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Neurotensin (NT) is distributed throughout the brain and gastrointestinal tract. Although the relationship between NT and matrix metalloproteinase-9 (MMP-9) activity in gastric cancer has not been reported, the elevation of MMP-9 and NT is reported in the breast, lung, prostate, and gastric cancer. The aim of our study is to investigate the relationship between NT and MMP-9 activity and the underlying signaling mechanism in gastric cancer cell lines. Commercial ELISA kits were used for estimation of NT and MMP-9 expression, and fluorescence resonance energy transfer (FRET) assay was used for measurement of MMP-9 activity. Cell migration and invasion were determined by wound healing and transwell assay. The expression of signaling proteins was measured by Western blotting. Our study reveals a positive correlation between increased plasma NT and MMP-9 activity in both of patient's serum and gastric cancer cell lines. A dose-dependent elevation of MMP-9 activity was observed by NT treatment in gastric cancer cells (MKN-1 and MKN-45) compared to untreated gastric cancer and normal epithelial cell (HFE-145). Moreover, NT-mediated migration and invasion were observed in gastric cancer cells unlike in normal cell. The signaling mechanism of NT in gastric cancer cells was confirmed in protein kinase C (PKC), extracellular-signal regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K) pathway. In addition, pretreatment of gastric cancer cells with NTR1 inhibitor SR48692 was shown to significantly inhibit the NT-mediated MMP-9 activity, cell invasion, and migration. Our finding illustrated NTR1 could be a possible therapeutic target for gastric cancer.

Huntington׳s disease (HD) is a neurodegenerative disorder caused by a mutation in the HTT gene (mHTT) encoding the protein huntingtin. An expansion in the gene׳s CAG repeat length renders a misfolded, dysfunctional protein with an abnormally long glutamine (Q) stretch at the N terminus that often incorporates into inclusion bodies and leads to neurodegeneration in many regions of the brain. HD is characterized by motor and cognitive decline as well as mood disorders, with depression being particularly common. Approximately 40% of the HD population suffers from depressive symptoms. Because these symptoms often manifest a decade or more prior to the knowledge that the person is at risk for the disease, a portion of the early depression in HD appears to be a consequence of the pathology arising from expression of the mutant gene. While the depression in HD patients is often treated with serotonin agonists, there is scant experimental evidence that the depression in HD responds well to these serotonin treatments or in a similar manner to how non-HD depression tends to respond. Additionally, at very early sub-threshold depression levels, abnormal changes in several neuronal populations are already detectable in HD patients, suggesting that a variety of brain structures may be involved. Taken together, the serotonin system is a viable candidate. However, at present there is limited evidence of the precise nuclei or circuits that play a role in HD depression. With this in mind, the current study was designed to control for the widespread brain neuropathology that occurs in HD and in transgenic mouse models of HD and focuses specifically on the influence of the midbrain dorsal raphe nucleus (DRN). The DRN provides the majority of the serotonin to the forebrain and exhibits cell loss in non-HD depression. Therefore, we employed a viral vector delivery system to investigate whether the over-expression of mHTT in the DRN׳s ventral sub-nuclei alone is sufficient to produce depressive-like behaviors. Wildtype mice were injected with an adeno-associated virus (AAV2/1) encoding HTT containing either a pathogenic (N171-82Q) or control (N171-16Q) CAG repeat length into the ventral DRN and depressive-like behaviors and motor behaviors were assessed for 12 weeks post-surgery. Quantitative PCR and immunohistochemistry (IHC) verified positive transduction in the ventral aspects of the DRN, including the ventral sub-nucleus (DRv) and interfascicular sub-nucleus (DRif). IHC demonstrated microgliosis in and around the injection site and mHTT-positive inclusions in serotonin-producing neurons and a small percentage of astrocytes in animals injected with N171-82Q compared to controls. Moreover, N171-82Q injected mice showed a 75% reduction in cells that stained positive for the serotonin synthesis enzyme, tryptophan hydroxylase-2 (TPH2) compared to controls (p<0.05). Despite mHTT-mediated pathology in the DRv and DRif, no significant changes in depressive-like behavior were detected. Consequently, we conclude that 12 weeks of N171-82Q expression in the ventral sub-nuclei of the DRN of wildtype mice causes characteristic disease-related cellular neuropathology but is not sufficient to elicit depressive-like behaviors. Ongoing studies are investigating whether a larger injection volume that transfects a larger percentage of the DRN and/or a longer time course of mHTT expression might elicit depressive-like behaviors. Moreover, mHTT expression in other regions of the brain, such as the hippocampal dentate gyrus and/or the frontal cortex might be necessary to elicit HD depression. Together, these results may prove helpful in addressing which therapeutic and/or pharmacological strategies might be most efficacious when treating depressive symptomology in patients suffering from HD.

Genes coding for the serotonergic pathway have been associated with depressive disorders. However, these associations have rarely been tested in acute coronary syndrome (ACS) patients vulnerable to depression. This study aimed to investigate whether polymorphisms of serotonin transporter (5-HTT) and serotonin 2a receptor (5-HTR2a) genes are associated with occurrence of depressive disorder in ACS. 969 patients with recently developed ACS were recruited at baseline, and 711 were followed 1 year thereafter. Depressive disorder was diagnosed according to DSM-IV criteria, and analysed as an outcome at baseline (prevalence), and follow up (incidence and persistence). Genotypes were ascertained for 5-HTTLPR, STin2 VNTR, 5-HTR2a 102T/C, and 5-HTR2a 1438A/G. Logistic regression models were used to investigate associations. The 5-HTTLPR s/s genotype was independently associated with depressive disorder prevalence and persistence following ACS, but no significant associations were found with the other polymorphisms. ACS patients with the 5-HTTLPR s allele are thus potentially susceptible to depressive disorder in the early phase after ACS, and with its persistence over the subsequent year.

Background. Hepatic angiosarcoma is a rare and aggressive tumor that often presents at an advanced stage with nonspecific symptoms. Objective. To report a case of primary hepatic angiosarcoma in an otherwise healthy man with normal liver function tests two months prior to presenting with a short period of jaundice that progressed to fulminant hepatic failure. Methods. Case report and review of literature. Conclusion. This case illustrates the rapidity of progression to death after the onset of symptoms in a patient with hepatic angiosarcoma. Research on early diagnostic strategies and newer therapies are needed to improve prognosis in this rare and poorly understood malignancy with limited treatment options.

Magnetic Particle Imaging (MPI) is a novel non-invasive biomedical imaging modality that uses safe magnetite nanoparticles as tracers. Controlled synthesis of iron oxide nanoparticles (NPs) with tuned size-dependent magnetic relaxation properties is critical for the development of MPI. Additional functionalization of these NPs for other imaging modalities (e.g. MRI and fluorescent imaging) would accelerate screening of the MPI tracers based on their in vitro and in vivo performance in pre-clinical trials. Here, we conjugated two different types of poly-ethylene-glycols (NH2-PEG-NH2 and NH2-PEG-FMOC) to monodisperse carboxylated 19.7 nm NPs by amide bonding. Further, we labeled these NPs with Cy5.5 near infra-red fluorescent (NIRF) molecules. Bi-functional PEG (NH2-PEG-NH2) resulted in larger hydrodynamic size (∼98 nm vs. ∼43 nm) of the tracers, due to inter-particle crosslinking. Formation of such clusters impacted the multimodal imaging performance and pharmacokinetics of these tracers. We found that MPI signal intensity of the tracers in blood depends on their plasmatic clearance pharmacokinetics. Whole body mice MPI/MRI/NIRF, used to study the biodistribution of the injected NPs, showed primary distribution in liver and spleen. Biodistribution of tracers and their clearance pathway was further confirmed by MPI and NIRF signals from the excised organs where the Cy5.5 labeling enabled detailed anatomical mapping of the tracers.in tissue sections. These multimodal MPI tracers, combining the strengths of each imaging modality (e.g. resolution, tracer sensitivity and clinical use feasibility) pave the way for various in vitro and in vivo MPI applications.

We demonstrate that nano-hydrophobicity, which governs the biological aggressiveness of nanoparticles, is determined by the outermost regions of surface ligands. We have also successfully modulated nano-hydrophobicity using systematic surface ligand modifications and built the first computational model of nano-hydrophobicity.

Huntington's disease (HD) is an inherited genetic disorder, characterized by cognitive dysfunction and abnormal body movements called chorea. Quinolinic acid (QA) is an endogenous metabolite of tryptophan in the kynurenine pathway. QA-induced alterations are similar to the symptoms of HD patients. Physical exercise has beneficial effects on the brain functions. Exercise increases production of neurotrophic factors in the brain and improves learning ability and memory function. In the present study, we investigated the effects of treadmill exercise short-term memory on QA-induced HD rats in relation with cell proliferation. For the induction of Huntington's animal model, 2 μL of 100 nmol QA was intrastriatal injected into the rats. The rats in the treadmill exercise groups were forced to run on a treadmill for 30 min once a day, five times a week for 2 weeks. Step-down avoidance test was conducted for the determination of short-term memory. Cell proliferation in the hippocampal dentate gyrus was determined by 5-bromo-2'-deoxyuridine (BrdU) and doublecortin (DCX) immunohistochemistry. Western blot for brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) were performed. In the present results, treadmill exercise alleviated QA-induced short-term memory impairment in HD rats. Treadmill exercise increased cell proliferation in the hippocampal dentate gyrus through enhancing BDNF expression in the HD rats. These results revealed that treadmill exercise is effective for the symptom improvement in the HD patients.

<h4>Background</h4>Social-stress mouse model, based on the resident-intruder paradigm was used to simulate features of human post-traumatic stress disorder (PTSD). The model involved exposure of an intruder (subject) mouse to a resident aggressor mouse followed by exposure to trauma reminders with rest periods. C57BL/6 mice exposed to SJL aggressor mice exhibited behaviors suggested as PTSD-in-mouse phenotypes: intermittent freezing, reduced locomotion, avoidance of the aggressor-associated cue and apparent startled jumping. Brain tissues (amygdala, hippocampus, medial prefrontal cortex, septal region, corpus striatum and ventral striatum) from subject (aggressor exposed: Agg-E) and control C57BL/6 mice were collected at one, 10 and 42 days post aggressor exposure sessions. Transcripts in these brain regions were assayed using Agilent's mouse genome-wide arrays.<h4>Results</h4>Pathways and biological processes associated with differentially regulated genes were mainly those thought to be involved in fear-related behavioral responses and neuronal signaling. Expression-based assessments of activation patterns showed increased activations of pathways related to anxiety disorders (hyperactivity and fear responses), impaired cognition, mood disorders, circadian rhythm disruption, and impaired territorial and aggressive behaviors. In amygdala, activations of these pathways were more pronounced at earlier time-points, with some attenuation after longer rest periods. In hippocampus and medial prefrontal cortex, activation patterns were observed at later time points. Signaling pathways associated with PTSD-comorbid conditions, such as diabetes, metabolic disorder, inflammation and cardiac infarction, were also significantly enriched. In contrast, signaling processes related to neurogenesis and synaptic plasticity were inhibited.<h4>Conclusions</h4>Our data suggests activations of behavioral responses associated with anxiety disorders as well as inhibition of neuronal signaling pathways important for neurogenesis, cognition and extinction of fear memory. These pathways along with comorbid-related signaling pathways indicate the pervasive and multisystem effects of aggressor exposure in mice, potentially mirroring the pathologic conditions of PTSD patients.

<h4>Background</h4>The brain, spinal cord, and neural retina comprise the central nervous system (CNS) of vertebrates. Understanding the regulatory mechanisms that underlie the enormous cell-type diversity of the CNS is a significant challenge. Whole-genome mapping of DNase I-hypersensitive sites (DHSs) has been used to identify cis-regulatory elements in many tissues. We have applied this approach to the mouse CNS, including developing and mature neural retina, whole brain, and two well-characterized brain regions, the cerebellum and the cerebral cortex.<h4>Results</h4>For the various regions and developmental stages of the CNS that we analyzed, there were approximately the same number of DHSs; however, there were many DHSs unique to each CNS region and developmental stage. Many of the DHSs are likely to mark enhancers that are specific to the specific CNS region and developmental stage. We validated the DNase I mapping approach for identification of CNS enhancers using the existing VISTA Browser database and with in vivo and in vitro electroporation of the retina. Analysis of transcription factor consensus sites within the DHSs shows distinct region-specific profiles of transcriptional regulators particular to each region. Clustering developmentally dynamic DHSs in the retina revealed enrichment of developmental stage-specific transcriptional regulators. Additionally, we found reporter gene activity in the retina driven from several previously uncharacterized regulatory elements surrounding the neurodevelopmental gene Otx2. Identification of DHSs shared between mouse and human showed region-specific differences in the evolution of cis-regulatory elements.<h4>Conclusions</h4>Overall, our results demonstrate the potential of genome-wide DNase I mapping to cis-regulatory questions regarding the regional diversity within the CNS. These data represent an extensive catalogue of potential cis-regulatory elements within the CNS that display region and temporal specificity, as well as a set of DHSs common to CNS tissues. Further examination of evolutionary conservation of DHSs between CNS regions and different species may reveal important cis-regulatory elements in the evolution of the mammalian CNS.

<h4>Unlabelled</h4>Ezetimibe inhibits intestinal cholesterol absorption and lowers low-density lipoprotein cholesterol. Uncontrolled studies have suggested that it reduces liver fat as estimated by ultrasound in nonalcoholic steatohepatitis (NASH). Therefore, we aimed to examine the efficacy of ezetimibe versus placebo in reducing liver fat by the magnetic resonance imaging-derived proton density-fat fraction (MRI-PDFF) and liver histology in patients with biopsy-proven NASH. In this randomized, double-blind, placebo-controlled trial, 50 patients with biopsy-proven NASH were randomized to either ezetimibe 10 mg orally daily or placebo for 24 weeks. The primary outcome was a change in liver fat as measured by MRI-PDFF in colocalized regions of interest within each of the nine liver segments. Novel assessment by two-dimensional and three-dimensional magnetic resonance elastography was also performed. Ezetimibe was not significantly better than placebo at reducing liver fat as measured by MRI-PDFF (mean difference between the ezetimibe and placebo arms -1.3%, P = 0.4). Compared to baseline, however, end-of-treatment MRI-PDFF was significantly lower in the ezetimibe arm (15%-11.6%, P < 0.016) but not in the placebo arm (18.5%-16.4%, P = 0.15). There were no significant differences in histologic response rates, serum alanine aminotransferase and aspartate aminotransferase levels, or longitudinal changes in two-dimensional and three-dimensional magnetic resonance elastography-derived liver stiffness between the ezetimibe and placebo arms. Compared to histologic nonresponders (25/35), histologic responders (10/35) had a significantly greater reduction in MRI-PDFF (-4.35 ± 4.9% versus -0.30 ± 4.1%, P < 0.019).<h4>Conclusions</h4>Ezetimibe did not significantly reduce liver fat in NASH. This trial demonstrates the application of colocalization of MRI-PDFF-derived fat maps and magnetic resonance elastography-derived stiffness maps of the liver before and after treatment to noninvasively assess treatment response in NASH.

<h4>Rationale</h4>While brain serotonin (5-HT) function is implicated in gene-by-environment interaction (GxE) impacting the vulnerability-resilience continuum in neuropsychiatric disorders, it remains elusive how the interplay of altered 5-HT synthesis and environmental stressors is linked to failure in emotion regulation.<h4>Objective</h4>Here, we investigated the effect of constitutively impaired 5-HT synthesis on behavioral and neuroendocrine responses to unpredictable chronic mild stress (CMS) using a mouse model of brain 5-HT deficiency resulting from targeted inactivation of the tryptophan hydroxylase-2 (Tph2) gene.<h4>Results</h4>Locomotor activity and anxiety- and depression-like behavior as well as conditioned fear responses were differentially affected by Tph2 genotype, sex, and CMS. Tph2 null mutants (Tph2(-/-)) displayed increased general metabolism, marginally reduced anxiety- and depression-like behavior but strikingly increased conditioned fear responses. Behavioral modifications were associated with sex-specific hypothalamic-pituitary-adrenocortical (HPA) system alterations as indicated by plasma corticosterone and fecal corticosterone metabolite concentrations. Tph2(-/-) males displayed increased impulsivity and high aggressiveness. Tph2(-/-) females displayed greater emotional reactivity to aversive conditions as reflected by changes in behaviors at baseline including increased freezing and decreased locomotion in novel environments. However, both Tph2(-/-) male and female mice were resilient to CMS-induced hyperlocomotion, while CMS intensified conditioned fear responses in a GxE-dependent manner.<h4>Conclusions</h4>Our results indicate that 5-HT mediates behavioral responses to environmental adversity by facilitating the encoding of stress effects leading to increased vulnerability for negative emotionality.

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington's disease.

Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and ZIPK have diverse, but predominantly distinct regulatory functions in vascular SMC and that ROCK1-mediated activation of ZIPK is not involved in most of these functions.

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.

We describe a case of severe neonatal anemia with kernicterus caused by compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1-null human. The phenotype of severe nonspherocytic hemolytic anemia, jaundice, hepatosplenomegaly, and marked erythroblastosis is more severe than that present in congenital dyserythropoietic anemia type IV as a result of dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found that human KLF1 acts like mouse Klf1 to coordinate expression of many genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell-cycle regulators, and blood group antigens. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. We also identify new roles for KLF1 in autophagy, global transcriptional control, and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.

<h4>Purpose</h4>Previous studies suggest a potential therapeutic role for mTOR inhibition in lymphoid malignancies. This single-center phase I/II study was designed to test the safety and efficacy of the mTOR inhibitor everolimus in combination with HyperCVAD chemotherapy in relapsed/refractory acute lymphoblastic leukemia (ALL).<h4>Experimental design</h4>Twenty-four patients were treated; 15 received everolimus 5 mg/day and 9 received 10 mg/day with HyperCVAD.<h4>Results</h4>The median age of patients was 25 years (range, 11-64) and median number of prior treatments was 2 (range, 1-7). Grade 3 mucositis was the dose-limiting toxicity and the maximum tolerated everolimus dose was 5 mg/day. Responses included complete remission (CR) in 6 patients (25%), CR without platelet recovery (CRp) in 1 (4%), and CR without recovery of counts (CRi) in 1 (4%), for an overall response rate of 33%. In addition, partial response (PR) was noted in 2 patients (8%). Seven of 11 patients treated in first salvage achieved CR/CRp (64%). The median OS was 29 weeks for patients in first salvage versus 15 weeks for patients in second salvage and beyond (P ≤ 0.001). A response was noted in 5 of 10 (50%) heavily pretreated T-ALL patients (median of 4 prior salvage regimens). Everolimus significantly inhibited phosphorylation of S6RP, but this did not correlate with response. No significant decreases in p4EBP1 and pAkt levels were noted. Responders had higher everolimus dose-adjusted area under the curve (P = 0.025) and lower clearance (P = 0.025) than nonresponders.<h4>Conclusions</h4>The combination of HyperCVAD and everolimus is well tolerated and moderately effective in relapsed ALL, specifically T-ALL.

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome.

We have developed a novel plasma protein analysis platform with optimized sample preparation, chromatography, and MS analysis protocols. The workflow, which utilizes chemical isobaric mass tag labeling for relative quantification of plasma proteins, achieves far greater depth of proteome detection and quantification while simultaneously having increased sample throughput than prior methods. We applied the new workflow to a time series of plasma samples from patients undergoing a therapeutic, "planned" myocardial infarction for hypertrophic cardiomyopathy, a unique human model in which each person serves as their own biologic control. Over 5300 proteins were confidently identified in our experiments with an average of 4600 proteins identified per sample (with two or more distinct peptides identified per protein) using iTRAQ four-plex labeling. Nearly 3400 proteins were quantified in common across all 16 patient samples. Compared with a previously published label-free approach, the new method quantified almost fivefold more proteins/sample and provided a six- to nine-fold increase in sample analysis throughput. Moreover, this study provides the largest high-confidence plasma proteome dataset available to date. The reliability of relative quantification was also greatly improved relative to the label-free approach, with measured iTRAQ ratios and temporal trends correlating well with results from a 23-plex immunoMRM (iMRM) assay containing a subset of the candidate proteins applied to the same patient samples. The functional importance of improved detection and quantification was reflected in a markedly expanded list of significantly regulated proteins that provided many new candidate biomarker proteins. Preliminary evaluation of plasma sample labeling with TMT six-plex and ten-plex reagents suggests that even further increases in multiplexing of plasma analysis are practically achievable without significant losses in depth of detection relative to iTRAQ four-plex. These results obtained with our novel platform provide clear demonstration of the value of using isobaric mass tag reagents in plasma-based biomarker discovery experiments.

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)(1)-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling.

<h4>Background</h4>The movement of mobile elements among species by horizontal transposon transfer (HTT) influences the evolution of genomes through the modification of structure and function. Helitrons are a relatively new lineage of DNA-based (class II) transposable elements (TEs) that propagate by rolling-circle replication, and are capable of acquiring host DNA. The rapid spread of Helitrons among animal lineages by HTT is facilitated by shuttling in viral particles or by unknown mechanisms mediated by close organism associations (e.g. between hosts and parasites).<h4>Results</h4>A non-autonomous Helitron independently annotated as BmHel-2 from Bombyx mori and the MITE01 element from Ostrinia nubilalis was predicted in the genomes of 24 species in the insect Order Lepidoptera. Integrated Helitrons retained ≥ 65% sequence identity over a 250 bp consensus, and were predicted to retain secondary structures inclusive of a 3'-hairpin and a 5'-subterminal inverted repeat. Highly similar Hel-2 copies were predicted in the genomes of insects and associated viruses, which along with a previous documented case of real-time virus-insect cell line transposition suggests that this Helitron has likely propagated by HTT.<h4>Conclusions</h4>These findings provide evidence that insect virus may mediate the HTT of Helitron-like TEs. This movement may facilitate the shuttling of DNA elements among insect genomes. Further sampling is required to determine the putative role of HTT in insect genome evolution.

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.

<h4>Background</h4>Atherosclerosis severity-independent alterations in DNA methylation, a reversible and highly regulated DNA modification, have been detected in aortic atheromas, thus supporting the hypothesis that epigenetic mechanisms participate in the pathogenesis of atherosclerosis. One yet unaddressed issue is whether the progression of atherosclerosis is associated with an increase in DNA methylation drift in the vascular tissue. The purpose of the study was to identify CpG methylation profiles that vary with the progression of atherosclerosis in the human aorta.<h4>Methods</h4>We interrogated a set of donor-matched atherosclerotic and normal aortic samples ranging from histological grade III to VII, with a high-density (>450,000 CpG sites) DNA methylation microarray.<h4>Results</h4>We detected a correlation between histological grade and intra-pair differential methylation for 1,985 autosomal CpGs, the vast majority of which drifted towards hypermethylation with lesion progression. The identified CpG loci map to genes that are regulated by known critical transcription factors involved in atherosclerosis and participate in inflammatory and immune responses. Functional relevance was corroborated by crossing the DNA methylation profiles with expression data obtained in the same human aorta sample set, by a transcriptome-wide analysis of murine atherosclerotic aortas and from available public databases.<h4>Conclusions</h4>Our work identifies for the first time atherosclerosis progression-specific DNA methylation profiles in the vascular tissue. These findings provide potential novel markers of lesion severity and targets to counteract the progression of the atheroma.

<h4>Background</h4>Vein graft stenosis after coronary artery bypass grafting (CABG) is common. Identifying genes associated with vein graft stenosis after CABG could reveal novel mechanisms of disease and discriminate patients at risk for graft failure. We hypothesized that genome-wide association would identify these genes.<h4>Methods</h4>We performed a genome-wide association study on a subset of patients presenting for cardiac catheterization for concern of ischemic heart disease, who also underwent CABG and subsequent coronary angiography after CABG for clinical indications (n = 521). Cases were defined as individuals with ≥50% stenosis in any vein graft on any cardiac catheterization, and controls were defined as those who did not have vein graft stenosis on any subsequent cardiac catheterization. Multivariable logistic regression was used to assess the association between single nucleotide polymorphisms (SNPs) and vein graft stenosis.<h4>Results</h4>Sixty-nine percent of patients had vein graft failure after CABG. Seven SNPs were significantly associated with vein graft stenosis, including intronic SNPs in the genes PALLD (Rs6854137, P = 3.77 × 10(-6)), ARID1B (Rs184074, P = 5.97 × 10(-6)), and TMEM123 (Rs11225247, P = 8.25 × 10(-6)); and intergenic SNPs near the genes ABCA13 (Rs10232860, P = 4.54 × 10(-6)), RMI2 (Rs9921338, P = 6.15 × 10(-6)), PRM2 (Rs7198849, P = 7.27 × 10(-6)), and TNFSF4 (Rs17346536, P = 9.33 × 10(-6)).<h4>Conclusions</h4>We have identified novel genetic variants that may predispose to risk of vein graft failure after CABG, many within biologically plausible pathways. These polymorphisms merit further investigation, as they could assist in stratifying patients with multi-vessel coronary artery disease, which could lead to alterations in management and revascularization strategy.

Chlorapatite can be considered a potential biomaterial for orthopaedic applications. Its use as plasma-sprayed coating could be of interest considering its thermal properties and particularly its ability to melt without decomposition unlike hydroxyapatite. Chlorapatite (ClA) was synthesized by a high-temperature ion exchange reaction starting from commercial stoichiometric hydroxyapatites (HA). The ClA powder showed similar characteristics as the original industrial HA powder, and was obtained in the monoclinic form. The HA and ClA powders were plasma-sprayed using a low-energy plasma spraying system with identical processing parameters. The coatings were characterized by physical-chemical methods, i.e. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy, including distribution mapping of the main phases detected such as amorphous calcium phosphate (ACP), oxyapatite (OA), and HA or ClA. The unexpected formation of oxyapatite in ClA coatings was assigned to a side reaction with contaminating oxygenated species (O2, H2O). ClA coatings exhibited characteristics different from HA, showing a lower content of oxyapatite and amorphous phase. Although their adhesion strength was found to be lower than that of HA coatings, their application could be an interesting alternative, offering, in particular, a larger range of spraying conditions without formation of massive impurities.

Biomimetic calcium phosphate coatings have been developed for bone regeneration and repair because of their biocompatibility, osteoconductivity, and easy preparation. They can be rendered osteoinductive by incorporating an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2), into the crystalline lattice work in physiological situations. The biomimetic calcium phosphate coating enables a controlled, slow and local release of BMP-2 when it undergoes cell mediated coating degradation induced by multinuclear cells, such as osteoclasts and foreign body giant cells, which mimics a physiologically similar release mode, to achieve sustained ectopic or orthotopic bone formation. Therefore, biomimetic calcium phosphate coatings are considered to be a promising delivery vehicle for osteogenic agents. In this review, we present an overview of biomimetic calcium phosphate coatings including their preparation techniques, physico-chemical properties, potential as drug carrier, and their pre-clinical application both in ectopic and orthotopic animal models. We briefly review some features of hydroxyapatite coatings and their clinical applications to gain insight into the clinical applications of biomimetic calcium phosphate coatings in the near future.

The present contribution has originated from a critical biomedical engineering issue e.g., loosening of metallic prostheses fixed with poly(methyl methylacrylate) (PMMA) bone cement especially in the case of hip joint replacement which ultimately forces the patient to undergo a revision surgery. Subsequently surgeons invented a cementless fixation technology introducing a bioactive hydroxyapatite (HAp) coating to the metallic implant surface. A wide variety of different coating methods have been developed to make the HAp coating on metallic implants more reliable; of which ultimately the plasma spraying method has been commercially accepted. However, the story was not yet finished at all, as many questions were raised regarding coating adherence, stability and bio-functionality in both in vitro and in vivo environments. Moreover, it has been now realized that the conventional high power plasma spraying (i.e. conventional atmospheric plasma spraying, CAPS) coating method creates many disadvantages in terms of phase impurity; reduced porosity limiting osseointegration and residual stresses which ultimately lead to inadequate mechanical properties and delamination of the coating. Further, poor crystallinity of HAp deposited by CAPS accelerates the rate of bioresorption, which may cause poor adhesion due to quick mass loss of HAp coatings. Therefore, in the present work a very recently developed method e.g. low power microplasma spraying method was utilized to coat HAp on SS316L substrates to minimize the aforementioned problems associated with commercial CAPS HAp coatings. Surgical grade SS316L has been chosen as the substrate material because it is more cost effective than Ti6Al4V and CoCrMo alloys.

Human umbilical cord blood (HUCB) cell therapies have shown promising results in reducing brain infarct volume and most importantly in improving neurobehavioral function in rat permanent middle cerebral artery occlusion, a model of stroke. In this study, we examined the gene expression profile in neurons subjected to oxygen-glucose deprivation (OGD) with or without HUCB treatment and identified signaling pathways (Akt/MAPK) important in eliciting HUCB-mediated neuroprotective responses. Gene chip microarray analysis was performed using RNA samples extracted from the neuronal cell cultures from four experimental groups: normoxia, normoxia+HUCB, OGD, and OGD+HUCB. Both quantitative RT-PCR and immunohistochemistry were carried out to verify the microarray results. Using the Genomatix software program, promoter regions of selected genes were compared to reveal common transcription factor-binding sites and, subsequently, signal transduction pathways. Under OGD condition, HUCB cells significantly reduced neuronal loss from 68% to 44% [one-way ANOVA, F(3, 16)=11, p=0.0003]. Microarray analysis identified mRNA expression of Prdx5, Vcam1, CCL20, Alcam, and Pax6 as being significantly altered by HUCB cell treatment. Inhibition of the Akt pathway significantly abolished the neuroprotective effect of HUCB cells [one-way ANOVA, F(3, 11)=8.663, p=0.0031]. Our observations show that HUCB neuroprotection is dependent on the activation of the Akt signaling pathway that increases transcription of the Prdx5 gene. We concluded that HUCB cell therapy would be a promising treatment for stroke and other forms of brain injury by modifying acute gene expression to promote neural cell protection.

