Gene Literature Dashboard

Given the importance of deregulated phosphoinositide (PI) signaling in leukemic hematopoiesis, genes coding for proteins that regulate PI metabolism may have significant and as yet unappreciated roles in leukemia. We performed a targeted knockdown (KD) screen of PI modulator genes in human acute myeloid leukemia (AML) cells and identified candidates required to sustain proliferation or prevent apoptosis. One of these, the lipid kinase phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A) regulates cellular levels of phosphatidylinositol-5-phosphate (PtsIns5P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). We found PIP4K2A to be essential for the clonogenic and leukemia-initiating potential of human AML cells, and for the clonogenic potential of murine MLL-AF9 AML cells. Importantly, PIP4K2A is also required for the clonogenic potential of primary human AML cells. Its KD results in accumulation of the cyclin-dependent kinase inhibitors CDKN1A and CDKN1B, G₁ cell cycle arrest and apoptosis. Both CDKN1A accumulation and apoptosis were partially dependent on activation of the mTOR pathway. Critically, however, PIP4K2A KD in normal hematopoietic stem and progenitor cells, both murine and human, did not adversely impact either clonogenic or multilineage differentiation potential, indicating a selective dependency that we suggest may be the consequence of the regulation of different transcriptional programs in normal versus malignant cells. Thus, PIP4K2A is a novel candidate therapeutic target in myeloid malignancy.

Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been used to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. PL inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that PL directly inhibited binding of Stat3 to its phosphotyrosyl peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as Janus kinases, Src kinase family members or receptor tyrosine kinases. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient-derived xenografts. PL's antitumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 has a role.

<h4>Background</h4>In an earlier study, we hypothesized that genomic segments with different sequence organization patterns (OPs) might display functional specificity despite their similar GC content. Here we tested this hypothesis by dividing the human genome into 100 kb segments, classifying these segments into five compositional groups according to GC content, and then characterizing each segment within the five groups by oligonucleotide counting (k-mer analysis; also referred to as compositional spectrum analysis, or CSA), to examine the distribution of sequence OPs in the segments. We performed the CSA on the entire DNA, i.e., its coding and non-coding parts the latter being much more abundant in the genome than the former.<h4>Results</h4>We identified 38 OP-type clusters of segments that differ in their compositional spectrum (CS) organization. Many of the segments that shared the same OP type were enriched with genes related to the same biological processes (developmental, signaling, etc.), components of biochemical complexes, or organelles. Thirteen OP-type clusters showed significant enrichment in genes connected to specific gene-ontology terms. Some of these clusters seemed to reflect certain events during periods of horizontal gene transfer and genome expansion, and subsequent evolution of genomic regions requiring coordinated regulation.<h4>Conclusions</h4>There may be a tendency for genes that are involved in the same biological process, complex or organelle to use the same OP, even at a distance of ~ 100 kb from the genes. Although the intergenic DNA is non-coding, the general pattern of sequence organization (e.g., reflected in over-represented oligonucleotide "words") may be important and were protected, to some extent, in the course of evolution.

The vast majority of the mammalian genome is transcribed giving rise to many different types of noncoding RNAs. Among them, long noncoding RNAs are the most numerous and functionally versatile class. Indeed, the lncRNA repertoire might be as rich as the proteome. LncRNAs have emerged as key regulators of gene expression at multiple levels. They play important roles in the regulation of development, differentiation and maintenance of cell identity and they also contribute to disease. In this review, we present recent advances in the biology of lncRNAs in muscle development and differentiation. We will also discuss the contribution of lncRNAs to muscle disease with a particular focus on Duchenne and facioscapulohumeral muscular dystrophies.

I was honored to be awarded the Casey Holter Essay Prize in 2013 by the Society for Research into Hydrocephalus and Spina Bifida. The purpose of the prize is to encourage original thinking in a way to improve the care of individuals with spina bifida and hydrocephalus. Having kept this purpose in mind, I have chosen the title: Neural stem cells, are they the hope of a better life for patients with fetal-onset hydrocephalus? The aim is to review and discuss some of the most recent and relevant findings regarding mechanisms leading to both hydrocephalus and abnormal neuro/gliogenesis. By looking at these outcome studies, it is hoped that we will recognize the potential use of neural stem cells in the treatment of hydrocephalus, and so prevent the disease or diminish/repair the associated brain damage.

<h4>Background</h4>As the population ages, it is increasingly important to use effective short cognitive tests for suspected dementia. We aimed to review systematically brief cognitive tests for suspected dementia and report on their validation in different settings, to help clinicians choose rapid and appropriate tests.<h4>Methods</h4>Electronic search for face-to-face sensitive and specific cognitive tests for people with suspected dementia, taking ≤ 20 minutes, providing quantitative psychometric data.<h4>Results</h4>22 tests fitted criteria. Mini-Mental State Examination (MMSE) and Hopkins Verbal Learning Test (HVLT) had good psychometric properties in primary care. In the secondary care settings, MMSE has considerable data but lacks sensitivity. 6-Item Cognitive Impairment Test (6CIT), Brief Alzheimer's Screen, HVLT, and 7 Minute Screen have good properties for detecting dementia but need further validation. Addenbrooke's Cognitive Examination (ACE) and Montreal Cognitive Assessment are effective to detect dementia with Parkinson's disease and Addenbrooke's Cognitive Examination-Revised (ACE-R) is useful for all dementias when shorter tests are inconclusive. Rowland Universal Dementia Assessment scale (RUDAS) is useful when literacy is low. Tests such as Test for Early Detection of Dementia, Test Your Memory, Cognitive Assessment Screening Test (CAST) and the recently developed ACE-III show promise but need validation in different settings, populations, and dementia subtypes. Validation of tests such as 6CIT, Abbreviated Mental Test is also needed for dementia screening in acute hospital settings.<h4>Conclusions</h4>Practitioners should use tests as appropriate to the setting and individual patient. More validation of available tests is needed rather than development of new ones.

<h4>Background</h4>In coronary artery disease (CAD), circulating angiogenic factors have been seen to increase, possibly as a response to ischaemia. Regular physical activity (PA) is recommended for prevention and treatment of CAD, but more research is needed to optimise PA regimes. We investigated the effect of home-based high frequency exercise (HFE) on angiogenic cytokines and cardiac markers in patients with stable CAD.<h4>Design</h4>This was a randomised case-control study<h4>Methods</h4>Sixty-two patients, with stable CAD, were randomised to HFE (n = 33), (aerobic exercise 70% of max, 30 min, five times/week and resistance exercise three times/week), performed at home, or usual lifestyle (control, n = 29). After eight weeks, percutaneous coronary intervention (PCI) was performed in both groups, and the HFE group continued another six months of exercise. Serum vascular endothelial growth factor (VEGF) and stromal derived factor-1 (SDF-1), plasma N-terminal-brain natriuretic peptide (NT-proBNP), high-sensitive troponin T (TnT) and copeptin were analysed.<h4>Results</h4>Data are presented as median (25(th), 75(th) percentile) of relative changes (%) from baseline. Values of p are given for the difference between the HFE and controls. HFE decreased circulating VEGF levels, before PCI (-5% (-15%, -2%)), while VEGF levels increased in the control group (5% (-3%, 20%) p = 0.004). A significant difference in VEGF remained at three months post-PCI (HFE (-1%(-12%, 5%), control (7% (0%, 14%), p = 0.04), but not at six months after PCI. SDF-1, NT-proBNP, TnT and copeptin levels did not differ significantly. In addition, VEGF levels were positively correlated to NT pro-BNP.<h4>Conclusions</h4>Home-based HFE decreased circulating VEGF in patients with stable CAD, suggesting a reduced ischaemic burden. HFE does not increase markers of cardiac dysfunction, suggesting that it is a safe therapeutic intervention in these patients.

Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.

Proline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) is a proto-oncogene that modulates estrogen receptor (ER) signaling. PELP1 expression is upregulated in breast cancer, contributes to therapy resistance, and is a prognostic marker of poor survival. In a subset of breast tumors, PELP1 is predominantly localized in the cytoplasm and PELP1 participates in extranuclear signaling by facilitating ER interactions with Src and phosphoinositide 3-kinase (PI3K). However, the mechanism by which PELP1 extranuclear actions contributes to cancer progression and therapy resistance remains unclear. In this study, we discovered that PELP1 cross-talked with the serine/threonine protein kinase mTOR and modulated mTOR signaling. PELP1 knockdown significantly reduced the activation of mTOR downstream signaling components. Conversely, PELP1 overexpression excessively activated mTOR signaling components. We detected the presence of the mTOR signaling complex proteins in PELP1 immunoprecipitates. mTOR-targeting drugs (rapamycin and AZD8055) significantly reduced proliferation of PELP1-overexpressed breast cancer cells in both in vitro and in vivo xenograft tumor models. MCF7 cells that uniquely retain PELP1 in the cytoplasm showed resistance to hormonal therapy and mTOR inhibitors sensitized PELP1cyto cells to hormonal therapy in xenograft assays. Notably, immunohistochemical studies using xenograft tumors derived from PELP1 overexpression model cells showed increased mTOR signaling and inhibition of mTOR rendered PELP1-driven tumors to be highly sensitive to therapeutic inhibition. Collectively, our data identified the PELP1-mTOR axis as a novel component of PELP1 oncogenic functions and suggest that mTOR inhibitor(s) will be effective chemotherapeutic agents for downregulating PELP1 oncogenic functions.

We recently demonstrated direct evidence of increased monoamine oxidase (MAO) activity in the brain of a simian immunodeficiency virus (SIV) model of human immunodeficiency virus (HIV)-associated central nervous system (CNS) disease, consistent with previously reported dopamine deficits in both SIV and HIV infection. In this study, we explored potential mechanisms behind this elevated activity. MAO B messenger RNA was highest in macaques with the most severe SIV-associated CNS lesions and was positively correlated with levels of CD68 and GFAP transcripts in the striatum. MAO B messenger RNA also correlated with viral loads in the CNS of SIV-infected macaques and with oxidative stress. Furthermore, in humans, striatal MAO activity was elevated in individuals with HIV encephalitis, compared with activity in HIV-seronegative controls. These data suggest that the neuroinflammation and oxidative stress caused by SIV infection in the CNS may provide the impetus for increased transcription of MAO B and that MAO, and more broadly, oxidative stress, have significant potential as therapeutic targets in CNS disease due to HIV.

Ubiquitination of misfolded proteins, a common feature of many neurodegenerative diseases, is mediated by different lysine (K) residues in ubiquitin and alters the levels of toxic proteins. In Huntington disease, polyglutamine expansion causes N-terminal huntingtin (Htt) to misfold, inducing neurodegeneration. Here we report that shorter N-terminal Htt fragments are more stable than longer fragments and find differential ubiquitination via K63 of ubiquitin. Aging decreases proteasome-mediated Htt degradation, at the same time increasing K63-mediated ubiquitination and subsequent Htt aggregation in HD knock-in mice. The association of Htt with the K48-specific E3 ligase, Ube3a, is decreased in aged mouse brain. Overexpression of Ube3a in HD mouse brain reduces K63-mediated ubiquitination and Htt aggregation, enhancing its degradation via the K48 ubiquitin-proteasome system. Our findings suggest that aging-dependent Ube3a levels result in differential ubiquitination and degradation of Htt fragments, thereby contributing to the age-related neurotoxicity of mutant Htt.

No abstract available.

<h4>Purpose</h4>The aim of this study was to characterize Korean patients with Fanconi anemia (FA), which is a rare but very challenging genetic disease.<h4>Methods</h4>The medical records of 12 FA patients diagnosed at Chonnam National University Hospital from 1991 to 2012 were retrospectively reviewed.<h4>Results</h4>The median age at diagnosis was 6.2 years. All patients showed evidence of marrow failure and one or more physical stigmata. Chromosome breakage tests were positive in 9 out of 11 available patients. The median follow-up duration was 69.5 months. The Kaplan-Meier (KM) survival of all patients was 83.3% at 10 years and 34.7% at 20 years, respectively. Seven patients underwent 9 stem cell transplantations (SCTs). Among them, 5 were alive by the end of the study. Ten-year KM survival after SCT was 71.4% with a median follow-up of 3.4 years. All 5 patients treated with supportive treatment alone died of infection or progression at the median age of 13.5 years, except for one with short follow-up duration. Acute leukemia developed in 2 patients at 15.4 and 18.1 years of age. Among 6 patients who are still alive, 3 had short stature and 1 developed insulin-dependent diabetes mellitus.<h4>Conclusion</h4>We provide information on the long-term outcomes of FA patients in Korea. A nation-wide FA registry that includes information of the genotypes of Korean patients is required to further characterize ethnic differences and provide the best standard of care for FA patients.

Simple sequence repeats (SSRs) are not only applied as genetic markers in evolutionary studies but they also play an important role in gene regulatory activities. Efficient identification of conserved and exclusive SSRs through cross-species comparison is helpful for understanding the evolutionary mechanisms and associations between specific gene groups and SSR motifs. In this paper, we developed an online cross-species comparative system and integrated it with a tag cloud visualization technique for identifying potential SSR biomarkers within fourteen frequently used model species. Ultraconserved or exclusive SSRs among cross-species orthologous genes could be effectively retrieved and displayed through a friendly interface design. Four different types of testing cases were applied to demonstrate and verify the retrieved SSR biomarker candidates. Through statistical analysis and enhanced tag cloud representation on defined functional related genes and cross-species clusters, the proposed system can correctly represent the patterns, loci, colors, and sizes of identified SSRs in accordance with gene functions, pattern qualities, and conserved characteristics among species.

<h4>Objective</h4>The growing demand for enhanced esthetic appearance has led to great developments in bleaching products. The exposure of hard tissues of the tooth to bleaching agents can affect the roughness of the enamel surface. The freshly bleached enamel surface exposed to various surface treatments such as fluoride and other remineralizing agents have been assessed in this study. The aim of this experimental study was to compare the effect of Casein Phosphopeptide-Amorphous Calcium Phosphate with Fluoride (MI Paste Plus) and Remin Pro on the enamel surface roughness after bleaching.<h4>Materials and methods</h4>Thirty enamel samples of sound human permanent molars were prepared for this study. After initial roughness measurement with profilometer, the samples were exposed to 37% carbamide peroxide bleaching agent 20 minutes twice, and randomly divided into three groups of ten. In group 1, a CPP-ACPF containing paste (MI Paste Plus) and in group 2, Remin Pro were applied to the teeth during a 15 day period for 5 minutes, twice a day. Samples of group 3 (control) were immersed in artificial saliva for 15 days. The roughness of all samples were measured at the beginning, after bleaching and after the study intervention and statistically analyzed.<h4>Results</h4>The surface roughness significantly increased in all groups following bleaching, and then it showed a decrease after application of both Remin Pro and CPP-ACPF in comparison to using bleaching agent (P<0.005). The surface roughness after using Remin Pro and CPP-ACPF was statistically similar to each other (P>0.05).<h4>Conclusion</h4>There was no difference between surface roughness of MI Paste Plus and Remin Pro groups. Also the surface roughness was decreased compared to the initial enamel surface roughness.

Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion of a polyglutamine repeats close to the N-terminal of the huntingtin protein. Although unequivocal genetic tests have been available for about 20 years, current pharmacological treatments do not prevent or slow down disease progression. Recent basic research identified potential novel drug targets for the treatment of Huntington disease. However, there are clear challenges in translating these discoveries into treatment strategies for these patients. The following is a brief discussion of these challenges using our recent experience as an example.

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.

Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, in which mutant huntingtin is expressed in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models was not associated with classical astrogliosis, but was associated with decreased Kir4.1 K(+) channel functional expression, leading to elevated in vivo striatal extracellular K(+), which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K(+), ameliorated aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K(+) homeostasis, revealing astrocytes and Kir4.1 channels as therapeutic targets.

Exposure to moderate levels of ethanol during brain development has a number of effects on social behavior but the molecular mechanisms that mediate this are not well understood. Gaining a better understanding of these factors may help to develop therapeutic interventions in the future. Zebrafish offer a potentially useful model in this regard. Here, we introduce a zebrafish model of moderate prenatal ethanol exposure. Embryos were exposed to 20mM ethanol for seven days (48hpf-9dpf) and tested as adults for individual social behavior and shoaling. We also tested their basal anxiety with the novel tank diving test. We found that the ethanol-exposed fish displayed reductions in social approach and shoaling, and an increase in anxiety in the novel tank test. These behavioral differences corresponded to differences in hrt1aa, slc6a4 and oxtr expression. Namely, acute ethanol caused a spike in oxtr and ht1aa mRNA expression, which was followed by down-regulation at 7dpf, and an up-regulation in slc6a4 at 72hpf. This study confirms the utility of zebrafish as a model system for studying the molecular basis of developmental ethanol exposure. Furthermore, it proposes a putative developmental mechanism characterized by ethanol-induced OT inhibition leading to suppression of 5-HT and up-regulation of 5-HT1A, which leads, in turn, to possible homeostatic up-regulation of 5-HTT at 72hpf and subsequent imbalance of the 5-HT system.

Methyl-mercury (MeHg) is a potent neuroendocrine disruptor that impairs reproductive processes in fish. The objectives of this study were to (1) characterize transcriptomic changes induced by MeHg exposure in the female largemouth bass (LMB) hypothalamus under controlled laboratory conditions, (2) investigate the health and reproductive impacts of MeHg exposure on male and female largemouth bass (LMB) in the natural environment, and (3) identify MeHg-associated gene expression patterns in whole brain of female LMB from MeHg-contaminated habitats. The laboratory experiment was a single injection of 2.5 μg MeHg/g body weight for 96 h exposure. The field survey compared river systems in Florida, USA with comparably lower concentrations of MeHg (Wekiva, Santa Fe, and St. Johns Rivers) in fish and one river system with LMB that contained elevated concentrations of MeHg (St. Marys River). Microarray analysis was used to quantify transcriptomic responses to MeHg exposure. Although fish at the high-MeHg site did not show overt health or reproductive impairment, there were MeHg-responsive genes and pathways identified in the laboratory study that were also altered in fish from the high-MeHg site relative to fish at the low-MeHg sites. Gene network analysis suggested that MeHg regulated the expression targets of neuropeptide receptor and steroid signaling, as well as structural components of the cell. Disease-associated gene networks related to MeHg exposure, based upon expression data, included cerebellum ataxia, movement disorders, and hypercalcemia. Gene responses in the CNS are consistent with the documented neurotoxicological and neuroendocrine disrupting effects of MeHg in vertebrates.

In this study we report the largest descriptive cohort of congenital antithrombin (AT) deficiency in children, its clinical presentation, molecular basis and genotype-phenotype correlation. Paediatric patients diagnosed with AT deficiency at two tertiary care children's hospitals over a 10-year period were retrospectively reviewed. SERPINC1 gene sequencing was offered to subjects who did not already have the test performed. Molecular modelling and stability simulations were performed for the novel mutations identified. Twenty-nine subjects from 18 pedigrees were identified. Mean age (± standard deviation) at diagnosis and mean duration of follow-up were 8.4 (± 6.6 years and 6.6 (± 5.7 years respectively. Most recent mean AT activity and AT antigen levels (n = 20) were 0.5 (± 0.0) iu/ml and 0.6 (± 0.1) iu/ml respectively. Ten subjects were diagnosed secondary to low AT activity measured following venous thrombo-embolism (VTE). All 10 subjects had additional risk factors at the time of VTE. None of the 19 subjects diagnosed with AT deficiency in the setting of positive family history have had VTE with 7.4 (± 5.8) years follow-up. Mutation analysis has been completed on 19 subjects from 16 pedigrees. Nine unique mutations, including 4 novel mutations were identified.

Disturbed sleep and disrupted circadian rhythms are a common feature of psychiatric disorders, and many groups have postulated an association between genetic variants in circadian clock genes and psychiatric disorders. Using summary data from the association analyses of the Psychiatric Genomics Consortia (PGC) for schizophrenia, bipolar disorder and major depressive disorder, we evaluated the evidence that common SNPs in genes encoding components of the molecular clock influence risk to psychiatric disorders. Initially, gene-based and SNP P-values were analyzed for 21 core circadian genes. Subsequently, an expanded list of genes linked to control of circadian rhythms was analyzed. After correcting for multiple comparisons, none of the circadian genes were significantly associated with any of the three disorders. Several genes previously implicated in the etiology of psychiatric disorders harbored no SNPs significant at the nominal level of P < 0.05, and none of the the variants identified in candidate studies of clock genes that were included in the PGC datasets were significant after correction for multiple testing. There was no evidence of an enrichment of associations in genes linked to control of circadian rhythms in human cells. Our results suggest that genes encoding components of the molecular clock are not good candidates for harboring common variants that increase risk to bipolar disorder, schizophrenia, or major depressive disorder.

<h4>Background</h4>Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC.<h4>Methods</h4>The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations.<h4>Results</h4>A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy.<h4>Conclusions</h4>High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.

Variation in the serotonin transporter gene (5-HTT; SERT; SLC6A4) has been suggested to pharmacogenetically drive interindividual differences in antidepressant treatment response. In the present analysis, a 'pharmaco-epigenetic' approach was applied by investigating the influence of DNA methylation patterns in the 5-HTT transcriptional control region on antidepressant treatment response. Ninety-four patients of Caucasian descent with major depressive disorder (MDD) (f = 61) were analysed for DNA methylation status at nine CpG sites in the 5-HTT transcriptional control region upstream of exon 1A via direct sequencing of sodium bisulfite treated DNA extracted from blood cells. Patients were also genotyped for the functional 5-HTTLPR/rs25531 polymorphisms. Clinical response to treatment with escitalopram was assessed by intra-individual changes of HAM-D-21 scores after 6 wk of treatment. Lower average 5-HTT methylation across all nine CpGs was found to be associated with impaired antidepressant treatment response after 6 wk (p = 0.005). This effect was particularly conferred by one individual 5-HTT CpG site (CpG2 (GRCh37 build, NC_000017.10 28.563.102; p = 0.002). 5-HTTLPR/rs25531 haplotype was neither associated with 5-HTT DNA methylation nor treatment response. This analysis suggests that DNA hypomethylation of the 5-HTT transcriptional control region - possibly via increased serotonin transporter expression and consecutively decreased serotonin availability - might impair antidepressant treatment response in Caucasian patients with MDD. This pharmaco-epigenetic approach could eventually aid in establishing epigenetic biomarkers of treatment response and thereby a more personalized treatment of MDD.

Huntington's disease (HD) is a fatal, progressive neurodegenerative disease with an autosomal dominant inheritance, characterized by chorea, involuntary movements of the limbs and cognitive impairments. Since identification of the HD gene in 1993, tremendous progress has been made in identifying underlying mechanisms involved in HD pathogenesis and progression, and in developing and testing molecular therapeutic targets, using cell and animal models of HD. Recent studies have found that mutant Huntingtin (mHtt) interacts with Dynamin-related protein 1 (Drp1), causing excessive fragmentation of mitochondria, leading to abnormal mitochondrial dynamics and neuronal damage in HD-affected neurons. Some progress has been made in developing molecules that can reduce excessive mitochondrial fission while maintaining both the normal balance between mitochondrial fusion and fission, and normal mitochondrial function in diseases in which excessive mitochondrial fission has been implicated. In this article, we highlight investigations that are determining the involvement of excessive mitochondrial fission in HD pathogenesis, and that are developing inhibitors of excessive mitochondrial fission for potential therapeutic applications.

<h4>Background</h4>Genome-wide association studies have identified hundreds of genetic variants associated with specific cancers. A few of these risk regions have been associated with more than one cancer site; however, a systematic evaluation of the associations between risk variants for other cancers and lung cancer risk has yet to be performed.<h4>Methods</h4>We included 18023 patients with lung cancer and 60543 control subjects from two consortia, Population Architecture using Genomics and Epidemiology (PAGE) and Transdisciplinary Research in Cancer of the Lung (TRICL). We examined 165 single-nucleotide polymorphisms (SNPs) that were previously associated with at least one of 16 non-lung cancer sites. Study-specific logistic regression results underwent meta-analysis, and associations were also examined by race/ethnicity, histological cell type, sex, and smoking status. A Bonferroni-corrected P value of 2.5×10(-5) was used to assign statistical significance.<h4>Results</h4>The breast cancer SNP LSP1 rs3817198 was associated with an increased risk of lung cancer (odds ratio [OR] = 1.10; 95% confidence interval [CI] = 1.05 to 1.14; P = 2.8×10(-6)). This association was strongest for women with adenocarcinoma (P = 1.2×10(-4)) and not statistically significant in men (P = .14) with this cell type (P het by sex = .10). Two glioma risk variants, TERT rs2853676 and CDKN2BAS1 rs4977756, which are located in regions previously associated with lung cancer, were associated with increased risk of adenocarcinoma (OR = 1.16; 95% CI = 1.10 to 1.22; P = 1.1×10(-8)) and squamous cell carcinoma (OR = 1.13; CI = 1.07 to 1.19; P = 2.5×10(-5)), respectively.<h4>Conclusions</h4>Our findings demonstrate a novel pleiotropic association between the breast cancer LSP1 risk region marked by variant rs3817198 and lung cancer risk.

<h4>Background</h4>Allogeneic mesenchymal precursor cells (MPCs) injected during left ventricular assist device (LVAD) implantation may contribute to myocardial recovery. This trial explores the safety and efficacy of this strategy.<h4>Methods and results</h4>In this multicenter, double-blind, sham-procedure controlled trial, 30 patients were randomized (2:1) to intramyocardial injection of 25 million MPCs or medium during LVAD implantation. The primary safety end point was incidence of infectious myocarditis, myocardial rupture, neoplasm, hypersensitivity reaction, and immune sensitization (90 days after randomization). Key efficacy end points were functional status and ventricular function while temporarily weaned from LVAD support (90 days after randomization). Patients were followed up until transplant or 12 months after randomization, whichever came first. Mean age was 57.4 (±13.6) years, mean left ventricular ejection fraction was 18.1%, and 66.7% were destination therapy LVADs. No safety events were observed. Successful temporary LVAD weaning was achieved in 50% of MPC and 20% of control patients at 90 days (P=0.24); the posterior probability that MPCs increased the likelihood of successful weaning was 93%. At 90 days, 3 deaths (30%) occurred in control patients, and none occurred in MPC patients. Mean left ventricular ejection fraction after successful wean was 24.0% (MPC=10) and 22.5% (control=2; P=0.56). At 12 months, 30% of MPC patients and 40% of control patients were successfully temporarily weaned from LVAD support (P=0.69), and 6 deaths (30%) occurred in MPC patients. Donor-specific HLA sensitization developed in 2 MPC and 3 control patients and resolved by 12 months.<h4>Conclusions</h4>In this preliminary trial, administration of MPCs appeared to be safe, and there was a potential signal of efficacy. Future studies will evaluate the potential for higher or additional doses to enhance the ability to wean LVAD recipients off support.<h4>Clinical trial registration url</h4>http://www.clinicaltrials.gov. Unique identifier: NCT01442129.

