Gene Literature Dashboard

<h4>Purpose</h4>Iron is essential for oxygen transport and oxidative metabolism; however, elevated iron stores can trigger overproduction of reactive oxygen species and induce DNA damage. Little is known about the association between body iron stores and glioma risk. This study examined the associations of iron levels measured in toenails and genetic variants linked to body iron stores with risk of glioma in a clinic-based case-control study.<h4>Methods</h4>Samples were collected a median of 24 days following glioma diagnosis in the cases (10th-90th percentile, range: 10-44 days). Nail iron levels were measured in 300 cases and 300 controls using neutron activation analysis. A total of 24 genetic variants associated with iron status were genotyped in 622 cases and 628 controls. Logistic regression was used to estimate odds ratios (OR) and 95 % confidence intervals (CI) for glioma risk according to toenail iron and the examined genotypes.<h4>Results</h4>No association was observed between toenail iron and glioma risk when restricting to cases with nails collected within ~3 weeks of diagnosis (OR = 0.93; 95 % CI 0.46, 1.87 comparing those with high (≥14 μg/g) vs. low (<6 μg/g) iron levels). In contrast, an inverse association with increasing iron was observed after restricting to cases with a delay of 3 weeks or greater (OR = 0.42; 95 % CI 0.19, 0.95), reflecting potentially insidious effects of advancing disease on iron levels among the cases. No associations were observed for any of the examined genetic variants.<h4>Conclusion</h4>The results do not support a role for body iron stores as a determinant of glioma risk.

This review summarizes recent efforts to create vascularized bone tissue in vitro and in vivo through the use of cell-based therapy approaches. The treatment of large and recalcitrant bone wounds is a serious clinical problem, and in the United States approximately 10% of all fractures are complicated by delayed union or non-union. Treatment approaches with the use of growth factor and gene delivery have shown some promise, but results are variable and clinical complications have arisen. Cell-based therapies offer the potential to recapitulate key components of the bone-healing cascade, which involves concomitant regeneration of vasculature and new bone tissue. For this reason, osteogenic and vasculogenic cell types have been combined in co-cultures to capitalize on the function of each cell type and to promote heterotypic interactions. Experiments in both two-dimensional and three-dimensional systems have provided insight into the mechanisms by which osteogenic and vasculogenic cells interact to form vascularized bone, and these approaches have been translated to ectopic and orthotopic models in small-animal studies. The knowledge generated by these studies will inform and facilitate the next generation of pre-clinical studies, which are needed to move cell-based orthopaedic repair strategies into the clinic. The science and application of cytotherapy for repair of large and ischemic bone defects is developing rapidly and promises to provide new treatment methods for these challenging clinical problems.

Growth cones integrate a remarkably complex concert of chemical cues to guide axons to their appropriate destinations. Recent work suggests that integrins contribute to axon guidance by interacting with a wide range of extracellular molecules including axon guidance molecules, by mechanisms that are not fully understood. Here, we describe an interaction between integrins and netrin-1 in growth cones that contributes to growth cone collapse. Our data show that netrin-1 causes growth cone collapse in a substratum-specific manner and is integrin-dependent. Netrin-1 causes collapse of cultured chick dorsal root ganglion (DRG) growth cones extending on high levels of laminin-1 (LN) but not growth cones extending on low levels of LN or on fibronectin. Blocking integrin function significantly decreases netrin-induced growth cone collapse on high LN. Netrin-1 and integrins interact on growth cones; netrin-1 causes integrin activation, a conformational shift to a high ligand-affinity state. Netrin-1 directly binds to integrin α3 and α6 peptides, further suggesting a netrin-integrin interaction. Interestingly, our data reveal that netrin-1 increases growth cone levels of cAMP in a substratum-specific manner and that netrin-induced growth cone collapse requires increased cAMP in combination with integrin activation. Manipulations that either decrease cAMP levels or integrin activation block netrin-induced collapse. These results imply a common mechanism for growth cone collapse and novel interactions between integrins, netrin-1 and cAMP that contribute to growth cone guidance.

Ovatodiolide was a unique macrocyclic diterpenoid isolated from the traditional Chinese medicinal herb Anisomeles indica. The present study attempted to examine the ovatodiolide effects on dendritic cell (DC) maturation and immuno-stimulatory activities. The effects of ovatodiolide on DC surface molecule expression, cytokine production, and capacity to induce T-cell differentiation were examined in ovalbumin (OVA)/thymic stromal lymphopoietin (TSLP)-stimulated DCs. Ovatodiolide attenuated the expression of DC surface molecules CD80, CD86, histocompatibility complex (MHC) class II, and Th2 subset of CD4(+) T cells co-stimulatory molecule-OX40 ligand production. Additionally, ovatodiolide suppressed the CD4(+) T cells proliferation, and production of inflammatory cytokines interleukin (IL)-4, IL-5, and tumor necrosis factor (TNF)-α. This study may be useful to develop ovatodiolide as a therapeutic adjuvant.

<h4>Background</h4>Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.<h4>Methods</h4>HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).<h4>Results</h4>An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum.<h4>Conclusions</h4>HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.

To gain insight into the hepatohistological alterations in noninjured rat liver, Nerium oleander (N.O.) leaves extract was injected intramuscularly to induce an acute phase reaction (APR). Histopathological changes were studied after 3, 12, and 24 h time course of sterile muscle abscess. Tissue integrity and any infiltration of inflammatory cells in the liver were investigated by Hematoxylin and Eosin and ED1 peroxidase stainings. The administration of N.O. leaves extract (10 mL/kg) in H & E stained sections showed a general vacuolization of cytoplasm resulting loss of polarity with prominent nucleoli after 3 h of induction. At 12 h, eccentric nuclei were also observed in the sections. Marked infiltration of leucocytes with predominate macrophages was also found after 24 h as seen by ED1 positive staining. In the present study, a possible relationship between serum hepcidin and total iron level was also investigated in vivo. An early increase of hepcidin and total iron level (3 h) with a maximum at 12 h (P < 0.01; P < 0.001) was observed. These changes indicate that sterile muscle abscess may induce APR resulting in hepatic damage which is evident with the recruitment of inflammatory cells into the organ.

A mathematical pharmacokinetic/anti-drug-antibody (PK/ADA) model was constructed for quantitatively assessing immunogenicity for therapeutic proteins. The model is inspired by traditional pharmacokinetic/pharmacodynamic (PK/PD) models, and is based on the observed impact of ADA on protein drug clearance. The hypothesis for this work is that altered drug PK contains information about the extent and timing of ADA generation. By fitting drug PK profiles while accounting for ADA-mediated drug clearance, the model provides an approach to characterize ADA generation during the study, including the maximum ADA response, sensitivity of ADA response to drug dose level, affinity maturation rate, time lag to observe an ADA response, and the elimination rate for ADA-drug complex. The model also provides a mean to estimate putative concentration-time profiles for ADA, ADA-drug complex, and ADA binding affinity-time profile. When simulating ADA responses to various drug dose levels, bell-shaped dose-response curves were generated. The model contains simultaneous quantitative modeling and provides estimation of the characteristics of therapeutic protein drug PK and ADA responses in vivo. With further experimental validation, the model may be applied to the simulation of ADA response to therapeutic protein drugs in silico, or be applied in subsequent PK/PD models.

There has been an increasing interest to develop new types of stimuli-responsive drug delivery vehicles with high drug loading and controlled release properties for chemotherapeutics. An acid-labile poly(ethylene oxide)-block-polyphosphoester-graft-PTX drug conjugate (PEO-b-PPE-g-PTX G2) degradable, polymeric paclitaxel (PTX) conjugate containing ultra-high levels of PTX loading is improved significantly, in this second-generation development, which involves connection of each PTX molecule to the polymer backbone via a pH-sensitive β-thiopropionate linkage. The PEO-b-PPE-g-PTX G2 forms well-defined nanoparticles in an aqueous solution, by direct dissolution into water, with a number-averaged hydrodynamic diameter of 114 ± 31 nm, and exhibits a PTX loading capacity as high as 53 wt%, with a maximum PTX concentration of 0.68 mg mL(-1) in water (vs 1.7 μg mL(-1) for free PTX). The PEO-b-PPE-g-PTX G2 shows accelerated drug release under acidic conditions (≈50 wt% PTX released in 8 d) compared with neutral conditions (≈20 wt% PTX released in 8 d). Compared to previously reported polyphosphoester-based PTX drug conjugates, PEO-b-PPE-g-PTX G1 without the β-thiopropionate linker, the PEO-b-PPE-g-PTX G2 shows pH-triggered drug release property and 5- to 8-fold enhanced in vitro cytotoxicity against two cancer cell lines.

Stem cells play an essential role in embryonic development, cell differentiation and tissue regeneration. Tissue homeostasis in adults is maintained by adult stem cells resident in the niches of different tissues. As one kind of adult stem cell, epidermal stem cells have the potential to generate diversified types of progeny cells in the skin. Although its biology is still largely unclarified, epidermal stem cells are widely used in stem cell research and regenerative medicine given its easy accessibility and pluripotency. Despite the same genome, cells within an organism have different fates due to the epigenetic regulation of gene expression. In this review, we will briefly discuss the current understanding of epigenetic modulation in epidermal stem cells.

The mammalian target of rapamycin (mTOR) inhibitors, a set of promising potential anti-cancer agents, has shown response variability among individuals. This study aimed to identify novel biomarkers and mechanisms that might influence the response to Rapamycin and Everolimus. Genome-wide association (GWA) analyses involving single nucleotide polymorphisms (SNPs), mRNA, and microRNAs microarray data were assessed for association with area under the cytotoxicity dose response curve (AUC) of two mTOR inhibitors in 272 human lymphoblastoid cell lines (LCLs). Integrated analysis among SNPs, expression data, microRNA data and AUC values were also performed to help select candidate genes for further functional characterization. Functional validation of candidate genes using siRNA screening in multiple cell lines followed by MTS assays for the two mTOR inhibitors were performed. We found that 16 expression probe sets (genes) that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors. One hundred and twenty seven and one hundred SNPs had P < 10(-4), while 8 and 10 SNPs had P < 10(-5) with Rapamycin and Everolimus AUC, respectively. Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line. Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs. In summary, this study identified genes and a microRNA that might contribute to response to mTOR inhibitors.

GLI pathogenesis-related 1 (GLIPR1) was previously identified as an epigenetically regulated tumor suppressor in prostate cancer and, conversely, an oncoprotein in glioma. More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms' tumor. Here we investigated GLIPR1 expression in metastatic melanoma cell lines and tissue. GLIPR1 was variably expressed in metastatic melanoma cells, and transcript levels correlated with degree of GLIPR1 promoter methylation in vitro. Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro. Immunohistochemical studies of melanoma tissue microarrays showed moderate to high staining for GLIPR1 in 50% of specimens analyzed. GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

<h4>Background</h4>Anemia in patients with cancer who are undergoing active therapy is commonly encountered and may worsen quality of life in these patients. The effect of blood transfusion is often temporary and may be associated with serious adverse events. Erythropoiesis-stimulating agents are not effective in 30%-50% of patients and may have a negative effect on overall survival.<h4>Aims</h4>To assess the efficacy and feasibility of intravenous iron therapy in patients with cancer who have non-iron-deficiency anemia and who are undergoing treatment with chemotherapy without the use of erythropoiesis-stimulating agents.<h4>Methods</h4>Adult patients with solid cancers and non-iron-deficiency anemia were included. Ferric sucrose at a dose of 200 mg was given in short intravenous infusions weekly for a total of 12 weeks. Hemoglobin level was measured at baseline, every 3 weeks, and 2 weeks after the last iron infusion (week 14). Adverse events related to intravenous iron were prospectively reported.<h4>Results</h4>Of 25 patients included, 19 (76.0%) completed at least three iron infusions and 14 (56.0%) finished the planned 12 weeks of therapy. The mean hemoglobin level of the 25 patients at baseline was 9.6 g/dL (median, 9.9 g/dL; range, 6.9 g/dL 10.9 g/dL). The mean change in hemoglobin level for the 15 patients who completed at least 9 treatments was 1.7 g/dL (median, 1.1 g/dL; range, -1.9 g/dL to 3.2 g/dL); it reached 2.1 g/dL (median, 1.3 g/dL; range, -0.2 g/dL to 4.6 g/dL; P = 0.0007) for the 14 patients who completed all 12 weekly treatments. Five (20.0%) patients were transfused and considered as treatment failures. No treatment-related adverse events were reported.<h4>Conclusion</h4>Intravenous iron treatment alone is safe and may reduce blood transfusion requirements and improve hemoglobin level in patients with cancer who are undergoing anticancer therapy. Further randomized studies are needed to confirm these findings.

<h4>Objectives</h4>Osteoarthritis (OA) is the most common form of arthritis with a clear genetic component. To identify novel loci associated with hip OA we performed a meta-analysis of genome-wide association studies (GWAS) on European subjects.<h4>Methods</h4>We performed a two-stage meta-analysis on more than 78,000 participants. In stage 1, we synthesised data from eight GWAS whereas data from 10 centres were used for 'in silico' or 'de novo' replication. Besides the main analysis, a stratified by sex analysis was performed to detect possible sex-specific signals. Meta-analysis was performed using inverse-variance fixed effects models. A random effects approach was also used.<h4>Results</h4>We accumulated 11,277 cases of radiographic and symptomatic hip OA. We prioritised eight single nucleotide polymorphism (SNPs) for follow-up in the discovery stage (4349 OA cases); five from the combined analysis, two male specific and one female specific. One locus, at 20q13, represented by rs6094710 (minor allele frequency (MAF) 4%) near the NCOA3 (nuclear receptor coactivator 3) gene, reached genome-wide significance level with p=7.9×10(-9) and OR=1.28 (95% CI 1.18 to 1.39) in the combined analysis of discovery (p=5.6×10(-8)) and follow-up studies (p=7.3×10(-4)). We showed that this gene is expressed in articular cartilage and its expression was significantly reduced in OA-affected cartilage. Moreover, two loci remained suggestive associated; rs5009270 at 7q31 (MAF 30%, p=9.9×10(-7), OR=1.10) and rs3757837 at 7p13 (MAF 6%, p=2.2×10(-6), OR=1.27 in male specific analysis).<h4>Conclusions</h4>Novel genetic loci for hip OA were found in this meta-analysis of GWAS.

Iron is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with excessive bone resorption. Iron uptake by osteoclast precursors via the transferrin cycle increases mitochondrial biogenesis, reactive oxygen species production, and activation of cAMP response element-binding protein, a critical transcription factor downstream of receptor activator of NF-κB-ligand-induced calcium signaling. These changes are required for the differentiation of osteoclast precursors to mature bone-resorbing osteoclasts. However, the molecular mechanisms regulating cellular iron metabolism in osteoclasts remain largely unknown. In this report, we provide evidence that Steap4, a member of the six-transmembrane epithelial antigen of prostate (Steap) family proteins, is an endosomal ferrireductase with a critical role in cellular iron utilization in osteoclasts. Specifically, we show that Steap4 is the only Steap family protein that is up-regulated during osteoclast differentiation. Knocking down Steap4 expression in vitro by lentivirus-mediated short hairpin RNAs inhibits osteoclast formation and decreases cellular ferrous iron, reactive oxygen species, and the activation of cAMP response element-binding protein. These results demonstrate that Steap4 is a critical enzyme for cellular iron uptake and utilization in osteoclasts and, thus, indispensable for osteoclast development and function.

OBJECTIVE We sought to identify predictors of diabetes diagnosed before hemochromatosis. RESEARCH DESIGN AND METHODS We studied these 16 variables in 159 nonscreening hemochromatosis probands with HFE C282Y homozygosity: age; sex; BMI; diabetes reports in first-degree family members (dichotomous); heavy ethanol consumption; cigarette smoking; elevated serum alanine aminotransferase/aspartate aminotransferase levels; nonalcoholic fatty liver; chronic viral hepatitis; cirrhosis; hand arthropathy; iron removed by phlebotomy; and positivity for HLA-A*01, B*08; A*03, B*07; and A*03, B*14 haplotypes. We performed univariable and multivariable analyses. RESULTS Twenty-three probands (14.5%) had diabetes; 19 were men. Each of the 23 probands had type 2 diabetes. Mean BMI was greater in probands with diabetes (31.7 ± 8.5 [SD] kg/m(2) vs. 27.6 ± 5.1 kg/m(2); P = 0.032). Reports of any first-degree family member with diabetes were more prevalent in probands with than in probands without diabetes (69.6 vs. 17.6%; P < 0.0001). In probands with diabetes, the odds ratio (OR) of maternal diabetes was 6.7 (95% CI 2.3-19.7; P = 0.0005) and of sibling diabetes was 11.7 (3.0-45.5; P = 0.0004). In a logistic regression model, predictors of diabetes at hemochromatosis diagnosis in 159 probands were diabetes reports in family members (OR 8.5 [95% CI 2.9-24.8]; P < 0.0001) and BMI (1.1 [1.0-1.2]; P = 0.049). This model explained 26.0% of total deviance contributing to diabetes. CONCLUSIONS In nonscreening hemochromatosis probands with HFE C282Y homozygosity, a heritable factor(s) increases the risk of diabetes diagnosed before hemochromatosis.

Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular etiology remains undiagnosed in the majority of cases. Through whole-exome sequencing, we identified recessive nonsense and splicing mutations in FBXL4 segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis, and severe mtDNA depletion in muscle. We show that FBXL4 is an F-box protein that colocalizes with mitochondria and that loss-of-function and splice mutations in this protein result in a severe respiratory chain deficiency, loss of mitochondrial membrane potential, and a disturbance of the dynamic mitochondrial network and nucleoid distribution in fibroblasts from affected individuals. Expression of the wild-type FBXL4 transcript in cell lines from two subjects fully rescued the levels of mtDNA copy number, leading to a correction of the mitochondrial biochemical deficit. Together our data demonstrate that mutations in FBXL4 are disease causing and establish FBXL4 as a mitochondrial protein with a possible role in maintaining mtDNA integrity and stability.

Whole-exome sequencing and autozygosity mapping studies, independently performed in subjects with defective combined mitochondrial OXPHOS-enzyme deficiencies, identified a total of nine disease-segregating FBXL4 mutations in seven unrelated mitochondrial disease families, composed of six singletons and three siblings. All subjects manifested early-onset lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy consistently associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures. A wide range of other multisystem features were variably seen, including dysmorphism, skeletal abnormalities, poor growth, gastrointestinal dysmotility, renal tubular acidosis, seizures, and episodic metabolic failure. Mitochondrial respiratory chain deficiency was present in muscle or fibroblasts of all tested individuals, together with markedly reduced oxygen consumption rate and hyperfragmentation of the mitochondrial network in cultured cells. In muscle and fibroblasts from several subjects, substantially decreased mtDNA content was observed. FBXL4 is a member of the F-box family of proteins, some of which are involved in phosphorylation-dependent ubiquitination and/or G protein receptor coupling. We also demonstrate that FBXL4 is targeted to mitochondria and localizes in the intermembrane space, where it participates in an approximately 400 kDa protein complex. These data strongly support a role for FBXL4 in controlling bioenergetic homeostasis and mtDNA maintenance. FBXL4 mutations are a recurrent cause of mitochondrial encephalomyopathy onset in early infancy.

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.

Bacteria respond to different small molecules that are produced by other neighboring bacteria. These molecules, called autoinducers, are classified as intraspecies (i.e., molecules produced and perceived by the same bacterial species) or interspecies (molecules that are produced and sensed between different bacterial species). AI-2 has been proposed as an interspecies autoinducer and has been shown to regulate different bacterial physiology as well as affect virulence factor production and biofilm formation in some bacteria, including bacteria of clinical relevance. Several groups have embarked on the development of small molecules that could be used to perturb AI-2 signaling in bacteria, with the ultimate goal that these molecules could be used to inhibit bacterial virulence and biofilm formation. Additionally, these molecules have the potential to be used in synthetic biology applications whereby these small molecules are used as inputs to switch on and off AI-2 receptors. In this review, we highlight the state-of-the-art in the development of small molecules that perturb AI-2 signaling in bacteria and offer our perspective on the future development and applications of these classes of molecules.

The cells expressing the T674I point mutant of FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα) in hypereosinophilics syndrome (HES) are resistant to imatinib and some second-generation tyrosine kinase inhibitors (TKIs). There is a desperate need to develop therapy to combat this acquired drug resistance. DCC-2036 has been synthesized as a third-generation TKI to combat especially the Bcr-Abl T315I mutant in chronic myeloid leukemia. This study evaluated the effect of DCC-2036 on FIP1L1-PDGFRα-positive cells, including the wild type (WT) and the T674I mutant. The in vitro effects of DCC-2036 on the PDGFRα signal pathways, proliferation, cell cycling and apoptosis of FIP1L1-PDGFRα-positive cells were investigated, and a nude mouse xenograft model was employed to assess the in vivo antitumor activity. We found that DCC-2036 decreased the phosphorylated levels of PDGFRα and its downstream targets without apparent effects on total protein levels. DCC-2036 inhibited proliferation, and induced apoptosis with MEK-dependent up-regulation of the pro-apoptotic protein Bim in FIP1L1-PDGFRα-positive cells. DCC-2036 also exhibited in vivo antineoplastic activity against cells with T674I FIP1L1-PDGFRα. In summary, FIP1L1-PDGFRα-positive cells are sensitive to DCC-2036 regardless of their sensitivity to imatinib. DCC-2036 may be a potential compound to treat imatinib-resistant HES.

Several intravenous iron complexes are available for the treatment of iron deficiency anemia (IDA). Iron dextran (DEX) is associated with an elevated risk of potentially serious anaphylactic reactions, whereas others must be administered in several small infusions to avoid labile iron reactions. Ferric carboxymaltose (FCM) is a nondextran intravenous iron which can be administered in high single doses. A randomized, open label, and multicenter comparison of FCM to DEX in adults with IDA and baseline hemoglobin of ≤11.0 g/dL was conducted. A total of 160 patients were in the safety population (FCM n = 82; DEX n = 78). Adverse events, including immune system disorders (0% in FCM versus 10.3% in DEX, P = 0.003) and skin disorders (7.3% in FCM versus 24.4% in DEX, P = 0.004), were less frequently observed in the FCM group. A greater portion of patients in the FCM group experienced a transient, asymptomatic decrease in phosphate compared to patients in the DEX group (8.5% in FCM versus 0% in DEX, P = 0.014). In the FCM arm, the change in hemoglobin from baseline to the highest observed level was 2.8 g/dL, whereas the DEX arm displayed a change of 2.4 g/dL (P = 0.20). Treatment of IDA with FCM resulted in fewer hypersensitivity-related reactions than DEX.

Activation of the immune system during pregnancy is believed to lead to psychiatric and neurological disorders in the offspring, but the molecular changes responsible are unknown. Polyinosinic:polycytidylic acid (poly(I:C)) is a viral-mimetic double-stranded RNA complex which activates Toll-Like-Receptor-3 and can activate the metabolism of tryptophan through the oxidative kynurenine pathway to compounds that modulate activity of glutamate receptors. The aim was to determine whether prenatal administration of poly(I:C) affects the expression of neurodevelopmental proteins in the offspring and whether such effects were mediated via the kynurenine pathway. Pregnant rats were treated with poly(I:C) during late gestation and the offspring were allowed to develop to postnatal day 21 (P21). Immunoblotting of the brains at P21 showed decreased expression of sonic hedgehog, a key protein in dopaminergic neuronal maturation. Expression of α-synuclein was decreased, while tyrosine hydroxylase was increased. Disrupted in Schizophrenia-1 (DISC-1) and 5-HT2C receptor levels were unaffected, as were the dependence receptors Unc5H1, Unc5H3 and Deleted in Colorectal Cancer (DCC), the inflammation-related transcription factor NFkB and the inducible oxidative enzyme cyclo-oxygenase-2 (COX-2). An examination of embryo brains 5 h after maternal poly(I:C) showed increased expression of GluN2B, with reduced doublecortin and DCC but no change in NFkB. Despite altered protein expression, there were no changes in the kynurenine pathway. The results show that maternal exposure to poly(I:C) alters the expression of proteins in the embryos and offspring which may affect the development of dopaminergic function. The oxidation of tryptophan along the kynurenine pathway is not involved in these effects.

Genetic variants within the serotonin transporter gene (5-HTTLPR) impact the neurobiology and risk for anxiety-related behaviours. There are also gender differences in the prevalence of anxiety-related behaviours. Although numerous studies have investigated the influence of 5-HTTLPR genotype on the neural systems involved in emotional regulation, none have investigated how these effects are modulated by gender and anxiety. We investigated this issue using two complementary region of interest-based structural neuroimaging approaches (voxel-based morphometry and Freesurfer) in 138 healthy individuals categorized into 'no anxiety' and 'subclinical anxiety' groups based on the Hamilton Rating Scale for Anxiety (HAM-A). Preliminarily, using anxiety as a continuous variable, we found a significant interaction effect of genotype by gender on anxiety. Females homozygous for the Short allele showed the highest HAM-A scores and males the lowest. In addition, a three-way significant interaction among genotype, gender and anxiety category was found for the right amygdala volume. Post hoc tests revealed that homozygous females carrying the Short variant with a subclinical anxiety condition had larger volume. The reported interaction effects demonstrate that gender strongly modulates the relationship between 5-HTTLPR genotype and subclinical expression of anxiety acting on amygdala, one region of the emotional neural network specifically involved in the anxiety-like behaviours.

Several clinical trials have shown anti-CD3 treatment to be a promising therapy for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3(+) regulatory T cells (Tregs) are likely to be involved, but through unknown mechanistic pathways. We profiled the transcriptional consequences in CD4(+) Tregs and conventional T cells (Tconvs) in the first hours and days after anti-CD3 treatment of NOD mice. Anti-CD3 treatment led to a transient transcriptional response, terminating faster than most Ag-induced responses. Most transcripts were similarly induced in Tregs and Tconvs, but several were differential, in particular, those encoding the IL-7R and transcription factors Id2/3 and Gfi1, upregulated in Tregs but repressed in Tconvs. Because IL-7R was a plausible candidate for driving the homeostatic response of Tregs to anti-CD3, we tested its relevance by supplementation of anti-CD3 treatment with IL-7/anti-IL-7 complexes. Although ineffective alone, IL-7 significantly improved the rate of remission induced by anti-CD3. Four anti-human CD3 mAbs exhibited the same differential effect on IL-7R expression in human as in mouse cells, suggesting that the mechanism also underlies therapeutic effect in human cells, and perhaps a rationale for testing a combination of anti-CD3 and IL-7 for the treatment of recent-onset human type 1 diabetes. Thus, systems-level analysis of the response to anti-CD3 in the early phase of the treatment demonstrates different responses in Tregs and Tconvs, and provides new leads to a mechanistic understanding of its mechanism of action in reverting recent-onset diabetes.

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.

To explore the response of β globin locus with established chromatin domains upon their exposure to new transcriptional environments, we transferred the chromatin-packaged β globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroblasts into an adult transcriptional environment. Distinct globin expression patterns were observed. In hESC-derived erythroblasts where both ε and γ globin were active and marked by similar chromatin modifications, ε globin was immediately silenced upon transfer, whereas γ globin continued to be expressed for months, implying that different transcriptional environments were required for their continuing expression. Whereas β globin was silent both in hESCs and in hESC-derived erythroblasts, β globin was only activated upon transfer from hESCs, but not in the presence of dominant γ globin transferred from hESC-derived erythroblasts, confirming the competing nature of γ versus β globin expression. With time, however, silencing of γ globin occurred in the adult transcriptional environment with concurrent activation of β-globin, accompanied by a drastic change in the epigenetic landscape of γ and β globin gene regions without apparent changes in the transcriptional environment. This switching process could be manipulated by overexpression or downregulation of certain transcription factors. Our studies provide important insights into the interplay between the transcription environment and existing chromatin domains, and we offer an experimental system to study the time-dependent human globin switching.

