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Cidofovir (1(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, CDV) is a potent inhibitor of orthopoxvirus DNA replication. Prior studies have shown that, when CDV is incorporated into a growing primer strand, it can inhibit both the 3'-to-5' exonuclease and the 5'-to-3' chain extension activities of vaccinia virus DNA polymerase. This drug can also be incorporated into DNA, creating a significant impediment to trans-lesion DNA synthesis in a manner resembling DNA damage. CDV and deoxycytidine share a common nucleobase, but CDV lacks the deoxyribose sugar. The acyclic phosphonate bears a hydroxyl moiety that is equivalent to the 3'-hydroxyl of dCMP and permits CDV incorporation into duplex DNA. To study the structural consequences of inserting CDV into DNA, we have used (1)H NMR to solve the solution structures of a dodecamer DNA duplex containing a CDV molecule at position 7 and of a control DNA duplex. The overall structures of both DNA duplexes were found to be very similar. We observed a decrease of intensity (>50%) for the imino protons neighboring the CDV (G6, T8) and the cognate base G18 and a large chemical shift change for G18. This indicates higher proton exchange rates for this region, which were confirmed using NMR-monitored melting experiments. DNA duplex melting experiments monitored by circular dichroism revealed a lower T(m) for the CDV DNA duplex (46 °C) compared to the control (58 °C) in 0.2 M salt. Our results suggest that the CDV drug is well accommodated and stable within the dodecamer DNA duplex, but the stability of the complex is less than that of the control, suggesting increased dynamics around the CDV.

<h4>Introduction</h4>Estrogen forms a complex with the estrogen receptor (ER) that binds to estrogen response elements (EREs) in the promoter region of estrogen-responsive genes, regulates their transcription, and consequently mediates physiological or tumorigenic effects. Thus, sequence variants in EREs have the potential to affect the estrogen-ER-ERE interaction. In this study, we examined the hypothesis that genetic variations of EREs are associated with breast cancer development.<h4>Methods</h4>This case-control study involved 815 patients of Asian descent with incident breast cancer and 821 healthy female controls. A total of 13,737 ERE sites in the whole genome predicted by a genome-wide computational algorithm were blasted with single-nucleotide polymorphism (SNP) sequences. Twenty-one SNPs located within 2,000 bp upstream or within introns 1 and 2 of putative genes and with a minor allele frequency greater than 5% were identified and genotyped. Frequencies of SNPs were compared between cases and controls to identify SNPs associated with cancer susceptibility.<h4>Results</h4>A significant combined effect of rs12539530, an ERE SNP in intron 2 of NRCAM which codes for a cell adhesion molecule, and SNPs of ESR1, the gene coding for ER, on breast cancer risk was found. Interestingly, this combined effect was more significant in women who had experienced a longer period of lifetime estrogen exposure, supporting a hormonal etiology of this SNP in breast tumorigenesis.<h4>Conclusions</h4>Our findings provide support for a role of genetic variation in ERE-ESR1 in determining susceptibility of breast cancer development.

<h4>Background</h4>Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem.<h4>Methods</h4>A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology.<h4>Results</h4>Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.<h4>Conclusions</h4>Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.

Malaria parasites are subjected to high levels of oxidative stress during their development inside erythrocytes and the ability of the parasite to defend itself against this assault is critical to its survival. Therefore, Plasmodium possesses an effective antioxidant defense system that could potentially be used as a target for the development of inhibitor-based therapy. We have identified an unusual peroxiredoxin protein that localizes to the nucleus of Plasmodium falciparum and have renamed it PfnPrx (PF10_0268, earlier called MCP1). Our work reveals that PfnPrx has a broad specificity of substrate being able to utilize thioredoxin and glutaredoxin as reductants and having the ability to reduce simple and complex peroxides. Intriguingly, chromatin immunoprecipitation followed by deep sequencing reveals that the enzyme associates with chromatin in a genome-wide manner with a slight enrichment in coding regions. Our results represent the first description of a dedicated chromatin-associated peroxiredoxin and potentially represent an ingenious way by which the parasite can survive the highly oxidative environment within its human host.

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT), and damage to the gastrointestinal (GI) tract plays a critical role in amplifying systemic disease. Intestinal stem cells (ISCs) play a pivotal role not only in physiological tissue renewal but also in regeneration of the intestinal epithelium after injury. In this study, we have discovered that pretransplant conditioning regimen damaged ISCs; however, the ISCs rapidly recovered and restored the normal architecture of the intestine. ISCs are targets of GVHD, and this process of ISC recovery was markedly inhibited with the development of GVHD. Injection of Wnt agonist R-spondin1 (R-Spo1) protected against ISC damage, enhanced restoration of injured intestinal epithelium, and inhibited subsequent inflammatory cytokine cascades. R-Spo1 ameliorated systemic GVHD after allogeneic BMT by a mechanism dependent on repair of conditioning-induced GI tract injury. Our results demonstrate for the first time that ISC damage plays a central role in amplifying systemic GVHD; therefore, we propose ISC protection by R-Spo1 as a novel strategy to improve the outcome of allogeneic BMT.

Gene expression profiles of human ameloblastoma microdissected cells were characterized with the purpose of identifying genes and their protein products that could be targeted as diagnostic and prognostic markers as well as for potential therapeutic interventions. Five formalin-fixed, decalcified, paraffin-embedded samples of ameloblastoma were subjected to laser capture microdissection, linear mRNA amplification, and hybridization to oligonucleotide human 41,000 RNA arrays and compared with universal human reference RNA, to determine the gene expression signature. Assessment of the data by Significance Analysis of Microarrays (SAM) and cluster analysis showed that 38 genes were highly expressed (two-fold increase) in all samples, while 41 genes were underexpressed (two-fold reduction). Elements of the sonic hedgehog pathway and Wingless type MMTV integration site family were validated by immunohistochemistry. We have identified the expression of multiple genes and protein products that could serve as potential diagnostic, prognostic, and therapeutic targets.

Genome-wide association study (GWAS) consortia and collaborations formed to detect genetic loci for common phenotypes or investigate gene-environment (G*E) interactions are increasingly common. While these consortia effectively increase sample size, phenotype heterogeneity across studies represents a major obstacle that limits successful identification of these associations. Investigators are faced with the challenge of how to harmonize previously collected phenotype data obtained using different data collection instruments which cover topics in varying degrees of detail and over diverse time frames. This process has not been described in detail. We describe here some of the strategies and pitfalls associated with combining phenotype data from varying studies. Using the Gene Environment Association Studies (GENEVA) multi-site GWAS consortium as an example, this paper provides an illustration to guide GWAS consortia through the process of phenotype harmonization and describes key issues that arise when sharing data across disparate studies. GENEVA is unusual in the diversity of disease endpoints and so the issues it faces as its participating studies share data will be informative for many collaborations. Phenotype harmonization requires identifying common phenotypes, determining the feasibility of cross-study analysis for each, preparing common definitions, and applying appropriate algorithms. Other issues to be considered include genotyping timeframes, coordination of parallel efforts by other collaborative groups, analytic approaches, and imputation of genotype data. GENEVA's harmonization efforts and policy of promoting data sharing and collaboration, not only within GENEVA but also with outside collaborations, can provide important guidance to ongoing and new consortia.

Protein controlled iron homeostasis is essential for maintaining appropriate levels and availability of metal within cells. Recently, two iron chaperones have been discovered that direct metal within two unique pathways: (1) mitochondrial iron-sulfur (Fe-S) cluster assembly and (2) within the ferritin iron storage system. Although structural and functional details describing how these iron chaperones operate are emerging, both share similar iron binding affinities and metal-ligand site structures that enable them to bind and release Fe2+ to specific protein partners. Molecular details related to iron binding and delivery by these chaperones will be explored within this review.

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification.

Huntington's disease (HD) is an autosomal dominantly inherited progressive neurodegenerative disease. The exact sequel of events finally resulting in neurodegeneration is only partially understood and there is no established protective treatment so far. Some lines of evidence speak for the contribution of oxidative stress to neuronal tissue damage. The fumaric acid ester dimethylfumarate (DMF) is a new disease modifying therapy currently in phase III studies for relapsing-remitting multiple sclerosis. DMF potentially exerts neuroprotective effects via induction of the transcription factor "nuclear factor E2-related factor 2" (Nrf2) and detoxification pathways. Thus, we investigated here the therapeutic efficacy of DMF in R6/2 and YAC128 HD transgenic mice which mimic many aspects of HD and are characterized by an enhanced generation of free radicals in neurons. Treatment with DMF significantly prevented weight loss in R6/2 mice between postnatal days 80-90. At the same time, DMF treatment led to an attenuated motor impairment as measured by the clasping score. Average survival in the DMF group was 100.5 days vs. 94.0 days in the placebo group. In the histological analysis on day 80, DMF treatment resulted in a significant preservation of morphologically intact neurons in the striatum as well as in the motor cortex. DMF treatment resulted in an increased Nrf2 immunoreactivity in neuronal subpopulations, but not in astrocytes. These beneficial effects were corroborated in YAC128 mice which, after one year of DMF treatment, also displayed reduced dyskinesia as well as a preservation of neurons. In conclusion, DMF may exert beneficial effects in mouse models of HD. Given its excellent side effect profile, further studies with DMF as new therapeutic approach in HD and other neurodegenerative diseases are warranted.

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.

<h4>Background</h4>Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington's disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.<h4>Methodology/principal findings</h4>We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V(L)12.3, turnover of soluble mHDx-1 in living cells is blocked.<h4>Conclusions/significance</h4>These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.

Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy. Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. The goal of this study was to unravel new clues on the biological pathways leading to HIBM by proteomic comparison. Muscle cultures and biopsies were analyzed by two dimensional gel electrophoresis (2-DE) and the same biopsy extracts by isobaric tag for relative and absolute quantitation (iTRAQ). Proteins that were differentially expressed in all HIBM specimens versus all controls in each analysis were identified by mass spectrometry. The muscle cultures 2-DE analysis yielded 41 such proteins, while the biopsies 2-DE analysis showed 26 differentially expressed proteins. Out of the 400 proteins identified in biopsies by iTRAQ, 41 showed altered expression. In spite of the different nature of specimens (muscle primary cultures versus muscle biopsies) and of the different methods applied (2D gels versus iTRAQ) the differentially expressed proteins identified in each of the three analyses where related mainly to the same pathways, ubiquitination, stress response and mitochondrial processes, but the most robust cluster (30%) was assigned to cytoskeleton and sarcomere organization. Taken together, these findings indicate a possible novel function of GNE in the muscle filamentous apparatus that could be involved in the pathogenesis of HIBM.

MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.

Bone is a hierarchically structured composite consisting of a protein phase (type I collagen) and a mineral phase (carbonated apatite). The objective of this study was to investigate the hierarchical structure of mineral in mature bone. A method to completely deproteinize bone without altering the original structure is developed, and the completion is confirmed by protein analysis techniques. Stereoscopy and field emission electron microscopy are used to examine the structural features from submillimeter- to micrometer- to nanometer-length scales of bovine femur cancellous bone. Stereoscopic images of fully deproteinized and demineralized bovine femur cancellous bone samples show that fine trabecular architecture is unaltered and the microstructural features are preserved, indicating the structural integrity of mineral and protein constituents. SEM revealed that bone minerals are fused together and form a sheet-like structure in a coherent manner with collagen fibrils. Well-organized pore systems are observed at varying hierarchical levels. Mineral sheets are peeled off and folded after compressive deformation, implying strong connection between individual crystallites. Results were compared with commercially available heat-deproteinized bone (Bio-Oss(®)), and evidence showed consistency in bone mineral structure. A two-phase interpenetrating composite model of mature bone is proposed and discussed.

In this study, STa peptide of enterotoxigenic Escherichia coli K99(+) was purified and successfully covalently cross-linked to modified bovine serum albumin after thorough evaluation of three different hapten-carrier conjugation protocols. Dimethyformamide (DMF) based STa-conjugation protocol demonstrated higher biological activity (10×10(6) STa Total Mouse Units [MU]) and 100% conjugation efficiency. A range of conjugation ratio of 4-12 STa molecules per one molecule of BSA was achieved and confirmed by matrix-assisted laser desorption ionization-time of flight/mass spectroscopy (MALDI-TOF/MS). This conjugate was used for immunization of ten rabbits for STa antibody production. A high antibody binding titer (10(6)) against STa was obtained with a neutralization capacity of 3×10(4) STa MUs/ml serum. These levels of high STa binding and neutralizing antibodies titers propose the potential use of this conjugate for the development of immunotherapeutic reagents and/or STa-based vaccine against ETEC K99(+).

High mobility group N (HMGN) is a family of intrinsically disordered nuclear proteins that bind to nucleosomes, alters the structure of chromatin and affects transcription. A major unresolved question is the extent of functional specificity, or redundancy, between the various members of the HMGN protein family. Here, we analyze the transcriptional profile of cells in which the expression of various HMGN proteins has been either deleted or doubled. We find that both up- and downregulation of HMGN expression altered the cellular transcription profile. Most, but not all of the changes were variant specific, suggesting limited redundancy in transcriptional regulation. Analysis of point and swap HMGN mutants revealed that the transcriptional specificity is determined by a unique combination of a functional nucleosome-binding domain and C-terminal domain. Doubling the amount of HMGN had a significantly larger effect on the transcription profile than total deletion, suggesting that the intrinsically disordered structure of HMGN proteins plays an important role in their function. The results reveal an HMGN-variant-specific effect on the fidelity of the cellular transcription profile, indicating that functionally the various HMGN subtypes are not fully redundant.

Large alterations in transcription accompany neurodegeneration in polyglutamine (polyQ) diseases. These pathologies manifest both general polyQ toxicity and mutant protein-specific effects. In this study, we report that the fat tumour suppressor gene mediates neurodegeneration induced by the polyQ protein Atrophin. We have monitored early transcriptional alterations in a Drosophila model of Dentatorubral-pallidoluysian Atrophy and found that polyQ Atrophins downregulate fat. Fat protects from neurodegeneration and Atrophin toxicity through the Hippo kinase cascade. Fat/Hippo signalling does not provoke neurodegeneration by stimulating overgrowth; rather, it alters the autophagic flux in photoreceptor neurons, thereby affecting cell homeostasis. Our data thus provide a crucial insight into the specific mechanism of a polyQ disease and reveal an unexpected neuroprotective role of the Fat/Hippo pathway.

The distribution of lipoprotein(a) [Lp(a)] levels can differ dramatically across diverse racial/ethnic populations. The extent to which genetic variation in LPA can explain these differences is not fully understood. To explore this, 19 LPA tagSNPs were genotyped in 7,159 participants from the Third National Health and Nutrition Examination Survey (NHANES III). NHANES III is a diverse population-based survey with DNA samples linked to hundreds of quantitative traits, including serum Lp(a). Tests of association between LPA variants and transformed Lp(a) levels were performed across the three different NHANES subpopulations (non-Hispanic whites, non-Hispanic blacks, and Mexican Americans). At a significance threshold of p<0.0001, 15 of the 19 SNPs tested were strongly associated with Lp(a) levels in at least one subpopulation, six in at least two subpopulations, and none in all three subpopulations. In non-Hispanic whites, three variants were associated with Lp(a) levels, including previously known rs6919246 (p = 1.18 × 10(-30)). Additionally, 12 and 6 variants had significant associations in non-Hispanic blacks and Mexican Americans, respectively. The additive effects of these associated alleles explained up to 11% of the variance observed for Lp(a) levels in the different racial/ethnic populations. The findings reported here replicate previous candidate gene and genome-wide association studies for Lp(a) levels in European-descent populations and extend these findings to other populations. While we demonstrate that LPA is an important contributor to Lp(a) levels regardless of race/ethnicity, the lack of generalization of associations across all subpopulations suggests that specific LPA variants may be contributing to the observed Lp(a) between-population variance.

Cells communicate with each other and the outside world through surface receptors, which need to be tightly regulated to prevent both overstimulation and receptor desensitization. Understanding the processes involved in the homeostatic control of cell surface receptors is essential, but we are not alone in trying to regulate these receptors. Viruses, as the ultimate host pathogens, have co-evolved over millions of years and have both pirated and adapted host genes to enable viral pathogenesis. K3 and K5 (also known as MIR1 and MIR2) are viral ubiquitin E3 ligases from Kaposi's Sarcoma Associated Herpesvirus (KSHV) which decrease expression of a number of cell surface receptors and have been used to interrogate cellular processes and improve our understanding of ubiquitin-mediated receptor endocytosis and degradation. In this review, we summarize what has been learned from the study of these viral genes and emphasize their role in elucidating the complexity of ubiquitin in receptor regulation.

<h4>Background</h4>Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.<h4>Methods</h4>We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.<h4>Results</h4>DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.<h4>Conclusions</h4>This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.

<h4>Background</h4>There are limited published data defining complete pleural fluid analysis, echocardiographic characteristics, or the presence or absence of ascites on sonographic or CT imaging in patients with hepatic hydrothorax.<h4>Methods</h4>We reviewed pleural fluid analysis and radiographic, sonographic, and echocardiographic findings in 41 consecutive patients with hepatic hydrothorax referred to the Pleural Procedure Service for thoracentesis.<h4>Results</h4>Ascites was detected on sonographic or CT imaging in 38 of 39 patients (97%). Diastolic dysfunction was found in 11 of 21 patients (52%). Contrast echocardiography with agitated saline demonstrated an intrapulmonary shunt in 18 of 23 cases (78%). Solitary hepatic hydrothorax had a median pleural fluid pH of 7.49 (fifth to 95th percentile, 7.40-7.57), total protein level of 1.5 g/dL (0.58-2.34), and lactate dehydrogenase (LDH) level of 65 IU/L (36-138). The median pleural fluid/serum protein ratio and pleural LDH/upper limit of normal serum LDH ratio were 0.25 (0.10-0.43) and 0.27 (0.14-0.57), respectively. The median absolute neutrophil count (ANC) was 26 cells/μL (1-230). Only a single patient had a protein discordant exudate despite 83% of patients receiving diuretics. When comparing solitary hepatic hydrothorax and spontaneous bacterial pleuritis, there was no statistically significant difference among pleural fluid total protein (P = .99), LDH (P = .33), and serum albumin (P = .47). ANC was higher in patients with spontaneous bacterial pleuritis (P < .0001).<h4>Conclusions</h4>Hepatic hydrothorax virtually always presents with ascites that is detectable on sonographic or CT imaging. The development of an "exudate" from diuretic therapy is a rare phenomenon in hepatic hydrothorax. In contrast, diastolic dysfunction and intrapulmonary shunting are common in patients with hepatic hydrothorax. There was no statistically significant change in pleural fluid parameters with spontaneous bacterial pleuritis, except an increased ANC.

Huntington's disease (HD) is a hereditary, progressively degenerative and fatal brain disorder classified as a rare, or 'orphan', disease. HD is caused by the extension of trinucleotide repeats encoding a stretch of glutamine residues at the amino-terminal end of the large huntingtin (HTT) protein. Since the discovery of the mutated HTT gene in 1993, the mechanisms by which the mutant HTT protein induces neurodegeneration remain poorly understood and no disease-modifying therapy is currently available. Several functional approaches combining different experimental models and experimental technologies have been used to shed some light on the mechanisms underlying this disease. This review presents these functional approaches, highlights their potential and limitations.

<h4>Background</h4>A typical pathological feature of Alzheimer's disease (AD) is the appearance in the brain of senile plaques made up of β-amyloid (Aβ) and neurofibrillary tangles. AD is also associated with an abnormal accumulation of some metal ions, and we have recently shown that one of these, aluminum (Al), plays a relevant role in affecting Aβ aggregation and neurotoxicity.<h4>Methodology</h4>In this study, employing a microarray analysis of 35,129 genes, we investigated the effects induced by the exposure to the Aβ(1-42)-Al (Aβ-Al) complex on the gene expression profile of the neuronal-like cell line, SH-SY5Y.<h4>Principal findings</h4>The microarray assay indicated that, compared to Aβ or Al alone, exposure to Aβ-Al complex produced selective changes in gene expression. Some of the genes selectively over or underexpressed are directly related to AD. A further evaluation performed with Ingenuity Pathway analysis revealed that these genes are nodes of networks and pathways that are involved in the modulation of Ca(2+) homeostasis as well as in the regulation of glutamatergic transmission and synaptic plasticity.<h4>Conclusions and significance</h4>Aβ-Al appears to be largely involved in the molecular machinery that regulates neuronal as well as synaptic dysfunction and loss. Aβ-Al seems critical in modulating key AD-related pathways such as glutamatergic transmission, Ca(2+) homeostasis, oxidative stress, inflammation, and neuronal apoptosis.

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMV(KS)EGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n = 3 per time point) and infected (EGFP(+)) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP(+) cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.

<h4>Background</h4>Measles remains a serious vaccine preventable cause of mortality in developing nations. Vietnam is aiming to achieve the level of immunity required to eliminate measles by maintaining a high coverage of routine first vaccinations in infants, routine second vaccinations at school entry and supplementary local campaigns in high-risk areas. Regular outbreaks of measles are reported, during 2005-2009.<h4>Methods</h4>National measles case-based surveillance data collected during 2005-June 2009 was analyzed to assess the epidemiological trend and risk factors associated with measles outbreak in Vietnam.<h4>Results</h4>Of the 36,282 measles suspected cases reported nationwide, only 7,086 cases were confirmed through laboratory examination. Although cyclical outbreaks occurred between 2005 and 2009, there was no definite trend in measles outbreaks during these periods. Overall, 2438 of measles confirmed cases were among children ≤5 years and 3068 cases were among people ≥16 years. The distribution with respect to gender skewed towards male (3667 cases) significant difference was not observed (P= 0.1693). Unsurprisingly, 4493 of the confirmed cases had no history of vaccination (X(2) <0.01). The northern and highland regions were identified as the main endemic foci and the spatial distribution changed with time. The occurrence of cases, in a considerable proportion of vaccinated population, is not only a reflection of the high vaccination coverage in Vietnam but also portrays a possibility of less than 100% vaccine efficacy. More so, in order to prevent measles in adults, high-risk groups must be identified and catch-up for selected groups selected.<h4>Conclusions</h4>This study therefore reinforces the need for continued improvement of surveillance system and to probe into the possible role of changes in age-distribution of cases if the effective control of measles is to be achieved.

<h4>Aims/hypothesis</h4>Obesity is a major risk factor for type 2 diabetes. Recent genome-wide association (GWA) studies have identified multiple loci robustly associated with BMI and risk of obesity. However, information on their associations with type 2 diabetes is limited. Such information could help increase our understanding of the link between obesity and type 2 diabetes. We examined the associations of 12 obesity susceptibility loci, individually and in combination, with risk of type 2 diabetes in the population-based European Prospective Investigation of Cancer (EPIC) Norfolk cohort.<h4>Methods</h4>We genotyped 12 SNPs, identified by GWA studies of BMI, in 20,428 individuals (aged 39-79 years at baseline) with an average follow-up of 12.9 years, during which 729 individuals developed type 2 diabetes. A genetic predisposition score was calculated by adding the BMI-increasing alleles across the 12 SNPs. Associations with incidence of type 2 diabetes were examined by logistic regression models.<h4>Results</h4>Of the 12 SNPs, eight showed a trend with increased risk of type 2 diabetes, consistent with their BMI-increasing effects. Each additional BMI-increasing allele in the genetic predisposition score was associated with a 4% increased odds of developing type 2 diabetes (OR 1.041, 95% CI 1.005-1.078; p = 0.02). Adjustment for BMI completely abolished the association with incident type 2 diabetes (OR 1.003, 95% CI 0.967-1.039; p = 0.89).<h4>Conclusions/interpretation</h4>The genetic predisposition to obesity leads to increased risk of developing type 2 diabetes, which is completely mediated by its obesity-predisposing effect.

<h4>Background</h4>The plastic monomer and plasticizer bisphenol A (BPA), used for manufacturing polycarbonate plastic and epoxy resins, is produced at over 2.5 million metric tons per year. Concerns have been raised that BPA acts as an endocrine disruptor on both developmental and reproductive processes and a large body of evidence suggests that BPA interferes with estrogen and thyroid hormone signaling. Here, we investigated BPA effects during embryonic development using the zebrafish and Xenopus models.<h4>Results</h4>We report that BPA exposure leads to severe malformations of the otic vesicle. In zebrafish and in Xenopus embryos, exposure to BPA during the first developmental day resulted in dose-dependent defects in otolith formation. Defects included aggregation, multiplication and occasionally failure to form otoliths. As no effects on otolith development were seen with exposure to micromolar concentrations of thyroid hormone, 17-ß-estradiol or of the estrogen receptor antagonist ICI 182,780 we conclude that the effects of BPA are independent of estrogen receptors or thyroid-hormone receptors. Na+/K+ ATPases are crucial for otolith formation in zebrafish. Pharmacological inhibition of the major Na+/K+ ATPase with ouabain can rescue the BPA-induced otolith phenotype.<h4>Conclusions</h4>The data suggest that the spectrum of BPA action is wider than previously expected and argue for a systematic survey of the developmental effects of this endocrine disruptor.

<h4>Introduction</h4>The eMERGE (electronic MEdical Records and GEnomics) Network is an NHGRI-supported consortium of five institutions to explore the utility of DNA repositories coupled to Electronic Medical Record (EMR) systems for advancing discovery in genome science. eMERGE also includes a special emphasis on the ethical, legal and social issues related to these endeavors.<h4>Organization</h4>The five sites are supported by an Administrative Coordinating Center. Setting of network goals is initiated by working groups: (1) Genomics, (2) Informatics, and (3) Consent & Community Consultation, which also includes active participation by investigators outside the eMERGE funded sites, and (4) Return of Results Oversight Committee. The Steering Committee, comprised of site PIs and representatives and NHGRI staff, meet three times per year, once per year with the External Scientific Panel.<h4>Current progress</h4>The primary site-specific phenotypes for which samples have undergone genome-wide association study (GWAS) genotyping are cataract and HDL, dementia, electrocardiographic QRS duration, peripheral arterial disease, and type 2 diabetes. A GWAS is also being undertaken for resistant hypertension in ≈ 2,000 additional samples identified across the network sites, to be added to data available for samples already genotyped. Funded by ARRA supplements, secondary phenotypes have been added at all sites to leverage the genotyping data, and hypothyroidism is being analyzed as a cross-network phenotype. Results are being posted in dbGaP. Other key eMERGE activities include evaluation of the issues associated with cross-site deployment of common algorithms to identify cases and controls in EMRs, data privacy of genomic and clinically-derived data, developing approaches for large-scale meta-analysis of GWAS data across five sites, and a community consultation and consent initiative at each site.<h4>Future activities</h4>Plans are underway to expand the network in diversity of populations and incorporation of GWAS findings into clinical care.<h4>Summary</h4>By combining advanced clinical informatics, genome science, and community consultation, eMERGE represents a first step in the development of data-driven approaches to incorporate genomic information into routine healthcare delivery.

Minerals are essential and toxic elements that have an impact on human health. Although we have learned a tremendous amount about the metabolism, biological roles, and health effects of minerals with the tools of biochemistry, cell biology, and molecular genetics, there are gaps in our knowledge of mineral biology that will benefit from new approaches. Forward genetics, whereby variations in phenotypes are mapped to natural genetic variation in the genome, has been successfully used to increase our understanding of many biologically important traits but has not yet been used extensively for mineral metabolism. In addition, the well-appreciated existence of interactions between minerals justifies a broader, systems approach to the study of mineral metabolism, i.e., ionomics. This short review will explain the value of forward genetics and ionomics as tools for exploring mammalian mineral metabolism.