No abstract available.

Cloud computing is changing the ways software is developed and managed in enterprises, which is changing the way of doing business in that dynamically scalable and virtualized resources are regarded as services over the Internet. Traditional manufacturing systems such as supply chain management (SCM), customer relationship management (CRM), and enterprise resource planning (ERP) are often developed case by case. However, effective collaboration between different systems, platforms, programming languages, and interfaces has been suggested by researchers. In cloud-computing-based systems, distributed resources are encapsulated into cloud services and centrally managed, which allows high automation, flexibility, fast provision, and ease of integration at low cost. The integration between physical resources and cloud services can be improved by combining Internet of things (IoT) technology and Software-as-a-Service (SaaS) technology. This study proposes a new approach for developing cloud-based manufacturing systems based on a four-layer SaaS model. There are three main contributions of this paper: (1) enterprises can develop their own cloud-based logistic management information systems based on the approach proposed in this paper; (2) a case study based on literature reviews with experimental results is proposed to verify that the system performance is remarkable; (3) challenges encountered and feedback collected from T Company in the case study are discussed in this paper for the purpose of enterprise deployment.

Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of frontotemporal dementia (FTD), a predominantly behavioral disease, and amyotrophic lateral sclerosis (ALS), a disease of motor neurons. The primary objectives of this review are to highlight the clinical heterogeneity associated with C9ORF72 pathogenic expansion and identify potential molecular mechanisms underlying selective vulnerability of distinct neural populations. The proposed mechanisms by which C9ORF72 expansion causes behavioral and motor neuron disease highlight the emerging role of impaired RNA and protein homeostasis in a spectrum of neurodegeneration and strengthen the biological connection between FTD and ALS.

<h4>Introduction</h4>To date, a growing number of advanced anticancer nanomedicines (e.g., Doxil(®), Lipoxal(®), DepoCyte(®)) have entered into different phases of clinical trials. However, most of these medicaments fail to differentiate between diseased and normal cells. They also do not have capability of real time monitoring of disease status trough on-demand imaging/sensing of target molecule(s). Multifunctional nanomedicines and theranostics can resolve such limitations, while formulation of these advanced seamless systems appear to involve various sophisticated process, exploiting several bioconjugations.<h4>Methods</h4>Recent works upon multifunctional nanomedicines for simultaneous imaging and therapy of cancer have been systematically reviewed, focusing on surface modification and application of advanced nanobiomaterials.<h4>Results</h4>Ultimate therapy of malignancies, as complex systems, demands implementation of seamless nanosystems (NSs) that can specifically target the cancerous cells and smartly deliver the anticancer agent(s) into the desired target site. Engineering of such NSs requires in-situ coordination of various technologies (e.g., synthesis, surface modification and bioconjugation) in order to achieve improved pharmacokinetics and pharmacodynamics outcomes.<h4>Conclusion</h4>Seamless multimodal NSs have potential to simultaneously target and monitor the tumor cells through homing and imaging/sensing devices and deliver the therapeutic agents. However, to achieve superior pharmacokinetics with maximal efficacy and minimal side effects, these advanced NSs need to become much more intelligent to sense the disease condition and liberate therapeutics on demand.

At least 52 genetic loci were associated with obesity-related traits. However, little is known about the genetic basis of obesity among children. This study aims to test whether 10 polymorphisms in obesity-related genes methionine sulfoxide reductase A (MSRA), transcription factor AP-2 beta (TFAP2B), melanocortin 4 receptor (MC4R), neurexin 3 (NRXN3), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), transmembrane protein 18 (TMEM18), homolog of S. cerevisiae Sec16 (SEC16B), homeobox B5 (HOXB5) and olfactomedin 4 (OLFM4) are associated with the risk of obesity in Portuguese children. A total of 730 children aging from 6 to 12 years old, recruited randomly from public schools in Portugal, were analysed. Anthropometric measurements were obtained and children were classified into three phenotypic groups, normal weight (n=256), overweight (n=320) and obese (n=154), according to the International Obesity Task Force cutoffs. Polymorphisms were genotyped by allelic discrimination TaqMan assays. The MC4R rs12970134 polymorphism was nominally associated with body mass index (BMI) (P=0.035), BMI Z-score (P=0.043) and waist circumference (P=0.020), and borderline associated with weight (P=0.053). Near nominal associations were also found for the PPARGC1A rs8192678 polymorphism with weight (P=0.061), and for the MSRA rs545854 polymorphism with BMI (P=0.055) and BMI Z-score (P=0.056). Furthermore, logistic regression showed that MC4R rs12970134 and TFAP2B rs987237 were nominally, respectively, associated (P=0.029) and borderline associated (P=0.056) with the obese phenotype. This study highlighted the possible association of MC4R, PPARGC1A, MSRA and TFAP2B polymorphisms with several obesity-related traits in a sample of Portuguese children. The two significant associated TFAP2B rs987237 and MC4R rs12970134 polymorphisms showed an opposite direction of effect to that in the original reports.

Treatment of calvarial defects has remained a challenge in reconstruction surgery, especially because of infection at these sites. We produced a bactericidal biomaterial for treating infected bone defects by using calcium phosphate bone cement mixed with antibiotics. We evaluated the usefulness of this material mixed with the antibiotic vancomycin in a cranium-infected rat model. The concentration of vancomycin used was 5.0 wt%, as reported in our previous study. In order to establish the rat model, a cranium defect (diameter, 5 mm) was made that was infected with methicillin-resistant Staphylococcus aureus (MRSA). Thirty-six rats were divided into 6 groups depending on whether an autologous graft or bone cement with or without antibiotic was used for the defect. After 1 and 4 weeks, abscess formation was checked, tissue bacterial counts were determined, and pathological examination was performed. At both 1 and 4 weeks, no MRSA was detected on tissue bacterial culture or pathological examination in groups that received bone cement with antibiotics. In groups that received bone cement without antibiotic, MRSA was detected, and the bone cement had compromised and disintegrated into several slices. In conclusion, bone cement that contains antibiotics appears to be effective not only for reconstruction in cases of cranial defect, but also in terms of preventing infection.

Diabetes mellitus (DM) is a complex progressive disease characterized by hyperglycemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral, and peripheral arterial trees. Oxidative stress is reported in diabetic patients. We investigated the hemostatic functions and oxidative stress in streptozotocin (STZ)-induced diabetic rats and the effects of warfarin and L-carnitine on those parameters. Forty male Sprague-Dawley rats were divided into four groups: control, DM, and DM received warfarin or L-carnitine. In all rats, blood glucose, insulin, hemoglobin A1c (HbA1c), fibrinogen, factor VII (FVII), plasminogen activator inhibitor-1 (PAI-1), fibrin degradation products (FDP), protein C, antithrombin III (ATIII), malondialdehydes (MDA), and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione) were measured. Also, prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation time, and platelet aggregation were evaluated. In diabetic rats, plasma glucose, HbA1c, MDA, fibrinogen, FVII, FDP, PAI-1, and platelet aggregation increased while insulin, PT, aPTT, coagulation time, protein C, ATIII, and antioxidants decreased. Warfarin administration to diabetic rats decreased FVII and FDP and increased PT, aPTT, and coagulation time with no effect on MDA, antioxidants, PAI-1, protein C, ATIII, and platelet aggregation. On the other hand, L-carnitine decreased fibrinogen, FVII, FDP, PAI-1, MDA, and platelet aggregation and increased PT, aPTT, coagulation time, protein C, ATIII, and antioxidants in diabetic rats. Therefore, we concluded that hyperglycemia plays an important role in hypercoagulation state and oxidative stress in STZ-induced DM. While L-carnitine improves oxidative stress and decreases the hypercoagulation state in DM, warfarin normalizes the hypercoagulation state with no effect on oxidative stress.

<h4>Background</h4>Copy number variation (CNV), a source of genetic diversity in mammals, has been shown to underlie biological functions related to production traits. Notwithstanding, there have been few studies conducted on CNVs using next generation sequencing at the population level.<h4>Results</h4>Illumina NGS data was obtained for ten Holsteins, a dairy cattle, and 22 Hanwoo, a beef cattle. The sequence data for each of the 32 animals varied from 13.58-fold to almost 20-fold coverage. We detected a total of 6,811 deleted CNVs across the analyzed individuals (average length = 2732.2 bp) corresponding to 0.74% of the cattle genome (18.6 Mbp of variable sequence). By examining the overlap between CNV deletion regions and genes, we selected 30 genes with the highest deletion scores. These genes were found to be related to the nervous system, more specifically with nervous transmission, neuron motion, and neurogenesis. We regarded these genes as having been effected by the domestication process. Further analysis of the CNV genotyping information revealed 94 putative selected CNVs and 954 breed-specific CNVs.<h4>Conclusions</h4>This study provides useful information for assessing the impact of CNVs on cattle traits using NGS at the population level.

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.

<h4>Objective</h4>Pseudoxanthoma elasticum is an inherited metabolic disorder resulting from ABCC6 gene mutations. It is characterized by progressive calcification and fragmentation of elastic fibers in the skin, retina, and the arterial wall. Despite calcium accumulation in the arteries of patients with pseudoxanthoma elasticum, functional consequences remain unknown. In the present study, we investigated arterial structure and function in Abcc6(-/-) mice, a model of the human disease.<h4>Approach and results</h4>Arterial calcium accumulation was evaluated using alizarin red stain and atomic absorption spectrometry. Expression of genes involved in osteochondrogenic differentiation was measured by polymerase chain reaction. Elastic arterial properties were evaluated by carotid echotracking. Vascular reactivity was evaluated using wire and pressure myography and remodeling using histomorphometry. Arterial calcium accumulation was 1.5- to 2-fold higher in Abcc6(-/-) than in wild-type mice. Calcium accumulated locally leading to punctuate pattern. Old Abcc6(-/-) arteries expressed markers of both osteogenic (Runx2, osteopontin) and chondrogenic lineage (Sox9, type II collagen). Abcc6(-/-) arteries displayed slight increase in arterial stiffness and vasoconstrictor tone in vitro tended to be higher in response to phenylephrine and thromboxane A2. Pressure-induced (myogenic) tone was significantly higher in Abcc6(-/-) arteries than in wild type. Arterial blood pressure was not significantly changed in Abcc6(-/-), despite higher variability.<h4>Conclusions</h4>Scattered arterial calcium depositions are probably a result of osteochondrogenic transdifferentiation of vascular cells. Lower elasticity and increased myogenic tone without major changes in agonist-dependent contraction evidenced in aged Abcc6(-/-) mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis in the long term.

Protein tyrosine phosphorylation is thought to be important for regulation of the proliferation, differentiation, and rapid turnover of intestinal epithelial cells (IECs). The role of protein tyrosine phosphatases in such homeostatic regulation of IECs has remained largely unknown, however. Src homology 2-containing protein tyrosine phosphatase (Shp2) is a ubiquitously expressed cytoplasmic protein tyrosine phosphatase that functions as a positive regulator of the Ras-mitogen-activated protein kinase (MAPK) signaling pathway operative downstream of the receptors for various growth factors and cytokines, and it is thereby thought to contribute to the regulation of cell proliferation and differentiation. We now show that mice lacking Shp2 specifically in IECs (Shp2 CKO mice) develop severe colitis and die as early as 3 to 4 weeks after birth. The number of goblet cells in both the small intestine and colon of Shp2 CKO mice was markedly reduced compared with that for control mice. Furthermore, Shp2 CKO mice showed marked impairment of both IEC migration along the crypt-villus axis in the small intestine and the development of intestinal organoids from isolated crypts. The colitis as well as the reduction in the number of goblet cells apparent in Shp2 CKO mice were normalized by expression of an activated form of K-Ras in IECs. Our results thus suggest that Shp2 regulates IEC homeostasis through activation of Ras and thereby protects against the development of colitis.

Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the "chromatin codes") remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles--we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions.

The Fanconi anemia group C protein (FANCC) is one of the several proteins that comprise the Fanconi anemia (FA) network involved in genomic surveillance. FANCC is mainly cytoplasmic and has many functions, including apoptosis suppression through caspase-mediated proteolytic processing. Here, we examined the role of FANCC proteolytic fragments by identifying their binding partners. We performed a yeast two-hybrid screen with caspase-mediated FANCC cleavage products and identified the dependence receptor uncoordinated-5A (UNC5A) protein. Here, we show that FANCC physically interacts with UNC5A, a pro-apoptotic dependence receptor. FANCC interaction occurs through the UNC5A intracellular domain, specifically via its death domain. FANCC modulates cell sensitivity to UNC5A-mediated apoptosis; we observed reduced UNC5A-mediated apoptosis in the presence of FANCC and increased apoptosis in FANCC-depleted cells. Our results show that FANCC interferes with UNC5A's functions in apoptosis and suggest that FANCC may participate in developmental processes through association with the dependence receptor UNC5A.

Neurological conditions, such as Alzheimer's disease and stroke, represent a prevalent group of devastating illnesses with few treatments. Each of these diseases or conditions is in part characterized by the dysregulation of many genes, including those that code for microRNAs (miRNAs) and histone deacetylases (HDACs). Recently, a complex relationship has been uncovered linking miRNAs and HDACs and their ability to regulate one another. This provides a new avenue for potential therapeutics as the ability to reinstate a careful balance between miRNA and HDACs has lead to improved outcomes in a number of in vitro and in vivo models of neurological conditions. In this review, we will discuss recent findings on the interplay between miRNAs and HDACs and its implications for pathogenesis and treatment of neurological conditions, including amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease and stroke.

Systemic mining of cancer exoproteome/secretome has emerged as a pivotal strategy for delineation of molecular pathways with mechanistic importance in cancer development, as well as the discovery of diagnostic/prognostic biomarkers. Although major advances in diagnostic and therapeutic management of colorectal cancer have been underscored in the last decade, this cancer still remains the second leading cause of cancer-related deaths in the developed world. Despite previous studies on deciphering the colorectal cancer exoproteome, such studies lack adequate depth and robustness due to technological limitations. Here, using a well-established LC-MS/MS method on an LTQ-Orbitrap mass spectrometer, we extensively delineated the exoproteome of 12 colon cancer cell lines. In total, 2979 non-redundant proteins were identified with a minimum of two peptides, of which ~62% were extracellular or cell membrane-bound, based on prediction software. To further characterize this dataset and identify clinical opportunities, first, we investigated overrepresented molecular concepts of interest via enrichment map analysis and second, we demonstrated translational importance of certain proteins, such as olfactomedin-4 and kallikrein-related peptidases-6 and -10, by investigating their expression levels in patient tissues and/or fluids. Overall, the present study details a comprehensive colorectal cancer exoproteome dataset, and may be used as future platform for biomarker discovery, and hypothesis-generating studies.<h4>Biological significance</h4>This article represents one of the most extensive and comprehensive proteomic datasets regarding the secreted/extracellular proteome of colorectal cancer cell lines. The reported datasets may form a platform for a plethora of future, discovery-based or hypothesis-generating studies, attempting to either delineate putative cancer biomarkers for CRC, or elucidate questions of mechanistic importance (e.g. investigation of deregulated pathways for CRC progression).

Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in a tumorigenicity assay, iron deprivation suppressed wild-type p53-dependent tumor growth, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but may also help predict and improve outcomes in iron-deprivation-based chemotherapy.

Neurodegenerative diseases in general and specifically late-onset Alzheimer's disease (LOAD) involve a genetically complex and largely obscure ensemble of causative and risk factors accompanied by complex feedback responses. The advent of "high-throughput" transcriptome investigation technologies such as microarray and deep sequencing is increasingly being combined with sophisticated statistical and bioinformatics analysis methods complemented by knowledge-based approaches such as Bayesian Networks or network and graph analyses. Together, such "integrative" studies are beginning to identify co-regulated gene networks linked with biological pathways and potentially modulating disease predisposition, outcome, and progression. Specifically, bioinformatics analyses of integrated microarray and genotyping data in cases and controls reveal changes in gene expression of both protein-coding and small and long regulatory RNAs; highlight relevant quantitative transcriptional differences between LOAD and non-demented control brains and demonstrate reconfiguration of functionally meaningful molecular interaction structures in LOAD. These may be measured as changes in connectivity in "hub nodes" of relevant gene networks (Zhang etal., 2013). We illustrate here the open analytical questions in the transcriptome investigation of neurodegenerative disease studies, proposing "ad hoc" strategies for the evaluation of differential gene expression and hints for a simple analysis of the non-coding RNA (ncRNA) part of such datasets. We then survey the emerging role of long ncRNAs (lncRNAs) in the healthy and diseased brain transcriptome and describe the main current methods for computational modeling of gene networks. We propose accessible modular and pathway-oriented methods and guidelines for bioinformatics investigations of whole transcriptome next generation sequencing datasets. We finally present methods and databases for functional interpretations of lncRNAs and propose a simple heuristic approach to visualize and represent physical and functional interactions of the coding and non-coding components of the transcriptome. Integrating in a functional and integrated vision coding and ncRNA analyses is of utmost importance for current and future analyses of neurodegenerative transcriptomes.

Huntington's disease (HD) is a late-onset and progressive neurodegenerative disorder that is caused by aggregation of mutant huntingtin protein which contains expanded-polyglutamine. The molecular chaperones modulate the aggregation in early stage and known for the most potent protector of neurodegeneration in animal models of HD. Over the past decades, a number of studies have demonstrated molecular chaperones alleviate the pathogenic symptoms by polyQ-mediated toxicity. Moreover, chaperone-inducible drugs and anti-aggregation drugs have beneficial effects on symptoms of disease. Here, we focus on the function of molecular chaperone in animal models of HD, and review the recent therapeutic approaches to modulate expression and turn-over of molecular chaperone and to develop anti-aggregation drugs.

Remodeling of the local chromatin structure is essential for the regulation of gene expression. While a number of biochemical and bioimaging experiments suggest decondensed chromatin structures are associated with transcription, a direct visualization of DNA and transcriptionally active RNA polymerase II (RNA pol II) at super-resolution is still lacking. Here we investigate the structure of chromatin isolated from HeLa cells using binding activatable localization microscopy (BALM). The sample preparation method preserved the structural integrity of chromatin. Interestingly, BALM imaging of the chromatin spreads revealed the presence of decondensed chromatin as gap structures along the spreads. These gaps were enriched with phosphorylated S5 RNA pol II, and were sensitive to the cellular transcriptional state. Taken together, we could visualize the decondensed chromatin regions together with active RNA pol II for the first time using super-resolution microscopy.

<h4>Background</h4>Glycyl-tRNA synthetase (GARS) is an aminoacyl-tRNA synthetase (ARS) that links the amino acid glycine to its corresponding tRNA prior to protein translation and is one of three bifunctional ARS that are active within both the cytoplasm and mitochondria. Dominant mutations in GARS cause rare forms of Charcot-Marie-Tooth disease and distal spinal muscular atrophy.<h4>Case presentation</h4>We report a 12-year old girl who presented with clinical and biochemical features of a systemic mitochondrial disease including exercise-induced myalgia, non-compaction cardiomyopathy, persistent elevation of blood lactate and alanine and MRI evidence of mild periventricular leukomalacia. Using exome sequencing she was found to harbor compound heterozygous mutations within the glycyl-tRNA synthetase (GARS) gene; c.1904C > T; p.Ser635Leu and c.1787G > A; p.Arg596Gln. Each mutation occurred at a highly conserved site within the anticodon binding domain.<h4>Conclusion</h4>Our findings suggest that recessive mutations in GARS may cause systemic mitochondrial disease. This phenotype is distinct from patients with previously reported dominant mutations in this gene, thereby expanding the spectrum of disease associated with GARS dysregulation.

<h4>Background</h4>In both beef and dairy cattle, the majority of early embryo loss occurs within the first 14 days following insemination. During this time-period, embryos are completely dependent on their maternal uterine environment for development, growth and ultimately survival, therefore an optimum uterine environment is critical to their survival. The objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high (HF) or low fertility (LF) (following four rounds of artificial insemination (AI)) using the Affymetrix® 23 K Bovine Gene Chip.<h4>Results</h4>Conception rates for each of the four rounds of AI were within a normal range: 70-73.3%. Microarray analysis of endometrial tissue collected on day 7 of the estrous cycle detected 419 differentially expressed genes (DEG) between HF (n = 6) and LF (n = 6) animals. The main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. DEG included, FST, SLC45A2, MMP19, FADS1 and GALNT6.<h4>Conclusions</h4>This study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. Differentially expressed genes are potential candidate genes for the identification of genetic variation influencing cow fertility, which may be incorporated into future breeding programmes.

Array-based comparative genomic hybridization (aCGH) is a powerful technique for detecting gene copy number variation. It is generally considered to be robust and convenient since it measures DNA rather than RNA. In the current study, we combine copy number estimates from four different platforms (Agilent 44 K, NimbleGen 385 K, Affymetrix 500 K and Illumina Human1Mv1_C) to compute a reliable, high-resolution, easy to understand output for the measure of copy number changes in the 60 cancer cells of the NCI-DTP (the NCI-60). We then relate the results to gene expression. We explain how to access that database using our CellMiner web-tool and provide an example of the ease of comparison with transcript expression, whole exome sequencing, microRNA expression and response to 20,000 drugs and other chemical compounds. We then demonstrate how the data can be analyzed integratively with transcript expression data for the whole genome (26,065 genes). Comparison of copy number and expression levels shows an overall medium high correlation (median r = 0.247), with significantly higher correlations (median r = 0.408) for the known tumor suppressor genes. That observation is consistent with the hypothesis that gene loss is an important mechanism for tumor suppressor inactivation. An integrated analysis of concurrent DNA copy number and gene expression change is presented. Limiting attention to focal DNA gains or losses, we identify and reveal novel candidate tumor suppressors with matching alterations in transcript level.

Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

Epigenome-wide studies of DNA methylation using blood-derived DNA from cancer patients are complicated by the heterogeneity of cell types within blood and the associated cell lineage specification of DNA methylation signatures. Here, we applied a novel set of analytic approaches to assess the association between cancer case-status and DNA methylation adjusted for leukocyte variation using blood specimens from three case-control cancer studies (bladder: 223 cases, 205 controls; head and neck: 92 cases, 92 controls; and ovarian: 131 cases, 274 controls). Using previously published data on leukocyte-specific CpG loci and a recently described approach to deconvolute subject-specific blood composition, we performed an epigenome-wide analysis to examine the association between blood-based DNA methylation patterns and each of the three aforementioned solid tumor types adjusted for cellular heterogeneity in blood. After adjusting for leukocyte profile in our epigenome-wide analysis, the omnibus association between case-status and methylation was significant for all three studies (bladder cancer: P = 0.047; HNSCC: P = 0.013; ovarian cancer: P = 0.0002). Subsequent analyses revealed that CpG sites associated with cancer were enriched for transcription factor binding motifs involved with cancer-associated pathways. These results support the existence of cancer-associated DNA methylation profiles in the blood of solid tumor patients that are independent of alterations in normal leukocyte distributions. Adoption of the methods developed here will make it feasible to rigorously assess the influence of variability of normal leukocyte profiles when investigating cancer related changes in blood-based epigenome-wide association studies.

Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.

Lgr4 and Lgr5 are known markers of adult and embryonic tissue stem cells in various organs. However, whether Lgr4 and Lgr5 are important for embryonic development remains unclear. To study their functions during intestinal crypt, skin and kidney development we now generated mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). E16.5 Lgr4KO mice displayed complete loss of Lgr5+/Olfm4+intestinal stem cells, compromised Wnt signaling and impaired proliferation and differentiation of gut epithelium. Similarly, E16.5 Lgr4KO mice showed reduced basal cell proliferation and hair follicle numbers in the developing skin, as well as dilated kidney tubules and ectatic Bowman׳s spaces. Although Lgr4KO and Lgr5KO mice both died perinatally, Lgr5 deletion did not compromise embryonic development of gut, kidney or skin. Concomitant deletion of Lgr4 and Lgr5 did not prevent perinatal lethality, in contrast to a previous report that suggested rescue of Lgr5 KO perinatal lethality by a hypomorphic Lgr4 mutant. While the double deletion did not further promote the phenotypes observed in Lgr4KO intestines, impaired kidney cell proliferation, reduced epidermal thickness, loss of Lgr5+follicular epithelium and impaired hair follicle development were only observed in Lgr4/5dKO mice. This supports complementary functions of both receptors. Our findings clearly establish the importance of Lgr4 and Lgr5 during embryonic gut, skin and kidney development, with a dominant role of Lgr4.

The epithelial cells of the mammary gland develop primarily after birth and undergo surges of hormonally regulated proliferation, differentiation, and apoptosis during both puberty and pregnancy. Thus, the mammary gland is a useful model to study fundamental processes of development and adult tissue homeostasis, such as stem and progenitor cell regulation, cell fate commitment, and differentiation. Long noncoding RNAs (lncRNAs) are emerging as prominent regulators of these essential processes, as their extraordinary versatility allows them to modulate gene expression via diverse mechanisms at both transcriptional and post-transcriptional levels. Not surprisingly, lncRNAs are also aberrantly expressed in cancer and promote tumorigenesis by disrupting vital cellular functions, such as cell cycle, survival, and migration. In this review, we first broadly summarize the functions of lncRNAs in mammalian development and cancer. Then we focus on what is currently known about the role of lncRNAs in mammary gland development and breast cancer. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.

Blocking TLR4/peroxiredoxin (Prx6) signaling is proposed to be a novel therapeutic strategy for ischemic stroke because extracellular Prx6 released from ischemic cells may act as an endogenous ligand for TLR4 and initiate destructive immune responses in ischemic brain. Our previous studies showed that ligustilide (LIG) exerted antineuroinflammatory and neuroprotective effects against ischemic insult, but the underlying mechanisms remain unclear. This study investigated whether the TLR4/Prx6 pathway is involved in the protective effect of LIG against postischemic neuroinflammation and brain injury induced by transient middle cerebral artery occlusion (MCAO) in rats. Intraperitoneal LIG administration (20 and 40 mg/kg/day) at reperfusion onset after MCAO resulted in a reduction of brain infarct size and improved neurological outcome over 72 h. LIG-induced neuroprotection was accompanied by improvement of neuropathological alterations, including neuron loss, astrocyte and microglia/macrophage activation, neutrophil and T-lymphocyte invasion, and regulation of inflammatory mediators expression. Moreover, LIG significantly inhibited the expression and extracellular release of Prx6 and activation of TLR4 signaling, reflected by decreased TLR4 expression, extracellular signal-regulated kinase 1/2 phosphorylation, and transcriptional activity of NF-κB and signal transducer and activator of transcription 3 in the ischemic brain. Our results demonstrate that LIG may provide an early and direct neuroprotection by inhibiting TLR4/Prx6 signaling and subsequent immunity and neuroinflammation after cerebral ischemia. These findings support the translational potential of blocking TLR4/Prx6 signaling for the treatment of ischemic stroke.

