAK in vitro and in vivo were examined by quantitative real-time polymerase chain reaction and invasion assays.<h4>Results</h4>CRC patients whose tumors displayed high TWEAK expression had a statistically significantly higher overall survival and a disease-free advantage over those with a low TWEAK expression. In in vitro invasion assays, alterations in TWEAK expression in CRC cell lines inversely modulated their invasive ability. By means of integrated genomics, we identified RKIP as a downstream effector in TWEAK-mediated invasion inhibition. Knockout of RKIP expression in HCT116 cells by short hairpin RNA (shRKIP) resulted in increased invasiveness. Clinically, RKIP and TWEAK mRNA expression showed strong positive correlations in CRC patient samples.<h4>Conclusions</h4>Our results implicate TWEAK as a key regulator of CRC invasion, and it appears to be a useful prognostic factor for patients with CRC.

Unilateral hypoxic-ischemia results in the frequent occurrence of interictal spikes, and occasionally sustained ictal discharges accompanied by a reduction in paired-pulse inhibition within the non-lesioned dentate gyrus. To elucidate the roles of serotonin (5-hydroxytryptamine [5-HT]) in an epileptogenic insult, we investigated the changes in 5-HT receptors and serotonin transporter (5-HTT) immunoreactivities within the lesioned and contralateral hippocampus following unilateral hypoxic-ischemia. During epileptogenic periods following hypoxic-ischemia, both 5-HT(1A) and 5HT(1B) receptor immunoreactivities were decreased within the lesioned and the non-lesioned hippocampus. However, 5-HTT immunoreactivity was transiently increased within the hippocampus bilaterally. These findings indicate that alteration of the 5-HT system results in a "diaschisis" pattern, and may contribute to neuronal death and the development of emotional disorders in epileptic patients accompanied by psychological stress.

Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. The most characteristic structural feature of this disease is neurodegeneration accompanied by gliosis in the striatum. BDNF has been proposed to protect striatal neurons from degeneration, because it is an important survival factor for these neurons from development to adulthood. Considering the extensive gliosis and the survival effects of BDNF, we constructed an adenovirus to express a BDNF cDNA in astrocyte cells using a promoter of the glial fibrillary acidic protein gene. Cells stably transfected in vitro with a BDNF cDNA driven by this promoter expressed BDNF and responded to external stimuli increasing BDNF production. When the vector was applied into the striata of mice transgenic for HD, long-term expression of the transgene was observed, associated with a delay of onset of the motor phenotype of the R6/2 HD transgenic mice. The present data indicate that the striatal expression of BDNF is a potential adjuvant for the treatment of HD.

Red meat consumption has been positively associated with colorectal cancer; however, the biological mechanism underlying this relationship is not understood. Red meat is a major source of iron, which may play a role in colorectal carcinogenesis via increased crypt cell proliferation, cytotoxicity, and endogenous N-nitrosation. In a nested case-control study within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we prospectively evaluated multiple iron exposure parameters, including dietary intake and serum measures of iron, ferritin, transferrin, total iron binding capacity (TIBC), and unsaturated iron binding capacity (UIBC) in relation to incident colorectal adenoma in 356 cases and 396 matched polyp-free controls. We also investigated variation in eight key genes involved in iron homeostasis in relation to colorectal adenoma in an additional series totaling 1,126 cases and 1,173 matched controls. We observed a positive association between red meat intake and colorectal adenoma [OR comparing extreme quartiles (OR(q4-q1)) = 1.59, 95% CI = 1.02-2.49, P(trend) = 0.03]. Serum TIBC and UIBC were inversely associated with colorectal adenoma (OR(q4-q1) = 0.57, 95% CI = 0.37-0.88, P(trend) = 0.03; and OR(q4-q1) = 0.62, 95% CI = 0.40-0.95, P(trend) = 0.04, respectively). Colorectal adenoma was not associated with serum ferritin, iron, or transferrin saturation or with polymorphisms in genes involved in iron homeostasis. Serum TIBC and UIBC, parameters that have a reciprocal relationship with overall iron load, were inversely related to colorectal adenoma, suggesting that individuals with lower iron status have a reduced risk of developing colorectal adenoma.

Primary cilia regulate polarized protein trafficking in photoreceptors, which are dynamic and highly compartmentalized sensory neurons of retina. The ciliary protein Cep290 modulates cilia formation and is frequently mutated in syndromic and non-syndromic photoreceptor degeneration. However, the underlying mechanism of associated retinopathy is unclear. Using the Cep290 mutant mouse rd16 (retinal degeneration 16), we show that Cep290-mediated photoreceptor degeneration is associated with aberrant accumulation of its novel interacting partner Rkip (Raf-1 kinase inhibitory protein). This effect is phenocopied by morpholino-mediated depletion of cep290 in zebrafish. We further demonstrate that ectopic accumulation of Rkip leads to defective cilia formation in zebrafish and cultured cells, an effect mediated by its interaction with the ciliary GTPase Rab8A. Our data suggest that Rkip prevents cilia formation and is associated with Cep290-mediated photoreceptor degeneration. Furthermore, our results indicate that preventing accumulation of Rkip could potentially ameliorate such degeneration.

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5-E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

Maintaining a stable genome is one of the most important tasks of every living cell and the mechanisms ensuring it are similar in all of them. The events leading to changes in DNA sequence (mutations) in diploid cells occur one to two orders of magnitude more frequently than in haploid cells. The majority of those events lead to loss of heterozygosity at the mutagenesis marker, thus diploid-specific genome stability mechanisms can be anticipated. In a new global screen for spontaneous loss of function at heterozygous forward mutagenesis marker locus, employing three different mutagenesis markers, we selected genes whose deletion causes genetic instability in diploid Saccharomyces cerevisiae cells. We have found numerous genes connected with DNA replication and repair, remodeling of chromatin, cell cycle control, stress response, and in particular the structural maintenance of chromosome complexes. We have also identified 59 uncharacterized or dubious ORFs, which show the genome instability phenotype when deleted. For one of the strongest mutators revealed in our screen, ctf18Δ/ctf18Δ the genome instability manifests as a tendency to lose the whole set of chromosomes. We postulate that this phenomenon might diminish the devastating effects of DNA rearrangements, thereby increasing the cell's chances of surviving stressful conditions. We believe that numerous new genes implicated in genome maintenance, together with newly discovered phenomenon of ploidy reduction, will help revealing novel molecular processes involved in the genome stability of diploid cells. They also provide the clues in the quest for new therapeutic targets to cure human genome instability-related diseases.

Intercellular junctions promote homotypic cell to cell adhesion and transfer intracellular signals which control cell growth and apoptosis. Junctional adhesion molecule-A (JAM-A) is a transmembrane immunoglobulin located at tight junctions of normal epithelial cells of mammary ducts and glands. In the present paper we show that JAM-A acts as a survival factor for mammary carcinoma cells. JAM-A null mice expressing Polyoma Middle T under MMTV promoter develop significantly smaller mammary tumors than JAM-A positive mice. Angiogenesis and inflammatory or immune infiltrate were not statistically modified in absence of JAM-A but tumor cell apoptosis was significantly increased. Tumor cells isolated from JAM-A null mice or 4T1 cells incubated with JAM-A blocking antibodies showed reduced growth and increased apoptosis which paralleled altered junctional architecture and adhesive function. In a breast cancer clinical data set, tissue microarray data show that JAM-A expression correlates with poor prognosis. Gene expression analysis of mouse tumor samples showed a correlation between genes enriched in human G3 tumors and genes over expressed in JAM-A +/+ mammary tumors. Conversely, genes enriched in G1 human tumors correlate with genes overexpressed in JAM-A-/- tumors. We conclude that down regulation of JAM-A reduces tumor aggressive behavior by increasing cell susceptibility to apoptosis. JAM-A may be considered a negative prognostic factor and a potential therapeutic target.

The growth of many human breast tumors requires the proliferative effect of estrogen acting via the estrogen receptor α (ERα). ERα signaling is therefore a clinically important target for breast cancer prevention and therapeutics. Although extensively studied, the mechanism by which ERα promotes proliferation remains to be fully established. We observed an up-regulation of transcript encoding the pH-sensitive two-pore domain potassium channel KCNK5 in a screen for genes stimulated by 17β-estradiol (E2) in the ERα(+) breast cancer cell lines MCF-7 and T47D. KCNK5 mRNA increased starting 1 h after the onset of E2 treatment, and protein levels followed after 12 h. Estrogen-responsive elements are found in the enhancer region of KCNK5, and chromatin immunoprecipitation assays revealed binding of ERα to the KCNK5 enhancer in E2-treated MCF-7 cells. Cells treated with E2 also showed increases in the amplitude of pH-sensitive potassium currents, as assessed by whole-cell recordings. These currents are blocked by clofilium. Although confocal microscopy suggested that most of the channels are located in intracellular compartments, the increase in macroscopic currents suggests that E2 treatment increases the number of active channels at the cell surface. Application of small interfering RNA specific for KCNK5 decreased pH-sensitive potassium currents and also reduced the estrogen-induced proliferation of T47D cells. We conclude that E2 induces the expression of KCNK5 via ERα(+) in breast cancer cells, and this channel plays a role in regulating proliferation in these cell lines. KCNK5 may therefore represent a useful target for treatment, for example, of tamoxifen-resistant breast cancer.

<h4>Background</h4>Genetic factors and environmental factors are assumed to interactively influence the pathogenesis of anxiety disorders. Thus, a gene-environment interaction (G × E) study was conducted with respect to anxiety sensitivity (AS) as a promising intermediate phenotype of anxiety disorders.<h4>Method</h4>Healthy subjects (N = 363) were assessed for AS, childhood maltreatment (Childhood Trauma Questionnaire), and genotyped for functional serotonin transporter gene variants (5-HTTLPR/5-HTT rs25531). The influence of genetic and environmental variables on AS and its subdimensions was determined by a step-wise hierarchical regression and a multiple indicator multiple cause (MIMIC) model.<h4>Results</h4>A significant G × E effect of the more active 5-HTT genotypes and childhood maltreatment on AS was observed. Furthermore, genotype (LL)-childhood trauma interaction particularly influenced somatic AS subdimensions, whereas cognitive subdimensions were affected by childhood maltreatment only.<h4>Conclusions</h4>Results indicate a G × E effect of the more active 5-HTT genotypes and childhood maltreatment on AS, with particular impact on its somatic subcomponent.

Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications.

In this investigation, we have carried out an autosomal genome-wide linkage analysis to map genes associated with type 2 diabetes (T2D) and five quantitative traits of blood lipids including total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol, and triglycerides in a unique family-based cohort from the Sikh Diabetes Study (SDS). A total of 870 individuals (526 male/344 female) from 321 families were successfully genotyped using 398 polymorphic microsatellite markers with an average spacing of 9.26 cM on the autosomes. Results of non-parametric multipoint linkage analysis using S(all) statistics (implemented in Merlin) did not reveal any chromosomal region to be significantly associated with T2D in this Sikh cohort. However, linkage analysis for lipid traits using QTL-ALL analysis revealed promising linkage signals with p≤0.005 for total cholesterol, LDL cholesterol, and HDL cholesterol at chromosomes 5p15, 9q21, 10p11, 10q21, and 22q13. The most significant signal (p = 0.0011) occurred at 10q21.2 for HDL cholesterol. We also observed linkage signals for total cholesterol at 22q13.32 (p = 0.0016) and 5p15.33 (p = 0.0031) and for LDL cholesterol at 10p11.23 (p = 0.0045). Interestingly, some of linkage regions identified in this Sikh population coincide with plausible candidate genes reported in recent genome-wide association and meta-analysis studies for lipid traits. Our study provides the first evidence of linkage for loci associated with quantitative lipid traits at four chromosomal regions in this Asian Indian population from Punjab. More detailed examination of these regions with more informative genotyping, sequencing, and functional studies should lead to rapid detection of novel targets of therapeutic importance.

Genetic analysis of factors affecting risk to develop excessive ethanol drinking has been extensively studied in humans and animal models for over 20 years. However, little progress has been made in determining molecular mechanisms underlying environmental or non-genetic events contributing to variation in ethanol drinking. Here, we identify persistent and substantial variation in ethanol drinking behavior within an inbred mouse strain and utilize this model to identify gene networks influencing such "non-genetic" variation in ethanol intake. C57BL/6NCrl mice showed persistent inter-individual variation of ethanol intake in a two-bottle choice paradigm over a three-week period, ranging from less than 1 g/kg to over 14 g/kg ethanol in an 18 h interval. Differences in sweet or bitter taste susceptibility or litter effects did not appreciably correlate with ethanol intake variation. Whole genome microarray expression analysis in nucleus accumbens, prefrontal cortex and ventral midbrain region of individual animals identified gene expression patterns correlated with ethanol intake. Results included several gene networks previously implicated in ethanol behaviors, such as glutamate signaling, BDNF and genes involved in synaptic vesicle function. Additionally, genes functioning in epigenetic chromatin or DNA modifications such as acetylation and/or methylation also had expression patterns correlated with ethanol intake. In verification for the significance of the expression findings, we found that a histone deacetylase inhibitor, trichostatin A, caused an increase in 2-bottle ethanol intake. Our results thus implicate specific brain regional gene networks, including chromatin modification factors, as potentially important mechanisms underlying individual variation in ethanol intake.

Imbalance in the activity of striatal direct and indirect pathway neurons contributes to motor disturbances in several neurodegenerative diseases. In Huntington's disease (HD), indirect pathway [dopamine (DA) D2 receptor-expressing] medium-sized spiny neurons (MSNs) are believed to show earlier vulnerability than direct pathway MSNs. We examined synaptic activity and DA modulation in MSNs forming the direct and indirect pathways in YAC128 and BACHD mouse models of HD. To visualize the two types of MSNs, we used mice expressing enhanced green fluorescent protein under the control of the promoter for the DA D1 or D2 receptor. Experiments were performed in early symptomatic (1.5 months) and symptomatic (12 months) mice. Behaviorally, early symptomatic mice showed increased stereotypies while symptomatic mice showed decreased motor activity. Electrophysiologically, at the early stage, excitatory and inhibitory transmission onto D1-YAC128 and D1-BACHD MSNs were increased, while there was no change in D2 MSNs. DA modulation of spontaneous excitatory postsynaptic currents (sEPSCs) in slices was absent in YAC128 cells at the early stage, but was restored by treating the slices with the DA depleter tetrabenazine (TBZ). In BACHD mice TBZ restored paired-pulse ratios and a D1 receptor antagonist induced a larger decrease of sEPSCs than in D1-WT cells, suggesting increased DA tone. Finally, TBZ decreased stereotypies in BACHD mice. These results indicate that by reducing DA or antagonizing D1 receptors, increases in inhibitory and excitatory transmission in early phenotypic direct pathway neurons can be normalized. In symptomatic YAC128 mice, excitatory synaptic transmission onto D1 MSNs was decreased, while inhibitory transmission was increased in D2 MSNs. These studies provide evidence for differential and complex imbalances in glutamate and GABA transmission, as well as in DA modulation, in direct and indirect pathway MSNs during HD progression.

Serum tumor markers are useful to evaluate a cancer's response to treatment, for early detection of cancer relapse, and, in some cases, to diagnose malignancy. In this paper, we present two patients with significantly elevated serum tumor markers without evidence of malignant disease. An 18-year-old patient suffering from autoimmune hepatitis had markedly increased alpha-fetoprotein (aFP) levels (2,002 μg/L; normal <10 ug/L). Extensive imaging showed no signs of hepatocellular carcinoma or other cancer, and treatment with Prednisone led to rapid normalization of both liver enzymes and aFP. The second patient, a 60-year-old female with painless jaundice due to biliary stone disease, had very high serum levels of CA19-9 (18,000 kU/L, normal <27 kU/L). Liver biochemistry and serum CA19-9 concentration decreased to almost normal values (45 kU/L) after biliary stenting. These cases demonstrate that serum tumor markers can be elevated in benign disease and are therefore not appropriate to diagnose cancer.