The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of new radiotracers and novel radiolabeling procedures with the most prominent short-lived positron emitters carbon-11 and fluorine-18. Radiolabeling with these radionuclides represents a remarkable challenge. Special attention has to be paid to synthesis time and specific labeling techniques due to the short physical half life of the respective radionuclides ¹¹C (t(½) = 20.4 min) and ¹⁸F (t½) = 109.8 min). In the past, numerous transition metal-catalyzed reactions were employed in organic chemistry, even though only a handful of these coupling reactions were adopted in radiochemical practice. Thus, the implementation of modern synthesis methods like cross-coupling reactions offers the possibility to develop a wide variety of novel radiotracers. The introduction of catalysts based on transition metal complexes bears a high potential for rapid, efficient, highly selective and functional group-tolerating incorporation of carbon-11 and fluorine-18 into target molecules. This review deals with design, application and improvement of transition metal-mediated carbon-carbon as well as carbon-heteroatom cross-coupling reactions as a labeling feature with the focus on the preparation of radiolabeled compounds for molecular imaging.

We re-annotated the interacting partners of the neuronal scaffold protein DISC1 using a knowledge-based approach that incorporated recent protein interaction data and published literature to. This revealed two highly connected networks. These networks feature cellular function and maintenance, and cell signaling. Of potentially greatest interest was the novel finding of a high degree of connectivity between the DISC1 scaffold protein, linked to psychiatric illness, and huntingtin, the protein which is mutated in Huntington's disease. The potential link between DISC1, huntingtin and their interacting partners may open new areas of research into the effects of pathway dysregulation in severe neurological disorders.

No abstract available.

Antithrombin (AT) is the most important physiological inhibitor of coagulation proteases. It is activated by glycosaminoglycans such as heparin. Hereditary antithrombin deficiency is a rare disease that is mainly associated with venous thromboembolism. So far, more than 200 different mutations in the antithrombin gene (SERPINC1) have been described. The aim of our study was to characterise the molecular background in a large cohort of patients with AT deficiency. Mutation analysis was performed by direct sequencing of SERPINC1 in 272 AT-deficient patients. Large deletions were identified by multiplex PCR coupled with liquid chromatography or multiplex ligation-dependent probe amplification (MLPA) analysis. To predict the effect of SERPINC1 sequence variations on the pathogenesis of AT deficiency, in silico assessments, multiple sequence alignment, and molecular graphic imaging were performed. The mutation profile consisted of 59% missense, 10% nonsense, 8% splice site mutations, 15% small deletions/insertions/duplications, and 8% large deletions. Altogether 87 different mutations, including 42 novel mutations (22 missense and 20 null mutations), were identified. Of the novel missense mutations, nine are suspected to impair the conformational changes that are needed for AT activation, two to affect the central reactive loop or the heparin binding site, and six to impair the structural integrity of the molecule. Despite the heterogeneous background of AT deficiency, 10 AT variants occurred in multiple index patients. Characterisation of the SERPINC1 mutation profile in large cohorts of patients may help to further elucidate the pathogenesis of AT deficiency and to establish genotype-phenotype associations.

The receptor for advanced glycation end (RAGE) products is a multi-ligand receptor that belongs to the immunoglobulin superfamily of cell surface receptors, whose ligands are known to be upregulated in neuropathological conditions. RAGE upregulation has been described in neurodegenerative diseases, such as Alzheimer's disease, Creutzfeldt-Jakob's disease and Huntington's disease (HD). To analyze in detail the implication of RAGE in HD, we studied the immunohistochemical distribution of RAGE in the striatum of the R6/2 mouse model of HD, with particular attention to the neuronal subpopulations and their relative vulnerability to HD neurodegeneration. We show that RAGE immunoreactivity is evenly distributed to the cytoplasm of neurons in the wild type mouse, while it is finely granular in the cytoplasm of striatal neurons of R6/2 mouse. RAGE is distributed in 98% of spiny projection neurons, both in the normal mouse and in the R6/2. RAGE co-localizes with all of the striatal interneuron subsets both in the wild-type and in the R6/2 mouse. However, the intensity of RAGE immunoreactivity is significantly higher in the spiny neurons and in the PARV neurons of R6/2 mouse, whereas it is comparable between R6/2 and wild-type in the cholinergic and somatostatinergic interneurons. These data support the concept that RAGE is upregulated in the neurodegenerative process of HD, and suggests that its activation is related to the individual vulnerability of the striatal neuronal subtype.

Both common forms of diabetes have an inflammatory pathogenesis in which immune and metabolic factors converge on interleukin-1β as a key mediator of insulin resistance and β-cell failure. In addition to improving insulin resistance and preventing β-cell inflammatory damage, there is evidence of genetic association between diabetes and histone deacetylases (HDACs); and HDAC inhibitors (HDACi) promote β-cell development, proliferation, differentiation and function and positively affect late diabetic microvascular complications. Here we review this evidence and propose that there is a strong rationale for preclinical studies and clinical trials with the aim of testing the utility of HDACi as a novel therapy for diabetes.

Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is essential to coordinate the development of tissues such as the musculature and nervous system during normal embryonic development. One class of signaling proteins that regulate actin cytoskeletal rearrangement is the evolutionarily conserved CDM (C. elegansCed-5, human DOCK180, DrosophilaMyoblast city, or Mbc) family of proteins, which function as unconventional guanine nucleotide exchange factors for the small GTPase Rac. This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We identified and characterized the role of Drosophila Sponge (Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein. Our analysis shows Spg mRNA and protein is expressed in the visceral musculature and developing nervous system, suggesting a role for Spg in later embryogenesis. As maternal null mutants of spg die early in development, we utilized genetic interaction analysis to uncover the role of Spg in central nervous system (CNS) development. Consistent with its role in ELMO-dependent pathways, we found genetic interactions with spg and elmo mutants exhibited aberrant axonal defects. In addition, our data suggests Ncad may be responsible for recruiting Spg to the membrane, possibly in CNS development. Our findings not only characterize the role of a new DOCK family member, but help to further understand the role of signaling downstream of N-cadherin in neuronal development.

Serotonin (5-hydroxytryptamine, 5-HT) neurotransmission is negatively regulated by 5-HT1A autoreceptors on raphe neurons, and is implicated in mood disorders. Pet-1/FEV is an ETS transcription factor expressed exclusively in serotonergic neurons and is essential for serotonergic differentiation, although its regulation of 5-HT receptors has not yet been studied. Here, we show by electrophoretic mobility shift assay that recombinant human Pet-1/FEV binds directly to multiple Pet-1 elements of the human 5-HT1A receptor promoter to enhance its transcriptional activity. In luciferase reporter assays, mutational analysis indicated that while several sites contribute, the Pet-1 site at -1406 bp had the greatest effect on 5-HT1A promoter activity. To address the effect of Pet-1 on 5-HT1A receptor regulation in vivo, we compared the expression of 5-HT1A receptor RNA and protein in Pet-1 null and wild-type littermate mice. In the raphe nuclei of Pet-1-/- mice tryptophan hydroxylase 2 (TPH2) RNA, and 5-HT and TPH immunostaining were greatly reduced, indicating a deficit in 5-HT production. Raphe 5-HT1A RNA and protein levels were also reduced in Pet-1-deficient mice, consistent with an absence of Pet-1-mediated transcriptional enhancement of 5-HT1A autoreceptors in serotonergic neurons. Interestingly, 5-HT1A receptor expression was up-regulated in the hippocampus, but down-regulated in the striatum and cortex. These data indicate that, in addition to transcriptional regulation by Pet-1 in raphe neurons, 5-HT1A receptor expression is regulated indirectly by alterations in 5-HT neurotransmission in a region-specific manner that together may contribute to the aggressive/anxiety phenotype observed in Pet-1 null mice.

<h4>Background</h4>Polymorphisms in intron 15 of potassium voltage-gated channel, KQT-like subfamily member 1 (KCNQ1) gene have been associated with type II diabetes (T2D) in Japanese genome-wide association studies (GWAS). More recently a meta-analysis of European GWAS has detected a new independent signal associated with T2D in intron 11 of the KCNQ1 gene. The purpose of this investigation is to examine the role of these variants with T2D in populations of Asian Indian descent from India and the US.<h4>Methods</h4>We examined the association between four variants in the KCNQ1 gene with T2D and related quantitative traits in a total of 3,310 Asian Indian participants from two different cohorts comprising 2,431 individuals of the Punjabi case-control cohort from the Sikh Diabetes Study and 879 migrant Asian Indians living in the US.<h4>Results</h4>Our data confirmed the association of a new signal at the KCNQ1 locus (rs231362) with T2D showing an allelic odds ratio (OR) of 1.24 95%CI [1.08-1.43], p = 0.002 in the Punjabi cohort. A moderate association with T2D was also seen for rs2237895 in the Punjabi (OR 1.14; p = 0.036) and combined cohorts (meta-analysis OR 1.14; p = 0.018). Three-site haplotype analysis of rs231362, rs2237892, rs2237895 exhibited considerably stronger evidence of association of the GCC haplotype with T2D showing OR of 1.24 95%CI [1.00-1.53], p = 0.001, permutation p = 8 × 10-4 in combined cohorts. The 'C' risk allele carriers of rs2237895 had significantly reduced measures of HOMA-B in the US cohort (p = 0.008) as well as in combined cohort in meta-analysis (p = 0.009).<h4>Conclusions</h4>Our investigation has confirmed that the variation within the KCNQ1 locus confers a significant risk to T2D among Asian Indians. Haplotype analysis further suggested that the T2D risk associated with KCNQ1 SNPs may be derived from 'G' allele of rs231362 and 'C' allele of rs2237895 and this appears to be mediated through β cell function.

<h4>Background</h4>Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome.<h4>Methods</h4>We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays.<h4>Results</h4>Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%.We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer cell lines.<h4>Conclusions</h4>Deep transcriptional sequencing and analysis with targeted and spliced alignment methods can effectively identify TIC events across the genome in individual tissues. Prostate and reference samples exhibit a wide range of TIC events, involving more genes than estimated previously using ESTs. Tissue specificity of TIC events is correlated with expression patterns of the upstream gene. Some TIC events, such as MSMB-NCOA4, may play functional roles in cancer.

SHOX (short stature homeobox-containing gene) encodes a transcription factor implicated in skeletal development. SHOX haploinsufficiency has been demonstrated in Leri-Weill dyschondrosteosis (LWD), a skeletal dysplasia associated with disproportionate short stature, as well as in a variable proportion of cases with idiopathic short stature (ISS). In order to gain insight into the SHOX signalling pathways, we performed a yeast two-hybrid screen to identify SHOX-interacting proteins. Two transcription factors, SOX5 and SOX6, were identified. Co-immunoprecipitation assays confirmed the existence of the SHOX-SOX5 and SHOX-SOX6 interactions in human cells, whereas immunohistochemical studies demonstrated the coexpression of these proteins in 18- and 32-week human fetal growth plates. The SHOX homeodomain and the SOX6 HMG domain were shown to be implicated in the SHOX-SOX6 interaction. Moreover, different SHOX missense mutations, identified in LWD and ISS patients, disrupted this interaction. The physiological importance of these interactions was investigated by studying the effect of SHOX on a transcriptional target of the SOX trio, Agc1, which encodes one of the main components of cartilage, aggrecan. Our results show that SHOX cooperates with SOX5/SOX6 and SOX9 in the activation of the upstream Agc1 enhancer and that SHOX mutations affect this activation. In conclusion, we have identified SOX5 and SOX6 as the first two SHOX-interacting proteins and have shown that this interaction regulates aggrecan expression, an essential factor in chondrogenesis and skeletal development.

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.

<h4>Purpose</h4>Altered glycosylation has been associated with oncogenic potential. Relationships of blood types (where expression is due to glycosylation pattern) and HER2/neu (where expression arises due to altered glycosylation) and breast cancer-associated markers like estrogen receptor/progesterone receptor (ER/PR) were examined and related to outcomes in patients with breast cancer.<h4>Methods</h4>A population-based retrospective study of 426 surgical breast cancer patients examined relationships between (1) patient characteristics, (2) breast tumor characteristics, and (3) outcomes of women diagnosed at the same medical center over a 10-year period relative to specific molecules defined by glycosylation patterns (eg. blood group, HER2/neu) and (4) ER/PR status.<h4>Results</h4>Following stratification by blood group, subjects exhibited significant differences in tumor size with persons in blood groups A and B having greater numbers of tumors ≤ 2 cm and those with blood types AB and O having tumors >2 cm. After adjusting for age, disease stage, and treatment with trastuzumab, tamoxifen, or aromatase inhibitors, no significant differences were observed in 5-year overall and disease-free survival based on blood type grouping. Blood group B was over-represented among the breast cancer cohort compared to the reference population, while blood group AB was under-represented.<h4>Conclusion</h4>No significant differences were observed in overall and disease-free survival based on blood group. No correlation was noted between HER2/neu, ER or PR status, and blood group type. Among this cohort, HER2/neu positivity was less than 20% and correlated with a 5-year disease-free survival rate ≥ 75% and overall survival of >80% across all blood groups.

Sox6 belongs to the Sry (sex-determining region Y)-related high-mobility-group-box family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector.

The central role of the prion protein (PrP) in a family of fatal neurodegenerate diseases has garnered considerable research interest over the past two decades. Moreover, the role of PrP in neuronal development, as well as its apparent role in metal homeostasis, is increasingly of interest. The host-encoded form of the prion protein (PrP(C)) binds multiple copper atoms via its N-terminal domain and can influence brain copper and iron levels. The importance of PrP(C) to the regulation of brain metal homeostasis and metal distribution, however, is not fully understood. We therefore employed synchrotron-based X-ray fluorescence imaging to map the level and distributions of several key metals in the brains of mice that express different levels of PrP(C). Brain sections from wild-type, prion gene knockout (Prnp(-/-)) and PrP(C) over-expressing mice revealed striking variation in the levels of iron, copper, and even zinc in specific brain regions as a function of PrP(C) expression. Our results indicate that one important function of PrP(C) may be to regulate the amount and distribution of specific metals within the central nervous system. This raises the possibility that PrP(C) levels, or its activity, might regulate the progression of diseases in which altered metal homeostasis is thought to play a pathogenic role such as Alzheimer's, Parkinson's and Wilson's diseases and disorders such as hemochromatosis.

<h4>Background</h4>Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil.<h4>Methods</h4>Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection.<h4>Results</h4>GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity.<h4>Conclusion</h4>Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.

<h4>Background/aims</h4>Hepcidin (gene name HAMP), an IL-6-inducible acute phase peptide with antimicrobial properties, is the key negative regulator of iron metabolism. Liver is the primary source of HAMP synthesis, but it is also produced by other tissues such as kidney or heart and is found in body fluids such as urine or cerebrospinal fluid. While the role of hepcidin in biliary system is unknown, a recent study demonstrated that conditional gp130-knockout mice display diminished hepcidin levels and increased rate of biliary infections.<h4>Methods</h4>Expression and localization of HAMP in biliary system was analyzed by real time RT-PCR, in-situ hybridization, immunostaining and -blotting, while prohepcidin levels in human bile were determined by ELISA.<h4>Results</h4>Hepcidin was detected in mouse/human gallbladder and bile duct epithelia. Biliary HAMP is stress-inducible, in that it is increased in biliary cell lines upon IL-6 stimulation and in gallbladder mucosa of patients with acute cholecystitis. Hepcidin is also present in the bile and elevated prohepcidin levels were observed in bile of primary sclerosing cholangitis (PSC) patients with concurrent bacterial cholangitis compared to PSC subjects without bacterial infection (median values 22.3 vs. 8.9; p = 0.03). In PSC-cholangitis subjects, bile prohepcidin levels positively correlated with C-reactive protein and bilirubin levels (r = 0.48 and r = 0.71, respectively). In vitro, hepcidin enhanced the antimicrobial capacity of human bile (p<0.05).<h4>Conclusion</h4>Hepcidin is a stress-inducible peptide of the biliary epithelia and a potential marker of biliary stress. In the bile, hepcidin may serve local functions such as protection from bacterial infections.

This article reports a historical study of factors influencing the achievement of clinical preimplantation genetic diagnosis (PGD) in 1990, 22 years after its first demonstration in animals. During the 1970s, research on PGD continued in large farm animals, but serious interest in human PGD was not evident until 1986. First, interest in PGD during the 1970s waned with the advent of prenatal testing, which for gynaecologists was clinically more familiar, technically simpler and ethically less challenging than IVF. Indeed, IVF was viewed with widespread suspicion until the first IVF births in 1978. Second, interest in clinical PGD was stimulated by the UK Parliamentary reaction against human embryo research that greeted the Warnock Report in 1984. This hostility led scientists to initiate a pro-research campaign, further galvanized in 1985 by MP Enoch Powell's bid to ban such research. However, while Powell abhorred embryo research, he approved of PGD, a stance that divided the anti-research lobby. Accordingly, the campaigners for research emphasized that it was needed to achieve PGD. Powell demanded evidence of such projects and PGD research increased from 1986. It is concluded that UK political debates on embryo research played a critical role in stimulating the achievement of clinical PGD. Human pregnancies following preimplantation genetic diagnosis (PGD) for embryo sex were announced in 1990, 22 years after the technique was pioneered in animals. PGD in humans required not only technological advances, such as IVF and sensitive diagnostic tests, but also the motivation to develop and apply them. Our historical analysis shows that, although research on PGD continued in large farm animals during the 1970s, and techniques of the required sensitivity were developed on mouse embryo models, interest in clinical PGD was not evident until 1986. Two factors stimulated this sudden change in motivation. First, interest in PGD was depressed during the 1970s by the advent of prenatal diagnostic techniques, which for gynaecologists were clinically, technically and ethically less challenging than IVF. IVF was then regarded with a suspicion that only started to wane in the early 1980s following the first IVF births. Second, the UK Parliamentary reaction against human embryo research that greeted the Warnock Report in 1984 provided a positive stimulus to clinical PGD by prompting scientists to form a pro-research lobby, which was further galvanized in early 1985 by MP Enoch Powell's almost-successful bid to ban human embryo research. We show that while Powell abhorred embryo research, he approved of PGD, a stance that fractured the unity of the anti-research lobby. Accordingly, the pro-research lobby emphasized that embryo research was needed to achieve PGD. Powell demanded evidence of such projects, thereby, we argue, stimulating PGD research from 1986. Our evidence shows that UK political debates about PGD played a critical role in stimulating the achievement of PGD clinically.

Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC(50). This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biological setting.

Aggression can be modulated by both genetic and environmental factors. Here, we analyse how the serotonin transporter (5-HTT) genotype and the environmental situation in which a contest takes place shape the display of offensive aggression. Therefore, male wildtype, heterozygous, and homozygous 5-HTT knockout mice, which are known to differ in inborn levels of anxiety, were confronted three times with a docile opponent in one of three environmental situations: own territory, opponent's territory or neutral area. The main findings were: The frequency of approaching the contestant in order to gather information about him depended significantly on the venue but not on the genotype with lowest frequencies in the opponent's territory. The decision how quickly to attack the opponent was significantly influenced by the 5-HTT genotype but not by the venue: Homozygous 5-HTT knockout mice showed longest latencies. The sum of offensive aggression was significantly influenced by the 5-HTT genotype, the environmental situation, and a genotype by environment interaction. It is likely that, due to their varying genetic predisposition for anxiety, mice of the three genotypes were differentially affected by the aversiveness of the respective venue and the opponent's behaviour, which influenced their decision to display offensive aggression. As a consequence, the amount of aggression shown by homozygous 5-HTT knockout mice was influenced by the venue and the opponent's behaviour, whereas heterozygotes reacted only to the venue. Strikingly, wildtypes behaved always the same way, irrespective of venue and opponent.

<h4>Introduction</h4>Recombinant activated factor VII (rFVIIa, NovoSeven®) is injected intravenously for the treatment of haemophilia patients with inhibitory antibodies. In plasma, rFVIIa forms complexes with protease inhibitors, primarily antithrombin III (ATIII). The liver is believed to be involved in clearance of rFVIIa, however, it is not known whether the liver is also involved for the clearance of the rFVIIa-ATIII complex. In this study, we explored the fate of intravenously injected rFVIIa from plasma to the hepatic lysosomes.<h4>Materials and methods</h4>A novel method using magnetic chromatography was used to isolate catabolic organelle (CO) fractions from mouse liver following injection of superparamagnetic dextran (SPD)-coated iron oxide particles and rFVIIa. The effect of co-circulating SPD particles on rFVIIa pharmacokinetic (PK) parameters was evaluated by ELISA. Cryo-immuno transmission electron microscopy (TEM) was used to study hepatic distribution of SPD particles and rFVIIa. The isolated hepatic CO fractions were characterized using Western Blotting (WB).<h4>Results</h4>Cryo-immuno TEM of the liver confirmed hepatic co-localisation of SPD particles and rFVIIa in identical endosomes and lysosomes of both hepatocytes and Kupffer cells. SPD particles did not affect the PK parameters of rFVIIa. WB analysis of plasma and CO fractions detected rFVIIa as the full-length protein and also in high molecular weight (HMW) complexes with ATIII and α-2 macroglobulin (α-2M).<h4>Conclusions</h4>Following injection, both hepatocytes and Kupffer cells appeared to be involved in the hepatic clearance and metabolism of both full-length rFVIIa and rFVIIa in complex with at least two plasma protease inhibitors; ATIII and α-2M.

<h4>Background</h4>The analyzed values in the ICSH reference method for serum iron analysis are affected by non-transferrin(Tf)-bound iron such as ferritin. Also, non-Tf-bound plasma iron (iron citrate) is present in iron-overloaded specimens from patients with hemochromatosis, which was measured as serum iron in previous methods. We developed a specific determination method for serum transferrin-bound iron (serum t Fe) by high-performance liquid immunoaffinity chromatography (HPLAC), and compared it with the ICSH method and a fully automated (FA) method.<h4>Methods</h4>Tf and t Fe were isolated from interferents in serum by HPLC using an immunoaffinity column. The concentration of t Fe isolated was determined by a colorimetric reaction using a highly sensitive chromogen.<h4>Results</h4>Interferents, except iron saccharate (detected at 5%), do not affect t Fe determination. Within-run and between-run imprecisions were in the ranges of 0.2-0.4% and 0.4-1.0% CV. The results of the HPLAC method correlated well with those of the ICSH method (r=0.9993) and FA method (r=0.9984).<h4>Conclusions</h4>In contrast to the ICSH and FA methods for determining serum iron, the HPLAC method is simple, highly precise and specific for serum t Fe, which can contribute to the measurement of iron status.

The formation of organized nanocrystals that resemble enamel is crucial for successful enamel remineralization. Calcium, phosphate and fluoride ions, and amelogenin are important ingredients for the formation of organized hydroxyapatite (HAP) crystals in vitro. However, the effects of these remineralization agents on the enamel crystal morphology have not been thoroughly studied. The objective of this study was to investigate the effects of fluoride ions, supersaturation degree and amelogenin on the crystal morphology and organization of ex vivo remineralized human enamel. Extracted third molars were sliced thin and acid-etched to provide the enamel surface for immersion in different remineralization solutions. The crystal morphology and mineral phase of the remineralized enamel surface were analyzed by field emission-scanning electron microscopy, attenuated total reflection-Fourier transformed infrared and X-ray diffraction. The concentration of fluoride and the supersaturation degree of hydroxyapatite had significant effects on the crystal morphology and crystal organization, which varied from plate-like loose crystals to rod-like densely packed nanocrystal arrays. Densely packed arrays of fluoridated hydroxyapatite nanorods were observed under the following conditions: σ(HAP)=10.2±2.0 with 1.5±0.5 mg l(-1) fluoride and 40±10 μg ml(-1) amelogenin, pH 6.8±0.4. A phase diagram summarizes the conditions that form dense or loose hydroxyapatite nanocrystal structures. This study provides the basis for the development of novel dental materials for caries management.

The purpose of our study was to determine the relationship between mutant huntingtin (Htt) and mitochondrial dynamics in the progression of Huntington's disease (HD). We measured the mRNA levels of electron transport chain genes, and mitochondrial structural genes, Drp1 (dynamin-related protein 1), Fis1 (fission 1), Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), Opa1 (optric atrophy 1), Tomm40 (translocase of outermembrane 40) and CypD (cyclophilin D) in grade III and grade IV HD patients and controls. The mutant Htt oligomers and the mitochondrial structural proteins were quantified in the striatum and frontal cortex of HD patients. Changes in expressions of the electron transport chain genes were found in HD patients and may represent a compensatory response to mitochondrial damage caused by mutant Htt. Increased expression of Drp1 and Fis1 and decreased expression of Mfn1, Mfn2, Opa1 and Tomm40 were found in HD patients relative to the controls. CypD was upregulated in HD patients, and this upregulation increased as HD progressed. Significantly increased immunoreactivity of 8-hydroxy-guanosine was found in the cortical specimens from stage III and IV HD patients relative to controls, suggesting increased oxidative DNA damage in HD patients. In contrast, significantly decreased immunoreactivities of cytochrome oxidase 1 and cytochrome b were found in HD patients relative to controls, indicating a loss of mitochondrial function in HD patients. Immunoblotting analysis revealed 15, 25 and 50 kDa mutant Htt oligomers in the brain specimens of HD patients. All oligomeric forms of mutant Htt were significantly increased in the cortical tissues of HD patients, and mutant Htt oligomers were found in the nucleus and in mitochondria. The increase in Drp1, Fis1 and CypD and the decrease in Mfn1 and Mfn2 may be responsible for abnormal mitochondrial dynamics that we found in the cortex of HD patients, and may contribute to neuronal damage in HD patients. The presence of mutant Htt oligomers in the nucleus of HD neurons and in mitochondria may disrupt neuronal functions. Based on these findings, we propose that mutant Htt in association with mitochondria imbalance and mitochondrial dynamics impairs axonal transport of mitochondria, decreases mitochondrial function and damages neurons in affected brain regions of HD patients.

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

<h4>Background</h4>A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.<h4>Methodology/principal findings</h4>In this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated. Our results showed that forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth. On the other hand, inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis. Further results revealed that these Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj1 and specific commissural markers TAG1 and Pax7 was unchanged.<h4>Conclusions/significance</h4>We first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.

Pilicides are a class of compounds that attenuate virulence in Gram negative bacteria by blocking the chaperone/usher pathway in Escherichia coli. It has also been shown that compounds derived from the peptidomimetic scaffold that the pilicides are based on can prevent both Aβ aggregation and curli formation. To facilitate optimizations towards the different targets, a new synthetic platform has been developed that enables fast and simple introduction of various substituents in position C-7 on the peptidomimetic scaffold. Importantly, this strategy also enables introduction of previously unattainable heteroatoms in this position. Pivotal to the synthetic strategy is the synthesis of a C-7 bromomethyl substituted derivative of the ring-fused dihydrothiazolo 2-pyridone pilicide scaffold. From this versatile and reactive intermediate various heteroatom-linked substituents could be introduced on the scaffold including amines, ethers, amides and sulfonamides. In addition, carbon-carbon bonds could be introduced to the sp(3)-hybridized bromomethyl substituted scaffold by Suzuki-Miyaura cross couplings. Evaluation of the 24 C-7 substituted compounds in whole-bacterial assays provided important structure-activity data and resulted in the identification of a number of new pilicides with activity as good or better than those developed previously.