The electronic Medical Records and Genomics (eMERGE) (Phase I) network was established in 2007 to further genomic discovery using biorepositories linked to the electronic health record (EHR). In Phase II, which began in 2011, genomic discovery efforts continue and in addition the network is investigating best practices for implementing genomic medicine, in particular, the return of genomic results in the EHR for use by physicians at point-of-care. To develop strategies for addressing the challenges of implementing genomic medicine in the clinical setting, the eMERGE network is conducting studies that return clinically-relevant genomic results to research participants and their health care providers. These genomic medicine pilot studies include returning individual genetic variants associated with disease susceptibility or drug response, as well as genetic risk scores for common "complex" disorders. Additionally, as part of a network-wide pharmacogenomics-related project, targeted resequencing of 84 pharmacogenes is being performed and select genotypes of pharmacogenetic relevance are being placed in the EHR to guide individualized drug therapy. Individual sites within the eMERGE network are exploring mechanisms to address incidental findings generated by resequencing of the 84 pharmacogenes. In this paper, we describe studies being conducted within the eMERGE network to develop best practices for integrating genomic findings into the EHR, and the challenges associated with such work.

The endometrium contains a population of immune cells that undergo changes during implantation and pregnancy. The majority of these cells are uterine natural killer (uNK) cells; however, it is unclear how these cells interact with endometrial epithelial cells. Therefore, we investigated the paracrine effects of the uNK cell-secretion medium on the gene expression profile of endometrial epithelial cells in vitro through microarray analysis. Our results, which were verified by qRT-PCR and western blot, revealed that soluble factors from uNK cells alter the gene expression profiles of epithelial cells. The upregulated genes included interleukin-15 (IL-15) and interleukin-15 receptor alpha (IL-15RA), which result in a loop that stimulates uNK cell proliferation. In addition, vascular endothelial growth factor C (VEGF-C) and chemokine (C-X-C motif) ligand 10 (CXCL-10) were also determined to be upregulated in epithelial cells, which suggests that uNK cells work synergistically with epithelial cells to support implantation and pregnancy. In addition, oriental herbal medicines have been used to treat infertility since ancient times; however, we failed to find that Zi Dan Yin can regulate these endometrial paracrine effects.

<h4>Objective</h4>Remyelination in multiple sclerosis has been attributed to the presence of oligodendrocyte progenitor cells (OPCs) in brain parenchyma. However, the precise identity of these progenitors is poorly defined. Here, we characterized populations of OPCs in the adult human brain and examined their myelination capacity and profile of miRNAs. Comparisons were made with fetal OPCs and mature oligodendrocytes.<h4>Methods</h4>We isolated human adult and fetal (early-to-mid second trimester) OPCs from surgically resected brain tissues using O4-, A2B5-, and MOG-directed fluorescence activated cell sorting and transplanted them into dysmyelinated shiverer slices to examine their myelination capacity. We used qRT-PCR to analyze expression of selective miRNAs implicated in OPC biology.<h4>Results</h4>Three subsets of putative OPCs were identified in adult brains: (1) A2B5(+), (2) O4(low), and (3) A2B5(+)O4(high)MOG(+) progenitors. In comparison, fetal brains contained (1) A2B5(+), (2) O4(+), and (3) A2B5(+)O4(+) progenitors, but no MOG(+) cells. We demonstrate that like fetal OPCs, adult OPCs have the capacity to ensheathe cerebellar axons. However, adult OPCs exhibit low to undetectable expression of miRNAs that were highly expressed in O4-expressing fetal OPCs. Adult OPCs also express different miRNAs compared to mature oligodendrocytes.<h4>Interpretation</h4>We conclude that phenotypically distinct subsets of OPCs are present in adult human brain and these OPCs show differential miRNA expression compared to fetal OPCs and mature oligodendrocytes. These suggest that remyelination in adult brain may involve multiple populations of progenitors within the brain and that OPC differentiation in adulthood may be differentially regulated compared to development.

Microemulsion represents an attractive platform for fundamental and applied biomedical research because the emulsified droplets can serve as millions of compartmentalized micrometer-sized reactors amenable to high-throughput screening or online monitoring. However, establishing stable emulsions with surfactants that are compatible with biological applications remains a significant challenge. Motivated by the lack of commercially available surfactants suitable for microemulsion-based biological assays, this study describes the facile synthesis of a biocompatible fluorosurfactant with nonionic tris(hydroxymethyl)methyl (Tris) polar head groups. We have further demonstrated compatibility of the developed surfactant with diverse emulsion-based applications, including DNA polymeric nanoparticle synthesis, enzymatic activity assay, and bacterial or mammalian cell culture, in the setup of both double- and multiphases of emulsions.

Skeletal myogenesis has been and is currently under extensive study in both mammals and teleosts, with the latter providing a good model for skeletal myogenesis because of their flexible and conserved genome. Parallel investigations of muscle studies using both these models have strongly accelerated the advances in the field. However, when transferring the knowledge from one model to the other, it is important to take into account both their similarities and differences. The main difficulties in comparing mammals and teleosts arise from their different temporal development. Conserved aspects can be seen for muscle developmental origin and segmentation, and for the presence of multiple myogenic waves. Among the divergences, many fish have an indeterminate growth capacity throughout their entire life span, which is absent in mammals, thus implying different post-natal growth mechanisms. This review covers the current state of the art on myogenesis, with a focus on the most conserved and divergent aspects between mammals and teleosts.

A synthetic method to prepare a core-shell-structured Fe3O4@SiO2 as a safe nanovehicle for tumor cell targeting has been developed. Superparamagnetic iron oxide is encapsulated inside nonporous silica as the core to provide magnetic targeting. Carboxymethyl chitosan-folic acid (OCMCS-FA) synthesized through coupling folic acid (FA) with OCMCS is then covalently linked to the silica shell and renders new and improved functions because of the original biocompatible properties of OCMCS and the targeting efficacy of FA. Cellular uptake of the nanovehicle was assayed by confocal laser scanning microscope using rhodamine B (RB) as a fluorescent marker in HeLa cells. The results show that the surface modification of the core-shell silica nanovehicle with OCMCS-FA enhances the internalization of nanovehicle to HeLa cells which over-express the folate receptor. The cell viability assay demonstrated that Fe3O4@SiO2-OCMCS-FA nanovehicle has low toxicity and can be used as an eligible candidate for drug delivery system. These unique advantages make the prepared core-shell nanovehicle promising for cancer-specific targeting and therapy.

<h4>Background</h4>Clubfoot is a common congenital birth defect with complex inheritance patterns. Currently, the genetic and morphological basis of clubfoot is poorly understood. To identify genetic risk factors associated with clubfoot, we performed a genome-wide association study of common genetic variants.<h4>Methods</h4>The DNA of 396 isolated clubfoot patients and 1000 controls of European descent was genotyped for >600 000 single nucleotide polymorphisms (SNP) using the Affymetrix 6.0 array. Replication was performed with an independent cohort of 370 isolated clubfoot cases and 363 controls of European descent.<h4>Results</h4>Strongest evidence for an association of clubfoot was found with an intergenic SNP on chromosome 12q24.31 between NCOR2 and ZNF664 (rs7969148, OR=0.58, p=1.25×10⁻⁵) that was significant on replication (combined OR=0.63, p=1.90×10⁻⁷). Additional suggestive SNPs were identified near FOXN3, SORCS1 and MMP7/TMEM123 that also confirmed on replication.<h4>Conclusions</h4>Our study suggests a potential role for common genetic variation in several genes that have not previously been implicated in clubfoot pathogenesis.

<h4>Background</h4>Avian reovirus (ARV) is a member of the Orthoreovirus genus in the Reoviridae family. It is the etiological agent of several diseases, among which viral arthritis and malabsorption syndrome are the most commercially important, causing considerable economic losses in the poultry industry. Although a small but increasing number of reports have characterized some aspects of ARV infection, global changes in protein expression in ARV-infected host cells have not been examined. The current study used a proteomics approach to obtain a comprehensive view of changes in protein levels in host cells upon infection by ARV.<h4>Methodology and principal findings</h4>The proteomics profiles of DF-1 chicken fibroblast cells infected with ARV strain S1133 were analyzed by two-dimensional differential-image gel electrophoresis. The majority of protein expression changes (≥ 1.5 fold, p<0.05) occurred at 72 h post-infection. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 51 proteins with differential expression levels, including 25 that were upregulated during ARV infection and 26 that were downregulated. These proteins were divided into eight groups according to biological function: signal transduction, stress response, RNA processing, the ubiquitin-proteasome pathway, lipid metabolism, carbohydrate metabolism, energy metabolism, and cytoskeleton organization. They were further examined by immunoblotting to validate the observed alterations in protein expression.<h4>Conclusion/significance</h4>This is the first report of a time-course proteomic analysis of ARV-infected host cells. Notably, all identified proteins involved in signal transduction, RNA processing, and the ubiquitin-proteasome pathway were downregulated in infected cells, whereas proteins involved in DNA synthesis, apoptosis, and energy production pathways were upregulated. In addition, other differentially expressed proteins were linked with the cytoskeleton, metabolism, redox regulation, and stress response. These proteomics data provide valuable information about host cell responses to ARV infection and will facilitate further studies of the molecular mechanisms underlying ARV pathogenesis.

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

<h4>Background</h4>The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence.<h4>Methods</h4>A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet Vmax.<h4>Results</h4>The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity.<h4>Conclusion</h4>This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation.

Our understanding of genomic regulation was revolutionized by the discovery that the genome is pervasively transcribed, giving rise to thousands of mostly uncharacterized non-coding ribonucleic acids (ncRNAs). Long, ncRNAs (lncRNAs) have thus emerged as a novel class of functional RNAs that impinge on gene regulation by a broad spectrum of mechanisms such as the recruitment of epigenetic modifier proteins, control of mRNA decay and DNA sequestration of transcription factors. We review those lncRNAs that are implicated in differentiation and homeostasis of metabolic tissues and present novel concepts on how lncRNAs might act on energy and glucose homeostasis. Finally, the control of circadian rhythm by lncRNAs is an emerging principles of lncRNA-mediated gene regulation.

Breast cancer is the most frequent cancer affecting women. Methotrexate (MTX) is an antimetabolic drug that remains important in the treatment of breast cancer. Its efficacy is compromised by resistance in cancer cells that occurs through a variety of mechanisms. This study evaluated apoptotic cell death and cell cycle arrest induced by an MTX derivative (MTX diethyl ester [MTX(OEt)2]) and MTX(OEt)2-loaded lipid-core nanocapsules in two MTX-resistant breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231. The formulations prepared presented adequate granulometric profile. The treatment responses were evaluated through flow cytometry. Relying on the mechanism of resistance, we observed different responses between cell lines. For MCF-7 cells, MTX(OEt)2 solution and MTX(OEt)2-loaded lipid-core nanocapsules presented significantly higher apoptotic rates than untreated cells and cells incubated with unloaded lipid-core nanocapsules. For MDA-MB-231 cells, MTX(OEt)2-loaded lipid-core nanocapsules were significantly more efficient in inducing apoptosis than the solution of the free drug. S-phase cell cycle arrest was induced only by MTX(OEt)2 solution. The drug nanoencapsulation improved apoptosis induction for the cell line that presents MTX resistance by lack of transport receptors.

Recent evidences identify Human Papillomavirus (HPV) sperm infection as a possible cause of male and couple infertility. It acts through different mechanisms at various steps of human conception and early gestational development. We performed a systematic review to assess the role of HPV semen infection on male and couple infertility. Analysis of available and eligible data does not permit us to fund clear evidences about clinical impact of HPV infection on fertility, although sperm parameters impairment is the most widely recognized effect. Regarding biomolecular implications, the available data are often conflicting. More studies are required to define the role of HPV sperm infection in clinical practice. The great majority of evidences are obtained by in vitro studies and this fact represents a limitation for the clinical management of HPVDNA sperm infection. Understanding the biological significance of HPV-DNA semen infection could permit us to explain most of the idiopathic male and couple infertility, leading to a better management of infertile men and a better timing for sperm banking storage before ART cycles.

Bipolar disorder (BP) is a chronic psychiatric condition characterized by dynamic, pathological mood fluctuations from mania to depression. To date, a major challenge in studying human neuropsychiatric conditions such as BP has been limited access to viable central nervous system tissue to examine disease progression. Patient-derived induced pluripotent stem cells (iPSCs) now offer an opportunity to analyze the full compliment of neural tissues and the prospect of identifying novel disease mechanisms. We have examined changes in gene expression as iPSC derived from well-characterized patients differentiate into neurons; there was little difference in the transcriptome of iPSC, but BP neurons were significantly different than controls in their transcriptional profile. Expression of transcripts for membrane bound receptors and ion channels was significantly increased in BP-derived neurons compared with controls, and we found that lithium pretreatment of BP neurons significantly altered their calcium transient and wave amplitude. The expression of transcription factors involved in the specification of telencephalic neuronal identity was also altered. Control neurons expressed transcripts that confer dorsal telencephalic fate, whereas BP neurons expressed genes involved in the differentiation of ventral (medial ganglionic eminence) regions. Cells were responsive to dorsal/ventral patterning cues, as addition of the Hedgehog (ventral) pathway activator purmorphamine or a dorsalizing agent (lithium) stimulated expression of NKX2-1 (ventral identity) or EMX2 (dorsal) in both groups. Cell-based models should have a significant impact on our understanding of the genesis and therefore treatment of BP; the iPSC cell lines themselves provide an important resource for comparison with other neurodevelopmental disorders.

Epithelial ovarian cancer is the most deadly gynecological cancer around the world, with high morbidity in industrialized countries. Early diagnosis is key in reducing its morbidity rate. Yet, robust biomarkers, diagnostics, and animal models are still limited for ovarian cancer. This calls for broader omics and systems science oriented diagnostics strategies. In this vein, the domestic chicken has been used as an ovarian cancer animal model, owing to its high rate of developing spontaneous epithelial ovarian tumors. Chicken blood has thus been considered a surrogate reservoir from which cancer biomarkers can be identified. However, the presence of highly abundant proteins in chicken blood has compromised the applicability of proteomics tools to study chicken blood owing to a lack of immunodepletion methods. Here, we demonstrate that a combinatorial peptide ligand library (CPLL) can efficiently remove highly abundant proteins from chicken blood samples, consequently doubling the number of identified proteins. Using an integrated CPLL-1DGE-LC-MSMS workflow, we identified a catalog of 264 unique proteins. Functional analyses further suggested that most proteins were coagulation and complement factors, blood transport and binding proteins, immune- and defense-related proteins, proteases, protease inhibitors, cellular enzymes, or cell structure and adhesion proteins. Semiquantitative spectral counting analysis identified 10 potential biomarkers from the present chicken ovarian cancer model. Additionally, many human homologs of chicken blood proteins we have identified have been independently suggested as diagnostic biomarkers for ovarian cancer, further triangulating our novel observations reported here. In conclusion, the CPLL-assisted proteomic workflow using the chicken ovarian cancer model provides a feasible platform for translational research to identify ovarian cancer biomarkers and understand ovarian cancer biology. To the best of our knowledge, we report here the most comprehensive survey of the chicken blood proteome to date.

Invadopodia are protrusive, F-actin-driven membrane structures that are thought to mediate basement membrane transmigration during development and tumor dissemination. An understanding of the mechanisms regulating invadopodia has been hindered by the difficulty of examining these dynamic structures in native environments. Using an RNAi screen and live-cell imaging of anchor cell (AC) invasion in Caenorhabditis elegans, we have identified UNC-60A (ADF/cofilin) as an essential regulator of invadopodia. UNC-60A localizes to AC invadopodia, and its loss resulted in a dramatic slowing of F-actin dynamics and an inability to breach basement membrane. Optical highlighting indicated that UNC-60A disassembles actin filaments at invadopodia. Surprisingly, loss of unc-60a led to the accumulation of invadopodial membrane and associated components within the endolysosomal compartment. Photobleaching experiments revealed that during normal invasion the invadopodial membrane undergoes rapid recycling through the endolysosome. Together, these results identify the invadopodial membrane as a specialized compartment whose recycling to form dynamic, functional invadopodia is dependent on localized F-actin disassembly by ADF/cofilin.

Cyclin dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase involved in various biological functions during normal brain development and neurodegeneration. In brain, Cdk5 activity is specific to post-mitotic neurons, due to neuronal specific expression of its activator p35. The biological functions of Cdk5 have been ascribed to its cytoplasmic substrates, however not much is known in nucleus. Here, we show that nuclear transcription factor Sox6 is a direct nuclear target of Cdk5. Sox6 is expressed in Tuj1 positive neurons, suggesting that Sox6 is expressed in differentiating neurons. The expression of Sox6 is high in mitotic nuclei during embryonic day 12 (E12) and gradually decreases during development into adult. On the other hand, Cdk5 expression gradually increases during its development. We show that Sox6 is expressed in mitotic nuclei in embryonic day 12 (E12) and in migrating neurons of E16. Sox6 is phosphorylated in vivo. Sox6 was detected by phospho-Ser/Thr and phospho-Ser/Thr-Pro and MPM-2 (Mitotic protein #2) antibodies in brain. Furthermore, calf intestinal alkaline phosphatase (CIAP) digestion resulted in faster migration of Sox6 band. The GST-Sox6 was phosphorylated by Cdk5/p35. The mass spectrometry analysis revealed that Sox6 is phosphorylated at T119PER motif. We show that Sox6 steady state levels are regulated by Cdk5. Cdk5 knockout mice die in utero and Sox6 protein expression is remarkably high in Cdk5-/- brain, however, there is no change in mRNA expression, suggesting a post-translational regulation of Sox6 by Cdk5. Transfection of primary cortical neurons with WT Cdk5 reduced Sox6 levels, while dominant negative (DN) Cdk5 and p35 increased Sox6 levels. Thus, our results indicate that Cdk5 regulates Sox6 steady state protein level that has an important role in brain development and function.

HIV-1 replication is dependent on binding of the viral capsid to the host protein cyclophilin A (CypA). Interference with cyclophilin A binding, either by mutations in the HIV-1 capsid protein (CA) or by the drug cyclosporine A (CsA), inhibits HIV-1 replication in cell culture. Resistance to CsA is conferred by A92E or G94D substitutions in CA. The mutant viruses are also dependent on CsA for their replication. Interestingly, infection of some cell lines by these mutants is enhanced by CsA, while infection of others is not affected by the drug. The cells are thus termed nonpermissive and permissive, respectively, for infection by CsA-dependent mutants. The mechanistic basis for the cell type dependence is not well understood, but has been hypothesized to result from a dominant-acting host factor that blocks HIV-1 infection by a mechanism that requires CypA binding to the viral capsid. In an effort to identify a CypA-dependent host restriction factor, we adopted a strategy involving comparative gene expression analysis in three permissive and three non-permissive cell types. We ranked the genes based on their relative overexpression in non-permissive cell types compared to the permissive cell types. Based on specific selection criteria, 26 candidate genes were selected and targeted using siRNA in nonpermissive (HeLa) cells. Depletion of none of the selected candidate genes led to the reversal of CsA-dependent phenotype of the A92E mutant. Our data suggest that none of the 26 genes tested is responsible for the dependence of the A92E mutant on CsA. Our study provides gene expression data that may be useful for future efforts to identify the putative CypA-dependent HIV-1 restriction factor and in studies of other cell-specific phenotypes.

Oxidative stress plays a significant role in the pathogenesis of Alzheimer's disease (AD), a devastating disease of the elderly. The brain is more vulnerable than other organs to oxidative stress, and most of the components of neurons (lipids, proteins, and nucleic acids) can be oxidized in AD due to mitochondrial dysfunction, increased metal levels, inflammation, and β-amyloid (Aβ) peptides. Oxidative stress participates in the development of AD by promoting Aβ deposition, tau hyperphosphorylation, and the subsequent loss of synapses and neurons. The relationship between oxidative stress and AD suggests that oxidative stress is an essential part of the pathological process, and antioxidants may be useful for AD treatment.

Late-onset Alzheimer's disease (LOAD) is an age-related neurodegenerative disorder characterized by gradual loss of synapses and neurons, but its pathogenesis remains to be clarified. Neurons live in an environment constituted by neurons themselves and glial cells. In this review, we propose that the neuronal degeneration in the AD brain is partially caused by diverse environmental factors. We first discuss various environmental stresses and the corresponding responses at different levels. Then we propose some mechanisms underlying the specific pathological changes, in particular, hypothalamic-pituitary adrenal axis dysfunction at the systemic level; cerebrovascular dysfunction, metal toxicity, glial activation, and Aβ toxicity at the intercellular level; and kinase-phosphatase imbalance and epigenetic modification at the intracellular level. Finally, we discuss the possibility of developing new strategies for the prevention and treatment of LOAD from the perspective of environmental stress. We conclude that environmental factors play a significant role in the development of LOAD through multiple pathological mechanisms.

Transplantation of stem cells for the treatment of Huntington's disease (HD) garnered much attention prior to the turn of the century. Several studies using mesenchymal stem cells (MSCs) have indicated that these cells have enormous therapeutic potential in HD and other disorders. Advantages of using MSCs for cell therapies include their ease of isolation, rapid propagation in culture, and favorable immunomodulatory profiles. However, the lack of consistent neuronal differentiation of transplanted MSCs has limited their therapeutic efficacy to slowing the progression of HD-like symptoms in animal models of HD. The use of MSCs which have been genetically altered to overexpress brain derived neurotrophic factor to enhance support of surviving cells in a rodent model of HD provides proof-of-principle that these cells may provide such prophylactic benefits. New techniques that may prove useful for cell replacement therapies in HD include the use of genetically altering fate-restricted cells to produce induced pluripotent stem cells (iPSCs). These iPSCs appear to have certain advantages over the use of embryonic stem cells, including being readily available, easy to obtain, less evidence of tumor formation, and a reduced immune response following their transplantation. Recently, transplants of iPSCs have shown to differentiate into region-specific neurons in an animal model of HD. The overall successes of using genetically altered stem cells for reducing neuropathological and behavioral deficits in rodent models of HD suggest that these approaches have considerable potential for clinical use. However, the choice of what type of genetically altered stem cell to use for transplantation is dependent on the stage of HD and whether the end-goal is preserving endogenous neurons in early-stage HD, or replacing the lost neurons in late-stage HD. This review will discuss the current state of stem cell technology for treating the different stages of HD and possible future directions for stem-cell therapy in HD.

Basal-like breast carcinoma is characterized by poor prognosis and high intratumour heterogeneity. In an immortalized basal-like breast epithelial cell line, we identified two anticorrelated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic three-dimensional culture. The first contains multiple TGF-β-related genes including TGFBR3, whereas the second contains JUND and the basal-like marker KRT5. TGFBR3 and JUND interconnect through four negative-feedback loops to form a circuit that exhibits spontaneous damped oscillations in three-dimensional culture. The TGFBR3-JUND circuit is conserved in some premalignant lesions that heterogeneously express KRT5. The circuit depends on ECM engagement, as detachment causes a rewiring that is triggered by RPS6 dephosphorylation and maintained by juxtacrine tenascin C, which is critical for intraductal colonization of basal-like breast cancer cells in vivo. Intratumour heterogeneity need not stem from partial differentiation and could instead reflect dynamic toggling of cells between expression states that are not cell autonomous.

<h4>Objective</h4>To analyze the antithrombin-III (AT-III) activity in the plasma in relation to the serum albumin and total protein in preeclampsia and gestational hypertension.<h4>Study design</h4>The medical records of 139 patients who were diagnosed with gestational hypertension (n=33) and preeclampsia (n=106) were reviewed, and the relationships between the activity of AT-III and serum albumin or total protein were evaluated.<h4>Main outcome measures</h4>The plasma AT-III activity in gestational hypertension and preeclampsia was correlated with the serum albumin and TP levels.<h4>Results</h4>There were significant correlations between AT-III activity and albumin in gestational hypertension (r=0.504, p=0.003) and preeclampsia (r=0.343, p=0.003). There were also significant correlations between AT-III activity and TP in gestational hypertension (r=0.619, p=0.001) and preeclampsia (r=0.366, p=0.001). Regression coefficients between AT-III and albumin and between AT-III and TP in gestational hypertension (23.7 and 14.0, respectively) were significantly steeper than those in preeclampsia (14.6 and 9.6, respectively).<h4>Conclusions</h4>The plasma AT-III activity in gestational hypertension and preeclampsia was correlated with the serum albumin and TP levels. This suggests that AT-III activity is more likely to decrease in gestational hypertension than in preeclampsia.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by an increase in the number of polyglutamine residues in the huntingtin (Htt) protein. With the identification of the underlying basis of HD, therapies are being developed that reduce expression of the causative mutant Htt. RNA interference (RNAi) that seeks to selectively reduce the expression of such disease-causing agents is emerging as a potential therapeutic strategy for this and similar disorders. This study examines the merits of administering a recombinant adeno-associated viral (AAV) vector designed to deliver small interfering RNA (siRNA) that targets the degradation of the Htt transcript. The aim was to lower Htt levels and to correct the behavioral, biochemical, and neuropathological deficits shown to be associated with the YAC128 mouse model of HD. Our data demonstrate that AAV-mediated RNAi is effective at transducing greater than 80% of the cells in the striatum and partially reducing the levels (~40%) of both wild-type and mutant Htt in this region. Concomitant with these reductions are significant improvements in behavioral deficits, reduction of striatal Htt aggregates, and partial correction of the aberrant striatal transcriptional profile observed in YAC128 mice. Importantly, a partial reduction of both the mutant and wild-type Htt levels is not associated with any notable overt neurotoxicity. Collectively, these results support the continued development of AAV-mediated RNAi as a therapeutic strategy for HD.