Competitive adsorption of two-component solutions containing fibronectin (Fn) and albumin (Ab) on hydroxyapatite (HAp) nanocrystals was analyzed <i>in situ</i> using the quartz crystal microbalance with dissipation (QCM-D) technique. Adsorption of the one-component protein (Fn or Ab) and the two-component proteins adjusted to different molar ratios of Fn to Ab at a fixed Fn concentration was investigated. The frequency shift (Δ<i>f</i>; Hz) and the dissipation energy shift (Δ<i>D</i>) were measured with the QCM-D technique, and the viscoelastic changes of adlayers were evaluated by the saturated Δ<i>D</i>/Δ<i>f</i> value and the Voigt-based viscoelastic model. For the adsorption of the one-component protein, the Fn adlayer showed a larger mass and higher viscoelasticity than the Ab adlayer, indicating the higher affinity of Fn on HAp. For the adsorption of the two-component proteins, the viscoelastic properties of the adlayers became elastic with increase in Ab concentration, whereas the adsorption mass was similar to that of Fn in the one-component solution regardless of the Ab concentration. The specific binding mass of the Ab antibody to the adlayers increased with increase in Ab concentration, whereas that of the Fn antibody decreased. Therefore, Fn preferentially adsorbs on HAp and Ab subsequently interacts with the adlayers, indicating that the interfacial viscoelasticity of the adlayers was dominated by the interaction between Fn and Ab.

No abstract available.

Developed in the early 1990s, peptide nucleic acid (PNA) has emerged as a promising class of nucleic acid mimic because of its strong binding affinity and sequence selectivity toward DNA and RNA and resistance to enzymatic degradation by proteases and nucleases; however, the main drawbacks, as compared to other classes of oligonucleotides, are water solubility and biocompatibility. Herein we show that installation of a relatively small, hydrophilic (R)-diethylene glycol ("miniPEG", R-MP) unit at the γ-backbone transforms a randomly folded PNA into a right-handed helix. Synthesis of optically pure (R-MP)γPNA monomers is described, which can be accomplished in a few simple steps from a commercially available and relatively cheap Boc-l-serine. Once synthesized, (R-MP)γPNA oligomers are preorganized into a right-handed helix, hybridize to DNA and RNA with greater affinity and sequence selectivity, and are more water soluble and less aggregating than the parental PNA oligomers. The results presented herein have important implications for the future design and application of PNA in biology, biotechnology, and medicine, as well as in other disciplines, including drug discovery and molecular engineering.

Thrombotic events are common and potentially fatal complications in patients receiving hematopoietic stem cell transplantation (HSCT). Early diagnosis is crucial but remains controversial. In this study, we investigated the early alterations of hemostatic parameters in allogeneic HSCT recipients and determined their potential diagnostic values in transplantation-related thrombotic complications and other post-HSCT events. Results from 107 patients with allogeneic HSCT showed higher levels of plasma plasminogen activator inhibitor-1 (PAI-1), fibrinogen, and tissue-plasminogen activator (t-PA) and a lower level of plasma protein C after transplantation. No change was found for prothrombin time, antithrombin III, D: -dimer, and activated partial thromboplastin time following HSCT. Transplantation-related complications (TRCs) in HSCT patients were defined as thrombotic (n=8), acute graft-versus-host disease (aGVHD, n=45), and infectious (n=38). All patients with TRCs, especially the patients with thrombotic complications, presented significant increases in the mean and maximum levels of PAI-1 during the observation period. Similarly, a high maximum t-PA level was found in the thrombotic group. In contrast, apparent lower levels of mean and minimum protein C were observed in the TRC patients, especially in the aGVHD group. Therefore, the hemostatic imbalance in the early phase of HSCT, reflecting prothrombotic state and endothelial injury due to the conditioning therapy or TRCs, might be useful in the differential diagnosis of the thrombotic complication from other TRCs.

The multifunctional cytoprotective protein peroxiredoxin 6 (Prdx6) maintains cellular homeostasis and membrane integrity by regulating expression of intracellular reactive oxygen species (ROS) and phospholipid turnover. Using cells derived from targeted inactivation of Prdx6 gene or its depletion by RNA interference or aging, we showed that Prdx6 deficiency in cells evoked unfolded protein response (UPR), evidenced by increased expression or activation of proapoptotic factors, CHOP, ATF4, PERK, IRE-α and eIF2-α and by increased caspases 3 and 12 processing. Those cells displayed enhanced and sustained expression of endoplasmic reticulum (ER) stress-related chaperon proteins, Bip/glucose-regulated protein 78, calnexin, and calreticulin. Under cellular stress induced by hypoxia (1% O(2) or CoCl(2) treatment) or tunicamycin, Prdx6-deficient cells exhibited aberrant activation of ER stress-responsive genes/protein with higher expression of ROS, and died with apoptosis. Wild-type cells exposed to tunicamycin or hypoxia remained relatively insensitive with lower expression of ROS and ER-responsive genes than did Prdx6-deficient cells, but upregulation of ER stress responsive proteins or chaperones mimicked the UPR response of Prdx6-deficient or aging cells. Expression of Prdx6 blocked ER stress-induced deleterious signaling by optimizing physiologically aberrant expression of ER stress responsive genes/proteins in Prdx6-deficient cells or cells facing stressors, and rescued the cells from apoptosis. These findings demonstrate that impaired homeostasis and progression of pathogenesis in Prdx6-deficient lens epithelial cells or in aging cells should be blocked by a supply of Prdx6. The results provide a new molecular basis for understanding the etiology of several age-associated degenerative disorders, and potentially for developing antioxidant Prdx6-based therapeutics.

<h4>Background</h4>Calcium channels are essential in coupling action potential to signal transduction in cells. There are several types of calcium channels, which can be pharmacologically classified as L-, N-, P/Q-, R- and T-type. But molecular basis of R-type channels is less clearly understood compared the other channel types. Therefore the current study aims at understanding the molecular functions of R-type calcium channels by identifying interaction partners of the channel.<h4>Methods</h4>In order to do so, a yeast two hybrid (Y2H) screen, with carboxy terminus of α1 subunit of the channel, as the bait, was performed. G1 subunit of v-ATPase was identified as a putative interaction partner of human Ca(v)2.3 by using the Y2H screening. The interaction was confirmed by immunoprecipitation. To study the functional importance of the interaction, bafilomycin A(1), a potent and specific inhibitor of v-ATPase was used in patch-clamp recordings in Ca(v)2.3 stably-transfected HEK-293 cells (2C6) as well as in electroretinography of the isolated bovine retina expressing R-type Ca(2+) channels.<h4>Results</h4>G1 subunit of v-ATPase interacts with C-terminal tail of Ca(v)2.3 and bafilomycin A(1) reduces Ca(v)2.3 mediated calcium currents. Additionally peak I(Ca) is inhibited in retinal signal transduction when recorded as ERG b-wave.<h4>Conclusions</h4>The results suggest that v-ATPase interacts physically and also functionally with Ca(v)2.3. This is the first demonstration of association of Ca(v)2.3 C-terminus with a protein complex which is involved in transmembrane signalling.

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by striatal neurodegeneration, involving apoptosis. FK506, an inhibitor of calcineurin (or protein phosphatase 3, formerly known as protein phosphatase 2B), has shown neuroprotective effects in several cellular and animal models of HD. In the present study, we show the protective effects of FK506 in two striatal HD models, primary rat striatal neurons treated with 3-nitropropionic acid (3-NP) and immortalized striatal STHdh cells derived from HD knock-in mice expressing normal (STHdh(7/7)) or full-length mutant huntingtin (FL-mHtt) with 111 glutamines (STHdh(111/111)), under basal conditions and after exposure to 3-NP or staurosporine (STS). In rat striatal neurons, FK506 abolished 3-NP-induced increase in caspase-3 activation, DNA fragmentation/condensation and necrosis. Nevertheless, in STHdh(111/111) cells under basal conditions, FK506 did not prevent, in a significant manner, the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, or alter Bax/Bcl-2 ratio, but significantly reverted caspase-3 activation. In STHdh(111/111) cells treated with 0.3mM 3-NP or 25 nM STS, linked to high necrosis, exposure to FK506 exerted no significant effects on caspase-3 activation. However, treatment of STHdh(111/111) cells exposed to 10nM STS with FK506 effectively prevented cell death by apoptosis and moderate necrosis. The results suggest that FK506 may be neuroprotective against apoptosis and necrosis under mild cell death stimulus in the presence of FLmHtt.

Depression and anxiety are among the leading causes of societal burden. Abnormalities in 5-hydroxytryptamine (5-HT; serotonin) neurotransmission are known to be associated with depressive and anxiety symptoms. The rostral projections of brainstem dorsal (DRN) and median (MRN) raphe nuclei are the main sources of forebrain 5-HT. The expression, turnover and distribution of tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme in 5-HT biosynthesis in the DRN and MRN are complex, in keeping with the existence of different subpopulations of 5-HT neurons in this area. In the present study, we measured the expression of TPH2 mRNA in the DRN and MRN using in situ hybridization in three genetically modified mouse models, all relevant to depression and anxiety, and matched wild-type controls. Our results show quantitative modifications in TPH2 mRNA expression in the three main subregions of the DRN as well as the MRN in relation to changes in serotonergic, glutamatergic and endocannabinoid neurotransmission systems. Thus, there were significant decreases in TPH2 transcript levels in 5-HT transporter (5-HTT)-/- mutant mice, whereas increases were observed in the vesicular glutamate transporter 1 hemi knock out (VGLUT1+/-) and cannabinoid receptor 1 mutant (CB1R-/-) mice. Based on these findings, we suggest that TPH2 mRNA expression is under the influence of multiple messenger systems in relation to presynaptic and/or postsynaptic feedback control of serotonin synthesis that, 5-HTT, VGLUT1 and CB1R seem to be involved in these feedback mechanisms. Finally, our data are in line with previous reports suggesting that TPH2 activity within different raphe subregions is differentially regulated under specific conditions.

As early as one month of age, nonobese diabetic (NOD) mice feature pancreatic infiltration of autoreactive T lymphocytes, which destruct insulin-producing beta cells, producing autoimmune diabetes mellitus (T1D) within eight months. Thus, we hypothesized that during the development of T1D, the transcriptional modulation of immune reactivity genes may occur as thymocytes mature into peripheral T lymphocytes. The transcriptome of thymocytes and peripheral CD3⁺ T lymphocytes from prediabetic or diabetic mice analyzed through microarray hybridizations identified 2,771 differentially expressed genes. Hierarchical clustering grouped mice according to age/T1D onset and genes according to their transcription profiling. The transcriptional activity of thymocytes developing into peripheral T lymphocytes revealed sequential participation of genes involved with CD4⁺/CD8⁺ T-cell differentiation (Themis), tolerance induction by Tregs (Foxp3), and apoptosis (Fasl) soon after T-cell activation (IL4), while the emergence of T1D coincided with the expression of cytotoxicity (Crtam) and inflammatory response genes (Tlr) by peripheral T lymphocytes.

The increased use of older and/or marginal donor organs in liver transplantation over the last decade calls for strategies to minimize ischaemic reperfusion (I/R) injury to prevent early graft failure. Tacrolimus, a very potent and effective calcineurin inhibitor, was selected because of its ability to ameliorate I/R injury. A randomized, blinded, controlled single-centre trial of 26 liver transplant recipients was performed between February 2008 and December 2009. Donor organs were randomized to be perfused intraportally during liver transplantation with 1.5 l 5% albumin infusion containing either 20 ng/ml tacrolimus or placebo. The primary end point was liver function as assessed by aspartate transaminase (AST) or alanine transaminase (ALT) levels 6 days after transplantation. Treatment effectiveness was tested by transcriptome-wide analysis of biopsies. There was no difference in the primary end point, i.e. AST (IU/l) and ALT (IU/l) at day 6 after transplantation between groups. Furthermore, choleastatic parameters as well as parameters of liver synthesis were not different between groups. However, tacrolimus treatment suppressed inflammation and immune response in the transplanted liver on a genome-wide basis. Intrahepatic administration of tacrolimus did not result in a reduction of AST and ALT within the first week after transplantation.

<h4>Background</h4>Loss of heterozygosity at 18q, which includes the Deleted in Colorectal Cancer (DCC) gene, has been linked to many human cancers. However, it is unclear if loss of DCC is the specific underlying cause of these cancers. The Drosophila imaginal discs are excellent systems in which to study DCC function, as it is possible to model human tumors through the generation of somatic clones of cells bearing multiple genetic lesions. Here, these attributes of the fly system were utilized to investigate the potential tumor suppressing functions of the Drosophila DCC homologue frazzled (fra) during eye-antennal disc development.<h4>Results</h4>Most fra loss of function clones are eliminated during development. However, when mutant clone cells generated in the developing eye were rescued from death, partially differentiated eye cells were found outside of the normal eye field, and in extreme cases distant sites of the body. Characterization of these cells during development indicates that fra mutant cells display characteristics of invasive tumor cells, including increased levels of phospho-ERK, phospho-JNK, and Mmp-1, changes in cadherin expression, remodeling of the actin cytoskeleton, and loss of polarity. Mutation of fra promotes basement membrane degradation and invasion which are repressed by inhibition of Rho1 signaling. Although inhibition of JNK signaling blocks invasive phenotypes in some metastatic cancer models in flies, blocking JNK signaling inhibits fra mutant cell death, thereby enhancing the fra mutant phenotype.<h4>Conclusions</h4>The results of this investigation provide the first direct link between point mutations in fra/DCC and metastatic phenotypes in an animal model and suggest that Fra functions as an invasive tumor suppressor during Drosophila development.

<h4>Background</h4>Understanding the molecular biology of colorectal cancer (CRC) provides opportunities for effective personalised patient management. We evaluated whether chromosomal aberrations, mutations in the PI(3)K signalling pathway and the CpG-island methylator phenotype (CIMP) in primary colorectal tumours can predict liver metastases.<h4>Methods</h4>Formalin-fixed paraffin-embedded material from primary colorectal tumours of three different groups were investigated: patients with CRC without metastases (M0, n=39), patients who were treated with hyperthermal intraperitoneal chemotherapy for CRC metastases confined to the peritoneum (PM, n=46) and those who had isolated hepatic perfusion for CRC metastases confined to the liver (LM, n=48).<h4>Results</h4>All samples were analysed for DNA copy number changes, PIK3CA, KRAS, BRAF mutations, CIMP and microsatellite instability. The primary CRCs of the LM group had significantly higher frequency of amplified chromosome 20q (P=0.003), significantly fewer mutations in the PI(3)K signalling pathway (P=0.003) and fewer CIMP high tumours (P=0.05). There was a strong inverse correlation between 20q and the PI(3)K pathway mutations.<h4>Conclusion</h4>The development of CRC liver metastases is associated with amplification of chromosome 20q and not driven by mutations in the PI(3)K signalling pathway.

Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity.

The meiotic drive gene in Aedes aegypti is tightly linked with the sex determination locus on chromosome 1, and causes highly male-biased sex ratios. We prepared cDNA libraries from testes from the Ae. aegypti T37 strain (driving) and RED strain (non-driving), and used suppressive subtraction hybridization techniques to enrich for T37 testes-specific transcripts. Expressed sequence tags (ESTs) were obtained from a total of 2784 randomly selected clones from the subtracted T37 (subT37) library as well as the primary libraries for each strain (pT37 and pRED). Sequence analysis identified a total of 171 unique genes in the subT37 library and 299 unique genes among the three libraries. The majority of genes enriched in the subT37 library were associated with signal transduction, development, reproduction, metabolic process and cell cycle functions. Further, as observed with meiotic drive systems in Drosophila and mouse, a number of these genes were associated with signaling cascades that involve the Ras superfamily of regulatory small GTPases. Differential expression of several of these genes was verified in Ae. aegypti pupal testes using qRT-PCR. This study increases our understanding of testes gene expression enriched in adult males from the meiotic drive strain as well as insights into the basic testes transcriptome in Ae. aegypti.