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the huntingtin gene (HTT). The primary neuropathology of HD has been attributed to the preferential degeneration of medium spiny neurons (MSN) in the striatum. Reports on striatal neurogenesis have been a subject of debate; nevertheless, it should be considered as an endogenous attempt to repair the brain. The subventricular zone (SVZ) might offer a close-by region to supply the degenerated striatum with new cells. Previously, we have demonstrated that R6/2 mice, a widely used preclinical model representing an early onset HD, showed reduced olfactory bulb (OB) neurogenesis but induced striatal migration of neuroblasts without affecting the proliferation of neural progenitor cell (NPCs) in the SVZ. The present study revisits these findings, using a clinically more relevant transgenic rat model of late onset HD (tgHD rats) carrying the human HTT gene with 51 CAG repeats and mimicking many of the neuropathological features of HD seen in patients. We demonstrate that cell proliferation is reduced in the SVZ and OB of tgHD rats compared to WT rats. In the OB of tgHD rats, although cell survival was reduced, the frequency of neuronal differentiation was not altered in the granule cell layer (GCL) compared to the WT rats. However, an increased frequency of dopamenergic neuronal differentiation was noticed in the glomerular layer (GLOM) of tgHD rats. Besides this, we observed a selective proliferation of neuroblasts in the adjacent striatum of tgHD rats. There was no evidence for neuronal maturation and survival of these striatal neuroblasts. Therefore, the functional role of these invading neuroblasts still needs to be determined, but they might offer an endogenous alternative for stem or neuronal cell transplantation strategies.

Autism spectrum disorders (ASD) are clinically heterogeneous and biologically complex. In general it remains unclear, what biological factors lead to changes in the brains of autistic individuals. A considerable number of transcriptome analyses have been performed in attempts to address this question, but their findings lack a clear consensus. As a result, each of these individual studies has not led to any significant advance in understanding the autistic phenotype as a whole. Here, we report a meta-analysis of more than 1000 microarrays across twelve independent studies on expression changes in ASD compared to unaffected individuals, in both blood and brain tissues. We identified a number of known and novel genes that are consistently differentially expressed across three studies of the brain (71 samples in total). A subset of the highly ranked genes is suggestive of effects on mitochondrial function. In blood, consistent changes were more difficult to identify, despite individual studies tending to exhibit larger effects than the brain studies. Our results are the strongest evidence to date of a common transcriptome signature in the brains of individuals with ASD.

<h4>Background</h4>Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are widely prescribed to reduce cholesterol levels. Studies have suggested that statins are associated with reduced risk of liver cancer, but much of the evidence is from regions of the world with high liver cancer incidence rates. The current study examined the statins-liver cancer relationship in a low-rate region and examined the effects of preexisting liver disease and diabetes on that association.<h4>Methods</h4>A nested case-control study was conducted within the United Kingdom's Clinical Practice Research Datalink (CPRD). Persons diagnosed with primary liver cancer between 1988 and 2011 were matched to controls at a four-to-one ratio. Matches stratified on liver disease and on diabetes were also completed. Odds ratios (ORs) and 95% confidence intervals (CIs) for associations of statins with liver cancer were estimated using conditional logistic regression.<h4>Results</h4>In total, 1195 persons with primary liver cancer were matched to 4640 control patients. Statin use was associated with a statistically significantly reduced risk of liver cancer (ORadj = 0.55, 95% CI = 0.45 to 0.69), especially among current users (ORadj = 0.53, 95% CI = 0.42 to 0.66). The reduced risk was statistically significant in the presence (ORadj = 0.32, 95% CI = 0.17 to 0.57) and absence of liver disease (ORadj = 0.65, 95% CI = 0.52 to 0.81) and in the presence (ORadj = 0.30, 95% CI = 0.21 to 0.42) and absence of diabetes (ORadj = 0.66, 95% CI = 0.51 to 0.85).<h4>Conclusions</h4>In the current study in a low-rate area, statin use was associated with a statistically significantly reduced risk of liver cancer overall. Risk was particularly reduced among persons with liver disease and persons with diabetes, suggesting that statin use may be especially beneficial in persons at elevated risk of liver cancer.

Detecting in vivo transcription factor (TF) binding is important for understanding gene regulatory circuitries. ChIP-seq is a powerful technique to empirically define TF binding in vivo. However, the multitude of distinct TFs makes genome-wide profiling for them all labor-intensive and costly. Algorithms for in silico prediction of TF binding have been developed, based mostly on histone modification or DNase I hypersensitivity data in conjunction with DNA motif and other genomic features. However, technical limitations of these methods prevent them from being applied broadly, especially in clinical settings. We conducted a comprehensive survey involving multiple cell lines, TFs, and methylation types and found that there are intimate relationships between TF binding and methylation level changes around the binding sites. Exploiting the connection between DNA methylation and TF binding, we proposed a novel supervised learning approach to predict TF-DNA interaction using data from base-resolution whole-genome methylation sequencing experiments. We devised beta-binomial models to characterize methylation data around TF binding sites and the background. Along with other static genomic features, we adopted a random forest framework to predict TF-DNA interaction. After conducting comprehensive tests, we saw that the proposed method accurately predicts TF binding and performs favorably versus competing methods.

The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic machineries, cohesin and condensins, that ensure their faithful segregation during mitosis. Cohesin holds sister chromatids together immediately after their synthesis during S phase until the establishment of bipolar attachments to the mitotic spindle in metaphase. Condensins, on the other hand, attempt to "resolve" sister chromatids by counteracting cohesin. The products of the balancing acts of cohesin and condensins are metaphase chromosomes, in which two rod-shaped chromatids are connected primarily at the centromere. In anaphase, this connection is released by the action of separase that proteolytically cleaves the remaining population of cohesin. Recent studies uncover how this series of events might be mechanistically coupled with each other and intricately regulated by a number of regulatory factors.

Creating valid mouse models of slowly progressing human neurological diseases is challenging, not least because the short lifespan of rodents confounds realistic modelling of disease time course. With their large brains and long lives, sheep offer significant advantages for translational studies of human disease. Here we used normal and CLN5 Batten disease affected sheep to demonstrate the use of the species for studying neurological function in a model of human disease. We show that electroencephalography can be used in sheep, and that longitudinal recordings spanning many months are possible. This is the first time such an electroencephalography study has been performed in sheep. We characterized sleep in sheep, quantifying characteristic vigilance states and neurophysiological hallmarks such as sleep spindles. Mild sleep abnormalities and abnormal epileptiform waveforms were found in the electroencephalographies of Batten disease affected sheep. These abnormalities resemble the epileptiform activity seen in children with Batten disease and demonstrate the translational relevance of both the technique and the model. Given that both spontaneous and engineered sheep models of human neurodegenerative diseases already exist, sheep constitute a powerful species in which longitudinal in vivo studies can be conducted. This will advance our understanding of normal brain function and improve our capacity for translational research into neurological disorders.

<h4>Background</h4>To investigate potential cardiovascular and other effects of long-term pharmacological interleukin 1 (IL-1) inhibition, we studied genetic variants that produce inhibition of IL-1, a master regulator of inflammation.<h4>Methods</h4>We created a genetic score combining the effects of alleles of two common variants (rs6743376 and rs1542176) that are located upstream of IL1RN, the gene encoding the IL-1 receptor antagonist (IL-1Ra; an endogenous inhibitor of both IL-1α and IL-1β); both alleles increase soluble IL-1Ra protein concentration. We compared effects on inflammation biomarkers of this genetic score with those of anakinra, the recombinant form of IL-1Ra, which has previously been studied in randomised trials of rheumatoid arthritis and other inflammatory disorders. In primary analyses, we investigated the score in relation to rheumatoid arthritis and four cardiometabolic diseases (type 2 diabetes, coronary heart disease, ischaemic stroke, and abdominal aortic aneurysm; 453,411 total participants). In exploratory analyses, we studied the relation of the score to many disease traits and to 24 other disorders of proposed relevance to IL-1 signalling (746,171 total participants).<h4>Findings</h4>For each IL1RN minor allele inherited, serum concentrations of IL-1Ra increased by 0.22 SD (95% CI 0.18-0.25; 12.5%; p = 9.3 × 10(-33)), concentrations of interleukin 6 decreased by 0.02 SD (-0.04 to -0.01; -1.7%; p = 3.5 × 10(-3)), and concentrations of C-reactive protein decreased by 0.03 SD (-0.04 to -0.02; -3.4%; p = 7.7 × 10(-14)). We noted the effects of the genetic score on these inflammation biomarkers to be directionally concordant with those of anakinra. The allele count of the genetic score had roughly log-linear, dose-dependent associations with both IL-1Ra concentration and risk of coronary heart disease. For people who carried four IL-1Ra-raising alleles, the odds ratio for coronary heart disease was 1.15 (1.08-1.22; p = 1.8 × 10(-6)) compared with people who carried no IL-1Ra-raising alleles; the per-allele odds ratio for coronary heart disease was 1.03 (1.02-1.04; p = 3.9 × 10(-10)). Per-allele odds ratios were 0.97 (0.95-0.99; p = 9.9 × 10(-4)) for rheumatoid arthritis, 0.99 (0.97-1.01; p = 0.47) for type 2 diabetes, 1.00 (0.98-1.02; p = 0.92) for ischaemic stroke, and 1.08 (1.04-1.12; p = 1.8 × 10(-5)) for abdominal aortic aneurysm. In exploratory analyses, we observed per-allele increases in concentrations of proatherogenic lipids, including LDL-cholesterol, but no clear evidence of association for blood pressure, glycaemic traits, or any of the 24 other disorders studied. Modelling suggested that the observed increase in LDL-cholesterol could account for about a third of the association observed between the genetic score and increased coronary risk.<h4>Interpretation</h4>Human genetic data suggest that long-term dual IL-1α/β inhibition could increase cardiovascular risk and, conversely, reduce the risk of development of rheumatoid arthritis. The cardiovascular risk might, in part, be mediated through an increase in proatherogenic lipid concentrations.

<h4>Objectives</h4>Huntington's disease is a neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms that are caused by huntingtin gene (HTT) CAG trinucleotide repeat alleles of 36 or more units. A greater than expected prevalence of incompletely penetrant HTT CAG repeat alleles observed among individuals diagnosed with major depressive disorder raises the possibility that another mood disorder, bipolar disorder, could likewise be associated with Huntington's disease.<h4>Methods</h4>We assessed the distribution of HTT CAG repeat alleles in a cohort of individuals with bipolar disorder. HTT CAG allele sizes from 2,229 Caucasian individuals diagnosed with DSM-IV bipolar disorder were compared to allele sizes in 1,828 control individuals from multiple cohorts.<h4>Results</h4>We found that HTT CAG repeat alleles > 35 units were observed in only one of 4,458 chromosomes from individuals with bipolar disorder, compared to three of 3,656 chromosomes from control subjects.<h4>Conclusions</h4>These findings do not support an association between bipolar disorder and Huntington's disease.

One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homolog of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.

Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P31 using X-ray crystallography. Moreover, the locations of bound Sr(2+) and Cs(+) ions were identified by anomalous X-ray diffraction. The location of one Cs(+)-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na(+) (90 mM Na(+)/10 mM Cs(+)). From an activity assay using isothermal titration calorimetry, the bound Sr(2+) and Cs(+) ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs(+)-binding site provides important information that is useful for the design of artificial Cs(+)-binding sites that may be useful in the bioremediation of radioactive isotopes.

Autism spectrum disorders (ASDs) are genetically and clinically heterogeneous and lack effective medications to treat their core symptoms. Studies of syndromic ASDs caused by single gene mutations have provided insights into the pathophysiology of autism. Fragile X and Rett syndromes belong to the syndromic ASDs in which preclinical studies have identified rational targets for drug therapies focused on correcting underlying neural dysfunction. These preclinical discoveries are increasingly translating into exciting human clinical trials. Since there are significant molecular and neurobiological overlaps among ASDs, targeted treatments developed for fragile X and Rett syndromes may be helpful for autism of different etiologies. Here, we review the targeted pharmacological treatment of fragile X and Rett syndromes and discuss related issues in both preclinical studies and clinical trials of potential therapies for the diseases.

The main objective of this study was to map global gene expression in order to provide information about the populations of mRNA species participating in murine tooth development at 24 h intervals, starting at the 11th embryonic day (E11.5) up to the 7th post-natal day (P7). The levels of RNA species expressed during murine tooth development were mesured using a total of 58 deoxyoligonucleotide microarrays. Microarray data was validated using real-time RT-PCR. Differentially expressed genes (p < 0.05) were subjected to bioinformatic analysis to identify cellular activities significantly associated with these genes. Using ANOVA the microarray data yielded 4362 genes as being differentially expressed from the 11th embryonic day (E11.5) up to 7 days post-natal (P7), 1921 of these being genes without known functions. The remaining 2441 genes were subjected to further statistical analysis using a supervised procedure. Bioinformatic analysis results for each time-point studied suggests that the main molecular functions associated with genes expressed at the early pre-natal stages (E12.5-E18.5) were cell cycle progression, cell morphology, lipid metabolism, cellular growth, proliferation, senescence and apoptosis, whereas most genes expressed at post-natal and secretory stages (P0-P7) were significantly associated with regulation of cell migration, biosynthesis, differentiation, oxidative stress, polarization and cell death. Differentially expressed genes (DE) not described earlier during murine tooth development; Inositol 1, 4, 5-triphosphate receptor 3 (Itpr3), metallothionein 1(Mt1), cyclin-dependent kinase 4 (Cdk4), cathepsin D (Ctsd), keratin complex 2, basic, gene 6a (Krt2-6a), cofilin 1, non-muscle (Cfl1), cyclin 2 (Ccnd2), were verified by real-time RT-PCR.

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the malignant transformation of hematopoietic precursors to a pathogenic cell clone. Chromosomal band 11q23 harboring MLL (=mixed lineage leukemia) gene is known to be involved in rearrangements with variety of genes as activating partners of MLL in different AML subtypes. Overall, an unfavorable prognosis is associated with MLL abnormalities. Here we investigated an 11-month-old male presenting with hyperleukocytosis being diagnosed with AML subtype FAB-M5b. In banding cytogenetics a der(19)t(19;?)(q13.3;?) and del(Y)(q11.23) were found as sole aberrations. Molecular cytogenetics revealed that the MLL gene was disrupted and even partially lost due to a t(10;19;11)(p12.31;q13.31;q23.3), an MLL/MLLT10 fusion appeared, and the der(Y) was an asymmetric inverted duplication with breakpoints in Yp11.2 and Yq11.23. The patient got hematopoietic stem cell transplantation from his haploidentical mother. Still three months afterwards 15% of blasts were detected in bone marrow and later the patient was lost during follow-up. The present case highlights the necessity to exclude MLL rearrangements, even when there seems to be no actual hint from banding cytogenetics.

<h4>Unlabelled</h4>Few studies have investigated the impact of liver cirrhosis on dendritic cell function. The purpose of this study was to compare the activation and antigen-presentation capacity of monocyte-derived dendritic cells (MoDC) from cirrhotic patients (CIR) relative to healthy donors (HD). MoDC from CIR and HD were matured, phenotyped, irradiated and pulsed with 15mer peptides for two hepatocellular carcinoma-related antigens, alphafetoprotein and glypican-3, then co-cultured with autologous T-cells. Expanded T-cells were evaluated by interferon-gamma ELISPOT and intracellular staining. 15 CIR and 7 HD were studied. While CD14+ monocytes from CIR displayed enhanced M2 polarization, under MoDC-polarizing conditions, we identified no significant difference between HD and CIR in maturation-induced upregulation of co-stimulation markers. Furthermore, no significant differences were observed between CIR and HD in subsequent expansion of tumor antigen-specific IFNγ+ T-cells.<h4>Conclusion</h4>MoDCs isolated from cirrhotic individuals retain similar capacity for in vitro activation, maturation and antigen-presentation as those from healthy donors.

Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6(-/-) mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6(-/-) males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6(-/-) males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6(-/-) males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice.

<h4>Background</h4>Hepatocellular carcinoma (HCC) is on the rise and the sixth most common cancer worldwide. To combat HCC effectively research is directed towards its early detection and the development of targeted therapies. Given the fact that epidermal growth factor (EGF) is an important mitogen for hepatocytes we searched for disease regulated proteins to improve an understanding of the molecular pathogenesis of EGF induced HCC. Disease regulated proteins were studied by 2DE MALDI-TOF/TOF and a transcriptomic approach, by immunohistochemistry and advanced bioinformatics.<h4>Results</h4>Mapping of EGF induced liver cancer in a transgenic mouse model identified n = 96 (p < 0.05) significantly regulated proteins of which n = 54 were tumour-specific. To unravel molecular circuits linked to aberrant EGFR signalling diverse computational approaches were employed and this defined n = 7 key nodes using n = 82 disease regulated proteins for network construction. STRING analysis revealed protein-protein interactions of > 70% disease regulated proteins with individual proteins being validated by immunohistochemistry. The disease regulated network proteins were mapped to distinct pathways and bioinformatics provided novel insight into molecular circuits associated with significant changes in either glycolysis and gluconeogenesis, argine and proline metabolism, protein processing in endoplasmic reticulum, Hif- and MAPK signalling, lipoprotein metabolism, platelet activation and hemostatic control as a result of aberrant EGF signalling. The biological significance of the findings was corroborated with gene expression data derived from tumour tissues to evntually define a rationale by which tumours embark on intriguing changes in metabolism that is of utility for an understanding of tumour growth. Moreover, among the EGF tumour specific proteins n = 11 were likewise uniquely expressed in human HCC and for n = 49 proteins regulation in human HCC was confirmed using the publically available Human Protein Atlas depository, therefore demonstrating clinical significance.<h4>Conclusion</h4>Novel insight into the molecular pathogenesis of EGF induced liver cancer was obtained and among the 37 newly identified proteins several are likely candidates for the development of molecularly targeted therapies and include the nucleoside diphosphate kinase A, bifunctional ATP-dependent dihydroyacetone kinase and phosphatidylethanolamine-binding protein1, the latter being an inhibitor of the Raf-1 kinase.

<h4>Background</h4>Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis.<h4>Results</h4>Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2.<h4>Conclusions</h4>Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.

Hand, foot and mouth disease (HFMD) is a serious public health problem that has emerged over the past several decades. Pathogen detection by the Chinese national HFMD surveillance system has focused mainly on enterovirus 71 (EV71) and coxsackievirus A16 (CA16). Therefore, epidemiological information regarding the other causative enteroviruses is limited. To identify the pandemic enterovirus in Suzhou, Jiangsu province, China, clinical samples from patients with HFMD were collected from 2012 to 2013 and analyzed. The results revealed that CA16 was the most dominant HFMD pathogen in 2012, whereas CA6 and CA10 were the dominant pathogens in 2013. Phylogenetic analysis revealed that the C4a sub-genogroup of EV71 and the B1a and B1b sub-genogroups of CA16 continued to evolve and circulate in Suzhou. The CA6 strains were assigned to six genotypes (A-F) and the CA10 strains were assigned to seven genotypes (A-G), with clear geographical and temporal distributions. All of the CA6 strains in Suzhou belonged to genogroup F, and there were several lineages circulating in Suzhou. All of the CA10 strains in Suzhou belonged to genogroup G, and they had the same genetic origin. Co-infections of EV71/CA16 and CA6/CA10 were found in the samples, and bootscan analysis of 5'-untranslated regions (UTRs) revealed that some CA16 strains in Suzhou had genetic recombination with EV71. This property might allow CA16 to alter its evolvability and circulating ability. This study underscores the need for surveillance of CA6 and CA10 in the Yangtze River Delta and East China.

Convergence of multiple stromal cell types is required to develop a tumorigenic niche that nurtures the initial development of cancer and its dissemination. Although the immune and vascular systems have been shown to have strong influences on cancer, a growing body of evidence points to a role of the nervous system in promoting cancer development. This review discusses past and current research that shows the intriguing role of autonomic nerves, aided by neurotrophic growth factors and axon cues, in creating a favorable environment for the promotion of tumor formation and metastasis.

Uncontrolled bleeding and infection are the major causes of death and morbidity from traumatic wounds during military conflicts, disasters, and accidents. Because immediate treatment is critical to survival, it is desirable to have a lightweight and rapidly applicable bandage-one capable of delivering a hemostat that can quickly resolve bleeding while addressing infection over short and longer time frames. It is challenging to design thin film coatings capable of multidrug release, particularly when the drugs are quite different in nature (biologic versus small molecule, charged versus neutral) and the desired release profiles are different for each drug. Herein we have adopted a layer-by-layer film assembly technique to create a linear combination of two independently functional films capable of rapidly releasing thrombin within minutes while sustaining vancomycin elution for more than 24 h. By conjugating vancomycin to a hydrolytically degradable polyacid, poly(β-L-malic acid), we were able to create a robust thin film with loading and release kinetics that remain unaffected by the additional deposition of a thrombin-based film, demonstrating the possibility for future multitherapeutic films with independently tunable release kinetics.

<h4>Unlabelled</h4>Early detection of the highly aggressive malignancy cholangiocarcinoma (CCA) remains a challenge but has the potential to render the tumor curable by surgical removal. This study evaluates a biomarker panel for the diagnosis of CCA by DNA methylation analyses of biliary brush samples. The methylation status of 13 candidate genes (CDO1, CNRIP1, DCLK1, FBN1, INA, MAL, SEPT9, SFRP1, SNCA, SPG20, TMEFF2, VIM, and ZSCAN18) was investigated in 93 tissue samples (39 CCAs and 54 nonmalignant controls) using quantitative methylation-specific polymerase chain reaction. The 13 genes were further analyzed in a test series of biliary brush samples (15 CCAs and 20 nonmalignant primary sclerosing cholangitis controls), and the methylation status of the four best performing markers was validated (34 CCAs and 34 primary sclerosing cholangitis controls). Receiver operating characteristic curve analyses were used to evaluate the performance of individual biomarkers and the combination of biomarkers. The 13 candidate genes displayed a methylation frequency of 26%-82% in tissue samples. The four best-performing genes (CDO1, CNRIP1, SEPT9, and VIM) displayed individual methylation frequencies of 45%-77% in biliary brushes from CCA patients. Across the test and validation biliary brush series, this four-gene biomarker panel achieved a sensitivity of 85% and a specificity of 98%, with an area under the receiver operating characteristic curve of 0.944.<h4>Conclusion</h4>We report a straightforward biomarker assay with high sensitivity and specificity for CCA, outperforming standard brush cytology, and suggest that the biomarker panel, potentially in combination with cytological evaluation, may improve CCA detection, particularly among primary sclerosing cholangitis patients.

Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 and Thr305/Thr306 regulates kinase activity and modulates subcellular targeting and is critical for normal synaptic plasticity and learning and memory. Here, a mass spectrometry-based approach was used to identify Ca(2+)-dependent and -independent in vitro autophosphorylation sites in recombinant CaMKIIα and CaMKIIβ. CaMKII holoenzymes were then immunoprecipitated from subcellular fractions of forebrains isolated from either wild-type (WT) mice or mice with a Thr286 to Ala knock-in mutation of CaMKIIα (T286A-KI mice) and analyzed using the same approach in order to characterize in vivo phosphorylation sites in both CaMKII isoforms and identify CaMKII-associated proteins (CaMKAPs). A total of six and seven autophosphorylation sites in CaMKIIα and CaMKIIβ, respectively, were detected in WT mice. Thr286-phosphorylated CaMKIIα and Thr287-phosphorylated CaMKIIβ were selectively enriched in WT Triton-insoluble (synaptic) fractions compared to Triton-soluble (membrane) and cytosolic fractions. In contrast, Thr306-phosphorylated CaMKIIα and Ser315- and Thr320/Thr321-phosphorylated CaMKIIβ were selectively enriched in WT cytosolic fractions. The T286A-KI mutation significantly reduced levels of phosphorylation of CaMKIIα at Ser275 across all subcellular fractions and of cytosolic CaMKIIβ at Ser315 and Thr320/Thr321. Significantly more CaMKAPs coprecipitated with WT CaMKII holoenzymes in the synaptic fraction compared to that in the membrane fraction, with functions including scaffolding, microtubule organization, actin organization, ribosomal function, vesicle trafficking, and others. The T286A-KI mutation altered the interactions of multiple CaMKAPs with CaMKII, including several proteins linked to autism spectrum disorders. These data identify CaMKII isoform phosphorylation sites and a network of synaptic protein interactions that are sensitive to the abrogation of Thr286 autophosphorylation of CaMKIIα, likely contributing to the diverse synaptic and behavioral deficits of T286A-KI mice.

Prenatal maternal stress (PNMS) can impact a variety of outcomes in the offspring throughout childhood and persisting into adulthood as shown in human and animal studies. Many of the effects of PNMS on offspring outcomes likely reflect the effects of epigenetic changes, such as DNA methylation, to the fetal genome. However, no animal or human research can determine the extent to which the effects of PNMS on DNA methylation in human offspring is the result of the objective severity of the stressor to the pregnant mother, or her negative appraisal of the stressor or her resulting degree of negative stress. We examined the genome-wide DNA methylation profile in T cells from 34 adolescents whose mothers had rated the 1998 Québec ice storm's consequences as positive or negative (that is, cognitive appraisal). The methylation levels of 2872 CGs differed significantly between adolescents in the positive and negative maternal cognitive appraisal groups. These CGs are affiliated with 1564 different genes and with 408 different biological pathways, which are prominently featured in immune function. Importantly, there was a significant overlap in the differentially methylated CGs or genes and biological pathways that are associated with cognitive appraisal and those associated with objective PNMS as we reported previously. Our study suggests that pregnant women's cognitive appraisals of an independent stressor may have widespread effects on DNA methylation across the entire genome of their unborn children, detectable during adolescence. Therefore, cognitive appraisals could be an important predictor variable to explore in PNMS research.

<h4>Context</h4>DNA methylation has been proposed to play a critical role in many cellular and biological processes.<h4>Objective</h4>To examine the influence of Roux-en-Y gastric bypass (RYGB) surgery on genome-wide promoter-specific DNA methylation in obese patients. Promoters are involved in the initiation and regulation of gene transcription.<h4>Methods</h4>Promoter-specific DNA methylation in whole blood was measured in 11 obese patients (presurgery BMI >35 kg/m(2), 4 females), both before and 6 months after RYGB surgery, as well as once only in a control group of 16 normal-weight men. In addition, body weight and fasting plasma glucose were measured after an overnight fast.<h4>Results</h4>The mean genome-wide distance between promoter-specific DNA methylation of obese patients at six months after RYGB surgery and controls was shorter, as compared to that at baseline (p<0.001). Moreover, postsurgically, the DNA methylation of 51 promoters was significantly different from corresponding values that had been measured at baseline (28 upregulated and 23 downregulated, P<0.05 for all promoters, Bonferroni corrected). Among these promoters, an enrichment for genes involved in metabolic processes was found (n = 36, P<0.05). In addition, the mean DNA methylation of these 51 promoters was more similar after surgery to that of controls, than it had been at baseline (P<0.0001). When controlling for the RYGB surgery-induced drop in weight (-24% of respective baseline value) and fasting plasma glucose concentration (-16% of respective baseline value), the DNA methylation of only one out of 51 promoters (~2%) remained significantly different between the pre-and postsurgery time points.<h4>Conclusions</h4>Epigenetic modifications are proposed to play an important role in the development of and predisposition to metabolic diseases, including type II diabetes and obesity. Thus, our findings may form the basis for further investigations to unravel the molecular effects of gastric bypass surgery.<h4>Clinical trial</h4>ClinicalTrials.gov NCT01730742.

<h4>Background</h4>The first step in infection by Candida albicans is adhesion to host cells or implanted medical devices and this followed by hyphal growth and biofilm formation. Yeast-to-hyphal transition has long been identified as a key factor in fungal virulence. Following biofilm formation, C. albicans is usually less sensitive or insensitive to antifungals. Therefore, development of new antifungals with inhibitory action on adhesion, yeast-hyphal transition and biofilm formation by C. albicans is very necessary.<h4>Methods</h4>The effects of magnolol and honokiol on hypha growth were investigated using different induction media. Their inhibitory effects were determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide assay, and biofilm thickness and viability were observed by a confocal scanning laser microscope. Mammalian cells were used in adhesion assays. Genes related to hyphae development and cell adhesions were analyzed by real-time reverse transcription-polymerase chain reaction. The exogenous cyclic adenosine monophosphate was used to determine the mechanisms of action of magnolol and honokiol. Caenorhabditis elegans was used as an in vivo model to estimate the antifungal activities of magnolol and honokiol.<h4>Results and conclusions</h4>Magnolol and honokiol inhibited adhesion, the transition from yeast to hypha, and biofilm formation by C. albicans through the Ras1-cAMP-Efg1 pathway. Moreover, magnolol and honokiol prolonged the survival of nematodes infected by C. albicans. Magnolol and honokiol have potential inhibitory effects against biofilm formation by C. albicans.<h4>General significance</h4>This study provides useful information towards the development of new strategies to reduce the incidence of C. albicans biofilm-associated infection.

The two major goals of oral cancer chemoprevention efforts are the ability to segregate the high-risk patients and the identification of an effective pharmacologic agent that halts progression to invasive cancer. Considerable progress has recently been achieved in profiling invasive head and neck squamous cell carcinomas, particularly with the use of high-throughput technologies. A similar molecular characterization of potentially malignant oral epithelial lesions (OPML; leukoplakia and erythroplakia) is yet to be accomplished. It is postulated, though, that molecular profiling could lead to the discovery of novel markers of cancer risk that could also serve as potential targets for chemoprevention. In this perspective, we comment on the work by Izumchenko and colleagues that reports a high prevalence of NOTCH1 gain-of-function mutations in Chinese patients with OPMLs. Although additional studies are needed to validate the findings, the study is the first to link alterations in this gene in oral premalignancy. These findings could serve as a first prototype of a single gene mutation as a potential target in clinical chemoprevention setting.

<h4>Background</h4>Long chain acyl-CoA synthetases (ACSL) catalyze long-chain fatty acids (FA) conversion to acyl-CoAs. Temporal ACSL1 inactivation in mouse hearts (Acsl1(H-/-)) impaired FA oxidation and dramatically increased glucose uptake, glucose oxidation, and mTOR activation, resulting in cardiac hypertrophy. We used unbiased metabolomics and gene expression analyses to elucidate the cardiac cellular response to increased glucose use in a genetic model of inactivated FA oxidation.<h4>Methods and results</h4>Metabolomics analysis identified 60 metabolites altered in Acsl1(H-/-) hearts, including 6 related to glucose metabolism and 11 to cysteine and glutathione pathways. Concurrently, global cardiac transcriptional analysis revealed differential expression of 568 genes in Acsl1(H-/-) hearts, a subset of which we hypothesized were targets of mTOR; subsequently, we measured the transcriptional response of several genes after chronic mTOR inhibition via rapamycin treatment during the period in which cardiac hypertrophy develops. Hearts from Acsl1(H-/-) mice increased expression of several Hif1α-responsive glycolytic genes regulated by mTOR; additionally, expression of Scl7a5, Gsta1/2, Gdf15, and amino acid-responsive genes, Fgf21, Asns, Trib3, Mthfd2, were strikingly increased by mTOR activation.<h4>Conclusions</h4>The switch from FA to glucose use causes mTOR-dependent alterations in cardiac metabolism. We identified cardiac mTOR-regulated genes not previously identified in other cellular models, suggesting heart-specific mTOR signaling. Increased glucose use also changed glutathione-related pathways and compensation by mTOR. The hypertrophy, oxidative stress, and metabolic changes that occur within the heart when glucose supplants FA as a major energy source suggest that substrate switching to glucose is not entirely benign.

Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.

The limitations of the current prostate cancer (PCa) screening tests demands new biomarkers for early diagnosis of PCa. In this study, we aim to investigate serum autoantibody signatures as PCa specific biomarkers. PCa proteins were resolved by 2-DE and then transferred onto polyvinylidene difluoride membrane, which were subsequently incubated with either pooled serum from PCa patients or from normal controls. Mass spectrometry results have identified 18 antigens from 21 different 2-DE spots associated with PCa. Autoantibody response to antigens PRDX2, PRDX6 and ANXA11 in PCa patient's sera was confirmed using recombinant antigens. Further validation with an independent set of PCa patient's sera have shown relatively increased abundance of PRDX6 and ANXA11 antibodies in PCa patients. Formal concept analysis method was applied to assess whether the abundance of these autoantibodies could influence the classification of patients. However, sensitivity of the single antibody to discriminate prostate tumor and healthy controls varies from 70% to 80%, whereas combination of both PRDX6 and ANXA11 antibodies increased sensitivity to 90% for tumors and 100% for healthy controls. Therefore, we hereby report that the detection of these antibodies in PCa patient's serum in combination with the existing non-invasive diagnostic procedures may have significance in PCa diagnosis.<h4>Biological significance</h4>The present study aimed to investigate serum autoantibody signatures as new biomarkers for early diagnosis of prostate cancer (PCa). To investigate serum autoantibodies in patients with PCa, we used proteomics approach based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Total tissue proteins extracted from prostate were separated by 2-DE and then transferred onto polyvinylidene difluoride (PVDF) membrane, which were subsequently incubated with either pooled serum from PCa patients or from normal controls with no history for PCa. Proteomic analysis results have identified 18 antigens that showed antibody response specifically to cancer patient's serum. For validation experiments using recombinant antigens, confirmed autoantibody response to three antigens PRDX2, PRDX6 and ANXA11. Further validation using a second independent set of PCa patient's sera has shown relatively increased abundance of PRDX6 and ANXA11 antibodies specifically in PCa patients. Partition analysis of patients based on abundance of autoantibodies highlighted a combination of both PRDX6 and ANXA11 antibodies in serum with 90% sensitivity in case of tumors and 100% in case of healthy controls. Therefore, we hereby report that the detection of these antibodies in PCa patient's serum in combination with known markers may have significance in diagnosis of PCa with further validation in larger cohort of samples.

Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan- or oxaliplatin-specific resistance profiles, with non-reciprocal cross-resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance-associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models.

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150's exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5'-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5'-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.

Corticosterone is synthesized in the adrenal glands and is circulated throughout the body to perform regulatory functions in various tissues. The testis is known to synthesize and secrete testosterone and other androgens. We developed an accurate method to measure steroid content using liquid chromatography-mass spectrometry analysis. In the present study, significant levels of the precursor compounds of testosterone and corticosterone synthesis could be detected in rat testis using this method. After adrenalectomy, corticosterone remained in the blood and testicular tissue at approximately 1% of the amount present in the control testis. When the excised testicular tissue was washed and incubated with NADH, NADPH and progesterone, not only testosterone and its precursors but also 11-deoxycorticosterone and corticosterone were produced; the levels of 11-deoxycorticosterone and corticosterone increased with incubation time. The production rate of 11-deoxycorticosterone from progesterone was estimated to be approximately 1/20 that of 17-hydroxyprogesterone, and the corticosterone level was approximately 1/10 that of testosterone. These ratios coincided with those in the testicular tissue of the adrenalectomized rats, indicating that corticosterone was synthesized in the testis and not in the blood. A primary finding of this study was that corticosterone and testosterone were synthesized in a 1/10-20 ratio in the testis. It is concluded that corticosterone, which has various functions, such as the regulation of glycolysis and mediating spermatogenesis, is produced locally in the testis and that this the local production is convenient and functional to respond to local needs.

<h4>Objectives</h4>Human airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model.<h4>Methods</h4>Air-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively.<h4>Main results</h4>ALI cultures were readily infected by HRV. RNA-seq analysis of HRV infected ALI cultures identified sets of genes associated with asthma specific viral responses. These genes are related to inflammatory pathways, epithelial structure and remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1, MUC5AC, CDHR3), and novel ones that were identified for the first time in this study (e.g. CCRL1).<h4>Conclusions</h4>ALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.

Oral vaccines appear less effective in children in the developing world. Proposed biologic reasons include concurrent enteric infections, malnutrition, breast milk interference, and environmental enteropathy (EE). Rigorous study design and careful data management are essential to begin to understand this complex problem while assuring research subject safety. Herein, we describe the methodology and lessons learned in the PROVIDE study (Dhaka, Bangladesh). A randomized clinical trial platform evaluated the efficacy of delayed-dose oral rotavirus vaccine as well as the benefit of an injectable polio vaccine replacing one dose of oral polio vaccine. This rigorous infrastructure supported the additional examination of hypotheses of vaccine underperformance. Primary and secondary efficacy and immunogenicity measures for rotavirus and polio vaccines were measured, as well as the impact of EE and additional exploratory variables. Methods for the enrollment and 2-year follow-up of a 700 child birth cohort are described, including core laboratory, safety, regulatory, and data management practices. Intense efforts to standardize clinical, laboratory, and data management procedures in a developing world setting provide clinical trials rigor to all outcomes. Although this study infrastructure requires extensive time and effort, it allows optimized safety and confidence in the validity of data gathered in complex, developing country settings.

Histone deacetylases (HDACs) inhibitors are active in T-cell lymphoma and are undergoing pre-clinical and clinical testing in other neoplasms. Entinostat is an orally bioavailable class I HDAC inhibitor with a long half-life, which is under evaluation in haematological and solid tumour malignancies. To define the activity and biological effects of entinostat in B-cell lymphoma we studied its anti-tumour activity in several rituximab-sensitive or -resistant pre-clinical models. We demonstrated that entinostat is active in rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL) and primary tumour cells isolated from lymphoma patients (n = 36). Entinostat exposure decreased Bcl-XL (BCL2L1) levels and induced apoptosis in cells. In RSCL and RRCL, entinostat induced p21 (CDKN1A) expression leading to G1 cell cycle arrest and exhibited additive effects when combined with bortezomib or cytarabine. Caspase inhibition diminished entinostat activity in some primary tumour cells suggesting that entinostat has dual mechanisms-of-action. In addition, entinostat increased the expression of CD20 and adhesion molecules. Perhaps related to these effects, we observed a synergistic activity between entinostat and rituximab in a lymphoma-bearing severe combined immunodeficiency (SCID) mouse model. Our data suggests that entinostat is an active HDAC inhibitor that potentiates rituximab activity in vivo and supports its further clinical development in B-cell lymphoma.

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

Inflammation is a complex process involving distinct but overlapping biochemical and molecular events that are highly regulated. Pulsed electromagnetic field (PEMF) therapy is increasingly used to treat pain and edema associated with inflammation following surgery involving soft tissue. However, the molecular and cellular effects of PEMF therapy on pathways involved in the resolution of inflammation are poorly understood. Using cell culture lines relevant to trauma-induced inflammation of the skin (human dermal fibroblasts, human epidermal keratinocytes, and human mononuclear cells), we investigated the effect of PEMF on gene expression involved in the acute and resolution phases of inflammation. We found that PEMF treatment was followed by changes in the relative amount of messenger (m)RNAs encoding enzymes involved in heme catabolism and removal of reactive oxygen species, including an increase in heme oxygenase 1 and superoxide dismutase 3 mRNAs, in all cell types examined 2 hours after PEMF treatment. A relative increase in mRNAs encoding enzymes involved in lipid mediator biosynthesis was also observed, including an increase in arachidonate 12- and 15-lipoxygenase mRNAs in dermal fibroblasts and epidermal keratinocytes, respectively. The relative amount of both of these lipoxygenase mRNAs was elevated in mononuclear cells following PEMF treatment relative to nontreated cells. PEMF treatment was also followed by changes in the mRNA levels of several cytokines. A decrease in the relative amount of interleukin 1 beta mRNA was observed in mononuclear cells, similar to that previously reported for epidermal keratinocytes and dermal fibroblasts. Based on our results, we propose a model in which PEMF therapy may promote chronic inflammation resolution by mediating gene expression changes important for inhibiting and resolving inflammation.

Background and Aim. Although mercury is involved in several immunological diseases, nothing is known about its implication in celiac disease. Our aim was to evaluate blood and urinary levels of mercury in celiac patients. Methods. We prospectively enrolled 30 celiac patients (20 treated with normal duodenal mucosa and 10 untreated with duodenal atrophy) and 20 healthy controls from the same geographic area. Blood and urinary mercury concentrations were measured by means of flow injection inductively coupled plasma mass spectrometry. Enrolled patients underwent dental chart for amalgam fillings and completed a food-frequency questionnaire to evaluate diet and fish intake. Results. Mercury blood/urinary levels were 2.4 ± 2.3/1.0 ± 1.4, 10.2 ± 6.7/2.2 ± 3.0 and 3.7 ± 2.7/1.3 ± 1.2 in untreated CD, treated CD, and healthy controls, respectively. Resulting mercury levels were significantly higher in celiac patients following a gluten-free diet. No differences were found regarding fish intake and number of amalgam fillings. No demographic or clinical data were significantly associated with mercury levels in biologic samples. Conclusion. Data demonstrate a fourfold increase of mercury blood levels in celiac patients following a gluten-free diet. Further studies are needed to clarify its role in celiac mechanism.

Child psychiatric epidemiology has developed rapidly from descriptive, cross-sectional studies in the 1960s to the current large-scale prospective cohorts that unravel aetiological mechanisms. The objective of the study was to give an overview of epidemiological studies that have influenced child psychiatry. A chronological overview of selected major milestone studies was obtained to demonstrate the development of child psychiatric epidemiology, with a more in-depth discussion of findings and methodological issues exemplified in one cohort, the Generation R Study. Epidemiological studies have been successful in describing the frequency and course of child psychiatric problems. The high expectations that biological factors can be used to better explain, diagnose or predict child psychiatric problems have not been met. More ambitious large-scale child psychiatric cohort studies are needed, carefully applying genetics, neuroscience or other molecular research to better understand how the brain produces maladaptive behaviour. Progress will only be attained if the basic sciences are systematically integrated in cohorts with rigorous epidemiological designs rather than hurriedly inserted in child psychiatric studies.

Hemojuvelin (HJV), the coreceptor of the BMP-SMAD pathway that up-regulates hepcidin transcription, is a repulsive guidance molecule (RGMc) which undergoes a complex intracellular processing. Following autoproteolysis, it is exported to the cell surface both as a full-length and a heterodimeric protein. In vitro membrane HJV (m-HJV) is cleaved by the transmembrane serine protease TMPRSS6 to attenuate signalling and to inhibit hepcidin expression. In this study, we investigated the number and position of HJV cleavage sites by mutagenizing arginine residues (R), potential TMPRSS6 targets, to alanine (A). We analysed translation and membrane expression of HJV R mutants and the pattern of fragments they release in the culture media in the presence of TMPRSS6. Abnormal fragments were observed for mutants at arginine 121, 176, 218, 288 and 326. Considering that all variants, except HJV(R121A) , lack autoproteolytic activity and some (HJV(R176A) and HJV(R288A) ) are expressed at reduced levels on cell surface, we identified the fragments originating from either full-length or heterodimeric proteins and defined the residues 121 and 326 as the TMPRSS6 cleavage sites in both isoforms. Using the N-terminal FLAG-tagged HJV, we showed that residue 121 is critical also in the rearrangement of the N-terminal heterodimeric HJV. Exploiting the recently reported RGMb crystallographic structure, we generated a model of HJV that was used as input structure for all-atoms molecular dynamics simulation in explicit solvent. As assessed by in silico studies, we concluded that some arginines in the von Willebrand domain appear TMPRSS6 insensitive, likely because of partial protein structure destabilization.

<h4>Background</h4>MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. In this study, the role of miR-15a and miR-16-1 in gastric adenocarcinoma (GAC) was investigated.<h4>Methods</h4>The expression of miR-15a and miR-16-1 in cell lines and primary tumors was examined by miRNA qRT-PCR. Proliferative assays, colony formation, cell invasion and migration, flow cytometry analysis and in vivo study were performed by ectopic expression of miR-15a and miR-16-1. The putative target genes of miR-15a and miR-16-1 were explored by TargetScan and further validated.<h4>Results</h4>We found that miR-15a and miR-16-1 were down-regulated in GAC cell lines and primary tumor samples compared with normal gastric epithelium. Functional study demonstrated that ectopic expression of miR-15a and miR-16-1 suppressed cell proliferation, monolayer colony formation, invasion and migration, and xenograft formation in vivo. In addition, miR-15a and miR-16-1 induced G0/G1 cell cycle arrest which was further confirmed by Western blot and qRT-PCR of related cell cycle regulators. YAP1 was confirmed to be a functional target of miR-15a and miR-16-1 in GAC. YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in GAC cells. In clinical samples, YAP1 protein expression shows negative correlation with miR-15a and miR-16-1 expression.<h4>Conclusion</h4>In conclusion, targeting YAP1 by tumor suppressor miRNA miR-15a and miR-16-1 plays inhibitory effect and this might have a therapeutic potential in GAC.

<h4>Background</h4>The Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus Premier Pack has been designed to genotype 1936 gene variants thought to be essential for screening patients in personalized drug therapy. These variants include the cytochrome P450s (CYP450s), the key metabolizing enzymes, many other enzymes involved in phase I and phase II pharmacokinetic reactions, and signaling mediators associated with variability in clinical response to numerous drugs not only among individuals, but also between ethnic populations.<h4>Materials and methods</h4>We genotyped 600 Saudi individuals for 1936 variants on the DMET platform to evaluate their clinical potential in personalized medicine in ethnic Arabs.<h4>Results</h4>Approximately 49% each of the 437 CYP450 variants, 56% of the 581 transporters, 56% of 419 transferases, 48% of the 104 dehydrogenases, and 58% of the remaining 390 variants were detected. Several variants, such as rs3740071, rs6193, rs258751, rs6199, rs11568421, and rs8187797, exhibited significantly either higher or lower minor allele frequencies (MAFs) than those in other ethnic groups.<h4>Discussion</h4>The present study revealed some unique distribution trends for several variants in Arabs, which displayed partly inverse allelic prevalence compared to other ethnic populations. The results point therefore to the need to verify and ascertain the prevalence of a variant as a prerequisite for engaging it in clinical routine screening in personalized medicine in any given population.

This case study over time describes five years of experience with interventions to improve laboratory test utilization at an academic medical center. The high-frequency laboratory tests showing the biggest declines in order volume post intervention were serum albumin (36%) and erythrocyte sedimentation rate (17%). Introduction of restrictions for 170 high-cost send-out tests resulted in a 23% decline in order volume. Targeted interventions reduced mis-orders involving several "look-alike" tests: 1,25-dihydroxyvitamin D, 25-hydroxyvitamin D; manganese, magnesium; beta-2-glycoprotein, beta-2-microglobulin. Lastly, targeted alerts reduced duplicate orders of germline genetic testing and orders of hepatitis B surface antigen within 2 weeks of hepatitis B vaccination.

<h4>Background</h4>While it is known that acute respiratory illness (ARI) is caused by an array of viruses, less is known about co-detections and the resultant comparative symptoms and illness burden. This study examined the co-detections, the distribution of viruses, symptoms, and illness burden associated with ARI between December 2012 and March 2013.<h4>Methods</h4>Outpatients with ARI were assayed for presence of 18 viruses using multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) to simultaneously detect multiple viruses.<h4>Results</h4>Among 935 patients, 60% tested positive for a single virus, 9% tested positive for ≥1 virus and 287 (31%) tested negative. Among children (<18 years), the respective distributions were 63%, 14%, and 23%; whereas for younger adults (18-49 years), the distributions were 58%, 8%, and 34% and for older adults (≥50 years) the distributions were 61%, 5%, and 32% (P < 0.001). Co-detections were more common in children than older adults (P = 0.01), and less frequent in households without children (P = 0.003). Most frequently co-detected viruses were coronavirus, respiratory syncytial virus, and influenza A virus. Compared with single viral infections, those with co-detections less frequently reported sore throat (P = 0.01), missed fewer days of school (1.1 vs. 2 days; P = 0.04), or work (2 vs. 3 days; P = 0.03); other measures of illness severity did not vary.<h4>Conclusions</h4>Among outpatients with ARI, 69% of visits were associated with a viral etiology. Co-detections of specific clusters of viruses were observed in 9% of ARI cases particularly in children, were less frequent in households without children, and were less symptomatic (e.g., lower fever) than single infections.

<h4>Background</h4>The etiology of varicocele, a common cause of male factor infertility, remains unclear. Proteomic changes responsible for the underlying pathology of unilateral varicocele have not been evaluated. The objective of this prospective study was to employ proteomic techniques and bioinformatic tools to identify and analyze proteins of interest in infertile men with unilateral varicocele.<h4>Methods</h4>Spermatozoa from infertile men with unilateral varicocele (n=5) and from fertile men (control; n=5) were pooled in two groups respectively. Proteins were extracted and separated by 1-D SDS-PAGE. Bands were digested and identified on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Bioinformatic analysis identified the pathways and functions of the differentially expressed proteins (DEP).<h4>Results</h4>Sperm concentration, motility and morphology were lower, and reactive oxygen species levels were higher in unilateral varicocele patients compared to healthy controls. The total number of proteins identified were 1055, 1010 and 1042 in the fertile group, and 795, 713 and 763 proteins in the unilateral varicocele group. Of the 369 DEP between both groups, 120 proteins were unique to the fertile group and 38 proteins were unique to the unilateral varicocele group. Compared to the control group, 114 proteins were overexpressed while 97 proteins were underexpressed in the unilateral varicocele group. We have identified 29 proteins of interest that are involved in spermatogenesis and other fundamental reproductive events such as sperm maturation, acquisition of sperm motility, hyperactivation, capacitation, acrosome reaction and fertilization. The major functional pathways of the 359 DEP related to the unilateral varicocele group involve metabolism, disease, immune system, gene expression, signal transduction and apoptosis. Functional annotations showed that unilateral varicocele mostly affected small molecule biochemistry and post-translational modification proteins. Proteins expressed uniquely in the unilateral varicocele group were cysteine-rich secretory protein 2 precursor (CRISP2) and arginase-2 (ARG2).<h4>Conclusions</h4>The expression of these proteins of interest are altered and possibly functionally compromised in infertile men with unilateral varicocele. If validated, these proteins may lead to potential biomarker(s) and help better understand the mechanism involved in the pathophysiology of unilateral varicocele in infertile men.

Polymorphisms in the 5-HTT and BDNF genes are shown to affect their function at the molecular and serum level. Prior work has tried to correlate the polymorphisms with post-stroke depression (PSD), the results nevertheless remain indefinitive. A plausible reason accounting for the uncertainty relates to the small sample of each published trial. In this study, we have performed a comprehensive meta-analysis in order to evaluate the effects of 5-HTT and BDNF polymorphisms (5-HTTLPR, STin2 VNTR, 5-HTR2a 102 T/C, Val66Met) on genetic risk of PSD. Human case-control trials were identified by computer-assisted and manual searches. The article search was performed until October 2014. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the fixed effects meta-analysis to measure the effects 5-HTT and BDNF polymorphisms exerted on PSD. We also performed test of heterogeneity, test of publication bias, and sensitivity analysis to examine the reliability and stability of combined effects. 5-HTTLPR was clearly associated with genetic risk of PSD. The association seemed to be more pronounced in the homozygous model (OR = 0.34, 95% CI = 0.23-0.51, P(Q-test) = 0.63). Both the heterozygous model and the recessive model showed 50% decreased risk of PSD (OR = 0.50, 95% CI = 0.37-0.67, P(Q-test) = 0.91; OR = 0.50, 95% CI = 0.36-0.70, P(Q-test) = 0.43, respectively). Such significant association was also detected for Caucasian and Asian. These results were reliable and stable based on related analyses. Taken together, 5-HTTLPR polymorphism of the 5-HTT gene seems to protect against the occurrence of PSD. Small sample size for the polymorphisms within 5-HTT and BDNF genes may have caused underestimated associations, and a larger study is required to further assess the relations.

<h4>Background</h4>Thrombocytopenia has been shown to be the single most useful laboratory investigation for identifying subclinical cirrhosis of varying etiologies. However, alcohol per se can result in thrombocytopenia, and hence it is unclear whether platelet count can identify cirrhosis in patients who are alcoholic.<h4>Objectives</h4>To characterize the utility of clinical predictors, especially platelet count, for identifying the presence of cirrhosis in alcoholics. To develop a simple, objective model for identifying cirrhosis in alcoholics.<h4>Design</h4>Retrospective cohort study.<h4>Participants</h4>A total of 2,471 consecutive hospitalized patients with abnormal liver enzyme levels were screened, from which 272 patients with a history of recent and ongoing alcohol intake, negative diagnostic studies for alternative etiologies of chronic liver disease, and recent liver imaging with ultrasound or CT scan were included.<h4>Main measures</h4>Results of liver imaging and admission laboratory studies including liver enzymes, coagulation studies, and blood counts.<h4>Key results</h4>One hundred twenty-nine patients (47%) had cirrhosis based on imaging; 143 patients (53%) had no cirrhosis. A pre-sobriety platelet count (during ongoing alcohol intake) of less than 70*10(3) cells/mm(3) was effective for ruling in cirrhosis (positive likelihood ratio [LR] 6.8, 95% CI: 3.4, 14); platelet count greater than 200*10(3) was useful for ruling out cirrhosis in alcoholics (negative LR 0.18, 95% CI: 0.10, 0.35). Multivariate logistic regression analysis identified international normalized ratio (INR) (p < 0.01) and pre-sobriety platelet count (p < 0.01) as independent predictors of cirrhosis. A Model for identifying Cirrhosis in Alcoholic Liver Disease (MCALD) was developed using the INR and pre-sobriety platelet count; it had an area under the receiver operating characteristic curve of 0.89 and Hosmer-Lemeshow goodness of fit chi(2) (p value) of 8.9 (0.35) for predicting cirrhosis in alcoholics. A MCALD score > 5.5 corresponded to an increased likelihood of cirrhosis (LR: 6.5, 95% CI: 4.3, 11.0) and a MCALD score < 5.5 corresponded to decreased likelihood of cirrhosis in alcoholics (LR: 0.25, 95% CI: 0.19, 0.36). Sobriety platelet count (after alcohol abstinence) at a cutoff of 160*10(3) had positive LR of 7.9 (95% CI: 4.4, 14) and negative LR of 0.42 (95% CI: 0.34, 0.52) for predicting cirrhosis in alcoholics.<h4>Conclusions</h4>A simple model of platelet count and INR has good diagnostic accuracy for identifying cirrhosis in alcoholics.

A large economics literature has shown long term impacts of birth weight on adult outcomes, including IQ and earnings that are often robust to sibling or twin fixed effects. We examine potential mechanisms underlying these effects by incorporating findings from the genetics and neuroscience literatures. We use a sample of siblings combined with an "orchids and dandelions hypothesis", where the IQ of genetic dandelions is not affected by in utero nutrition variation but genetic orchids thrive under advantageous conditions and wilt in poor conditions. Indeed, using variation in three candidate genes related to neuroplasticity (APOE, BDNF, and COMT), we find substantial heterogeneity in the associations between birth weight and adult outcomes, where part of the population (i.e., "dandelions") is not affected by birth weight variation. Our results help uncover why birth weight affects adult outcomes.

<h4>Background</h4>Acute respiratory distress syndrome (ARDS) is a disease associated with a high mortality rate. The initial phase is characterized by induction of inflammatory cytokines and chemokines and influx of circulating inflammatory cells, including macrophages which play a pivotal role in the innate and adaptive immune responses to injury. Growing evidence points to phenotypic heterogeneity and plasticity between various macrophage activation states.<h4>Methods</h4>In this study, gene expression in alveolar macrophages and circulating leukocytes from healthy control subjects and patients with ARDS was assessed by mRNA microarray analysis.<h4>Results</h4>Both alveolar macrophages and circulating leukocytes demonstrated up-regulation of genes encoding chemotactic factors, antimicrobial peptides, chemokine receptors, and matrix metalloproteinases. Two genes, the pro-inflammatory S100A12 and the anti-inflammatory IL-1 decoy receptor IL-1R2 were significantly induced in both cell populations in ARDS patients, which was confirmed by protein quantification. Although S100A12 levels did not correlate with disease severity, there was a significant association between early plasma levels of IL-1R2 and APACHE III scores at presentation. Moreover, higher levels of IL-1R2 in plasma were observed in non-survivors as compared to survivors at later stages of ARDS.<h4>Conclusions</h4>These results suggest a hybrid state of alveolar macrophage activation in ARDS, with features of both alternative activation and immune tolerance/deactivation.. Furthermore, we have identified a novel plasma biomarker candidate in ARDS that correlates with the severity of systemic illness and mortality.

Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.

<h4>Background</h4>Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.<h4>Methodology/principal findings</h4>By repetitive RBV (100 μM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.<h4>Conclusions/significance</h4>These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.

<h4>Background</h4>The frequency of alcoholic liver disease (ALD), including alcoholic steatosis, hepatitis, and cirrhosis, varies significantly by ethnicity.<h4>Methods</h4>With the goal to assess the role of ethnicity in determining the age of onset and severity of ALD and to compare the risk factors for its progression among ethnic groups, we conducted a retrospective chart review of all patients with ALD who were admitted or were followed as outpatients at University of California Davis Medical Center between 2002 and 2010. After excluding HBsAg- and HIV-positive subjects, we reviewed the charts of 791 patients with ALD including 130 with alcoholic fatty liver, 154 with alcoholic hepatitis, and 507 with alcoholic cirrhosis.<h4>Results</h4>When controlling for all variables in the model, Hispanic patients presented at significantly 4 to 10 years younger ages than White/Caucasian patients, in each of the 3 disease severity categories, and the results were confirmed after excluding HCV Ab-/RNA-positive subjects. There were more obese Hispanic patients than White/Caucasian patients, whereas the proportion of patients with hepatitis C was significantly greater in African American subjects with alcoholic hepatitis, and the proportion of patients with diabetes mellitus was significantly lower in White/Caucasian subjects than in Hispanic subjects with cirrhosis. The proportion of subjects with severe alcoholic hepatitis was similar in Hispanic and White/Caucasian patients, but lower in African American subjects.<h4>Conclusions</h4>Ethnicity is a major factor affecting the age and severity of presentation of different subtypes of ALD.

Wilson's disease is an autosomal recessive disorder in which the liver does not properly release copper into bile, resulting in prominent copper accumulation in various tissues. Affected patients suffer from hepatic disorders and severe neurological defects. Experimental studies in mutant mice in which the copper-transporting ATPase gene (Atp7b) is disrupted revealed a drastic, time-dependent accumulation of hepatic copper that is accompanied by formation of regenerative nodes resembling cirrhosis. Therefore, these mice represent an excellent exploratory model for Wilson's disease. However, the precise time course in hepatic copper accumulation and its impact on other trace metals within the liver is yet poorly understood. We have recently established novel laser ablation inductively coupled plasma mass spectrometry protocols allowing quantitative metal imaging in human and murine liver tissue with high sensitivity, spatial resolution, specificity and quantification ability. By use of these techniques, we here aimed to comparatively analyse hepatic metal content in wild-type and Atp7b deficient mice during ageing. We demonstrate that the age-dependent accumulation of hepatic copper is strictly associated with a simultaneous increase in iron and zinc, while the intrahepatic concentration and distribution of other metals or metalloids is not affected. The same findings were obtained in well-defined human liver samples that were obtained from patients suffering from Wilson's disease. We conclude that in Wilson's disease the imbalances of hepatic copper during ageing are closely correlated with alterations in intrahepatic iron and zinc content.

G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs.

<h4>Objective</h4>To identify baseline characteristics of women with unexplained infertility to determine whether treatment with an aromatase inhibitor will result in a lower rate of multiple gestations than current standard ovulation induction medications.<h4>Design</h4>Randomized, prospective clinical trial.<h4>Setting</h4>Multicenter university-based clinical practices.<h4>Patient(s)</h4>A total of 900 couples with unexplained infertility.<h4>Intervention(s)</h4>Collection of baseline demographics, blood samples, and ultrasonographic assessments.<h4>Main outcome measure(s)</h4>Demographic, laboratory, imaging, and survey characteristics.<h4>Result(s)</h4>Demographic characteristics of women receiving clomiphene citrate (CC), letrozole, or gonadotropins for ovarian stimulation were very consistent. Their mean age was 32.2 ± 4.4 years and infertility duration was 34.7 ± 25.7 months, with 59% primary infertility. More than one-third of the women were current or past smokers. The mean body mass index (BMI) was 27 and mean antimüllerian hormone level was 2.6; only 11 women (1.3%) had antral follicle counts of <5. Similar observations were identified for hormonal profiles, ultrasound characterization of the ovaries, semen parameters, and quality of life assessments in both male and female partners.<h4>Conclusion(s)</h4>The cause of infertility in the couples recruited to this treatment trial is elusive, as the women were regularly ovulating and had evidence of good ovarian reserve both by basal FSH, antimüllerian hormone levels, and antral follicle counts; the male partners had normal semen parameters. The three treatment groups have common baseline characteristics, thereby providing comparable patient populations for testing the hypothesis that use of letrozole for ovarian stimulation can reduce the rates of multiples from that observed with gonadotropin and CC treatment.<h4>Clinical trial registration number</h4>NCT 01044862.