The cellular responses to steroids are mediated by 2 general mechanisms: genomic and rapid/nongenomic effects. Identification of the mechanisms underlying aldosterone (ALDO)'s rapid vs their genomic actions is difficult to study, and these mechanisms are not clearly understood. Recent data suggest that striatin is a mediator of nongenomic effects of estrogen. We explored the hypothesis that striatin is an intermediary of the rapid/nongenomic effects of ALDO and that striatin serves as a novel link between the actions of the mineralocorticoid and estrogen receptors. In human and mouse endothelial cells, ALDO promoted an increase in phosphorylated extracellular signal-regulated protein kinases 1/2 (pERK) that peaked at 15 minutes. In addition, we found that striatin is a critical intermediary in this process, because reducing striatin levels with small interfering RNA (siRNA) technology prevented the rise in pERK levels. In contrast, reducing striatin did not significantly affect 2 well-characterized genomic responses to ALDO. Down-regulation of striatin with siRNA produced similar effects on estrogen's actions, reducing nongenomic, but not some genomic, actions. ALDO, but not estrogen, increased striatin levels. When endothelial cells were pretreated with ALDO, the rapid/nongenomic response to estrogen on phosphorylated endothelial nitric oxide synthase (peNOS) was enhanced and accelerated significantly. Importantly, pretreatment with estrogen did not enhance ALDO's nongenomic response on pERK. In conclusion, our results indicate that striatin is a novel mediator for both ALDO's and estrogen's rapid and nongenomic mechanisms of action on pERK and phosphorylated eNOS, respectively, thereby suggesting a unique level of interactions between the mineralocorticoid receptor and the estrogen receptor in the cardiovascular system.

Induced pluripotent stem cells (iPSCs) show considerable promise for cell replacement therapies for Huntington's disease (HD). Our laboratory has demonstrated that tail-tip fibroblasts, reprogrammed into iPSCs via two adenoviruses, can survive and differentiate into neuronal lineages following transplantation into healthy adult rats. However, the ability of these cells to survive, differentiate, and restore function in a damaged brain is unknown. To this end, adult rats received a regimen of 3-nitropropionic acid (3-NP) to induce behavioral and neuropathological deficits that resemble HD. At 7, 21, and 42 days after the initiation of 3-NP or vehicle, the rats received intrastriatal bilateral transplantation of iPSCs. All rats that received 3-NP and vehicle treatment displayed significant motor impairment, whereas those that received iPSC transplantation after 3-NP treatment had preserved motor function. Histological analysis of the brains of these rats revealed significant decreases in optical densitometric measures in the striatum, lateral ventricle enlargement, as well as an increase in striosome size in all rats receiving 3-NP when compared with sham rats. The 3-NP-treated rats given transplants of iPSCs in the 7- or 21-day groups did not exhibit these deficits. Transplantation of iPSCs at the late-stage (42-day) time point did not protect against the 3-NP-induced neuropathology, despite preserving motor function. Transplanted iPSCs were found to survive and differentiate into region-specific neurons in the striatum of 3-NP rats, at all transplantation time points. Taken together, these results suggest that transplantation of adenovirus-generated iPSCs may provide a potential avenue for therapeutic treatment of HD.

Pyruvate kinase M2 (PKM2) is predominantly expressed in cancers, which is considered as a key regulator of the Warburg effect. In this study, HSP40 was identified as a novel binding partner of PKM2. HSP40-PKM2 association destabilized PKM2 protein through HSC70. In the presence of HSP40, PKM2 protein level and PKM2-mediated PDK1 expression were down-regulated. Moreover, HSP40 was involved in regulating glucose metabolism on PKM2 dependent way and at the mean time had an effect on mitochondrial oxygen respiration. In line with inhibition effect of HSP40 on glycolysis, the growth of cancer cells was inhibited by HSP40.Our data provided a new regulation mechanism of PKM2, which suggested a new therapeutic target for cancer therapy.

With the use of iTRAQ technique, a multifactorial comparative proteomic study can be performed. In this study, to obtain an overview of ethanol, CYP2E1 and gender effects on liver injury and gain more insight into the underlying molecular mechanism, mouse liver proteomes were quantitatively analyzed using iTRAQ under eight conditions including mice of different genders, wild type versus CYP2E1 knockout, and normal versus alcohol diet. A series of statistical and bioinformatic analyses were explored to simplify and clarify multifactorial comparative proteomic data. First, with the Principle Component analysis, six proteins, CYP2E1, FAM25, CA3, BHMT, HIBADH and ECHS1, involved in oxidation reduction, energy and lipid metabolism and amino acid metabolism, were identified as the most differentially expressed gene products across all of the experimental conditions of our chronic alcoholism model. Second, hierarchical clustering analysis showed CYP2E1 knockout played a primary role in the overall differential protein expression compared with ethanol and gender factors. Furthermore, pair-wise multiple comparisons have revealed that the only significant expression difference lied in wild-type and CYP2E1 knockout mice both treated with ethanol. Third, K-mean clustering analysis indicated that the CYP2E1 knockout had the reverse effect on ethanol induced oxidative stress and lipid oxidation. More importantly, IPA analysis of proteomic data inferred that the gene expressions of two upstream regulators, NRF2 and PPARα, regulated by chronic alcohol feeding and CYP2E1 knockout, are involved in ethanol induced oxidative stress and lipid oxidation. The present study provides an effectively comprehensive data analysis strategy to compare multiple biological factors, contributing to biochemical effects of alcohol on the liver. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with data set identifier of PXD000635.

Anxiety disorders are influenced by both environmental and genetic factors. A well-known example for gene x environment interactions in psychiatry is the low activity (s) allelic variant of the serotonin transporter (5-HTT) promoter polymorphism (5-HTTLPR) that in the context of stress increases risk for depression and post-traumatic stress disorder (PTSD). Previously, we observed robust anxiety-related phenotypes, such as an impairment in fear extinction, in 5-HTT knockout (5-HTT-/-) versus wild-type (5-HTT+/+) rats housed in open cages. Recently, housing conditions were changed from open cages to individually ventilated cages (IVC), which are associated with a high ventilation fold and noise. This switch in housing conditions prompted an unplanned 5-HTT gene x environment interaction study in our rats. The current study shows that lifetime stress by means of IVC cage housing abolished genotype differences in fear extinction between 5-HTT-/- and 5-HTT+/+ rats. Although this effect was not attributed specifically to either the 5-HTT+/+ or the 5-HTT-/- genotype, the findings are in agreement with the modulatory role of serotonin in the processing of environmental stimuli. Our findings also underline the possibility that housing conditions confound the interpretation of anxiety-related behaviours in rodents.

Transferrin receptor 2 (TFR2) is a transmembrane glycoprotein expressed in the liver and in the erythroid compartment, mutated in a form of hereditary hemochromatosis. Hepatic TFR2, together with HFE, activates the transcription of the iron-regulator hepcidin, while erythroid TFR2 is a member of the erythropoietin receptor complex. The TMPRSS6 gene, encoding the liver-expressed serine protease matriptase-2, is the main inhibitor of hepcidin and inactivation of TMPRSS6 leads to iron deficiency with high hepcidin levels. Here we evaluate the phenotype resulting from the genetic loss of Tmprss6 in Tfr2 total (Tfr2(-/-)) and liver-specific (Tfr2(LCKO)) knockout mice. Tmprss6(-/-)Tfr2(-/-) and Tmprss6(-/-)Tfr2(LCKO) mice have increased hepcidin levels and show iron-deficiency anemia like Tmprss6(-/-)mice. However, while Tmprss6(-/-)Tfr2(LCKO) are phenotypically identical to Tmprss6(-/-) mice, Tmprss6(-/-)Tfr2(-/-) mice have increased red blood cell count and more severe microcytosis than Tmprss6(-/-) mice. In addition hepcidin expression in Tmprss6(-/-)Tfr2(-/-) mice is higher than in the wild-type animals, but lower than in Tmprss6(-/-) mice, suggesting partial inhibition of the hepcidin activating pathway. Our results prove that hepatic TFR2 acts upstream of TMPRSS6. In addition Tfr2 deletion causes a relative erythrocytosis in iron-deficient mice, which likely attenuates the effect of over-expression of hepcidin in Tmprss6(-/-) mice. Since liver-specific deletion of Tfr2 in Tmprss6(-/-) mice does not modify the erythrocyte count, we speculate that loss of Tfr2 in the erythroid compartment accounts for the hematologic phenotype of Tmprss6(-/-)Tfr2(-/-) mice. We propose that TFR2 is a limiting factor for erythropoiesis, particularly in conditions of iron restriction.

Huntington's disease (HD) is a signature polyglutamine disorder. An enduring theory of HD pathogenesis has involved dysregulation of transcription. Indeed, transcriptional regulatory proteins can be modulated to overcome cardinal features of HD-modeled mice, and efforts to move these into human studies are ongoing. Here, we discuss a unifying hypothesis emerging from these studies, which is that HD represents the pathological disruption of evolutionarily conserved adaptive gene programs to counteract oxidative stress, mitochondrial dysfunction and accumulation of misfolded proteins. Transcriptional dyshomeostasis of adaptive genes is further exacerbated by repression of genes involved in normal synaptic activity or growth factor signaling.

Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic and antioxidant properties. We investigated the effect of all trans-retinoic acid (ATRA) at different stages of the neoplastic transformation using an in vitro model of breast cancer progression. This model was previously developed by treating the MCF-10F human normal breast epithelial cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: MCF-10F, normal stage; trMCF, transformed MCF-10F; bsMCF, invasive stage; and caMCF, tumorigenic stage. In 3D cultures, MCF-10F cells form tubules resembling the structures in the normal mammary gland. After treatment with estradiol, these cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with ATRA. After treatment with 10 or 1 µM ATRA, the trMCF clone 11 cells showed tubules in collagen; 10 and 43% of the structures were tubules in cells treated with 10 and 1 µM ATRA, respectively. Gene expression studies showed that 207 genes upregulated in transformed trMCF clone 11 cells were downregulated after 1 µM ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were downregulated in trMCF clone 11 were upregulated after 1 µM ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 1 µM ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after ATRA treatment. These results suggest that ATRA could be used as a chemopreventive agent to inhibit the progression of premalignant lesions of the breast.

Malleable biomaterials such as Herschel-Bulkley (H-B) fluids possess shear responsive rheological properties and are capable of self-assembly and viscoelastic recovery following mechanical disruption (e.g., surgical placement via injection or spreading). This study demonstrated that the addition of moderate molecular weight glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) (Mw = 15-30 kDa) and hyaluronic acid (HA) (Mw = 20-41 kDa) can be used to modify several rheological properties including consistency index (K), flow-behavior index (n), and yield stress (τy) of submicrometer hydroxyapatite (HAP) (Davg ≤ 200 nm) colloidal gels. GAG-HAP colloidal mixtures exhibited substantial polymer-particle synergism, likely due to "bridging" flocculation, which led to a synergistic increase in consistency index (KGAG-HAP ≥ KGAG + KHAP) without compromising shear-thinning behavior (n < 1) of the gel. In addition, GAG-HAP colloids containing high concentrations of HAP (60-80% w/v) exhibited substantial yield stress (τy ≥ 100 Pa) and viscoelastic recovery properties (G'recovery ≥ 64%). While rheological differences were observed between CS-HAP and HA-HAP colloidal gels, both CS and HA represent feasible options for future studies involving bone defect filling. Overall, this study identified mixture regions where rheological properties in CS-HAP and HA-HAP colloidal gels aligned with desired properties to facilitate surgical placement in non-load-bearing tissue-filling applications such as calvarial defects.

MHC class I (MHC I) antigen presentation is a ubiquitous process by which cells present endogenous proteins to CD8(+) T lymphocytes during immune surveillance and response. Hereditary hemochromatosis protein, HFE, is involved in cellular iron uptake but, while structurally homologous to MHC I, is unable to bind peptides. However, increasing evidence suggests a role for HFE in the immune system. Here, we investigated the impact of HFE on CD8(+) T-lymphocyte activation. Using transient HFE transfection assays in a model of APCs, we show that WT HFE (HFEWT ), but not C282Y-mutated HFE, inhibits secretion of MIP-1β from antigen-specific CD8(+) T lymphocytes. HFEWT expression also resulted in major decreases in CD8(+) T-lymphocyte activation as measured by 4-1BB expression. We further demonstrate that inhibition of CD8(+) T-lymphocyte activation was independent of MHC I surface levels, β2-m competition, HFE interaction with transferrin receptor, antigen origin, or epitope affinity. Finally, we identified the α1-2 domains of HFEWT as being responsible for inhibiting CD8(+) T-lymphocyte activation. Our data imply a new role for HFEWT in altering CD8(+) T-lymphocyte reactivity, which could modulate antigen immunogenicity.

<h4>Background</h4>Post-pneumonectomy bronchopleural fistulas (BPFs) are associated with high morbidity and mortality. Currently, since the management of BPFs is difficult to assess, the best therapeutic approach is prevention. Our objective was to evaluate the effects of adipose-derived stem cells (ASCs) on the healing of the bronchial stump in an experimental animal model.<h4>Methods</h4>Left pneumonectomy was performed in 37 Sprague-Dawley rats. Animals were randomly assigned to a control group (n = 13), an ASC group (n = 12), and an ASC plus Tissucol(®) group (ASCT) (n = 12). The ASCs and ASCTs were locally administered at the bronchial stump after surgical pneumonectomy. Animals were killed at 10 and 20 days. We analyzed histological changes and changes in the expression of relevant genes involved in wound repair in the bronchial stump.<h4>Results</h4>Two control animals, one animal from the ASC group, and one from the ASCT group died from early BPF. All the remaining animals had an adequate postoperative outcome. ASCs and ASCTs significantly decreased the necrosis and ulcerations of the bronchial stump at 10 and 20 days. ASCs significantly decreased mRNA expression of Igf1 at 10 days and Igf1, Tgfb1, Vegfa, and Col2a1 at 20 days, whereas there was increased expression of Agc1 and Col2a1 at 10 days and Sox6 at 20 days.<h4>Conclusions</h4>Our findings indicate that local ASCs protected the bronchial stump after pneumonectomy and induced local changes in gene expression related to their protective action. These results could lead to a potential new therapeutic modality for the prevention of BPF.

Teneurin-4 (Ten-4), a transmembrane protein, is expressed in the nervous systems and the mesenchymal tissues, including the cartilage. However, the Ten-4 function in cartilage development remains unknown. Here, we showed that Ten-4 is a novel regulator of chondrogenesis. In situ hybridization analysis revealed that Ten-4 was highly expressed in the mesenchymal condensation area of the mouse femur at embryonic day (E) 13.5, while its expression was decreased in the growth plate of the femur at E18.5. Using the cartilage-like pellet culture of human synovial mesenchymal cells, Ten-4 expression was induced and peaked 7 days after induction of differentiation, while a production of type II and X collagens was increased after Day 14. In the cartilage-like pellet, Ten-4 was highly expressed in the less differentiated region. In the chondrogenic cell line ATDC5, knockdown of Ten-4 expression significantly increased the alcian blue staining and expression levels of aggrecan and type II and X collagens. Further, an elevated expression of Sox6, Sox9, and Runx2 and an attenuation of the ERK activation were observed in the Ten-4-knockdown ATDC5 cells. These results suggested that Ten-4 suppresses chondrogenic differentiation and regulates the expression and activation of the key molecules for chondrogenesis.

<h4>Background</h4>The purpose of this manuscript is to describe the PhenX RISING network and the site experiences in the implementation of PhenX measures into ongoing population-based genomic studies.<h4>Methods</h4>Eighty PhenX measures were implemented across the seven PhenX RISING groups, thirty-three of which were used at more than two sites, allowing for cross-site collaboration. Each site used between four and 37 individual measures and five of the sites are validating the PhenX measures through comparison with other study measures. Self-administered and computer-based administration modes are being evaluated at several sites which required changes to the original PhenX Toolkit protocols. A network-wide data use agreement was developed to facilitate data sharing and collaboration.<h4>Results</h4>PhenX Toolkit measures have been collected for more than 17,000 participants across the PhenX RISING network. The process of implementation provided information that was used to improve the PhenX Toolkit. The Toolkit was revised to allow researchers to select self- or interviewer administration when creating the data collection worksheets and ranges of specimens necessary to run biological assays has been added to the Toolkit.<h4>Conclusions</h4>The PhenX RISING network has demonstrated that the PhenX Toolkit measures can be implemented successfully in ongoing genomic studies. The next step will be to conduct gene/environment studies.

To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX) were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid) (poly(γ-GA)), followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide)-g-monomethoxy poly(ethylene glycol) in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs) showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C) at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

<h4>Objective</h4>The aim of this study was to examine the association between ectopic fat and organ-specific insulin resistance (IR) in insulin-target organs in patients with nonalcoholic fatty liver disease (NAFLD).<h4>Methods</h4>Organ-specific IR in the liver (hepatic glucose production (HGP) × fasting plasma insulin (FPI) and suppression of HGP by insulin [%HGP]), skeletal muscle (insulin-stimulated glucose disposal [Rd]), and adipose tissue (suppression of FFA by insulin [%FFA]) was measured in 69 patients with NAFLD using a euglycemic hyperinsulinemic clamp with tracer infusion ([6,6-2H2]glucose). Liver fat, intramyocellular lipid (IMCL), and body composition were measured by liver biopsy, proton magnetic resonance spectroscopy, and bioelectrical impedance analysis, respectively.<h4>Results</h4>HGP × FPI was significantly correlated with Rd (r =  -0.57, P<0.001), %HGP with %FFA (r = 0.38, P<0.01), and Rd with %FFA (r = 0.27, P<0.05). Liver steatosis score was negatively associated with Rd (r =  -0.47, P<0.001) as well as with HGP × FPI (r = 0.43, P<0.001). Similarly, intrahepatic lipid was negatively associated with Rd (r =  -0.32, P<0.05). IMCL was not associated with Rd (r =  -0.16, P = 0.26). Fat mass and its percentage were associated with HGP × FPI (r = 0.50, P<0.001; r = 0.48, P<0.001, respectively) and Rd (r =  -0.59, P<0.001; r =  -0.52, P<0.001, respectively), but not with %FFA (r =  -0.21, P = 0.10; r =  -0.001, P = 0.99, respectively).<h4>Conclusion</h4>Unexpectedly, fat accumulation in the skeletal muscle and adipose tissue was not associated with organ-specific IR. Instead, liver fat was associated not only with hepatic IR but also with skeletal muscle IR, suggesting a central role of fatty liver in systemic IR and that a network exists between liver and skeletal muscle.

The continuously prolonged human lifespan is accompanied by increase in neurodegenerative diseases incidence, calling for the development of inexpensive blood-based diagnostics. Analyzing blood cell transcripts by RNA-Seq is a robust means to identify novel biomarkers that rapidly becomes a commonplace. However, there is lack of tools to discover novel exons, junctions and splicing events and to precisely and sensitively assess differential splicing through RNA-Seq data analysis and across RNA-Seq platforms. Here, we present a new and comprehensive computational workflow for whole-transcriptome RNA-Seq analysis, using an updated version of the software AltAnalyze, to identify both known and novel high-confidence alternative splicing events, and to integrate them with both protein-domains and microRNA binding annotations. We applied the novel workflow on RNA-Seq data from Parkinson's disease (PD) patients' leukocytes pre- and post- Deep Brain Stimulation (DBS) treatment and compared to healthy controls. Disease-mediated changes included decreased usage of alternative promoters and N-termini, 5'-end variations and mutually-exclusive exons. The PD regulated FUS and HNRNP A/B included prion-like domains regulated regions. We also present here a workflow to identify and analyze long non-coding RNAs (lncRNAs) via RNA-Seq data. We identified reduced lncRNA expression and selective PD-induced changes in 13 of over 6,000 detected leukocyte lncRNAs, four of which were inversely altered post-DBS. These included the U1 spliceosomal lncRNA and RP11-462G22.1, each entailing sequence complementarity to numerous microRNAs. Analysis of RNA-Seq from PD and unaffected controls brains revealed over 7,000 brain-expressed lncRNAs, of which 3,495 were co-expressed in the leukocytes including U1, which showed both leukocyte and brain increases. Furthermore, qRT-PCR validations confirmed these co-increases in PD leukocytes and two brain regions, the amygdala and substantia-nigra, compared to controls. This novel workflow allows deep multi-level inspection of RNA-Seq datasets and provides a comprehensive new resource for understanding disease transcriptome modifications in PD and other neurodegenerative diseases.

DNA microarray technologies are used extensively to profile the expression levels of thousands of genes under various conditions, yielding extremely large data-matrices. Thus, analyzing this information and extracting biologically relevant knowledge becomes a considerable challenge. A classical approach for tackling this challenge is to use clustering (also known as one-way clustering) methods where genes (or respectively samples) are grouped together based on the similarity of their expression profiles across the set of all samples (or respectively genes). An alternative approach is to develop biclustering methods to identify local patterns in the data. These methods extract subgroups of genes that are co-expressed across only a subset of samples and may feature important biological or medical implications. In this study we evaluate 13 biclustering and 2 clustering (k-means and hierarchical) methods. We use several approaches to compare their performance on two real gene expression data sets. For this purpose we apply four evaluation measures in our analysis: (1) we examine how well the considered (bi)clustering methods differentiate various sample types; (2) we evaluate how well the groups of genes discovered by the (bi)clustering methods are annotated with similar Gene Ontology categories; (3) we evaluate the capability of the methods to differentiate genes that are known to be specific to the particular sample types we study and (4) we compare the running time of the algorithms. In the end, we conclude that as long as the samples are well defined and annotated, the contamination of the samples is limited, and the samples are well replicated, biclustering methods such as Plaid and SAMBA are useful for discovering relevant subsets of genes and samples.

The BRAF (V600E) mutation in colorectal cancers that are microsatellite stable (MSS) confers a poor patient prognosis, whereas BRAF mutant microsatellite-unstable (MSI) colorectal cancers have an excellent prognosis. BRAF wild type cancers are typically MSS and display chromosomal instability (CIN). CIN has not been extensively studied on a genome-wide basis in relation to BRAF mutational status in colorectal cancer. BRAF mutant/MSS (BRAFmut/MSS) cancers (n = 33) and BRAF mutant/MSI (BRAFmut/MSI) cancers (n = 30) were compared for presence of copy number aberrations (CNAs) indicative of CIN, with BRAF wild type/MSS (BRAFwt/MSS) cancers (n = 18) using Illumina CytoSNP-12 arrays. BRAFmut/MSS and BRAFwt/MSS cancers showed comparable numbers of CNAs/cancer at 32.8 and 29.8 respectively. However, there were differences in patterns of CNA length between MSS cohorts, with BRAFmut/MSS cancers having significantly greater proportions of focal CNAs compared to BRAFwt/MSS cancers (p<0.0001); whereas whole chromosomal arm CNAs were more common in BRAFwt/MSS cancers (p<0.0001). This related to a reduced average CNA length in BRAFmut/MSS compared to BRAFwt/MSS cancers (20.7 Mb vs 33.4 Mb;p<0.0001); and a smaller average percent of CIN affected genomes in BRAFmut/MSS compared to BRAFwt/MSS cancers (23.9% vs 34.9% respectively). BRAFmut/MSI cancers were confirmed to have low CNA rates (5.4/cancer) and minimal CIN-affected genomes (average of 4.5%) compared to MSS cohorts (p<0.0001). BRAFmut/MSS cancers had more frequent deletion CNAs compared to BRAFwt/MSS cancers on 6p and 17q at loci not typically correlated with colorectal cancer, and greater amplification CNAs on 8q and 18q compared to BRAFwt/MSS cancers. These results indicate that comparable rates of CIN occur between MSS subgroups, however significant differences in their patterns of instability exist, with BRAFmut/MSS cancers showing a 'focal pattern' and BRAFwt/MSS cancers having a 'whole arm pattern' of CIN. This and the genomic loci more frequently affected in BRAFmut/MSS cancers provides further evidence of the biological distinctions of this important cancer subgroup.

The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice.

The homeostatic balance between oxidants and antioxidants in biological systems is known as redox balance, and is regulated by complex processes. Redox balance regulates many of the known cellular pathways and disease processes. The dysregulation of redox balance can lead to acute or long-term oxidative or reductive stresses that are associated with many of the abnormalities observed in cystic fibrosis (CF). Over the past 5 decades researchers have examined contributors to redox dysregulation, their molecular products, and their impact on ion transport, cell proliferation, inflammation, bacterial killing, and the metabolism of nucleic acids, proteins, and lipids in CF. CF patients exhibit elevated markers of oxidative stress when compared to non-CF healthy controls; however, whether the reported redox imbalance is sufficient to produce pathology has been controversial. In addition, comparisons between CF and non-CF disease controls have been lacking. To better understand the mechanisms which mediate the generation of oxidants and antioxidants in CF and the importance of their balance in effecting oxidative or reductive stress, we will review the determinants of redox balance in the blood, lumen, and cellular compartments. From the perspective of methodological application, we will focus on the approaches most often used to study oxidant and antioxidants in CF, including biochemical, proteomic, metabolomic, and lipidomic studies, with a discussion of the few transcriptomic analyses that predict changes in the expression of regulators of redox. Finally, we will discuss the utility of oxidants and antioxidants as biomarkers of disease and the use of antioxidant therapy in CF.

Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs) are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

Dexras1 and RHES, monomeric G proteins, are members of small GTPase family that are involved in modulation of pathophysiological processes. Dexras1 and RHES levels are modulated by hormones and Dexras1 expression undergoes circadian fluctuations. Both these GTPases are capable of modulating calcium ion channels which in turn can potentially modulate neurosecretion/hormonal release. These two GTPases have been reported to prevent the aberrant cell growth and induce apoptosis in cell lines. Present review focuses on role of these two monomeric GTPases and summarizes their role in pathophysiological processes.

Hepcidin is a 25-amino-acid peptide demonstrated to be the iron regulatory hormone capable of blocking iron absorption from the duodenum and iron release from macrophages. Mutations affecting hepcidin regulators or the hepcidin gene itself cause hemochromatosis, a common genetic disorder. Hepcidin is produced mainly by the liver, but many cells and tissues express low levels of the hormone. To determine the contribution of these hepcidin-producing tissues in body iron homeostasis, we have developed a new mouse model in which the hepcidin gene can be conditionally inactivated. Here we compare a liver-specific knockout (KO) mouse model with total KO mice. We show that the liver-specific KO mice fully recapitulate the severe iron overload phenotype observed in the total KO mice, with increased plasma iron and massive parenchymal iron accumulation. This result demonstrates that the hepatocyte constitutes the predominant reservoir for systemic hepcidin and that the other tissues are unable to compensate.