Photoreceptor cell death is the ultimate cause of vision loss in various retinal disorders, including retinal detachment (RD). Photoreceptor cell death has been thought to occur mainly through apoptosis, which is the most characterized form of programmed cell death. The caspase family of cysteine proteases plays a central role for inducing apoptosis, and in experimental models of RD, dying photoreceptor cells exhibit caspase activation; however, there is a paradox that caspase inhibition alone does not provide a sufficient protection against photoreceptor cell loss, suggesting that other mechanisms of cell death are involved. Recent accumulating evidence demonstrates that non-apoptotic forms of cell death, such as autophagy and necrosis, are also regulated by specific molecular machinery, such as those mediated by autophagy-related proteins and receptor-interacting protein kinases, respectively. Here we summarize the current knowledge of cell death signaling and its roles in photoreceptor cell death after RD and other retinal degenerative diseases. A body of studies indicate that not only apoptotic but also autophagic and necrotic signaling are involved in photoreceptor cell death, and that combined targeting of these pathways may be an effective neuroprotective strategy for retinal diseases associated with photoreceptor cell loss.

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

On September 11, 2001, one of the deadliest terrorist attacks in US history took place on American soil and people around the world were impacted in myriad ways. Building on prior literature which suggests individuals are more likely to purchase a gun for self-protection if they are fearful of being victimized, the authors hypothesized that the terrorist attacks of 9/11 would lead to an increase in gun carrying among US residents. At the same time, a line of research has shown that a polymorphism in the 5-HTT gene (i.e., 5-HTTLPR) interacts with environmental stressors to predict a range of psychopathologies and behaviors. Thus, it was hypothesized that 9/11 and 5-HTTLPR would interact to predict gun carrying. The results supported both hypotheses by revealing a positive association between 9/11 and gun carrying (b = .426, odds ratio = 1.531, standard error for b = .194, z = 2.196, p = .028) in the full sample of respondents (n = 15,052) and a statistically significant interaction between 9/11 and 5-HTTLPR in the prediction of gun carrying (b = -1.519, odds ratio = .219, standard error for b = .703, z = -2.161, p = .031) in the genetic subsample of respondents (n = 2,350). This is one of the first studies to find an association between 9/11 and gun carrying and, more importantly, is the first study to report a gene-environment interaction (GxE) between a measured gene and a terrorist attack.

Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.

The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates' ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.

Endometriosis-associated ovarian cancers demonstrate substantial morphological and genetic diversity. The transcription factor, hepatocyte nuclear factor (HNF)-1β, may be one of several key genes involved in the identity of ovarian clear cell carcinoma (CCC). The present study reviews a considerably expanded set of HNF-1β-associated genes and proteins that determine the pathophysiology of CCC. The current literature was reviewed by searching MEDLINE/PubMed. Functional interpretations of gene expression profiling in CCC are provided. Several important CCC-related genes overlap with those known to be regulated by the upregulation of HNF-1β expression, along with a lack of estrogen receptor (ER) expression. Furthermore, the genetic expression pattern in CCC resembles that of the Arias-Stella reaction, decidualization and placentation. HNF-1β regulates a subset of progesterone target genes. HNF-1β may also act as a modulator of female reproduction, playing a role in endometrial regeneration, differentiation, decidualization, glycogen synthesis, detoxification, cell cycle regulation, implantation, uterine receptivity and a successful pregnancy. In conclusion, the present study focused on reviewing the aberrant expression of CCC-specific genes and provided an update on the pathological implications and molecular functions of well-characterized CCC-specific genes.

There are currently more than 15 million preterm births each year. We propose that gene-environment interaction is a major contributor to preterm birth. To address this experimentally, we generated a mouse model with uterine deletion of Trp53, which exhibits approximately 50% incidence of spontaneous preterm birth due to premature decidual senescence with increased mTORC1 activity and COX2 signaling. Here we provide evidence that this predisposition provoked preterm birth in 100% of females exposed to a mild inflammatory insult with LPS, revealing the high significance of gene-environment interactions in preterm birth. More intriguingly, preterm birth was rescued in LPS-treated Trp53-deficient mice when they were treated with a combination of rapamycin (mTORC1 inhibitor) and progesterone (P4), without adverse effects on maternal or fetal health. These results provide evidence for the cooperative contributions of two sites of action (decidua and ovary) toward preterm birth. Moreover, a similar signature of decidual senescence with increased mTORC1 and COX2 signaling was observed in women undergoing preterm birth. Collectively, our findings show that superimposition of inflammation on genetic predisposition results in high incidence of preterm birth and suggest that combined treatment with low doses of rapamycin and P4 may help reduce the incidence of preterm birth in high-risk women.

Fetal alcohol spectrum disorders (FASD) results from ethanol exposure to the developing fetus and is the leading cause of mental retardation. FASD is associated with a broad range of neurobehavioral deficits which may be mediated by ethanol-induced neurodegeneration in the developing brain. An immature brain is more susceptible to ethanol neurotoxicity. We hypothesize that the enhanced sensitivity of the immature brain to ethanol is due to a limited capacity to alleviate cellular stress. Using a third trimester equivalent mouse model of ethanol exposure, we demonstrated that subcutaneous injection of ethanol induced a wide-spread neuroapoptosis in postnatal day 4 (PD4) C57BL/6 mice, but had little effect on the brain of PD12 mice. We analyzed the expression profile of genes regulating apoptosis, and the pathways of ER stress response (also known as unfolded protein response, UPR) and autophagy during these ethanol-sensitive and resistant periods (PD4 versus PD12) using PCR microarray. The expression of pro-apoptotic genes, such as caspase-3, was much higher on PD4 than PD12; in contrast, the expression of genes that regulate UPR and autophagy, such as atf6, atg4, atg9, atg10, beclin1, bnip3, cebpb, ctsb, ctsd, ctss, grp78, ire1α, lamp, lc3 perk, pik3c3, and sqstm1 was significantly higher on PD12 than PD4. These results suggest that the vulnerability of the immature brain to ethanol could result from high expression of pro-apoptotic proteins and a deficiency in the stress responsive system, such as UPR and autophagy.

Olanzapine is widely used in treating multiple domains of schizophrenia symptoms but induces serious metabolic side-effects. Recent evidence has showed that co-treatment of betahistine (a histaminergic H1 receptor agonist and H3 receptor antagonist) is effective for preventing olanzapine-induced weight gain/obesity, however it is not clear whether this co-treatment affects on the primary therapeutic receptor binding sites of olanzapine such as serotonergic 5-HT2A receptors (5-HT2AR) and dopaminergic D2 receptors (D2R). Therefore, this study investigated the effects of this co-treatment on 5-HT2AR, 5-HT transporter (5-HTT) and D2R bindings in various brain regions involved in antipsychotic efficacy. Female Sprague Dawley rats were administered orally (t.i.d.) with either olanzapine (1mg/kg), betahistine (2.7 mg/kg), olanzapine plus betahistine (O+B), or vehicle (control) for 2 weeks. Quantitative autoradiography was used to detect the density of [(3)H]ketanserin, [(3)H]paroxetine and [(3)H]raclopride binding site to 5-HT2AR, 5-HTT and D2R. Compared to the controls, olanzapine significantly decreased [(3)H]ketanserin bindings to 5-HT2AR in the prefrontal cortex, cingulate cortex, and nucleus accumbens. Similar changes in 5-HT2AR bindings in these nuclei were also observed in the O+B co-treatment group. Olanzapine also significantly decreased [(3)H]paroxetine binding to 5-HTT in the ventral tegmental area and substantia nigra, however, both olanzapine only and O+B co-treatment did not affect [(3)H]raclopride binding to D2R. The results confirmed the important role of 5-HT2AR in the efficacy of olanzapine, which is not influenced by the O+B co-treatment. Therefore, betahistine co-treatment would be an effective combination therapy to reduce olanzapine-induced weight gain side-effects without affecting olanzapine's actions on 5-HT2AR transmissions.

The ubiquitin-proteasome system (UPS) is the primary selective degradation system in the nuclei and cytoplasm of eukaryotic cells, required for the turnover of myriad soluble proteins. The hundreds of factors that comprise the UPS include an enzymatic cascade that tags proteins for degradation via the covalent attachment of a poly-ubiquitin chain, and a large multimeric enzyme that degrades ubiquitinated proteins, the proteasome. Protein degradation by the UPS regulates many pathways and is a crucial component of the cellular proteostasis network. Dysfunction of the ubiquitination machinery or the proteolytic activity of the proteasome is associated with numerous human diseases. In this review we discuss the contributions of the proteasome to human pathology, describe mechanisms that regulate the proteolytic capacity of the proteasome, and discuss strategies to modulate proteasome function as a therapeutic approach to ameliorate diseases associated with altered UPS function. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

The human genome encodes several thousand long non-protein coding transcripts>200 nucleotides in length, a subset of which were shown to play important roles in regulation of gene expression. We recently identified TINCR, a lncRNA required for induction of key differentiation genes in epidermal tissue, including genes mutated in human skin diseases characterized by disrupted epidermal barrier formation. High-throughput analyses of TINCR RNA- and protein-interactomes revealed TINCR interaction with differentiation mRNAs as well as the Staufen1 protein. TINCR, together with Staufen1, seems to stabilize a subset of mRNAs required for epidermal differentiation. Here, we discuss the emerging roles of Staufen1 and TINCR in the regulation of mammalian cell differentiation mediated by interaction with target mRNAs. We consider a role for TINCR as an epithelial-specific guide for targeting the Staufen1 protein to specific mRNAs, reflecting the increasing complexity of gene regulatory processes in mammalian cells and tissue.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded trinucleotide CAG repeat in the Huntingtin (Htt) gene. The molecular basis for the development and progression of HD is currently poorly understood. However, different DNA repair pathways have been implicated in both somatic expansion and disease progression. Embryonic stem cells provide a remarkable in vitro system to study HD and could have implications for understanding disease development and for therapeutic treatment. Here, we derive pluripotent stem cells from the mouse R6/1 HD model and demonstrate that repeated exposure to genotoxic agents inducing oxidative DNA damage gave a significant and dose dependent increase in somatic triplet expansion. Further investigation into specific steps of DNA repair revealed impaired double stranded break repair in exposed R6/1 cells, accompanied by the induction of apoptosis. We also found that differentiation status, and consequently DNA repair efficiency influenced somatic expansion. Our data underscore the importance of DNA damage and repair for the stability of the HD triplet in pluripotent stem cells.

Several candidate genes have been so far implicated in the pathogenesis of proliferative diabetic retinopathy (PDR) in subjects with type 2 diabetes. Since the principal pathogenetic mechanisms for diabetic retinopathy (DR) and PDR are different, the main pathogenetic mechanism in DR is increased vascular permeability, whereas in PDR the crucial pathogenetic mechanisms are fibrosis and neoangiogenesis. Due to that fact, different candidate genes are expected to be involved in the development of either DR or PDR. None of the candidate genes, however, can be fully and solely responsible for the development of PDR and for DR progression into PDR. Epigenetic mechanisms are expected to be involved in the pathogenesis of PDR as well. Gene polymorphisms responsible for PDR and epigenetic mechanisms responsible for PDR are reviewed in this paper.

Although the intensification of therapy for children with T-cell acute lymphoblastic leukemia (T-ALL) has substantially improved clinical outcomes, T-ALL remains an important challenge in pediatric oncology. Here, we report that the cooperative synergy between prostate apoptosis response factor-4 (Par-4) and THAP1 induces cell cycle and apoptosis regulator 1 (CCAR1) gene expression and cellular apoptosis in human T-ALL cell line Jurkat cells, CEM cells and primary cultured neoplastic T lymphocytes from children with T-ALL. Par-4 and THAP1 collaborated to activate the promoter of CCAR1 gene. Mechanistic investigations revealed that Par-4 and THAP1 formed a protein complex by the interaction of their carboxyl termini, and THAP1 bound to CCAR1 promoter though its zinc-dependent DNA-binding domain at amino terminus. Par-4/THAP1 complex and Notch3 competitively bound to CCAR1 promoter and competitively modulated alternative pre-mRNA splicing of CCAR1, which resulted in two different transcripts and played an opposite role in T-ALL cell survival. Despite Notch3 induced a shift splicing from the full-length isoform toward a shorter form of CCAR1 mRNA by splicing factor SRp40 and SRp55, Par-4/THAP1 complex strongly antagonized this inductive effect. Our finding revealed a mechanistic rationale for Par-4/THAP1-induced apoptosis in T-ALL cells that would be of benefit to develop a new therapy strategy for T-ALL.

Elucidating targets of physiological tumor metastasis suppressors can highlight key signaling pathways leading to invasion and metastasis. To identify downstream targets of the metastasis suppressor Raf-1 kinase inhibitory protein (RKIP/PEBP1), we utilized an integrated approach based upon statistical analysis of tumor gene expression data combined with experimental validation. Previous studies from our laboratory identified the architectural transcription factor and oncogene, high mobility group AT-hook 2 (HMGA2), as a target of inhibition by RKIP. Here we identify two signaling pathways that promote HMGA2-driven metastasis. Using both human breast tumor cells and an MMTV-Wnt mouse breast tumor model, we show that RKIP induces and HMGA2 inhibits expression of miR-200b; miR-200b directly inhibits expression of lysyl oxidase (LOX), leading to decreased invasion. RKIP also inhibits syndecan-2 (SDC2), which is aberrantly expressed in breast cancer, via downregulation of HMGA2; but this mechanism is independent of miR-200. Depletion of SDC2 induces apoptosis and suppresses breast tumor growth and metastasis in mouse xenografts. RKIP, LOX and SDC2 are coordinately regulated and collectively encompass a prognostic signature for metastasis-free survival in ER-negative breast cancer patients. Taken together, our findings reveal two novel signaling pathways targeted by the metastasis suppressor RKIP that regulate remodeling of the extracellular matrix and tumor survival.

Kcnq1, which encodes for the pore-forming α-subunit of a voltage-gated potassium channel, was identified as a gastrointestinal (GI) tract cancer susceptibility gene in multiple Sleeping Beauty DNA transposon-based forward genetic screens in mice. To confirm that Kcnq1 has a functional role in GI tract cancer, we created Apc(Min) mice that carried a targeted deletion mutation in Kcnq1. Results demonstrated that Kcnq1 is a tumor suppressor gene as Kcnq1 mutant mice developed significantly more intestinal tumors, especially in the proximal small intestine and colon, and some of these tumors progressed to become aggressive adenocarcinomas. Gross tissue abnormalities were also observed in the rectum, pancreas and stomach. Colon organoid formation was significantly increased in organoids created from Kcnq1 mutant mice compared with wild-type littermate controls, suggesting a role for Kcnq1 in the regulation of the intestinal crypt stem cell compartment. To identify gene expression changes due to loss of Kcnq1, we carried out microarray studies in the colon and proximal small intestine. We identified altered genes involved in innate immune responses, goblet and Paneth cell function, ion channels, intestinal stem cells, epidermal growth factor receptor and other growth regulatory signaling pathways. We also found genes implicated in inflammation and in cellular detoxification. Pathway analysis using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis confirmed the importance of these gene clusters and further identified significant overlap with genes regulated by MUC2 and CFTR, two important regulators of intestinal homeostasis. To investigate the role of KCNQ1 in human colorectal cancer (CRC), we measured protein levels of KCNQ1 by immunohistochemistry in tissue microarrays containing samples from CRC patients with liver metastases who had undergone hepatic resection. Results showed that low expression of KCNQ1 expression was significantly associated with poor overall survival.

<h4>Background</h4>Obesity is a public health concern. Yet the identification of adiposity-related genetic variants among United States (US) Hispanics, which is the largest US minority group, remains largely unknown.<h4>Objective</h4>To interrogate an a priori list of 47 (32 overall body mass and 15 central adiposity) index single-nucleotide polymorphisms (SNPs) previously studied in individuals of European descent among 3494 US Hispanic women in the Women's Health Initiative SNP Health Association Resource (WHI SHARe).<h4>Design</h4>Cross-sectional analysis of measured body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR) were inverse normally transformed after adjusting for age, smoking, center and global ancestry. WC and WHR models were also adjusted for BMI. Genotyping was performed using the Affymetrix 6.0 array. In the absence of an a priori selected SNP, a proxy was selected (r(2)0.8 in CEU).<h4>Results</h4>Six BMI loci (TMEM18, NUDT3/HMGA1, FAIM2, FTO, MC4R and KCTD15) and two WC/WHR loci (VEGFA and ITPR2-SSPN) were nominally significant (P<0.05) at the index or proxy SNP in the corresponding BMI and WC/WHR models. To account for distinct linkage disequilibrium patterns in Hispanics and further assess generalization of genetic effects at each locus, we interrogated the evidence for association at the 47 surrounding loci within 1 Mb region of the index or proxy SNP. Three additional BMI loci (FANCL, TFAP2B and ETV5) and five WC/WHR loci (DNM3-PIGC, GRB14, ADAMTS9, LY86 and MSRA) displayed Bonferroni-corrected significant associations with BMI and WC/WHR. Conditional analyses of each index SNP (or its proxy) and the most significant SNP within the 1 Mb region supported the possible presence of index-independent signals at each of these eight loci as well as at KCTD15.<h4>Conclusion</h4>This study provides evidence for the generalization of nine BMI and seven central adiposity loci in Hispanic women. This study expands the current knowledge of common adiposity-related genetic loci to Hispanic women.

MicroRNAs (miRNAs) are small 19- to 24-nt noncoding RNAs that have the capacity to regulate fundamental biological processes essential for cancer initiation and progression. In cancer, miRNAs may function as oncogenes or tumor suppressors. Here, we conducted global profiling for miRNAs in a cohort of stage 1 nonsmall cell lung cancers (n = 81) and determined that miR-486 was the most down-regulated miRNA in tumors compared with adjacent uninvolved lung tissues, suggesting that miR-486 loss may be important in lung cancer development. We report that miR-486 directly targets components of insulin growth factor (IGF) signaling including insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and phosphoinositide-3-kinase, regulatory subunit 1 (alpha) (PIK3R1, or p85a) and functions as a potent tumor suppressor of lung cancer both in vitro and in vivo. Our findings support the role for miR-486 loss in lung cancer and suggest a potential biological link to p53.

Huntington disease (HD) is a progressive neurodegenerative disorder caused by dominant polyglutamine (polyQ) expansion within the N terminus of huntingtin (Htt) protein. Abnormal metal accumulation in the striatum of HD patients has been reported for many years, but a causative relationship has not yet been established. Furthermore, if metal is indeed involved in HD, the underlying mechanism needs to be explored. Here using a Drosophila model of HD, wherein Htt exon1 with expanded polyQ (Htt exon1-polyQ) is introduced, we show that altered expression of genes involved in copper metabolism significantly modulates the HD progression. Intervention of dietary copper levels also modifies HD phenotypes in the fly. Copper reduction to a large extent decreases the level of oligomerized and aggregated Htt. Strikingly, substitution of two potential copper-binding residues of Htt, Met8 and His82, completely dissociates the copper-intensifying toxicity of Htt exon1-polyQ. Our results therefore indicate HD entails two levels of toxicity: the copper-facilitated protein aggregation as conferred by a direct copper binding in the exon1 and the copper-independent polyQ toxicity. The existence of these two parallel pathways converging into Htt toxicity also suggests that an ideal HD therapy would be a multipronged approach that takes both these actions into consideration.

<h4>Background</h4>Wilms tumor is the most common childhood renal tumor. Although the majority of patients with favorable histology Wilms tumor (FHWT) have good outcomes, some patients still experience disease recurrence and death from disease. The goal of the current study was to determine whether tumor-specific chromosome 1q gain is associated with event-free survival (EFS) and overall survival (OS) in patients with FHWT.<h4>Methods</h4>Unilateral FHWT samples were obtained from patients enrolled on National Wilms Tumor Study-4 and Pediatric Oncology Group Wilms Biology Study (POG 9046). 1q gain, 1p loss, and 16q loss were determined using multiplex ligation-dependent probe amplification.<h4>Results</h4>The 8-year EFS rate was 87% (95% confidence interval [95% CI], 82%-91%) for the entire cohort of 212 patients. Tumors from 58 of 212 patients (27%) displayed 1q gain. A strong relationship between 1q gain and 1p/16q loss was observed. The 8-year EFS rate was 76% (95% CI, 63%-85%) for patients with 1q gain and 93% (95% CI, 87%-96%) for those lacking 1q gain (P = .0024). The 8-year OS rate was 89% (95% CI, 78%-95%) for those with 1q gain and 98% (95% CI, 94%-99%) for those lacking 1q gain (P = .0075). Gain of 1q was not found to correlate with disease stage (P = .16). After stratification for stage of disease, 1q gain was associated with a significantly increased risk of disease recurrence (risk ratio estimate: 2.72; P = .0089).<h4>Conclusions</h4>Gain of 1q may provide a valuable prognostic marker with which to stratify therapy for patients with FHWT. A confirmatory study is necessary before this biomarker is incorporated into the risk stratification schema of future therapeutic studies.

<h4>Background</h4>Selective estrogen receptor modulators, such as tamoxifen, play a pivotal role in the treatment of luminal-type breast cancer. However, in clinical applications, nearly half of breast cancer patients are insensitive to tamoxifen, a small number of whom have early recurrence or disease progression when receiving tamoxifen. The underlying mechanism of this resistance has not been determined. ZNF703 is a novel oncogene in the 15% of breast cancers that harbor 8p12 amplifications. Therefore, the goal of our study was to explore the role of ZNF703 in tamoxifen resistance.<h4>Methodology/principal findings</h4>We used immunohistochemistry techniques to examine ZNF703 expression in stage I-III primary breast cancer specimens and found a positive expression rate of 91.3%. All patients were divided into either high or low ZNF703 expression groups. We found that high ZNF703 expression mainly occurred in ER+ and PR+ breast cancers. Furthermore, 4-hydroxytamoxifen had different modes of action in breast cancer cell lines with high or low ZNF703 expression. ZNF703 overexpression in MCF-7 breast cancer cells activated the Akt/mTOR signaling pathway, downregulated ERα, and reduced the antitumor effect of tamoxifen. Low-dose tamoxifen did not suppress, but rather, stimulated the growth of cells overexpressing ZNF703. ZNF703 knockdown in MDA-MB-134 and HCC1500 luminal B-type breast cancer cell lines by siRNA significantly decreased survival rates when cells were treated with tamoxifen. Furthermore, targeting ZNF703 with a mTOR inhibitor increased the inhibitory effects of tamoxifen in ZNF703-overexpressing cells.<h4>Conclusion/significance</h4>Our study suggests that ZNF703 expression levels may predict tamoxifen sensitivity. Tamoxifen should be administered with caution to those patients bearing tumors with ZNF703 overexpression. However, large clinical trials and prospective clinical studies are needed to verify these results.

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.

Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) have opposing but complementary functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore, regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal truncation products all bind to TGF-β1 and TGF-β2 with a similar but somewhat less affinity than they bind BMP-2; that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-β with greater affinity than do the truncation products; that FL-Spp24 inhibits TGF-β2 induced bone formation in vivo, but Spp14.5 does not; and that co-administration of FL-Spp24 or Spp14.5 with TGF-β2 in vivo is associated with a reduction in the amount of cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-dose TGF-β administration in vivo is associated with greater bone formation than high-dose TGF-β administration, and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-β activity or availability during bone growth and development. The similarities and differences of the interactions between Spp24 proteins and TGF-β compared to the interaction of the Spp24 proteins and BMPs have significant implications with respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal disorders.

<h4>Introduction</h4>Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC.<h4>Experimental design</h4>Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry.<h4>Results</h4>In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression.<h4>Conclusions</h4>Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management.

Mitochondria contain a 16.6 kb circular genome encoding 13 proteins as well as mitochondrial tRNAs and rRNAs. Copies of the genome are organized into nucleoids containing both DNA and proteins, including the machinery required for mtDNA replication and transcription. The transcription factor TFAM is critical for initiation of transcription and replication of the genome, and is also thought to perform a packaging function. Although specific binding sites required for initiation of transcription have been identified in the D-loop, little is known about the characteristics of TFAM binding in its nonspecific packaging state. In addition, it is unclear whether TFAM also plays a role in the regulation of nuclear gene expression. Here we investigate these questions by using ChIP-seq to directly localize TFAM binding to DNA in human cells. Our results demonstrate that TFAM uniformly coats the whole mitochondrial genome, with no evidence of robust TFAM binding to the nuclear genome. Our study represents the first high-resolution assessment of TFAM binding on a genome-wide scale in human cells.

<h4>Background</h4>Movement disorders, particularly those associated with basal ganglia disease, have a high rate of comorbid neuropsychiatric illness.<h4>Methods</h4>We consider the pathophysiological basis of the comorbidity between movement disorders and neuropsychiatric illness by 1) reviewing the epidemiology of neuropsychiatric illness in a range of hyperkinetic movement disorders, and 2) correlating findings to evidence from studies that have utilized modern neuroimaging techniques to investigate these disorders. In addition to diseases classically associated with basal ganglia pathology, such as Huntington disease, Wilson disease, the neuroacanthocytoses, and diseases of brain iron accumulation, we include diseases associated with pathology of subcortical white matter tracts, brain stem nuclei, and the cerebellum, such as metachromatic leukodystrophy, dentatorubropallidoluysian atrophy, and the spinocerebellar ataxias.<h4>Conclusions</h4>Neuropsychiatric symptoms are integral to a thorough phenomenological account of hyperkinetic movement disorders. Drawing on modern theories of cortico-subcortical circuits, we argue that these disorders can be conceptualized as disorders of complex subcortical networks with distinct functional architectures. Damage to any component of these complex information-processing networks can have variable and often profound consequences for the function of more remote neural structures, creating a diverse but nonetheless rational pattern of clinical symptomatology.

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin's tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin's tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin's tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.

Huntington's disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (Htt) protein. Here we report a genome-wide overexpression suppressor screen in which we identified 317 ORFs that ameliorate the toxicity of a mutant Htt fragment in yeast and that have roles in diverse cellular processes, including mitochondrial import and copper metabolism. Two of these suppressors encode glutathione peroxidases (GPxs), which are conserved antioxidant enzymes that catalyze the reduction of hydrogen peroxide and lipid hydroperoxides. Using genetic and pharmacological approaches in yeast, mammalian cells and Drosophila, we found that GPx activity robustly ameliorates Huntington's disease-relevant metrics and is more protective than other antioxidant approaches tested here. Notably, we found that GPx activity, unlike many antioxidant treatments, does not inhibit autophagy, which is an important mechanism for clearing mutant Htt. Because previous clinical trials have indicated that GPx mimetics are well tolerated in humans, this study may have important implications for treating Huntington's disease.

Executive function is a collection of cognitive processes essential for higher order mental function. Processes involved in executive function include, but are not limited to, working memory, attention, cognitive flexibility, and impulse control. These complex behaviors are largely mediated by prefrontal cortical function but are modulated by dopaminergic, noradrenergic, serotonergic, and cholinergic input. The ability of these neurotransmitter systems to modulate executive function allows for adaptation in cognitive behavior in response to changes in the environment. Because of the important role these neurotransmitter systems play in regulating executive function, changes in these systems can also have a grave impact on executive function. In addition, polymorphisms in genes associated with these neurotransmitters are associated with phenotypic differences in executive function. Understanding how these naturally occurring polymorphisms contribute to different executive function phenotypes will advance basic knowledge of cognition and potentially further understanding and treatment of mental illness that involve changes in executive function. In this review, we will examine the influence of dopamine, norepinephrine, serotonin, and acetylcholine on the following measures of executive function: attention, cognitive flexibility, and impulse control. We will also review the effects of polymorphisms in genes associated with these neurotransmitter systems on these measures of executive function.