<h4>Background</h4>Coping Effectively with Heart Failure (COPE-HF) is an ongoing randomized clinical trial funded by the National Institutes of Health to evaluate if a coping skills training (CST) intervention will result in improved health status and quality of life as well as reduced mortality and hospitalizations compared with a heart failure education (HFE) intervention.<h4>Methods and results</h4>Two hundred heart failure (HF) patients recruited from the Duke University Medical Center and the University of North Carolina Hospital system will be randomized to a CST intervention (16 weekly 30-minute telephone counseling sessions including motivational interviewing and individually tailored cognitive behavioral therapy) or to an HFE intervention (16 weekly 30-minute telephone sessions including education and symptom monitoring). Primary outcomes will include postintervention effects on HF biomarkers (B-type natriuretic peptide, ejection fraction) and quality of life, as well as long-term clinical outcomes (hospitalizations and death). Secondary analyses will include an evaluation of treatment effects across subpopulations, and potential mechanisms by which CST may improve clinical outcomes.<h4>Conclusions</h4>COPE-HF is a proof-of-concept study that should provide important insights into the health benefits of a CST intervention designed to enhance HF self-management, improve health behaviors, and reduce psychologic distress.

Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.

<h4>Background aims</h4>Bone marrow stromal cells (BMSC) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSC were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality.<h4>Methods</h4>Early passage BMSC prepared from marrow aspirates of seven healthy subjects were compared with three human embryonic stem cell (hESC) samples, CD34(+) cells from three healthy subjects and three fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35 000 probes.<h4>Results</h4>BMSC gene expression signatures of BMSC differed from those of hematopoietic stem cells (HSC), hESC and fibroblasts. Genes upregulated in BMSC were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The upregulated genes most probably belonged to pathways for integrin signaling, integrin-linked kinase (ILK) signaling, NF-E2-related factor-2 (NFR2)-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wingless-type MMTV integration site (Wnt/β catenin signaling. Among the most highly upregulated genes were structural extracellular matrix (ECM) proteins (α5 and β5 integrin chains, fibronectin and collagen type IIIα1 and Vα1) and functional EMC proteins [connective tissue growth factor (CTGF), transforming growth factor beta-induced protein (TGFBI) and A disintegrin and metalloproteinase (ADAM12)].<h4>Conclusions</h4>Global analysis of human BMSC suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects.

<h4>Background</h4>Injury to the peripheral branch of dorsal root ganglia (DRG) neurons prior to injury to the central nervous system (CNS) DRG branch results in the regeneration of the central branch. The exact mechanism mediating this regenerative trigger is not fully understood. It has been proposed that following peripheral injury, the intraganglionic inflammatory response by macrophage cells plays an important role in the pre-conditioning of injured CNS neurons to regenerate. In this study, we investigated whether the presence of macrophage cells is crucial for this type of regeneration to occur. We used a clodronate liposome technique to selectively and temporarily deplete these cells during the conditioning phase of DRG neurons.<h4>Results</h4>Retrograde and anterograde tracing results indicated that in macrophage-depleted animals, the regenerative trigger characteristic of pre-conditioned DRG neurons was abolished as compared to injury matched-control animals. In addition, depletion of macrophage cells led to: (i) a reduction in macrophage infiltration into the CNS compartment even after cellular repopulation, (ii) astrocyte up-regulation at rostral regions and down-regulation in brain derived neurotrophic factor (BDNF) concentration in the serum.<h4>Conclusion</h4>Activation of macrophage cells in response to the peripheral nerve injury is essential for the enhanced regeneration of ascending sensory neurons.

<h4>Objective</h4>Congenital dyserythropoietic anemia (CDA) is characterized by ineffective erythropoiesis, binuclearity of erythroid precursors and secondary hemochromatosis. Recently, the gene mutated in CDA type II (CDA II), SEC23B, was identified. All Israeli patients with CDA II are of North African (mainly Moroccan) Jewish descent. We investigated the molecular basis of CDA II in those patients.<h4>Methods</h4>Participants included 11 patients with CDA II from 8 apparently unrelated families. Clinical data were retrieved from medical files, and blood was collected for DNA analysis.<h4>Results</h4>The majority of patients (10/11) were homozygous for a common SEC23B mutation (E109K). Haplotype analysis revealed a common genetic background in all patients. One patient was a compound heterozygote for the E109K mutation and a novel mutation, T710M. All patients were transfusion independent, with increasing iron overload with age. We estimate the E109K mutation to be 2,400 years old, in line with Jewish migration history.<h4>Conclusions</h4>Most CDA II patients in Israel are of Moroccan Jewish origin and carry a common SEC23B mutation, E109K, the first to be described as a founder mutation causing CDA II. As previously suggested, carrying 2 missense mutations is associated with a relatively nonsevere phenotype.

An uncharacterized multisystemic mitochondrial cytopathy was diagnosed in two infants from consanguineous Palestinian kindred living in a single village. The most significant clinical findings were tubulopathy (hyperuricemia, metabolic alkalosis), pulmonary hypertension, and progressive renal failure in infancy (HUPRA syndrome). Analysis of the consanguineous pedigree suggested that the causative mutation is in the nuclear DNA. By using genome-wide SNP homozygosity analysis, we identified a homozygous identity-by-descent region on chromosome 19 and detected the pathogenic mutation c.1169A>G (p.Asp390Gly) in SARS2, encoding the mitochondrial seryl-tRNA synthetase. The same homozygous mutation was later identified in a third infant with HUPRA syndrome. The carrier rate of this mutation among inhabitants of this Palestinian isolate was found to be 1:15. The mature enzyme catalyzes the ligation of serine to two mitochondrial tRNA isoacceptors: tRNA(Ser)(AGY) and tRNA(Ser)(UCN). Analysis of amino acylation of the two target tRNAs, extracted from immortalized peripheral lymphocytes derived from two patients, revealed that the p.Asp390Gly mutation significantly impacts on the acylation of tRNA(Ser)(AGY) but probably not that of tRNA(Ser)(UCN). Marked decrease in the expression of the nonacylated transcript and the complete absence of the acylated tRNA(Ser)(AGY) suggest that this mutation leads to significant loss of function and that the uncharged transcripts undergo degradation.

Huntington's disease (HD) is a progressive, neurodegenerative movement disorder. Here, we used fast-scan cyclic voltammetry to measure dopamine release and uptake in striatal brain slices from R6/1 HD model mice. Peak dopamine release ([DA](max)) was significantly diminished in R6/1 mice (52% of wild-type at 24 weeks of age). Similarly, dopamine released per locally applied electrical stimulus pulse ([DA](p)), which is [DA](max) corrected for uptake and electrode performance, was also diminished in R6/1 mice (43% of wild-type by 24 weeks of age). Moreover, V(max), the maximum rate of dopamine uptake, obtained by modeling the stimulated release plots, was decreased at 16 and 24 weeks of age in R6/1 mice (51 and 48% of wild-type, respectively). Thus, impairments in both dopamine release and uptake appear to progress in an age-dependent manner in R6/1 mice.

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women. It is also associated with metabolic disturbances that place women at increased risk for obesity and type 2 diabetes. There is strong evidence for familial clustering of PCOS and a genetic predisposition. However, the gene(s) responsible for the PCOS phenotypes have not been elucidated. This two-phase family-based and case-control genetic study was designed to address the question of whether SNPs identified as susceptibility loci for obesity in genome-wide association studies (GWAS) are also associated with PCOS and elevated BMI. Members of 439 families having at least one offspring with PCOS were genotyped for 15 SNPs previously shown to be associated with obesity. Linkage and association with PCOS was assessed using the transmission/disequilibrium test (TDT). These SNPs were also analyzed in an independent case-control study involving 395 women with PCOS and 176 healthy women with regular menstrual cycles. Only one of these 15 SNPs (rs2815752 in NEGR1) was found to have a nominally significant association with PCOS (χ(2) = 6.11, P = 0.013), but this association failed to replicate in the case-control study. While not associated with PCOS itself, five SNPs in FTO and two in MC4R were associated with BMI as assessed with a quantitative-TDT analysis, several of which replicated association with BMI in the case-control cohort. These findings demonstrate that certain SNPs associated with obesity contribute to elevated BMI in PCOS, but do not appear to play a major role in PCOS per se. These findings support the notion that PCOS phenotypes are a consequence of an oligogenic/polygenic mechanism.

Genetic variants altering cis-regulation of normal gene expression (cis-eQTLs) have been extensively mapped in human cells and tissues, but the extent by which controlled, environmental perturbation influences cis-eQTLs is unclear. We carried out large-scale induction experiments using primary human bone cells derived from unrelated donors of Swedish origin treated with 18 different stimuli (7 treatments and 2 controls, each assessed at 2 time points). The treatments with the largest impact on the transcriptome, verified on two independent expression arrays, included BMP-2 (t = 2h), dexamethasone (DEX) (t = 24 h), and PGE₂ (t = 24 h). Using these treatments and control, we performed expression profiling for 18,144 RefSeq transcripts on biological replicates of the complete study cohort of 113 individuals (n(total) = 782) and combined it with genome-wide SNP-genotyping data in order to map treatment-specific cis-eQTLs (defined as SNPs located within the gene ± 250 kb). We found that 93% of cis-eQTLs at 1% FDR were observed in at least one additional treatment, and in fact, on average, only 1.4% of the cis-eQTLs were considered as treatment-specific at high confidence. The relative invariability of cis-regulation following perturbation was reiterated independently by genome-wide allelic expression tests where only a small proportion of variance could be attributed to treatment. Treatment-specific cis-regulatory effects were, however, 2- to 6-fold more abundant among differently expressed genes upon treatment. We further followed-up and validated the DEX-specific cis-regulation of the MYO6 and TNC loci and found top cis-regulatory variants located 180 kb and 250 kb upstream of the transcription start sites, respectively. Our results suggest that, as opposed to tissue-specificity of cis-eQTLs, the interactions between cellular environment and cis-variants are relatively rare (∼1.5%), but that detection of such specific interactions can be achieved by a combination of functional genomic approaches as described here.

The ATP-binding cassette multidrug resistance protein 8 (MRP8/ABCC11) mediates the excretion of anticancer drugs. ABCC11 mRNA and protein levels were enhanced by DEX (dexamethasone) and by PROG (progesterone) in MCF7 (progesterone receptor-(PR-) positive) but not in MDA-MB-231 (PR-negative) breast cancer cells. This suggested a PR-signaling pathway involvement in ABCC11 regulation. Nevertheless, pregnenolone-16α-carbonitrile (GR antagonist) and clotrimazole strongly and moderately decreased ABCC11 expression levels in Glucocortocoid Receptor-(GR-) and Pregnane X Receptor (PXR)-positive MCF7 cells but not in MDA-MB-231 cells (GR- and PXR-positive). Thus, GR-signaling pathway involvement could not be excluded in ABCC11 regulation in MCF7 cells. Furthermore, ABCC11 levels were positively correlated with the PR status of postmenopausal patient breast tumors from two independent cohorts. Thus, in the subclass of breast tumors (Estrogen Receptor-(ER-) negative/PR-positive), the elevated expression level of ABCC11 may alter the sensitivity to ABCC11 anticancer substrates, especially under treatment combinations with DEX.

Both external and internal exposure to ionizing radiation are strong risk factors for the development of thyroid tumors. Until now, the diagnosis of radiation-induced thyroid tumors has been deduced from a network of arguments taken together with the individual history of radiation exposure. Neither the histological features nor the genetic alterations observed in these tumors have been shown to be specific fingerprints of an exposure to radiation. The aim of our work is to define ionizing radiation-related molecular specificities in a series of secondary thyroid tumors developed in the radiation field of patients treated by radiotherapy. To identify molecular markers that could represent a radiation-induction signature, we compared 25K microarray transcriptome profiles of a learning set of 28 thyroid tumors, which comprised 14 follicular thyroid adenomas (FTA) and 14 papillary thyroid carcinomas (PTC), either sporadic or consecutive to external radiotherapy in childhood. We identified a signature composed of 322 genes which discriminates radiation-induced tumors (FTA and PTC) from their sporadic counterparts. The robustness of this signature was further confirmed by blind case-by-case classification of an independent set of 29 tumors (16 FTA and 13 PTC). After the histology code break by the clinicians, 26/29 tumors were well classified regarding tumor etiology, 1 was undetermined, and 2 were misclassified. Our results help shed light on radiation-induced thyroid carcinogenesis, since specific molecular pathways are deregulated in radiation-induced tumors.

<h4>Background</h4>Until recently, chromosomal translocations and fusion genes have been an underappreciated class of mutations in solid tumors. Next-generation sequencing technologies provide an opportunity for systematic characterization of cancer cell transcriptomes, including the discovery of expressed fusion genes resulting from underlying genomic rearrangements.<h4>Results</h4>We applied paired-end RNA-seq to identify 24 novel and 3 previously known fusion genes in breast cancer cells. Supported by an improved bioinformatic approach, we had a 95% success rate of validating gene fusions initially detected by RNA-seq. Fusion partner genes were found to contribute promoters (5' UTR), coding sequences and 3' UTRs. Most fusion genes were associated with copy number transitions and were particularly common in high-level DNA amplifications. This suggests that fusion events may contribute to the selective advantage provided by DNA amplifications and deletions. Some of the fusion partner genes, such as GSDMB in the TATDN1-GSDMB fusion and IKZF3 in the VAPB-IKZF3 fusion, were only detected as a fusion transcript, indicating activation of a dormant gene by the fusion event. A number of fusion gene partners have either been previously observed in oncogenic gene fusions, mostly in leukemias, or otherwise reported to be oncogenic. RNA interference-mediated knock-down of the VAPB-IKZF3 fusion gene indicated that it may be necessary for cancer cell growth and survival.<h4>Conclusions</h4>In summary, using RNA-sequencing and improved bioinformatic stratification, we have discovered a number of novel fusion genes in breast cancer, and identified VAPB-IKZF3 as a potential fusion gene with importance for the growth and survival of breast cancer cells.

<h4>Background</h4>Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans.<h4>Results</h4>97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species.<h4>Conclusions</h4>Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens.

<h4>Objective</h4>Severe drinking can cause serious morbidity and death. Because the serotonin transporter (5-HTT) is an important regulator of neuronal 5-HT function, allelic differences at that gene may modulate the severity of alcohol consumption and predict therapeutic response to the 5-HT(3) receptor antagonist, ondansetron.<h4>Method</h4>The authors randomized 283 alcoholics by genotype in the 5'-regulatory region of the 5-HTT gene (LL/LS/SS), with additional genotyping for another functional single-nucleotide polymorphism (T/G), rs1042173, in the 3'-untranslated region, in a double-blind controlled trial. Participants received either ondansetron (4 μg/kg twice daily) or placebo for 11 weeks, plus standardized cognitive-behavioral therapy.<h4>Results</h4>Individuals with the LL genotype who received ondansetron had a lower mean number of drinks per drinking day (-1.62) and a higher percentage of days abstinent (11.27%) than those who received placebo. Among ondansetron recipients, the number of drinks per drinking day was lower (-1.53) and the percentage of days abstinent higher (9.73%) in LL compared with LS/SS individuals. LL individuals in the ondansetron group also had a lower number of drinks per drinking day (-1.45) and a higher percentage of days abstinent (9.65%) than all other genotype and treatment groups combined. For both number of drinks per drinking day and percentage of days abstinent, 5'-HTTLPR and rs1042173 variants interacted significantly. LL/TT individuals in the ondansetron group had a lower number of drinks per drinking day (-2.63) and a higher percentage of days abstinent (16.99%) than all other genotype and treatment groups combined.<h4>Conclusions</h4>The authors propose a new pharmacogenetic approach using ondansetron to treat severe drinking and improve abstinence in alcoholics.

<h4>Background</h4>High birth weight is associated with adult body mass index (BMI). We hypothesized that birth weight and BMI may partly share a common genetic background.<h4>Objective</h4>The objective was to examine the associations of 12 established BMI variants in or near the NEGR1, SEC16B, TMEM18, ETV5, GNPDA2, BDNF, MTCH2, BCDIN3D, SH2B1, FTO, MC4R, and KCTD15 genes and their additive score with birth weight.<h4>Design</h4>A meta-analysis was conducted with the use of 1) the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk, Hertfordshire, Fenland, and European Youth Heart Study cohorts (n(max) = 14,060); 2) data extracted from the Early Growth Genetics Consortium meta-analysis of 6 genome-wide association studies for birth weight (n(max) = 10,623); and 3) all published data (n(max) = 14,837).<h4>Results</h4>Only the MTCH2 and FTO loci showed a nominally significant association with birth weight. The BMI-increasing allele of the MTCH2 variant (rs10838738) was associated with a lower birth weight (β ± SE: -13 ± 5 g/allele; P = 0.012; n = 23,680), and the BMI-increasing allele of the FTO variant (rs1121980) was associated with a higher birth weight (β ± SE: 11 ± 4 g/allele; P = 0.013; n = 28,219). These results were not significant after correction for multiple testing.<h4>Conclusions</h4>Obesity-susceptibility loci have a small or no effect on weight at birth. Some evidence of an association was found for the MTCH2 and FTO loci, ie, lower and higher birth weight, respectively. These findings may provide new insights into the underlying mechanisms by which these loci confer an increased risk of obesity.

Health-care information technology and genotyping technology are both advancing rapidly, creating new opportunities for medical and scientific discovery. The convergence of these two technologies is now facilitating genetic association studies of unprecedented size within the context of routine clinical care. As a result, the medical community will soon be presented with a number of novel opportunities to bring functional genomics to the bedside in the area of pharmacotherapy. By linking biological material to comprehensive medical records, large multi-institutional biobanks are now poised to advance the field of pharmacogenomics through three distinct mechanisms: (i) retrospective assessment of previously known findings in a clinical practice-based setting, (ii) discovery of new associations in huge observational cohorts, and (iii) prospective application in a setting capable of providing real-time decision support. This review explores each of these translational mechanisms within a historical framework.

Huntington disease (HD) results from CAG expansion in the huntingtin (HTT) gene. Although HD occurs worldwide, there are large geographic differences in its prevalence. The prevalence in populations derived from Europe is 10-100 times greater than in East Asia. The European general population chromosomes can be grouped into three major haplogroups (group of similar haplotypes): A, B and C. The majority of HD chromosomes in Europe are found on haplogroup A. However, in the East-Asian populations of China and Japan, we find the majority of HD chromosomes are associated with haplogroup C. The highest risk HD haplotypes (A1 and A2), are absent from the general and HD populations of China and Japan, and therefore provide an explanation for why HD prevalence is low in East Asia. Interestingly, both East-Asian and European populations share a similar low level of HD on haplogroup C. Our data are consistent with the hypothesis that different HTT haplotypes have different mutation rates, and geographic differences in HTT haplotypes explain the difference in HD prevalence. Further, the bias for expansion on haplogroup C in the East-Asian population cannot be explained by a higher average CAG size, as haplogroup C has a lower average CAG size in the general East-Asian population compared with other haplogroups. This finding suggests that CAG-tract size is not the only factor important for CAG instability. Instead, the expansion bias may be because of genetic cis-elements within the haplotype that influence CAG instability in HTT, possibly through different mutational mechanisms for the different haplogroups.

The incidence and severity of atherosclerotic vascular disease are increased in diabetic patients, in part because of increased production of reactive oxygen species (ROS). Previously, we found both increased atherosclerosis and arterial protein oxidation 6 months after streptozotocin-induced diabetes in monkeys fed an atherogenic diet, the pattern of which was indicative of redox-active transition metal involvement. The goal of this study was to determine if short-term (1 month) hyperglycemia increases oxidative stress and dysregulates iron metabolism before differences in atherosclerosis. Cynomolgus monkeys with preexisting atherosclerosis were stratified by dietary history and plasma lipids and received either streptozotocin (STZ-DM; n = 10) or vehicle (control; n = 10). One month after diabetes induction, blood and artery samples were collected. There were no differences in plasma lipoprotein cholesterol, arterial cholesterol, and atherosclerosis between control and STZ-DM. However, plasma lipid peroxides were elevated 137% (P < .01); arterial superoxide was increased 47% (P < .05); plasma ferritin, an indicator of whole-body iron stores, was 46% higher (P < .05); and iron deposition within aortic atherosclerotic lesions was more prevalent in STZ-DM compared with controls. Arterial levels of the antioxidant enzymes, superoxide dismutase, catalase, and heme oxygenase-1 were not higher in STZ-DM, although superoxide was higher, suggesting impaired antioxidant response. The increase in ROS before differences in atherosclerosis supports ROS as an initiating event in diabetic vascular disease. Further studies are needed to determine if increases in iron stores and arterial iron deposition promote hydroxyl radical formation from superoxide and accelerate diabetic vascular damage.

While much research has elucidated the neurobiology of fear learning, the neural systems supporting the generalization of learned fear are unknown. Using functional magnetic resonance imaging (fMRI), we show that regions involved in the acquisition of fear support the generalization of fear to stimuli that are similar to a learned threat, but vary in fear intensity value. Behaviorally, subjects retrospectively misidentified a learned threat as a more intense stimulus and expressed greater skin conductance responses (SCR) to generalized stimuli of high intensity. Brain activity related to intensity-based fear generalization was observed in the striatum, insula, thalamus/periacqueductal gray, and subgenual cingulate cortex. The psychophysiological expression of generalized fear correlated with amygdala activity, and connectivity between the amygdala and extrastriate visual cortex was correlated with individual differences in trait anxiety. These findings reveal the brain regions and functional networks involved in flexibly responding to stimuli that resemble a learned threat. These regions may comprise an intensity-based fear generalization circuit that underlies retrospective biases in threat value estimation and overgeneralization of fear in anxiety disorders.

Adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), is resistant to treatment. In this study, we examined the effects of a new inhibitor of deacetylase enzymes, LBH589, on ATLL cells. LBH589 effectively induced apoptosis in ATLL-related cell lines and primary ATLL cells and reduced the size of tumors inoculated in SCID mice. Analyses, including with a DNA microarray, revealed that neither death receptors nor p53 pathways contributed to the apoptosis. Instead, LBH589 activated an intrinsic pathway through the activation of caspase-2. Furthermore, small interfering RNA experiments targeting caspase-2, caspase-9, RAIDD, p53-induced protein with a death domain (PIDD) and RIPK1 (RIP) indicated that activation of RAIDD is crucial and an event initiating this pathway. In addition, LBH589 caused a marked decrease in levels of factors involved in ATLL cell proliferation and invasion such as CCR4, IL-2R and HTLV-1 HBZ-SI, a spliced form of the HTLV-1 basic zipper factor HBZ. In conclusion, we showed that LBH589 is a strong inducer of apoptosis in ATLL cells and uncovered a novel apoptotic pathway initiated by activation of RAIDD.

C282Y and H63D are two common variants of the hemochromatosis protein HFE. SH-SY5Y human neuroblastoma cells stably transfected to express either wild type HFE (WT-HFE), or the C282Y or H63D allele were analyzed for effect of expression of the mutant proteins on transcription of 14 enzymes involved in sphingolipid metabolism. Cells expressing the C282Y variant showed significant increases (>2-fold) in transcription of five genes and decreases in two compared to that seen for cells expressing WT-HFE, while cells expressing the H63D variant showed an elevation in transcription of one gene and a decrease in two. These changes were seen as alterations in ganglioside composition, cell surface binding by the binding subunit of cholera toxin, expression of sphingosine-kinase-1 and synthesis of sphingosine-1-phosphate. These changes may explain why C282Y-HFE is a risk factor for colon and breast cancer and possibly protective against Alzheimer's disease while H63D-HFE is a risk factor for neurodegenerative diseases.

Hepatic steatosis is one of the most common liver disorders in the general population. The main cause of hepatic steatosis is nonalcoholic fatty liver disease (NAFLD), representing the hepatic component of the metabolic syndrome, which is characterized by type 2 diabetes, obesity, and dyslipidemia. Insulin resistance and excess adiposity are considered to play key roles in the pathogenesis of NAFLD. Although the risk factors for NAFLD are well established, the genetic basis of hepatic steatosis is largely unknown. Here we review recent progress on genomic variants and their association with hepatic steatosis and discuss the potential impact of these genetic studies on clinical practice. Identifying the genetic determinants of hepatic steatosis will lead to a better understanding of the pathogenesis and progression of NAFLD.

An expanded polyglutamine tract (>37 glutamines) in the N-terminal region of huntingtin (htt) causes htt to accumulate in the nucleus, leading to transcriptional dysregulation in Huntington disease (HD). In HD knock-in mice that express full-length mutant htt at the endogenous level, mutant htt preferentially accumulates in the nuclei of striatal neurons, which are affected most profoundly in HD. The mechanism underlying this preferential nuclear accumulation of mutant htt in striatal neurons remains unknown. Here, we report that serine 16 (S16) in htt is important for the generation of small N-terminal fragments that are able to accumulate in the nucleus and form aggregates. Phosphorylation of N-terminal S16 in htt promotes the nuclear accumulation of small N-terminal fragments and reduces the interaction of N-terminal htt with the nuclear pore complex protein Tpr. Mouse brain striatal tissues show increased S16 phosphorylation and a decreased association between mutant N-terminal htt and Tpr. These findings provide mechanistic insight into the nuclear accumulation of mutant htt and the selective neuropathology of HD, revealing potential therapeutic targets for treating this disease.

SMAD4 is localized to chromosome 18q21, a frequent site for loss of heterozygosity in advanced stage colon cancers. Although Smad4 is regarded as a signaling mediator of the TGFβ signaling pathway, its role as a major suppressor of colorectal cancer progression and the molecular events underlying this phenomenon remain elusive. Here, we describe the establishment and use of colon cancer cell line model systems to dissect the functional roles of TGFβ and Smad4 inactivation in the manifestation of a malignant phenotype. We found that loss of function of Smad4 and retention of intact TGFβ receptors could synergistically increase the levels of VEGF, a major proangiogenic factor. Pharmacologic inhibition studies suggest that overactivation of the TGFβ-induced MEK-Erk and p38-MAPK (mitogen-activated protein kinase) auxiliary pathways are involved in the induction of VEGF expression in SMAD4 null cells. Overall, SMAD4 deficiency was responsible for the enhanced migration of colon cancer cells with a corresponding increase in matrix metalloprotease 9 enhanced hypoxia-induced GLUT1 expression, increased aerobic glycolysis, and resistance to 5'-fluoruracil-mediated apoptosis. Interestingly, Smad4 specifically interacts with hypoxia-inducible factor (HIF) 1α under hypoxic conditions providing a molecular basis for the differential regulation of target genes to suppress a malignant phenotype. In summary, our results define a molecular mechanism that explains how loss of the tumor suppressor Smad4 promotes colorectal cancer progression. These findings are also consistent with targeting TGFβ-induced auxiliary pathways, such as MEK-ERK, and p38-MAPK and the glycolytic cascade, in SMAD4-deficient tumors as attractive strategies for therapeutic intervention.