The synthesis, tautomerism and antibacterial activity of novel barbiturates is reported. In particular, 3-acyl and 3-carboxamidobarbiturates exhibited antibacterial activity, against susceptible and some resistant Gram-positive strains of particular interest is that these systems possess amenable molecular weight, rotatable bonds and number of proton-donors/acceptors for drug design as well as less lipophilic character, with physicochemical properties and ionic states that are similar to current antibiotic agents for oral and injectable use. Unfortunately, the reduction of plasma protein affinity by the barbituric core is not sufficient to achieve activity in vivo. Further optimization to reduce plasma protein affinity and/or elevate antibiotic potency is therefore required, but we believe that these systems offer unusual opportunities for antibiotic drug discovery.

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.

A series of mercaptoundecahydrododecaborate (B12H11SH(2-), BSH) bearing mono- and dicarboxyalkyl derivatives was prepared, characterized, and their reactivity towards amidation and esterification in DMF was evaluated. Symmetrical alkylation of BSH was achieved by treatment with primary haloalkyl carboxylic acids in aqueous acetonitrile to produce S,S-bis(carboxyalkyl)sulfonium-undecahydro-closo-dodecaborate tetramethylammonium salts. Unsymmetrically substituted sulfonium salts were obtained through a similar treatment of cyanoethylthioether-undecahydro-closo-dodecaborate tetramethylammonium salt with haloalkyl carboxylic acid. Selective removal of the remaining cyanoethyl group upon treatment with tetramethylammonium hydroxide yielded S-carboxyalkyl-thioether-undecahydro-closo-dodecaborate ditetramethylammonium salts. N,N'-dicyclohexylcarbodiimide (DCC) activated amidation of S,S-bis(carboxyalkyl)sulfonium-undecahydro-closo-dodecaborate or S-carboxyalkyl-thioether-undecahydro-closo-dodecaborate tetramethylammonium salts with propargylamine provided the opportunity to install terminal acetylene groups for further conjugation. These compounds acted as powerful building blocks for the synthesis of a broad range of 1,4-disubstituted 1,2,3-triazole products in high yields, utilizing the Cu(I)-mediated click cycloaddition reaction. The synthesis of BSH-lipid with a two-tailed moiety was also achieved, by esterification of S,S-bis(carboxyethyl)sulfoniumundecahydro-closo-dodecaborate(1-) tetramethylammonium salt with 1,2-O-distearoyl-sn-3-glycerol, which may prove useful in the liposomal boron delivery system. The bio-compatibility of the azide-alkyne click reaction was then utilized by performing this reaction in cell culture. The distribution of BSH in HeLa cells could be visualized by treating the cells first with a BSH-alkyne compound and then with Alexa Fluor 488(®) azide dye. The BSH-dye conjugate, which did not wash out, revealed the distribution of boron in the HeLa cells. Cytotoxicity assays of these BSH derivatives revealed that the synthesized BSH-conjugated triazoles possessed low cytotoxicity in HeLa cancer cells. Of these compounds, BSH conjugated triazole 15 induced a significant increase in the level of boron accumulation in HeLa cells.

Sex hormones have been implicated in neurite outgrowth, synaptogenesis, dendritic branching, myelination and other important mechanisms of neural plasticity. Here we review the evidence from animal experiments and human studies reporting interactions between sex hormones and the dominant neurotransmitters, such as serotonin, dopamine, GABA and glutamate. We provide an overview of accumulating data during physiological and pathological conditions and discuss currently conceptualized theories on how sex hormones potentially trigger neuroplasticity changes through these four neurochemical systems. Many brain regions have been demonstrated to express high densities for estrogen- and progesterone receptors, such as the amygdala, the hypothalamus, and the hippocampus. As the hippocampus is of particular relevance in the context of mediating structural plasticity in the adult brain, we put particular emphasis on what evidence could be gathered thus far that links differences in behavior, neurochemical patterns and hippocampal structure to a changing hormonal environment. Finally, we discuss how physiologically occurring hormonal transition periods in humans can be used to model how changes in sex hormones influence functional connectivity, neurotransmission and brain structure in vivo.

This study aimed to explore the influence of two genetic polymorphisms of the 5-hydroxytryptamine 1A receptor (5-HT1A) and solute carrier family 6, member 4 (SLC6A4) genes on the clinical symptoms and treatment resistance in Slovenian patients with schizophrenia. A total of 138 patients with schizophrenia were evaluated using the Positive and Negative Syndrome Scale, Brief Psychiatric Rating Scale, Clinical Global Impression, and Global Assessment of Functioning. Based on the selected criteria, 94 patients were included in the treatment-responsive and 44 in the treatment-resistant group. All subjects and 94 controls were genotyped for the 5-HT1A rs6295 and 5-HTTLPR polymorphisms. There were no statistically significant differences in the frequencies of these polymorphisms between the patients with schizophrenia and the control group and between the treatment-resistant and treatment-responsive group of schizophrenia patients. Polymorphisms rs6295 and 5-HTTLPR had an influence on the Global Assessment of Functioning scale score, while 5-HTTLPR also had an influence on the total score of the negative subscale within the Positive and Negative Syndrome Scale. Although we found no effect on progression toward the treatment-resistant schizophrenia, our data suggest that the rs6295 and 5-HTTLPR polymorphisms can influence some clinical symptoms in schizophrenia.

Until recently, only a little was understood about molecular mechanisms of the development of an intracranial aneurysm (IA). Recent advancements over the last decade in the field of genetics and molecular biology have provided us a wide variety of evidences supporting the notion that chronic inflammation is closely associated with the pathogenesis of IA development. In the field of genetics, large-scale Genome-wide association studies (GWAS) has identified some IA susceptible loci and genes related to cell cycle and endothelial function. Researches in molecular biology using human samples and animal models have revealed the common pathway of the initiation, progression, and rupture of IAs. IA formation begins with endothelial dysfunction followed by pathological remodeling with degenerative changes of vascular walls. Medical treatments inhibiting inflammatory cascades in IA development are likely to prevent IA progression and rupture. Statins and aspirin are expected to suppress IA progression by their anti-inflammatory effects. Decoy oligodeoxynucleotides (ODNs) inhibiting inflammatory transcription factors such as nuclear factor kappa-B (NF-κB) and Ets-1 are the other promising choice of the prevention of IA development. Further clarification of molecular mechanisms of the formation and progression of IAs will shed light to the pathogenesis of IA development and provide insight into novel diagnostic and therapeutic strategies for IAs.

Diverse subcellular localizations of the huntingtin-containing inclusion bodies are frequently suspected of reflecting crucial divisions between different cellular pathways contributing to the pathophysiology of Huntington's disease. Here, we use a panel of different N-terminal huntingtin fragments overexpressed in transfected neuronal and non-neuronal cells to demonstrate that it is the length of the N-terminal huntingtin fragments rather than a presence of any specific amino acid sequences that determines the ratio between the nuclear and cytoplasmic inclusion bodies. Importantly, the length of those fragments does also seem to strongly influence the folding of the aggregating huntingtin species, as indicated by the apparent differences in their accessibility for different antibodies directed against particular subdomains within the N-terminal part of huntingtin, although these differences do not correlate with the peptides' ability to efficiently aggregate within the cell nucleus. Furthermore, the relatively long huntingtin fragment containing 588 amino acids of the reference sequence shows intracellular behavior that is substantially different from that exhibited by its shorter counterparts (containing either 171, 120, 89 or 64 amino acids), as this rarely aggregating peptide is not only accumulating in cytoplasmic inclusions of slightly different morphology but is also most strongly affected by the FLAG-tagging procedure that unexpectedly induces (or enhances) autophagy-related processes. Together, our results reveal a significant heterogeneity of the huntingtin accumulation patterns that are observed at the cellular level. These patterns are not only strongly dependent on both the length and the amino acid composition of the N-terminal huntingtin peptides but also seem to engage different cellular mechanisms implicated in the pathogenesis of Huntington's disease, including the non-proteasomal degradation of potentially toxic huntingtin forms.

<h4>Background</h4>The prevalence of chronic diseases such as cancer, type 2 diabetes, metabolic syndrome (MetS), and cardiovascular disease increases with age in all populations. Epigenetic features are hypothesized to play important roles in the pathophysiology of age-associated diseases, but a map of these markers is lacking. We searched for genome-wide age-associated methylation signatures in peripheral blood of individuals at high risks for MetS by profiling 485,000 CpG sites in 192 individuals of Northern European ancestry using the Illumina HM450 array. Subjects (ages 6-85 years) were part of seven extended families, and 73% of adults and 32% of children were overweight or obese.<h4>Results</h4>We found 22,122 genome-wide significant age-associated CpG sites (P α=0.05 = 3.65 × 10(-7) after correction for multiple testing) of which 14,155 are positively associated with age while 7,967 are negatively associated. By applying a positional density-based clustering algorithm, we generated a map of epigenetic 'hot-spots' of age-associated genomic segments, which include 290 age-associated differentially methylated CpG clusters (aDMCs), of which 207 are positively associated with age. Gene/pathway enrichment analyses were performed on these clusters using FatiGO. Genes localized to both the positively (n = 241) and negatively (n = 16) age-associated clusters are significantly enriched in specific KEGG pathways and GO terms. The most significantly enriched pathways are the hedgehog signaling pathway (adjusted P = 3.96 × 10(-3)) and maturity-onset diabetes of the young (MODY) (adjusted P = 6.26 × 10(-3)) in the positive aDMCs and type I diabetes mellitus (adjusted P = 3.69 × 10(-7)) in the negative aDMCs. We also identified several epigenetic loci whose age-associated change rates differ between subjects diagnosed with MetS and those without.<h4>Conclusion</h4>We conclude that in a family cohort at high risk for MetS, age-associated epigenetic features enrich in biological pathways important for determining the fate of fat cells and for insulin production. We also observe that several genes known to be related to MetS show differential epigenetic response to age in individuals with and without MetS.

Apoptosis, or programmed cell death, is involved in numerous human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer, and is often confused with other types of cell death. Therefore strategies that enable visualized detection of apoptosis would be of enormous benefit in the clinic for diagnosis, patient management, and development of new therapies. In recent years, improved understanding of the apoptotic machinery and progress in imaging modalities have provided opportunities for researchers to formulate microscopic and macroscopic imaging strategies based on well-defined molecular markers and/or physiological features. Correspondingly, a large collection of apoptosis imaging probes and approaches have been documented in preclinical and clinical studies. In this review, we mainly discuss microscopic imaging assays and macroscopic imaging probes, ranging in complexity from simple attachments of reporter moieties to proteins that interact with apoptotic biomarkers, to rationally designed probes that target biochemical changes. Their clinical translation will also be our focus.

Clear cell renal cell carcinoma (ccRCC) is the major subtype of kidney cancer that is characterized by frequent inactivation of the von Hippel-Lindau (VHL) gene in 80-90% of the tumors. Recent reports using massive parallel sequencing technologies have discovered additional cancer driver genes. PBRM1 was found to be mutated in about 40% of ccRCC tumors, whereas BAP1 and SETD2 were each mutated in about 10-15% of ccRCC tumors. JARID1C and UTX, two histone H3 demethylases, were also found to harbor mutations in ccRCC, albeit at lower rates. ccRCC tumors display a high degree of intra-tumoral heterogeneity, with some mutations present in all cancer cells (ubiquitous), whereas others are subclonal. The VHL mutations were always ubiquitous in the tumors; PBRM1 mutations were also ubiquitous but to a lesser extent. On the contrary, mutations in BAP1, SETD2, JARID1C, and UTX were all subclonal, meaning that they were present in a subset of cancer cells in a tumor. The prognostic value of PBRM1 mutations in ccRCC is still controversial, whereas BAP1 mutations were tightly linked to worse clinical outcomes in multiple studies. The molecular functions of these newly identified cancer driver genes are discussed, and they were known readers, writers, or erasers of histone marks on histone H2 and H3 tails that are very close to each other, suggesting that these factors might functionally interact and affect common pathways. The studies on these newly identified tumor suppressors will shed light on ccRCC tumorigenesis and development, and will likely lead to development of novel therapeutic interventions for ccRCC patients.

Recent advances in genome-wide association studies are pointing towards a major role for voltage-gated ion channels in neuropsychiatric disorders and, in particular, bipolar disorder (BD). The phenotype of BD is complex, with symptoms during mood episodes and deficits persisting between episodes. We have tried to elucidate the common neurobiological mechanisms associated with ion channel signaling in order to provide a new perspective on the clinical symptoms and possible endophenotypes seen in BD patients. We propose a model in which the multiple variants in genes coding for ion channel proteins would perturb motivational circuits, synaptic plasticity, myelination, hypothalamic-pituitary-adrenal axis function, circadian neuronal rhythms, and energy regulation. These changes in neurobiological mechanisms would manifest in endophenotypes of aberrant reward processing, white matter hyperintensities, deficits in executive function, altered frontolimbic connectivity, increased amygdala activity, increased melatonin suppression, decreased REM latency, and aberrant myo-inositol/ATP shuttling. The endophenotypes result in behaviors of poor impulse control, motivational changes, cognitive deficits, abnormal stress response, sleep disturbances, and energy changes involving different neurobiological process domains. The hypothesis is that these disturbances start with altered neural circuitry during development, following which multiple environmental triggers may disrupt the neuronal excitability balance through an activity-dependent molecular process, resulting in clinical mood episodes.

The formation of visual circuitry is a multistep process that involves cell-cell interactions based on a range of molecular mechanisms. The correct implementation of individual events, including axon outgrowth and guidance, the formation of the topographic map, or the synaptic targeting of specific cellular subtypes, are prerequisites for a fully functional visual system that is able to appropriately process the information captured by the eyes. Cell adhesion molecules (CAMs) with their adhesive properties and their high functional diversity have been identified as key actors in several of these fundamental processes. Because of their growth-promoting properties, CAMs play an important role in neuritogenesis. Furthermore, they are necessary to control additional neurite development, regulating dendritic spacing and axon pathfinding. Finally, trans-synaptic interactions of CAMs ensure cell type-specific connectivity as a basis for the establishment of circuits processing distinct visual features. Recent discoveries implicating CAMs in novel mechanisms have led to a better general understanding of neural circuit formation, but also revealed an increasing complexity of their function. This review aims at describing the different levels of action for CAMs to shape neural connectivity, with a special focus on the visual system.

Alcoholism is a progressive brain disorder that is marked by increased sensitivity to the positive and negative reinforcing properties of ethanol, compulsive and habitual use despite negative consequences, and chronic relapse to alcohol drinking despite repeated attempts to reduce intake or abstain from alcohol. Emerging evidence from preclinical and clinical studies implicates serotonin (5-hydroxytryptamine; 5-HT) systems in the pathophysiology of alcohol dependence, suggesting that drugs targeting 5-HT systems may have utility in the treatment of alcohol use disorders. In this Review, we discuss the role of 5-HT systems in alcohol dependence with a focus on 5-HT interactions with neural circuits that govern all three stages of the addiction cycle. We attempt to clarify how 5-HT influences circuit function at these different stages with the goal of identifying neural targets for pharmacological treatment of this debilitating disorder.

<h4>Background</h4>Much of our understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. We know considerably less about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. Here, we investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops.<h4>Results</h4>We find that as animals grow, motoneurons increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity.<h4>Conclusions</h4>In the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals.

Immunoglobulin G (IgG)-based drugs are arguably the most successful class of protein therapeutics due in part to their remarkably long blood circulation. This arises from IgG interaction with the neonatal Fc receptor, FcRn. FcRn is the central regulator of IgG and albumin homeostasis throughout life and is increasingly being recognized as an important player in autoimmune disease, mucosal immunity, and tumor immune surveillance. Various engineering approaches that hijack or disrupt the FcRn-mediated transport pathway have been devised to develop long-lasting and non-invasive protein therapeutics, protein subunit vaccines, and therapeutics for treatment of autoimmune and infectious disease. In this review, we highlight the diverse biological functions of FcRn, emerging therapeutic opportunities, as well as the associated challenges of targeting FcRn for drug delivery and disease therapy.

<h4>Introduction</h4>Huntington's disease (HD) is an autosomal dominant disorder caused by an expanded CAG repeat (greater than 38) on the short arm of chromosome 4, resulting in loss and dysfunction of neurons in the neostriatum and cortex, leading to cognitive decline, motor dysfunction, and death, typically occurring 15 to 20 years after the onset of motor symptoms. Although an effective treatment for HD has remained elusive, current studies using transplants of bone-marrow-derived mesenchymal stem cells provides considerable promise. This study further investigates the efficacy of these transplants with a focus on comparing how passage number of these cells may affect subsequent efficacy following transplantation.<h4>Methods</h4>In this study, mesenchymal stem cells isolated from the bone-marrow of mice (BM MSCs), were labeled with Hoechst after low (3 to 8) or high (40 to 50) numbers of passages and then transplanted intrastriatally into 5-week-old R6/2 mice, which carries the N-terminal fragment of the human HD gene (145 to 155 repeats) and rapidly develops symptoms analogous to the human form of the disease.<h4>Results</h4>It was observed that the transplanted cells survived and the R6/2 mice displayed significant behavioral and morphological sparing compared to untreated R6/2 mice, with R6/2 mice receiving high passage BM MSCs displaying fewer deficits than those receiving low-passage BM MSCs. These beneficial effects are likely due to trophic support, as an increase in brain derived neurotrophic factor mRNA expression was observed in the striatum following transplantation of BM MSCs.<h4>Conclusion</h4>The results from this study demonstrate that BM MSCs hold significant therapeutic value for HD, and that the amount of time the cells are exposed to in vitro culture conditions can alter their efficacy.

The coating of fibrous polyester constructs with a layer of bioactive calcium phosphate (CP) is efficient to improve the potential use as bone tissue engineering scaffold. In this study, a fast procedure for the coating of electrospun poly(d,l-lactide) (PDLLA) fibers with a CP layer was optimized. The fiber surface was activated by immersion in demineralized water under ultrasonication. The resulting reactive groups served as nucleation points for CP precipitation, induced by alternate dipping of the samples in Ca(2+) and PO4 (3-) rich solutions. Variations in the conditions of the alternate dipping procedure, in particular the number of cycles, concentration and immersion time of both solutions, not only affected the degree of surface mineralization but also the type of deposited CP. For the current experimental conditions, in about 30 minutes either a slightly carbonated calcium deficient apatite (CDAp; Ca10-x-y (PO4 )6-x-y (HPO4 )y (CO3 )x (OH)2-x-y ) or a combination of apatite and dicalcium phosphate dihydrate (DCPD; CaHPO4 .2H2 O) was formed. The cell viability, adhesion, and proliferation of MC3T3-E1 cells on untreated samples were compared with samples coated with either an adequate amount of CDAp, an excess of CDAp or an excess of a combination of apatite and DCDP. After 7 days of culture the number of attached cells was significantly higher on all CP coated samples compared to the untreated PDLLA. In particular, the samples coated with an adequate amount of CDAp showed an exceedingly enhanced cell response with similar cell morphologies as the ones found on the positive control.

<h4>Introduction</h4>Given the overlapping modes of transmission of HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV), understanding the burden and relationship of these infections is critical for an effective response. Representative data on these infections among males who inject drugs (MWID), the key high-risk population for HIV in Vietnam, are currently lacking.<h4>Methods</h4>Data and stored specimens from Vietnam's 2009-2010 Integrated Biologic and Behavioral Survey, a cross-sectional study among high-risk populations, were used for this analysis. Plasma samples were tested for HIV, HBV, and HCV using commercial assays. A questionnaire was administered to provide demographic, behavior, and service-uptake information. Provincial-level analyses were conducted to profile MWID enrollees and to provide estimates on the prevalence of HIV, HBV, and HCV infection.<h4>Results</h4>Among 3010 MWID sampled across 10 provinces, the median (range) HIV prevalence was 28.1% (1.0%-55.5%). Median prevalence for current HBV infection (HBsAg+) was 14.1% (11.7%-28.0%), for previous exposure to HBV (total anti-HBc+) was 71.4% (49.9%-83.1%), and for current or past HCV infection (HCV Ag/Ab+) was 53.8% (10.9%-80.8%). In adjusted analysis, HBsAg+ (aOR: 2.09, 1.01-4.34) and HCV Ag/Ab+ (aOR: 19.58, 13.07-29.33) status were significantly associated with HIV infection; the association with total anti-HBc+ approached significance (aOR: 1.29, 0.99-1.68).<h4>Conclusion</h4>The prevalence and association between HIV, HBV, and HCV are high among MWID in Vietnam. These findings indicate the need for integrated policies and practice that for the surveillance, prevention, screening, and treatment of both HIV and viral hepatitis among MWID in Vietnam.

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.

Establishment and maintenance of the correct epigenetic code is essential for a plethora of physiological pathways and disturbed epigenetic patterns can provoke severe consequences, e.g. tumour formation. In recent years, epigenetic drugs altering the epigenome of tumours actively have been developed for anti-cancer therapies. However, such drugs could potentially also affect other physiological pathways and systems in which intact epigenetic patterns are essential. Amongst those, male fertility is one of the most prominent. Consequently, we addressed possible direct effects of two epigenetic drugs, decitabine and vorinostat, on both, the male germ line and fertility. In addition, we checked for putative transgenerational epigenetic effects on the germ line of subsequent generations (F1-F3). Parental adult male C57Bl/6 mice were treated with either decitabine or vorinostat and analysed as well as three subsequent untreated generations derived from these males. Treatment directly affected several reproductive parameters as testis (decitabine & vorinostat) and epididymis weight, size of accessory sex glands (vorinostat), the height of the seminiferous epithelium and sperm concentration and morphology (decitabine). Furthermore, after decitabine administration, DNA methylation of a number of loci was altered in sperm. However, when analysing fertility of treated mice (fertilisation, litter size and sex ratio), no major effect of the selected epigenetic drugs on male fertility was detected. In subsequent generations (F1-F3 generations) only subtle changes on reproductive organs, sperm parameters and DNA methylation but no overall effect on fertility was observed. Consequently, in mice, decitabine and vorinostat neither affected male fertility per se nor caused marked transgenerational effects. We therefore suggest that both drugs do not induce major adverse effects-in terms of male fertility and transgenerational epigenetic inheritance-when used in anti-cancer-therapies.

Curcumin, an ingredient of turmeric, exhibits a variety of biological activities such as anti-inflammatory, anti-atherosclerotic, anti-proliferative, anti-oxidant, anti-cancer and anti-metastatic. It is a highly pleiotropic molecule that inhibits cell proliferation and induces apoptosis in cancer cells. Despite its imperative biological activities, chemical instability, photo-instability and poor bioavailability limits its utilization as an effective therapeutic agent. Therefore, enhancing the bioavailability of curcumin may improve its therapeutic index for clinical setting. In the present study, we have conjugated curcumin with a biodegradable polymer Poly (D, L-lactic-co-glycolic acid) and evaluated its apoptotic potential in human colon carcinoma cells (HCT 116). The results show that curcumin-PLGA conjugate efficiently inhibits cell proliferation and cell survival in human colon carcinoma cells as compared to native curcumin. Additionally, curcumin conjugated with PLGA shows improved cellular uptake and exhibits controlled release at physiological pH as compared to native curcumin. The curcumin-PLGA conjugate efficiently activates the cascade of caspases and promotes intrinsic apoptotic signaling. Thus, the results suggest that conjugation potentiates the sustainability, anti-proliferative and apoptotic activity of curcumin. This approach could be a promising strategy to improve the therapeutic index of cancer therapy.

In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold). Three relative high abundant enzymes (XDH, LPL, and RNASE1) and cell division and proliferation protein (CREG1) may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.

The aim of the study was to determine whether chronic muscle overload has measurable effect on electrophysiological properties of motoneurons (MNs), and whether duration of this overload influences intensity of adaptations. The compensatory overload was induced in the rat medial gastrocnemius (MG) by bilateral tenotomy of its synergists (lateral gastrocnemius, soleus, and plantaris); as a result, only the MG was able to evoke the foot plantar flexion. To assure regular activation of the MG muscle, rats were placed in wheel-equipped cages and subjected to a low-level treadmill exercise. The intracellular recordings from MG motoneurons were made after 5 or 12 wk of the overload, and in a control group of intact rats. Some of the passive and threshold membrane properties as well as rhythmic firing properties were considerably modified in fast-type MNs, while remaining unaltered in slow-type MNs. The significant changes included a shortening of the spike duration and the spike rise time, an increase of the afterhyperpolarization amplitude, an increase of the input resistance, a decrease of the rheobase, and a decrease of the minimum current necessary to evoke steady-state firing. The data suggest higher excitability of fast-type MNs innervating the overloaded muscle, and a shift towards electrophysiological properties of slow-type MNs. All of the adaptations could be observed after 5 wk of the compensatory overload with no further changes occurring after 12 wk. This indicates that the response to an increased level of chronic activation of MNs is relatively quick and stable.

Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington's disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

The immune system and tumors are closely intertwined initially upon tumor development. During this period, tumors evolve to promote self-survival through immune escape, including by targeting crucial components involved in the presentation of antigens to the immune system in order to avoid recognition. Accordingly, components involved in MHC I presentation of tumor antigens are often mutated and down-regulated targets in tumors. On the other hand, the immune system has been shown to influence tumors through production of immunosuppressive cytokines, recruitment and polarization of cells favoring or impeding tumor escape or through production of anti-tumor cytokines promoting tumor rejection. We previously discovered that the hemochromatosis protein HFE, a negative regulator of iron absorption, dampens classical MHC I antigen presentation. In this study, we evaluated the impact of activated T lymphocytes purified from peripheral blood mononuclear cells (PBMC) on HFE expression in tumor cell lines. We co-cultured tumor cell lines from melanoma, lung, and kidney cancers with anti-CD3-activated PBMC and established that HFE expression is increased in tumor cell lines compared to healthy tissues, whilst being down-regulated significantly upon exposure to activated PBMC. HFE down-regulation was mediated by both CD4 and CD8 T lymphocytes, through production of soluble mediators, namely TNF and IFN-γ. These results suggest that the immune system may modulate tumor HFE expression in inflammatory conditions in order to regulate MHC I antigen presentation and promote tumor clearance.

Immunoglobulin E (IgE) is a central mediator of allergic (atopic) inflammation. Therapies directed against IgE can alleviate hay fever and allergic asthma. Genetic association studies have not yet identified novel therapeutic targets or pathways underlying IgE regulation. We therefore surveyed epigenetic associations between serum IgE concentrations and methylation at loci concentrated in CpG islands genome wide in 95 nuclear pedigrees, using DNA from peripheral blood leukocytes. We validated positive results in additional families and in subjects from the general population. Here we show replicated associations--with a meta-analysis false discovery rate less than 10(-4)--between IgE and low methylation at 36 loci. Genes annotated to these loci encode known eosinophil products, and also implicate phospholipid inflammatory mediators, specific transcription factors and mitochondrial proteins. We confirmed that methylation at these loci differed significantly in isolated eosinophils from subjects with and without asthma and high IgE levels. The top three loci accounted for 13% of IgE variation in the primary subject panel, explaining the tenfold higher variance found compared with that derived from large single-nucleotide polymorphism genome-wide association studies. This study identifies novel therapeutic targets and biomarkers for patient stratification for allergic diseases.

The aberrant expression of human microRNA-181a-1 (hsa-miR-181a) has been implicated in the pathogenesis of various cancers, serving as an oncogene or a tumor suppressor. However, the role of hsa-miR-181a in the pathogenesis of endometrial carcinoma (EC) and its clinical significance are unclear. This study aimed to search for the molecular targets of hsa-miR-181a using bioinformatic tools and then determine the expression levels of hsa-miR-181a in normal, hyperplasia, and EC samples from humans. To predict the targets of hsa-miR-181a, ten different algorithms were used, including miRanda-mirSVR, DIANA microT v5.0, miRDB, RNA22 v2, TargetMiner, TargetScan 6.2, PicTar, MicroCosm Targets v5, and miRWALK. Two algorithms, TarBase 6.0 and miRTarBase, were used to identify the validated targets of hsa-miR-181a-5p (a mature product of hsa-miR-181a), and the web-based Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.7 was used to provide biological functional interpretation of the validated targets of hsa-miR-181a-5p. A total of 78 formalin-fixed, paraffin-embedded tissue specimens from 65 patients and 13 healthy subjects were collected and examined, including normal endometrium (n=13), endometrial hyperplasia (n=18), and EC (37 type I and 10 type II EC cases). Our bioinformatic studies have showed that hsa-miR-181a might regulate a large number of target genes that are important in the regulation of critical cell processes, such as cell fate, cell survival, metabolism, and cell death. To date, 313 targets of hsa-miR-181a have been validated, and 22 of these targets are cancer genes. The precision of predictions by all the algorithms for hsa-miR-181a-1's targets was low. Many of these genes are involved in tumorigenesis of various cancers, including EC, based on the DAVID and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In comparison with normal endometrial tissue, the expression level of hsa-miR-181a was significantly increased in type I and type II EC (P<0.05), and type II EC exhibited a significant higher expression level of hsa-miR-181a than that in type I EC (P<0.05). In addition, there was a significant increase in the expression level of hsa-miR-181a in type II EC compared with endometrial hyperplasia (P<0.05). Taken together, these results suggest that hsa-miR-181a may serve as an oncogene in endometrial tumorigenesis and that hsa-miR-181a might be used as a new biomarker in the prediction of prognosis of EC in clinical practice. More functional and mechanistic studies are needed to validate the role of hsa-miR-181a in the development, progression, and metastasis of EC.

Iron is an essential component of erythropoiesis and its metabolism is tightly regulated by a variety of internal and external cues including iron storage, tissue hypoxia, inflammation and degree of erythropoiesis. There has been remarkable improvement in our understanding of the molecular mechanisms of iron metabolism past decades. The classical model of iron metabolism with iron response element/iron response protein (IRE/IRP) is now extended to include hepcidin model. Endogenous and exogenous signals funnel down to hepcidin via wide range of signaling pathways including Janus Kinase/Signal Transducer and Activator of Transcription 3 (JAK/STAT3), Bone Morphogenetic Protein/Hemojuvelin/Mothers Against Decapentaplegic Homolog (BMP/HJV/SMAD), and Von Hippel Lindau/Hypoxia-inducible factor/Erythropoietin (VHL/HIF/EPO), then relay to ferroportin, which directly regulates intra- and extracellular iron levels. The successful molecular delineation of iron metabolism further enhanced our understanding of common genetic and acquired disorder, hemochromatosis. The majority of the hereditary hemochromatosis (HH) patients are now shown to have mutations in the genes coding either upstream or downstream proteins of hepcidin, resulting in iron overload. The update on hepcidin centered mechanisms of iron metabolism and their clinical perspective in hemochromatosis will be discussed in this review.