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. Using resting-state fMRI (rs-fMRI) we investigated the functional integrity of resting-state networks (RSN) in HD. 17 HD and 19 matched control participants were examined at a 3 Tesla MR scanner. After controlling for structural degeneration by means of voxel-based morphometry, task-free rs-fMRI data were analyzed using Independent Component Analysis (ICA) and a dual-regression approach in the context of genetic and clinical parameters. Further, we evaluated HD-related differences in interregional connectivity between networks. RSN analysis showed a significant increase in intrinsic functional connectivity in the HD sample compared with controls, including the thalamus, striatum, prefrontal, premotor, and parietal maps. A subset of the Default Mode Network (DMN) was also affected. In the HD cohort, motor impairment correlated with higher network connectivity in mainly motor and parietal cortices. Deteriorating total functional capacity was additionally associated with higher connectivity in the striatum, thalamus, insular and frontal areas. This pattern of increased activity in intrinsic functional networks might suggest a reduced ability of intra-network differentiation with clinical disease progression in HD. Finally, results showed reduced long-range connectivity between parietal ICA components in HD compared to controls, indicating impaired functional coupling between interregional networks in HD. Our data demonstrates that functional connectivity is profoundly altered in HD, both within and between RSN. Rs-fMRI analysis may provide additional valuable insights into neuronal dysfunctions beyond HD-related structural degeneration and disruptions of functional circuits in HD.

Systemic autoimmune diseases (SADs) are a group of connective tissue diseases with diverse, yet overlapping, symptoms and autoantibody development. The etiology behind SADs is not fully elucidated, but a number of genetic and environmental factors are known to influence the incidence of SADs. Recent findings link dysregulation of Epstein-Barr virus (EBV) with SAD development. EBV causes a persistent infection with a tight latency programme in memory B-cells, which enables evasion of the immune defence. A number of immune escape mechanisms and immune-modulating proteins have been described for EBV. These immune modulating functions make EBV a good candidate for initiation of autoimmune diseases and exacerbation of disease progression. This review focuses on systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren's syndrome (SS) and sum up the existing data linking EBV with these diseases including elevated titres of EBV antibodies, reduced T-cell defence against EBV, and elevated EBV viral load. Together, these data suggest that uncontrolled EBV infection can develop diverse autoreactivities in genetic susceptible individuals with different manifestations depending on the genetic background and the site of reactivation.

Attachment to an abusive caregiver has wide phylogenetic representation, suggesting that animal models are useful in understanding the neural basis underlying this phenomenon and subsequent behavioral outcomes. We previously developed a rat model, in which we use classical conditioning to parallel learning processes evoked during secure attachment (odor-stroke, with stroke mimicking tactile stimulation from the caregiver) or attachment despite adversity (odor-shock, with shock mimicking maltreatment). Here we extend this model to mice. We conditioned infant mice (postnatal day (PN) 7-9 or 13-14) with presentations of peppermint odor and either stroking or shock. We used (14) C 2-deoxyglucose (2-DG) to assess olfactory bulb and amygdala metabolic changes following learning. PN7-9 mice learned to prefer an odor following either odor-stroke or shock conditioning, whereas odor-shock conditioning at PN13-14 resulted in aversion/fear learning. 2-DG data indicated enhanced bulbar activity in PN7-9 preference learning, whereas significant amygdala activity was present following aversion learning at PN13-14. Overall, the mouse results parallel behavioral and neural results in the rat model of attachment, and provide the foundation for the use of transgenic and knockout models to assess the impact of both genetic (biological vulnerabilities) and environmental factors (abusive) on attachment-related behaviors and behavioral development.

<h4>Background</h4>Deletion of the Williams-Beuren syndrome (WBS) critical region (WBSCR), at 7q11.23, causes a developmental disorder commonly characterized by hypersociability and excessive talkativeness and often considered the opposite behavioral phenotype to autism. Duplication of the WBSCR leads to severe delay in expressive language. Gene-dosage effects on language development at 7q11.23 have been hypothesized.<h4>Methods</h4>Molecular characterization of the WBSCR was performed by fluorescence in situ hybridization and high-resolution single-nucleotide polymorphism array in two individuals with severe autism enrolled in a genetic study of autism who showed typical WBS facial dysmorphism on systematic clinical genetic examination. The serotonin transporter promoter polymorphism (5-HTTLPR, locus SLC6A4) was genotyped. Platelet serotonin levels and urinary 6-sulfatoxymelatonin excretion were measured. Behavioral and cognitive phenotypes were examined.<h4>Results</h4>The two patients had common WBSCR deletions between proximal and medial low copy repeat clusters, met diagnostic criteria for autism and displayed severe impairment in communication, including a total absence of expressive speech. Both patients carried the 5-HTTLPR ss genotype and exhibited platelet hyperserotonemia and low melatonin production.<h4>Conclusions</h4>Our observations indicate that behaviors and neurochemical phenotypes typically associated with autism can occur in patients with common WBSCR deletions. The results raise intriguing questions about phenotypic heterogeneity in WBS and regarding genetic and/or environmental factors interacting with specific genes at 7q11.23 sensitive to dosage alterations that can influence the development of social communication skills. Thus, the influence of WBSCR genes on social communication expression might be dramatically modified by other genes, such as 5-HTTLPR, known to influence the severity of social communication impairments in autism, or by environmental factors, such as hyperserotonemia, given that hyperserotonemia is found in WBS associated with autism but not in WBS without autism. In this regard, WBS provides a potentially fruitful model with which to develop integrated genetic, cognitive, behavioral and neurochemical approaches to study genotype-phenotype correlations, possible gene-environment interactions and genetic background effects. The results underscore the importance of considering careful clinical and molecular genetic examination of individuals diagnosed with autism.

Producing gene fusions through genomic structural rearrangements is a major mechanism for tumor evolution. Therefore, accurately detecting gene fusions and the originating rearrangements is of great importance for personalized cancer diagnosis and targeted therapy. We present a tool, BreakTrans, that systematically maps predicted gene fusions to structural rearrangements. Thus, BreakTrans not only validates both types of predictions, but also provides mechanistic interpretations. BreakTrans effectively validates known fusions and discovers novel events in a breast cancer cell line. Applying BreakTrans to 43 breast cancer samples in The Cancer Genome Atlas identifies 90 genomically validated gene fusions. BreakTrans is available at http://bioinformatics.mdanderson.org/main/BreakTrans.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Despite their physiological importance, selective interactions between nuclear receptors (NRs) and their cofactors are poorly understood. Here, we describe a novel signature motif (F/YSXXLXXL/Y) in the developmental regulator BCL11A that facilitates its selective interaction with members of the NR2E/F subfamily. Two copies of this motif (named here as RID1 and RID2) permit BCL11A to bind COUP-TFs (NR2F1;NR2F2;NR2F6) and Tailless/TLX (NR2E1), whereas RID1, but not RID2, binds PNR (NR2E3). We confirmed the existence of endogenous BCL11A/TLX complexes in mouse cortex tissue. No interactions of RID1 and RID2 with 20 other ligand-binding domains from different NR subtypes were observed. We show that RID1 and RID2 are required for BCL11A-mediated repression of endogenous γ-globin gene and the regulatory non-coding transcript Bgl3, and we identify COUP-TFII binding sites within the Bgl3 locus. In addition to their importance for BCL11A function, we show that F/YSXXLXXL/Y motifs are conserved in other NR cofactors. A single FSXXLXXL motif in the NR-binding SET domain protein NSD1 facilitates its interactions with the NR2E/F subfamily. However, the NSD1 motif incorporates features of both LXXLL and FSXXLXXL motifs, giving it a distinct NR-binding pattern in contrast to other cofactors. In summary, our results provide new insights into the selectivity of NR/cofactor complex formation.

The beneficial effects of chronic and early pharmacological treatment with ethosuximide on epileptogenesis were studied in a genetic absence epilepsy model comorbid for depression. It was also investigated whether there is a critical treatment period and treatment length. Cortical excitability in the form of electrical evoked potentials, but also to cortico-thalamo-cortical network activity (spike-wave discharges, SWD and afterdischarges), white matter changes representing extra cortico-thalamic functions and depressive-like behavior were investigated. WAG/Rij rats received either ethosuximide for 2 months (post natal months 2-3 or 4-5), or ethosuximide for 4 months (2-5) in their drinking water, while control rats drank plain water. EEG measurements were made during treatment, and 6 days and 2 months post treatment. Behavioral test were also done 6 days post treatment. DTI was performed ex vivo post treatment. SWD were suppressed during treatment, and 6 days and 2 months post treatment in the 4 month treated group, as well as the duration of AD elicited by cortical electrical stimulation 6 days post treatment. Increased fractional anisotropy in corpus callosum and internal capsula on DTI was found, an increased P8 evoked potential amplitude and a decreased immobility in the forced swim test. Shorter treatments with ETX had no large effects on any parameter. Chronic ETX has widespread effects not only within but also outside the circuitry in which SWD are initiated and generated, including preventing epileptogenesis and reducing depressive-like symptoms. The treatment of patients before symptom onset might prevent many of the adverse consequences of chronic epilepsy.

The proper function of the nervous system depends on precise production and connection of distinct neurons and glia. Cell fate determination of neurons and glia is tightly controlled by complex gene expression regulation in the developing and adult nervous system. Emerging evidence has demonstrated the importance of noncoding microRNAs (miRNAs) in neural development and function. This review highlights current discoveries of miRNA functions in specifying neuronal and glial cell fate. We summarize the roles of miRNAs in expansion and differentiation of neural stem cells, specification of neuronal subtypes and glial cells, reprogramming of functional neurons from embryonic stem cells and fibroblasts, and left-right asymmetric organization of neuronal subtypes. Investigating the network of interactions between miRNAs and target genes will reveal new gene regulation machinery involved in tuning the cell fate decisions of neurons and glia.

According to the idea that the central serotonergic system has a modulatory function on behavior and personality in general, we aimed to highlight its association to habitual positive emotionality. In a placebo-controlled double-blind and randomized cross-over neuroendocrine challenge design (n = 72 healthy males) we investigated the association of the central serotonergic responsivity, 5-HTTLPR-genotype as well as their combined effects on positive emotionality. Regression analyses revealed an involvement of the serotonergic system in positive emotionality. There was, however, no direct association between positive emotionality and cortisol responses to S-citalopram; rather 5-HTTLPR-genotype showed an association (p < 0.05). That is, positive emotionality scores increased with the number of s-alleles carried by the individuals. Most notable was the moderating role of 5-HTTLPR-genotype (p < 0.05) on the association between acute serotonergic responsivity and positive emotionality. Indeed, this association was only found in ss-homozygotes, in which the acute responsivity of the serotonergic system additionally seems to contribute to the level of positive emotionality (r = 0.70, p < 0.05). The findings correspond to previous research demonstrating that the 5-HTTLPR is not only involved in the negative-emotional aspects of behavior and temperament, but is associated, moreover, with positive affectivity-supporting the assumption of its valence-neutrality. In addition, our data are in line with the idea of possible influences of the 5-HTTLPR-genotype on early neuronal development. They also indicate the need for further studies in order to clearly elucidate the role of the serotonergic system and its subcomponents in the regulation of positive emotionality.

Obesity has reached epidemic proportions globally and has become the cause of several major health risks worldwide. Presently, more than 100 loci have been related to obesity and metabolic traits in humans by genome-wide association studies. The complex genetic architecture behind obesity has triggered a need for the development of better animal models than rodents. The pig has emerged as a very promising biomedical model to study human obesity traits. In this study, we have characterized the expression patterns of six obesity-related genes, leptin (LEP), leptin receptor (LEPR), melanocortin 4 receptor (MC4R), fat mass and obesity associated (FTO), neuronal growth regulator 1 (NEGR)1 and adiponectin (ADIPOQ), in seven obesity-relevant tissues (liver; muscle; pancreas; hypothalamus; and retroperitoneal, subcutaneous and mesenteric adipose tissues) in two pig breeds (production pigs and Göttingen minipigs) that deviate phenotypically and genetically from each other with respect to obesity traits. We observe significant differential expression for LEP, LEPR and ADIPOQ in muscle and in all three adipose tissues. Interestingly, in pancreas, LEP expression is only detected in the fat minipigs. FTO shows significant differential expression in all tissues analyzed, and NEGR1 shows significant differential expression in muscle, pancreas, hypothalamus and subcutaneous adipose tissue. The MC4R transcript can be detected only in hypothalamus. In general, the expression profiles of the investigated genes are in accordance with those observed in human studies. Our study shows that both the differences between the investigated breeds and the phenotypic state with respect to obesity/leanness play a large role for differential expression of the obesity-related genes.

Haploinsufficiency of TCF4 causes Pitt-Hopkins syndrome (PTHS): a severe form of mental retardation with phenotypic similarities to Angelman, Mowat-Wilson and Rett syndromes. Genome-wide association studies have also found that common variants in TCF4 are associated with an increased risk of schizophrenia. Although TCF4 is transcription factor, little is known about TCF4-regulated processes in the brain. In this study we used genome-wide expression profiling to determine the effects of acute TCF4 knockdown on gene expression in SH-SY5Y neuroblastoma cells. We identified 1204 gene expression changes (494 upregulated, 710 downregulated) in TCF4 knockdown cells. Pathway and enrichment analysis on the differentially expressed genes in TCF4-knockdown cells identified an over-representation of genes involved in TGF-β signaling, epithelial to mesenchymal transition (EMT) and apoptosis. Among the most significantly differentially expressed genes were the EMT regulators, SNAI2 and DEC1 and the proneural genes, NEUROG2 and ASCL1. Altered expression of several mental retardation genes such as UBE3A (Angelman Syndrome), ZEB2 (Mowat-Wilson Syndrome) and MEF2C was also found in TCF4-depleted cells. These data suggest that TCF4 regulates a number of convergent signaling pathways involved in cell differentiation and survival in addition to a subset of clinically important mental retardation genes.

Colorectal cancer (CRC) is the most commonly diagnosed cancer in Europe. Because CRC is also a major cause of cancer-related deaths worldwide, a lot of research has been focused on the discovery and development of biomarkers to improve the diagnostic process and to predict treatment outcomes. Up till now only a few biomarkers are recommended by expert panels. Current TNM criteria, however, cause substantial under- and overtreatment of CRC patients. Consequently, there is a growing need for new and efficient biomarkers to ensure optimal treatment allocation. An ideal biomarker should be easily translated into clinical practice, to identify patients who can be spared from treatment or benefit from therapy, ultimately resulting in precision medicine in the future. In this review we aim to provide an overview of a number of frequently studied biomarkers in CRC and, at the same time, we will emphasize the challenges and controversies that withhold the clinical introduction of these biomarkers. We will discuss both prognostic and predictive markers of chemotherapy, aspirin therapy as well as overall therapy toxicity. Currently, only mutant KRAS, mutant BRAF, MSI and the Oncotype DX® Colon Cancer Assay are used in clinical practice. Other biomarker studies showed insufficient evidence to be introduced into clinical practice. Divergent patient selection criteria, absence of validation studies and a large number of single biomarker studies are possibly responsible. We therefore recommend that future studies focus on combining key markers, rather than analysing single markers, standardizing study protocols, and validate the results in independent study cohorts, followed by prospective clinical trials.

Clinical guidelines recommend concurrent treatment of anemia in end-stage renal disease with erythropoiesis-stimulating agents (ESAs) and iron. However, there are mixed data about optimal iron supplementation. To help address this gap, the relationship between iron markers and hemoglobin (Hb) response to ESA (Epoetin alfa) dose was examined. Electronic medical records of 1902 US chronic hemodialysis patients were analyzed over a 12-month period between June 2009 and June 2010. The analysis included patients who had at least one Hb value during each 4-week interval for four consecutive intervals (k - 2, k - 1, k, and k + 1; k is the index interval), received at least one ESA dose during intervals k - 1 or k, had at least one transferrin saturation (TSAT) value at interval k, and at least one ferritin value during intervals k - 2, k - 1, or k. Effect modification by TSAT and ferritin on Hb response was evaluated using the generalized estimating equations approach. Patients had a mean (standard deviation) age of 62 (15) years; 41% were Caucasian, 34% African American, 65% had hypertension, and 39% diabetes. Transferrin saturation, but not ferritin, had a statistically significant (P < 0.05) modifying effect on Hb response. Maximum Hb response was achieved when TSAT was 34%, with minimal incremental effect beyond these levels. Of the two standard clinical iron markers, TSAT should be used as the primary marker of the modifying effect of iron on Hb response to ESA. Long-term safety of iron use to improve Hb response to ESA warrants further study.

<h4>Background</h4>Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals.<h4>Results</h4>We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals.<h4>Conclusions</h4>These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.

High blood pressure (BP) is more prevalent and contributes to more severe manifestations of cardiovascular disease (CVD) in African Americans than in any other United States ethnic group. Several small African-ancestry (AA) BP genome-wide association studies (GWASs) have been published, but their findings have failed to replicate to date. We report on a large AA BP GWAS meta-analysis that includes 29,378 individuals from 19 discovery cohorts and subsequent replication in additional samples of AA (n = 10,386), European ancestry (EA) (n = 69,395), and East Asian ancestry (n = 19,601). Five loci (EVX1-HOXA, ULK4, RSPO3, PLEKHG1, and SOX6) reached genome-wide significance (p < 1.0 × 10(-8)) for either systolic or diastolic BP in a transethnic meta-analysis after correction for multiple testing. Three of these BP loci (EVX1-HOXA, RSPO3, and PLEKHG1) lack previous associations with BP. We also identified one independent signal in a known BP locus (SOX6) and provide evidence for fine mapping in four additional validated BP loci. We also demonstrate that validated EA BP GWAS loci, considered jointly, show significant effects in AA samples. Consequently, these findings suggest that BP loci might have universal effects across studied populations, demonstrating that multiethnic samples are an essential component in identifying, fine mapping, and understanding their trait variability.

<h4>Background</h4>The many attempts that have been made to identify genes for bipolar disorder (BD) have met with limited success, which may reflect an inadequacy of diagnosis as an informative and biologically relevant phenotype for genetic studies. Here we have explored aspects of personality as quantitative phenotypes for bipolar disorder through the use of the Temperament and Character Inventory (TCI), which assesses personality in seven dimensions. Four temperament dimensions are assessed: novelty seeking (NS), harm avoidance (HA), reward dependence (RD), and persistence (PS). Three character dimensions are also included: self-directedness (SD), cooperativeness (CO), and self-transcendence (ST).<h4>Methods</h4>We compared personality scores between diagnostic groups and assessed heritability in a sample of 101 families collected for genetic studies of BD. A genome-wide SNP linkage analysis was then performed in the subset of 51 families for which genetic data was available.<h4>Results</h4>Significant group differences were observed between BD subjects, their first-degree relatives, and independent controls for all but RD and PS, and all but HA and RD were found to be significantly heritable in this sample. Linkage analysis of the heritable dimensions produced several suggestive linkage peaks for NS (chromosomes 7q21 and 10p15), PS (chromosomes 6q16, 12p13, and 19p13), and SD (chromosomes 4q35, 8q24, and 18q12).<h4>Limitations</h4>The relatively small size of our linkage sample likely limited our ability to reach genome-wide significance in this study.<h4>Conclusions</h4>While not genome-wide significant, these results suggest that aspects of personality may prove useful in the identification of genes underlying BD susceptibility.

Nine new 3'-N-phenylsulfonyl docetaxel analogs were synthesized in good yields from the key intermediate N-phenylsulfonyl oxazolidine via a six-step route. These analogs were tested for anti-hepatitis B virus (HBV) activity in vitro. Compounds 3e, 3g and 3j showed more potent inhibitory activity against HBeAg secretion than the positive control lamivudine. Further extensive SAR and mechanistic studies will be reported in due course.

The extended major histocompatibility complex (xMHC) is the most gene-dense region of the genome and harbors a disproportionately large number of genes involved in immune function. The postulated role of infection in the causation of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) suggests that the xMHC may make an important contribution to the risk of this disease. We conducted association mapping across an approximately 4 megabase region of the xMHC using a validated panel of single nucleotide polymorphisms (SNPs) in childhood BCP-ALL cases (n=567) enrolled in the Northern California Childhood Leukemia Study (NCCLS) compared with population controls (n=892). Logistic regression analyses of 1,145 SNPs, adjusted for age, sex, and Hispanic ethnicity indicated potential associations between several SNPs and childhood BCP-ALL. After accounting for multiple comparisons, one of these included a statistically significant increased risk associated with rs9296068 (OR=1.40, 95% CI=1.19-1.66, corrected p=0.036), located in proximity to HLA-DOA. Sliding window haplotype analysis identified an additional locus located in the extended class I region in proximity to TRIM27 tagged by a haplotype comprising rs1237485, rs3118361, and rs2032502 (corrected global p=0.046). Our findings suggest that susceptibility to childhood BCP-ALL is influenced by genetic variation within the xMHC and indicate at least two important regions for future evaluation.

This study tested the 5-HTTLPR gene as a moderator in the relation between maternal unresponsiveness and child externalizing symptoms in a disadvantaged, predominantly Black sample of two hundred and one 2-year-old children and their mothers. Using a multimethod, prospective design, structural equation model analyses indicated that maternal unresponsiveness significantly predicted increases in externalizing symptoms 2 years later only for children possessing the LL genotype. Moderation was expressed in a "for better" or "for worse" form hypothesized in differential susceptibility theory. In examining why the risk posed by maternal unresponsiveness differed across the 5-HTTLPR polymorphism, mediated moderation analyses showed that children's angry reactivity to maternal negativity partly accounted for the greater susceptibility of homozygous L carriers to variations in maternal unresponsiveness.

Fibromyalgia (FM), characterized by chronic widespread pain, fatigue, and cognitive/mood disturbances, leads to reduced workplace productivity and increased healthcare expenses. To determine if acquired epigenetic/genetic changes are associated with FM, we compared the frequency of spontaneously occurring micronuclei (MN) and genome-wide methylation patterns in women with FM (n = 10) to those seen in comparably aged healthy controls (n = 42 (MN); n = 8 (methylation)). The mean (sd) MN frequency of women with FM (51.4 (21.9)) was significantly higher than that of controls (15.8 (8.5)) (χ (2) = 45.552; df = 1; P = 1.49 × 10(-11)). Significant differences (n = 69 sites) in methylation patterns were observed between cases and controls considering a 5% false discovery rate. The majority of differentially methylated (DM) sites (91%) were attributable to increased values in the women with FM. The DM sites included significant biological clusters involved in neuron differentiation/nervous system development, skeletal/organ system development, and chromatin compaction. Genes associated with DM sites whose function has particular relevance to FM included BDNF, NAT15, HDAC4, PRKCA, RTN1, and PRKG1. Results support the need for future research to further examine the potential role of epigenetic and acquired chromosomal alterations as a possible biological mechanism underlying FM.

Adaptive evolution has provided us with a unique set of characteristics that define us as humans, including morphological, physiological and cellular changes. Yet, natural selection provides no assurances that adaptation is without human health consequences; advantageous mutations will increase in frequency so long as there is a net gain in fitness. As such, the current incidence of human disease can depend on previous adaptations. Here, I review genome-wide and gene-specific studies in which adaptive evolution has played a role in shaping human genetic disease. In addition to the disease consequences of adaptive phenotypes, such as bipedal locomotion and resistance to certain pathogens, I review evidence that adaptive mutations have influenced the frequency of linked disease alleles through genetic hitchhiking. Taken together, the links between human adaptation and disease highlight the importance of their combined influence on functional variation within the human genome and offer opportunities to discover and characterize such variation.

For advanced treatment of diseases such as cancer, multicomponent, multifunctional nanoparticles hold great promise. In the current study we report the synthesis of a complex nanoparticle (NP) system with dual drug loading as well as diagnostic properties. To that aim we present a methodology where chemically modified poly(lactic-co-glycolic) acid (PLGA) polymer is formulated into a polymer-lipid NP that contains a cytotoxic drug doxorubicin (DOX) in the polymeric core and an anti-angiogenic drug sorafenib (SRF) in the lipidic corona. The NP core also contains gold nanocrystals (AuNCs) for imaging purposes and cyclodextrin molecules to maximize the DOX encapsulation in the NP core. In addition, a near-infrared (NIR) Cy7 dye was incorporated in the coating. To fabricate the NP we used a microfluidics-based technique that offers unique NP synthesis conditions, which allowed for encapsulation and fine-tuning of optimal ratios of all the NP components. NP phantoms could be visualized with computed tomography (CT) and near-infrared (NIR) fluorescence imaging. We observed timed release of the encapsulated drugs, with fast release of the corona drug SRF and delayed release of a core drug DOX. In tumor bearing mice intravenously administered NPs were found to accumulate at the tumor site by fluorescence imaging.

Damage of oligodendrocytes after ischemia has negative impact on white matter integrity and neuronal function. In this work, we explore whether Netrin-1 (NT-1) overexpression facilitates white matter repairing and remodeling. Adult CD-1 mice received stereotactic injection of adeno-associated virus carrying NT-1 gene (AAV-NT-1). One week after gene transfer, mice underwent 60 minutes of middle cerebral artery occlusion. The effect of NT-1 on neural function was evaluated by neurobehavioral tests. Proliferated oligodendrocyte progenitor cells (OPCs), newly matured oligodendrocytes, and remyelination were semi-quantified by immunohistochemistry. The role of NT-1 in oligodendrogenesis was further explored by examining specific NT-1 receptors and their function. Netrin-1 overexpression was detected in neurons and astrocytes 2 weeks after AAV-NT-1 gene transfer and significantly improved the neurobehavioral outcomes compared with the control (P<0.05). In comparison with the control, proliferated OPCs, newly matured oligodendrocytes, and remyelination were greatly increased in the ipsilateral hemisphere of AAV-NT-1-transduced mice. Furthermore, both NT-1 receptors deleted in colorectal carcinoma and UNC5H2 were expressed on OPCs whereas only UNC5H2 was expressed in myelinated axons. Our study indicated that NT-1 promoted OPC proliferation, differentiation, and increased remyelination, suggesting that NT-1 is a promising factor for white matter repairing and remodeling after ischemia.

Approximately 1 in 14 men and women during their lifetime will be diagnosed with lung cancer, which is the leading cause of cancer-related mortality in the world. As of January 1, 2008, there were about 373,500 men and women living with lung cancer in the United States. Fewer than 60,000 of these are estimated to be alive by January 2013, reflecting a poor overall 5-year relative survival rate of under 16 %. With metastatic cancer, the overall 5-year survival is meager 4 %. On the other hand, the overall five-year survival is over 50 % when the cancer is still in the localized stage. However, unfortunately, more than half of cases of lung cancer are diagnosed at an advanced stage Howlader et al. (2010). Cancer metastasis, the single most critical prognostic factor, is still poorly understood and a highly complex phenomenon. The most common sites of lung cancer metastasis are the lymph nodes, liver, adrenals, brain and bones. The gastrointestinal (GI) tract is an exceptionally rare site of metastasis; with only a handful of cases reported in the literature Centeno et al. (Lung Cancer, 18: 101-105, 1997); Hirasaki et al. (World J Gastroenterol, 14: 5481-5483, 2008); Carr and Boulos (Br J Surg, 83: 647, 1996); Otera et al. (Eur Respir Rev, 19: 248-252, 2010); Antler et al. (Cancer, 49: 170-172, 1982); Fujiwara et al. (Gen Thorac Cardiovasc Surg, 59: 748-752, 2011); Stinchcombe et al. (J Clin Oncol, 24: 4939-4940, 2006); John et al. (J Postgrad Med, 48: 199-200, 2002); Carroll and Rajesh (Eur J Cardiothorac Surg, 19: 719-720, 2001); Brown et al. (Dis Colon Rectum, 23: 343-345, 1980). We report three cases of non-small cell (squamous cell) lung cancer with GI tract metastasis-two in the colon and one in the jejunum. Then we present a review of literature exploring various theories of metastasis, as an attempt to understand the reason of preferential tumor metastasis.

As recent studies highlight the importance of alternative mechanisms in the control of bone turnover, new therapeutic approaches can be envisaged for bone diseases and periodontitis-induced bone loss. Recently, it has been shown that Fluoxetine and Venlafaxine, serotonin re-uptake inhibitors commonly used as antidepressants, can positively or negatively affect bone loss in rat models of induced periodontitis. Serotonin is a neurotransmitter that can be found within specific nuclei of the central nervous system, but can also be produced in the gut and be sequestered inside platelet granules. Although it is known to be mainly involved in the control of mood, sleep, and intestinal physiology, recent evidence has pointed at far reaching effects on bone metabolism, as a mediator of the effects of Lrp5, a membrane receptor commonly associated with Wnt canonical signaling and osteoblast differentiation. Deletion of Lrp5 in mice lead to increased expression of Tryptophan Hydroxylase 1, the gut isoform of the enzyme required for serotonin synthesis, thus increasing serum levels of serotonin. Serotonin, in turn, could bind to HTR1B receptors on osteoblasts and stop their proliferation by activating PKA and CREB.Although different groups have reported controversial results on the existence of an Lrp5-serotonin axis and the action of serotonin in bone remodeling, there is convincing evidence that serotonin modulators such as SSRIs can affect bone turnover. Consequently, the effects of this drug family on periodontal physiology should be thoroughly explored.