<h4>Aims</h4>Sinonasal intestinal-type adenocarcinoma (ITAC) is an uncommon neoplasm morphologically similar to colorectal adenocarcinoma, with a well-recognized association with occupational exposure to wood or leather dusts. Here, we analyse several gene products with pivotal roles in tumorigenesis, including p53, p16, deleted in colon cancer (DCC), retinoblastoma, adenomatous polyposis coli, β-catenin, E-cadherin and CD10, and discuss their relation to clinical behaviour and to similar pathways in colorectal adenocarcinomas.<h4>Methods and results</h4>Immunohistochemical analysis of 62 ITACs was conducted on a tissue microarray. Aberrant expression of p53 and p16 were the most commonly observed alterations (61.3% and 64.5% of cases, respectively). Analysis according to the histological subtype showed that p53 overexpression was less frequent in mucinous ITACs (35.3% versus 71.1%, P = 0.018), while loss of DCC and E-cadherin were observed more frequently in this subtype (76.5% versus 31.1%, P=0.002 and 82.4% versus 31.1%, P<0.001, respectively). No correlation was found between the aberrant expression of these and clinical behaviour while mucinous adenocarcinomas had a significantly worse prognosis, with shorter disease-free interval and overall survival (P=0.005 and P<0.001, respectively).<h4>Conclusions</h4>Mucinous ITACs appear to follow a distinct molecular pathway(s) from the non-mucinous variants, and pursue an aggressive clinical behaviour.

Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal β-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic β-type globin promoters but not to the adult β-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1β, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult β-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult β-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic β-type globin genes through coordinated epigenetic chromatin modifications.

Yeast prions are self-propagating protein conformations that transmit heritable phenotypes in an epigenetic manner. The recently identified yeast prion [SWI(+)] is an alternative conformation of Swi1, a component of the evolutionarily conserved SWI/SNF chromatin-remodeling complex. Formation of the [SWI(+)] prion results in a partial loss-of-function phenotype for Swi1. The amino-terminal region of Swi1 is dispensable for its normal function but is required for [SWI(+)] formation and propagation; however, the precise prion domain (PrD) of Swi1 has not been elucidated. Here, we define the minimal Swi1 PrD as the first 37 amino acids of the protein. This region is extremely asparagine rich but, unexpectedly, contains no glutamine residues. This unusually small prion domain is sufficient for aggregation, propagation, and transmission of the [SWI(+)] prion. Because of its unusual size and composition, the Swi1 prion domain defined here has important implications for describing and identifying novel prions.

Characterized bacteria, unlike eukaryotes and some archaea, initiate replication bidirectionally from a single replication origin contained within a circular or linear chromosome. We constructed Escherichia coli cells with two WT origins separated by 1 Mb in their 4.64-Mb chromosome. Productive bidirectional replication initiated synchronously at both spatially separate origins. Newly replicated DNA from both origins was segregated sequentially as replication progressed, with two temporally and spatially separate replication termination events. Replication initiation occurred at a cell volume identical to that of cells with a single WT origin, showing that initiation control is independent of cellular and chromosomal oriC concentration. Cells containing just the ectopic origin initiated bidirectional replication at the expected cell mass and at the normal cellular location of that region. In all strains, spatial separation of sister loci adjacent to active origins occurred shortly after their replication, independently of whether replication initiated at the normal origin, the ectopic origin, or both origins.

Macrophages are critical players in both innate and adaptive immunity. While the exogenous signaling events leading to the terminal differentiation of macrophages from monocytes have been studied extensively, the underlying intracellular transcriptional mechanisms remain poorly understood. Here we report that the homeobox transcription factor VentX plays a pivotal role in human macrophage terminal differentiation and proinflammatory function. Our study showed that VentX expression was upregulated upon human primary monocyte-to-macrophage differentiation induced by cytokines such as M-CSF, GM-CSF, and IL-3. Moreover, ablation of VentX expression in primary monocytes profoundly impaired their differentiation to macrophages, and ectopic expression of VentX in a myeloid progenitor cell line triggered its differentiation with prominent macrophage features. Further analysis revealed that VentX was pivotal for the proinflammatory response of terminally differentiated macrophages. Mechanistically, VentX was found to control expression of proteins key to macrophage differentiation and activation, including M-CSF receptor. Importantly, preliminary analysis of gene expression in leukocytes from patients with autoimmune diseases revealed a strong correlation between levels of VentX and those of proinflammatory cytokines. Our results provide mechanistic insight into the crucial roles of VentX in macrophage differentiation and proinflammatory activation and suggest that dysregulation of VentX may play a role in the pathogenesis of autoimmune diseases.

<h4>Objective</h4>Physiological and pharmacological studies indicate that altered brain serotonin (5-HT) activity could contribute to a susceptibility to develop appetitive and behavioral alterations that are characteristic of bulimia nervosa (BN).<h4>Method</h4>Eight individuals recovered from BN (REC BN) and eight healthy control women were scanned with [11C]DASB and positron emission tomography imaging of the 5-HT transporter (5-HTT). Logan graphical analysis was applied, and parametric binding potential (BP(nondisplaceable (ND)) ) images were generated. Voxel-by-voxel t-tests and a region of interest (ROI) analysis were conducted.<h4>Results</h4>REC BN had significantly lower [11C]DASB BP(ND) in midbrain, superior and inferior cingulate and significantly higher [11C]DASB BP(ND) in anterior cingulate and superior temporal gyrus in the voxel-based analysis. ROI analysis indicated lower [11C]DASB BP(ND) in midbrain (p = .07), containing the dorsal raphe, in REC BN, consistent with our earlier studies.<h4>Discussion</h4>These preliminary findings of a small-scale study confirm and extend previous data suggesting that ill and recovered BN have altered 5-HTT measures, which potentially contribute to BN symptomatology and/or differential responses to medication.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of huntingtin (HTT). Relatively little attention has been directed to the genomic features of the antisense strand at the HD locus, though the presence of a transcript from this strand has been suggested by a survey of the entire transcriptome and the existence of several EST tags. In this study, we identified huntingtin antisense (HTTAS), a natural antisense transcript at the HD repeat locus that contain the repeat tract. HTTAS is 5' capped, poly (A) tailed and contains three exons, alternatively spliced into HTTAS_v1 (exons 1 and 3) and HTTAS_v2 (exons 2 and 3). Exon 1 includes the repeat. HTTAS_v1 has a weak promoter, and is expressed at low levels in multiple tissue types and throughout the brain. Reporter assays indicate that while efficient promoter activity requires a short repeat, repeat expansion reduces promoter efficiency. Consistent with the reporter assays, levels of HTTAS_v1 are reduced in human HD frontal cortex. In cell systems, overexpression of HTTAS_v1 specifically reduces endogenous HTT transcript levels, while siRNA knockdown of HTTAS_v1 increases HTT transcript levels. Minigene constructs of the HD locus confirm the regulatory effect of HTTAS_v1 on HTT, and demonstrate that the effect is dependent on repeat length and is at least partially Dicer dependent. Together, these findings provide strong evidence for the existence of a gene antisense to HTT, with properties that include regulation of HTT expression.

Parkinson's disease (PD) is a progressive extrapyramidal motor disorder. Pathologically, this disease is characterized by the selective dopaminergic (DAergic) neuronal degeneration in the substantia nigra. Correcting the DA deficiency in PD with levodopa (L-dopa) significantly attenuates the motor symptoms; however, its effectiveness often declines, and L-dopa-related adverse effects emerge after long-term treatment. Nowadays, DA receptor agonists are useful medication even regarded as first choice to delay the starting of L-dopa therapy. In advanced stage of PD, they are also used as adjunct therapy together with L-dopa. DA receptor agonists act by stimulation of presynaptic and postsynaptic DA receptors. Despite the usefulness, they could be causative drugs for valvulopathy and nonmotor complication such as DA dysregulation syndrome (DDS). In this paper, physiological characteristics of DA receptor familyare discussed. We also discuss the validity, benefits, and specific adverse effects of pharmaceutical DA receptor agonist.

Collagen is the fundamental structural protein, comprising 25-35% of the total body protein, its rod-like triple helix providing support in many tissues. Our laboratory has synthesised 113 Toolkit peptides, each 63 residues long, covering the entirety of the homotrimeric helix sequence of collagen II and collagen III. These are used primarily to investigate protein-collagen interactions, from which biomedical applications are under development. Upon increasing the temperature of a Toolkit peptide solution, a novel low temperature transition (LTT) as well as a broadening of the helix unfolding higher temperature transition (HTT) was observed. Here, we hypothesized that unfolding of imperfect helices can account for the LTT. Peptides of various purities were isolated by HPLC or gel filtration, and their unfolding measured by polarimetry, CD, and DSC. The resulting temperature transitions were fitted to a kinetic unfolding equation, allowing comparison of the data, and explanation of the observed melting curve complexity as due to peptide imperfections. Finally, using a mathematical model, this data can be replicated by setting a parameter that quantifies the mutual stabilization conferred by helices on each side of a peptide defect within a triple helix.

In colorectal cancer, the functional impact of proteins from different signaling pathways varies between tumor center and tumor front. Our objective was to identify differential protein expression profiles between the tumor center and the tumor front of colorectal cancer. Twenty proteins from different signaling pathways (epidermal growth factor receptor [EGFR], phosphorylated extracellular signal regulated kinase [pERK], receptor for hyalouronic acid mediated motility [RHAMM], Raf-1 kinase inhibitor protein [RKIP], β-catenin, E-cadherin, phosphorylated AK transforming [pAKT], p16, p21, Ki-67, B-cell Lymphoma-2 [BCL2], vascular endothelial growth factor, apoptosis protease activating factor 1 [APAF-1], mucin1 [MUC1], ephrin B2 receptor [EphB2], matrix metalloproteinase 7 [MMP7], phosphorylated mothers against decapentaplegic 2 [pSMAD2], caudal type homeobox transcription factor 2 [CDX2], Laminin5γ2, and mammalian sterile 20-like kinase 1 [MST1]) involved in colorectal cancer progression were studied immunohistochemically on 220 well-characterized patients using a multiple-punch tissue microarray including 437 and 430 samples from the tumor center and the invasive front, respectively. Mean expression between the tumor center and the tumor front varied statistically significantly for pSMAD2, pERK, Raf-1 kinase inhibitor protein, E-cadherin, pAKT, BCL2, vascular endothelial growth factor, EphB2, matrix metalloproteinase 7, CDX2, Laminin5γ2, MST1, and APAF-1. Overexpression of pAKT, BCL2, vascular endothelial growth factor, APAF-1, pERK, EphB2, Raf-1 kinase inhibitor protein, CDX2, E-cadherin, MST1 (P < .001 each), and pSMAD2 (P = .002) was more frequently observed in the tumor center, whereas matrix metalloproteinase 7 and Laminin5γ2 (P < .001 each) overexpression was associated with the invasive front. In multivariate analysis, vascular endothelial growth factor (P < .001), Raf-1 kinase inhibitor protein (P = .009), and Laminin5γ2 (P < .001) were the most relevant proteins with the multimarker phenotypes positive/positive/negative and negative/negative/positive being most discriminating between the tumor center and the tumor front. Moreover, the combination negative/negative/positive vascular endothelial growth factor/Raf-1 kinase inhibitor protein/Laminin5γ2 at the tumor front was associated with vascular/lymphatic invasion (P = .014), distant metastasis (P = .019), higher tumor grade (P < .001), and poorer survival (P = .05). Our findings show that, in colorectal cancer progression, vascular endothelial growth factor overexpression seems to play a role in the tumor center, whereas Laminin5γ2-positivity combined with Raf-1 kinase inhibitor protein loss is associated with tumor invasion at the front.

Progressive mitochondrial failure is tightly associated with the onset of many age-related human pathologies. This tight connection results from the double-edged sword of mitochondrial respiration, which is responsible for generating both ATP and ROS, as well as from risks that are inherent to mitochondrial biogenesis. To prevent and treat these diseases, a precise understanding of the mechanisms that maintain functional mitochondria is necessary. Mitochondrial protein quality control is one of the mechanisms that protect mitochondrial integrity, and increasing evidence implicates the cytosolic ubiquitin/proteasome system (UPS) as part of this surveillance network. In this review, we will discuss our current understanding of UPS-dependent mitochondrial protein degradation, its roles in diseases progression, and insights into future studies.

<h4>Background</h4>The association between body iron stores and coronary heart disease (CHD) was inconsistent. We sought to explore this association in Chinese Han population and further examine the association of the variations in hemochromatosis (HFE) gene and CHD risk.<h4>Methods</h4>We conducted a case-control study including 1334 CHD patients and 1334 age- and sex-frequency matched controls. The plasma ferritin levels were measured by enzyme linked immunosorbent assay. Genotypes of the tagging single nucleotide polymorphisms (tagSNPs) were determined by TaqMan SNP allelic discrimination.<h4>Results</h4>The plasma ferritin levels in CHD cases (197.9μg/L [2.7-932.9μg/L]) were higher than those in controls (179.9μg/L [21.1-878.2μg/L]; P=0.028). The odds ratios (ORs) across the tertiles of plasma ferritin levels were 1.0 (reference), 0.93 (0.76-1.13), and 1.23 (1.02-1.48; P for trend=0.028). Adjustment for the traditional risk factors attenuated the associations to null (P for trend=0.22). Compared with the TT genotype of tagSNP rs9366637, subjects with C allele had higher risk of CHD (OR=1.35 for TC and 1.76 for CC; P=0.001 and <0.001 respectively). After adjustment for the conventional risk factors the results remained unchanged. We did not find significantly different plasma ferritin levels among different genotypes of rs9366637 (P=0.52).<h4>Conclusions</h4>The plasma ferritin levels were not significantly associated with CHD risk. However, the SNP rs9366637 in HFE gene was associated with higher CHD risk in Chinese Han population. The underlie mechanism remained to be elucidated in further studies.

A series of analogues were synthesized by optimizing the structure of papaverine. The in vitro PDE10A binding affinity (IC(50)) values for these new analogues were measured; for compounds that have IC(50) value less than 60 nM for PDE10A, the binding affinities (IC(50) value) for PDE3A and PDE3B were tested. Of these analogues, compounds 6a, 6b, 6n, 8b, 8c and 11 displayed relatively higher PDE10A potency with IC(50) value in the range of 28-60 nM. The most potent compound 1-(4-(2-(2-fluoroethoxy)ethoxy)-3-methoxybenzyl)-6,7-dimethoxyisoquinoline (8c) has the IC(50) value of 28 ± 1.2 nM for PDE10A, 2200 ± 437 nM for PDE3A and 2520 ± 210 nM for PDE3B. Compared to papaverine, compound 8c displayed similar PDE10A potency but improved selectivity to PDE10A versus PDE3A and PDE3B. To identify high potent PDE10A inhibitor, further optimization of the structures of these analogues is necessary.

Two hundred and twenty Escherichia coli isolates from 314 Mediterranean water buffalo calves less than 4 weeks old affected by severe diarrhoea with a lethal outcome were characterized for the presence of the virulence factors LT, ST, Stx1, Stx2, haemolysins, intimin, CNF1, CNF2, CDT-I, CDT-II, CDT-III, CDT-IV, and F17-related fimbriae (F17a, F17b, F17c, F17d). The prevalence of ETEC, STEC and NTEC were 1.8%, 6.8% and 20.9%, respectively. The ETEC isolates were all LT-positive and ST-negative. The STEC isolates were all Stx and intimin-positive, with Stx1 (80%) more frequent than Stx2 (27%). The NTEC isolates were all CNF and Hly-positive, with CNF2 (83%) more frequent than CNF1 (22%). Susceptibility assays to 11 antimicrobials displayed high rates of resistance (>30%) to antimicrobials tested. These data show that the most prevalent strains in diarrhoeic water buffalo calves were NTEC, mostly CNF2 and HlyA-positive, with strong associations CNF2/CDT-III and CNF2/F17c.