Many neurodegenerative disorders are linked to irreversible protein aggregation, a process that usually comes along with toxicity and serious cellular damage. However, it is emerging that protein aggregation can also serve for physiological purposes, as impressively shown for prions. While the aggregation of this protein family was initially considered exclusively toxic in mammalians organisms, it is now almost clear that many other proteins adopt prion-like attributes to rationally polymerize into higher order complexes with organized physiologic roles. This implies that cells can tolerate at least in some measure the accumulation of inherently dangerous protein aggregates for functional profit. This review summarizes currently known strategies that living organisms adopt to preserve beneficial aggregation, and to prevent the catastrophic accumulation of toxic aggregates that frequently accompany neurodegeneration.

Background. Even if various pathophysiological events have been proposed as explanations, the putative cause of sudden hearing loss remains unclear. Objectives. To investigate and to reveal associations (if any) between the main iron-related gene variants and idiopathic sudden sensorineural hearing loss. Study Design. Case-control study. Materials and Methods. A total of 200 sudden sensorineural hearing loss patients (median age 63.65 years; range 10-92) were compared with 400 healthy control subjects. The following genetic variants were investigated: the polymorphism c.-8CG in the promoter of the ferroportin gene (FPN1; SLC40A1), the two isoforms C1 and C2 (p.P570S) of the transferrin protein (TF), the amino acidic substitutions p.H63D and p.C282Y in the hereditary hemochromatosis protein (HFE), and the polymorphism c.-582AG in the promoter of the HEPC gene, which encodes the protein hepcidin (HAMP). Results. The homozygous genotype c.-8GG of the SLC40A1 gene revealed an OR for ISSNHL risk of 4.27 (CI 95%, 2.65-6.89; P = 0.001), being overrepresented among cases. Conclusions. Our study indicates that the homozygous genotype FPN1 -8GG was significantly associated with increased risk of developing sudden hearing loss. These findings suggest new research should be conducted in the field of iron homeostasis in the inner ear.

The fatality of avian influenza A(H7N9) infection in humans was over 30%. To identify human genetic susceptibility to A(H7N9) infection, we performed a genome-wide association study (GWAS) involving 102 A(H7N9) patients and 106 heavily-exposed healthy poultry workers, a sample size critically restricted by the small number of human A(H7N9) cases. To tackle the stringent significance cutoff of GWAS, we utilized an artificial imputation program SnipSnip to improve the association signals. In single-SNP analysis, one of the top SNPs was rs13057866 of LGALS1. The artificial imputation (AI) identified three non-genotyped causal variants, which can be represented by three anchor/partner SNP pairs rs13057866/rs9622682 (AI P = 1.81 × 10(-7)), rs4820294/rs2899292 (2.13 × 10(-7)) and rs62236673/rs2899292 (4.25 × 10(-7)) respectively. Haplotype analysis of rs4820294 and rs2899292 could simulate the signal of a causal variant. The rs4820294/rs2899292 haplotype GG, in association with protection from A(H7N9) infection (OR = 0.26, P = 5.92 × 10(-7)) correlated to significantly higher levels of LGALS1 mRNA (P = 0.050) and protein expression (P = 0.025) in lymphoblast cell lines. Additionally, rs4820294 was mapped as an eQTL in human primary monocytes and lung tissues. In conclusion, functional variants of LGALS1 causing the expression variations are contributable to the differential susceptibility to influenza A(H7N9).

<h4>Purpose</h4>Colorectal cancer (CRC) is one of the major health problems worldwide and is often diagnosed at late stage. There is growing body of evidence in early detection of this disease with novel screening modalities to reduce compliance and increase specificity of available methods. The aim of current review is to give an overview on currently available screening methods (e.g., fecal occult blood testing (FOBT), flexible sigmoidoscopy, and colonoscopy), with their own merits and disadvantages, and new genetic/epigenetic/protein markers, as novel screening modalities.<h4>Result</h4>There are several serum and fecal biomarkers that can predict CRC and polyps. Overall sensitivities for detection by fecal DNA markers ranged from 53 to 87%, while a panel of serum protein markers provides a sensitivity/specificity up to 85% for CRC. In particular, DNA methylation markers (e.g., SEPT9, SFRP2, and ALX4), circulating microRNAs (e.g., microRNA21), SNPs in microRNAs binding site (e.g., rs4596 located within a target region of the predicted miR-518a-5p and miR-527), protein markers (e.g., carcinoembryonic antigen, N-methyltransferase), or microsatellites instability in tumors with deficient mismatch repair of some genes are among the most interesting and promising biomarkers.<h4>Conclusion</h4>Increasing evidence supports the use of combined fecal and serum biomarkers with sigmoidoscopy and colonoscopy screening in order to maximize the benefits and reduce the number of false-positive tests and patients undergoing invasive methods, which in turn could overcome the limitations of the current screening methods for early detection of CRC and adenomas.

<h4>Objective</h4>To investigate the association between VNTR polymorphisms of DRD4, DAT1 and 5-HTT genes and obesity.<h4>Material and methods</h4>Peripheral blood samples of 234 obese (BMI ≥ 30) and 148 healthy individuals (BMI ≤ 25) were objected to PCR to detect the VNTR of the 2nd intron of 5-HTT, 3rd exon of DRD4 and 3'UTR of DAT1 genes.<h4>Results</h4>The association between obesity and genotype distributions of 5-HTT, DAT1 and DRD4 genes and between obesity and distributions of allele frequencies were tested by Chi Square (χ(2)) test and were not found statistically significant. BMI values for genotype of obese and morbidly obese (BMI > 40) individuals were analyzed by Kruskal-Wallis and not found statistically significant differences between BMI values for the most frequent genotypes of 5-HTT, DAT1 and DRD4 genes.<h4>Conclusions</h4>As a conclusion, there was no association between 5-HTT, DAT1 and DRD4 genes VNTR polymorphisms and obesity.

Low back pain is the most common musculoskeletal problem and the single most common cause of disability, often attributed to degeneration of the intervertebral disc. Lack of effective treatment is directly related to our limited understanding of the pathways responsible for maintaining disc health. While transcriptional analysis has permitted initial insights into the biology of the intervertebral disc, complete proteomic characterization is required. We therefore employed liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) protein/peptide separation and mass spectrometric analyses to characterize the protein content of intervertebral discs from skeletally mature wild-type mice. A total of 1360 proteins were identified and categorized using PANTHER. Identified proteins were primarily intracellular/plasma membrane (35%), organelle (30%), macromolecular complex (10%), extracellular region (9%). Molecular function categorization resulted in three distinct categories: catalytic activity (33%), binding (molecule interactions) (29%), and structural activity (13%). To validate our list, we confirmed the presence of 14 of 20 previously identified IVD-associated markers, including matrix proteins, transcriptional regulators, and secreted proteins. Immunohistochemical analysis confirmed distinct localization patterns of select protein with the intervertebral disc. Characterization of the protein composition of healthy intervertebral disc tissue is an important first step in identifying cellular processes and pathways disrupted during aging or disease progression.

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is an autosomal dominantly-inherited disease that produces progressive problems with movement. It is caused by the expansion of an area of CAG repeats in a coding region of ATXN3. The number of repeats is inversely associated with age at disease onset (AO) and is significantly associated with disease severity; however, the degree of CAG expansion only explains 50 to 70% of variance in AO. We tested two SNPs, rs709930 and rs910369, in the 3' UTR of ATXN3 gene for association with SCA3/MJD risk and with SCA3/MJD AO in an independent cohort of 170 patients with SCA3/MJD and 200 healthy controls from mainland China. rs709930 genotype frequencies were statistically significantly different between patients and controls (p = 0.001, α = 0.05). SCA3/MJD patients carrying the rs709930 A allele and rs910369 T allele experienced an earlier onset, with a decrease in AO of approximately 2 to 4 years. The two novel SNPs found in this study might be genetic modifiers for AO in SCA3/MJD.

<h4>Background</h4>As measurement of arterial oxygen saturation (SpO2) is common in the delivery room, target SpO2 ranges allow clinicians to titrate oxygen therapy for preterm infants in order to achieve saturation levels similar to those seen in normal term infants in the first minutes of life. However, the influence of the onset of ventilation and the timing of cord clamping on systemic and cerebral oxygenation is not known.<h4>Aim</h4>We investigated whether the initiation of ventilation, prior to, or after umbilical cord clamping, altered systemic and cerebral oxygenation in preterm lambs.<h4>Methods</h4>Systemic and cerebral blood-flows, pressures and peripheral SpO2 and regional cerebral tissue oxygenation (SctO2) were measured continuously in apnoeic preterm lambs (126±1 day gestation). Positive pressure ventilation was initiated either 1) prior to umbilical cord clamping, or 2) after umbilical cord clamping. Lambs were monitored intensively prior to intervention, and for 10 minutes following umbilical cord clamping.<h4>Results</h4>Clamping the umbilical cord prior to ventilation resulted in a rapid decrease in SpO2 and SctO2, and an increase in arterial pressure, cerebral blood flow and cerebral oxygen extraction. Ventilation restored oxygenation and haemodynamics by 5-6 minutes. No such disturbances in peripheral or cerebral oxygenation and haemodynamics were observed when ventilation was initiated prior to cord clamping.<h4>Conclusion</h4>The establishment of ventilation prior to umbilical cord clamping facilitated a smooth transition to systemic and cerebral oxygenation following birth. SpO2 nomograms may need to be re-evaluated to reflect physiological management of preterm infants in the delivery room.

<h4>Purpose</h4>Adoptive transfer of Epstein-Barr virus (EBV)-specific and cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) genetically modified to express a chimeric antigen receptor (CAR) induces objective tumor responses in clinical trials. In vivo expansion and persistence of these cells are crucial to achieve sustained clinical responses. We aimed to develop an off-the-shelf whole-cell vaccine to boost CAR-redirected virus-specific CTLs in vivo after adoptive transfer. As proof of principle, we validated our vaccine approach by boosting CMV-specific CTLs (CMV-CTLs) engineered with a CAR that targets the GD2 antigen.<h4>Experimental design</h4>We generated the whole-cell vaccine by engineering the K562 cell line to express the CMV-pp65 protein and the immune stimulatory molecules CD40L and OX40L. Single-cell-derived clones were used to stimulate CMV-CTLs in vitro and in vivo in a xenograft model. We also assessed whether the in vivo boosting of CAR-redirected CMV-CTLs with the whole-cell vaccine enhances the antitumor responses. Finally, we addressed potential safety concerns by including the inducible safety switch caspase9 (iC9) gene in the whole-cell vaccine.<h4>Results</h4>We found that K562-expressing CMV-pp65, CD40L, and OX40L effectively stimulate CMV-specific responses in vitro by promoting antigen cross-presentation to professional antigen-presenting cells (APCs). Vaccination also enhances antitumor effects of CAR-redirected CMV-CTLs in xenograft tumor models. Activation of the iC9 gene successfully induces growth arrest of engineered K562 implanted in mice.<h4>Conclusions</h4>Vaccination with a whole-cell vaccine obtained from K562 engineered to express CMV-pp65, CD40L, OX40L and iC9 can safely enhance the antitumor effects of CAR-redirected CMV-CTLs.

5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS generation in A549 cells. Collectively, this SILAC study quantitatively evaluates the proteomic response to treatment with DMXAA that helps to globally identify the potential molecular targets and elucidate the underlying mechanism of DMXAA in the treatment of NSCLC.

<h4>Unlabelled</h4>Non-alcoholic fatty liver disease has an increasing prevalence in Western countries, affecting up to 20% of the population.<h4>Objective</h4>The aim of this project was to systematically review and summarise the genetic association studies that investigate possible genetic influences that confer susceptibility to non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.<h4>Design</h4>The MEDLINE and SCOPUS databases were searched to identify candidate gene studies on histologically diagnosed non-alcoholic fatty liver disease.<h4>Results</h4>A total of 85 articles have been summarised and categorised on the basis of the general pathway each candidate gene is involved in, including lipid metabolism, lipoprotein processing, cholesterol synthesis, glucose homoeostasis, inflammatory response, protection against oxidative stress and whole body metabolism.<h4>Conclusions</h4>The main findings demonstrate a small but consistent association of PNPLA3 with non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Genetic association studies have investigated general disease susceptibility, histological characteristics, severity and progression. However, further study is required to better elucidate the genetic factors influencing fatty liver disease.

Converging lines of evidence have identified genetic interactions between the serotonin transporter (SERT) gene and ITGB3, which encodes the β3 subunit that forms the αIIbβ3 and αvβ3 integrin receptor complexes. Here we examine the consequences of haploinsufficiency in the mouse integrin β3 subunit gene (Itgb3) on SERT function and selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRI) effectiveness in vivo. Biochemical fractionation studies and immunofluorescent staining of murine brain slices reveal that αvβ3 receptors and SERTs are enriched in presynaptic membranes from several brain regions and that αvβ3 colocalizes with a subpopulation of SERT-containing synapses in raphe nuclei. Notably, we establish that loss of a single allele of Itgb3 in murine neurons is sufficient to decrease 5-HT uptake by SERT in midbrain synaptosomes. Pharmacological assays to elucidate the αvβ3-mediated mechanism of reduced SERT function indicate that decreased integrin β3 subunit expression scales down the population size of active SERT molecules and, as a consequence, lowers the effective dose of SSRIs. These data are consistent with the existence of a subpopulation of SERTs that are tightly modulated by integrin αvβ3 and significantly contribute to global SERT function at 5-HT synapses in the midbrain. Importantly, our screen of a normal human population for single nucleotide polymorphisms in human ITGB3 identified a variant associated with reductions in integrin β3 expression levels that parallel our mouse findings. Thus, polymorphisms in human ITGB3 may contribute to the differential responsiveness of select patients to SSRIs.

The recovery of motor function in rats is inhibited following contusion spinal cord injury (cSCI). However, the mechanism of tumour necrosis factor α (TNF-α) in motor function after cSCI associated with peroxiredoxin 6 (PRDX6) remains unknown. We randomly divided rats into four groups: sham, cSCI, vector and lentivirus mediating TNF-α RNA interference (TNF-α-RNAi-LV) group. The Basso, Beattie, Bresnahan (BBB) scale was used to evaluate motor function. Real-time quantitative PCR (qRT-PCR) and western blotting were used to detect the expression of TNF-α and PRDX6, which were located in neurons using immunohistochemistry (IHC) and immunofluorescence. Subsequently, lentiviral-mediated TNF-α was used to determine the role of TNF-αand the relationship of PRDX6 and TNF-α in cSCI. After cSCI, the motor capability of hind limbs disappeared and was followed by recovery of function. IHC analysis indicated that TNF-α and PRDX6 were primarily located in spinal cord neurons. TNF-α interference significantly improved neural behaviour and increased expression of PRDX6. Our study suggests that inhibition of TNF-α can promote the recovery of motor function. The underlying mechanism of TNF-α-promoted motor function may be connected with the up-regulation of PRDX6. This provides a new strategy or target for the clinical treatment of SCI in future.

Selective macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. Here, we demonstrate that Huntingtin, the Huntington disease gene product, functions as a scaffold protein for selective macroautophagy but it is dispensable for non-selective macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with Lys-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress is attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which releases ULK1 from negative regulation by mTOR. Our data uncover an important physiological function of Huntingtin and provide a missing link in the activation of selective macroautophagy in metazoans.

<h4>Background</h4>Trauma-induced coagulopathy (TIC) is a disorder of the blood clotting process that occurs soon after trauma injury. A diagnosis of TIC on admission is associated with increased mortality rates, increased burdens of transfusion, greater risks of complications and longer stays in critical care. Current diagnostic testing follows local hospital processes and normally involves conventional coagulation tests including prothrombin time ratio/international normalized ratio (PTr/INR), activated partial prothrombin time and full blood count. In some centres, thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are standard tests, but in the UK they are more commonly used in research settings.<h4>Objectives</h4>The objective was to determine the diagnostic accuracy of thromboelastography (TEG) and rotational thromboelastometry (ROTEM) for TIC in adult trauma patients with bleeding, using a reference standard of prothrombin time ratio and/or the international normalized ratio.<h4>Search methods</h4>We ran the search on 4 March 2013. Searches ran from 1970 to current. We searched The Cochrane Library, MEDLINE (OvidSP), EMBASE Classic and EMBASE, eleven other databases, the web, and clinical trials registers. The Cochrane Injuries Group's specialised register was not searched for this review as it does not contain diagnostic test accuracy studies. We also screened reference lists, conducted forward citation searches and contacted authors.<h4>Selection criteria</h4>We included all cross-sectional studies investigating the diagnostic test accuracy of TEG and ROTEM in patients with clinically suspected TIC, as well as case-control studies. Participants were adult trauma patients in both military and civilian settings. TIC was defined as a PTr/INR reading of 1.2 or greater, or 1.5 or greater.<h4>Data collection and analysis</h4>We piloted and performed all review stages in duplicate, including quality assessment using the QUADAS-2 tool, adhering to guidance in the Cochrane Handbook for Diagnostic Test Accuracy Reviews. We analysed sensitivity and specificity of included studies narratively as there were insufficient studies to perform a meta-analysis.<h4>Main results</h4>Three studies were included in the final analysis. All three studies used ROTEM as the test of global haemostatic function, and none of the studies used TEG. Tissue factor-activated assay EXTEM clot amplitude (CA) was the focus of the accuracy measurements in blood samples taken near to the point of admission. These CAs were not taken at a uniform time after the start of the coagulopathic trace; the time varied from five minutes, to ten minutes and fifteen minutes. The three included studies were conducted in the UK, France and Afghanistan in both civilian and military trauma settings. In two studies, median Injury Severity Scores were 12, inter-quartile range (IQR) 4 to 24; and 22, IQR 12 to 34; and in one study the median New Injury Severity Score was 34, IQR 17 to 43.There were insufficient included studies examining each of the three ROTEM CAs at 5, 10 and 15 minutes to make meta-analysis and investigation of heterogeneity valid. The results of the included studies are thus reported narratively and illustrated by a forest plot and results plotted on the receiver operating characteristic (ROC) plane.For CA5 the accuracy results were sensitivity 70% (95% CI 47% to 87%) and specificity 86% (95% CI 82% to 90%) for one study, and sensitivity 96% (95% CI 88% to 100%) and specificity 58% (95% CI 44% to 72%) for the other.For CA10 the accuracy results were sensitivity 100% (95% CI 94% to 100%) and specificity 70% (95% CI 56% to 82%).For CA15 the accuracy results were sensitivity 88% (95% CI 69% to 97%) and specificity 100% (95% CI 94% to 100%).No uninterpretable ROTEM study results were mentioned in any of the included studies.Risk of bias and concerns around applicability of findings was low across all studies for the patient and flow and timing domains. However, risk of bias and concerns around applicability of findings for the index test domain was either high or unclear, and the risk of bias for the reference standard domain was high. This raised concerns around the interpretation of the sensitivity and specificity results of the included studies, which may be misleading.<h4>Authors' conclusions</h4>We found no evidence on the accuracy of TEG and very little evidence on the accuracy of ROTEM. The value of accuracy estimates are considerably undermined by the small number of included studies, and concerns about risk of bias relating to the index test and the reference standard. We are unable to offer advice on the use of global measures of haemostatic function for trauma based on the evidence on test accuracy identified in this systematic review. This evidence strongly suggests that at present these tests should only be used for research. We consider more thoroughly what this research could be in the Discussion section.

A 55-year-old man with a history of diabetes mellitus, hypertension and hypercholesterolaemia developed increasing peripheral oedema over the course of several months. He was found to have nephrotic range proteinuria (15.7 g/24 h). His renal ultrasound scan was normal and the autoimmune screen was negative. His renal biopsy demonstrated evidence of membranous glomerulonephritis and increased iron deposition. At this juncture, a serum ferritin was checked which showed an initial value 933 µg/L with transferrin saturation at 96.6%. A subsequent liver biopsy also showed evidence of iron overload but without fibrotic changes. Genetic studies including C282Y HFE, ferroportin and DMT1 studies were also negative. He was subsequently treated with interval venesection which was associated with significant symptomatic and biochemical evidence of improvement in oedema and proteinuria.

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells.

A chiral perylene diimide building block has been prepared based on an amine derivative of the amino acid L-phenylalanine. Detailed studies were carried out into the self-assembly behaviour of the material in solution and the solid state using UV/Vis, circular dichroism (CD) and fluorescence spectroscopy. For the charged building block BTPPP, the molecular chirality of the side chains is translated into the chiral supramolecular structure in the form of right-handed helical aggregates in aqueous solution. Temperature-dependent UV/Vis studies of BTPPP in aqueous solution showed that the self-assembly behaviour of this dye can be well described by an isodesmic model in which aggregation occurs to generate short stacks in a reversible manner. Wide-angle X-ray diffraction studies (WXRD) revealed that this material self-organises into aggregates with π-π stacking distances typical for π-conjugated materials. TEM investigations revealed the formation of self-assembled structures of low order and with no expression of chirality evident. Differential scanning calorimetry (DSC) and polarised optical microscopy (POM) were used to investigate the mesophase properties. Optical textures representative of columnar liquid-crystalline phases were observed for solvent-annealed samples of BTPPP. The high solubility, tunable self-assembly and chiral ordering of these materials demonstrate their potential as new molecular building blocks for use in the construction of chiro-optical structures and devices.

We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate.

Nonalcoholic fatty liver (NAFL) is an emerging global epidemic which progresses to nonalcoholic steatohepatitis (NASH) and cirrhosis in a subset of subjects. Various reviews have focused on the etiology, epidemiology, pathogenesis and treatment of NAFLD. This review highlights specifically the triggers implicated in disease progression from NAFL to NASH. The integrating role of genes, dietary factors, innate immunity, cytokines and gut microbiome have been discussed.

The oxidative stress response is a cellular defense mechanism that protects cells from oxidative damage and cancer development. The exact molecular mechanism by which reactive oxygen species (ROS) contribute to DNA damage and increase genome instability in prostate cancer merits further investigation. Here, we aimed to determine the effects of NKX3.1 loss on antioxidant defense in response to acute and chronic inflammation in an in vitro model. Oxidative stress-induced DNA damage resulted in increased H2AX((S139)) phosphorylation (a hallmark of DNA damage), along with the degradation of the androgen receptor (AR), p53 and NKX3.1, upon treatment with conditioned medium (CM) obtained from activated macrophages or H2O2. Furthermore, the expression and stability of SIRT1 were increased by CM treatment but not by H2O2 treatment, although the level of ATM((S1981)) phosphorylation was not changed compared with controls. Moreover, the deregulated antioxidant response resulted in upregulation of the pro-oxidant QSCN6 and the antioxidant GPX2 and downregulation of the antioxidant GPX3 after CM treatment. Consistently, the intracellular ROS level increased after chronic treatment, leading to a dose-dependent increase in the ability of LNCaP cells to tolerate oxidative damage. These data suggest that the inflammatory microenvironment is a major factor contributing to DNA damage and the deregulation of the oxidative stress response, which may be the underlying cause of the increased genetic heterogeneity during prostate tumor progression.

<h4>Background</h4>Neurofibromatosis type-1 (NF1) is a complex neurogenetic disorder characterised by the development of benign and malignant tumours of the peripheral nerve sheath (MPNSTs). Whilst biallelic NF1 gene inactivation contributes to benign tumour formation, additional cellular changes in gene structure and/or expression are required to induce malignant transformation. Although few molecular profiling studies have been performed on the process of progression of pre-existing plexiform neurofibromas to MPNSTs, the integrated analysis of copy number alterations (CNAs) and gene expression is likely to be key to understanding the molecular mechanisms underlying NF1-MPNST tumorigenesis. In a pilot study, we employed this approach to identify genes differentially expressed between benign and malignant NF1 tumours.<h4>Results</h4>SPP1 (osteopontin) was the most differentially expressed gene (85-fold increase in expression), compared to benign plexiform neurofibromas. Short hairpin RNA (shRNA) knockdown of SPP1 in NF1-MPNST cells reduced tumour spheroid size, wound healing and invasion in four different MPNST cell lines. Seventy-six genes were found to exhibit concordance between CNA and gene expression level.<h4>Conclusions</h4>Pathway analysis of these genes suggested that glutathione metabolism and Wnt signalling may be specifically involved in NF1-MPNST development. SPP1 is associated with malignant transformation in NF1-associated MPNSTs and could prove to be an important target for therapeutic intervention.

The aim of this experimantal study which is applied on rats, is to determine the differences on the clotting factors over the application of low and high intraabdominal pressure (IAP) values in different periods of time in carbon dioxide (CO2) pneumoperitoneum. Thirty rats were randomized into five groups (n = 6): a control group (Group K) and 1 h and 6 mm Hg IAP (Group A), 2 h and 6 mm Hg IAP (Group B), 1 h and 12 mm Hg IAP (Group C) and 2 h and 12 mm Hg IAP were created with CO2 pneumoperitoneum (Group D). At the end of the experiment, plasma samples taken from subjects and fibrinogen, FII (prothrombin), FV, FVII, FVIII, FIX, FX, FXI, FXII, von willebrand's factor (vWF), ristocetin cofactor, protein C, protein S, antithrombin III (AT III) levels are studied. There were statistically significant differences in the mean levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, and protein S between the groups. A hypercoagulable state occurred with the following: increase in the coagulation parameters compared to the control group; increase in FVII in the group only Group C; decrease in AT III in all groups compared to the control group; decrease in protein C in the group only XII Group D compared to control group; decrease in protein S in all groups except group D compared to control group. CO2 insufflation predisposes to thromboembolic events both by inducing coagulation factors and by suppressing the fibrinolytic system contrary to the controversies in the literature.

The ubiquitously expressed plasma membrane Na(+)-H(+) exchanger NHE1 is a 12 transmembrane-spanning protein that directs important cell functions such as homeostatic intracellular volume and pH control. The 315 amino acid cytosolic tail of NHE1 binds plasma membrane phospholipids and multiple proteins that regulate additional, ion-translocation independent functions. This review focuses on NHE1 structure/function relationships, as well as the role of NHE1 in kidney proximal tubule functions, including pH regulation, vectorial Na(+) transport, cell volume control and cell survival. The implications of these functions are particularly critical in the setting of progressive, albuminuric kidney diseases, where the accumulation of reabsorbed fatty acids leads to disruption of NHE1-membrane phospholipid interactions and tubular atrophy, which is a poor prognostic factor for progression to end stage renal disease. This review amplifies the vital role of the proximal tubule NHE1 Na(+)-H(+) exchanger as a kidney cell survival factor.

<h4>Objective</h4>Spontaneous preterm birth (SPTB) is a complex condition that is likely a final common pathway with multiple possible causes. We hypothesized that a comprehensive classification system appropriately could group women with similar STPB causes and could provide an explanation, at least in part, for the disparities in SPTB that are associated with race and gestational age at delivery.<h4>Study design</h4>This was a planned analysis of a multicenter, prospective study of singleton SPTBs. Women with SPTB at <34 weeks' gestation were included. We defined 9 potential SPTB phenotypes based on clinical data: infection/inflammation, maternal stress, decidual hemorrhage, uterine distention, cervical insufficiency, placental dysfunction, premature rupture of the membranes, maternal comorbidities, and familial factors. Each woman's condition was evaluated for each phenotype. Delivery gestational age was compared between those with and without each phenotype. Phenotype profiles were also compared between women with very early (20.0-27.9 weeks' gestation) SPTB vs those with early SPTB (28.0-34.0 weeks' gestation) and between African American and white women. Statistical analysis was by t test and χ(2) test, as appropriate.<h4>Results</h4>The phenotyping tool was applied to 1025 women with SPTBs who delivered at a mean 30.0 ± 3.2 (SD) weeks' gestation. Of these, 800 women (78%) had ≥2 phenotypes. Only 43 women (4.2%) had no phenotypes. The 281 women with early SPTBs were more likely to have infection/inflammation, decidual hemorrhage, and cervical insufficiency phenotypes (all P ≤ .001). African American women had more maternal stress and cervical insufficiency but less decidual hemorrhage and placental dysfunction compared with white women (all P < .05). Gestational age at delivery decreased as the number of phenotypes that were present increased.<h4>Conclusion</h4>Precise SPTB phenotyping classifies women with SPTBs and identifies specific differences between very early and early SPTB and between African American and white women.

<h4>Objective</h4>Women who sell sex and use drugs have dual risks for HIV infection. Despite increasing reports of drug use among female sex workers (FSW) in Vietnam, FSW HIV interventions remain focused mainly on sexual risk reduction. We assessed the impact of drug use and inconsistent condom use on HIV infection among FSW in Vietnam, which few studies have quantified.<h4>Methods</h4>We surveyed 5298 women aged ≥18 years who had sold sex in the past month from ten geographically dispersed provinces. We performed multivariate logistic regression on data from provinces with high (≥10%) or low (<10%) HIV prevalence among FSW.<h4>Results</h4>Compared to FSW who never used illicit drugs, the odds of HIV infection among FSW who had ever injected drugs and those who reported non-injection drug use were 3.44 (CI 2.32-5.09) and 1.76 (CI 1.14-2.71), respectively, in high-prevalence provinces. FSW who always used condoms with clients had lower odds of HIV infection than those who did not (AOR=0.71; CI 0.52-0.98). In low-prevalence provinces lifetime injection drug use (AOR 22.05, CI 12.00-40.49), but not non-injecting drug use or inconsistent condom use, was significantly associated with HIV infection.<h4>Conclusions</h4>Because injection drug use and inconsistent condom use were key risk factors for HIV infection in high-prevalence provinces, drug injection risk reduction should be as much a focus of HIV prevention as sexual risk reduction. Where HIV prevalence remains low in FSW, a more general emphasis on harm reduction for all drug users will benefit FSW.