<h4>Objective</h4>We measured the levels of mutant huntingtin (mtHtt) and total huntingtin (tHtt) in blood leukocytes from Prospective Huntington At-Risk Observational Study (PHAROS) subjects at 50% risk of carrying the Huntington disease mutation using a homogeneous time-resolved fluorescence (HTRF) assay to assess its potential as a biomarker.<h4>Methods</h4>Peripheral blood mononuclear cells from consenting PHAROS subjects were analyzed by HTRF using antibodies that simultaneously measured mtHtt and tHtt. mtHtt levels were normalized to tHtt, double-stranded DNA, or protein and analyzed according to cytosine-adenine-guanine repeat length (CAGn), demographics, predicted time to clinical onset or known time since clinical onset, and available clinical measures.<h4>Results</h4>From 363 assayed samples, 342 met quality control standards. Levels of mtHtt and mt/tHtt were higher in 114 subjects with expanded CAG repeats (CAG ≥ 37) compared with 228 subjects with nonexpanded CAG repeats (CAG <37) (p < 0.0001). Analysis of relationships to predicted time to onset or to phenoconversion suggested that the HTRF signal could mark changes during the Huntington disease prodrome or after clinical onset.<h4>Conclusions</h4>The HTRF assay can effectively measure mtHtt in multicenter sample sets and may be useful in trials of therapies targeting huntingtin.

<h4>Motivation</h4>Despite the central role of diseases in biomedical research, there have been much fewer attempts to automatically determine which diseases are mentioned in a text-the task of disease name normalization (DNorm)-compared with other normalization tasks in biomedical text mining research.<h4>Methods</h4>In this article we introduce the first machine learning approach for DNorm, using the NCBI disease corpus and the MEDIC vocabulary, which combines MeSH® and OMIM. Our method is a high-performing and mathematically principled framework for learning similarities between mentions and concept names directly from training data. The technique is based on pairwise learning to rank, which has not previously been applied to the normalization task but has proven successful in large optimization problems for information retrieval.<h4>Results</h4>We compare our method with several techniques based on lexical normalization and matching, MetaMap and Lucene. Our algorithm achieves 0.782 micro-averaged F-measure and 0.809 macro-averaged F-measure, an increase over the highest performing baseline method of 0.121 and 0.098, respectively.<h4>Availability</h4>The source code for DNorm is available at http://www.ncbi.nlm.nih.gov/CBBresearch/Lu/Demo/DNorm, along with a web-based demonstration and links to the NCBI disease corpus. Results on PubMed abstracts are available in PubTator: http://www.ncbi.nlm.nih.gov/CBBresearch/Lu/Demo/PubTator .

Mycophenolic acid has played an important role in treating immunosuppression and autoimmune diseases. Nevertheless, the agent needs a high dosage in treatment, following some side effects. To tackle this problem, in this study, mycophenolic acid-glucosamine conjugate (MGC), modified by 2-glucosamine, was synthesized to achieve kidney targeting and improved drug efficacy with a lower dosage. (1)H NMR, (13)C NMR and HRMS spectroscopy were used to verify the conjugate whose stability was good in vitro. The transport of MGC by human proximal renal tubular epithelial HK-2 cells was temperature-, time-, concentration-dependent and saturable, suggesting the involvement of carrier-mediated uptake. In addition, the cellular uptake of MGC dropped substantially with the inhibition of megalin receptor. The specific tissue distribution indicated the commendable renal-targeting capability of MGC. The concentration of MGC was improved in the kidney except for other tissues, about 6.76 times higher than that of MPA. Further, the bioavailability of MGC in plasma decreased as compared with mycophenolic acid. Moreover, therapeutic effect of MGC was enhanced significantly compared with MPA in the acute kidney injury model. All the findings suggested the potential of mycophenolic acid-glucosamine conjugate in kidney targeted drug delivery.

<h4>Background</h4>Vitamin C is associated with a lower risk of coronary heart disease possibly due to its anti-oxidative effects, beneficial effects on endothelial function and importance in collagen synthesis. The sodium-dependent vitamin C transporter 2 is responsible for the transport of vitamin C into various cells and malfunction of this protein leads to reduced vitamin C in tissue, including the arterial wall. We tested the hypothesis that candidate variations rs6139591 and rs1776964 in the gene coding for sodium-dependent vitamin C transporter 2 are associated with development of acute coronary syndrome.<h4>Design</h4>In the Danish Diet, Cancer and Health cohort study, we performed a case-cohort study among 57,053 subjects aged 50-64 years.<h4>Results</h4>During a mean follow-up period of 6.4 years, we identified 936 cases and randomly selected a sub-cohort (n = 1,580) with full information on genotypes and covariates. Using Cox proportional hazard models, we found that women with the rs6139591 TT genotype and a lower than median dietary vitamin C intake had a higher risk of acute coronary syndrome compared with those with the CC genotype (adjusted HR 5.39, 95% confidence interval, 2.01-14.50). We also observed a not as strong but positive although inconsistent association for women at a higher than median intake of vitamin C rich food. For the rs1776964 polymorphism, we found a higher risk (adjusted HR 3.45, 95% CI, 1.16-10.28) among TT-homozygous women with higher than median vitamin C intake compared with the CC genotype and low vitamin C intake. Among men, weaker and non-significant associations were observed for both polymorphisms.<h4>Conclusion</h4>Genetic variation in the sodium-dependent vitamin C transporter 2 is associated with risk of incident acute coronary syndrome in women. The genotype effects may not be fully compensated by a higher intake of vitamin C rich food.

The aim of this study is to test the hypothesis that the newly synthesized poly(δ-valerolactone)/poly(ethylene glycol)/poly(δ-valerolactone) (VEV) copolymer grafted with folic acid would impart targetability and further enhance the anti-tumor efficacy of doxorubicin (DOX). Here, folic acid conjugated VEV (VEV-FOL) was synthesized by a modified esterification method and characterized using IR and NMR. DOX loaded VEV-FOL micelles were synthesized using a novel solvent evaporation method and were obtained with a mean diameter of 97 nm with high encapsulation efficiency and sustained in vitro release profile. Comparative studies of polymer micelles with and without folate for cellular uptake and cytotoxicity were done on folate receptor-positive breast cancer cell line, MDAMB231. The intracellular uptake tests showed significant increase in folate micellar uptake when compared to non-folate-mediated micelles. MTT assay followed by apoptosis assays clearly indicated that folate decorated micelles showed significantly better cytotoxicity (IC50 = 0.014 µM) and efficiency to induce apoptosis than other treated groups. Moreover, a significant G2/M arrest was induced by DOX loaded VEV-FOL micelles at a concentration where free drug failed to show any activity. Thus, our results show that the folic acid-labeled VEV copolymer is a promising biomaterial with controlled and sustainable tumor targeting ability for anticancer drugs which can open new frontiers in the area of targeted chemotherapy.

<h4>Background</h4>Opioidergic SLP (sustained ligand-activated preconditioning) induced by 3-5 days of opioid receptor (OR) agonism induces persistent protection against ischemia-reperfusion (I-R) injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium.<h4>Methodology/principal findings</h4>Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%). Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des), natriuretic peptides (Nppa,Nppb) and stress-signaling elements (Csda,Ptgds). Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3), cytokines (Il1b,Il6,Tnf) and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3), together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1) and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid). Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia), which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2) and other forms of stress (Xirp1,Ankrd1,Clu), and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1) and Txnip.<h4>Conclusions</h4>Protection via SLP is associated with transcriptional repression of inflammation/immunity, up-regulation of sarcomeric elements and natriuretic peptides, and modulation of cell stress, growth and development, while conventional protective molecules are unaltered.

Previous reports from our laboratory have shown that a bifunctional agent obtained by conjugating a photosensitizer (HPPH) to a cyanine dye (CD) can be used for fluorescence image-guided treatment of tumor by photodynamic therapy (PDT). However, the resulting HPPH-CD conjugate showed a significant difference between the tumor-imaging and therapeutic doses. It was demonstrated that the singlet oxygen ( (1) O 2 (*), a key cytotoxic agent in PDT) produced by the conjugate upon excitation of the HPPH moiety was partially quenched by the CD-moiety; this resulted in a reduced PDT response when compared to HPPH-PDT under similar treatment parameters. To improve the therapeutic potential of the conjugate, we synthesized a series of dual functional agents in which one or two HPPH moieties were separately conjugated to three different dyes (Cypate, modified IR820 or modified IR783). The newly synthesized conjugates were compared with our lead compound HPPH-CD in terms of photophysical properties, in vitro and in vivo PDT efficacy, tumor uptake and imaging potential. Among the analogs investigated, the conjugate, in which two HPPH moieties were linked to the modified IR820 produced enhanced tumor uptake and tumor contrast in both Colon 26 (a murine Colon carcinoma) and U87 (a human glioblastoma) cell lines. The long-term PDT efficacy (cure) of this conjugate in BALB/c mice, bearing Colon 26 tumors was also enhanced; however, its efficacy in Nude mice bearing U87 tumors was slightly reduced. It was also found that in all the conjugates the singlet oxygen generation and, consequently, PDT efficacy were compromised by a competing pathway, whereby an electronic excitation of HPPH, the energy donor, is deactivated through an electronic excitation energy transfer (Forster Resonance Energy Transfer, FRET) to the CD fluorophore, the energy acceptor, resulting in overall reduction of the singlet oxygen production. Conjugates with increased FRET showed reduced singlet oxygen production and PDT efficacy. Among the conjugates investigated, the bifunctional agent in which two HPPH moieties were linked to the benzoindole-based cyanine dye 11 showed superiority over the lead candidate 9 (mono HPPH-cyanine dye).

<h4>Significance</h4>Ion channels control membrane potential, cellular excitability, and Ca(++) signaling, all of which play essential roles in cellular functions. The regulation of ion channels enables cells to respond to changing environments, and post-translational modification (PTM) is one major regulation mechanism.<h4>Recent advances</h4>Many PTMs (e.g., S-glutathionylation, S-nitrosylation, S-palmitoylation, S-sulfhydration, etc.) targeting the thiol group of cysteine residues have emerged to be essential for ion channels regulation under physiological and pathological conditions.<h4>Critical issues</h4>Under oxidative stress, S-glutathionylation could be a critical PTM that regulates many molecules. In this review, we discuss S-glutathionylation-mediated structural and functional changes of ion channels. Criteria for testing S-glutathionylation, methods and reagents used in ion channel S-glutathionylation studies, and thiol modification crosstalk, are also covered. Mechanotransduction, and S-glutathionylation of the mechanosensitive KATP channel, are discussed.<h4>Future directions</h4>Further investigation of the ion channel S-glutathionylation, especially the physiological significance of S-glutathionylation and thiol modification crosstalk, could lead to a better understanding of the thiol modifications in general and the ramifications of such modifications on cellular functions and related diseases.

The oncoprotein Bcr-Abl, the causative agent of chronic myeloid leukemia (CML), requires homo-oligomerization via a coiled-coil domain to function [Bartram, C. R.; et al. Nature 1983, 306 (5940), 277-280; and Zhao, X.; et al. Nat. Struct. Biol. 2002, 9(2), 117-120]. While tyrosine kinase inhibitors (TKIs) have shown great efficacy as treatment options for CML, their use may cause an acquisition of mutations in the tyrosine kinase domain, which prevent TKI binding and lead to a loss in activity [Woessner, D. W.; et al. Cancer J. 2011, 17(6), 477-486]. Previously, we have shown that a rationally modified coiled-coil domain (CC(mut3)) can disrupt this oligomerization, inhibit proliferation, and induce apoptosis in CML cells [Dixon, A. S.; et al. Mol. Pharmaceutics 2012, 9(1), 187-195]. Here, we show that using the most recently approved TKI, ponatinib (Iclusig), in combination with CC(mut3) allows a dose reduction of ponatinib and increased therapeutic efficacy in vitro measured by reduction in kinase activity, induction of apoptosis via caspase-3/7 and 7-AAD/Annexin V assays, and reduced transformative ability measured by a colony forming assay. The combination was effective not only in cells containing wild-type Bcr-Abl (K562, Ba/F3-p210) but also cells with Bcr-Abl containing the T315I mutation (Ba/F3-p210-T315I). In addition, we report for the first time the ability of CC(mut3) alone to inhibit the T315I mutant form of Bcr-Abl. This novel combination may prove to be more potent than single agent therapies and should be further explored for clinical use.

Here we sought to identify transcription factors that induce ADAMTS5, a crucial proteinase for osteoarthritis development. Exhaustive comparison of the genomic sequences of human, macaque, and mouse ADAMTS5 genes revealed that the proximal 1.4 kb of the 5'-end-flanking regions containing several consensus motifs was highly conserved. Among putative transcription factors for these motifs, NF-κB family member RelA/p65 most strongly stimulated the promoter activity. In the ADAMTS5 gene, there were three NF-κB binding motifs, in which deletion, mutagenesis, and tandem repeat analyses of the luciferase assay identified the core responsive elements of RelA/p65 to be -896/-887 and -424/-415 bp with specific bindings. The endogenous Adamts5 expression in ATDC5 cells was increased by RelA/p65 overexpression and decreased by knockdown through its siRNA. The expression was also inhibited by the Rela deletion through Cre transfection in primary articular chondrocytes from Rela(fl/fl) mice. In the ex vivo culture of femoral head cartilage from mesenchymal cell-specific Rela knock-out (Prx1-Cre;Rela(fl/fl)) mice, aggrecanolysis was significantly lower than that in the Rela(fl/fl) cartilage. Finally, in the experimental mouse osteoarthritis model, ADAMTS5 and RelA were co-localized in chondrocytes of degraded articular cartilage. We conclude that RelA/p65 is a potent transcriptional activator of ADAMTS5 in chondrocytes during osteoarthritis development.

A series of schizonepetin derivatives have been designed and synthesized in order to obtain potent antivirus agents. The antiviral activity against HSV-1 and influenza virus H3N2 as well as the cytotoxicity of these derivatives was evaluated by using cytopathic effect (CPE) inhibition assay in vitro. Compounds M2, M4, M5 and M34 showed higher inhibitory activity against HSV-1 virus with the TC50 values being in micromole. Compounds M28, M33, and M35 showed higher inhibitory activity against influenza virus H3N2 with their TC50 values being 96.4, 71.0 and 75.4 μM, respectively. Preliminary biological activity evaluation indicated that the anti-H3N2 and anti-HSV-1 activities improved obviously through the introduction of halogen into the structure of schizonepetin.

Primary human dendritic cells (DC) were used to explore the inflammatory effectors, including cytokines and microRNAs, regulated by Helicobacter pylori. In a 48 h ex-vivo co-culture system, both H. pylori B38 and B45 strains activated human DCs and promoted a strong inflammatory response characterized by the early production of pro-inflammatory TNFα and IL-6 cytokines, followed by IL-10, IL-1β, and IL-23 secretion. IL-23 was the only cytokine dependent on the cag pathogenicity island status of the bacterial strains. DC activation and cytokine production were accompanied by an early miR-146a upregulation followed by a strong miR-155 induction, which mainly controlled TNFα production. These results pave the way for further investigations into the nature of H. pylori antigens and the subsequently activated signaling pathways involved in the inflammatory response to H. pylori infection, the deregulation of which may likely contribute to gastric lymphomagenesis.

<h4>Objectives</h4>Serotonergic genes are associated with suicidal behavior; this association has not been tested in stroke survivors, however. In this study, we investigated whether serotonin transporter (5-HTT) and serotonin 2a receptor (5-HTR2a) genes were associated with suicidal ideation at 2 weeks and at 1 year after stroke.<h4>Methods</h4>A total of 286 patients were evaluated 2 weeks after stroke, and 222 (78%) participated in a follow-up evaluation one year later. Suicidal ideation was ascertained using the "suicidal thoughts" item of the Montgomery and Asberg Depression Rating Scale. Associations between suicidal ideation and 5-HTTLPR, STin2 VNTR, 5-HTR2a 1438A/G, and 5-HTR2a 102T/C genotypes were estimated using logistic regression models, and gene-gene interactions were investigated using the generalized multifactor dimensionality reduction method after adjustment for potential covariates, including depression.<h4>Results</h4>The 5-HTR2a 1438 A/A genotype was significantly associated with suicidal ideation 2 weeks after stroke, before and after statistical adjustment. The 5-HTTLPR short allele was associated with suicidal ideation 2 weeks after stroke, although the significance of this finding was not evident after adjustment. No significant associations were observed between any genotype and suicidal ideation one year after stroke. No significant gene-gene interactions were detected.<h4>Conclusions</h4>Individuals with the 5-HTR2a 1438 A/A genotype were more susceptible to suicidal ideation 2 weeks after stroke. Careful evaluation and management are indicated for those with increased genetic vulnerability, particularly during the acute phase of stroke.

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used 'reference plasma sample', a pool of plasma from 10 healthy individuals from Indian population in the age group of 25-60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by ≥ 2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.

Acupuncture is an alternative therapy to induce ovulation in women with polycystic ovary syndrome (PCOS), but there is no study reporting the live birth rate following ovulation induction by acupuncture or its potential as an adjuvant treatment to clomiphene citrate (CC). We assess the efficacy of acupuncture with or without CC in achieving live births among 1000 PCOS women in Mainland China. This paper reports the methodology of an ongoing multicenter randomized controlled trial. The randomization scheme is coordinated through the central mechanism and stratified by the participating sites. Participants will be randomized into one of the four treatment arms: (A) true acupuncture and CC, (B) control acupuncture and CC, (C) true acupuncture and placebo CC, and (D) control acupuncture and placebo CC. To ensure the quality and integrity of the trial we have developed a unique multinational team of investigators and Data and Safety Monitoring Board. Up to the end of April 2013, 326 subjects were recruited. In conclusion, the success of this trial will allow us to evaluate the additional benefit of acupuncture beyond the first line medicine for fertility treatment in PCOS women in an unbiased manner.

<h4>Unlabelled</h4>Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells.<h4>Conclusion</h4>Genistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5' partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in-frame to six N-terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAF(V) (600E) mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.

An apical tuft, which is observed in a wide range of embryos/larvae of marine invertebrates, is composed of a group of cilia that are longer and less motile than the abundant lateral cilia covering the rest of the embryonic surface. Although the apical tuft has been thought to function as a sensory organ, its molecular composition and roles are poorly understood. Here, we identified a glutathione transferase theta (GSTT) as an abundant and specific component of the apical tuft in sea urchin embryos. The expression of GSTT mRNA increases and becomes limited to the animal plate of the mesenchyme blastula, gastrula, and prism larva. Electron microscopy and tandem mass spectrometry demonstrated that the apical tuft contains almost every axonemal component for ciliary motility. Low concentrations of an inhibitor of glutathione transferase bromosulphophthalein (BSP) induce bending of apical tuft, suggesting that GSTT regulates motility of apical tuft cilia. Embryos treated with BSP swim with normal velocity and trajectories but show less efficiency of changing direction when they collide with an object. These results suggest that GSTT in the apical tuft plays an important role in the mechanical reception for the motility regulation of lateral motile cilia in sea urchin embryos.

Antisense oligonucleotides are powerful tools to regulate gene expression in cells and model organisms. However, a transfection or microinjection is typically needed for efficient delivery of the antisense agent. We report the conjugation of multiple HIV TAT peptides to a hairpin-protected antisense agent through a light-cleavable nucleobase caging group. This conjugation allows for the facile delivery of the antisense agent without a transfection reagent, and photochemical activation offers precise control over gene expression. The developed approach is highly modular, as demonstrated by the conjugation of folic acid to the caged antisense agent. This enabled targeted cell delivery through cell-surface folate receptors followed by photochemical triggering of antisense activity. Importantly, the presented strategy delivers native oligonucleotides after light-activation, devoid of any delivery functionalities or modifications that could otherwise impair their antisense activity.

We previously found that phosphatidylcholine-specific phospholipase C (PC-PLC) was a key inducing element of atherosclerosis, and might negatively regulate human umbilical vein endothelial cell (HUVEC) autophagy. To further investigate the mechanism of PC-PLC action, we initially identified phosphatidylethanolamine binding protein 1 (PEBP1) as a binding partner of PC-PLC by using mass spectrometry (MS, MALDI-TOF/TOF). We found that PEBP1 positively regulated PC-PLC activity in HUVECs, and inhibition of PC-PLC by its inhibitor D609 suppressed PEBP1 expression dramatically. Moreover, both PC-PLC and PEBP1 negatively regulated HUVEC autophagy independently of mammalian target of rapamycin (mTOR). Furthermore, the PEBP1 level was elevated during the development of atherosclerosis, while D609 significantly decreased the upregulated PEBP1 level in apoE(-/-) mice.

Most bacterial pathogens require iron to grow and colonize host tissues. The Gram-negative bacterium Salmonella enterica serovar Typhimurium causes a natural systemic infection of mice that models acute and chronic human typhoid fever. S. Typhimurium resides in tissues within cells of the monocyte lineage, which limit pathogen access to iron, a mechanism of nutritional immunity. The primary ferric iron import system encoded by Salmonella is the siderophore ABC transporter FepBDGC. The Fep system has a known role in acute infection, but it is unclear whether ferric iron uptake or the ferric iron binding siderophores enterobactin and salmochelin are required for persistent infection. We defined the role of the Fep iron transporter and siderophores in the replication of Salmonella in macrophages and in mice that develop acute followed by persistent infections. Replication of wild-type and iron transporter mutant Salmonella strains was quantified in cultured macrophages, fecal pellets, and host tissues in mixed- and single-infection experiments. We show that deletion of fepB attenuated Salmonella replication and colonization within macrophages and mice. Additionally, the genes required to produce and transport enterobactin and salmochelin across the outer membrane receptors, fepA and iroN, are needed for colonization of all tissues examined. However, salmochelin appears to be more important than enterobactin in the colonization of the spleen and liver, both sites of dissemination. Thus, the FepBDGC ferric iron transporter and the siderophores enterobactin and salmochelin are required by Salmonella to evade nutritional immunity in macrophages and cause persistent infection in mice.

Bullying is a serious problem for schools, parents, and public-policymakers alike. Bullying creates risks of health and social problems in childhood, but it is unclear if such risks extend into adulthood. A large cohort of children was assessed for bullying involvement in childhood and then followed up in young adulthood in an assessment of health, risky or illegal behavior, wealth, and social relationships. Victims of childhood bullying, including those that bullied others (bully-victims), were at increased risk of poor health, wealth, and social-relationship outcomes in adulthood even after we controlled for family hardship and childhood psychiatric disorders. In contrast, pure bullies were not at increased risk of poor outcomes in adulthood once other family and childhood risk factors were taken into account. Being bullied is not a harmless rite of passage but throws a long shadow over affected people's lives. Interventions in childhood are likely to reduce long-term health and social costs.

Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.

With the advent of deep sequencing technology, a variety of miRNA length and sequence variants, termed isomiRNAs (isomiRs), have been discovered. However, the functional roles of these commonly detected isomiRs remain unknown. In this paper, we demonstrated that miRNAs regulate the expression of the HTT gene, whose mutation leads to Huntington's disease (HD), a hereditary degenerative disorder. Specifically, we validated the interactions of canonical miRNAs, miR-137, miR-214, and miR-148a, with the HTT 3'UTR using a luciferase assay. Moreover, we applied synthetic miRNA mimics to examine whether a slight shifting of miRNA seed regions might alter the regulation of the HTT transcript. We also examined miR-137, miR-214, and miR-148a isomiRs and showed the activity of these isoforms on reporter constructs bearing appropriate sequences from the HTT 3'UTR. Hence, we demonstrated that certain 5'-end variants of miRNAs might be functional for the regulation of the same targets as canonical miRNAs.

The senescence-accelerated mouse (SAMP8) strain exhibits decreased learning and memory and increased amyloid beta (Aβ) peptide accumulation at 12 months. To detect differences in gene expression in SAMP8 mice, we used a control mouse that was a 50% cross between SAMP8 and CD-1 mice and which showed no memory deficits (50% SAMs). We then compared gene expression in the hippocampus of 4- and 12-month-old SAMP8 and control mice using Affymetrix gene arrays. At 12 months, but not at 4 months, pathway analysis revealed significant differences in the long term potentiation (6 genes), phosphatidylinositol signaling (6 genes), and endocytosis (10 genes) pathways. The changes in long term potentiation included mitogen-activated protein kinase (MAPK) signaling (N-ras, cAMP responsive element binding protein [CREB], protein phosphatase inhibitor 1) and Ca-dependent signaling (inositol triphosphate [ITP] receptors 1 and 2 and phospholipase C). Changes in phosphatidylinositol signaling genes suggested altered signaling through phosphatidylinositol-3-kinase, and Western blotting revealed phosphorylation changes in serine/threonine protein kinase AKT and 70S6K. Changes in the endocytosis pathway involved genes related to clathrin-mediated endocytosis (dynamin and clathrin). Endocytosis is required for receptor recycling, is involved in Aβ metabolism, and is regulated by phosphatidylinositol signaling. In summary, these studies demonstrate altered gene expression in 3 SAMP8 hippocampal pathways associated with memory formation and consolidation. These pathways might provide new therapeutic targets in addition to targeting Aβ metabolism itself.

The importance of genetics and epigenetic changes in the pathogenesis of non alcoholic fatty liver disease (NAFLD) has been increasingly recognized. Adiponectin has a central role in regulating glucose and lipid metabolism and controlling inflammation in insulin-sensitive tissues and low adiponectin levels have been linked to NAFLD. APPL1 and APPL2 are adaptor proteins that interact with the intracellular region of adiponectin receptors and mediate adiponectin signaling and its effects on metabolism. The aim of our study was the evaluation of a potential association between variants at APPL1 and APPL2 loci and NAFLD occurrence. The impact on liver damage and hepatic steatosis severity has been also evaluated. To this aim allele frequency and genotype distribution of APPL1- rs3806622 and -rs4640525 and APPL2-rs 11112412 variants were evaluated in 223 subjects with clinical diagnosis of NAFLD and compared with 231 healthy subjects. The impact of APPL1 and APPL2 SNPs on liver damage and hepatic steatosis severity has been also evaluated. The minor-allele combination APPL1-C/APPL2-A was associated with an increased risk of NAFLD (OR = 2.50 95% CI 1.45-4.32; p<0.001) even after adjustment for age, sex, body mass index, insulin resistance (HOMA-IR), triglycerides and adiponectin levels. This allele combination carrier had higher plasma alanine aminotransferase levels (Diff = 15.08 [7.60-22.57] p = 0.001) and an increased frequency of severe steatosis compared to the reference allele combination (OR = 3.88; 95% CI 1.582-9.531; p<0.001). In conclusion, C-APPL1/A-APPL2 allele combination is associated with NAFLD occurrence, with a more severe hepatic steatosis grade and with a reduced adiponectin cytoprotective effect on liver.

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome that is characterized by pure red-cell aplasia and associated physical deformities. It has been proven that defects of ribosomal proteins can lead to this disease and that RPS19 is the most frequently mutated gene in DBA patients. Previous studies suggest that p53-dependent genes and pathways play important roles in RPS19-deficient embryos. However, whether there are other vital factors linked to DBA has not been fully clarified. In this study, we compared the whole genome RNA-Seq data of zebrafish embryos injected with RPS19 morpholino (RPS19 MO), RPS19 and p53 morpholino simultaneously (RPS19+p53 MO) and control morpholino (control). We found that genes enriched in the functions of hematological systems, nervous system development and skeletal and muscular disorders had significant differential expression in RPS19 MO embryos compared with controls. Co-inhibition of p53 partially alleviates the abnormalities for RPS19-deficient embryos. However, the hematopoietic genes, which were down-regulated significantly in RPS19 MO embryos, were not completely recovered by the co-inhibition of p53. Furthermore, we identified the genome-wide p53-dependent and -independent genes and pathways. These results indicate that not only p53 family members but also other factors have important impacts on RPS19-deficient embryos. The detection of potential pathogenic genes and pathways provides us a new paradigm for future research on DBA, which is a systematic and complex hereditary disease.