As an alternative technique for calcium phosphate coating on titanium alloys, we propose to functionalize the metal surface with anionic bath containing chlorides of palladium or silver as activators. This new deposition route has several advantages such as controlled conditions, applicability to complex shapes, no adverse effect of heating, and cost effectiveness. A mixture of hydroxyapatite and calcium phosphate hydrate is deposited on the surface of Ti-6Al-4V. Calcium phosphate coating is built faster compared with the one by Simulated Body Fluid. Cell morphology and density are comparable to the control one; and the results prove no toxic compound is released into the medium during the previous seven days of immersion. Moreover, the cell viability is comparable with cells cultivated with the virgin medium. These experimental treatments allowed producing cytocompatible materials potentially applicable to manufacture implantable devices for orthopedic and oral surgeries.

The currently recognized principal forms of periodontitis-chronic and aggressive-lack an unequivocal, pathobiology-based foundation. We explored whether gingival tissue transcriptomes can serve as the basis for an alternative classification of periodontitis. We used cross-sectional whole-genome gene expression data from 241 gingival tissue biopsies obtained from sites with periodontal pathology in 120 systemically healthy nonsmokers with periodontitis, with available data on clinical periodontal status, subgingival microbial profiles, and serum IgG antibodies to periodontal microbiota. Adjusted model-based clustering of transcriptomic data using finite mixtures generated two distinct clusters of patients that did not align with the current classification of chronic and aggressive periodontitis. Differential expression profiles primarily related to cell proliferation in cluster 1 and to lymphocyte activation and unfolded protein responses in cluster 2. Patients in the two clusters did not differ with respect to age but presented with distinct phenotypes (statistically significantly different whole-mouth clinical measures of extent/severity, subgingival microbial burden by several species, and selected serum antibody responses). Patients in cluster 2 showed more extensive/severe disease and were more often male. The findings suggest that distinct gene expression signatures in pathologic gingival tissues translate into phenotypic differences and can provide a basis for a novel classification.

<h4>Objective</h4>To examine the interactions between genetic predisposition and consumption of fried food in relation to body mass index (BMI) and obesity.<h4>Design</h4>Prospective cohort study.<h4>Setting</h4>Health professionals in the United States.<h4>Participants</h4>9623 women from the Nurses' Health Study, 6379 men from the Health Professionals Follow-up Study, and a replication cohort of 21,421 women from the Women's Genome Health Study.<h4>Main outcome measure</h4>Repeated measurement of BMI over follow-up.<h4>Results</h4>There was an interaction between fried food consumption and a genetic risk score based on 32 BMI-associated variants on BMI in both the Nurses' Health Study and Health Professionals Follow-up Study (P ≤ 0.001 for interaction). Among participants in the highest third of the genetic risk score, the differences in BMI between individuals who consumed fried foods four or more times a week and those who consumed fried foods less than once a week amounted to 1.0 (SE 0.2) in women and 0.7 (SE 0.2) in men, whereas the corresponding differences were 0.5 (SE 0.2) and 0.4 (SE 0.2) in the lowest third of the genetic risk score. The gene-diet interaction was replicated in the Women's Genome Health Study (P<0.001 for interaction). Viewed differently, the genetic association with adiposity was strengthened with higher consumption of fried foods. In the combined three cohorts, the differences in BMI per 10 risk alleles were 1.1 (SE 0.2), 1.6 (SE 0.3), and 2.2 (SE 0.6) for fried food consumption less than once, one to three times, and four or more times a week (P<0.001 for interaction); and the odds ratios (95% confidence intervals) for obesity per 10 risk alleles were 1.61 (1.40 to 1.87), 2.12 (1.73 to 2.59), and 2.72 (2.12 to 3.48) across the three categories of consumption (P=0.002 for interaction). In addition, the variants in or near genes highly expressed or known to act in the central nervous system showed significant interactions with fried food consumption, with the FTO (fat mass and obesity associated) variant showing the strongest result (P<0.001 for interaction).<h4>Conclusion</h4>Our findings suggest that consumption of fried food could interact with genetic background in relation to obesity, highlighting the particular importance of reducing fried food consumption in individuals genetically predisposed to obesity.

<h4>Objective</h4>We describe a novel congenital motor neuron disease with early demise due to respiratory insufficiency with clinical overlap with spinal muscular atrophy with respiratory distress (SMARD) type 1 but lacking a mutation in the IGHMBP2 gene.<h4>Methods</h4>Exome sequencing was used to identify a de novo mutation in the LAS1L gene in the proband. Pathogenicity of the mutation was validated using a zebrafish model by morpholino-mediated knockdown of las1l.<h4>Results</h4>We identified a de novo mutation in the X-linked LAS1L gene in the proband (p.S477N). The mutation is in a highly conserved region of the LAS1L gene predicted to be deleterious by bioinformatic analysis. Morpholino-based knockdown of las1l, the orthologous gene in zebrafish, results in early lethality and disruption of muscle and peripheral nerve architecture. Coinjection of wild-type but not mutant human RNA results in partial rescue of the phenotype.<h4>Conclusion</h4>We report a patient with a SMARD phenotype due to a mutation in LAS1L, a gene important in coordinating processing of the 45S pre-rRNA and maturation of the large 60S ribosomal subunit. Similarly, the IGHMB2 gene associated with SMARD type 1 has been suggested to have an important role in ribosomal biogenesis from its role in processing the 45S pre-rRNA. We propose that disruption of ribosomal maturation may be a common pathogenic mechanism linking SMARD phenotypes caused by both IGHMBP2 and LAS1L.

The multifunctional molecule netrin-1 is upregulated in various malignancies and has recently been presented as a major general player in tumorigenesis leading to tumor progression and maintenance in various animal models. However, there is still a lack of clinico-epidemiological data related to netrin-1 expression. Therefore, the aim of our study was to elucidate the association of netrin-1 expression and patient survival in brain metastases since those constitute one of the most limiting factors for patient prognosis. We investigated 104 brain metastases cases for netrin-1 expression using in-situ hybridization and immunohistochemistry with regard to clinical parameters such as patient survival and MRI data. Our data show that netrin-1 is strongly upregulated in most cancer subtypes. Univariate analyses revealed netrin-1 expression as a significant factor associated with poor patient survival in the total cohort of brain metastasis patients and in sub-entities such as non-small cell lung carcinomas. Interestingly, many cancer samples showed a strong nuclear netrin-1 signal which was recently linked to a truncated netrin-1 variant that enhances tumor growth. Nuclear netrin-1 expression was associated with poor patient survival in univariate as well as in multivariate analyses. Our data indicate both total and nuclear netrin-1 expression as prognostic factors in brain metastases patients in contrast to other prognostic markers in oncology such as patient age, number of brain metastases or Ki67 proliferation index. Therefore, nuclear netrin-1 expression constitutes one of the first reported molecular biomarkers for patient survival in brain metastases. Furthermore, netrin-1 may constitute a promising target for future anti-cancer treatment approaches in brain metastases.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3' exons of neurotrophic tyrosine kinase receptor type 1 (NTRK1, encoding TrkA) are fused to 5' exons of the genes that are highly expressed in neuronal tissues, neurofascin (NFASC) and brevican (BCAN). The fusions preserved both the transmembrane and kinase domains of NTRK1 in frame. NTRK1 is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked NTRK1 expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated NTRK1-pathway activity. Lentiviral transduction of the NFASC-NTRK1 fusion gene in NIH 3T3 cells increased proliferation in vitro, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.

Glycated hemoglobin A1c (HbA1c) is used as a measure of glycemic control and also as a diagnostic criterion for diabetes. To discover novel loci harboring common variants associated with HbA1c in East Asians, we conducted a meta-analysis of 13 genome-wide association studies (GWAS; N = 21,026). We replicated our findings in three additional studies comprising 11,576 individuals of East Asian ancestry. Ten variants showed associations that reached genome-wide significance in the discovery data set, of which nine (four novel variants at TMEM79 [P value = 1.3 × 10(-23)], HBS1L/MYB [8.5 × 10(-15)], MYO9B [9.0 × 10(-12)], and CYBA [1.1 × 10(-8)] as well as five variants at loci that had been previously identified [CDKAL1, G6PC2/ABCB11, GCK, ANK1, and FN3KI]) showed consistent evidence of association in replication data sets. These variants explained 1.76% of the variance in HbA1c. Several of these variants (TMEM79, HBS1L/MYB, CYBA, MYO9B, ANK1, and FN3K) showed no association with either blood glucose or type 2 diabetes. Among individuals with nondiabetic levels of fasting glucose (<7.0 mmol/L) but elevated HbA1c (≥6.5%), 36.1% had HbA1c <6.5% after adjustment for these six variants. Our East Asian GWAS meta-analysis has identified novel variants associated with HbA1c as well as demonstrated that the effects of known variants are largely transferable across ethnic groups. Variants affecting erythrocyte parameters rather than glucose metabolism may be relevant to the use of HbA1c for diagnosing diabetes in these populations.

O-GlcNAcylation is an important post-translational modification of proteins and is known to regulate a number of pathways involved in cellular homeostasis. This involves dynamic and reversible modification of serine/threonine residues of different cellular proteins catalyzed by O-linked N-acetylglucosaminyltransferase and O-linked N-acetylglucosaminidase in an antagonistic manner. We report here that decreasing O-GlcNAcylation enhances the viability of neuronal cells expressing polyglutamine-expanded huntingtin exon 1 protein fragment (mHtt). We further show that O-GlcNAcylation regulates the basal autophagic process and that suppression of O-GlcNAcylation significantly increases autophagic flux by enhancing the fusion of autophagosome with lysosome. This regulation considerably reduces toxic mHtt aggregates in eye imaginal discs and partially restores rhabdomere morphology and vision in a fly model for Huntington disease. This study is significant in unraveling O-GlcNAcylation-dependent regulation of an autophagic process in mediating mHtt toxicity. Therefore, targeting the autophagic process through the suppression of O-GlcNAcylation may prove to be an important therapeutic approach in Huntington disease.

Uncontrolled transforming growth factor-β (TGFβ) signaling promotes aggressive metastatic properties in late-stage breast cancers. However, how TGFβ-mediated cues are directed to induce tumorigenic events is poorly understood, particularly given that TGFβ has clear tumor suppressing activity in other contexts. Here, we demonstrate that the transcriptional regulators TAZ and YAP (TAZ/YAP), key effectors of the Hippo pathway, are necessary to promote and maintain TGFβ-induced tumorigenic phenotypes in breast cancer cells. Interactions between TAZ/YAP, TGFβ-activated SMAD2/3, and TEAD transcription factors reveal convergent roles for these factors in the nucleus. Genome-wide expression analyses indicate that TAZ/YAP, TEADs, and TGFβ-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by TAZ/YAP, TEAD, and TGFβ, such as the novel targets NEGR1 and UCA1, are necessary for maintaining tumorigenic activity in metastatic breast cancer cells. Nuclear TAZ/YAP also cooperate with TGFβ signaling to promote phenotypic and transcriptional changes in nontumorigenic cells to overcome TGFβ-repressive effects. Our work thus identifies cross-talk between nuclear TAZ/YAP and TGFβ signaling in breast cancer cells, revealing novel insight into late-stage disease-driving mechanisms.

Functional integrity of mitochondria is critical for optimal cellular physiology. A suite of conserved mitochondrial proteases known as intramitochondrial quality control represents one of the mechanisms assuring normal mitochondrial function. We previously demonstrated that ATP-independent metalloprotease Oma1 mediates degradation of hypohemylated Cox1 subunit of cytochrome c oxidase and is active in cytochrome c oxidase-deficient mitochondria. Here we show that Oma1 is important for adaptive responses to various homeostatic insults and preservation of normal mitochondrial function under damage-eliciting conditions. Changes in membrane potential, oxidative stress, or chronic hyperpolarization lead to increased Oma1-mediated proteolysis. The stress-triggered induction of Oma1 proteolytic activity appears to be associated with conformational changes within the Oma1 homo-oligomeric complex, and these alterations likely involve C-terminal residues of the protease. Substitutions in the conserved C-terminal region of Oma1 impair its ability to form a labile proteolytically active complex in response to stress stimuli. We demonstrate that Oma1 genetically interacts with other inner membrane-bound quality control proteases. These findings indicate that yeast Oma1 is an important player in IM protein homeostasis and integrity by acting in concert with other intramitochondrial quality control components.

An endophytic fungus Phomopsis liquidambaris CBR-15, was isolated from Cryptolepis buchanani Roem. (Asclepiadaceae) and identified by its characteristic culture morphology and molecular analysis of the ITS region of rDNA and intervening 5.8S rRNA gene. The impact of different culture media on biosynthesis of antimicrobial metabolites was tested by disc diffusion assay. Polyketide synthase gene (PKS) of the endophytic fungus was investigated using three pairs of degenerate primers LC1-LC2c, LC3-LC5c and KS3-KS4c by PCR. TLC-bioautography method was employed to detect the antimicrobial metabolites. Antimicrobial metabolites fractionated with ethyl acetate extract showed significant antimicrobial activity against the test bacteria and fungi. Biosynthesis of antimicrobial metabolites was optimum as depicted by zone of inhibition from ethyl acetate extract cultured in potato dextrose broth. Strain CBR-15 was identified as Phomopsisliquidambaris and PKS genes of the fungus were amplified with LC3-LC5c and KS3-KS4c sets of degenerate primers. These findings suggest that endophytic P.liquidambaris CBR-15 harbor iterative type I fungal PKS gene domain which indicates the biosynthetic potential of endophytic fungi as producers of natural antimicrobial metabolites. The study also demonstrates the utilization and optimization of different culture media which best supports for the biosynthesis of the antimicrobial metabolites from P.liquidambaris.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT). One prominent target of the mutant huntingtin protein (mhtt) is the mitochondrion, affecting its morphology, distribution, and function. Thus, mitochondria have been suggested as potential therapeutic targets for the treatment of HD. Olesoxime, a cholesterol-like compound, promotes motor neuron survival and neurite outgrowth in vitro, and its effects are presumed to occur via a direct interaction with mitochondrial membranes (MMs). We examined the properties of MMs isolated from cell and animal models of HD as well as the effects of olesoxime on MM fluidity and cholesterol levels. MMs isolated from brains of aged Hdh Q111/Q111 knock-in mice showed a significant decrease in 1,6-diphenyl-hexatriene (DPH) anisotropy, which is inversely correlated with membrane fluidity. Similar increases in MM fluidity were observed in striatal STHdh Q111/Q111 cells as well as in MMs isolated from brains of BACHD transgenic rats. Treatment of STHdh cells with olesoxime decreased the fluidity of isolated MMs. Decreased membrane fluidity was also measured in olesoxime-treated MMs isolated from brains of HD knock-in mice. In both models, treatment with olesoxime restored HD-specific changes in MMs. Accordingly, olesoxime significantly counteracted the mhtt-induced increase in MM fluidity of MMs isolated from brains of BACHD rats after 12 months of treatment in vivo, possibly by enhancing MM cholesterol levels. Thus, olesoxime may represent a novel pharmacological tool to treat mitochondrial dysfunction in HD.

CaO-P2O5-SiO2-B2O3-ZnO bioactive glasses were prepared via an optimized sol-gel method. The current investigation was focused on producing novel zinc based calcium phosphoborosilicate glasses and to evaluate their mechanical, rheological, and biocompatible properties. The morphology and composition of these glasses were studied using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The particle size, mechanical and flexural strength was also determined. Furthermore, the zeta potential of all the glasses were determined to estimate their flocculation tendency. The thermal analysis and weight loss measurements were carried out using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) respectively. For assessing the in-vitro bioactive character of synthesized glasses, the ability for apatite formation on their surface upon their immersion in simulated body fluid (SBF) was checked using SEM and pH measurements. MTS assay cytotoxicity assay and live-dead cell viability test were conducted on J774A.1 cells murine macrophage cells for different glass concentrations.

FSH is a key hormone in the regulation of follicular development. Together with the EGF network, these molecules mediate oocyte maturation and competence in preparation for the action of LH. FSH isoforms regulate distinct biological pathways and have specific effects on granulosa cell function and maturation of the ovarian follicle. Their dynamic interactions occur during the follicular cycle; short-living forms are predominant in the pre-ovulatory phase, whereas long-acting molecules characterize the luteal-follicular transition. Recombinant FSH (rFSH) molecules have a reduced number of isoforms and are less acidic, with a shorter half-life. We have investigated sequential stimulation, comparing hFSH + rFSH, vs. rFSH alone and hFSH alone for the entire stimulation phase. Sequential stimulation leads to an E2 per MII oocyte ratio that is much lower than is seen during treatment with the two drugs individually. Although there is a positive tendency in favor of the sequential treatment, there was no significant difference in pregnancy rates, even taking frozen embryos into consideration. The cumulus cell transcriptome varies considerably between the treatments, although with no clear significance. When comparing pregnant vs. non-pregnant patients, in general a decrease in mRNA expression can be observed in the pregnant patients, especially in expression of folic acid receptor 1 and ovostatin 2. This indicates that material has been transferred from CC to the oocyte. However, a common observation in the literature is that variations in the transcriptome of the cumulus cells are highly dependent upon the patient genotype; the potential for applying this strategy as a basis for selecting embryos is, at the very least, questionable.

Recent studies have used a variety of analytical methods to identify genes targeted by selection in high-altitude populations located throughout the Tibetan Plateau. Despite differences in analytic strategies and sample location, hypoxia-related genes, including EPAS1 and EGLN1, were identified in multiple studies. By applying the same analytic methods to genome-wide SNP information used in our previous study of a Tibetan population (n = 31) from the township of Maduo, located in the northeastern corner of the Qinghai-Tibetan Plateau (4200 m), we have identified common targets of natural selection in a second geographically and linguistically distinct Tibetan population (n = 46) in the Tuo Tuo River township (4500 m). Our analyses provide evidence for natural selection based on iHS and XP-EHH signals in both populations at the p<0.02 significance level for EPAS1, EGLN1, HMOX2, and CYP17A1 and for PKLR, HFE, and HBB and HBG2, which have also been reported in other studies. We highlight differences (i.e., stratification and admixture) in the two distinct Tibetan groups examined here and report selection candidate genes common to both groups. These findings should be considered in the prioritization of selection candidate genes in future genetic studies in Tibet.

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

Exercise is beneficial for a variety of age-related disorders. However, the molecular mechanisms mediating the beneficial adaptations to exercise in older adults are not well understood. The aim of the current study was to utilize a dual approach to characterize the genetic and metabolic adaptive pathways altered by exercise in veteran athletes and age-matched untrained individuals. Two groups of 50-60 year old males: competitive cyclists (athletes, n = 9; VO2peak 59.1±5.2 ml·kg(-1)·min(-1); peak aerobic power 383±39 W) and untrained, minimally active individuals (controls, n = 8; VO2peak 35.9±9.7 ml·kg(-1)·min(-1); peak aerobic power 230±57 W) were examined. All participants completed an acute bout of submaximal endurance exercise, and blood and urine samples pre- and post-exercise were analyzed for gene expression and metabolic changes utilizing genome-wide DNA microarray analysis and NMR spectroscopy-based metabolomics, respectively. Our results indicate distinct differences in gene and metabolite expression involving energy metabolism, lipids, insulin signaling and cardiovascular function between the two groups. These findings may lead to new insights into beneficial signaling pathways of healthy aging and help identify surrogate markers for monitoring exercise and training load.

Precise regulation of gene expression during biological processes, including development, is often achieved by combinatorial action of multiple transcription factors. The mechanisms by which these factors collaborate are largely not known. We have shown previously that Isl1, a Lim-Homeodomain transcription factor, and Pou4f2, a class IV POU domain transcription factor, co-regulate a set of genes required for retinal ganglion cell (RGC) differentiation. Here we further explore how these two factors interact to precisely regulate gene expression during RGC development. By GST pulldown assays, co-immunoprecipitation, and electrophoretic mobility shift assays, we show that Isl1 and Pou4f2 form a complex in vitro and in vivo, and identify the domains within these two proteins that are responsible for this interaction. By luciferase assay, in situ hybridization, and RNA-seq, we further demonstrate that the two factors contribute quantitatively to gene expression in the developing RGCs. Although each factor alone can activate gene expression, both factors are required to achieve optimal expression levels. Finally, we discover that Isl1 and Pou4f2 can interact with other POU and Lim-Homeodomain factors respectively, indicating the interactions between these two classes of transcription factors are prevalent in development and other biological processes.

The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also specifically impact alternative splicing.

The localization of signaling molecules such as G protein-coupled receptors (GPCRs) to primary cilia is essential for correct signal transduction. Detailed studies over the past decade have begun to elucidate the diverse sequences and trafficking mechanisms that sort and transport GPCRs to the ciliary compartment. However, a systematic analysis of the pathways required for ciliary targeting of multiple GPCRs in different cell types in vivo has not been reported. Here we describe the sequences and proteins required to localize GPCRs to the cilia of the AWB and ASK sensory neuron types in Caenorhabditis elegans. We find that GPCRs expressed in AWB or ASK utilize conserved and novel sequences for ciliary localization, and that the requirement for a ciliary targeting sequence in a given GPCR is different in different neuron types. Consistent with the presence of multiple ciliary targeting sequences, we identify diverse proteins required for ciliary localization of individual GPCRs in AWB and ASK. In particular, we show that the TUB-1 Tubby protein is required for ciliary localization of a subset of GPCRs, implying that defects in GPCR localization may be causal to the metabolic phenotypes of tub-1 mutants. Together, our results describe a remarkable complexity of mechanisms that act in a protein- and cell-specific manner to localize GPCRs to cilia, and suggest that this diversity allows for precise regulation of GPCR-mediated signaling as a function of external and internal context.

The pathogenesis of Sjögren's syndrome (SS) likely involves complex interactions between genes and the environment. While the candidate gene approach has been previously used to identify several genes associated with SS, two recent large-scale genome-wide association studies (GWAS) have implicated many more loci as genetic risk factors. Of particular relevance, was the significant association of SS with additional immune-related genes including IL12A, BLK, and CXCR5. GWAS has also uncovered other loci and suggestive gene associations in SS, but none are related to genes encoding salivary or lacrimal components, secretion machinery and neuronal proteins involved in innervations of the glands, respectively. In this review, we discuss these genetic findings with particular attention paid to the genes identified, the strength of associations, and how the SS-associated genes compare to what has been discovered previously in systemic lupus erythematosus (SLE). We also summarize the potential impact of these associated gene products on NFκB and immune pathways and describe how this new information might be integrated further for identifying clinical subsets and understanding the pathogenesis of SS.

Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma-infected mice 5days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin (5-HT) and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and 5-HT metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/β-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

To improve the understanding of the enriched functions of proteins and to identify potential biomarkers in human breast cancer, the present study constructed a differentially expressed protein profile by screening immunohistochemistry maps of human breast cancer proteins. A total of 1,688 proteins were found to be differentially expressed in human breast cancer, including 773 upregulated and 915 downregulated proteins. Of these proteins, secreted and membrane proteins were screened and clustered, and more enriched biological functions and pathways were presented in the upregulated protein profiles. Furthermore, altered serum levels of peroxiredoxin (PRDX)2, PRDX6, cathepsin (CTS)B and CTSD were detected by ELISA assay. The present study provides a novel global mapping of potential breast cancer biomarkers that could be used as background to identify the altered pathways in human breast cancer, as well as potential cancer targets.

Seven years have passed since the initial report of the generation of induced pluripotent stem cells (iPSCs) from adult human somatic cells, and in the intervening time the field of neuroscience has developed numerous disease models using this technology. Here, we review progress in the field and describe both the advantages and potential pitfalls of modeling neurodegenerative and neurodevelopmental diseases using this technology. We include tables with information on neural differentiation protocols and studies that developed human iPSC lines to model neurological diseases. We also discuss how one can: investigate effects of genetic mutations with iPSCs, examine cell fate-specific phenotypes, best determine the specificity of a phenotype, and bring in vivo relevance to this in vitro technique.

The localization of mRNAs within axons and dendrites allows neurons to manipulate protein levels in a time and location dependent manner and is essential for processes such as synaptic plasticity and axon guidance. However, an essential step in the process of mRNA localization, the decision to traffic to dendrites and/or axons, remains poorly understood. Here we show that Myosin Va and actin filaments are necessary for the dendritic localization of the mRNA binding protein Staufen 1 and of mRNA encoding the microtubule binding protein Map2. Blocking the function or expression of Myosin Va or depolymerizing actin filaments leads to localization of Staufen 1 and of Map2 mRNA in both axons and dendrites. Furthermore, interaction with Myosin Va plays an instructive role in the dendritic localization of Hermes 1, an RNA binding protein. Wild-type Hermes 1 localizes to both axons and dendrites, whereas Hermes 1 fused with a Myosin Va binding peptide, localizes specifically to dendrites. Thus, our results suggest that targeting of mRNAs to the dendrites is mediated by a mechanism that is dependent on actin and Myosin Va.

<h4>Background</h4>To investigate the implication of ribonucleotide reductase M2 (RRM2) in the carcinogenesis of uterine cervix and its relationship with clinicopathological characteristics and prognosis of cancer patients.<h4>Methodology and principal findings</h4>The impact of RRM2 on cell viability was investigated in SiHa cervical cancer cells after RRM2 knockdown and the addition of cisplatin, which induces inter- and intra-strand DNA crosslinks. RRM2 immunoreactivity was evaluated by semi-quantitative H score among 29 normal, 30 low-grade dysplasia, 30 high-grade dysplasia and 103 invasive cancer tissue specimens of the uterine cervix, using tissue microarrays. RRM2 was then correlated with the clinicopathological variables of cervical cancer and patient survival. A greater toxic effect on cell viability using cisplatin was reflected by the greater reduction in RRM2 protein expression in SiHa cells. The RRM2 expression in cancer tissues was higher than that in high-grade dysplasia, low-grade dysplasia or normal cervical tissues. RRM2 upregulation was correlated with deep stromal invasion, large tumors and parametrial invasion and predicted poor survival.<h4>Conclusions</h4>RRM2 is a new molecular marker for the diagnosis and clinical outcomes of cervical cancer. It is involved in cervical carcinogenesis and predicts poor survival, and may be a potential therapeutic target including in cisplatin treatment.

Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.