The assembly of MHC class I molecules is governed by stringent endoplasmic reticulum (ER) quality control mechanisms. MHC class I heavy chains that fail to achieve their native conformation in complex with β2-microglobulin (β2m) and peptide are targeted for ER-associated degradation. This requires ubiquitination of the MHC class I heavy chain and its dislocation from the ER to the cytosol for proteasome-mediated degradation, although the cellular machinery involved in this process is unknown. Using an siRNA functional screen in β2m-depleted cells, we identify an essential role for the E3 ligase HRD1 (Synoviolin) together with the E2 ubiquitin-conjugating enzyme UBE2J1 in the ubiquitination and dislocation of misfolded MHC class I heavy chains. HRD1 is also required for the ubiquitination and degradation of the naturally occurring hemochromatosis-associated HFE-C282Y mutant, which is unable to bind β2m. In the absence of HRD1, misfolded HLA-B27 accumulated in cells with a normal MHC class I assembly pathway, and HRD1 depletion prevented the appearance of low levels of cytosolic unfolded MHC I heavy chains. HRD1 and UBE2J1 associate in a complex together with non-β2m bound MHC class I heavy chains, Derlin 1, and p97 and discriminate misfolded MHC class I from conformational MHC I-β2m-peptide heterotrimers. Together these data support a physiological role for HRD1 and UBE2J1 in the homeostatic regulation of MHC class I assembly and expression.

The induction of the iron-regulatory peptide hepcidin by proinflammatory cytokines is thought to result in the withholding of iron from invading pathogens. Hfe and transferrin receptor 2 (Tfr2) are involved in the homeostatic regulation of hepcidin and their disruption causes hereditary hemochromatosis (HH). To determine whether either Hfe or Tfr2 is involved in the inflammatory pathway regulating hepcidin, we analyzed the effect of inflammation in 3 mouse models of HH. The inflammatory response and indicators of iron homeostasis were measured in wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice injected with lipopolysaccharide (LPS). The administration of LPS significantly reduced serum iron in wild-type and Hfe(-/-) mice, with smaller reductions in Tfr2(-/-) and Hfe(-/-)/Tfr2(-/-) mice. Low basal levels of hepcidin in the Hfe(-/-)/Tfr2(-/-) mice were increased in response to LPS, but remained significantly lower than in the other strains of mice. These results suggest that despite the absence of Hfe and Tfr2, hepcidin is responsive to inflammation; however, the low basal expression and subsequent low levels of circulating hepcidin are insufficient to reduce serum iron effectively. This suggests that in HH, the iron-withholding response to invading pathogens may be inadequate, and this is especially the case in the absence of both Hfe and Tfr2.

The CAG repeat expansions that occur in translated regions of specific genes can cause human genetic disorders known as polyglutamine (poly-Q)-triggered diseases. Huntington's disease and spinobulbar muscular atrophy (SBMA) are examples of these diseases in which underlying mutations are localized near other trinucleotide repeats in the huntingtin (HTT) and androgen receptor (AR) genes, respectively. Mutant proteins that contain expanded polyglutamine tracts are well-known triggers of pathogenesis in poly-Q diseases, but a toxic role for mutant transcripts has also been proposed. To gain insight into the structural features of complex triplet repeats of HTT and AR transcripts, we determined their structures in vitro and showed the contribution of neighboring repeats to CAG repeat hairpin formation. We also demonstrated that the expanded transcript is retained in the nucleus of human HD fibroblasts and is colocalized with the MBNL1 protein. This suggests that the CAG repeats in the HTT mRNA adopt ds-like RNA conformations in vivo. The intracellular structure of the CAG repeat region of mutant HTT transcripts was not sufficiently stable to be protected from cleavage by an siRNA targeting the repeats and the silencing efficiency was higher for the mutant transcript than for its normal counterpart.

<h4>Objective</h4>The aim of the present study was to determine if the electroporation-mediated gene transfer of SOX trio enhances the chondrogenic potential of adipose stem cells (ASCs).<h4>Design</h4>ASCs were transfected with SOX trio genes using an electroporation technique and cultured for 3 weeks. The pellets were analyzed for DNA and glycosaminoglycan (GAG) analysis, and the gene and protein expression of SOX-5, SOX-6, SOX-9, type 1 collagen (COL1Al), type 2 collagen (COL2Al) and type 10 collagen (COL10A1) using real-time PCR and Western blot analysis. Further in vivo studies were carried out by subcutaneous transplantation of pellets in severe combined immunodeficiency (SCID) mice for 3 weeks.<h4>Results</h4>The gene transfer efficiency was high (approximately 70%). Transfected ASCs showed high expression of corresponding genes after 21 days, and each SOX protein was detected in ASCs transfected with the corresponding gene. The chondrogenic differentiation of ASCs, as demonstrated by GAG levels and Safranin-O staining, showed significant enhancement when SOX trio were co-transfected, while subsets with single gene transfer of SOX-5, -6, or -9 did not show significant elevation. SOX trio co-transfection enhanced COL2A1 mRNA, but did not increase COL1A1 and COL10A1 mRNA. Type II collagen protein dramatically increased, and type X collagen decreased with co-transfection of the SOX trio. When pellets were implanted in the subcutaneous pouch of SCID mice for 3 weeks, ASCs co-transfected with SOX trio demonstrated abundant proteoglycan, significantly reduced mineralization.<h4>Conclusion</h4>The electroporation-mediated transfection of SOX trio greatly enhances chondrogenesis from ASCs, while decreasing hypertrophy.

<h4>Background</h4>Hepatocellular carcinoma (HCC) is the fifth most common neoplasia in the world. In the past, treatment of advanced HCC with conventional antineoplastic drugs did not result in satisfactory outcomes: recently, in this patient population the oral multikinase inhibitor sorafenib has been able to induce a statistically significant improvement of overall survival. Similarly to other anti-angiogenic drugs employed in other tumour types, also sorafenib seldom induces the dimensional tumour shrinking usually observed with conventional cytotoxic drugs: data gathered from studies carried out with sorafenib and other competitors under development do not report any complete response in HCV-induced HCC.<h4>Case presentation</h4>An 84-year old man with a long-lasting history of chronic HCV hepatitis was referred to our Institution for an ultrasonography investigation of a focal hepatic lesion. To better characterize the liver disease and clearly define the diagnosis of the focal hepatic lesion, the patient was hospitalized in our department. Laboratory and instrumental investigations confirmed the clinical picture of HCV-related liver cirrhosis and identified a hepatic lesion of about 6 cm featuring infiltrating HCC with thrombosis of the portal trunk. Due to the advanced stage of the disease, therapy with sorafenib 400 mg bid was started. Right from one month after the treatment was started, a reduction of alpha-fetoprotein level was observed which, by the third month, turned down within the normal limits. In addition the CT scan showed 50% reduction of the neoplastic lesion along with canalization of the portal trunk. At the sixth month the normalization of the alpha-fetoprotein level at the lower limit of normality was confirmed and the MRI showed complete disappearance of the neoplasia. In addition a reduction of a metallo-proteinase serum level was observed. At the twelfth month a further MRI confirmed complete response had been maintained. At present the patient is in a follow-up program to evaluate the duration of the complete response.<h4>Conclusions</h4>This case is worth mentioning since, to the best of our knowledge, it represents the first evidence of complete response to sorafenib in an elderly patient with advanced HCV-related HCC.

Ikaros is associated with both gene transcriptional activation and repression in lymphocytes. Ikaros acts also as repressor of human γ-globin (huγ-) gene transcription in fetal and adult erythroid cells. Whether and eventually, how Ikaros can function as a transcriptional activator in erythroid cells remains poorly understood. Results presented herein demonstrate that Ikaros is a developmental-specific activator of huγ-gene expression in yolk sac erythroid cells. Molecular analysis in primary cells revealed that Ikaros interacts with Gata-1 and favors Brg1 recruitment to the human β-globin Locus Control Region and the huγ-promoters, supporting long-range chromatin interactions between these regions. Additionally, we demonstrate that Ikaros contributes to transcription initiation and elongation of the huγ-genes, since it is not only required for TBP and RNA Polymerase II (Pol II) assembly at the huγ-promoters but also for conversion of Pol II into the elongation-competent phosphorylated form. In agreement with the latter, we show that Ikaros interacts with Cyclin-dependent kinase 9 (Cdk9), which contributes to efficient transcription elongation by phosphorylating the C-terminal domain of the large subunit of Pol II on Serine 2, and favours Cdk9 recruitment to huγ-promoters. Our results show that Ikaros exerts dual functionality during gene activation, by promoting efficient transcription initiation and elongation.

Background. Transcatheter arterial lipiodol chemoembolization (TACE) can be used in cirrhotic patients with hepatocellular carcinoma to avoid tumor progression before transplantation. Objective. To evaluate the efficacy and safety of TACE used as a bridge to liver transplantation. Methods. TACE was performed in 30 cirrhotic patients with hepatocellular carcinoma. Milan criteria were used to select patients for transplant. Patients had a good or moderately impaired liver function, no arterioportal fistulae, and a good portal perfusion. Results. 48 TACE were performed in 30 patients. Before transplantation, 4 patients were dropped off the list due to tumor extension or liver failure. Complete necrosis of the tumor was observed in 11 patients and partial necrosis in 15 patients. After transplantation, 6 patients died and tumor recurrence was observed in 5 patients with a tumor beyond Milan criteria or no response to TACE. Conclusion. TACE is useful as a bridge to liver transplantation in a selected group of cirrhotic patients with hepatocellular carcinoma. A combined therapeutic approach before surgery might improve the prognosis in these patients.

<h4>Purpose</h4>For decades of years, hundreds of candidate gene-based association studies explored the relationship between single nucleotide polymorphisms (SNPs) and hepatocellular carcinoma (HCC). There was no systematic review summarized the results of these association studies of candidate SNPs and HCC to date. In order to summarize the results of the association studies, we conducted a concise systematic review.<h4>Methods</h4>By searching Pubmed database before October 2010, we reviewed all the association studies about candidate SNPs and HCC. If the eligible study number on a given SNP was more than three, we conducted a meta-analysis. We reported here only the overall positive-association results with statistical significance and evaluated the reliability of the associations by using false-positive report probability (FPRP) analysis and the Venice guidelines on genetic epidemiology studies.<h4>Results</h4>Six SNPs of five genes (rs1800562 of HFE, rs17868323 and rs11692021 of UGT1A7, rs2279744 of MDM2, rs1143627 of IL-1B, and rs4880 of MnSOD) showed overall significant associations with HCC. The eligible number of the studies varied from three to nine. Two SNPs (rs1800562 of HFE and rs2279744 of MDM2) passed the FPRP threshold (FPRP < 0.20). According to the Venice guidelines, the associations between the two SNPs (rs1800562 and rs2279744) and HCC were of moderate evidence.<h4>Conclusions</h4>Two SNPs (rs1800562 of HFE and rs2279744 of MDM2) were associated with HCC with moderate epidemiological evidence and deserve further study and additional biological and clinical assessment.

The human chemokine lymphotactin (Ltn) is a remarkable protein that interconverts between two unrelated native state structures in the condensed phase. It is possible to shift the equilibrium toward either conformation with selected sequence substitutions. Previous results have shown that a disulfide-stabilized variant preferentially adopts the canonical chemokine fold (Ltn10), while a single amino acid change (W55D) favors the novel Ltn40 dimeric structure. Selective noncovalent adduct protein probing (SNAPP) is a recently developed method for examining solution phase protein structure. Herein, it is demonstrated that SNAPP can easily recognize and distinguish between the Ltn10 and Ltn40 states of lymphotactin in aqueous solution. The effects of organic denaturants, acid, and disulfide bond reduction and blocking were also examined using SNAPP for the CC3, W55D, and wild type proteins. Only disulfide reduction was shown to significantly perturb the protein, and resulted in considerably decreased adduct formation consistent with loss of tertiary/secondary structure. Cold denaturation experiments demonstrated that wild-type Ltn is the most temperature sensitive of the three proteins. Examination of the higher charge states in all experiments, which are presumed to represent transition state structures between Ltn-10 and Ltn-40, reveals increased 18C6 attachment relative to the more folded structures. This observation is consistent with increased competitive intramolecular hydrogen bonding, which may guide the transition. Experiments examining the gas phase structures revealed that all three proteins can be structurally distinguished in the gas phase. In addition, the gas phase experiments enabled identification of preferred adduct binding sites.

This study's goal was to assess the safety of tumor-targeted interleukin-12 (ttIL-12) when administered by electrogenetherapy in C3H/HeJ mice by identifying an initial safe dose for human dose escalation schemes, toxicity target organs, markers of toxicity, and toxicity reversibility. Tumor-free mice receiving two doses of 0.45% NaCl, 1 μg ttIL-12 DNA in 0.45% NaCl or 5 μg ttIL-12 DNA in 0.45% NaCl, 10 days apart combined with low-intensity electroporation were compared with non-treatment controls over time. All mice had blood cell counts, serum chemistry profiles, plasma interleukin-12 and IFNγ determinations, necropsy and multi-organ histopathology. Mild treatment-associated changes included electroporation-associated muscle changes that resolved by 30 days; decreased total white blood cell counts and infectious disease in the 5 μg ttIL-12 group, but not in the 1 μg group, and liver changes in ttIL-12 groups that correlated with alanine transaminase levels and resolved by 30 days. Dystrophic cardiac calcification seen in older, 5 μg ttIL-12-treated mice was the only serious toxicity. Based on these results and the lack of any effect on wound healing when combined with surgery, low-intensity electrogenetherapy with ttIL-12 appears to be safe and well tolerated.

<h4>Background & aims</h4>Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors due to their conversion into postmitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SCs), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiologic ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4).<h4>Methods</h4>Transgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ER(T2)).<h4>Results</h4>Notch1 signaling was found to be activated in intestinal SCs. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into postmitotic goblet cells, concomitant with loss of SCs (Olfm4(+), Lgr5(+), and Ascl2(+)). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut.<h4>Conclusions</h4>Notch signaling in SCs and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SCs. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice.

Low expression of the human serotonin transporter (5-HTT) gene presumably interacts with stressful life events enhancing susceptibility for affective disorders. 5-Htt knockout (KO) mice display an anxious phenotype, and behavioural differences compared to wild-type (WT) mice are exacerbated after repeated loser experience in a resident-intruder stress paradigm. To assess whether genotype-dependent and stress-induced behavioural differences are reflected in alterations of neuronal morphology in limbic areas, we studied dendritic length and complexity of pyramidal neurons in the anterior cingulate and infralimbic cortices (CG, IL), hippocampus CA1 region, and of pyramidal neurons and interneurons in the lateral (La) and basolateral (BL) amygdaloid nuclei in Golgi-Cox-stained brains of male WT and 5-Htt KO control and loser mice. Spine density was analysed for IL apical and amygdaloid apical and basal pyramidal neuron dendrites. While group differences were absent for parameters analysed in CG, CA1 and amygdaloid interneurons, pyramidal neurons in the IL displayed tendencies to shorter and less spinous distal apical dendrites in 5-Htt KO controls, and to extended proximal dendrites in WT losers compared to WT controls. In contrast, spine density of several dendritic compartments of amygdaloid pyramids was significantly higher in 5-Htt KO mice compared to WT controls. While a tendency to increased spine density was observed in the same dendritic compartments in WT after stress, changes were lacking in stressed compared to control 5-Htt KO mice. Our findings indicate that disturbed 5-HT homeostasis results in alterations of limbic neuronal morphology, especially in higher spinogenesis in amygdaloid pyramidal neurons. Social stress leads to similar but less pronounced changes in the WT, and neuroplasticity upon stress is reduced in 5-Htt KO mice.

<h4>Background & aims</h4>Pathologists participating in the National Institutes of Health-sponsored Biliary Atresia Research Consortium (BARC) developed and then evaluated a standardized system for histologic reporting of liver biopsies from infants with cholestasis.<h4>Methods</h4>A set of 97 anonymous liver biopsy samples was sent to 10 pathologists at BARC centers. A semiquantitative scoring system that had 16 histologic features was developed and then used by the pathologists, who had no knowledge of clinical history, imaging results, or laboratory data. Interobserver agreement was evaluated statistically. Agreement on scoring of each feature and on the pathologists' diagnosis, compared with the final clinical diagnosis, was evaluated by using weighted kappa statistics.<h4>Results</h4>There was moderate to substantial interobserver agreement in identification of bile plugs in ducts, giant-cell transformation, extramedullary hematopoiesis, and bile duct proliferation. The pathologists' diagnosis of obstruction in clinically proven cases of biliary atresia (BA) ranged from 79%-98%, with a positive predictive value of 90.7%. Histologic features that best predicted BA, on the basis of logistic regression, included bile duct proliferation, portal fibrosis, and absence of sinusoidal fibrosis (each P<.0001).<h4>Conclusions</h4>The BARC histologic assessment system identified features of liver biopsies from cholestatic infants, with good interobserver agreement, that might be used in diagnosis and determination of prognosis. The system diagnosed BA with a high level of sensitivity and identified infants with biliary obstruction with reasonable interobserver agreement. However, distinguishing between BA and disorders such as total parenteral nutrition-associated liver disease and alpha(1)-antitrypsin deficiency is not possible without adequate clinical information.

Thiopurines including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine are effective anticancer agents with remarkable success in clinical practice, especially in effective treatment of acute lymphoblastic leukemia (ALL). (S)G is understood to act as a DNA hypomethylating agent in ALL cells, however, the underlying mechanism leading to global cytosine demethylation remains unclear. Here we report that (S)G treatment results in reactivation of epigenetically silenced genes in T leukemia cells. Bisulfite genomic sequencing revealed that (S)G treatment universally elicited demethylation in the promoters and/or first exons of the genes that were reactivated. (S)G treatment also attenuated the expression of histone lysine-specific demethylase 1 (LSD1), thereby stimulating lysine methylation of the DNA methylase DNMT1 and triggering its degradation via the ubiquitin-proteasomal pathway. Taken together, our findings reveal a previously uncharacterized but vital mechanistic link between (S)G treatment and DNA hypomethylation.

SDS3 is a key component of the histone deacetylase (HDAC)-dependent Sin3A co-repressor complex, serving to maintain its HDAC activity. Here, we report both exogenous and endogenous functional interaction between deubiquitinating enzyme USP17 and human SDS3 by MALDI-TOF-MS, co-immunoprecipitation assay, and GST pull-down assay. In this study, we demonstrated that SDS3 readily undergoes endogenous polyubiquitination, which is associated specifically with Lys-63-branched polyubiquitin chains and not with Lys-48-branched polyubiquitin chains. Further, we also demonstrated that USP17 specifically deubiquitinates Lys-63-linked ubiquitin chains from SDS3 and regulates its biological functions. The deubiquitinating activity of USP17 on SDS3 negatively regulates SDS3-associated HDAC activity. The constitutive expression of USP17 and its substrate SDS3 was involved in the inhibition of anchorage-independent tumor growth and blocks cell proliferation, leading to apoptosis in cervical carcinoma cells. Furthermore, we showed that USP17 and SDS3 mutually interact with each other to regulate cancer cell viability. These data support the possibility that SDS3, being a substrate of USP17, may play an important role in developing a novel therapeutic means to inhibit specific HDAC activities in cancer.

The molecular mechanisms controlling sex determination and differentiation in zebrafish (Danio rerio) are largely unknown. A genome-wide analysis may provide comprehensive insights into the processes involved. The mRNA expression in zebrafish gonads has been fairly well studied, but much less data on the corresponding protein expression are available, although the proteins are considered to be more relevant markers of gene function. Because mRNA and protein abundances rarely correlate well, mRNA profiles need to be complemented with the information on protein expression. The work presented here analyzed the proteomes of adult zebrafish gonads by a multidimensional protein identification technology, generating the to-date most populated lists of proteins expressed in mature zebrafish gonads. The acquired proteomics data partially confirmed existing transcriptomics information for several genes, including several novel transcripts. However, disagreements between mRNA and protein abundances were often observed, further stressing the necessity to assess the expression on different levels before drawing conclusions on a certain gene's expression and function. Several gene groups expressed in a sexually dimorphic way in zebrafish gonads were identified. Their potential importance for gonad development and function is discussed. The data gained in the current study provide a basis for further work on elucidating processes occurring during zebrafish development with use of high-throughput proteomics.

<h4>Background</h4>Inadequate and inappropriate complementary feeding are major factors contributing to excess morbidity and mortality in young children in low resource settings. Animal source foods in particular are cited as essential to achieve micronutrient requirements. The efficacy of the recommendation for regular meat consumption, however, has not been systematically evaluated.<h4>Methods/design</h4>A cluster randomized efficacy trial was designed to test the hypothesis that 12 months of daily intake of beef added as a complementary food would result in greater linear growth velocity than a micronutrient fortified equi-caloric rice-soy cereal supplement. The study is being conducted in 4 sites of the Global Network for Women's and Children's Health Research located in Guatemala, Pakistan, Democratic Republic of the Congo (DRC) and Zambia in communities with toddler stunting rates of at least 20%. Five clusters per country were randomized to each of the food arms, with 30 infants in each cluster. The daily meat or cereal supplement was delivered to the home by community coordinators, starting when the infants were 6 months of age and continuing through 18 months. All participating mothers received nutrition education messages to enhance complementary feeding practices delivered by study coordinators and through posters at the local health center. Outcome measures, obtained at 6, 9, 12, and 18 months by a separate assessment team, included anthropometry; dietary variety and diversity scores; biomarkers of iron, zinc and Vitamin B12 status (18 months); neurocognitive development (12 and 18 months); and incidence of infectious morbidity throughout the trial. The trial was supervised by a trial steering committee, and an independent data monitoring committee provided oversight for the safety and conduct of the trial.<h4>Discussion</h4>Findings from this trial will test the efficacy of daily intake of meat commencing at age 6 months and, if beneficial, will provide a strong rationale for global efforts to enhance local supplies of meat as a complementary food for young children.<h4>Trial registration</h4>NCT01084109.

Little is known about endogenous estrogen receptor β (ERβ) gene targets in human breast cancer. We reported that estradiol (E(2)) induces nuclear respiratory factor-1 (NRF-1) transcription through ERα in MCF-7 breast cancer cells. Here we report that 4-hydroxytamoxifen (4-OHT), with an EC(50) of ~1.7 nM, increases NRF-1 expression by recruiting ERβ, cJun, cFos, CBP, and RNA polymerase II to and dismissing NCoR from the NRF1 promoter. Promoter deletion and transient transfection studies showed that the estrogen response element (ERE) is essential and that an adjacent AP-1 site contributes to maximal 4-OHT-induced NRF-1 transcription. siRNA knockdown of ERβ revealed that ERβ inhibits basal NRF-1 expression and is required for 4-OHT-induced NRF-1 transcription. An AP-1 inhibitor blocked 4-OHT-induced NRF-1 expression. The 4-OHT-induced increase in NRF-1 resulted in increased transcription of NRF-1 target CAPNS1 but not CYC1, CYC2, or TFAM despite increased NRF-1 coactivator PGC-1α protein. The absence of TFAM induction corresponds to a lack of Akt-dependent phosphorylation of NRF-1 with 4-OHT treatment. Overexpression of NRF-1 inhibited 4-OHT-induced apoptosis and siRNA knockdown of NRF-1 increased apoptosis, indicating an antiapoptotic role for NRF-1. Overall, NRF-1 expression and activity is regulated by 4-OHT via endogenous ERβ in MCF-7 cells.

<h4>Purpose</h4>Epidemiological and experimental studies have revealed that exposure to ultraviolet B (UVB) light can induce cataractogenesis. The objective of this study was to determine gene expression changes in human lens epithelial cells in response to UVB exposure and identify factors that can be involved in UVB-induced cataractogenesis.<h4>Methods</h4>SV40 T-antigen-transformed human lens epithelial cells (SRA01/04) were irradiated at various UVB-energy levels (10-80 mJ/cm²) and checked for viability. An irradiation condition of 30 mJ/cm² was adopted for transcriptome analysis. Total RNAs isolated from UVB-exposed and unexposed cells at 12 h and 24 h after UVB exposure were examined for global gene expression changes using Affymetrix Human Gene 1.0 ST array. mRNA levels of specific genes were examined by RT-PCR and real-time PCR, and protein levels in the conditioned media were assayed by ELISA. To examine mRNA expression in human lens, primary cultured human lens epithelial (HLE) cells were prepared from surgically removed lens epithelium, and used for UVB-irradiation and expression analysis. Effects of certain gene products on SRA01/04 cell metabolism were examined using commercially available recombinant proteins.<h4>Results</h4>Expression of most the genes analyzed was essentially unchanged (between 0.5 and 2.0 fold) in UVB-irradiated cells compared to non-irradiated cells at both 12 and 24 h after UVB exposure. Sixty one and 44 genes were upregulated more than twofold by UVB exposure at 12 h and 24 h, respectively. Emphasis was placed on genes encoding extracellular proteins, especially growth factors and cytokines. A total of 18 secreted protein genes were upregulated more than twofold at either or both time points. Amphiregulin (AREG) and growth differentiation factor 15 (GDF15) were chosen because of their higher upregulation and novelty, and their upregulation was confirmed in SRA01/04 cells using RT-PCR and real-time PCR analysis. AREG and GDF15 protein levels in conditioned media significantly increased at all UVB-energy points at 24 h, while they were scarcely detectable at 12 h. AREG and GDF15 mRNA levels were also significantly upregulated in UVB-irradiated primary cultured HLE cells compared with the corresponding control culture. AREG significantly stimulated ³H-thymidine and ³H-leucine uptake in SRA01/04 cells as did a positive control epidermal growth factor (EGF). Recombinant GDF15 did not stimulate ³H-thymidine incorporation at any concentration tested, but significantly stimulated ³H-leucine uptake. RT-PCR analysis demonstrated that primary cultured HLE and SRA01/04 cells expressed not only epidermal growth factor receptor (EGFR) mRNA but also transforming growth factor β receptors (TGFBR1 and TGFBR2) mRNAs.<h4>Conclusions</h4>These results indicate that AREG and GDF15 produced in response to UVB exposure can affect the growth and protein synthesis of lens epithelial cells, suggesting that they have autocrine and paracrine roles related to pathological changes of lens tissue during long-term UVB exposure.

<h4>Background</h4>As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.<h4>Methodology/principal findings</h4>A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated.<h4>Conclusions/significance</h4>These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

Hypertension and diabetes mellitus are important risk factors for chronic kidney disease (CKD). We previously showed that the C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with myocardial infarction. The purpose of the present study was to examine an association of rs6929846 of BTN2A1 with CKD in individuals with or without hypertension or diabetes mellitus, thereby contributing to the personalized prevention of CKD in such individuals separately. The study population comprised 7,542 unrelated individuals, including 2,289 subjects with CKD [estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m(2)] and 5,253 controls (eGFR ≥60 ml/min/1.73 m(2)) with or without hypertension or diabetes mellitus. The Chi-square test, a multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that the C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with CKD in normotensive individuals, in diabetic individuals and in individuals with hypertension and diabetes mellitus, or without either condition, with the T allele representing a risk factor for CKD. Stratification of subjects based on hypertension or diabetes mellitus may thus be important in order to achieve personalized prevention of CKD with the use of genetic information.