<h4>Unlabelled</h4>Stress and negative moods, which are thought to be partly mediated by reduced brain serotonin function, often increase emotional eating in dieting women (restrainers). Because the short (S) allele polymorphism in the serotonin transporter gene (5-HTTLPR) is associated with serotonin dysfunction, S allele compared to long (L) allele 5-HTTLPR genotypes may be more susceptible to stress-induced emotional eating. Consequently, serotonin challenge via tryptophan (TRP)-rich protein hydrolysate (TPH) may alleviate stress-induced emotional eating particularly in S/S allele carriers. We tested whether acute stress affects emotional eating in women with high or low dietary restraints depending on their 5-HTTLPR genotype and TPH intake. Nineteen female subjects who were homozygous for the short-allele 5-HTTLPR genotype (S'/S'=S/L(G), L(G)/L(G): restrainers vs. nonrestrainers) and 23 female subjects who were homozygous for the long-allele 5-HTTLPR genotype (L'/L'=L(A)/L(A): restrainers vs. nonrestrainers) were tested in a double-blind, placebo-controlled crossover study of stress-induced emotional eating following intake of TPH or a placebo. TPH intake significantly increased the plasma TRP/large neutral amino acid ratio (P<.0001) in the L'/L' group (70%) compared to the S'/S' group (30%). TPH reduced food intake in both groups, but in the L'/L' group, it also reduced stress-induced negative mood (P=.037) and the desire for sweet, high-fat foods (P=.011) regardless of dietary restraint.<h4>Conclusions</h4>Since TPH caused a greater increase in the plasma TRP/large neutral amino acid ratio in the L'/L' group compared to S'/S' group, the exclusive beneficial effects of L'/L' genotype may be due to enhanced brain 5-HT function.

<h4>Background</h4>Influenza outbreaks in the childcare setting are a significant cause of excess winter morbidity. This study explored methods of follow up and sample collection for a proposed randomised controlled trial of influenza vaccination in children attending childcare.<h4>Methods</h4>The study was conducted in four Sydney childcare centres during 2007. Healthy children aged 6-59 months eligible for vaccination were recruited in two centres, with another two acting as controls. Data on influenza-like illness (ILI: ≥37.8°C plus at least one respiratory symptom) occurrence were collected weekly. In those children with an ILI, parents were asked to collect nasal swabs and send via surface mail for viral polymerase chain reaction. Vaccine efficacy (VE) for ILI was estimated overall and for subgroups aged 6-23 and 24-59 months using the formula VE = 1 - relative risk (RR).<h4>Results</h4>Sixty-three per cent (151/238) of eligible children had parents give consent. Sixty-three children received influenza vaccine and 88 participated as controls. Of 26 specimens returned, a virus was detected in 18 (69%); none with influenza. Two symptomatic children had positive near-patient influenza tests in general practice (one a vaccine failure). The RR with 95% confidence interval in all children and those aged 6-23 months were less than one, 0.56 (0.32-1.02) and 0.46 (0.15-1.45), respectively.<h4>Conclusions</h4>This study demonstrated the feasibility and utility of parent-collected and mailed respiratory specimens for VE research in the childcare setting. Two-thirds of parent-collected swabs proved positive for at least one virus. Finding ways to reduce reluctance of parents to submit samples could improve the representativeness of samples collected and the power of the study. No evidence was found for influenza VE, but point estimates were in the direction of protection.

<h4>Background</h4>An essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism Caenorhabditis elegans.<h4>Results</h4>Here, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners.<h4>Conclusions</h4>These results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization.

Forming synaptic connections of the appropriate strength between specific neurons is crucial for constructing neural circuits to control behavior. A recent paper in Neural Development describes the use of a synapse-specific label in Caenorhabditis elegans to implicate local UNC-6/netrin signaling in this developmental process. Thus, as well as their well known roles in cell migration and axon guidance, UNC-6/netrin signals distinguish an appropriate synaptic partner from other potential targets.

Much attention has been given to the problem of creating reliable multiple sequence alignments in a model incorporating substitutions, insertions, and deletions. Far less attention has been paid to the problem of optimizing alignments in the presence of more general rearrangement and copy number variation. Using Cactus graphs, recently introduced for representing sequence alignments, we describe two complementary algorithms for creating genomic alignments. We have implemented these algorithms in the new "Cactus" alignment program. We test Cactus using the Evolver genome evolution simulator, a comprehensive new tool for simulation, and show using these and existing simulations that Cactus significantly outperforms all of its peers. Finally, we make an empirical assessment of Cactus's ability to properly align genes and find interesting cases of intra-gene duplication within the primates.

Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCerSyn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected.

Polysaccharide sidechains attached to proteins play important roles in cell-cell and receptor-ligand interactions. Variation in the carbohydrate component has been extensively studied for the iron transport protein transferrin, because serum levels of the transferrin isoforms asialotransferrin + disialotransferrin (carbohydrate-deficient transferrin, CDT) are used as biomarkers of excessive alcohol intake. We conducted a genome-wide association study to assess whether genetic factors affect CDT concentration in serum. CDT was measured in three population-based studies: one in Switzerland (CoLaus study, n = 5181) and two in Australia (n = 1509, n = 775). The first cohort was used as the discovery panel and the latter ones served as replication. Genome-wide single-nucleotide polymorphism (SNP) typing data were used to identify loci with significant associations with CDT as a percentage of total transferrin (CDT%). The top three SNPs in the discovery panel (rs2749097 near PGM1 on chromosome 1, and missense polymorphisms rs1049296, rs1799899 in TF on chromosome 3) were successfully replicated , yielding genome-wide significant combined association with CDT% (P = 1.9 × 10(-9), 4 × 10(-39), 5.5 × 10(-43), respectively) and explain 5.8% of the variation in CDT%. These allelic effects are postulated to be caused by variation in availability of glucose-1-phosphate as a precursor of the glycan (PGM1), and variation in transferrin (TF) structure.

Upon activation, CD4(+) T cells release cytokines, chemokines, and other soluble factors that influence the kinetics of HIV-1 replication in macrophages (M). In this article, we show that activation of human primary T cells suppresses the early stages of HIV-1 replication in human primary Mφ by downregulating the main cellular receptor for the virus CD4. The secreted factors responsible for this effect have a molecular mass greater than conventional cytokines, are independent of Th1 or Th2 polarization, and are not IFN-γ, IL-16, RANTES, or macrophage inhibitory factor, as revealed by cytokine array analysis and neutralization assays. CD4 downregulation is entirely posttranslational and involves serine phosphorylation of CD4 and its targeting to an intracellular compartment destined for acidification and degradation. CD4 downregulation is dependent on the activities of both protein kinase C and NF-κB as well as the proteasomes. Using high-resolution liquid chromatography-tandem mass spectrometry analysis in conjugation with label-free protein quantitation software, we found that proteins that promote Mφ adherence and spreading, such as attractin, fibronectin, and galectin-3-binding protein, were significantly overrepresented in the activated T cell supernatant fractions. These results reveal the existence of previously unreported anti-HIV-1 proteins, released by activated T cells that downregulate CD4 expression, and are of fundamental importance to understand the kinetics of HIV infection in vivo.

Observational studies of human health and disease (basic, clinical and epidemiological) are vulnerable to methodological problems -such as selection bias and confounding- that make causal inferences problematic. Gene-disease associations are no exception, as they are commonly investigated using observational designs. A rich body of knowledge exists in medicine and epidemiology on the assessment of causal relationships involving personal and environmental causes of disease; it includes seminal causal criteria developed by Austin Bradford Hill and more recently applied directed acyclic graphs (DAGs). However, such knowledge has seldom been applied to assess causal relationships in clinical genetics and genomics, even in studies aimed at making inferences relevant for human health. Conversely, incorporating genetic causal knowledge into clinical and epidemiological causal reasoning is still a largely unexplored area.As the contribution of genetics to the understanding of disease aetiology becomes more important, causal assessment of genetic and genomic evidence becomes fundamental. The method we develop in this paper provides a simple and rigorous first step towards this goal. The present paper is an example of integrative research, i.e., research that integrates knowledge, data, methods, techniques, and reasoning from multiple disciplines, approaches and levels of analysis to generate knowledge that no discipline alone may achieve.

<h4>Introduction</h4>The C allele of the nonsynonymous Arg265His (rs3733591) variant of SLC2A9 confers risk for gout in Han Chinese, Solomon Island and Japanese samples, with a stronger role in tophaceous gout. There is no evidence for an association with gout in Caucasian populations. In the present study, we tested rs3733591 for association with gout in New Zealand (NZ) Māori, Pacific Island and Caucasian samples.<h4>Methods</h4>Rs3733591 was genotyped across gout patients (n = 229, 232 and 327 NZ Māori, Pacific Island and Caucasian samples, respectively) and non-gout controls (n = 343, 174 and 638 Māori, Pacific Island and Caucasian samples, respectively). Further Caucasian sample sets consisting of 67 cases and 4,712 controls as well as 153 cases and 6,969 controls were obtained from the Framingham Heart Study and the Atherosclerosis Risk in Communities study, respectively. The Polynesian samples were analyzed according to Eastern and Western Polynesian ancestry.<h4>Results</h4>No evidence for risk conferred by the C allele of rs3733591 with gout was found in the sample sets of NZ Māori (odd ratio (OR) = 0.98, P = 0.86), Eastern Polynesians (OR = 0.99, P = 0.92), Western Polynesians (OR = 1.16, P = 0.36) or combined Caucasians (OR = 1.15, P = 0.13). The C allele was significantly overrepresented in Māori tophaceous cases compared to cases without tophi (OR = 2.21, P = 0.008), but not in the other ancestral groupings.<h4>Conclusions</h4>Noting that our study's power was limited for detecting weak genetic effects, we were unable to replicate associations of rs3733591 with gout in Eastern Polynesian, Western Polynesian and Caucasian samples. However, consistent with a previous study of Han Chinese and Solomon Island populations, our data suggest that rs3733591 could be a marker of severe gout in some populations. Our results also suggest that the effect of this variant is population-specific, further confirming population heterogeneity regarding the association of SLC2A9 with gout.

Minocycline (7-dimethylamino-6-dimethyl-6-deoxytetracycline) is a second-generation tetracycline that can cross the blood-brain barrier and has anti-inflammatory and neuroprotective effects. The potential of minocycline as a drug for treating Huntington's disease has been studied; however, the molecular mechanism underlying the neuroprotective properties of minocycline remains elusive. In this study, we tested the hypothesis that a principal cellular target of minocycline is Apaf-1, a key protein in the formation of the apoptosome, a multiprotein complex involved in caspase activation. Minocycline binds to Apaf-1, as shown by nuclear magnetic resonance spectroscopy, and inhibits apoptosome activity in vitro and in ex vivo models. As a consequence, minocycline-treated cells as well as Apaf-1 knock-out cells are resistant to the development of mutant huntingtin-dependent protein aggregation.

This paper will consider the right not to know in the context of psychiatric disorders. It will outline the arguments for and against acquiring knowledge about the results of genetic testing for conditions such as breast cancer and Huntington's disease, and examine whether similar considerations apply to disclosing to clients the results of genetic testing for psychiatric disorders such as depression and Alzheimer's disease. The right not to know will also be examined in the context of the diagnosis of psychiatric disorders that are associated with stigma or for which there is no effective treatment.

<h4>Background/aims</h4>OVE26 (OVE) mice provide a valuable model of advanced diabetic nephropathy (DN), but they take 8 months to develop moderate interstitial fibrosis and reduced glomerular filtration rate (GFR). The aim of this project was to produce a more rapid and advanced model of DN.<h4>Methods</h4>Uninephrectomy was applied to OVE and FVB mice at 2 months of age. Albuminuria, GFR, glomerulosclerosis, interstitial fibrosis, gene expression and monocyte infiltration were evaluated as a function of diabetes and uninephrectomy.<h4>Results</h4>Albuminuria, monocyte infiltration, mesangial matrix expansion and renal fibrosis were greatly accelerated in uninephrectomized mice. DN was more advanced 10 weeks after uninephrectomy than in untreated OVE mice at 8 months of age. Uninephrectomy had almost no effect on these characteristics in non-diabetic mice. Microarray studies indicated that the accelerated fibrosis and cell infiltration in nephrectomized OVE mice were accompanied by corresponding gene expression changes in canonical pathways for fibrosis and inflammation.<h4>Conclusion</h4>Uninephrectomy greatly accelerates all features of diabetic renal damage. This procedure provides a 10-week period after surgery to examine very large changes in the pathology of DN. The model may be particularly useful for testing new therapies and for analysis of the progression of albuminuria and fibrosis in DN.

<h4>Background</h4>Correlations between Educational Attainment (EA) and measures of cognitive performance are as high as 0.8. This makes EA an attractive alternative phenotype for studies wishing to map genes affecting cognition due to the ease of collecting EA data compared to other cognitive phenotypes such as IQ.<h4>Methodology</h4>In an Australian family sample of 9538 individuals we performed a genome-wide association scan (GWAS) using the imputed genotypes of ∼2.4 million single nucleotide polymorphisms (SNP) for a 6-point scale measure of EA. Top hits were checked for replication in an independent sample of 968 individuals. A gene-based test of association was then applied to the GWAS results. Additionally we performed prediction analyses using the GWAS results from our discovery sample to assess the percentage of EA and full scale IQ variance explained by the predicted scores.<h4>Results</h4>The best SNP fell short of having a genome-wide significant p-value (p = 9.77×10(-7)). In our independent replication sample six SNPs among the top 50 hits pruned for linkage disequilibrium (r(2)<0.8) had a p-value<0.05 but only one of these SNPs survived correction for multiple testing--rs7106258 (p = 9.7*10(-4)) located in an intergenic region of chromosome 11q14.1. The gene based test results were non-significant and our prediction analyses show that the predicted scores explained little variance in EA in our replication sample.<h4>Conclusion</h4>While we have identified a polymorphism chromosome 11q14.1 associated with EA, further replication is warranted. Overall, the absence of genome-wide significant p-values in our large discovery sample confirmed the high polygenic architecture of EA. Only the assembly of large samples or meta-analytic efforts will be able to assess the implication of common DNA polymorphisms in the etiology of EA.

Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in zinc finger protein 644 isoform 1 (ZNF644) was identified as being related to the phenotype of this family. After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations (I587V, R680G, C699Y, 3'UTR+12 C>G, and 3'UTR+592 G>A) in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE). Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form.

Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. Here we report a similar phenotype for the telomeric partner of HP1, HOAP, and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, Sxl(Pe), is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (Sxl(Pm)) in both sexes. HOAP mutants show inappropriate Sxl(Pe) firing in males and the concomitant inappropriate splicing of Sxl(Pm)-derived transcripts, while females show premature firing of Sxl(Pe). HP1 mutants, by contrast, display Sxl(Pm) splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with Sxl(Pe) sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl(Pe) are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at Sxl(Pe).

The majority of glutamatergic synapses formed onto principal neurons of the mammalian central nervous system are associated with dendritic spines. Spines are tiny protuberances that house the proteins that mediate the response of the postsynaptic cell to the presynaptic release of glutamate. Postsynaptic signals are regulated by an ion channel signaling cascade that is active in individual dendritic spines and involves voltage-gated calcium (Ca) channels, small conductance (SK)-type Ca-activated potassium channels, and NMDA-type glutamate receptors. Pharmacological studies using the toxin SNX-482 indicated that the voltage-gated Ca channels that signal within spines to open SK channels belong to the class Ca(V)2.3, which is encoded by the Alpha-1E pore-forming subunit. In order to specifically test this conclusion, we examined the effects of SNX-482 on synaptic signals in acute hippocampal slices from knock-out mice lacking the Alpha-1E gene. We find that in these mice, application of SNX-482 has no effect on glutamate-uncaging evoked synaptic potentials and Ca influx, indicating that that SNX-482 indeed acts via the Alpha-1E-encoded Ca(V)2.3 channel.

In this study the "green chemistry" use of the biosurfactant surfactin for the synthesis of calcium phosphate using the reverse microemulsion technique was demonstrated. Calcium phosphates are bioactive materials that are a major constituent of human teeth and bone tissue. A reverse microemulsion technique with surfactin was used to produce nanocrystalline brushite particles. Structural diversity (analyzed by SEM and TEM) resulted from different water to surfactin ratios (W/S; 250, 500, 1000 and 40,000). The particle sizes were found to be in the 16-200 nm range. Morphological variety was observed in the as-synthesized microemulsions, which consisted of nanospheres (~16 nm in diameter) and needle-like (8-14 nm in diameter and 80-100 nm in length) noncalcinated particles. However, the calcinated products included nanospheres (50-200 nm in diameter), oval (~300 nm in diameter) and nanorod (200-400 nm in length) particles. FTIR and XRD analysis confirmed the formation of brushite nanoparticles in the as-synthesized products, while calcium pyrophosphate was produced after calcination. These results indicate that the reverse microemulsion technique using surfactin is a green process suitable for the synthesis of nanoparticles.