A significant association between HFE gene mutations and the HLA-A*03-B*07 and HLA-A*29-B*44 haplotypes has been reported in the Spanish population. It has been proposed that these mutations are probably connected with Celtic and North African ancestry, respectively. We aimed to find the possible ancestral association between HLA alleles and haplotypes associated with the HFE gene (C282Y and H63D) mutations in 214 subjects from Antioquia, Colombia. These were 18 individuals with presumed hereditary hemochromatosis ("HH") and 196 controls. The HLA-B*07 allele was in linkage disequilibrium (LD) with C282Y, while HLA-A*23, A*29, HLA-B*44, and B*49 were in LD with H63D. Altogether, our results show that, although the H63D mutation is more common in the Antioquia population, it is not associated with any particular HLA haplotype, whereas the C282Y mutation is associated with HLA-A*03-B*07, this supporting a northern Spaniard ancestry.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of dopamine-generating cells in the substantia nigra (SN). Acupuncture stimulation results in an enhanced survival of dopaminergic neurons in the SN in Parkinsonism animal models. The present study investigated changes in gene expression profiles measured using whole transcript array in the SN region related to the inhibitory effects of acupuncture in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism model. In this model, acupuncture stimulation at GB34 and LR3 attenuated the decrease in tyrosine hydroxylase in the SN region; stimulation at non-acupoints did not suppress this decrease. Gene array analysis revealed that 22 (10 annotated genes: Cdh1, Itih2, Mpzl2, Rdh9, Serping1, Slc6a13, Slc6a20a, Slc6a4, Tph2, and Ucma) probes that were up-regulated in MPTP animals relative to controls were exclusively down-regulated by acupuncture stimulation. In addition, 17 (two annotated genes: 4921530L21Rik and Gm13931) probes that were down-regulated in MPTP animals compared to controls were exclusively up-regulated by acupuncture stimulation. These findings indicate that the 39 probes (12 annotated genes) affected by MPTP and acupuncture may be responsible for the inhibitory effects of acupuncture on degeneration-related gene expression in the SN following damage induced by MPTP intoxication.

Regulatory T cells (Treg cells) express members of the tumor-necrosis factor (TNF) receptor superfamily (TNFRSF), but the role of those receptors in the thymic development of Treg cells is undefined. We found here that Treg cell progenitors had high expression of the TNFRSF members GITR, OX40 and TNFR2. Expression of those receptors correlated directly with the signal strength of the T cell antigen receptor (TCR) and required the coreceptor CD28 and the kinase TAK1. The neutralization of ligands that are members of the TNF superfamily (TNFSF) diminished the development of Treg cells. Conversely, TNFRSF agonists enhanced the differentiation of Treg cell progenitors by augmenting responsiveness of the interleukin 2 receptor (IL-2R) and transcription factor STAT5. Costimulation with the ligand of GITR elicited dose-dependent enrichment for cells of lower TCR affinity in the Treg cell repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated the development of Treg cells. Thus, expression of members of the TNFRSF on Treg cell progenitors translated strong TCR signals into molecular parameters that specifically promoted the development of Treg cells and shaped the Treg cell repertoire.

Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.

<h4>Background</h4>KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness.<h4>Methods</h4>We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer.<h4>Results</h4>KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility.<h4>Conclusions</h4>Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it may represent a novel target for biomarker development and a novel therapeutic target for breast cancer.

modENCODE was a 5year NHGRI funded project (2007-2012) to map the function of every base in the genomes of worms and flies characterizing positions of modified histones and other chromatin marks, origins of DNA replication, RNA transcripts and the transcription factor binding sites that control gene expression. Here we describe the Drosophila modENCODE datasets and how best to access and use them for genome wide and individual gene studies.

<h4>Objective</h4>To comparatively evaluate traditional liver tests and fetuin A as predictors of cardiometabolic risk, we studied associations between serum alanine transaminase (ALT), γ-glutamyl transferase (GGT), aspartate aminotransferase (AST) and fetuin-A and anthropometric, metabolic, and cardiovascular parameters cross-sectionally at baseline, and prospectively, after 2-years of follow-up.<h4>Research design and methods</h4>616 randomly enrolled young healthy participants in the Cyprus Metabolism Study, including all 93 subjects who participated in the follow-up study 2 years after baseline assessment, were included in this study.<h4>Results</h4>In the cross-sectional study, serum ALT and GGT were strongly correlated with anthropometric, cardiovascular, and metabolic variables, while serum AST was only correlated with waist circumference and waist-to-hip ratio. Fetuin-A was correlated with anthropometric variables, systolic blood pressure (SBP), insulin, and homeostasis model of assessment-insulin resistance (HOMA-IR) in the unadjusted model. In the fully adjusted model, both serum ALT and GGT levels remained positively correlated with total and low-density lipoprotein (LDL) cholesterol. GGT levels also remained correlated with triglycerides. ALT levels remained strongly positively correlated with insulin (r=0.17, p<.0001) and HOMA-IR (r=0.16, p=0.0001). Serum fetuin-A levels were no longer significantly correlated with any variables. Prospectively, ALT and GGT were predictors of anthropometric variables and LDL cholesterol, while baseline levels of AST and fetuin-A were not predictors of any variables at 2-year follow-up.<h4>Conclusions</h4>We confirmed associations of ALT and GGT levels but failed to demonstrate an independent association between fetuin-A and cardiometabolic risk factors in young healthy men. Traditional liver tests (LFTs) are thus better than fetuin-A predictors of metabolic risk factors cross-sectionally and prospectively in young healthy adults.

<h4>Purpose</h4>This pilot study was aimed to establish techniques for assessing and observing trends in endothelial function, antioxidant status and vascular compliance in newly diagnosed HFE haemochromatosis during the first year of venesection.<h4>Patients/methods</h4>Untreated newly diagnosed HFE haemochromatosis patients were tested for baseline liver function, iron indices, lipid profile, markers of endothelial function, anti-oxidant status and vascular compliance. Following baseline assessment, subjects attended at 6-weeks and at 3, 6, 9 and 12-months for follow-up studies.<h4>Results</h4>Ten patients were recruited (M=8, F=2, mean age=51 years). Venesection significantly increased high density lipoproteins at 12-months (1.25 mmol/L vs. 1.37 mmol/L, p=0.01). However, venesection did not significantly affect lipid hydroperoxides, intracellular and vascular cell adhesion molecules or high sensitivity C-reactive protein (0.57 μmol/L vs. 0.51 μmol/L, p=0.45, 427.4 ng/ml vs. 307.22 ng/ml, p=0.54, 517.70 ng/ml vs. 377.50 ng/ml, p=0.51 and 290.75 μg/dL vs. 224.26 μg/dL, p=0.25). There was also no significant effect of venesection on anti-oxidant status or pulse wave velocity (9.65 m/s vs. 8.74 m/s, p=0.34).<h4>Conclusions</h4>Venesection significantly reduced high density lipoproteins but was not associated with significant changes in endothelial function, anti-oxidant status or vascular compliance. Larger studies using this established methodology are required to clarify this relationship further.

Hepatocellular carcinoma is one of the leading causes of death by cancer worldwide. Prognosis of hepatocellular carcinoma is determined by characteristics of the tumor and the surrounding cirrhotic liver. Several molecular signatures reflecting tumor biology and derived from tumor analyses predict early tumor recurrence and survival. In contrast, molecular signatures from cirrhotic non-tumor samples are enriched in immunity/inflammation related genes and could predict late tumor recurrence. Moreover, combination of clinical, pathological, and molecular features may refine prognosis prediction in these patients. Finally, molecular signatures from both tumor and non-tumor tissues will be helpful in the future to guide treatments in different clinical settings.

Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.

<h4>Background</h4>Immediately after renal transplantation, patients experience rapid and significant improvement of their clinical conditions and undergo considerable systemic and cellular modifications. However, some patients present a slow recovery of the renal function commonly defined as delayed graft function (DGF). Although clinically well characterized, the molecular mechanisms underlying this condition are not totally defined, thus, we are currently missing specific clinical markers to predict and to make early diagnosis of this event.<h4>Methods</h4>We investigated, using a pathway analysis approach, the transcriptomic profile of peripheral blood mononuclear cells (PBMC) from renal transplant recipients with DGF and with early graft function (EGF), before (T0) and 24 hours (T24) after transplantation.<h4>Results</h4>Bioinformatics/statistical analysis showed that 15 pathways (8 up-regulated and 7 down-regulated) and 11 pathways (5 up-regulated and 6 down-regulated) were able to identify DGF patients at T0 and T24, respectively. Interestingly, the most up-regulated pathway at both time points was NLS-bearing substrate import into nucleus, which includes genes encoding for several subtypes of karyopherins, a group of proteins involved in nucleocytoplasmic transport. Signal transducers and activators of transcription (STAT) utilize karyopherins-alpha (KPNA) for their passage from cytoplasm into the nucleus. In vitro functional analysis demonstrated that in PBMCs of DGF patients, there was a significant KPNA-mediated nuclear translocation of the phosphorylated form of STAT3 (pSTAT3) after short-time stimulation (2 and 5 minutes) with interleukin-6.<h4>Conclusions</h4>Our study suggests the involvement, immediately before transplantation, of karyopherin-mediated nuclear transport in the onset and development of DGF. Additionally, it reveals that karyopherins could be good candidates as potential DGF predictive clinical biomarkers and targets for pharmacological interventions in renal transplantation. However, because of the low number of patients analyzed and some methodological limitations, additional studies are needed to validate and to better address these points.

Brush-shaped amphiphilic block copolymers were conjugated with a monoclonal antibody against CD105 (i.e., TRC105) and a macrocyclic chelator for (64)Cu-labeling to generate multifunctional theranostic unimolecular micelles. The backbone of the brush-shaped amphiphilic block copolymer was poly(2-hydroxyethyl methacrylate) (PHEMA) and the side chains were poly(L-lactide)-poly(ethylene glycol) (PLLA-PEG). The doxorubicin (DOX)-loaded unimolecular micelles showed a pH-dependent drug release profile and a uniform size distribution. A significantly higher cellular uptake of TRC105-conjugated micelles was observed in CD105-positive human umbilical vein endothelial cells (HUVEC) than nontargeted micelles due to CD105-mediated endocytosis. In contrast, similar and extremely low cellular uptake of both targeted and nontargeted micelles was observed in MCF-7 human breast cancer cells (CD105-negative). The difference between the in vivo tumor accumulation of (64)Cu-labeled TRC105-conjugated micelles and that of nontargeted micelles was studied in 4T1 murine breast tumor-bearing mice, by serial positron emission tomography (PET) imaging and validated by biodistribution studies. These multifunctional unimolecular micelles offer pH-responsive drug release, noninvasive PET imaging capability, together with both passive and active tumor-targeting abilities, thus making them a desirable nanoplatform for cancer theranostics.

Huntington's disease (HD) is a neurological degenerative disease and quinolinic acid (QA) has been used to establish HD model in animals through the mechanism of excitotoxicity. Yet the specific pathological changes and the underlying mechanisms are not fully elucidated. We aimed to reveal the specific morphological changes of different striatal neurons in the HD model. Sprague-Dawley (SD) rats were subjected to unilaterally intrastriatal injections of QA to mimic the HD model. Behavioral tests, histochemical and immunhistochemical stainings as well as Western blots were applied in the present study. The results showed that QA-treated rats had obvious motor and cognitive impairments when compared with the control group. Immunohistochemical detection showed a great loss of NeuN+ neurons and Darpp32+ projection neurons in the transition zone in the QA group when compared with the control group. The numbers of parvalbumin (Parv)+ and neuropeptide Y (NPY)+ interneurons were both significantly reduced while those of calretinin (Cr)+ and choline acetyltransferase (ChAT)+ were not changed notably in the transition zone in the QA group when compared to the controls. Parv+, NPY+ and ChAT+ interneurons were not significantly increased in fiber density while Cr+ neurons displayed an obvious increase in fiber density in the transition zone in QA-treated rats. The varicosity densities of Parv+, Cr+ and NPY+ interneurons were all raised in the transition zone after QA treatment. In conclusion, the present study revealed that QA induced obvious behavioral changes as well as a general loss of striatal projection neurons and specific morphological changes in different striatal interneurons, which may help further explain the underlying mechanisms and the specific functions of various striatal neurons in the pathological process of HD.

The health benefits of fish oil enriched with high omega-3 polyunsaturated fatty acids (n-3 PUFA) are widely documented. Fish oil as dietary supplements, however, show moderate clinical efficacy, highlighting an immediate scope of systematic in vitro feedback. Our transcriptomic study was designed to investigate the genomic shift of murine brains fed on fish oil enriched diets. A customized fish oil enriched diet (FD) and standard lab diet (SD) were separately administered to two randomly chosen populations of C57BL/6J mice from their weaning age until late adolescence. Statistical analysis mined 1,142 genes of interest (GOI) differentially altered in the hemibrains collected from the FD- and SD-fed mice at the age of five months. The majority of identified GOI (∼ 40%) encodes proteins located in the plasma membrane, suggesting that fish oil primarily facilitated the membrane-oriented biofunctions. FD potentially augmented the nervous system's development and functions by selectively stimulating the Src-mediated calcium-induced growth cascade and the downstream PI3K-AKT-PKC pathways. FD reduced the amyloidal burden, attenuated oxidative stress, and assisted in somatostatin activation-the signatures of attenuation of Alzheimer's disease, Parkinson's disease, and affective disorder. FD induced elevation of FKBP5 and suppression of BDNF, which are often linked with the improvement of anxiety disorder, depression, and post-traumatic stress disorder. Hence we anticipate efficacy of FD in treating illnesses such as depression that are typically triggered by the hypoactivities of dopaminergic, adrenergic, cholinergic, and GABAergic networks. Contrastingly, FD's efficacy could be compromised in treating illnesses such as bipolar disorder and schizophrenia, which are triggered by hyperactivities of the same set of neuromodulators. A more comprehensive investigation is recommended to elucidate the implications of fish oil on disease pathomechanisms, and the result-driven repositioning of fish oil utilization may revitalize its therapeutic efficacy.

Lysosomal membrane permeabilization is an essential step in TRAIL-induced apoptosis of liver cancer cell lines. TRAIL-induced lysosomal membrane permeabilization is mediated by the multifunctional sorting protein PACS-2 and repressed by the E3 ligases cIAP-1 and cIAP-2. Despite the opposing roles for PACS-2 and cIAPs in TRAIL-induced apoptosis, an interaction between these proteins has yet to be examined. Herein, we report that cIAP-1 and cIAP-2 confer TRAIL resistance to hepatobiliary cancer cell lines by reducing PACS-2 levels. Under basal conditions, PACS-2 underwent K48-linked poly-ubiquitination, resulting in PACS-2 proteasomal degradation. Biochemical assays showed cIAP-1 and cIAP-2 interacted with PACS-2 in vitro and co-immunoprecipitation studies demonstrated that the two cIAPs bound PACS-2 in vivo. More importantly, both cIAP-1 and cIAP-2 directly mediated PACS-2 ubiquitination in a cell-free assay. Single c-Iap-1 or c-Iap-2 gene knock-outs in mouse hepatocytes did not lead to PACS-2 accumulation. However, deletion of both cIAP-1 and cIAP-2 reduced PACS-2 ubiquitination, which increased PACS-2 levels and sensitized HuH-7 cells to TRAIL-induced lysosomal membrane permeabilization and apoptosis. Correspondingly, deletion of cIAPs sensitized wild-type, but not PACS-2-deficient hepatocarcinoma cells or Pacs-2-/- mouse hepatocytes to TRAIL-induced apoptosis. Together, these data suggest cIAPs constitutively downregulate PACS-2 by polyubiquitination and proteasomal degradation, thereby restraining TRAIL-induced killing of liver cancer cells.

The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity.

All cases of Huntington's disease (HD) are caused by mutant huntingtin protein (mhtt), yet the molecular mechanisms that link mhtt to disease symptoms are not fully elucidated. Given glycogen synthase kinase-3 (GSK3) is implicated in several neurodegenerative diseases as a molecular mediator of neuronal decline and widely touted as a therapeutic target, we investigated GSK3 in cells expressing mhtt, brains of R6/1 HD mice and post-mortem human brain samples. Consistency in data across the two models and the human brain samples indicate decreased GSK3 signalling contributes to neuronal dysfunction in HD. Inhibitory phosphorylation of GSK3 (pGSK3) was elevated in mhtt cells and this appeared related to an overall energy metabolism deficit as the mhtt cells had less ATP and inhibiting ATP production in control cells expressing non-pathogenic htt with paraquat also increased pGSK3. pGSK3 was increased and ATP levels decreased in the frontal cortex and striatum of R6/1 mice and levels of cortical pGSK3 inversely correlated with cognitive function of the mice. Consistent with decreased GSK3 activity in the R6/1 mouse brain, β-catenin levels were increased and phosphorylation of collapsin response mediator protein-2 (CRMP2) decreased in the frontal cortex where inhibitory phosphorylation of GSK3 was the greatest. pGSK3 was predominantly undetectable in HD and healthy control human brain samples, but levels of total GSK3 were decreased in the HD-affected frontal cortex and this correlated with decreased pCRMP2. Thus, disruptions to cortical GSK3 signalling, possibly due to localized energy metabolism deficits, appear to contribute to the cognitive symptoms of HD.

<h4>Background</h4>Current technology permits an unbiased massive analysis of somatic genetic alterations from tumor DNA as well as the generation of individualized mouse xenografts (Avatar models). This work aimed to evaluate our experience integrating these two strategies to personalize the treatment of patients with cancer.<h4>Methods</h4>We performed whole-exome sequencing analysis of 25 patients with advanced solid tumors to identify putatively actionable tumor-specific genomic alterations. Avatar models were used as an in vivo platform to test proposed treatment strategies.<h4>Results</h4>Successful exome sequencing analyses have been obtained for 23 patients. Tumor-specific mutations and copy-number variations were identified. All samples profiled contained relevant genomic alterations. Tumor was implanted to create an Avatar model from 14 patients and 10 succeeded. Occasionally, actionable alterations such as mutations in NF1, PI3KA, and DDR2 failed to provide any benefit when a targeted drug was tested in the Avatar and, accordingly, treatment of the patients with these drugs was not effective. To date, 13 patients have received a personalized treatment and 6 achieved durable partial remissions. Prior testing of candidate treatments in Avatar models correlated with clinical response and helped to select empirical treatments in some patients with no actionable mutations.<h4>Conclusion</h4>The use of full genomic analysis for cancer care is encouraging but presents important challenges that will need to be solved for broad clinical application. Avatar models are a promising investigational platform for therapeutic decision making. While limitations still exist, this strategy should be further tested.

Excitable tissues rely on junctional membrane complexes to couple cell surface signals to intracellular channels. The junctophilins have emerged as a family of proteins critical in coordinating the maturation and maintenance of this cellular ultrastructure. Within skeletal and cardiac muscle, junctophilin 1 and junctophilin 2, respectively, couple sarcolemmal and intracellular calcium channels. In neuronal tissue, junctophilin 3 and junctophilin 4 may have an emerging role in coupling membrane neurotransmitter receptors and intracellular calcium channels. These important physiological roles are highlighted by the pathophysiology which results when these proteins are perturbed, and a growing body of literature has associated junctophilins with the pathogenesis of human disease.

Brain wiring is a highly intricate process in which trillions of neuronal connections are established. Its initial phase is particularly crucial in establishing the general framework of neuronal circuits. During this early step, differentiating neurons extend axons, which reach their target by navigating through a complex environment with extreme precision. Research in the past 20 years has unraveled a vast and complex array of chemotropic cues that guide the leading tip of axons, the growth cone, throughout its journey. Tight regulation of these cues, and of their receptors and signaling pathways, is necessary for the high degree of accuracy required during circuit formation. However, little is known about the nature of regulatory molecules or mechanisms fine-tuning axonal cue response. Here we review recent, and somewhat fragmented, research on the possibility that microRNAs (miRNAs) could be key fine-tuning regulatory molecules in axon guidance. miRNAs appear to shape long-range axon guidance, fasciculation and targeting. We also present several lines of evidence suggesting that miRNAs could have a compartmentalized and differential action at the cell soma, and within axons and growth cones.

<h4>Aim</h4>To evaluate the ability of interleukin (IL)-15 to control T cell functions through its influence on CD30 and OX40 expressing cells in Celiac Disease (CD). In peripheral blood (PB), by examining the expression of OX40 in conventional effectors cells and T cells with a phenotypic specialization of regulatory cells [CD4+CD25high forkhead box protein 3 (Foxp3)+], and the co stimulation of IFN-γ and IL-4 production within CD30 and OX40 positive subsets of T cells. At the duodenal mucosa, by assessing the expression of CD30 and OX40 in intraepithelial (IE) and lamina propria (LP) lymphocytes (IEL, LPL).<h4>Patients and methods</h4>PB and duodenal mucosal biopsies were obtained from 38 patients with classic CD (Cel) and 38 healthy controls (HC). Analysis of cell surface and/or intracellular antigens was performed in anti-CD3-treated PB mononuclear cells (PBMC) before and after treatment with recombinant IL-15 (rIL-15), and in IE and LP cellular suspensions prepared from duodenal biopsies pre-treated with/without rIL-15.<h4>Results</h4>A subpopulation of CD3+OX40+ T blasts was induced in Cel and HC by a 3days treatment of PBMC with anti-CD3 and decreased its size thereafter, regardless of the presence of rIL-15. However, the addition of rIL-15 to T blasts distinctively induced the survival of T cells with a regulatory phenotype that expresses OX40 antigen in Cel (p<0.05). Celiac patients showed higher frequencies of IFN-γ-producing CD3+CD30+ blasts before and after treatment with rIL-15 (p<0.05, vs. HC). IL-15 increased the frequencies of CD3+CD30+ LPL (HC: p<0.05, Cel: p<0.05) but not of CD3+OX40+ LPL, and CD30 or OX40 positive IEL.<h4>Conclusions</h4>The distinctive control of OX40+ cells with a T regulatory phenotype mediated by the influence of IL-15 comes out as new function of this cytokine in the context of CD. The higher production of IFN-γ by a subpopulation of peripheral CD3+CD30+ cells contributes to the type I biased immune response.

Hemochromatosis is an inherited genetic disorder of iron metabolism which can also occur as a secondary result of iron-overload. It leads to organ damage such as cardiomyopathy, liver cirrhosis, hypogonadism, and diabetes. This paper discusses a case of secondary hemochromatosis associated with repeated transfusions, presenting as asymptomatic hypoparathyroidism and subclinical hypothyroidism with multiple organ involvement. The 29-year-old female, who had severe aplastic anemia, received multiple transfusions totaling approximately 1,400 units of red blood cells over 15 years. During her routine laboratory examination, hypocalcemia was detected with decreased intact parathyroid hormone and increased thyroid stimulating hormone. Serum ferritin, iron, and total iron binding capacity had increased to 27,583.03 ng/mL, 291 µg/dL, and 389 µg/dL, respectively. She had unusually bronze skin and computed tomography revealed iron deposition in the thyroid, liver, and heart. Multiorgan involvement as seen in this case is rare in hemochromatosis associated with secondary transfusions. To the best of the author's knowledge, this is the first case report in Korea of hypoparathyroidism and subclinical hypothyroidism due to iron deposition in the parathyroid and thyroid gland.

The current report presents a case of <i>de novo</i> acute myeloid leukemia (AML) in a 32-year-old male. Cytogenetic analysis showed that the karyotype of the bone marrow cells was as follows: 46,XY,t(11;22)(q23;q11.2)[13]/46,X,-Y,+10,t(11;22)(q23;q11.2)[7]/47,XY,+10,t(11;22)(q23;q11.2)[1]/46,XY[1]. Fluorescence in situ hybridization analysis using a mixed lineage leukemia (MLL)-specific probe showed a split in the MLL gene. Reverse transcription polymerase chain reaction (PCR) analysis demonstrated an MLL-septin 5 (SEPT5) fusion transcript in the patient. Nucleotide sequencing analysis of the PCR product confirmed the fusion between the MLL exon 9 and SEPT5 exon 3, and the product was 521 bp in length. The present study reviewed the clinical and molecular features of the AML with an MLL-SEPT5 fusion gene.

<h4>Significance</h4>Iron is the most abundant transition metal in biology and an essential cofactor for many cellular enzymes. Iron homeostasis impairment is also a component of peripheral neuropathies.<h4>Recent advances</h4>During the past years, much effort has been paid to understand the molecular mechanism involved in maintaining systemic iron homeostasis in mammals. This has been stimulated by the evidence that iron dyshomeostasis is an initial cause of several disorders, including genetic and sporadic neurodegenerative disorders.<h4>Critical issues</h4>However, very little has been done to investigate the physiological role of iron in peripheral nervous system (PNS), despite the development of suitable cellular and animal models.<h4>Future directions</h4>To stimulate research on iron metabolism and peripheral neuropathy, we provide a summary of the knowledge on iron homeostasis in the PNS, on its transport across the blood-nerve barrier, its involvement in myelination, and we identify unresolved questions. Furthermore, we comment on the role of iron in iron-related disorder with peripheral component, in demyelinating and metabolic peripheral neuropathies.

<h4>Objective</h4>Genome-wide association studies have identified many obesity/body mass index (BMI)-associated loci in Europeans and East Asians. Since then, a large number of studies have investigated the role of BMI-associated loci in the development of type 2 diabetes (T2D). However, the results have been inconsistent. The objective of this study was to investigate the associations of eleven obesity/BMI loci with T2D risk and explore how BMI influences this risk.<h4>Methods</h4>We retrieved published literature from PubMed and Embase. The pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated using fixed- or random-effect models.<h4>Results</h4>In the meta-analysis of 42 studies for 11 obesity/BMI-associated loci, we observed a statistically significant association of the FTO rs9939609 polymorphism (66 425 T2D cases/239 689 normoglycaemic subjects; P = 1·00 × 10(-41) ) and six other variants with T2D risk (17 915 T2D cases/27 531 normoglycaemic individuals: n = 40 629-130 001; all P < 0·001 for SH2B1 rs7498665, FAIM2 rs7138803, TMEM18 rs7561317, GNPDA2 rs10938397, BDNF rs925946 and NEGR1 rs2568958). After adjustment for BMI, the association remained statistically significant for four of the seven variants (all P < 0·05 for FTO rs9939609, SH2B1 rs7498665, FAIM2 rs7138803, GNPDA2 rs10938397). Subgroup analysis by ethnicity demonstrated similar results.<h4>Conclusions</h4>This meta-analysis indicates that several BMI-associated variants are significantly associated with T2D risk. Some variants increase the T2D risk independent of obesity, while others mediate this risk through obesity.

Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders.

An estimated 30-40% of patients with chronic hepatitis C have elevated serum iron, transferrin saturation, and ferritin levels. Clinical data suggest that iron is a co-morbidity factor for disease progression following HCV infection. Iron is essential for a number of fundamental metabolic processes in cells and organisms. Mammalian iron homeostasis is tightly regulated and this is maintained through the coordinated action of sensory and regulatory networks that modulate the expression of iron-related proteins at the transcriptional and/or posttranscriptional levels. Disturbances of iron homeostasis have been implicated in infectious disease pathogenesis. Viruses, similarly to other pathogens, can escape recognition by the immune system, but they need iron from their host to grow and spread. Hepcidin is a 25-aa peptide, present in human serum and urine and represents the key peptide hormone, which modulates iron homeostasis in the body. It is synthesized predominantly by hepatocytes and its mature form is released in circulation. In this review, we discuss recent advances in the exciting crosstalk of molecular mechanisms and cell signaling pathways by which iron and hepcidin production influences HCV-induced liver disease.