<h4>Background</h4>Because species-specific gene expression is driven by species-specific regulation, understanding the relationship between sequence and function of the regulatory regions in different species will help elucidate how differences among species arise. Despite active experimental and computational research, relationships among sequence, conservation, and function are still poorly understood.<h4>Results</h4>We compared transcription factor occupied segments (TFos) for 116 human and 35 mouse TFs in 546 human and 125 mouse cell types and tissues from the Human and the Mouse ENCODE projects. We based the map between human and mouse TFos on a one-to-one nucleotide cross-species mapper, bnMapper, that utilizes whole genome alignments (WGA). Our analysis shows that TFos are under evolutionary constraint, but a substantial portion (25.1% of mouse and 25.85% of human on average) of the TFos does not have a homologous sequence on the other species; this portion varies among cell types and TFs. Furthermore, 47.67% and 57.01% of the homologous TFos sequence shows binding activity on the other species for human and mouse respectively. However, 79.87% and 69.22% is repurposed such that it binds the same TF in different cells or different TFs in the same cells. Remarkably, within the set of repurposed TFos, the corresponding genome regions in the other species are preferred locations of novel TFos. These events suggest exaptation of some functional regulatory sequences into new function. Despite TFos repurposing, we did not find substantial changes in their predicted target genes, suggesting that CRMs buffer evolutionary events allowing little or no change in the TFos - target gene associations. Thus, the small portion of TFos with strictly conserved occupancy underestimates the degree of conservation of regulatory interactions.<h4>Conclusion</h4>We mapped regulatory sequences from an extensive number of TFs and cell types between human and mouse using WGA. A comparative analysis of this correspondence unveiled the extent of the shared regulatory sequence across TFs and cell types under study. Importantly, a large part of the shared regulatory sequence is repurposed on the other species. This sequence, fueled by turnover events, provides a strong case for exaptation in regulatory elements.

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that acts as a ligand for five G-protein coupled receptors (S1P1-5) whose downstream effects are implicated in a variety of important pathologies including sickle cell disease, cancer, inflammation, and fibrosis. The synthesis of S1P is catalyzed by sphingosine kinase (SphK) isoforms 1 and 2, and hence, inhibitors of this phosphorylation step are pivotal in understanding the physiological functions of SphKs. To date, SphK1 and 2 inhibitors with the potency, selectivity, and in vivo stability necessary to determine the potential of these kinases as therapeutic targets are lacking. Herein, we report the design, synthesis, and structure-activity relationship studies of guanidine-based SphK inhibitors bearing an oxadiazole ring in the scaffold. Our studies demonstrate that SLP120701, a SphK2-selective inhibitor (Ki = 1 μM), decreases S1P levels in histiocytic lymphoma (U937) cells. Surprisingly, homologation with a single methylene unit between the oxadiazole and heterocyclic ring afforded a SphK1-selective inhibitor in SLP7111228 (Ki = 48 nM), which also decreased S1P levels in cultured U937 cells. In vivo application of both compounds, however, resulted in contrasting effect in circulating levels of S1P. Administration of SLP7111228 depressed blood S1P levels while SLP120701 increased levels of S1P. Taken together, these compounds provide an in vivo chemical toolkit to interrogate the effect of increasing or decreasing S1P levels and whether such a maneuver can have implications in disease states.

Antisense oligonucleotides (ASOs) are single stranded, backbone modified nucleic acids, which mediate cleavage of complementary RNA by directing RNase H cleavage in cell culture and in animals. It has generally been accepted that the single stranded state in conjunction with the phosphorothioate modified backbone is necessary for cellular uptake and transport to the active compartment. Herein, we examine the effect of using hairpin structured ASOs to (1) determine if an ASO agent requires a single stranded conformation for efficient RNA knock down, (2) use a fluorophore-quencher labeled ASO to evaluate which moieties the ASO interacts with in cells and examine if cellular distribution can be determined with such probes, and (3) evaluate if self-structured ASOs can improve allele selective silencing between closely related huntingtin alleles. We show that hairpin shaped ASOs can efficiently down-regulate RNA in vitro, but potency correlates strongly negatively with increasing stability of the hairpin structure. Furthermore, self-structured ASOs can efficiently reduce huntingtin mRNA in the central nervous system of mice.

The X chromosome constitutes a unique genomic environment because it is present in one copy in males, but two copies in females. This simple fact has motivated several theoretical predictions with respect to how standing genetic variation on the X chromosome should differ from the autosomes. Unmasked expression of deleterious mutations in males and a lower census size are expected to reduce variation, while allelic variants with sexually antagonistic effects, and potentially those with a sex-specific effect, could accumulate on the X chromosome and contribute to increased genetic variation. In addition, incomplete dosage compensation of the X chromosome could potentially dampen the male-specific effects of random mutations, and promote the accumulation of X-linked alleles with sexually dimorphic phenotypic effects. Here we test both the amount and the type of genetic variation on the X chromosome within a population of Drosophila melanogaster, by comparing the proportion of X linked and autosomal trans-regulatory SNPs with a sexually concordant and discordant effect on gene expression. We find that the X chromosome is depleted for SNPs with a sexually concordant effect, but hosts comparatively more SNPs with a sexually discordant effect. Interestingly, the contrasting results for SNPs with sexually concordant and discordant effects are driven by SNPs with a larger influence on expression in females than expression in males. Furthermore, the distribution of these SNPs is shifted towards regions where dosage compensation is predicted to be less complete. These results suggest that intrinsic properties of dosage compensation influence either the accumulation of different types of trans-factors and/or their propensity to accumulate mutations. Our findings document a potential mechanistic basis for sex-specific genetic variation, and identify the X as a reservoir for sexually dimorphic phenotypic variation. These results have general implications for X chromosome evolution, as well as the genetic basis of sex-specific evolutionary change.

<h4>Introduction</h4>Despite implementation of the biological passport to detect erythropoietin abuse, a need for additional biomarkers remains. We used a proteomic approach to identify novel serum biomarkers of prolonged erythropoiesis-stimulating agent (ESA) exposure (Darbepoietin-α) and/or aerobic training.<h4>Trial design</h4>Thirty-six healthy young males were randomly assigned to the following groups: Sedentary-placebo (n = 9), Sedentary-ESA (n = 9), Training-placebo (n = 10), or Training-ESA (n = 8). They were treated with placebo/Darbepoietin-α subcutaneously once/week for 10 weeks followed by a 3-week washout period. Training consisted of supervised biking 3/week for 13 weeks at the highest possible intensity. Serum was collected at baseline, week 3 (high dose Darbepoietin-α), week 10 (reduced dose Darbepoietin-α), and after a 3-week washout period.<h4>Methods</h4>Serum proteins were separated according to charge and molecular mass (2D-gel electrophoresis). The identity of proteins from spots exhibiting altered intensity was determined by mass spectrometry.<h4>Results</h4>Six protein spots changed in response to Darbepoietin-α treatment. Comparing all 4 experimental groups, two protein spots (serotransferrin and haptoglobin/haptoglobin related protein) showed a significant response to Darbepoietin-α treatment. The haptoglobin/haptoglobin related protein spot showed a significantly lower intensity in all subjects in the training-ESA group during the treatment period and increased during the washout period.<h4>Conclusion</h4>An isoform of haptoglobin/haptoglobin related protein could be a new anti-doping marker and merits further research.<h4>Trial registration</h4>ClinicalTrials.gov NCT01320449.

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.

Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

<h4>Background</h4>Persistent aminotransferase elevations are common in human immunodeficiency virus (HIV)-infected patients on antiretroviral therapy (ART), including those without hepatitis B or C coinfection, but their clinical significance is unknown.<h4>Methods</h4>HIV-infected adults with aminotransferase levels elevated above the upper limit of normal for ≥6 months while receiving ART, and without chronic viral hepatitis or other known causes of chronic liver disease, underwent a detailed metabolic assessment and liver biopsy.<h4>Results</h4>Sixty-two HIV-infected subjects completed the study. Forty (65%) had clinically significant liver pathology, including 34 (55%) with nonalcoholic steatohepatitis (NASH) and 11 (18%) with bridging fibrosis, 10 of whom also had NASH. Nonspecific abnormalities alone were seen in 22 (35%) subjects, including mild steatosis, mild to moderate inflammation, and evidence of drug adaptation. Insulin resistance, obesity, and the presence of either of 2 minor alleles in the PNPLA3 gene were significantly associated with increased risk of NASH and fibrosis. NASH and/or fibrosis were not associated with duration of HIV infection or ART, specific antiretroviral drugs, history of opportunistic infection, immune status, or duration of aminotransferase elevation.<h4>Conclusions</h4>HIV-infected adults with chronic aminotransferase elevations while receiving ART have a high rate of liver disease. Noninvasive testing can help identify liver disease in such patients, but liver biopsy is necessary to definitively identify those at risk for liver disease progression and complications. Longitudinal follow-up of this cohort will better characterize the natural history of aminotransferase elevations in this population and identify noninvasive biomarkers of liver disease progression.

Ionizing radiation is a potent activator of the tumor suppressor gene p53, which itself regulates the transcription of genes involved in canonical pathways such as the cell cycle, DNA repair and apoptosis as well as other biological processes like metabolism, autophagy, differentiation and development. In this study, we performed a meta-analysis on gene expression data from different in vivo and in vitro experiments to identify a signature of early radiation-responsive genes which were predicted to be predominantly regulated by p53. Moreover, we found that several genes expressed different transcript isoforms after irradiation in a p53-dependent manner. Among this gene signature, we identified novel p53 targets, some of which have not yet been functionally characterized. Surprisingly, in contrast to genes from the canonical p53-regulated pathways, our gene signature was found to be highly enriched during embryonic and post-natal brain development and during in vitro neuronal differentiation. Furthermore, we could show that for a number of genes, radiation-responsive transcript variants were upregulated during development and differentiation, while radiation non-responsive variants were not. This suggests that radiation exposure of the developing brain and immature cortical neurons results in the p53-mediated activation of a neuronal differentiation program. Overall, our results further increase the knowledge of the radiation-induced p53 network of the embryonic brain and provide more evidence concerning the importance of p53 and its transcriptional targets during mouse brain development.

The actin-binding protein profilin-1 (Pfn1) inhibits tumor growth and yet is also required for cell proliferation and survival, an apparent paradox. We previously identified Ser-137 of Pfn1 as a phosphorylation site within the poly-l-proline (PLP) binding pocket. Here we confirm that Ser-137 phosphorylation disrupts Pfn1 binding to its PLP-containing ligands with little effect on actin binding. We find in mouse xenografts of breast cancer cells that mimicking Ser-137 phosphorylation abolishes cell cycle arrest and apoptotic sensitization by Pfn1 and confers a growth advantage to tumors. This indicates a previously unrecognized role of PLP binding in Pfn1 antitumor effects. Spatial restriction of Pfn1 to the nucleus or cytoplasm indicates that inhibition of tumor cell growth by Pfn1 requires its nuclear localization, and this activity is abolished by a phosphomimetic mutation on Ser-137. In contrast, cytoplasmic Pfn1 lacks inhibitory effects on tumor cell growth but rescues morphological and proliferative defects of PFN1 null mouse chondrocytes. These results help reconcile seemingly opposed cellular effects of Pfn1, provide new insights into the antitumor mechanism of Pfn1, and implicate Ser-137 phosphorylation as a potential therapeutic target for breast cancer.

Real and simulated microgravity induce a variety of changes in human cells. Most importantly, changes in the cytoskeleton have been noted, and studies on microtubules have shown that they are gravisensitive. This study focuses on the effects of short-term real microgravity on gene expression, protein content, and cytoskeletal structure of human chondrocytes. We cultivated human chondrocytes, took them along a parabolic flight during the 24th Deutsches Zentrum für Luft- und Raumfahrt Parabolic (DLR) Flight Campaign, and fixed them after the 1st and the 31st parabola. Immunofluorescence microscopy revealed no changes after the 1st parabola, but disruptions of β-tubulin, vimentin, and cytokeratin networks after the 31st parabola. No F-actin stress fibers were detected even after 31 parabolas. Furthermore, mRNA and protein quantifications after the 31st parabola showed a clear up-regulation of cytoskeletal genes and proteins. The mRNAs were significantly up-regulated as follows: TUBB, 2-fold; VIM, 1.3-fold; KRT8, 1.8-fold; ACTB, 1.9-fold; ICAM1, 4.8-fold; OPN, 7-fold; ITGA10, 1.5-fold; ITGB1, 1.2-fold; TGFB1, 1.5-fold; CAV1, 2.6-fold; SOX9, 1.7-fold; BMP-2, 5.3-fold. However, SOX5 (-25%) and SOX6 (-28%) gene expression was decreased. Contrary, no significant changes in gene expression levels were observed during vibration and hypergravity experiments. These data suggest that short-term microgravity affects the gene expression of distinct proteins. In contrast to poorly differentiated follicular thyroid cancer cells or human endothelial cells, chondrocytes only exert moderate cytoskeletal alterations. The up-regulation of BMP-2, TGF-β1, and SOX9 in chondrocytes may play a key role in preventing cytoskeletal alterations.

The incidence of opportunistic non-tuberculous mycobacteria (NTM) infections has increased considerably in the past decades causing an array of infections, including respiratory and soft-tissue infections. NTM are ubiquitous and can be found in numerous environments, including households and water plants. However, NTM have not been reported to be associated with the healthy human oral microbiome. Since the oral cavity and upper respiratory track are the main ports of entry of microorganisms into the human body, elucidating NTM diversity and prevalence will assist in the assessment of the potential risks of infection elicited by these opportunistic pathogens. Here, we report the identification of a 'non-tuberculous mycobacteriome' in healthy individuals. We employed a modified DNA extraction procedure in conjunction with mycobacterial-specific primers to screen niches in the oral cavity (buccal mucosa and dental plaque) and upper respiratory tract (nostrils and oropharynx) of 10 healthy subjects. A total of 50 prevalent operational taxonomic units sequenced on MiSeq (Illumina) using 16S rRNA V3-V4 region were detected across all screened niches, showing the presence of diverse NTM communities. NTM DNA was detected in the nostrils of all 10 subjects, in buccal mucosa of 8 subjects, in the oropharynx of 7 subjects, and in the dental plaques of 5 subjects. Results from quantitative PCR showed each individual harbored 10(3)-10(4) predicted NTM per each screened niche. The modification of standard DNA isolation methods to increase sensitivity toward mycobacterial species represents an important step to advance the knowledge of the oral as well as the overall human microbiome. These findings clearly reveal for the first time that healthy individuals harbor a 'non-tuberculous mycobacteriome' in their oral cavity and upper respiratory tract and may have important implications in our understanding of infections caused by NTM.

No abstract available.

<h4>Purpose</h4>Activation of proto-oncogenes and inactivation of tumour suppressor genes are the major genetic alterations involved in carcinogenesis. The increase in methylation at the promoter region of a tumour suppressor gene can lead to gene inactivation, selecting cells with proliferative advantage. Thus, promoter hypermethylation is considered a marker in a variety of malignant tumours, including oral cavity.<h4>Experimental design</h4>The methylation pattern of eight genes was evaluated in 40 oral cavity squamous cell carcinomas (OSCCs) and 40 saliva samples from healthy individuals by Q-MSP. Different combinations of genes were also assessed in order to identify gene panels that could better distinguish between OSCC and saliva samples.<h4>Results</h4>CCNA1, DAPK, DCC and TIMP3 methylation were highly specific for being found in the OSCC samples. Moreover, the combination of these genes improved detection when compared with single markers, reaching values of 92.5% for sensitivity and specificity (when using the panel CCNA1, DCC, TIMP3). Moreover, DAPK, DCC and TIMP3 were hypermethylated in nearly 90% of clinically T1 and T2 cases.<h4>Conclusion</h4>The pursuing of this panel of hypermethylated genes is an important tool for the detection of individuals with OSCC. Moreover, the identification of these markers in early stages of OSCC shows the feasibility of using the panel on saliva as possible biomarkers for early diagnosis. The lack of association between the methylation status of these genes and clinical characteristics shows that they are able to distinguish OSCC cases irrespective of social and clinical factors (gender, age, human papillomavirus (HPV) status, clinical stage, vascular embolisation and perineural invasion).

Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. The complications related to the usage of pluripotent embryonic stem cells (ESCs) in human medicine, particularly ESC isolation and histoincompatibility were bypassed with induced pluripotent stem cell (iPSC) technology. The human iPSCs can be used for studying embryogenesis, disease modeling, drug testing and regenerative medicine. iPSCs can be diverted to different cell lineages using small molecules and growth factors. In this review we have focused on iPSC differentiation towards cardiac and neuronal lineages. Moreover, we deal with the use of iPSCs in regenerative medicine and modeling diseases like myocardial infarction, Timothy syndrome, dilated cardiomyopathy, Parkinson's, Alzheimer's and Huntington's disease. Despite the promising potential of iPSCs, genome contamination and low efficacy of cell reprogramming remain significant challenges.

Deubiquitylating enzymes (DUBs), act downstream of ubiquitylation. As such, these post-post-translational modifiers function as the final arbitrators of a protein substrate's ubiquitylation status, thus regulating its fate. In most instances, DUBs moderate the absolute level of a substrate, its locality or activity, rather than being an "all-or-none" phenomenon. Yet, disruption of this quantitative regulation can produce dramatic qualitative differences. The ubiquitin-specific protease 9X (USP9X/FAM) is a substrate-specific DUB, which displays an extraordinarily high level of sequence conservation from Drosophila to mammals. It is primarily the recent revelations of USP9X's pivotal role in human cancers, both as oncogene or tumour suppressor, in developmental disorders including intellectual disability, epilepsy, autism and developmental delay that has led to a subsequent re-examination of its molecular and cellular functions. Results from experimental animal models have implicated USP9X in neurodegeneration, including Parkinson's and Alzheimer's disease, as well as autoimmune diseases. In this review, we describe the current and accumulated knowledge on the molecular, cellular and developmental aspects of USP9X function within the context of the biological consequences during normal development and disease.

Serotonin transporter gene variants are known to interact with stressful life experiences to increase chances of developing affective symptoms, and these same variants have been shown to influence amygdala reactivity to affective stimuli in non-psychiatric populations. The impact of these gene variants on affective neurocircuitry in anxiety and mood disorders has been studied less extensively. Utilizing a triallelic assay (5-HTTLPR and rs25531) to assess genetic variation linked with altered serotonin signaling, this fMRI study investigated genetic influences on amygdala and anterior insula activity in 50 generalized anxiety disorder patients, 26 of whom also met DSM-IV criteria for social anxiety disorder and/or major depressive disorder, and 39 healthy comparison subjects. A Group x Genotype interaction was observed for both the amygdala and anterior insula in a paradigm designed to elicit responses in these brain areas during the anticipation of and response to aversive pictures. Patients who are S/L(G) carriers showed less activity than their L(A)/L(A) counterparts in both regions and less activity than S/L(G) healthy comparison subjects in the amygdala. Moreover, patients with greater insula responses reported higher levels of intolerance of uncertainty, an association that was particularly pronounced for patients with two LA alleles. A genotype effect was not established in healthy controls. These findings link the serotonin transporter gene to affective circuitry findings in anxiety and depression psychopathology and further suggest that its impact on patients may be different from effects typically observed in healthy populations.

In the adult the interface between the cerebrospinal fluid and the brain is lined by the ependymal cells, which are joined by gap junctions. These intercellular connections do not provide a diffusional restrain between the two compartments. However, during development this interface, initially consisting of neuroepithelial cells and later radial glial cells, is characterized by "strap" junctions, which limit the exchange of different sized molecules between cerebrospinal fluid and the brain parenchyma. Here we provide a systematic study of permeability properties of this inner cerebrospinal fluid-brain barrier during mouse development from embryonic day, E17 until adult. Results show that at fetal stages exchange across this barrier is restricted to the smallest molecules (286Da) and the diffusional restraint is progressively removed as the brain develops. By postnatal day P20, molecules the size of plasma proteins (70 kDa) diffuse freely. Transcriptomic analysis of junctional proteins present in the cerebrospinal fluid-brain interface showed expression of adherens junctional proteins, actins, cadherins and catenins changing in a development manner consistent with the observed changes in the permeability studies. Gap junction proteins were only identified in the adult as was claudin-11. Immunohistochemistry was used to localize at the cellular level some of the adherens junctional proteins of genes identified from transcriptomic analysis. N-cadherin, β - and α-catenin immunoreactivity was detected outlining the inner CSF-brain interface from E16; most of these markers were not present in the adult ependyma. Claudin-5 was present in the apical-most part of radial glial cells and in endothelial cells in embryos, but only in endothelial cells including plexus endothelial cells in adults. Claudin-11 was only immunopositive in the adult, consistent with results obtained from transcriptomic analysis. These results provide information about physiological, molecular and morphological-related permeability changes occurring at the inner cerebrospinal fluid-brain barrier during brain development.

In this review, we discuss molecular and cellular mechanisms important for the function of neuronal progenitors during development, revealed by their perturbation in different cortical malformations. We focus on a class of neuronal progenitors, radial glial cells (RGCs), which are renowned for their unique morphological and behavioral characteristics, constituting a key element during the development of the mammalian cerebral cortex. We describe how the particular morphology of these cells is related to their roles in the orchestration of cortical development and their influence on other progenitor types and post-mitotic neurons. Important for disease mechanisms, we overview what is currently known about RGC cellular components, cytoskeletal mechanisms, signaling pathways and cell cycle characteristics, focusing on how defects lead to abnormal development and cortical malformation phenotypes. The multiple recent entry points from human genetics and animal models are contributing to our understanding of this important cell type. Combining data from phenotypes in the mouse reveals molecules which potentially act in common pathways. Going beyond this, we discuss future directions that may provide new data in this expanding area.

Combinations of anticancer agents may have synergistic anti-tumor effects, but enhanced hematological toxicity often limit their clinical use. We examined whether "microarray profiles" could be used to compare early molecular responses following a single dose of agents administered individually with that of the agents administered in a combination. We compared the mRNA responses within bone marrow of Sprague-Dawley rats after a single 30 min treatment with topotecan at 4.7 mg/kg or oxaliplatin at 15 mg/kg alone to that of sequentially administered combination therapy or vehicle control for 1, 6, and 24 h. We also examined the histopathology of the bone marrow following all treatments. Drug-related histopathological lesions were limited to bone marrow hypocellularity for animals dosed with either agent alone or in combination. Lesions had an earlier onset and higher incidence for animals given topotecan alone or in combination with oxaliplatin. Severity increased from mild to moderate when topotecan was administered prior to oxaliplatin compared with administering oxaliplatin first. Notably, six patterns of co-expressed genes were detected at the 1 h time point that indicate regulatory expression of genes that are dependent on the order of the administration. These results suggest alterations in histone biology, chromatin remodeling, DNA repair, bone regeneration, and respiratory and oxidative phosphorylation are among the prominent pathways modulated in bone marrow from animals treated with an oxaliplatin/topotecan combination. These data also demonstrate the potential for early mRNA patterns derived from target organs of toxicity to inform toxicological risk and molecular mechanisms for agents given in combination.

No abstract available.

Olfactomedin 4 (OLFM4) has been demonstrated to serve an important function in tumor progression. This study aims to analyze the correlation between OLFM4 expression and clinicopathological features and the prognostic significance of OLFM4 in the context of smoking status of non-small cell lung cancer (NSCLC) patients. A total of 218 NSCLC patients, who were histopathologically diagnosed from 2001 to 2013, were reviewed in the study. OLFM4 expression was analyzed by immunohistochemical staining of tissue samples. The association of OLFM4 with clinicopathological parameters was evaluated. Overall survival and disease-specific survival were evaluated by Kaplan-Meier survival analysis. Immunohistochemical analyses showed that OLFM4 was highly expressed in 64.2% of NSCLC patients. OLFM4 expression level in NSCLC lesions was strongly correlated with pathologic grade (P = .017), lymph node metastasis (P = .012), peritumor intravascular cancer emboli (P = .03), and smoking status (P < .001). Kaplan-Meier survival curves showed that, among smoking patients, those with low OLFM4 expression had shorter survival time (overall survival and disease-specific survival) than those with high OLFM4 (P < .05). Conclusively, although low OLFM4 expression is not an independent prognostic biomarker, it might indicate worse prognosis for smoking patients with NSCLC, thereby identifying patients who might benefit from targeting OLFM4 therapy.

Sorafenib confers a survival benefit for patients with advanced hepatocellular carcinoma (HCC) and Child-Pugh (CP) A liver cirrhosis. At present, limited data exists with regard to the safety and efficacy of sorafenib in treating CP-B HCC patients. The present study describes the use of sorafenib in patients with HCC and CP-A or -B cirrhosis. Clinical data was obtained from patients with HCC who were treated with sorafenib at the Department of Clinical and Experimental Medicine, Second University of Naples (Naples, Italy) and were analyzed retrospectively in terms of tumor response, tolerance and survival. The treatment outcomes were analyzed according to the respective CP status. The adverse events (AEs) were graded using the Common Terminology Criteria for Adverse Events, version 3.0, and the tumor response was assessed according to the Response Evaluation Criteria in Solid Tumors, version 1.2. In total, 26 patients received sorafenib at 400 mg twice daily. The median age was 69 years (range, 58-81 years) and the ratio of males to females was 18:8. Overall, 15 patients were infected with the hepatitis C virus (HCV), eight with HBV and three were co-infected with HCV/HBV. In total, 20 (77%) patients presented with an underlying CP-A (CP-A5 and CP-A6) cirrhosis and six (23%) with CP-B (CP-B7). Previous treatments included surgery (n=4), transarterial chemoembolization (n=5) and percutaneous ethanol injection or radiofrequency interstitial thermal ablation (n=12). A partial response was observed in three patients (12%), a stable disease lasting at least 12 weeks in 13 patients (50%) and a progression of disease in 10 patients (38%). The median overall survival (OS) time was 7.4 months [95% confidence interval (CI), 3.2-11.6) and the median progression-free survival (PFS) time was 3.7 months (95% CI, 1.9-5.5). The median OS and PFS times differed between patients with CP-A and CP-B, with a trend (P=0.06) toward a worse outcome in those with CP-B, although this was not statistically significant. The CP-A and CP-B groups experienced a similar incidence in the majority of AEs. A reduction in dose was required in 59% of the patients. The CP-A5, CP-A6 and CP-B7 patients tolerated sorafenib similarly, and derived comparable clinical and survival benefits.

<h4>Rationale</h4>Vortioxetine is a novel multimodal antidepressant that is a 5-HT1B receptor partial agonist, a 5-HT1A receptor agonist, an inhibitor of the serotonin (5-HT) transporter, and a 5-HT1D, 5-HT3, and 5-HT7 receptor antagonist in vitro. In vivo studies have shown that vortioxetine enhances levels of 5-HT and desensitizes 5-HT1A autoreceptors.<h4>Objectives</h4>The aim of the present study was to investigate the effects of acute and long-term administration of vortioxetine on the terminal 5-HT1B receptor and the tonic activation of 5-HT1A receptor in the rat hippocampus.<h4>Methods</h4>These receptors were assessed following vortioxetine administration acutely or subcutaneously using minipumps for 14 days. These studies were carried out using in vivo electrophysiological recording, microiontophoresis, and stimulation of the ascending 5-HT fibers.<h4>Results</h4>Vortioxetine enhanced the inhibitory effect of the stimulation of the 5-HT bundle at a high, but not low frequency and reversed the inhibitory effect of the 5-HT1B receptor agonist CP 94253. These results indicate that this compound acted as a 5-HT1B receptor partial agonist. Vortioxetine inhibited 5-HT reuptake but did not dampen the sensitivity of postsynaptic 5-HT1A receptors on pyramidal neurons. Long-term administration of vortioxetine and escitalopram (both at 5 mg/kg/day) induced an increase of tonic activation of the 5-HT1A receptors in CA3 pyramidal neurons, resulting in an increase in 5-HT transmission. In addition, vortioxetine decreased the function of terminal 5-HT1B autoreceptor following its sustained administration.<h4>Conclusions</h4>Desensitization of 5-HT1B autoreceptor and an increase of tonic activation of 5-HT1A receptors in the hippocampus may contribute to the antidepressant effect of vortioxetine.

During surgical procedures, abdominal swabs are routinely used to adsorb blood from the operation field and for the retention of tissues and organs. Due to the material characteristics, abdominal swabs exhibit a slight procoagulant activity, which is usually desirable and mostly harmless. However, during cardiac surgery with heart-lung machine (HLM) support, abnormal clot formation may result in life-threatening thromboembolic complications. Therefore, a simple clotting test (SCT) allowing in vitro detection of abdominal swabs with elevated hypercoagulant potency in the presence of heparinized human blood was developed and validated. In order to establish a SCT, heparinized human blood from 100 donors was incubated with five different cotton abdominal swabs for 30 min at 37 °C and then macroscopically analyzed. In a second study, 10 other swabs were screened with the established SCT (n=11) to confirm its suitability. Scanning electron microscopy, measurements of activated clotting times and thrombin-antithrombin were further performed. In the SCT, the results are dichotomized as negative (no detectable blood clot) and positive (blood clot formation). In the first study, three of the five tested abdominal swabs exhibited hypercoagulant potency in at least 25% of the donors. Calculations using the binomial distribution showed that blood of 11 donors is needed for routine testing with the SCT, which was confirmed in the second study using another 10 swabs. The established SCT can be used for detection of abdominal swabs with an elevated procoagulant potency, thereby minimizing the risk of thromboembolic complications during cardiac surgery with HLM support.

Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.

Multiple sclerosis (MS) is a debilitating chronic inflammatory disease of the nervous system that affects approximately 2.3 million individuals worldwide, with higher prevalence in females, and a strong genetic component. While over 200 MS susceptibility loci have been identified in GWAS, the underlying mechanisms whereby they contribute to disease susceptibility remains ill-defined. Forward genetics approaches using conventional laboratory mouse strains are useful in identifying and functionally dissecting genes controlling disease-relevant phenotypes, but are hindered by the limited genetic diversity represented in such strains. To address this, we have combined the powerful chromosome substitution (consomic) strain approach with the genetic diversity of a wild-derived inbred mouse strain. Using experimental allergic encephalomyelitis (EAE), a mouse model of MS, we evaluated genetic control of disease course among a panel of 26 consomic strains of mice inheriting chromosomes from the wild-derived PWD strain on the C57BL/6J background, which models the genetic diversity seen in human populations. Nineteen linkages on 18 chromosomes were found to harbor loci controlling EAE. Of these 19 linkages, six were male-specific, four were female-specific, and nine were non-sex-specific, consistent with a differential genetic control of disease course between males and females. An MS-GWAS candidate-driven bioinformatic analysis using orthologous genes linked to EAE course identified sex-specific and non-sex-specific gene networks underlying disease pathogenesis. An analysis of sex hormone regulation of genes within these networks identified several key molecules, prominently including the MAP kinase family, known hormone-dependent regulators of sex differences in EAE course. Importantly, our results provide the framework by which consomic mouse strains with overall genome-wide genetic diversity, approximating that seen in humans, can be used as a rapid and powerful tool for modeling the genetic architecture of MS. Moreover, our data represent the first step towards mechanistic dissection of genetic control of sexual dimorphism in CNS autoimmunity.