<h4>Background</h4>Systems biological approach of molecular connectivity map has reached to a great interest to understand the gene functional similarities between the diseases. In this study, we developed a computational framework to build molecular connectivity maps by integrating mutated and differentially expressed genes of neurological and psychiatric diseases to determine its relationship with aging.<h4>Results</h4>The systematic large-scale analyses of 124 human diseases create three classes of molecular connectivity maps. First, molecular interaction of disease protein network generates 3632 proteins with 6172 interactions, which determines the common genes/proteins between diseases. Second, Disease-disease network includes 4845 positively scored disease-disease relationships. The comparison of these disease-disease pairs with Medical Subject Headings (MeSH) classification tree suggests 25% of the disease-disease pairs were in same disease area. The remaining can be a novel disease-disease relationship based on gene/protein similarity. Inclusion of aging genes set showed 79 neurological and 20 psychiatric diseases have the strong association with aging. Third and lastly, a curated disease biomarker network was created by relating the proteins/genes in specific disease contexts, such analysis showed 73 markers for 24 diseases. Further, the overall quality of the results was achieved by a series of statistical methods, to avoid insignificant data in biological networks.<h4>Conclusions</h4>This study improves the understanding of the complex interactions that occur between neurological and psychiatric diseases with aging, which lead to determine the diagnostic markers. Also, the disease-disease association results could be helpful to determine the symptom relationships between neurological and psychiatric diseases. Together, our study presents many research opportunities in post-genomic biomarkers development.

The potential for mitochondrial (mt) DNA mutation accumulation during antiretroviral therapy (ART), and preferential accumulation in patients with lipoatrophy compared with control participants, remains controversial. We sequenced the entire mitochondrial genome, both before ART and after ART exposure, in 29 human immunodeficiency virus (HIV)-infected Swiss HIV Cohort Study participants initiating a first-line thymidine analogue-containing ART regimen. No accumulation of mtDNA mutations or deletions was detected in 13 participants who developed lipoatrophy or in 16 control participants after significant and comparable ART exposure (median duration, 3.3 and 3.7 years, respectively). In HIV-infected persons, the development of lipoatrophy is unlikely to be associated with accumulation of mtDNA mutations detectable in peripheral blood.

Dlx5, a homeobox transcription factor, plays a key role in the development of many organ systems. It is a candidate gene for human split-hand/split-foot type 1 malformation associated with sensorineural hearing loss. A deletion of one of its enhancers has been implicated in human craniofacial defects/hearing loss and it has also been associated with autism. However, little is known of how Dlx5 exerts its regulatory effects. We identified direct targets of Dlx5 in the mouse inner ear by gene expression profiling wild-type and Dlx5 null otic vesicles from embryonic stages E10 and E10.5. Four hundred genes were differentially expressed. We examined the genomic DNA sequences in the promoter regions of these genes for (i) previously described Dlx5 binding sites, (ii) novel 12 bp long motifs with a canonical homeodomain element shared by two or more genes and (iii) 100% conservation of these motifs in promoters of human orthologs. Forty genes passed these filters, 12 of which are expressed in the otic vesicle in domains that overlap with Dlx5. Chromatin immunoprecipitation using a Dlx5 antibody confirmed direct binding of Dlx5 to promoters of seven of these (Atbf1, Bmper, Large, Lrrtm1, Msx1, Ebf1 and Lhx1) in a cell line over-expressing Dlx5. Bmper and Lrrtm1 were up-regulated in this cell line, further supporting their identification as targets of Dlx5 in the inner ear and potentially in other organs. These direct targets support a model in which Bmp signaling is downstream of Dlx5 in the early inner ear and provide new insights into how the Dlx5 regulatory cascade is initiated.

<h4>Background</h4>Heterozygotes for the p.Cys282Tyr (C282Y) mutation of the HFE gene do not usually express a hemochromatosis phenotype. Apart from the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele, other rare HFE mutations can be found in trans on chromosome 6.<h4>Design and methods</h4>We performed molecular investigation of the genes implicated in hereditary hemochromatosis in six patients who presented with iron overload but were simple heterozygotes for the HFE C282Y mutation at first genetic testing. Functional impairment of new variants was deduced from computational methods including molecular modeling studies.<h4>Results</h4>We identified four rare HFE mutant alleles, three of which have not been previously described. One mutation is a 13-nucleotide deletion in exon 6 (c.1022_1034del13, p.His341_Ala345 > LeufsX119), which is predicted to lead to an elongated and unstable protein. The second one is a substitution of the last nucleotide of exon 2 (c.340G > A, p.Glu114Lys) which modifies the relative solvent accessibility in a loop interface. The third mutation, p.Arg67Cys, also lies in exon 2 and introduces a destabilization of the secondary structure within a loop of the α1 domain. We also found the previously reported c.548T > C (p.Leu183Pro) missense mutation in exon 3. No other known iron genes were mutated. We present an algorithm at the clinical and genetic levels for identifying patients deserving further investigation. Conclusions Our results suggest that additional mutations in HFE may have a clinical impact in C282Y carriers. In conjunction with results from previously described cases we conclude that an elevated transferrin saturation level and elevated hepatic iron index should indicate the utility of searching for further HFE mutations in C282Y heterozygotes prior to other iron gene studies.

The formation and plasticity of dendritic spines and synapses, which are poorly understood on a molecular level, are critical for cognitive functions, such as learning and memory. The adaptor protein containing a PH domain, PTB domain, and leucine zipper motif (APPL1) is emerging as a critical regulator of various cellular processes in non-neuronal cells, but its function in the nervous system is not well understood. Here, we show that APPL1 localizes to dendritic spines and synapses and regulates the development of these structures in hippocampal neurons. Knockdown of endogenous APPL1 using siRNA led to a significant decrease in the number of spines as well as synapses and this defect could be rescued by expression of siRNA-resistant APPL1. Expression of exogenous APPL1 increased the spine and synaptic density and the amount of surface GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Deletion of the C-terminal phosphotyrosine binding domain of APPL1, which binds the serine/threonine kinase Akt, resulted in a significant decrease in the spine and synaptic density, suggesting a role for Akt in regulating the development of these structures. Consistent with this, knockdown of Akt with siRNA or expression of dominant negative Akt led to a dramatic decrease in spine and synapse formation. In addition, APPL1 increased the amount of active Akt in spines and synapses and the effects of APPL1 on these structures were dependent on Akt, indicating that Akt is an effector of APPL1 in the regulation of these processes. Moreover, APPL1 signaling modulates spine and synapse formation through p21-activated kinase (PAK). Thus, our results indicate that APPL1 signaling through Akt and PAK is critical for spine and synaptic development and point to a role for APPL1 and its effectors in regulating cognitive function.

The papilla of the mammalian kidney must tolerate greatly varying degrees of hyperosmotic stress during urine concentration and depending on whole organism hydration state. To identify proteome adaptations supporting cell function and survival in such a harsh environment we compared the proteome of a) the hyperosmotic renal papilla with that of adjacent iso-osmotic cortex tissue and b) the renal papilla of diuretic versus that of anti-diuretic mice. Though functionally distinct the papilla is in close physical proximity to the renal cortex, an iso-osmotic region. Proteomic differences between the papilla and cortex of C57BL6 mice were identified using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry. We found 37 different proteins characteristic of the cortex and 16 proteins over-represented in the papilla. Regional specificity was confirmed by Western blot and further substantiated by immunohistochemistry for selected proteins. Proteins that are characteristic of the renal papilla include αB crystallin, Hsp beta-1, Hsp90, 14-3-3 protein, glutathione S-transferase, aldose reductase, actin and tropomyosin. Gene ontology analysis confirmed a significant increase in molecular functions associated with protein chaperoning and cell stabilization. Proteins over-represented in the cortex were largely related to routine metabolism. During antidiuresis 15 different proteins changed significantly while 18 different proteins changed significantly during diuresis relative to normally hydrated controls. Changes were confirmed by Western blot for selected proteins. Proteins that are significantly altered by diuretic state are associated with cell structure (actin, tubulin), signaling (Rho GDP dissociation inhibitor, abhydrolase domain-containing protein 14B), chaperone functioning (Hsp beta-1, αB crystallin, T complex protein-1) and anti-oxidant functions (α-enolase, GAPDH and LDH). Taken together our study reveals that specific proteins involved in protein folding, cytoskeletal stabilization, antioxidant responses, and stress signaling contribute greatly to the unique hyperosmotic stress resistant phenotype of the kidney papilla.

The central serotonergic (5-HT) system is closely involved in regulating various mental functions such as mood and emotion. In this system, the serotonin transporter (5-HTT) and the 5-HT(1A) receptor play important roles in the pathophysiology and treatment of mood and anxiety disorders. However, only a few integrated databases have considered the intraindividual relationship between pre- and postsynaptic serotonergic transmission. In the present study, we constructed a database of 5-HTT and 5-HT(1A) receptors using positron emission tomography (PET) with [¹¹C]DASB and [¹¹C]WAY100635, respectively. Seventeen healthy young men participated in this study. After anatomic standardization of original images, BP(ND) was calculated on a voxel-by-voxel basis using reference tissue methods. The highest binding to 5-HTT was observed in the dorsal raphe nucleus, striatum, and thalamus; moderate binding, in the insula and cingulate cortex; and very low binding, in the cerebral neocortex. In contrast, the highest binding to 5-HT(1A) receptors was seen in the hippocampal regions, insula, neocortical regions, and dorsal raphe nucleus, and very low binding was found in the thalamus and basal ganglia. These distribution patterns were in agreement with those reported in human postmortem studies and previous PET investigations. In addition, exploratory analysis indicated significant negative correlations between the BP(ND) values with both radiotracers in certain regions of the brain, such as the cingulate, insula, and frontal, temporal and parietal cortices (Pearson's correlation, P < 0.05). These databases facilitate the understanding of the regional distribution of serotonergic neurotransmission function in the living human brain and the pathophysiology of various neuropsychiatric disorders.

The most common form of hemochromatosis is caused by mutations in the HFE gene. Rare forms of the disease are caused by mutations in other genes. We present a patient with hyperferritinemia and iron overload, and facial flushing. Magnetic resonance imaging was performed to measure hepatic iron overload, and a molecular study of the genes involved in iron metabolism was undertaken. The iron overload was similar to that observed in HFE hemochromatosis, and the patient was double heterozygous for two novel mutations, c.-20G>A and c.718A>G (p.K240E), in the HFE and ferroportin (FPN1 or SLC40A1) genes, respectively. Hyperferritinemia and facial flushing improved after phlebotomy. Two of the patient's children were also studied, and the daughter was heterozygous for the mutation in the SLC40A1 gene, although she did not have hyperferritinemia. The patient presented a mild iron overload phenotype probably because of the two novel mutations in the HFE and SLC40A1 genes.

There is currently much interest in clinical applications of therapeutic hypothermia. Hypothermia can be a consequence of hypometabolism. We have recently established a procedure for the induction of a reversible deep hypometabolic state in mice using 5'-adenosine monophosphate (5'-AMP) in conjunction with moderate ambient temperature. The current study aims at investigating the impact of this technology at the gene expression level in a major metabolic organ, the liver. Our findings reveal that expression levels of the majority of genes in liver are not significantly altered by deep hypometabolism. However, among those affected by hypometabolism, more genes are differentially upregulated than downregulated both in a deep hypometabolic state and in the early arousal state. These altered gene expression levels during 5'-AMP induced hypometabolism are largely restored to normal levels within 2 days of the treatment. Our data also suggest that temporal control of circadian genes is largely stalled during deep hypometabolism.

The aggregation of polyglutamine containing protein sequences is implicated in a family of familial neurodegenerative diseases, the expanded CAG repeat diseases. While the cellular aggregation process undoubtedly depends on the flux and local environment of these proteins, their intrinsic physical properties and folding/aggregation propensities must also contribute to their cellular behavior. Here we describe a series of methods for determining mechanistic details of the spontaneous aggregation of polyQ-containing sequences, including the identification and structural examination of aggregation intermediates.

This paper compares the remineralization of human natural caries and artificial caries-like dentin lesions treated with a novel whisker-reinforced experimental composite resin (ART composite) with a resin-modified glass ionomer cement (RM-GIC) as control. Ten molars with moderate natural dentin caries were prepared (N). Artificial caries-like dentin lesions were prepared in occlusal dentin of 10 caries-free molars and demineralized at pH 4.3 for 48 h (A). The cavities were restored with ART composite or RM-GIC. All restored teeth were sliced into 120 μm sections. Transverse microradiography combined with digital image analysis was performed to analyze the change in mineral density at the same position in the specimens before and after 4 and 8 weeks remineralization/demineralization treatment. The mean percent remineralization ± standard deviation after 4 and 8 weeks are: N with ART composite, 27 ± 9 and 46 ± 14, respectively; N with RM-GIC, 18 ± 6 and 36 ± 11, respectively; A with ART composite, 48 ± 9 and 66 ± 11, respectively; A with RM-GIC, 50 ± 13 and 62 ± 11, respectively. There was a significant difference between the ART composite and RM-GIC for the remineralization of natural caries (P<0.05). For both restoratives there were significant differences between the remineralization of natural and artificial caries (P<0.001). The ART composite and RM-GIC remineralized natural and artificial caries differently, most likely due to differences in the microstructure and composition of the caries dentin.

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.

We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants.

<h4>Objective</h4>We investigated associations of early pregnancy maternal vitamin D concentrations with differential gene expression and post-transcription regulation.<h4>Method</h4>Plasma 25-hydroxyvitamin D (25[OH]D) was measured among participants of a nested case-control study. Participants with low (<25.5 ng/ml) and high (≥31.7 ng/ml) 25[OH]D were identified among controls. Peripheral blood messenger RNA (mRNA) (N = 21) and microRNA (miRNA) (N = 13) expression studies were conducted among participants with low and high 25[OH]D concentrations. Differential expression between low/high groups were evaluated using Student's t-test, fold change, and SAM comparisons. We further investigated functions and functional relationships of differentially expressed mRNAs and targets of differentially expressed miRNAs.<h4>Results</h4>Three hundred and five genes (299 upregulated and 6 downregulated) and 11 miRNAs (10 downregulated and 1 upregulated) were differentially expressed among participants with low 25[OH]D compared with those who had high 25[OH]D. Genes that participate in a wide range of cellular functions, including organ and system development (e.g. angiogenesis), inflammation and metabolic processes (e.g. carbohydrate/lipid metabolism), as well as miRNAs that target these genes were differentially expressed among women with low 25[OH]D compared with those with high 25[OH]D.<h4>Conclusion</h4>Early pregnancy plasma 25[OH]D concentrations are associated with maternal peripheral blood gene expression and post-transcription regulation.

Some studies have found that donor-recipient gender mismatch predicts posttransplant outcomes but whether this is independent of donor quality is unknown. To evaluate the association between gender mismatch and graft loss, 11 508 females (F) and 16 714 males (M) who underwent liver transplant from March 1, 2002 to December 31, 2007 were studied. Of 11 donor characteristics, clinically relevant differences between F and M donors were median age (47 vs. 39 years), height (165 vs. 178 cm) and proportion dying of stroke (59 vs. 35%) (p < 0.001 for all). The donor risk index was significantly lower for F than M donors (1.3 vs. 1.6, p < 0.001). Recipients of gender-mismatched grafts had an 11% higher risk of graft loss (p < 0.001). Compared to M→M donor-recipient-matched transplants in univariable analysis, F→M mismatch was associated with a 17% increased risk of graft loss (95% CI = 1.11-1.24, p < 0.001), whereas M→F mismatch was not (HR = 1.02; 95% CI = 0.96-1.09; p = 0.46). However, adjustment for significant recipient and donor factors eliminated the association between F→M mismatch and graft loss (HR = 0.95; 95% CI = 0.89-1.02; p = 0.18). In conclusion, donor quality differs significantly between female and male donors-female donors are older, shorter and die more frequently of stroke-and gender differences in donor quality, rather than gender mismatch are predictive of graft loss.

<h4>Background</h4>Gastric bypass surgery is a highly effective therapy for long-term weight loss in severely obese patients, but carries significant perioperative risks including infection, wound dehiscence, and leaks from staple breakdown. Iron status can affect immune function and wound healing, thus may influence peri-operative complications. Common mutations in the HFE gene, the gene responsible for the iron overload disorder hereditary hemochromatosis, may impact iron status.<h4>Methods</h4>We analyzed 1064 extremely obese Caucasian individuals who underwent open and laparoscopic Roux-n-Y gastric bypass surgery at the Geisinger Clinic. Serum iron, ferritin, transferrin, and iron binding capacity were measured pre-operatively. All patients had intra-operative liver biopsies and were genotyped for the C282Y and H63D mutations in the HFE gene. Associations between surgical complications and serum iron measures, HFE gene status, and liver iron histology were determined.<h4>Results</h4>We found that increased serum iron and transferrin saturation were present in patients with any post-operative complication, and that increased serum ferritin was also increased in patients with major complications. Increased serum transferrin saturation was also associated with wound complications in open RYGB, and transferrin saturation and ferritin with prolonged lengths of stay. The presence of 2 or more HFE mutations was associated with overall complications as well as wound complications in open RYGB. No differences were found in complication rates between those with stainable liver iron and those without.<h4>Conclusion</h4>Serum iron status and HFE genotype may be associated with complications following RYGB surgery in the extremely obese.

<h4>Purpose</h4>We review recent advances in the biologic understanding and treatment of childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), identify therapeutically challenging subgroups, and suggest future directions of research.<h4>Methods</h4>A review of English literature on childhood acute leukemias from the past 5 years was performed.<h4>Results</h4>Contemporary treatments have resulted in 5-year event-free survival rates of approximately 80% for childhood ALL and almost 60% for pediatric AML. The advent of high-resolution genome-wide analyses has provided new insights into leukemogenesis and identified many novel subtypes of leukemia. Virtually all ALL and the vast majority of AML cases can be classified according to specific genetic abnormalities. Cooperative mutations involved in cell differentiation, cell cycle regulation, tumor suppression, drug responsiveness, and apoptosis have also been identified in many cases. The development of new formulations of existing drugs, molecularly targeted therapy, and immunotherapies promises to further advance the cure rates and improve quality of life of patients.<h4>Conclusion</h4>The application of new high-throughput sequencing techniques to define the complete DNA sequence of leukemia and host normal cells and the development of new agents targeted to leukemogenic pathways promise to further improve outcome in the coming decade.

The synthesis of (4R,5R)-streptopyrrolidine (1), (4S,5R)-streptopyrrolidine (2) (4R,5S)-streptopyrrolidine (3) and (4S,5S)-streptopyrrolidine (4) have been achieved in a concise and highly efficient manner via a highly stereoselective aldol type reaction with the trimethylsilyl enolate of ethyl acetate and Lewis acid mediated lactamization as the key reactions in ≈42% yield over six steps starting from D-phenylalanine and L-phenylalanine, respectively. The absolute configuration of the natural product was shown to be (4S,5S) by comparing its spectral and analytical data with the reported values.

Excessive nitrosative and oxidative stress is thought to trigger cellular signaling pathways leading to neurodegenerative conditions. Such redox dysregulation can result from many cellular events, including hyperactivation of the N-methyl-D-aspartate-type glutamate receptor, mitochondrial dysfunction, and cellular aging. Recently, we and our colleagues have shown that excessive generation of free radicals and related molecules, in particular nitric oxide species (NO), can trigger pathological production of misfolded proteins, abnormal mitochondrial dynamics (comprised of mitochondrial fission and fusion events), and apoptotic pathways in neuronal cells. Emerging evidence suggests that excessive NO production can contribute to these pathological processes, specifically by S-nitrosylation of specific target proteins. Here, we highlight examples of S-nitrosylated proteins that regulate misfolded protein accumulation and mitochondrial dynamics. For instance, in models of Parkinson's disease, these S-nitrosylation targets include parkin, a ubiquitin E3 ligase and neuroprotective molecule, and protein-disulfide isomerase, a chaperone enzyme for nascent protein folding. S-Nitrosylation of protein-disulfide isomerase may also be associated with mutant Cu/Zn superoxide dismutase toxicity in amyotrophic lateral sclerosis. Additionally, in models of Alzheimer's disease, excessive NO generation leads to the formation of S-nitrosylated dynamin-related protein 1 (forming SNO-Drp1), which contributes to abnormal mitochondrial fragmentation and resultant synaptic damage.

Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25 amino acids. This hormone binds to ferroportin, an iron exporter expressed on the cell surface, and facilitates its degradation. Here we investigated if hepcidin is a substrate for SOPT1 and SOPT2 and if the hormone has any intracellular function in RPE. Hepcidin inhibited competitively the uptake of deltorphin II (a synthetic oligopeptide substrate for SOPT1) and DADLE (a synthetic oligopeptide substrate for SOPT2) with IC50 values in the range of 0.4-1.7 μM. FITC-hepcidin was taken up into RPE, and this uptake was inhibited by deltorphin II and DADLE. The entry of FITC-hepcidin into cells was confirmed by flow cytometry. Incubation of RPE with hepcidin decreased the levels of ferroportin mRNA. This effect was not a consequence of hepcidin-induced ferroportin degradation because excessive iron accumulation in RPE, which is expected to occur in these cells as a result of ferroportin degradation, did not decrease but instead increased the levels of ferroportin mRNA. This study reveals for the first time a novel intracellular function for hepcidin other than its established cell surface action on ferroportin.

Anemia of chronic disorders is a very important phenomenon and iron is a crucial factor of this complex process. To better understand this process and its influence on some other factors we have built a mathematical model of the human body iron homeostasis, which possibly most exactly would reflect the metabolism of iron in the case of anemia and inflammation. The model has been formulated in the language of Petri net theory, which allows for its simulation and precise analysis. The obtained results of the analysis of the model's behavior, concerning the influence of anemia and inflammation on the transferrin receptors, and hepcidin concentration changes are the valuable complements to the knowledge following from clinical research. This analysis is one of the first attempts to investigate properties and behavior of a not fully understood biological system on a basis of its Petri net based model.

Statins are believed to exert beneficial effects against cardiovascular disease beyond correction of dyslipidemia. There are however still very sparse data on how individual statins interact with the production of vasoactive eicosanoids and nitric oxide (NO) in human vascular endothelial cells. Here we have determined how fluvastatin affects the mRNA expression of genes associated with vascular reactivity as well as the formation of two major vasodilators, prostacyclin (PGI2) and NO, in human endothelial cells. Also, the influence of fluvastatin on arterial resistance was assessed in isolated small arteries. We show that the promoter activity of prostacyclin synthase (PTGIS), the mRNA expression of PTGIS and endothelial nitric oxide synthase (eNOS), and the production of PGI2 and NO are significantly induced by fluvastatin. Also, strong rapid dilatation ex vivo was observed, with the equal contribution of PGI2 and NO. Our findings in cell culture experiments and in isolated human arteries indicate that fluvastatin-evoked endothelium-derived vasodilator production may confer protection of the endothelial cells via both acute and long-term effects of fluvastatin treatment. If these effects take place in vivo, we suggest a protective pleiotropic role of fluvastatin on the cardiovascular system, particularly at the level of the vascular endothelium, to ameliorate the process of atherogenesis and in the acute manner to reduce vascular tone.

A novel short-step methodology for the synthesis in good yields of functionalized coumarins has been developed starting from an activated precursor, the N-hydroxysuccinimide ester of O-acetylsalicylic acid. The procedure is based on a tandem C-acylation-cyclization process under mild reaction conditions. The structure of 3-methoxycarbonyl-4-hydroxy coumarin has been established by X-ray diffraction analysis and its geometry was compared with optimized parameters by means of DFT calculations.

<h4>Background</h4>The p53 tumor suppressor protein is involved in a complicated regulatory network, mediating expression of ~1000 human genes. Recent studies have shown that many p53 in vivo binding sites (BSs) reside in transposable repeats. The relationship between these BSs and functional p53 response elements (REs) remains unknown, however. We sought to understand whether the p53 REs also reside in transposable elements and particularly in the most-abundant Alu repeats.<h4>Results</h4>We have analyzed ~160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements. In addition, using a position weight matrix approach, we found ~400,000 potential p53 BSs in Alu elements genome-wide. Importantly, these putative BSs are located in the same regions of Alu repeats as the functional p53 REs - namely, in the vicinity of Boxes A/A' and B of the internal RNA polymerase III promoter. Earlier nucleosome-mapping experiments showed that the Boxes A/A' and B have a different chromatin environment, which is critical for the binding of p53 to DNA. Here, we compare the Alu-residing p53 sites with the corresponding Alu consensus sequences and conclude that the p53 sites likely evolved through two different mechanisms - the sites overlapping with the Boxes A/A' were generated by CG → TG mutations; the other sites apparently pre-existed in the progenitors of several Alu subfamilies, such as AluJo and AluSq. The binding affinity of p53 to the Alu-residing sites generally correlates with the age of Alu subfamilies, so that the strongest sites are embedded in the 'relatively young' Alu repeats.<h4>Conclusions</h4>The primate-specific Alu repeats play an important role in shaping the p53 regulatory network in the context of chromatin. One of the selective factors responsible for the frequent occurrence of Alu repeats in introns may be related to the p53-mediated regulation of Alu transcription, which, in turn, influences expression of the host genes.

Huntington's disease (HD) and spinocerebellar ataxias (SCAs) are autosomal-dominant neurodegenerative disorders. HD is caused by polyglutamine (polyQ) expansion in the amino-terminal region of a protein huntingtin (Htt) and primarily affects medium spiny striatal neurons (MSN). Many SCAs are caused by polyQ-expansion in ataxin proteins and primarily affect cerebellar Purkinje cells. The reasons for neuronal dysfunction and death in HD and SCAs remain poorly understood and no cure is available for the patients. Our laboratory discovered that mutant huntingtin, ataxin-2 and ataxin-3 proteins specifically bind to the carboxy-terminal region of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1), an intracellular Ca(2+) release channel. Moreover, we found that association of mutant huntingtin or ataxins with IP(3)R1 causes sensitization of IP(3)R1 to activation by IP(3) in planar lipid bilayers and in neuronal cells. These results suggested that deranged neuronal Ca(2+) signaling might play an important role in pathogenesis of HD, SCA2 and SCA3. In support of this idea, we demonstrated a connection between abnormal Ca(2+) signaling and neuronal cell death in experiments with HD, SCA2 and SCA3 transgenic mouse models. Additional data in the literature indicate that abnormal neuronal Ca(2+) signaling may also play an important role in pathogenesis of SCAl, SCA5, SCA6, SCA14 and SCA15/16. Based on these results I propose that IP(3)R and other Ca(2+) signaling proteins should be considered as potential therapeutic targets for treatment of HD and SCAs.

<h4>Background</h4>SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined.<h4>Results</h4>We show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells.<h4>Conclusions</h4>We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.