Intricate, dynamic, and absolutely unavoidable ageing affects cells and organisms through their entire lifetime. Driven by diverse mechanisms all leading to compromised cellular functions and finally to death, this process is a challenge for researchers. The molecular mechanisms, the general rules that it follows, and the complex interplay at a molecular and cellular level are yet little understood. Here, we present our results showing a connection between the linker histones, the higher-order chromatin structures, and the process of chronological lifespan of yeast cells. By deleting the gene for the linker histone in Saccharomyces cerevisiae we have created a model for studying the role of chromatin structures mainly at its most elusive and so far barely understood higher-order levels of compaction in the processes of yeast chronological lifespan. The mutant cells demonstrated controversial features showing slower growth than the wild type combined with better survival during the whole process. The analysis of the global chromatin organization during different time points demonstrated certain loss of the upper levels of chromatin compaction in the cells without linker histone. The results underlay the importance of this histone for the maintenance of the chromatin loop structures during ageing.

Autoimmune hepatitis (AIH) is an inflammatory condition of the liver that has a multitude of clinical presentations from chronic hepatitis to acute fulminant hepatitis. AIH diagnosis is typically suspected after ruling out other causes of hepatitis (such as vial hepatitis, hemochromatosis, Wilson's disease, and primary biliary cirrhosis) through serological tests and by findings of high titers of certain autoantibodies (ANA and anti-SMA for type 1 AIH and anti-LKM-1 for type 2 AIH). AIH like most inflammatory conditions is associated with increased ferritin levels (acute-phase reactant) but typically near-normal transferrin saturation. The presence of excessive ferritin level in absence of high-transferrin saturation helps differentiate secondary iron overload from hemochromatosis where transferrin saturation is typically high. We herein describe a case of AIH that presented with high ferritin levels and transferrin saturation suggesting a diagnosis of hemochromatosis and needed arduous work-up to arrive at accurate diagnosis of AIH.

Functional outcomes for long-term survivors of acute liver failure (ALF) are not well characterized. The aim of this prospective study was to determine health-related quality of life in long-term adult ALF survivors. Acute Liver Failure Study Group registry participants completed the Centers for Disease Control and Prevention Health-Related Quality of Life 14 and Short Form 36 (SF-36) questionnaires at 1- and/or 2-year follow-up study visits. Responses were compared among ALF subgroups and to those for available general US population controls. Among the 282 adult ALF patients, 125 had undergone liver transplantation (LT), whereas 157, including 95 acetaminophen overdose (APAP) patients and 62 non-APAP patients, were spontaneous survivors (SSs). APAP SS patients reported significantly lower general health scores and more days of impaired mental and physical health, activity limitations due to poor health, pain, depression, and anxiety in comparison with the other groups (P ≤ 0.001). There were no significant differences in coma grade or in the use of mechanical ventilation or intracranial pressure monitoring among the patient groups during their ALF hospitalization, but APAP SSs had significantly higher rates of psychiatric disease and substance abuse (P < 0.001). In comparison with the general US population, a greater proportion of the combined SS patients reported fair or poor health and ≥14 days of impaired physical/mental health and activity limitations due to poor health. In addition, a greater proportion of LT recipients reported ≥14 days of impaired physical/mental health. Similar results were observed with the SF-36 across the 3 ALF subgroups and in comparison with population controls. In conclusion, long-term adult survivors of ALF reported significantly lower quality of life scores than US population controls. Furthermore, APAP SS patients reported the lowest quality of life scores, possibly because of higher rates of premorbid psychiatric and substance abuse disorders.

Huntingtin is a ubiquitous cytoplasmic protein. Mutant huntingtin causes Huntington's disease and its intranuclear inclusion is associated with cytotoxicity. Nuclear localization of normal huntingtin is detected in the oocyte up to 2.5 days post coitum. Therefore, huntingtin is expected to reside in the nucleus even before fertilization. The present study determined normal huntingtin distribution during spermatogenesis. Testicles from an adult male Sprague-Dawley rat were stained with anti-huntingtin antibody and nuclear counterstaining was performed with 4',6-diamidino-2-phenylindole. Concerning nuclear localization, huntingtin was detected in the spermatids, whereas predominant cytoplasmic localization of it was evident in the spermatogonia. Between the primary and secondary spermatocytes, huntingtin appeared to be delocalized in the nuclei when meiosis occurred. The findings provide evidence that normal huntingtin is transported to the nuclear compartment during the meiotic stage of spermatogenesis.

Thrombin is the protease involved in blood coagulation. Its deregulation can lead to hemostatic abnormalities, which range from subtle subclinical to serious life-threatening coagulopathies, i.e., during septicemia. Additionally, thrombin plays important roles in many (patho)physiological conditions that reach far beyond its well-established role in stemming blood loss and thrombosis, including embryonic development and angiogenesis but also extending to inflammatory processes, complement activation, and even tumor biology. In this review, we will address thrombin's broad roles in diverse (patho)physiological processes in an integrative way. We will also discuss thrombin as an emerging major target for novel therapies.

<h4>Background</h4>Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) region of the Huntingtin (HTT) gene. The clinical features of HD are characterized by cognitive, psychological, and motor deficits. Molecular instability, a core component in neurological disease progression, can be comprehensively evaluated through longitudinal transcriptomic profiling. Development of animal models amenable to longitudinal examination enables distinct disease-associated mechanisms to be identified.<h4>Results</h4>Here we report the first longitudinal study of transgenic monkeys with genomic integration of various lengths of the human HTT gene and a range of polyQ repeats. With this unique group of transgenic HD nonhuman primates (HD monkeys), we profiled over 47,000 transcripts from peripheral blood collected over a 2 year timespan from HD monkeys and age-matched wild-type control monkeys.<h4>Conclusions</h4>Messenger RNAs with expression patterns which diverged with disease progression in the HD monkeys considerably facilitated our search for transcripts with diagnostic or therapeutic potential in the blood of human HD patients, opening up a new avenue for clinical investigation.

Spider silks have been a focus of research for almost two decades due to their outstanding mechanical and biophysical properties. Recent advances in genetic engineering have led to the synthesis of recombinant spider silks, thus helping to unravel a fundamental understanding of structure-function-property relationships. The relationships between molecular composition, secondary structures and mechanical properties found in different types of spider silks are described, along with a discussion of artificial spinning of these proteins and their bioapplications, including the role of silks in biomineralization and fabrication of biomaterials with controlled properties.

Psychiatric disorders are caused by perturbed molecular pathways that affect brain circuitries. The identification of specific biosignatures that are the result of altered pathway activities in major depression, bipolar disorder and schizophrenia can contribute to a better understanding of disease etiology and aid in the implementation of diagnostic assays. In the present study we identified disease-specific protein biosignatures in cerebrospinal fluid of depressed (n: 36), bipolar (n: 27) and schizophrenic (n: 35) patients using the Reverse Phase Protein Microarray technology. These biosignatures were able to stratify patient groups in an objective manner according to cerebrospinal fluid protein expression patterns. Correct classification rates were over 90%. At the same time several protein sets that play a role in neuronal growth, proliferation and differentiation (NEGR1, NPDC1), neurotransmission (SEZ6) and protection from oxidative damage (GPX3) were able to distinguish diseased from healthy individuals (n: 35) indicating a molecular signature overlap for the different psychiatric phenotypes. Our study is a first step toward implementing a psychiatric patient stratification system based on molecular biosignatures. Protein signatures may eventually be of use as specific and sensitive biomarkers in clinical trials not only for patient diagnostic and subgroup stratification but also to follow treatment response.

<h4>Background</h4>This descriptive study of the abdominal fat transcriptome takes advantage of two experimental lines of meat-type chickens (Gallus domesticus), which were selected over seven generations for a large difference in abdominal (visceral) fatness. At the age of selection (9 wk), the fat line (FL) and lean line (LL) chickens exhibit a 2.5-fold difference in abdominal fat weight, while their feed intake and body weight are similar. These unique avian models were originally created to unravel genetic and endocrine regulation of adiposity and lipogenesis in meat-type chickens. The Del-Mar 14K Chicken Integrated Systems microarray was used for a time-course analysis of gene expression in abdominal fat of FL and LL chickens during juvenile development (1-11 weeks of age).<h4>Results</h4>Microarray analysis of abdominal fat in FL and LL chickens revealed 131 differentially expressed (DE) genes (FDR≤0.05) as the main effect of genotype, 254 DE genes as an interaction of age and genotype and 3,195 DE genes (FDR≤0.01) as the main effect of age. The most notable discoveries in the abdominal fat transcriptome were higher expression of many genes involved in blood coagulation in the LL and up-regulation of numerous adipogenic and lipogenic genes in FL chickens. Many of these DE genes belong to pathways controlling the synthesis, metabolism and transport of lipids or endocrine signaling pathways activated by adipokines, retinoid and thyroid hormones.<h4>Conclusions</h4>The present study provides a dynamic view of differential gene transcription in abdominal fat of chickens genetically selected for fatness (FL) or leanness (LL). Remarkably, the LL chickens over-express a large number of hemostatic genes that could be involved in proteolytic processing of adipokines and endocrine factors, which contribute to their higher lipolysis and export of stored lipids. Some of these changes are already present at 1 week of age before the divergence in fatness. In contrast, the FL chickens have enhanced expression of numerous lipogenic genes mainly after onset of divergence, presumably directed by multiple transcription factors. This transcriptional analysis shows that abdominal fat of the chicken serves a dual function as both an endocrine organ and an active metabolic tissue, which could play a more significant role in lipogenesis than previously thought.

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

<h4>Background</h4>Glutathione-S-transferase gene (GST) polymorphisms can result in variable ability of these enzymes to remove electrophilic substrates. We investigated whether the GSTP1 Val105 and GSTM1 deletion polymorphisms modify the lead-cognitive function association.<h4>Methods</h4>We used repeated measures analysis to compare the association between cumulative lead biomarkers-bone lead measured using K-shell X-Ray Fluorescence-and Mini-Mental State Exam (MMSE) score by GST variants, adjusted for covariates, among Normative Aging Study participants, a Boston-based prospective cohort of men. We had complete data for 698 men (providing 1292 observations) for GSTM1 analyses and 595 men (providing 1142 observations) for GSTP1 analyses.<h4>Results</h4>A 15μg/g higher tibia lead concentration (interquartile range of tibia lead) was associated with a 0.24 point decrement in MMSE score among GSTP1 Val105 variant carriers, which was significantly stronger than the association among men with only wild-type alleles (p=0.01). The association among GSTP1 Val105 carriers was comparable to that of 3 years of age in baseline MMSE scores. The association between tibia lead and MMSE score appeared progressively steeper in participants with increasingly more GSTP1 Val105 alleles. A modest association between tibia lead and lower MMSE score was seen among participants with the GSTM1 deletion polymorphism. Neither of the glutathione S-transferase variants was independently associated with cognitive function, nor with lead biomarker measures. The results pertaining to patella lead were similar to those observed for tibia lead.<h4>Conclusion</h4>Our results suggest that the GSTP1 Val105 polymorphism confers excess susceptibility to the cognitive effects of cumulative lead exposure.

Runs of homozygosity (ROH) may play a role in complex diseases. In the current study, we aimed to test if ROHs are linked to the risk of autism and related language impairment. We analyzed 546,080 SNPs in 315 Han Chinese affected with autism and 1,115 controls. ROH was defined as an extended homozygous haplotype spanning at least 500 kb. Relative extended haplotype homozygosity (REHH) for the trait-associated ROH region was calculated to search for the signature of selection sweeps. Totally, we identified 676 ROH regions. An ROH region on 11q22.3 was significantly associated with speech delay (corrected p = 1.73×10(-8)). This region contains the NPAT and ATM genes associated with ataxia telangiectasia characterized by language impairment; the CUL5 (culin 5) gene in the same region may modulate the neuronal migration process related to language functions. These three genes are highly expressed in the cerebellum. No evidence for recent positive selection was detected on the core haplotypes in this region. The same ROH region was also nominally significantly associated with speech delay in another independent sample (p = 0.037; combinatorial analysis Stouffer's z trend = 0.0005). Taken together, our findings suggest that extended recessive loci on 11q22.3 may play a role in language impairment in autism. More research is warranted to investigate if these genes influence speech pathology by perturbing cerebellar functions.

Carotid sinus hypersensitivity has a high prevalence in the elderly and is a possible cause of falls. In carotid sinus hypersensitivity, external triggers cause sudden reductions in blood pressure, leading to dizziness or syncope, resulting in falls. Turning of the head is considered an important example of such an external trigger in everyday life, wherein rotation of the neck is thought to manipulate the hypersensitive carotid sinus. However, direct evidence for this is lacking. The aim of this study was to investigate the effects of head turning in elderly with carotid sinus hypersensitivity. We performed a prospective, observational study in 105 elderly patients who visited a geriatric falls clinic in a university teaching hospital and in 25 community dwelling healthy elderly subjects. Continuous measurements of blood pressure and heart rate (Finapres) were performed before, during, and after head turning. Head turning-induced hypotension was defined as a drop in systolic blood pressure of at least 20 mmHg during head turning. Carotid sinus hypersensitivity was examined with carotid sinus massage. We also tested for two other common geriatric hypotensive syndromes, orthostatic hypotension and post prandial hypotension, using active standing and a meal test. All three hypotensive syndromes were defined using consensus definitions. Head turning resulted in hypotension in 39% of patients (mean systolic blood pressure drop 36 mm Hg) and in 44% of the healthy elderly, irrespective of the direction of the head movement. Carotid sinus hypersensitivity was associated with head-turning induced hypotension (OR= 3.5, 95% CI= 1.48 to 8.35). We conclude that head turning is indeed an important cause of sudden drops in blood pressure in elderly with carotid sinus hypersensitivity.

Netrins are a family of proteins involved in cell migration and axon guidance during embryogenesis. The different functions and mechanisms of action of this family of proteins have been better characterized with the study of the netrin-1. They are chemotropic and act as a bifunctional regulator of neuron migration. Apart from its role in the central nervous system, researchers have proven that netrin-1 plays a role in the development and formation of non-neural tissue, thus netrin-1 is involved in regulation of cancers, cardiovascular diseases, kidney diseases and other diseases. Concerning the cardio-vascular realm, netrin-1 promotes angiogenesis and accelerates atherosclerosis, protects the heart against ischemia-reperfusion injury and reduces the infarct size. These findings make the neuroimmune guidance cue netrin-1 an important therapeutic target. This work seeks to review the subject based on studies that have been conducted over the past decade to identify the perspectives and extent of the research on this protein in the field of cardiology.

Proximal interstitial 6q deletion involving Single-minded 1 (SIM1) gene causes a syndromic form of obesity mimicking Prader-Willi syndrome. In addition to obesity, Prader-Willi syndrome includes several other endocrinopathies, such as hypothyroidism, growth hormone deficiency, and hypogonadotropic hypogonadism. The endocrine phenotype of interstitial 6q deletion remains largely unknown, although clinical similarities between Prader-Willi syndrome and interstitial 6q deletion suggest endocrine abnormalities also may contribute to the interstitial 6q deletion phenotype. This report describes the endocrine phenotype in a propositus with the Prader-Willi-like syndrome associated with an interstitial 6q deletion including the SIM1 gene. Detailed endocrine evaluation of the propositus during childhood and adolescence revealed hypopituitarism, though initial endocrine evaluations during infancy were unremarkable. Our patient raises the possibility that hypopituitarism may be part of the phenotype, especially short stature, caused by interstitial 6q deletion. SIM1 plays an important role in the development of neuroendocrine lineage cells, implicating SIM1 haploinsufficiency in the pathophysiology of hypopituitarism seen in our propositus. Early identification of endocrine abnormalities can improve clinical outcome by allowing timely introduction of hormone replacement therapy. Hence, we suggest that detailed endocrine evaluation and longitudinal endocrine follow up be performed in individuals with proximal interstitial 6q deletion involving SIM1.

Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions, including Alzheimer's disease (AD), Parkinson's disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases, the underlying cause of iron mis-metabolism is known, while in others, our understanding is, at best, incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggests that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron-modulating proteins, dysfunction of a specific pathway or selective absence of iron-modulating protein(s) in systemic organs has provided important insights into the maintenance of iron homeostasis within the brain. Here, we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies which are aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.

<h4>Background/aims</h4>The aims of this study were to validate the newly developed version of the Addenbrooke's Cognitive Examination (ACE-III) against standardised neuropsychological tests and its predecessor (ACE-R) in early dementia.<h4>Methods</h4>A total of 61 patients with dementia (frontotemporal dementia, FTD, n = 33, and Alzheimer's disease, AD, n = 28) and 25 controls were included in the study.<h4>Results</h4>ACE-III cognitive domains correlated significantly with standardised neuropsychological tests used in the assessment of attention, language, verbal memory and visuospatial function. The ACE-III also compared very favourably with its predecessor, the ACE-R, with similar levels of sensitivity and specificity.<h4>Conclusion</h4>The results of this study provide objective validation of the ACE-III as a screening tool for cognitive deficits in FTD and AD.

B-cell lymphoma 2 (Bcl-2) family proteins regulate survival, mitochondria morphology dynamics and metabolism in many cell types including neurons. Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat tract in the IT15 gene that encodes for the protein huntingtin (htt). In vitro and in vivo models of HD and HD patients' tissues show abnormal mitochondrial function and increased cell death rates associated with alterations in Bcl-2 family protein expression and localization. This review aims to draw together the information related to Bcl-2 family protein alterations in HD to decipher their potential role in mutated htt-related cell death and mitochondrial dysfunction.

Disorders of human communication abilities can be classified into speech and language disorders. Speech disorders (e.g., dyspraxia) affect the sound generation and sequencing, while language disorders (e.g., dyslexia and specific language impairment, or SLI) are deficits in the encoding and decoding of language according to its rules (reading, spelling, grammar). The diagnosis of such disorders is often complicated, especially when a patient presents more than one disorder at the same time. The present review focuses on these challenges. We have combined data available from the literature with an in silico approach in an attempt to identify putative miRNAs that may have a key role in dyspraxia, dyslexia and SLI. We suggest the use of new miRNAs, which could have an important impact on the three diseases. Further, we relate those miRNAs to the axon guidance pathway and discuss possible interactions and the role of likely deregulated proteins. In addition, we describe potential differences in expressional deregulation and its role in the improvement of diagnosis. We encourage experimental investigations to test the data obtained in silico.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is an amphetamine derivative widely abused by young adults. Although many studies have reported that relatively high doses of MDMA deplete serotonin (5-HT) content and decrease the availability of serotonin transporters (5-HTT), limited evidence is available as to the adaptive mechanisms taking place in gene expression levels in the brain following a dosing regimen of MDMA comparable to human consumption. In order to further clarify this issue, we used quantitative PCR to study the long-term changes induced by acute administration of MDMA (5 mg/kg × 3) in the expression of genes related to serotonergic and dopaminergic systems, as well as those related to cellular toxicity in the cortex, hippocampus, striatum, and brain stem of rats. Seven days after MDMA administration, we found a significantly lower expression of the 5-HTT (Slc6a4) and the vesicular monoamine transporter (Slc18a2) genes in the brain stem area. In the hippocampus, monoamine oxidase B (Maob) and tryptophan hydroxylase 2 (Tph2) gene expressions were increased. In the striatum, tyrosine hydroxylase (Th) expression was decreased, and a lower expression of α-synuclein (Snca) was observed in the cortex. In contrast, no significant changes were observed in the genes considered to be biomarkers of toxicity including the glial fibrillary acidic protein (Gfap) and the heat-shock 70 kD protein 1A (Hspa1a) in any of the structures assayed. These results suggest that MDMA promotes adaptive changes in genes related to serotonergic and dopaminergic functionality, but not in genes related to neurotoxicity.

Central obesity, measured by waist circumference (WC) or waist-hip ratio (WHR), is a marker of body fat distribution. Although obesity disproportionately affects minority populations, few studies have conducted genome-wide association study (GWAS) of fat distribution among those of predominantly African ancestry (AA). We performed GWAS of WC and WHR, adjusted and unadjusted for BMI, in up to 33,591 and 27,350 AA individuals, respectively. We identified loci associated with fat distribution in AA individuals using meta-analyses of GWA results for WC and WHR (stage 1). Overall, 25 SNPs with single genomic control (GC)-corrected p-values<5.0 × 10(-6) were followed-up (stage 2) in AA with WC and with WHR. Additionally, we interrogated genomic regions of previously identified European ancestry (EA) WHR loci among AA. In joint analysis of association results including both Stage 1 and 2 cohorts, 2 SNPs demonstrated association, rs2075064 at LHX2, p = 2.24×10(-8) for WC-adjusted-for-BMI, and rs6931262 at RREB1, p = 2.48×10(-8) for WHR-adjusted-for-BMI. However, neither signal was genome-wide significant after double GC-correction (LHX2: p = 6.5 × 10(-8); RREB1: p = 5.7 × 10(-8)). Six of fourteen previously reported loci for waist in EA populations were significant (p<0.05 divided by the number of independent SNPs within the region) in AA studied here (TBX15-WARS2, GRB14, ADAMTS9, LY86, RSPO3, ITPR2-SSPN). Further, we observed associations with metabolic traits: rs13389219 at GRB14 associated with HDL-cholesterol, triglycerides, and fasting insulin, and rs13060013 at ADAMTS9 with HDL-cholesterol and fasting insulin. Finally, we observed nominal evidence for sexual dimorphism, with stronger results in AA women at the GRB14 locus (p for interaction = 0.02). In conclusion, we identified two suggestive loci associated with fat distribution in AA populations in addition to confirming 6 loci previously identified in populations of EA. These findings reinforce the concept that there are fat distribution loci that are independent of generalized adiposity.

The netrin axon guidance genes have previously been implicated in fertility in C. elegans and in vertebrates. Here we show that adult Drosophila lacking both netrin genes, NetA and NetB, have fertility defects in both sexes together with an inability to fly and reduced viability. NetAB females produce fertilized eggs at a much lower rate than wild type. Oocyte development and ovarian innervation are unaffected in NetAB females, and the reproductive tract appears normal. A small gene, hog, that resides in an intron of NetB does not contribute to the NetAB phenotype. Restoring endogenous NetB expression rescues egg-laying, but additional genetic manipulations, such as restoration of netrin midline expression and inhibition of cell death have no effect on fertility. NetAB males induce reduced egg-laying in wild type females and display mirror movements of their wings during courtship. Measurement of courtship parameters revealed no difference compared to wild type males. Transgenic manipulations failed to rescue male fertility and mirror movements. Additional genetic manipulations, such as removal of the enabled gene, a known suppressor of the NetAB embryonic CNS phenotype, did not improve the behavioral defects. The ability to fly was rescued by inhibition of neuronal cell death and pan-neural NetA expression. Based on our results we hypothesize that the adult fertility defects of NetAB mutants are due to ovulation defects in females and a failure to properly transfer sperm proteins in males, and are likely to involve multiple neural circuits.

Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles.

Gemcitabine is one of the most widely used drugs for the treatment of advanced Non-small cell lung cancer (NSCLC), but modest objective response rate of patients to gemcitabine makes it necessary to identify novel biomarkers for patients who can benefit from gemcitabine-based therapy and to improve the effect of clinical therapy. In this work, 3 NSCLC cell lines displaying different sensitivities to gemcitabine were applied for mRNA and microRNA (miR) expression chips to figure out the biomarkers for gemcitabine sensitivity. Genes whose expression increased dramatically in sensitive cell lines were mainly enriched in cell adhesion (NRP2, CXCR3, CDK5R1, IL32 and CDH2) and secretory granule (SLC11A1, GP5, CD36 and IGF1), while genes with significantly upregulated expression in resistant cell line were mainly clustered in methylation modification (HIST1H2BF, RAB23 and TP53) and oxidoreductase (TP53I3, CYP27B1 and SOD3). The most intriguing is the activation of Wnt/β-catenin signaling in gemcitabine resistant NSCLC cell lines. The miR-155, miR-10a, miR-30a, miR-24-2* and miR-30c-2* were upregulated in sensitive cell lines, while expression of miR-200c, miR-203, miR-885-5p, miR-195 and miR-25* was increased in resistant cell line. Genes with significantly altered expression and putatively mediated by the expression-changed miRs were mainly enriched in chromatin assembly (MAF, HLF, BCL2, and IGSF3), anti-apoptosis (BCL2, IGF1 and IKBKB), protein kinase (NRP2, PAK7 and CDK5R1) (all the above genes were upregulated in sensitive cells) and small GTPase mediated signal transduction (GNA13, RAP2A, ARHGAP5 and RAB23, down-regulated in sensitive cells). Our results might provide potential biomarkers for gemcitabine sensitivity prediction and putative targets to overcome gemcitabine resistance in NSCLC patients.

<h4>Background</h4>The relevance of recurrent molecular abnormalities in cytogenetically normal (CN) acute myeloid leukemia (AML) was recently acknowledged by the inclusion of molecular markers such as NPM1, FLT3, and CEBPA as a complement to cytogenetic information within both the World Health Organization and the European Leukemia Net classifications. Mitochondrial metabolism is different in cancer and normal cells. A novel cytosolic type 2-hydroxybutyrate dehydrogenase, BDH2, originally named DHRS6, plays a physiological role in the cytosolic utilization of ketone bodies, which can subsequently enter mitochondria and the tricarboxylic acid cycle. Moreover, BDH2 catalyzes the production of 2, 3-DHBA during enterobactin biosynthesis and participates in 24p3 (LCN2)-mediated iron transport and apoptosis.<h4>Results</h4>We observed that BDH2 expression is an independent poor prognostic factor for CN-AML, with an anti-apoptotic role. Patients with high BDH2 expression have relatively shorter overall survival (P = 0.007) and a low complete response rate (P = 0.032). BDH2-knockdown (BDH2-KD) in THP1 and HL60 cells increased the apoptosis rate under reactive oxygen species stimulation. Decrease inducible survivin, a member of the inhibitors of apoptosis family, but not members of the Bcl-2 family, induced apoptosis via a caspase-3-independent pathway upon BDH2-KD.<h4>Conclusions</h4>BDH2 is a novel independent poor prognostic marker for CN-AML, with the role of anti-apoptosis, through surviving.

<h4>Background</h4>In patients with chronic hepatitis C (CHC), obesity is involved in the pathogenesis of insulin resistance, fatty liver disease and progression of fibrosis. The objective of this study was to compare a normoglucidic low-calorie diet (NGLCD) with a low-fat diet (LFD) among participants with CHC. Aimed to measure the impact of dietary changes in reduction of insulin resistance, obesity but also in steatosis and fibrosis.<h4>Methods</h4>Randomized, controlled trial in three medical centers with assessments at baseline, 6 months and 12 months. Participants were patients over 35 years with chronic hepatitis C (n = 120) with BMI over 25 kg/m². We evaluated the effects of NGLCD vs. LFD in weight management and metabolic improvement. The primary endpoint was to measure the impact of dietary changes through nutritional intervention in reversibility of insulin resistance, obesity, steatosis, and fibrosis. We performed anthropometric measurements, fasting glucose profile, serum lipids, liver profile, blood count at baseline, 6 and 12 months. Steatosis was evaluated using ultrasonographic criteria. Liver fibrosis was non-invasively assessed.<h4>Results</h4>After 6 and 12 months of intervention, both groups had a significant decrease in caloric consumption. At 6 months, weight loss was greater in the NGLCD group (-5.02 ± 3.43 kg vs. -4.1 ± 2.6 kg; p = 0.002) compared to the LFD group. At 1-year, however, weight loss was similar in both groups (-3.9 ± 3.3 kg vs. -3.1 ± 2.6 kg; p = 0.139). At 12 months, fasting plasma glucose, fasting plasma insulin, and HOMA-IR had significant improvements in both groups. With both diets aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT) decreased with significant differences; also there were significant improvements in AST/ALT ratio, Forns fibrosis index. The two diets were associated with reduction of both the prevalence and the severity of steatosis (all p < 0.001). At 12 months, total cholesterol, HDL-cholesterol, triglycerides improved in both groups (all p < 0.05).<h4>Conclusions</h4>The present study establishes the benefits of low-calorie diet and low-fat diet in management of patients with hepatitis C regarding improvement of insulin resistance, steatosis and also fibrosis.Overweight or obese patients with CHC undergoing a lifestyle intervention (specific dietary intervention and physical activity) for 1-year had significant improvements in body weight, lipid and hepatic profile.<h4>Trial registration</h4>PNCI2-3343/41008/2007.