<h4>Introduction</h4>Citric acid-polyethylene glycol-citric acid (CPEGC) triblock dendrimers can serve as potential delivery systems.<h4>Methods</h4>In this investigation, CPEGC triblock dendrimers were synthesized and then imidazole groups were conjugated onto the surface of the G1, G2 and G3 of the obtained dendrimers. In order to study the type of the interactions between the functionalized dendrimers and a drug molecule, Naproxen which contains acidic groups, was examined as a hydrophobic drug in which the interactions would be of the electrostatic kind between its acidic groups and the lone pair electrons of nitrogen atom in imidazole groups. The quantity of the trapped drug and also the amount of its release were measured with UV spectrometric method in pH 1, 7.4 and 10. The average diameter of the nanocarriers was measured by Dynamic Light Scattering (DLS) technique.<h4>Results</h4>The size range of particles was determined to be 16-50 nm for different generations. The rate of the release increased in pH=10 in all generations due to the increases in Naproxen solubility and the hydrolysis of the esteric bonds in the mentioned pH. The results showed that the amount of the trapped drug increased with the increase in the generation of the dendrimer and pH.<h4>Conclusion</h4>Based on our findings, we suggest CPEGC triblock dendrimers possess great potential to be used as drug/gene delivery system.

<h4>Background</h4>MicroRNAs (miRNAs) are small, noncoding oligonucleotides with an important role in posttranscriptional regulation of gene expression at the level of translation and mRNA degradation. Recent studies have revealed that miRNAs play important roles in a variety of biological processes, such as cell proliferation, neuronal differentiation, developmental timing, synapse function, and neurogenesis. A single miRNA can target hundreds of mRNA transcripts for either translation repression or degradation, but the function of many human miRNAs is not known.<h4>Methods</h4>miRNA array analysis was performed on the prefrontal cortex of 27 individual human cases (14 alcoholics and 13 matched controls). Target genes for differentially expressed miRNAs were predicted using multiple target prediction algorithms and a consensus approach, and predicted targets were matched against differentially expressed mRNAs from the same samples. Over- and under-representation analysis was performed using hypergeometric probability and z-score tests.<h4>Results</h4>Approximately 35 miRNAs were significantly up-regulated in the alcoholic group compared with controls. Target prediction showed a large degree of overlap with our published cDNA microarray data. Functional classification of the predicted target genes of the regulated miRNAs includes apoptosis, cell cycle, cell adhesion, nervous system development, and cell-cell signaling.<h4>Conclusions</h4>These data suggest that the reduced expression of genes in human alcoholic cases may be because of the up-regulated miRNAs. Cellular processes fundamental to neuronal plasticity appear to represent major targets of the suggested miRNA regulation.

<h4>Background</h4>Proteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.<h4>Results</h4>The best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.<h4>Conclusions</h4>In spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.

Sensory afferent transmission and associated spinal reflexes are normally inhibited by serotonin (5-HT) derived from the brain stem. Spinal cord injury (SCI) that eliminates this 5-HT innervation leads to a disinhibition of sensory transmission and a consequent emergence of unusually long polysynaptic excitatory postsynaptic potentials (EPSPs) in motoneurons. These EPSPs play a critical role in triggering long polysynaptic reflexes (LPRs) that initiate muscles spasms. In the present study we examined which 5-HT receptors modulate the EPSPs and whether these receptors adapt to a loss of 5-HT after chronic spinal transection in rats. The EPSPs and associated LPRs recorded in vitro in spinal cords from chronic spinal rats were consistently inhibited by 5-HT(1B) or 5-HT(1F) receptor agonists, including zolmitriptan (5-HT(1B/1D/1F)) and LY344864 (5-HT(1F)), with a sigmoidal dose-response relation, from which we computed the 50% inhibition (EC(50)) and potency (-log EC(50)). The potencies of 5-HT receptor agonists were highly correlated with their binding affinity to 5-HT(1B) and 5-HT(1F) receptors, and not to other 5-HT receptors. Zolmitriptan also inhibited the LPRs and general muscle spasms recorded in vivo in the awake chronic spinal rat. The 5-HT(1B) receptor antagonists SB216641 and GR127935 and the inverse agonist SB224289 reduced the inhibition of LPRs by 5-HT(1B) agonists (zolmitriptan). However, when applied alone, SB224289, SB216641, and GR127935 had no effect on the LPRs, indicating that 5-HT(1B) receptors do not adapt to chronic injury, remaining silent, without constitutive activity. The reduction in EPSPs with zolmitriptan unmasked a large glycine-mediated inhibitory postsynaptic current (IPSC) after SCI. This IPSC and associated chloride current reversed at -73 mV, slightly below the resting membrane potential. Zolmitriptan did not change motoneuron properties. Our results demonstrate that 5-HT(1B/1F) agonists, such as zolmitriptan, can restore inhibition of sensory transmission after SCI without affecting general motoneuron function and thus may serve as a novel class of antispastic drugs.

We describe a case of Negativicoccus succinicivorans bacteremia in an adult man with hemochromatosis and acute pancreatitis. Conventional phenotypic tests and commercial identification systems failed to definitively identify the tiny anaerobic Gram-negative coccus isolated from two sets of blood cultures. The bacterium was identified by 16S rRNA gene sequencing and analysis using the SmartGene Integrated Database Network System software. This is the first published report of the recovery of this organism from a patient with invasive infection.

The yeast kinase Yck2 tethers to the cytoplasmic surface of the plasma membrane through dual palmitoylation of its C-terminal Cys-Cys dipeptide, mediated by the Golgi-localized palmitoyl-transferase Akr1. Here, the Yck2 palmitoylation signal is found to consist of three parts: 1) a 10-residue-long, conserved C-terminal peptide (CCTP) that includes the C-terminal Cys-Cys dipeptide; 2) the kinase catalytic domain (KD); and mapping between these two elements; and 3) a 176-residue-long, poorly conserved, glutamine-rich sequence. The CCTP, which contains the C-terminal cysteines as well as an important Phe-Phe dipeptide, likely serves as an Akr1 recognition element, because CCTP mutations disrupt palmitoylation within a purified in vitro palmitoylation system. The KD contribution appears to be complex with roles for both KD activity (e.g., Yck2-mediated phosphorylation) and structure (e.g., Akr1 recognition elements). KD and CCTP mutations are strongly synergistic, suggesting that, like the CCTP, the KD may also participate at the Yck2-Akr1 recognition step. The long, glutamine-rich domain, which is located between the KD and CCTP, is predicted to be intrinsically disordered and may function as a flexible, interdomain linker, allowing a coupled interaction of the KD and CCTP with Akr1. Multipart palmitoylation signals may prove to be a general feature of this large class of palmitoylation substrates. These soluble proteins have no clear means of accessing membranes and thus may require active capture out of the cytoplasm for palmitoylation by their membrane-localized transferases.

This review describes the properties of some rare eukaryotic chaperones that each assist in the folding of only one target protein. In particular, we describe (1) the tubulin cofactors, (2) p47, which assists in the folding of collagen, (3) α-hemoglobin stabilizing protein (AHSP), (4) the adenovirus L4-100 K protein, which is a chaperone of the major structural viral protein, hexon, and (5) HYPK, the huntingtin-interacting protein. These various-sized proteins (102-1,190 amino acids long) are all involved in the folding of oligomeric polypeptides but are otherwise functionally unique, as they each assist only one particular client. This raises a question regarding the biosynthetic cost of the high-level production of such chaperones. As the clients of faithful chaperones are all abundant proteins that are essential cellular or viral components, it is conceivable that this necessary metabolic expenditure withstood evolutionary pressure to minimize biosynthetic costs. Nevertheless, the complexity of the folding pathways in which these chaperones are involved results in error-prone processes. Several human disorders associated with these chaperones are discussed.

In the peripheral nervous system, Schwann cells (SCs) promote nerve regeneration by the secretion of trophic support molecules and the establishment of a supportive growth matrix. Elucidating factors that promote SC outgrowth following nerve injury is an important strategy for improving nerve regeneration. We identified the Netrin-1 receptors, Deleted in Colorectal Cancer (DCC) and Uncoordinated (Unc)5H2 as SC receptors that influence nerve regeneration by respectively promoting or inhibiting SC outgrowth. Significantly, we show both DCC and Unc5H2 receptors are distributed within SCs. In adult nerves, DCC is localized to the paranodes and Schmidt-Lantermann incisures of myelinating SCs, as well as along unmyelinated axons. After axotomy, DCC is prominently expressed in activated SCs at the regenerating nerve front. In contrast, Unc5H2 receptor is robustly distributed in myelinating SCs of the intact nerve and it is found at low levels in the SCs of the injury site. Local in vivo DCC siRNA mRNA knockdown at the growing tip of an injured nerve impaired SC activation and, in turn, significantly decreased axon regeneration. This forced DCC inhibition was associated with a dramatic reciprocal upregulation of Unc5H2 in the remaining SCs. Local Unc5H2 knockdown at the injury site, however, facilitated axon regrowth, indicating it has a role as an intrinsic brake to peripheral nerve regeneration. Our findings demonstrate that in adult peripheral nerves, SCs respond to DCC and Unc5H2 signaling, thereby promoting or hindering axon outgrowth and providing a novel mechanism for SC regulation during nerve regeneration.

The in vivo mechanisms that coordinate the timing of axon growth and guidance are not well understood. In the Caenorhabditis elegans hermaphrodite specific neurons (HSNs), the lin-4 microRNA controls the stage of axon initiation independent of the UNC-40 and SAX-3 ventral guidance receptors. lin-4 loss-of-function mutants exhibit marked delays in axon outgrowth, while lin-4 overexpression leads to precocious growth in the L3 larval stage. Here, we show that loss of the POU transcription factor UNC-86 not only results in penetrant ventral axon growth defects in in the HSNs, but also causes processes to extend in the L1, three stages earlier than wild-type. This temporal shift is not dependent on UNC-40 or SAX-3, and does not require the presence of lin-4. We propose that unc-86(lf) HSN axons are misguided due to the temporal decoupling of axon initiation and ventral guidance responses.

No abstract available.

HD (Huntington's disease) is characterized by dysfunction and death of striatal MSNs (medium-sized spiny neurons). Excitotoxicity, transcriptional dysregulation and mitochondrial abnormalities are among the mechanisms that are proposed to play roles in HD pathogenesis. To determine the extent of cell-autonomous effects of mhtt (mutant huntingtin) protein on vulnerability to excitotoxic insult in MSNs in vivo, we measured the number of degenerating neurons in response to intrastriatal injection of QA (quinolinic acid) in presymptomatic and symptomatic transgenic (D9-N171-98Q, also known as DE5) mice that express mhtt in MSNs but not in cortex. After QA, the number of degenerating neurons in presymptomatic DE5 mice was not significantly different from the number in WT (wild-type) controls, suggesting the early, increased vulnerability to excitotoxicity demonstrated in other HD mouse models has a largely non-cell-autonomous component. Conversely, symptomatic DE5 mice showed significantly fewer degenerating neurons relative to WT, implying the resistance to excitotoxicity observed at later ages has a primarily cell-autonomous origin. Interestingly, mitochondrial complex II respiration was enhanced in striatum of symptomatic mice, whereas it was reduced in presymptomatic mice, both relative to their age-matched controls. Consistent with the QA data, MSNs from symptomatic mice showed decreased NMDA (N-methyl-d-aspartate) currents compared with age-matched controls, suggesting that in addition to aging, cell-autonomous mechanisms mitigate susceptibility to excitotoxicity in the symptomatic stage. Also, symptomatic DE5 mice did not display some of the electrophysiological alterations present in other HD models, suggesting that blocking the expression of mhtt in cortical neurons may restore corticostriatal function in HD.

<h4>Background</h4>Colorectal cancer (CRC) is a major health concern worldwide, and recently becomes the most common cancer in Asia. The case collection of this study is one of the largest sets of CRC in Asia, and serves as representative data for investigating genomic differences between ethnic populations. We took comprehensive and high-resolution approaches to compare the clinicopathologic and genomic profiles of microsatellite instability (MSI) vs. microsatellite stability (MSS) in Taiwanese sporadic CRCs.<h4>Methods</h4>1,173 CRC tumors were collected from the Taiwan population, and sequencing-based microsatellite typing assay was used to determine MSI and MSS. Genome-wide SNP array was used to detect CN alterations in 16 MSI-H and 13 MSS CRCs and CN variations in 424 general controls. Gene expression array was used to evaluate the effects of CN alterations, and quantitative PCR methods were used to replicate the findings in independent clinical samples.<h4>Results</h4>These 1,173 CRC tumors can be classified into 75 high-frequency MSI (MSI-H) (6.4%), 96 low-frequency MSI (8.2%) and 1,002 MSS (85.4%). Of the 75 MSI-H tumors, 22 had a BRAF mutation and 51 showed MLH1 promoter hypermethylation. There were distinctive differences in the extent of CN alterations between CRC MSS and MSI-H subtypes (300 Mb vs. 42 Mb per genome, p-value < 0.001). Also, chr7, 8q, 13 and 20 gains, and 8p and 18 losses were frequently found in MSS but not in MSI-H. Nearly a quarter of CN alterations were smaller than 100 kb, which might have been missed in previous studies due to low-resolution technology. 514 expressed genes showed CN differences between subtypes, and 271 of them (52%) were differentially expressed.<h4>Conclusions</h4>Sporadic CRCs with MSI-H displayed distinguishable clinicopathologic features, which differ from those of MSS. Genomic profiling of the two types of sporadic CRCs revealed significant differences in the extent and distribution of CN alterations in the cancer genome. More than half of expressed genes showing CN differences can directly contribute to their expressional diversities, and the biological functions of the genes associated with CN changes in sporadic CRCs warrant further investigation to establish their possible clinical implications.

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzyme's ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(-)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3-activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

The aim of this study was to investigate the effect of berberine and evodiamine on serotonin transporter (5-HTT) expression and then test how allelic variations previously identified in the promoter region could modulate that effect in the serotonergic neuronal cell line RN46A. Both berberine and evodiamine, alone and in combination, increased 5-HTT mRNA and protein expression significantly across the various alleles. When tested against the S, XS(11), L(G), L(A), XL(17), and XL(18) alleles, respectively, 100 μM berberine increased 5-HTT promoter activities by 67%, 128.7%, 106.9%, 100.4%, 26.2% and 82%, 2 μM evodiamine increased 5-HTT promoter activities by 216.7%, 81.6%, 305.6%, 181.5%, 175.3% and 102.2%. Berberine and evodiamine increased 5-HTT promoter activity differently depending on the genetic variation of the 5-HTTLPR polymorphism. This study has provided a convincing example of how herbal compounds influence the expression of one of the most intensively studied psychiatric candidate genes, the serotonin transporter.

How are excitatory (glutamatergic) and inhibitory (GABAergic) synapses established? Do distinct molecular mechanisms direct differentiation of glutamatergic and GABAergic synapses? In the brain, glutamatergic and GABAergic synaptic connections are formed with specific patterns. To establish such precise synaptic patterns, neurons pass through multiple checkpoints during development, such as cell fate determination, cell migration and localization, axonal guidance and target recognition, and synapse formation. Each stage offers key molecules for neurons/synapses to obtain glutamatergic or GABAergic specificity. Some mechanisms are based on intrinsic systems to induce gene expression, whereas others are based on extrinsic systems mediated by cell-cell or axon-target interactions. Recent studies indicate that specific formation of glutamatergic and GABAergic synapses is controlled by the expression or activation of different sets of molecules during development. In this review, the authors outline stages critical to the determination of glutamatergic or GABAergic specificity and describe molecules that act as determinants of specificities in each stage, with a particular focus on the synapse formation stage. They also discuss possible mechanisms underlying glutamatergic and GABAergic synapse formation via synapse-type specific synaptic organizers.

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.

Mutations in distal S6 were shown to significantly alter the stability of the open state of Ca(V)2.3 (Raybaud, A., Baspinar, E. E., Dionne, F., Dodier, Y., Sauvé, R., and Parent, L. (2007) J. Biol. Chem. 282, 27944-27952). By analogy with K(V) channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in Ca(V)2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a -50 mV shift in the activation potential and a -20 mV shift in its charge-voltage relationship as compared with Ca(V)2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔG(interact) > 2 kcal mol(-1)) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔG(interact) ≈6 kcal mol(-1) suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated Ca(V) channel.