<h4>Background/objectives</h4>Recent large-scale genome-wide association studies have identified multiple loci robustly associated with BMI, predominantly in European ancestry (EA) populations. However, associations of these loci with obesity and related traits have not been well described in Chinese Hans. This study aimed to investigate whether BMI-associated loci are, individually and collectively, associated with adiposity-related traits and obesity in Chinese Hans and whether these associations are modified by physical activity (PA).<h4>Subjects/methods</h4>We genotyped 28 BMI-associated single nucleotide polymorphisms (SNPs) in a population-based cohort including 2,894 unrelated Han Chinese. Genetic risk score (GRS), EA and East Asian ancestry (EAA) GRSs were calculated by adding BMI-increasing alleles based on all, EA and EAA identified SNPs, respectively. Interactions of GRS and PA were examined by including the interaction-term in the regression model.<h4>Results</h4>Individually, 26 of 28 SNPs showed directionally consistent effects on BMI, and associations of four loci (TMEM18, PCSK1, BDNF and MAP2K5) reached nominal significance (P<0.05). The GRS was associated with increased BMI, trunk fat and body fat percentages; and increased risk of obesity and overweight (all P<0.05). Effect sizes (0.11 vs. 0.17 kg/m2) and explained variance (0.90% vs. 1.45%) of GRS for BMI tended to be lower in Chinese Hans than in Europeans. The EA GRS and EAA GRS were associated with 0.11 and 0.13 kg/m2 higher BMI, respectively. In addition, we found that PA attenuated the effect of the GRS on BMI (Pinteraction = 0.022).<h4>Conclusions</h4>Our observations suggest that the combined effect of obesity-susceptibility loci on BMI tended to be lower in Han Chinese than in EA. The overall, EA and EAA GRSs exert similar effects on adiposity traits. Genetic predisposition to increased BMI is attenuated by PA in this population of Han Chinese.

<h4>Objectives</h4>To investigate the effects of the urinary metabolite profiles of background exposure to the atmospheric pollutants polycyclic aromatic hydrocarbon (PAH) and Framingham risk score (FRS), which assesses an individual's cardiovascular disease risk, on heart rate variability (HRV).<h4>Methods</h4>The study conducted from April to May 2011 in Wuhan, China, included 1978 adult residents with completed questionnaires, physical examinations, blood and urine samples, and 5-min HRV indices (including SD of all normal to normal intervals (SDNN), root mean square successive difference (rMSSD), low frequency (LF), high frequency (HF) and their ratio (LF/HF), and total power) obtained from 3-channel Holter monitor. 12 urinary PAH metabolites were measured by gas chromatography-mass spectrometry. FRS was calculated by age, sex, lipid profiles, blood pressure, diabetes and smoking status. Linear regression models were constructed after adjusting for potential confounders.<h4>Results</h4>Elevated total concentration of hydroxynaphthalene (ΣOHNa) was significantly associated, in a dose-responsive manner, with decreased SDNN and LF/HF (ptrend=0.014 and 0.007, respectively); elevated total concentration of hydroxyfluorene (ΣOHFlu) was significantly associated with reduced SDNN, LF and LF/HF (ptrend=0.027, 0.003, and <0.0001, respectively); and elevated total concentration of all PAH metabolites (ΣOH-PAHs) was associated with decreased LF and LF/HF (ptrend=0.005 and <0.0001, respectively). Moreover, increasing quartiles of FRS were significantly associated with decreased HRV indices, except LF/HF (all ptrend<0.0001). Interestingly, individuals in low-risk subgroups had greater decreases in SDNN, LF and LF/HF in relation to ΣOH-PAHs, ΣOHNa and ΣOHFlu than those in high-risk subgroups (all p<0.05).<h4>Conclusions</h4>Environmental PAH exposure may differentially affect HRV based on individual coronary risk profiles.

The miR-34 family was originally found to be a direct target of p53 and is a group of putative tumor suppressors. Surprisingly, mice lacking all mir-34 genes show no increase in cancer formation by 18 months of age, hence placing the physiological relevance of previous studies in doubt. Here, we report that mice with prostate epithelium-specific inactivation of mir-34 and p53 show expansion of the prostate stem cell compartment and develop early invasive adenocarcinomas and high-grade prostatic intraepithelial neoplasia, whereas no such lesions are observed after inactivation of either the mir-34 or p53 genes alone by 15 months of age. Consistently, combined deficiency of p53 and miR-34 leads to acceleration of MET-dependent growth, self-renewal, and motility of prostate stem/progenitor cells. Our study provides direct genetic evidence that mir-34 genes are bona fide tumor suppressors and identifies joint control of MET expression by p53 and miR-34 as a key component of prostate stem cell compartment regulation, aberrations in which may lead to cancer.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis.

Autophagy is the cellular process by which proteins, macromolecules, and organelles are targeted to and degraded by the lysosome. Given that neurodegenerative diseases involve the production of misfolded proteins that cannot be degraded by the protein quality-control systems of the cell, the autophagy pathway is now the focus of intense scrutiny, because autophagy is primarily responsible for maintaining normal cellular proteostasis in the central nervous system (CNS). Huntington's disease (HD) is an inherited CAG-polyglutamine repeat disorder, resulting from the production and accumulation of misfolded huntingtin (Htt) protein. HD shares key features with common neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD) and, thus, belongs to a large class of disorders known as neurodegenerative proteinopathies. Multiple independent lines of research have documented alterations in autophagy function in HD, and numerous studies have demonstrated a potential role for autophagy modulation as a therapeutic intervention. In this review, we consider the evidence for autophagy dysfunction in HD, and delineate different targets and mechanistic pathways that might account for the autophagy abnormalities detected in HD. We assess the utility of autophagy modulation as a treatment modality in HD, and suggest guidelines and caveats for future therapy development directed at the autophagy pathway in HD and related disorders.

With accumulating research on the interconnections among different types of genomic regulations, researchers have found that multidimensional genomic studies outperform one-dimensional studies in multiple aspects. Among many sources of multidimensional genomic data, The Cancer Genome Atlas (TCGA) provides the public with comprehensive profiling data on >30 cancer types, making it an ideal test bed for conducting and comparing different analyses. In this article, the analysis goal is to apply several existing methods and associate multidimensional genomic measurements with cancer outcomes in particular prognosis, with special focus on the predictive power of genomic signatures. We exploit clinical data and four types of genomic measurement including mRNA gene expression, DNA methylation, microRNA and copy number alterations for breast invasive carcinoma, glioblastoma multiforme, acute myeloid leukemia and lung squamous cell carcinoma collected by TCGA. To accommodate the high dimensionality, we extract important features using Principal Component Analysis, Partial Least Squares and Least Absolute Shrinkage and Selection Operator (Lasso), which are representative of dimension reduction and variable selection techniques and have been extensively adopted, and fit Cox survival models with combined important features. We calibrate the predictive power of each type of genomic measurement for the prognosis of four cancer types and find that the results vary across cancers. Our analysis also suggests that for most of the cancers in our study and the adopted methods, there is no substantial improvement in prediction when adding other genomic measurement after gene expression and clinical covariates have been included in the model. This is consistent with the findings that molecular features measured at the transcription level affect clinical outcomes more directly than those measured at the DNA/epigenetic level.

Heme constitutes 95% of functional iron in the human body, as well as two-thirds of the average person's iron intake in developed countries. Hence, a wide range of epidemiological studies have focused on examining the association of dietary heme intake, mainly from red meat, with the risks of common diseases. High heme intake is associated with increased risk of several cancers, including colorectal cancer, pancreatic cancer and lung cancer. Likewise, the evidence for increased risks of type-2 diabetes and coronary heart disease associated with high heme intake is compelling. Furthermore, recent comparative metabolic and molecular studies of lung cancer cells showed that cancer cells require increased intracellular heme biosynthesis and uptake to meet the increased demand for oxygen-utilizing hemoproteins. Increased levels of hemoproteins in turn lead to intensified oxygen consumption and cellular energy generation, thereby fueling cancer cell progression. Together, both epidemiological and molecular studies support the idea that heme positively impacts cancer progression. However, it is also worth noting that heme deficiency can cause serious diseases in humans, such as anemia, porphyrias, and Alzheimer's disease. This review attempts to summarize the latest literature in understanding the role of dietary heme intake and heme function in diverse diseases.

Some N-[(N-benzoyldehydrophenylalalnyl)glycinyl/cysteinyl]-N,N'-dicyclohexylurea analogs (3a-3o) were synthesized by conjugating different substituted N-benzoyldehydrophenylalanyl glycines/cysteines (differing substitutions on benzylidene ring) and dicyclohexyl carbodiimide (DCC) using base as a catalyst. The synthesized compounds were characterized and evaluated for biological activities. Compounds 3a and 3h with unsubstituted dehydrophenylalanyl glycinyl/cysteinyl moiety exhibited moderate anti-inflammatory and analgesic activities. Compound 3j bearing 4-hydroxy substitution on benzylidene ring of dehydrophenylalanyl cysteinyl moiety displayed potent antimicrobial and antioxidant activities. The results obtained from docking studies on compound 3j with penicillin binding protein and protease supported the results.

We evaluated clinicopathological factors affecting survival and recurrence after initial hepatectomy in non-B non-C (NBNC) hepatocellular carcinoma (HCC) patients with comparison to hepatitis B or C virus, paying attention to relationship between alcohol consumption and histopathological findings. The medical records on the 201HCC patients who underwent initial hepatectomy between January 2000 and April 2013 were retrospectively reviewed. NBNC patients had higher prevalence of hypertension (47.4%), diabetes mellitus (35.5%), alcohol consumption (>20 g/day) (61.8%), and preserved liver function than hepatitis B or C patients. The 5-year survival rate of NBNC patients (74.1%) was significantly better than hepatitis B (49.1%) or C (65.0%) patients (NBNC versus B, P = 0.031). Among the NBNC patients, there was no relationship between alcohol consumption and clinicopathological findings including nonalcoholic fatty liver disease activity score (NAS). However, the 5-year OS and RFS rates in the alcohol-unrelated NBNC patients tend to be better than in the alcohol-related. By multivariate analysis, independent factors for OS in NBNC patients were Child-Pugh B/C, intrahepatic metastasis (im), and extrahepatic recurrence. NBNC patients, who were highly associated with lifestyle-related disease and preserved liver function, had significantly better prognosis compared to hepatitis B/C patients; however, there was no association between alcohol consumption and histopathological findings.

Introduction. Prenatal programming secondary to maternal protein restriction renders an inherent susceptibility to neural compromise in neonates and any addition of glucocorticosteroids results in further damage. This is an investigation of consequent global gene activity due to effects of antenatal steroid therapy on a protein restriction mouse model. Methods. C57BL/6N pregnant mice were administered control or protein restricted diets and subjected to either 100  μ g/Kg of dexamethasone sodium phosphate with normosaline or normosaline alone during late gestation (E10-E17). Nontreatment groups were also included. Brain samples were collected on embryonic day 17 and analyzed by mRNA microarray analysis. Results. Microarray analyses presented 332 significantly regulated genes. Overall, neurodevelopmental genes were overrepresented and a subset of 8 genes allowed treatment segregation through the hierarchical clustering method. The addition of stress or steroids greatly affected gene regulation through glucocorticoid receptor and stress signaling pathways. Furthermore, differences between dexamethasone-administered treatments implied a harmful effect during conditions of high stress. Microarray analysis was validated using qPCR. Conclusion. The effects of antenatal steroid therapy vary in fetuses according to maternal-fetal factors and environmental stimuli. Defining the key regulatory networks that signal either beneficial or damaging corticosteroid action would result in valuable adjustments to current treatment protocols.

Colorectal cancer (CRC), which has high prevalence in Saudi Arabia and worldwide, needs better understanding by exploiting the latest available cytogenetic microarrays. We used biopsy tissue from consenting colorectal cancer patients to extract DNA and carry out microarray analysis using a CytoScan HD platform from Affymetrix. Patient specific comparisons of tumor-normal pairs were carried out. To find out the high probability key players, we performed Genomic Identification of Significant Targets in Cancer analysis and found 144 genes to form the list of driver genes. Of these, 24 genes attained high GISTIC scores and suggest being significantly associated with CRC. Loss of heterozygosity and uniparental disomy were found to affect 9 genes and suggest different mechanisms associated with CRC in every patient. Here we present the details of the methods used in carrying out the above analyses. Also, we provide some additional data on biomarker analysis that would complement the findings.

Kinetics studies of dNTP analogues having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase β that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T•A and G•C) or wrong (T•G and G•T). Brønsted plots of the catalytic rate constant (log(kpol)) versus pKa4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from -0.6 to -2.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme-primer-template complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step.

There is intense interest in the role of vitamin D in the development of asthma and allergies. However, studies differ on whether a higher vitamin D intake or status in pregnancy or at birth is protective against asthma and allergies. To address this uncertainty, the Vitamin D Antenatal Asthma Reduction Trial (VDAART) was developed. VDAART is a randomized, double-blind, placebo-controlled trial of vitamin D supplementation in pregnant women to determine whether prenatal supplementation can prevent the development of asthma and allergies in women's offspring. A secondary aim is to determine whether vitamin D supplementation can prevent the development of pregnancy complications, such as preeclampsia, preterm birth, and gestational diabetes. Women were randomized to the treatment arm of 4000IU/day of vitamin D3 plus a daily multivitamin that contained 400IU of vitamin D3 or the placebo arm of placebo plus a multivitamin that contained 400IU daily of vitamin D3. Women who were between the gestational ages of 10 and 18 weeks were randomized from three clinical centers across the United States - Boston Medical Center, Washington University in St. Louis, and Kaiser Permanente Southern California Region (San Diego, CA). Supplementation took place throughout pregnancy. Monthly monitoring of urinary calcium to creatinine ratio was performed in addition to medical record review for adverse events. Offspring are being evaluated quarterly through questionnaires and yearly during in-person visits until the 3rd birthday of the child. Ancillary studies will investigate neonatal T-regulatory cell function, maternal vaginal flora, and maternal and child intestinal flora.

The mammalian neocortex is a sheet of cells covering the cerebrum that provides the structural basis for the perception of sensory inputs, motor output responses, cognitive function, and mental capacity of primates. Recent discoveries promote the concept that increased cortical surface size and thickness in phylogenetically advanced species is a result of an increased generation of neurons, a process that underlies higher cognitive and intellectual performance in higher primates and humans. Here, we review some of the advances in the field, focusing on the diversity of neocortical progenitors in different species and the cellular mechanisms of neurogenesis. We discuss recent views on intrinsic and extrinsic molecular determinants, including the role of epigenetic chromatin modifiers and microRNA, in the control of neuronal output in developing cortex and in the establishment of normal cortical architecture.

<h4>Background</h4>Snail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE.<h4>Methods</h4>ARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments.<h4>Results</h4>Snail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells.<h4>Conclusions</h4>This study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer.

Delivered dialysis dose by continuous renal replacement therapies (CRRT) depends on circuit efficacy, which is influenced in part by the anticoagulation strategy. We evaluated the association of anticoagulation strategy used on solute clearance efficacy, circuit longevity, bleeding complications, and mortality. We analyzed data from 1740 sessions 24 h in length among 244 critically ill patients, with at least 48 h on CRRT. Regional citrate, heparin, or saline flushes was variably used to prevent or attenuate filter clotting. We calculated delivered dose using the standardized Kt/Vurea . We monitored filter efficacy by calculating effluent urea nitrogen/blood urea nitrogen ratios. Filter longevity was significantly higher with citrate (median 48, interquartile range [IQR] 20.3-75.0 hours) than with heparin (5.9, IQR 8.5-27.0 hours) or no anticoagulation (17.5, IQR 9.5-32 hours, P < 0.0001). Delivered dose was highest in treatments where citrate was employed. Bleeding complications were similar across the three groups (P = 0.25). Compared with no anticoagulation, odds of death was higher with the heparin use (odds ratio [OR] 1.82, 95% confidence interval [CI] 1.02-3.32; P = 0.033), but not with citrate (OR 1.02 95% CI 0.54-1.96; P = 0.53). Relative to heparin or no anticoagulation, the use of regional citrate for anticoagulation in CRRT was associated with significantly prolonged filter life and increased filter efficacy with respect to delivered dialysis dose. Rates of bleeding complications, transfusions, and mortality were similar across the three groups. While these and other data suggest that citrate anticoagulation may offer superior technical performance than heparin or no anticoagulation, adequately powered clinical trials comparing alternative anticoagulation strategies should be performed to evaluate overall safety and efficacy.

The prevalence of osteoporosis and the incidence of age-related fragility fracture vary by ethnicity. There is greater than 10-fold variation in fracture probabilities between countries across the world. Mineral and bone metabolism are intimately interlinked, and both are known to exhibit patterns of daily variation, known as the diurnal rhythm (DR). Ethnic differences are described for Ca and P metabolism. The importance of these differences is described in detail between select ethnic groups, within the USA between African-Americans and White-Americans, between the Gambia and the UK and between China and the UK. Dietary Ca intake is higher in White-Americans compared with African-Americans, and is higher in White-British compared with Gambian and Chinese adults. Differences are observed also for plasma 25-hydroxy vitamin D, related to lifestyle differences, skin pigmentation and skin exposure to UVB-containing sunshine. Higher plasma 1,25-dihydroxy vitamin D and parathyroid hormone are observed in African-American compared with White-American adults. Plasma parathyroid hormone is also higher in Gambian adults and, in winter, in Chinese compared with White-British adults. There may be ethnic differences in the bone resorptive effects of parathyroid hormone, with a relative skeletal resistance to parathyroid hormone observed in some, but not all ethnic groups. Renal mineral excretion is also influenced by ethnicity; urinary Ca (uCa) and urinary P (uP) excretions are lower in African-Americans compared with White-Americans, and in Gambians compared with their White-British counterparts. Little is known about ethnic differences in the DR of Ca and P metabolism, but differences may be expected due to known differences in lifestyle factors, such as dietary intake and sleep/wake pattern. The ethnic-specific DR of Ca and P metabolism may influence the net balance of Ca and P conservation and bone remodelling. These ethnic differences in Ca, P and the bone metabolism may be important factors in the variation in skeletal health.

During acute viral infections such as influenza, humans often experience not only transient fever, but also prolonged fatigue or depressive feelings with a decrease in social activity for days or weeks. These feelings are thought to be due to neuroinflammation in the brain. Recent studies have suggested that chronic neuroinflammation is a precipitating event of various neurological disorders, but the mechanism determining the duration of neuroinflammation has not been elucidated. In this study, neuroinflammation was induced by intraperitoneal injection of polyriboinosinic:polyribocytidylic acid (poly I:C), a Toll-like receptor-3 agonist that mimics viral infection in male Sprague-Dawley rats, and then investigated how the neuroinflammation shift from acute to the chronic state. The rats showed transient fever and prolonged suppression of spontaneous activity for several days following poly I:C injection. NS-398, a cyclooxygenase-2 inhibitor, completely prevented fever, but did not improve spontaneous activity, indicating that suppression of spontaneous activity was not induced by the arachidonate cascade that generated the fever. The animals overexpressed interleukin (IL)-1β and IL-1 receptor antagonist (IL-1ra) in the brain including the cerebral cortex. Blocking the IL-1 receptor in the brain by intracerebroventricular (i.c.v.) infusion of recombinant IL-1ra completely blocked the poly I:C-induced suppression of spontaneous activity and attenuated amplification of brain interferon (IFN)-α expression, which has been reported to produce fatigue-like behavior by suppressing the serotonergic system. Furthermore, i.c.v. infusion of neutralizing antibody for IL-1ra prolonged recovery from suppression of spontaneous activity. Our findings indicated that IL-1β is the key trigger of neuroinflammation and that IL-1ra prevents the neuroinflammation entering the chronic state.

Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3'UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community.

Recently, Pérez-Montero and colleagues (Developmental cell, 26: 578-590, 2013) described the occurrence of a new histone H1 variant (dBigH1) in Drosophila. The presence of unusual acidic amino acid patches at the N-terminal end of dBigH1 is in contrast to the arginine patches that exist at the N- and C-terminal domains of other histone H1-related proteins found in the sperm of some organisms. This departure from the strictly lysine-rich composition of the somatic histone H1 raises a question about the true definition of its protein members. Their minimal essential requirements appear to be the presence of a lysine- and alanine-rich, intrinsically disordered C-terminal domain, with a highly helicogenic potential upon binding to the linker DNA regions of chromatin. In metazoans, specific targeting of these regions is further achieved by a linker histone fold domain (LHFD), distinctively different from the characteristic core histone fold domain (CHFD) of the nucleosome core histones.

Protein-Protein Interaction (PPI) extraction is an important task in the biomedical information extraction. Presently, many machine learning methods for PPI extraction have achieved promising results. However, the performance is still not satisfactory. One reason is that the semantic resources were basically ignored. In this paper, we propose a multiple-kernel learning-based approach to extract PPIs, combining the feature-based kernel, tree kernel and semantic kernel. Particularly, we extend the shortest path-enclosed tree kernel (SPT) by a dynamic extended strategy to retrieve the richer syntactic information. Our semantic kernel calculates the protein-protein pair similarity and the context similarity based on two semantic resources: WordNet and Medical Subject Heading (MeSH). We evaluate our method with Support Vector Machine (SVM) and achieve an F-score of 69.40% and an AUC of 92.00%, which show that our method outperforms most of the state-of-the-art systems by integrating semantic information.

Cell-based therapies are a viable option for the long-term treatment of Huntington's disease (HD), which is characterized by progressive neurodegeneration predominately in the striatum and cortex. Current research focuses on genetic suppression of the mutant huntingtin (mHTT) gene and cell replacement therapy of the lost cells in HD. As we discuss here, the recent development of induced pluripotent stem (iPS) cells technology demonstrated the potential of cell-based therapy in rodent models. It was shown that iPSCs were capable of differentiating into lost neurons in HD and stem cell grafts can improve motor deficiency in HD rodent models. Altogether, these findings have shown great promise for developing the foundation of the cell-based therapy.

The Enhanced Permeability and Retention (EPR) effect is extensively used in drug delivery research. Taking into account that EPR is a highly variable phenomenon, we have here set out to evaluate if contrast-enhanced functional ultrasound (ceUS) imaging can be employed to characterize EPR-mediated passive drug targeting to tumors. Using standard fluorescence molecular tomography (FMT) and two different protocols for hybrid computed tomography-fluorescence molecular tomography (CT-FMT), the tumor accumulation of a ~10 nm-sized near-infrared-fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) was evaluated in CT26 tumor-bearing mice. In the same set of animals, two different ceUS techniques (2D MIOT and 3D B-mode imaging) were employed to assess tumor vascularization. Subsequently, the degree of tumor vascularization was correlated with the degree of EPR-mediated drug targeting. Depending on the optical imaging protocol used, the tumor accumulation of the polymeric drug carrier ranged from 5 to 12% of the injected dose. The degree of tumor vascularization, determined using ceUS, varied from 4 to 11%. For both hybrid CT-FMT protocols, a good correlation between the degree of tumor vascularization and the degree of tumor accumulation was observed, within the case of reconstructed CT-FMT, correlation coefficients of ~0.8 and p-values of <0.02. These findings indicate that ceUS can be used to characterize and predict EPR, and potentially also to pre-select patients likely to respond to passively tumor-targeted nanomedicine treatments.

The ubiquitination of proteins is a post-translational modification that was first described as a means to target misfolded or unwanted proteins for degradation by the proteasome. It is now appreciated that the ubiquitination of proteins also serves as a mechanism to modify protein function and cellular functions such as protein trafficking, cell signaling, DNA repair, chromatin modifications, cell-cycle progression and cell death. The ubiquitination of proteins occurs through the hierarchal transfer of ubiquitin from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme and finally to an E3 ubiquitin ligase that transfers the ubiquitin to its target protein. It is the final E3 ubiquitin ligase that confers the substrate specificity for ubiquitination and is the focus of this review. Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells undergo mitotic proliferation and expansion of the diploid spermatogonial population, differentiate into spermatocytes and progress through two meiotic divisions to produce haploid spermatids that proceed through a final morphogenesis to generate mature spermatozoa. The ubiquitination of proteins in the cells of the testis occurs in many of the processes required for the progression of mature spermatozoa. Since it is the E3 ubiquitin ligase that recognizes the target protein and provides the specificity and selectivity for ubiquitination, this review highlights known examples of E3 ligases in the testis and the differing roles that they play in maintaining functional spermatogenesis.

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm with an extremely poor prognosis. Maintaining ATLL cells in vitro is difficult and little is known about how they maintain themselves or grow in patients. Elucidating the interaction between ATLL cells and surrounding host factors might therefore provide important insights into pathophysiology. We cultured primary ATLL cells in various culture conditions using IL-2, IL-4 and feeder cells, and established two cell lines dependent on IL-2, IL-4 and a follicular dendritic cell-derived cell line, HK, in which OX40-ligand was induced. Our study indicates the importance of microenvironment in the homeostasis of ATLL.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an autosomal recessive inborn error of metabolism that leads to the impaired mitochondrial fatty acid β-oxidation of short chain fatty acids. It is heterogeneous in clinical presentation including asymptomatic in most patients identified by newborn screening. Multiple mutations have been identified in patients; however, neither clear genotype-phenotype relationships nor a good correlation between genotype and current biochemical markers for diagnosis has been identified. The definition and pathophysiology of this deficiency remain unclear. To better understand this disorder at a global level, quantitative alterations in the mitochondrial proteome in SCAD deficient mice were examined using a combined proteomics approach: two-dimensional gel difference electrophoresis (2DIGE) followed by protein identification with MALDI-TOF/TOF and iTRAQ labeling followed by nano-LC/MALDI-TOF/TOF. We found broad mitochondrial dysfunction in SCAD deficiency. Changes in the levels of multiple energy metabolism related proteins were identified indicating that a more complex mechanism for development of symptoms may exist. Affected pathways converge on disorders with neurologic symptoms, suggesting that even asymptomatic individuals with SCAD deficiency may be at risk to develop more severe disease. Our results also identified a pattern associated with hepatotoxicity implicated in mitochondrial dysfunction, fatty acid metabolism, decrease of depolarization of mitochondria and mitochondrial membranes, and swelling of mitochondria, demonstrating that SCAD deficiency relates more directly to mitochondrial dysfunction and alteration of fatty acid metabolism. We propose several candidate molecules that may serve as markers for recognition of clinical risk associated with this disorder.