<h4>Rationale</h4>Left ventricular (LV) mass and related phenotypes are heritable, important predictors of cardiovascular disease, particularly in hypertensive individuals.<h4>Objective</h4>Identify genetic predictors of echocardiographic phenotypes in hypertensive families.<h4>Methods and results</h4>A multistage genome-wide association study (GWAS) was conducted in hypertensive-ascertained black families (HyperGEN, stage I; GENOA, stage II); findings were replicated in HyperGEN white families (stage III). Echocardiograms were collected using a common protocol, and participants were genotyped with the Affymetrix Genome-Wide Human SNP 6.0 Array. The following were analyzed using mixed models adjusted for ancestry: in stages I and II, 1258 and 989 blacks, respectively; and in stage III, 1316 whites. Phenotypes included LV mass, LV internal dimension (LVID), wall thicknesses (posterior [PWT] and intraventricular septum [IVST]), and relative wall thickness (RWT). In stage I, 5 single nucleotide polymorphisms (SNPs) had P≤10(-6). In stage II, 1 SNP (rs1436109; NCAM1 intron 1) replicated with the same phenotype (PWT, P=0.025) in addition to RWT (P=0.032). In stage III, rs1436109 was associated with RWT (P=5.47×10(-4)) and LVID (P=1.86×10(-4)). Fisher combined probability value for all stages was RWT=3.80×10(-9), PWT=3.12×10(-7), IVST=8.69×10(-7), LV mass=2.52×10(-3), and LVID=4.80×10(-4).<h4>Conclusions</h4>This GWAS conducted in hypertensive families identified a variant in NCAM1 associated with LV wall thickness and RWT. NCAM is upregulated during the remodeling period of hypertrophy to heart failure in Dahl salt-sensitive rats. Our initial screening in hypertensive blacks may have provided the context for this novel locus.

Environmental estrogen mimics, including metalloestrogens that can activate estrogen receptor-alpha (ERα), may contribute to breast cancer risk. However, the underlying mechanisms through which these molecular mimics activate the ERα are generally poorly understood. With concern to this important question, we investigated whether intracellular calcium may mediate the cross-talk between signaling pathways that activate ERα and the ligand-binding domain of ERα. MCF-7 cells treated with EGF, ATP, extracellular calcium, or caffeine to increase intracellular calcium triggered a rapid recruitment of ERα to estrogen-responsive promoters and stimulated expression of estrogen-responsive genes including pS2, complement C3, and progesterone receptor. Induction was blocked by an antiestrogen but also by the chelation of intracellular calcium. Treatment with extracellular calcium also increased the growth of MCF-7 cells through an ER-dependent mechanism. We found that EGF and extracellular calcium activated the C-terminus of ERα and the activation was blocked by the antiestrogen. Mechanistic investigations identified four potential sites on the solvent-accessible surface of the ERα ligand-binding domain as important for calcium activation of the receptor. Taken together, our results suggest that calcium mediates the cross-talk between ERα-activating signaling pathways and the ligand-binding domain of ERα providing a potential explanation for the ability of certain environmental metalloestrogens to activate the receptor.

<h4>Purpose</h4>Several ocular diseases involve the iris, notably including oculocutaneous albinism, pigment dispersion syndrome, and exfoliation syndrome. To screen for candidate genes that may contribute to the pathogenesis of these diseases, genome-wide iris gene expression patterns were comparatively analyzed from mouse models of these conditions.<h4>Methods</h4>Iris samples from albino mice with a Tyr mutation, pigment dispersion-prone mice with Tyrp1 and Gpnmb mutations, and mice resembling exfoliation syndrome with a Lyst mutation were compared with samples from wild-type mice. All mice were strain (C57BL/6J), age (60 days old), and sex (female) matched. Microarrays were used to compare transcriptional profiles, and differentially expressed transcripts were described by functional annotation clustering using DAVID Bioinformatics Resources. Quantitative real-time PCR was performed to validate a subset of identified changes.<h4>Results</h4>Compared with wild-type C57BL/6J mice, each disease context exhibited a large number of statistically significant changes in gene expression, including 685 transcripts differentially expressed in albino irides, 403 in pigment dispersion-prone irides, and 460 in exfoliative-like irides.<h4>Conclusions</h4>Functional annotation clusterings were particularly striking among the overrepresented genes, with albino and pigment dispersion-prone irides both exhibiting overall evidence of crystallin-mediated stress responses. Exfoliative-like irides from mice with a Lyst mutation showed overall evidence of involvement of genes that influence immune system processes, lytic vacuoles, and lysosomes. These findings have several biologically relevant implications, particularly with respect to secondary forms of glaucoma, and represent a useful resource as a hypothesis-generating dataset.

Central nervous system myelination requires recognition and signalling processes between neuronal axons and oligodendrocytes. Complex cellular rearrangements occur in myelination-competent oligodendrocytes requiring spatio-temporal control mechanisms. Although the molecular repertoire is becoming increasingly transparent, the signalling mechanisms governing myelination initiation are only poorly understood. The non-receptor tyrosine kinase Fyn has been implicated in axon-glial signal transduction and in several cellular processes required for oligodendrocyte maturation and myelination. Here, we review oligodendroglial Fyn signalling and discuss the role of Fyn in axon-glia interaction mediating myelination.

Bioactive hydroxyapatite (HA) with addition of silicon (Si) in the crystal structure (silicon-doped hydroxyapatite (SiHA)) has become a highly attractive alternative to conventional HA in bone replacement owing to the significant improvement in the in vivo bioactivity and osteoconductivity. Nanometre-scaled SiHA (nanoSiHA), which closely resembles the size of bone mineral, has been synthesized in this study. Thus, the silicon addition provides an extra chemical cue to stimulate and enhance bone formation for new generation coatings, and the next stage in metallic implantation design is to further improve cellular adhesion and proliferation by control of cell alignment. Topography has been found to provide a powerful set of signals for cells and form contact guidance. Using the recently developed novel technique of template-assisted electrohydrodynamic atomization (TAEA), patterns of pillars and tracks of various dimensions of nanoSiHA were achieved. Modifying the parameters of TAEA, the resolution of pattern structures was controlled, enabling the topography of a substrate to be modified accordingly. Spray time, flow rate and distance between the needle and substrate were varied to improve the pattern formation of pillars and tracks. The 15 min deposition time provided the most consistent patterned topography with a distance of 50 mm and flow rate of 4 µl min(-1). A titanium substrate was patterned with pillars and tracks of varying widths, line lengths and distances under the optimized TAEA processing condition. A fast bone-like apatite formation rate was found on nanoSiHA after immersion in simulated body fluid, thus demonstrating its high in vitro bioactivity. Primary human osteoblast (HOB) cells responded to SiHA patterns by stretching of the filopodia between track and pillar, attaching to the apex of the pillar pattern and stretching between two. HOB cells responded to the track pattern by elongating along and between the track, and the length of HOB cells was proportional to the gaps between track patterns, but this relationship was not observed on the pillar patterns. The study has therefore provided an insight for future design of next generation implant surfaces to control and guide cellular responses, while TAEA patterning provides a controllable technique to provide topography to medical implants.

Huntington disease results from an expanded polyglutamine region in the N terminus of the huntingtin protein. HD pathology is characterized by neuronal degeneration and protein inclusions containing N-terminal fragments of mutant huntingtin. Structural information is minimal, though it is believed that mutant huntingtin polyglutamine adopts β structure upon conversion to a toxic form. To this end, we designed mammalian cell expression constructs encoding compact β variants of Htt exon 1 N-terminal fragment and tested their ability to aggregate and induce toxicity in cultured neuronal cells. In parallel, we performed molecular dynamics simulations, which indicate that constructs with expanded polyglutamine β-strands are stabilized by main-chain hydrogen bonding. Finally, we found a correlation between the reactivity to 3B5H10, an expanded polyglutamine antibody that recognizes a compact β rich hairpin structure, and the ability to induce cell toxicity. These data are consistent with an important role for a compact β structure in mutant huntingtin-induced cell toxicity.

The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family.

We describe the use of dynamic combinatorial chemistry (DCC) to identify ligands for the stem-loop structure located at the exon 10-5'-intron junction of Tau pre-mRNA, which is involved in the onset of several tauopathies including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). A series of ligands that combine the small aminoglycoside neamine and heteroaromatic moieties (azaquinolone and two acridines) have been identified by using DCC. These compounds effectively bind the stem-loop RNA target (the concentration required for 50% RNA response (EC(50)): 2-58 μM), as determined by fluorescence titration experiments. Importantly, most of them are able to stabilize both the wild-type and the +3 and +14 mutated sequences associated with the development of FTDP-17 without producing a significant change in the overall structure of the RNA (as analyzed by circular dichroism (CD) spectroscopy), which is a key factor for recognition by the splicing regulatory machinery. A good correlation has been found between the affinity of the ligands for the target and their ability to stabilize the RNA secondary structure.

Small heterodimer partner (SHP, NR0B2) is a unique orphan nuclear receptor that contains the dimerization and a putative ligand-binding domain, but lacks the conserved DNA binding domain. SHP exerts its physiological function as an inhibitor of gene transcription through physical interaction with multiple nuclear receptors and transcriptional factors. SHP is a critical transcriptional regulator affecting diverse biological functions, including bile acid, cholesterol and lipid metabolism, glucose and energy homeostasis, and reproductive biology. Recently, we and others have demonstrated that SHP is an epigenetically regulated transcriptional repressor that suppresses the development of liver cancer. In this review, we summarize recent major findings regarding the role of SHP in cell proliferation, apoptosis, and DNA methylation, and discuss recent progress in understanding the function of SHP as a tumor suppressor in the development of liver cancer. Future study will be focused on identifying SHP associated novel pro-oncogenes and anti-oncogenes in liver cancer progression and applying the knowledge gained on SHP in liver cancer prevention, diagnosis and treatment.

<h4>Background</h4>Oxidized low-density lipoprotein (oxLDL) uptake by macrophages plays an important role in foam cell formation. It has been suggested the presence of heterogeneous subsets of macrophage, such as M1 and M2, in human atherosclerotic lesions. To evaluate which types of macrophages contribute to atherogenesis, we performed cDNA microarray analysis to determine oxLDL-induced transcriptional alterations of each subset of macrophages.<h4>Results</h4>Human monocyte-derived macrophages were polarized toward the M1 or M2 subset, followed by treatment with oxLDL. Then gene expression levels during oxLDL treatment in each subset of macrophages were evaluated by cDNA microarray analysis and quantitative real-time RT-PCR. In terms of high-ranking upregulated genes and functional ontologies, the alterations during oxLDL treatment in M2 macrophages were similar to those in nonpolarized macrophages (M0). Molecular network analysis showed that most of the molecules in the oxLDL-induced highest scoring molecular network of M1 macrophages were directly or indirectly related to transforming growth factor (TGF)-β1. Hierarchical cluster analysis revealed commonly upregulated genes in all subset of macrophages, some of which contained antioxidant response elements (ARE) in their promoter regions. A cluster of genes that were specifically upregulated in M1 macrophages included those encoding molecules related to nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) signaling pathway. Quantitative real-time RT-PCR showed that the gene expression of interleukin (IL)-8 after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 cells. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment.<h4>Conclusions</h4>The present study demonstrated transcriptional alterations in polarized macrophages during oxLDL treatment. The data suggested that oxLDL uptake may affect TGF-β1- and NF-κB-mediated functions of M1 macrophages, but not those of M0 or M2 macrophages. It is likely that M1 macrophages characteristically respond to oxLDL.

Many human aneuploidy syndromes have unique phenotypic consequences, but in most instances it is unclear whether these phenotypes are attributable to alterations in the dosage of specific genes. In human trisomy 13, there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. Using partial trisomy cases, we mapped this trait to chromosomal band 13q14; by examining the genes in this region, two microRNAs, miR-15a and -16-1, appear as top candidates for the elevated HbF levels. Indeed, increased expression of these microRNAs in primary human erythroid progenitor cells results in elevated fetal and embryonic hemoglobin gene expression. Moreover, we show that a direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus we demonstrate how the developmental regulation of a clinically important human trait can be better understood through the genetic and functional study of aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB may be important therapeutic targets to increase HbF levels in patients with sickle cell disease and β-thalassemia.

In the peripheral nervous system, target tissues control the final size of innervating neuronal populations by producing limited amounts of survival-promoting neurotrophic factors during development. However, it remains largely unknown if the same principle works to regulate the size of neuronal populations in the developing brain. Here we show that neurotrophin signaling mediated by the TrkB receptor controls striatal size by promoting the survival of developing medium-sized spiny neurons (MSNs). Selective deletion of the gene for the TrkB receptor in striatal progenitors, using the Dlx5/6-Cre transgene, led to a hindpaw-clasping phenotype and a 50% loss of MSNs without affecting striatal interneurons. This loss resulted mainly from increased apoptosis of newborn MSNs within their birthplace, the lateral ganglionic eminence. Among MSNs, those expressing the dopamine receptor D2 (DRD2) were most affected, as indicated by a drastic loss of these neurons and specific down-regulation of the DRD2 and enkephalin. This specific phenotype of mutant animals is likely due to preferential TrkB expression in DRD2 MSNs. These findings suggest that neurotrophins can control the size of neuronal populations in the brain by promoting the survival of newborn neurons before they migrate to their final destinations.

No abstract available.

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to prevent the development of obesity in rodent models. Here, we examined the effect of EGCG on markers of fat oxidation in high fat-fed C57bl/6J mice. High fat-fed mice treated with 0.32% dietary EGCG for 16 weeks had reduced body weight gain and final body weight (19.2% and 9.4%, respectively) compared to high fat-fed controls. EGCG-treatment decreased fasting blood glucose, plasma insulin, and insulin resistance by 18.5%, 25.3%, and 33.9%, respectively. EGCG treatment also reduced markers of obesity-related fatty liver disease in high fat-fed mice. Gene expression analysis of skeletal muscle showed that EGCG increased mRNA levels of nuclear respiratory factor (nrf)1, medium chain acyl coA decarboxylase (mcad), uncoupling protein (ucp)3, and peroxisome proliferator responsive element (ppar)α by 1.4-1.9-fold compared to high fat-fed controls. These genes are all related to mitochondrial fatty acid oxidation. In addition, EGCG increased fecal excretion of lipids in high fat-fed mice. In summary, it appears that EGCG modulates body weight gain in high fat-fed mice both by increasing the expression of genes related fat oxidation in the skeletal muscle and by modulating fat absorption from the diet.

No abstract available.

The minisequencing method is a cost-effective tool to study single-nucleotide polymorphisms in human disease. For this reason, a novel polymerase chain reaction multiplex SNaPshot reaction has been developed that targets 10 autosomal mutations in genes, or regions near to them, reported to be involved in iron metabolism: TMPRSS6, TF, and HFE. To validate this multiplex, 284 samples from unrelated women from the Spanish population were tested at a fertile age. The novel polymerase chain reaction multiplex SNaPshot reaction developed is a very simple, sensitive, and low-cost approach, and therefore will be useful as a tool of clinic diagnosis in iron metabolism alterations and to replicate results obtained from genome-wide linkage analysis.

The present study examined the association between a measure of sociopathy and 5-HTTLPR genotype in a sample of individuals from Project MATCH, a multi-center alcohol treatment trial. 5-HTTLPR, an insertion-deletion polymorphism in SLC6A4, the gene encoding the serotonin transporter protein, results in functionally distinct long (L) and short (S) alleles. The S allele has been associated with a variety of psychiatric disorders and symptoms including alcohol dependence, but it is unknown whether 5-HTTLPR increases the risk for co-morbid sociopathy among those with alcohol dependence. Eight hundred sixty-two subjects diagnosed with alcohol dependence completed the California Psychological Inventory, a psychological assessment that includes a measure of socialization, which was used as a proxy measure of sociopathy. Subjects were genotyped for the insertion-deletion polymorphism, as well as a single nucleotide polymorphism (A→G) that is located in the inserted region. Regression analysis revealed that after controlling for age, which was negatively related to socialization score, 5-HTTLPR genotype interacted with sex to determine socialization score (P < 0.001). Males with the L'L' genotype (i.e. those homozygous for the L(A) allele) had lower socialization scores (i.e. greater sociopathy) than males who were carriers of the S' allele (P = 0.03). In contrast, women with the S'S' genotype had lower socialization scores than women with two L' alleles (P = 0.002) and tended to have lower Socialization Index of the California Psychological Inventory scores than women with one copy of the L' allele (P = 0.07). Among individuals with alcohol use disorders, the tri-allelic 5-HTTLPR polymorphism had opposite effects on socialization scores in men than women. The basis for this finding is unknown, but it may have implications for sub-typing alcoholics.

Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G-D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G-D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein.

<h4>Background</h4>Patients with a personal or familial history of thromboembolism are considered at higher risk for thromboembolic disease after knee arthroplasty. While it remains unclear why some patients develop deep vein thrombosis (DVT) or pulmonary embolism (PE) despite similar operative procedures and the same prophylactic regimen, we presume one explanation would be genetic predisposition.<h4>Questions/purposes</h4>We determined the frequency of 12 factors including antithrombin III activity, prothrombin gene mutations, and the presence of phospholipid antibodies in a high-risk patient cohort and compared those findings with the known prevalence in the population at large.<h4>Patients and methods</h4>Patients identified preoperatively as having a personal or familial history of DVT and/or PE were referred for hemostatic serum and genetic tests, including % antithrombin III activity (ATIII), protein C and protein S activities, APC resistance, Factor V gene (Leiden) mutations, prothrombin gene mutations, lupus anticoagulant antibody presence, cardiolipin antibody presence, phosphatidyl antibody presence, β2-glycoprotein antibody presence, and serum homocysteine and lipoprotein(a) levels The frequencies of varying abnormalities were identified and compared to the prevalence reported in the literature.<h4>Results</h4>Forty-three of 1944 patients undergoing knee arthroplasty had a history of DVT or PE. Sixteen of 43 (37%) patients had an abnormality and eight of these (19%) had two or more abnormalities. The frequency of nine of the 12 tests appeared to be greater in this cohort than in the population at large.<h4>Conclusions</h4>Patients with a personal or familial history of DVT or PE appear to have a high frequency of hereditary prothrombotic abnormalities. Preoperative evaluation by a hematologist may be warranted in patients with a personal or familial history of DVT or PE as the postoperative anticoagulation protocols may be altered and identification of these abnormalities may affect a patient's risk for other disease states.<h4>Level of evidence</h4>Level IV, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.

<h4>Objective</h4>We sought to obtain insight into possible mechanisms underlying preeclampsia using genomewide transcriptional profiling in decidua basalis.<h4>Study design</h4>Genomewide transcriptional profiling was performed on decidua basalis tissue from preeclamptic (n = 37) and normal (n = 58) pregnancies. Differentially expressed genes were identified and merged into canonical pathways and networks.<h4>Results</h4>Of the 26,504 expressed transcripts detected, 455 were differentially expressed (P < .05; false discovery rate, P < .1). Both novel (ARL5B, SLITRK4) and previously reported preeclampsia-associated (PLA2G7, HMOX1) genes were identified. Pathway analysis revealed that tryptophan metabolism, endoplasmic reticulum stress, linoleic acid metabolism, notch signaling, fatty acid metabolism, arachidonic acid metabolism, and NRF2-mediated oxidative stress response were overrepresented canonical pathways.<h4>Conclusion</h4>In the present study single genes, canonical pathways, and gene-gene networks that are likely to play an important role in the pathogenesis of preeclampsia have been identified. Future functional studies are needed to accomplish a greater understanding of the mechanisms involved.

Injectable reverse thermal gels have great potentials as biomaterials for tissue engineering and drug delivery. However, most existing gels lack functional groups that can be modified with biomolecules that can guide cell/material interactions. We created an amine-functionalized ABA block copolymer, poly(ethylene glycol)-poly(serinol hexamethylene urethane), or ESHU. This reverse thermal gel consists of a hydrophobic block (B): poly(serinol hexamethylene urethane) and a hydrophilic block (A): poly(ethylene glycol). The polymer was characterized by GPC, FTIR and (1)H FTNMR. Rheological study demonstrated that ESHU solution in phosphate-buffered saline initiated phase transition at 32 °C and reached maximum elastic modulus at 37 °C. The in vitro degradation tests performed in PBS and cholesterol esterase solutions revealed that the polymer was hydrolyzable and the presence of cholesterol esterase greatly accelerated the hydrolysis. The in vitro cytotoxicity tests carried out using baboon smooth muscle cells demonstrated that ESHU had good cytocompatibility with cell viability indistinguishable from tissue culture treated polystyrene. Subcutaneous implantation in rats revealed well tolerated accurate inflammatory response with moderate ED-1 positive macrophages in the early stages, which largely resolved 4 weeks post-implantation. We functionalized ESHU with a hexapeptide, Ile-Lys-Val-Ala-Val-Ser (IKVAVS), which gelled rapidly at body temperature. We expect this new platform of functionalizable reverse thermal gels to provide versatile biomaterials in tissue engineering and regenerative medicine.

<h4>Objective</h4>To determine the frequency and character of arthropathy in hereditary hemochromatosis (HH) and to investigate the relationship between this arthropathy, nodal interphalangeal osteoarthritis, and iron load.<h4>Methods</h4>Participants were recruited from the community by newspaper advertisement and assigned to diagnostic confidence categories for HH (definite/probable or possible/unlikely). Arthropathy was determined by use of a predetermined clinical protocol, radiographs of the hands of all participants, and radiographs of other joints in which clinical criteria were met.<h4>Results</h4>An arthropathy considered typical for HH, involving metacarpophalangeal joints 2-5 and bilateral specified large joints, was observed in 10 of 41 patients with definite or probable HH (24%), all of whom were homozygous for the C282Y mutation in the HFE gene, while only 2 of 62 patients with possible/unlikely HH had such an arthropathy (P=0.0024). Arthropathy in definite/probable HH was more common with increasing age and was associated with ferritin concentrations>1,000 μg/liter at the time of diagnosis (odds ratio 14.0 [95% confidence interval 1.30-150.89], P=0.03). A trend toward more episodes requiring phlebotomy was also observed among those with arthropathy, but this was not statistically significant (odds ratio 1.03 [95% confidence interval 0.99-1.06], P=0.097). There was no significant association between arthropathy in definite/probable HH and a history of intensive physical labor (P=0.12).<h4>Conclusion</h4>An arthropathy consistent with that commonly attributed to HH was found to occur in 24% of patients with definite/probable HH. The association observed between this arthropathy, homozygosity for C282Y, and serum ferritin concentrations at the time of diagnosis suggests that iron load is likely to be a major determinant of arthropathy in HH and to be more important than occupational factors.

<h4>Objectives</h4>Genome-wide association meta-analyses have recently identified multiple loci associated with blood pressure. We sought to validate previously identified blood pressure loci by replication in a single large homogeneous population-based cohort and to identify new genome-wide significant loci using both conventional and expression-guided approaches.<h4>Methods</h4>We examined the associations of 18 single-nucleotide polymorphisms (SNPs) with genome-wide significance (P < 5.0 × 10⁻⁸, 'primary'), and 13 suggestive SNPs (5.0 × 10⁻⁸ < P < 5.6 × 10⁻⁵, 'secondary'), all from previously established genome-wide association studies, with self-reported blood pressure in 23 019 women from the Women's Genome Health Study. We then targeted for replication 12 gene expression-associated SNPs (eSNPs) that were also previously associated with blood pressure phenotypes.<h4>Results</h4>Using these replication strategies, we found confirmatory evidence for 13/18 primary SNPs, 3/13 secondary SNPs, and 4/12 eSNPs in the Women's Genome Health Study. Meta-analysis combining the Women's Genome Health Study results with prior study results revealed one previously unrecognized blood pressure locus with genome-wide significance: a BLK-GATA4-adjacent region (P = 3.2 × 10⁻⁸).<h4>Conclusion</h4>In this analysis, conventional and eSNP-guided strategies were complementary and illustrate two ways for extending initial genome-wide association results for discovery of new genes involved in human disease. Using this strategy, we report a newly identified blood pressure locus, BLK-GATA4, that may further understanding of the complex genetic pathways regulating blood pressure.

<h4>Background and objective</h4>Angiotensin-converting enzyme inhibitors (ACEi) are the first selected drugs for hypertensive patients because of its protective properties against heart and kidney diseases. Persistent cough is a common adverse reaction associated with ACEi, which can bind to the treatment cessation, but its etiology remains an unresolved issue. The most accepted mechanism is that the inhibition of ACEi increases kinins levels, resulting in the activation of proinflammatory mechanisms and nitric oxide generation. However, relatively little is known about the genetic susceptibility to ACEi-induced cough in hypertensive patients.<h4>Methods</h4>We carried out a monogenic association analysis of 39 polymorphisms and haplotypes in genes encoding key proteins related to ACEi activity with the occurrence of ACEi-induced cough. We also carried out a digenic association analysis and investigated the existence of epistatic interactions between the analyzed polymorphisms using a logistic regression procedure. Finally, we investigated the predictive value of the identified associations for ACEi-induced cough.<h4>Results</h4>We found that genetic polymorphisms in MME [rs2016848, P=0.002, odds ratio (OR)=1.795], BDKRB2 (rs8012552, P=0.012, OR=1.609), PTGER3 (rs11209716, P=0.002, OR=0.565), and ACE (rs4344) genes are associated with ACEi-related cough. For the latter, the effect is sex specific, having a protective effect in males (P=0.027, OR=0.560) and increasing the risk in females (P=0.031, OR=1.847). In addition, genetic interactions between peptidases involved in kinins levels (CPN1 and XPNPEP1) and proteins related to prostaglandin metabolism (PTGIS and PTGIR) strongly modify the risk of ACEi-induced cough presentation (0.102≤OR≤0.384 for protective combinations and 2.732≤OR≤7.216 for risk combinations).<h4>Conclusion</h4>These results are consistent with the hypothesis that the mechanism of cough is related to the accumulation of bradykinin, substance P, and prostaglandins.

The mesenchymal elements of the intestinal lamina propria reviewed here are the myofibroblasts, fibroblasts, mural cells (pericytes) of the vasculature, bone marrow-derived stromal stem cells, smooth muscle of the muscularis mucosae, and smooth muscle surrounding the lymphatic lacteals. These cells share similar marker molecules, origins, and coordinated biological functions previously ascribed solely to subepithelial myofibroblasts. We review the functional anatomy of intestinal mesenchymal cells and describe what is known about their origin in the embryo and their replacement in adults. As part of their putative role in intestinal mucosal morphogenesis, we consider the intestinal stem cell niche. Lastly, we review emerging information about myofibroblasts as nonprofessional immune cells that may be important as an alarm system for the gut and as a participant in peripheral immune tolerance.

Olfactomedin 4 (OLFM4) is a member of olfactomedin domain-containing protein family. Human OLFM4 is preferentially expressed in the gastrointestinal tract (stomach, small intestine and colon), prostate, and bone marrow. Recent studies demonstrate that OLFM4 is involved in the establishment and/or development of some types of malignancies, especially in gastrointestinal cancers. Induction of OLFM4 in cancer cells has a novel antiapoptotic action and promotes proliferation of cancer cells. OLFM4 regulates cell cycle and promotes S phase transition in proliferation of cancer cells. In addition, OLFM4 is associated with cancer adhesion and metastasis. In this minireview, we mainly focus on the OLFM4 expression and its biological significances in tumor differentiation and progression as well as the contributions of OLFM4 to tumorigenesis.

Type 1 (non-neuronopathic) Gaucher disease was the first lysosomal storage disorder for which an effective enzyme replacement therapy was developed and it has become a prototype for treatments for related orphan diseases. There are currently four treatment options available to patients with Gaucher disease, nevertheless, almost 25% of Type 1 Gaucher patients do not gain timely access to therapy because of delays in diagnosis after the onset of symptoms. Diagnosis of Gaucher disease by enzyme testing is unequivocal, but the rarity of the disease and nonspecific and heterogeneous nature of Gaucher disease symptoms may impede consideration of this disease in the differential diagnosis. To help promote timely diagnosis and optimal management of the protean presentations of Gaucher disease, a consensus meeting was convened to develop algorithms for diagnosis and disease management for Gaucher disease.