Tregs play a central role in modulating FcεRI-dependent MC effector functions in the course of the allergic response. Cellular interaction depends on the constitutive expression of OX40 on Tregs and the OX40L counterpart on MCs. Study of OX40L signaling on MCs is hampered by the need of a highly purified molecule, which triggers OX40L specifically. We now report that sOX40 mimics the physiological activity of Treg interaction by binding to activated MCs. When treated with sOX40, activated MCs showed decreased degranulation and Ca(++) influx, whereas PLC-γ2 phosphorylation remained unaffected. Once injected into experimental animals, sOX40 not only located within the endothelium but also in parenchyma, where it could be found in close proximity and apparently bound to MCs. This soluble molecule triggers MC-OX40L without the requirement of Tregs, thus allowing study of OX40L signaling pathways in MCs and in other OX40L-expressing cell populations. Importantly, as sOX40 inhibits MC degranulation, it may provide an in vivo therapeutic tool in allergic disease.

Pancreatic cancer is one of the leading causes of cancer-related deaths, for which serological biomarkers are urgently needed. Most discovery-phase studies focus on the use of one biological source for analysis. The present study details the combined mining of pancreatic cancer-related cell line conditioned media and pancreatic juice for identification of putative diagnostic leads. Using strong cation exchange chromatography, followed by LC-MS/MS on an LTQ-Orbitrap mass spectrometer, we extensively characterized the proteomes of conditioned media from six pancreatic cancer cell lines (BxPc3, MIA-PaCa2, PANC1, CAPAN1, CFPAC1, and SU.86.86), the normal human pancreatic ductal epithelial cell line HPDE, and two pools of six pancreatic juice samples from ductal adenocarcinoma patients. All samples were analyzed in triplicate. Between 1261 and 2171 proteins were identified with two or more peptides in each of the cell lines, and an average of 521 proteins were identified in the pancreatic juice pools. In total, 3479 nonredundant proteins were identified with high confidence, of which ∼ 40% were extracellular or cell membrane-bound based on Genome Ontology classifications. Three strategies were employed for identification of candidate biomarkers: (1) examination of differential protein expression between the cancer and normal cell lines using label-free protein quantification, (2) integrative analysis, focusing on the overlap of proteins among the multiple biological fluids, and (3) tissue specificity analysis through mining of publically available databases. Preliminary verification of anterior gradient homolog 2, syncollin, olfactomedin-4, polymeric immunoglobulin receptor, and collagen alpha-1(VI) chain in plasma samples from pancreatic cancer patients and healthy controls using ELISA, showed a significant increase (p < 0.01) of these proteins in plasma from pancreatic cancer patients. The combination of these five proteins showed an improved area under the receiver operating characteristic curve to CA19.9 alone. Further validation of these proteins is warranted, as is the investigation of the remaining group of candidates.

Natural products with interesting biological properties and structural diversity have often served as valuable lead drug candidates for the treatment of various human diseases. Largazole, isolated from the marine cyanobacterium Symploca sp. has exhibited potent inhibitory activity against many cancer cell lines. Besides, it shows remarkable selectivity between transformed and nontransformed cells, which is the main disadvantage of other antitumor natural products such as paclitaxel and actinomycin D. Due to its potential as a potent and selective anticancer drug candidate, a great deal of attention has been focused on largazole and its analogues. It is the aim of this review to highlight synthetic aspects of largazole and its analogues as well as their preliminary structure-activity relationship studies.

Understanding how individual cells behave inside living systems will help enable new diagnostic tools and cellular therapies. Superparamagnetic iron oxide particles can be used to label cells and theranostic capsules for noninvasive tracking using MRI. Contrast changes from superparamagnetic iron oxide are often subtle relative to intrinsic sources of contrast, presenting a detection challenge. Here, we describe a versatile postprocessing method, called Phase map cross-correlation Detection and Quantification (PDQ), that automatically identifies localized deposits of superparamagnetic iron oxide, estimating their volume magnetic susceptibility and magnetic moment. To demonstrate applicability, PDQ was used to detect and characterize superparamagnetic iron oxide-labeled magnetocapsules implanted in porcine liver and suspended in agarose gel. PDQ was also applied to mouse brains infiltrated by MPIO-labeled macrophages following traumatic brain injury; longitudinal, in vivo studies tracked individual MPIO clusters over 3 days, and tracked clusters were corroborated in ex vivo brain scans. Additionally, we applied PDQ to rat hearts infiltrated by MPIO-labeled macrophages in a transplant model of organ rejection. PDQ magnetic measurements were signal-to-noise ratio invariant for images with signal-to-noise ratio > 11. PDQ can be used with conventional gradient-echo pulse sequences, requiring no extra scan time. The method is useful for visualizing biodistribution of cells and theranostic magnetocapsules and for measuring their relative iron content.

The expression of small, non-coding RNA or microRNAs (miR), is frequently deregulated in human cancer, but how these pathways affect disease progression is still largely elusive. Here, we report on a miR, miR-296, which is progressively lost during tumor progression and correlates with metastatic disease in colorectal, breast, lung, gastric, parathyroid, liver and bile ducts cancers. Functionally, miR-296 controls a global cell motility gene signature in epithelial cells by transcriptionally repressing the cell polarity-cell plasticity module, Scribble (Scrib). In turn, loss of miR-296 causes aberrantly increased and mislocalized Scrib in human tumors, resulting in exaggerated random cell migration and tumor cell invasiveness. Re-expression of miR-296 in MDA-MB231 cells inhibits tumor growth in vivo. Finally, miR-296 or Scrib levels predict tumor relapse in hepatocellular carcinoma patients. These data identify miR-296 as a global repressor of tumorigenicity and uncover a previously unexplored exploitation of Scrib in tumor progression in humans.

Psoriasis is well known as a chronic inflammatory dermatosis. The disease affects persons of all ages and is a burden worldwide. Psoriasis is associated with various diseases such as arthritis. The disease is characterized by well-demarcated lesions on the skin of the elbows and knees. Various genetic and environmental factors are related to the pathogenesis of psoriasis. In order to identify enzymes that are potential therapeutic targets for psoriasis, we utilized a computational approach, combining microarray analysis and protein interaction prediction. We found 6,437 genes (3,264 upregulated and 3,173 downregulated) that have significant differences in expression between regions with and without lesions in psoriasis patients. We identified potential candidates through protein-protein interaction predictions made using various protein interaction resources. By analyzing the hub protein of the networks with metrics such as degree and centrality, we detected 32 potential therapeutic candidates. After filtering these candidates through the ENZYME nomenclature database, we selected 5 enzymes: DNA helicase (RUVBL2), proteasome endopeptidase complex (PSMA2), nonspecific protein-tyrosine kinase (ZAP70), I-kappa-B kinase (IKBKE), and receptor protein-tyrosine kinase (EGFR). We adopted a computational approach to detect potential therapeutic targets; this approach may become an effective strategy for the discovery of new drug targets for psoriasis.

The aim of this study was to investigate the possible influence of hemochromatosis gene mutations (HFE-C282Y and H63D) and transferrin gene C2 variant (TF-C2) on susceptibility to schizophrenia and schizoaffective disorder and/or age at first hospital admission. Genotyping was performed in 176 Croatian patients and 171 non-psychiatric Croatian controls using PCR-RFLP analyses. Regarding the H63D mutation, allele and genotype frequencies reached boundary statistical significance. Other allele and genotype distributions were not significantly different between two groups. We also analyzed age at first hospital admission as a continuous variable using the non-parametric Mann-Whitney U-test and Kruskal-Wallis test, and multiple regression analysis. The results of these tests were negative. We concluded that investigated HFE mutations and TF-C2 variant are not high-risk genetic variants for schizophrenia/schizoaffective disorder in our population. Also our data do not support their impact on age at onset of the first psychotic symptoms.

Mirror movements (MM) are involuntary movements of one side of the body that accompany and mirror intentional movements on the opposite side. Physiological MM can occur during normal childhood development, probably owing to corpus callosum immaturity. Pathological congenital MM may be clinically isolated or part of a complex congenital syndrome, including Kallmann syndrome, Klippel-Feil syndrome, and congenital hemiplegia. Congenital isolated MM are usually familial. Recently, heterozygous mutations of the DCC gene, with autosomal dominant inheritance, were shown to cause some cases of MM. The pathogenesis of congenital MM may involve (i) abnormal interhemispheric inhibition between the two motor cortices; (ii) functional alteration of motor planning and motor execution; and/or (iii) abnormal persistence of the ipsilateral corticospinal tract. Fundamental and clinical research is providing novel insights into the complex underlying molecular pathways, and recent experimental work has identified several mechanisms that may mediate the motor network dysfunction. In this review, we analyze clinical, genetic, neurophysiologic, and neuroimaging data on congenital MM, and discuss how this knowledge may improve our understanding of bimanual motor control.

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β-β associations found in radixin-adhesion molecule complexes. We also revealed direct interactions between the MyTH4-FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.

Huntington's disease (HD) is a progressive neurodegenerative disorder for which there is no effective disease modifying treatment. Following-on from studies in HD animal models, histone deacetylase (HDAC) inhibition has emerged as an attractive therapeutic option. In parallel, several reports have demonstrated a role for histone deacetylase 6 (HDAC6) in the modulation of the toxicity caused by the accumulation of misfolded proteins, including that of expanded polyglutamine in an N-terminal huntingtin fragment. An important role for HDAC6 in kinesin-1 dependent transport of brain-derived neurotrophic factor (BDNF) from the cortex to the striatum has also been demonstrated. To elucidate the role that HDAC6 plays in HD progression, we evaluated the effects of the genetic depletion of HDAC6 in the R6/2 mouse model of HD. Loss of HDAC6 resulted in a marked increase in tubulin acetylation throughout the brain. Despite this, there was no effect on the onset and progression of a wide range of behavioural, physiological, molecular and pathological HD-related phenotypes. We observed no change in the aggregate load or in the levels of soluble mutant exon 1 transprotein. HDAC6 genetic depletion did not affect the efficiency of BDNF transport from the cortex to the striatum. Therefore, we conclude that HDAC6 inhibition does not modify disease progression in R6/2 mice and HDAC6 should not be prioritized as a therapeutic target for HD.

The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC(50) of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.

β-N-Oxalyl-L-α,β-diaminopropionic acid (l-ODAP) an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist activates protein kinase C in white leghorn chick brain. The current study focuses on the protein kinase C downstream signaling targets associated with L-ODAP excitotoxicity in SK-N-MC human neuroblastoma cells and white leghorn male chick (Gallus domesticus) brain extracts. L-ODAP treatment in SK-N-MC cells (1.5 mM) and chicks (0.5 mg/g body weight) results in a decreased expression and increased phosphorylation of phosphatidylehthanolamine-binding protein 1 (PEBP1) up to 4 h which however, returns to normal by 8 h. D-ODAP, the non-toxic enantiomer however, did not affect PEBP1 levels in either chick brain or SK-N-MC cells. Decreased PEBP1 expression correlated with subsequent activation of Raf-1, MEK and ERK signaling components of the mitogen-activated protein kinase cascade and nuclear translocation of hypoxia inducible factor-1α (HIF-1α) in chick brain nuclear extracts and SK-N-MC cells. SK-N-MC cells over-expressing PEBP1 inhibited nuclear translocation of HIF-1α when treated with l-ODAP, indicating that down-regulation of PEBP1 is responsible for HIF-1α stabilization and nuclear localization. Excitotoxicity of L-ODAP may thus be the result of phosphorylation and down-regulation of PEBP1, a crucial signaling protein regulating diverse signaling cascades. L-ODAP induced convulsions and seizures in chicks could be the result of a hypoxic insult to brain.

Genome-wide association studies have identified several polymorphisms that appear to be on hypertension-susceptible regions. We performed the current replication study in order to evaluate the association of these loci with hypertension in healthy Japanese males and then examined the combined effect of 12 independent variants. Overall, 735 Japanese men from two independent cohorts were recruited. Association with hypertension was assessed in 16 polymorphisms on 12 genes and 12 were chosen to evaluate the combined impact. Polymorphisms on the COMT, ATP2B1, CYP11A1 and the CSK genes were confirmed to be associated with hypertension and blood pressure (BP). Current findings also replicated previous results for the CYP11B2 and PTGIS genes. Although there were no significant associations found for other variants, our results suggested there was a combined impact for 12 loci. Individuals carrying more risk alleles had a higher risk of hypertension (P for the slope=0.002). Blood pressures also increased in conjunction with an increasing risk allele score (P for trend=7.84 × 10(-6) and 1.85 × 10(-5) for SBP and DBP, respectively). Our results confirmed the associations between hypertension or blood pressure and four gene variants. We also found a significant combined effect of the 12 gene loci.

Tea (Camellia sinensis, Theaceae) has been shown to have obesity preventive effects in laboratory studies. We hypothesized that dietary epigallocatechin-3-gallate (EGCG) could reverse metabolic syndrome in high fat-fed obese C57bl/6J mice, and that these effects were related to inhibition of pancreatic lipase (PL). Following treatment with 0.32% EGCG for 6 weeks, a 44% decrease in body weight (BW) gain in high fat-fed, obese mice (P < 0.01) was observed compared to controls. EGCG treatment increased fecal lipid content by 29.4% (P < 0.05) compared to high fat-fed control, whereas in vitro, EGCG dose-dependently inhibited PL (IC(50) = 7.5 µmol/l) in a noncompetitive manner with respect to substrate concentration. (-)-Epicatechin-3-gallate exhibited similar inhibitory activity, whereas the nonester-containing (-)-epigallocatechin did not. In conclusion, EGCG supplementation reduced final BW and BW gain in obese mice, and some of these effects may be due to inhibition of PL by EGCG.

Staufens (Stau) are RNA-binding proteins involved in mRNA transport, localization, decay and translational control. The Staufen 1 (Stau1) isoform was recently identified as necessary for the protein synthesis-dependent late phase long-term potentiation (late-LTP) and for the maintenance of mature dendritic spines and synaptic activity in hippocampal CA1 pyramidal cells, strongly suggesting a role of mRNA regulation by Stau1 in these processes. However, the causal relationship between these impairments in synaptic function (spine shape and basal synaptic activity) and plasticity (late-LTP) remains unclear. Here, we determine that the effects of Stau1 knockdown on spine shape and size are mimicked by blocking NMDA receptors (or elevating extracellular Mg2+) and that Stau1 knockdown in the presence of NMDA receptor blockade (or high Mg2+) has no further effect on spine shape and size. Moreover, the effect of Stau1 knockdown on late-LTP cannot be explained by these effects, since when tested in normal medium, slice cultures that had been treated with high Mg2+ (to impair NMDA receptor function) in combination with a control siRNA still exhibited late-LTP, while siRNA to Stau1 was still effective in blocking late-LTP. Our results indicate that Stau1 involvement in spine morphogenesis is dependent on ongoing NMDA receptor-mediated plasticity, but its effects on late-LTP are independent of these changes. These findings clarify the role of Stau1-dependent mRNA regulation in physiological and morphological changes underlying long-term synaptic plasticity in pyramidal cells.

<h4>Background</h4>Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA.<h4>Results</h4>We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization.<h4>Conclusions</h4>The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes.