Accurate measurements of the abundances, synthesis rates and degradation rates of cellular proteins are critical for understanding how cells and organisms respond to changes in their environments. Over the past two decades, there has been increasing interest in the use of mass spectrometry for proteomic analysis. In many systems, however, protein diversity as well as cell and tissue heterogeneity limit the usefulness of mass spectrometry-based proteomics. As a result, researchers have had difficulty in systematically identifying proteins expressed within specified time intervals, or low abundance proteins expressed in specific tissues or in a few cells in complex microbial systems. In this review, we present recently-developed tools and strategies that probe these two subsets of the proteome: proteins synthesized during well-defined time intervals--temporally resolved proteomics--and proteins expressed in predetermined cell types, cells or cellular compartments--spatially resolved proteomics--with a focus on chemical and biological mass spectrometry-based methodologies.

<h4>Background</h4>Ensiling is a well-known method for preserving green biomasses through anaerobic production of organic acids by lactic acid bacteria. In this study, wheat straw is subjected to ensiling in combination with hydrothermal treatment as a combined pretreatment method, taking advantage of the produced organic acids.<h4>Results</h4>Ensiling for 4 weeks was accomplished in a vacuum bag system after addition of an inoculum of Lactobacillus buchneri and 7% w/w xylose to wheat straw biomass at 35% final dry matter. Both glucan and xylan were preserved, and the DM loss after ensiling was less than 0.5%. When comparing hydrothermally treated wheat straw (170, 180 and 190°C) with hydrothermally treated ensiled wheat straw (same temperatures), several positive effects of ensiling were revealed. Glucan was up-concentrated in the solid fraction and the solubilisation of hemicellulose was significantly increased. Subsequent enzymatic hydrolysis of the solid fractions showed that ensiling significantly improved the effect of pretreatment, especially at the lower temperatures of 170 and 180°C. The overall glucose yields after pretreatments of ensiled wheat straw were higher than for non-ensiled wheat straw hydrothermally treated at 190°C, namely 74-81% of the theoretical maximum glucose in the raw material, which was ~1.8 times better than the corresponding yields for the non-ensiled straw pretreated at 170 or 180°C. The highest overall conversion of combined glucose and xylose was achieved for ensiled wheat straw hydrothermally treated at 180°C, with overall glucose yield of 78% and overall conversion yield of xylose of 87%.<h4>Conclusions</h4>Ensiling of wheat straw is shown to be an effective pre-step to hydrothermal treatment, and can give rise to a welcomed decrease of process temperature in hydrothermal treatments, thereby potentially having a positive effect on large scale pretreatment costs.

No abstract available.

Most adults affected with hereditary hemochromatosis are homozygous for a single point mutation of HFE (p.Cys282Tyr). Apart from the compound heterozygous state for the p.Cys282Tyr mutant and the widespread p.His63Asp variant allele, other rare HFE mutations can be found in trans and may have clinical impact. In the present report we describe the structural and functional consequences of a new mutation, namely the p.Arg226Gly which was inherited in trans with the p.Cys282Tyr allele in a patient affected with a mild iron overload. Because the R226G substitution is located in the vicinity of the normal Cys225S-S282Cys disulfide bond we initially investigated the structure of the variant by molecular dynamics techniques in order to estimate the effect of the mutation on the global structure of HFE domain α3. We found that the solvation free energy, hydrophobicity and formation of salt bridges are slightly modified with the global secondary structure of the α3 domain being conserved. In a previous paper, we demonstrated that the Q283P substitution leads to the loss of the normal Cys225S-S282Cys disulfide bridge. Similar to the Q283P substitution, the R226G substitution does not substitute a residue directly involved in the formation of the disulfide bridge. However, unlike the p.Gln283Pro variant which destroyed the normal disulfide bridge, the R226G mutation does not affect the normal Cys225S-S282Cys bridge. Furthermore based on cell line studies we clearly show that the mutation does not prevent cell surface localization, β2-microglobulin association and binding to transferrin receptor 1. This new compound heterozygous phenotype is very close to those of the C282Y/H63D compound heterozygous patients who display the biochemical hemochromatosis phenotype but with lower body iron stores than C282Y homozygotes. Our results do not exclude unknown genetic and/or metabolic factors that may act synergistically to increase the ferritin level.

Recurrent pregnancy loss (RPL) is a complex, multifactorial condition. Inherited thrombophilia is the leading cause of thromboembolism and is associated with an increased risk of RPL. The aims of the current study were to investigate the effects of polymorphisms in hemostasis-related genes antithrombin (SERPINC1), thrombomodulin (THBD), tissue factor pathway inhibitor (TFPI), factor V, factor II and annexin A5 (ANXA5), involved in reproductive failure in 94 RPL cases with two or more consecutive pregnancy losses prior to 20weeks of pregnancy and 169 healthy controls who had at least one term delivery and no history of pregnancy loss. The genotypes of SERPINC1 G786A, THBD C1418T, TFPI T-33C, factor V G1628A, factor II A19911G and ANXA5 G76A were assayed by the Sequenom MassARRAY system. Genotype and allele frequencies for SERPINC1 (rs2227589), TFPI (rs8176592), factor V (rs6020), factor II (rs3136516) and ANXA5 (rs113588187) in cases and controls were similar. The distribution of THBD C1418T allele showed significant differences between RPL cases and healthy controls (odds ratio (OR): 1.58, 95%, confidence interval (CI): 1.05-2.39, P=0.027). In univariate logistic regression analyses, carriers of THBD 1418T allele (CT+TT) had an increased risk of RPL (OR: 1.83, 95%, CI: 1.10-3.06, P=0.020). This indicated that THBD 1418T allele was associated with increasing the risk of RPL.

<h4>Background</h4>Chronic exposure to arsenic in drinking water is associated with increased risk of type 2 diabetes mellitus (T2DM) but the underlying molecular mechanism remains unclear.<h4>Objectives</h4>This study evaluated the interaction between single nucleotide polymorphisms (SNPs) in genes associated with diabetes and arsenic exposure in drinking water on the risk of developing T2DM.<h4>Methods</h4>In 2009-2011, we conducted a follow up study of 957 Bangladeshi adults who participated in a case-control study of arsenic-induced skin lesions in 2001-2003. Logistic regression models were used to evaluate the association between 38 SNPs in 18 genes and risk of T2DM measured at follow up. T2DM was defined as having a blood hemoglobin A1C level greater than or equal to 6.5% at follow-up. Arsenic exposure was characterized by drinking water samples collected from participants' tubewells. False discovery rates were applied in the analysis to control for multiple comparisons.<h4>Results</h4>Median arsenic levels in 2001-2003 were higher among diabetic participants compared with non-diabetic ones (71.6 µg/L vs. 12.5 µg/L, p-value <0.001). Three SNPs in ADAMTS9 were nominally associated with increased risk of T2DM (rs17070905, Odds Ratio (OR)  = 2.30, 95% confidence interval (CI) 1.17-4.50; rs17070967, OR = 2.02, 95%CI 1.00-4.06; rs6766801, OR = 2.33, 95%CI 1.18-4.60), but these associations did not reach the statistical significance after adjusting for multiple comparisons. A significant interaction between arsenic and NOTCH2 (rs699780) was observed which significantly increased the risk of T2DM (p for interaction = 0.003; q-value = 0.021). Further restricted analysis among participants exposed to water arsenic of less than 148 µg/L showed consistent results for interaction between the NOTCH2 variant and arsenic exposure on T2DM (p for interaction  = 0.048; q-value = 0.004).<h4>Conclusions</h4>These findings suggest that genetic variation in NOTCH2 increased susceptibility to T2DM among people exposed to inorganic arsenic. Additionally, genetic variants in ADAMTS9 may increase the risk of T2DM.

This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background.

To date, it is widely recognized that Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) can exert considerable anti-tumor effects regarding many types of cancers. The prolonged use of NSAIDs is highly associated with diverse side effects. Therefore, tailoring down the NSAID application onto individual patients has become a necessary and relevant step towards personalized medicine. This study conducts the systemsbiological approach to construct a molecular model (NSAID model) containing a cyclooxygenase (COX)-pathway and its related signaling pathways. Four cancer hallmarks are integrated into the model to reflect different developmental aspects of tumorigenesis. In addition, a Flux-Comparative-Analysis (FCA) based on Petri net is developed to transfer the dynamic properties (including drug responsiveness) of individual cellular system into the model. The gene expression profiles of different tumor-types with available drug-response information are applied to validate the predictive ability of the NSAID model. Moreover, two therapeutic developmental strategies, synthetic lethality and microRNA (miRNA) biomarker discovery, are investigated based on the COX-pathway. In conclusion, the result of this study demonstrates that the NSAID model involving gene expression, gene regulation, signal transduction, protein interaction and other cellular processes, is able to predict the individual cellular responses for different therapeutic interventions (such as NS-398 and COX-2 specific siRNA inhibition). This strongly indicates that this type of model is able to reflect the physiological, developmental and pathological processes of an individual. The approach of miRNA biomarker discovery is demonstrated for identifying miRNAs with oncogenic and tumor suppressive functions for individual cell lines of breast-, colon- and lung-tumor. The achieved results are in line with different independent studies that investigated miRNA biomarker related to diagnostics of cancer treatments, therefore it might shed light on the development of biomarker discovery at individual level. Particular results of this study might contribute to step further towards personalized medicine with the systemsbiological approach.

Variously substituted 2-hydroxy-N-(arylalkyl)benzamides were prepared and screened for antiproliferative and cytotoxic activity in cancer cell lines in vitro. Five compounds, out of 33 showed single-digit micromolar IC50 values against several human cancer cell lines. One of the most potent compounds N-((R)-1-(4-chlorophenylcarbamoyl)-2-phenylethyl)-5-chloro-2-hydroxybenzamide (6k) reduced proliferation and induced apoptosis in the melanoma cell line G361 in a dose-dependent manner, as shown by decrease in 5-bromo-2'-deoxyuridine incorporation and increase in several apoptotic markers, including subdiploid population increase, activation of caspases and site-specific poly-(ADP-ribose)polymerase (PARP) cleavage.

DNAzymes are synthetic, single-stranded, catalytic nucleic acids that bind and cleave target mRNA in a sequence-specific manner, and have been explored for genotherapeutics. One bottleneck restricting their application is the lack of an efficient delivery system. As an inorganic nanomaterial with potentially wide application, nano-hydroxyapatite particles (nHAP) have attracted increasing attention as new candidates for nonviral vectors. In this study, we developed an nHAP-based delivery system and explored its cellular uptake mechanisms, intracellular localization, and biological effects. Absorption of arginine-modified nanohydroxyapatite particles (Arg-nHAP) and DZ1 (latent membrane protein 1 [LMP1]-targeted) reached nearly 100% efficiency under in vitro conditions. Using specific inhibitors, cellular uptake of the Arg-nHAP/DZ1 complex was shown to be mediated by the energy-dependent endocytosis pathway. Further, effective intracellular delivery and nuclear localization of the complex was confirmed by confocal microscopy. Biologically, the complex successfully downregulated the expression of LMP1 in nasopharyngeal carcinoma cells. In a mouse tumor xenograft model, the complex was shown to be delivered efficiently to tumor tissue, downregulating expression of LMP1 and suppressing tumor growth. These results suggest that Arg-nHAP may be an efficient vector for nucleic acid-based drugs with potential clinical application.

<h4>Background</h4>Androgen-deprivation therapy (ADT) is standard treatment for locally advanced or metastatic prostate cancer (PCa). Many patients develop castration resistance (castration-resistant PCa [CRPC]) after approximately 2-3 yr, with a poor prognosis. The molecular mechanisms underlying CRPC progression are unclear.<h4>Objective</h4>To undertake quantitative tumour transcriptome profiling prior to and following ADT to identify functionally important androgen-regulated pathways or genes that may be reactivated in CRPC.<h4>Design, setting, and participants</h4>RNA sequencing (RNA-seq) was performed on tumour-rich, targeted prostatic biopsies from seven patients with locally advanced or metastatic PCa before and approximately 22 wk after ADT initiation. Differentially regulated genes were identified in treatment pairs and further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on cell lines and immunohistochemistry on a separate CRPC patient cohort. Functional assays were used to determine the effect of pathway modulation on cell phenotypes.<h4>Outcome measurements and statistical analysis</h4>We searched for gene expression changes affecting key cell signalling pathways that may be targeted as proof of principle in a CRPC in vitro cell line model.<h4>Results and limitations</h4>We identified ADT-regulated signalling pathways, including the Wnt/β-catenin signalling pathway, and observed overexpression of β-catenin in a subset of CRPC by immunohistochemistry. We validated 6 of 12 (50%) pathway members by qRT-PCR on LNCaP/LNCaP-AI cell RNAs, of which 4 (67%) demonstrated expression changes consistent with RNA-seq data. We show that the tankyrase inhibitor XAV939 (which promotes β-catenin degradation) reduced androgen-independent LNCaP-AI cell line growth compared with androgen-responsive LNCaP cells via an accumulation of cell proportions in the G0/G1 phase and reduction in the S and G2/M phases. Our biopsy protocol did not account for tumour heterogeneity, and pathway inhibition was limited to pharmacologic approaches.<h4>Conclusions</h4>RNA-seq of paired PCa samples revealed ADT-regulated signalling pathways. Proof-of-principle inhibition of the Wnt/β-catenin signalling pathway specifically delays androgen-independent PCa cell cycle progression and proliferation and warrants further investigation as a potential target for therapy for CRPC.

<h4>Objectives</h4>Genome wide association studies have shown 32 loci to influence BMI in European-American adults but replication in other studies is inconsistent and may be attributed to gene-by-age effects. The aims of this study were to determine if the influence of the summed risk score of these 32 loci (GRS) on BMI differed across age from birth to 40 years, and to determine if additive genetic effects other than those in the GRS differed by age.<h4>Methods</h4>Serial measures of BMI were calculated at 0, 1, 3, 6, 9, 12, 18, and 28 months, and 4, 7, 11, 15, 19, 23, 30, and 40 years for 1,176 (605 females, 571 males) European-American participants in the Fels Longitudinal Study. SOLAR was used for genetic analyses.<h4>Results</h4>GRS was significant (P < 0.05) at ages: 6, 9 months, 4-15 years, and 23-40 years. Remaining additive genetic effects independently influenced BMI (P < 5.3 × 10(-5) , 0.40 < h(2) < 0.76). Some genetic correlations between ages were not significant. Differential GRS effects did not retain significance after multiple comparisons adjustments.<h4>Conclusions</h4>While well-known BMI variants do not appear to have significant differential effects, other additive genes differ over the lifespan.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder involving both upper motor neurons (UMN) and lower motor neurons (LMN). Enormous research has been done in the past few decades in unveiling the genetics of ALS, successfully identifying at least fifteen candidate genes associated with familial and sporadic ALS. Numerous studies attempting to define the pathogenesis of ALS have identified several plausible determinants and molecular pathways leading to motor neuron degeneration, which include oxidative stress, glutamate excitotoxicity, apoptosis, abnormal neurofilament function, protein misfolding and subsequent aggregation, impairment of RNA processing, defects in axonal transport, changes in endosomal trafficking, increased inflammation, and mitochondrial dysfunction. This review is to update the recent discoveries in genetics of ALS, which may provide insight information to help us better understanding of the disease neuropathogenesis.

Thyroid paraganglioma is an extremely rare tumor and frequently mistaken for other thyroid neoplasms. Increased awareness of its potential presentation in thyroid and its characteristic features is essential for avoiding diagnostic and therapeutic pitfalls. We describe here three additional cases of primary thyroid paraganglioma and analyze their clinical findings and pathological characteristics. Patients included two women and one man presenting with asymptomatic thyroid nodules. Radiological examinations were nonspecific and none had been diagnosed correctly before surgery. On intraoperative frozen section consultation they were all misdiagnosed as carcinomas, either primary or metastatic. However, the permanent sections showed features consistent with paraganglioma. Of note, two cases displayed extension into adjacent thyroid tissues, one of which exhibited increased mitotic activity, confluent tumor necrosis and vascular invasion. Immunohistochemically, the neoplastic chief cells expressed chromogranin, synaptophysin, neuron-specific enolase and CD56, whereas the sustentacular cells were highlighted by S100 protein. All three patients were well with normal hormone secretion, without local recurrence or distant metastasis at last follow-up (range 10-47 months). We further reviewed the literature to summarize the characteristics of this distinctive entity. Albeit being very rare, paraganglioma should be included in the differential diagnosis of hypervascular thyroidal neoplasms. Accurate diagnosis relies on the histopathogical findings and adjunctive immunohistochemcial studies. To date, all the reported cases have pursued a benign course. Although atypical features seem to have no association with clinical behavior, long time postoperative surveillance with biochemical screening of hormone secretion, cervical ultrasonography and whole-body CT scan is recommended.

Autophagy, a process of self-digestion of the cytoplasm and organelles through which cellular components are recycled for reuse or energy production, is an evolutionarily conserved response to metabolic stress found in eukaryotes from yeast to mammals. It is noteworthy that autophagy is also associated with various pathophysiologic conditions in which this cellular process plays either a cytoprotective or cytopathic role in response to a variety of stresses such as metabolic, inflammatory, neurodegenerative, and therapeutic stress. It is now generally believed that modulating the activity of autophagy through targeting specific regulatory molecules in the autophagy machinery may impact disease processes, thus autophagy may represent a new pharmacologic target for drug development and therapeutic intervention of various human disorders. Induction or inhibition of autophagy using small molecule compounds has shown promise in the treatment of diseases such as cancer. Depending on context, induction or suppression of autophagy may exert therapeutic effects via promoting either cell survival or death, two major events targeted by therapies for various disorders. A better understanding of the biology of autophagy and the pharmacology of autophagy modulators has the potential for facilitating the development of autophagy-based therapeutic interventions for several human diseases.

Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

In postnatal skin the transcription factor Sox2 is expressed in the dermal papilla (DP) of guard/awl/auchene hair follicles and by mechanosensory Merkel cells in the touch domes of guard hairs. To investigate the consequences of Sox2 ablation in skin we deleted Sox2 in DP cells via Blimp1Cre and in Merkel cells via K14Cre. Loss of Sox2 from the DP did not inhibit hair follicle morphogenesis or establishment of the dermis and hypodermis. However, Sox2 expression in the DP was necessary for postnatal maintenance of awl/auchene hair follicles. Deletion of Sox2 via K14Cre resulted in a decreased number of Merkel cells but had no effect on other epithelial compartments or on the dermis. The reduced number of Merkel cells did not affect the number or patterning of guard hairs, nerve density or the interaction of nerve cells with the touch domes. We conclude that Sox2 is a marker of two distinct lineages in the skin and regulates the number of differentiated cells in the case of the Merkel cell lineage and hair follicle type in the case of the DP.

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals.

<h4>Background</h4>The etiology and pathology of anxiety disorders involve both genetic and environmental influences. Adverse working conditions may contribute to the development of anxiety. The serotonin transporter-linked polymorphic region (5-HTTLPR) has been implicated in stress sensitivity. Therefore, we investigated the potential interplay between 5-HTTLPR and job-related risk factors in the prediction of the occurrence of anxiety.<h4>Methods</h4>We conducted a prospective study using the first two waves of a Swedish population-based cohort. At Wave I, 1585 individuals without anxiety, depression or dysthymia who were active in the labor market during both waves were included. Information on job demands, skill discretion, decision authority and social climate was collected at Wave I. After a three year interval, the presence of anxiety disorders was determined at Wave II. All 1585 participants were genotyped for 5-HTTLPR. Both additive and multiplicative models were considered in examining the potential interaction between 5-HTTLPR and adverse working conditions on the development of anxiety.<h4>Results</h4>Anxiety was associated with high job demands but not with 5-HTTLPR. An interaction was observed between 5-HTTLPR and high job demands among females. Individuals with 5-HTTLPR high expression genotype (LL) developed anxiety disorders more frequently when exposed to high job demands compared to 'LS/SS' carriers.<h4>Limitations</h4>A limited number of participants developed anxiety.<h4>Conclusions</h4>High job demands predict the development of anxiety. The 5-HTT polymorphism has a moderating effect on the relationship between high job demands and anxiety among females.

<h4>Background</h4>Wilson disease is rare, found in 3 of 100,000 people (0.03%). Ceruloplasmin is often ordered to evaluate liver enzyme elevations. Because Wilson disease often presents before middle-age, the American Association for the Study of Liver Disease recommends screening patients between the ages of 3 and 55 years with liver abnormalities of uncertain cause. We evaluate guideline adherence and the clinical and economic impact of current clinical use of ceruloplasmin.<h4>Methods</h4>We reviewed all ceruloplasmin measurements at a clinical laboratory that serves a large primary care network, specialty clinics, and a 600-bed tertiary referral center between January 1, 2003, and December 12, 2011.<h4>Results</h4>Ceruloplasmin was measured 5325 times in 5023 unique patients, resulting in 8 (0.16%) new Wilson disease diagnoses. Ceruloplasmin's positive predictive value was 8.4% (95% confidence interval, 7.7-9.3) and false-positive rate was 98.1% (95% confidence interval, 96.2-99.1). A total of 1109 ceruloplasmin levels (20.8%) were ordered in the 1066 patients aged more than 55 years (none with Wilson disease). A "shotgun" approach to liver disease diagnosis was found: Ceruloplasmin was ordered on the same day as hepatitis B (81.0%), hepatitis C (76.0%), autoimmune hepatitis (75.1%), and hemochromatosis (73.1%). Of 424 positive ceruloplasmin results, 91% were not pursued further.<h4>Conclusions</h4>Guideline adherence restricts ceruloplasmin use to a population with a higher pre-test probability of Wilson disease: patients with chronic liver disease aged 3 to 55 years who have been tested for common causes of liver disease. The majority of the serum ceruloplasmin was measured in patients not indicated by the guidelines, resulting in poor test performance and wasted healthcare resources. Our data on ceruloplasmin use could serve as a touchstone for broader discussions on rational clinical decision making.

Dimethyl sulfoxide (DMSO) is frequently utilized as a solvent in toxicological and pharmaceutical investigations. It is therefore important to establish the cellular and molecular targets of DMSO in order to differentiate its intrinsic effects from those elicited by a compound of interest. We performed a genome-wide functional screen in Saccharomyces cerevisiae to identify deletion mutants exhibiting sensitivity to 1% DMSO, a concentration standard to yeast chemical profiling studies. We report that mutants defective in Golgi/ER transport are sensitive to DMSO, including those lacking components of the conserved oligomeric Golgi (COG) complex. Moreover, strains deleted for members of the SWR1 histone exchange complex are hypersensitive to DMSO, with additional chromatin remodeling mutants displaying a range of growth defects. We also identify DNA repair genes important for DMSO tolerance. Finally, we demonstrate that overexpression of histone H2A.Z, which replaces chromatin-associated histone H2A in a SWR1-catalyzed reaction, confers resistance to DMSO. Many yeast genes described in this study have homologs in more complex organisms, and the data provided is applicable to future investigations into the cellular and molecular mechanisms of DMSO toxicity.

Huntington's disease (HD) is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt). Although both Htt and the HD pathogenic mutation (mHtt) are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC) expansion, differentiation and induction experiments using huntingtin knock-out (KO) and mutant huntingtin knock-in (Q111) mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities.

<h4>Objective</h4>We performed comprehensive proteomic analyses of articular cartilage by using the isobaric tags for relative and absolute quantitation (iTRAQ) method, and searched for candidate biomarkers for osteoarthritis (OA).<h4>Methods</h4>Articular cartilage was collected from patients with OA or femoral neck fracture for the control group. Molecular variations were detected by the iTRAQ method, and quantitative analyses were performed by western blot.<h4>Results</h4>Using the iTRAQ method, we identified 76 proteins with different expression levels in OA patients and the control group. Among these proteins, we selected LECT2 (leukocyte cell-derived chemotaxin-2), BAALC (brain and acute leukemia, cytoplasmic), and PRDX6 (peroxiredoxin-6), which had not been reported as biomarkers for OA.<h4>Conclusions</h4>Use of these proteins in combination with conventional OA biomarkers may better reflect the grade and prognosis of OA.

Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness.

The discovery of corticotropin-releasing factor (CRF) or CRH defining the upper regulatory arm of the hypothalamic-pituitary-adrenal (HPA) axis, along with the identification of the corresponding receptors (CRFRs 1 and 2), represents a milestone in our understanding of central mechanisms regulating body and local homeostasis. We focused on the CRF-led signaling systems in the skin and offer a model for regulation of peripheral homeostasis based on the interaction of CRF and the structurally related urocortins with corresponding receptors and the resulting direct or indirect phenotypic effects that include regulation of epidermal barrier function, skin immune, pigmentary, adnexal, and dermal functions necessary to maintain local and systemic homeostasis. The regulatory modes of action include the classical CRF-led cutaneous equivalent of the central HPA axis, the expression and function of CRF and related peptides, and the stimulation of pro-opiomelanocortin peptides or cytokines. The key regulatory role is assigned to the CRFR-1α receptor, with other isoforms having modulatory effects. CRF can be released from sensory nerves and immune cells in response to emotional and environmental stressors. The expression sequence of peptides includes urocortin/CRF→pro-opiomelanocortin→ACTH, MSH, and β-endorphin. Expression of these peptides and of CRFR-1α is environmentally regulated, and their dysfunction can lead to skin and systemic diseases. Environmentally stressed skin can activate both the central and local HPA axis through either sensory nerves or humoral factors to turn on homeostatic responses counteracting cutaneous and systemic environmental damage. CRF and CRFR-1 may constitute novel targets through the use of specific agonists or antagonists, especially for therapy of skin diseases that worsen with stress, such as atopic dermatitis and psoriasis.

The congenital dyserythropoietic anemias (CDAs) are hereditary disorders characterized by distinct morphologic abnormalities of marrow erythroblasts. The unveiling of the genes mutated in the major CDA subgroups (I-CDAN1 and II-SEC23B) has now been completed with the recent identification of the CDA III gene (KIF23). KIF23 encodes mitotic kinesin-like protein 1, which plays a critical role in cytokinesis, whereas the cellular role of the proteins encoded by CDAN1 and SEC23B is still unknown. CDA variants with mutations in erythroid transcription factor genes (KLF1 and GATA-1) have been recently identified. Molecular diagnosis of CDA is now possible in most patients.

<h4>Objective</h4>Varus thrust visualized during walking is associated with a greater medial knee load and an increased risk of medial knee osteoarthritis (OA) progression. Little is known about how varus thrust presence determined by visual observation relates to quantitative gait kinematic data. We hypothesized that varus thrust presence is associated with greater knee frontal plane dynamic movement during the stance phase of gait.<h4>Methods</h4>Participants had knee OA in at least one knee. Trained examiners assessed participants for varus thrust presence during ambulation. Frontal plane knee motion during ambulation was captured using external passive reflective markers and an 8-camera motion analysis system. To examine the cross-sectional relationship between varus thrust and frontal plane knee motion, we used multivariable regression models with the quantitative motion measures as dependent variables and varus thrust (present/absent) as predictor; models were adjusted for age, gender, body mass index (BMI), gait speed, and knee static alignment.<h4>Results</h4>236 persons [mean BMI: 28.5 kg/m(2) (standard deviation (SD) 5.5), mean age: 64.9 years (SD 10.4), 75.8% women] contributing 440 knees comprised the study sample. 82 knees (18.6%) had definite varus thrust. Knees with varus thrust had greater peak varus angle and greater peak varus angular velocity during stance than knees without varus thrust (mean differences 0.90° and 6.65°/s, respectively). These patterns remained significant after adjusting for age, gender, BMI, gait speed, and knee static alignment.<h4>Conclusion</h4>Visualized varus thrust during walking was associated with a greater peak knee varus angular velocity and a greater peak knee varus angle during stance phase of gait.