Sepsis is a harmful hyper-inflammatory state characterized by overproduction of cytokines. Removal of these cytokines using an extracorporeal device is a potential therapy for sepsis. We are developing a cytokine adsorption device (CAD) filled with porous polymer beads which efficiently depletes middle-molecular weight cytokines from a circulating solution. However, removal of one of our targeted cytokines, tumor necrosis factor (TNF), has been significantly lower than other smaller cytokines. We addressed this issue by incorporating anti-TNF antibodies on the outer surface of the beads. We demonstrated that covalent immobilization of anti-TNF increases overall TNF capture from 55% (using unmodified beads) to 69%. Passive adsorption increases TNF capture to over 99%. Beads containing adsorbed anti-TNF showed no significant loss in their ability to remove smaller cytokines, as tested using interleukin-6 (IL-6) and interleukin-10 (IL-10). We also detail a novel method for quantifying surface-bound ligand on a solid substrate. This assay enabled us to rapidly test several methods of antibody immobilization and their appropriate controls using dramatically fewer resources. These new adsorbed anti-TNF beads provide an additional level of control over a device which previously was restricted to nonspecific cytokine adsorption. This combined approach will continue to be optimized as more information becomes available about which cytokines play the most important role in sepsis.

Metabolism in tumors deviates significantly from that of normal tissues. Increasingly, the underlying aberrant metabolic pathways are being considered as novel targets for cancer therapy. Denoted "metabolic targeting", small molecule drugs are under investigation for focused inhibition of key metabolic steps that are utilized by tumors, since such inhibitors should harbor minimal toxicity towards surrounding normal tissues. This review will examine the primary biochemical pathways that tumors harness to enhance their bioenergetic capacity, which in turn, help their rapid proliferation and metastasis within the host. It is hoped that "metabolite-mimetic" drugs can be utilized to interfere with metabolic flux pathways active within the tumor, and across tumor-microenvironment boundary. In fact, the major pathways of mammalian metabolism, i.e., the carbohydrate, amino-acid, and fatty-acid metabolic pathways have been examined as putative targets for drug development, with some drug candidates advancing to phase II/III stages. In this regard, glucose metabolism, i.e., the glycolytic pathway - that predominates the bio-energetic flux in tumors, and the associated mitochondrial metabolism have received the most attention as suitable "druggable" targets, focused either at the pathway enzymes or at the plasma-membrane-bound metabolite transporters. Outlined in this review are pre-clinical studies that have led to the discovery of promising drug candidates to target tumor-metabolic flux, and ensuing patents, with descriptions of the biochemical rationale for the combinatorial strategy of a particular metabolic pathway-drug candidate pair.

Propagation of a species requires periodic cell renewal to avoid clonal extinction. Sexual reproduction and the separation of germ cells from the soma provide a mechanism for such renewal, but are accompanied by an apparently mandatory ageing of the soma. Data obtained during the last decade suggest that a division of labour exists also between cells of vegetatively reproducing unicellular organisms, leading to the establishment of a soma-like and germ-like lineage with distinct fitness and longevity characteristics. This division of labour in both bacteria and yeast entails segregation of damaged and aggregated proteins such that the germ-like lineage is kept free of damage to the detriment of the soma-like lineage. In yeast, this spatial protein quality control (SQC) encompasses a CCT-chaperonin-dependent translocation and merging of cytotoxic protein aggregates. This process is regulated by Sir2, a protein deacetylase that modulates the rate of ageing in organisms ranging from yeast to worms and flies. Recent data also demonstrate that SQC is intimately integrated with the machinery establishing proper cell polarity and that this machinery is required for generating a soma-like and germ-like lineage in yeast. Deciphering the details of the SQC network may increase our understanding of the development of age-related protein folding disorders and shed light on the selective forces that paved the way for polarity and lineage-specific ageing to evolve.

Late onset is a common hallmark character of numerous disorders including human neurodegenerative maladies such as Huntington's, Parkinson's and Alzheimer's diseases. Why these diseases manifest in aged individuals and why distinct disorders share strikingly similar emergence patterns were until recently unsolved enigmas. During the past decade, invertebrate-based studies indicated that the insulin/IGF signalling pathway (IIS) mechanistically links neurodegenerative-associated toxic protein aggregation and ageing; yet, until recently it was unclear whether this link is conserved from invertebrates to mammals. Recent studies performed in Alzheimer's mouse models indicated that ageing alteration by IIS reduction slows the progression of Alzheimer's-like disease, protects the brain and mitigates the behavioural, pathological and biochemical impairments associated with the disease. Here, we review these novel studies and discuss the potential of ageing alteration as a therapeutic approach for the treatment of late onset neurodegeneration.

In this paper, we describe using Synthesis-View, a new method of presenting complex genetic data, to revisit results of a study from the BioVU Vanderbilt DNA databank. BioVU is a biorepository of DNA samples coupled with de-identified electronic medical records (EMR). In the Ritchie et al. study ~10,000 BioVU samples were genotyped for 21 SNPs that were previously associated with 5 diseases: atrial fibrillation, Crohn Disease, multiple sclerosis, rheumatoid arthritis, and type 2 diabetes. In the proof-of-concept study, the 21 tests of association replicated previous findings where sample size provided adequate power. The majority of the BioVU results were originally presented in tabular form. Herein we have revisited the results of this study using Synthesis-View. The Synthesis-View software tool visually synthesizes the results of complex, multi-layered studies that aim to characterize associations between small numbers of single-nucleotide polymorphisms (SNPs) and diseases and/or phenotypes, such as the results of replication and meta-analysis studies. Using Synthesis-View with the data of the Ritchie et al. study and presenting these data in this integrated visual format demonstrates new ways to investigate and interpret these kinds of data. Synthesis-View is freely available for non-commercial research institutions, for full details see https://chgr.mc.vanderbilt.edu/synthesisview.

An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system.

We examined the involvement of peroxiredoxin 6 (Prdx 6) in providing chemoprotection against cisplatin cytotoxicity in SKOV-3 ovarian cancer cells. Treatment of SKOV-3 cells with cisplatin-induced cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and apoptosis mediated by proteolytically activated caspase 3 and 9. Overexpression of Prdx 6 protein or exposure to N-acetylcysteine (NAC) reversed the apoptotic effect of cisplatin by reducing ROS levels and suppressing the caspase signaling pathway. These results indicate that targeting Prdx 6 may sensitize cancer cells to ROS-producing therapeutic treatments, such as anticancer drugs and radiation.

<h4>Background</h4>Wyman W. Lai, MD, MPH, and Victoria L. Vetter, MD, MPH. The Pediatric Heart Network (PHN), funded under the U.S. National Institutes of Health-National Heart, Lung, and Blood Institute (NIH-NHLBI), includes two Clinical Research Skills Development (CRSD) Cores, which were awarded to The Children's Hospital of Philadelphia and to the Morgan Stanley Children's Hospital of New York-Presbyterian. To provide information on how to develop a clinical research career to a larger number of potential young investigators in pediatric cardiology, the directors of these two CRSD Cores jointly organized a one-day seminar for fellows and junior faculty from all of the PHN Core sites. The participants included faculty members from the PHN and the NHLBI. The day-long seminar was held on April 29, 2009, at the NHLBI site, immediately preceding the PHN Steering Committee meeting in Bethesda, MD.<h4>Methods</h4>the goals of the seminar were 1) to provide fellows and early investigators with basic skills in clinical research 2) to provide a forum for discussion of important research career choices 3) to introduce attendees to each other and to established clinical researchers in pediatric cardiology, and 4) to publish a commentary on the future of clinical research in pediatric cardiology.<h4>Results</h4>the following chapters are compilations of the talks given at the 2009 PHN Clinical Research Skills Development Seminar, published to share the information provided with a broader audience of those interested in learning how to develop a clinical research career in pediatric cardiology. The discussions of types of clinical research, research skills, career development strategies, funding, and career management are applicable to research careers in other areas of clinical medicine as well.<h4>Conclusions</h4>the aim of this compilation is to stimulate those who might be interested in the research career options available to investigators.

Hereditary hemochromatosis (HH) refers to several inherited disorders of iron metabolism leading to tissue iron overload. Classical HH is associated with mutations in HFE (C282Y homozygotes or C282Y/H63D compound heterozygotes) and is almost exclusively found in populations of northern European descent. Non-HFE-associated HH is caused by mutations in other recently identified genes involved in iron metabolism. Hepcidin is an iron regulatory hormone that inhibits ferroportin-mediated iron export from enterocytes and macrophages. Defective hepcidin gene expression or function may underlie most forms of HH. Target organs and tissues affected by HH include the liver, heart, pancreas, joints, and skin, with cirrhosis and diabetes mellitus representing late signs of disease in patients with markedly elevated liver iron concentration. Recently, we have encountered the rare representation of this disease of the oral cavity associated with generalized burning sensation of the tongue. The diagnosis was established accidently, from the lab investigations, otherwise the patient was healthy and free from classical signs and symptoms of the disease. The patient was adequately treated by phlebotomy. To conclude, all patients with a chief complaint of burning sensation of the oral cavity and tongue should be adequately screened for hereditary hemochromatosis to prevent the associated mortality and morbidity with the hemochromatosis.

During nervous system development, axons generate branches to connect with multiple synaptic targets. As with axon growth and guidance, axon branching is tightly controlled in order to establish functional neural circuits, yet the mechanisms that regulate this important process are less well understood. Here, we review recent advances in the study of several common branching processes in the vertebrate nervous system. By focusing on each step in these processes we illustrate how different types of branching are regulated by extracellular cues and neural activity, and highlight some common principles that underlie the establishment of complex neural circuits in vertebrate development.

Despite advances in surgery, radiation therapy, and chemotherapy, patients with cancer have a poor prognosis. Sustained aberrant tumor angiogenesis and metastasis is a major obstacle for effective cancer treatment. Just a few years ago, few would argue that one of the key success stories of the modern cancer medicine were the anti-angiogenic drugs targeting the vascular endothelial growth factor (VEGF) signaling pathway approved by FDA. This initial success inspired many researchers to search for new anti-angiogenic targets and drugs with the hope that one day, anti-angiogenic therapy might really become the panacea for cancer patients. Unfortunately, the limited clinical benefits achieved with anti-angiogenic drugs conflicts with the widely accepted notion that angiogenesis is a key event in tumor progression. Emerging data indicate that unique characteristics of the tumor vasculature within the tumor microenvironment may hold the key for success of anti-angiogenic therapy. In particular, the molecular and cellular alterations that sustain aberrant tumor angiogenesis in the face of angiogenic inhibitors represents novel targets for rationally designing and improving current anti-angiogenic strategies.

The cancer stem cell model was described for hematologic malignancies in 1997 and since then evidence has emerged to support it for many solid tumors as well, including colon cancer. This model proposes that certain cells within the tumor mass are pluripotent and capable of self-renewal and have an enhanced ability to initiate distant metastasis. The cancer stem cell model has important implications for cancer treatment, since most current therapies target actively proliferating cells and may not be effective against the cancer stem cells that are responsible for recurrence. In recent years great progress has been made in identifying markers of both normal and malignant colon stem cells. Proteins proposed as colon cancer stem cell markers include CD133, CD44, CD166, ALDH1A1, Lgr5, and several others. In this review we consider the evidence for these proteins as colon cancer stem cell markers and as prognostic indicators of colon cancer survival. Additionally, we discuss potential functions of these proteins and the implications this may have for development of therapies that target colon cancer stem cells.

<h4>Background/aim</h4>Color Doppler examination of changes in hepatic venous waveforms is being evaluated as a means of prediction of severity of portal hypertension and presence of esophageal varices. Normal hepatic venous waveform shows a triphasic pattern. In cirrhosis, this pattern changes to a biphasic or monophasic pattern. We aimed to study the sensitivity of loss of normal hepatic venous waveforms in predicting large varices in a cross-sectional analysis.<h4>Materials and methods</h4>All patients, admitted or attending the outpatient department, with a diagnosis of cirrhosis were included in the study. All patients were subjected to oesophagogastroduodenoscopy and Color Doppler examination, and waveform patterns in hepatic vein were recorded. The sensitivity and specificity of changes in waveform in detecting large varices were studied.<h4>Results</h4>A total of 51 cases were examined. Triphasic waves were seen in 4 (7.8%) cases, biphasic in 26 (51%) cases, and monophasic in 21 (41.2%) cases. Small varices were seen in 30 (58.8%) cases and large varices in 21 (41.2%) cases. The sensitivity of loss of the triphasic wave pattern in detecting significant varices (Grade 3 or 4) was very high (95.23%) and negative predictive value was also high (75%). Severity of liver disease as indicated by Child-Pugh and MELD scores did not correlate with changes in hepatic venous waveforms.<h4>Conclusion</h4>Loss of triphasic hepatic venous waveform is highly sensitive in predicting significant varices in patients with cirrhosis.

Alcohol dependence remains among the most common substance abuse problems worldwide, and compulsive alcohol consumption is a significant public health concern. Alcohol is an addictive drug that alters brain function through interactions with multiple neurotransmitter systems. These neurotransmitter systems mediate the reinforcing effects of alcohol. Specifically, the serotonergic system is important in mediating alcohol reward, preference, dependence, and craving. In this review chapter, we first discuss the serotonin system as it relates to alcoholism, and then outline interactions between this system and other neurotransmitter systems. We emphasize the serotonin transporter and its possible role in alcoholism, then present several serotonergic receptors and discuss their contribution to alcoholism, and finally assess the serotonin system as a target for pharmacotherapy, with an emphasis on current and potential treatments.

The molecular mechanisms and genetic programs required for cancer metastasis are sometimes overlapping, but components are clearly distinct from those promoting growth of a primary tumor. Every sequential, rate-limiting step in the sequence of events leading to metastasis requires coordinated expression of multiple genes, necessary signaling events, and favorable environmental conditions or the ability to escape negative selection pressures. Metastasis suppressors are molecules that inhibit the process of metastasis without preventing growth of the primary tumor. The cellular processes regulated by metastasis suppressors are diverse and function at every step in the metastatic cascade. As we gain knowledge into the molecular mechanisms of metastasis suppressors and cofactors with which they interact, we learn more about the process, including appreciation that some are potential targets for therapy of metastasis, the most lethal aspect of cancer. Until now, metastasis suppressors have been described largely by their function. With greater appreciation of their biochemical mechanisms of action, the importance of context is increasingly recognized especially since tumor cells exist in myriad microenvironments. In this chapter, we assemble the evidence that selected molecules are indeed suppressors of metastasis, collate the data defining the biochemical mechanisms of action, and glean insights regarding how metastasis suppressors regulate tumor cell communication to-from microenvironments.

Advanced high-throughput molecular cytogenetic analysis has enabled the identification of small chromosomal rearrangements, and two or more concurrently occurring chromosomal rearrangements have been identified using this technique. A girl with severe psychomotor developmental delay associated with an uncertain abnormality (detected by conventional karyotyping) in chromosome 10q had a sudden stroke at the age of 35 months. Laboratory and radiographic examinations revealed antithrombin (AT) deficiency and a patent foramen ovale (PFO). Two concurrent chromosomal aberrations, inverted duplication and deletion in the 10q26 region and a microdeletion in the 1q24.2q25.2 region including the AT gene (SERPINC1), were identified by microarray-based comparative genomic hybridization analysis. Both chromosomal aberrations were found to be of paternal origin. This study described the concurrence of chromosomal rearrangements involving two chromosomes, and estimated the frequency of two or more chromosomal aberrations as 2-4%.

No abstract available.

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.

<h4>Introduction</h4>Experiments were designed to investigate the effects of ethyl pyruvate (EP) in a murine model of hind-limb ischemia-reperfusion (IR) injury.<h4>Methods</h4>C57BL6 mice underwent 90 minutes of unilateral ischemia followed by 24 hours of reperfusion using two treatment protocols. For the preischemic treatment (pre-I) protocol, mice (n=6) were given 300 mg/kg EP before ischemia, followed by 150 mg/kg of EP just before reperfusion and at 6 hours and 12 hours after reperfusion. In a postischemic treatment (post-I) protocol, mice (n=7) were treated with 300 mg/kg EP at the end of the ischemic period, then 15 minutes later, and 2 hours after reperfusion and 150 mg/kg of EP at 4 hours, 6 hours, 10 hours, 16 hours, and 22 hours after reperfusion. Controls mice for both protocols were treated with lactated Ringers alone at time intervals identical to EP. Skeletal muscle levels of adenosine triphosphate (ATP), interleukin-1β, keratinocyte chemoattractant protein, and thrombin antithrombin-3 complex were measured. Skeletal muscle architectural integrity was assessed microscopically.<h4>Results</h4>ATP levels were higher in mice treated with EP compared with controls under the both treatment protocols (p=0.02). Interleukin-1β, keratinocyte chemoattractant protein, thrombin antithrombin-3 complex (p<0.05), and the percentage of injured fibers (p<0.0001) were significantly decreased in treated versus control mice under the both protocols.<h4>Conclusion</h4>Muscle fiber injury and markers of tissue thrombosis and inflammation were reduced, and ATP was preserved with EP in pre-I and post-I protocols. Further investigation of the efficacy of EP to modulate IR injury in a larger animal model of IR injury is warranted.

There has been an explosion of interest in studying gene-environment interactions (GxE) as they relate to the development of psychopathology. In this article, I review different methodologies to study gene-environment interaction, providing an overview of methods from animal and human studies and illustrations of gene-environment interactions detected using these various methodologies. Gene-environment interaction studies that examine genetic influences as modeled latently (e.g., from family, twin, and adoption studies) are covered, as well as studies of measured genotypes. Importantly, the explosion of interest in gene-environment interactions has raised a number of challenges, including difficulties with differentiating various types of interactions, power, and the scaling of environmental measures, which have profound implications for detecting gene-environment interactions. Taking research on gene-environment interactions to the next level will necessitate close collaborations between psychologists and geneticists so that each field can take advantage of the knowledge base of the other.

CXCL12/CXCR4 signaling is critical for cortical interneuron migration and their final laminar distribution. No information is yet available on CXCR7, a newly defined CXCL12 receptor. Here we demonstrated that CXCR7 regulated interneuron migration autonomously, as well as nonautonomously through its expression in immature projection neurons. Migrating cortical interneurons coexpressed Cxcr4 and Cxcr7, and Cxcr7(-/-) and Cxcr4(-/-) mutants had similar defects in interneuron positioning. Ectopic CXCL12 expression and pharmacological blockade of CXCR4 in Cxcr7(-/-) mutants showed that both receptors were essential for responding to CXCL12 during interneuron migration. Furthermore, live imaging revealed that Cxcr4(-/-) and Cxcr7(-/-) mutants had opposite defects in interneuron motility and leading process morphology. In vivo inhibition of Gα(i/o) signaling in migrating interneurons phenocopied the interneuron lamination defects of Cxcr4(-/-) mutants. On the other hand, CXCL12 stimulation of CXCR7, but not CXCR4, promoted MAP kinase signaling. Thus, we suggest that CXCR4 and CXCR7 have distinct roles and signal transduction in regulating interneuron movement and laminar positioning.

Although converging epidemiological evidence links exposure to stressful life events with increased risk for affective spectrum disorders, there is extraordinary interindividual variability in vulnerability to adversity. The environmentally moderated penetrance of genetic variation is thought to play a major role in determining who will either develop disease or remain resilient. Research on genetic factors in the aetiology of disorders of emotion regulation has, nevertheless, been complicated by a mysterious discrepancy between high heritability estimates and a scarcity of replicable gene-disorder associations. One explanation for this incongruity is that at least some specific gene effects are conditional on environmental cues, i.e. gene-by-environment interaction (G × E) is present. For example, a remarkable number of studies reported an association of variation in the human serotonin (5-HT) transporter gene (SLC6A4, 5-HTT, SERT) with emotional and cognitive traits as well as increased risk for depression in interaction with psychosocial adversity. The results from investigations in non-human primate and mouse support the occurrence of G × E interaction by showing that variation of 5-HTT function is associated with a vulnerability to adversity across the lifespan leading to unfavourable outcomes resembling various neuropsychiatric disorders. The neural and molecular mechanisms by which environmental adversity in early life increases disease risk in adulthood are not known but may include epigenetic programming of gene expression during development. Epigenetic mechanisms, such as DNA methylation and chromatin modification, are dynamic and reversible and may also provide targets for intervention strategies (see Bountra et al., Curr Top Behav Neurosci, 2011). Animal models amenable to genetic manipulation are useful in the identification of molecular mechanisms underlying epigenetic programming by adverse environments and individual differences in resilience to stress. Therefore, deeper insight into the role of epigenetic regulation in the process of neurodevelopmental programmes is likely to result in early diagnosis of affective spectrum disorders and will contribute to the design of innovative treatments targeting neural pathways that foster resilience.

<h4>Objective</h4>DCC is the receptor for netrin, a protein that guides axon migration of developing neurons across the body's midline. Mutations in the DCC gene were recently identified in 2 families with congenital mirror movements (MM). The objective was to study clinical and genetic characteristics of 3 European families with MM and to test whether this disorder is genetically homogeneous.<h4>Methods</h4>We studied 3 MM families with a total of 13 affected subjects. Each patient had a standardized interview and neurologic examination, focusing on the phenomenology and course of the MM. The severity of MM was also assessed. Molecular analysis of DCC was performed in the index cases. In addition, linkage analysis of the DCC locus was performed in a large French family.<h4>Results</h4>The clinical expression and course of MM were very similar in all the affected subjects, regardless of DCC mutational status. However, slight intersubject variability in the severity of MM was noted within each family. Onset always occurred in infancy or early childhood, and MM did not deteriorate over time. Motor disability due to MM was mild and restricted to activities that require independent movements of the 2 hands. We found a novel mutation in the DCC gene in an Italian family with MM associated with abnormal ipsilateral corticospinal projection. The DCC locus was excluded in the French family.<h4>Conclusion</h4>DCC has a crucial role in the development of corticospinal tracts in humans. Congenital MM is genetically heterogeneous, despite its clinical homogeneity.

Anorexia nervosa (AN) and bulimia nervosa (BN) are disorders characterized by aberrant patterns of feeding behavior, weight regulation, and disturbances in attitudes and perceptions toward body weight and shape. Several lines of evidence nominate disturbances of serotonin (5-HT) pathways as playing a role in the pathogenesis and pathophysiology of AN and BN. For example, 5-HT pathways are known to contribute to the modulation of a range of behaviors commonly seen in individuals with AN and BN. New technology using brain imaging with radioligands offers the potential for understanding previously inaccessible brain 5-HT neurotransmitter function and its dynamic relationship with human behaviors. Recent studies using positron emission tomography and single photon emission computed tomography with 5-HT-specific radioligands have consistently shown 5-HT(1A) and 5-HT(2A) receptor and 5-HT transporter alterations in AN and BN in cortical and limbic structures, which may be related to anxiety, behavioral inhibition, and body image distortions. These disturbances are present when subjects are ill and persist after recovery, suggesting that these may be traits that are independent of the state of the illness. Effective treatments for AN and BN have been elusive. A better understanding of neurobiology is likely to be important for developing specific and more powerful therapies for these often chronic and deadly disorders.

<h4>Context</h4>Concerns regarding the risks associated with estrogen and progesterone to manage menopausal symptoms have resulted in its declining use and increased interest in nonhormonal treatments with demonstrated efficacy for hot flashes.<h4>Objective</h4>To determine the efficacy and tolerability of 10 to 20 mg/d escitalopram, a selective serotonin reuptake inhibitor, in alleviating the frequency, severity, and bother of menopausal hot flashes.<h4>Design, setting, and patients</h4>A multicenter, 8-week, randomized, double-blind, placebo-controlled, parallel group trial that enrolled 205 women (95 African American; 102 white; 8 other) between July 2009 and June 2010.<h4>Intervention</h4>Women received 10 to 20 mg/d of escitalopram or a matching placebo for 8 weeks.<h4>Main outcome measures</h4>Primary outcomes were the frequency and severity of hot flashes assessed by prospective daily diaries at weeks 4 and 8. Secondary outcomes were hot flash bother, recorded on daily diaries, and clinical improvement (defined as hot flash frequency ≥50% decrease from baseline).<h4>Results</h4>Mean (SD) daily hot flash frequency was 9.78 (5.60) at baseline. In a modified intent-to-treat analysis that included all randomized participants who provided hot flash diary data, the mean difference in hot flash frequency reduction was 1.41 (95% CI, 0.13-2.69) fewer hot flashes per day at week 8 among women taking escitalopram (P < .001), with mean reductions of 4.60 (95% CI, 3.74-5.47) and 3.20 (95% CI, 2.24-4.15) hot flashes per day in the escitalopram and placebo groups, respectively. Fifty-five percent of women in the escitalopram group vs 36% in the placebo group reported a decrease of at least 50% in hot flash frequency (P = .009) at the 8-week follow-up. Reductions in hot flash severity scores were significantly greater in the escitalopram group (-0.52; 95% CI, -0.64 to -0.40 vs -0.30; 95% CI, -0.42 to -0.17 for placebo; P < .001). Race did not significantly modify the treatment effect (P = .62). Overall discontinuation due to adverse events was 4% (7 in the active group, 2 in the placebo group). Three weeks after treatment ended, women in the escitalopram group reported a mean 1.59 (95% CI, 0.55-2.63; P = .02) more hot flashes per day than women in the placebo group.<h4>Conclusion</h4>Among healthy women, the use of escitalopram (10-20 mg/d) compared with placebo resulted in fewer and less severe menopausal hot flashes at 8 weeks of follow-up.<h4>Trial registration</h4>clinicaltrials.gov Identifier: NCT00894543.

In Huntington's disease (HD), the expansion of polyglutamine (polyQ) repeats at the N terminus of the ubiquitous protein huntingtin (htt) leads to neurodegeneration in specific brain areas. Neurons degenerating in HD develop synaptic dysfunctions. However, it is unknown whether mutant htt impacts synaptic function in general. To investigate that, we have focused on the nerve terminals of motor neurons that typically do not degenerate in HD. Here, we have studied synaptic transmission at the neuromuscular junction of transgenic mice expressing a mutant form of htt (R6/1 mice). We have found that the size and frequency of miniature endplate potentials are similar in R6/1 and control mice. In contrast, the amplitude of evoked endplate potentials in R6/1 mice is increased compared to controls. Consistent with a presynaptic increase of release probability, synaptic depression under high-frequency stimulation is higher in R6/1 mice. In addition, no changes were detected in the size and dynamics of the recycling synaptic vesicle pool. Moreover, we have found increased amounts of the synaptic vesicle proteins synaptobrevin 1,2/VAMP 1,2 and cysteine string protein-α, and the SNARE protein SNAP-25, concomitant with normal levels of other synaptic vesicle markers. Our results reveal that the transgenic expression of a mutant form of htt leads to an unexpected gain of synaptic function. That phenotype is likely not secondary to neurodegeneration and might be due to a primary deregulation in synaptic protein levels. Our findings could be relevant to understand synaptic toxic effects of proteins with abnormal polyQ repeats.

Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells. Although each pathway is governed by its own set of factors, they all contain Rab GTPases that serve as master regulators. In this review, we discuss how Rabs can regulate virtually all steps of membrane traffic from the formation of the transport vesicle at the donor membrane to its fusion at the target membrane. Some of the many regulatory functions performed by Rabs include interacting with diverse effector proteins that select cargo, promoting vesicle movement, and verifying the correct site of fusion. We describe cascade mechanisms that may define directionality in traffic and ensure that different Rabs do not overlap in the pathways that they regulate. Throughout this review we highlight how Rab dysfunction leads to a variety of disease states ranging from infectious diseases to cancer.

Gastric cancer is one of the leading causes of cancer-related deaths worldwide, although the incidence has gradually decreased in many Western countries. Two main gastric cancer histotypes, intestinal and diffuse, are recognised. Although most of the described genetic alterations have been observed in both types, different genetic pathways have been hypothesized. Genetic and epigenetic events, including 1q loss of heterozygosity (LOH), microsatellite instability and hypermethylation, have mostly been reported in intestinal-type gastric carcinoma and its precursor lesions, whereas 17p LOH, mutation or loss of E-cadherin are more often implicated in the development of diffuse-type gastric cancer. In this review, we summarize the sometimes contradictory findings regarding those markers which influence the progression of gastric adenocarcinoma.

The monoaminergic neuron, in particular the dopaminergic neuron, is central to mediating the hedonic and addictive properties of drugs of abuse. The effects of amphetamine (AMPH) and cocaine (COC), for example, depend on the ability to increase dopamine in the synapse, by effects on either the plasma membrane transporter DAT or the vesicular transporter for monoamine storage, VMAT2. The potential role of DAT as a target for AMPH and COC has been reviewed extensively. Here, we present VMAT2 as a target that enables the rewarding and addictive actions of these drugs, based on imaging, neurochemical, biochemical, cell biological, genetic, and immunohistochemical evidence. The presence of VMAT2 in noradrenergic, serotoninergic, histaminergic, and potentially trace aminergic neurons invites consideration of a wider role for aminergic neurotransmission in AMPH and COC abuse and addiction.

There is considerable evidence that alterations in striatal medium-sized spiny neurons (MSSNs) giving rise to the direct (D1 receptor-expressing) and indirect (D2 receptor-expressing) pathways differentially contribute to the phenotype of Huntington's disease (HD). To determine how each subpopulation of MSSN is functionally affected, we examined spontaneous excitatory postsynaptic currents (sEPSCs) and dopamine (DA) modulation in two HD mouse models, the YAC128 and the BACHD (a bacterial-artificial chromosome). These mice also expressed enhanced green fluorescent protein (EGFP) under the control of the promoter for either DA D1 or D2 receptors to identify neurons. In early symptomatic YAC128 and BACHD mice, glutamate transmission was increased in both D1 and D2 MSSNs, but in different ways. D1 cells displayed increased sEPSC frequencies and decreased paired-pulse ratios (PPRs) while D2 cells displayed larger evoked glutamate currents but no change in sEPSC frequencies or PPRs. D1 receptor modulation of sEPSCs was absent in D1-YAC128 cells at the early symptomatic stage but was restored by treating the slices with tetrabenazine. In contrast, in fully symptomatic YAC128 mice, glutamate transmission was decreased specifically in D1 cells, and D1 receptor modulation was normal in D1-YAC128 cells. Behaviorally, early symptomatic mice showed increased stereotypies that were decreased by tetrabenazine treatment. Together, these studies support differential imbalances in glutamate and DA transmission in direct and indirect pathway MSSNs. Stereotypic behavior at an early stage could be explained by increased glutamate activity and DA tone in direct pathway neurons, whereas hypokinesia at later stages could result from reduced input onto these neurons.

Endocannabinoids (eCBs) are retrograde neurotransmitters that modulate the function of many types of synapses. The presence of eCBs, their CB1 receptor (CB1R), and metabolizing enzymes at embryonic and early postnatal periods have been linked to developmental processes such as neuronal proliferation, differentiation, and migration, axon guidance, and synaptogenesis. Here, we demonstrate the presence of a functional eCB system in the developing visual system and the role of CB1R during axon growth and retinothalamic development. Pharmacological treatment of retinal explants and primary cortical neuron cultures with ACEA, a selective CB1R agonist, induced a collapse of the growth cone (GC). Furthermore the application of AM251, a CB1R inverse agonist, to the neuronal cultures increased the surface area of GC. In vivo, intraocular injection of ACEA diminished retinal projection growth, while AM251 promoted growth and caused aberrant projections. In addition, compared with their wild-type littermates, CB1R-deficient adult mice revealed a lower level of eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus. Finally, we found that pharmacological modulation of CB1R affected the trafficking of Deleted in colorectal cancer (DCC) receptor to the plasma membrane in a PKA-dependent manner. Moreover, pharmacological inhibition or genetic inactivation of DCC abolished the CB1R-induced reorganization of the GC. Overall, these findings establish a mechanism by which the CB1R influences GC behavior and nervous system development in concerted action with DCC.

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells.

<h4>Background</h4>Several telemedicine-based eHealth programs exist, but patient-focused personalized decision support (PDS) is usually lacking. We evaluated the acceptance, efficiency, and cost-effectiveness of telemedicine-assisted PDS in routine outpatient diabetes care.<h4>Methods</h4>Data are derived from the Diabetiva® program of the German health insurance company BKK TAUNUS. Diabetiva offers telemedicine-based outpatient health care in combination with PDS generated by the Karlsburg Diabetes Management System, KADIS®. This retrospective analysis is based on data from the first year of running KADIS-based PDS in routine diabetes care. Participants were insured persons diagnosed with diabetes and cardiovascular diseases. For final analysis, patients were grouped retrospectively as users or nonusers according to physician acceptance or not (based on questionnaires) of the KADIS-based PDS.<h4>Results</h4>A total of 538 patients participated for more than one year in the Diabetiva program. Of these patients, 289 had complete data sets (two continuous glucose monitoring measurements, two or more hemoglobin A1c (HbA1c) values, and a signed questionnaire) and were included in the final data analysis. Of the physicians, 74% accepted KADIS-based PDS, a rate that was clearly related to HbA1c at the beginning of the observation. If KADIS-based PDS was accepted, HbA1c decreased by 0.4% (7.1% to 6.7%). In contrast, rejection of KADIS-based PDS resulted in an HbA1c increase of 0.5% (6.8% to 7.3%). The insurance company revealed an annual cost reduction of about 900 € per participant in the Diabetiva program.<h4>Conclusions</h4>KADIS-based PDS in combination with telemedicine has high potential to improve the outcome of routine outpatient diabetes care.

The small subunit (SSU) processome is a 2.2-MDa ribonucleoprotein complex involved in the processing, assembly, and maturation of the SSU of eukaryotic ribosomes. The identities of many of the factors involved in SSU biogenesis have been elucidated over the past 40 years. However, as our understanding increases, so do the number of questions about the nature of this complicated process. Cataloging the components is the first step toward understanding the molecular workings of a system. This review will focus on how identifying components of ribosome biogenesis has led to the knowledge of how these factors, protein and RNA alike, associate with one another into subcomplexes, with a concentration on the small ribosomal subunit. We will also explore how this knowledge of subcomplex assembly has informed our understanding of the workings of the ribosome synthesis system as a whole.

Infrared, Raman, and nuclear resonant vibrational (NRVS) spectroscopies have been used to address the Fe-H bonding in trans-HFe(CO) iron hydride compound, HFe(CO)(dppe)2, dppe = 1,2-bis(diphenylphosphino)ethane. H and D isotopomers of the compound, with selective substitution at the metal-coordinated hydrogen, have been considered in order to address the Fe-H/D stretching and bending modes. Experimental results are compared to the normal mode analysis by density functional theory (DFT). The results are that (i) the IR spectrum does not clearly show Fe-H stretching or bending modes; (ii) Fe-H stretching modes are clear but weak in the Raman spectrum, and Fe-H bending modes are weak; (iii) NRVS 57Fe spectroscopy resolves Fe-H bending clearly, but Fe-H or Fe-D stretching is above its experimentally resolved frequency range. DFT calculations (with no scaling of frequencies) show intensities and peak locations that allow unambiguous correlations between observed and calculated features, with frequency errors generally less than 15 cm(-1). Prospects for using these techniques to unravel vibrational modes of protein active sites are discussed.

We describe a case of heparin binding site Arg79Cys mutation in the gene encoding antithrombin, SERPINC1, in a Korean patient with hereditary antithrombin (AT) deficiency. The patient was a 34-year-old Korean man who presented with deep vein thrombosis (DVT) in his right leg without precipitating factors. On outpatient evaluation, coagulation tests without anticoagulation revealed a decreased AT III activity level at 48%, but normal AT III antigen level at 103%, indicating type II AT deficiency. Family studies revealed that his father (62 years of age) had decreased AT activity (48%) but had normal AT antigen levels (116%), indicating that the proband had a paternally inherited type II AT deficiency. Direct sequencing of the SERPINC1 gene in the patient and his father revealed a heterozygotic missense mutation, a cytosine to thymine substitution at nucleotide position 235 in exon 2 of the SERPINC1 gene (p.Arg79Cys). To our knowledge, this is the first report of Arg79Cys heterozygote mutation in family members with venous thromboembolism.

Infiltration of the liver by hematologic malignancies is an uncommon cause of liver failure. B-Cell chronic lymphocytic leukemia (cll) is a usually indolent disease that may infiltrate the liver, but based on a review of the literature, has never been reported to induce acute liver failure. Here, we describe the case of a 78-year-old woman with acute liver failure secondary to infiltration with cll being unresponsive to chemotherapy and causing death. This case is notable because of its atypical presentation and ultimate poor prognosis.

The Alzheimer's disease (AD)-associated amyloid-β protein precursor (AβPP) is cleaved by α-, β-, and presenilin (PS)/γ-secretases through sequential regulated proteolysis. These proteolytic events control the generation of the pathogenic amyloid-β (Aβ) peptide, which excessively accumulates in the brains of individuals afflicted by AD. A growing number of additional proteins cleaved by PS/γ-secretase continue to be discovered. Similarly to AβPP, most of these proteins are type-I transmembrane proteins involved in vital signaling functions regulating cell fate, adhesion, migration, neurite outgrowth, or synaptogenesis. All the identified proteins share common structural features, which are typical for their proteolysis. The consequences of the PS/γ-secretase-mediated cleavage on the function of many of these proteins are largely unknown. Here, we review the current literature on the proteolytic processing mediated by the versatile PS/γ-secretase complex. We begin by discussing the steps of AβPP processing and PS/γ-secretase complex composition and localization, which give clues to how and where the processing of other PS/γ-secretase substrates may take place. Then we summarize the typical features of PS/γ-secretase-mediated protein processing. Finally, we recapitulate the current knowledge on the possible physiological function of PS/γ-secretase-mediated cleavage of specific substrate proteins.

Osteoporosis is in 60-80% a hereditary disease with a characteristic multifactorial pathogenesis during which the effects of many "weak" genes interact with external factors. To date, most information relating to the correlations between genes and bone parameter variability (density, quality and metabolism) has been provided by association studies of candidate genes for osteoporosis. The best known genes related to bone density have been identified as the genes for the vitamin D, estrogen and calcitonin receptor, LRP5 and LRP6. The genes for IL-1alpha and osteoprotegerin are responsible for the parameters of bone remodeling. Recently discovered genes related to bone phenotype include identified genes for hypolactasia, tetrafolate reductase and ALDH7A1. Bone size and dimensions are probably partially controlled by the PLCL1 gene. Candidate genes for osteoporosis probably also determine the production of calciotropic hormones (PTH, sex steroids) and even some extra-osseous phenotypes (inflammation, immunity, susceptibility to malignancies). On the contrary, genes that determine extra-osseous parameters (e.g. lipoprotein levels) are associated with the bone phenotype (the gene for ApoE is related to bone density). Association studies, though, have serious limitations. Among others, these include the influence of linkage disequilibrium associated with the close proximity of the identified genes within DNA, which may be one of the causes of false positive results. In children, where building of the skeleton is influenced predominantly by external factors (nutrition, physical activity), the relationship between candidate genes and bone mass is less close than in adults. This overview deals with the physiology and sexual differentiation of pubertal bone. It discusses the importance of identifying candidate genes in the prevention and targeted treatment of osteoporosis (pharmacogenetics) as well as the application of the FRAX (WHO) program in the ten-year prediction of fractures in osteopenic patients.

No abstract available.

To explore the association of polymorphism in the serotonin transporter gene and the susceptibility to alcohol dependence in Yunnan Han population, PCR and DNA sequencing techniques were used to detect 5-HTT-linked promoter region (5-HTTLPR). One hundred and eighteen alcohol dependent patients as case group and 214 normal people as control group were employed in this study. Significant differences in genotype frequencies were present between case group and control group of 5-HTTLPR (P<0.05). The proportion of L/L and L/S genotype was significantly smaller in case group than that was in control group (OR=0.581, P=0.026). No significant association was observed in allelic frequencies, which differed in different ethnic groups. In conclusion, 5-HTTLPR polymorphism may be associated with alcohol dependent patients, and the genotype L/L or L/S may be a genetic factor that is responsible for decreasing susceptibility of alcohol dependence in Yunnan Han population.

Sox9 is a direct transcriptional activator of cartilage-specific extracellular matrix genes and has essential roles in chondrogenesis. Mutations in or around the SOX9 gene cause campomelic dysplasia or Pierre Robin Sequence. However, Sox9-dependent transcriptional control in chondrogenesis remains largely unknown. Here we identify Wwp2 as a direct target of Sox9. Wwp2 interacts physically with Sox9 and is associated with Sox9 transcriptional activity via its nuclear translocation. A yeast two-hybrid screen using a cDNA library reveals that Wwp2 interacts with Med25, a component of the Mediator complex. The positive regulation of Sox9 transcriptional activity by Wwp2 is mediated by the binding between Sox9 and Med25. In zebrafish, morpholino-mediated knockdown of either wwp2 or med25 induces palatal malformation, which is comparable to that in sox9 mutants. These results provide evidence that the regulatory interaction between Sox9, Wwp2 and Med25 defines the Sox9 transcriptional mechanisms of chondrogenesis in the forming palate.

<h4>Objective</h4>To investigate the effects of Yiqi Chutan Recipe (YCR, a Chinese herbal prescription for invigorating qi and removing phlegm) on the growth and metastasis of tumor, survival time, and the expressions of peroxiredoxin (PRDX-1 and PRDX-6) in tumor tissue of C57BL/6J mice bearing Lewis lung carcinoma.<h4>Methods</h4>Lewis lung carcinoma cells were transplanted to 90 C57BL/6J mice receiving preconditioning for inducing Pi-deficiency syndrome and divided into three groups treated respectively with saline, high dose YCR (3.0 g/kg) and low dose YCR (1.0 g/kg) once a day via gastric infusion. Besides, a group of 30 healthy mice simply received tumor cell transplantation was set up for controls. Ten mice selected from each group were sacrificed 21 days later, the size, weight and lung metastasis foci of tumor in mice were measured, and expressions of PRDX-1 and PRDX-6 in tumor tissue were detected using immunohistochemical method. The survival time of the remained 20 mice in each groups was observed.<h4>Results</h4>Tumor size, weight and the numbers of lung metastatic foci were (1.14 +/- 0.30) cm3, (0.83 +/- 0.26) g, (6.20 +/- 2.53) foci in the high dose YCR treated group, which were significantly lower than those in the control group [(2.83 +/- 0.35) cm3, (2.08 +/- 0.28) g, and (8.60 +/- 1.84) foci] respectively, also lower than those in the saline treated group [(2.29 +/- 0.49) cm3, (1.67 +/- 0.33) g and (8.70 +/- 2.00) foci]. The median survival time in the three groups, in above order, were 29.00 +/- 0.89 days, 22.00 +/- 0.75 days and 21.00 +/- 0.53 days; the average survival time in them 29.60 +/- 0.53 days, 22.90 +/- 0.50 days and 20.95 +/- 0.44 days; the PRDX-1 expression were 0.15 +/- 0.03, 0.52 +/- 0.07 and 0.61 +/- 0.09; and the PRDX-6 expression were 0.12 +/- 0.02, 0.43 +/- 0.06 and 0.56 +/- 0.07, all showed significant difference in comparing the indices in the high dose treated group with those in the control group and in the saline treated group (P < 0.05 or P < 0.01). The tumor growth inhibition rate was 50.30% in the high dose YCR group with life prolongation rate of 41.29%, all better than those in the low dose YCR treated group (P < 0.05).<h4>Conclusions</h4>YCR can remarkably inhibit the growth and metastasis of Lewis lung carcinoma in mice with Pi-asthenia syndrome, prolong their survival period, and its mechanism is possibly related to the reduction of over expressed PRDX-1 and PRDX-6.

<h4>Objectives</h4>Non-alcoholic Steatohepatitis (NASH) is a cause of cirrhosis. Detecting its preventable causes could be useful in decreasing the disease and its complications. This study aimed to assess the prevalence of NASH in adult population living in Isfahan city as a sample of Iranian population.<h4>Methods</h4>Blood samples of 2030 individuals referring to Dr. Shariati Hospital for routine health checkup were collected. All individuals with high liver enzymes [aspartate aminotransferase (AST) and/or Alanine aminotransferease (ALT)] were included in the study. Known causes of altered liver enzymes were excluded. Second blood samples were obtained 6 months later and those cases with persistent enzyme elevation were surveyed with ultrasonography.<h4>Results</h4>In the first survey, 234 individuals (11.5 %) had ALT levels of more than 40 U/L. By rechecking samples after 6 months, 50% of all high serum ALT individuals remained at high level. After conducting complementary biochemical tests, 3.3% of the population was considered to have NASH; 20% of NASH cases did not have any abnormal findings in ultrasonography. Hypercholesterolemia was the most coexisting risk factor in NASH cases. We found a reverse correlation between ALT level and age (P = 0.02).<h4>Conclusions</h4>The prevalence of NASH was comparable with other countries. According to the significantl correlation between ALT level and age, preventive interactions in younger individuals would decrease the incidence of NASH.

Profiling of serum and plasma proteins has substantial relevance to the discovery of circulating disease biomarkers. However, the extreme complexity and vast dynamic range of protein abundance in serum and plasma present a formidable challenge for protein analysis. Thus, integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomic analysis of serum or plasma. In this chapter, we describe an orthogonal multidimensional intact-protein analysis system (IPAS) (Wang et al., Mol Cell Proteomics 4:618-625, 2005) coupled with protein tagging (Faca et al., J Proteome Res 5:2009-2018, 2006) to profile the serum and plasma proteomes quantitatively, which we have applied in our biomarker discovery studies (Katayama et al., Genome Med 1:47, 2009; Faca et al., PLoS Med 5:e123, 2008; Zhang et al. Genome Biol 9:R93, 2008).

The covalent attachment of small ubiquition-like modifier (SUMO) polypeptides, or sumoylation, is an important regulator of the functional properties of many proteins. Among these are many proteins implicated in human diseases including cancer and Huntington's, Alzheimer's, and Parkinson's diseases, as well as spinocerebellar ataxia 1 and amyotrophic lateral sclerosis. The results of two more recent studies identify two additional human disease-associated proteins that are sumoylated, amyloid precursor protein (APP), and lamin A. APP sumoylation modulates Aβ peptide levels, suggesting a potential role in Alzheimer's disease, and decreased lamin A sumoylation due to mutations near its SUMO site has been implicated in causing some forms of familial dilated cardiomyopathy.

There has been considerable promise and hope that pharmacogenomics will optimize existing treatments for major depression, as well as identify novel targets for drug discovery. Immediately after the sequencing of the human genome, there was much hope that tremendous progress in pharmacogenomics would rapidly be achieved. In the past 10 years this initial enthusiasm has been replaced by a more sober optimism, as we have gone a long way towards the goal of guiding therapeutics based on genomics. While the effort to translate discovery to clinical applications is ongoing, we now have a vast body of knowledge as well as a clear direction forward. This article will provide a critical appraisal of the state of the art in the pharmacogenomics of depression, both in terms of pharmacodynamics and pharmacokinetics.

It has been more than 17 years since the causative mutation for Huntington's disease was discovered as the expansion of the triplet repeat in the N-terminal portion of the Huntingtin (HTT) gene. In the intervening time, researchers have discovered a great deal about Huntingtin's involvement in a number of cellular processes. However, the role of Huntingtin in the key pathogenic mechanism leading to neurodegeneration in the disease process has yet to be discovered. Here, we review the body of knowledge that has been uncovered since gene discovery and include discussions of the HTT gene, CAG triplet repeat expansion, HTT expression, protein features, posttranslational modifications, and many of its known protein functions and interactions. We also highlight potential pathogenic mechanisms that have come to light in recent years.

Huntington's disease (HD) is caused by a triplet repeat expansion in the IT15 gene on chromosome 4 encoding huntingtin. Gene mutations are found in about 99% of cases, with symptoms and signs suggestive of HD. This implies the existence of other causes of this syndrome, and, in recent years, several other distinct genetic disorders have been identified that can present with a clinical picture indistinguishable from HD, termed HD-like (HDL) syndromes. So far, four genes associated with HDL syndromes have been identified, including the prion protein gene (HDL1), the junctophilin 3 gene (HDL2) and, the gene encoding the TATA box-binding protein (HDL4). In addition, a single family with a recessively inherited HD phenocopy, the exact genetic basis of which is currently unknown (HDL3), has been described. These disorders, however, account for only a small proportion of HDL cases, and the list of HDL genes and conditions is set to grow. In this article, we review the currently identified HD phenocopy disorders and discuss clinical clues to facilitate further investigations. We will concentrate on the four so-called HDL syndromes mentioned above. Other genetic choreatic syndromes such as dentatorubral-pallidoluysian atrophy, neuroferritinopathy, pantothenate kinase-associated neurodegeneration, and chorea-acanthocytosis are also briefly discussed.

This article reviews the history of widespread exchanges of genetic segments initiated over 3 billion years ago, to be part of their life style, by sphero-protoplastic cells, the ancestors of archaea, prokaryota, and eukaryota. These primordial cells shared a hostile anaerobic and overheated environment and competed for survival. "Coexist with, or subdue and conquer, expropriate its most useful possessions, or symbiose with it, your competitor" remain cellular life's basic rules. This author emphasizes the role of viruses, both in mediating cell fusions, such as the formation of the first eukaryotic cell(s) from a united crenarchaeon and prokaryota, and the transfer of host cell genes integrated into viral (phages) genomes. After rising above the Darwinian threshold, rigid rules of speciation and vertical inheritance in the three domains of life were established, but horizontal gene transfers with or without cell fusions were never abolished. The author proves with extensive, yet highly selective documentation, that not only unicellular microorganisms, but the most complex multicellular entities of the highest ranks resort to, and practice, cell fusions, and donate and accept horizontally (laterally) transferred genes. Cell fusions and horizontally exchanged genetic materials remain the fundamental attributes and inherent characteristics of the living matter, whether occurring accidentally or sought after intentionally. These events occur to cells stagnating for some 3 milliard years at a lower yet amazingly sophisticated level of evolution, and to cells achieving the highest degree of differentiation, and thus functioning in dependence on the support of a most advanced multicellular host, like those of the human brain. No living cell is completely exempt from gene drains or gene insertions.

Helicases are motor proteins that catalyze the unwinding of duplex nucleic acids in an ATP-dependent manner. They are involved in almost all the nucleic acid transactions. In the present study, we report a comprehensive analysis of helicase gene family in human and its comparison with homologs in model organisms. The human genome encodes for 95 non-redundant helicase proteins, of which 64 are RNA helicases and 31 are DNA helicases. 57 RNA helicases are validated based on annotations and occurrence of conserved helicase signature motifs. These include 14 DExH and 37 DExD subfamily members, six other members such as U5.snRNP, ATR-X, Suv3, FANCJ, and two of superkiller viralicidic activity 2-like helicases. 31 DNA helicases are also identified, which include RecQ, MCM and RuvB-like helicases. Finding a set of helicases in human and almost similar sequences in model organisms suggests that the "core" members of helicase gene family are highly conserved throughout evolution. The present study gives an overview of members of RNA and DNA helicases encoded by the human genome along with their conserved motifs, phylogeny and homologs in model organisms. The study on comparing these homologs will spread light on the organization and complexity of helicase gene family in model organisms. The comprehensive analysis of human helicases presented in this study will further provide an invaluable resource for elaborate biological research on these helicases.

Recent studies have demonstrated that hemichannels, which form gap junctions when paired from apposing cells, may serve additional roles when unpaired including cell adhesion and paracrine communication. Hemichannels in mammals are formed by connexins or pannexins, while in insects they are formed by pannexin homologues termed innexins. The formation of functional gap junctions by insect innexins has been established, although their ability to form functional nonjunctional hemichannels has not been reported. Here the characteristics of nonjunctional hemichannels were examined in three lepidopteran cell types, two cell lines (High Five and Sf9) and explanted hemocytes from Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Selective fluorescent dye uptake by hemichannels was observed in a significant minority of cells, using fluorescence microscopy and flow cytometry. Carbenoxelone, an inhibitor of mammalian junctions, disrupted dye uptake, while flufenamic acid and mefloquine did not. The presence of Ca(2+) and Mg(2+) in the media increased hemichannel activity. Additionally, lipopolysaccharide, a stimulator of immune activity in lepidopterans, decreased dye uptake. These results demonstrate for the first time the activity of nonjunctional hemichannels in insect cells, as well as pharmacological tools to manipulate them. These results will facilitate the further examination of the role of innexins and nonjunctional hemichannels in insect cell biology, including paracrine signaling, and comparative studies of mammalian pannexins and insect innexins.

ETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5'-GGA(A/T)-3'. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells.

Metastasis is the major cause of thyroid cancer-related death. However, little is known about the genes involved in the metastatic spread of thyroid carcinomas. We have created a mouse that spontaneously develops metastatic follicular thyroid carcinoma (FTC). This mouse harbors a targeted mutation (denoted TRβPV) in the thyroid hormone receptor β gene (Thrb(PV/PV) mice). Our recent studies show that the highly elevated level of thyroid stimulating hormone (TSH) in Thrb(PV/PV) mice promotes proliferation of thyroid tumor cells, but requires the collaboration of the oncogenic action of TRβPV to empower the tumor cells to undergo distant metastasis. To uncover genes destined to drive the metastatic process, we used cDNA microarrays to compare the genomic expression profile of laser capture microdissected thyroid tumor lesions of Thrb(PV/PV) mice with that of hyperplastic thyroid cells of wild-type mice having elevated TSH induced by treatment with the anti-thyroid drug propylthiouracil (WT-PTU mice). Analyses of microarray data indicated that the expressions of 150 genes were significantly altered between Thrb(PV/PV) and WT-PTU mice (87 genes had higher expression and 63 genes had lower expression in Thrb(PV/PV) mice than in WT-PTU mice). Thirty-six percent of genes with altered expression function as key regulators in metastasis. The remaining genes were involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/extracellular matrix signaling. The present studies have uncovered novel genes responsible for the metastatic spread of FTC and, furthermore, have shown that the metastatic process of thyroid cancer requires effective collaboration among genes with diverse cellular functions. Importantly, the present studies indicate that the tumor cells in the primary lesions are endowed with the genes destined to promote metastasis. Thus, our study has provided new insights into the understanding of the metastatic spread of human thyroid cancer.