Lung cancer is the leading cause of cancer-related mortality in the United States. Epigenetic alterations, including DNA methylation, histone modifications, and noncoding RNA expression, have been reported widely in the literature to play a major role in the genesis of lung cancer. The goal of this review is to summarize the common epigenetic changes associated with lung cancer to give some clarity to its etiology, and to provide an overview of the potential translational applications of these changes, including applications for early detection, diagnosis, prognostication, and therapeutics.

Sulfated derivatives of Artemisia sphaerocephala polysaccharide (ASP) with high degree of substitution (DS) were synthesized using 4-dimethylaminopyridine (DMAP)/dimethylcyclohexylcarbodiimide (DCC) as catalyst. Size exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS) results showed a decrease in fractal dimension (df) values of sulfated ASP (SASP). Compared to ASP and SASP with low DS (0.51-1.01), SASPcata2 exhibited an internal structure between rigid rod and random coil with a DS of 1.24. DS had greater influence on its conformation in aqueous solution. Circular dichroism (CD), methylene blue (MB) and Congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results confirmed that the degradation of ASP and SO3H groups improved significantly the stiffness of the chains due to the electrostatic effect. Furthermore, antioxidant experiments revealed that high DS could enhance the scavenging activities of radicals and reducing power of SASP in vitro. The extended chain conformation was beneficial to enhance the biological activity of sulfated polysaccharides.

Humans have developed the perception, production and processing of sounds into the art of music. A genetic contribution to these skills of musical aptitude has long been suggested. We performed a genome-wide scan in 76 pedigrees (767 individuals) characterized for the ability to discriminate pitch (SP), duration (ST) and sound patterns (KMT), which are primary capacities for music perception. Using the Bayesian linkage and association approach implemented in program package KELVIN, especially designed for complex pedigrees, several single nucleotide polymorphisms (SNPs) near genes affecting the functions of the auditory pathway and neurocognitive processes were identified. The strongest association was found at 3q21.3 (rs9854612) with combined SP, ST and KMT test scores (COMB). This region is located a few dozen kilobases upstream of the GATA binding protein 2 (GATA2) gene. GATA2 regulates the development of cochlear hair cells and the inferior colliculus (IC), which are important in tonotopic mapping. The highest probability of linkage was obtained for phenotype SP at 4p14, located next to the region harboring the protocadherin 7 gene, PCDH7. Two SNPs rs13146789 and rs13109270 of PCDH7 showed strong association. PCDH7 has been suggested to play a role in cochlear and amygdaloid complexes. Functional class analysis showed that inner ear and schizophrenia-related genes were enriched inside the linked regions. This study is the first to show the importance of auditory pathway genes in musical aptitude.

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.

The effect of blockade of either 5-hydroxytryptamine (5-HT)/serotonin transporter (SERT) with citalopram or the organic cation transporter 3 (OCT3)/plasma membrane monoamine transporter (PMAT) with decynium-22 (D-22) on spontaneous and evoked release of 5-HT in the nucleus tractus solitarius (NTS) was investigated in rat brainstem slices treated with gabazine. 5-HT release was measured indirectly by changes in the frequency and amplitude of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) [in the presence of tetrodotoxin (TTX)] and evoked EPSCs. Blockade of 5-HT3 receptors with granisetron reduced, whereas the 5-HT3 agonist phenylbiguanide increased, the frequency of mEPSCs. 5-HT decreased mEPSC frequency at low concentrations and increased frequency at high concentrations. This inhibition was blocked by the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635), which was ineffective on its own, whereas the excitation was reversed by granisetron. The addition of citalopram or D-22 caused inhibition, which was prevented by 5-HT1A blockade. Thus, in the NTS, the spontaneous release of 5-HT is able to activate 5-HT3 receptors, but not 5-HT1A receptors, as the release in their vicinity is removed by uptake. The ineffectiveness of corticosterone suggests that the low-affinity, high-capacity transporter is PMAT, not OCT3. For evoked 5-HT release, only D-22 caused an increase in the amplitude of EPSCs, with a decrease in the paired pulse ratio, and increased the number of spontaneous EPSCs after 20-Hz stimulation. Thus, for the evoked release of 5-HT, the low-affinity, high-capacity transporter PMAT, but not 5-HT transporter (5-HTT)/SERT, is important in the regulation of changes in 5-HT extracellular concentration.

Thiacremonone (2, 4-dihydroxy-2, 5-dimethyl-thiophene-3-one) is an antioxidant substance as a novel sulfur compound generated from High-Temperature-High-Pressure-treated garlic. Peroxiredoxin 6 (PRDX6) is a member of peroxidases, and has glutathione peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Several studies have demonstrated that PRDX6 stimulates lung cancer cell growth via an increase of glutathione peroxidase activity. A docking model study and pull down assay showed that thiacremonone completely fits on the active site (cys-47) of glutathione peroxidase of PRDX6 and interacts with PRDX6. Thus, we investigated whether thiacremonone inhibits cell growth by blocking glutathione peroxidase of PRDX6 in the human lung cancer cells, A549 and NCI-H460. Thiacremonone (0-50 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decrease of xIAP, cIAP and Bcl2 expression. Thiacremonone further inhibited glutathione peroxidase activity in lung cancer cells. However, the cell growth inhibitory effect of thiacremonone was not observed in the lung cancer cells transfected with mutant PRDX6 (C47S) and in the presence of dithiothreitol and glutathione. In an allograft in vivo model, thiacremonone (30 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression and glutathione peroxidase activity, but increased expression of cleaved caspase-3, -8, -9, Bax, p21 and p53. These data indicate that thiacremonone inhibits tumor growth via inhibition of glutathione peroxidase activity of PRDX6 through interaction. These data suggest that thiacremonone may have potentially beneficial effects in lung cancer.

<h4>Background</h4>Increasing evidence indicated plasma D-dimer could be regarded as a marker in cancers, however, its role in gastric cancer is still largely unknown.<h4>Methods</h4>Plasma D-dimer levels were measured by enzyme linked fluorescent immunoassays and evaluated by receiver operating characteristic (ROC) curves for peritoneal dissemination in gastric cancer and healthy subjects. The overall survival (OS) characteristics were determined using Kaplan-Meier and Cox regression analyses.<h4>Results</h4>The average of the plasma D-dimer levels for gastric cancer patients was significantly higher than the healthy subjects. A Spearman correlation analysis showed that plasma D-dimer levels correlated with the depth of invasion, lymph node metastasis, peritoneal dissemination, distant metastasis, tumor size and TNM stage. The mean plasma D-dimer level was 2.20 ± 1.51 µg/mL in peritoneal dissemination patients and 1.01 ± 0.79 µg/mL in non-peritoneal dissemination patients (P<0.001). Additionally, the mean plasma D-dimer concentration in patients alive at the final follow-up evaluation was 0.79 ± 0.72 µg/mL,which was significantly lower than the amounts determined for the deceased patients (1.36 ± 1.13 µg/mL) (P<0.001). The AUC of D-dimer was 0.833 (95%CI: 0.780-0.885). At a cut-off value of 1.465 µg/mL, the D-dimer measurement had a sensitivity of 78.00%, a specificity of 83.76% and an accuracy of 82.59%. The median OS was 48.10 months (95% CI: 43.88-52.31) in patients with plasma D-dimer levels less than 1.465 µg/mL and 22.39 months (95% CI: 16.95-27.82) in patients with plasma D-dimer levels exceeding 1.465 µg/mL (log-rank test, P<0.001). Importantly, plasma D-dimer levels exceeding 1.465 µg/mL were significantly associated with poor OS, as determined using a multivariate Cox regression analysis (hazard ratio [HR], 2.28; 95%CI: 1.36-3.81; P = 0.002).<h4>Conclusions</h4>Plasma D-dimer levels are increased in gastric cancer patients and may be a valuable biomarker for peritoneal dissemination, with high D-dimer levels predicting poor outcomes for gastric cancer patients.

Compelling evidence suggests that the early interaction between porcine circovirus 2 (PCV-2) and the innate immune system is the key event in the pathogenesis of Post-Weaning Multisystemic Wasting Syndrome (PMWS). Furthermore, PCV2 has been detected in bone-marrow samples, potentially enabling an easy spread and reservoir for the virus. To assess the gene-expression differences induced by an in-vitro PCV2b infection in different three different myeloid innate immune cell subsets generated from the same animal, we used the Agilent Porcine Gene Expression Microarray (V2). Alveolar macrophages (AMØs), monocyte-derived dendritic cells (MoDCs) and bone-marrow cells (BMCs) were generated from each animal, and challenged with a UK-isolate of a PCV2 genotype b-strain at a MOI of 0.5. Remarkably, analysis showed a highly distinct and cell-type dependent response to PCV2b challenge. Overall, MoDCs showed the most marked response to PCV2b challenge in vitro and revealed a key role for TNF in the interaction with PCV2b, whereas only few genes were affected in BMCs and AMØs. These observations were further supported by an enrichment of genes in the downstream NF-κB Signalling pathway as well as an up regulation of genes with pro-apoptotic functions post-challenge. PCV2b challenge increases the expression of a large number of immune-related and pro-apoptotic genes mainly in MoDC, which possibly explain the increased inflammation, granulomatous inflammation and lymphocyte depletion seen in PMWS-affected pigs.

Bipolar disorder (BD) is a common and highly heritable mental illness and genome-wide association studies (GWAS) have robustly identified the first common genetic variants involved in disease aetiology. The data also provide strong evidence for the presence of multiple additional risk loci, each contributing a relatively small effect to BD susceptibility. Large samples are necessary to detect these risk loci. Here we present results from the largest BD GWAS to date by investigating 2.3 million single-nucleotide polymorphisms (SNPs) in a sample of 24,025 patients and controls. We detect 56 genome-wide significant SNPs in five chromosomal regions including previously reported risk loci ANK3, ODZ4 and TRANK1, as well as the risk locus ADCY2 (5p15.31) and a region between MIR2113 and POU3F2 (6q16.1). ADCY2 is a key enzyme in cAMP signalling and our finding provides new insights into the biological mechanisms involved in the development of BD.

The Chinese crocodile lizard Shinisaurus crocodilurus is a critically endangered species, listed in Appendix II of CITES. Its populations and habitat in China have undergone significant changes in recent years. Understanding the genetic variability and phylogeography of this species is very important for successful conservation. In this study, samples were taken from 11 wild ponds and two captive populations in China. We sequenced mitochondrial CYTB, partial ND6, and partial tRNA-Glu and genotyped 10 microsatellite loci. Our analyses of these data showed low genetic variability, no strong isolation caused by distance, and a lack of a phylogeographic structure in this species. Based on our results, the basal divergence between two clades of S. crocodilurus in China may have been caused by the formation of the Pearl River system. We found a population expansion in one of these clades. Microsatellite analysis indicated the presence of three clusters, separated by significant genetic differences. We found that most individuals in the two captive populations were from the Luokeng (Guangdong) and Guangxi wild source populations, respectively.

Striatal dopamine D2 receptor (D2R) relies upon G protein- and β-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gαi-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of β-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.

The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggregation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute significantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both all-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.

Non-alcohol-related fatty liver disease (NAFLD) encompasses a wide spectrum, ranging from steatosis alone to steatohepatitis and fibrosis. Presence of steatohepatitis and fibrosis are key hallmarks of disease progression. Previous studies have demonstrated an association between hepatocyte p21 expression and fibrosis stage in NAFLD. The aim of this study is to investigate the association between the variants of CDKN1A, which encodes p21, and disease progression in NAFLD. To this end, the relation between CDKN1A polymorphism and liver fibrosis was studied in 2 cohorts of biopsy-proven NAFLD patients from UK (n = 323) and Finland (n = 123). Genotyping was performed using DNA isolated from lymphocytes collected at the time of liver biopsy. The findings of the UK cohort were tested in the Finnish cohort. Both the UK and Finnish cohorts were significantly different from each other in basic demographics. In the UK cohort, rs762623, of the 6 SNPs across CDKN1A tested, was significantly associated with disease progression in NAFLD. This association was confirmed in the Finnish cohort. Despite the influence on fibrosis development, SNPs across CDKN1A did not affect the progression of liver fibrosis. In conclusion, CDKN1A variant rs762623 is associated with the development but not the propagation of progressive liver disease in NAFLD.

Evidence from the various sources indicates alterations in 5-HT2C receptor functions in anxiety, depression and suicide, and other stress-related disorders treated with antidepressant drugs. Although the notion of a 5-HT2C receptor desensitization following antidepressant treatments is rather well anchored in the literature, this concept is mainly based on in vitro assays and/or behavioral assays (hypolocomotion, hyperthermia) that have poor relevance to anxio-depressive disorders. Our objective herein is to provide a comprehensive overview of the studies that have assessed the effects of antidepressant drugs on 5-HT2C receptors. Relevant molecular (second messengers, editing), neurochemical (receptor binding and mRNA levels), physiological (5-HT2C receptor-induced hyperthermia and hormone release), behavioral (5-HT2C receptor-induced changes in feeding, anxiety, defense and motor activity) data are summarized and discussed. Setting the record straight about drug-induced changes in 5-HT2C receptor function in specific brain regions should help to determine which pharmacotherapeutic strategy is best for affective and anxiety disorders.

Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte-endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease.

Iron-overload disorders owing to genetic misregulation of iron acquisition are referred to as hereditary hemochromatosis (HH). The most prevalent genetic iron overload disorder in Caucasians is caused by mutations in the HFE gene, an atypical MHC class I molecule. Recent studies classified HFE/Hfe-HH as a liver disease with the primarily failure in the production of the liver iron hormone hepcidin in hepatocytes. Inadequate hepcidin expression signals for excessive iron absorption from the diet and iron deposition in tissues causing multiple organ damage and failure. This review focuses on the molecular actions of the HFE/Hfe and hepcidin in maintaining systemic iron homeostasis and approaches undertaken so far to combat iron overload in HFE/Hfe-HH. In the light of the recent investigations, novel roles of extra-hepatocytic Hfe are discussed raising a question to the relevance of the multipurpose functions of Hfe for the understanding of HH-associated pathologies.

In nanomedicine, the hydrophobic nature of paclitaxel has favored its incorporation into many nanoparticle formulations for anti-cancer chemotherapy. At lower doses taxanes are reported to elicit anti-angiogenic responses. In the present study, the facile synthesis, development and characterization of a new lipase-labile docetaxel prodrug is reported and shown to be an effective anti-angiogenic agent in vitro and in vivo. The Sn 2 phosphatidylcholine prodrug was stably incorporated into the lipid membrane of α(v)β₃-integrin targeted perfluorocarbon (PFC) nanoparticles (α(v)β₃-Dxtl-PD NP) and did not appreciably release during dissolution against PBS buffer or plasma over three days. Overnight exposure of α(v)β₃-Dxtl-PD NP to plasma spiked with phospholipase enzyme failed to liberate the taxane from the membrane until the nanoparticle integrity was compromised with alcohol. The bioactivity and efficacy of α(v)β₃-Dxtl-PD NP in endothelial cell culture was as effective as Taxol(®) or free docetaxel in methanol at equimolar doses over 96 hours. The anti-angiogenesis effectiveness of α(v)β₃-Dxtl-PD NP was demonstrated in the Vx2 rabbit model using MR imaging of angiogenesis with the same α(v)β₃-PFC nanoparticle platform. Nontargeted Dxtl-PD NP had a similar MR anti-angiogenesis response as the integrin-targeted agent, but microscopically measured decreases in tumor cell proliferation and increased apoptosis were detected only for the targeted drug. Equivalent dosages of Abraxane(®) given over the same treatment schedule had no effect on angiogenesis when compared to control rabbits receiving saline only. These data demonstrate that α(v)β₃-Dxtl-PD NP can reduce MR detectable angiogenesis and slow tumor progression in the Vx2 model, whereas equivalent systemic treatment with free taxane had no benefit.

We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) cocultured with adult liver flukes (Opisthorchis viverrini) for 6 to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increases in secretion of proinflammatory cytokines, costimulating protein, adhesion molecules, and chemotactic chemokines relative to untreated controls. In contrast, secretion of the CD40 ligand, interleukin 16, and macrophage inflammatory protein 1β decreased. Proteomic analysis revealed that expression of 48 proteins was significantly altered in PBMCs stimulated with O. viverrini. Annexin A1 (ANXA1) was selected for further study, and immunoblotting showed upregulation of ANXA1 expression in PBMCs after 12 and 24 h coculture with liver flukes. In an in vivo study, transcription and translation of ANXA1 significantly increased in livers of hamsters infected with O. viverrini at 21 days and from 3 months onwards compared to normal controls. Interestingly, immunohistochemistry revealed that ANXA1 was present not only in the cytoplasm of inflammatory cells but also in the cytoplasm of cholangiocytes, which are in close contact with the parasite and its excretory/secretory products in the biliary system. Expression of ANXA1 increased with time concomitant with bile duct enlargement, bile duct formation, and epithelial cell proliferation. In conclusion, several cytokines/chemokines secreted by PBMCs and upregulation of ANXA1 in PBMCs and biliary epithelial cells might have a role in host defense against O. viverrini infection and tissue resolution of inflammation.

Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV) in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV) using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white+ transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w+ silencing during development. This P element dependent silencing (PDS) can be modified by the haplo-suppressors/triplo-enhancers, Su(var)205 and Su(var)3-7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1) that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white+ in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1), phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV) at w(m4), indicating a previously unknown common silencing mechanism between the two.

To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator--Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T(c) mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.

The quality of follicular oocytes depends on interactions with surrounding granulosa cells. Development of molecular techniques and methods enables better understanding of processes underlying mammalian reproduction on cellular level. The success in reproductive biology and medicine in different species depends on reliable assessment of oocyte and embryo viability which presently mainly bases on embryo morphology. Although successful pregnancies have been achieved using this approach, its precision still should be improved and completed with other, more objective, and accurate assessment strategies. Global profiling of gene expression in follicular cumulus cells using microarrays is continuously leading to the establishment of new biomarkers which can be used to select oocytes with highest developmental potential. Even more potential applications and greater precision could be achieved using next generation sequencing (NGS) of granulosa and cumulus cell RNA (RNA-seq). However, due to the high cost, this method is not used as frequently as microarrays at the moment. In any case, high-throughput technologies offer the possibilities and advantages in ovarian somatic cell analysis on scale that has not been noted so far. The aim of this work is to present current directions and examples of global molecular profiling of granulosa cells and underline its impact on reproductive biology and medicine.

Integrons are one of the genetic elements involved in the acquisition of antibiotic resistance. The aim of the present research is to investigate the presence of integrons in commensal Escherichia coli (E. coli) strains, isolated from pigs at different stages of production system and from the environment in an Argentinian farm. Five sows postpartum and five randomly chosen piglets from each litter were sampled by rectal swabs. They were sampled again at day 21 and at day 70. Environmental samples from the farm were also obtained. E. coli containing any integron class or combination of both integrons was detected by polymerase chain reaction in 100% of sows and in piglets at different stages of production: farrowing pen stage 68.1%;, weaning 60%, and growing/finishing 85.8%, showing an increase along the production system. From environmental samples 78.4% of E. coli containing any integron class was detected. We conclude that animals and farm environment can act as reservoirs for potential spread of resistant bacteria by means of mobile genetic elements as integrons, which has a major impact on production of food animals and that can reach man through the food chain, constituting a problem for public health.

Circulating tumor cells (CTCs) are becoming promising biomarkers in several cancers, such as colon, prostate, and breast carcinomas. Independent research groups have reported a correlation between CTC numbers and patient prognosis. Even more, the development of personalized medicine gives physicians impetus to utilize the advancement of molecular characterization of CTCs. This review introduces a new technique, CTCscope, and compares it with the current methods of CTCs detection, with particular emphasis on cancer research, and discusses the future application of this new method from bench to bed-side.

The process of axonal transport serves to move components over very long distances on microtubule tracks in order to maintain neuronal viability. Molecular motors - kinesin and dynein - are essential for the movement of neuronal cargoes along these tracks; defects in this pathway have been implicated in the initiation or progression of some neurodegenerative diseases, suggesting that this process may be a key contributor in neuronal dysfunction. Recent work has led to the identification of some of the motor-cargo complexes, adaptor proteins, and their regulatory elements in the context of disease proteins. In this review, we focus on the assembly of the amyloid precursor protein, huntingtin, mitochondria, and the RNA-motor complexes and discuss how these may be regulated during long-distance transport in the context of neurodegenerative disease. As knowledge of these motor-cargo complexes and their involvement in axonal transport expands, insight into how defects in this pathway contribute to the development of neurodegenerative diseases becomes evident. Therefore, a better understanding of how this pathway normally functions has important implications for early diagnosis and treatment of diseases before the onset of disease pathology or behavior.

The process in which nanostructured surfaces mediate cell adhesion is not well understood, and may be indirect (via protein adsorption) or direct. We prepared Sr-doped hydroxyapatite (Sr1-HA) 3D nanorods (with interrod spacing of 67.3 ± 3.8, 95.7 ± 4.2, and 136.8 ± 8.7 nm) and 2D nanogranulate patterned coatings on titanium. Employing the coatings under the same surface chemistry and roughness, we investigated the indirect/direct role of Sr1-HA nanotopographies in the regulation of osteoblast adhesion in both serum-free and serum-containing Dulbecco's Modified Eagle/Ham's Medium. The results reveal that the number of adherent cells, cell-secreted anchoring proteins (fibronectin, vitronectin, and collagen), vinculin and focal adhesion kinase (FAK) denoted focal adhesion (FA) contact, and gene expression of vinculin, FAK, and integrin subunits (α2, α5, αv, β1, and β3), undergo significant changes in the inter-nanorod spacing and dimensionality of Sr1-HA nanotopographies in the absence of serum; they are significantly enhanced on the <96 nm spaced nanorods and more pronounced with decreasing interrod spacing. However, they are inhibited on the >96 nm spaced nanorods compared to nanogranulated 2D topography. Although the adsorption of fibronectin and vitronectin from serum are higher on 136.8 ± 8.7 nm spaced nanorod patterned topography than nanogranulated topography, cell adhesion is inhibited on the former compared to the latter in the presence of serum, further suggesting that reduced cell adhesion is independent of protein adsorption. It is clearly indicated that 3D nanotopography can directly modulate cell adhesion by regulating integrins, which subsequently mediate anchoring proteins' secretion and FA formation rather than via protein adsorption.

<h4>Aims</h4>Vascular perfusion may be impaired in primary open-angle glaucoma (POAG); thus, we evaluated a panel of markers in vascular tone-regulating genes in relation to POAG.<h4>Methods</h4>We used Illumina 660W-Quad array genotype data and pooled P-values from 3108 POAG cases and 3430 controls from the combined National Eye Institute Glaucoma Human Genetics Collaboration consortium and Glaucoma Genes and Environment studies. Using information from previous literature and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we compiled single-nucleotide polymorphisms (SNPs) in 186 vascular tone-regulating genes. We used the 'Pathway Analysis by Randomization Incorporating Structure' analysis software, which performed 1000 permutations to compare the overall pathway and selected genes with comparable randomly generated pathways and genes in their association with POAG.<h4>Results</h4>The vascular tone pathway was not associated with POAG overall or POAG subtypes, defined by the type of visual field loss (early paracentral loss (n=224 cases) or only peripheral loss (n=993 cases)) (permuted P≥0.20). In gene-based analyses, eight were associated with POAG overall at permuted P<0.001: PRKAA1, CAV1, ITPR3, EDNRB, GNB2, DNM2, HFE, and MYL9. Notably, six of these eight (the first six listed) code for factors involved in the endothelial nitric oxide synthase activity, and three of these six (CAV1, ITPR3, and EDNRB) were also associated with early paracentral loss at P<0.001, whereas none of the six genes reached P<0.001 for peripheral loss only.<h4>Discussion</h4>Although the assembled vascular tone SNP set was not associated with POAG, genes that code for local factors involved in setting vascular tone were associated with POAG.

<h4>Background</h4>There is compelling evidence of a genetic foundation of patient-reported quality of life (QOL). Given the rapid development of substantial scientific advances in this area of research, the current paper updates and extends reviews published in 2010.<h4>Objectives</h4>The objective was to provide an updated overview of the biological pathways, candidate genes, and molecular markers involved in fatigue, pain, negative (depressed mood) and positive (well-being/happiness) emotional functioning, social functioning, and overall QOL.<h4>Methods</h4>We followed a purposeful search algorithm of existing literature to capture empirical papers investigating the relationship between biological pathways and molecular markers and the identified QOL domains.<h4>Results</h4>Multiple major pathways are involved in each QOL domain. The inflammatory pathway has the strongest evidence as a controlling mechanism underlying fatigue. Inflammation and neurotransmission are key processes involved in pain perception, and the catechol-O-methyltransferase (COMT) gene is associated with multiple sorts of pain. The neurotransmitter and neuroplasticity theories have the strongest evidence for their relationship with depression. Oxytocin-related genes and genes involved in the serotonergic and dopaminergic pathways play a role in social functioning. Inflammatory pathways, via cytokines, also play an important role in overall QOL.<h4>Conclusions</h4>Whereas the current findings need future experiments and replication efforts, they will provide researchers supportive background information when embarking on studies relating candidate genes and/or molecular markers to QOL domains. The ultimate goal of this area of research is to enhance patients' QOL.

<h4>Background</h4>The prognosis of patients with advanced biliary tract cancer (BTC) is extremely poor and only a few standard treatments are available for this condition. We performed a phase I trial to investigate the safety, immune response and anti-tumor effect of vaccination with three peptides derived from cancer-testis antigens.<h4>Methods</h4>This study was conducted as a phase I trial. Nine patients with advanced BTC who had unresectable tumors and were refractory to standard chemotherapy were enrolled. Three HLA-A*2402 restricted epitope peptides-cell division cycle associated 1 (CDCA1), cadherin 3 (CDH3) and kinesin family member 20A (KIF20A)-were administered subcutaneously, and the adverse events and immune response were assessed. The clinical effects observed were the tumor response, progression-free survival (PFS) and overall survival (OS).<h4>Results</h4>The three-peptide vaccination was well-tolerated up to a dose of 3 mg per peptide (9 mg total). No grade 3 or 4 adverse events were observed after vaccination. Peptide-specific T cell immune responses were observed in all patients and stable disease was observed in 5 of 9 patients. The median PFS and OS were 3.4 and 9.7 months. The Grade 2 injection site reaction and continuous vaccination after PD judgment appeared to be prognostic of OS.<h4>Conclusions</h4>Multiple-peptide vaccination was well tolerated and induced peptide-specific T-cell responses.<h4>Trial registration</h4>This study was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR000003229).