Huntington disease (HD) is caused by polyglutamine expansion in the huntingtin (HTT) protein. Huntingtin-interacting protein 14 (HIP14), one of 23 DHHC domain-containing palmitoyl acyl transferases (PATs), binds to HTT and robustly palmitoylates HTT at cysteine 214. Mutant HTT exhibits reduced palmitoylation and interaction with HIP14, contributing to the neuronal dysfunction associated with HD. In this study, we confirmed that, among 23 DHHC PATs, HIP14 and its homolog DHHC-13 (HIP14L) are the two major PATs that palmitoylate HTT. Wild-type HTT, in addition to serving as a palmitoylation substrate, also modulates the palmitoylation of HIP14 itself. In vivo, HIP14 palmitoylation is decreased in the brains of mice lacking one HTT allele (hdh+/-) and is further reduced in mouse cortical neurons treated with HTT antisense oligos (HTT-ASO) that knockdown HTT expression by ∼95%. Previously, it has been shown that palmitoylation of DHHC proteins may affect their enzymatic activity. Indeed, palmitoylation of SNAP25 by HIP14 is potentiated in vitro in the presence of wild-type HTT. This influence of HTT on HIP14 activity is lost in the presence of CAG expansion. Furthermore, in both brains of hdh+/- mice and neurons treated with HTT-ASO, we observe a significant reduction in palmitoylation of endogenous SNAP25 and GluR1, synaptic proteins that are substrates of HIP14, suggesting wild-type HTT also influences HIP14 enzymatic activity in vivo. This study describes an important biochemical function for wild-type HTT modulation of HIP14 palmitoylation and its enzymatic activity.

Here we report the first example of using β-galactosidase to trigger the formation of cell compatible, supramolecular nanofibers, which ultimately may lead to a new approach for the development of soft nanotechnology.

Cancer development involves multiple genetic changes, which can occur in tumor suppressor genes and lead to loss of function in a recessive manner. Recent findings have identified a novel tumor suppressor gene named GRIM-19. Similar to what has been observed for other known tumor suppressor proteins such as p53, GRIM-19 gene mutations and loss of protein expression have been observed in several tumor types. In this review, we perform a detailed description on the current understanding of GRIM-19 function in carcinogenesis.

<h4>Background</h4>Three-dimensional regenerative tissue with large bulk generally requires blood perfusion through a vascular network to maintain its viability, and one promising approach is induction of neovascular growth from the recipient bed into the tissue. To induce ingrowth of a vascular network, it is necessary to furnish the regenerative tissue with a scaffold structure for neovasculature and a delivery system for an angiogenic growth factor. As such a scaffold structure, the present study created novel hydrogel materials by chemically cross-linking alkali-treated collagen (AlCol) with trisuccinimidyl citrate (TSC).<h4>Materials and methods</h4>Many prototypes, consisting of several concentrations of TSC and AlCol, were implanted into the subfascial space of the rat rectus muscle, and 7 days later, the implanted materials were excised for histological analysis. Cross-sections were stained and neovascular development in the materials was evaluated by measuring vessel density, length and number of joints and branches.<h4>Results</h4>Significant ingrowth of vascularized granulation was observed in some materials, which surpassed the angiogenic ability of Matrigel(TM). Further, combination with basic fibroblast growth factor (bFGF) significantly increased the vascular formation in these gels.<h4>Conclusions</h4>The TSC-AlCol gel functioned as a favorable scaffold for neovascular formation and also as a reservoir for controlled delivery of bFGF.

The complex patterns of neuronal wiring in the adult nervous system depend on a series of guidance events during neural development that establish a framework on which functional circuits can be built. In this subject collection, the cellular and molecular mechanisms that underlie neuronal guidance are considered from several perspectives, ranging from how cytoskeletal dynamics within extending neuronal growth cones steer axons, to how guidance cues influence synaptogenesis. We introduce here some basic topics to frame the more detailed reviews in following articles, including the cellular strategies that define basic themes governing neuronal wiring throughout life, an enumeration of the molecular cues and receptors known to play key guidance roles during neural development, and an overview of the signaling mechanisms that transduce guidance information into growth-cone steering.

The zebrafish, Danio rerio, has been established as an excellent vertebrate model for the study of developmental biology and gene function. It also has proven to be a valuable model to study human diseases. Here, we reviewed recent publications using zebrafish to study the pathology of human neurodegenerative diseases including Parkinson's, Huntington's, and Alzheimer's. These studies indicate that zebrafish genes and their human homologues have conserved functions with respect to the etiology of neurodegenerative diseases. The characteristics of the zebrafish and the experimental approaches to which it is amenable make this species a useful complement to other animal models for the study of pathologic mechanisms of neurodegenerative diseases and for the screening of compounds with therapeutic potential.

Postmenopausal osteoporosis is characterized by an imbalance of bone resorption exceeding bone formation, resulting in a net loss of bone mineral density (BMD). Estrogen deficiency is known to promote bone resorption. However, the causative factors that impair bone formation have not been identified. Women after menopause experience not only estrogen deficiency but also iron accumulation as a result of cessation of menstruation. In this study we investigated whether increased iron plays a role in osteoporosis. By growing primary mouse osteoclast and osteoblast progenitor cells as well as immortalized cell lines in the presence of iron, we found that increased iron had minimal effects on osteoclast cell differentiation. Interestingly, iron, particularly in its inorganic form, and to a lesser extent ferritin and transferrin all suppressed alkaline phosphatase (ALP) activities in osteoblasts. Moreover, iron downregulated mRNA levels of several other osteoblastogenic markers such as Runx2, osterix, osteopontin, and osteocalcin. To further show that this in vitro finding is relevant to the in vivo condition, we demonstrated that iron-accumulated mice with intact ovaries exhibited a significant decrease in BMD. Although iron inhibited preosteoblast cell differentiation, it did enhance preosteoblast cell proliferation, as evidenced by increased cell growth and expression of cell cycle regulator genes such as CDK4, CDK6, cyclin D1, and cyclin D3 and G(2) /M phase cell population. Taken together, our results suggest that increased iron could be a factor that slows down bone formation in postmenopausal women.

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.

<h4>Purpose</h4>GPR91, a succinate receptor, is expressed in retinal ganglion cells and induces vascular endothelial growth factor (VEGF) expression. RPE also expresses VEGF, but whether this cell expresses GPR91 is not known. Excessive iron is also proangiogenic, and hemochromatosis is associated with iron overload. Therefore, we examined the expression and iron-dependent regulation of GPR91 in the RPE.<h4>Methods</h4>GPR91 expression was examined by RT-PCR and immunohistochemistry. Hemochromatosis mice, cytomegalovirus (CMV) infection of retina, expression of CMV-US2 in RPE, and exposure of RPE to ferric ammonium citrate (FAC) were used to examine the iron-dependent regulation of GPR91 expression. VEGF expression was quantified by qPCR. Knockdown of GPR91 in ARPE-19 cells was achieved with shRNA.<h4>Results</h4>GPR91 was expressed in RPE but only in the apical membrane. Retinal expression of GPR91 was higher in hemochromatosis (Hfe(-/-)) mice than in wild-type (WT) mice. Primary RPE cells from Hfe(-/-) mice had increased GPR91 expression compared with WT RPE cells. Iron accumulation in cells induced by CMV infection, expression of CMV-US2, or treatment with FAC increased GPR91 expression. VEGF expression in the Hfe(-/-) mouse retina was increased at ages younger than 18 months, but the expression was downregulated at older ages. The involvement of GPR91 in succinate-induced expression of VEGF in RPE cells was confirmed with GPR91-specific shRNA.<h4>Conclusions</h4>GPR91 is expressed in the RPE with specific localization to the apical membrane, indicating that succinate in the subretinal space serves as the GPR91 agonist. Excessive iron in the retina and RPE enhances GPR91 expression; however, VEGF expression does not always parallel GPR91 expression.

Hepatocellular carcinoma (HCC) is an aggressive malignancy of the liver and occurs most often in the setting of chronic liver disease. The most common acquired causes for this are chronic viral hepatitis infections (mostly HBV and HCV), and alcohol. Other causes include nonalcoholic fatty liver disease-related nonalcoholic steatohepatitis, autoimmune liver disease, and biliary diseases. In addition, certain heritable diseases like hemochromatosis and α-1-antitrypsin deficiency can also lead to HCC. Therefore, prevention of HCC can be achieved by preventing and controlling these problems. For treatment, curative modalities are surgical resection and liver transplantation. However, most patients are not candidates for these surgical maneuvers, and outcomes are poor. New therapeutic developments have brought some improvement with both local and systemic disease control.

A 1-yr-old Japanese male infant developed hepatitis-associated aplastic anemia (AA), and anti-thymocyte globulin (ATG) plus cyclosporine A (CsA) was administered without any appreciable effects. Laboratory examination of the patient's serum obtained before therapy revealed various autoantibodies, such as PA-IgG, anti-platelets, anti-single-stranded DNA (ssDNA), and anti-double-stranded DNA (dsDNA) antibodies (Abs) in addition to anti-DRS-1 Abs and anti-moesin Abs, both of which are known to be detectable in approximately 40% of all patients presenting with AA. He was therefore treated with 17.5 mg/kg/d rituximab 5.5 months after ATG/CsA therapy. The same rituximab therapy was repeated three times once a month thereafter. His neutrophil counts started to increase 50 d after the first rituximab therapy and he achieved a complete remission at 16 months after the last rituximab administration. All of the autoantibodies including anti-ssDNA, dsDNA, DRS-1, and moesin became undetectable when he attained the remission. Anti-CD20 monoclonal antibody therapy may be effective in a subset of patients with AA characterized by the presence of autoantibodies.

Huntington's disease (HD) is the most common inherited neurodegenerative disease and is characterized by uncontrolled excessive motor movements and cognitive and emotional deficits. The mutation responsible for HD leads to an abnormally long polyglutamine (polyQ) expansion in the huntingtin (Htt) protein, which confers one or more toxic functions to mutant Htt leading to neurodegeneration. The polyQ expansion makes Htt prone to aggregate and accumulate, and manipulations that mitigate protein misfolding or facilitate the clearance of misfolded proteins tend to slow disease progression in HD models. This article will focus on HD and the evidence that it is a conformational disease.

Mitochondria are cytoplasmic organelles responsible for life and death. Extensive evidence from animal models, postmortem brain studies of and clinical studies of aging and neurodegenerative diseases suggests that mitochondrial function is defective in aging and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Several lines of research suggest that mitochondrial abnormalities, including defects in oxidative phosphorylation, increased accumulation of mitochondrial DNA defects, impaired calcium influx, accumulation of mutant proteins in mitochondria, and mitochondrial membrane potential dissipation are important cellular changes in both early and late-onset neurodegenerative diseases. Further, emerging evidence suggests that structural changes in mitochondria, including increased mitochondrial fragmentation and decreased mitochondrial fusion, are critical factors associated with mitochondrial dysfunction and cell death in aging and neurodegenerative diseases. This paper discusses research that elucidates features of mitochondria that are associated with cellular dysfunction in aging and neurodegenerative diseases and discusses mitochondrial structural and functional changes, and abnormal mitochondrial dynamics in neurodegenerative diseases. It also outlines mitochondria-targeted therapeutics in neurodegenerative diseases.

Receptor-recognized forms of α₂ -macroglobulin (α₂ M) bind to cell surface-associated GRP78 and initiate pro-proliferative and anti-apoptotic signaling. Ligation of GRP78 with α₂ M also upregulates TFII-I, which binds to the GRP78 promoter and enhances GRP78 synthesis. In addition to its transcriptional functions, cytosolic TFII-I regulates agonist-induced Ca(2+) entry. In this study we show that down regulation of TFII-I gene expression by RNAi profoundly impairs its cell surface expression and anti-apoptotic signaling as measured by significant reduction of GRP78, Bcl-2, and cyclin D1 in 1-Ln and DU-145 human prostate cancer cells stimulated with α₂ M. In contrast, this treatment significantly increases levels of the pro-apoptotic proteins p53, p27, Bax, and Bak and causes DNA fragmentation. Furthermore, down regulation of TFII-I expression activates agonist-induced Ca(2+) entry. In plasma membrane lysates p-PLCγ1, TRPC3, GRP78, MTJ1, and caveolin co-immunoprecipitate with TFII-I suggesting multimeric complexes of these proteins. Consistent with this hypothesis, down regulating TFII-I, MTJ1, or GRP78 expression by RNAi greatly attenuates cell surface expression of TFII-I. In conclusion, we demonstrate that not only does cell surface GRP78 regulate apoptosis, but it also regulates Ca(2+) homeostasis by controlling cell surface localization of TFII-I.

Imaging studies have shown that serum concentrations of the selective serotonin reuptake inhibitor citalopram correlate with serotonin transporter (5-HTT) occupancy in vivo. In patients with major depressive disorders treated with citalopram, 80% 5-HTT occupancy was considered to be necessary for maximal therapeutic effects, which requires citalopram serum concentrations of at least 50 ng/mL. The aim of this study was to compare treatment outcome in patients with citalopram serum concentrations greater than and less than 50 ng/mL after 7 days of treatment. This study included inpatients with acute major depressive disorder according to International Classification of Disease, 10th Revision who were treated with citalopram. In weekly intervals, the severity of depression was assessed with the 17-item Hamilton Depression Rating Scale (HAMD-17), and serum concentrations of citalopram were measured from baseline until week 5. Fifty-five patients were eligible for this analysis. After 7 days of treatment, 19 patients showed citalopram serum concentrations of 50 ng/mL or greater; 36 patients had lower concentrations. Patients at greater than the 50-ng/mL threshold had (i) lower mean HAMD-17 sum scores from day 7 to end point (P ≤ 0.018 for each analysis); (ii) a more pronounced HAMD-17 decrease (P ≤ 0.019 for each analysis), and (iii) 23 days' shorter duration of hospitalization (P = 0.033) than patients with levels of citalopram less than 50 ng/mL. As regards adverse effects, both patient groups were not significantly different. Despite therapeutic doses, a significant number of patients had serum concentrations less than 50 ng/mL, and these were associated with an unfavorable treatment outcome; therapeutic drug monitoring is recommended to optimize dosing citalopram in the early phase of treatment.

Netrins are secreted proteins that were first identified as guidance cues, directing cell and axon migration during neural development. Subsequent findings have demonstrated that netrins can influence the formation of multiple tissues, including the vasculature, lung, pancreas, muscle and mammary gland, by mediating cell migration, cell-cell interactions and cell-extracellular matrix adhesion. Recent evidence also implicates the ongoing expression of netrins and netrin receptors in the maintenance of cell-cell organisation in mature tissues. Here, we review the mechanisms involved in netrin signalling in vertebrate and invertebrate systems and discuss the functions of netrin signalling during the development of neural and non-neural tissues.

High doses of T3 are mitogenic in liver, causing hyperplasia that has numerous differences from the compensatory regeneration induced by partial hepatectomy (PH). T3 binds to the thyroid hormone receptor (TR), which directly regulates transcription, while PH acts indirectly through signal transduction pathways. We therefore carried out a proteomic analysis to compare early effects of the two treatments. Transcriptome analysis by DNA microarray also confirmed the observed proteomic changes, demonstrating that they were caused by transcriptional regulation. Among the differentially expressed proteins, many are directly or indirectly involved in energy metabolism and response to oxidative stress. Several enzymes of lipid metabolism (e.g., Acaa2, Acads, Hadh, and Echs1) were differentially regulated by T3. In addition, altered expression levels of several mitochondrial proteins (e.g., Hspa9, Atp5b, Cps1, Glud1, Aldh2, Ak2, Acads) demonstrated the known increase of mitochondrial biogenesis mediated by T3. The present results provide insights in changes in metabolic balance occurring following T3-stimulation and define a basis for dissecting the molecular pathways of hepatocyte hyperplasia.

In parallel with our growing understanding of the molecular pathways underlying colorectal neoplasia, significant advances have been made in the treatment of colorectal cancer (CRC). For the past few decades, 5-fluorouracil-based therapy has been the cornerstone of adjuvant therapy. More recently, additional cytotoxic drugs and molecular-targeted therapies have provided additional clinical benefit in certain patient populations. Unfortunately, overall survival remains about 45%. Notably, our understanding of why certain patients do or do not respond to treatment remains limited. Thus, as therapeutic options for CRC continue to expand, there is now an even greater imperative to identify reliable biomarkers that have the potential to predict prognosis as well as response to chemotherapy. In this review, we will summarize the current status of such molecular prognostic and predictive biomarkers in CRC and assess their usefulness in tailoring therapeutic options.

<h4>Background and aims</h4>To determine genome-wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori-induced gastric carcinogenesis.<h4>Methods</h4>Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer-related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation-specific PCR method for four genes (MOS, DCC, CRK, and PTPN6).<h4>Results</h4> The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation-specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01).<h4>Conclusions</h4> Genome-wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori-induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation-specific PCR method.