An important challenge in drug discovery and disease prognosis is to predict genes that are preferentially expressed in one or a few tissues, i.e. showing a considerably higher expression in one tissue(s) compared to the others. Although several data sources and methods have been published explicitly for this purpose, they often disagree and it is not evident how to retrieve these genes and how to distinguish true biological findings from those that are due to choice-of-method and/or experimental settings. In this work we have developed a computational approach that combines results from multiple methods and datasets with the aim to eliminate method/study-specific biases and to improve the predictability of preferentially expressed human genes. A rule-based score is used to merge and assign support to the results. Five sets of genes with known tissue specificity were used for parameter pruning and cross-validation. In total we identify 3434 tissue-specific genes. We compare the genes of highest scores with the public databases: PaGenBase (microarray), TiGER (EST) and HPA (protein expression data). The results have 85% overlap to PaGenBase, 71% to TiGER and only 28% to HPA. 99% of our predictions have support from at least one of these databases. Our approach also performs better than any of the databases on identifying drug targets and biomarkers with known tissue-specificity.

Hepatitis C virus (HCV) induced liver disease is the leading indication for liver transplantation (LTx). Reinfection and accelerated development of fibrosis is a universal phenomenon following LTx. The molecular events that lead to fibrosis following HCV infection still remains poorly defined. In this study, we determined microRNA (miRNA) and mRNA expression profiles in livers from chronic HCV patients and normals using microarrays. Using Genego software and pathway finder we performed an interactive analysis to identify target genes that are modulated by miRNAs. 22 miRNAs were up regulated (>2 fold) and 35 miRNAs were down regulated (>2fold) compared to controls. Liver from HCV patients demonstrated increased expression of 306 genes (>3 fold) and reduced expression of 133 genes (>3 fold). Combinatorial analysis of the networks modulated by the miRNAs identified regulation of the phospholipase C pathway (miR200c, miR20b, and miR31through cellular proto-oncogene tyrosine-protein kinase Src (cSrc)), response to growth factors and hormones (miR141, miR107 and miR200c through peroxisome proliferator-activated receptor alpha and extracellular-signal-regulated kinases, and regulation of cellular proliferation (miR20b, miR10b, and miR141 through cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 p21). Real time PCR (RT-PCR) validation of the miRNA in HCV infected livers demonstrated a 3.3 ±0.9 fold increase in miR200c. In vitro transfection of fibroblasts with miR200c resulted in a 2.2 fold reduction in expression of tyrosine-protein phosphatase non-receptor type 13 or FAS associated phosphatase 1 (FAP-1) and 2.3 fold increase in expression of cSrc. miR200c transfection resulted in significant increases in expression of collagen and fibroblast growth factor (2.8 and 3.4 fold, p<0.05). Therefore, we propose that HCV induced increased expression of miR200c can down modulate the expression of FAP1, a critical regulator of Src and MAP kinase pathway that play an important role in the production of fibrogenic growth factors and development of fibrosis.

<h4>Background</h4>Cancer is a broad group of genetic diseases which account for millions of deaths worldwide each year. Cancers are classified by various clinical, pathological and molecular methods, but even within a well-characterized disease, there is a significant inter-patient variability in survival, response to treatment, and other parameters. Especially in molecular level, tumours of the same category can appear significantly dissimilar due to complex combinations of genetic aberrations leading to a similar malignancy. We extended the current classification methods by studying tumour heterogeneity at pathway level.<h4>Methods</h4>We computed the rate of alterations in 1994 pathways and 2210 tumours consisting of eight different cancers. Using gene set enrichment analysis, each sample was computed a pathway aberration profile that reflected its molecular state. The profiles were analysed together to infer the characteristic aberration rates for each pathway within each cancer. Subgroups of tumours defined by similar pathway aberrations were identified using clustering analyses. The pathway aberration and gene expression profiles of the subgroups were consecutively compared across all eight cancer types to search for similar tumours crossing the standard classification.<h4>Results</h4>We identified pathways and processes that were common to all cancers as well as traits that are unique to a cancer type or closely related cancers. Studying the gene expression patterns within the pathway context suggested potential alteration mechanisms. Clustering analysis revealed five clinically relevant subgroups of tumours in four cancers that exhibited significant differences in survival compared to others. The cross-cancer analysis of the subgroups resulted in the identification of tumours that shared potentially significant alterations.<h4>Conclusions</h4>This study represents the first effort to extend the molecular characterizations towards pathway level descriptions across the family of cancers. In addition to providing a proof-of-concept for single sample pathway aberration analysis in this context, we present a comprehensive pathway aberration dataset that can be used to study pathway aberration patterns within or across cancers. Significant similarities between subgroups of different cancers on pathway and gene expression levels provide interesting hypotheses for understanding variable drug response, or transferring treatments across diseases by identifying common druggable pathways or genes, for example.

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and β-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and β-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, β-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas β-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the β-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and β-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and β-III-tubulin during the neural differentiation of H9 cells.

For this literature review, medical literature data bases were searched for studies on patient compliance after genetic risk assessment. The review focused on conditions where secondary or tertiary preventive options exist, namely cancer syndromes (BRCA-related cancer, HNPCC/colon cancer), hemochromatosis, thrombophilia, smoking cessation, and obesity. As a counterpart, patient compliance was assessed regarding medication adherence and medical advice in some of the most epidemiologically important conditions (including high blood pressure, metabolic syndrome, and coronary heart disease) after receiving medical advice based on nongenetic risk information or a combination of genetic and nongenetic risk information. In the majority of studies based on genetic risk assessments, patients were confronted with predictive rather than diagnostic genetic profiles. Most of the studies started from a knowledge base around 10 years ago when DNA testing was at an early stage, limited in scope and specificity, and costly. The major result is that overall compliance of patients after receiving a high-risk estimate from genetic testing for a given condition is high. However, significant behavior change does not take place just because the analyte is "genetic." Many more factors play a role in the complex process of behavioral tuning. Without adequate counseling and guidance, patients may interpret risk estimates of predictive genetic testing with an increase in fear and anxiety.

<h4>Summary</h4>This paper estimates the risk of bacterial meningitis following surgery between 1996 and 2009 in Denmark. We conducted two retrospective nationwide cohort studies; first by linking notified bacterial meningitis cases to the National Patient Registry to see how many had undergone a surgical procedure; second, we scrutinized notified bacterial meningitis cases to see if the clinician suspected a surgical procedure to be the aetiology. We found that ear, nose and throat surgery had an 11-fold, and neurosurgery a sevenfold, increased risk compared to the reference group in the first 10 days following surgery. Streptococcus pneumoniae was the pathogen most often involved. Operation procedures involving penetration of dura mater was associated with increased risk for post-operative bacterial meningitis. In absolute numbers we found few bacterial meningitis cases after surgery; however, patients undergoing certain surgical procedures are at-risk and should be considered when national vaccination guidelines are revised.

Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG·CAG triplet repeats in the 3' untranslated region of the DMPK1 gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG·CAG triplet-repeat instability is not fully understood. Herein, induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington's disease patient fibroblasts. We isolated 41 iPSC clones from DM1 fibroblasts, all showing different CTG·CAG repeat lengths, thus demonstrating somatic instability within the initial fibroblast population. During propagation of the iPSCs, the repeats expanded in a manner analogous to the expansion seen in somatic cells from DM1 patients. The correlation between repeat length and expansion rate identified the interval between 57 and 126 repeats as being an important length threshold where expansion rates dramatically increased. Moreover, longer repeats showed faster triplet-repeat expansion. However, the overall tendency of triplet repeats to expand ceased on differentiation into differentiated embryoid body or neurospheres. The mismatch repair components MSH2, MSH3 and MSH6 were highly expressed in iPSCs compared with fibroblasts, and only occupied the DMPK1 gene harboring longer CTG·CAG triplet repeats. In addition, shRNA silencing of MSH2 impeded CTG·CAG triplet-repeat expansion. The information gained from these studies provides new insight into a general mechanism of triplet-repeat expansion in iPSCs.

Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.

Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

The non-canonical amino acid labeling techniques BONCAT (bioorthogonal non-canonical amino acid tagging) and FUNCAT (fluorescent non-canonical amino acid tagging) enable the specific identification and visualization of newly synthesized proteins. Recently, these techniques have been applied to neuronal systems to elucidate protein synthesis dynamics during plasticity, identify stimulation-induced proteomes and subproteomes and to investigate local protein synthesis in specific subcellular compartments. The next generation of tools and applications, reviewed here, includes the development of new tags, the quantitative identification of newly synthesized proteins, the application of NCAT to whole animals, and the ability to genetically restrict NCAT labeling. These techniques will enable not only improved detection but also allow new scientific questions to be tackled.

RNA is an important yet vastly underexploited target for small molecule chemical probes or lead therapeutics. Small molecules have been used successfully to modulate the function of the bacterial ribosome, viral RNAs and riboswitches. These RNAs are either highly expressed or can be targeted using substrate mimicry, a mainstay in the design of enzyme inhibitors. However, most cellular RNAs are neither highly expressed nor have a lead small molecule inhibitor, a significant challenge for drug discovery efforts. Herein, I describe the design of small molecules targeting expanded repeating transcripts that cause myotonic muscular dystrophy (DM). These test cases illustrate the challenges of designing small molecules that target RNA and the advantages of targeting repeating transcripts. Lastly, I discuss how small molecules might be more advantageous than oligonucleotides for targeting RNA.

Navigation of retinal projections towards their targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we present in vitro and in vivo evidences that the cannabinoid receptor 2 (CB2R) is expressed along the retino-thalamic pathway and exerts a modulatory action on axon guidance. These effects are specific to CB2R since no changes were observed in mice where the gene coding for this receptor was altered (cnr2 (-/-)). The CB2R induced morphological changes observed at the growth cone are PKA dependent and require the presence of the netrin-1 receptor, Deleted in Colorectal Cancer. Interfering with endogenous CB2R signalling using pharmacological agents increased retinal axon length and induced aberrant projections. Additionally, cnr2 (-/-) mice showed abnormal eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus (dLGN) indicating CB2R's implication in retinothalamic development. Overall, this study demonstrates that the contribution of endocannabinoids to brain development is not solely mediated by CB1R, but also involves CB2R.

13-lined ground squirrels, Ictidomys tridecemlineatus, are obligate hibernators that transition annually between summer homeothermy and winter heterothermy - wherein they exploit episodic torpor bouts. Despite cerebral ischemia during torpor and rapid reperfusion during arousal, hibernator brains resist damage and the animals emerge neurologically intact each spring. We hypothesized that protein changes in the brain underlie winter neuroprotection. To identify candidate proteins, we applied a sensitive 2D gel electrophoresis method to quantify protein differences among forebrain extracts prepared from ground squirrels in two summer, four winter and fall transition states. Proteins that differed among groups were identified using LC-MS/MS. Only 84 protein spots varied significantly among the defined states of hibernation. Protein changes in the forebrain proteome fell largely into two reciprocal patterns with a strong body temperature dependence. The importance of body temperature was tested in animals from the fall; these fall animals use torpor sporadically with body temperatures mirroring ambient temperatures between 4 and 21°C as they navigate the transition between summer homeothermy and winter heterothermy. Unlike cold-torpid fall ground squirrels, warm-torpid individuals strongly resembled the homeotherms, indicating that the changes observed in torpid hibernators are defined by body temperature, not torpor per se. Metabolic enzymes were largely unchanged despite varied metabolic activity across annual and torpor-arousal cycles. Instead, the majority of the observed changes were cytoskeletal proteins and their regulators. While cytoskeletal structural proteins tended to differ seasonally, i.e., between summer homeothermy and winter heterothermy, their regulatory proteins were more strongly affected by body temperature. Changes in the abundance of various isoforms of the microtubule assembly and disassembly regulatory proteins dihydropyrimidinase-related protein and stathmin suggested mechanisms for rapid cytoskeletal reorganization on return to euthermy during torpor-arousal cycles.

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

The inositol-depletion hypothesis proposes that lithium attenuates phosphatidylinositol signaling. Knockout (KO) mice of two genes (IMPA1 or Slc5a3), each encoding for a protein related to inositol metabolism, were studied in comparison with lithium-treated mice. Since we previously demonstrated that these KO mice exhibit a lithium-like neurochemical and behavioral phenotype, here we searched for pathways that may mediate lithium's/the KO effects. We performed a DNA-microarray study searching for pathways affected both by chronic lithium treatment and by the KO of each of the genes. The data were analyzed using three different bioinformatics approaches. We found upregulation of mitochondria-related genes in frontal cortex of lithium-treated, IMPA1 and Slc5a3 KO mice. Three out of seven genes differentially expressed in all three models, Cox5a, Ndufs7, and Ndufab, all members of the mitochondrial electron transfer chain, have previously been associated with bipolar disorder and/or lithium treatment. Upregulation of the expression of these genes was verified by real-time PCR. To further support the link between mitochondrial function and lithium's effect on behavior, we determined the capacity of chronic low-dose rotenone, a mitochondrial respiratory chain complex I inhibitor, to alter lithium-induced behavior as measured by the forced-swim and the amphetamine-induced hyperlocomotion paradigms. Rontenone treatment counteracted lithium's effect on behavior, supporting the proposition suggested by the bioinformatics analysis for a mitochondrial function involvement in behavioral effects of lithium mediated by inositol metabolism alterations.The results provide support for the notion that mitochondrial dysfunction is linked to bipolar disorder and can be ameliorated by lithium. The phenotypic similarities between lithium-treated wild-type mice and the two KO models suggest that lithium may affect behavior by altering inositol metabolism.

This study exploited Blackface lambs that varied in their resistance to the abomasal nematode parasite, Teladorsagia circumcincta. Infection of these lambs over 3 months identified susceptible (high adult worm count, high faecal egg count and low IgA antibody) and resistant animals that had excluded all parasites. Previous work had shown that susceptibility and resistance is dependent on the differential immune response to the parasite, which occurs within the abomasal (gastric) lymph node (ALN) that drains the site of infection. The Affymetrix ovine gene array was used to interrogate the transcriptome of the ALN to identify genes and physiological pathways associated with resistance. We used a bovine RT-qPCR array of 84 genes to validate the gene array, and also report digital gene expression analysis on the same tissues, reanalysed using the Oar v3.1 sheep genome assembly. These analyses identified Humoral Immune Response, Protein Synthesis, Inflammatory Response and Hematological System Development and Function as the two top-ranked networks associated with resistance. Central genes within these networks were IL4, IL5, IL13RA2 and in particular IL13, which confirmed that differential activation of Th2 polarized responses is critical to the resistance phenotype. Furthermore, in resistant sheep there was up-regulation of genes linked to control and suppression of inflammation. The identity of differentially-expressed chemokines and receptors in the resistant and susceptible sheep also begins to explain the cellular nature of the host response to infection. This work will greatly help in the identification of candidate genes as potential selectable markers of genetic resistance.

<h4>Objectives</h4>We explain why traits of interest to behavioral scientists may have a genetic architecture featuring hundreds or thousands of loci with tiny individual effects rather than a few with large effects and why such an architecture makes it difficult to find robust associations between traits and genes.<h4>Methods</h4>We conducted a genome-wide association study at 2 sites, Harvard University and Union College, measuring more than 100 physical and behavioral traits with a sample size typical of candidate gene studies. We evaluated predictions that alleles with large effect sizes would be rare and most traits of interest to social science are likely characterized by a lack of strong directional selection. We also carried out a theoretical analysis of the genetic architecture of traits based on R.A. Fisher's geometric model of natural selection and empirical analyses of the effects of selection bias and phenotype measurement stability on the results of genetic association studies.<h4>Results</h4>Although we replicated several known genetic associations with physical traits, we found only 2 associations with behavioral traits that met the nominal genome-wide significance threshold, indicating that physical and behavioral traits are mainly affected by numerous genes with small effects.<h4>Conclusions</h4>The challenge for social science genomics is the likelihood that genes are connected to behavioral variation by lengthy, nonlinear, interactive causal chains, and unraveling these chains requires allying with personal genomics to take advantage of the potential for large sample sizes as well as continuing with traditional epidemiological studies.

In this essay, we describe a new era of public health research in which prevention science principles are combined with genomic science to produce gene × intervention (G × I) research. We note the roles of behavioral and molecular genetics in risk and protective mechanisms for drug use and psychopathology among children and adolescents, and the results of first-generation genetically informed prevention trials are reviewed. We also consider the need for second-generation research that focuses on G × I effects on mediators or intermediate processes. This research can be used to further understanding of etiological processes, to identify individual differences in children's and adolescents' responses to risk, and to increase the precision of prevention programs. We note the caveats about using genetic data to select intervention participants.

Haematopoietic cell transplantation (HCT) survivors are at increased risk for developing congestive heart failure (CHF), primarily due to pre-HCT exposure to anthracyclines. We examined the association between the development of CHF after HCT and polymorphisms in 16 candidate genes involved in anthracycline metabolism, iron homeostasis, anti-oxidant defence, and myocardial remodelling. A nested case-control study design was used. Cases (post-HCT CHF) were identified from 2950 patients who underwent HCT between 1988 and 2007 at City of Hope and had survived ≥1 year. This cohort formed the sampling frame for selecting controls (without CHF) matched on: age, race/ethnicity, cumulative anthracycline exposure, stem cell source (allogeneic, autologous), and length of follow-up. Seventy-seven cases with pre-HCT germline DNA and 178 controls were genotyped. Multivariate analysis revealed that the odds of CHF was higher in females [Odds Ratio (OR) = 2·9, P < 0·01], individuals with pre-HCT chest radiation (OR = 4·7, P = 0·05), hypertension (OR = 2·9, P = 0·01), and with variants of genes coding for the NAD(P)H-oxidase subunit RAC2 (rs13058338, 7508T→A; OR = 2·8, P < 0·01), HFE (rs1799945, 63C→G; OR = 2·5, P = 0·05) or the doxorubicin efflux transporter ABCC2 (rs8187710, 1515G→A; OR = 4·3, P < 0·01). A combined (clinical and genetic) CHF predictive model performed better [area under the curve (AUC), 0·79] than the genetic (AUC = 0·67) or the clinical (AUC = 0·69) models alone.

Lupus nephritis is an immune complex GN that develops as a frequent complication of SLE. The pathogenesis of lupus nephritis involves a variety of pathogenic mechanisms. The extrarenal etiology of systemic lupus is based on multiple combinations of genetic variants that compromise those mechanisms normally assuring immune tolerance to nuclear autoantigens. This loss of tolerance becomes clinically detectable by the presence of antinuclear antibodies. In addition, nucleic acids released from netting or apoptotic neutrophils activate innate and adaptive immunity via viral nucleic acid-specific Toll-like receptors. Therefore, many clinical manifestations of systemic lupus resemble those of viral infection. In lupus, endogenous nuclear particles trigger IFN-α signaling just like viral particles during viral infection. As such, dendritic cells, T helper cells, B cells, and plasma cells all contribute to the aberrant polyclonal autoimmunity. The intrarenal etiology of lupus nephritis involves antibody binding to multiple intrarenal autoantigens rather than the deposition of circulating immune complexes. Tertiary lymphoid tissue formation and local antibody production add to intrarenal complement activation as renal immunopathology progresses. Here we provide an update on the pathogenic mechanisms that lead to lupus nephritis and provide the rationale for the latest and novel treatment strategies.

<h4>Introduction</h4>Antithrombin (AT) deficiency is associated with an increasing risk of thrombosis.<h4>Materials and methods</h4>15 unrelated patients with AT deficiency defined by thrombophilic assays were recruited and detailed clinical information about patients, focusing on the personal and family history of thromboembolism (TE), were recorded. Mutation analysis was performed by direct sequencing of an AT gene (SERPINC1) in the patients and their family members.<h4>Results</h4>A total of 15 heterozygous causative mutations, each being identified in one family, were identified. Five mutations (33.3%) were reported here for the first time, including three null mutations (Ser36X, Lys70X and Try307X) and two missense mutations (Phe123Cys and Leu340Phe) probably impairing the structural integrity and stability of protein based on the AT structural analysis. Of the 15 patients, 33.3% (5/15) had additional risk factors and only one patient presented with additional genetic alteration causing an early onset of thrombosis. Fourteen patients (93.9%) suffered from multisite recurrent thrombotic episodes after a first episode of thrombosis. 93.3% of the patients experienced deep vein thrombosis (DVT) and 40.0% presented with mesenteric venous thrombosis (MVT). In addition, both venous and arterial thrombosis was present in two unrelated patients. 51.0% subjects with AT deficiency in the 15 unrelated pedigrees experienced TE events.<h4>Conclusions</h4>Prophylactic anticoagulation may be suggested in AT-deficient patients to avoid the recurrent and multisite thrombosis. The association of primary MVT and AT deficiency is highlighted.

Unimolecular micelles formed by dendritic amphiphilic block copolymers poly(amidoamine)-poly(L-lactide)-b-poly(ethylene glycol) conjugated with anti-CD105 monoclonal antibody (TRC105) and 1,4,7-triazacyclononane-N, N', N-triacetic acid (NOTA, a macrocyclic chelator for (64)Cu) (abbreviated as PAMAM-PLA-b-PEG-TRC105) were synthesized and characterized. Doxorubicin (DOX), a model anti-cancer drug, was loaded into the hydrophobic core of the unimolecular micelles formed by PAMAM and PLA via physical encapsulation. The unimolecular micelles exhibited a uniform size distribution and pH-sensitive drug release behavior. TRC105-conjugated unimolecular micelles showed a CD105-associated cellular uptake in human umbilical vein endothelial cells (HUVEC) compared with non-targeted unimolecular micelles, which was further validated by cellular uptake in CD105-negative MCF-7 cells. In 4T1 murine breast tumor-bearing mice, (64)Cu-labeled targeted micelles exhibited a much higher level of tumor accumulation than (64)Cu-labeled non-targeted micelles, measured by serial non-invasive positron emission tomography (PET) imaging and confirmed by biodistribution studies. These unimolecular micelles formed by dendritic amphiphilic block copolymers that synergistically integrate passive and active tumor-targeting abilities with pH-controlled drug release and PET imaging capabilities provide the basis for future cancer theranostics.

<h4>Objectives</h4>This study sought to determine whether the extent of late gadolinium enhancement (LGE) can provide additive prognostic information in patients with a nonischemic dilated cardiomyopathy (NIDC) with an indication for implantable cardioverter-defibrillator (ICD) therapy for the primary prevention of sudden cardiac death (SCD).<h4>Background</h4>Data suggest that the presence of LGE is a strong discriminator of events in patients with NIDC. Limited data exist on the role of LGE quantification.<h4>Methods</h4>The extent of LGE and clinical follow-up were assessed in 162 patients with NIDC prior to ICD insertion for primary prevention of SCD. LGE extent was quantified using both the standard deviation-based (2-SD) method and the full-width half-maximum (FWHM) method.<h4>Results</h4>We studied 162 patients with NIDC (65% male; mean age: 55 years; left ventricular ejection fraction [LVEF]: 26 ± 8%) and followed up for major adverse cardiac events (MACE), including cardiovascular death and appropriate ICD therapy, for a mean of 29 ± 18 months. Annual MACE rates were substantially higher in patients with LGE (24%) than in those without LGE (2%). By univariate association, the presence and the extent of LGE demonstrated the strongest associations with MACE (LGE presence, hazard ratio [HR]: 14.5 [95% confidence interval (CI): 6.1 to 32.6; p < 0.001]; LGE extent, HR: 1.15 per 1% increase in volume of LGE [95% CI: 1.12 to 1.18; p < 0.0001]). Multivariate analyses showed that LGE extent was the strongest predictor in the best overall model for MACE, and a 7-fold hazard was observed per 10% LGE extent after adjustments for patient age, sex, and LVEF (adjusted HR: 7.61; p < 0.0001). LGE quantitation by 2-SD and FWHM both demonstrated robust prognostic association, with the highest MACE rate observed in patients with LGE involving >6.1% of LV myocardium.<h4>Conclusions</h4>LGE extent may provide further risk stratification in patients with NIDC with a current indication for ICD implantation for the primary prevention of SCD. Strategic guidance on ICD therapy by cardiac magnetic resonance in patients with NIDC warrants further study.

Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate.

Childhood maltreatment (CM) has estimated prevalence among Western societies between 10% and 15%. As CM associates with increased risk of several psychiatric disorders, early age of illness onset, increased comorbidity and negative clinical outcome, it imposes a major public health, social and economic impact. Although the clinical consequences of CM are well characterized, a major challenge remains to understand how negative early-life events can affect brain function over extended periods of time. We review here both animal and human studies indicating that the epigenetic mechanism of DNA methylation is a crucial mediator of early-life experiences, thereby maintaining life-long neurobiological sequelae of CM, and strongly determining psychopathological risk.

<h4>Background</h4>Several studies have identified multiple obesity-associated loci mainly in European populations. However, their contribution to obesity in other ethnicities such as Mexicans is largely unknown. The aim of this study was to examine 26 obesity-associated single-nucleotide polymorphisms (SNP) in a sample of Mexican mestizos.<h4>Methods</h4>9 SNPs in biological candidate genes showing replications (PPARG, ADRB3, ADRB2, LEPR, GNB3, UCP3, ADIPOQ, UCP2, and NR3C1), and 17 SNPs in or near genes associated with obesity in first, second and third wave GWAS (INSIG2, FTO, MC4R, TMEM18, FAIM2/BCDIN3, BDNF, SH2B1, GNPDA2, NEGR1, KCTD15, SEC16B/RASAL2, NPC1, SFRF10/ETV5, MAF, PRL, MTCH2, and PTER) were genotyped in 1,156 unrelated Mexican-Mestizos including 683 cases (441 obese class I/II and 242 obese class III) and 473 normal-weight controls. In a second stage we selected 12 of the SNPs showing nominal associations with obesity, to seek associations with quantitative obesity-related traits in 3 cohorts including 1,218 Mexican Mestizo children, 945 Mexican Mestizo adults, and 543 Indigenous Mexican adults.<h4>Results</h4>After adjusting for age, sex and admixture, significant associations with obesity were found for 6 genes in the case-control study (ADIPOQ, FTO, TMEM18, INSIG2, FAIM2/BCDIN3 and BDNF). In addition, SH2B1 was associated only with class I/II obesity and MC4R only with class III obesity. SNPs located at or near FAIM2/BCDIN3, TMEM18, INSIG2, GNPDA2 and SEC16B/RASAL2 were significantly associated with BMI and/or WC in the combined analysis of Mexican-mestizo children and adults, and FTO locus was significantly associated with increased BMI in Indigenous Mexican populations.<h4>Conclusions</h4>Our findings replicate the association of 8 obesity-related SNPs with obesity risk in Mexican adults, and confirm the role of some of these SNPs in BMI in Mexican adults and children.

Blockade of the serotonin reuptake transporter (5-HTT), using fluoxetine, has been identified as a potential therapeutic target for preventing and, importantly, reversing pulmonary hypertension (PH). This study utilized synchrotron radiation microangiography to determine whether fluoxetine could prevent or reverse endothelial dysfunction and vessel rarefaction, which underpin PH. PH was induced by a single injection of monocrotaline (MCT; 60 mg kg(-1)). Following MCT administration, rats received daily injections of either saline or fluoxetine (MCT+Fluox; 10 mg kg(-1)) for three weeks. A third group of rats also received the fluoxetine regime, but only three weeks after MCT (MCT+FluoxDelay). Control rats received daily injections of saline. Pulmonary microangiography was performed to assess vessel branching density and visualize dynamic changes in vessel diameter following (i) acute fluoxetine or (ii) acetylcholine, sodium nitroprusside, BQ-123 (ET-1A receptor blocker) and L-NAME (NOS inhibitor). Monocrotaline induced PH that was inevitably terminal. `Delayed' treatment of fluoxetine (MCT+FluoxDelay) was unable to reverse the progression of PH. Early fluoxetine treatment pre-PH (i.e. MCT+Fluox) attenuated but did not completely prevent vascular remodeling, vessel rarefaction and an increase in pulmonary pressure, and it did not prevent pulmonary endothelial dysfunction. Interestingly, fluoxetine treatment did counter-intuitively prevent the onset of right ventricular hypertrophy. Using synchrotron radiation microangiography, selective blockade of the serotonin reuptake transporter alone is highlighted as not being sufficient to prevent pulmonary endothelial dysfunction, which is the primary instigator for the inevitable onset of vascular remodeling and vessel rarefaction. Accordingly, potential therapeutic strategies should aim to target multiple pathways to ensure an optimal outcome.