The Q175 knockin mouse model of Huntington's disease (HD) carries a CAG trinucleotide expansion of the human mutant huntingtin allele in its native mouse genomic context and recapitulates the genotype more closely than transgenic models. In this study we examined the progression of changes in intrinsic membrane properties and excitatory and inhibitory synaptic transmission, using whole cell patch-clamp recordings of medium-sized spiny neurons (MSNs) in the dorsolateral striatum and cortical pyramidal neurons (CPNs) in layers 2/3 of the primary motor cortex in brain slices from heterozygous (Q175(+/-)) and homozygous (Q175(+/+)) mice. Input resistance in MSNs from Q175(+/+) and Q175(+/-) mice was significantly increased compared with wild-type (WT) littermates beginning at 2 mo. Furthermore, the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) was significantly reduced in MSNs from Q175(+/+) and Q175(+/-) mice compared with WTs beginning at 7 mo. In contrast, the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and IPSC-to-EPSC ratios were increased in MSNs from Q175(+/+) mice beginning at 2 mo. Morphologically, significant decreases in spine density of MSNs from Q175(+/-) and Q175(+/+) mice occurred at 7 and 12 mo. In CPNs, sIPSC frequencies and IPSC-to-EPSC ratios were significantly increased in Q175(+/-) mice compared with WTs at 12 mo. There were no changes in intrinsic membrane properties or morphology. In summary, we show a number of alterations in electrophysiological and morphological properties of MSNs in Q175 mice that are similar to other HD mouse models. However, unlike other models, CPN inhibitory activity is increased in Q175(+/-) mice, indicating reduced cortical excitability.

Repeat pregnancies with different perinatal outcomes minimize underlying maternal genetic diversity and provide unique opportunities to investigate nongenetic risk factors and epigenetic mechanisms of pregnancy complications. We investigated gestational diabetes mellitus (GDM)-related differential DNA methylation in early pregnancy peripheral blood samples collected from women who had a change in GDM status in repeat pregnancies. Six study participants were randomly selected from among women who had 2 consecutive pregnancies, only 1 of which was complicated by GDM (case pregnancy) and the other was not (control pregnancy). Epigenome-wide DNA methylation was profiled using Illumina HumanMethylation 27 BeadChips. Differential Identification using Mixture Ensemble and false discovery rate (<10%) cutoffs were used to identify differentially methylated targets between the 2 pregnancies of each participant. Overall, 27 target sites, 17 hypomethylated (fold change [FC] range: 0.77-0.99) and 10 hypermethylated (FC range: 1.01-1.09), were differentially methylated between GDM and control pregnancies among 5 or more study participants. Novel genes were related to identified hypomethylated (such as NDUFC1, HAPLN3, HHLA3, and RHOG) or hypermethylated sites (such as SEP11, ZAR1, and DDR). Genes related to identified sites participated in cell morphology, cellular assembly, cellular organization, cellular compromise, and cell cycle. Our findings support early pregnancy peripheral blood DNA methylation differences in repeat pregnancies with change in GDM status. Similar, larger, and repeat pregnancy studies can enhance biomarker discovery and mechanistic studies of GDM.

We report a broader than previously appreciated clinical spectrum for hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and a potential pathogenic mechanism for DNA methyltransferase (DNMT1) mutations. The clinical presentations and genetic characteristics of nine newly identified HSAN1E kinships (45 affected subjects) were investigated. Five novel mutations of DNMT1 were discovered; p.C353F, p.T481P, p.P491L, p.Y524D and p.I531N, all within the target-sequence domain, and two mutations (p.T481P, p.P491L) arising de novo. Recently, HSAN1E has been suggested as an allelic disorder of autosomal dominant cerebellar ataxia, deafness and narcolepsy. Our results indicate that all the mutations causal for HSAN1E are located in the middle part or N-terminus end of the TS domain, whereas all the mutations causal for autosomal dominant cerebellar ataxia, deafness and narcolepsy are located in the C-terminus end of the TS domain. The impact of the seven causal mutations in this cohort was studied by cellular localization experiments. The binding efficiency of the mutant DNMT proteins at the replication foci and heterochromatin were evaluated. Phenotypic characterizations included electromyography, brain magnetic resonance and nuclear imaging, electroencephalography, sural nerve biopsies, sleep evaluation and neuropsychometric testing. The average survival of HSAN1E was 53.6 years. [standard deviation = 7.7, range 43-75 years], and mean onset age was 37.7 years. (standard deviation = 8.6, range 18-51 years). Expanded phenotypes include myoclonic seizures, auditory or visual hallucinations, and renal failure. Hypersomnia, rapid eye movement sleep disorder and/or narcolepsy were identified in 11 subjects. Global brain atrophy was found in 12 of 14 who had brain MRI. EEGs showed low frequency (delta waves) frontal-predominant abnormality in five of six patients. Marked variability in cognitive deficits was observed, but the majority of patients (89%) developed significant cognitive deficit by the age of 45 years. Cognitive function decline often started with personality changes and psychiatric manifestations. A triad of hearing loss, sensory neuropathy and cognitive decline remains as the stereotypic presentation of HSAN1E. Moreover, we show that mutant DNMT1 proteins translocate to the cytoplasm and are prone to form aggresomes while losing their binding ability to heterochromatin during the G2 cell cycle. Our results suggest mutations in DNMT1 result in imbalanced protein homeostasis through aggresome-induced autophagy. This mechanism may explain why mutations in the sole DNA maintenance methyltransferase lead to selective central and peripheral neurodegeneration.

Well-defined unnatural dipeptide-alcohols based on a cis-2,5-disubstitued pyrrolidine backbone were synthesized from commercially available starting materials meso-diethyl-2,5-dibromoadipate, (S)-(-)-1-phenylethylamine, and phenylalaninol. The structures of these unnatural dipeptide-alcohols are supported by HRMS, 1H- and 13C-NMR spectroscopy. These unnatural dipeptide-alcohols can act as building blocks for peptidomimetics.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in huntingtin (htt) protein. Dysregulation of brain iron homeostasis, oxidative stress and neurodegeneration are consistent features of the HD phenotype. Therefore, environmental factors that exacerbate oxidative stress and iron dysregulation may potentiate HD. Iron supplementation in the human population is common during infant and adult-life stages. In this study, iron supplementation in neonatal HD mice resulted in deterioration of spontaneous motor running activity, elevated levels of brain lactate and oxidized glutathione consistent with increased energetic dysfunction and oxidative stress, and increased striatal and motor cortical neuronal atrophy, collectively demonstrating potentiation of the disease phenotype. Oxidative stress, energetic, and anatomic markers of degeneration were not affected in wild-type littermate iron-supplemented mice. Further, there was no effect of elevated iron intake on disease outcomes in adult HD mice. We have demonstrated an interaction between the mutant huntingtin gene and iron supplementation in neonatal HD mice. Findings indicate that elevated neonatal iron intake potentiates mouse HD and promotes oxidative stress and energetic dysfunction in brain. Neonatal-infant dietary iron intake level may be an environmental modifier of human HD.

While most temperate broad-leaved tree species form ectomycorrhizal (EM) symbioses, a few species have arbuscular mycorrhizas (AM). It is not known whether EM and AM tree species differ systematically with respect to fine root morphology, fine root system size and root functioning. In a species-rich temperate mixed forest, we studied the fine root morphology and biomass of three EM and three AM tree species from the genera Acer, Carpinus, Fagus, Fraxinus, and Tilia searching for principal differences between EM and AM trees. We further assessed the evidence of convergence or divergence in root traits among the six co-occurring species. Eight fine root morphological and chemical traits were investigated in root segments of the first to fourth root order in three different soil depths and the relative importance of the factors root order, tree species and soil depth for root morphology was determined. Root order was more influential than tree species while soil depth had only a small effect on root morphology All six species showed similar decreases in specific root length and specific root area from the 1st to the 4th root order, while the species patterns differed considerably in root tissue density, root N concentration, and particularly with respect to root tip abundance. Most root morphological traits were not significantly different between EM and AM species (except for specific root area that was larger in AM species), indicating that mycorrhiza type is not a key factor influencing fine root morphology in these species. The order-based root analysis detected species differences more clearly than the simple analysis of bulked fine root mass. Despite convergence in important root traits among AM and EM species, even congeneric species may differ in certain fine root morphological traits. This suggests that, in general, species identity has a larger influence on fine root morphology than mycorrhiza type.

<h4>Introduction</h4>Depression and psychosis are two of the most severe neuropsychiatric symptoms (NPS) in dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). Both NPS have negative effects on cognitive performance and life expectancy. The current study aimed to investigate and compare monoaminergic etiologies between both neurodegenerative conditions, given the lack of an efficient pharmacological treatment until present.<h4>Methods</h4>Eleven behaviorally relevant brain regions of the left frozen hemisphere of 10 neuropathologically confirmed AD patients with/without depression (AD + D/-D; 5 were psychotic within AD + D), 10 confirmed DLB patients, all of whom were depressed (DLB + D; 5 psychotic patients), and, finally, 10 confirmed control subjects were regionally dissected. All patients were retrospectively assessed before death using the Behavioral Pathology in Alzheimer's Disease Rating Scale (Behave-AD) and Cornell Scale for Depression in Dementia amongst others. The concentrations of dopamine (DA), serotonin (5-HT), (nor)adrenaline and respective metabolites, i.e. 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), 5-hydroxy-3-indoleacetic acid (5-HIAA), and, 3-methoxy-4-hydroxyphenylglycol (MHPG), were determined using reversed-phase high-performance liquid chromatography with electrochemical detection.<h4>Results</h4>DLB subjects had the overall lowest monoamine and metabolite concentrations regarding 33 out of 41 significant monoaminergic group alterations. Moreover, MHPG levels were significantly decreased in almost 8 out of 11 brain regions of DLB- compared to AD patients. We also observed the lowest 5-HT and 5-HIAA levels, and 5-HIAA/5-HT turnover ratios in DLB + D compared to AD + D subjects. Additionally, a 4- and 7-fold increase of DOPAC/DA and HVA/DA turnover ratios, and, a 10-fold decrease of thalamic DA levels in DLB + D compared to AD + D patients and control subjects was noticed. Regarding psychosis, hippocampal DA levels in the overall DLB group significantly correlated with Behave-AD AB scores. In the total AD group, DA levels and HVA/DA ratios in the amygdala significantly correlated with Behave-AD AB scores instead.<h4>Conclusions</h4>Monoaminergic neurotransmitter alterations contribute differently to the pathophysiology of depression and psychosis in DLB as opposed to AD, with a severely decreased serotonergic neurotransmission as the main monoaminergic etiology of depression in DLB. Similarly, psychosis in DLB might, in part, be etiologically explained by dopaminergic alterations in the hippocampus, whereas in AD, the amygdala might be involved.

Two novel series of unsymmetrically substituted 1,2,4-oxadiazole viz., R.Ox.C(*)C n compounds are synthesized and characterized. An optically active, (S)-(+)-methyl 3-hydroxy-2-methylpropionate is used to introduce a chiral center in the molecule. A biphenyl moiety prepared by Suzuki coupling reaction is directly attached to the oxadiazole core at C-5 position. Investigations for the phase behavior revealed that the series with a benzyl group on one end of the oxadiazole core exhibits an 1D orthogonal smectic-A phase while the second series with dodecyl flexible end chain shows orthogonal smectic-A and tilted chiral smectic-C (SmC*) phases over a wide range of temperatures. The smectic-C phase exhibits ferroelectric (FE) polarization switching. The mesomorphic thermal stabilities of these compounds are discussed in the domain of the symmetry and the flexibility of the alkyloxy end chain length attached to the chiral center.

This quality improvement project was inspired as an answer to a problem that many fellow psychiatric trainees had been struggling with while on-call covering the old age mental health hospital which includes a specialist dementia ward. The issue was that decisions around ceilings of care for patients were often not discussed or at least recorded in the electronic notes and as a result when reviewing deteriorating patients out of hours trainees would find themselves without any guidance on the treating medics opinion on what was in the best interests of the patient. This led to situations where unnecessary transfers to the acute hospital would occur overnight which could have been avoided with more consistent planning. Prior to initiating the changes it was recorded that nine out of 47 inpatients had documented decisions on ceiling of care of treatment in the consultant's ward round entries. Next policies from acute hospitals were reviewed, opinions were discussed in departmental meetings, and eventually there was agreed a change in procedure with the consultant on the dementia ward around resuscitation and ceiling of care status and consistent recording of this. Following the intervention there was seen an improvement in the recording of decisions around treatment and transfer of patients on the dementia ward of 80% (4/5) fully compliant with new criteria and then 71% (5/7) in successive cycles. Further communication both with relevant professionals on the old age ward and with the trainees on the on-call rota will be necessary to sustain any change but the centralised recording of resuscitation status and ceiling of care in the ward round entries have provided much more guidance than was previously available. In the future it may be possible to spread this policy throughout the entire old age mental health unit.

<h4>Background</h4>Dementia is a costly disease. People with dementia, their families, and their friends are affected on personal, emotional, and financial levels. Prior work has shown that the "Partners in Dementia Care" (PDC) intervention addresses unmet needs and improves psychosocial outcomes and satisfaction with care.<h4>Objective</h4>We examined whether PDC reduced direct Veterans Health Administration (VHA) health care costs compared with usual care.<h4>Design</h4>This study was a cost analysis of the PDC intervention in a 30-month trial involving five VHA medical centers.<h4>Participants</h4>Study subjects were veterans (N = 434) 50 years of age and older with dementia and their caregivers at two intervention (N = 269) and three comparison sites (N = 165).<h4>Interventions</h4>PDC is a telephone-based care coordination and support service for veterans with dementia and their caregivers, delivered through partnerships between VHA medical centers and local Alzheimer's Association chapters.<h4>Main measures</h4>We tested for differences in total VHA health care costs, including hospital, emergency department, nursing home, outpatient, and pharmacy costs, as well as program costs for intervention participants. Covariates included caregiver reports of veterans' cognitive impairment, behavior problems, and personal care dependencies. We used linear mixed model regression to model change in log total cost post-baseline over a 1-year follow-up period.<h4>Key results</h4>Intervention participants showed higher VHA costs than usual-care participants both before and after the intervention but did not differ significantly regarding change in log costs from pre- to post-baseline periods. Pre-baseline log cost (p ≤ 0.001), baseline cognitive impairment (p ≤ 0.05), number of personal care dependencies (p ≤ 0.01), and VA service priority (p ≤ 0.01) all predicted change in log total cost.<h4>Conclusions</h4>These analyses show that PDC meets veterans' needs without significantly increasing VHA health care costs. PDC addresses the priority area of care coordination in the National Plan to Address Alzheimer's Disease, offering a low-cost, structured, protocol-driven, evidence-based method for effectively delivering care coordination.

<h4>Purpose</h4>The transcription factor Fos-related antigen-1 (Fra-1) has been described to affect the morphology, motility and invasive potential of breast cancer cells. Since tumor cell adhesion plays an essential role in the metastatic process, especially for extravasation from blood vessels, we investigated the influence of Fra-1 on breast cancer cell interactions with the endothelium.<h4>Methods</h4>Using Fra-1-overexpressing MCF7 [weakly invasive, estrogen receptor (ER)-positive] and MDA MB231 (strongly invasive, ER-negative) cells, we performed dynamic cell flow adhesion assays on surfaces coated with E-selectin or with human pulmonary microvascular endothelial cells.<h4>Results</h4>We found a significant increased adhesion of Fra-1-overexpressing MCF7 cells to E-selectin but also to activate endothelial cells, whereas the MDA MB231 cell line showed moderate enhanced cell rolling and tethering on both coated surfaces. These different adhesion behaviors corresponded to an up-regulation of various adhesion-related proteins such as CD44 and integrin α5 in Fra-1-overexpressing MCF7 cells measured by microarray analysis and flow cytometry in comparison with no deregulation of key adhesion molecules observed in Fra-1-overexpressing MDA MB231 cells. In line with these results and based on cDNA microarray data of breast cancer patients (n = 197), high Fra-1 expression significantly correlates with shorter overall survival and higher rate of lung metastasis in ER-positive breast cancer patients (n = 130), but has no impact on the prognosis of patients with ER-negative tumors.<h4>Conclusion</h4>Thus, in addition to its pro-invasive and pro-migratory effect, Fra-1 might influence the metastatic potential of breast cancer cells by changing the expression of adhesion molecules, resulting in increased adherence to endothelial cells under flow conditions.

Perturbations in autophagy and apoptosis are associated with cancer development. XIAP and cIAP1 are two members of the inhibitors of apoptosis protein family whose expression is elevated in different cancers. Here we report that XIAP and cIAP1 induce autophagy by upregulating the transcription of Beclin 1, an essential autophagy gene. The E3 ubiquitin ligase activity of both proteins activates NFκB signalling, leading to the direct binding of p65 to the promoter of Beclin 1 and to its transcriptional activation. This mechanism may be relevant in cancer cells, since we found increased levels of autophagy in different B-cell lymphoma-derived cell lines where XIAP is overexpressed and pharmacological inhibition of XIAP in these cell lines reduced autophagosome biogenesis. Thus, the chemotherapy resistance associated with XIAP and cIAP1 overexpression observed in several human cancers may be, at least in part, due to the Beclin 1-dependent autophagy activation by IAPs described in this study. In this context, the disruption of this increased autophagy might represent a valuable pharmacological tool to be included in combined anti-neoplastic therapies.

Mitochondria provide numerous essential functions for cells and their dysfunction leads to a variety of diseases. Thus, obtaining a complete mitochondrial proteome should be a crucial step toward understanding the roles of mitochondria. Many mitochondrial proteins have been identified experimentally but a complete list is not yet available. To fill this gap, methods to computationally predict mitochondrial proteins from amino acid sequence have been developed and are widely used, but unfortunately, their accuracy is far from perfect. Here we describe MitoFates, an improved prediction method for cleavable N-terminal mitochondrial targeting signals (presequences) and their cleavage sites. MitoFates introduces novel sequence features including positively charged amphiphilicity, presequence motifs, and position weight matrices modeling the presequence cleavage sites. These features are combined with classical ones such as amino acid composition and physico-chemical properties as input to a standard support vector machine classifier. On independent test data, MitoFates attains better performance than existing predictors in both detection of presequences and in predicting their cleavage sites. We used MitoFates to look for undiscovered mitochondrial proteins from 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants). MitoFates predicts 1167 genes to have at least one isoform with a presequence. Five-hundred and eighty of these genes were not annotated as mitochondrial in either UniProt or Gene Ontology. Interestingly, these include candidate regulators of parkin translocation to damaged mitochondria, and also many genes with known disease mutations, suggesting that careful investigation of MitoFates predictions may be helpful in elucidating the role of mitochondria in health and disease. MitoFates is open source with a convenient web server publicly available.

The regulation of dendritic branching is critical for sensory reception, cell-cell communication within the nervous system, learning, memory, and behavior. Defects in dendrite morphology are associated with several neurologic disorders; thus, an understanding of the molecular mechanisms that govern dendrite morphogenesis is important. Recent investigations of dendrite morphogenesis have highlighted the importance of gene regulation at the posttranscriptional level. Because RNA-binding proteins mediate many posttranscriptional mechanisms, we decided to investigate the extent to which conserved RNA-binding proteins contribute to dendrite morphogenesis across phyla. Here we identify a core set of RNA-binding proteins that are important for dendrite morphogenesis in the PVD multidendritic sensory neuron in Caenorhabditis elegans. Homologs of each of these genes were previously identified as important in the Drosophila melanogaster dendritic arborization sensory neurons. Our results suggest that RNA processing, mRNA localization, mRNA stability, and translational control are all important mechanisms that contribute to dendrite morphogenesis, and we present a conserved set of RNA-binding proteins that regulate these processes in diverse animal species. Furthermore, homologs of these genes are expressed in the human brain, suggesting that these RNA-binding proteins are candidate regulators of dendrite development in humans.

<h4>Objective</h4>Test the hypothesis that greater baseline peak external knee adduction moment (KAM), KAM impulse, and peak external knee flexion moment (KFM) during the stance phase of gait are associated with baseline-to-2-year medial tibiofemoral cartilage damage and bone marrow lesion progression, and cartilage thickness loss.<h4>Methods</h4>Participants all had knee OA in at least one knee. Baseline peak KAM, KAM impulse, and peak KFM (normalized to body weight and height) were captured and computed using a motion analysis system and six force plates. Participants underwent MRI of both knees at baseline and 2 years later. To assess the association between baseline moments and baseline-to-2-year semiquantitative cartilage damage and bone marrow lesion progression and quantitative cartilage thickness loss, we used logistic and linear regressions with generalized estimating equations (GEE), adjusting for gait speed, age, gender, disease severity, knee pain severity, and medication use.<h4>Results</h4>The sample consisted of 391 knees (204 persons): mean age 64.2 years (SD 10.0); BMI 28.4 kg/m(2) (5.7); 156 (76.5%) women. Greater baseline peak KAM and KAM impulse were each associated with worsening of medial bone marrow lesions, but not cartilage damage. Higher baseline KAM impulse was associated with 2-year medial cartilage thickness loss assessed both as % loss and as a threshold of loss, whereas peak KAM was related only to % loss. There was no relationship between baseline peak KFM and any medial disease progression outcome measures.<h4>Conclusion</h4>Findings support targeting KAM parameters in an effort to delay medial OA disease progression.

In this study we investigated how the networks mediating respiratory and locomotor drives to lumbar motoneurons interact and how this interaction is modulated in relation to periodic variations in blood pressure (Mayer waves). Seven decerebrate cats, under neuromuscular blockade, were used to study central respiratory drive potentials (CRDPs, usually enhanced by added CO2) and spontaneously occurring locomotor drive potentials (LDPs) in hindlimb motoneurons, together with hindlimb and phrenic nerve discharges. In four of the cats both drives and their voltage-dependent amplification were absent or modest, but in the other three, one or other of these drives was common and the voltage-dependent amplification was frequently strong. Moreover, in these three cats the blood pressure showed marked periodic variation (Mayer waves), with a slow rate (periods 9-104 s, mean 39 ± 17 SD). Profound modulation, synchronized with the Mayer waves was seen in the occurrence and/or in the amplification of the CRDPs or LDPs. In one animal, where CRDPs were present in most cells and the amplification was strong, the CRDP consistently triggered sustained plateaux at one phase of the Mayer wave cycle. In the other two animals, LDPs were common, and the occurrence of the locomotor drive was gated by the Mayer wave cycle, sometimes in alternation with the respiratory drive. Other interactions between the two drives involved respiration providing leading events, including co-activation of flexors and extensors during post-inspiration or a locomotor drive gated or sometimes entrained by respiration. We conclude that the respiratory drive in hindlimb motoneurons is transmitted via elements of the locomotor central pattern generator. The rapid modulation related to Mayer waves suggests the existence of a more direct and specific descending modulatory control than has previously been demonstrated.

<h4>Background</h4>Excessive and inappropriate immune responses are the hallmark of several autoimmune disorders, including the inflammatory bowel diseases (IBD): Crohn's disease (CD) and ulcerative colitis (UC). A complex etiology involving both environmental and genetic factors influences IBD pathogenesis. The role of microRNAs (miRNAs), noncoding RNAs involved in regulating numerous biological processes, to IBD pathology, in terms of initiation and progression, remains ill-defined. In the present study, we evaluated the relationship between colon, peripheral blood, and saliva whole miRNome expression in IBD patients and non-inflammatory bowel disease (non-IBD) controls to identify miRNAs that could discriminate CD from UC. Quantitative real-time PCR (qRT-PCR) was used to validate and assess miRNA expression.<h4>Results</h4>Microarray analysis demonstrated that upwards of twenty six miRNAs were changed in CD and UC colon biopsies relative to the non-IBD controls. CD was associated with the differential expression of 10 miRNAs while UC was associated with 6 miRNAs in matched colon tissues. CD was associated with altered expression of 6 miRNAs while UC was associated with 9 miRNAs in whole blood. Expression of miR-101 in CD patients and miR-21, miR-31, miR-142-3p, and miR-142-5p in UC patients were altered in saliva.<h4>Conclusions</h4>Our results suggest that there is specific miRNA expression patterns associated with UC versus CD in three separate tissue/body fluids (colon, blood, and saliva). Further, the aberrant miRNA expression profiles indicate that miRNAs may be contributory to IBD pathogenesis, or at least reflect the underlying inflammation. Scrutinizing miRNA expression in saliva and blood samples may be beneficial in monitoring or diagnosing disease in IBD patients. A panel of miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-146a, and miR-375) may be used as markers to identify and discriminate between CD and UC.

Neutrophil extracellular traps (NETs) are involved in bacterial killing as well as autoimmunity, because NETs contain proteases, bactericidal peptides, DNA and ribonucleoprotein. NETs are formed via a novel type of cell death called NETosis. NETosis is distinct from apoptosis, but it resembles necrosis in that both membranes are not intact so that they allow intracellular proteins to leak outside of the cells. Removal of NETs and neutrophils undergoing NETosis by phagocytes and its subsequent response are not completely clarified, as compared with the response after removal of either apoptotic or necrotic neutrophils by phagocytes. How neutrophil density in peripheral blood is kept within a certain range is important for health and disease. Although the studies on severe congenital neutropenia and benign ethnic neutropenia have provided unbiased views on it, the studies are rather limited to human neutropenia, and mice with a mutation of mouse counterpart gene often fail to exhibit neutropenia. Degranulation plays a critical role in bactericidal action. The recent studies revealed that it is also involved in immunomodulation, pain control and estrous cycle control. N1 and N2 are representative of neutrophil subpopulations. The dichotomy holds true in patients or mice with severe trauma or cancer, providing the basis of differential roles of neutrophils in diseases.

Sustained and controlled pellets are considered as one of the ideal dosage forms. Due to the large coverage area of pellets, loaded drugs can be absorbed completely in the body and bioavailability is improved correspondingly. Coated pellets-containing tablet is a special oral formulation consisting of various pellets with different release rate. Desired rate of drug release rate can be achieved by adjusting the proportion of pellets. However, this formulation faces strict requirements in the process of preparation. Several factors will influence release behavior of tablets, including pellet cores, coating, and tabletting. Therefore, these factors will be investigated sufficiently in this review to provide valuable information for manufacturing process.

Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.

The enteric nervous system (ENS) is sometimes called the "second brain" because of the diversity of neuronal cell types and complex, integrated circuits that permit the ENS to autonomously regulate many processes in the bowel. Mechanisms supporting ENS development are intricate, with numerous proteins, small molecules, and nutrients that affect ENS morphogenesis and mature function. Damage to the ENS or developmental defects cause vomiting, abdominal pain, constipation, growth failure, and early death. Here, we review molecular mechanisms and cellular processes that govern ENS development, identify areas in which more investigation is needed, and discuss the clinical implications of new basic research.

Therapies targeting cancer metastasis are challenging owing to the complexity of the metastatic process and the high number of effectors involved. Although tumour hypoxia has previously been associated with increased aggressiveness as well as resistance to radio- and chemotherapy, the understanding of a direct link between the level and duration of hypoxia and the individual steps involved in metastasis is still missing. Using live imaging in a chick embryo model, we have demonstrated that the exposure of neuroblastoma cells to 1% oxygen for 3 days was capable of (1) enabling cell migration towards blood vessels, (2) slowing down their velocity within blood vessels to facilitate extravasation and (3) promoting cell proliferation in primary and secondary sites. We have shown that cells do not have to be hypoxic anymore to exhibit these acquired capabilities as a long-term memory of prior hypoxic exposure is kept. Furthermore, non-hypoxic cells can be influenced by neighbouring hypoxic preconditioned cells and be entrained in the metastatic progression. The acquired aggressive phenotype relies on hypoxia-inducible factor (HIF)-dependent transcription of a number of genes involved in metastasis and can be impaired by HIF inhibition. Altogether, our results demonstrate the need to consider both temporal and spatial tumour heterogeneity because cells can 'remember' an earlier environment and share their acquired phenotype with their close neighbours. As a consequence, it is necessary to monitor the correct hypoxic markers to be able to predict the consequences of the cells' history on their behaviour and their potential response to therapies.

Mutations in KDM5C are an important cause of X-linked intellectual disability in males. KDM5C encodes a histone demethylase, suggesting that alterations in chromatin landscape may contribute to disease. We used primary patient cells and biochemical approaches to investigate the effects of patient mutations on KDM5C expression, stability and catalytic activity. We report and characterize a novel nonsense mutation, c.3223delG (p.V1075Yfs*2), which leads to loss of KDM5C protein. We also characterize two KDM5C missense mutations, c.1439C>T (p.P480L) and c.1204G>T (p.D402Y) that are compatible with protein production, but compromise stability and enzymatic activity. Finally, we demonstrate that a c.2T>C mutation in the translation initiation codon of KDM5C results in translation re-start and production of a N-terminally truncated protein (p.M1_E165del) that is unstable and lacks detectable demethylase activity. Patient fibroblasts do not show global changes in histone methylation but we identify several up-regulated genes, suggesting local changes in chromatin conformation and gene expression. This thorough examination of KDM5C patient mutations demonstrates the utility of examining the molecular consequences of patient mutations on several levels, ranging from enzyme production to catalytic activity, when assessing the functional outcomes of intellectual disability mutations.

Since its discovery and isolation, exogenous insulin has dramatically changed the outlook for patients with diabetes. However, even when patients strictly follow an insulin regimen, serious complications can result as patients experience both hyperglycemic and hypoglycemic states. Several chemically or genetically modified insulins have been developed that tune the pharmacokinetics of insulin activity for personalized therapy. Here, we demonstrate a strategy for the chemical modification of insulin intended to promote both long-lasting and glucose-responsive activity through the incorporation of an aliphatic domain to facilitate hydrophobic interactions, as well as a phenylboronic acid for glucose sensing. These synthetic insulin derivatives enable rapid reversal of blood glucose in a diabetic mouse model following glucose challenge, with some derivatives responding to repeated glucose challenges over a 13-h period. The best-performing insulin derivative provides glucose control that is superior to native insulin, with responsiveness to glucose challenge improved over a clinically used long-acting insulin derivative. Moreover, continuous glucose monitoring reveals responsiveness matching that of a healthy pancreas. This synthetic approach to insulin modification could afford both long-term and glucose-mediated insulin activity, thereby reducing the number of administrations and improving the fidelity of glycemic control for insulin therapy. The described work is to our knowledge the first demonstration of a glucose-binding modified insulin molecule with glucose-responsive activity verified in vivo.