<h4>Objective</h4>The purpose of this study was to determine the ex vivo ability of dual-energy dual-source CT (DSCT) with additional tin filtration to differentiate among five groups of human renal stone types.<h4>Materials and methods</h4>Forty-three renal stones of 10 types were categorized into five primary groups on the basis of effective atomic numbers, which were calculated as the weighted average of the atomic numbers of constituent atoms. Stones were embedded in porcine kidneys and placed in a 35-cm water phantom. Dual-energy DSCT scans were performed at 80 and 140 kV with and without tin filtration of the 140-kV beam. The CT number ratio, defined as the ratio of the CT number of a given material in the low-energy image to the CT number of the same material in the high-energy image, was calculated on a volumetric voxel-by-voxel basis for each stone. Statistical analysis was performed, and receiver operating characteristic (ROC) curves were plotted to compare the difference in CT number ratio with and without tin filtration, and to measure the discrimination among stone groups.<h4>Results</h4>The CT number ratio of non-uric acid stones increased on average by 0.17 (range, 0.03-0.36) with tin filtration. The CT number ratios for non-uric acid stone groups were not significantly different (p > 0.05) between any of the two adjacent groups without tin filtration. Use of the additional tin filtration on the high-energy x-ray tube significantly improved the separation of non-uric acid stone types by CT number ratio (p < 0.05). The area under the ROC curve increased from 0.78 to 0.84 without fin filtration and to 0.89-0.95 with tin filtration.<h4>Conclusion</h4>Our results showed better separation among different stone types when additional tin filtration was used on dual-energy DSCT. The increased spectral separation allowed a five-group stone classification scheme. Some overlapping between particular stone types still exists, including brushite and calcium oxalate.

Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(-) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(-) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(-) cell lines. Differentially methylated loci between HPV(+) and HPV(-) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(-) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(-) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(-) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.

This article reports the effects of heating temperature and composition of nanoparticle multilayer films on the morphology, stability, and optical property of gold nanoisland films prepared by nanoparticle self-assembly/heating method. First, nanoparticle-polymer multilayer films are prepared by the layer-by-layer assembly. Nanoparticle multilayer films are then heated at temperature ranging from 500 °C to 625 °C in air to induce an evaporation of organic matters from the films. During the heating process, the nanoparticles on the solid surface undergo coalescence, resulting in the formation of nanostructured gold island arrays. Characterization of nanoisland films using atomic force microscopy and UV-vis spectroscopy suggests that the morphology and stability of gold island films change when different heating temperatures are applied. Stable gold nanoisland thin film arrays can only be obtained after heat treatments at or above 575 °C. In addition, the results show that the use of nanoparticles with different sizes produces nanoisland films with different morphologies. Multilayer films containing smaller gold nanoparticles tend to produce more monodisperse and smaller island nanostructures. Other variables such as capping ligands around nanoparticles and molecular weight of polymer linkers are found to have only minimal effects on the structure of island films. The adsorption of streptavidin on the biotin-functionalized nanoisland films is studied for examining the biosensing capability of nanoisland arrays.

<h4>Background</h4>High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis.<h4>Methods</h4>Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls.<h4>Results</h4>One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects.<h4>Conclusions</h4>HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.

To identify a group of transcription factors required for chondrogenesis, several researchers tried to detect a chondrocyte-specific enhancer element of Col2a1 gene. Benoit de Crombrugghe's group finally found out 48bp in the first intron of Col2a1 gene as a chondrocyte-specific enhancer element, and moreover they also concluded that binding of homodimer of Sox9 and homo-or heterodimer of Sox5÷Sox6 to this element is indispensable for Col2a1 transcription in chodrocytes. Furthermore, mouse genetic approaches revealed that Sox9, Sox5 and Sox6 are required for chondrogenesis, leading to conclusion that these Sox transcription factors are master regulators in chondrogenesis. Recent studies showed that p300÷CBP, Trap230 (med12) , Wwp2, and Med25 are components of transcriptional machinery of Sox9 in chondrogenesis.

<h4>Background</h4>Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity.<h4>Results</h4>In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy.<h4>Conclusions</h4>These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER(T2)-R26R(YFP) mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.

Striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed in striatal projection neurons, the neuronal population most affected in Huntington's disease. Here, we examined STEP expression and phosphorylation, which regulates its activity, in N-terminal exon-1 and full-length mutant huntingtin mouse models. R6/1 mice displayed reduced STEP protein levels in the striatum and cortex, whereas its phosphorylation was increased in the striatum, cortex, and hippocampus. The early increase in striatal STEP phosphorylation levels correlated with a deregulation of the protein kinase A pathway, and decreased calcineurin activity at later stages further contributes to an enhancement of STEP phosphorylation and inactivation. Accordingly, we detected an accumulation of phosphorylated ERK2 and p38, two targets of STEP, in R6/1 mice striatum at advanced stages of the disease. Activation of STEP participates in excitotoxic-induced cell death. Because Huntington's disease mouse models develop resistance to excitotoxicity, we analyzed whether decreased STEP activity was involved in this process. After intrastriatal quinolinic acid (QUIN) injection, we detected higher phosphorylated STEP levels in R6/1 than in wild-type mice, suggesting that STEP inactivation could mediate neuroprotection in R6/1 striatum. In agreement, intrastriatal injection of TAT-STEP increased QUIN-induced cell death. R6/2, Tet/HD94, and Hdh(Q7/Q111) mice striatum also displayed decreased STEP protein and increased phosphorylation levels. In Tet/HD94 mice striatum, mutant huntingtin transgene shutdown reestablished STEP expression. In conclusion, the STEP pathway is severely downregulated in the presence of mutant huntingtin and may participate in compensatory mechanisms activated by striatal neurons that lead to resistance to excitotoxicity.

In response to physiological stimuli, skeletal muscle alters its myofiber composition to significantly affect muscle performance and metabolism. This process requires concerted regulation of myofiber-specific isoforms of sarcomeric and calcium regulatory proteins that couple action potentials to the generation of contractile force. Here, we identify Sox6 as a fast myofiber-enriched repressor of slow muscle gene expression in vivo. Mice lacking Sox6 specifically in skeletal muscle have an increased number of slow myofibers, elevated mitochondrial activity, and exhibit down-regulation of the fast myofiber gene program, resulting in enhanced muscular endurance. In addition, microarray profiling of Sox6 knockout muscle revealed extensive muscle fiber-type remodeling, and identified numerous genes that display distinctive fiber-type enrichment. Sox6 directly represses the transcription of slow myofiber-enriched genes by binding to conserved cis-regulatory elements. These results identify Sox6 as a robust regulator of muscle contractile phenotype and metabolism, and elucidate a mechanism by which functionally related muscle fiber-type specific gene isoforms are collectively controlled.

Saturated free fatty acids (FFA) induce hepatocyte lipoapoptosis, a key mediator of liver injury in nonalcoholic fatty liver disease (NAFLD). Lipoapoptosis involves the upregulation of the BH3-only protein PUMA, a potent pro-apoptotic protein. Given that dysregulation of hepatic microRNA expression has been observed in NAFLD, we examined the role of miRNA in regulating PUMA expression during lipotoxicity. By in silico analysis, we identified two putative binding sites for miR-296-5p within the 3' untranslated region (UTR) of PUMA mRNA. Enforced miR-296-5p levels efficiently reduced PUMA protein expression in Huh-7 cells, while antagonism of miR-296-5p function increased PUMA cellular levels. Reporter gene assays identified PUMA 3'UTR as a direct target of miR-296-5p. The saturated FFA, palmitate, repressed miR-296-5p expression; and Huh-7 cells were sensitized to palmitate-induced lipotoxicity by antagonism of miR-296-5p function using a targeted locked nucleic acid (LNA). Finally, miR-296-5p was reduced in liver samples from nonalcoholic steatohepatitis (NASH) patients compared with patients with simple steatosis (SS) or controls. Also miR-296-5p levels inversely varied with PUMA mRNA levels in human liver specimens. Our results implicate miR-296-5p in the regulation of PUMA expression during hepatic lipoapoptosis. We speculate that enhancement of miR-296-5p expression may represent a novel approach to minimize apoptotic damage in human fatty liver diseases.

Chromosome structure is dynamically regulated during cell division, and this regulation is dependent, in part, on condensin. The localization of condensin at chromosome arms is crucial for chromosome partitioning during anaphase. Condensin is also enriched at kinetochores but its precise role and loading machinery remain unclear. Here we show that fission yeast (Schizosaccharomyces pombe) kinetochore proteins Pcs1 and Mde4--homologues of budding yeast (Saccharomyces cerevisiae) monopolin subunits and known to prevent merotelic kinetochore orientation--act as a condensin 'recruiter' at kinetochores, and that condensin itself may act to clamp microtubule binding sites during metaphase. In addition to the regional recruitment factors, overall condensin association with chromatin is governed by the chromosomal passenger kinase Aurora B. Aurora-B-dependent phosphorylation of condensin promotes its association with histone H2A and H2A.Z, which we identify as conserved chromatin 'receptors' of condensin. Condensin phosphorylation and its deposition onto chromosome arms reach a peak during anaphase, when Aurora B kinase relocates from centromeres to the spindle midzone, where the separating chromosome arms are positioned. Our results elucidate the molecular basis for the spatiotemporal regulation of mitotic chromosome architecture, which is crucial for chromosome partitioning.

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted at two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders.

We study the segregation of two long chains from parallel but randomly twisted start conformations under good solvent conditions using Monte Carlo simulations to mimic chromatin segregation after replication in eukaryotic cells in the end of prophase. To measure the segregation process, we consider the center-of-mass separation between the two chains and the average square distance between the monomers which were connected before segregation starts. We argue that segregation is dominated by free diffusion of the chains, assuming that untwisting can be achieved by Rouse-like fluctuations on the length scale of a twisted loop. Using scaling analysis, we find that chain dynamics is in very good agreement with the free diffusion hypothesis, and segregation dynamics follows this scaling nearly. Long chains, however, show retardation effects that can be described by a new (to us) dynamical exponent, which is slightly larger than the dynamical exponent for Rouse-like diffusion. Our results indicate that nearly free diffusion of chains during a timescale of a few Rouse-times can lead to segregation of chains. A main obstacle during segregation by free diffusion is random twists between daughter strands. We have calculated the number of twists formed by the daughter strands in the start conformations, which turns out to be rather low and increases only with the square-root of the chain length.

Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones.

Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal serrated polyps and is associated with an increased colorectal cancer (CRC) risk. The mixture of distinct precursor lesion types and malignancies in HPS provides a unique model to study the canonical pathway and a proposed serrated CRC pathway in humans. To establish which CRC pathways play a role in HPS and to obtain new support for the serrated CRC pathway, we assessed the molecular characteristics of polyps (n = 84) and CRCs (n = 19) in 17 patients with HPS versus control groups of various sporadic polyps (n = 59) and sporadic microsatellite-stable CRCs (n = 16). In HPS and sporadic polyps, APC mutations were exclusively identified in adenomas, whereas BRAF mutations were confined to serrated polyps. Six of 19 HPS CRCs (32%) were identified in a serrated polyp. Mutation analysis performed in the CRC and the serrated component of these lesions showed identical BRAF mutations. One HPS CRC was located in an adenoma, both components harboring an identical APC mutation. Overall, 10 of 19 HPS CRCs (53%) carried a BRAF mutation versus none in control group CRCs (P = 0.001). Six BRAF-mutated HPS CRCs (60%) were microsatellite unstable owing to MLH1 methylation. These findings provide novel supporting evidence for the existence of a predominant serrated CRC pathway in HPS, generating microsatellite-stable and microsatellite-instable CRCs.

Autophagy is a regulated process that can be involved in the elimination of intracellular microorganisms and in antigen presentation. Some in vitro studies have shown an altered autophagic response in hepatitis C virus infected hepatocytes. The present study aimed at evaluating the autophagic process in the liver of chronic hepatitis C (CHC) patients. Fifty-six CHC patients and 47 control patients (8 with nonalcoholic steatohepatitis or alcoholic liver disease, 18 with chronic heptatitis B virus infection, and 21 with no or mild liver abnormalities at histological examination) were included. Autophagy was assessed by means of electron microscopy and microtubule-associated protein light chain 3 immunoblotting. Using light chain 3 immunoblotting, the form present on autophagic vesicle (light chain 3-II) was significantly higher in CHC patients than in controls (P < 0.05). Using quantitative electron microscopy analysis, the median number of autophagic vesicles observed in hepatocytes from CHC patients was sixfold higher than in overall controls (P < 0.001). In contrast, there was no difference between CHC patients and controls in the number of mature lysosomes with electron-dense contents arguing in favor of a lack of fusion between autophagosome and lysosome. Neither genotype nor viral load influenced the autophagy level. In conclusion, autophagy is altered in hepatocytes from CHC patients, likely due to a blockade of the last step of the autophagic process.

Netrins are guidance cues involved in neural connectivity. We have shown that the netrin-1 receptor DCC (deleted in colorectal cancer) is involved in the functional organization of the mesocorticolimbic dopamine (DA) system. Adult mice with a heterozygous loss-of-function mutation in dcc exhibit changes in indexes of DA function, including DA-related behaviors. These phenotypes are only observed after puberty, a critical period in the maturation of the mesocortical DA projection. Here, we examined whether dcc heterozygous mice exhibit structural changes in medial prefrontal cortex (mPFC) DA synaptic connectivity, before and after puberty. Stereological counts of tyrosine-hydroxylase (TH)-positive varicosities were increased in the cingulate 1 and prelimbic regions of the pregenual mPFC. dcc heterozygous mice also exhibited alterations in the size, complexity, and dendritic spine density of mPFC layer V pyramidal neuron basilar dendritic arbors. Remarkably, these presynaptic and postsynaptic partner phenotypes were not observed in juvenile mice, suggesting that DCC selectively influences the extensive branching and synaptic differentiation that occurs in the maturing mPFC DA circuit at puberty. Immunolabeling experiments in wild-type mice demonstrated that DCC is segregated to TH-positive fibers innervating the nucleus accumbens, with only scarce DCC labeling in mPFC TH-positive fibers. Netrin had an inverted target expression pattern. Thus, DCC-mediated netrin-1 signaling may influence the formation/maintenance of mesocorticolimbic DA topography. In support of this, we report that dcc heterozygous mice exhibit a twofold increase in the density of mPFC DCC/TH-positive varicosities. Our results implicate DCC-mediated netrin-1 signaling in the establishment of mPFC DA circuitry during puberty.

Identification of complex molecular networks underlying common human phenotypes is a major challenge of modern genetics. In this study, we develop a method for network-based analysis of genetic associations (NETBAG). We use NETBAG to identify a large biological network of genes affected by rare de novo CNVs in autism. The genes forming the network are primarily related to synapse development, axon targeting, and neuron motility. The identified network is strongly related to genes previously implicated in autism and intellectual disability phenotypes. Our results are also consistent with the hypothesis that significantly stronger functional perturbations are required to trigger the autistic phenotype in females compared to males. Overall, the presented analysis of de novo variants supports the hypothesis that perturbed synaptogenesis is at the heart of autism. More generally, our study provides proof of the principle that networks underlying complex human phenotypes can be identified by a network-based functional analysis of rare genetic variants.

Lhx6 and Lhx8 transcription factor coexpression in early-born MGE neurons is required to induce neuronal Shh expression. We provide evidence that these transcription factors regulate expression of a Shh enhancer in MGE neurons. Lhx6 and Lhx8 are also required to prevent Nkx2-1 expression in a subset of pallial interneurons. Shh function in early-born MGE neurons was determined by genetically eliminating Shh expression in the MGE mantle zone (MZ). This mutant had reduced SHH signaling in the overlying progenitor zone, which led to reduced Lhx6, Lhx8, and Nkx2-1 expression in the rostrodorsal MGE and a preferential reduction of late-born somatostatin(+) and parvalbumin(+) cortical interneurons. Thus, Lhx6 and Lhx8 regulate MGE development through autonomous and nonautonomous mechanisms, the latter by promoting Shh expression in MGE neurons, which in turn feeds forward to promote the developmental program of the rostrodorsal MGE.

European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate macrophage activation influence the ability of Toxoplasma to establish a chronic infection.