Elevations in hepatic iron content occur with aging and physiological stressors, which may promote oxidative injury to the liver. Since dysregulation of the iron regulatory hormone, hepcidin, can cause iron accumulation, our goal was to characterize the regulation of hepcidin in young (6 mo) and old (24 mo) Fischer 344 rats exposed to environmental heat stress. Liver and blood samples were taken in the control condition and after heating. Hepcidin expression did not differ between young and old rats in the control condition, despite higher levels of hepatic iron and IL-6 mRNA in the latter. Following heat stress, pSTAT3 increased in both groups, but C/EBPα and hepcidin mRNA increased only in old rats. Despite this, serum iron decreased in both age groups 2 h after heat stress, suggesting hepcidin-independent hypoferremia in the young rats. The differential regulation of hepcidin between young and old rats after hyperthermia may be due to the enhanced expression of C/EBPα protein in old rats. These data support the concept of "inflammaging" and suggest that repeated exposures to stressors may contribute to the development of anemia in older individuals.

Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragment-based Huntington's disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3-trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171-82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD.

No abstract available.

Meta-analysis is a commonly used approach to increase the sample size for genome-wide association searches when individual studies are otherwise underpowered. Here, we present a meta-analysis procedure to estimate the heterogeneity of the quantitative trait variance attributable to genetic variants using Levene's test without needing to exchange individual-level data. The meta-analysis of Levene's test offers the opportunity to combine the considerable sample size of a genome-wide meta-analysis to identify the genetic basis of phenotypic variability and to prioritize single-nucleotide polymorphisms (SNPs) for gene-gene and gene-environment interactions. The use of Levene's test has several advantages, including robustness to departure from the normality assumption, freedom from the influence of the main effects of SNPs, and no assumption of an additive genetic model. We conducted a meta-analysis of the log-transformed body mass index of 5892 individuals and identified a variant with a highly suggestive Levene's test P-value of 4.28E-06 near the NEGR1 locus known to be associated with extreme obesity.

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.

The human endogenous retrovirus family HERV-K(HML-2) Rec protein is an RNA transport factor that enhances nuclear export of intron-containing retroviral transcripts. Using the yeast two-hybrid approach, we have newly identified human Staufen-1 as a Rec-interacting protein. The interaction was confirmed by coimmunoprecipitation experiments, and the relevant site in Staufen-1 has been mapped to double-stranded RNA binding domain 4 (RBD4). Staufen-1 is in several aspects functionally related to retroviral RNA transport proteins. It binds mRNAs and targets its ribonuclear cargo to polysomes for efficient translation. We observed an accumulation of Staufen-1 in the nucleus of Rec-expressing cells and colocalization in the nucleoli as well as in the cytoplasm. Overexpression of Staufen-1 resulted in a 5-fold enhancement in nuclear export and/or translation of unspliced HERV-K(HML-2) viral RNAs in the presence of Rec and its Rec-responsive element (RcRE) binding site together with a clear increase in virus production. Staufen-1 was previously shown to interact with the Gag protein of HIV-1, promoting Gag oligomerization and RNA encapsidation. We demonstrate here that Staufen-1 also binds to the Gag protein of HERV-K(HML-2). Under stress conditions, Rec colocalizes with Staufen-1 in stress granules in cells that express viral RNA but not in mRNA-decay-related processing bodies. Our results suggest a new role for Staufen-1 as a cellular Rec and HERV-K(HML-2) Gag cofactor.

The Alzheimer's Disease Neuroimaging Initiative (ADNI) is an ongoing, longitudinal, multicenter study designed to develop clinical, imaging, genetic, and biochemical biomarkers for the early detection and tracking of Alzheimer's disease (AD). The study aimed to enroll 400 subjects with early mild cognitive impairment (MCI), 200 subjects with early AD, and 200 normal control subjects; $67 million funding was provided by both the public and private sectors, including the National Institute on Aging, 13 pharmaceutical companies, and 2 foundations that provided support through the Foundation for the National Institutes of Health. This article reviews all papers published since the inception of the initiative and summarizes the results as of February 2011. The major accomplishments of ADNI have been as follows: (1) the development of standardized methods for clinical tests, magnetic resonance imaging (MRI), positron emission tomography (PET), and cerebrospinal fluid (CSF) biomarkers in a multicenter setting; (2) elucidation of the patterns and rates of change of imaging and CSF biomarker measurements in control subjects, MCI patients, and AD patients. CSF biomarkers are consistent with disease trajectories predicted by β-amyloid cascade (Hardy, J Alzheimers Dis 2006;9(Suppl 3):151-3) and tau-mediated neurodegeneration hypotheses for AD, whereas brain atrophy and hypometabolism levels show predicted patterns but exhibit differing rates of change depending on region and disease severity; (3) the assessment of alternative methods of diagnostic categorization. Currently, the best classifiers combine optimum features from multiple modalities, including MRI, [(18)F]-fluorodeoxyglucose-PET, CSF biomarkers, and clinical tests; (4) the development of methods for the early detection of AD. CSF biomarkers, β-amyloid 42 and tau, as well as amyloid PET may reflect the earliest steps in AD pathology in mildly symptomatic or even nonsymptomatic subjects, and are leading candidates for the detection of AD in its preclinical stages; (5) the improvement of clinical trial efficiency through the identification of subjects most likely to undergo imminent future clinical decline and the use of more sensitive outcome measures to reduce sample sizes. Baseline cognitive and/or MRI measures generally predicted future decline better than other modalities, whereas MRI measures of change were shown to be the most efficient outcome measures; (6) the confirmation of the AD risk loci CLU, CR1, and PICALM and the identification of novel candidate risk loci; (7) worldwide impact through the establishment of ADNI-like programs in Europe, Asia, and Australia; (8) understanding the biology and pathobiology of normal aging, MCI, and AD through integration of ADNI biomarker data with clinical data from ADNI to stimulate research that will resolve controversies about competing hypotheses on the etiopathogenesis of AD, thereby advancing efforts to find disease-modifying drugs for AD; and (9) the establishment of infrastructure to allow sharing of all raw and processed data without embargo to interested scientific investigators throughout the world. The ADNI study was extended by a 2-year Grand Opportunities grant in 2009 and a renewal of ADNI (ADNI-2) in October 2010 through to 2016, with enrollment of an additional 550 participants.

Erythropoiesis is the process by which progenitors for red blood cells are produced and terminally differentiate. In all vertebrates, two morphologically distinct erythroid lineages (primitive, embryonic, and definitive, fetal/adult) form successively within the yolk sac, fetal liver, and marrow and are essential for normal development. Red blood cells have evolved highly specialized functions in oxygen transport, defense against oxidation, and vascular remodeling. Here we review key features of the ontogeny of red blood cell development in mammals, highlight similarities and differences revealed by genetic and gene expression profiling studies, and discuss methods for identifying erythroid cells at different stages of development and differentiation.

In previous work, we developed novel antibacterial hybrid coatings based on dextran containing dispersed Ag NPs (~5 nm, DEX-Ag) aimed to offer dual protection against two of the most common complications associated with implant surgery, infections and rejection of the implant. However, their blood-material interactions are unknown. In this study, we assess the hemocompatibility and biocompatibility of DEX-Ag using fresh blood and two cell lines of the immune system, monocytes (THP-1 cells) and macrophages (PMA-stimulated THP-1 cells). Glass, polyurethane (PU) and bare dextran (DEX) were used as reference surfaces. PU, DEX and DEX-Ag exhibited non-hemolytic properties. Relative to glass (100%), platelet attachment on PU, DEX and DEX-Ag was 15%, 10% and 34%, respectively. Further, we assessed cell morphology and viability, pro-inflammatory cytokines expression (TNF-α and IL-1β), pro-inflammatory eicosanoid expression (Prostaglandin E2, PGE2) and release of reactive oxygen species (ROS, superoxide and H2O2) following incubation of the cells with the surfaces. The morphology and cell viability of THP-1 cells were not affected by DEX-Ag whereas DEX-Ag minimized spreading of PMA-stimulated THP-1 cells and caused a reduction in cell viability (16% relative to other surfaces). Although DEX-Ag slightly enhanced release of ROS, the expression of pro-inflammatory cytokines remained minimal with similar levels of PGE2, as compared to the other surfaces studied. These results highlight low toxicity of DEX-Ag and hold promise for future applications in vivo.

The emergence of functional neuronal connectivity in the developing cerebral cortex depends on neuronal migration. This process enables appropriate positioning of neurons and the emergence of neuronal identity so that the correct patterns of functional synaptic connectivity between the right types and numbers of neurons can emerge. Delineating the complexities of neuronal migration is critical to our understanding of normal cerebral cortical formation and neurodevelopmental disorders resulting from neuronal migration defects. For the most part, the integrated cell biological basis of the complex behavior of oriented neuronal migration within the developing mammalian cerebral cortex remains an enigma. This review aims to analyze the integrative mechanisms that enable neurons to sense environmental guidance cues and translate them into oriented patterns of migration toward defined areas of the cerebral cortex. We discuss how signals emanating from different domains of neurons get integrated to control distinct aspects of migratory behavior and how different types of cortical neurons coordinate their migratory activities within the developing cerebral cortex to produce functionally critical laminar organization.

The prevalence of severe obesity, defined as body mass index (BMI) ≥ 35.0 kg/m(2), is rising rapidly. Given the disproportionately high health burden and healthcare costs associated with this condition, understanding the underlying aetiology, including predisposing genetic factors, is a biomedical research priority. Previous studies have suggested that severe obesity represents an extreme tail of the population BMI variation, reflecting shared genetic factors operating across the spectrum. Here, we sought to determine whether a panel of 32 known common obesity-susceptibility variants contribute to severe obesity in patients (n = 1,003, mean BMI 48.4 ± 8.1 kg/m(2)) attending bariatric surgery clinics in two European centres. We examined the effects of these 32 common variants on obesity risk and BMI, both as individual markers and in combination as a genetic risk score, in a comparison with normal-weight controls (n = 1,809, BMI 18.0-24.9 kg/m(2)); an approach which, to our knowledge, has not been previously undertaken in the setting of a bariatric clinic. We found strong associations with severe obesity for SNP rs9939609 within the FTO gene (P = 9.3 × 10(-8)) and SNP rs2815752 near the NEGR1 gene (P = 3.6 × 10(-4)), and directionally consistent nominal associations (P<0.05) for 12 other SNPs. The genetic risk score associated with severe obesity (P = 8.3 × 10(-11)) but, within the bariatric cohort, this score did not associate with BMI itself (P = 0.264). Our results show significant effects of individual BMI-associated common variants within a relatively small sample size of bariatric patients. Furthermore, the burden of such low-penetrant risk alleles contributes to severe obesity in this population. Our findings support that severe obesity observed in bariatric patients represents an extreme tail of the population BMI variation. Moreover, future genetic studies focused on bariatric patients may provide valuable insights into the pathogenesis of obesity at a population level.

Structured gene annotations are a foundation upon which many bioinformatics and statistical analyses are built. However the structured annotations available in public databases are a sparse representation of biological knowledge as a whole. The rate of biomedical data generation is such that centralized biocuration efforts struggle to keep up. New models for gene annotation need to be explored that expand the pace at which we are able to structure biomedical knowledge. Recently, online games have emerged as an effective way to recruit, engage and organize large numbers of volunteers to help address difficult biological challenges. For example, games have been successfully developed for protein folding (Foldit), multiple sequence alignment (Phylo) and RNA structure design (EteRNA). Here we present Dizeez, a simple online game built with the purpose of structuring knowledge of gene-disease associations. Preliminary results from game play online and at scientific conferences suggest that Dizeez is producing valid gene-disease annotations not yet present in any public database. These early results provide a basic proof of principle that online games can be successfully applied to the challenge of gene annotation. Dizeez is available at http://genegames.org.

Research conducted during the past 30 years has increased our understanding of the mechanisms involved in colorectal cancer initiation and development. The findings have demonstrated the existence of at least three pathways: chromosomal instability, microsatellite instability and CpG island methylator phenotype. Importantly, new studies have shown that inflammation and microRNAs contribute to colorectal carcinogenesis. Recent data have demonstrated that several genetic and epigenetic changes are important in determining patient prognosis and survival. Furthermore, some of these mechanisms are related to patients' response to drugs, such as aspirin, which could be used for both chemoprevention and treatment in specific settings. Thus, in the near future, we could be able to predict disease behavior based on molecular markers found on tumors, and direct the best treatment options for patients.

Epidemiological and clinical studies have shown that children exposed to adverse experiences are at increased risk for the development of depression, anxiety disorders, and posttraumatic stress disorder (PTSD). A history of child abuse and maltreatment increases the likelihood of being subsequently exposed to traumatic events or of developing PTSD as an adult. The brain is highly plastic during early life and encodes acquired information into lasting memories that normally subserve adaptation. Translational studies in rodents showed that enduring sensitization of neuronal and neuroendocrine circuits in response to early life adversity are likely risk factors of life time vulnerability to stress. Hereby, the hypothalamic-pituitary-adrenal (HPA) axis integrates cognitive, behavioral, and emotional responses to early-life stress and can be epigenetically programed during sensitive windows of development. Epigenetic mechanisms, comprising reciprocal regulation of chromatin structure and DNA methylation, are important to establish and maintain sustained, yet potentially reversible, changes in gene transcription. The relevance of these findings for the development of PTSD requires further studies in humans where experience-dependent epigenetic programing can additionally depend on genetic variation in the underlying substrates which may protect from or advance disease development. Overall, identification of early-life stress-associated epigenetic risk markers informing on previous stress history can help to advance early diagnosis, personalized prevention, and timely therapeutic interventions, thus reducing long-term social and health costs.

Excipients having self-assembling properties are less explored in the field of dry powder inhalation (DPI) technology. An amphiphilic lipopolymer system was developed using stearic acid (SA) and branched polyethyleneimine (BPEI) (1800 Dalton), at different proportions by covalent conjugation. A molecular dynamic (MD) simulation tool was employed for predicting the carrier behavior in a polar in vivo condition. The structural characterization was carried out using nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared (FTIR) spectroscopy. The physical nature of the lipopolymer was analyzed by differential scanning calorimetry. Determination of zeta potential and diameter of the micelles showed existence of cationic particles in the nano size range when a lower number of primary amino groups of BPEI was grafted with SA. The rifampicin (RIF)-loaded lipopolymer was also formulated further into spray-dried microparticles. Powder X-ray diffraction (PXRD) studies revealed that the RIF API (active pharmaceutical ingredient) exists as molecular dispersion in spray-dried microparticles. Topological analysis of the spray-dried nanomicelle was carried out using scanning electron microscopy (SEM). A large population of the drug-carrying particles were found to be under the inhalable size range (fine particle fraction 67.88% ± 3%). In vitro drug release kinetics from spray-dried nanomicelles were carried out at lung fluid pH.

<h4>Purpose</h4>To investigate differences in the biological features of the most immunoexpressed prostate cancer (PC) profiles (PSA+, PSMA+) according to the RKIP.<h4>Methods</h4>19 PC with dominant Gleason grade ≥ 8 were studied. Expression of PSA, PSMA, RKIP, Raf-1, MEK-1, ERK-1, ERK-2, p-Akt (T308), p-Akt (S473), NF- κ B p50, and NF- κ Bp65 were detected immunohistochemically.<h4>Results</h4>. Loss of RKIP in the most immunoexpressed PC (PSA+, PSMA+) profile was associated with increased levels of PSA and PSMA expression. Intensities of immunoreactions to PSA and PSMA were higher in cancer cells negative for RKIP (12.51 ± 1.6 and 34.95 ± 1.92) compared to those positive for RKIP (4.68 ± 1.11 and 28.56 ± 0.91). In parallel, missing RKIP expression in PC patients with PSA+, PSMA+ profile was connected with increased components of both Raf-1/MEK/ERK and NF- κ B (p65/p50), whereas Akt is activated independently of RKIP.<h4>Conclusions</h4>Although characterized by the same (PSA+, PSMA+) profile, PC phenotype missing the RKIP related to invasive potential and greater biological aggressiveness reflected in overexpression of components of Raf-1/MEK/ERK and NF- κ B (p65/p50) in which Akt is activated independently of RKIP. Taking into account the PC phenotypes according to RKIP among PSA-PSMA profiles may improve distinguishing them from cancers that will become more aggressive and therefore adapt the therapeutic strategies in those patients.

Hepatocellular carcinoma (HCC) is a deadly and emerging disease leading to death in Asian countries. High hepatitis B virus (HBV) load and chronic hepatitis B (CHB) infection increase the risk of developing HCC. HBV is a DNA virus that can integrate DNA into host genome thereby increase the yield of transactivator protein HBxAg that may deregulate many pathways involving in metabolism of cells. Several monogenic and polygenic risk factors are also involved in HCC development. This review summarizes the mechanism involved in HCC development and discusses some promising therapies to make HCC curative.

Bone development depends on environmental, nutritional and hormonal factors. Yet, an ordered and timed activation of genes and their associated molecular pathways are central for the growth and development of healthy bones. The correct expression of genes depends on both cis- and trans-regulatory elements. Of these, the elusive role of chromatin ultrastructure is just beginning to become appreciated. Changes in the higher-order structure of chromatin are affecting the expression of genes in response to intrinsic and environmental signals. Cohesin and condensin are members of the structural maintenance of chromosome (SMC) family of protein complexes, which mediate higher-order chromatin structure by tethering distinct regions of chromatin either inter- or intra-molecularly. In recent years, SMCs had been identified for their function in the regulation of gene expression and developmental processes, whereas malfunction of cohesin or condensin has an impact on human health. However, little is known about the specific roles of SMC complexes in bone development and their possible effect on bone health. Here, we review studies that suggest an intimate link between SMCs and bone development, as well as a plausible effect, direct or indirect, on the bone health. We describe genetic syndromes associated with SMCs with distinctive bone phenotypes and identify links between SMCs and bone-related molecular pathways. Future studies of the relationship between SMCs and bone development will reveal new understandings of both the cellular and molecular roles of SMC complexes and provide new insights into the growth and developmental processes in the bone.

Organocatalyzed enantioselective allylation of 2-iodocyclohexenone followed by methylation and oxy-Cope rearrangement delivered enantiomerically enriched 2-methyl 3-allyl cyclohexanone, which engaged in acid-catalyzed Robinson annulation to give the bicyclic enone. Subsequent elaboration of the pendant allyl group into an α-diazo β-keto ester set the stage for Rh-mediated cyclization to deliver the tricyclic A-B-C core of the Veratrum alkaloids.

<h4>Background</h4>Most eukaryotic species represent stable karyotypes with a particular diploid number. B chromosomes are additional to standard karyotypes and may vary in size, number and morphology even between cells of the same individual. For many years it was generally believed that B chromosomes found in some plant, animal and fungi species lacked active genes. Recently, molecular cytogenetic studies showed the presence of additional copies of protein-coding genes on B chromosomes. However, the transcriptional activity of these genes remained elusive. We studied karyotypes of the Siberian roe deer (Capreolus pygargus) that possess up to 14 B chromosomes to investigate the presence and expression of genes on supernumerary chromosomes.<h4>Results</h4>Here, we describe a 2 Mbp region homologous to cattle chromosome 3 and containing TNNI3K (partial), FPGT, LRRIQ3 and a large gene-sparse segment on B chromosomes of the Siberian roe deer. The presence of the copy of the autosomal region was demonstrated by B-specific cDNA analysis, PCR assisted mapping, cattle bacterial artificial chromosome (BAC) clone localization and quantitative polymerase chain reaction (qPCR). By comparative analysis of B-specific and non-B chromosomal sequences we discovered some B chromosome-specific mutations in protein-coding genes, which further enabled the detection of a FPGT-TNNI3K transcript expressed from duplicated genes located on B chromosomes in roe deer fibroblasts.<h4>Conclusions</h4>Discovery of a large autosomal segment in all B chromosomes of the Siberian roe deer further corroborates the view of an autosomal origin for these elements. Detection of a B-derived transcript in fibroblasts implies that the protein coding sequences located on Bs are not fully inactivated. The origin, evolution and effect on host of B chromosomal genes seem to be similar to autosomal segmental duplications, which reinforces the view that supernumerary chromosomal elements might play an important role in genome evolution.

Axon navigation through the developing body of an embryo is a challenging and exquisitely precise process. Axonal processes within the nervous system harbor extremely complicated internal regulatory mechanisms that enable each of them to respond to environmental cues in a unique way, so that every single neuron has an exact stereotypical localization and axonal projection pattern. Receptors and adhesion molecules expressed on axonal membranes will determine their guidance properties. Axon guidance is thought to be controlled to a large extent through transcription factor codes. These codes would be responsible for the deployment of specific guidance receptors and adhesion molecules on axonal membranes to allow them to reach their targets. Although families of transcriptional regulators as well as families of guidance molecules have been conserved across evolution, their relationships seem to have developed independently. This review focuses on the midline and the neuromuscular system in both vertebrates and Drosophila in which such relationships have been particularly well studied.

Many signal transduction cascades are initiated by transmembrane receptors with the presence or absence and abundance of receptors dictating cellular responsiveness. We provide a validated array of quantitative reverse transcription polymerase chain reaction (qRT-PCR) reagents for high-throughput profiling of the presence and relative abundance of transcripts for 194 transmembrane receptors in the human genome. We found that the qRT-PCR array had greater sensitivity and specificity for the detected receptor transcript profiles compared to conventional oligonucleotide microarrays or exon microarrays. The qRT-PCR array also distinguished functional receptor presence versus absence more accurately than deep sequencing of adenylated RNA species by RNA sequencing (RNA-seq). By applying qRT-PCR-based receptor transcript profiling to 40 human cell lines representing four main tissues (pancreas, skin, breast, and colon), we identified clusters of cell lines with enhanced signaling capabilities and revealed a role for receptor silencing in defining tissue lineage. Ectopic expression of the interleukin-10 (IL-10) receptor-encoding gene IL10RA in melanoma cells engaged an IL-10 autocrine loop not otherwise present in this cell type, which altered signaling, gene expression, and cellular responses to proinflammatory stimuli. Our array provides a rapid, inexpensive, and convenient means for assigning a receptor signature to any human cell or tissue type.

Proper circuit connectivity is critical for nervous system function. Connectivity derives from the interaction of two interdependent modules: synaptic specificity and synaptic assembly. Specificity involves both targeting of neurons to specific laminar regions and the formation of synapses onto defined subcellular areas. In this review, we focus discussion on recently elucidated molecular mechanisms that control synaptic specificity and link them to synapse assembly. We use these molecular pathways to underscore fundamental cell biological concepts that underpin, and help explain, the rules governing synaptic specificity.

The Hedgehog signaling is a determinant pathway for tumor progression. However, while inhibition of the Hedgehog canonical pathway-Patched-Smoothened-Gli-has proved efficient in human tumors with activating mutations in this pathway, recent clinical data have failed to show any benefit in other cancers, even though Sonic Hedgehog (SHH) expression is detected in these cancers. Cell-adhesion molecule-related/down-regulated by Oncogenes (CDON), a positive regulator of skeletal muscle development, was recently identified as a receptor for SHH. We show here that CDON behaves as a SHH dependence receptor: it actively triggers apoptosis in the absence of SHH. The pro-apoptotic activity of unbound CDON requires a proteolytic cleavage in its intracellular domain, allowing the recruitment and activation of caspase-9. We show that by inducing apoptosis in settings of SHH limitation, CDON expression constrains tumor progression, and as such, decreased CDON expression observed in a large fraction of human colorectal cancer is associated in mice with intestinal tumor progression. Reciprocally, we propose that the SHH expression, detected in human cancers and previously considered as a mechanism for activation of the canonical pathway in an autocrine or paracrine manner, actually provides a selective tumor growth advantage by blocking CDON-induced apoptosis. In support of this notion, we present the preclinical demonstration that interference with the SHH-CDON interaction triggers a CDON-dependent apoptosis in vitro and tumor growth inhibition in vivo. The latter observation qualifies CDON as a relevant alternative target for anticancer therapy in SHH-expressing tumors.

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺) and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

Bladder cancer is the fourth most common cancer in men in the United States, and its recurrence rate is highest among all malignancies. The unmet need for improved strategies for early detection, treatment, and monitoring of the progression of this disease continues to translate into high mortality and morbidity. The quest for advanced diagnostic, therapeutic, and prognostic approaches for bladder cancer is a high priority, which can be achieved by understanding the molecular mechanisms of the initiation and progression of this malignancy. Aberrant DNA methylation in single or multiple cancer-related genes/loci has been found in human bladder tumors and cancer cell lines, and urine sediments, and correlated with many clinicopathological features of this disease, including tumor relapse, muscle-invasiveness, and survival. The present review summarizes the published research on aberrant DNA methylation in connection with human bladder cancer. Representative studies are highlighted to set forth the current state of knowledge, gaps in the knowledgebase, and future directions in this prime epigenetic field of research. Identifying the potentially reversible and 'drugable' aberrant DNA methylation events that initiate and promote bladder cancer development can highlight biological markers for early diagnosis, effective therapy and accurate prognosis of this malignancy.

Angiogenesis is an important mechanism of tumor development, growth and metastasis in hepatocellular carcinoma (HCC). The poor prognosis of HCC patients has been associated with a failure to detect recurrences following surgery. In the present study, we investigated the association between the patient characteristics and the expression of angiogenic genes to identify early biomarkers of HCC. A comprehensive angiogenic gene expression profile was obtained by paired TaqMan gene array analysis of primary HCC nodules and adjacent non-HCC liver tissue from 12 patients. A total of 14 genes were found to be differentially expressed in HCC liver nodules (>2-fold change); the genes encoding collagen type XVα1, IVα1 and IVα2 were upregulated and the genes associated with vessel growth, neuropilin 2 (<i>NRP2</i>) and lymphatic vessel endothelial hyaluronan receptor-1 (<i>LYVE-1</i>) were downregulated. The histopathological analysis revealed that the evolution of HCC nodules from well to poorly differentiated was associated with a 5-fold decrease in <i>LYVE-1</i> expression, reaching its lowest level early during the transition. The significance of this gene as a biomarker of postoperative survival was demonstrated by a 2-fold decrease in overall survival (OS) rates in the low expression group compared to the high expression group. The multivariate and univariate Cox regression analyses identified <i>LYVE-1</i> expression as a significant independent prognostic parameter of OS [hazard ratio (HR)=3.067; 95% confidence interval (CI): 1.507-6.273; P=0.0021]. Thus, the results of this study suggested that <i>LYVE-1</i> expression may constitute a novel early biomarker of postoperative survival in HCC patients.

Free radicals are formed as a result of cellular processes and play a key role in predisposition to and development of numerous diseases and of premature aging. Recently, we reported the syntheses of a number of novel phenolic antioxidants for possible application in food industry. In the present study, analyses of the cellular processes and molecular gene expression effects of some of the novel antioxidants in normal human tissues and in cancer cells were undertaken. Results indicated that whereas the examined antioxidants showed no effects on morphology and gene expression of normal human oral and gingival epithelial tissues, they exerted a profound cell killing effect on breast cancer cells, including on chemotherapy-resistant breast cancer cells and on oral squamous carcinoma cells. Among the tested antioxidants, N-decyl-N-(3-methoxy-4-hydroxybenzyl)-3-(3,4-dihydroxyphenyl) propanamide and N-decyl-N-(3,5-dimethoxy-4-hydroxybenzyl)-3-(3,4-dihydroxyphenyl) propanamide were the most promising, with excellent potential for cancer treatment. Moreover, our gene expression databases can be used as a roadmap for future analysis of mechanisms of antioxidant action.

Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.