Hollyhock (Althaea rosea (Linn.) Cavan) belongs to Althaea genus, Malvaceae family, is a perennial garden plant distributed throughout the world in warm temperate and tropical regions, which was used as anti-diabetes ingredient in traditional Chinese medicine. In the process of our research, ethanolic extract of hollyhock flower (HFE) was found to decrease serum triglyceride and glucose levels significantly in KK-A(y) mice after oral administration for 8weeks. Meanwhile, gene expressions on AMPK, IRS2, PI3K, AKT and GLUT4 in liver were remarkably up-regulated. Three new dihydroflavonol glycosides, named as roseaflavanonolosides A (1), B (2), and C (3), together with two known ones were obtained from HFE. Their structures were elucidated by chemical and spectroscopic methods. Hepatic cell glucose uptake experiment was performed using 2-NBDG as a glucose uptake indicator. At the dosage of 20μg/mL for 1-5, the glucose uptake increasing level was nearly 30%-40% in HepG2 cells. We partly revealed the glucose metabolism regulation effect may relate to dihydroflavonols in hollyhock flower.

Genes encoding proteins with antioxidant properties may influence susceptibility to endometrial hyperplasia (EH) and endometrial carcinoma (ECa). Patients with EH (n = 89), EH concurrent with ECa (n = 76), ECa (n = 186), and healthy controls (n = 1110) were genotyped for five polymorphic variants in the genes involved in metabolism of lipoproteins (APOE Cys112Arg and Arg158Cys), iron (HFE Cys282Tyr and His63Asp), and catecholamines (COMT Val158Met). Patients and controls were matched by ethnicity (all Caucasians), age, body mass index (BMI), and incidence of hypertension and diabetes. The frequency of the APOE E 2 allele (158Cys) was higher in patients with EH + ECa than in controls (P = 0.0012, P(Bonferroni) = 0.018, OR = 2.58, 95% CI 1.49-4.45). The APOE E 4 allele (112Arg) was more frequently found in patients with EH than in controls and HFE minor allele G (63Asp) had a protective effect in the ECa group, though these results appeared to be nonsignificant after correction for multiple comparisons. The results of the study indicate that E 2 allele might be associated with concurrent occurrence of EH and ECa.

Global fish production from aquaculture has rapidly grown over the past decades, and grass carp shares the largest portion. However, hemorrhagic disease caused by grass carp reovirus (GCRV) results in tremendous loss of grass carp (Ctenopharyngodon idella) industry. During the past years, development of molecular biology and cellular biology technologies has promoted significant advances in the understanding of the pathogen and the immune system. Immunoprophylaxis based on stimulation of the immune system of fish has also got some achievements. In this review, authors summarize the recent progresses in basic researches on GCRV; viral nucleic acid sensors, high-mobility group box proteins (HMGBs); pattern recognition receptors (PRRs), Toll-like receptors (TLRs) and retinoic acid inducible gene I- (RIG-I-) like receptors (RLRs); antiviral immune responses induced by PRRs-mediated signaling cascades of type I interferon (IFN-I) and IFN-stimulated genes (ISGs) activation. The present review also notices the potential applications of molecule genetic markers. Additionally, authors discuss the current preventive and therapeutic strategies (vaccines, RNAi, and prevention medicine) and highlight the importance of innate immunity in long term control for grass carp hemorrhagic disease.

Data from high throughput experiments often produce far more results than can ever appear in the main text or tables of a single research article. In these cases, the majority of new associations are often archived either as supplemental information in an arbitrary format or in publisher-independent databases that can be difficult to find. These data are not only lost from scientific discourse, but are also elusive to automated search, retrieval and processing. Here, we use the nanopublication model to make scientific assertions that were concluded from a workflow analysis of Huntington's Disease data machine-readable, interoperable, and citable. We followed the nanopublication guidelines to semantically model our assertions as well as their provenance metadata and authorship. We demonstrate interoperability by linking nanopublication provenance to the Research Object model. These results indicate that nanopublications can provide an incentive for researchers to expose data that is interoperable and machine-readable for future use and preservation for which they can get credits for their effort. Nanopublications can have a leading role into hypotheses generation offering opportunities to produce large-scale data integration.

<h4>Background</h4>Long non-coding RNAs (lncRNA) play an important role in carcinogenesis; knowledge on lncRNA expression in renal cell carcinoma is rudimental. As a basis for biomarker development, we aimed to explore the lncRNA expression profile in clear cell renal cell carcinoma (ccRCC) tissue.<h4>Results</h4>Microarray experiments were performed to determine the expression of 32,183 lncRNA transcripts belonging to 17,512 lncRNAs in 15 corresponding normal and malignant renal tissues. Validation was performed using quantitative real-time PCR in 55 ccRCC and 52 normal renal specimens. Computational analysis was performed to determine lncRNA-microRNA (MiRTarget2) and lncRNA-protein (catRAPID omics) interactions. We identified 1,308 dysregulated transcripts (expression change >2-fold; upregulated: 568, downregulated: 740) in ccRCC tissue. Among these, aberrant expression was validated using PCR: lnc-BMP2-2 (mean expression change: 37-fold), lnc-CPN2-1 (13-fold), lnc-FZD1-2 (9-fold), lnc-ITPR2-3 (15-fold), lnc-SLC30A4-1 (15-fold), and lnc-SPAM1-6 (10-fold) were highly overexpressed in ccRCC, whereas lnc-ACACA-1 (135-fold), lnc-FOXG1-2 (19-fold), lnc-LCP2-2 (2-fold), lnc-RP3-368B9 (19-fold), and lnc-TTC34-3 (314-fold) were downregulated. There was no correlation between lncRNA expression with clinical-pathological parameters. Computational analyses revealed that these lncRNAs are involved in RNA-protein networks related to splicing, binding, transport, localization, and processing of RNA. Small interfering RNA (siRNA)-mediated knockdown of lnc-BMP2-2 and lnc-CPN2-1 did not influence cell proliferation.<h4>Conclusions</h4>We identified many novel lncRNA transcripts dysregulated in ccRCC which may be useful for novel diagnostic biomarkers.

<h4>Background</h4>Rotavirus infections are a major cause of diarrhea in children in both developed and developing countries. Rotavirus genetics, patient immunity, and environmental factors are thought to be related to the severity of acute diarrhea due to rotavirus in infants and young children. The objective of this study was to provide a correlation between rotavirus genotypes, clinical factors and degree of severity of acute diarrhea in children under 5 years old in Surabaya, Indonesia.<h4>Methods</h4>A cross-sectional study was conducted in children aged 1-60 months with acute diarrhea hospitalized in Soetomo Hospital, Surabaya, Indonesia from April to December 2013. Rotavirus in stool specimens was identified by ELISA and genotyping (G-type and P-type) using multiplex reverse transcription PCR. Severity was measured using the Ruuska and Vesikari scoring system. The clinical factors were investigated included patient's age (months), hydration, antibiotic administration, nutritional state, co-bacterial infection and co-viral infection.<h4>Results</h4>A total of 88 children met the criteria; 80.7% were aged 6-24 months, watery diarrhea was the most common type (77.3%) and 73.6% of the subjects were co-infected with bacteria, of which pathogenic Escherichia coli was the most common (42.5%). The predominant VP7 genotyping (G-type) was G2 (31.8%) and that of VP4 genotyping (P-type) was P[4] (31.8%). The predominant rotavirus genotype was G2P[4] (19.3%); G1P[4] and G9P[4] were uncommon with a prevalence of 4.5%. There were significant differences between the common genotype and uncommon genotype with respect to the total severity score of diarrhea (p <0.05). G3, G4 and G9 were significantly correlated with severe diarrhea (p = 0.009) in multivariate analyses and with frequency of diarrhea (>10 times a day) (p = 0.045) in univariate analyses, but there was no significant correlation between P typing and severity of diarrhea. For combination genotyping of G and P, G2P[4] was significantly correlated with severe diarrhea in multivariate analyses (p = 0.029).<h4>Conclusions</h4>There is a correlation between rotavirus genotype and severity of acute diarrhea in children. Genotype G2P[4] has the highest prevalence. G3, G4, G9 and G2P[4] combination genotype were found to be associated with severe diarrhea.

The purpose of this study is to determine the relationship between personality, the serotonin transporter (5HTT) and monoamine oxidase A (MAO-A) polymorphisms and the severity of climacteric and depressive symptoms in postmenopausal women. The study involved 272 healthy postmenopausal women from Poland. This survey-based study was performed using the following: the Beck Depression Inventory for depressive symptoms, the Blatt-Kupperman Menopausal Index and the Neuroticism-Extroversion-Openness-Five Factor Inventory for personality. A polymerase chain reaction was employed to identify the DNA polymorphisms. The women were aged 55.4 ± 5.5 years on average. Significant correlations were proved between the allele frequency of the 30-bp variable-number tandem repeat (VNTR) polymorphism in the MAO-A promoter region and the incidence of depressive symptoms in the women analysed (p ≤ 0.05), as well as between the severity of climacteric symptoms in the postmenopausal women and the allele frequency of the polymorphism in the 5HTT gene (the 5HTT 's' variant) (p ≤ 0.05). There was a significant correlation between the severity of climacteric and depressive symptoms (p < 0.001). (1) The severity of climacteric and depressive symptoms depends on personality traits. (2) Personality traits are biologically determined, and the level of their expression is associated with the 5HTT polymorphism. (3) Identification of homogeneous groups of women having predispositions to depressive and severe climacteric symptoms may help to implement early prevention programmes for this group of recipients.

Mammary gland is an exocrine and sebaceous gland made up of branching network of ducts that end in alveoli. Milk is synthesized in the alveoli and secreted into alveolar lumen. Mammary gland represents an ideal system for the study of organogenesis that undergoes successive cycles of pregnancy, lactation and involution. To gain insights on the molecular events that take place in pubertal and lactating mammary gland, we have identified 43 differentially expressed proteins in mammary tissue of heifer (non-lactating representing a virgin mammary gland), and lactating buffaloes (Bubalus bubalis) by 2D-difference gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty one proteins were upregulated during lactation whereas 8 proteins were upregulated in heifer mammary gland significantly (p<0.05). Bioinformatics analyses of the identified proteins showed that a majority of the proteins are involved in metabolic processes. The differentially expressed proteins were validated by real-time PCR and Western blotting. We observed differential expressions of certain new proteins including EEF1D, HSPA5, HSPD1 and PRDX6 during lactation which have not been reported before. The differentially expressed proteins were mapped to available biological pathways and networks involved in lactation. This study signifies the importance of some proteins which are preferentially expressed during lactation and in heifer mammary gland.<h4>Biological significance</h4>This work is important because we have generated information in water buffalo (B. bubalis) for the first time which is the major milk producing animal in Indian Subcontinent. Out of a present production of 133milliontons of milk produced in India, contribution of buffalo milk is around 54%. Its physiology is somewhat different from the lactating cows. Buffalo milk composition varies from cow milk in terms of higher fat and total solid content, which confers an advantage in preparation of specialized cheese, curd and other dairy products. Being a major milk producing animal in India it is highly essential to understand the lactation associated proteins in the mammary gland of buffalo. In the present investigation our attempt has been to identify new protein evidences which are expressed in lactating buffalo mammary gland and have not been reported before. The findings reported in the present study will help in understanding the lactation biology of buffalo mammary gland in particular and the mammary gland biology in general.

<h4>Background</h4>Adrenal glands are essential endocrine organs composed of two embryological distinct tissues. Morphological changes during their development are well described, but less understood with regard to their molecular mechanisms. To identify proteins and pathways, which drive the initial steps of the specification of the endocrine function of the adrenal gland, rat's adrenal glands were isolated at different embryonic days (E): E14, E16, E18, E19 and postnatal day 1 (P1).<h4>Results</h4>The alteration of the proteome during the stages E16, E19 and P1 was investigated by combining two dimensional gel electrophoresis and mass spectrometric analysis. Out of 594 excised protein spots, 464 spots were identified, resulting in 203 non-redundant proteins. The ontogenic classification of the identified proteins according to their molecular function resulted in 10 different categories, whereas the classification of their biological processes resulted in 19 different groups. This gives an insight into the complex mechanisms underlying adrenal gland development. Interestingly, the expression of retinoic acid pathway proteins was decreased during the development of the adrenal gland, suggesting that this pathway is only important at early stages. On the other hand, key proteins of the cholesterol synthesis increased their expression significantly at E19 revealing the initiation of the endocrine specialization of the adrenal glands.<h4>Conclusions</h4>This study presents the first comprehensive wide proteome analysis of three different stages of embryonic adrenal gland development. The identified proteins, which were expressed in early stages of development, will shed light on the molecular mechanisms underlying embryonic development of the adrenal gland.

<h4>Background</h4>In community-acquired pneumonia host inflammatory response against the causative microorganism is necessary for infection resolution. However an excessive response can have deleterious effects. In addition to antimicrobial effects, macrolide antibiotics are known to possess immunomodulatory properties.<h4>Methods</h4>A prospective study was performed on 52 admitted patients who developed an inadequate response after 72 hours of antibiotic treatment - non-responders community-acquired pneumonia - (blood and bronchoalveolar lavage), and two control groups: 1) community-acquired pneumonia control (blood) and 2) non-infection control (blood and bronchoalveolar lavage). Cytokine profiles (interleukin (IL)-6, IL-8, IL-10), tumour necrosis factor α and clinical outcomes were assessed.<h4>Results</h4>Non-responders patients treated with macrolide containing regimens showed significantly lower levels of IL-6 and TNF-α in bronchoalveolar lavage fluid and lower IL-8 and IL-10 in blood than those patients treated with non-macrolide regimens. Clinical outcomes showed that patients treated with macrolide regimens required fewer days to reach clinical stability (p < 0.01) and shorter hospitalization periods (p < 0.01).<h4>Conclusions</h4>After 72 hours of antibiotic effect, patients who received macrolide containing regimens exhibited lower inflammatory cytokine levels in pulmonary and systemic compartments along with faster stabilization of infectious parameters.

<h4>Background</h4>Polyunsaturated fatty acids (PUFAs) are nutrients necessary for life. The liver is the essential metabolic center, which aids in maintaining health via diverse biological actions. In the present work, a proteomics study was conducted with an aim to provide new insights into PUFA-regulated hepatic protein expression in apoE-knockout mice. Additionally, we investigated how n-3 PUFAs influence cytokine-challenge by using HepG2 cells as a model.<h4>Results</h4>Through the proteomic analysis using 2-dimensional electrophoresis and mass spectrometry, we found that 28, 23, 14, and 28 hepatic proteins were up-regulated at least a two-fold difference in intensity compared with the control group in mice treated with the docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, and linoleic acid, respectively. In contrast, 12 hepatic proteins were down-regulated with a ratio value of less than 0.5 compared to their control counterparts by these four fatty acids. All of the altered proteins were then sorted according to their biochemical properties related to metabolism, redox stress/inflammation, enzymatic reactions, and miscellaneous functions. The results provide evidence that PUFAs may act as either pro-inflammatory or anti-inflammatory agents. Cytokine-challenged HepG2 cells were used to reveal the anti-inflammatory function of n-3 PUFAs. The results showed that interleukin (IL)-1β combined with IL-6 induced C-reactive protein (CRP) mRNA expression and its protein secretion by HepG2 cells. The CRP promoter activity was significantly increased in the IL-6-treated cells, whereas IL-1β alone had no effect. However, IL-1β and IL-6 acted synergistically to further enhance CRP promoter activities. Furthermore, n-3 PUFAs inhibited nuclear factor-κB (NF-κB) activation and the phosphorylation of the nuclear signal transducer and activator of transcription 3 (STAT3) during cytokine-induced CRP production.<h4>Conclusion</h4>This study indicates that PUFAs induced changes in the hepatic protein profile in vivo. Furthermore, n-3 PUFAs exert their anti-inflammatory properties through differential molecular mechanisms in hepatic cells. These results provide novel information regarding the roles of PUFAs in the liver at the tissue and cellular levels.

Avascular necrosis of head of femur is a rare presentation in Dyskeratosis Congenita (DC). Aplastic anaemia, increased susceptibility to infections and fatal pulmonary complications often complicate the clinical scenario in these patients leading to short life span and difficulty in performing surgical interventions. The diagnosis initially and subsequent management by total hip replacement posed a great challenge. To our knowledge this is the first case report of any intervention in form of total hip Arthroplasty done in DC. At 5 years of follow up the patient is asymptomatic with no radiographic signs of loosening or lysis.

The unfolded protein response (UPR) occurs following the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and orchestrates an intricate balance between its prosurvival and apoptotic arms to restore cellular homeostasis and integrity. However, in certain neurodegenerative diseases, the apoptotic arm of the UPR is enhanced, resulting in excessive neuronal cell death and disease progression, both of which can be overcome by modulating the UPR. Here, we describe a novel crosstalk between glucocorticoid receptor signaling and the apoptotic arm of the UPR, thus highlighting the potential of glucocorticoid therapy in treating neurodegenerative diseases. Several glucocorticoids, but not mineralocorticoids, selectively antagonize ER stress-induced apoptosis in a manner that is downstream of and/or independent of the conventional UPR pathways. Using GRT10, a novel selective pharmacological modulator of glucocorticoid signaling, we describe the importance of the transrepression arm of the glucocorticoid signaling pathway in protection against ER stress-induced apoptosis. Furthermore, we also observe the protective effects of glucocorticoids in vivo in a Drosophila model of Huntington's disease (HD), wherein treatment with different glucocorticoids diminished rhabdomere loss and conferred neuroprotection. Finally, we find that growth differentiation factor 15 has an important role downstream of glucocorticoid signaling in antagonizing ER stress-induced apoptosis in cells, as well as in preventing HD-mediated neurodegeneration in flies. Thus, our studies demonstrate that this novel crosstalk has the potential to be effectively exploited in alleviating several neurodegenerative disorders.

Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.

Heritability is one of the strongest risk factors of prostate cancer, emphasizing the importance of the genetic contribution towards prostate cancer risk. To date, 86 established prostate cancer risk variants have been identified by genome-wide association studies (GWAS). To determine if these risk variants are located near genes that interact together in biological networks or pathways contributing to prostate cancer initiation or progression, we generated gene sets based on proximity to the 86 prostate cancer risk variants. We took two approaches to generate gene lists. The first strategy included all immediate flanking genes, up- and downstream of the risk variant, regardless of distance from the index variant, and the second strategy included genes closest to the index GWAS marker and to variants in high LD (r2 ≥0.8 in Europeans) with the index variant, within a 100 kb window up- and downstream. Pathway mapping of the two gene sets supported the importance of the androgen receptor-mediated signaling in prostate cancer biology. In addition, the hedgehog and Wnt/β-catenin signaling pathways were identified in pathway mapping for the flanking gene set. We also used the HaploReg resource to examine the 86 risk loci and variants high LD (r2 ≥0.8) for functional elements. We found that there was a 12.8 fold (p = 2.9 x 10-4) enrichment for enhancer motifs in a stem cell line and a 4.4 fold (p = 1.1 x 10-3) enrichment of DNase hypersensitivity in a prostate adenocarcinoma cell line, indicating that the risk and correlated variants are enriched for transcriptional regulatory motifs. Our pathway-based functional annotation of the prostate cancer risk variants highlights the potential regulatory function that GWAS risk markers, and their highly correlated variants, exert on genes. Our study also shows that these genes may function cooperatively in key signaling pathways in prostate cancer biology.

Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.

Plasmacytoid dendritic cells (pDCs) were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs) containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs). The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation.

<h4>Objectives</h4>To evaluate hepatic fat fraction on dual- and triple-echo gradient-recalled echo MRI sequences in healthy children.<h4>Materials and methods</h4>We retrospectively reviewed the records of children in a medical check-up clinic from May 2012 to November 2013. We excluded children with abnormal laboratory findings or those who were overweight. Hepatic fat fraction was measured on dual- and triple-echo sequences using 3T MRI. We compared fat fractions using the Wilcoxon signed rank test and the Bland-Altman 95% limits of agreement. The correlation between fat fractions and clinical and laboratory findings was evaluated using Spearman's correlation test, and the cut-off values of fat fractions for diagnosing fatty liver were obtained from reference intervals.<h4>Results</h4>In 54 children (M:F = 26:28; 5-15 years; mean 9 years), the dual fat fraction (0.1-8.0%; median 1.6%) was not different from the triple fat fraction (0.4-6.5%; median 2.7%) (p = 0.010). The dual- and triple-echo fat fractions showed good agreement using a Bland-Altman plot (-0.6 ± 2.8%). Eight children (14.8%) on dual-echo sequences and six (11.1%) on triple-echo sequences had greater than 5% fat fraction. From these children, six out of eight children on dual-echo sequences and four out of six children on triple-echo sequences had a 5-6% hepatic fat fraction. When using a cut-off value of a 6% fat fraction derived from a reference interval, only 3.7% of children were diagnosed with fatty liver. There was no significant correlation between clinical and laboratory findings with dual and triple-echo fat fractions.<h4>Conclusions</h4>Dual fat fraction was not different from triple fat fraction. We suggest a cut-off value of a 6% fat fraction is more appropriate for diagnosing fatty liver on both dual- and triple-echo sequences in children.

DNA-templated self-assembly is an emerging strategy for generating functional supramolecular systems, which requires the identification of potent multi-point binding ligands. In this line, we recently showed that bis-functionalized guanidinium compounds can interact with ssDNA and generate a supramolecular complex through the recognition of the phosphodiester backbone of DNA. In order to probe the importance of secondary interactions and to identify side groups that stabilize these DNA-templated self-assemblies, we report herein the implementation of a dynamic combinatorial approach. We used an in situ fragment assembly process based on reductive amination and tested various side groups, including amino acids. The results reveal that aromatic and cationic side groups participate in secondary supramolecular interactions that stabilize the complexes formed with ssDNA.

The expression of Ferroportin (Fpn) was examined at different time points in rats following focal cerebral ischemia treated with or without the traditional Chinese medicine Naotaifang. Initially, rats were randomly divided into 2, 6, 12, 24 and 72 h groups following middle cerebral artery occlusion (MCAO) and the mRNA and protein level of Fpn was detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT‑PCR) at the above time points. Secondly, the rats were randomly divided into five groups as follows: Sham surgery group, model group, low‑dose group (3 g/kg NTE), medium dose group (9 g/kg NTE) and the high‑dose group (27 g/kg NTE). After 3 days of corresponding therapy by intragastric administration once a day, the regional cerebral ischemia model was reproduced by the MCAO suture method. On the third day, the neurological behavior of the rats was analyzed by neurobehavioral assessment. Fpn in the hippocampal CA2 region was measured by immunohistochemistry and the mRNA level of Fpn was detected by RT‑PCR. Expression of Fpn in the hippocampal CA2 region reached a peak 12 h after surgery (P<0.05, compared with the model group). The high‑dose group (27 g/kg NTE) exhibited a lower neurological behavior score (P<0.05) and a higher level of expression of Fpn at the mRNA and protein level compared with the sham surgery group and model group (P<0.05). Dysregulation of intracellular iron balance is possibly a new mechanism underlying cerebral ischemia. NTE can protect the neuronal population in the hippocampal CA2 region by adjusting the expression of Fpn to balance iron levels following cerebral ischemia.

The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

<h4>Introduction</h4>Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE type I) or dysfunction (C1-INH-HAE type II) is a rare disease characterized by recurrent episodes of edema with an estimated frequency of 1:50,000 in the global population without racial or gender differences. In this study we present the results of a nationwide survey of C1-INH-HAE patients referring to 17 Italian centers, the Italian network for C1-INH-HAE, ITACA.<h4>Methods</h4>Italian patients diagnosed with C1-INH-HAE from 1973 to 2013 were included in the study. Diagnosis of C1-INH-HAE was based on family and/or personal history of recurrent angioedema without urticaria and on antigenic and/or functional C1-INH deficiency.<h4>Results</h4>983 patients (53% female) from 376 unrelated families were included in this survey. Since 1973, 63 (6%) patients diagnosed with C1-INH-HAE died and data from 3 patients were missing when analysis was performed. Accordingly, the minimum prevalence of HAE in Italy in 2013 is 920:59,394,000 inhabitants, equivalent to 1:64,935. Compared to the general population, patients are less represented in the early and late decades of life: men start reducing after the 5(th) decade and women after the 6(th). Median age of patients is 45 (IQ 28-57), median age at diagnosis is 26 years (IQ 13-41). C1-INH-HAE type 1 are 87%, with median age at diagnosis of 25 (13-40); type 2 are 13% with median age at diagnosis of 31 (IQ 16-49). Functional C1INH is ≤50% in 99% of patients. Antigen C1INH is ≤50% in 99% of type 1. C4 is ≤50% in 96% of patients. The chance of having C1-INH-HAE with C4 plasma levels >50% is < 0.05.<h4>Conclusion</h4>This nationwide survey of C1-INH-HAE provides for Italy a prevalence of 1:64,935. C1-INH-HAE patients listed in our database have a shorter life expectancy than the general population. An increased awareness of the disease is needed to reduce this discrepancy. Measurement of C4 antigen can exclude diagnosis of C1-INH-HAE with an accuracy > 95%. This parameter should be therefore considered for initial screening in differential diagnosis of angioedema.

<h4>Background</h4>Finding a noninvasive method to predict liver fibrosis using inexpensive and easy-to-use markers is important.<h4>Objectives</h4>We aimed to clarify whether NX-des-γ-carboxyprothrombin (NX-DCP) could become a new noninvasive model to predict liver fibrosis in hepatitis C virus (HCV) related liver disease.<h4>Patients and methods</h4>We performed a prospective cohort study on a consecutive group of 101 patients who underwent liver biopsy for HCV-related liver disease at Kobe University Hospital. Laboratory measurements were performed on the same day as the biopsy. Factors associated with significant fibrosis (F3-4) were assessed by multivariate analyses. A comparison of predictive ability between multivariate factors and abovementioned noninvasive models was also performed.<h4>Results</h4>Increase in serum NX-DCP was significantly related to increase in fibrosis stage (P = 0.006). Moreover, NX-DCP was a multivariate factor associated with the presence of significant fibrosis F 3-4 (median 21 of F0-2 group vs. median 22 of F3-4 group with P = 0.002). The AUC of NX-DCP showed no significant differences compared with those of the AST-to-platelet ratio index (APRI), modified-APRI, the Göteborg University Cirrhosis Index (GUCI), the Lok index, the Hui score, cirrhosis discriminating score (CDS) and the Pohl score (P > 0.05).<h4>Conclusions</h4>NX-DCP correlated positively with fibrosis stage and could discriminate well between HCV-related patients with or without significant fibrosis. Moreover, NX-DCP had a similar predictive ability to the abovementioned models, and thereby could be a new noninvasive prediction tool for fibrosis.

<h4>Background</h4>Nanoparticles have been extensively investigated for targeted delivery of anticancer drugs. Since the folate receptor is universally over-expressed on the tumor cell membrane, folic acid is often used to modify the fate of nanoparticles in biologicals.<h4>Methods</h4>To fabricate targetable nanoparticles, folic acid was conjugated to a pullulan backbone and poly(DL-lactide-co-glycolide) (PLGA) (abbreviated as FAPuLG) was conjugated. KB cells and NIH3T3-cell-bearing mice were prepared to prove folate receptor targeting of FAPuLG nanoparticles.<h4>Results and discussion</h4>Nanoparticles of FAPuLG copolymer that self-assembled in water were small with diameters <200 nm. Doxorubicin (DOX) as a model drug was incorporated into the FAPuLG nanoparticles that were used to treat folate receptor over-expressing KB human carcinoma cells. Fluorescence microscopy revealed that DOX-incorporated FAPuLG nanoparticles induced strong red fluorescence in the KB cells in the absence of folic acid. However, fluorescence intensity was decreased by blocking folate receptors. Antitumor activity of FAPuLG nanoparticles against KB cells in vitro was also decreased by blocking folate receptors. In animal study using near-infrared dye-conjugated FAPuLG nanoparticles, fluorescence intensity was significantly higher at KB solid tumor than that of NIH3T3.<h4>Conclusions</h4>The results indicate that FAPuLG nanoparticles can target the folate receptor of tumor cells. FAPuLG nanoparticles are a promising candidate for active targeting of anticancer agents.

Microsatellite instability (MSI) is associated with the prognosis in several cancers and is used for determination of the chemotherapy regimen in stage II colon cancer in the National Comprehensive Cancer Network guideline. However, the association between MSI and the prognosis of gastric cancer remains unclear. PubMed database was searched until January 2014 using MeSH terms and key words to identify the studies evaluating MSI and prognosis of gastric cancer and the references were manually searched. The main outcome was the overall survival rate and the subordinate outcome was the association between high-frequency MSI (MSI-H) and clinicopathological characteristics. Eight studies met the inclusion criteria and the majority of data were collected retrospectively. There were 1,976 patients, 431 of which were MSI-H patients, with a range of 11.68-33.82%. Four studies used the National Cancer Institute panel to define MSI-H, the other four had microsatellite markers ranging 2-11. Significant associations were found in three studies and the overall summary estimate was hazard ratio, 0.63 (95% confidence interval, 0.52-0.77), with no evidence of inter-study heterogeneity (I²=0.0%). MSI-H patients were identified to have a tendency to have less lymph node (LN) metastasis, superficial tumor invasion and to be intestinal type. In conclusion, MSI-H gastric cancers have an improved prognosis, accompanied with reduced risk of LN metastasis, tumor invasion and mortality.

<h4>Background</h4>In this study, clustering was performed using a bitmap representation of HIV reverse transcriptase and protease sequences, to produce an unsupervised classification of HIV sequences. The classification will aid our understanding of the interactions between mutations and drug resistance. 10,229 HIV genomic sequences from the protease and reverse transcriptase regions of the pol gene and antiretroviral resistant related mutations represented in an 82-dimensional binary vector space were analyzed.<h4>Results</h4>A new cluster representation was proposed using an image inspired by microarray data, such that the rows in the image represented the protein sequences from the genotype data and the columns represented presence or absence of mutations in each protein position.The visualization of the clusters showed that some mutations frequently occur together and are probably related to an epistatic phenomenon.<h4>Conclusion</h4>We described a methodology based on the application of a pattern recognition algorithm using binary data to suggest clusters of mutations that can easily be discriminated by cluster viewing schemes.